Mouse Sperm Capacitation Research Articles

Using an Extracellular Flux Analyzer to Measure Changes in Glycolysis and Oxidative Phosphorylation during Mouse Sperm Capacitation

Using an Extracellular Flux Analyzer to Measure Changes in Glycolysis and Oxidative Phosphorylation during Mouse Sperm Capacitation

Kinetic Model of the Action of 17α-Ethynylestradiol on the Capacitation of Mouse Sperm, Monitored by HPLC-MS/MS

17α-Ethynylestradiol (EE2), a synthetic estrogen used in contraceptive pills, is resistant to hepatic degradation and is excreted in the urine. It is chemically stable and has a negative impact on the endocrine system. The aim of this work was to mathematically describe the possible interaction of EE2 (200, 20, and 2 μg/L) with sperm estrogen receptors during sperm maturation, which is called capacitation. The concentrations of the unbound EE2 remaining in capacitating medium during 180 min of sperm capacitation were determined at 30 min intervals by high performance liquid chromatography with tandem mass spectrometric detection (HPLC-MS/MS) and the data obtained (relative concentrations Bt) were subjected to kinetic analysis. The suggested kinetic schema was described by the system of differential equations with the optimization of rate constants used to calculate the theoretical Bt values. Optimal parameters (overall rate constants K1–K5 and molar ratio n) were determined by searching the minimum of absolute values of the difference between theoretical and experimental Bt values. These values were used for the design of the theoretical B(t) curves which fit to experimental points. The proposed kinetic model assumes the formation of an unstable adduct between EE2 and the receptor in cytoplasm, which acts as an autocatalytic agent and gradually decomposes.

MRP4-mediated cAMP efflux is essential for mouse spermatozoa capacitation.

Mammalian spermatozoa must undergo biochemical and structural changes to acquire the capacity for fertilization, in a process known as capacitation. Activation of PKA enzymes is essential for capacitation, and thus cAMP levels are tightly regulated during this process. Previously, we demonstrated that during capacitation, bovine spermatozoa extrude cAMP through multidrug resistance-associated protein 4 (MRP4, also known as ABCC4), which regulates intracellular levels of the nucleotide and provides cAMP to the extracellular space. Here, we report the presence of functional MRP4 in murine spermatozoa, since its pharmacological inhibition with MK571 decreased levels of extracellular cAMP. This also produced a sudden increase in PKA activity, with decreased tyrosine phosphorylation at the end of capacitation. Blockade of MRP4 inhibited induction of acrosome reaction, hyperactivation and in vitro fertilization. Moreover, MRP4 inhibition generated an increase in Ca2+ levels mediated by PKA, and depletion of Ca2+ salts from the medium prevented the loss of motility and phosphotyrosine inhibition produced by MK571. These results were supported using spermatozoa from CatSper Ca2+ channel knockout mice. Taken together, these results suggest that cAMP efflux via MRP4 plays an essential role in mouse sperm capacitation.This article has an associated First Person interview with the first author of the paper.

Capacitation of mouse sperm is modulated by gamma-aminobutyric acid (GABA) concentration.

In mammals, ejaculated sperm acquire their fertilizing ability during migration through the female reproductive tract, which secretes several factors that contribute to sperm capacitation. Gamma-aminobutyric acid (GABA) is a well-known neurotransmitter in the central nervous system, but additionally enhances the sperm acrosome reaction in the rat and cow. However, the detailed effects of GABA concentration on sperm function remain unclear. In this study, we detected the presence of the GABA type A receptor (GABA A) in mouse epididymal sperm by western blot analysis and in the sperm acrosome by immunocytochemistry. We also investigated the effects of GABA on sperm fertilizing ability. We found that GABA facilitated the tyrosine phosphorylation of sperm proteins, which is an index of sperm capacitation. GABA also promoted the acrosome reaction, which was suppressed by a selective GABA A receptor antagonist. We then found that the effective GABA concentration for the acrosome reaction corresponds to sperm concentration, but we did not detect any marked effect of GABA on sperm motility using a computer-assisted sperm analysis system. Using immunohistochemistry, we also detected GABA expression in the epithelia of the mouse uterus and oviduct. Furthermore, we found that the mRNA levels of glutamate decarboxylase (Gad), which generates GABA from L-glutamate, were higher in the oviduct than in the uterus, and that Gad mRNA levels were higher at estrus than at the diestrus stage. These results indicate that the GABA concentration can act as a modulator of the acrosome reaction and sperm capacitation in the female reproductive tract.

Sperm capacitation is associated with phosphorylation of the testis-specific radial spoke protein Rsph6a†.

Mammalian sperm undergo a series of biochemical and physiological changes collectively known as capacitation in order to acquire the ability to fertilize. Although the increase in phosphorylation associated with mouse sperm capacitation is well established, the identity of the proteins involved in this signaling cascade remains largely unknown. Tandem mass spectrometry (MS/MS) has been used to identify the exact sites of phosphorylation and to compare the relative extent of phosphorylation at these sites. In the present work, we find that a novel site of phosphorylation on a peptide derived from the radial spoke protein Rsph6a is more phosphorylated in capacitated mouse sperm. The Rsph6a gene has six exons, five of which are conserved during evolution in flagellated cells. The exon containing the capacitation-induced phosphorylation site was found exclusively in eutherian mammals. Transcript analyses revealed at least two different testis-specific splicing variants for Rsph6a.Rsph6a mRNA expression was restricted to spermatocytes. Using antibodies generated against the Rsph6a N-terminal domain, western blotting and immunofluorescence analyses indicated that the protein remains in mature sperm and localizes to the sperm flagellum. Consistent with its role in the axoneme, solubility analyses revealed that Rsph6 is attached to cytoskeletal structures. Based on previous studies in Chlamydomonas reinhardtii, we predict that Rsph6 participates in the interaction between the central pair of microtubules and the surrounding pairs. The findings that Rsph6a is more phosphorylated during capacitation and is predicted to function in axonemal localization make Rsph6a a candidate protein mediating signaling processes in the sperm flagellum.

Lysine acetylation modulates mouse sperm capacitation

Mammalian sperm are unable to fertilize the egg immediately after ejaculation. To gain fertilization competence, they need to undergo a series of modifications inside the female reproductive tract, known as capacitation. Capacitation involves several molecular events such as phosphorylation cascades, hyperpolarization of the plasma membrane and intracellular Ca2+ changes, which prepare the sperm to develop two essential features for fertilization competence: hyperactivation and acrosome reaction. Since sperm cells lack new protein biosynthesis, post-translational modification of existing proteins plays a crucial role to obtain full functionality. Here, we show the presence of acetylated proteins in murine sperm, which increase during capacitation. Pharmacological hyperacetylation of lysine residues in non-capacitated sperm induces activation of PKA, hyperpolarization of the sperm plasma membrane, CatSper opening and Ca2+ influx, all capacitation-associated molecular events. Furthermore, hyperacetylation of non-capacitated sperm promotes hyperactivation and prepares the sperm to undergo acrosome reaction. Together, these results indicate that acetylation could be involved in the acquisition of fertilization competence of mammalian sperm.

Disruption of protein kinase A localization induces acrosomal exocytosis in capacitated mouse sperm

Protein kinase A (PKA) is a broad-spectrum Ser/Thr kinase involved in the regulation of several cellular activities. Thus, its precise activation relies on being localized at specific subcellular places known as discrete PKA signalosomes. A-Kinase anchoring proteins (AKAPs) form scaffolding assemblies that play a pivotal role in PKA regulation by restricting its activity to specific microdomains. Because one of the first signaling events observed during mammalian sperm capacitation is PKA activation, understanding how PKA activity is restricted in space and time is crucial to decipher the critical steps of sperm capacitation. Here, we demonstrate that the anchoring of PKA to AKAP is not only necessary but also actively regulated during sperm capacitation. However, we find that once capacitated, the release of PKA from AKAP promotes a sudden Ca2+ influx through the sperm-specific Ca2+ channel CatSper, starting a tail-to-head Ca2+ propagation that triggers the acrosome reaction. Three-dimensional super-resolution imaging confirmed a redistribution of PKA within the flagellar structure throughout the capacitation process, which depends on anchoring to AKAP. These results represent a new signaling event that involves CatSper Ca2+ channels in the acrosome reaction, sensitive to PKA stimulation upon release from AKAP.

TRPV1: a channel for mammalian sperm thermotaxis?

A number of studies have proposed that mammalian spermatozoa are guided within the oviduct towards the fertilization site by thermotaxis. Thermotaxis relies on a temperature gradient within the oviduct originated by a drop of temperature occurring at the utero-tubal junction during ovulation (1,2). This temperature gradient allows capacitated rabbit, bull, mouse and human spermatozoa to orientate their swim away from the utero-tubal junction, towards the warmer temperature, where the oocyte awaits (3-5). Based on empirical proofs, Bahat et al. (2012) (6) determined that spermatozoa can sense temperature changes as little as 0.0006oC and change the direction of swimming towards the warmer temperature. This discovery leads to the intriguing questions of what is the cellular machinery conferring such extraordinary sensitivity to the spermatozoa.

Novel Sperm and Gonadotropin-releasing Hormone-based Recombinant Fusion Protein: Achievement of 100% Contraceptive Efficacy by Co-immunization of Male and Female Mice.

Improvements in long-term female contraception can be achieved by vaccinating with sperm-derived proteins. Here, recombinant proteins comprising either (i) N- (amino acid residues 1-80) or C- (amino acid residues 76-126) terminal fragments of mouse sperm protein 17 (Sp17) fused to the promiscuous T non-B cell epitope of tetanus toxoid (TT), amino acid residues 830-844 followed by di-lysine linker (KK) (TT-KK-Sp17N or TT-KK-Sp17C , respectively) or (ii) mouse equatorin (amino acid residues 21-185) fused to the T non-B cell epitope of bovine RNase (amino acid residues 94-104) were expressed in Escherichia coli. Immunization of female FVB/J mice, using alum as an adjuvant, led to the generation of high antibody titers against the above proteins. Antibodies against both N- and C-terminal fragments of Sp17 reacted with the entire capacitated mouse spermatozoa, whereas those against equatorin reacted exclusively with the equatorial region. Despite the reactivity of all immune sera, only sera from mice immunized with TT-KK-Sp17N and TT-KK-Sp17C significantly reduced mouse in vitro fertilization. Mating studies of the immunized females with un-immunized male mice revealed the highest infertility in the TT-KK-Sp17C -immunized group. In an attempt to further boost the immune response, the C-terminal fragment of Sp17 was expressed as fusion protein with a tandem repeat of gonadotropin-releasing hormone (GnRH) (Sp17C -GnRH2 ). Immunization of both male and female mice with Sp17C -GnRH2 led to higher contraceptive efficacy compared to mice immunized with TT-KK-Sp17C . Interestingly, mating studies wherein partners were both immunized with Sp17C -GnRH2 showed a complete failure of female mice to conceive. Thus, immunization of both males and females with Sp17C -GnRH2 has the potential to increase contraceptive efficacy. Mol. Reprod. Dev. 83: 1048-1059, 2016. © 2016 Wiley Periodicals, Inc.

Tyrosine phosphorylation of dihydrolipoamide dehydrogenase as a potential cadmium target and its inhibitory role in regulating mouse sperm motility.

Cadmium (Cd) is reported to reduce sperm motility and functions. However, the molecular mechanisms of Cd-induced toxicity remain largely unknown, presenting a major knowledge gap in research on reproductive toxicology. In the present study, we identified a candidate protein, dihydrolipoamide dehydrogenase (DLD), which is a post-pyruvate metabolic enzyme, exhibiting tyrosine phosphorylation in mouse sperm exposed to Cd both in vivo and in vitro. Immunoprecipitation assay demonstrated DLD was phosphorylated in tyrosine residues without altered expression after Cd treatment, which further confirmed our identified result. However, the tyrosine phosphorylation of DLD did not participate in mouse sperm capacitation and Bovine Serum Albumin (BSA) effectively prevented the tyrosine phosphorylation of DLD. Moreover, Cd-induced tyrosine phosphorylation of DLD lowered its dehydrogenase activity and meanwhile, Nicotinamide Adenine Dinucleotide Hydrogen (NADH) content, Adenosine Triphosphate (ATP) production and sperm motility were all inhibited by Cd. Interestingly, when the tyrosine phosphorylation of DLD was blocked by BSA, the decrease of DLD activity, NADH and ATP content as well as sperm motility was also suppressed simultaneously. These results suggested that Cd-induced tyrosine phosphorylation of DLD inhibited its activity and thus suppressed the tricarboxylic acid (TCA) cycle, which resulted in the reduction of NADH and hence the ATP production generated through oxidative phosphorylation (OPHOXS). Taken together, our results revealed that Cd induced DLD tyrosine phosphorylation, in response to regulate TCA metabolic pathway, which reduced ATP levels and these negative effects led to decreased sperm motility. This study provided new understanding of the mechanisms contributing to the harmful effects of Cd on the motility and function of spermatozoa.

Carbonic anhydrases and their functional differences in human and mouse sperm physiology

Fertilization is a key reproductive event in which sperm and egg fuse to generate a new individual. Proper regulation of certain parameters (such as intracellular pH) is crucial for this process. Carbonic anhydrases (CAs) are among the molecular entities that control intracellular pH dynamics in most cells. Unfortunately, little is known about the function of CAs in mammalian sperm physiology. For this reason, we re-explored the expression of CAI, II, IV and XIII in human and mouse sperm. We also measured the level of CA activity, determined by mass spectrometry, and found that it is similar in non-capacitated and capacitated mouse sperm. Importantly, we found that CAII activity accounts for half of the total CA activity in capacitated mouse sperm. Using the general CA inhibitor ethoxyzolamide, we studied how CAs participate in fundamental sperm physiological processes such as motility and acrosome reaction in both species. We found that capacitated human sperm depend strongly on CA activity to support normal motility, while capacitated mouse sperm do not. Finally, we found that CA inhibition increases the acrosome reaction in capacitated human sperm, but not in capacitated mouse sperm.

Artificially Generated Male-Specific Rabbit Antibody Against DBY-Multi-Epitope Fusion Protein And Its Immunoreactive Examination

Abstract DEAD (Asp-Glu-Ala-Asp) box polypeptide 3, Y-linked (DBY or Ddx3y) is a candidate gene for male-specific antigen. The DBY gene detected in capacitated mouse sperm codes putative ATP-dependent RNA helicase. The objective was to produce anti-predicted DBY multi-epitope fusion protein antibody, which could be used to determine male specificity of DBY. Epitope prediction is to aid the design of molecules that can mimic structure and function of a genuine epitope, is a useful tool in protein molecule design. This study predicted the DBY epitopes, prepared rabbit poloclonal antibody against DBY multi-epitope fusion protein, then investigated its immunoreactivity. The fusion protein used as the antigen consisted of three regions of DBY with greatest divergence from other family members, cloned together in-frame (with a His tag to facilitate purification). The resulting antibody recognized both the DBY1-2-3 fusion protein and an endogenous DBY protein of the same size. Furthermore, DBY protein was present (Western blot) in testis, male mouse splenocytes and brain, whereas a weaker band was present in the female brain and splenocytes, and finally, ovary produced only a barely visible protein band. Optical density of DBY protein was higher for males versus corresponding tissues from females. Finally, positive signals of DBY1-2-3 antibody were present on only ~60% of mature murine sperm (based on immunofluorescent staining and flow cytometry), in accordance with the expected proportion of Y-bearing sperm. We hypothesized that our antibodies recognized a specific epitope present in subpopulations of mouse sperm. Therefore, we concluded that anti-DBY1-2-3 antibody could be an alternative way of producing antibodies to DBY protein. Furthermore, this novel DBY antibody against a multi-epitope artificial antigen has potential for both investigating male-specific binding of DBY and as a new method of sex selection.

PKA-dependent phosphorylation of LIMK1 and Cofilin is essential for mouse sperm acrosomal exocytosis

Mammalian sperm must acquire their fertilizing ability after a series of biochemical modifications in the female reproductive tract collectively called capacitation to undergo acrosomal exocytosis, a process that is essential for fertilization. Actin dynamics play a central role in controlling the process of exocytosis in somatic cells as well as in sperm from several mammalian species. In somatic cells, small GTPases of the Rho family are widely known as master regulators of actin dynamics. However, the role of these proteins in sperm has not been studied in detail. In the present work we characterized the participation of small GTPases of the Rho family in the signaling pathway that leads to actin polymerization during mouse sperm capacitation. We observed that most of the proteins of this signaling cascade and their effector proteins are expressed in mouse sperm. The activation of the signaling pathways of cAMP/PKA, RhoA/C and Rac1 is essential for LIMK1 activation by phosphorylation on Threonine 508. Serine 3 of Cofilin is phosphorylated by LIMK1 during capacitation in a transiently manner. Inhibition of LIMK1 by specific inhibitors (BMS-3) resulted in lower levels of actin polymerization during capacitation and a dramatic decrease in the percentage of sperm that undergo acrosomal exocytosis. Thus, we demonstrated for the first time that the master regulators of actin dynamics in somatic cells are present and active in mouse sperm. Combining the results of our present study with other results from the literature, we have proposed a working model regarding how LIMK1 and Cofilin control acrosomal exocytosis in mouse sperm.

In vivo exposure to 17β-estradiol triggers premature sperm capacitation in cauda epididymis

Estrogens play a crucial role in spermatogenesis and estrogen receptor α knock-out male mice are infertile. It has been demonstrated that estrogens significantly increase the speed of capacitation in vitro; however this may lead to the reduction of reproductive potential due to the decreased ability of these sperm to undergo the acrosome reaction. To date the in vivo effect of estrogens on the ability of sperm to capacitate has not been investigated. Therefore, in this study, we exposed mice (n=24) to 17β-estradiol (E2) at the concentration of 20 ng/ml either during puberty from the fourth to seventh week of age (n=8), or continuously from birth for a period of 12 weeks (n=8) at which age the animals from both groups were killed. The capacitation status of epididymal and testicular sperm was analysed by tyrosine phosphorylation (TyrP) antibody (immunofluorescence and western blot) and chlortetracycline (CTC) assay. According to our results, in vivo exposure to increased E2 concentrations caused premature sperm capacitation in the epididymis. The effect of E2, however, seems reversible because after the termination of the exposure premature epididymal sperm capacitation is decreased in animals treated during puberty. Furthermore the changes in epididymal sperm capacitation status detected by TyrP and CTC positively correlate with plasma levels of E2 and the expression of the estrogen-dependent trefoil factor 1 (Tff1) gene in testicular tissue. Therefore, our data implicate that in vivo exposure to E2 under specific conditions leads to the premature capacitation of mouse sperm in epididymis with a potential negative impact on the sperm reproductive fitness in the female reproductive tract.

Mouse Sperm Membrane Potential Hyperpolarization Is Necessary and Sufficient to Prepare Sperm for the Acrosome Reaction

Mammalian sperm are unable to fertilize the egg immediately after ejaculation; they acquire this capacity during migration in the female reproductive tract. This maturational process is called capacitation and in mouse sperm it involves a plasma membrane reorganization, extensive changes in the state of protein phosphorylation, increases in intracellular pH (pH(i)) and Ca(2+) ([Ca(2+)](i)), and the appearance of hyperactivated motility. In addition, mouse sperm capacitation is associated with the hyperpolarization of the cell membrane potential. However, the functional role of this process is not known. In this work, to dissect the role of this membrane potential change, hyperpolarization was induced in noncapacitated sperm using either the ENaC inhibitor amiloride, the CFTR agonist genistein or the K(+) ionophore valinomycin. In this experimental setting, other capacitation-associated processes such as activation of a cAMP-dependent pathway and the consequent increase in protein tyrosine phosphorylation were not observed. However, hyperpolarization was sufficient to prepare sperm for the acrosome reaction induced either by depolarization with high K(+) or by addition of solubilized zona pellucida (sZP). Moreover, K(+) and sZP were also able to increase [Ca(2+)](i) in non-capacitated sperm treated with these hyperpolarizing agents but not in untreated cells. On the other hand, in conditions that support capacitation-associated processes blocking hyperpolarization by adding valinomycin and increasing K(+) concentrations inhibited the agonist-induced acrosome reaction as well as the increase in [Ca(2+)](i). Altogether, these results suggest that sperm hyperpolarization by itself is key to enabling mice sperm to undergo the acrosome reaction.

SNARE Complex Phosphorylation Shows Dynamic Changes During Mouse Sperm Capacitation.

SNARE Complex Phosphorylation Shows Dynamic Changes During Mouse Sperm Capacitation.

In Vivo Exposition to 17B-Estradiol Cause Premature Capacitation of Epididymal Mouse Sperm.

Estrogens play an important role both in female and male reproductive system. In males, estrogens influence process of spermatogenesis and estrogen receptor alpha knock-out mice are infertile. Increasing evidence suggests that compounds with estrogen activity also significantly influence sperm physiological processes including capacitation. It has been demonstrated that in vitro estrogens significantly increase a speed of capacitation process however this leads in vitro to a reproductive potential reduction. During in vivo experiments, the situation could be different and in vivo exposition to estrogens and subsequent analysis of sperm capacitation was required. Therefore, in presented study, we investigated effect of in vivo exposition to 17B-estradiol (E2) in concentration 20 ng/ml on sperm capacitation in vitro. In vivo exposure to E2 was either during puberty between 3 and 8 weeks of age or postnataly for 12 weeks until animals were sacrificed. According to our results, in vivo exposition to E2 causes premature sperm capacitation already in the epididymis. The effect of E2 seems however reversible, since the end of the exposition leads to reduction in the number of capacitated epididymal sperm. After subsequent in vitro capacitation, the differences between control and experimental groups were mainly caused by initial difference between the numbers of capacitated sperm at the start of the capacitation. Therefore, our data implicates that in vivo exposition to E2 leads to premature capacitation of mouse sperm in epididymis with a potential negative impact on sperm reproductive fitness in female reproductive tract. (The work was supported by Grants of the Ministry of Education of the Czech Republic Nos. VC 1M06011 and VZ 0021620828, the Grant Agency of the Czech Republic No. 523/08/H064 and by the Institutional Research Supports AVOZ 50520701 and SVV-2012-265 206.)

Sperm arylsulfatase A binds to mZP2 and mZP3 glycoproteins in a nonenzymatic manner

We have shown previously that sperm surface arylsulfatase A (ASA) of mouse, pig, and human is involved in sperm-egg zona pellucida (ZP) binding. By treating capacitated mouse sperm with A23187 to induce the acrosome reaction, we demonstrated by immunoblotting that ASA also existed in the acrosomal content and on the inner acrosomal membrane. Since mZP2 and mZP3 are known as sperm receptors, whereas mZP1 as a cross-linker of mZP2/mZP3, we determined whether purified ASA bound to mZP2 and mZP3 selectively. The three mZP glycoproteins were purified from solubilized ovarian ZP by size exclusion column chromatography. Immuno-dot blot analyses revealed that purified sperm ASA bound to mZP2 at the highest level followed by mZP3, whereas the binding of ASA to mZP1 was minimal. The results confirmed the physiological significance of sperm ASA in the ZP binding process. The binding of ASA to mZP2 and mZP3 was, however, not dependent on the active site pocket amino acids, Cys69, Lys123, and Lys302, which are pertinent to the capturing of an arylsulfate substrate, since ASA mutant with Ala substitution at these three residues still bound to mZP2 and mZP3. The availability of the active site pocket of ASA bound to the ZP suggested that ASA would still retain enzymatic activity, which might be important for subsequent sperm penetration through the ZP.

The association between CDC42 and caveolin-1 is involved in the regulation of capacitation and acrosome reaction of guinea pig and mouse sperm

In the mammalian sperm, the acrosome reaction (AR) is considered to be a regulated secretion that is an essential requirement for physiological fertilization. The AR is the all-or-nothing secretion system that allows for multiple membrane fusion events. It is a Ca(2)(+)-regulated exocytosis reaction that has also been shown to be regulated by several signaling pathways. CDC42 has a central role in the regulated exocytosis through the activation of SNARE proteins and actin polymerization. Furthermore, the lipid raft protein caveolin-1 (CAV1) functions as a scaffold and guanine nucleotide dissociation inhibitor protein for CDC42, which is inactivated when associated with CAV1. CDC42 and other RHO proteins have been shown to localize in the acrosome region of mammalian sperm; however, their relationship with the AR is unknown. Here, we present the first evidence that CDC42 and CAV1 could be involved in the regulation of capacitation and the AR. Our findings show that CDC42 is activated early during capacitation, reaching an activation maximum after 20 min of capacitation. Spontaneous and progesterone-induced ARs were inhibited when sperm were capacitated in presence of secramine A, a specific CDC42 inhibitor. CAV1 and CDC42 were co-immunoprecipitated from the membranes of noncapacitated sperm; this association was reduced in capacitated sperm, and our data suggest that the phosphorylation (Tyr14) of CAV1 by c-Src is involved in such reductions. We suggest that CDC42 activation is favored by the disruption of the CAV1-CDC42 interaction, allowing for its participation in the regulation of capacitation and the AR.

SPINK3 modulates mouse sperm physiology through the reduction of nitric oxide level independently of its trypsin inhibitory activity

Serine protease inhibitor Kazal-type (SPINK3)/P12/PSTI-II is a small secretory protein from mouse seminal vesicle which contains a KAZAL domain and shows calcium (Ca(2+))-transport inhibitory (caltrin) activity. This molecule was obtained as a recombinant protein and its effect on capacitated sperm cells was examined. SPINK3 inhibited trypsin activity in vitro while the fusion protein GST-SPINK3 had no effect on this enzyme activity. The inactive GST-SPINK3 significantly reduced the percentage of spermatozoa positively stained for nitric oxide (NO) with the specific probe DAF-FM DA and NO concentration measured by Griess method in capacitated mouse sperm; the same effect was observed when sperm were capacitated under low Ca(2+) concentration, using either intracellular (BAPTA-AM) or extracellular Ca(2+) (EDTA) chelators. The percentage of sperm showing spontaneous and progesterone-induced acrosomal reaction was significantly lower in the presence of GST-SPINK3 compared to untreated capacitated spermatozoa. Interestingly, this decrease was overcome by the exogenous addition of the NO donors, sodium nitroprusside (SNP), and S-nitrosoglutathione (GSNO). Phosphorylation of sperm proteins in tyrosine residues was partially affected by GST-SPINK3, however, only GSNO was able to reverse this effect. Sperm progressive motility was not significantly diminished by GST-SPINK3 or BAPTA-AM but enhanced by the addition of SNP. This is the first report that demonstrates that SPINK3 modulates sperm physiology through a downstream reduction of endogenous NO concentration and independently of SPINK3 trypsin inhibitory activity.