PHD Thesis 1994

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ACTIONS OF PENTOXIFYLLINE ON SPERMATOZOA KINEMATICS,
THE ACROSOME REACTION AND SPERM-ZONA INTERACTION
A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY
DEPARTMENT OF BIOLOGY AND BIOCHEMISTRY,
BRUNEL UNIVERSITY, UXBRIDGE, UNITED KINGDOM
BY
MOSES PAUL
FERTILITY LABORATORY, DEPARTMENT OF CHEMICAL PATHOLOGY

ROYAL POSTGRADUATE MEDICAL SCHOOL, QUEEN CHARLOTTE'S
AND HAMMERSMITH HOSPITALS, GOLDHAWK ROAD, LONDON
1994
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Thesis title Actions of pentoxifylline on spermatozoa

kinematics : the acrosome reaction and sperm-
zona interaction.
Author Paul, Moses.
Awarding Brunel University

Institution
Year of Award 1994
Qualification PhD thesis Qualification doctoral

name Level
Keywords Assisted reproduction Human physiology

Biochemistry Human physiology Biochemistry
BL Ref. Nos.
ThOS Persistent ID uk.bl.ethos.241598
ILS catalogue number 7125998 Shelfmark DX184829
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ABSTRACT
In many assisted reproduction procedures, the drug Pentoxifylline (PF) is
used to enhance spermatozoa (sperm) motion. However, no thorough study has
examined under what circumstances PF might be useful, nor what concentration is
most appropriate, nor exactly what parameters of motion (and other characteristics of
sperm) are affected by PF. Therefore, the overall objective was to answer these
questions. The thesis then proceeds to study the effect of PF on the acrosome
reaction and sperm-zona pellucida binding.
This study revealed that the optimum conditions to produce the maximum
stimulation with PF were one hour incubation at 37 0C and the optimal concentration
was 6 mM PF/L in semen and 2.8 mM PF/L in suspensions of sperm. Results also
showed a significant enhancement in curvilinear velocity and lateral head
displacement. However, PF did not affect the percentage of motile sperm. It further
demonstrated that each sample of sperm responded to varying degree of
enhancement, with 1 in 10 samples not responding to PF stimulation.
Washing alone produced an increase in motion characteristics in the
control samples. However, suspensions of sperm that had been stimulated with PF
and then had the drug removed by washing showed a significant reduction in the
sperm motion characteristics.
PF alone did not affect the proportion of sperm that had undergone the
acrosome reaction; however, in the presence of Ionophore A23187, it significantly
reduced the proportion of sperm that had undergone the acrosome reaction. Sperm in
the presence of PF had a significantly increased tendency to bind to the zona
pellucida; however, sperm pretreated with PF, which was then subsequently removed
by washing, showed a decreased tendency to bind to the zona pellucida.
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ACKNOWLEDGEMENTS
I wish to express my sincere thanks to Dr Kevin Lindsay for his guidance,
advice, wisdom and encouragement throughout this project which made this research
a memorable experience. I am grateful to Kevin for allowing me to use the research
facilities at the Fertility Laboratory. Special thanks goes to the members of the
laboratory, Mrs Ivy Floyd and Mr Robert Swan, for technical support in routine
seminology and for putting-up with my constant harassment in asking for semen
samples.
A special word of thanks goes to Professor John Sumpter for his
intellectual guidance, constructive criticism, unfailing enthusiasm and inspiration
which enabled me to carry out this research.
I take this opportunity to thank the staff of Electron Microscopy Unit, Dr
Tim Ryder and Miss Margaret Mobberley, for their assistance in cutting of blocks,
staining, mounting of specimens and for teaching me how to use the Electron
Microscope.
I am greatly indebted to Mr Vic Robinson, Consultant Obstetrician and
Gynaecologist of Hillingdon Hospital, who initially allowed me to embark on this
project and without his support and encouragement this course of study may not have
taken off the ground. I am sincerely grateful for this.
Finally, I wish to express my gratitude and thanks to my wife Hiroko for
her patience, understanding and encouragement, and advice on statistical
manipulation of data. Also to my 2½ year old son Shimon for his tolerance as he was
ignored while I worked on this project.
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TABLE OF CONTENTS
Title page 1
British Library, UK filing 2
Doctorate Award 3
Abstract 4
Acknowledgements 5
Table of contents 6
List of Tables 14
List of Figures 16
Dedication 18
Abbreviations 19
Chapter 1: GENERAL INTRODUCTION 20
1.1 Spermatogenesis 20
1.2 Sperm structure 22
1.3 Oogenesis 27
1.4 Gamete Transport 29

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1.5 Fertilization 31
1.6 Infertility 34
1.7 Methods to study sperm motility 38
1.8 Inducers of sperm motility 43
1.9 Pentoxifylline 45
1.10 Aim of study 47
Chapter 2: GENERAL MATERIALS AND METHODS 48
2.1 Equipment 48
2.2 Chemicals 49
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2.3 Sample selection and Sperm assessment 51
2.4 Sperm suspension preparation 52
2.5 CASA methodology 54
2.6 Stability of Pentoxifylline to external factors 61
2.7 Statistical analysis of experimental data 65
Chapter 3 INVESTIGATIONS INTO FACTORS
AFFECTING SPERM MOTILITY 67
3.1 Introduction 67
3.2 Effect of incubation temperature on sperm
motility when Pentoxifylline is present 69

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3.3 Effect of incubation time on sperm motility
when Pentoxifylline is present 72
3.4 Effect of Heparin on sperm motility when
Pentoxifylline is present 77
3.5 Effect of Percoll on sperm motility 80
3.6 Effect of centrifugation on sperm motility 85
3.7 Comparison between discontinuous Percoll
gradient and 'swim-up' methods of sperm
suspension preparation 87
3.8 Conclusions 89
Chapter 4 ACTION OF PENTOXIFYLLINE ON SEMEN 90

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4.1 Introduction 90
4.2 Materials and Methods 91
4.3 Statistical Analysis and calculations 93
4.4 Results 95
4.5 Discussion 102
Chapter 5 ACTION OF PENTOXIFYLLINE ON SUSPENSIONS OF
SPERMATOZOA AND EFFECTS AFTER ITS
REMOVAL BY WASHING 106
5.1 Introduction 106
5.3 Statistical analysis and calculations 111

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5.4 Results 113
5.5 Discussion 124
Chapter 6 THE ACROSOME REACTION RESPONSE
TO PENTOXIFYLLINE CHALLENGE 130
6.3 Statistical Analysis 139
6.4 Results 140
6.5 Discussion 152
Chapter 7 THE EFFECT OF PENTOXIFYLLINE ON THE BINDING
OF SPERMATOZOA TO THE ZONA PELLUCIDA 159

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7.3 Statistical analysis 172
7.4 Results 173
7.5 Discussion 179
Chapter 8 GENERAL DISCUSSION 185
Appendix A EHBS chemical composition 195
Appendix B Action of PF on semen - The number of sperm
analyzed per PF conc. group 196
Appendix C Action of PF on suspensions of sperm -
The number of sperm analyzed per PF conc. group 196
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REFERENCES 197
Papers published based on this thesis 240
1. Actions of pentoxifylline directly on semen 241
2. The paradoxical effects of pentoxifylline on the binding of
Spermatozoa to the human zona pellucida 247
3. Factors affecting pentoxifylline stimulation of sperm kinematics
in suspensions. 253
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LIST OF TABLES
Table 2.1 Parameter setting of Celltrack/s CASA 57
Table 2.2 CASA reproducibility 59
Table 2.3 Comparison between live and taped CASA
measurements 60
Table 2.4 Stability of stock PF to freezing for 6 weeks 62
Table 2.5 Stability of stock PF to freezing for 10 weeks 63
Table 2.6 Stability of stock PF to thawing at 560 C 64
Table 3.1 The effects of incubation temperature on sperm
motion after treatment with Pentoxifylline 71
Table 3.2 Effect of PF on VCL and ALH values of sperm
incubated at two different temperatures 72
Table 3.3 The effects of incubation times on sperm motion after
treatment of sperm samples with Pentoxifylline 75
Table 3.4 The effect of heparin on sperm motion after
treatment of sperm samples with Pentoxifylline 79
Table 3.5 The effect of Percoll on sperm motion characteristics 81
Table 3.6 The effect of centrifugation on sperm motion characteristics 86
Table 3.7 Comparison between Percoll gradient and
'swim-up' separation methods 88
Table 4.1 Design of experiments and number of samples
per group 91
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Table 4.2 Kinematic responses of sperm to various concentrations
of Pentoxifylline 96
Table 4.3 Maximum recovery of motile sperm when treated with
various concentrations of PF 98
Table 5.1 Kinematic responses of sperm in suspension
to various concentrations of Pentoxifylline 114
Table 5.2 Residual effects of PF after its removal by washing 120
Table 6.1 The effect of PF on the acrosome reaction 141
Table 6.2 The effect of PF+IP on the acrosome reaction 142
Table 6.3 The acrosome reaction of controls evaluated by TEM 144
Table 6.4 Motion characteristics of sperm used in the AR experiment 149
Table 6.5 Comparison of methodology condition used in evaluating AR155
Table 7.1 Effect of FITC labelling of sperm on their ability
to bind to intact-zona 173
Table 7.2 Evaluation of cutting the intact-zona into equal halves 174
Table 7.3 Percentage of sperm bound to intact-zona using
control sperm labelled with FITC 175
PF-pretreated sperm labelled with FITC 176
Table 7.5 Effect of PF on sperm-hemizona binding 177
Table 7.6 Comparison of percentage of sperm binding to
intact-zona and hemizona 178
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LIST OF FIGURES
Figure 1.1 Diagrammatic representation of a human sperm 23
Figure 3.1a The effect of incubation time on VCL of PF-treated sperm 75
Figure 3.1b The effect of incubation time on ALH of PF-treated sperm 76
Figure 3.1c The effect of incubation time on LIN of PF-treated sperm 76
Figure 3.2a The effect of Percoll and its removal by washing on VCL 82
Figure 3.2b The effect of Percoll and its removal by washing on ALH 83
Figure 3.2c The effect of Percoll and its removal by washing on MOT% 83
Figure 3.2d The effect of Percoll and its removal by washing on LIN 84
Figure 4.1a Effect of different concentrations of PF on VCL 97
Figure 4.1b Effect of different concentrations of PF on VSL 97
Figure 4.1c Effect of different concentrations of PF on ALH 98
Figure 4.1d Effect of different concentrations of PF on manual count 99
Figure 4.2 Effect of different concentrations of PF on sperm - SI 1 100
Figure 4.3a Rise in VCL in individual patient's sperm
in response to 6 mM PF/L 101
Figure 4.3b Rise in ALH in individual patient's sperm
Figure 5.1 Flow diagram of experimental protocol 111
Figure 5.2a Effect of different concentrations of PF on VCL
on sperm in suspension 115
Figure 5.2b Effect of different concentrations of PF on ALH
on sperm in suspension 115
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Figure 5.3 Effect of different concentrations of PF on sperm
in suspension - SI 2 116
Figure 5.4a Rise in VCL in individual patient's sperm suspension
Figure 5.4b Rise in ALH in individual patient's sperm suspension
Figure 5.5a Persistent effects of PF on VCL - Percoll gradient method 121
Figure 5.5b Persistent effects of PF on ALH - Percoll gradient method 121
Figure 5.6a Persistent effects of PF on VCL - 'swim-up' method 123
Figure 5.6b Persistent effects of PF on ALH - 'swim-up' method 123
Figure 6.1 Flow diagram of experimental protocol 139
Figure 6.2 CTC stain - Effect of PF, PF+IP & IP on the AR 143
Figure 6.3 Electron micrograph of sperm head x 12 000 magnification
showing various stages of the human sperm AR 146
Figure 6.4 Effect of PF, PF+IP & IP on MOT% 150
Figure 6.5 Effect of PF, PF+IP & IP on VCL 150
Figure 6.6 Effect of PF, PF+IP & IP on ALH 151
Figure 7.1 Flow diagram of sperm-zona binding 168
Figure 8.1 Flow diagram of Conclusions 194
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DEDICATION
To my wife Hiroko and our son Shimon
for their love, understanding, and
patience
If a man will begin with certainties, he shall end in doubts;
but if he will be content to begin with doubts,
he shall end in certainties.
Francis Bacon 1561-1626
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ABBREVIATIONS
ALH Lateral head displacement

AR Acrosome reaction
CASA Computer assisted sperm analysis
cAMP Cyclic adenosine monophosphate
CTC Chlortetracycline
EHBS Earles-Hepes balanced salt
FITC-PNA Fluorescein isothiocyanate conjugated Arachis hypogea agglutinin
FITC-PSA Fluorescein isothiocyanate conjugated Pisum sativum agglutinin
FSH Follicle stimulating hormone
HSA Human serum albumin
IP Ionophore A23187
LIN Linearity
LH Luteinizing hormone
MOT Motility
NS Not significant
PBS Phosphate buffered saline
PF Pentoxifylline
ROS Reactive oxygen species
RT Room temperature
SEM Standard error of mean
SI Stimulation index
Sperm Spermatozoon/Spermatozoa
TEM Transmission electron microscopy
VCL Curvilinear velocity
VSL Straight line velocity
ZP Zona Pellucida
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CHAPTER 1
GENERAL INTRODUCTION
The roots of education are bitter, but the fruit is sweet.
Aristotle 384-322 BC
Human spermatozoa (sperm) are highly specialised cells with a haploid
number of chromosomes that must display several disparate properties such as cell
recognition, movement, secretion and membrane fusion, if they are to successfully
participate in the reproductive process. Consequently, the fertilizing potential of a
sperm will not depend on any single aspect of its biochemistry and physiology, but
rather on the consolidation of several independent components, each of which
contributes to the general functional competence of the cell.
1.1 Spermatogenesis
The process of gametogenesis in the male occurs within the seminiferous
tubules of the testes, resulting in the production of sperm. It consists of two phases,
spermatogenesis and spermiogenesis (Dadoune and Demoulin, 1993).
Spermatogenic activity requires an adequate concentration of testosterone, an
androgen that is produced by the Leydig cells when they are stimulated by luteinizing
hormone (LH) (Roberts et al., 1991). Sertoli cells regulated by follicle stimulating
hormone (FSH) play a crucial role in regulation of spermatogenesis (Matsumoto and
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Bremner, 1987). By mitotic (proliferative) and meiotic (reductive) divisions, millions of
spermatids are produced, each with an haploid number of chromosomes. Meiosis
ensures the biological necessity of evolution through the introduction of controlled
variability by the process of cross-over.
Just before the first meiotic division, primary spermatocytes replicate their
DNA and contain twice the normal amount (4N). In the diplotene stage of meiosis,
the sister chromatids of homologous chromosomes that are linked by chiasmata,
exchange their chromosomal material by cross-over. After the first meiotic division,
each secondary spermatocyte contains an haploid number of chromosomes, but the
total amount of DNA in each daughter spermatocyte is equal to that of a normal
somatic cell (2N), since each chromosome is in a double structure. During the
second meiotic division, each double-structured chromosome divides, so that each
daughter cell, a spermatid (1N), contains 23 chromosomes. The possible number of
recombinations of the 23 chromosomes in man is enormous (Egozcue et al., 1983;
De Braekeleer and Dao, 1991).
1.1.1 Spermiogenesis
The spermatids complete their development into sperm by undergoing
structural changes that involve extensive nuclear and cytoplasmic reorganisation.
The nucleus condenses and becomes the sperm head; the two centrioles give rise to
the flagellum or axial filament; part of the Golgi apparatus becomes the acrosome;
and the mitochondria concentrates into a sheath located between two centrioles
(Holstein, 1976).
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1.2 SPERM STRUCTURE
1.2.1 Sperm
A typical human sperm (as shown in Fig. 1.1) is a complex and highly
specialized cell composed of a head and tail or flagellum (Mann, 1964; reviewed by
Fawcett, 1975; Yanagimachi, 1981; reviewed by Zamboni, 1992). The head is a
flattened oval measuring four to six microns in length, two to four microns in width,
and 0.5 to 1.5 microns in thickness. The total length of the sperm is about 60
microns.
1.2.2 Sperm head
The head of a human sperm is occupied mostly by the nucleus and the
acrosome, with a small amount of cytoplasm and some cytoskeletal components.
The nucleus consists of dense chromatin matrix carrying the haploid genome. The
major nuclear protein associated with sperm DNA is protamine, which is relatively
small highly basic protein rich in arginine and cysteine (Grimes, 1986).
The acrosome is a membranous structure that sits as a cap over the nucleus,
occupying three-fourths of the anterior part of the sperm head. It is an organelle that
originates from the Golgi complex in spermatids, and contains hydrolytic enzymes
that may facilitate sperm penetration of the zona pellucida and fusion with plasma
membrane of the oocyte to achieve fertilization (Yanagimachi, 1988).
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1.2.3 Capacitation
Capacitation was first described, independently, by Austin and Chang in
1951. They observed that sperm must reside within the Fallopian tubes (oviduct) for
some time before ovulation to acquire fertilizing ability. Capacitation can be defined
as a process that provides a sperm with the ability to undergo physiological
modification that requires a relatively high concentration of extracellular Ca2+ (Stocks,
1990), such that sperm-oocyte fusion can occur. In the mouse, it has been
demonstrated that an increase in flagellar activity known as hyperactivation is a
characteristic feature of capacitation (Fraser, 1977). Hyperactivation is characterized
by episodic, wide amplitude or "whiplashing" beating of the flagellum. Therefore,
sperm movement is characterized by periods of nonprogression ("dancing")
interrupted by a brief linear motion ("dashing") with the sperm head exhibiting an
erratic figure of eight motion in human (Burkman, 1984).
1.2.4 Acrosome
The acrosome is a vesicular structure, a lysosome-like organelle (Allison and
Hartree, 1970), lying beneath the plasma membrane of the head. It can be divided
morphologically into anterior and posterior segments, called the acrosomal cap and
the equatorial segment, respectively. The acrosome contains a variety of
glycoproteins, glycolipids and a large array of hydrolytic enzymes. The following
enzymes are reported to be present: - hyaluronidase, acrosin, proacrosin, esterase,
neuraminidase, collagenase, phosphatase, phospholipase A, β-N-
acetylglucosaminidase, arylsulfatase and arylamindase (McRorie and Williams,
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1974; Stambaugh and Smith, 1976; Meizel, 1984). These enzymes may be
associated with the membranes or contained within the acrosome. The equatorial
segment forms a band that approximately overlies the equator of the head of
spatulate sperm.
1.2.5 Acrosome reaction
The acrosome reaction (acrosomal exocytosis) is initiated by multiple fusion
between the plasma membrane and the outer acrosomal membrane, resulting in
vesiculation that is largely confined to the acrosomal cap region (Piko, 1969; Barros
et al., 1967; Bedford et al., 1978). The acrosome reaction causes the acrosomal
contents to be externalized and the inner acrosomal membranes to become the
limiting membrane of the anterior sperm head. Two main functions are served by the
acrosome reaction:- it gives the sperm the capability to, firstly, penetrate through the
zona pellucida of the oocyte and, secondly, to fuse with the oocyte plasma
membrane.
1.2.6 Sperm Flagellum
The flagellum of human sperm (Eddy, 1988; Satir, 1979; Linck, 1979) consist
of four segments: the connecting piece (neck), the middle piece, the principal piece
and the end piece. The human sperm flagellum is about 55 microns in length. A
narrow neck links the sperm head to the flagellum. The sperm flagellum, which is
more than one micron in diameter in the connecting piece segment, tapers
progressively towards its posterior tip. The connecting piece marks the beginning of
the axial filament complex, or axoneme, which forms the core of the flagellum. The
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axoneme consists of nine microtubular doublets, circularly arranged to form a
cylinder around and connected to the central pair of single microtubule by dynein
arms. This "nine plus two" arrangement extends the full length of the flagellum. The
forward progressive motile force necessary for the sperm to reach the oocyte and
achieve fertilization is provided by the flagellum (Gibbons, 1979; Ishijima, 1990). The
flagellar movements result from sliding of the axonemal microtubules alongside one
another. The coordination of the sliding movements between the peripheral doublets
and central singlets lead to creating flagellar waves. Besides an intact plasma
membrane, flagellar motility requires an adequate supply of adenosine triphosphate
(reviewed by Zamboni, 1992).
1.2.7 Sperm motility
Sperm motility is an expression of its viability and structural integrity, and is
necessary for transportation through the female tract to the site of fertilization (Katz
and Drobnis, 1990). The mechano-chemical mechanisms responsible for sperm
motion are complex and incompletely understood. The processes involved are
intrinsic and extrinsic to the sperm (Hoskin, 1979). Intrinsic processes include the
sperm metabolic activity, the structure of its axoneme, membrane integrity, and
transport phenomena. Extrinsic factors include substrate availability, ionic signals
and the physical properties of the sperm microenvironment (Tash and Means, 1983).
It has been reported (David et al., 1981) that sperm show four important aspects in
their movement: 1) flagellar beating occurs in the transverse plane of the head and
always to the same side; 2) flagellar beating and rotation are synchronised; 3)
rotation occurs when the flagellar wave has reached a point about 20-25 microns
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distal to the neck of the sperm; 4) the velocity of wave propagation on the flagellum is
highly correlated with the velocity of cell progression. On average, freshly ejaculated
sperm swim at 75 μM/sec (Blasco et al., 1984)
1.3 OOGENESIS
The ovaries are derived from the germinal ridge during embryogenesis and
descend into the pelvis in fetal life. During the fetal period, primordial cells, or
oogonia, proliferate within the cortex of the fetal ovaries and subsequently become
surrounded by epithelial cells to form primary follicles.
1.3.1 Oocyte maturation
In the ovaries (Szöllösi, 1993), each primary oocyte undergoes two
specialized nuclear divisions that result in the formation of four cells containing half
the number of chromosomes. In the first stage of meiosis, the primary oocyte is
actively synthesizing DNA and protein in preparation for entering prophase. The
DNA content doubles during prophase as each chromosome replicates. Each
chromosome pair is attracted to its homologous mate to form a tetrad; chromosomes
of the same parental origin are connected to one another by their centromeres. The
members of the tetrad come to lay side by side. Before separation, the homologous
pairs of chromosomes exchange genetic material by a process known as crossing-
over, which accounts for most of the qualitative differences between the resulting
gametes. The subsequent meiotic stages distribute the members of the tetrad to the
daughter cells so that each cell receives the haploid number of chromosomes. At
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telophase, one secondary oocyte and a polar body have been formed which are no
longer genetically identical, since the members of the chromosomal pairs, and parts
of chromosomes, may have been exchanged.
Cytoplasmic organisation of the oocyte occurs during the final stages of
oocyte maturation and is regulated by steroids (Jones, 1990; Törnell et al., 1991).
Meiotic and cytoplasmic maturations are stimulated by the luteinizing hormone (LH)
surge. The Golgi apparatus of the oocyte synthesizes lysosomal-like granules that
migrate towards the surface and may be gathered into clusters or scattered
individually in the subcortical ooplasm (Gulyas, 1980). New and distinctive proteins
are synthesized (Wassarman, 1983) and this activity prepares the ooplasm for
fertilization.
Structural changes in the zona pellucida occur at the ultrastructural level
during oocyte maturation (Tesarik et al., 1988). During follicular growth, mitotic
activity of a single layer of follicular cells surrounding the oocyte results in an
increase of 3-5 layers. The outermost layers of follicular cells form the granulosa
cells, which differentiate into the cumulus oophorus. The oocyte synthesizes and
secretes a proteoglycan-like substance between the ooplasm and the innermost
follicular cells, forming the zona pellucida. It is a relatively thick, mesh-like
interconnecting filament that surrounds the oocyte, separating it from the follicle
(Greve and Wassarman, 1985). The mouse zona pellucida is composed of three
sulphated glycoproteins; ZP1, ZP2, ZP3 (Bleil and Wassarman, 1980a, 1980b). ZP3
induces the sperm acrosome reaction and mediates the initial binding of sperm to
the oocyte. ZP2 acts as a secondary sperm receptor. ZP2 and ZP3 exist as dimers in
long filaments that appear to be cross-linked by ZP1. ZP2 along with ZP3, is
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biochemically modified after fertilization to provide the postfertilization block to
polyspermy (Wassarman, 1988a).
In the human ovaries, there are between 100,000 and 400,000 primary
oocytes present at puberty (Byrd and Wolf, 1984). These germ cells are arrested as
primary oocytes at the diplotene stage of the meiotic prophase until just before they
are ovulated. The matured ovum, approximately 100 to 150 μM in diameter, is
released from the ovary at the secondary oocyte stage; the second stage of meiotic
division is triggered in the oviduct by the entry of the sperm.
1.4 GAMETE TRANSPORT
The human sperm, about 60 microns in length, must travel through some 30-
40 centimetre of male and female tract before reaching the point where fertilization
occurs, the oviduct (Harper, 1988). Although over twenty million sperm per millilitre
are produced by a fertile man, only one sperm is required for fertilization. Before the
sperm can acquire this fertilizing capacity, they undergo a series of changes in the
male and female reproductive tracts. These changes are called maturation in the
male tract (Moore, 1983), and capacitation and activation in the female tract. The
entire process of maturation in the male tract is crucially dependent upon adequate
stimulation of the epididymis by LH and the testosterone produced by Leydig cells
(Roberts et al., 1991).
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1.4.1 Sperm transport in the male
The sperm produced by spermatogenesis are initially completely immotile and
are transported passively from the seminiferous tubules to the rete testis which acts
as a reservoir (Bedford, 1975). Ten to twenty ductuli efferentes connect the rete
testis to the single, long, highly convoluted duct called the epididymis (Robaire and
Hermo, 1988). Ciliary activity of the luminal epithelium, contractile activities of the
smooth muscular elements of the efferent duct wall, and the flow of secretions from
the testis all contribute to the movement of sperm at this stage. During sperm
passage through the epididymis, they acquire the ability to swim progressively and
the capacity to fertilise (Bedford et al., 1973). The sperm pass from the epididymis
into the vas deferens as a very densely packed mass, the movement being due to
the muscular activity of the epididymis and vas deferens. During the process of
ejaculation, mature sperm with seminal fluid are transported out through the urethra.
1.4.2 Sperm transport in the female
The sperm deposited in the vagina travel through the cervical mucus before
reaching the oviduct (Fox and Fox, 1971). Thus, sperm motility is of prime
importance. The cervical canal has very thick connective tissue walls which are lined
by many thick crypts. Sperm, after gaining entry into the cervical mucus, are found
lodged in these cervical crypts, from which they are subsequently released to
continue their journey into the uterus (Elstein et al., 1972; Harper, 1988). Sperm
motility in the uterus is controlled by uterine contractions rather than by the sperm
flagellar activity. In goats and cattle, it has been shown that at the uterotubal junction,
certain selective filtering of sperm occurs, presumably to permit the fittest to survive
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and fertilize the ova in the ampullar region of the oviduct, while the dead sperm are
denied entry (Mattner, 1968).
The oviduct, through which the sperm need to travel before reaching the site
of fertilization, can be pictured as a tube (ampulla) of five to eight centimetres long,
internal diameter ranging from one millimetre to one centimetre, with a funnel like
structure (the infundibulum) at the end. The lumen of the ampulla is wider at the
infundibular end than at the ampullar-isthmic junction. Motile sperm reaching the
ampullar-isthmic junction are carried by the currents of the ampullar fluids secreted
by the oviductal secretory epithelium, assisted by the oviductal ciliated epithelium
and by the flagellar activity of the sperm (Suarez et al., 1990). Fertilization is thought
to occur in the oviduct nearer to the infundibulum (Harper, 1988).
1.5 FERTILIZATION
Fertilization of the oocyte by sperm, the means by which sexual reproduction
takes place in all multicellular organisms, is fundamental to propagation. In both
mammals and non-mammals, the pathway that leads to fusion of an oocyte with a
single sperm consists of many steps that occur in a mandatory order. This sequence
of steps in the mouse oocyte includes species-specific cellular recognition,
intracellular and intercellular membrane fusions, and enzyme catalysed modifications
of cellular investments (Wassarman, 1987; Overstreet and Cross, 1988).
Fertilization, whether occurring in vivo or in vitro, proceeds in a fixed pathway:-
sperm capacitation, penetration through oocyte investments, acrosome reaction,
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binding and penetration of the zona pellucida, fusion of the gametes, and resulting in
the development of the pronucleus (Wassarman, 1988a; Kopf, 1990; Crozet, 1993).
1.5.1 Sperm penetration through oocyte investments
The capacitated sperm can move through the cumulus and corona radiata
cells through the action of hyaluronidase, which hydrolyses and depolymerizes the
intercellular hyaluronic acid matrix (Yanagimachi, 1988). Only capacitated sperm
can penetrate through cumulus cells while sperm that have lost the acrosome cap
through acrosome reaction appear to stick to the cumulus surface (Cummins et al.,
1986).
1.5.2 Sperm receptors
After passing through the cumulus mass, the capacitated sperm attach to the
zona pellucida via receptors on the surface of the oocyte and proteins present on
sperm outer membranes (Wassarman, 1988b). The primary site for sperm-zona
interaction is the zona pellucida (Tesarik, 1989; Shabanowitz and O' Rand, 1988a;
1988b) which is a highly differentiated acellular structure rich in carbohydrate
residues that may play the key role in sperm-zona binding (Ahuja, 1985; Henderson
et al., 1988; Fraser and Ahuja, 1988; Mori et al., 1993). The receptor on the mouse
oocyte zona pellucida has been identified as ZP3, a glycoprotein of molecular weight
83 kDa (Bleil and Wassarman, 1980a; 1983; Wassarman, 1988c; 1990). They further
showed that ZP3 acts as the acrosome reaction inducer, causing the sperm to
undergo the acrosome reaction, and it also mediates the initial binding of sperm to
the zona via O-linked oligosaccharide side chains. ZP2 acts as a secondary sperm
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receptor. Similarly, the human zona pellucida is shown to contain the ZP3 receptor
(Shabanowitz and O' Rand, 1988a; 1988b). The human genes that encode ZP3
receptor have been cloned and sequenced using mouse ZP3 cDNA as a probe
(Chamberlin and Dean, 1990). Chamberlin and Dean (1990) demonstrated that there
is a high degree of conservation between the coding regions of the human ZP3 and
mouse ZP3; both have unusually short 5' and 3' untranslated regions and both
contain a single open reading frame that is 74% identical. Further, recent sequencing
studies on the human ZP2 genes has shown that the sequences of its coding
regions are 70% identical with those of the mouse ZP2 (Liang and Dean, 1993).
1.5.3 Sperm-oocyte fusion
The acrosome-reacted sperm 'drills' through the zona pellucida to enter the
perivitelline space. The equatorial segment of the sperm head attaches to the
plasma membrane of the oocyte and this process activates the oocyte (Gaddum-
Rosse, 1985). The 'activated' oocyte completes its second meiotic division; 23
double-stranded chromosomes split at their centromeres, and chromatids separate
to oocyte or second polar body. This process results in a haploid number of
chromosomes and a haploid amount of DNA in the oocyte. Fusion occurs between
the sperm plasma membrane and the oocyte plasma membrane, gradually
incorporating the sperm into the ooplasm. When the sperm nucleus is incorporated
into the oocyte cytoplasm, it undergoes a series of transformations such as nuclear
envelope disintegration, reduction of the disulphide bonds of DNA-associated
protamines, chromatin decondensation and replacement of the sperm specific
protamines by histones leading to the formation of the male and female pronuclei
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from the sperm and oocyte chromatin, respectively. The final event of fertilization
process involves the reorganization and pairing of maternal and paternal
chromosomes and formation of the zygote (Yanagimachi, 1988). The gamete fusion
also triggers the oocyte to initiate the cortical reaction and the 'block to polyspermy'.
1.5.4 Cortical reaction and the block to polyspermy
The human oocyte relies primarily on the 'zona reaction' to control polyspermy
(Plachot and Mandelbaum, 1990). The cortical granules, membrane bound
lysosome-like organelles 200 to 600 nm in diameter, are released into the
perivitelline space between the plasma membrane and zona pellucida (Gulyas,
1980). The granules contain various hydrolytic enzymes such as proteinases and
peroxidase. In the golden hamster, cortical granules induce the loss of sperm
receptor activity in the zona pellucida so that binding and penetration of
supplemental sperm is blocked (Barros and Yanagimachi, 1971; Wolf and Hamada,
1977).
1.6 INFERTILITY
1.6.1 Male-related infertility
Infertility is often defined as the inability to produce a pregnancy within one
year of regular sexual intercourse without any contraceptive measures being
adopted (Menning, 1980). It affects approximately 15% of couples and it is estimated
that the man is subfertile in 40 to 50% of these infertile couples (reviewed by
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Oehninger et al., 1992). The World Health Organisation (WHO, 1992) has
recommended that the normal parameters of semen are as follows:-
Volume 2.0 mL
pH 7.2-7.8
Sperm density 20x106 sperm/mL
Total sperm count 40x106 sperm/mL
Motility 50% with forward progression
Morphology 30% with normal morphology
Viability 75% live
White cells <1x106 /mL
MAR test <10% sperm with adherent particles
The male-related subfertility group contains those men whose semen values
is sub-optimal according to these WHO criteria. Acosta et al. (1988) have suggested
that male infertility may be indicated when the basic semen analysis reveals the
following values:-
Sperm density <20x106 /mL
Motility <40% motile
Morphology <14% normal forms
Total recovery of motile sperm
after separation techniques <10x106/mL
Male-related infertility (Irianni and Coddington, 1992; Fisch and Lipshultz,
1992) can be categorized into five groups:-
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1) Testicular causes - factors affecting spermatogenesis
2) Post-testicular causes - obstructive problems in ducts and sexual dysfunction
3) Pre-testicular causes - hypothalamic or pituitary disorders
4) Genitourinary infections
5) Immunologic causes
1.6.1.1 Medical treatment of male infertility
To treat subfertility, men have been orally treated with clomiphene citrate and
tamoxifen (Vermeulen and Comhaire, 1978), which increased pituitary
gonadotrophin secretion, leading to an increase in sperm density (in some cases),
but there was no concomitant improvement in sperm motility. Subfertile men with
persistently low sperm motility have been treated with human chorionic gonadotropin
(hCG), which did produce some improvement in sperm motility (Misurale et al.,
1969). However, the failure of gonadotropin therapy is due to the induction of
antibodies against hCG (Sokol et al., 1980). A limited number of subfertile men have
been treated with testosterone, but the main obstacle to this therapy is that the liver
metabolises the testosterone before it reaches the target cells (Johnsen et al., 1974).
Treatment of subfertile men orally with Pentoxifylline is controversial (see section
1.9.2). Thus, to date, no oral therapy has been successful in stimulating
spermatogenesis in situ, in order to increase the quality and quantity of sperm
produced by subfertile men. Several new pharmacological compounds are currently
under clinical evaluation for their ability to improve the fertility of certain group of
patients. Most of these investigations are Phase-One studies.
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A different approach is to treat the sperm produced by such men, rather than
treat the men themselves. The aim here is to enhance the quality of sperm
possessing sub-optimal characteristics in vitro with chemical inducers (Hammitt et
al., 1989; Oehninger and Alexander, 1991) such as caffeine (Moussa, 1983; Rees et
al., 1990), Pentoxifylline (Aparicio, 1980b, Tesarik et al., 1992a), or 2-
deoxyadenosine (Aitken et al., 1986) before insemination. Although the mode of
action of chemical inducers on sperm motion is not clearly understood, nevertheless
their potential contribution as a stimulant is significant.
1.6.2 Female related infertility
Infertility (reviewed by: Corsan and Kemmann, 1991; Breckwoldt et al., 1993)
in females can be caused by:-
1) Hypothalamic-pituitary failure - reduced or absent pituitary gonadotropin release
(follicle stimulating hormone & luteinizing hormone).
2) Hypothalamic-pituitary dysfunction - menstrual cycle disturbances, including
luteal phase insufficiency, anovulatory cycles or amenorrhoea.
3) Ovarian failure - with no evidence of ovarian estrogen production and with
elevated follicle stimulating hormone levels.
4) Congenital or acquired genital tract disorder - anatomical disorders of the genital
tract.
5) Endometriosis
6) Obstruction of the tubules
7) Infection of the uterus
8) Immunological reaction
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1.6.3 Unexplained infertility
In the 15% of all couples who seek assistance in conceiving, there is a
proportion in whom no definite cause of infertility can be ascertained; semen quality
fulfils the criteria for normality, and no defect in the woman's reproductive system can
be shown. Two possible reasons have been suggested (Templeton et al., 1990): 1)
defective transport of gametes to the normal site of fertilization and, 2) failure of
fertilization due to defective gamete function.
1.7 METHODS TO STUDY SPERM MOTILITY
Although sperm were first observed more than 300 years ago, the concept of
semen analysis is relatively new. In 1929, Macomber and Sanders were the first to
do sperm counts in man and to look at the differences between fertile and infertile
groups. Their fertile group, which contained men who had fathered children, had
sperm count over 60 million per millilitre. Macleod (1950, 1951) was the first to look
at semen quality by comparing volume, sperm density, proportion of motile sperm,
quality of motility and proportion of sperm with normal morphology. These studies set
the foundation for modern seminology.
1.7.1 Manual assessment of semen quality
Largely, seminology involved looking at sperm with an optical microscope and
deducing the various parameters. The results of such analyzes were subjective and
operator dependant. Various studies (Bartoov, 1980; Badenoch et al., 1990; Davis
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and Boyers, 1992) have reported significant variability in the subjective estimates of
sperm progression and percent motility, within a single observer and between two
observers. Therefore, it is difficult to obtain a precise and accurate results with the
use of traditional semen analysis methods. The problems of subjective analysis were
recognised by the World Health Organisation (WHO, 1987), and led to the
introduction of standards that were published in its laboratory manuals. It
recommended a simplified motility grading system, based on a standard method of
sperm counting, and standardization of other measured parameters. However, these
measures have not resolved the problem of subjectivity, which has consequently
lead people to seek methods that can measure sperm parameters objectively.
1.7.2 Development of techniques to assess semen quality
Spectrophotometry (Sokoloski et al., 1977), an indirect method of measuring
sperm speeds in suspension, and laser-Doppler velocimetry (Jouannet et al., 1977),
which measure changes in light reflectance by moving sperm, were developed, but
unfortunately these methods do not provide information on individual sperm and
therefore did not gain popularity.
Direct methods, such as photographic ones involving visual assessment of
swimming speeds of individual sperm in real time using microscopic grids and
stopwatch, were introduced (Harvey, 1960). These included time-exposure
photomicrography, multiple-exposure photography and cinemicrography, and
included both manual and computer-assisted analysis of the results.
Time-exposure photography (Overstreet, 1979) involves photographing for
one second the moving sperm using dark-field illumination. Motile sperm produce a
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dark track on a film negative, while immotile sperm appear as overexposed sperm
heads, and stationary sperm are blurred or appear with multiple tail images. Using
this technique, specific movement characteristics can be studied and the velocity
calculated by measuring the straight line track of the motile sperm. However, this
method is labour intensive and unsuitable for routine work.
Multiple-exposure photography was introduced by Troll and Goldzieher
(1962), and involves photographing sperm samples twice, two seconds apart. The
negative of one picture was superimposed on the positive of the second, allowing an
estimate of sperm velocity to be determined during those two seconds. The problem
was accurate superimposition of the positive and negative, which proved to be
tedious. Although this method was modified by Makler (1980a), it never gained
popularity.
Cinemicrography is a 'movie' of sperm motion (Zorgniotti et al., 1958) taken
under constant illumination with multiple film frames exposed in rapid succession.
This method was further modified to record the images on video cassette tapes
(Morales, 1988). Motile sperm occupy different positions on each frame. Frame by
frame analysis allowed an accurate identification and estimation of motile and
nonmotile sperm velocity and trajectory. Although frame by frame analysis done
manually were labour intensive and laborious, it could be computerised.
Computerisation technique (Pedigo et al., 1989) promised to revolutionize the study
of sperm behaviour, because it could dramatically improve our ability to easily and
objectively quantify motion.
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1.7.3 Computer-aided sperm analysis (CASA)
As the name implies, this technique requires the appropriate equipment to
visualise and digitize the static and dynamic images of sperm in a sample, and a
video recorder to record this on magnetic tapes.
Briefly, the setup consists of :-
1) a temperature controlled specimen stage and imaging area
2) an optical magnification system
3) a video camera and video cassette recorder
4) a monitor to view analog and digital images
5) a computer system with software for image digitization and mathematical
analysis of sperm tracks
6) a keyboard and printer for data input and output
The image in CASA video camera (CCD, charge-coupled device) is
generated when individual picture elements, or pixel, is activated as light strikes the
CCD array (reviewed by: Boyers et al., 1989, Davis and Katz, 1989; Davis and
Boyers, 1992). Each activated pixel produces a voltage proportional to the intensity
of the light striking it, and this is encoded into a complex analog signal. These
different voltages can be used to activate a pixel in a monitor, thus creating an image
on the screen for visual inspection, or they can be encoded as numbers (digitization),
to be used by the CASA.
In sperm motion analysis (Davis et al., 1992), the first step is to identify
digitized sperm heads from non-sperm images; this can be achieved by setting the
size range acceptable as a sperm head. Motion is determined by calculating the

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centroid of each digitized head in all video frames and calculating the trajectories of
all sperm heads across all video frames. The centroid for sperm heads in each video
frame is calculated as a simple average of the x, y coordinates of the pixel making up
the object's image. If the centroid does not move from the set value between frames,
it is considered nonmotile. Employing mathematical calculations, CASA can provide
the following information:- motility%, count, number of cells meeting the tracking
requirements, straight line velocity, curvilinear velocity, lateral head displacement,
and average path velocity. Recently, due to improvements in the software, additional
information like beat-cross frequency, circularity, mean angular displacement,
maximum amplitude head oscillation and basic head oscillation, can also be provided
by a CASA.
The problems in CASA (Boyers et al., 1989; Bendvold and Aanesen, 1990;
Olds-Clarke et al., 1990) are image jitters, apparent motion, Doppler shift and bump
& cross. Image jitters are produced from fluctuations in the size, shape and
luminosity of a sperm image, and are more serious in slow-moving sperm than in
fast-moving sperm. Apparent motion is an artifact of the video interlacing process
that can be reduced by adjusting the magnification of the sperm heads compared
with their real size. Doppler shift is caused by the video camera scanning technique,
and can be reduced by insuring that the sperm move randomly with respect to the
orientation of the video array. Bump & cross is the result of two or more sperm
images occupying the same space simultaneously; it can be reduced by decreasing
the concentration of the sperm present in a sperm suspension being analyzed.
In a study conducted by Mathur (1986), he was able to show that a
computerised analysis of sperm swimming motion is a reliable and rapid technique

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for evaluating semen samples, and that it offered more discrimination than routine
semen analysis done manually. Manual methods provide little information on sperm
motion characteristics. Therefore, the goals of computerised sperm analysis are: 1) to
identify the motion characteristics of sperm that will reliably discriminate between non-
fertile and fertile males; 2) to use those motion characteristics to detect the earliest
signs of altered reproductive potential; and 3) to relate those parameters to the
physiology of sperm in vivo. Sperm motility is believed to be the most important
characteristic for evaluating the fertility potential of ejaculated sperm. CASA
generated information has shown that fertilization rate in vitro correlates with sperm
motility (Chan et al., 1989; Fetterolf and Rogers, 1990; Check et al., 1990; Liu et al.,
1991) and that the technique can be used to assess sperm hyperactivation (Mack et
al., 1989; Burkman, 1991).
1.8 INDUCERS OF SPERM MOTILITY
There are many published reports suggesting that specific chemical agents
can stimulate sperm motility or increase the fertilizing ability. These agents include
caffeine (Garbers et al., 1971a; Traub et al., 1982), 2-deoxyadenosine (Aitken et al.,
1986), Pentoxifylline (Aparicio, 1980b), theophylline (Garbers et al., 1971b; Loughlin
and Agarwal, 1992), platelet-activating factor (Ricker et al., 1989), relaxin (Essig et
al., 1982; Colon et al., 1986), progesterone (Mbizvo et al., 1990), prostaglandins E
(Colon et al., 1986), 3-Isobutyl-1-methylxanthine, IBMX, (Jiang et al., 1984), cyclic
AMP (De Turner et al., 1978) and kallikrein (Schill, 1982a; Sato and Schill, 1987). In
addition, combinations of inducing agents have been used to potentiate sperm

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motility, including calcium & creatine phosphate (Fakih et al., 1986), IBMX plus 2-
deoxyadenosine, and caffeine plus 2-deoxyadenosine (Aitken et al., 1986).
Furthermore, chemicals like lanthanum and α-chlorohydrin have been shown to
inhibit sperm motility (Gwatkin, 1985).
The effect of Pentoxifylline, a phosphodiesterase inhibitor, has recently been
tested in patients with male-factor infertility (Yovich et al., 1990). The results of this
work suggested there was an improvement in in vitro fertilization when suspensions
of sperm were treated with 3.6 mM PF/L. The choice of 3.6 mM PF/L is empirical. PF
is also known to depress the production of superoxide anions by the human sperm
(Gavella et al., 1991). The superoxide anions are detrimental to sperm. Furthermore,
PF has been shown to improve sperm motion characteristics in both
normozoospermic and asthenozoospermic semen samples (Tesarik et al., 1992a).
Hammitt et al. (1989) have shown that caffeine, Pentoxifylline and 2-
deoxyadenosine significantly increased sperm motility in cryopreserved human
semen. Their study also looked at the effect of cAMP, relaxin, adenosine, kallikrein
and calcium in sperm motility, but none of these chemicals was found to be a
significant motility stimulant.
Caffeine (Rees et al., 1990) has been shown to increase lateral head
displacement of sperm when a sperm suspension was incubated with 6 mM
caffeine/L. The rate of glycolysis of these sperm increased by over 40%. Between 3
and 6 mM caffeine/L, when added directly to semen, showed a good stimulatory
effect on the percentage motility, an effect that was statistically significant (Moussa,
1983).
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1.9 PENTOXIFYLLINE
1.9.1 Chemical structure
Pentoxifylline, C18 H18 N4 O3, (oxpentifylline), a phosphodiesterase inhibitor of
the methylxanthine group, has a molecular weight of 278.3, a melting point of 1050 C,
and a solubility of 77 mg/mL in water. It was first synthesized in the laboratory by
Mohler et al. (1966). The structure of Pentoxifylline is shown in figure 1.2.
Fig.1.2 Chemical structure of Pentoxifylline
1.9.2 Effects of Pentoxifylline in humans
Pentoxifylline is an orally active haemorheological agent used for the
treatment of peripheral vascular disease, cerebrovascular disease and several other
conditions involving defective regional microcirculation. Pentoxifylline acts by
improving the oxygen supply to ischaemic areas, at least in part by increasing red
cell deformability and reducing blood viscosity, with consequent improvement in
blood flow through the nutritive microcirculation (Muller et al., 1981; Ward et al.,
1987). Extensive open and placebo-controlled clinical trials have shown that
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Pentoxifylline, orally given as 300 to 1200 mg/day for at least 6 weeks, results in
good to excellent therapeutic response in 60 to 100% of patients with peripheral
vascular disorders. In vitro studies using human and bovine platelets have shown
that Pentoxifylline raises cAMP levels and inhibits membrane-bound
phosphodiesterase. These changes activate protein kinase that catalyses the
phosphorylation of membrane protein by ATP, resulting in inhibition of platelet
aggregation tendencies (Stefanovich, 1978).
Aparicio et al. (1980a) showed that asthenozoospermic men, when orally
treated with 400 to 1200 mg Pentoxifylline/day for two to 12 months, produced
semen with significant increases in sperm concentration and motility. In a similar
study by Shen et al. (1991), it was demonstrated that when PF was orally given to
asthenozoospermic men for three months, the sperm motility significantly increased
but sperm concentration did not increase. However, patients with oligozoospermia
showed no improvement in sperm motility or in the conception rate (Schill, 1982b).
Therefore, the current treatment of male factor infertility with orally administered PF
is still rather controversial.
Quite a few studies have been published on the use of PF to increase sperm
motion characteristics in vitro, prior to use of the sperm for assisted reproductive
techniques. The results of these studies are discussed in relation to my own results
in the following chapters.
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1.10 AIM OF STUDY
Although PF is already widely used to enhance sperm motility in Fertility
Units, the techniques used to prepare the sperm prior to the treatment with PF are
not standardized. It is obviously possible (even likely) that the techniques used might
affect the magnitude of the response to PF. Therefore, one aim of this project was to
standardize the incubation temperature and the length of incubation of PF in sperm
suspensions, and any other factors that may affect sperm motion.
As evidenced from section 1.6, there are a proportion of patients with infertility
problems for whom PF stimulation of sperm motion characteristics may assist in the
treatment. Therefore this project attempts to define the beneficial (or non-beneficial
as the case may be) effect of Pentoxifylline on semen and in sperm suspensions,
and to determine exactly what dose and conditions are optimum. Investigations were
also carried out to study the effect of drug removal by washing, prior to use of the
sperm for fertilization which is the normal procedure carried out in Fertility Units. The
possible effect of Pentoxifylline on the acrosome reaction, a prerequisite to
fertilization, was also studied. Finally, to complete the story, the action of
Pentoxifylline on sperm-zona binding was examined.
[Please note: When I embarked on this project about four years ago, not much was
known about the effects of PF on sperm kinematics, the acrosome reaction and
sperm-zona interactions. Since then a number of studies had been carried out and
published by various research groups on the subject. The results of these studies
are discussed together with my own results in the following chapters.]
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CHAPTER 2
GENERAL MATERIALS AND METHODS
Imagination is more important than knowledge.
Albert Einstein 1879-1955
2.1 EQUIPMENT
2.1.1 Celltrak/s Motion Analyzer
Motion Analysis VP110 (Motion Analysis Corp., Santa Rosa, USA) was used
to analyze all sperm motility characteristics. The analyzer consists of an Olympus
BH-2 microscope fitted with a TI-23A CCD camera (NEC, Japan) and a heating
stage supplied by Motion Analysis Corp. The analyzer was also connected to a
Panasonic NV-W1 video recorder (Panasonic, Japan) to record sperm motility video
images on tapes that could be used as permanent experimental records or stored for
analysis at a future date.
2.1.2 Fluorescence Microscope
The fluorescence microscope used was a Diapan Large Laboratory
Microscope (Reichert, Austria) fitted with fluorescent equipment for incident light
excitation. The incident light was provided by a HBO-50 mercury vapour burner. The
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fluorescent equipment consisted of a 0515 dichroic mirror with a BG12 excitation
filter and a barrier filter (1.5 mm OG1 + 1 mm GG9). The objective used for low
magnification work was a Fluor 16, giving a total magnification of 200-times, while
for higher magnification work the objective used was an Iris 40, giving a total
magnification of 500-times.
2.2 CHEMICALS
All general chemicals were of Analar grade and obtained from BDH (Poole,
Dorset, UK.) or Sigma (Poole, Dorset, UK.) unless otherwise stated. Fine chemicals
and special chemicals of tissue culture grade were obtained from Sigma. Earles
medium was obtained as 10x concentrate from Gibco Ltd (Paisley, Scotland).
Gentamycin sulphate BP was supplied by Gibco. Fresenius Water for injection was
obtained from FL (Manufacturing) Ltd (Basingstoke, UK). Gas mixture was supplied
by British Oxygen Company, UK.
2.2.1 Earles-Hepes balanced salt solution preparation
The medium used throughout this study for preparation of sperm suspensions
and Pentoxifylline (PF) solution was modified Earles-Hepes balanced salt1 (EHBS)
solution. The medium was made from Earles 10x concentrate reconstituted with
water for injection, supplemented with 20 mM N-(2-hydroxyethyl) piperazine-N-
ethanesulphonic acid, 20 mg/L gentamycin, 0.125 mM sodium pyruvate and with the
1
EHBS chemical composition is given in Appendix A.
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osmolality adjusted to 280-285 mOsmol/L, gassed (5% CO2 in air) and pH
maintained at 7.4. It was filtered through 0.2 micron filters (Gelman Sciences, USA)
and stored at 40 C. Before use, a consistent source of human serum albumin (HSA,
Zenalb 20, Bioproducts Laboratory, UK) was added to the culture medium to give a
final concentration equivalent to 10% serum. This medium was more robust to
potential pH changes from atmospheric effects, while providing sufficient nutrients to
support the viability of sperm in the culture medium.
2.2.2 Preparation of discontinuous Percoll gradient
Percoll (Pharmacia, UK) is a stable, non-toxic colloidal suspension of silica
particles of diameter 15-30 nm coated with polyvinylpyrrolididone (PVP). The density
of 1.13 g/mL and low osmolality of <25 mOs/kg H2O are exploited to select and
separate viable cells from non-viable cells. It is also easy to make isotonic solution.
To harvest motile sperm, generally, a 2-layer gradient of 80 and 40% Percoll was
used.
The stock solution of 80% Percoll was prepared by adding 80 mL Percoll to
10 mL Earles 10x concentrate plus 10 mL 0.25 mM sodium bicarbonate. The
working solution of 80% Percoll was supplemented with 10% HSA. The 40% Percoll
solution was prepared by diluting the 80% stock solution with an equal volume of
EHBS (without HSA) and supplementing it with 10% HSA.
The 40/80% discontinuous Percoll gradient was prepared by placing 2 mL
80% working Percoll in a tube and layering on the top with 2 mL 40% working
Percoll, carefully and slowly so as not to disturb the interface.
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2.2.3 Preparation of Pentoxifylline
Fresh stock of PF was prepared by dissolving 0.1392 mg in 10 mL of EHBS
medium (without HSA) giving a concentration of 50 mM/L. The stock was aliquoted
into 2 mL portions and frozen at -200 C until required. 10 to 50 mM PF/L was made
by diluting the stock with the appropriate volume of EHBS medium. The prepared
working solution was kept at room temperature and discarded at the end of the day.
Fresh working solution was prepared every day, either from frozen stock or from
fresh stock solution.
2.3 SAMPLE SELECTION AND SPERM ASSESSMENT
Semen samples were obtained from patients attending the Fertility Clinic at
Queen Charlotte's and Hammersmith Hospitals. All samples used had normal count
(>20x106 per millilitre) and motility (50% forward progressive, >25% rapid
progressive) in accordance with WHO guidelines (1992). Subjects were requested to
abstain from sexual activity for at least three days before producing the sample.
Samples were obtained by masturbation and were allowed to liquefy at room
temperature before routine semen analysis was performed in accordance with WHO
guidelines (1992).
The volume and pH (with pH paper) of the sample were measured.
The viscosity of the semen was assessed by drawing up 100 μL in a pipette and
expelling 10 μL onto a microscopic glass slide. The viscosity was classified as
normal, semi-mucoid or mucoid. Mucoid samples were not used in any experiments.
The expelled drop of semen was covered with a coverslip, and the general
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appearance and morphology were assessed using a light microscope. Semen
samples with >50% abnormal morphology of sperm and samples with >20% white
cells were not included in any experiments. The motilities of the sperm were
subjectively assessed by examining several fields and classified into total and
progressive motility. All samples were tested for antisperm antibodies by mixed
antiglobulin reaction (MAR) test and samples showing >10% of sperm bound to red
cells were excluded from the study. A sperm count was done by diluting the semen
1:20 with formal saline and pipetting 10 μL onto a Neubauer chamber and counting
the cells under 200x magnification with a light microscope.
2.4 SPERM SUSPENSION PREPARATION
2.4.1 Centrifugation migration method
Liquefied semen was mixed with 2x its volume of EHBS medium and centrifuged at
500g for five minutes. The supernatant was removed and 1.5 mL of fresh medium
was added to the pellet and thoroughly mixed. The sample was centrifuged and the
supernatant discarded. The sperm pellet was layered, carefully and gently so as not to
disturbed it, with 1 to 1.5 mL medium and placed in a humidified 37 0 C incubator to
allow motile sperm to migrate into the overlying medium. At the end of an hour
incubation, 0.5 to 1.0 mL of the supernatant was removed, taking care not to disturb
the lower layer. The sperm concentration was assessed by taking 20 μL and heat
treating at 560 C and counting the cells with a Neubauer haemocytometer. A sperm
suspension of five to 10 million per millilitre was obtained by adjusting the suspension
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with medium.
2.4.2 Migration centrifugation method
After liquefaction, about 1 mL of semen was layered with 1 to 1.5 mL EHBS
medium, minimising disturbance at the interface. The tubes were incubated in a
humidified incubator at 370C. At the end of an hour incubation, 0.5 to 1.0 mL of the
supernatant was removed, taking care not to disturb the lower layer. The sperm
concentration was assessed by taking 20 μL and heat treating at 560 C and counting
the cells with a Neubauer haemocytometer. A sperm suspension of 5 to 10 million
per millilitre was obtained by adjusting the suspension with medium.
2.4.3 Percoll gradient method
Upto 2 mL of liquefied semen was layered gently onto the prepared 40/80%
Percoll gradients (section 2.2.2). The tubes were gently placed in a bench top
centrifuge and centrifuged at 500 g for 20 minutes. The tubes were then gently
removed and the three top layers were aspirated, leaving behind a small pellet at the
bottom of the tube. Any remaining Percoll was washed off by adding 5 mL EHBS to
the pellet and thoroughly mixing and re-centrifuging at 600 g for 10 minutes. The
supernatant was discarded. The pellet was suspended in 0.8 to 1.5 mL medium.
The sperm concentration was assessed by taking 20 μL and heat treated at 56 0 C
and the cells counted with a Neubauer haemocytometer. A sperm suspension of 5
to 10 million per millilitre was obtained by adjusting the suspension with medium.
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2.5 CASA METHODOLOGY
Sperm motion analysis was carried out on a Celltrack/s Motion Analyzer. Five
microlitres of each sample was pipetted onto a prewarmed Thoma chamber (Weber
Scientific, Teddington, UK) with a depth of 20 ± 0.2 μm (manufacturer's
specification). This was placed on the heated stage (370C) of the Olympus BH-2
microscope and incubated for 3 minutes before analyzing under pseudo-dark field
illumination with 5x objective, numerical aperture 0.12 (Watson, Falmouth). In
accordance with WHO guidelines (1992), three to six fields were examined and at
least 100 cells were counted and 50 cells were tracked. The total number of motile
and tracked cells, and the average values (tracked cells) of the following CASA
parameters were recorded: motility %, curvilinear velocity, straight-line velocity,
linearity and lateral head displacement.
2.5.1 CASA calibration and parameter setting
Frame rate - the number of pictures per second that are taken to sample the motion
of the moving sperm. In clinical evaluation of semen samples, a value of 60 or 30
frames/sec is commonly used.
Duration of data capture - is specified in frames, commonly set at 60.
Minimum path length - is the minimum number of frames for an individual cell to be
considered a 'valid' path. If a cell appears for less than this number of frames (due to
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it swimming into or out of the field of view), it is not counted as a cell at all (either
motile or non-motile).
Minimum motile speed - user-definable threshold speed by which motile cells are
distinguished from non-motile cells. This threshold is based on the average VSL for
each path.
Maximum burst speed - the value should be set slightly higher than any maximum
expected speed of the sperm to be tracked. For human sperm in semen, a value of
250 to 400 microns/sec is recommended.
Distance scale factor - this number relates the internal video units of pixels to the
units of microns, which is calibrated with a scaling grid by the user.
Camera aspect ratio - different kinds of video cameras have slightly different
horizontal to vertical scaling ratios, which is corrected for in this correction value.
ALH smoothing factor - is the number used in computing the Mean Path for each
sperm. The larger the number, the smoother the mean path. Recommended figure
is 7.
Centroid X search neighborhood - is the value used in the image processing front-
end to distinguish separate objects from each other. The recommended setting is 4
pixels for single edge detection.
Centroid Y search neighborhood - is the value used in the image processing front-
end to distinguish separate objects from each other. The recommended setting is 2
pixels for all normal use
Centroid cell size minimum - is the setting that allows one to discard objects in the
video field that are smaller than this specified size. It is calibrated by the machine.
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The recommended value for normal clinical uses, where there are between 20 and
40 cells in the microscopic view, is 2 units
Centroid cell size maximum - is the setting that allows one to discard objects in the
video field that are larger than this specified size. It is calibrated by the machine. The
recommended value for normal clinical uses, where there are between 20 and 40
cells in the microscopic view, is 6 to 8 units
Path maximum interpolation - The local gaps in the paths are filled in with linearly
interpolated data to bridge the gap. Once centroids are computed, the pathfinder
searches through time and space to connect centroids into valid paths. The Path
Maximum Interpolation instructs the pathfinder to 'bridge gaps' in possible paths
where the data disappears for one or more frames. Recommended value is 0 or 1
frame for normal clinical use.
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The Motion Analyzer was calibrated and set as follows: -
Table 2.1 Parameter setting of Celltrack/s CASA
System Parameter Parameter Units
Frame Rate 60 frames/sec

Duration of Data capture 60 frames
Distance scale factor 1.7875 μm/pixel
Camera aspect ratio 1.0210 -
Minimum path length 59 frames
Minimum motile speed 10 μm/s
Maximum Burst speed 500 μm/s
ALH smoothing factor 7 frames
Cell range 1-5 pixels
Depth of sample 20 μm
Cent. X search neighbourhood 4 pixels
Cent. Y search neighbourhood 2 pixels
Path max. interpolation 1 frame
2.5.2 CASA terminology
There is a standard terminology (WHO, 1992) for parameters measured by CASA
systems:-
VCL - curvilinear velocity, μm/s. Time-average velocity of a sperm head along its
actual curvilinear trajectory, as perceived in two dimensions under the microscope.
VSL - straight-line velocity, μm/s. Time-average velocity of a sperm head along the
straight line between its first detected position and its last position.
LIN - linearity. The linearity of a curvilinear trajectory. The ratio of VSL:VCL.
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ALH - amplitude of lateral head displacement, μm. Magnitude of lateral displacement
of a sperm head about its spatial average trajectory. It is expressed as an average of
such displacement.
VAP - average path velocity, μm/s. It is the time-average velocity of a sperm head
along its spatial average trajectory, computed and smoothed by the computer.
STR - straightness. The linearity of spatial average path. The ratio of VSL : VAP.
WOB - wobble. The measure of oscillation of actual trajectory about its spatial
average path, the ratio of VAP to VCL.
BCF - beat/cross frequency, beat/s. It is the time -average rate at which the curvilinear
sperm trajectory crosses its average path trajectory.
MAD - mean angular displacement, degrees. The time-average of absolute values of
the instantaneous turning angle of the sperm head along its curvilinear trajectory.
MOT - motility, %. It is the ratio of motile cells to non-motile cells expressed as a
percentage.
Total cells analyzed - It is the total cells examined and counted that satisfied the set
criteria (section 2.5.1).
Cells tracked - It is the number of tracked cells meeting the set criteria.
In this project the following parameters were measured by the Celltrack/s
Motion Analyzer:- VSL, VCL, LIN, ALH and MOT %.
2.5.3 Evaluation of CASA reproducibility
Since CASA was the main equipment used in measurement of sperm motility,
it was essential to find out how reliable the data obtained using it were. To do this, a
sperm suspension was prepared by the Percoll gradient method (section 2.4.3) of
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concentration between 3 and 15 million per mL. Each sample was measured twice
by CASA (section 2.5), and each time a new Thoma slide was prepared. The results
(Table 2.2) were assessed by Paired t-test and Pearson's correlation, r.
Table 2.2 CASA reproducibility - Mean values of 30 samples
CASA 1st 2nd Correlation p value

parameter Analysis Analysis (r)
VSL - μm/s 62.5 ± 2.3 61.1 ± 2.22 0.81 0.0001
VCL - μm/s 119 ± 2.97 118 ± 2.92 0.96 0.0001
LIN 54 ± 1.64 54 ± 1.65 0.77 0.0001
ALH - μm 4.8 ± 0.12 4.8 ± 0.12 0.91 0.0001
Mean ± SEM; Paired t-test showed no significant mean difference between 1st and
2nd analysis.
The results showed that there was no significant mean difference between the
first and second readings, as shown by t-test. The Pearson's correlation between 1st
and 2nd analysis was highly significantly (p<0.0001) for all CASA parameters. It
follows, therefore, that the CASA system was very reliable and the repeatability of
results was consistent. Hence, only single CASA measurements were taken in the
experiments reported in chapters 3 to 7.
2.5.4 Comparison of CASA measurements between live and taped samples
In certain experiments, it was necessary to video tape the sperm motion and
subsequently play back the tape and analyze the sperm motion characteristics in a
Celltrack/s Analyzer. Therefore, it was essential to investigate if there was any
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difference between images analyzed from live and taped samples. 17 sperm
suspension samples were prepared by the migration centrifugation method (as
described in section 2.4.2) to a final concentration between 5 and 10 million sperm
per millilitre. Each live sample was measured by a Celltrack/s analyzer (Section 2.5)
and simultaneously the images were recorded on a video tape using a video
recorder. The tapes were analyzed at a later date. The results (Table 2.3) obtained
showed a non-parametric distribution; therefore, significant median differences were
tested by Wilcoxon signed rank test. The precision between the two sets of data was
assessed by F-test.
Table 2.3 Comparison between live and taped CASA measurements
Parameter Live Taped Pearson's Wilcoxon

sample sample correlatio signed
F-test
n rank
p value
VSL - μm/s 52 52 0.95 NS NS

42-61 46-63
VCL - μm/s 96 102 0.97 <0.01 NS

83-117 88-124
LIN 53 53 0.87 NS NS
43-60 42-57
ALH - μm 4.5 5.0 0.97 <0.05 NS

3.7-5.4 4.1-5.9
MOT -% 91 93 0.96 <0.001 NS

79-95 83-96
Median Values with 25th and 75th centile; NS=Not Significant
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The results showed there were some significant median differences between
samples analyzed in the live state compared with analysis of taped sperm motion.
VCL showed a significant increase of 6% (p<0.01) for taped analysis compared with
live sample analysis, and a highly significant positive correlation (r = 0.97) between
the two sets of data (p<0.001). Similarly, ALH showed a significant increase of 11%
(p<0.05) for taped analysis, and a highly significant positive correlation (r = 0.97)
between the two sets of data (p<0.001). MOT% also showed a significant increase
(p<0.001) for taped analysis, again with a highly significant positive correlation (r =
0.96) between the two sets of data (p<0.01). VSL and LIN showed no significant
change. This appeared to suggest that analysis of sperm characteristics done by
using taped images have raised CASA values, with different parameters being
affected to varying extent. These increased values obtained from taped data may be
due to the inherent problem of the electronics involved in image capture. The image
of each moving sperm was captured on a moving tape, while for the live sample
analysis, the sample was stationary. The results of this study showed that, in any
studies, all analysis must be done either on live samples or on taped samples and
that the two methods of data collection should not be mixed in any one study. F-test
showed that the precision between live and taped analyzes were the same.
Considering this information, all data collections from CASA studies were done on
tapes in chapter 6; the results for all other studies were obtained from live recordings.
2.6 STABILITY OF PENTOXIFYLLINE TO EXTERNAL FACTORS
Most published papers appear to suggest that PF must be prepared fresh.
This for a routine laboratory is inconvenient and would lead to wastage of chemicals.
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I decided, therefore, to test the effect of freeze-thaw and the shelf-life of a frozen
stock solution of PF.
2.6.1 Effect of freezing stock for 6 and 10 weeks
A solution of PF at 50 mM/L was prepared in EHBS medium and aliquoted
into small vials of 2mL and stored at -200 C. At the end of six or ten weeks, the
stock was thawed at room temperature (23 ± 2) 0 C and 20 mM PF/L working
solutions prepared. A fresh working solution of 20 mM PF/L was also prepared
(section 2.2.3). 50 μL of PF working solution was added to 0.5 mL sperm
suspension, prepared by Percoll gradient method (section 2.4.3), at a concentration
between 5 and 10 million per mL. The suspension was mixed and incubated at 370 C
for 1 hour before CASA measurements were taken. The results were assessed by
Paired t-test.
Table 2.4 Stability of stock PF to freezing for 6 weeks.
CASA Control 2mM PF/L 2mM PF/L p Value

parameters Freshly Froz. stock
prep.
VSL - μm/s 53 ± 2.07 64 ± 2.93 62 ± 2.63 0.64
VCL - μm/s 99 ± 2.24 122 ± 2.25 121 ± 2.47 0.91
LIN 53 ± 1.55 55 ± 2.51 53 ± 2.21 0.54
ALH - μm 4.3 ± 0.08 5.0 ± 0.16 5.1 ± 0.15 0.66
MOT -% 89 ± 4.43 89 ± 4.92 88 ± 5.91 0.75
Mean ± SEM; n=10
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Table 2.5 Stability of stock PF to freezing for 10 weeks.

prep.
VSL - μm/s 51.0 ± 2.26 57 ± 2.79 58 ± 2.58 0.91
VCL - μm/s 94 ± 2.84 115 ± 3.58 116 ± 3.14 0.70
LIN 54 ± 1.57 52 ± 1.40 52 ± 1.26 0.93
ALH - μm 4.1 ± 0.12 5.0 ± 0.14 5.0 ± 0.13 0.90
MOT - % 85 ± 3.86 87 ± 2.48 89 ± 2.31 0.63
Mean ± SEM; n=12
The results (Tables 2.4 and 2.5) show that, when compared, PF prepared
freshly and from frozen stock stimulate sperm motion characteristics to a similar
extent. The results showed that there were no significant differences between the
working solution prepared from frozen stock and the fresh working solution for any of
the parameters.
2.6.2 Effect of heat on freeze- thawing
A solution of PF at 50 mM/L was prepared in EHBS medium and aliquoted in
small vials of 2 mL and stored at -200 C At the end of two weeks, the stock was
thawed at 560 C in a water bath and a 20 mM PF/L working solution prepared. A
fresh working solution of 20 mM PF/L was also prepared. 50 μL of PF working
solution was added to 0.5 mL sperm suspension, prepared by Percoll method
(section 2.4.3), of concentration between 5 and 10 million per mL. The suspension
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was mixed and incubated at 370 C for 1 hour before CASA measurements were
done. The results were analyzed by Paired t-test.
Table 2.6 Stability of stock PF to thawing at 560C.

prep.
VSL - μm/s 56 ± 2.38 65 ± 1.94 65 ± 2.07 0.93
VCL - μm/s 105 ± 3.05 127 ± 2.51 125 ± 2.21 0.68
LIN 54 ± 1.29 53 ± 1.48 54 ± 1.63 0.69
ALH - μm 4.5 ± 0.10 5.4 ± 0.13 5.2 ± 0.10 0.34
MOT - % 92 ± 1.46 92 ± 1.41 93 ± 1.26 0.36
Mean ± SEM; n=16
The results (Table 2.6) show that, when compared, PF prepared freshly and
from frozen stock thawed at 560 C stimulated sperm motion characteristics to a
similar extent. The results showed that there were no significant differences between
the working solution prepared from frozen stock thawed at 56 0 C and the fresh
working solution for any of the parameters.
2.6.3 Conclusions
The above results showed that PF could be prepared as a 50 mM PF/L stock
solution and frozen up to 10 weeks before use. The frozen stock can be thawed at
560 C without any detrimental effect. It also showed that PF was a relatively stable
compound between -200 and +560 C in solution. Hence, PF stock solutions for the
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experiments described in chapters 4 to 7 were routinely prepared and frozen for a
maximum of six weeks.
2.7 STATISTICAL ANALYSIS OF EXPERIMENTAL DATA
One purpose of statistical analysis is to reduce a mass of data into a more
compact form that highlights general trends and show relationships between
variables. The chief objective is to provide quantitative (and therefore, hopefully
objective) way of distilling the essential features from the data (Godfrey, 1985a,
Godfrey, 1985b).
Continuous data, assessed by histogram, which show symmetrical distribution
about a central value, with increasing rarity of occurrence as distance above or
below this central value increased, is considered under these circumstances to fit a
Normal or Gaussian distribution (Sokal and Rohlf, 1981). The mean and median
value is usually equal. Any departure from normal frequency distribution is
considered skewed distribution, where one tail of the curve is drawn out more than
the other and the mean is not equal to the median value. A non-normal distribution
can sometimes be converted to a Normal distribution by applying various types of
transformation (Gladen et al., 1991; Bland and Altman, 1986). The importance of
knowing the type of distribution is valuable in deciding (a) what type of statistical test
can be applied and (b) can confirm or reject certain underlying hypotheses about the
nature of the factors affecting the phenomenon studied.
In this project, all experimental data were initially assessed by histograms.
Data that conformed to a Gaussian distribution was then analyzed by one way
Analysis of Variance (one-way ANOVA). An Analysis of Variance answers the
question whether there are differences among the population means of the groups
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being compared, but it does not pinpoint which population, if any, differ from the
others (Godfrey, 1985b). Null hypothesis testing for significance of difference was
done, using Paired t-test (or Two sample t-test, depending on the design of the
experiment) and the significance of difference was taken at p<0.05. The average
response value quoted in Tables and Figures was mean ± standard error mean
(SEM).
Data that did not conform to a Gaussian distribution were analyzed by non-
parametric tests, which were more robust in handling the skewed data than
parametric tests. These sets of data were analyzed by Kruskal-Wallis (analogous to
one-way ANOVA) test. Null hypothesis testing for significance of difference was done
by using Wilcoxon signed-rank test (analogous to Paired t-test) or Mann-Whitney test
(analogous to Two sample t-test) and the significance of difference was taken at
p<0.05. The average response value quoted in Tables and Figures was the median
and the distribution was from lower quartile (25th percentile) to 3rd quartile (75th
percentile).
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CHAPTER 3
INVESTIGATIONS INTO FACTORS AFFECTING

SPERMATOZOA MOTILITY
For the scientific acquisition of knowledge is almost
as tedious as a routine acquisition of wealth.
Eric Linklater b1899
3.1 INTRODUCTION
Spermatozoa (sperm) motility is an important factor in determining the
fertilization rate in man. Therefore, much work has been done in investigating sperm
motility in sperm suspensions that are invariably prepared with culture media (Quinn
et al., 1985; Liu et al., 1988; Muggleton-Harris et al., 1990; Yovich et al., 1990; Ing et
al., 1991). However, there is very little information in the way of published works to
support the various methods currently used to prepare sperm suspensions. This
project investigates what factors, if any, may affect sperm motion characteristics and,
based on the results, aims to standardise the method of sperm preparation.
In examining the influence of Pentoxifylline (PF) on sperm stimulation (Yovich
et al., 1990; Sikka et al., 1991), all authors have used different incubation times
and temperatures to show the beneficial effect of PF. Thus, there was no systematic
approach in studying the incubation time or temperature in all these published
works.
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The discontinuous Percoll gradient method of separating motile sperm from
non-motile sperm has been promoted by Gorus and Pipeleers (1981). It was said to
be a rapid, simple procedure and easily applicable in routine seminology work. It has
also been shown (Aitken et al., 1988) that Percoll prevents sperm being damaged by
reactive oxygen species (ROS) during sperm preparation techniques. Yet, very little
is known about its adverse effect, if any, on sperm motility.
When sperm stimulants are added to a sperm suspension, or when sperm
suspensions are prepared, centrifugation is generally the method used in washing
and preparing the sperm (Yovich et al., 1990) and in removing residual Percoll after
Percoll gradient separation techniques (Pickering et al., 1989). It is surprising to
know how little is known about the effect of centrifugation on human sperm motility.
In the specialised treatment of infertility, procedures such as Gamete
Intrafallopian Transfer (GIFT), In vitro fertilization - Embryo Transfer (IVF-ET) and
Pronuclear stage tubal transfer (PROST) all involves the removal of oocytes from the
ovary. The aspirated oocytes are suspended in culture medium (usually EHBS
medium) containing heparin that has been used in flushing out the oocytes from the
ovary. Heparin prevents blood clotting in the extraction of the oocytes. However,
there were no published studies on the effect of heparin on oocytes or sperm.
In summary, most of the conditions used in the preparation of sperm were
empirically chosen, and therefore it lacks standardisation. This part of my project
was devised to investigate the ideal optimal of incubation with Pentoxifylline, and to
determine at what temperature this optimal effect on sperm motion characteristics
can be obtained. Also, I studied the effect of centrifugation and presence of heparin
in culture medium on sperm motion. Percoll is becoming increasingly popular in

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separating motile from non-motile sperm and it is, therefore, important to know the
effects of Percoll on sperm motion characteristics. Furthermore, different research
groups use different methods of sperm suspensions preparation, therefore it is
essential to know if these different techniques induce different motion characteristics
in these preparations.
3.1.1 Material Selection
All specimens used in sections 3.2 to 3.7 had normal count (>20x106 per
millilitre) and motility (50% total, >25% progressive) as defined by WHO (1992). The
samples were processed as described in section 2.3.
3.1.2 Earles-Hepes balanced salt solution preparation
All Earles-Hepes (EHBS) medium used in section 3.2 to 3.7 were prepared as
described in section 2.2.1
3.2 EFFECT OF INCUBATION TEMPERATURE ON SPERM
MOTILITY WHEN PENTOXIFYLLINE IS PRESENT
3.2.1 Materials and Methods
A total of 23 semen samples were examined and the method used for
preparation of the sperm suspensions was the discontinuous Percoll gradient

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method, as described in section 2.4.3. The prepared sperm suspensions, of
concentration between 5 to 10 million per mL, were divided into 4 portions of 0.5 mL
each. The 1st and 3rd portion were treated as controls, and had 50 μL EHBS
medium added to them, whereas the 2nd and 4th portion were mixed with 50 μL of
20 mM PF/L (final concentration 2 mM PF/L). Preparation of PF as described in
section 2.2.3. The 1st and 2nd samples were incubated at room temperature (RT=23
to 250 C) for 30 minutes. Samples 3 and 4 were incubated at 370 C for 30 minutes.
At the end of the incubation period, all samples were analyzed by CASA (section 2.5)
and the results of VSL, VCL, LIN, ALH and motility % were recorded. The data
produced were normally distributed, and therefore they were analyzed by Analysis
of Variance followed by Paired t-test to check statistical significance between groups
(section 2.7).
3.2.2 Results and Discussion
Table 3.1 shows the results obtained from control sperm samples incubated
either at RT or 370 C temperature, and shows no significant change in motion
characteristics as assessed by t-test. The effect of temperature on VSL, LIN, and
MOT % was not significant. Although the VCL value had risen from 124 μm/s at RT
to 129 μm/s at 370 C, this increase is not significant. Similarly, the control ALH value
rose from 5.2 at RT to 5.5 μm/s at 370 C (p<0.1), which was statistically insignificant.
Therefore, it can be concluded that motion characteristics of control sperm samples
as measured by VSL, VCL, LIN, and ALH were not affected by the incubation
temperatures used.
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However, when PF was added to sperm samples, the picture appears to
change somewhat, as shown in Table 3.1. Statistically, incubation temperature has
no influence on VSL, LIN, and MOT % of sperm samples treated with 2 mM PF/L.
However, VCL and ALH were influenced by incubation temperature. The VCL of 139
μm/s at RT had increased to 149 μm/s at 370 C, which was significant at the p<0.01
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level, and for ALH the value had risen from 6.0 μm at RT to 6.4 μm at 370 C
(p<0.05), overall increases of about 15% above their respective controls. These
significant changes are illustrated more specifically in Table 3.2.
Table 3.2 Effect of PF on VCL and ALH values of sperm
incubated at two different temperatures
% Increase in response to PF above control
CASA Parameter Incubation at RT Incubation at 370 C
VCL - μm/sec 124  139 = 12% 129  149 = 16%
ALH - μm 5.2  6.0 = 15% 5.5  6.4 = 19%
It can be seen that, for both motion parameters, PF caused an increase of
about 15% at both temperatures.
It was apparent from this study that incubation temperature had some
influence on some of the sperm motion characteristics in the presence of PF, but not
in its absence. In the interest of standardization, all experiments were carried out at
370 C.
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3.3 EFFECT OF INCUBATION TIME ON SPERM MOTILITY
WHEN PENTOXIFYLLINE IS PRESENT
A total of 19 semen samples were examined and the method used for
preparation of sperm suspension was the discontinuous Percoll gradient method as
described in section 2.4.3. Aliquots of prepared sperm suspension of 0.5 mL were
used. CASA measurements were taken and recorded which acted as control (0
time). The prepared samples were mixed with 50μL 20 mM PF/L (final
concentration 2 mM PF/L), the PF having been prepared as described in section
2.2.3 and incubated at 370 C. At intervals of 30, 60, 90, 120, 150, and 180 minutes
after addition of PF, CASA measurements were taken and the results recorded. The
data were normally distributed and the appropriate statistical tests were done to
analyze the results.
When PF was added to sperm samples, the length of incubation time appears
to be a major factor in influencing sperm motion parameters, as shown in Table 3.3.
Analysis of Variance on the values for VCL, ALH and LIN over the whole range of
incubation times (from 0 to 180 minutes) showed highly significant effects (p<0.001)
compared with the control. For example, the VCL (Fig. 3.1a) was 123 μm/s at 0
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time but rose to 152 μm/s after 60 minutes incubation and after that rose further, to
159 μm/s at 90 minutes, when it plateaued out, indicating that the maximum effect of
temperature had been attained. The ALH rose, in parallel with
the change in VCL, to 6.9 μm/s at 90 minutes and thereafter remained unchanged
(Fig. 3.1b). The increase in VCL and ALH induced a decrease in LIN, as evidenced
by Fig. 3.1c, this change being highly significant (p<0.001). The LIN value dropped
very dramatically from 49 at 0 time to 34 at 60 minutes and then plateaued from 90
minutes onwards. This appeared to suggest that the sperm head moved more
vigorously from side to side, making bigger wave-like movements with increased
flagellar amplitude and wavelength, simultaneously making less forward progressive
movement. The VSL fell initially, but then rose a little, although not back to the
control value.
Kay et al. (1993) showed that washed sperm from cryopreserved semen
exhibited hyperactivation when incubated with 3.6 mM PF/L. On examination of the
data, they showed that the maximum stimulation occurred between 15 and 75
minutes of incubation. This is consistent with my findings reported above. They (Kay
et al., 1993) also showed that PF treatment increased the VCL & ALH, but
decreased the LIN, which again is consistent with my findings.
Hyperactivation of sperm in rodents has been described by Yanagimachi et
al. (1970) (Fraser, 1977) as a whiplash figure of eight pattern of movement. It
produces large amplitude of proximal waves by the flagellum, resulting in high
amplitude lateral head displacements. The pattern of flagellar beating causes the
sperm head to move more rapidly without any net gain in forward momentum. This
hyperactivation model of rodent sperm also applies to human sperm, as was shown
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by Burkman (1984). My results of human sperm that had been incubated with PF up
to 3 hours appear to indicate that the activity of the sperm is consistent with motion in
the hyperactive state.
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3.4 EFFECT OF HEPARIN ON SPERM MOTILITY
WHEN PENTOXIFYLLINE IS PRESENT
3.4.1 Materials and Method
A total of 23 semen samples were used, and each sample split into two
portions of semen. Both portions of semen were separated by the discontinuous
Percoll gradient method, as described in section 2.4.3. The pellet obtained from the
1st portion of semen was suspended in Earles-Hepes medium containing 5000 iu
sodium Heparin/L (Minihep, Leo Laboratories Ltd, UK). This prepared sperm
suspension was divided into 2 portions of 0.5 mL each. One portion served as a
control, therefore 50 μL of EHBS medium was added, while the other portion was
mixed with 50 μL 20 mM PF/L (final concentration 2 mM PF/L), prepared as
described in section 2.2.3.
The pellet from the 2nd portion of semen was suspended in EHBS medium,
and this suspension divided into 2 portions of 0.5 mL each. One portion served as
a control, therefore 50 μL of EHBS medium was added, while the other portion was
mixed with 50 μL 20 mM PF/L (final concentration 2 mM/L).
All samples were incubated for 30 minutes at 370 C before analysis by CASA.
The results of VSL, VCL, LIN, ALH and motility % of sperm were recorded. The data
produced were normally distributed and the appropriate statistical tests were done to
test the significance of any differences.
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Table 3.4 summarises the effects of heparin, in the presence and absence of
PF, on sperm motion characteristics. In the control samples, heparin had no
influence on sperm motility parameters like VSL, VCL, LIN, ALH, and MOT %, as
evidenced by the p values. Addition of 2 mM PF/L to sperm suspensions containing
heparin in the culture medium made no difference to the motion characteristics, as
shown by the mean values in Table 3.4. In light of this information, when there was
surplus EHBS medium (with heparin) to clinical requirement in the Fertility
Laboratory, rather than disposing of the media, it was used for research work on this
project reported in chapter five.
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3.5 EFFECT OF PERCOLL ON SPERM MOTILITY
A total of 11 semen samples were used and the method used for preparation
of sperm suspension was the centrifugation migration method as described in
section 2.4.1. The prepared sperm suspensions were divided into 2 portions of 0.5
mL each per tube. The 1st tube served as the control and to the second tube, 50 μL
of Percoll was added. Both tubes were incubated at 370 C for 30 minutes before
CASA measurements were taken and the results recorded.
The second tube containing Percoll was then mixed with 4 times its volume of
EHBS medium and centrifuged at 600g for 5 minutes. The supernatant was removed
and the pellet was thoroughly mixed with 0.4 mL of EHBS medium. CASA
measurements were taken and the results recorded as "1st wash". This procedure
was repeated until 6 sets of washed results were obtained from the Percoll treated
sample.
Assessment of data showed that they were not of Normal distribution;
therefore, a non-parametric approach was taken. Table 3.5 shows the median values
with lower and upper quartile value. Kruskal-Wallis analysis of data from control to
wash 6 showed that there was statistical significance at p<0.05
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for VSL, VCL, LIN, ALH and MOT %, as indicated in Table 3.5. The significance of
difference from control values was tested by Wilcoxon signed rank test.
The immediate effect of Percoll on sperm was to depress all CASA
parameters except LIN and MOT %. The effect on VCL parameter was highly
significant at p<0.01. The increase in VCL value from control to wash 1 was not
significant but from wash 3 to wash 6, it was significant. The data appear to suggest
an upward trend in value from wash 1 to wash 6, as is shown graphically in figure
3.2a.
The gradual increase in ALH value from control to wash 6 (Fig.3.2b) follows a
similar pattern to that shown by the VCL parameter, the most significant change in
ALH value being at wash 6 (p<0.01) (Table 3.5). The Pearson's correlation between
VCL and ALH was r = 0.98 (p<0.0001), indicating a very high positive correlation.
The results in Table 3.5 show a progressive decrease in MOT % from control
to wash 6. This trend is graphically shown in Fig. 3.2c. The effect of Percoll removal
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on LIN from Wash 2 to Wash 4 was to depress the values (p<0.05) and the trend is
graphically shown in Fig. 3.2d. Percoll removal by washing did not affect VSL
parameter.
Although the initial effect of Percoll on sperm motion parameters was to
depress all values, the subsequent process of multiple washing to remove added
Percoll induced a decrease in overall sperm MOT %, but increased the curvilinear
velocity and lateral head displacement of sperm. The centrifugation
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experiment in Section 3.6 will demonstrate that the apparent rise in VCL and ALH
values was not due to centrifugation. The apparent cause for this rise must then be
connected with washing. This phenomenon of washing effect is the subject of
investigation in the second part of chapter 5 and its connection with frequent
changes in culture medium.
In this context, it is of interest to note an ultrastructural study by Barthelemy et
al. (1992) demonstrated that Percoll had no deleterious effects on sperm when
assessed by transmission electron microscopy. And it is reassuring to know that
when Percoll was injected into rabbit's ovaries, histopathological assessment
showed there was no observable cellular response in the ovaries (Arora et al., 1994).
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3.6 EFFECT OF CENTRIFUGATION ON SPERM MOTILITY
A total of 11 semen samples were used and the method used for preparation
of sperm suspension was the centrifugation migration method as described in
section 2.4.1. The prepared sperm suspension was divided into 2 portions of 0.5 mL
each per tube. The 1st tube served as the control and the second tube was the test.
CASA measurements were taken and the results of VSL, VCL, LIN, ALH, & motility
% were recorded.
The second tube was then subjected to centrifugation at 600g for 5 minutes.
The tube was removed and the contents were thoroughly mixed before CASA
measurements were taken and the results recorded as "1st Spin".
The above procedure was repeated until 4 centrifugation results were
obtained from the test sample. The data were normally distributed and Paired t-test
were done to analyze the results.
The results of centrifugation and its effect on sperm motion characteristics are
summarised in Table 3.6. Analysis of Variance on VSL, VCL, LIN and ALH, over the
whole range of spins from control to 4th spin, showed that statistically there were no
significant effects on the total of 11 samples examined, except MOT %, where there
was a significant drop in motility effect (p<0.05) compared with the control value.
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This result was consistent with the findings of Alvarez et al. (1993). They
showed that there was a decline in percentage motility of sperm that had undergone
centrifugation. Their study also showed that, if following centrifugation, a second
'swim-up' was performed, the proportion of motile sperm recovered was significantly
decreased. They postulated that the decreased recovery of sperm was due to
sub-lethal membrane damage induced by centrifugation.
In this study, centrifuging the same sample four times, did not statistically
affect the sperm motion characteristics adversely. The MOT % remained constant
from spin 1 to spin 4. As sperm motility is a function of overall cell integrity (Eddy,
1988), that there was no membrane damage due to centrifuging.
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3.7 COMPARISON BETWEEN DISCONTINUOUS PERCOLL GRADIENT AND
'SWIM-UP' METHODS OF SPERM SUSPENSION PREPARATION
A total of 30 samples were used for the preparation of sperm suspensions by
the isotonic discontinuous Percoll gradient method, as described in section 2.4.3.
Another 30 samples were used for the preparation of sperm suspensions by the
migration centrifugation method, as described in section 2.4.2.
The 60 sperm suspensions were analyzed by CASA (section 2.5) and the
results of VSL, VCL, LIN, ALH and motility % were recorded. The data produced
were normally distributed, and therefore they were analyzed by Analysis of Variance
followed by Two sample t-test to assess any statistical significance between the two
methods of separating motile sperm.
The results of the two methods of preparing sperm suspensions and their
effects on motion characteristics are summarised in Table 3.7. The results showed
that there was significant difference between the two methods. VCL, LIN and ALH
values were significantly different at p<0.0001. The motion characteristics of sperm
separated by Percoll exhibited higher VCL (24%) and ALH (23%) values compared
with sperm separated by the 'swim-up' technique.
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The LIN value was reduced by 13% in the Percoll gradient separation. The changes
in VSL and MOT% were not significant.
The results showed that the sub-population of sperm separated by the two
methods are different. Using the Percoll gradient method, abnormal sperm, cell
debris and low density sperm (irrespective of motility) were retained on the top of the
Percoll layer and between the 40/80 interface, whereas sperm of similar density
(irrespective of motility) to the Percoll pass through it to collect at the bottom of the
tube as a pellet. This preselection of sperm produces a sub-population rather than a
representative portion of the motile sperm, and therefore it was possible that this
fraction may have motion characteristics different from 'swim-up' preparations. In the
'swim-up' technique, only motile cells can migrate into the upper layer. Therefore, it
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is essential that the appropriate method is chosen for the preparation of sperm
suspensions; the two methods are not interchangeable. In light of this information,
the method of sperm separation was decided at the outset of each study.
3.8 CONCLUSION
The above studies on factors affecting sperm motion characteristics appears
to suggest that on the issue of standardization of method in studying sperm motion,
sperm suspensions incubated at 370 C and for one hour in the presence of a sperm
stimulant produces optimal results, and hence this procedure was employed in the
studies reported in chapters 4 to 7, unless otherwise stated. The reason why 60
minutes incubation time was chosen, rather than 90 minutes incubation, which
appeared to be the peak response, was that the changes in VCL and ALH values at
60 and 90 minutes were not significantly different from one another. Secondly, the
one hour incubation time fitted well within laboratory working conditions, i.e. it was
advantageous for practical reason. The presence of heparin and centrifugation of
sperm have statistically no significant impact on sperm motion characteristics.
Separation of sperm by the Percoll gradient method produced a preparation with
significantly different motion parameters from the 'swim-up' separation technique and
therefore it is essential to define the method of sperm separation in any studies.
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CHAPTER 4
ACTION OF PENTOXIFYLLINE ON SEMEN
A man may learn wisdom even from a foe.

Aristophanes c. 444-380 BC
4.1 INTRODUCTION
Pentoxifylline (PF), a phosphodiesterase inhibitor in the methylxanthine group,
is well documented to stimulate spermatozoa (sperm) motility, and is used clinically
(Yovich et al., 1990; Sikka and Hellstrom, 1991). Many published works (Aparico et
al., 1980b; Yovich et al., 1990; Sikka and Hellstrom, 1990; Tesarik et al., 1992a)
have all reported that PF, although it does not increase the number of motile sperm
does, however, improve sperm motion in preselected sub-populations of sperm.
Preselection is usually performed either by the 'swim-up' technique (Ing et al., 1991)
or by using the isotonic discontinuous Percoll gradient method (Iizuka et al., 1988).
Hammitt et al. (1989) have reported that when PF was added to cryopreserved
human semen rather than preselected sperm, there was increased stimulation of
motion characteristics of sperm. The concentration of PF chosen for their study was
empirically selected to be 3.6 mM PF/L, which may not be the optimal dose for
maximum stimulation. Sikka and Hellstrom (1990) observed that motion of washed,
cryopreserved sperm was significantly stimulated in a dose-dependent manner with
PF. Their drug concentrations ranged from 0.1 to 10 mM/L, and the peak response
was at 3 mM PF/L after a 3 hour incubation (250 C).

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Most reported studies on the action of PF have been on a selected population
of sperm enriched in highly motile cells. However, patients whose semen parameters
are sub-optimal may benefit if PF, when added to semen (before selection), could be
shown to increase recovery rate of sperm with enhanced motion characteristics.
Increased recovery and enhancement of sperm motility would be advantageous for
oligozoospermic and asthenozoospermic patients undergoing either IVF or GIFT
treatment, or any other form of assisted conception. The purpose of this study,
therefore, was to examine PF effects directly on semen over a range of PF
concentrations. A further aim was to determine the optimal PF concentration
required to achieve maximum enhancement of sperm motion characteristics.
4.2 MATERIALS AND METHODS
Semen samples were obtained from 37 normal individuals attending the Fertility
Clinic at Queen Charlotte's and Hammersmith Hospitals. All samples used had
normal count (>20x106 per millilitre) and motility (>50% forward progressive, >25%
rapid progressive) as defined by WHO (1992). Subjects were requested to abstain
from sexual activity for at least 3 days before producing the sample. Samples were
obtained by masturbation and were allowed to liquefy at room temperature before
routine semen analyses were performed in accordance with WHO (1992) guidelines
as described in section 2.3.
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Table 4.1 Design of experiments and number of samples per group
Groups Final concentration of Pentoxifylline in Total number

semen of samples
mM/L
1 0 3 6 12 12
2 0 4 5 6 7 8 12
3 0 8 9 10 11 12 5
4 0 1 2 3 4 8
The patients were grouped into four blocks as shown in Table 4.I. The
number of samples used per PF concentration is given in Tables 4.2 and 4.3. The
reason for this block design was to cover the whole range of PF concentration from 1
to 12 mM/L, and since a single semen specimen could not be separated into enough
aliquots to cover the entire range under investigation. Semen from each patient was
divided into 4, 5 or 6 aliquots of 0.5 mL each, depending on the volume produced
and into which group they were randomly allocated. One aliquot from each patient
was treated as a control and the rest were exposed to PF.
The first aliquot of semen was treated as a control, to which 0.5 mL of Earles-
Hepes (EHBS) media (containing 10% albumin) was added. Preparation of EHBS as
detailed in section 2.2.1. An equal volume of EHBS/10% containing 2 to 24 mM PF/L
was added to the rest of the samples to give final concentrations of 1 to 12 mM PF/L.
Preparation of PF was as described in section 2.2.3.
The tubes containing control and test samples were mixed and incubated at
370 C in humidified air. At the end of a one hour incubation period (incubation time
based on the study reported in section 3.3), tubes were gently overlaid with one mL
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of Earles-Hepes/10% HSA media (as described in section 2.4.2) and incubated for a
further one hour at 370 C. At the end of this incubation, 0.4 mL of supernatant was
removed, taking care not to disturb the lower layer. 0.2 mL of the aspirated
supernatant was analyzed by a Celltrack/s Motion Analyzer as described in section
2.5 and the results of VSL, VCL, LIN, ALH, and MOT % were recorded 2. The
remaining suspension was treated with 10 μl of 50% formalin in saline to kill the
sperm and a manual count of sperm performed using a Neubauer haemocytometer.
4.3 Statistical Analysis and Calculations
In view of the non-Gaussian distribution of the data, a non-parametric
approach was taken. Medians were used as an average response to PF. Significant
median differences from control values were tested by the Mann-Whitney test.
In view of varying effects of PF on different CASA parameters, the overall
effect of PF on sperm was studied by calculating a 'Stimulation Index'. The
Stimulation Index 1 (SI 1) was calculated as follows: each median value of each
group was subtracted from the median value of the control and the percentage
change at each PF concentration was calculated for each parameter of interest (in
this study it was VSL, VCL, ALH and manual sperm count). The SI was an
unweighted addition of all the parameters at each PF concentration and represents
the total response of sperm to PF effect. A graph of SI 1 against PF concentration
was plotted and the best curve fitted with a quadratic equation.
1
The total number of cells analyzed, motile cells counted and the number of cells tracked per PF
concentration group were recorded, and these values are reported in Appendix B.
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The individual sample responses to PF stimulation were examined by
calculating the percentage change of CASA parameter for each patient as follows:
the difference between each patient's CASA parameter value at 6 mM PF/L and the
control value was divided by the control value and multiplied by 100%. The
percentage change was plotted against the patient number.
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4.4 RESULTS
4.4.1 Kinematics of Sperm response to PF challenge
Sperm showed a significant increase in some of their motion parameters
when exposed to concentrations of PF above 3 mM/L, as is summarised in Table
4.2. Curvilinear velocity (VCL), straight line velocity (VSL) and manual sperm count
showed a parabolic curve fit when plotted against PF concentration, while lateral
head displacement (ALH) was concentration-dependent up to 12 mM/L in a linear
fashion. The peak VCL response occurred around 8 mM/L (p<0.001; Fig.4.1a) and
was 18% increase (calculated from the best fit curve; range 8 to 28%) above the
control value. Median VSL showed a similar pattern with the peak response
occurring at a concentration of 7 mM/L (Fig.4.1b). The VSL in the presence of 7 mM
PF/L was 11% (range was from 1% to 22%) above the control value. PF produced a
concentration - dependent enhancement in median ALH (Fig.4.1c) up to a
concentration of 12 mM/L. Regression analysis showed a linear relationship with a
positive correlation of 0.93 (p<0.0001). Sperm recovery (Table 4.3), as reflected by
the median manual sperm count from the top layer of culture media, was highest in
the presence of 5 mM PF/L (calculated from best fit curve, Fig.4.1d). At this PF
concentration, the recovery was 36% (the range was from -36 to 204%) higher than
the untreated group. The recovery of motile sperm from semen that had been treated
with over 10 mM PF/L was lower than the control value by about 20%. The
percentage of motile sperm was not influenced by Pentoxifylline.
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Table 4.2 Kinematic responses of sperm to various concentrations of Pentoxifylline
PF conc. VSL VCL LIN ALH MOT No. of

mM/L μm/s μm/s μm % samples
0 59 104 55 4.3 89 37
49-63 93-114 53-58 3.9-4.8 76-95
1 53 101 51 4.5 92 8
43-62 87-113 45-56 3.8-5.0 85-94
2 56 100 56 4.4 95 8
49-64 94-108 49-61 3.8-4.7 90-96
3 60 113b 54 4.9b 91 20
52-64 102-121 52-57 4.3-5.2 85-95
4 60 115b 54 4.9b 91 20
54-66 99-118 53-57 4.5-5.1 89-93
5 64b 115b 54 4.8 93 12
57-66 106-119 51-57 4.1-5.0 82-97
6 63b 119a 55 4.9a 91 24
56-66 110-123 51-59 4.5-5.3 81-94
7 62 118a 55 4.8b 93 12
56-67 107-121 51-58 4.5-5.3 87-97
8 62 120a 55 5.0a 91 15
56-71 108-126 51-61 4.6-5.5 82-95
9 56 114 49 5.1b 92 5
46-66 105-133 43-55 4.6-5.6 84-93
10 59 116b 51 5.2b 88 5
51-69 110-137 47-55 4.8-5.8 80-94
11 60 120b 51 5.3b 85 5
51-65 108-134 47-55 4.7-5.9 64-99
12 60 119b 51 5.3a 89 17
52-66 113-133 48-56 4.7-5.7 80-92
Median values with first and third quartile value; significant median differences from control
shown by p values at a<0.01; b <0.05.
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4.4.2 Effect of PF on motion characteristics of sperm
The overall effect of PF on sperm motion characteristics was studied by
calculating the "Stimulation Index 1". The SI 1 was calculated from VCL, ALH, VSL
and manual count as explained in section 4.3. A graph of SI 1 against PF was plotted
and the curve fitted by employing a quadratic equation. The graph of SI 1 (Fig.4.2)
showed that as the concentration of PF increased, the beneficial effect on sperm
increased proportionally, reaching a maximum at 6 mM/L and thereafter any further
increase in the concentration of PF produced a pronounced decline in the beneficial
effect.
4.4.3 Observations on individual responses to 6 mM PF/L

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A subset of 24 matched-pair samples was recruited from group 1 and 2.
CASA parameters VCL and ALH were examined more closely as the effect of PF
was more pronounced on these parameters. Although sperm from the majority of
individuals showed a maximum response at a concentration of 6 mM PF/L, there
was marked inter-individual variation to PF challenge (Fig.4.3a and 4.3b). The
semen of two individuals (~10% of the number analyzed) showed little or no
detectable response to PF challenge. The variation in percentage rise in VCL and
ALH ranged from 0% (patient number 21) to about 50% for VCL and about 45% for
ALH (patient number 19), as shown in Figures 4.3a & 4.3b. Pearson's correlation
between the percentage increases in VCL and ALH was 0.81 (p<0.0001).
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4.5 DISCUSSION
Most studies on the effect of PF on sperm motion characteristics report on a
fixed concentration of PF done in suspensions of sperm. However, in this study I
report the effect of PF, using concentrations ranging from 1 to 12 mM/L, on sperm
motion characteristics done directly on semen. The results obtained in this study
indicate that the optimal PF concentration required to obtain the maximum beneficial
effect on sperm enhancement was 6 mM/L when the PF was added directly to
semen, this result being derived from the Stimulation Index. Thus, SI is an useful
mathematical tool in discovering a total maximum effect when a chemical compound
has varying degrees of effect on different parameters within a sample. The peak
response on VCL was at 8 mM/L (p<0.01), while for VSL it was at 7 mM/L. The
response of ALH was proportional to PF concentration, describing a linear
relationship (r=0.93, p<0.0001). At any concentration of PF, there was a
considerable variation in sperm response. By examining a subset of 24 matched pair
samples, it was observed that the responses of sperm to 6 mM PF/L show an
increase which ranged from 0% to over 40% for both VCL and ALH parameters. In
addition, I found that approximately 1 in 10 patient's sperm does not respond to PF
challenge, a result consistent with other results from my related projects (section
5.4.1).
Moohan et al. (1993) reported a large variability in the response of human
sperm in suspensions to PF challenge (the response varied from 0 to above 40%),
and was thus similar to this study in which PF was added directly to semen. It is,
therefore, very important to do preliminary testing with PF before selecting the

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appropriate drug concentration for clinical application. PF has been shown (Fuse et
al., 1993; Kay et al., 1993; Tesarik et al., 1992a) to increase the VCL and ALH of
sperm in suspension. Tesarik et al. (1992a) also reported a variability in the degree
of increase in VCL after treatment with PF. An increase in the speed (defined as
time-average velocity of the sperm head along its average trajectory) of motile sperm
may play an important role in enhancing in vitro fertilization. Holt et al. (1985)
showed that increased sperm speed correlates with increased in vitro fertilization and
improved sperm penetrating capability in the zona-free hamster egg assay. This
beneficial effect of PF was further supported by Yovich et al. (1990), who showed
that the drug improved the fertilization rate in cases of severe male factor infertility. It
has also been reported (Tesarik and Mendoza, 1993) that sperm treated with PF
showed improved fertilizing ability in patients with acrosome reaction insufficiency.
However, in contrast, Tournaye et al. (1993a) have reported that PF-treated sperm
showed no therapeutic advantage in IVF for male factor infertility in cases with
previous fertilization failure.
The ALH showed a concentration-dependent linear increase (Fig.4.1c) as the
PF concentration increased up to 12 mM/L, but there was no corresponding increase
in either VCL or VSL at the higher concentrations
(>8 mM/L). This appeared to suggest that the sperm head moved more vigorously
from side to side, making bigger wavelike movements, presumably with increased
flagellar amplitude and wavelength, but simultaneously making less forward
progressive movement. Kay et al. (1993) showed that washed sperm from
cryopreserved semen exhibited 28% hyperactivation at 75 minutes when incubated
with 3.6 mM PF/L, with the maximum stimulation occurring between 15 and 75
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minutes of incubation. Hyperactivation has also been shown (Mbizvo et al., 1993) in
a suspension of cryopreserved sperm in the presence of 3 mM PF/L.
Hyperactivation of rodent sperm has been described (Yanagimachi et al., 1970;
Fraser, 1977) as a whiplash figure-of-eight pattern of movement. It produces large
amplitude proximal waves by the flagellum, resulting in high amplitude lateral head
displacements. The patterns of flagellar beating causes the sperm head to move
more rapidly without any net gain in forward momentum. This hyperactivation model
of rodent sperm may also apply to human sperm (Burkman, 1984; Robertson et al.,
1988; Mortimer and Mortimer, 1990). The results of this study showed that when
semen was incubated with a high concentration of PF, the sperm exhibited
characteristics consistent with hyperactive-like motion.
This study showed that when PF was added directly to semen before any
'swim-up' procedure was performed, incubated for an hour and subsequently
subjected to the 'swim-up' process, there was increased recovery of motile cells. The
effect of an optimal concentration of PF (ie. 6 mM/L) on motile sperm results in
changes to movement characteristics, with increased forward movement
accompanied, presumably, by intensification of flagellar beat and increased lateral
head displacement. The net effect was increased recovery of motile sperm from
semen. Clinically, this could be beneficial in cases of patients with impaired sperm
motility in semen. Using conventional methodology to obtain sufficient motile sperm
for artificial insemination can produce low yield. However, these results demonstrate
that the addition of PF to semen may improve the recovery of motile sperm, which
could be useful in some male factor patients; for example, it has been shown that
the in vitro fertilization rate increased in patients with severely abnormal sperm (<4%
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normal) when the number of sperm added was increased 2 to 10-fold (Oehninger et
al., 1988). Furthermore, addition of PF to poor quality semen directly may also
decrease the time required to prepare the sperm suspensions using the current
methodology.
[Please note: A paper based on the data from this chapter has been accepted by Human
Reproduction to be published in 1995; 10:2 ]
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CHAPTER 5
ACTION OF PENTOXIFYLLINE ON SUSPENSIONS OF SPERMATOZOA

AND EFFECTS AFTER ITS REMOVAL BY WASHING
Knowledge itself is power.
Francis Bacon 1561-1626
5.1 INTRODUCTION
Spermatozoa must have the ability to move to achieve fertilization in the
oviduct. Active movement of the sperm is due to forces generated by the flagellar
activity (Dresdner et al., 1981; Kratz et al., 1989). Sperm flagellar undulation
generates active forces and torques along the sperm body, resulting in forward
movement. However, the generation of force and propulsion by the sperm depends
upon its environment and the availability of ATP. ATP is the intracellular constituent
necessary for sperm motility (Calamera et al., 1982) and its presence reflects some
of the functional capability of the gamete (Calamera et al., 1986). Conversion of ATP
to cAMP is catalysed by adenylate cyclase and elevated concentrations of cAMP
could be translated into increased flagellar movement (Tash and Means, 1982).
However, phosphodiesterase converts cAMP to AMP, and Perreault and Rogers
(1982b) showed that an inhibition of phosphodiesterase, raises the concentrations of
cAMP in sperm.
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There are a number of chemicals that can stimulate sperm motility, as is
discussed in section 1.8. Pentoxifylline (PF), a phosphodiesterase inhibitor, is the
most popular drug used clinically in enhancing sperm motility in suspension.
Increased sperm velocity is closely associated with fertility (Milligan et al., 1980).
Hence, it has been reported (Yovich et al., 1990) that in severe male factor infertility,
when 3.6 mM PF/L was added to the sperm suspension and incubated, followed by
subsequent removal of the PF by washing before insemination, PF improved the
fertilization rate. In another study by Tesarik et al. (1992a), also using 3.6 mM PF/L, it
was shown that when the drug was added to a sperm suspension, there were
increased sperm motion characteristics and the maximum motion was attained within
10 minutes of incubation with PF. Further, the activity persisted for at least two hours
after drug removal. They also showed that PF does not improve the percentage of
motile sperm.
Most of the studies have empirically chosen to use 3.6 mM PF/L to achieve a
stimulative effect in sperm suspension. In this study, the first aim was to find the
optimal concentration of PF required to produce a maximum increase in sperm
motion characteristics of sperm in suspension. It is a common practice in IVF
laboratories, after sperm have been exposed to PF, to remove the drug by washing
prior to use of the sperm suspension for fertilization, in order to minimise the risk to
embryo development (Tournaye et al., 1993b). Thus, the second aim was to
examine the effect of drug removal by washing, to discover if the stimulation induced
by PF was retained after removal of the drug.
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5.2 MATERIAL AND METHODS
5.2.1 Effects of PF on sperm suspensions
Semen samples were obtained from 22 normal individuals and processed as
described in section 2.3. Sperm suspensions were prepared as detailed in section
2.4.3, using the Percoll gradient method. Preparation of Earles-Hepes balanced salt
(EHBS) medium was as stated in section 2.2.1. Pentoxifylline was prepared as
described in section 2.2.3.
Each prepared sperm suspension was divided into 6 aliquots of 0.5 mL. One
aliquot was treated as the control, while the remaining aliquots were treated as test.
To each test aliquot, 50 μL of the appropriate PF concentration was added, to give a
final concentration range of 1 to 5 mM PF/L. The control contained 50 μL EHBS/10%
HSA medium. All tubes were mixed thoroughly and incubated at 370 C for 1 hour. At
the end of incubation period, CASA measurements were taken as described in
section 2.5 and the results of VSL, VCL, LIN, ALH, and MOT% were recorded3.
The decision to examine the PF concentration range from 1 to 5 mM/L was
based on a pilot study done on 11 sperm suspension samples that were exposed to
PF concentrations ranging from 1 to 10 mM/L at 1 mM PF/L intervals. Concentrations
of 5 mM PF/L and above appeared to be detrimental to sperm and resulted in
decreased MOT %, VCL and ALH (data not presented); 10 mM PF/L being the most
detrimental to sperm and 5 mM PF/L being the least. Concentrations between 1 and
1
The total number of cells analyzed, motile cells counted and the number of cells tracked per PF
concentration group were recorded, and these values are reported in Appendix C
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5 mM/L appeared to stimulate sperm motion, hence the choice of this concentration
range.
5.2.2 Effects of removal of PF by washing on subsequent
sperm motion characteristics
In this case, I used both methods (Percoll gradient method and 'swim-up'
method) to prepare the sperm suspensions. The decision to examine both methods
of sperm suspension preparation was based on information gained in section 3.7
which showed that these two methods produced different sub-population of sperm.
The sperm suspensions were treated with PF, incubated and sperm motion
characteristics determined, before the sperm were washed (to remove the PF) and
motion characteristics determined a second time. The object was to determine the
effects, if any, of washing the sperm. The exact procedure is outlined as a flow
diagram in Fig. 5.1
5.2.2.1 Series A - Separation of sperm by Percoll gradient method
described in section 2.3. Semen samples were separated by the isotonic Percoll
gradient method as detailed in section 2.4.3. The pellet so obtained was used in the
centrifugation migration method (section 2.4.1) to produce a population of highly
motile sperm. The supernatant was aliquoted into 3 parts of 0.5 mL each. One
aliquot was treated as the control, with nothing being added, and CASA
measurements were taken. The second aliquot was mixed with 50 μL of EHBS/10%
HSA and the third aliquot was treated with 50 μL of 30 mM PF/L, to give a final
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concentration of 3 mM PF/L. The second and third samples were mixed and
incubated at 370 C in humidified air. At the end of a one hour incubation period, the
tubes were removed, and CASA measurements were taken. The tubes were then
mixed with 4 mL EHBS/10% HSA, centrifuged and the supernatant discarded. This
wash was repeated. At the end of second wash, the pellet was suspended in EHBS
medium to give a concentration of between 5 and 10 million cells per millilitre. These
final sperm suspensions were analyzed by a Celltrack/s Motion Analyzer as
described in section 2.5 and the results of VSL, VCL, LIN, ALH and MOT% were
recorded.
5.2.2.2 Series B - Separation of sperm by migration centrifugation method
described in section 2.3. Semen samples were separated by the migration
centrifugation method as described in section 2.4.2. The sperm suspensions were
separated into 3 aliquots of 0.5 mL each and treated as described in section 5.2.2.1
above. At the end of second wash, the sperm suspensions were analyzed by
Celltrack/s Motion Analyzer as described in section 2.5 and the results of VSL, VCL,
LIN, ALH and MOT% were recorded.
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5.3 Statistical analysis and calculations
5.3.1 EFFECT OF PF ON SPERM SUSPENSIONs
Presentation of data by histograms showed that the data were not of
Gaussian distribution, and therefore, a non-parametric approach was taken to
analyze the data, as described in section 2.7. Medians were used as an average
response to PF. Significant median differences from control values were tested by
Wilcoxon signed-rank test.
The Stimulation Index 2 (SI 2) was calculated as follows: each median value
of each group was subtracted from the median value of the control and the
percentage change at each PF concentration was calculated for each parameter of
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interest (in this study it was VSL, VCL, ALH and MOT%). The SI was an unweighted
addition of all the parameters at each PF concentration and represents the total
response of sperm to PF effect. A graph of SI 2 against PF concentration was
plotted and the best curve fitted with a quadratic equation.
The individual sample responses to PF stimulation were examined by
calculating the percentage change of CASA parameter for each patient as follows:
the difference between each patient's CASA parameter value at 3 mM PF/L and the
control value was divided by the control value and multiplied by 100%. The
percentage change was plotted against the patient number.
5.3.2 REMOVAL OF PF BY WASHING
Histograms of data showed that the data were of normal distribution, and
therefore a parametric approach was taken to analyze the data, as described in
section 2.7. Significant mean differences were tested by the Paired t-test.
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5.4 RESULTS
5.4.1 RESPONSE OF SPERM SUSPENSIONS TO PF CHALLENGE
-----------------------------------------------------------------------------------------------
5.4.1.1 Kinematics of the response of sperm to PF challenge
Table 5.1 shows the summary of median values with first and third centiles.
There was a significant increase in both VCL and ALH parameters from a
concentration of 1 mM PF/L onwards. VCL (Fig 5.2a) and ALH (Fig 5.2b) showed
significant elevations in response to PF, which were best described by a parabolic
curve. The peak VCL response was at 3 mM PF/L (p<0.001) and was 12% increase
(calculated from best fit curve) above the control value, with a range from 122 to 145
microns/sec (25th to 75th centile). Median ALH showed a similar pattern, with the
peak response occurring at about 3 mM PF/L (p<0.001). This was 16% (calculated
from best fit curve) above the control value, with a range from 5.4 to 6.1 microns
(25th to 75th centile). The relationship between VCL and ALH was a positive
correlation of r=0.92 (p<0.0001). There was no significant change in VSL, LIN and
MOT % parameters.
5.4.1.2 Effect of PF on motion characteristics of sperm in suspension
The effect of PF on sperm motion characteristics was studied by calculating
the "Stimulation Index 2". The SI 2 was calculated from VCL, ALH, VSL and MOT%
as explained in section 5.3.1. A graph of SI 2 against PF was plotted and the curve
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fitted by employing a quadratic equation. The graph of SI 2 (Fig 5.3) showed that, as
the concentration of PF increased, the beneficial effect on sperm increased
proportionally, reaching a maximum at 2.8 ± 0.2 mM PF/L, and after that any further
increase in concentration of PF appeared to produce a pronounced decline in any
beneficial effect.
5.4.1.3 Observations on responses of individual samples to 3 mM PF/L
The effect of PF on twenty-two matched pair samples was examined more
closely by calculating the percentage change. Although the CASA parameters VCL
and ALH showed maximum responses at about 3 mM PF/L, there was marked inter-
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individual variation to PF (Fig 5.4a and Fig 5.4b). The sperm of two individuals
(~10% of the number analyzed) showed no detectable response to PF. The variation
in percentage rise in VCL and ALH ranged from 0% (patients 14 and 19) to about
35% for VCL and about 40% for ALH. Pearson's correlation between VCL and ALH
percentage change was r=0.83 (p<0.0001).
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5.4.2 RESIDUAL EFFECTS ON SPERM AFTER REMOVAL OF PF BY WASHING
-------------------------------------------------------------------------------------------------------------
5.4.2.1 Sperm obtained by the Percoll gradient method
The results are summarised in Table 5.2. The Analysis of Variance of CASA
parameters VSL, VCL, LIN and ALH all indicated significant changes, whereas
MOT% showed no significant change.
The PF treatment caused a significant 20% increase in VCL (p<0.0001), as is
shown in Fig.5.5a, and a 19% increase in ALH (p<0.0001) (Fig.5.5b), in response to
3 mM PF/L. There were significant decreases in VSL (p<0.001) and LIN (p<0.0001),
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and did not affect motility %. In general these effects persisted after removal of the
PF by washing; VCL (p<0.0001) and ALH (p<0.0001) remained higher than the
control values, LIN (p<0.05) remained lower than the control value, but the effect of
PF on VSL was lost after its removal. Motility% remained unaffected.
Surprisingly, washing had quite pronounced effects on the control samples;
that is, those samples which had not been treated with PF. It led to a significant
increase, by 13%, in VCL (p<0.0001) (Fig. 5.5a) and by 11% in ALH (p<0.0001) (Fig.
5.5b) from the '0' time control values, but did not affect VSL, LIN or motility%. There
was a significant difference between PF-treated & washed samples and the control
washed samples for the VCL and ALH parameters at p<0.01, and for VSL at the
p<0.05 level.
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5.4.2.2 Sperm obtained by the 'swim-up' method
Table 5.2 shows the residual effects on sperm separated by 'swim-up' after
removal of PF. The Analysis of Variance of CASA parameters VSL, VCL, LIN, and
ALH all indicated significant changes.
When sperm were incubated with 3 mM PF/L, the VCL increased by 20%
(p<0.0001) from the control value, as is shown in Fig.5.6a. The ALH showed a
similar pattern to the VCL, as is shown in Fig. 5.6b. The ALH increased by 23%
(p<0.0001) from the control value. The VSL increased significantly (p<0.01),
whereas LIN decreased (p<0.0.001). Motility% remained unaffected. In general,
these effects persisted after removal of the PF by washing; VCL (p<0.0001) and ALH
(p<0.0001) remained higher, and LIN (p<0.01) remained lower, than the control
values, but the effect of PF on VSL was lost after its removal. Motility% decreased by
11% (p<0.0001).
Washing had quite pronounced effects on the control samples, consistent with
those observed when the sperm was prepared by the Percoll gradient method. After
washing, the VCL was 6% higher (p<0.0001) and ALH 7% higher (p<0.0001)
compared with pre-wash values. In contrast, both the LIN and MOT% decreased
(p<0.0001 in both cases). The VSL remained unaffected. In general, the effects of
washing were similar to those in response to PF, albeit somewhat less pronounced.
There was no significant difference between PF-treated & washed samples and the
control washed samples for VCL, ALH, VSL, LIN and MOT% parameters.
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5.5 DISCUSSION
The results of this study showed that when sperm suspensions were
incubated with PF, it resulted in significant increases in sperm motion characteristics.
The optimal concentration of PF that produced the maximum stimulation was found
to be 2.8 ± 0.2 mM PF/L, as calculated from the SI. However, there was great
variability in individual responses to PF stimulation.
There is a body of evidence (Sikka & Hellstrom, 1991; Tesarik et al., 1992a;
Lewis et al., 1993; Moohan et al., 1993; Fuse et al., 1993) showing that PF elevates
sperm motion characteristics in sperm separated by the 'swim-up' technique. My
results are consistent with these findings; however, there were essential differences.
All previous studies used different concentrations of PF (probably empirically chosen)
and different incubation times and temperatures, making comparisons difficult. For
example, Sikka & Hellstrom (1990) reported that 3 mM PF/L significantly stimulated
sperm motion characteristics in washed sperm maintained at 25 o C, with the peak
response occurring at three hours incubation, whereas in the study by Lewis et al.
(1993), it was shown that in the presence of 3.6 mM PF/L, VCL was significantly
enhanced after 15 minutes of incubation at 370 C and remained high for up to 240
minutes. Elevated sperm motion characteristics were achieved when sperm
suspensions were incubated for 2 hours at room temperature with 5 mM PF/L (Fuse
et al., 1993), while Moohan et al. (1993) showed that the maximum response was
commonly observed at a PF concentration of 2 mM/L when incubated at 370 C for 30
minutes.
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My paired-controlled study showed that the maximum enhancement of sperm
motion characteristics was attained at 2.8 ± 0.2 mM PF/L (Fig. 5.3) when the sperm
were incubated at 370 C for 1 hour. The incubation temperature and time were based
on information obtained from the studies reported in section 3.2 and 3.3. Although
the VCL parameter showed a maximum rise at 3 mM PF/L (Fig.5.2a), whereas the
maximum ALH occurred at less than 3 mM PF/L (Fig.5.2b), the total effect
(assessed by the SI 2) showed that the actual maximum elevation was at 2.8 ± 0.2
mM PF/L. Thus, SI is a useful mathematical tool in discovering a total maximum
effect when a chemical compound has varying degrees of effect on different
parameters of the same sample.
Consistent with other studies (Moohan et al., 1993), this study also shows a
wide range of individual patients' responses to PF. Of the 22 individuals examined,
the VCL and ALH parameters (Fig. 5.4 a and b) of 2 patients did not show any
detectable response to PF challenge (~10% of patients), consistent with findings in
section 4.3. Four patients had a VCL increase of over 30% while 3 patients had an
ALH increase of over 30%. As suggested in section 4.4, preliminary testing with PF
is an important issue if the drug is to be used most efficaciously.
Treatment of sperm with PF, whether prepared by 'swim-up' or Percoll
gradient, caused some motion parameters to increase significantly, others to
decrease significantly, and others to remain unchanged. When the PF was
subsequently removed by washing, in some cases the effect was "washed away"
i.e. was lost (true of VSL in both types of separated sperm) but in others it remained
(VCL, LIN are good examples). Sometimes, when the effect remained, it was not so
pronounced i.e. some of the effect, but not all, was washed away (VCL and ALH in
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'swim-up' sperm, for example), but in other cases none of the effect was lost (VCL
and ALH in the Percoll gradient sperm, for example). However, washing alone had
quite pronounced effects on sperm motion characteristics, with increases in VCL and
ALH. These findings may appear to contradict the results of others who have
reported that the effects of PF remain after washing off' of the drug. For example,
Tesarik4 et al. (1992a) showed that the PF effect persisted for at least 3 hours after
drug removal. In another study, Hammitt3 et al. (1989) reported that although PF
significantly increased sperm velocity, however, after drug removal by washing, the
linearity and velocity of stimulated and untreated sperm were similar. Detailed
examination of the data in Hammitt et al. (1989) study revealed that the velocity of
control sperm was elevated with time, reaching a maximum at 1 hour post-wash, and
declining after that. Hence, the apparent persistence of the stimulating effects of PF
may have been more to do with the process of washing which increased the motion
characteristics of the untreated, control sperm than it was with any effects of PF
persisting in the treated sperm. Kay2 et al. (1993) showed that after PF removal by
washing, hyperactivation remained high for up to 1 hour, but returned to control
values after 3 hours. Examination of the data of Kay et al. (1993) on the effects of PF
removal, also reveals that the motion characteristics of the control, untreated sperm
values had risen after the wash when compared with before the wash. Thus, the
results of these two studies (Hammitt et al., 1989; Kay et al., 1993) are consistent
with the present study, and it is interesting to note that the authors of these two
4 3
'swim-up' separation Percoll gradient separation
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papers did not make any comment on the significance in the increase in the motion
parameters of the control, untreated sperm after they had been washed.
Results from the studies reported in sections 3.5 and 3.6 lead one to reject
the hypothesis that either the Percoll or centrifugation processes may affect sperm.
This unexpected phenomenon of the increase in sperm motion characteristics of the
control, untreated sperm after washing may be due to some factors being present in
the culture medium which enhance sperm activity or the presence of some factors in
sperm suspensions which inhibit activity until removed by washing.
Discrete subpopulations of sperm have been shown to produce sudden burst
of reactive oxygen species (ROS) (Aitken and Clarkson, 1987; 1988; Aitken et al.,
1989). Further, leucocytes present in semen have also been shown to generate ROS
(Kessopoulou et al., 1992; Aitken et al., 1992; Weese et al., 1993). These reactive
oxygen species also originate from the cellular components of the sperm (Iwasaki
and Gagnon, 1992) generated by the centrifugation process (Aitken and Clarkson,
1988). ROS, as well as lipid peroxides, have been shown to have deleterious effects
on sperm motility (Jones et al., 1979). Besides ROS, there are other factors, like
cytotoxic end-products of lipid peroxidation, which are present in semen and which
can cause irreversible loss of sperm motility (Selley et al., 1991; Aitken et al., 1993).
Further, decapacitation factors (DF), which are glycoproteins present on the
surface of sperm, can probably cause inhibition of sperm motility and the acrosome
reaction (Oliphant et al., 1985; Fraser and Ahuja, 1988; Fraser et al., 1990). When
preparing a 'swim-up' preparation of sperm, all these factors might diffuse into the
overlaid layer of culture medium. It follows that washing of sperm suspensions by
centrifugation, discarding the supernatant and replacing it with fresh medium,

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removes the toxic effects of these factors (ROS and DF), thereby preventing sperm
motility being inhibited.
In the washing procedures, after centrifugation, the supernatant was removed
and replaced with fresh culture media that contained 10% HSA. In a study conducted
by De Lamirande & Gagnon (1991), it was shown that serum stimulated motility of
sperm in suspensions in a dose-dependent manner, but that this effect varied from
one sperm population to another. Additional support that serum albumin is
implicated in maintaining sperm motility comes from a study done by Hammitt et al.
(1990). They showed that different types of proteins (preovulatory donor serum,
Cohn Fr. V human serum albumin and highly purified HSA) possess an equal ability
to support sperm motion characteristics. Thus the replacement of culture medium5 in
the wash procedures I used might have contributed to the increase in sperm motility.
However, the reasons given above do not fully explain why Percoll separated
sperm (and PF-treated) retained a higher increase in motion characteristics
compared with sperm (and PF-treated) obtained by the 'swim-up' method. A
possible reason could be that in Percoll separation, sperm were separated based on
density of the cell, irrespective of their motility, while in the 'swim-up' separation only
the progressive motile sperm swim into the upper layer of the culture media, with
immotile and poorly motile sperm remaining at the semen/culture media interface.
The resulting differences between these two methods of separation leads to the
conclusion that different subpopulation of sperm were selected by the two methods.
It is plausible that one subpopulation may respond to stimulation differently from a
5
contains serum albumin
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different subpopulation. As this study shows, this might indeed have been the case.
The results in Table 5.2 show that sperm separated by Percoll exhibit higher motion
characteristic values (control - VCL=123 ± 3.86, ALH=5.3 ± 0.18 and when treated
with PF, VCL=147 ± 3.53, ALH=6.3 ± 0.15) compared with sperm separated by
'swim-up' (control - VCL=104 ± 1.69, ALH=4.4 ± 0.06 and when treated with PF,
VCL= 125 ± 1.99, ALH=5.4 ± 0.09). This observation was further supported by the
study reported in section 3.7, where motion characteristics of Percoll separated
samples were significantly different from those of 'swim-up' separated sperm.
In conclusion, sperm motion characteristics were elevated in the presence of
PF and the maximum response was obtained at 2.8 ± 0.2 mM PF/L, although
different subpopulations of sperm responded differently to PF challenge. This
increased motion was reduced by washing; however, the removal of various 'factors'
in the culture medium probably masked the true reduction in stimulated sperm
samples and increased the motion values of the control samples. In the future, it
may be worth investigating further the influence of various 'factors' on sperm motion
characteristics.
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CHAPTER 6
The ACROSOME REACTION RESPONSE TO
PENTOXIFYLLINE CHALLENGE
Science is organised knowledge.
Herbert Spencer 1820-1903
6.1 INTRODUCTION
Leading up to fertilization, the spermatozoa must undergo a complex cascade
of events prior to union with the oocytes. Thus, the acrosome reaction that occurs as
a consequence of sperm capacitation is an indispensable prerequisite for sperm
passage through the zona pellucida and for its fusion with the oolemma. The
acrosome reaction (AR) is an exocytotic event that involves the fusion and
vesiculation of the outer acrosomal membrane and the surrounding plasma
membrane, and culminates in the dispersal and release of the acrosomal contents.
Adequate supplies of free Ca2+, Na+, K+ and energy substrates all play key roles in
the acrosome reaction (Fraser, 1992).
The site of the acrosome reaction during fertilization has been studied in vitro
in many laboratory animals. In the mouse, the AR occurs on the zona pellucida
surface (Florman and Storey, 1982; Wassarman, 1987). In the guinea pig, it appears
to occur at a distance from the zona pellucida, probably during sperm passage
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through the cumulus (Huang et al., 1981). The hamster represents an intermediate
situation in which both acrosome-intact as well as acrosome-reacted sperm bind to
the zona pellucida (Cummins and Yanagimachi, 1982, 1986). In the marsupial egg,
which lacks follicular cells, the AR may occur on the surface of the zona pellucida
(Rodger and Bedford, 1982), whereas in the sea urchin, the AR is initiated by a
fructose sulphate polymer moiety of the jelly coat that is thought to be equivalent to
the extracellular matrix of the cumulus oophorus of the mammalian oocyte (Lopo,
1983). The AR of human sperm is a calcium-dependent, exocytotic process and is
thought to be induced by the zona pellucida and occurs at the zona pellucida,
although sperm which have already undergone the AR can bind to the zona
pellucida (Cross et al., 1988).
There are several inducers that can initiate the acrosome reaction.
Physiological agents including albumin, zona pellucida extracts (Cross et al., 1988;
Bielfeld et al., 1994), follicular fluid (Zinaman et al., 1989; Mortimer and Camenzind,
1989) neuraminidase, glycosaminoglycans, catecholamine, prostaglandins,
oestrogen and progesterone (Uhler et al., 1992) are all known to stimulate the
acrosome reaction (Meizel, 1985; Yanagimachi, 1988; Meizel et al., 1990). Chemical
substances like Ionophore A23187 are also known to induce the acrosome reaction
(De Jonge et al., 1989; Cummins et al., 1991). Other chemical substances known to
induce the AR are Ionomycin and lysophosphatidycholine (reviewed by Wolf, 1989).
There is also a suggestion that Pentoxifylline may modify the sperm head surface,
making it more permeable and resulting in enhanced AR (Tesarik et al., 1992b).
Physical agents that are known to inhibit the AR include freeze-thaw, temperature
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shock and hyperosmotic medium (reviewed by Wolf, 1989). Cryopreservation has
also been shown to damage the acrosome (McLaughlin et al., 1993).
The AR in human sperm can be detected by employing electron transmission
microscopy (TEM) or using conventional optical microscopy. The TEM (Bartoov et
al., 1980) method is labourious, time-consuming and requires an expensive
instrument, and hence is beyond routine application. Therefore, light microscope
techniques based on staining the acrosome with histological stains were developed;
one such method is the triple stain technique (Talbot and Chacon, 1981).
Subsequently, fluorescein labelled dyes were developed to study the AR (Blasak et
al., 1982; Cross and Meizel, 1989). Methods now available for the study of the
acrosome reaction includes fluorescein isothiocyanate conjugated Pisum sativum
(pea) agglutinin (FITC-PSA) (Cross et al., 1986; Mendoza et al., 1992), fluorescein
isothiocyanate conjugated Arachis hypogea (peanut) agglutinin (FITC-PNA)
(Mortimer et al., 1987; 1990), chlortetracycline-UV (CTC) (Lee et al., 1987), and use
of monoclonal antibodies (Coddington et al., 1990; Zhang et al., 1990; Parinaud et
al., 1993)
The present study was undertaken, firstly, to evaluate the fluorescent staining
techniques currently available and to adapt one of them for routine use. Secondly, to
study the effect of PF on the acrosome reaction. Finally, the effect of PF plus
Ionophore A23187 on the acrosome reaction.
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6.2.1 Preparation of materials
described in section 2.3. Sperm suspensions were prepared as described in section
2.4.2, using the migration centrifugation method.
Earles-Hepes balanced salt (EHBS) medium was prepared as described in
section 2.2.1. Pentoxifylline was prepared as described in section 2.2.3.
6.2.2 Methods of acrosome evaluation
There are number of ways in which the acrosome reaction can be evaluated.
In this study, I used the fluorescent stain. I evaluated the PSA, PNA, and CTC
staining methods for their efficiency, speed, reliability and ease under laboratory
conditions. Incubation with Ionophore A23187 was used as a positive control of the
acrosome reaction, and the vital stain bis-benzamide (Mortimer et al., 1990) was
used to assess the live:dead ratio.
6.2.2.1 Ionophore A23187 challenge
The influx of calcium ions across the sperm membranes is generally
considered to be one of the steps in the acrosome reaction, which occurs once the
sperm are fully capacitated (Fraser, 1981; Aitken et al., 1984; Fraser and McDermott,
1985). The divalent cation transporting agent, Ionophore (IP) A23187, can be used to
induce the acrosome reaction, in a manner similar to that which occurs under
physiological conditions (Suarez et al., 1986). This property has been exploited and
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ionophore-treated samples were used as positive controls in my acrosome
evaluation studies.
The method used, was similar to that described by De Jonge et al. (1989) and
Cummins et al. (1991). In the pilot study, one millilitre of prepared sperm suspension
containing about 10-15 million sperm per mL was divided into two aliquots; one
aliquot was challenged with ionophore A23187 (final concentration 10 μM/L) in
DMSO and the other aliquot, the control, was treated with 10% DMSO. Samples
were incubated at 370 C for 30 minutes before the staining procedure was carried
out.
6.2.2.2 Staining using Vital stain
Cell viability was assessed using the method described by DasGupta et al.
(1993). A stock solution of Hoechst bis-benzamide 33258 (Sigma) was made by
dissolving 100 mg of the dye in Fresenius water for injection. The prepared stock
solution was aliquoted into smaller portions and frozen until required. Before use, the
frozen stock was thawed and diluted to 1:1000 in protein-free EHBS medium and
then further diluted 1:100 in the sperm suspension (final concentration of dye in
sperm suspensions was 1 μg/mL). The suspension was incubated for 5 minutes at
370 C and subsequently washed with 3 mL 2% polyvinylpyrrolidone (PVP 40, Sigma)
in PBS buffer before centrifugation at 800g for 10 minutes. The resulting pellet was
resuspended in EHBS medium to obtain a concentration of about 10 million sperm
per mL. The suspension was then ready for lectin or CTC staining.
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6.2.2.3 Staining using fluorescein isothiocyanate conjugated Pisum
sativum (pea) agglutinin (FITC-PSA stain)
FITC-PSA binds to α-methyl mannoside residues localized within the
acrosome (Cross et al., 1986); therefore, FITC-PSA will label the acrosome contents
of suitably permeabilized sperm. The method used for staining was similar to that
described by Cross et al. (1986) and Hoshi et al. (1993). A microscopic slide was
smeared evenly with sperm suspension and air dried for 30 minutes in a 37 0 C
incubator. The sperm were then permeabilized in 95% ethanol for 30 minutes,
washed twice with distilled water and air dried. The dried, smeared slide was
covered with 50 μL of FITC-PSA stain (100 μg/mL in distilled water) and left at room
temperature for an hour in the dark. The stained slides were mounted with 10 μL of
0.22 M 1,4 diazabycyclo [2,2,2] - octane/L (DABCO, Sigma) in glycerol plus
phosphate - buffered saline (9:1) and covered with 22x26 mm coverslips. DABCO
suspension delays fluorescence decay. The slides were examined at 200 times
magnification within 24 hours using a fluorescence microscope, set up as described
in section 2.1.2.
6.2.2.4 Staining using fluorescein isothiocyanate conjugated Arachis
hypogea (peanut) agglutinin (FITC-PNA stain)
FITC-PNA lectin binds to β - D-galactosyl residues localized on the outer
acrosomal membrane. The method used for staining was similar to that described by
Mortimer et al. (1990). A microscopic slide was smeared evenly with sperm
suspension and air dried for 30 minutes in a 370 C incubator. The dried, smeared
slide was covered with 50 μL of FITC-PNA stain (100 μg/mL in distilled water) and
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left at room temperature for an hour in the dark. The stained slides were mounted
with 10 μL of 0.22 M 1,4 diazabycyclo [2,2,2] - octane/L (DABCO, Sigma) in
glycerol plus phosphate - buffered saline (9:1) and covered with 22x26 mm
coverslips. DABCO suspension delays fluorescence decay and the slides were
examined at 200-times magnification within 24 hours using a fluorescence
microscope set up as described in section 2.1.2.
6.2.2.5 Staining using chlortetracycline (CTC) stain
The CTC fluorescent staining method used was similar to that described by
Lee et al. (1987) and DasGupta et al. (1993). CTC solution was prepared fresh each
day and contained 750 μM CTC/L (Sigma) in 130 mM Nacl/L, 5 mM cysteine/L and
20 mM Tris-Hcl/L, producing a final pH of 7.8. The prepared solution was stored in
the dark at 40 C until required. 100 μL of the prepared sperm suspension containing
about 5-10 million sperm per mL was added to 100 μL of CTC solution and mixed
thoroughly. The cells were fixed by adding 10 μL of 10% paraformaldehyde in 0.5
M/L Tris-Hcl, final pH 7.4, and mixed.
Microscopic slides were prepared by placing 10 μL of the suspension on the
glass, mixing with a drop of DABCO, and gently pressing down the cover slip to
remove excess mixture. The prepared slides were stored in the dark, allowing the
sperm to settle down, and the slides were read at 200-times magnification the
following day using a fluorescence microscope set up as described in section 2.1.2.
6.2.3 Transmission Electron Microscopy (TEM)
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0.5 mL of sperm suspension in an Eppendoff vial was mixed with 1.0 mL of
3% glutaraldehyde in 0.1M cacodylate buffer (pH 7.2-7.4) and left to fix for 1-2 hours
at room temperature. At the end of the fixation period, the fixed sperm were
precipitated by centrifugation and the supernatant removed. 1 mL of 0.1M cacodylate
buffer was added to the pellet and the sample was then sent for TEM.
Blocking, cutting and staining were performed by the staff of the Electron
Microscopy Unit. The pellet was dehydrated through a graded series of increasing
concentrations of ethyl alcohol and then embedded in Araldite resin. Ultra-thin
sections (<100 nm) were cut with a diamond knife in an ultra-microtome. The
sections were mounted on copper grids, stained with uranyl acetate and lead citrate,
and allowed to air dry.
The sections were examined with a Hitachi HU12A Transmission Electron
Microscope (TEM) at 12,000-times magnification. All grids were viewed blind i.e. I
was unaware of the cohort of origin. 100 consecutive sperm were individually
assessed with regard to head shape and the state of the acrosome. The following
criteria were used to classify the sperm (Stock, 1990):
Group 1 consisted of acrosome-intact sperm i.e. the acrosomal cap was in place
and appeared normal.
Group 2 consisted of acrosome-reacting sperm i.e. the acrosomal cap was still in
place but appeared swollen, less electron dense, with vesicle formation, and it was
beginning to show signs of ballooning out.
Group 3 consisted of acrosome-reacted sperm i.e. the acrosomal cap was absent or
only scanty remnants of the acrosomal membranes were seen.
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Group 4 consisted of miscellaneous cells i.e. sperm showing abnormal morphology,
or where the sectioning of the sperm cell was at inclined plane and hence it could not
be classified.
A total of 100 cells in Group 1 to Group 3 was counted.
6.2.4 The Acrosome Reaction evaluation experiment
Prepared sperm suspensions were divided into 5 aliquots:- two aliquots
served as negative and positive controls of 0.2 mL each; and three aliquots served
as test samples of 0.4 mL each, as described in the flow diagram of the experimental
protocol (Fig. 6.1). 20 μL of EHBS buffer was added to the negative control sample.
40 μL of the appropriate PF concentration (10, 30, 50 mM PF/L) was added to test
samples to give a final concentration of 1, 3, 5 mM PF/L. All tubes were mixed
thoroughly and incubated at 370 C for 1 hour. At the end of incubation period, 0.2 mL
of each test sample was removed for video taping and for staining with vital and CTC
stain. The motion characteristics6 were recorded on video tapes for 2 minutes using
5 different fields employing a Celltrack/s analyzer (the tapes were analyzed at a later
date). 5 μL of Ionophore A23187 (final concentration 10 μM/L) was added to the
remaining 0.2 mL test sample; similarly, 5 μL of Ionophore A23187 (final
concentration 10 μM/L) was added to the positive control. All tubes were then
incubated for a further 30 minutes at 370 C before video taping and staining with vital
stain (section 6.2.2.2) and CTC stain (section 6.2.2.5). The slides were read by
counting the cells in 10 to 15 fields covering the whole slide until 100 sperm were
6
In light of the information gained from the studies reported in section 2.5.4, all CASA
measurements were done on tapes.
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counted. The results of total, partial, non-reacted and dead cells were expressed as
percentages of the total cells counted.
CASA parameters measured from the video tapes were VSL, VCL, LIN, ALH,
and MOT %. Of the 15 samples, 2 samples that had very good recovery of motile
sperm were randomly selected for Transmission Electron Microscopy study of
positive and negative controls to confirm that the AR was inducible with IP A23187.
6.3 Statistical Analysis
All data generated were assessed by histograms. Data were of normal
distribution, therefore a parametric approach was taken to analyze the data as
described in section 2.7. Significant mean differences were tested by the Paired t-
test.
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6.4 RESULTS
6.4.1 Conclusions of the pilot AR staining study
A pilot AR evaluation study was performed on 6 samples employing FITC-
PNA, FITC-PSA, and CTC staining with the AR induced by IP A23187. Although the
staining of sperm with FITC-PNA and FITC-PSA produced good fluorescence, the
decay to UV light was rapid, making it very difficult to differentiate between reacted
and non-reacted sperm. These two staining techniques also produced a high
variability in the number of sperm which appeared to have undergone the acrosome
reaction when repeated measurements were made.
CTC staining procedure produced good fluorescence, and no visual decay of
the fluorescence to UV light, which lasted for at least 7 days, was observed.
Differentiating between reacted and non-reacted cells were easy. Results obtained
from repeated measurements of the 6 samples were consistent. Hence, CTC
staining was the method of choice for the experiments described in this chapter.
6.4.2 Evaluation of the AR by CTC fluorescent staining
6.4.2.1 Effect of vital staining
The number of dead cells, as assessed by vital staining constituted less than
3% of all cells, as shown in Tables 6.1 and 6.2.
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6.4.2.2 Effect of Ionophore A23187 on the Acrosome Reaction
The positive control samples that had been induced by ionophore A23187 to
undergo the AR showed that 22% (p<0.0001) of the sperm were acrosome-reacted
compared with 8% of the controls which had undergone spontaneous AR (Table
6.1). This approximated to a 3-fold increase. Further, 7% of the sperm in the positive
control had undergone a partial acrosome reaction, compared with 4% in the
control (p<0.01).
6.4.2.3 Effect of PF on the Acrosome Reaction
Pentoxifylline, at 1, 3 or 5 mM /L, did not affect the proportion of sperm that
had undergone either total or partial AR, as shown in Table 6.1.
Table 6.1 The effect of PF on the acrosome reaction
Control +ve 1 mM PF/L 3 mM PF/L

5 mM PF/L
Control
d
Total Reacted -% 8 ± 1.10 22 ± 1.67 7 ± 1.15 10 ± 1.33 9 ± 1.00
Partial Reacted - % 4 ± 0.48 7b ± 1.01 4 ± 0.78 5 ± 0.70 4 ± 0.63
Non-Reacted - % 86 ± 0.97 69d ± 1.83 87 ± 1.61 83 ± 1.26 84 ± 1.28
Dead Cells - % 2 ± 0.43 2 ± 0.50 2 ± 0.39 2 ± 0.56 2 ± 0.46
N=15; Mean values ± SEM
Paired t-test showed significant mean differences between the +ve control values and the control
values at the following p values: a<0.05; b<0.01; c<0.001; d<0.0001
Analysis of Variance showed that there were no significant mean differences between the PF-treated
and the control samples.
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6.4.2.4 Effect of PF + !P A23187 on the Acrosome Reaction
In the presence of IP, PF-treated7 sperm showed a significant (p<0.0001)
decrease in the proportion which had undergone the AR compared with the positive
control values, is as shown in Table 6.2. Also the proportion which had undergone
the partial AR was significantly decreased at 1 mM PF/L (p<0.05) compared with the
positive control value. Thus, the proportion of non-reacted sperm was higher in the
PF-treated samples (p<0.001) compared with the positive control.
Table 6.2 The effect of PF+IP A23187 on the acrosome reaction
+ve Control 1 mM PF/L 3 mM PF/L 5 mM PF/L

(+ IP) + IP + IP + IP
Total Reacted -% 22 ± 1.67 12d ± 1.42 13d ± 1.11 11d ± 1.72
Partial Reacted - % 7 ± 1.01 5a ± 0.66 6 ± 0.55 6 ± 0.70
Non-Reacted - % 69 ± 1.83 80d ± 1.74 79c ± 1.23 81d ± 1.75
Dead Cells -% 2 ± 0.50 3 ± 0.37 2 ± 0.47 2 ± 0.64
N=15; Mean ± SEM

Paired t-test showed significant mean differences between the PF+IP samples and the +ve control at
the following p values: a<0.05; b<0.01; c<0.001; d<0.0001
7
1, 3 or 5 mM PF/L
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6.4.2.5 Comparison between PF treatment and PF+IP treatment on the AR
The comparison between the PF-treated sperm and PF+IP treated sperm
showed that the proportion which had undergone the AR was higher in the second
group, as is shown in Fig. 6.2. There was a 71% (p<0.01) greater number of sperm
that had undergone the AR at 1 mM PF/L + IP 23187 compared with 1 mM PF/L.
Similarly, there was a significantly higher proportion (30%) which had undergone the
AR at 3 mM PF/L + IP A23187 compared with 3 mM PF/L (p<0.01). However, there
was no significant difference in the number of sperm that had undergone the AR
between the samples treated with 5 mM PF/L + IP A23187 and 5 mM PF/L alone
(Fig. 6.2). The proportion of sperm that had undergone a partial AR were not
significantly different when these two groups (PF and PF+IP treated) were
compared. Also, the proportion of non-reacted sperm in these two groups were not
significantly different (i.e.differences were never greater than 12%).
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6.4.3 Evaluation of the AR by TEM in the negative & positive controls
TEM analysis of the AR showed that 5% of the sperm in the positive
control had the AR induced, compared with 2% which had spontaneously undergone
the AR in the negative control group. This approximated to about 3-fold increase.
There was no significant change in the proportion which had undergone the partial
AR between the two groups. Electron micrographs (Fig. 6.3a-d) show the various
stages of the acrosome reaction.
Fig. 6.3a shows a normal sperm head with intact acrosomal cap.
Fig. 6.3b&c shows acrosome-reacting sperm - they are swollen and beginning to
balloon out - (b), while (c) shows the vesicle formation.
Fig. 6.3d shows a sperm head after it had undergone the AR.
Table 6.3 The acrosome reaction of controls evaluated by TEM
Control - n1 / n2 +ve Control - n1 / n2
Total Reacted - % 1/2; x=2 4/5; x=5
Partial Reacted - % 3/4; x=4 3/2; x=3
Non-Reacted - % 96/94; x=95 93/93; x=93
Miscellaneous Cells 49/36 44/60
n1 / n2 represents sample 1 and 2 with mean. +ve control contains IP A23187

Miscellaneous Cells (numbers) = composed of abnormal shaped sperm counted but not included in
the percentage calculation.
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Fig 6.3 Electron Micrograph of sperm head x 18 000 magnification showing
various stages of the human sperm acrosome reaction

Bar = 2.5 μm
Fig 6.3a: Intact acrosome
Fig 6.3b: Swollen acrosomal matrix
Fig 6.3c: Raptured acrosome cap with remnants
Fig 6.3d: Acrosome cap is absent
1 = Outer acrosomal membrane
2 = Swollen outer acrosomal membrane with early stage of vesicle formation
3 = Vesicles
4 = Inner acrosomal membrane
5 = Remnants of outer acrosomal membrane
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6.4.4 Motion characteristics of sperm used in the AR experiment
The results are summarised in Table 6.4.
6.4.4.1 Effect of Ionophore (+ve control)
In the presence of Ionophore A23187 (IP), overall motility was significantly
reduced (p<0.05) compared with control values (Fig.6.4). ALH showed a similar
significant reduction (P<0.01), although there were no significant changes in VSL,
VCL and LIN.
6.4.4.2 Effect of PF
The results in Table 6.4 also indicate that in the presence of 1, 3, or 5 mM
PF/L, there were significant changes in VSL, VCL, and ALH, as expected. VCL
(Fig.6.5) and ALH (Fig.6.6) reached peak values (p<0.0001, compared with control
values) at 3 mm PF/L, when they were 16% and 12% higher than their respective
control values. The values of VCL at 1 and 5 mM/L were significantly increased (p
<0.0001) from the control value. The ALH value at 1 mM PF/L (p<0.05) and at 5 mM
PF/L (p<0.01) was also significantly raised from the control value. The VSL was
significantly higher at 1 mM PF/L (p<0.001), 3 mM PF/L (p<0.01) and at 5 mM PF/L
(p<0.0001) than control value. There were no significant changes in LIN and MOT %
when compared with the control values.
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6.4.4.3 Effect of PF+IP
The results in Table 6.4 shows that when IP A23187 was added to PF-treated
sperm (PF+IP), all sperm motion characteristics declined significantly compared
with those of PF-treated sperm. The MOT% decrease (Fig.6.4) at 1 mM PF/L was
12% (p<0.01) and at 3 mM PF/L was 20% (p<0.001), while the biggest decrease of
37% (p<0.001) occurred at 5 mM PF/L. Analysis of Variance showed that MOT% of
PF+IP samples was significantly reduced compared with the control MOT%.
The VCL (Fig.6.5) at 1 mM PF/L showed a drop of 7% (p<0.05), at 3 mM
PF/L a drop of 14% (p<0.01) and at 5 mM PF/L the drop was 13% (p<0.02). Analysis
of Variance showed that the VCL of PF+IP samples was not significantly different
from the control VCL value.
The ALH (Fig.6.6) of PF+IP samples showed a drop of 4% at 1 mM PF/L, a
significant drop of 8% (p<0.05) at 3 mM PF/L and at 5 mM PF/L the drop was 6%.
Similarly, the VSL of PF+IP samples showed a 25 % (p<0.05) decline in value at 1
and 3 mM PF/L while at 5 mM PF/L the drop was 35% (p<0.01). The linearity of
PF+IP samples showed a decrease of 13% (p<0.05) at 3 mM PF/L while at 5 mM
PF/L the drop was 24% (p<0.01) compared with PF-treated samples.
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6.5 DISCUSSION
The CTC staining procedure produced good fluorescence, and no visual
decay of the fluorescence to UV light was observed. Differentiating between reacted
and non-reacted cells was easy. The repeated measurements were consistent.
Hence, CTC staining was the method of choice for the experiments described in this
chapter.
The TEM analysis of the negative control samples showed that lower
proportion of sperm had undergone the AR than the fluorescent staining method
because, it is probable, that in the TEM analysis greater proportion of sperm were
classified as abnormal under 12 000-times magnification compared with the 200-
times magnification used in the fluorescent technique. At 12 000- times
magnification, greater details of cell ultrastructure are seen, leading me to classify
any sperm that does not appear normal as abnormal and the acrosome-reacted
sperm can be visually identified. However, under 200-times magnification, sperm
cannot be discriminated between normal and abnormal, and furthermore,
classification of the acrosome-reacted sperm is based on external fluorescent
appearance. TEM analysis of the AR showed that sperm treated with IP A23187
had a 3-fold increase in the number of acrosome-reacted cells compared with the
control sample, a result consistent with results from the CTC staining technique.
The study also demonstrated that PF increases sperm motion characteristics,
but that this enhancement was significantly reduced in the presence of Ionophore
A23187. De Jonge et al. (1989) showed that Ionophore A23187 could induce the AR
and that a high concentration of IP A23187 (>40 μM) had a deleterious effect on
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sperm motility %. This observation is supported by the findings of White et al.
(1990), who showed that a high concentration of IP A23187 significantly reduced
sperm motility. My results are consistent with these studies, in which the positive
control sample, which was treated with Ionophore A23187, showed a 3-fold increase
(p<0.001) in the proportion of sperm that had undergone the AR together with a 7%
reduction (p<0.05) in MOT %. This demonstrates that even at very low concentration
of IP A23187 (10 μM), adverse effects on sperm motility occur. This study also
showed that sperm treated with 5 mM PF/L, and in the presence of IP A23187, has
a highly significant further reduction in MOT % compared with both the control and
the PF-treated sperm, but that there was no corresponding significant change in VSL
and VCL, although there was a significant change in LIN and ALH compared with the
control values. This would suggest that a subpopulation/subset of sperm were more
labile/sensitive to chemicals. These more sensitive sperm are immobilized (thereby
accounting for the decrease in motility), but still move their heads vigorously (thereby
accounting for the increase in the ALH value). Similarly, at a lower concentration of
PF (i.e. 1 and 3 mM/L), in the presence of Ionophore A23187, similar effects on MOT
% were observed, but to a lesser extent. This would indicate a dose-response
relationship; the concentration of PF (in the presence of IP A23187) was proportional
to the reduction in sperm MOT %. A highly significant negative correlation between
PF concentration in the presence of a constant concentration of IP A23187 and MOT
% reduction was found: r = -0.98 (p<0.02).
It has been demonstrated (Tesarik et al., 1992b; Tesarik and Mendoza, 1993;
Carver-Ward et al., 1994; Tasdemir et al., 1993) that PF does not, by itself, induce
the acrosome reaction. My results agree with this conclusion, by showing that at 1, 3
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and 5 mM PF/L, there was no significant change in the number of acrosome-reacted
sperm.
However, when sperm were treated with PF, and then exposed to Ionophore
A23187, the Ionophore was less effective at stimulating the AR than it was when the
sperm were not treated previously with PF. This finding contradicts the current view
of the effects of PF+IP on the AR. Several authors (Tesarik and Mendoza, 1993;
Carver-Ward et al., 1994; Tasdemir et al., 1993) have shown that sperm treated with
PF, and challenged subsequently with Ionophore A23187, had undergone increased
induced acrosome reactions. This apparent discrepancy could be due simply to the
culture medium used. Table 6.5 shows the different methodologies used by the
above three groups of authors and myself. Other than some minor differences in the
methodology, the major difference between the studies was the culture medium
used. Tesarik and Mendoza, (1993) used B2 medium, Carver-Ward et al., (1994)
and Tasdemir et al., (1993) used Human Tubal Fluid (HFT) medium, whereas I have
used Earles medium containing Hepes (EHBS). I suspect that there was some
chemical interaction between the Hepes, PF and Ionophore A23187, giving rise to
some unknown chemical molecule (or molecules). It is possible that this chemical
molecule(s) may have inhibited the induction of the AR. This hypothesis requires
verification.
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Table 6.5 Comparison of the experimental conditions used in

various studies to evaluate the AR
Conditions Tesarik & Carver-Ward Tasdemir Myself

Mendoza, 1993 etal., 1994 etal., 1993 1994
Total number of samples 18 20 33 15
Sperm separation Percoll gradient 'swim-up' 'swim-up' 'swim-up'
Incubation time (min) :

PF 30 30 60 60
IP 30 45 60 30
Conc. of PF - mM/L 3.6 3.6 7.2 3.0
Conc. of IP A23187 - μM 10 10 10 10
Incubation buffer B2 HFT/BSA HFT/BSA EHBS/HSA

+ Hepes
Method of detection PSA Flow PSA CTC

cytometric
Result (%) :
Control 10 10 8 8
IP induced 14 23 27 22
PF induced 11 10 8 10
PF+IP induced 48 29 32 13
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A survey of the literature (Perreault and Rogers, 1982; Rogers and Perreault,
1990, Kay et al., 1993; Pang et al., 1993) appears to suggest that phosphodiesterase
inhibitors not only enhance sperm motility by inhibiting cAMP breakdown, but may
also be implicated in other sperm functions like capacitation, hyperactivation and the
AR, and that all of these might be modulated by cAMP. The prevailing hypothesis
(Tesarik et al., 1992b; Tesarik and Mendoza, 1993) suggest that PF sensitizes the
complex physiological acrosome on the sperm head. Chemical stimuli like Ionophore
A23187 which induce the AR may increase membrane permeability towards ionic
calcium influx by acting through the Na+/Ca2+ antiporter (Roldan and Harrison, 1990;
Fraser and McDermott, 1992). Increased calcium levels increase the intra-acrosomal
pH by acting via a Ca2+-dependent ATPase, and this in turn activates proacrosin to
acrosin (Meizel, 1984). This leads to fusion between the plasma and outer
acrosomal membranes, culminating in exocytosis of acrosomal contents
(Yanagimachi, 1981; Fraser, 1984; Hinrichsen-Kohane et al., 1984; Langlais and
Roberts, 1985). The role of channels, antiporters or ATPases in the regulation of
calcium is still a controversial issue. Different lines of evidence appear to suggest
that mammalian sperm do not have voltage-operated channels. The reasons
attributed for this assumption are based on the slowly emerging following facts:- (a)
changes in membrane potential do not trigger an AR; (b) specific antagonists of
voltage-operated Ca2+-channels do not inhibit Ca2+ uptake; and (c) treatment of
sperm with different Ca2+-channel antagonists do not prevent the acrosome reaction
(Roldan and Harrison, 1990). The lack of evidence for ion channels in sperm has
resulted in people postulating that the major mechanism regulating Ca 2+ influx at the
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time of the AR might be a Na+/Ca2+ antiporter. However, studies in mouse sperm
have shown that the AR can be inhibited by some Ca++-channel antagonist (Fraser
and McIntyre, 1989).
Recent studies (Fraser et al., 1993) demonstrate that ions other than Ca 2+
may also be involved in acrosomal exocytosis. In mouse sperm, Na+ is implicated in
the AR via the Na+-H+ exchange mechanism. It is thought that, at the time when the
mouse sperm comes in contact with the zona pellucida, there occurs an influx of Na +
into the fertilizing sperm, causing a rise in intracellular pH that in turns opens Ca 2+-
channels to allow the influx of Ca2+, resulting in the acrosome reaction.
An increasing body of recent evidence shows that cholesterol (lipid) may be
involved in acrosomal exocytosis (reviewed by Benoff, 1993). Phospholipid constitute
about 60 to 75% of sperm lipids (Scott, 1973). Lipid modifications on the sperm head
undoubtedly play a fundamental role in the acrosome reaction, since extensive
membrane alterations are involved. Although the exact mechanism of the role played
by lipid has not been addressed, electron microscopic studies have shown changes
in sterol and anionic phospholipid distribution in human sperm plasma membranes
during in vitro capacitation. For example, the cholesterol concentration is
preferentially reduced within the plasma membrane domain overlying the acrosomal
cap during capacitating incubations (Tesarik and Flechon, 1986).
In conclusion, there are a number of hypotheses to account for the molecular
mechanisms leading to the acrosome reaction, but no clear evidence to support any
of these hypotheses. My results, to recollect, showed that PF had no significant
effect on the acrosome reaction, and that PF treatment inhibited the subsequent
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effect of Ionophore A23187 in enhancing the acrosome reaction. CASA
measurements show that PF+IP greatly affects sperm motility. In the treatment of
male factor infertility, it would be advantageous to the patient if it could be
demonstrated that sperm treated with stimulant were positively correlated with
increased acrosomal exocytosis at the site of sperm-oocyte fusion, which could lead
to an improvement in fertilization rate.
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CHAPTER 7
THE EFFECT OF PENTOXIFYLLINE ON THE BINDING OF
SPERMATOZOA TO THE ZONA PELLUCIDA
Sciences may be learned by rote, but wisdom not.
Lawrence Sterne 1713-1768
7.1 INTRODUCTION
The beginning of a new individual involves the fusion of a spermatozoon and
oocyte. Therefore, the process of fertilization is fundamental to the maintenance of
life in both plants and animals. In the past two decades, the understanding of the
biology and chemistry of mammalian fertilization has led to improvements in the
treatment and diagnosis of infertility. Although the sperm can be brought to the
vicinity of the oocyte, unless the sperm binds and proceeds with the penetration of
the zona pellucida (zona), there cannot be a new creation of life. Thus,
understanding the fundamental mechanisms in sperm-zona interactions, which
represent a complex sequence of mutually linked events, is crucial. Sperm binding
and penetration of the oocyte depend on a finely tuned balance between the actions
of different types of cells and intercellular matrices (Tesarik and Testart, 1989).
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Binding to, and penetration of, the oocyte by the capacitated sperm is
species-specific (Wassarman, 1987). After the cumulus mass and corona radiata,
the zona is the next barrier the sperm has to cross in the process of fertilization.
Ultrastructural studies (Sathananthan et al., 1982) in the human have shown that
sperm attached to the zona have intact acrosomes and that their plasma membranes
in contact with the zona are often swollen before they undergo the acrosome
reaction, which is a prerequisite for fertilization. Cross et al. (1988) showed that
acrosome-reacted and acrosome-intact sperm are both equally effective in initiating
binding to the zona in human. The zona pellucida in mouse oocytes is a sulphated
glycoprotein composed of several families of glycoproteins: ZP1(200 kDa), ZP2 (120
kDa), and ZP3 (83 kDa) (Bleil and Wassarman, 1980a). ZP3 has been identified as
the prime sperm receptor and inducer of the sperm acrosome reaction, while ZP2
has been shown to be a secondary sperm receptor (Bleil and Wassarman, 1980b,
Wassarman, 1988a). Recently, the genes encoding mouse ZP2 and ZP3 have been
characterized and sequenced (Kinloch et al., 1988; Kinloch and Wassarman, 1989;
Lunsford et al., 1990). Examination of zona by scanning electron microscope has
shown that the outer surface of the zona has a fenestrated, lattice-like appearance,
whereas the inner surface appears particulated (Greve and Wassarman, 1985).
Similarly, in the human, the zona pellucida consists of ZP1 (90-110 kDa), ZP2 (64-
78 kDa), and ZP3 (57-73 kDa) (Shabanowitz and O' Rand, 1988a; 1988b); these are
significantly smaller than the molecular weights of their mouse counterparts. ZP3 has
been cloned and sequenced using mouse ZP3 cDNA as a probe (Chamberlin and
Dean, 1990; van Duin et al., 1992). It has been demonstrated that there is high
degree of conservation between the coding regions of human ZP3 and mouse ZP3,
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and similarly with human ZP2 and mouse ZP2 (Liang and Dean, 1993). Recently, the
presence of steroid receptors on the membranes of human sperm was identified,
and their mechanism of action partially characterised (reviewed by Revelli et al.,
1994). The importance of steroid receptors lies on the fact that steroids have been
shown to induce the acrosome reaction, and preserve sperm viability and motility.
However, it is yet to be shown if these steroid receptors play a part in the sperm-
zona interactions.
In some cases, infertility may be caused by the failure of the sperm to interact
with the oocyte, and the reason for this problem could lie with the sperm, or the
oocyte, or both. Thus, understanding sperm-zona interaction could assist and
improve the treatment in some cases of infertility. Pentoxifylline, a
phosphodiesterase inhibitor, has been shown to increase sperm motion
characteristics (chapters four and five ) and it would be interesting to find out what
effect, if any, the drug may have on sperm-zona pellucida binding.
Evidence presented in chapter 6 showed that PF does not induce the
acrosome reaction, yet there are studies (Yovich et al., 1990; Sikka and Hellstrom,
1991) showing that PF treatment of sperm was correlated with an improvement in
the fertilization rate in in vitro fertilization programmes. One possible explanation is
that PF enhances sperm-zona interaction. Therefore, the present study was
undertaken to investigate the effect of PF on sperm-zona binding.
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To study the effect of PF on the binding of sperm to the zona pellucida, two
methods were used. The first method is called the intact-zona assay, and uses
whole human oocytes while the second method is called the hemizona assay, and
uses bisected human oocyte.
7.2.1 Rationale of intact-zona binding assay
The large variability in the number of sperm binding to any one intact-zona,
together with the lack of sufficient oocytes to overcome this variability statistically,
has led to a particular experimental design being adopted for this study. In this
design, a single intact zona is exposed to both control sperm and test sperm
simultaneously, with the two types of sperm competing for binding sites on the zona.
The two sperm populations can be differentiated by labelling either the control or the
test sperm with a fluorescent stain. In the first set of test experiments, the control
sperm were labelled. In the second set of test experiments, the PF-treated sperm
were labelled. The reason for doing this was to examine the effect of interaction
between PF and fluorescent stain on the binding of sperm to the intact-zona and also
to examine the effect that the fluorescent labelling process might have on the
binding. Using this experimental approach, PF must be removed by washing the
sperm (now called PF-pretreated) prior to co-incubation with the intact-zona, to
ensure that the control population of sperm is not exposed to the drug. Preliminary
experiments were done to examine the effect of fluorescent stain on binding.
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7.2.1.1 Rationale of hemizona binding assay
Owing to the large variability in the number of sperm binding to any one intact-
zona, the hemizona assay were developed, which uses bisected oocytes. In my
experiments, one half of the zona was used as the control and the other matching
half was used as the test hence fluorescent staining was not required and the PF-
treated sperm were not washed. Preliminary experiments were done to assess the
effect of cutting the oocytes into equal halves on sperm binding.
7.2.2 Oocytes
Human oocytes (usually treated like gold dust!), from the in vitro fertilization
program of the Royal Postgraduate Medical School, London, that had been exposed
to sperm but failed to fertilize, were kindly donated towards this study. These
oocytes were non-viable, and had no developmental potential. Any residual potential
was nullified by storage for 5 days or greater at 40 C. It has been shown that low
temperature causes the depolymerisation of major structural protein of microtubules
in the oocytes (Osborn and Moor, 1984), making the oocytes non-viable. Individual
oocytes were kept in 1 mL EHBS culture medium (preparation of media as described
in section 2.2.1) in capped tubes at 40 C until required. They were kept like this for
up to three weeks before being discarded. [Ethical and legal implication ensures that
the use of virgin oocytes for experimental research is severely restricted, although
this would be the best material for this study].
7.2.3 Preparation of intact-zona

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Each oocyte (now called intact-zona for the purpose of this study) was
examined with an inverted microscope (Olympus CK, Olympus, Japan) and any with
more than five sperm bound to it were rejected. Selected intact-zona was then
washed in EHBS medium three times. Washing was done by placing a drop of 0.2
mL EHBS medium in a culture dish (Falcon 3002, Beckon Dickson, UK) and
aspirating the intact-zona in and out of the EHBS medium several times, using a
finely drawn glass pipette to dislodge any loosely adherent sperm. The process was
repeated three times with fresh medium. The intact-zona was then transferred to 0.5
mL fresh medium in a culture dish and kept at room temperature (230 C) until
required.
7.2.4 Dissection of intact-zona for hemizona assay
Prepared intact-zona was examined with a dissecting microscope (Olympus
SZ60, Olympus, Japan) at 60 times magnification. Each intact-zona was cut into
equal hemispheres by Dr KS Lindsay with a 25G needle attached to a syringe,
using the sharp edge of the needle as the knife. Great care was taken to cut into
equal halves, and any portion that did not appear to be a complete half zona was
rejected. Each half zona in 0.5 mL EHBS medium was kept in a separate labelled
culture dish at room temperature (230 C) until required.
7.2.5 Preparation of sperm suspension
Semen samples were obtained from normal individuals and processed as

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described in section 2.3. Sperm suspensions of about 8 million sperm per millilitre
were prepared as described in section 2.4.2 using the migration centrifugation
method. Sperm suspensions for control experiments were diluted with EHBS
medium to obtain a concentration of about 4 million sperm per mL.
7.2.6 Preparation of Fluorescein isothiocyanate (FITC) stain
and labelling of sperm
The technique of fluorescein labelling of sperm was modified from the method
of Parrish and Foote (1985) as described by Liu et al. (1988). 1 mg fluorescein
isothiocyanate (FITC, Sigma, UK) was dissolved in 0.1 mL of 0.1 M potassium
hydroxide and diluted to 5 mL with Dulbecco phosphate-buffered saline (Bio-
Whittaker, USA) containing D-glucose and sodium pyruvate. The fluorochrome
solution was stored at 40 C in a plastic tube wrapped in aluminum foil for up to 1
week.
Sperm pellets were prepared using 0.5 mL of the 8 million sperm/mL
suspensions by centrifuging them at 600g for 5 minutes and discarding the
supernatant. The pellet was suspended in 150 μL of the fluorochrome solution and
incubated at 370 C for 15 minutes. At the end of the incubation period, the sperm
were recovered by centrifuging at 600g for 5 minutes and discarding the supernatant.
The pellet was resuspended in 5 mL of EHBS medium and the centrifugation
process repeated. The resulting fluorescein labelled pellet was resuspended in
EHBS medium
to obtain a concentration of approximately 6 million sperm per mL. This labelled
sperm suspension was kept at 370 C in the dark until required.

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7.2.7 Preparation of PF-pretreated sperm
Sperm suspensions were divided into 3 parts of 0.5 mL each. Each aliquot
was exposed to 50 μL of 1, 30 or 50 mM PF/L (final concentration was 0.1, 3, or 5
mM PF/L). Preparation of PF was as described in section 2.2.3. [Please note: The
choice of 0.1 mM PF/L was based on results from a related project which showed
that sperm exposed to 0.1 mM PF/L had greater motility after 20 hours incubation
compared with sperm that were exposed to 1 mM PF/L (Moohan et al., 1993)]. After
a 1 hour incubation at 370 C, 4 mL of EHBS medium was added to the tubes, which
were mixed thoroughly, centrifuged at 600g for 5 minutes and the supernatant
discarded. The process was repeated to remove any excess PF present in the
sperm suspension. The resulting PF-pretreated pellet was resuspended in fresh
EHBS medium to obtain a sperm concentration of about 4 million per mL and kept at
370 C until required.
7.2.8 Effect of FITC stain labelling on sperm motility
A pilot evaluation study using 6 sperm samples was done to determine
whether labelling of sperm with FITC stain would affect sperm motion characteristics.
Each sperm suspensions was divided into 4 portions of 0.5 mL each and separate
sperm pellets prepared. The first pellet was treated as a control, to which was added
300 μL EHBS medium, while to pellets 2, 3, and 4 were added 150, 300, 500 μL of
FITC stain and the labelling carried out as described in section 7.2.6. The labelled
sperm suspension was then analysed using a Celltrack/s Motion Analyser and the
following motion characteristics were measured: - VSL, VCL, LIN, ALH, and MOT %.
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The results obtained showed that all motion characteristics were significantly
(p<0.05) depressed when 300 and 500 μL of FITC stain was added. For example,
the VCL value was depressed by 30 and 43% with 300 and 500 μL of FITC stain,
respectively. Similarly, the ALH value was decreased by 36 and 51% with 300 and
500 μL of FITC stain, respectively. However, the addition of 150 μL of FITC stain to
the sperm pellet did not affect any of the CASA parameters. In light of this
information, all labelling of sperm pellets was done with 150 μL of FITC stain.
7.2.9 Intact-zona binding assay - methodology
The principle of the method used, was similar to that described by Burkman et
al. (1988; 1990) and Franken et al.(1989a,1989b) but modified as shown in Fig 7.1.
100 μL of sperm suspension was added to an intact-zona in 500 μL of EHBS
medium in a culture dish and covered. The mixture in the culture dish was mixed
gently and incubated at 370 C overnight (20 hours). At the end of the incubation
period, the intact-zona was removed with a finely drawn glass pipette and washed 3
times in EHBS medium. The washing was done as described in section 7.2.2. The
washed intact-zona was removed with an Eppendorf Varipette 4710 pipette
(Eppendorf-N-Gmbh, Hamburg, Germany) in 0.5 μL EHBS medium and placed in a
microwell produced by PFTE (Teflon)
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coating on a disposable-diagnostic slide (Muratech Scientific, UK). The intact-zona
was solubilized on the slide by adding 0.5 μL of 1 M HCL and mixed with a 21G
needle. At this concentration of acid, the sperm were intact and undamaged visually.
The slide was covered with a coverslip and the edges sealed with nail varnish and
stored in the dark until it was read within 24 hours.
All the sperm present in the microwell were systematically counted from left to
right field under a light microscope set at 200-times magnification. The total number
of sperm (unlabelled and labelled) present was recorded. Labelled sperm were
examined under a fluorescence microscope (section 2.1.1) at 450 to 490 nm
wavelength, 200-times magnification. All the labelled (fluorescent green) sperm
present in the microwell were manually re-counted and recorded. The difference
between the total and labelled sperm value was equal to the number of unlabelled
sperm bound to the intact-zona, and enabled a ratio of labelled : unlabelled sperm to
be determined.
7.2.10 Hemizona binding assay - methodology
The methodology used for hemizona assay was essentially the same as
described above, and is shown in Fig. 7.1. However, instead of using intact-zona,
bisected zona were used, the sperm were unlabelled, and PF-treated sperm were
not washed. One half of the cut zona was exposed to control sperm and the other
matching half was exposed to test sperm.
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7.2.11 Effect of FITC labelled sperm on intact-zona binding
Sperm suspensions were prepared as described in section 7.2.4. Each sperm
suspension was divided into 2 parts of 0.5 mL each. The first aliquot was treated as
a control (non-labelled) and the second aliquot of sperm was labelled with FITC stain
as described in section 7.2.6. The motion characteristics were measured using
CASA. Two intact-zona were selected and prepared as described in section 7.2.3.
To the first intact-zona, initially in 0.5 mL of EHBS medium in a culture dish, 50 μL
of control sperm suspension was added. The second intact-zona received 50 μL of
labelled sperm suspension. The intact-zona binding assay was carried out as
described in section 7.2.9. All the unlabelled and labelled sperm were counted and
the percentage of sperm binding to the intact-zona was calculated.
7.2.12 Assessment of cutting the intact-zona into equal halves
The intact-zona was selected and prepared as described in section 7.2.3 and
was cut into 2 equal halves as described in section 7.2.4. Sperm suspensions of 4
million sperm/mL concentration were prepared as described in section 7.2.5. To
each hemizona, initially in 0.5 mL EHBS medium in a culture dish, 50 μL of sperm
suspension was added, both hemizona receiving the same sample sperm
preparation. The hemizona binding assay was carried out as described in section
7.2.10. All sperm on each slide were counted and the percentage of sperm binding to
the hemizona was calculated.
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7.2.13 Effect of PF on intact-zona binding
Sperm suspensions, either control or PF-treated were prepared as described
in section 7.2.5. Each sperm suspension was divided into 5 parts of 0.5 mL each.
The first aliquot was treated as a control and was labelled with 150 μL of FITC stain
as described in section 7.2.6. Aliquots 2, 3 and 4 were treated with PF (final
concentration of 0.1, 3, and 5 mM PF/L) as described in section 7.2.7. Aliquot 5,
which served as a positive control to show the effect of PF, was exposed to 30 mM
PF/L (final concentration 3 mM PF/L). At the end of an hour incubation at 37 0 C,
CASA measurements were recorded.
Three intact-zona were selected and prepared as described in section 7.2.3.
To the first intact-zona, initially in 0.5 mL of EHBS medium in a culture dish, 50 μL of
labelled-control sperm was added followed by 50 μL of 0.1 mM PF/L pretreated
sperm. This process was repeated to intact-zona 2 and 3, using 3 and 5 mM PF/L
pretreated sperm. The intact-zona binding assay was carried out as described in
section 7.2.9. All the labelled and total sperm on each slide were counted and the
percentage of sperm bound to the intact-zona was calculated.
[Please Note: In the second set of experiments, the PF-pretreated sperm were
labelled with FITC stain.]
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7.2.14 Effect of PF on hemizona binding
The hemizona were prepared as described in section 7.2.4. Sperm
suspensions of 4 million sperm/mL were prepared as described in section 7.2.5, and
divided into 2 aliquots. One aliquot was treated as a control and received 50 μL of
EHBS medium. 50 μL of 30 mM PF/L (final concentration was 3 mM PF/L) was
added to the second aliquot, mixed thoroughly and incubated at 370 C. At the end of
an hour incubation, the CASA measurements were taken. 50 μL of the control sperm
suspension was added to one half of the hemizona while the other matching half
zona received 50 μL of the PF-treated sperm. The hemizona binding assay was
carried out as described in section 7.2.10. All sperm on each slide were counted
under a light microscope and the percentage of sperm binding to the hemizona was
calculated.
7.3 Statistical Analysis
Histograms of data showed that the percentage of sperm bound to the zona
were of normal distribution, therefore a parametric approach was taken to analyze
the data, as is described in section 2.7. Significant mean differences between assay
were tested by Two sample t-test and Paired t-tests.
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7.4 RESULTS
7.4.1 Effect of FITC labelling of sperm on their ability to bind to intact-zona
The results (Table 7.1) showed that FITC stain labelling of sperm had no
significant effect on the binding to the intact-zona. 50% of the labelled and 50% of
the non-labelled sperm were bound to the intact-zona. The results also showed that
there were great variability in the number of sperm bound to the different intact-zona;
for example, the range was from 85 to 306 sperm per intact-zona.
Table 7.1 Effect of FITC labelling of sperm on their ability to

bind to intact-zona
Intact-zona No. Total No. of No. of labelled % of labelled

sperm bound sperm bound sperm bound
1 232 121 52
2 180 96 53
3 781 306 48
4 562 257 46
5 288 153 53
6 171 85 50
Mean % of labelled sperm bound to intact-zona 50 ± 1.2
N=6; Mean ± sem
The motion characteristics of sperm used in the above assay were all consistent with
control values of previous studies.
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7.4.2 Assessment of cutting the intact-zona into equal halves
The results (Table 7.2) showed that after cutting the intact-zona into two
halves, approximately equal number of sperm were bound to each hemizona. 50% of
the sperm bound to the 1st half and 50% to the 2nd half of the intact-zona. Again, the
results showed a great variability in the number of sperm bound to the different
hemizona; for example, the range was from 124 to 873 sperm per hemizona.
Table 7.2 Evaluation of cutting the intact-zona into equal halves
Hemizona No. Total No. of No. of sperm % of sperm

st
sperm bound bound to 1 half bound to 1st half
1 342 183 54
2 268 124 46
3 588 285 48
4 897 461 51
5 1638 873 53
6 662 323 49
7 1207 618 51
8 587 277 47
Mean % of sperm bound to 1st half - hemizona 50 ± 1.4
N=8; Mean ± sem
The motion characteristics of sperm used in the above assay were all consistent with control
values of previous studies.
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7.4.3 Effect of PF on intact-zona binding using control sperm labelled with
FITC The calculated percentages of sperm binding to the intact-zona are
summarised in Table 7.3. The results showed that PF significantly inhibited
(p=0.0001) sperm binding to intact-zona at 0.1, 3, & 5 mM/L compared with the
control. However, the results also showed that there were no significant differences
among the various PF concentrations in the degree of inhibition of sperm binding to
the intact-zona, which averaged 23%. Therefore, there were no significant
differences in the percentage of labelled control sperm binding to the intact-zona at
the various PF concentrations, which averaged 77%. Consistent with previous
experiments, the number of sperm bound to the intact-zona varied from 65 to 530
sperm per intact-zona (data not shown).

control sperm labelled with FITC
Concentration of Pentoxifylline
0.1 mM/L 3 mM/L 5 mM/L
Control sperm - % 75 ± 3.0 79 ± 2.3 76 ± 5.2
PF-treated sperm - % 25a ± 3.0 21a ± 2.3 24a ± 5.2
N=8; Mean ± SEM; Significant mean difference from control values shown
by p value at a<0.0001
The motion characteristics of sperm used in the above experiment were analyzed by Paired
t-test and showed a significant difference (p=0.05) between the control and the positive
control, as expected. For example, the VCL was raised by 28% and the ALH was raised by
36% in the PF-treated sperm.
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7.4.4 Effect of PF on intact-zona binding using PF-pretreated
sperm labelled with FITC
The calculated percentages of sperm binding to the intact-zona are
summarised in Table 7.4. The results showed that PF significantly inhibited
(p=0.0001) sperm binding to intact-zona at 0.1, 3, & 5 mM/L compared with the
control. However, the results also showed that there were no significant differences
among the various PF concentrations in the degree of inhibition of sperm binding to
the intact-zona, which averaged 15%. Therefore, there were no significant
differences in the percentage of control sperm binding to the intact-zona at the
various PF concentrations, which averaged 85%. Consistent with previous
experiments, the number of sperm bound to the intact-zona varied from 82 to 509
sperm per intact-zona (data not shown).

PF-pretreated sperm labelled with FITC
0.1 mM/L 3 mM/L 5 mM/L
Control sperm - % 87 ± 0.8 83 ± 1.6 85 ± 1.6
PF-treated sperm - % 13a ± 0.8 17a ± 1.6 15a ± 1.6
Mean ± SEM; N=8; Significant mean differences from control values shown
The motion characteristics of sperm used in the above experiment were analysed by Paired
t-test and showed a significant difference (p=0.05) between the control and the positive
control as expected. For example, the VCL was raised by 24% and the ALH was raised by
26% in the PF-treated sperm.
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7.4.5 Effect of PF on sperm-hemizona binding
The calculated percentages of sperm binding to the hemizona are
summarised in Table 7.5. The results showed that PF significantly enhanced
(p=0.001) sperm binding to hemizona. In the presence of 3 mM PF/L, 60% of the
PF-treated sperm were bound to the hemizona compared with 40% of the control
sperm. Consistent with previous experiments, the number of sperm bound to the
hemizona varied from 56 to 1340 sperm per hemizona (data not shown)
Table 7.5 Effect of PF on sperm-hemizona binding
Hemizona number % of control sperm % of PF-treated sperm

bound to hemizona bound to hemizona
1 41 59
2 41 59
3 40 60
4 39 61
5 41 59
6 45 55
7 31 69
8 42 58
Mean % bound 40 ± 1.4 60a ± 1.4
N=8; Mean ± SEM; Significant mean differences from control values shown
The motion characteristics of sperm used in the above experiment were analysed by Paired
t-test and showed a significant difference (p=0.05) between the control and PF-treated
sperm, as expected. For example, the VCL was raised by 21% and the ALH was raised by
23% in PF-treated sample.
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7.4.6 Comparison between the intact-zona binding
and hemizona binding assays
At all three concentrations of PF, PF-pretreated sperm had a very much
reduced (4-fold) chance (p<0.0001) of binding to the intact-zona compared to the
control sperm (Table 7.6). In the hemizona assay, sperm in the presence of 3 mM
PF/L had a 50% greater chance (p<0.0001) of binding to the hemizona, compared to
the control sperm.
Table 7.6 Comparison of percentage of sperm binding to intact-zona

and hemizona
Methodology
Control 0.1 mM/L 3.0 mM/L 5.0 mM/L
Intact-zona -% 80 ± 2.3 19a ± 2.2 19a ± 2.5 20a ± 2.8

N=48 N=16 N=16 N=16
Hemizona -% 40 ± 2.3 _ 60a ± 2.5 _

N=8 N=8
Mean ± SEM; Significant mean differences from the control values shown
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7.5 DISCUSSION
The results obtained in this study show that labelling sperm with 150 μL of 0.2
mg FITC/mL stain had no detectable adverse effect on sperm binding to intact-zona,
a finding consistent with that of Liu et al. (1988). However, sperm that had been
stained with 300 and 500 μL of FITC showed a significant reduction in motion
characteristics. Therefore, it is probable that staining with FITC, even at a lower
concentration, may have subtle effects on sperm that might have implications for the
sperm-zona binding. Sperm that had been challenged with PF, which was then
removed by washing before the binding assay, showed a decreased ability to bind
with intact-zona compared with the control sperm. However, in the presence of 3 mM
PF, sperm showed a significantly increased ability to bind to the hemizona compared
with the control sperm. The wide variation in the number of sperm bound to different
zona in the same treatment group was similar to the results reported by Liu et al.
(1990).
Most studies (Franken et al., 1989b; Liu et al., 1990; Liu and Baker, 1992;
1994a; 1994b) on sperm-zona interaction cite the number of sperm bound to each
zona as greater than 100 (if there are more than 100 sperm present on the zona)
rather than counting them precisely. Technically, it is very difficult to accurately count
sperm tightly attached to the zona, as the sperm tend to bind in a 3-dimensional
manner on the surface of the intact-zona, which is then examined with a
microscope in 2 dimensions. Further, binding occurs in clusters and clumping occurs.
In my study, the problem of accurately counting the bound sperm has been
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overcome by solubilising the zona in acid, which does not appear to damage the
sperm integrity. Solubilization also assist in dispersing the tightly bound sperm in
clumps around the microwell, making it easier to count, although the counting is
tedious. This may account for the high number of sperm counted (as many as 1340)
in this study. It is probable that the high numbers of sperm attached to the zona may
also be attributed at least partially to 'non-specific' binding to the zona on both sides
(outer coat and inner coat) in the hemizona assay.
One of the main difficulties in the human sperm-zona interaction studies is
obtaining sufficient number of oocytes to overcome statistically the high variability in
the number of sperm binding to any one zona. Thus, the hemizona assay appeared
to be a solution. Each half zona possesses the same binding characteristics, thus
overcoming the variability between control and test experiments. However, the main
drawback to hemizona assay is the problem of cutting an oocyte into approximately
equal halves which requires micro-manipulation. Therefore, in order to study a range
of PF concentrations (0.1, 3.0, 5.0 mM PF/L) on sperm-zona interaction, I used the
intact-zona method with some modification. In the first set of experiments, the control
sperm were labelled with FITC stain, whereas in the second set of experiments, the
PF-treated sperm were labelled. It is thought that the advantages of this
procedure would be that it takes into account any effect of staining on the ability of
sperm to bind, and it would also demonstrate if there was any interaction between
PF and FITC stain that would affect the sperm-zona interaction. Results from this
study showed that when control sperm were labelled with FITC stain, the average
binding of PF-treated (across the concentrations range) sperm was 23% compared
with 15% when PF-treated sperm were labelled with FITC stain.
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The comparison between intact-zona and hemizona shows that in the
presence of PF, which occurs only in the hemizona assay, there is increased binding
of PF-treated sperm. From previous studies reported in this thesis (chapters four and
five), we know that PF enhances sperm motility, and therefore this might have
influenced binding to the zona. Just by random chance, a fast-moving sperm has a
greater chance of contact with the zona than a slow-moving sperm. A survey of the
literature shows that there has been no direct study reported on the effect of PF on
sperm binding, either to human intact-zona or to hemizona, except the single study
reported by Kaskar et al. (1994). Their study involved the use of teratozoospermic (>
40% abnormal sperm) samples, prepared by swim-up, and co-incubated with
human hemizona in the presence of 4 mM PF/L for 4 hours. The conclusion from
their study was that PF stimulates sperm motility in teratozoospermic samples, but
that after 4 hour incubation in the presence of human hemizona, there was no
significant difference in the amount of binding of sperm to hemizona between the
control and PF-treated sperm, i.e. the increase in sperm motility caused by
exposure to PF was not correlated to the degree of sperm-hemizona binding. These
findings are at variance with the results reported in this study, which show that
sperm-hemizona binding was increased by 50% in the presence of 3 mM PF/L.
The possible explanation for the difference in results could lie in the concentration of
PF used and the limitations of method used in counting sperm bound to the zona.
From the dose-response studies reported in chapter 5, it has been shown that 2.8
mM PF/L is the optimal concentration to stimulate sperm motion characteristics.
Higher concentrations of PF may have deleterious effects on sperm motion
characteristics. Therefore, the concentration of 4 mM used by Kaskar et al. (1994)

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might have contributed to their failure to show that PF enhances sperm-hemizona
binding. Another factor that might have contributed to the lack of PF effect on sperm-
hemizona binding reported by Kaskar et al. (1994) could be the presence of a large
number of abnormal sperm in the semen which could have migrated up into the
overlaid medium during the preparation of sperm suspensions. It has been
demonstrated (Liu and Baker, 1992; 1994b) that when sperm suspensions
containing a very low percentage of normal sperm are used in intact-zona assay,
lower numbers of sperm were bound to the zona. The use of different sub-
populations of sperm may have contributed to the difference in results between the
two studies. In my study, all the samples were normal, as defined by WHO
guidelines (1992).
From the findings reported in chapter 5, washing would have removed some
of the stimulation of sperm motion characteristics induced by PF. Therefore, one
would expect the binding of the control and PF-pretreated samples to be nearly
similar in the intact-zona assay, where the PF is removed by washing. However, the
results from my study of sperm binding to intact-zona indicate that PF-pretreated
sperm binds much less well to the intact-zona than the control sperm that had not
been exposed to PF. To explain this phenomenon, it requires knowledge on both the
mechanisms of sperm-zona binding and on what changes are imposed on the sperm
by PF treatment, and which remained even after the drug was removed by washing,
before we can understand how PF reduces the binding ability of sperm.
The chronology of fertilization has been extensively studied in the mouse
(Wassarman, 1987, 1988; reviewed by: Fraser and Ahuja, 1988; Yanagimachi, 1988;
Wassarman, 1990; Dean, 1992). The following stages are thought to occur in mouse
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sperm-zona interaction:- (1) The sperm initially associate with the zona at the surface
of the zona pellucida. This relatively loose, nonspecific association is called
attachment. (2) The attached sperm then form a relatively tenacious, species-specific
adhesion with the zona that is referred to as binding. (3) Bound sperm then complete
acrosome exocytosis in preparation for penetration of the zona pellucida and fusion
with the oocyte oolemma. A number of hypotheses (Yanagimachi, 1994) have been
proposed to explain the mechanism of sperm penetration into the zona pellucida,
namely, (a) the mechanical hypothesis and (b) the enzymatic hypothesis. The
mechanical hypothesis suggests that the sperm movement into the intact-zona
investment is purely mechanical, with the acrosome enzymes playing no part in the
penetration. According to this hypothesis, the sole function of the AR is to expose the
perforatorium, which is the inner acrosomal membrane of the sperm head. This
sharply pointed perforatorium cuts the zona as the sperm is in hyperactive state with
its flagellum beating vigorously. Electron micrographs support this view
(Yanagimachi, 1988). The enzymatic hypothesis suggests that the large variety of
acrosomal enzymes present in the acrosome are involved in every step of sperm
penetration into the oocyte. Sperm motility is of secondary importance. The
acrosomal enzymes (e.g. acrosin) modulate the sperm entry, assisted by various
sperm receptors (ZP3, ZP2) on the oocyte (Wassarman, 1990) and other
carbohydrate determinants present on the surface of the sperm (Ahuja, 1985; Fraser
and Ahuja, 1988). As mentioned earlier in this chapter, the human sperm receptor
has been cloned and sequenced by Chamberlin and Dean (1990), and a high degree
of similarity shown to exist between the human and mouse sperm receptors.
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I think it is likely that, in the human, sperm-zona interaction uses both
mechanical (stage 1 for attachment) and enzymatic (stage 2 for binding) processes
to penetrate the zona. If one imagines that the zona is like multi-layers of mesh made
of glycoproteins (fibrillogranular strands), then the free swimming sperm get loosely
attached, leading to mechanical binding. This transient binding is possibly assisted
by the highly motile and hyperactivated sperm, with its "whiplash" movement. Up
to this stage the process is presumably reversible. It can be shown in sperm-zona
binding assays that sperm bound to zona can be removed if the zona is washed
vigorously. The loose binding, in all probability, is followed by enzymatic penetration
into the zona, a process involving the acrosome reaction, and sperm receptor, ZP3.
This scenario may explain why, in the presence of PF (with increases in VCL and
ALH of sperm), there was increased binding of sperm to the zona in the hemizona
assay. In the intact-zona study, however, PF (where the drug effect was reduced
by washing) inhibited binding, possibly due to alteration in the molecular structure of
the sperm head. The FITC labelling and the centrifugation process may have
sensitised the surface membrane of the sperm head, leading to changes in its
chemical structure. The subsequent exposure to PF may have increased the
resistance to binding with the zona pellucida. This requires further investigation.
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CHAPTER 8
GENERAL DISCUSSION
Philosophy is nothing but discretion.
John Seldon 1584-1654
When trying to advance our knowledge of human spermatozoa, several
factors must be borne in mind. The semen, fairly viscous in nature, consists of a
heterogeneous population of sperm suspended in seminal fluid that is rich in acid
phosphatase, lysozymes, citric acid, fructose, prostaglandins, zinc and magnesium.
A typical ejaculate may consist of mature, various stages of immature, abnormal and
dead sperm cells. In addition, there may be leucocytes present. The motion
characteristics of this heterogeneous population of sperm in semen can vary from
zero to about 70% overall motility. As alluded to in previous chapters, sperm motility
plays a crucial role in the process of procreation. However, 15% of all couples will
experience primary or secondary infertility at some during their reproductive lives
(Menning, 1980; reviewed by Skakkebaek et al., 1994). In approximately 50% of all
these cases, the man is subfertile. It is probable that in a proportion of the male
factor cases, the fundamental cause of infertility is defective sperm motion.
Therefore, over the years, attempts have been made to improve the motility or the
fertilizing ability of sperm by using pharmacological agents (reviewed by Lanzafame
et al., 1994 and Tournaye et al., 1994a) like Pentoxifylline (PF) and caffeine.
Recently, other inducers like platelet-activating factor (PAF) have been shown to
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stimulate sperm motility (Calvo et al., 1989; Ricker at al., 1989; Krausz et al., 1994)
and possibly increase the fertilizing potential of sperm (Angle et al., 1993).
Nearly a decade ago, computer-aided sperm analysis was introduced for
semen analysis in basic and clinical applications. It was thought that CASA would
overcome many of the limitations (section 1.7.1) of visual semen analysis (Davis and
Boyers, 1992; Bartoov et al., 1993) but, however, in the course of time it has become
apparent that the cost of the computer technology and laboratory staff's resistance
has severely limited its applicability (Davis and Katz, 1993). In addition, computer
technology itself introduced problems like image jitters, apparent motion and bump &
cross (discussed in section 1.7.2), which became apparent when CASA systems
were used in research. In spite of its pitfalls, CASA has been shown to provide useful
information in the diagnosis and treatment of subfertile patients (Katz and Overstreet,
1981; Chan et al., 1989; Fetterolf and Rogers, 1990; Check et al., 1990; Liu et al.,
1991).
In this project, I undertook to study the effects of PF on sperm motion
characteristics, employing computer-aided sperm analysis, and assessed its effects
on the acrosome reaction and sperm-zona pellucida interactions. Although CASA
can generate data on individual sperm tracks, I have used the average values by
tracking more than 50 sperm per sample. The advantage of this method is that more
samples can be analyzed and it will represent the whole sample. However, tracking
many individual sperm is time consuming, and therefore only a limited number of
samples can be assessed in the control and test experiments. Because of this
limitation, currently most of the published studies are based on the average CASA
values (Check et al., 1990; Tesarik et al., 1992a; Fuse et al., 1993).
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A survey of the literature showed that basic information on PF, such as the
optimal dose and optimal incubation time, was lacking. So, in chapters two and
three, I set out to investigate how these basic factors would influence sperm motion
characteristics in the presence and absence of PF. The results showed that the
conditions under which PF could induce optimal stimulation were an incubation for
one hour at the temperature of 370 C. It was also found that centrifuging the sperm
suspension four times did not statistically affect the sperm motion characteristics
adversely, a result consistent with the findings of Alvarez et al. (1993). The use of
Percoll in discontinuous Percoll gradient for the preparation of motile sperm in
suspension showed that the immediate effect of Percoll on sperm was to depress all
CASA parameters except LIN and MOT%, but subsequent washing to remove any
remaining Percoll in the sperm suspension showed that there was an increase in the
value of VCL and ALH accompanied with a decrease in MOT%. Further
investigations, reported in chapter five, showed a similar effect and it is hypothesized
that the removal of decapacitation factors and reactive oxygen species which
presumably accompany frequent changes of fresh culture medium may have
contributed to the increase in these motion values. In the study involving comparison
between Percoll gradient and 'swim-up' sperm separation, it was found that the
population of sperm isolated by the two methods were essentially different. In the
Percoll gradient method, separation is based on density, irrespective of sperm
motility, whereas in the 'swim-up' technique, only the progressive motile cells that
could swim into the overlaid culture medium were collected. Experimental results
indicated that sperm separated by the Percoll gradient method showed higher values
in VCL, LIN, and ALH when compared with sperm separated by 'swim-up'. Thus, the
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two methods of separation represent different sub-populations/sub-sets of sperm
exhibiting different motion characteristics. It thus becomes very important when
undertaking any studies on sperm motility that the method of sperm separation is
standardised at the outset.
Sperm stimulant has been demonstrated to improve sperm motion
characteristics (Aparico et al., 1980b; Yovich et al., 1990; Tesarik et al., 1992a).
Investigation of the optimal concentration of PF that would result in maximum
stimulation of sperm in semen formed the basis of the study reported in chapter 4.
The results gathered from the study showed that, in semen, the optimal
concentration of PF that produced the maximum response was 6 mM PF/L. The
figure of 6 mM/L was obtained from the appropriate Stimulation Index (section 4.3),
although individual CASA parameters peaked at different concentrations of PF.
Hence, Stimulation Index is a useful mathematical tool in discovering a total
maximum effect when a chemical compound has varying degrees of effect on
different parameters in the same sample. PF produced a significant increase in VSL,
VCL, and ALH, but no significant change in MOT% or LIN. The recovery of motile
sperm after 370 C incubation for one hour with 6 mM PF/L was 36% above the
control group. On examining a subset of 24 matched pairs of samples, there was
considerable variation (0 to 40%) in response of the sperm to PF challenge. This
might be because different semen samples contain different proportion of the various
sub-population/sub-sets of sperm, only some of which respond to PF stimulation.
This variation in the degree of response of different semen samples was consistent
with the results of other studies (Tesarik et al., 1992a; Moohan et al., 1993).
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De Turner et al. (1978) demonstrated that a four-hour exposure of sperm in
suspensions to cyclic adenosine monophosphate (cAMP) increased the duration of
sperm activity and significantly improved the percentage of sperm with forward
progressive movement. Subsequently, Tash and Means (1982) were able to
demonstrate that the addition of 10 μM cAMP to sperm in suspensions, produced a
2.5-fold increase in the proportion of motile sperm. In addition, the wave amplitude of
sperm movement was increased, which would facilitate an increase in forward
velocity. The authors were further able to show that the addition of the cAMP
inhibitor, protein kinase inhibitor (PKI), blocked the effects of cAMP on sperm motility.
It is thought that PF exerts its stimulating influence on sperm motion by inhibiting
cAMP phosphosdiesterase (Garbers et al., 1971b; Stefanovich, 1973), thereby
increasing intracellular cAMP concentrations. An increase in intracellular cAMP
concentration has been reported to increase sperm motility (Calamera et al., 1982;
Calamera et al., 1986) with enhancement of endogenous adenosine triphosphate
(ATP) utilization. ATP produced in sperm mitochondria is the source of energy for
sperm motion. In contrast, a study by Makler et al. (1980b) showed that addition of
10 to 1000 μg of cAMP directly to semen did not have any effect on sperm motion
parameters as assessed by multiple exposure photography.
The theme of chapter 5 was to find the optimal concentration of PF that would
produce a maximum elevation in sperm motion characteristics of sperm in
suspensions, and to assess if this increase was maintained after washing. A survey
of the literature (Sikka and Hellstrom, 1991; Lewis et al., 1993; Moohan et al., 1993;
Fuse et al., 1993) showed that different research groups used different
concentrations of PF to demonstrate the beneficial effect of PF on sperm

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suspensions, and that this ranged from 2.0 to 5.0 mM PF/L, and appeared to be
chosen empirically. The results obtained in this study showed the maximum increase
in sperm motion characteristics occurred at 2.8 ± 0.2 mM PF/L, as calculated from
the appropriate Simulation Index (section 5.3), although individual CASA parameters
peaked at different concentrations of PF. Significant increases were seen in VCL and
ALH, but there were no significant changes in VSL, LIN, or MOT%. On examining
responses of individual samples of sperm suspensions to 3 mM PF/L challenge, it
revealed that there was considerable inter-individual variation in the increase in VCL
and ALH, ranging from 0% to approximately 40%. These results were consistent
with the findings in chapter 4 where sperm in semen, rather than in media, were
challenged with PF. Nevertheless, the stimulation of sperm motion characteristics by
PF was reduced by washing. These findings are in contrast to the results obtained
by Tesarik et al. (1992a), who have reported that the stimulation of sperm by PF
remained for two hours after washing. For reasons already discussed in section 5.5,
the maintenance of raised sperm motion characteristics in the control samples might
be due to removal of reactive oxygen species produced by the sperm (Aitken, 1994)
and the removal of decapacitation factors which presumably accompanies frequent
changes of culture media involved in the wash procedures.
This project has so far demonstrated that PF stimulated sperm motion
characteristic; however, does the enhancement of sperm motion correlate with the
acrosome reaction? This question formed the basis of the research reported in
chapter 6. The results obtained from the study showed that PF stimulation of sperm
motion characteristics does not correlate with the acrosome reaction. There was no
significant increase in the proportion of sperm that had undergone the acrosome
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reaction after treatment with PF, a result consistent with the results of other
researchers (Tesarik et al., 1992b; Tasdemir et al., 1993; Carver-Ward et al., 1994).
However, a very recent study by Gearon et al. (1994) showed that 3.6 mM PF/L was
a potent inducer of the acrosome reaction in sperm which had been prepared by the
Percoll gradient method. Studies conducted in this project have shown that sperm
separated by the Percoll gradient method were a sub-population with higher sperm
motion characteristics than sperm separated by 'swim-up' method, a factor that might
have contributed to the difference in result reported by Gearon et al. (1994)
compared to all other studies (Tesarik et al., 1992b; Tasdemir et al., 1993; Carver-
Ward et al., 1994), in which the sperm have been prepared by the 'swim-up' method.
As alluded to in section 6.5, the mechanism of the acrosome reaction is still not fully
understood (Zaneveld et al., 1993).
Pentoxifylline has been shown to improve the fertilising ability of sperm in
some cases of infertility (Yovich et al., 1990; Tesarik and Mendoza, 1993). This issue
was the subject matter of investigation in chapter 7, where the effect of PF on sperm-
zona pellucida binding was studied in order to answer the question: does PF
treatment improve sperm competence to fertilize oocyte? The results obtained
showed that sperm treated with PF and subsequently washed with culture medium to
remove the drug showed a decreased ability to bind with zona pellucida. However,
sperm in the presence of 3 mM PF/L showed increased ability to bind with zona
pellucida. Kaskar et al. (1994) demonstrated that sperm from teratozoospermic
(>40% abnormal sperm) patients, in the presence of 4 mM PF/L, had increased
sperm motion characteristics, but that there was no corresponding increased sperm
binding with zona pellucida. The possible explanation for their apparently different
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result could lie in the different populations of sperm used; in their study, the semen
came from teratozoospermic patients, whereas in my study, all samples were
normozoospermic. Fertilization is one of the most complex forms of cellular
interactions, involving not merely the surface contact between gametes, but also
encompassing other cellular processes (Garbers, 1989) like the acrosome reaction
(Liu and Baker, 1994b). The various hypothesis to explain sperm-zona pellucida
interaction are discussed in section 7.5.
An interesting observation was made from the studies reported in chapters six
and seven which involved labelling of sperm with fluorescent stain. Although labelling
of sperm with a low concentration of the stain showed no adverse effect on the
motion characteristics, with higher concentration, there was a significant decrease in
motion characteristics. This may imply that the use of the stain in labelling sperm
could introduce subtle changes which are not easily detectable. Therefore, caution
has to be exercised in its application.
Considering the above discussion, the use of PF as a sperm stimulant to
enhance sperm motility to treat patients with subfertility needs careful consideration.
The studies in this project show that not all sperm samples respond to PF
stimulation, and hence there is a need to do preliminary testing. It may be necessary
that the use of PF be tailored to each individual patients. Indiscriminate use of PF in
treatment of subfertility does not improve either sperm motion characteristics or
fertilization, a view strongly supported by a recently published paper (Tournaye et al.,
1994b).
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In conclusion (Fig. 8.1), Pentoxifylline stimulates sperm motion
characteristics, although the degree of stimulation can vary from sample to sample,
and it does not appear to promote the acrosome reaction. However, in the presence
of this drug there was increased sperm-zona binding. Proposed research for the
future would include:-
1) Studying the effect of PF directly on semen in asthenozoospermic
patients.
2) Investigating the effect of PF on multiple sperm samples from the
same individual but obtained over a period.
3) The effect of Hepes in culture medium on the Acrosome Reaction
inducible by Ionophore A23187 in the presence of Pentoxifylline.
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Appendix A
Chemical composition of Earles-Hepes balanced salt (EHBS) solution
Components mg/L
------------------ --------
NaCl 6800
KCl 400
MgSO47H2O 200
NaH2PO42H2O 158
CaCl22H2O 264
NaHCO3 2200
Glucose 1000
Phenol red 10
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Data from this thesis was used to publish the following papers:-
1. Actions of pentoxifylline directly on semen
2. The paradoxical effects of pentoxifylline on the binding of
Spermatozoa to the human zona pellucida
3. Factors affecting pentoxifylline stimulation of sperm kinematics
in suspensions.
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PHD Thesis 1994

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1

ACTIONS OF PENTOXIFYLLINE ON SPERMATOZOA KINEMATICS,

THE ACROSOME REACTION AND SPERM-ZONA INTERACTION

A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY

DEPARTMENT OF BIOLOGY AND BIOCHEMISTRY,

BRUNEL UNIVERSITY, UXBRIDGE, UNITED KINGDOM

FERTILITY LABORATORY, DEPARTMENT OF CHEMICAL PATHOLOGY

Thesis title Actions of pentoxifylline on spermatozoa

Author Paul, Moses.

Awarding Brunel University

Year of Award 1994

Qualification PhD thesis Qualification doctoral

Keywords Assisted reproduction Human physiology

In many assisted reproduction procedures, the drug Pentoxifylline (PF) is

used to enhance spermatozoa (sperm) motion. However, no thorough study has

examined under what circumstances PF might be useful, nor what concentration is

reaction and sperm-zona pellucida binding.

showed a significant enhancement in curvilinear velocity and lateral head

demonstrated that each sample of sperm responded to varying degree of

enhancement, with 1 in 10 samples not responding to PF stimulation.

Washing alone produced an increase in motion characteristics in the

sperm motion characteristics.

acrosome reaction; however, in the presence of Ionophore A23187, it significantly

the presence of PF had a significantly increased tendency to bind to the zona

by washing, showed a decreased tendency to bind to the zona pellucida.

I wish to express my sincere thanks to Dr Kevin Lindsay for his guidance,

a memorable experience. I am grateful to Kevin for allowing me to use the research

A special word of thanks goes to Professor John Sumpter for his

intellectual guidance, constructive criticism, unfailing enthusiasm and inspiration

which enabled me to carry out this research.

I take this opportunity to thank the staff of Electron Microscopy Unit, Dr

I am greatly indebted to Mr Vic Robinson, Consultant Obstetrician and

Gynaecologist of Hillingdon Hospital, who initially allowed me to embark on this

taken off the ground. I am sincerely grateful for this.

Finally, I wish to express my gratitude and thanks to my wife Hiroko for

her patience, understanding and encouragement, and advice on statistical

ignored while I worked on this project.

British Library, UK filing 2

Chapter 1: GENERAL INTRODUCTION 20

1.2 Sperm structure 22

1.4 Gamete Transport 29

1.7 Methods to study sperm motility 38

1.8 Inducers of sperm motility 43

1.10 Aim of study 47

Chapter 2: GENERAL MATERIALS AND METHODS 48

2.3 Sample selection and Sperm assessment 51

2.4 Sperm suspension preparation 52

2.5 CASA methodology 54

2.6 Stability of Pentoxifylline to external factors 61

2.7 Statistical analysis of experimental data 65

Chapter 3 INVESTIGATIONS INTO FACTORS

AFFECTING SPERM MOTILITY 67

3.2 Effect of incubation temperature on sperm

motility when Pentoxifylline is present 69

3.3 Effect of incubation time on sperm motility

when Pentoxifylline is present 72

3.4 Effect of Heparin on sperm motility when

3.5 Effect of Percoll on sperm motility 80

3.6 Effect of centrifugation on sperm motility 85

3.7 Comparison between discontinuous Percoll

gradient and 'swim-up' methods of sperm

Chapter 4 ACTION OF PENTOXIFYLLINE ON SEMEN 90