Nuevos Conocimientos Sobre La (In) Fertilidad Masculina - La Importancia Del NO

1/3/2021 Nuevos conocimientos sobre la (in) fertilidad masculina: la importancia del NO
Br J Pharmacol. 2015 Mar; 172 (6): 1455–1467. PMCID: PMC4369256

Publicado en línea el 2 de julio de 2014 doi: 10.1111 / bph.12675 PMID: 24601995
Nuevos conocimientos sobre la (in) fertilidad masculina: la

importancia del NO
B Buzadzic , 1 M Vucetic , 1 A Jankovic , 1 A Stancic , 1 A Korac , 2 B Korac , 1 y V Otasevic 1
1Department of Physiology, Institute for Biological Research ‘Sinisa Stankovic’, University of Belgrade,
Belgrade, Serbia
2Faculty of Biology, Institute of Zoology and Center for Electron Microscopy, University of Belgrade, Belgrade,
Serbia
Correspondence, Vesna Otasevic, Department of Physiology, Institute for Biological Research ‘Sinisa
Stankovic’, University of Belgrade, Bulevar despota Stefana 142, 11060 Belgrade, Serbia. E-mail:
vesna@ibiss.bg.ac.rs
Received 2013 Oct 31; Revised 2014 Jan 31; Accepted 2014 Mar 3.
Copyright © 2015 The British Pharmacological Society
Resumen
La infertilidad es un problema global que va en aumento, especialmente durante la última década.
Actualmente, la infertilidad afecta aproximadamente al 10-15% de la población mundial. La frecuencia
y el origen de las diferentes formas de infertilidad varían. Se ha demostrado que las especies reactivas
de oxígeno y nitrógeno (ROS y RNS) están involucradas en la etiología de la infertilidad,
especialmente la infertilidad masculina. Se han diseñado varias estrategias para eliminar o disminuir la
producción de ROS y RNS en los espermatozoides, en particular durante la fertilización in vitro. Sin
embargo, en los últimos años se ha demostrado que los espermatozoides producen de forma natural una
variedad de ROS / RNS, incluido el radical anión superóxido (O 2 ⋅−), peróxido de hidrógeno y NO.
Estas especies reactivas, en particular el NO, son esenciales en la regulación de la capacidad de los
espermatozoides y la reacción del acrosoma, dos procesos que deben ser adquiridos por los
espermatozoides para alcanzar el potencial de fertilización. Además, se ha demostrado recientemente
que la función mitocondrial se correlaciona positivamente con el potencial y la calidad de la
fertilización del esperma humano y que los precursores de NO y NO aumentan la motilidad de los
espermatozoides al aumentar la producción de energía en las mitocondrias. Revisaremos el nuevo
vínculo entre la regulación redox impulsada por el NO de los espermatozoides y la infertilidad en este
documento. Se hará especial hincapié en la posible implementación de nuevas sustancias activas redox
que modulan el contenido de NO en los espermatozoides para aumentar la fertilidad y promover la
concepción.
Artículos vinculados
Este artículo es parte de una sección temática sobre farmacología de los gasotransmisores. Para ver los
otros artículos de esta sección, visite http://dx.doi.org/10.1111/bph.2015.172.issue-6
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4369256/ 1/24
Palabras clave: óxido nítrico, infertilidad, espermatozoides, fertilización in vitro, mitocondrias,

regulación redox, capacitación, reacción acrosómica, motilidad.
Introducción
La infertilidad es uno de los problemas médicos más graves del mundo. La prevalencia de la
infertilidad va en aumento, especialmente durante la última década. A nivel mundial, una de cada seis a
siete parejas en todo el mundo tiene actualmente alguna dificultad con la concepción (Sharma et al .,
2013 ). La infertilidad generalmente se define como la incapacidad para concebir después de 1 año de
relaciones sexuales regulares sin protección, pero en las parejas en las que la pareja femenina tiene> 35
años, la infertilidad se diagnostica después de una incapacidad para concebir durante 6 meses (Cooper
et al ., 2010).). Aunque la frecuencia y el origen de la infertilidad varían, se ha establecido que casi el
40% de los problemas relacionados con la infertilidad son atribuibles a un factor masculino, otro 40%
debido a un factor femenino y el 20% son el resultado de factores masculinos y femeninos combinados
( Sharlip et al ., 2002 ).
Durante la última década, se han producido avances significativos en el diagnóstico y tratamiento de

los trastornos reproductivos. Sin embargo, algunos de los factores causales que subyacen a la
infertilidad solo pueden superarse con éxito con el uso de tecnologías de reproducción asistida. Varias
técnicas, como in vitroLa fertilización (FIV), la inyección intracitoplasmática de espermatozoides, la
inseminación intrauterina con el semen de una pareja o un donante, el reemplazo de embriones
congelados y la donación de óvulos, se han desarrollado para tratar la infertilidad. Desde 1978, cuando
nació el primer bebé de FIV, las técnicas utilizadas en la FIV han avanzado de forma espectacular; de
hecho, la tasa de éxito del procedimiento actual basada en los embarazos clínicos es del 25 al 50%, lo
que muestra claramente que los avances en la medicina general y reproductiva ocurren
simultáneamente. Sin embargo, los patrones de estilo de vida modernos, como los factores sociales y
ambientales, explican parte del aumento en la prevalencia de la infertilidad. Por estos motivos, una de
las orientaciones biomédicas más importantes en la actualidad es el desarrollo de nuevos enfoques
terapéuticos para la mejora de la fecundación, tanto bajo métodos de reproducción natural como
asistida.
Una extensa investigación en infertilidad ha confirmado que la producción excesiva de especies

reactivas de oxígeno y nitrógeno (ROS y RNS), es decir, estrés oxidativo, está involucrada en la
etiología de la infertilidad, especialmente la infertilidad masculina (MacLeod, 1943 ; Aitken y
Clarkson, 1987 ; Iwasaki y Gagnon, 1992 ; Sukcharoen et al ., 1996 ; Agarwal y Allamaneni, 2004).
Todos estos informes han asociado ROS / RNS, generados tanto de forma exógena como endógena,
con varios aspectos de la infertilidad masculina, como la disminución de la motilidad de los
espermatozoides, la morfología anormal y la disminución de la penetración de los espermatozoides y
los óvulos. El mecanismo de acción de ROS y RNS implica la peroxidación lipídica de la membrana
plasmática de los espermatozoides, que es altamente susceptible al daño oxidativo debido a grandes
cantidades de ácidos grasos poliinsaturados, al afectar la fluidez y movilidad de la membrana (Alvarez
y Storey, 1995 ; Baker et al . , 1996 ). Además, ROS y RNS pueden dañar el axonema del esperma y
alterar la función mitocondrial, el ADN, el ARN y las proteínas (de Lamirande y Gagnon, 1992 ;
Agarwal y Prabakaran, 2005 ).
Por lo tanto, durante muchos años, ROS y RNS se han considerado dañinos y se diseñaron varias
estrategias para eliminar o disminuir su producción, especialmente durante la FIV, cuando los
espermatozoides están expuestos a concentraciones de oxígeno más altas que in vivo . Algunas de las
estrategias utilizadas incluyen la suplementación de los medios de cultivo con antioxidantes (vitamina
E y agentes reductores de disulfuro), la eliminación de células productoras de ROS (principalmente
leucocitos y espermatozoides dañados) mediante gradiente de densidad para la preparación de
espermatozoides e incubación en baja tensión de O 2 y iluminación (de Lamirande y Gagnon, 1992 ;
Aitken et al ., 1996 ; Irvine, 1996 ; Parinaud et al ., 1997). Sin embargo, en contraste con la
participación de ROS y RNS en la infertilidad masculina, se ha demostrado que la ingesta
suplementaria de antioxidantes en humanos mejora las funciones reproductivas solo en casos
seleccionados (Dawson et al ., 1992 ; Kessopoulou et al ., 1995 ). Además, algunos de los estudios no
han informado cambios en los parámetros del semen o la fertilidad masculina después de la
administración oral prolongada de antioxidantes (Hughes et al ., 1998 ; Rolf et al ., 1999 ). Estos datos
han suscitado preocupación y debate sobre la eficacia de la administración oral de antioxidantes como
terapia para la infertilidad masculina.
La base del "fracaso" de la terapia antioxidante en el tratamiento de la infertilidad masculina es que se

necesitan ROS y RNS en concentraciones fisiológicas pequeñas y controladas para el funcionamiento
normal de los espermatozoides. Por lo tanto, ROS / RNS debe mantenerse en niveles apropiados para
asegurar una función fisiológica. Específicamente, se ha demostrado que como producto del
metabolismo normal en las células germinales --espermatozoides y ovocitos-- se produjeron pequeñas
cantidades de ROS y RNS, y estas especies reactivas, cuando se producen en concentraciones
fisiológicas controladas, funcionan como mensajeros secundarios que regulan cruciales procesos
reproductivos, especialmente en los espermatozoides (de Lamirande et al ., 1997 ; Leclerc et al .,
1997). Específicamente, se ha demostrado que el NO y el radical anión superóxido (O 2 ⋅− ) son
esenciales en la regulación de la capacidad espermática y la reacción acrosómica (AR), dos procesos
necesarios para que los espermatozoides adquieran potencial de fertilización (de Lamirande et al .,
1997 ; Leclerc et al ., 1997 ). También se ha demostrado que ROS y RNS estimulan la motilidad
hiperactivada de los espermatozoides (Suzuki et al ., 1997 ) y la unión a la zona pelúcida (ZP) (Aitken
et al ., 1989 ), enfatizando que los eventos de fertilización espermática esenciales son sensibles al
redox.
Es importante señalar que el principal sitio de producción y el objetivo de ROS y RNS son las
mitocondrias. En consecuencia, mediante la producción de ROS / RNS y la activación posterior de
varias vías de señalización dependientes de redox, las mitocondrias están involucradas en la regulación
de las funciones de los espermatozoides. Además, se ha demostrado que a través de la producción de
ATP, las mitocondrias regulan la espermatogénesis, la diferenciación, la AR, la fusión de ovocitos y la
fertilización (Agarwal et al ., 2008 ). Además, a través de la señalización de Ca 2+ , las mitocondrias
regulan la hiperactivación de los espermatozoides, la actividad flagelar durante los eventos que
conducen a la fertilización y el metabolismo energético (Publicover et al ., 2008).). La importancia de
las mitocondrias en el funcionamiento de los espermatozoides se fortaleció con evidencia reciente que
muestra que la función mitocondrial está correlacionada positivamente con el potencial y la calidad de
la fertilización del esperma humano (Kasai et al ., 2002 ; Marchetti et al ., 2004 ; Gallon et al ., 2006 ;
Otasevic et al ., 2006 ; Otasevic et al ., 2002 ; Marchetti et al ., 2004 ; Gallon et al ., 2006 ; Otasevic et
al ., 2006) . al ., 2013 ). Por el contrario, las mitocondrias son el sitio principal para la focalización de
ROS y RNS. De acuerdo con esta observación, durante la última década se han hecho evidentes
múltiples efectos reguladores del NO sobre el funcionamiento de las mitocondrias (Nisoli et al ., 2003 ;
Petrović et al.., 2005; 2008; 2010a, b,,,; Vucetic et al ., 2011 ). En nuestro trabajo reciente, hemos
demostrado que el NO logra efectos beneficiosos sobre la función de los espermatozoides a través de la
mejora del estado funcional de las mitocondrias de los espermatozoides (Otasevic et al ., 2013 ). Por
estas razones, la participación del NO en las mitocondrias de los espermatozoides y la función de los
espermatozoides merece una mayor consideración y se analiza con más detalle en esta revisión.
La revisión actual ha abordado la infertilidad desde el aspecto de la regulación redox. Nos centramos
en la (in) fertilidad masculina y la regulación redox mediada por NO de eventos cruciales de
fertilización de espermatozoides. Se hizo especial hincapié en la posible implementación de nuevas
sustancias activas redox que, mediante la modulación del contenido de NO en los espermatozoides,
podrían aumentar la fertilidad masculina y promover la concepción.
NO como molécula de señalización: una descripción histórica

El NO representa un gas diatómico simple que funciona como una molécula de señalización celular en
células de mamíferos, controlando funciones fundamentales en el sistema cardiovascular, cerebro,
defensa del huésped, inmunidad, sistema reproductivo, sistema gastrointestinal y tejidos secretores
(Alderton et al ., 2001 ; Govers and Oess, 2004 ). La primera descripción del NO estuvo relacionada
con el descubrimiento de la nitroglicerina (NG) en 1847 por el científico italiano Sobrero (Marsh y
Marsh, 2000). Unos años más tarde, la NG se utilizó para tratar los dolores de cabeza, mientras que
desde 1870 se ha utilizado en el tratamiento de la angina de pecho. Un siglo después, se demostró que
NG activa la GC soluble (sGC), además de aumentar la producción de cGMP. La mayor contribución al
descubrimiento de los efectos del NO fue el descubrimiento del factor relajante derivado del endotelio
(EDRF) por Furchgott y Zawadzki ( 1980 ). Siete años después, Ignarro et al . (1987a, b,) mostraron
que EDRF es realmente NO, y un año después el grupo de Moncada (Palmer et al ., 1988 ) informó que
las células de mamíferos sintetizan endógenamente NO a partir de -arginina. La importancia de este
descubrimiento quedó demostrada con el nombramiento de NO como la 'molécula del año' por el editor
de Science en 1992. En 1998, los científicos que participaron en el descubrimiento del NO y definieron
el papel del NO en el sistema cardiovascular. sistema fueron galardonados con el Premio Nobel de
Medicina.
SIN síntesis
El NO se sintetiza en prácticamente todas las células de mamíferos mediante la oxidación de -
arginina por una familia de isoformas de NOS (Moncada et al ., 1989 ). Hasta ahora se han identificado
tres isoformas distintas de NOS (véase Alexander et al ., 2013 ), como sigue: neuronal (nNOS), que se
describió originalmente en tejido neuronal; endotelial (eNOS), que se describió originalmente en
células endoteliales; y NOS inducible (iNOS), que se identificó originalmente en macrófagos
(Moncada et al ., 1991 ; Alderton et al ., 2001). Todas las isoformas de NOS de mamíferos
caracterizadas son proteínas que contienen hemo que son diméricas en condiciones nativas con una
masa molecular monomérica de 126-160 kDa. Las isoformas de NOS muestran diferentes afinidades
por la calmodulina, mientras que nNOS y eNOS son dependientes de Ca 2+ -calmodulina e iNOS es
casi independiente de Ca 2+ -calmodulina. Otra diferencia entre las isoformas NOS está relacionada
con el nivel de producción de NO porque se ha demostrado que iNOS produce niveles más altos de NO
(μM-mM) y permanece activo durante un período de tiempo más largo en comparación con nNOS y
eNOS (concentraciones de NO de nM- μM) (Moncada et al ., 1991 ; Alderton et al ., 2001 ; Buzadzic
et al ., 2006; Otasevic et al ., 2011 ). Todas las isoformas de NOS pueden encontrarse unidas a las
mitocondrias o dentro de ellas, en cuyo caso la NO sintasa se conoce como la cuarta isoforma NOS
mitocondrial (mtNOS) (Kobzik et al ., 1995 ; Bates et al ., 1996 ; Ghafourifar y Richter, 1999 ). Sin
embargo, la existencia de mtNOS es controvertida y la caracterización completa de mtNOS aún está en
progreso.
Mecanismos por los que actúa el NO

En general, se ha demostrado que el NO exhibe sus efectos a través de dos vías de señalización
diferentes de la siguiente manera: señalización dependiente de cGMP y señalización independiente de
cGMP (señalización clásica y no clásica respectivamente) (Martínez-Ruiz et al ., 2011 ; Otasevic et al.
., 2011 ). La señalización clásica implica la activación de sGC, la generación de cGMP y la activación
subsiguiente de enzimas específicas dependientes de cGMP (proteína quinasas, canales y
fosfodiesterasas). La sensibilidad de la sGC al NO se encuentra dentro del rango nanomolar (Martínez-
Ruiz et al ., 2011). La señalización no clásica se produce a través de la modificación covalente
postraduccional de las proteínas diana, es decir, S-nitrosilación, S-glutatión y nitración de tirosina
(Mieyal et al ., 2008 ; Souza et al ., 2008 ; Lima et al ., 2010 ) . Esta señalización no clásica se rige por
concentraciones de NO superiores a las necesarias para la activación de sGC. La S-nitrosilación de las

proteínas y tioles está mediada no solo por NO, sino también por otras especies de NO, complejos
metal-NO, peroxinitrito o nitrito (Otasevic et al ., 2011). Casi todas las clases de proteínas, incluidos
los receptores, las enzimas y los factores de transcripción, son objetivos de la S-nitrosilación. Por el
contrario, las modificaciones de proteínas por nitración de tirosina, que están mediadas principalmente
por peroxinitrito, potencialmente dan como resultado alteración, pérdida o ganancia de función, pero
no se ha demostrado la reversibilidad y, por lo tanto, no se puede hacer una asignación clara a la
regulación redox.
Es importante destacar específicamente el NO que actúa de manera independiente de cGMP, en el que

el NO interactúa con otras hemoproteínas, como la citocromo c oxidasa (Cleeter et al ., 1994 ). Esta vía
independiente de cGMP es una vía menos clásica pero igualmente importante para el NO, que podría
tener implicaciones importantes para la respiración y el metabolismo celular (Brookes et al ., 2002 ;
Erusalimsky y Moncada, 2007 ; Vucetic et al ., 2011 ).
Isoformas NOS en espermatozoides

Los espermatozoides humanos expresan las tres isoformas de NOS, que se encuentran en la cabeza y /
o en las regiones del flagelo de los espermatozoides (Herrero et al ., 2003 ; de Lamirande y Lamothe,
2009 ; de Lamirande et al ., 2009 ). Se ha demostrado que en condiciones de capacitación, la actividad
de las NOS aumenta de manera dependiente del tiempo (de Lamirande y Lamothe, 2009 ; de
Lamirande et al ., 2009 ). Además, recientemente se ha demostrado que la presencia y actividad de los
espermatozoides NOS dependen de la madurez de las células germinales masculinas (Roessner et al .,
2010). La correlación entre la actividad de la NOS y la madurez de los espermatozoides subyace
claramente al papel fisiológico que juega el NO en los espermatozoides. A menudo es muy difícil
definir una función regulada exclusivamente por una isoforma NOS específica. Sin embargo, Table1
resume la función específica reportada, hasta ahora, que cada isoforma NOS juega en la regulación de
eventos importantes para la funcionalidad de los espermatozoides.
tabla 1
Función específica de las isoformas NOS en la regulación de eventos importantes para la
funcionalidad de los espermatozoides.
Isoforma Localización (Lee y Cheng, 2008 ) Función

eNOS Células germinales, células de Sertoli, Control de la unión estrecha en los testículos, apoptosis de
células de Leydig, miofibroblastos, células germinales, motilidad de los espermatozoides y
células endoteliales en vasos reacción acrosómica inducida por el líquido folicular,
sanguíneos, cabeza y pieza intermedia espermatogénesis, regulación de la erección del pene
del espermatozoide (Burnett et al ., 1996 ; Zini et al ., 1996 ; 1998 ; 1999 ;
O'Bryan et al ., 1998 ; Revelli et al ., 1999 ; Lee y Cheng,
2008 )
nNOS Células de Sertoli, acrosoma, células Motilidad de los espermatozoides, regulando la erección
de Leydig, miofibroblastos, células del pene.
endoteliales en vasos sanguíneos,
(de Lamirande et al ., 1984 ; Burnett et al ., 1996 ; Schaad
cabeza y pieza intermedia del
et al ., 1996 ; Donnelly et al ., 1997 )
espermatozoide
iNOS Células de Leydig, células de Sertoli, Control de la unión estrecha en los testículos, apoptosis de
células germinales, cabeza y pieza células germinales, fertilización y diferentes aspectos de la
intermedia del espermatozoide interacción espermatozoide-óvulo durante la fertilización,
apoptosis de células germinales y número de
espermatozoides.
(Lue et al ., 2003 ; Yang et al ., 2005 ; Vera et al ., 2006 ;
Lee y Cheng, 2008 )
TnNOS Células de Leydig Esteroidogénesis
(Wang et al ., 2002 )
Abrir en una ventana separada
TnNOS, nNOS específico de testículo.
Específicamente, se sabe que el NO está involucrado en la regulación de la motilidad, viabilidad,

hiperactivación, capacitación, AR y fusión con el ovocito de los espermatozoides (Figura 1) (Herrero et
al ., 1999; 2003; de Lamirande y Lamothe, 2009 ; de Lamirande et al ., 2009 ; Kothari et al ., 2010 ;
Doshi et al ., 2012 ).
Figura 1
NO regula las funciones de los espermatozoides. A niveles fisiológicos, el NO y el RNS regulan los
eventos de fertilización espermática esenciales. A niveles suprafisiológicos, el NO logra efectos
perjudiciales sobre la función de los espermatozoides mostrada anteriormente. PLCγ1, fosfolipasa γ1
específica de fosfoinosítido.
Efectos del NO sobre los efectos fertilizantes de los espermatozoides
Movilidad, morfología y viabilidad de los espermatozoides

La motilidad de los espermatozoides es un parámetro crucial de la calidad del semen (Wang et al .,
2012 ). En consecuencia, una causa principal de infertilidad masculina es la disminución de la
motilidad de los espermatozoides, y los hombres con espermatozoides poco móviles o inmóviles suelen
ser estériles (Turner, 2006 ). El primer informe que mostró un papel regulador del NO en la motilidad
de los espermatozoides provino de Hellstrom et al . ( 1994 ), quienes determinaron los efectos de los
compuestos liberadores de NO sobre la movilidad y viabilidad de los espermatozoides. Mostraron
(Hellstrom et al ., 1994) que el nitroprusiato de sodio liberador de NO (SNP) en concentraciones bajas
(25-100 nM) es beneficioso para el mantenimiento del movimiento y la viabilidad de los
espermatozoides humanos después de la descongelación. En contraste, varios informes han mostrado
una disminución en la motilidad progresiva de los espermatozoides en presencia de concentraciones
altas de NO puro (25-125 μM) y SNP (0.25-2.5 mM), o S-nitroso-N-acetilpenicilamina (12-600 μM ) y
3-morfolinosidnonimina (100-125 μM) (Herrero et al ., 1994 ; Rosselli et al ., 1995 ; Weinberg et al .,
1995 ; Nobunaga et al ., 1996). Está claro que, como en otros sistemas, el NO ejerce una acción
bimodal sobre la motilidad de los espermatozoides; específicamente, las concentraciones bajas de NO
aumentan la motilidad de los espermatozoides, mientras que las concentraciones altas de NO reducen la
motilidad de los espermatozoides (Weinberg et al ., 1995 ; Herrero y Gagnon, 2001 ; Miraglia et al .,
2011 ; Doshi et al ., 2012 ). De acuerdo con este hallazgo, se han informado niveles altos de NO en el
semen de hombres infértiles con disminución de la motilidad del esperma (Rosselli et al ., 1995 ;
Perera et al ., 1996 ; Balercia et al ., 2004). Además, se ha demostrado que la producción de nitrito por
los hombres astenozoospérmicos es menor que la de los hombres normozoospérmicos, lo que indica
que una disminución del NO endógeno influye en la motilidad del esperma y, en consecuencia, en el
potencial de fertilización del esperma (Lewis et al ., 1996 ; Bolaños et al . , 2008 ). Por consiguiente, la
adición de N G -nitro- -arginina metil éster ( -NAME) al medio de esperma-incubando reduce
significativamente la movilidad del esperma humano (Donnelly et al ,. 1997 ; Yeoman et al ., 1998 ).
Miraglia y col . ( 2011 ) informaron que el NO influye en la motilidad de los espermatozoides a través
de la activación de sGC, la posterior síntesis de cGMP y la activación de proteína quinasas
dependientes de cGMP. Además, nuestros datos recientes sugieren que el NO aumenta la motilidad de
los espermatozoides al aumentar la producción de energía que se origina en el compartimento
mitocondrial (Otasevic et al ., 2013 ). En consecuencia, los niveles altos de NO en los espermatozoides
detectados en hombres infértiles están relacionados con una disminución del metabolismo de los
espermatozoides (Doshi et al ., 2012 ).
Moreover, NO contributes to normal sperm morphology, which has been shown to facilitate the
accurate prediction of fertility status and pregnancy outcomes during IVF (Revelli et al., 2001).
Nevertheless, similar to sperm motility, it has been shown that in high concentrations NO induces
atypical sperm morphology (Wu et al., 2004). Sperm viability is also affected by NO. Specifically,
researchers have reported that viability of sperm in the presence of 10−4 M SNP is significantly lower
than sperm without this treatment (Doshi et al., 2012).
Sperm capacitation
Motile and morphologically mature human spermatozoa are not fertile immediately after ejaculation,
but rather achieve fertilizing ability during transition in the female genital tract. Sperm first undergoes
a complex and timely series of biochemical and physiological changes, which is termed capacitation
(Chang, 1951; Austin, 1952). In fact, capacitation is the final maturation process that ensures that only
fertile spermatozoa are able to reach, bind to and penetrate the oocyte (Baker and Aitken, 2004).
Membrane and metabolic changes that occur during capacitation confer upon the spermatozoa an
ability to gain hyperactive motility, interact with the oocyte ZP, undergo an AR and initiate oocyte
plasma membrane fusion (Baker and Aitken, 2004).
The involvement of NO in capacitation was first demonstrated in human spermatozoa incubated with
low concentrations of NO-releasing compounds (Zini et al., 1995). Specifically, it has been shown that
NO donors (with different potencies) accelerate capacitation, while NOS inhibitors significantly
decrease this process (Herrero et al., 1999). These data clearly demonstrate that endogenous NO is
necessary for spermatozoa to display full fertilizing ability. Furthermore, it was subsequently shown
that this NO-dependent redox control appears crucial for capacitation triggered by different agents,
such as albumin, progesterone and fetal cord serum ultrafiltrate, which modulate the same actions
independently of the inducer (O'Flaherty et al., 2006; de Lamirande and O'Flaherty, 2008).
At the molecular level, capacitation involves a cascade of events (an efflux of cholesterol, fluxes of
bicarbonate, and a rise in cAMP, Ca2+ and intracellular pH) that is initiated by ROS and RNS produced
by spermatozoa (de Lamirande et al., 1997; 2009; Herrero et al., 1999; 2003; O'Flaherty et al., 2006; de
Lamirande and O'Flaherty, 2008; de Lamirande and Lamothe, 2009). Specifically, it has been shown
that NO and O2⋅− are generated in spermatozoa from the beginning of capacitation and control the
increase in cAMP and most of the known serine/threonine phosphorylation of PKA substrates, tyrosine
phosphorylation of fibrous sheath proteins as well as tyrosine nitration of proteins. Moreover, it has
been shown that NO is needed over the course of capacitation and more than one NOS is probably
involved in this process (de Lamirande and Lamothe, 2009).
Hyperactivation
Hyperactivation coincides with the onset of capacitation and represents a subcategory of capacitation.
In non-hyperactive spermatozoa, low-amplitude flagellar movements are associated with slow, linear
movements of the cell. In contrast, hyperactive spermatozoa exhibit high amplitude asymmetric
flagellar movements and non-linear motility. In the female genital tract, hyperactivated spermatozoa
exhibit less stagnation in the oviductal epithelium and the surrounding viscous mucus and a propulsive
force to penetrate the cells of the oocyte (Miraglia et al., 2007; Kothari et al., 2010). Results supporting
the role of NO in the initiation of hyperactivation are well accepted. Similar to the NO effects on
‘regular’ sperm motility, it has been shown that NO concentrations < 1 μM increase sperm
hyperactivation, while NO in concentrations > 1 μM decreases sperm-hyperactivated motility (Lewis
et al., 1996; Miraglia et al., 2011; Otasevic et al., 2013).
AR
When hyperactivated motility has drowned the capacitated spermatozoa to the ovum, sperm binds to
the ZP and begins to bypass this barrier by the exocytotic release of proteolytic enzymes in a process
referred to as the AR. The AR encompasses the release of proteolytic enzymes from the acrosome cap
of the spermatozoa that create a pore in the extracellular matrix of the ZP and allows spermatozoa to
bind appropriately, penetrate the barrier and fuse with the oocyte. At the molecular level, the AR shares
a significant overlap with molecular events of capacitation, such as phosphorylation of similar tyrosine
residues in proteins, an influx of Ca2+, activities of AC, cAMP and PKA (de Lamirande and
O'Flaherty, 2008). Similar to capacitation, reactive species, including NO, stimulate the AR by
activating AC that triggers downstream target molecules to initiate the exocytotic event (Zini et al.,
1995).
The fact that NO participates in ZP binding, as well as in the AR has been well characterized (Herrero
and Gagnon, 2001). Treatment of human spermatozoa with low concentrations of SNP in the
capacitation medium increases the number of spermatozoa bound to hemizona (Sengoku et al., 1998),
and increases sperm affinity for the ZP. In contrast, it has been demonstrated that addition of various
concentrations of -NAME to capacitated spermatozoa do not affect ZP binding, but significantly
inhibit the fusion effect (Francavilla et al., 2000). Similarly, NO-releasing compounds induce the AR in
human spermatozoa in a concentration-dependent manner, whereas haemoglobin, as a NO scavenger,
prevents the AR in spermatozoa capacitated by human follicular fluid (Revelli et al., 1999).
NO and sperm mitochondria

Mitochondria are essential in the regulation of a variety of cellular activities, including ATP synthesis,
intracellular calcium homeostasis and apoptosis (Duchen, 2000). In sperm cells, mitochondria supply
sperm with energy for several purposes (Figure 2), and thus play important roles in spermatogenesis,
differentiation, motility, AR, oocyte fusion and fertilization (Agarwal et al., 2008). In addition to the
generation of ATP to supply the above sperm fertilization processes, other functions of mitochondria in
sperm, such as Ca2+ signalling and protein synthesis, have been documented (Gur and Breitbart, 2006;
Publicover et al., 2008) (Figure 2). Several studies have confirmed that mitochondrial functionality
positively correlates with human sperm fertilizing potential and quality (Kasai et al., 2002; Marchetti
et al., 2004; Gallon et al., 2006; Otasevic et al., 2013). The major determinants of sperm mitochondrial
functionality are the activity and expression of components of the electron transport chain (ETC)
(Ruiz-Pesini et al., 1998; Hüttemann et al., 2003) as well as mitochondrial membrane potential (MMP)
(Marchetti et al., 2004; Gallon et al., 2006; Otasevic et al., 2013) Accordingly, any disruption of
mitochondrial ETC components or MMP is associated with decreased sperm motility, reduced sperm
fertilization potential and male sterility (Marchetti et al., 2002; 2004; Rossato, 2008; Otasevic et al.,
2013).
Figure 2
Importance of mitochondria in regulation of sperm functions. Mitochondria supply spermatozoa with

energy (ATP) for vital sperm-fertilizing processes. In addition, through production of ROS and RNS and
subsequent activation of various redox-dependent signalling pathways, mitochondria are involved in the
regulation of sperm functions. Other roles of mitochondria in spermatozoa, such as Ca2+ signalling and
protein synthesis, have also been presented.
NO has been shown to affect the mitochondria on several levels, as follows: regulates mitochondrial
respiration; biogenesis, remodelling and increasing O2 and substrate supply to the mitochondria (Nisoli
et al., 2003; Petrović et al., 2005; 2008; 2010a,b; Vasilijevic et al., 2010; Vucetic et al., 2011). Current
data show that NO can adversely influence the mitochondrial function of sperm cells depending on the
NO level. A growing amount of data have shown that higher levels of NO (μM) inhibit respiration in
sperm mitochondria (Rosselli et al., 1995; Weinberg et al., 1995), while iNOS-derived high (mM) NO
levels have been shown to induce mitochondrial hyperpolarization, cytochrome c release and sperm
cell death (Mishra and Shaha, 2005). Low concentrations of NO (nM) increase sperm motility and this
has been shown to be strongly associated with energy production in mitochondria (Hellstrom et al.,
1994). Our very recent work revealed that NO improved the functional status of sperm mitochondria
and sperm quality (Otasevic et al., 2013). The functional state of sperm mitochondria, as indicated by
the percentage of positive MitoTracker®Green FM (Molecular Probes, Inc., Eugene, OR, USA) sperm
cells and the expression of components of ETC, was significantly higher in sperm samples with higher
NO concentration. These data were positively correlated with sperm motility, a major indicator of
semen quality, and clearly showed that NO improved the fertilizing potential of spermatozoa.
Therapeutic applications
To date, the majority of trials oriented to NO-dependent improvement of sperm function have been
performed with a precursor of NO synthesis, -arginine (Scibona et al., 1994; Aydin et al., 1995; Patel
et al., 1998; Stanislavov et al., 2009). In addition to -arginine per se, the products of its metabolism
play significant roles in the regulation of fundamental cellular processes and physiological functions
(Vasilijevic et al., 2007; Stancic et al., 2012). However, many of the positive effects of -arginine in
male fertility disorders (improvement in the sperm count and motility in oligospermic and
asthenospermic patients) have been proven to be mediated through increased biosynthesis of NO
(Scibona et al., 1994; Aydin et al., 1995; Srivastava et al., 2000). Stanislavov et al. (2009) reported that
supplementation of subfertile men with a combination of pycnogenol and -arginine aspartate in a
double-blind, placebo-controlled, crossover study significantly increased sperm volume, concentration,
motility and % normal morphology through the antioxidative effect of pycnogenol, induction of eNOS
and consequent endogenous production of NO in spermatozoa.
However, important progress in -arginine research suggests that this amino acid and its metabolites
play a more complex role in the regulation of whole body energy homeostasis, and thus dietary
supplementation of -arginine must be taken with vigilance. When supplemented in appropriate
physiological doses, the duration and intake frequency of -arginine shows multiple beneficial effects;
however, with supplementation in high, supra-physiological doses (Luiking et al., 1998; Grimble,
2007) and over a long-term period (Park, 1993; Tome et al., 1999), -arginine can exert some side
effects (nausea, gastrointestinal discomfort, diarrhoea, and changes in electrolytes in the blood,
including potassium). In addition, there are a few pathophysiological states in which -arginine must be
applied with caution, such as myocardial infarction (Schulman et al., 2006), asthma (Takano et al.,
1998), viral infections (Tankersley, 1964), cancer (Yeatman et al., 1991; Grossie, 1996), and renal and
liver diseases (Tome et al., 1999; Appleton, 2002). Bearing in mind all of these findings and the fact
that infertility currently affects one in seven couples worldwide (NB, many experts in the field predict
this could double over the next decade), it is clear that novel strategies to treat sterility must be
developed (Table 2).
Table 2
List of redox-active substances in clinical trials or commercially available that ameliorate
sperm-fertilizing capacity by (at least partially) modulating the NO-signalling pathway
Compound Effect
-arginine Increases sperm volume, concentration, motility and Scibona et al., 1994; Aydin
morphology by increasing eNOS activity et al., 1995; Srivastava et
al., 2000
Superoxide Improves the functional status of sperm mitochondria, and Otasevic et al., 2013
dismutase thus the fertilizing potential of spermatozoa, by increasing NO
mimic, M40403 bioavailability
Sildenafil Improves erectile function through inhibition of cGMP- Schwartz and Kloner, 2010
(Viagra™) dependent phosphodiesterase
Increase velocity, capacitation and amplitude of lethal head Lefièvre et al., 2000
displacement in spermatozoa (mechanism unclear)
Tadalafil Improves erectile function through inhibition of cGMP- Schwartz and Kloner, 2010
(Cialis™) dependent phosphodiesterase
Vardenafil Improves erectile function through inhibition of cGMP- Schwartz and Kloner, 2010
(Levitra™) dependent phosphodiesterase
Ginsenoside Re Improves sperm motility through induction of NOS activity Zhang et al., 2006
(increase of NO production)
Open in a separate window
Accordingly, our recent data shed light on the development of new pharmacological strategies to treat
male infertility using novel, safe and effective redox-active substances (Otasevic et al., 2013). Due to
the important implications of the redox status in male factor infertility, we examined how changes in
the sperm redox milieu affect sperm functionality. To modulate the sperm cell redox state, sperm
incubation medium was supplemented with the superoxide dismutase (SOD) mimic, M40403, which is
representative of the Mn II pentaazamacrocyclic class of complexes (Figure 3). This is a potent low
molecular mass SOD mimic with marked protective effects in inflammation, stroke, atherosclerosis and
hypertension (Salvemini et al., 2002; Vallance and Leiper, 2002). The main advantage of M40403 is
high selectivity for O2⋅–, lack of peroxidase/catalase activity and lack of reactivity with NO. Our results
have shown that M40403 restores the population of sperm with functionally active mitochondria, thus
stimulating sperm mitochondrial activity and subsequent sperm quality. The SOD mimic positively
affected another component of the functional status of sperm mitochondria, that is, increased mRNA
expression of the nucleus encoded subunits of both complexes I and IV of the ETC, while subunits
encoded by the mitochondrial genome were restored to the control level. These data undoubtedly
showed improvement of functional status of sperm mitochondria, and thus the fertilizing potential of
spermatozoa by M40403, while definitive proof of improvements in sperm-fertilizing potential was
shown by the finding that the mimic increased sperm motility, a crucial parameter of semen quality.

Figure 3
Structure of M40403, a SOD mimic.
This is the very first time that M40403 has been shown to improve the molecular basis of sperm
mitochondrial function, as well as increase sperm motility, and thus has a positive effect on the
functionality of spermatozoa. Although the precise mechanism of this M40403-acting remains to be
elucidated, the increase in NO content in spermatozoa after treatment with M40403 implies the
involvement of NO in the observed beneficial effects of this drug. This seems quite plausible
considering that NO affects mitochondria on several levels and that NO and its precursors increase
sperm motility by increasing energy production in the mitochondrial compartment (Hellstrom et al.,
1994). In addition, M40403 increased eNOS gene expression, which strongly indicates that an increase
in the NO level after M40403 may depend, at least partly, on drug-elicited induction of eNOS and an
increase in endogenous NO production. Thus, in future studies designed to define the precise
mechanism underlying the M40403-driven increase in the population of sperm with active
mitochondria and motile spermatozoa, the involvement of NO warrants further consideration (Figure 4
).

Figure 4
Strategies to modify NO concentration in spermatozoa. As a substrate for NOSs, -arginine increases NO

production. In contrast, the SOD mimic removes O2⋅−, and by decreasing the bioavailability of O2− for
interaction with NO, the amount of NO consequently increases.
Nevertheless, what is certain and clearly shown by this study is that the redox modulator, M40403, is a
promising pharmacological tool for the improvement of sperm function during assisted fertilization and
for the treatment of infertile states accompanied by mitochondrial impairments and/or a disturbed
sperm redox state.
In addition, another potentially important fact that could be significant for determination of NO
bioavailability and roles that NO plays in male (in)fertility is NOS uncoupling (Alderton et al., 2001).
That is, it is known that in a number of pathological conditions NOS enzymatic activity becomes
uncoupled, resulting in the production of O2⋅– instead of NO, and leading to a decrease in NO
bioavailability. A number of potential mechanisms are responsible for uncoupling of NOSs, although
the most consistent evidence suggests it is due to tetrahydrobiopterin deficiency (Kietadisorn et al.,
2012). NOS uncoupling also occurs when there is lack of other NOS cofactors, substrate – -arginine,
when they are dephosphorylated or redistributed to the cytosolic fraction. Thus, this could be
potentially very important from the aspect of male infertility because, theoretically, modulation of NOS
uncoupling (by providing their substrate, cofactors, or stabilizing their activity) could be an attractive
therapeutic approach to increase NO synthesis and consequently improve sperm functions.
In line with this, it has been shown that when eNOS activity is uncoupled, treatments with redox-active
substances, that is, redox changes in endothelium, can substantially ameliorate NO production and thus
achieve beneficial effects in cardiovascular system (Visioli and Hagen, 2011). Specifically, it was
shown that vitamin C facilitates eNOS-derived NO synthesis by maintaining/increasing intracellular
tetrahydrobiopterin in a highly reduced state, necessary for NOS activity, thereby promoting or
maintaining eNOS coupling in endothelium (Heller et al., 1999; Carr et al., 2000; Huang et al., 2000).
In addition, GSH and stimulators of GSH synthesis, such as (R)-α-lipoic acid, markedly increase eNOS
activity by promoting/preserving eNOS phosphorylation, especially at the S1176 residue (Visioli et al.,
2002; Suh et al., 2004), whereas polyphenols increase endothelial NO synthesis markedly, but the
underlying mechanisms are still elusive. Based on these data obtained in vascular dysfunction, there is
compelling rationale to use these substances to increase NO production in spermatozoa, at least in
conditions that could lead to uncoupled activity of NOSs. In addition, a recently reported link between
vascular dysfunction and various forms of infertility (Visioli and Hagen, 2011) further strengthens the
rationale for the use of these molecules to increase bioavailability of NO in spermatozoa.
Conclusions and perspectives

Although infertility is a common global problem, treatment is sometimes ineffective, and the number
of couples seeking infertility services has increased dramatically over the last decade. Such an increase
in infertility rates clearly indicates the need for the development of new strategies to treat sterility.
It has become evident that redox regulation in reproductive biology, that is, regulation driven by ROS
and RNS, represents an inevitable new tool for studying fundamental fertilization processes.
Accordingly, the present article dealt with the problem of sterility from the perspective of redox
regulation. We aimed here not only to review the known facts of the multiple roles that NO plays in
spermatozoa and integrating different aspects of the physiological act, but also to open new fields for
future research of its complex action in male infertility, emphasizing it as a potentially useful strategy
for prevention and management of infertility. The ubiquitous involvement of NO in the regulation of
crucial sperm-fertilizing events may have huge clinical implications in developing new therapeutic
strategies to treat fertilization disorders in men.
In this regard, use of new potent substances that modulate sperm redox state by selective
production/removal of certain ROS/RNS could be a new power tool to increase male fertility and
promote conception.
Acknowledgments
This work was supported by the COST BM1005 action and by the Ministry of Education, Science and
Technological Development of the Republic of Serbia, Grant Nos. 173054 and 173055. We would like
to acknowledge that the drug/molecular target nomenclature used in this work conforms to BJP's
Concise Guide to Pharmacology (Alexander et al., 2013).
Glossary
AR acrosome reaction
EDRF endothelial-derived relaxing factor
eNOS endothelial NOS
ETC electron transport chain
iNOS inducible NOS
IVF in vitro fertilization

G
-NAME N -nitro- -arginine methyl ester
MMP mitochondrial membrane potential
mtNOS mitochondrial NOS
NG nitroglycerine
nNOS neuronal NOS
O2⋅− superoxide anion radical
RNS reactive nitrogen species
ROS reactive oxygen species
sGC soluble GC
SNP sodium nitroprusside
SOD superoxide dismutase
ZP zona pellucida
Conflict of interest
The authors have no conflicts of interest to declare.
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Nuevos Conocimientos Sobre La (In) Fertilidad Masculina - La Importancia Del NO

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1/3/2021 Nuevos conocimientos sobre la (in) fertilidad masculina: la importancia del NO

Br J Pharmacol. 2015 Mar; 172 (6): 1455–1467. PMCID: PMC4369256

Nuevos conocimientos sobre la (in) fertilidad masculina: la

Copyright © 2015 The British Pharmacological Society

Palabras clave: óxido nítrico, infertilidad, espermatozoides, fertilización in vitro, mitocondrias,

Durante la última década, se han producido avances significativos en el diagnóstico y tratamiento de

Una extensa investigación en infertilidad ha confirmado que la producción excesiva de especies

La base del "fracaso" de la terapia antioxidante en el tratamiento de la infertilidad masculina es que se

NO como molécula de señalización: una descripción histórica

Mecanismos por los que actúa el NO

concentraciones de NO superiores a las necesarias para la activación de sGC. La S-nitrosilación de las

Es importante destacar específicamente el NO que actúa de manera independiente de cGMP, en el que

Isoformas NOS en espermatozoides

Isoforma Localización (Lee y Cheng, 2008 ) Función

Abrir en una ventana separada

TnNOS, nNOS específico de testículo.

Específicamente, se sabe que el NO está involucrado en la regulación de la motilidad, viabilidad,

Efectos del NO sobre los efectos fertilizantes de los espermatozoides

Movilidad, morfología y viabilidad de los espermatozoides

NO and sperm mitochondria

Importance of mitochondria in regulation of sperm functions. Mitochondria supply spermatozoa with

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Structure of M40403, a SOD mimic.

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Strategies to modify NO concentration in spermatozoa. As a substrate for NOSs, -arginine increases NO

Conclusions and perspectives

EDRF endothelial-derived relaxing factor

eNOS endothelial NOS

ETC electron transport chain

iNOS inducible NOS

IVF in vitro fertilization

MMP mitochondrial membrane potential

mtNOS mitochondrial NOS

nNOS neuronal NOS

O2⋅− superoxide anion radical

RNS reactive nitrogen species

ROS reactive oxygen species

SNP sodium nitroprusside

SOD superoxide dismutase

1996;55:935–941. [PubMed] [Google Scholar]

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