In vitro Capacitation of Ejaculated Rabbit Spermatozoa

Prior to fertilization mammalian spermatozoa undergo physiological changes in the female reproductive tract. These changes are collectively known as capacitation. In essence capacitation is a further differentiation that the sperm cell must acquire beyond the maturational changes that it undergoes in the epididymis. Although capacitation in vitro has been easily achieved in rodents, its accomplishment in the rabbit is inconsistent and difficult. We report here successful in vitro capacitation of ejaculated rabbit sperm, assessed by in vitro and in vivo fertilization of rabbit ova. Sperm were used from pooled ejaculates collected from bucks of proven fertility since sperm collected from individual bucks resulted in significant differences in fertilization levels. Conditions favoring in vitro capacitation were: (1) extended incubation time of 12 h, (2) addition of 20% heated rabbit serum to incubation medium, and (3) an atmosphere of 5% CO2, 8% O2, and 87% N2 during incubation. In vitro capacitation of sperm under these conditions resulted in 67% fertilization compared to 89% for control sperm capacitated for 13 h in the uterus.     

Physiology of spermatozoa at high dilution rates: the influence of seminal plasma

Extensive dilution of spermatozoa, as occurs during flow-cytometric sperm sorting, can reduce their motility and viability. These effects may be minimized by the use of appropriate dilution and collection media, containing balanced salts, energy sources, egg yolk and some protein. Dilution and flow-cytometric sorting of spermatozoa, which involves the removal of seminal plasma, also destabilizes sperm membranes leading to functional capacitation. This membrane destabilization renders the spermatozoa immediately capable of fertilization in vitro, or in vivo after deposition close to the site of fertilization, but shortens their lifespan, resulting in premature death if the cells are deposited in the female tract distant from the site of fertilization or are held in vitro at standard storage temperatures. This functional capacitation can be reversed in boar spermatozoa by inclusion of seminal plasma in the medium used to collect the cells from the cell sorter and, consequently, reduces their in vitro fertility. It has yet to be determined whether seminal plasma would have similar effects on flow cytometrically sorted spermatozoa of other species, and what its effects might be on the in vivo fertility of flow sorted boar.     

Induction of buffalo (Bubalus bubalis ) sperm capacitation and acrosome reaction in the excised reproductive tract of hamsters

A study was conducted on the induction of buffalo sperm capacitation and acrosome reaction in the excised reproductive tract of hamsters at the estrogen- and progesterone-dominated stages of estrus. The percentages of the maximum capacitation and acrosome reaction were significatly (P < 0.01) higher for spermatozoa incubated in the uterus with oviducts of estrogen dominated hamsters compared with those incubated in BWW medium in a test tube (64.6%, 60.2%; 16.2%, 14.7%). Buffalo spermatozoa incubated in the uterus and oviducts of progesterone-dominated hamsters showed significantly (P < 0.01) lower capacitation and acrosome reaction rates than those incubated in the uterus and oviducts of estrogen-dominated hamsters (34.8%, 34.3%: 64.6%, 60.2%). The percentage of capacitation and acrosome reaction in spermatozoa were significantly (P < 0.01) more when incubated in the uterus plus oviducts than without the oviduct irrespective of whether the reproduct tract of hamster was estrogen- or progesterone-dominated. The time for the onset of maximum capacitation and acrosome reaction was reduced from 12 to 10 h when the spermatozoa were incubated in the hamster reproductive tract rather than in BWW medium in test tubes. The significance of the results in relation to hormonal regulation of sperm capaciation and acrosome reaction are also discussed.     

Ovarian programming of gamete progression and maturation in the female genital tract

Whilst the rate of displacement and migration of sperm cells in the female reproductive tract of rodents, farm animals and humans has attracted attention for at least 50 years, the overriding purpose of sperm transport has not always been kept in focus. This report is concerned with spermatozoa that can penetrate the egg investments and promote formation of a zygote, judgements involving a surgical approach and subsequent phase-contrast microscopy. A minimum period of 6–8 hours was required for such spermatozoa to be established in the oviducts in sheep and cows mated at the onset of oestrus. Sperm were then arrested in the caudal 12 cm of the isthmus for 17–18 hours or more until just before the moment of ovulation, when they were activated and displaced onwards to the site of fertilization at the ampullary-isthmic junction. The time-scale of these events differs in pigs as a result of the intra-uterine site of ejaculation and the 40-hour interval between the onset of oestrus and ovulation, but the pre-ovulatory sequestering of viable spermatozoa in the caudal tip of the oviduct is conspicuous for 36 hours or more. This function of the oviduct appears to be under local control from ovarian follicular hormones and, as judged by sperm motility and membranous changes, so does the process of capacitation. Completion of capacitation is interpreted as a peri-ovulatory event.     

A possible mechanism of action for fertilization promoting peptide,a TRH-related tripeptide that promotes capacitation and fertilizing ability in mammalian spermatozoa

Fertilization promoting peptide (FPP), a tripeptide structurally related to thyrotrophin releasing hormone (TRH), has been shown to stimulate capacitation and fertilizing ability in both mouse and human spermatozoa, but the mechanisms of action involved in these responses are currently unknown. In the present study utilizing epididymal mouse spermatozoa, we have compared the ability of FPP, TRH, and pyroglutamylphenylalanineprolineamide (an uncharged structurally related tripeptide found in seminal plasma) to stimulate capacitation. At 50 nM, the mean concentration of FPP found in human seminal plasma, only FPP produced a significant response. This suggests that if a receptor is involved, it is one distinct from the TRH receptor. A significant response to FPP required the presence of extracellular Ca²⁺, with 90 μm Ca²⁺ being sufficient to support a stimulation of capacitation. The addition of FPP to suspensions at later stages of capacitation indicated that the nature of the response changed, such that addition of FPP to capacitated suspensions inhibited spontaneous acrosome reactions; however, FPP-treated, cells were still able to undergo acrosomal exocytosis in response to progesterone, a physiological agonist of acrosomal exocytosis. Because earlier studies had identified a similar capacitation-related change in response to adenosine, being stimulatory early in capacitation and inhibitory later in capacitation, we investigated the possibility that FPP and adenosine might be acting via the same pathway. The combination of FPP plus adenosine, whether used at low, non-stimulatory concentrations or high, maximally-stimulatory concentrations, was more effective in promoting capacitation than either compound used individually. As observed with FPP, addition of adenosine to capacitated cells inhibited spontaneous acrosome loss but did not inhibit exocytosis in response to progesterone. This suggests that the two molecules are affecting a common pathway. Since adenosine, acting via specific cell surface receptors, can stimulate fertilizing ability and adenylate cyclase activity in uncapacitated cells and then inhibit enzyme activity in capacitated cells, we propose that FPP may act by modulating the adenylate cyclase/cyclic AMP signal transduction pathway. In vivo, FPP, which would contact spermatozoa at ejaculation and probably remain bound to cells for some time, could stimulate capacitation as the spermatozoa ascend the female tract; adenosine, present in seminal plasma and the female tract, could either augment FPP's action or replace it if FPP is lost from the cell surface. We therefore suggest that FPP and adenosine, by modulating adenylate cyclase activity to promote capacitation but inhibit spontaneous acrosomal exocytosis, may provide an endogenous mechanism that helps to optimize the fertilizing potential of the few sperm cells that reach the site of fertilization in vivo. © 1996 Wiley-Liss, Inc.     

Proteins implicated in sperm capacitation

Spermatozoa after being deposited in the female reproductive tract spend a considerable time in this foreign environment prior to fertilization of the oocyte. Chang and Austin independently observed1,2 that this time spent by the spermatozoa in the female tract is not consequential but a necessary event in the life cycle of the male gamete, and Austin2 first called this maturation period of spermatozoa as 'Capacitation'. Ever since, attempts have been made to understand and unravel the molecular mechanism of capacitation. Based on the results obtained so far, it is clear that capacitation is guided by novel signal transduction pathways influencing varied aspects of spermatozoa. Capacitation could be, thus, defined as the cumulative molecular, cellular and physiological changes that occur in spermatozoa in the female reproductive tract to achieve the final competence to fertilize the oocyte. This review is structured so as to first understand the key features of capacitation and then to survey the players which bring about these changes during capacitation.     

The “switching on” of mammalian spermatozoa: Molecular events involved in promotion and regulation of capacitation

Following the discovery of mammalian sperm capacitation and its fundamental importance for the acquisition of fertilizing potential, it has gradually become possible to identify some specific molecules and molecular events that play pivotal roles in the “switching on” of spermatozoa. These are discussed in the context of the promotion and regulation of capacitation, emphasizing differences between commonly used conditions in vitro and the environment in vivo where spermatozoa normally undergo capacitation. Although typical culture media used in vitro do support capacitation, they do not prevent capacitated cells from undergoing spontaneous acrosome reactions and so losing fertilizing potential. This is not a problem in vitro, but could be in vivo where few spermatozoa reach the site of fertilization. Several small molecules, known to be present in vivo, have been shown in vitro to bind to spermatozoa and to regulate capacitation, first accelerating capacitation and then inhibiting spontaneous acrosome reactions, by regulating cAMP production. Since spermatozoa would contact these molecules during and after ejaculation, it is plausible that they serve a similar function in vivo. The mechanisms whereby the presence or absence of decapacitation factors might alter plasma membrane architecture and so alter functionality of a number of membrane‐associated enzymes involved in capacitation are also considered. Finally, several unresolved issues relating to events during capacitation are discussed. Mol. Reprod. Dev. 77: 197–208, 2010. © 2009 Wiley‐Liss, Inc.     

Identification of the molecular chaperone, heat shock protein 1 (chaperonin 10), in the reproductive tract and in capacitating spermatozoa in the male mouse

Mammalian spermatozoa must undergo epididymal maturation in the male reproductive tract and capacitation in the female tract before acquiring the ability to fertilize an oocyte. Previous studies from our laboratory have demonstrated a causal relationship between capacitation-associated surface phosphotyrosine expression and the ability of mouse spermatozoa to recognize the oocyte and engage in sperm-zona pellucida interaction. Our previous analyses of the surface phosphoproteome of capacitated murine spermatozoa identified two molecular chaperones, heat shock protein (HSP) D1 and HSP90B1, with well-characterized roles in protein folding and the assemblage of multimeric protein complexes. The expression of these chaperones was restricted to the rostral aspect of the sperm head, in an ideal position to mediate sperm-zona pellucida interaction. Herein, we report the characterization of an additional chaperone in this location, HSPE1 (chaperonin 10; HSP10). This chaperone was identified using a coimmunoprecipitation strategy employing HSPD1 as bait. The putative interaction between HSPE1 and HSPD1 was supported by reciprocal immunoprecipitation and colocalization studies, which demonstrated the coordinated appearance of both proteins on the surface of the sperm head during capacitation. However, the surface exposure of the protein was lost upon induction of acrosomal exocytosis, as would be expected of a protein potentially involved in sperm-zona pellucida interaction. Collectively, these data invite speculation that a number of molecular chaperones are involved in modification of the sperm surface during capacitation to render these cells functionally competent to engage the process of fertilization.     

Sperm transport in the female reproductive tract of the brushtail possum, Trichosurus vulpecula, following superovulation and artificial insemination

This study investigated sperm transport following superovulation and artificial insemination (AI) in the common brushtail possum, Trichosurus vulpecula. Females were superovulated by treatment with 15 IU pregnant mare serum gonadotrophin (PMSG) then 4 mg luteinizing hormone (LH) 78 h later. Inseminations were performed 27 h after LH (4 million motile spermatozoa/uterus). At 1.5, 3, 6, 9 and 12 h after AI (n=5 per group), females were euthanised and reproductive tracts removed for examination and flushed for sperm. No ovulations had occurred by 1.5 h, but 20% of animals had ovulated by 3 or 6 h, and 80% by 9 or 12 h. The mean numbers of spermatozoa recovered ranged from 249 to 275x10(3) in the uterus; 16-51x10(3) in the isthmus; 8-11x10(3) in the middle segment; and 6-16x10(3) in the ampulla at 1.5, 3 and 6 h after AI. Sperm numbers in all regions decreased at later times (P<0.05) except the isthmus, where 100x10(3) sperm were recovered by 12 h. Highly motile thumbtack sperm (a putative indicator of capacitation in marsupials), were recovered from the isthmus (20%), middle segment (50%) and ampulla (90%) at all sampling times, but not from the uterus. The epithelium of the oviduct segments contained mucus-secreting and ciliated cells and peak secretory activity was observed in the ampulla at 6 h. At 3, 6 and 12 h, many spermatozoa were found in epithelial folds within the isthmus. The present study has provided basic information on sperm transport and storage events within the female reproductive tract of T. vulpecula following superovulation and AI. It is concluded that this model may be useful to better understand pre-fertilization sperm maturation events in the possum, which could facilitate the development of IVF technology.     

Sperm transport and survival in the mare

Following the deposition of semen in the mares uterus, spermatozoa must be transported to the site of fertilization, be maintained in the female tract until ovulation occurs, and be prepared to fertilize the released ovum. Sperm motility, myometrial contractions, and a spontaneous post-mating uterine inflammation are important factors for the transport and survival of spermatozoa in the mares reproductive tract. Fertilizable sperm are present in the oviduct within 4 hours after insemination. At this time, the uterus is the site of a hostile inflammatory environment. Our data suggest that spermatozoa trigger an influx of polymorphonuclear neutrophils (PMNs) into the uterine lumen via activation of complement. Furthermore, seminal plasma appears to have a modulatory effect on the post-mating inflammation through its suppressive effect on PMN chemotaxis and migration. Spermatozoa that safely have reached the oviduct can be stored in a functional state for several days, but prolonged sperm storage in the female tract is not required for capacitation and fertilization in the horse. The caudal isthmus has been proposed as a sperm reservoir in the mare. The pattern of sperm transport and survival of spermatozoa in the mares reproductive tract are different between fertile and subfertile stallions, between fertile and some infertile mares, and between fresh and frozen-thawed semen. Possible explanations for these differences include a selective phagocytosis of damaged or dead spermatozoa, impaired myometrial activity in subfertile mares, bio-physiological changes of spermatozoa during cryopreservation, and the removal of seminal plasma during cryopreservation of equine semen.     

Mammalian sperm chemotaxis and its association with capacitation

Much progress has been made in recent years in establishing mammalian sperm chemotaxis and understanding sperm capacitation. Thus far, chemotaxis to follicular fluid has been established by a variety of means in human and mouse spermatozoa. It was found that only a small fraction of a given sperm population (averaging around 10%) is chemotactically responsive and that this fraction constitutes capacitated (ripe) spermatozoa. Both the chemotactic responsiveness and the capacitated state are transient (with a lifetime of 50 min to 4 h) and they occur only once in the sperm's lifetime. It has been proposed that the role of sperm chemotaxis in mammals (at least in humans) is selective recruitment of capacitated spermatozoa for fertilizing the egg, and that the role of the continuous replacement of chemotactic/capacitated spermatozoa is to prolong the time during which capacitated spermatozoa are available in the female reproductive tract. The sperm chemoattractants have not been identified, but they appear to be heat‐stable peptides. Although the molecular mechanism and the in vivo location of sperm chemotaxis are not known, a number of possible mechanisms and locations are discussed. Dev. Genet. 25:87–94, 1999. © 1999 Wiley‐Liss, Inc.     

Sperm transport and survival in the mare: a review

After the deposition of semen in the mare's uterus, spermatozoa must be transported to the site of fertilization, be maintained in the female tract until ovulation occurs, and be prepared to fertilize the released ovum. Sperm motility, myometrial contractions, and a spontaneous post-mating uterine inflammation are important factors for the transport and survival of spermatozoa in the mare's reproductive tract. Fertilizable sperm are present in the oviduct within 4 h after insemination. At this time, the uterus is the site of a hostile inflammatory environment. Our data suggest that spermatozoa trigger an influx of polymorphonuclear neutrophils (PMNs) into the uterine lumen via activation of complement. Furthermore, semen plasma appears to have a modulatory effect on the post-mating inflammation through its suppressive effect on PMN chemotaxis and migration. Spermatozoa that safely have reached the oviduct can be stored in a functional state for several days, but prolonged sperm storage in the female tract is not required for capacitation and fertilization in the horse. The caudal isthmus has been proposed as a sperm reservoir in the mare. The pattern of sperm transport and survival of spermatozoa in the mare's reproductive tract are different between fertile and subfertile stallions, between fertile and some infertile mares, and between fresh and frozen/thawed semen. Possible explanations for these differences include a selective phagocytosis of damaged or dead spermatozoa, impaired myometrial activity in subfertile mares, bio-physiological changes in spermatozoa during cryopreservation, and the removal of semen plasma during cryopreservation of equine semen.     

Role of signaling pathways in regulating the capacitation of mammalian spermatozoa.

Mammalian testicular spermatozoa are immotile and incompetent for fertilization. They acquire motility during epididymal maturation and fertilizing ability during a second phase of maturation in the female reproductive tract, termed as capacitation. Capacitation was discovered independently by Austin and Cang in early 1950s and was defined as the obligate period of residency of spermatozoa in the female reproductive tract, which confers on the spermatozoa the ability to fertilize an oocyte. Over the years, the definition of capacitation has changed and it has been recognized as a complex phenomenon, which is correlated with changes associated with the spermatozoa in the female tract. These alterations in metabolism, intracellular ion concentration, membrane fluidity, intracellular pH, cAMP concentration and concentration of reactive oxygen species, ultimately make the spermatozoa fertilization-competent. The molecular basis of capacitation is poorly understood despite the fact that it is an important event preceding fertilization. This review presents our current understanding of the signaling events involved in the process of capacitation.     

Bicarbonate Induces Membrane Reorganization and CBR1 and TRPV1 Endocannabinoid Receptor Migration in Lipid Microdomains in Capacitating Boar Spermatozoa

Mammalian spermatozoa acquire full fertilizing ability only after a morphofunctional maturation called "capacitation." During this process the high level of bicarbonate present within the upper female genital tract or in culture medium induces a marked reorganization of sperm membranes characterized by a biphasic behavior: In a few minutes, it promotes membrane phospholipid scrambling preliminary to the apical translocation of sterol that, 2-4?h later, enables spermatozoa to recognize zona pellucida after albumin-mediated cholesterol extraction. In the present research it was demonstrated that spermatozoa incubated with bicarbonate in protein-free media underwent a marked reorganization of lipid microdomains present in a detergent-resistant membrane fraction (DRM) isolated by ultracentrifugation on sucrose density gradient. In fact, bicarbonate exposed sperm (ES) cells, compared with ejaculated spermatozoa (nonexposed sperm [nES] cells), displayed an increase in protein DRM content and, in particular, in Cav-1 and CD55, markers of caveolae and lipid rafts, as well in acrosin-2, a marker of the outer acrosomal membrane (OAM). Moreover, the amount of certain proteins involved in capacitation, such as the endocannabinoid system receptors cannabinoid receptor type 1 (CBR1) and transient receptor potential cation channel 1 (TRPV1), increased in DRM obtained from ES. These data allow us to hypothesize that sperm membrane reorganization takes place even in the absence of extracellular proteins; that not only the plasma membrane but also the OAM participate in this process; and that important molecules playing a key role in inside-out signaling, such as the endocannbinoid receptors TRPV1 and CBR1, are involved in this event, with potentially important consequences on sperm function.     

Identification of capacitation in boar spermatozoa by chlortetracycline staining

The functional status of boar spermatozoa undergoing capacitation in vitro was investigated. Two fluorescent stains were used: chlortetracycline (CTC) and a FITC-conjugated lectin (FITC-PSA). The first has been used for the direct identification of the capacitated boar spermatozoa, while the second, based on the identification of capacitated spermatozoa by their ability to undergo zona-induced acrosome reaction (AR), was used to confirm and validate the CTC assay in this species. Spermatozoa obtained from 5 different boars was washed and incubated under capacitating conditions. Aliquots of spermatozoa were collected at 0, 90 and 180 min of incubation and then stained with CTC or FITC-PSA. After CTC staining, 3 different fluorescent patterns were observed: Pattern A with the fluorescence uniformly distributed on the sperm head, Pattern B with the fluorescence concentrated in the post-acrosomial region, and Pattern C with the fluorescence concentrated in the acrosomial region. The percentage of spermatozoa displaying fluorescent Pattern A decreased throughout the incubation while that of spermatozoa with Pattern C showed a concomitant progressive increase. Pattern B fluorescence remained unchanged throughout the maturation period. Exposure to zonae pellucidae (ZP) brought back the levels of Pattern C fluorescence to basal values. Since only the capacitated spermatozoa are believed to react to ZP, this observation together with the rising incidence of Pattern C throughout maturation suggests that fluorescence in the acrosomial region identifies capacitated spermatozoa. The analysis of acrosome integrity carried out with FITC-PSA showed that the proportion of zona-induced AR was nearly the same as that of spermatozoa displaying Pattern C, thus confirming that CTC staining is suitable for the detection of boar sperm capacitation. In the second part of this study, CTC was used to investigate the effects of sperm origin and storage on the capacitation process. Our finding demonstrates that capacitation kinetics show wide variations in sperm samples derived from different boars; moreover, capacitation is also affected by sperm storage. While fresh semen showed a progressive increase in capacitated spermatozoa, ranging from low levels at the beginning of the culture to 46% at the end of incubation, the refrigerated semen had a relatively high percentage of capacitated spermatozoa at the beginning of culture, but this proportion increased only slightly during the following 90 to 180 min of treatment. These data indicate that CTC can be used to identify capacitated boar spermatozoa, and, because of its rapid and easy execution, it can be used routinely to identify the optimal capacitation time for different sperm samples.     

Nitric oxide regulates human sperm capacitation and protein-tyrosine phosphorylation in vitro.

The aim of the present study was to investigate whether the generation of nitric oxide by human spermatozoa is associated with human sperm capacitation and with the tyrosine phosphorylation of sperm proteins. Human spermatozoa were capacitated in the presence or absence of nitric oxide-releasing compounds or nitric oxide synthase inhibitors, and then the percentage of acrosome loss induced by human follicular fluid or by calcium ionophore was determined. The presence of the nitric oxide-releasing compounds primed spermatozoa to respond earlier to human follicular fluid whereas nitric oxide synthase inhibitors decreased the percentage of acrosome reaction. Moreover, nitric oxide modulated tyrosine phosphorylation of sperm proteins. A tight correlation between capacitation and tyrosine phosphorylation regulated by nitric oxide was observed. Results indicate that nitric oxide is involved in human sperm capacitation and emphasize the importance of oxidoreduction reactions in the fine control of sperm physiology.     

Bovine oviductal epithelial cells: their cell culture and applications in studies for reproductive biology

Epithelial cells of the mammalian oviduct play an important role in reproductive and developmental events that occur there. Oviductal epithelial cells from several mammalian species can be isolated and cultured in serum or serum-free medium in vitro and cell culture of bovine oviductal epithelial cells (BOEC) has been described by many investigators. Cultured BOEC show a wide variety of secretory activities and these secretory factors may influence early embryonic development or sperm function. Monolayer cultures of BOEC have been widely used for in vitro co-culture of bovine preimplantation embryos. The use of BOEC co-culture systems has improved embryonic development in nearly all the studies conducted. In addition, interaction of bovine spermatozoa with BOEC, in a similar manner to that observed for spermatozoa in vivo, induced specific changes in sperm capacitation and consequently improved the fertilizing capacity of bovine spermatozoa in vitro. Thus co-culture systems with BOEC may not only offer an excellent model for studying the mechanisms of capacitation and acrosome reaction of bovine spermatozoa but also provide a useful tool for the improvement of embryo development in vitro.     

Boar spermatozoa in the oviduct

In the pig, a functional tubal sperm reservoir (SR) is established before ovulation to ensure availability of suitable numbers of viable spermatozoa for fertilization. The boar's large ejaculate is split: most spermatozoa are delivered in a sperm-rich fraction (SRF) followed by a post-SRF fraction containing increasing amounts of the spermadhesin PSP-I/PSP-II-rich seminal vesicle secretion. This heterodimer acts as leukocyte chemoattractant both in vitro and in vivo, contributing to the phagocytosis of those spermatozoa not reaching the SR. Sequential ejaculate deposition of marked spermatozoa and SR screening showed that most spermatozoa in the SR arose from the fortuitous PSP-poor, first portion of the SRF fraction, escaping phagocytosis and replenishing the SR within 2-3 h. The SR-sperm numbers diminish gradually in relation to ovulation, spermatozoa being continuously redistributed toward the upper isthmus. In vitro, only uncapacitated spermatozoa bind to epithelial explants, suggesting that the SR influences sperm capacitation. In vivo, most viable spermatozoa--usually harbored in the deep furrows in the pre- or peri-ovulatory SR during spontaneous standing estrus--are uncapacitated, but capacitation significantly increases after ovulation. Pre-/peri-ovulatory SR spermatozoa promptly capacitate in vitro when exposed to the effector bicarbonate, an influence that can be reversed by co-incubation with SR fluid or its component hyaluronan. Fluid collected from the ampullar segment (rich in bicarbonate) induces capacitation in vitro. In conclusion, the lack of massive sperm capacitation in the SR and the diverse individual response to capacitation shown by tubal spermatozoa would relate both to the insurance of full sperm viability before ovulation and the presence of spermatozoa at different stages of capacitation in the upper oviduct, thus maximizing the chances of normal fertilization.     

Anandamide induces sperm release from oviductal epithelia through nitric oxide pathway in bovines

Mammalian spermatozoa are not able to fertilize an egg immediately upon ejaculation. They acquire this ability during their transit through the female genital tract in a process known as capacitation. The mammalian oviduct acts as a functional sperm reservoir providing a suitable environment that allows the maintenance of sperm fertilization competence until ovulation occurs. After ovulation, spermatozoa are gradually released from the oviductal reservoir in the caudal isthmus and ascend to the site of fertilization. Capacitating-related changes in sperm plasma membrane seem to be responsible for sperm release from oviductal epithelium. Anandamide is a lipid mediator that participates in the regulation of several female and male reproductive functions. Previously we have demonstrated that anandamide was capable to release spermatozoa from oviductal epithelia by induction of sperm capacitation in bovines. In the present work we studied whether anandamide might exert its effect by activating the nitric oxide (NO) pathway since this molecule has been described as a capacitating agent in spermatozoa from different species. First, we demonstrated that 1 μM NOC-18, a NO donor, and 10 mM L-Arginine, NO synthase substrate, induced the release of spermatozoa from the oviductal epithelia. Then, we observed that the anandamide effect on sperm oviduct interaction was reversed by the addition of 1 μM L-NAME, a NO synthase inhibitor, or 30 μg/ml Hemoglobin, a NO scavenger. We also demonstrated that the induction of bull sperm capacitation by nanomolar concentrations of R(+)-methanandamide or anandamide was inhibited by adding L-NAME or Hemoglobin. To study whether anandamide is able to produce NO, we measured this compound in both sperm and oviductal cells. We observed that anandamide increased the levels of NO in spermatozoa, but not in oviductal cells. These findings suggest that anandamide regulates the sperm release from oviductal epithelia probably by activating the NO pathway during sperm capacitation.     

Crosstalk between protein kinase A and C regulates phospholipase D and F-actin formation during sperm capacitation

Mammalian spermatozoa should reside in the female reproductive tract for a certain time before gaining the ability to fertilize. During this time, the spermatozoa undergo a series of biochemical processes collectively called capacitation. We recently demonstrated that actin polymerization is a necessary step in the cascade leading to capacitation. We demonstrate here for the first time a role for phospholipase D (PLD) in the induction of actin polymerization and capacitation in spermatozoa. The involvement of PLD is supported by specific inhibition of F-actin formation during sperm capacitation by PLD inhibitors and the stimulation of fast F-actin formation by exogenous PLD or phosphatidic acid (PA). Moreover, PLD activity is enhanced during capacitation before actin polymerization. Protein kinase A (PKA), known to be active in sperm capacitation, and protein kinase C (PKC), involved in the acrosome reaction, can both activate PLD and actin polymerization. We suggest that PKA- and PKC-dependent signal transduction pathways can potentially lead to PLD activation; however, under physiological conditions, actin polymerization depends primarily on PKA activity. Activation of PKA during capacitation causes inactivation of phospholipase C, and as a result, PKC activation is prevented. It appears that PKA activation promotes sperm capacitation whereas early activation of PKC during capacitation would jeopardize this process.