BIOLOGY OF REPRODUCTION (2014) 90(1):3, 112

Published online before print 20 November 2013.

DOI 10.1095/biolreprod.112.105809

Regulated Wnt/Beta-Catenin Signaling Sustains Adult Spermatogenesis in Mice1

Genevieve E. Kerr,4 Julia C. Young,4,5 Katja Horvay,4 Helen E. Abud,3,4 and Kate L. Loveland2,3,4,5
4
5

Department of Anatomy and Developmental Biology, Monash University, Clayton, Victoria, Australia
Department of Biochemistry, Monash University, Clayton, Victoria, Australia

The importance of Wnt signaling for postnatal testis function

has been previously studied in several mouse models, with
chronic pathway disruption addressing its function in Sertoli
cells and in postmeiotic germ cells. While chronic beta-catenin
deletion in Sertoli cells does not profoundly affect testis
development, new data indicate that Wnt signaling is required
at multiple stages of spermatogenesis. We used two mouse
models that allow acute disruption of Wnt signaling to explore
the importance of regulated Wnt pathway activity for normal
germ cell development in adult male mice. Short-term induction
of mutations in Adenomatous polyposis coli (Apc) and betacatenin (Ctnnbl), which increase and decrease Wnt signaling
levels, were generated in AhCre Apcfl/fl and AhCre Ctnnb1fl/fl
mice, respectively. Each exhibited a distinct phenotype of
disrupted spermatogenesis that was evident within 24 h and
persisted for up to 4 days. Outcomes included germ cell
apoptosis and rapid loss and altered blood-testis barrier protein
distribution and morphology. The functional significance of
nuclear localized beta-catenin protein in spermatocytes and
round spermatids, indicative of active Wnt signaling, was
highlighted by the profound loss of postmitotic germ cells in
both models. Developmentally regulated Wnt signaling mediators identified through transcriptional profiling of wild-type and
AhCre Ctnnb1fl/fl mouse testes identified Wnt receptors (e.g.,
Fzd4) and ligands (e.g., Wnt3, Wnt3a, Wnt5b, Wnt7a, and
Wnt8b). This demonstration that Wnt signaling control is
essential for adult spermatogenesis supports the growing
understanding that its disruption may underpin certain cases of
male infertility.
APC, germ cells, mouse models, spermatids, spermatocytes, testis

INTRODUCTION
Wnt signaling is a highly conserved pathway that is
essential for diverse processes during development and in
adult tissue homeostasis [1]. Perturbations of Wnt signaling can
result in birth defects, cancer, and other diseases [2]. The
intracellular localization of b-catenin, the central mediator of
canonical Wnt signaling, is considered to be indicative of Wnt
pathway-activation status [3, 4]. Its nuclear localization is
regarded as a hallmark of active Wnt signaling due to the
1
Supported by National Health and Medical Research Council of
Australia (545916 to K.L. and 3236507 to K.L. and H.A.).
2
Correspondence: Kate L. Loveland, Departments of Anatomy &
Developmental Biology and Biochemistry & Molecular Biology,
Building 76 level 2, Monash University, Wellington Rd., Clayton,
Victoria 3168, Australia. E-mail: Kate.loveland@monash.edu
3
These authors contributed equally to this work.

Received: 25 October 2012.

First decision: 5 December 2012.
Accepted: 13 November 2013.
2014 by the Society for the Study of Reproduction, Inc.
eISSN: 1529-7268 http://www.biolreprod.org
ISSN: 0006-3363

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capacity of b-catenin to regulate target gene transcription, but

b-catenin also has a role in cell adhesion at the plasma
membrane [5, 6]. In the absence of a Wnt ligand, cytoplasmic
b-catenin is sequestered by a destruction complex that contains
adenomatous polyposis coli (APC), Axin, the protein kinase
casein kinase 1 (CK1), and glycogen synthase kinase 3
(GSK3); the latter phosphorylates and targets b-catenin for
proteasomal degradation [4]. Rapid pathway activation occurs
when a Wnt ligand binds to membrane-associated receptor
complexes comprising Frizzled (Fzd) family members and a
low-density lipoprotein receptor (LDLR)-related protein
(LRP). This causes dissociation of the destruction complex to
enable first cytoplasmic accumulation of b-catenin, then its
translocation into the nucleus to complex with T-cell factor/
lymphoid enhancer binding factor (TCF/LEF) transcription
factors to activate target genes (reviewed in [6]). Other Wntactivated pathways that are independent of b-catenin, such as
the planar cell polarity pathway, are described as noncanonical;
these rely on distinct receptors and activate different signaling
mediators, including JNKs [7]. Their activation has been
shown to provoke alternative, and even contrasting, cellular
responses to canonical Wnt signaling, for example in blood cell
development [8]. However, many key developmental outcomes
are driven by canonical Wnt signaling, and in our initial
observations of mice in which this pathway was acutely
disrupted for studies on gut stem cell biology [9], we noticed
that male fertility was reduced. This outcome led to the present
study examining the hypothesis that canonical Wnt signaling is
integral to normal testis function in adults.
The seminiferous epithelium of the mammalian testis
constitutes a highly organized unit within which spermatogenesis is tightly regulated by interacting signaling networks of
hormones and growth factors that are produced and processed
by the surrounding epithelial and interstitial compartments [10
13]. During spermatogenesis, generations of spermatogonial
stem cells, present at the basement membrane of the
seminiferous tubules, undergo a progression of developmental
steps involving mitosis, meiosis, and differentiation to produce
spermatozoa that are released into the seminiferous tubule
lumen [14]. These processes are supported by the somatic
Sertoli cells that surround the germ cells and form the
epithelium essential for their normal maturation [12]. Each
generation of germ cells develops in parallel with subsequent
generations in a highly ordered, cyclic manner. These distinct
and invariant cohorts are divided into 12 successive stages of
germ cell associations in mice that collectively represent the
cycle of the adult seminiferous epithelium [14].
Wnt signaling is required for events in fetal life that
underpin adult fertility. Wnt 4 is essential for normal male fetal
reproductive tract development, acting downstream of Sry but
upstream of Sox9 and desert hedgehog [15]. Wnt 4 mutant
testes show compromised Sertoli cell differentiation due to the
action of Wnt 4 within the early genital ridge [15]. Wnt7a is
similarly required, as its deletion inhibits Mullerian duct
regression and causes retention of female reproductive tract

ABSTRACT

KERR ET AL.

signaling is perturbed. Considered in light of our additional

new data that identify the Wnt pathway components present in
the developing and adult testis, we can provide a more robust
understanding of the importance and mechanisms of Wnt
signaling in the adult mouse testis.

tissues in adult males, which can impede sperm passage at

ejaculation [16]. Constitutive b-catenin activation in the
Mullerian duct mesenchyme also leads to Mullerian duct
retention [17], highlighting the importance of balanced
pathway signaling in fetal life for normal fertility.
Several studies have recently established the contribution of
regulated Wnt signaling within Sertoli cells to male fertility
using mouse models that activate or block Wnt signaling
(Supplemental Table S1; all Supplemental Data is available
online at www.biolreprod.org). Expression of a constitutively
active b-catenin isoform in Sertoli cells that begins during
embryogenesis under the control of AMH-Cre results in germ
cell depletion by Postnatal Day 0, reduced levels of the Sertoli
cell identity markers Sox9 and AMH from Embryonic Day
13.5, and formation of Sertoli cell tumors after 8 mo of age [18,
19]. Similarly, postnatal expression of activated b-catenin in
Sertoli cells under the control of AMHR2-Cre results in
infertility, seminiferous tubule degeneration, progressive germ
cell loss due to apoptosis, and increased AMH expression [20,
21]. The activation of Wnt signaling via mutation of Apc also
leads to impaired germ cell development as well as loss of
Sertoli cell apical extensions and blood-testis barrier integrity
[22]. In contrast, deletion of b-catenin in Sertoli cells causes no
phenotypic defects at the embryonic or postnatal stages [18, 22,
23]. These studies collectively indicate that the constitutive
activation of Wnt signaling in Sertoli cells causes spermatogenic defects, while deletion of b-catenin does not affect the
capacity for Sertoli cells to support germ line development.
A limited number of studies have explored the importance
of b-catenin selectively within germ cells. Expression of the
stable, active form of b-catenin in primordial germ cell
promoter TNAP-Cre-expressing cells and cell lineages results
in male and female germ cell deficiency [24], while haploid
germ cell b-catenin deletion (Prm1-Cre) causes reduced sperm
count, increased germ cell apoptosis, and impaired fertility,
attributed by the study authors to the role of b-catenin in cell
adhesion [25]. The role of Wnt signaling per se, however, was
not examined in the latter study. The noncanonical ligand
Wnt5a has been demonstrated to promote spermatogonial stem
cell maintenance in culture independent of b-catenin; however,
in vivo evidence of active Wnt signaling in spermatogonia has
yet to be provided [26]. A murine stem cell spermatogonia celllike line, C18-4, exhibited features of canonical Wnt signaling,
responding to Wnt3a with proliferation and TOPFlash
activities [27]. In postmitotic germ cells, an inactivating
mutation in naked cuticle 1, a Wnt pathway inhibitor, was
associated with increased nuclear b-catenin in elongating
spermatids and defective adult spermatogenesis [28].
These findings present an emerging picture in which Wnt
ligands act at multiple sites to influence testis development and
contribute to the functional integrity of the adult seminiferous
epithelium. However, there is minimal information available
concerning the identity of Wnt ligands within the testis, their
sites of impact, and their specific influence on spermatogenic
progression in vivo. Thus, while existing evidence shows that
control of Wnt signaling levels is essential for Sertoli cell
support of spermatogenesis, the absence of canonical Wnt
signaling in Sertoli cells appears to have minimal effects on
testis development [18, 22, 23], and the specific roles of Wnt
signaling in germ cells remains to be defined.
In the present study, we show that b-catenin-dependent Wnt
signaling is active in germ cells of the adult testis and use two
models of acutely altered Wnt signaling in which a profound
impact is observed on germ line cells following disruption to
the normal levels of Wnt signaling. These findings identify
particular germ cell types that are affected when canonical Wnt

MATERIALS AND METHODS

Animals and Tissue Collection

Histological Analysis
Loss of germ cells by 5 days after BNF injection was measured for mice of
both mutant strains by analysis of hematoxylin-eosin (H&E) sections. Three 5lm sections, separated from each other by at least 150 lm, were chosen from
one testis per animal. Scans of whole-testis sections were captured using an
Aperio Scanscope XT (Aperio Technologies) and analyzed using ImageScope
software (Aperio Technologies) by placing a grid over the section, which
created lines 500 lm apart. Tubules touching these lines were evaluated. The
most mature germ cell type present in each tubule cross section was recorded
using standard histological criteria [34]. Ninety to 550 tubules were scored per
animal, and the proportion for each animal of all tubules containing elongating
spermatids, round spermatids, pachytene spermatocytes, leptotene/zygotene
spermatocytes, or spermatogonia or lacking germ cells (i.e., Sertoli cell only)
was calculated. The incidence of cellular apoptosis within the seminiferous
epithelium was quantified by determining the proportion of rounded tubule
cross sections with any terminal deoxynucleotidyl transferase dUTP nick end
labeling (TUNEL) positive cells. Between 50 and 250 tubules were counted per
animal, and three to four animals were analyzed per genotype. To quantify
differences in cell proliferation, anti-PCNA-stained sections were examined.
All tubule cross sections contained proliferating cell nuclear antigen (PCNA)positive cells, but their distribution was altered by tissue shrinkage in samples
exhibiting loss of more mature germ cell types. Thus, we recorded the
proportion of each seminiferous tubule periphery that lacked PCNA-positive
cells in 2050 rounded tubule cross sections per animal, from four to five
animals per genotype [35]. The incidence of undifferentiated spermatogonia
was quantified by counting the number of Plzf-positive cells per rounded tubule
cross section. Ten to forty seminiferous tubules were counted per animal for
three to four animals per genotype. A Student t-test was performed to determine
statistical significance between groups, with P , 0.05 determining significance.

Immunohistochemistry
Immunohistochemistry was performed using established protocols [9].
Slides were dewaxed, dehydrated, and incubated in 0.5% H2O2 in methanol for
30 min. After subsequent rehydration, slides were heated for 10 min in a

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Postnatal wild-type Swiss mice were obtained from Monash University

Central Animal Services. Investigations conformed to the National Health and
Medical Research Council/Commonwealth Scientific and Industrial Research
Organisation/Australian Agricultural Council Code of Practice for the Care and
Use of Animals for Experimental Purposes and were approved by the Monash
Animal Research Platform Committee on Ethics in Animal Experimentation.
Conditional deletion of b-catenin and Apc in the testes was achieved by
breeding Ctnnb1fl/fl mice [29] (obtained from J. Huelsken, ISREC, Switzerland)
and Apcfl/fl mice with mice containing the AhCre transgene (obtained from A.
Clarke, Cardiff, U.K.) as previously described for intestinal studies [9, 30]. In
this system, Cre recombinase is under the control of the cytochrome P4501A1
(CYP1A1) promoter element, and mice containing this transgene are referred to
as AhCre mice [30]. Expression of Cre recombinase in AhCre mice is inducible
by administration of b-napthoflavone (BNF), which binds to cytoplasmic aryl
hydrocarbon receptors that are then transported into the nucleus, where they
bind DNA response elements in the CYP1A1 promoter and activate
transcription [30, 31]. Cre recombinase expression is induced in the intestine
and several other tissues following administration of BNF (80 mg/kg) to AhCre
mice (Supplemental Fig. S1) [30]. Aryl hydrocarbon receptors that can
efficiently bind BNF are present in mice from C57 and CBA genetic
backgrounds [32] in many tissues including the seminiferous tubules and
interstitial cells of the testis [33]. Control and test mice at 7 wk of age were
given daily i.p. injections of BNF, and animals were killed for collection of
testes on the following day; the number of injections varied between one and
four. Testes were fixed immediately after collection in Bouin fixative for 5 h,
dehydrated through a graded ethanol series (70%100%), embedded in
paraffin, and sectioned at 5 lm onto Superfrost Plus 2 slides (Lomb Scientific).
Control samples bearing one floxed allele of the gene were treated
simultaneously with test samples throughout.

Wnt SIGNALING IN THE POSTNATAL MOUSE TESTIS

spermatogonia, pachytene spermatocytes, and step 18 round spermatids; and
GDS2390 [37]: Male Germ Cells (http://www.ncbi.nlm.nih.gov/sites/
GDSbrowser?accGDS2390) transcriptional analysis of 11 time points from
Postnatal Day 0 through to Day 54 of whole developing mouse testis using the
Affymetrix GeneChip system. From these records, the expression values for
each mouse Wnt pathway component were graphed using GraphPad Prism 5
software.

pressure cooker in 10 mM citrate buffer (pH 6.0) and blocked with CAS block
(Invitrogen) for 1 h. Sections were incubated overnight in a humid chamber
with primary antibodies at 48C diluted in PBS with 0.2% bovine serum albumin
(BSA). Antibodies were used to detect b-catenin (610154; 1:500; BD
Transduction Laboratories), PCNA (M0879; 1:800; Dako), Plzf (sc-22839;
1:800; Santa Cruz), Vasa (Ab13840; 1:400; Abcam), Sox9 (sc20095; 1:200;
Santa Cruz), Connexin43 (3512; 1:300; Cell Signaling), AMH (sc6886; 1:500;
Santa Cruz), and smooth muscle actin (A2547; 1:2000; Sigma). For detection
of the primary antibodies, anti-mouse, anti-rabbit, or anti-goat horseradish
peroxidase secondary antibodies (1:200, all from Invitrogen) in PBS with 0.2%
BSA were applied to each section for 1 h at room temperature. Peroxidase
activity was detected with a diaminobenzidine liquid kit (Dako). Sections were
counterstained with hematoxylin, dehydrated, and mounted under glass cover
slips. Images were captured on a Zeiss Axioimager microscope running
Version Rel4.7 Software (Carl Zeiss). TUNEL assays were performed using an
ApopTag Peroxidase In Situ Apoptosis Detection Kit S7100 (Millipore)
according to the manufacturers instructions.

RESULTS
Perturbation of Wnt Signaling Results in Germ Cell Loss by
Apoptosis

Quantitative Reverse Transcription PCR (qRT-PCR) Array

and Axin2 qRT-PCR Analysis
Total RNA was extracted from decapsulated wild-type Swiss mouse testes
at Postnatal Days 7, 14, and 28 and at 7 wk (adult) using TRIzol reagent
(Invitrogen). The resulting RNA was then DNAseI-treated (Qiagen on-column
kit; Qiagen), repurified using Qiagen RNeasy columns (as per the
manufacturers instructions), and quantified using a Nanodrop 2000 spectrophotometer (Thermo Scientific). Testis RNA from two mice was pooled at each
age, and two independent pooled samples for each age were assayed.
Complementary DNA was synthesized using the RT2 first strand kit
(SABiosciences, Qiagen) from 400 ng total pooled testis RNA, and the qPCR
reactions were performed in RT2 qPCR Master Mix (SABiosciences); both
were performed according to the manufacturers instructions. An RT2 Profiler
PCR array specific for Wnt signaling pathway components (SABiosciences
array PAMM-043) was used to assess transcript levels in the testis. The arrays
were loaded by automated liquid handling (Qiagen CAS 1200 liquid handing
instrument) and run on an ABI7900 PCR machine at the Gandel Trust
Sequencing Centre (Monash Health Translation Precinct). Raw Ct values were
analysed using the SAB PCR Array Data Analysis Web Portal online analysis
software (http://pcrdataanalysis.sabiosciences.com/pcr/arrayanalysis.php),
where Ct, DCt, 2 0 (-Avg.(DCt)), fold change, and fold regulation were
autogenerated. All array internal quality controls were similarly calculated to
indicate an absence of genomic contamination, optimal reverse transcription
efficiency, and high sensitivity. The independent array data sets were calculated
as within acceptable limits for comparison across arrays (SABiosciences online
analysis software). Age series data are presented as fold change relative to the
Postnatal Day 7 values (set to 1). Additional arrays were performed in the same
manner to measure changes in Wnt pathway components and signaling
outcomes following acute (2-day) disruption of b-catenin in adult testes using
AhCre Ctnnb1fl/fl, as compared to littermate controls. These data are presented
as the fold change of experimental samples relative to controls run
simultaneously. Two independent sample pairs were analyzed. Axin 2 was
assayed separately from the same RNA kept as four independent samples
following cDNA preparation with 100 U Superscript III reverse transcriptase
and random hexamer primers (Applied Biosystems). Axin2 (accession number
NM_015732.4) forward (5 0 -GCAGCTCAGCAAAAAGGGAAAT-3 0 ) and
reverse (5 0 - TACATGGGGAGCACTGTCTCGT-3 0 ) primers were used for
PCR performed at 958C for 10 min, with 45 cycles of amplification at 958C for
15 sec, and then 628C for 30 sec, on a 7900HT real-time system (Applied
Biosystems, Gandel Charitable Trust Sequencing Centre, Monash Institute for
Medical Research). Technical triplicates were performed for each sample, with
values taken from the average. Relative standard curve analysis, performed
with SDS2.3 software (Applied Biosystems), was used to quantify sample data.
Standard curves for each primer set were generated from an equal mix of the
adult testis RNA samples. Axin2 data for each sample were normalized to 18S
values (accession number NR_003278) with forward (5 0 -GTAACCCGTT
GAACCCCATT-3 0 ) and reverse (5 0 - CCATCCAATCGGTAGTAGCG-3 0 )
primers.

Geoprofiles Data Collection

The expression graphs for each gene were generated by downloading
publicly available transcript profile records from the Entrez website. The GEO
Data-Set records downloaded were GDS605 [36]: spermatogenesis and testis
developmental time course (MG-U74A) (http://www.ncbi.nlm.nih.gov/geo/
gds/ gds_browse.cgi?gds_605), microarray analysis on Affymetrix MG-430 2.0
chips on four spermatic subpopulations, including type A and type B

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To analyze the physiological consequences of acutely

altering canonical Wnt signaling levels in the adult mouse
testis, we compared the effect of elevating canonical Wnt
signaling by mutation of the negative regulator, Apc, with the
outcome of blocking Wnt signaling through mutation of the
central pathway mediator, b-catenin. This was achieved using
the transgenic mouse line, AhCre, in which Cre recombinase
expression, directed by the inducible CYP1A1 promoter,
becomes transcriptionally activated in the presence of BNF,
as observed following systemic exposure in the intestine and in
several other tissues [30]. As this system has not been
thoroughly investigated in the testis, we first performed control
experiments to examine whether BNF exposure, AhCre, and
floxed alleles of b-catenin and Apc individually caused
changes in the testis. In nearly all cases, no defects in terms
of overall testis structure and presence of germ cell types were
observed in these controls (Supplemental Fig. S1, AI).
Occasional tubule cross sections from Apcfl/fl animals showed
some germ cell loss (Supplemental Fig. S1I). We also tested
the ability of the AhCre transgene to mediate recombination in
the testis by crossing these mice to Rosa26Reporter (R26R)
mice [38] (Supplemental Fig. S1). Some b-galactosidase
activity, signified by blue staining, was observed in testes of
AhCreR26R mice that had not been treated with BNF,
indicative of some AhCre transgene leakiness in the testis.
Staining was observed in interstitial cells and occasionally in
germ and somatic cells. Following treatment with BNF,
however, b-galactosidase activity, indicative of Cre function,
was readily observed in all spermatogenic and Sertoli cells,
confirming the utility of the AhCre allele for manipulating
genes within the testis seminiferous epithelium (Supplemental
Fig. S1, M and N).
Prior to initial analysis of BNF-induced Cre recombination
mutant phenotypes (Fig. 1), background phenotypes were also
detected in AhCre Ctnnb1fl/fl and AhCre Apc fl/fl testes prior to
induction with BNF, again suggesting some leakiness of AhCre
activity (Supplemental Fig. S1, J and K). Analysis of testes
from a cohort of AhCre Ctnnb1fl/fl and of AhCre Apcfl/fl mice
without induction indicated various degrees of leakiness, with
some tubules showing germ cell loss (Supplemental Fig. S1, L
and O). Phenotypes were drastically enhanced in AhCre
Ctnnb1fl/fl and AhCre Apcfl/fl testes after BNF induction (Figs.
2E and 3E), becoming nearly uniform throughout the testis
between samples.
We first analyzed the effects of disrupting Wnt signaling in
testes of AhCre Ctnnb1fl/fl and AhCre Apcfl/fl adult mice 1 day
after exposure to BNF. TUNEL analysis revealed a statistically
significant increase in the frequency of apoptotic cells in both
AhCre Ctnnb1fl/fl and AhCre Apcfl/fl mutant testes compared to
that of controls (Fig. 1). Cells undergoing apoptosis were
identified by their morphology and position within the tubule
as germ cells, often appearing in clusters typical of apoptosis
within germ cell syncytia. Tubule cross sections with apoptotic
cells were evenly distributed throughout each testis section. In

KERR ET AL.

Apcfl/ testes, some tubule cross sections contained only Sertoli

cells (Fig. 1, arrowheads), which was interpreted as arising
from the known hypomorphic functionality of the Apc floxed
allele [39].

visualized with Sox9 throughout both control and mutant

samples (Fig. 2, J and K). However, an obvious elevation in
AMH staining, typically a feature of immature Sertoli cells,
was observed in some cross sections; this was particularly
evident in areas of extensive germ cell loss (Supplemental Fig.
S2, E and F). Connexin43 localization was used to interrogate
organization of the blood-testis barrier, which is normally
localized in Sertoli cell junctions close to the basement
membrane [41, 42] and uniformly observed in control samples.
However, this pattern was missing in many cross sections of
mutant testis tubules, demonstrating that the underlying
structure of the blood-testis barrier was perturbed following
acute reduction of b-catenin (Fig. 2M compared with control in
Fig. 2L). Peritubular myoid cells, as detected by immunostaining for smooth muscle actin, appeared grossly normal in
mutants (Supplemental Fig. S2, C and D). Mutant testes
showed a higher proportion of apoptotic cells evident from
more abundant TUNEL- and Casp3-positive cells (Supplemental Fig. S2, GJ), but no other obvious phenotypes were
evident following inspection of seven individual samples per
mutant genotype.

Acute Mutation of b-Catenin Results in Spermatocyte and

Spermatid Depletion
To determine the outcomes of reduced Wnt signaling in the
adult testis, we examined testes from AhCre Ctnnb1fl/fl mice
after 4 days of BNF treatment. Seminiferous tubules from these
mutant mice consistently contained reduced numbers of
postmitotic germ cell types, both spermatocytes and spermatids
(Fig. 2, B, D, and E), compared to controls (Fig. 2, A, C, and
E). The proportion of tubules containing elongating spermatids
was drastically lower in mutants compared to those of controls
(26.71% 6 6.22% in AhCreCtnnb1f/lfl, 97.15% 6 0.84% in
controls; Fig. 2E). In contrast, the proportion of tubules
displaying round spermatids as the most mature germ cell type
increased from 2.46% 6 0.84% in controls to 56.36% 6
5.48% in AhCre Ctnnb1fl/fl animals. In these mutant testes,
13.61% 6 1.86% of tubules contained pachytene spermatocytes and 2.59% 6 1.13% zygotene or leptotene spermatocytes
as the most mature germ cell type, and 0.68% 6 0.33% of
tubules contained only Sertoli cells (Fig. 2E). Some seminiferous tubules displayed Sertoli cell vacuolization (black
arrowheads), a feature typically associated with germ cell loss
[40]. Immunohistochemical analyses revealed no statistically
significant change in the extent of PCNA-positive staining in
AhCre Ctnnb1fl/fl samples (Fig. 2, F, G, N, and O). The
appearance of Vasa-positive cells was reduced in the mutants,
reinforcing the observation of an overall decrease in germ cell
numbers (Fig. 2, H and I). However, there was no change in the
occurrence of Plzf-positive stained cells, suggesting that
undifferentiated spermatogonia were maintained (Fig. 2P and
Supplemental Fig. S2, A and B). Uniform basal positioning of
Sertoli cell nuclei, a characteristic of mature Sertoli cells, was

Acute Mutation of Apc Results in Perturbed

Spermatogenesis
The outcomes of acute Wnt signaling activation were
examined in AhCre Apc fl/fl mouse testes following 4 days of
BNF treatment. These samples displayed a predominant
absence of postmitotic mature germ cell types (Fig. 3, AD);
however, there was a difference in the general pattern of
germ cell loss compared to that evident in b-catenin mutants.
Seminiferous tubules from AhCre Apcfl/fl mice contained
reduced numbers of spermatocytes and spermatids (Fig. 3, B,
D, and E) compared to controls (Fig. 3, A, C, and E). The
proportion of tubules containing elongating spermatids
decreased from 92.86% 6 0.95% in controls to 35.11% 6
9.97% in AhCreApcfl/fl animals (Fig. 3E). The proportion of
tubules containing round spermatids as the most mature germ
4

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FIG. 1. Perturbation of Wnt signaling results in acute germ cell loss and apoptosis in two distinguishable patterns. TUNEL analysis marks apoptotic cells
by brown staining in hematoxylin-stained sections of control Ctnnb1fl/ (A) and test AhCre Ctnnb1fl/fl (B, C) and control Apcfl/ (E) and test AhCre Apcfl/fl
(F, G) mouse testes 1 day after BNF injection. Proportion of seminferous tubules with TUNEL-positive cells in Ctnnb1 testes (D) and Apc testes (H); 50250
rounded tubules per animal and three to four animals per genotype counted. Bars 50 lm. Arrowhead indicates tubule cross section containing only
Sertoli cells. A Student t-test was performed to determine statistical significance between groups, with *P , 0.05 determining significance. Error bars show
the SEM.

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FIG. 2. Defective spermatogenesis following blockage of Wnt signaling in AhCre Ctnnb1fl/fl mice after 4 days of BNF exposure. AD) H&E-stained
sections from control Ctnnb1fl/(A, C) compared to mutant AhCre Ctnnb1fl/fl (B, D) adult testes revealed predominant loss of postmeiotic germ cell types
in mutants. E) Quantification of germ cell loss depicts the percentage of seminiferous tubules with the most mature germ cell type present in tubule cross
sections, identified as elongating spermatids (ES), round spermatids (RS), pachytene spermatocytes (PS), leptotene/zygote spermatocytes (L/Z), or
spermatogonia (Spg) and tubules with no germ cells (Sertoli cell-only tubules [SO]). Ninety to three hundred tubules counted per animal, seven animals
per genotype. White bars show control Ctnnb1fl/ BNF, gray bars show intermediate AhCre Ctnnb1fl/fl  BNF, and black bars show test AhCre Ctnnb1fl/fl
BNF samples. Accompanying AhCre Ctnnb1fl/fl  BNF analysis is presented in Supplemental Figure S1. Representative immunostaining for PCNA
identified proliferative cells in control Ctnnb1fl/ (F) compared to mutant Ahcre Ctnnb1fl/fl (G) samples. Immunohistochemical identification of the germ
cell marker, Vasa, in control (H) and mutant (I) samples; the Sertoli cell nuclear marker, Sox9, in control (J) and mutant (K) testes; and the blood-testis
barrier marker, connexin43, in control (L) and mutant (M) samples. Images represent observations from examining a total of seven animals per genotype.
Bars 50 lm. Arrowhead indicates tubules with Sertoli cell vacuolization. The distribution of proliferating cells was quantified (N, O) by determining the
proportion of each seminiferous tubule periphery containing PCNA-positive cells for four animals per genotype. N) The average outcome measured for
each animal in control (white) versus test (grey) samples. These data are presented for each individual animal in O to show the specific distribution pattern
of PCNA-positive cells (open circles, Ctnnb1fl/ ; black squares, AhCre Ctnnb1fl/fl). The distribution of undifferentiated spermatogonia (P) was measured as
the number of Plzf-positive cells per seminferous tubule cross section; colors as for N. Error bars show SEM. *P , 0.05. NS, not significant.

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FIG. 3. Defective spermatogenesis following elevation of Wnt signaling in AhCre Apcfl/fl mice after 4 days of BNF exposure. AD) Adult testes from
Apcfl/ (A, C) compared to mutant AhCre Apcfl/fl (B, D) mice revealed loss of postmitotic germ cell types. E) Quantification of germ cell loss depicts the
percentage of seminiferous tubules with the most mature germ cell type present in tubule cross sections identified as elongating spermatids (ES), round
spermatids (RS), pachytene spermatocytes (PS), leptotene/zygote spermatocytes (L/Z), or spermatogonia (Spg) and tubules with no germ cells (Sertoli cellonly tubules [SO]) . Ninety to three hundred tubules counted per animal, seven animals per genotype. White bars show control Apcfl/ BNF, gray bars
show intermediate AhCre Apcfl/fl  BNF, and black bars show test AhCre Apcfl/fl BNF samples. Accompanying AhCre Apcfl/fl  BNF analysis is presented
in Supplemental Figure S1. Representative immunostaining for PCNA identified proliferative cells in control Apcfl/ (F) compared to mutant AhCre Apcfl/fl
(G) samples. Immunohistochemical identification of the germ cell marker, Vasa, in control (H) and mutant (I) samples; the Sertoli cell nuclear marker,
Sox9, in control (J) and mutant (K) testes; and the blood-testis barrier marker, connexin43, in control (L) and mutant (M) samples. Images represent
observations from examining a total of seven animals per genotype. Bars 50 lm. Arrowhead indicates tubules with Sertoli cell vacuolization. The
distribution of proliferating cells was quantified as in Figure 2 (N, O) for four animals per genotype. N) The average outcome for each animal in control
(white) versus test (grey) samples. Data shown for each individual animal (O); open circles, Apcfl/; black squares, AhCre Apcfl/fl. Distribution of
undifferentiated spermatogonia (P) was measured as number of Plzf-positive cells per seminferous tubule cross section; colors as for N. Error bars show
SEM. *P , 0.05. NS, not significant.

most mature germ cell type, and 4.00% 6 2.90% of tubules

contained only Sertoli cells (Fig. 2E).
In AhCreApcfl/fl animals, there was an overall reduction in
PCNA- (Fig. 3, F, G, N, and O) and Vasa-positive cells;

cell type was 4.81% 6 0.60% in controls and 30.45% 6

8.82% in AhCreApcfl/fl animals. In these mutants, 23.59% 6
5.17% of tubules contained pachytene spermatocytes and
6.31% 6 4.73% zygotene or leptotene spermatocytes as the
6

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Wnt SIGNALING IN THE POSTNATAL MOUSE TESTIS

we performed pathway-focused transcript profiling of whole

mouse testis at four postnatal ages: Day 7, when the only germ
cells present are spermatogonia and all somatic cell types are
proliferating; Day 14, when spermatogonia and early spermatocytes are present, Sertoli cells have ceased proliferation,
and some peritubular and Leydig cells are proliferating and
differentiating; Day 28, when spermatogonia, spermatocytes,
round spermatids, and maturing somatic cells are all present;
adult, when all germ cell types and mature somatic cells are
present (Fig. 5). Transcripts encoding several Wnt pathway
components were present at low levels on Days 7 and 14 but
were detected at higher levels at Day 28, an interval that
overlaps with the first appearance of pachytene spermatocytes
and round spermatids. These included several Wnt ligands
(Wnt1, Wnt2, Wnt3, Wnt3a, Wnt5b, Wnt7a, Wnt8a, and Wnt
8b) (Fig. 5, A and B), Wnt receptors (Lrp5, Fzd4, and Fzd6;
Fig. 5E), and known Wnt signaling targets (Sox17, Btrc, Lef1,
and Dkk1; Fig. 5, C and D). We observed Axin2 at the highest
level at Day 7, gradually decreasing through to adulthood,
suggesting it is a Wnt signaling target in mitotic germ cells or
in somatic cells (Fig. 5F).

however, this was quite variable between tubules, with some

tubules containing very few germ cells (Fig. 3, H and I). There
was no difference in the frequency of Plzf-positive cells
between tubule cross sections of different genotypes (Fig. 3P
and Supplemental Fig. S3, A and B), in agreement with the
histological quantification data demonstrating that the more
mature germ cells were lost in mutant samples. Although Sox9positive nuclei retained a basal position in all Sertoli cells (Fig.
3, J and K), the greater intensity of AMH staining in some
sections (Supplementary Fig. 3, E and F) and the areas of
diffuse Connexin 43 staining (Fig. 3, L and M) were similar to
those observed in AhCre Ctnnb1fl/fl samples, indicating altered
functionality of these Sertoli cells. Peritubular myoid cells also
appeared unaffected in mutants (Supplemental Fig. S3, C and
D). Mutant testes also showed a higher proportion of apoptotic
cells evident from more abundant TUNEL- and Casp3-positive
cells (Supplemental Fig. S3, GJ).
Nuclear b-Catenin Identifies Active Canonical Wnt
Signaling in Postmitotic Germ Cells of the Adult Mouse
Testis

Having identified the requirement for controlled Wnt

signaling in adult testis, we undertook Wnt-pathway transcript
profiling of AhCre Ctnnb1fl/fl testes to predict which Wnt
signaling components may be present in postmitotic germ cells.
The phenotypes of AhCre Ctnnb1fl/fl mice after 2 and 4 days of
BNF treatment were similar (compare Figs. 2 and 6). Further
histological analysis was also performed on this tissue to
confirm the phenotype similarities (Supplemental Fig. S4). We
reasoned that transcripts present at reduced levels after 2 days
of BNF treatment may be present in spermatocytes and
spermatids, while transcripts that increased would be present in
spermatogonia and somatic cells. Two day-induced AhCre
Ctnnb1fl/fl mutant testes contained higher levels of Wnt1 and
Wnt5a transcripts compared to controls but lower levels of
Wnt3, Wnt3a, Wnt5b, Wnt7a, Wnt8b, Wnt9a and Wnt16.
Wnt5b, Wnt7a and Wnt 8b levels increased with testis age but
were lower in AhCre Ctnnb1fl/fl mutants, suggesting these
ligands are produced by postmitotic germ cells. In contrast,
Wnt5a levels declined with postnatal age but were higher in
AhCre Ctnnb1fl/fl mutants; this ligand most likely is made in
spermatogonia and/or somatic cells. Messenger RNAs encoding the receptors Fzd1, Fzd2, and Fzd3 were higher in mutants
compared to control counterparts, while Fzd4 was lower. Other
Wnt signaling-related transcripts, including Dkk1, Dvl1, Pitx2,
Slc9a3r1, Ctbp2, and Wif1, were lower in mutants, while Nkd1
was higher. The low levels of Axin2 identified in the
transcriptional profiles of spermatocytes and spermatids
(GDS2098; Supplemental Fig. S5) and in adult testes
(Supplemental Fig. S5) suggest that this common indicator of
Wnt pathway activation is not produced in postmitotic germ
cells. These data identify several specific Wnt ligands,
receptors, and associated proteins as being part of the Wnt
signaling pathway that may contribute to maintenance of
spermatogenesis (Fig. 7).
DISCUSSION
Our interrogation of the AhCre Ctnnb1fl/fl and AhCre Apc fl/fl
mutants has identified two overlapping but distinct phenotypes,
demonstrating that precise control of Wnt signaling is essential
for multiple steps in normal spermatogenesis. This finding
correlates with the growing understanding that cellular

Identification of Wnt Signaling Components in the

Developing Testis
To provide information about which Wnt signaling
components are present in developing and adult mouse testes,
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Histology and Transcriptional Profiling of Day 2

Postinduction AhCre b-catfl/fl Mice

Nuclear localization of b-catenin is a recognized hallmark of

active canonical Wnt signaling [3, 4]. We investigated the
subcellular localization of b-catenin in adult mouse testes using
a well-characterized antibody and immunohistochemistry
protocol that was previously optimized for detecting nuclear
b-catenin in intestinal cells [4], recording b-catenin localization
at each stage of the seminiferous epithelium cycle (Fig. 4, A
E). No signal was evident in spermatogonia. Staining
associated with the nuclear envelope was detected in stage
VI pachytene spermatocytes, and this gradually increased to a
more intense nuclear signal in stage IXXII spermatocytes. The
most prominent nuclear staining was visible in stage IVIII
round spermatids. A cytoplasmic signal was visible in Sertoli
cells at all stages, with the staining strongest in the basal region
(Fig. 4, AF). These data demonstrate that Wnt signaling is
active in postmitotic germ cells of the adult mouse testis.
Expression and localization of b-catenin was also investigated at several stages of postnatal testis development.
Although cytoplasmic and plasma membrane-localized bcatenin was evident in all germ cell subtypes, germ cell
nuclear staining was not detected at Postnatal Days 0 (in
gonocytes), 5, or 10 (in spermatogonia) but was present from
Day 20 in round spermatids from the time of their first
appearance [43] (Fig. 4, GK). Nuclear localization was
observed in some Sertoli cells up to Day 10, corresponding
with the period in which they proliferate, but not at Day 20,
when they are postmitotic and considered to be mature [44]
(Fig. 4, G and H). This temporal pattern of b-catenin nuclear
localization in the testis overlaps with that previously described
using the TCF/LEF-lacZ mouse [26] and the BAT-gal
transgenic [45] to identify sites of active b-catenin signaling,
and highlights the prominent nuclear accumulation of b-catenin
in round spermatids that is also evident in the adult testis.
These data indicate that the pathway is robustly active in
immature Sertoli cells, but that the extent or nature of this alters
as these cells cease mitosis and differentiate to form the germ
cell niche that supports full spermatogenesis throughout adult
life.

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FIG. 4. Nuclear localization of b-catenin indicates active canonical Wnt signaling in postmeiotic germ cells. Immunostaining showed b-catenin in adult
mouse testis sections. Brown staining indicates localization of b-catenin on hemotoxylin-counterstained sections. AE) b-Catenin was detected in the
cytoplasm of some spermatogonia, spermatocytes, and spermatids, and in the nucleus of stage VI spermatocytes onward and in the nucleus of all round
spermatids. F) Schematic diagram summarizing b-catenin localization in the adult mouse testis with brown shading. b-Catenin localization was also
examined at Postnatal Days 0 (G), 5 (H), 10 (I), 20 (J), and 35 (K). Primary antibody was omitted in negative controls (insets). G, gonocytes; Sg,
spermatogonia; LS, leptotene spermatocytes; ZS, zygotene spermatocytes; PS, pachytene spermatocytes; RS, round spermatids; ES, elongating spermatid;
SN, Sertoli cell nuclei. Bars 20 lm.

spermatogenic stages. This result is in accord with our

identification of nuclear b-catenin in spermatocytes and
spermatids. While another group examining the localization
of b-catenin did not report a nuclear signal in germ cells [18],
our study utilized a protocol previously optimized to identify a
nuclear signal in intestinal stem cells [4, 9] and agrees with
another publication [45]. We summarize that differences in
tissue handling and antibody affinity underlie these different
outcomes. Active Wnt signaling has also been identified in
round spermatids of the adult testis in BAT-gal transgenic mice
[45]. Our results are also consistent with the recent report that
chronic deletion of b-catenin in spermatids causes spermatogenic failure [25], with the reported cellular lesion being loss of
Sertoli cell-germ cell adhesions at the apical ectoplasmic
specialization. We saw no obvious evidence of spermatogonial

differences in Wnt signaling thresholds specify physiological

and pathological outcomes, including proliferation and differentiation during embryonic development and in adult tissue
homeostasis. Importantly, we also demonstrated the use of the
AhCre model to analyze gene function in the testis. The AhCre
allows investigation of mice that have matured into adulthood
before induction of complete expression of Cre recombinase.
However, this Cre allele does display some leakiness and
mediates recombination in both somatic and germ cell
compartments, which needs to be considered when interpreting
phenotypes.
The predominant phenotype in AhCre Ctnnb1fl/fl mice
arising from BNF administration to facilitate widespread Cre
activation was a loss of postmitotic germ cells, indicating that
b-catenin-mediated Wnt signaling is vital in the later
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Wnt SIGNALING IN THE POSTNATAL MOUSE TESTIS

FIG. 6. Phenotypic analysis of defective spermatogenesis in AhCre Ctnnb1fl/fl mice 2 days post-BNF injection. Sections from control Ctnnb1fl/ (A) were
compared to mutant AhCre Ctnnb1fl/fl (B); H&E staining (A, B) revealed loss of postmitotic germ cell types in mutant testes after 2 days of BNF treatment.
Bars 50 lm. Images illustrate one representative sample of a total of seven animals per genotype. Transcriptional profiling of Wnt ligands (C), receptors
(D), and other (E) signaling components in whole AhCre Ctnnb1fl/fl mouse testis compared to control Ctnnb1fl/ mouse testes using the Wnt signaling
mouse qRT-PCR array. Graphs show fold change of mutant testis transcript values compared to a paired control testis analyzed simultaneously, and results
from each pair are plotted as a single point. Mean values are indicated by grey bars but absent from genes where replicates did not show a similar result.

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FIG. 5. Transcriptional profiling of key Wnt signaling components in whole Swiss mouse testes at Postnatal Days 7, 14, and 28 and adult (Week 7). Data
acquired from biological duplicates using a Wnt signaling mouse qRT-PCR array are presented in functional groupings separated into high- and low-fold
changes compared to Day 7 (AE). Axin2 data acquired separately using qRT-PCR are presented as the average relative transcript levels from four
independent samples at each age (F).

KERR ET AL.

spermatids at increasing levels in germ cells progressing from

round to elongating spermatids [28]. A protein bearing
homology to the known Wnt signaling antagonist family
Dickkopf, DkkL1, is present in the testis [46, 47] and appears
during spermatogenesis initially in developing spermatocytes
and then later localizes to the acrosome of mature sperm heads
[47]. These findings provide further demonstration of the need
for precise regulation of Wnt function in postmitotic germ
cells.
Control of Wnt signaling within somatic cells of the adult
testis is also crucial to fertility. Selective introduction into
Sertoli cells of an inactivating mutation in Apc caused
premature germ cell depletion and loss of Sertoli cell apical
extensions and blood-testis barrier integrity [22]. AhCre Apcfl/fl
mice used in the present study differ from previous models
because they reveal defects observed after only 2 days of
disrupted Apc function. This phenotype appears similar to the
previously reported APCck8 [22] mice, suggesting the phenotype reported may reflect changes in Sertoli cell function. In the
context of Sertoli cell development, nuclear b-catenin was
observed in a subset of Sertoli cells prior to Postnatal Day 10
but not at later ages when they have ceased mitosis and
assumed the differentiated phenotype required to sustain

or somatic cell loss in the models generated for the present

study. In addition, several groups have previously deleted bcatenin selectively in Sertoli cells and reported an absence of
defects [18, 22, 23]. These data collectively indicate that the
mutant b-catenin phenotype presented here is due to a germ
cell-specific role for this protein. The short-term time course of
genetic manipulation used in the present study highlights the
acute importance of Wnt signaling for maintaining spermatogenesis. These models add to the growing body of evidence
from analysis of genetically modified mice that delineate
targets and consequences of Wnt actions in the testis
(summarized in Supplemental Table S1 and discussed further
below).
Aberrant up-regulation of Wnt signaling results in a
phenotype subtly distinct from that observed in AhCre
Ctnnb1fl/fl animals, with acute loss of Apc in AhCre Apcfl/fl
mutant mice leading to germ cell loss at a wider range of
stages, but again it is the postmitotic germ cells that appear to
be directly affected. Two factors capable of mediating intrinsic
control of Wnt signaling in spermatogenesis have been
identified in these cells. Mice bearing a nonfunctional form
of the Wnt antagonist naked cuticle 1 (Nkd1) have reduced
fertility, with the Nkd1 transcript shown to be present in
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FIG. 7. Summary of deduced sites of production for Wnt signaling components in the adult mouse testes. Data were derived from (1) publications
documenting transcript or protein localization, (2) GeoProfiles data sets GDS2390 and GDS605 (in Supplemental Figs. S5.1 and S5.2), and (3) our array
data including concordance between testis-development profiles and mutant mouse models.

Wnt SIGNALING IN THE POSTNATAL MOUSE TESTIS

[26] and BAT-gal transgenic mice [45] and with a separate

report by Tanwar et al. [22]. The nuclear localization of bcatenin reported here clearly establishes that canonical Wnt
signaling is active in postmeiotic germ cells of the testis, but
we predict that the transcriptional outcomes will be cell typespecific during spermatogenesis. Identification of key Wnt
targets will provide valuable clues to what key molecules are
required to sustain spermatogenesis in men and could offer new
avenues for fertility control when testis-specific signaling
outcomes are discovered.

spermatogenesis. In previous reports describing the outcomes

of Sertoli cell-specific constitutive Wnt signaling activation,
spermatogenesis was initiated and was generally normal, but
there was progressively increased production of immature
Sertoli cell makers and seminiferous tubule degradation with
age [18, 20, 21].
Our unbiased evaluation of transcripts relating to Wnt
signaling improves our understanding of where and when Wnt
activities are important within developing and adult mouse
testes. To propose a model of cell-specific production of Wnt
components during spermatogenesis, we have incorporated
information from previous publications with three independent
lines of transcriptional profiling evidence: (1) Our own qRTPCR profiling of normal mouse development, (2) Geoprofiles
data from Affymetrix arrays of normal mouse testis development and from isolated germ cell subtypes [36, 37], and (3) our
qRT-PCR profiling of adult mice with acute disruption to Wnt
signaling. This is summarized in Figure 7. Our findings could
aid in development of strategies to introduce germ cell-specific
conditional mutations with key alleles to refine our understanding of how regulated Wnt signaling controls germ line
survival, interaction with somatic cells, and maturation.
Our own data reinforce other independent analyses to
demonstrate that levels of certain Wnt signaling components
are modulated during spermatogenesis, enabling particular
ligands and receptors to be targeted in future analyses of human
infertility and testicular dysgenesis. For example, while
inactivation of b-catenin function in mouse spermatids was
recently shown to cause spermatogenic failure [25], we can
now propose that Frzd4 and Lrp5 are involved. This finding
also highlights the value of examining male germline
development to determine what dynamic features of Wnt
signaling govern cellular differentiation. There is a switch in
the synthesis of two receptor subunits between Lrp6 transcription in spermatogonia and Lrp5 in spermatids. The functional
significance of this is not known. Gene expression at each
spermatogenic stage is governed by distinct drivers, and these
may promote synthesis of functionally redundant receptors to
sustain ongoing Wnt activity throughout spermatogenesis;
alternatively, there may be a functional difference between the
two receptors that reflects their predominant expression in
distinct germ cell types. Overall, there are now several lines of
functional data demonstrating that spermatocytes and spermatids require regulated Wnt signaling, and the transcriptional
profiles presented here identify pathway components that are
involved in these and other cells of the testis.
An important outcome from this analysis is the emerging
understanding of which indicators of active Wnt signaling are
appropriate to consider during spermatogenic development.
Conventionally, Axin2, TCF/LEF, and nuclear b-catenin are
associated with cells in which Wnt target genes that promote
cell proliferation are activated [6, 48]. An important exception
are Paneth cells in the intestinal epithelium, which, unlike their
close neighbors, the crypt base columnar stem cells, require
canonical Wnt pathway activation for differentiation, even
though both cell types do display TCF/LEF and nuclear bcatenin and depend on canonical Wnt signaling [49, 50]. In the
publically accessible transcriptional profiles relating to male
germline differentiation (Supplemental Fig. S5C), there is
evidence of only low-level Axin2 transcription, confirmed by
our independent examination of this transcript in the
developing testis (Fig. 5F), suggesting that Axin2 is not a
target of Wnt signaling in postmeiotic germline cells. In
contrast, the Lef1 mRNA is up-regulated in spermatocytes
compared to other cell types (Supplemental Fig. S5J); this is in
agreement with data from studies using TCF/LEF-LacZ mice

ACKNOWLEDGMENT
The authors thank Derk ten Berge and Ans van Pelt for helpful input
regarding this manuscript and Helen Lescesen for assistance with mouse
colony management and genotyping.

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