Inhibition of sperm capacitation and fertilizing capacity by adjudin is mediated by chloride and its channels in humans.

STUDY QUESTION: Does adjudin disrupt chloride ion (Cl⁻) ion transport function in human sperm and impede sperm capacitation and fertilizing ability in vitro? SUMMARY ANSWER: In this study the results indicate that adjudin is a potent blocker of Cl⁻ channels: disrupting Cl⁻ ion transport function results in a decline in sperm capacitation and fertilizing ability in humans in vitro. WHAT IS KNOWN ALREADY: Although our previous studies have demonstrated that adjudin exerts its effect by disrupting sertoli-germ cell adhesion junctions, most notably apical ectoplasmic specialization by targeting testin and actin filament bundles that disrupts the actin-based cytoskeleton in sertoli cells, it remains unclear whether adjudin impedes Cl⁻ ion transport function in the human sperm. STUDY DESIGN, SIZE AND DURATION: Semen samples were obtained from 45 fertile men (aged 25-32). Spermatozoa were isolated from the semen in the human tube fluid (HTF) medium by centrifugation through a discontinuous Percoll gradient, and incubated with adjudin at 10 nM-10 µM and/or other reagents under capacitating conditions for 0-5 h. PARTICIPANTS/MATERIALS, SETTING, METHODS: We evaluated the effect of adjudin and different reagents on sperm functions with which they were incubated at 37 °C. Sperm motility and hyperactivation were analyzed by a computer-assisted sperm analysis (CASA) system. Sperm capacitation and the acrosome reaction were assessed by chlortetracycline fluorescence staining. Sperm fertilizing ability was evaluated by sperm penetration of zona-free hamster egg assay, and cellular cAMP levels in spermatozoa were quantified by the EIA kit. The proteins tyrosine, serine and threonine phosphorylation in the presence or absence of adjudin were analyzed by means of a immunodetection of spermatozoa, especially, compared the effect of adjudin on sperm hyperactivation and capacitation in the complete HTF medium with the Cl⁻-deficient HTF medium as well as the various Cl⁻ channel blockers. MAIN RESULTS AND THE ROLE OF CHANCE: Adjudin significantly inhibited sperm hyperactivation but not sperm motility. Adjudin-induced inhibition of sperm capacitation was reversible, and it was found to block the rhuZP3ß- and progesterone-induced acrosome reaction in a dose-dependent manner. Adjudin also blocked sperm penetration of zona-free hamster eggs, and significantly inhibited both forskolin-activated transmembrane adenylyl cyclase and soluble adenylyl cyclase activities leading to a significant decline in the cellular cAMP levels in human spermatozoa. Adjudin failed to reduce sperm protein tyrosine phosphorylation but it did prevent sperm serine and threonine protein phosphorylation. Interestingly, adjudin was found to exert its inhibitory effects on sperm capacitation and capacitation-associated events only in the complete Cl⁻-HTF medium but not Cl⁻-deficient medium, illustrating the likely involvement of Cl⁻. Adjudin inhibits the fertility capacity of human sperm is mediated by disrupting chloride ion and its transport function. LIMITATIONS, REASONS FOR CAUTION: This study has examined the effect of adjudin only on human sperm capacitation and fertilizing ability in vitro and thus has some limitations. Further investigations in vivo are needed to confirm adjudin is a potent male contraceptive. WIDER IMPLICATIONS OF THE FINDINGS: Our studies demonstrated that adjudin inhibition of capacitation is reversible and its toxicity is low, opening the door for the examination of adjudin as a mediator of male fertility control. Adjudin may be a safe, efficient and reversible male antifertility agent and applicable to initial clinical trials of adjudin as a male antifertility agent in humans. STUDING FUNDING/COMPETING INTEREST(S): This work was supported by the National Basic Research Program of China (2006CB504002), the Nature Science Foundation of China (Nos. 81000244 and 81170554), Zhejiang Project of Science and Technology (2011C23046), the Nature Science Fund of Zhejiang province (Nos.Y2100058 and Y2090236), the key Science and Technology Innovation Team of Zhejiang Province (No.2012R10048-07) and the National Institutes of Health (NICHD U54 HD029990 project 5), USA. The authors declare no conflict of interest.

Secretory phospholipase A2 group IID Is involved in progesterone-induced acrosomal exocytosis of human spermatozoa.

Phospholipase A2 (PLA(2)) plays a major role during acrosomal exocytosis (AE) in mammalian spermatozoa, but the identity of PLA(2) subtypes present in spermatozoa remains elusive. This study explored whether secretory PLA(2) Group IID (sPLA(2)-IID) isoform is present in human spermatozoa and whether it is involved in AE. Localization and expression of sPLA(2)-IID in human spermatozoa were explored by immunofluorescence staining and Western blot analysis. Occurrence of AE was evaluated by triple staining, and arachidonic acid (AA) levels were quantified by gas chromatography-mass spectrometry. Sperm motion parameters and hyperactivation were analyzed by computer-assisted sperm analysis. sPLA(2)-IID was localized in the postacrosomal region of the head and the midpiece of tail in human sperm. A 16-kd protein band was detected by Western blotting in sperm extracts. Progesterone-induced AE was significantly inhibited in a concentration-dependent manner using a sPLA(2)-IID neutralizing antibody. The increase in AA levels seen during progesterone-stimulated exocytosis was significantly abrogated by the antibody. The sPLA(2)-IID antibody significantly inhibited hyperactivation, sperm curvilinear velocity, and amplitude of lateral head displacement, but it did not affect the proportion of motile sperm. In conclusion, sPLA(2)-IID is present at the head and midpiece in the human sperm, and activation of such sPLA(2)-IID seems to be involved in AE. Therefore, sPLA(2)-IID isoform plays a functional role during the AE in human sperm.

The decline of fertility in male uremic patients is correlated with low expression of the cystic fibrosis transmembrane conductance regulator protein (CFTR) in human sperm.

BACKGROUND: The present study was designed to investigate the possible association between infertility of male uremic patients and expression of the cystic fibrosis transmembrane conductance regulator (CFTR) protein in their sperm. METHODS: Semen was collected and analyzed. Serum levels of FSH, LH and testosterone were measured by radioimmunoassay. The sperm CFTR expressions of 21 uremic patients and 15 renal transplant patients were measured and compared with those of 32 healthy and 33 infertile men. RESULTS: Only 9 ± 5.9% of sperm from uremic patients expressed CFTR, significantly less than those of the renal transplant patients (29 ± 14.3%, P< 0.001), the infertile men (42 ± 20.7%, P< 0.001) and the healthy men (51 ± 20.5%, P< 0.001). Furthermore, significantly fewer sperm from renal transplant patients expressed CFTR than those of the infertile men (P< 0.05) and the healthy men (P< 0.01). LH levels in uremic patients were significantly higher than in all other groups, whereas FSH levels in uremic patients were only significantly higher than in infertile and healthy men. There was no significant difference in testosterone level among the four categories. CONCLUSIONS: Sperm CFTR expression is depressed in uremic patients but recovers to some degree after renal transplant along with some improvement in fertility, indicating a 'reversible' change. These results suggest that the CFTR expression rate in sperm is correlated with the decline of uremic patients' fertility, and may be considered as a potential marker to assess the fertility of male uremic patients.

Defective CFTR-dependent CREB activation results in impaired spermatogenesis and azoospermia.

Cystic fibrosis (CF) is the most common life-limiting recessive genetic disease among Caucasians caused by mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) with over 95% male patients infertile. However, whether CFTR mutations could affect spermatogenesis and result in azoospermia remains an open question. Here we report compromised spermatogenesis, with significantly reduced testicular weight and sperm count, and decreased cAMP-responsive element binding protein (CREB) expression in the testes of CFTR knockout mice. The involvement of CFTR in HCO(3) (-) transport and the expression of the HCO(3) (-) sensor, soluble adenylyl cyclase (sAC), are demonstrated for the first time in the primary culture of rat Sertoli cells. Inhibition of CFTR or depletion of HCO(3) (-) could reduce FSH-stimulated, sAC-dependent cAMP production and phosphorylation of CREB, the key transcription factor in spermatogenesis. Decreased CFTR and CREB expression are also observed in human testes with azoospermia. The present study reveals a previously undefined role of CFTR and sAC in regulating the cAMP-CREB signaling pathway in Sertoli cells, defect of which may result in impaired spermatogenesis and azoospermia. Altered CFTR-sAC-cAMP-CREB functional loop may also underline the pathogenesis of various CF-related diseases.

Aquaporin3 is a sperm water channel essential for postcopulatory sperm osmoadaptation and migration.

In the journey from the male to female reproductive tract, mammalian sperm experience a natural osmotic decrease (e.g., in mouse, from ~415 mOsm in the cauda epididymis to ~310 mOsm in the uterine cavity). Sperm have evolved to utilize this hypotonic exposure for motility activation, meanwhile efficiently silence the negative impact of hypotonic cell swelling. Previous physiological and pharmacological studies have shown that ion channel-controlled water influx/efflux is actively involved in the process of sperm volume regulation; however, no specific sperm proteins have been found responsible for this rapid osmoadaptation. Here, we report that aquaporin3 (AQP3) is a sperm water channel in mice and humans. Aqp3-deficient sperm show normal motility activation in response to hypotonicity but display increased vulnerability to hypotonic cell swelling, characterized by increased tail bending after entering uterus. The sperm defect is a result of impaired sperm volume regulation and progressive cell swelling in response to physiological hypotonic stress during male-female reproductive tract transition. Time-lapse imaging revealed that the cell volume expansion begins at cytoplasmic droplet, forcing the tail to angulate and form a hairpin-like structure due to mechanical membrane stretch. The tail deformation hampered sperm migration into oviduct, resulting in impaired fertilization and reduced male fertility. These data suggest AQP3 as an essential membrane pathway for sperm regulatory volume decrease (RVD) that balances the "trade-off" between sperm motility and cell swelling upon physiological hypotonicity, thereby optimizing postcopulatory sperm behavior.

CFTR is essential for sperm fertilizing capacity and is correlated with sperm quality in humans.

BACKGROUND: Our previous studies have demonstrated the cystic fibrosis transmembrane conductance regulator (CFTR) is important for capacitation and male fertility in mouse and guinea pig spermatozoa. However, the exact function of CFTR on human sperm fertilizing capacity, and correlation with sperm quality has not been established. The present study may shed light on some unexplained male infertility, and on a possible new method for diagnosis of male infertility and strategy for male contraception. METHODS: To assess the effect of CFTR on human sperm fertilizing capacity, we examined sperm capacitation and the acrosome reaction using chlortetracycline staining, analyzed sperm hyperactivation by computer-assisted semen analysis (CASA), measured intracellular cAMP levels using ElA and evaluated sperm penetration of zona-free hamster eggs assay in fertile men. The percentage of spermatozoa expressing CFTR from fertile, healthy and infertile men (mainly teratospermic, asthenoteratospermic, asthenospermic and oligospermic) was conducted by indirect immunofluorescence staining. RESULTS: Progesterone significantly facilitated human sperm capacitation and ZP3 triggered the acrosome reaction, both were significantly inhibited by CFTR inhibitor-172 (CFTRinh-172; 10 nM-1 microM) in a dose-dependent manner. The presence of 100 nM CFTRinh-172 markedly depressed intracellular cAMP levels, sperm hyperactivation and sperm penetration of zona-free hamster eggs. In addition, the percentage of spermatozoa expressing CFTR in the fertile men was significantly higher than healthy and infertile men categories (P < 0.01). CONCLUSIONS: CFTR is essential for human sperm fertilizing capacity and the impairment of CFTR expression in spermatozoa is correlated with a reduction of sperm quality. These results suggest that defective expression of CFTR in human sperm may lead to the reduction of sperm fertilizing capacity.

HongrES1, a cauda epididymis-specific protein, is involved in capacitation of guinea pig sperm.

Capacitation requires removal of proteins secreted by the cauda epididymis. Previously, we isolated and cloned the HongrES1 gene from rat cauda epididymis and found that it was exclusively expressed there. Here we report that HongrES1 mRNA is also expressed in the guinea pig cauda epididymis using Northern blot analysis, and the molecular weight of its cognate protein is approximately 48 kDa by Western blot analysis. Therefore, we investigated whether HongrES1 was involved in regulation of sperm capacitation in guinea pig. The results show that HongrES1 antisera (HA) significantly enhances sperm capacitation with maximal stimulation at a dilution of 1:500. Capacitation was reversed when capacitated spermatozoa were re-exposed to HongrES1 protein (HP, 0.25 microg/ml). In other words, HP acted as a decapacitation factor. HA accelerated the onset of capacitation and promoted a sperm hyperactivated motility response. Sperm capacitation was accelerated by HA stimulation of extracellular calcium influx while HP prevented extracellular calcium from influxing. Indirect immunofluorescence staining finds HP localized over the acrosomal anterior region of the sperm head, which exfoliates gradually during capacitation incubation, and completely disappeared after the acrosome reaction. Thus, HongrES1 expressed by the cauda epididymis is a novel molecule that regulates the physiology of guinea pig sperm prior to fertilization.

Activation of GABAA receptor/Cl- channel and capacitation in rat spermatozoa: HCO3- and Cl- are essential.

This study was designed to determine whether HCO(3)(-) and Cl(-) are required for the activation of the GABA(A) receptor/Cl(-) channel (GBRC) by GABA and the subsequent capacitation of rat sperm. Spermatozoa from adult Sprague Dawley rats were incubated in four different media: modified complete rat fertilization medium (mRFM), Cl(-)-deficient (Cl(-)-DF) mRFM, HCO(3)(-)-DF mRFM, and Cl(-)-DF HCO(3)(-)-DF mRFM, with or without GBRC agonists (GABA and progesterone) or GBRC antagonists (bicuculline and picrotoxin) for 0-6 h under capacitating conditions. Sperm capacitation and hyperactivation were assessed by chlortetracycline staining and computer-assisted sperm analysis, respectively. The results showed that GABA added to the mRFM accelerated capacitation and hyperactivation, followed by increase in the acrosome reaction, reaching maximum value after 5 h. Progesterone also accelerated sperm capacitation and hyperactivation. Bicuculline and picrotoxin, antagonists of GABA, blocked the effects of both GABA and progesterone acceleration of sperm capacitation and hyperactivation. Sperm capacitation required both Cl(-) and HCO(3)(-). These results indicate that activation of GBRC may contribute to sperm capacitation and hyperactivation, and that both HCO(3)(-) and Cl(-) are essential. This is the first report of a close relationship between HCO(3)(-)/Cl(-) transport and the activation of GBRC in rat sperm capacitation and hyperactivation.

Cl- is required for HCO3- entry necessary for sperm capacitation in guinea pig: involvement of a Cl-/HCO3- exchanger (SLC26A3) and CFTR.

Our previous study demonstrated the involvement of cystic fibrosis transmembrane conductance regulator (CFTR) in transporting bicarbonate that is necessary for sperm capacitation; however, whether its involvement is direct or indirect remains unclear. The present study investigated the possibility of a Cl-/HCO3- exchanger (solute carrier family 26, number 3 [SLC26A3]) operating with CFTR during guinea pig sperm capacitation. Incubating sperm in media with various concentrations of Cl- resulted in varied percentages of capacitated sperm in a concentration-dependent manner. Depletion of Cl-, even in the presence of HCO3-, abolished sperm capacitation and vice versa, indicating the involvement of both anions in the process. Capacitation-associated HCO3--dependent events, including increased intracellular pH, cAMP production, and protein tyrosine phosphorylation, also depend on Cl- concentrations. Similar Cl- dependence and inhibitor sensitivity were observed for sperm-hyperactivated motility and for sperm-egg fusion. The expression and localization of CFTR and SLC26A3 were demonstrated using immunostaining and Western blot analysis. Taken together, our results indicate that Cl- is required for the entry of HCO3- that is necessary for sperm capacitation, implicating the involvement of SLC26A3 in transporting HCO3-, with CFTR providing the recycling pathway for Cl-.

Cystic fibrosis transmembrane conductance regulator is vital to sperm fertilizing capacity and male fertility.

Cystic fibrosis transmembrane conductance regulator (CFTR) is an anion channel, mutations of which cause cystic fibrosis, a disease characterized by defective Cl(-) and HCO(3)(-) transport. Although >95% of all CF male patients are infertile because of congenital bilateral absence of the vas deferens (CBAVD), the question whether CFTR mutations are involved in other forms of male infertility is under intense debates. Here we report that CFTR is detected in both human and mouse sperm. CFTR inhibitor or antibody significantly reduces the sperm capacitation, and the associated HCO(3)(-)-dependent events, including increases in intracellular pH, cAMP production and membrane hyperpolarization. The fertilizing capacity of the sperm obtained from heterozygous CFTR mutant mice is also significantly lower compared with that of the wild-type. These results suggest that CFTR in sperm may be involved in the transport of HCO(3)(-) important for sperm capacitation and that CFTR mutations with impaired CFTR function may lead to reduced sperm fertilizing capacity and male infertility other than CBAVD.

Acrosome reaction induced by recombinant human zona pellucida 3 peptides rhuZP3a22 approximately 176 and rhuZP3b177 approximately 348 and their mechanism.

To explore the biological characteristics of the recombinant zona pellucida 3 (ZP3) peptides of rhuZP3a22 approximately 176 and rhuZP3b177 approximately 348, we examined whether rhuZP3a22 approximately 176 or rhuZP3b177 approximately 348 trigger the acrosome reaction (AR) of human spermatozoa and we investigated the underlying mechanism. The assessment of AR was performed using chlortetracycline staining. The intracellular free calcium concentration ([Ca2+]i) in Fura-2/AM-loaded human sperm was monitored with a spectrofluorophotometer. We found that rhuZP3a22 approximately 176 and rhuZP3b177 approximately 348 were capable of eliciting AR at different concentrations. With the addition of either peptide, the [Ca2+]i level was raised to a peak with or without a plateau. The AR could be inhibited by pertussis toxin (PTX), EGTA, and pimozide (a T-type calcium channel blocker), whereas verapamil was less effective in this regard. The results of the present study suggest that peptides rhuZP3a22 approximately 176 and rhuZP3b177 approximately 348 have a role similar to human ZP3, and that the mechanism of the response to the peptides involves influx of calcium, the G protein pathway, and a T-type calcium channel.

Critical role of CFTR in uterine bicarbonate secretion and the fertilizing capacity of sperm.

Cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP-activated Cl- channel expressed in a wide variety of epithelial cells, mutations of which are responsible for hallmark defective Cl- and HCO3- secretion seen in cystic fibrosis (CF). However, the physiological role of CFTR in reproductive tracts is far from understood although infertility has been observed in CF patients of both sexes. Previously we have demonstrated the expression of CFTR in the female reproductive tract and the involvement of CFTR in mediating anion secretion by the endometrium. Our recent results show that endometrial epithelial cells possess a cAMP-activated HCO3- transport mechanism, which could be impaired with channel blockers known to block CFTR or antisense against CFTR. Co-culture of sperm with CFTR antisense-treated endometrial cells or HCO3- secretion-defective CF epithelial cells resulted in reduced sperm capacitation and egg-fertilizing ability. Addition of HCO3- to the culture media and transfection of wild-type CFTR into CF cells rescued the fertilizing capacity of sperm. Immunostaining and Western blot revealed that CFTR is expressed in rodent sperm and intracellular measurement of pH during sperm capacitation indicated that the entry of HCO3- into sperm could be inhibited by CFTR inhibitor. These results are consistent with a critical role of CFTR in controlling uterine HCO3- secretion and sperm fertilizing capacity, suggesting that CFTR may be a potential target for post-meiotic regulation of fertility.

[Immunogenicity of recombinant human zona pellucida-3 peptides expressed in E. coli and efficacy of their antisera to inhibit in vitro human sperm-egg binding].

The present study was aimed to analyze the immunogenicity of recombinant human zona pellucida-3 peptides (r-huZP3a(22 approximately 176) and r-huZP3b(177 approximately 348)) expressed in E. coli through immunizing rabbits, and to evaluate the efficacy of their polyclonal antisera against r-huZP3a(22 approximately 176) and r-huZP3b(177 approximately 348) to inhibit in vitro human sperm-egg binding respectively. Male New Zealand rabbits were immunized using r-huZP3a(22 approximately 176) or r-huZP3b(177 approximately 348) as antigen respectively, which was purified through an improved method of preparative gel polyacryulamide gel electrophoresis. The antibody response level of r-huZP3a(22 approximately 176) or r-huZP3b(177 approximately 348) immunogen in rabbits was determined by ELISA using mouse ZP3-5 (amino acid sequence(137 approximately 150) being completely conserved with huZP3(138 approximately 151) sequence) and specific huZP3-14 (amino acid sequence(327 approximately 340)) synthetic peptides as coating antigens respectively. The immunoreactivity and specificity of the anti-r-huZP3a(22 approximately 176) and anti-r-huZP3b(177 approximately 348) antisera with each r-huZP3 peptides, were tested by immunoblot and immunohistochemistry (using native huZP and human ovary section) respectively. A competitive hemizona assay (HZA) was used to evaluate the efficacy of the antisera against r-huZP3a(22 approximately 176) and r-huZP3b(177 approximately 348) to inhibit in vitro human sperm-egg binding. Both r-huZP3 peptides were able to induce higher antibody titers in rabbits. Each antiserum could specifically recognize or bind to each target r-huZP3 peptide expressed in E. coli and native human ZP in vitro. The antisera also inhibited sperm-egg binding in the HZA. These results show that r-huZP3a(22 approximately 176) and r-huZP3b(177 approximately 348) are of strong immunogenicity. They can be used to develop a kit for detecting whether there are autoantibodies to zona pellucida in unexplained infertile women, and their antisera might be useful tools for determining minimal B-cell epitope sequences of several known huZP3 epitope peptides.

GABA, progesterone and zona pellucida activation of PLA2 and regulation by MEK-ERK1/2 during acrosomal exocytosis in guinea pig spermatozoa.

We investigated whether GABA activates phospholipase A2 (PLA2) during acrosomal exocytosis, and if the MEK-ERK1/2 pathway modulates PLA2 activation initiated by GABA, progesterone or zona pellucida (ZP). In guinea pig spermatozoa prelabelled with [14C]arachidonic acid or [14C]choline chloride, GABA stimulated a decrease in phosphatidylcholine (PC), and release of arachidonic acid and lysoPC, during exocytosis. These lipid changes are indicative of PLA2 activation and appear essential for exocytosis since inclusion of aristolochic acid (a PLA2 inhibitor) abrogated them, along with exocytosis. GABA activation of PLA2 seems to be mediated, at least in part, by diacylglycerol (DAG) and protein kinase C since inclusion of the DAG kinase inhibitor R59022 enhanced PLA2 activity and exocytosis stimulated by GABA, whereas exposure to staurosporine decreased both. GABA-, progesterone- and ZP-induced release of arachidonic acid and exocytosis were prevented by U0126 and PD98059 (MEK inhibitors). Taken together, our results suggest that PLA2 plays a fundamental role in agonist-stimulated exocytosis and that MEK-ERK1/2 are involved in PLA2 regulation during this process.

Progesterone primes zona pellucida-induced activation of phospholipase A2 during acrosomal exocytosis in guinea pig spermatozoa.

We investigated, using guinea-pig spermatozoa as a model, whether phospholipase A2 (PLA2) is involved in progesterone or zona pellucida (ZP)-stimulated acrosomal exocytosis, if progesterone enhances ZP-induced activation of PLA2, and mechanisms underlying PLA2 regulation. Spermatozoa were capacitated and labeled in low Ca2+ medium with [14C]choline chloride or [14C]arachidonic acid, washed, and then exposed to millimolar Ca2+ and progesterone and/or ZP. Each agonist stimulated decrease of phosphatidylcholine (PC) and release of arachidonic acid and lysoPC, indicative of PLA2 activation. Aristolochic acid (a PLA2 inhibitor) abrogated lipid changes and exocytosis, indicating that these lipid changes are essential for exocytosis. Exposure of spermatozoa to submaximal concentrations of both progesterone and ZP resulted in a synergistic increase of arachidonic acid and lysoPC releases, and exocytosis, suggesting that, under natural conditions, both agonists interact to bring about acrosomal exocytosis. Progesterone-induced PLA2 activation appears to be mediated by a GABA(A)-like receptor, because bicuculline (a GABA(A) receptor antagonist) blocked arachidonic acid release and exocytosis. In agreement with this, GABA mimicked progesterone actions. ZP-induced activation of PLA2 seemed to be transduced via G(i) proteins because pertussis toxin blocked arachidonic acid release and acrosomal exocytosis. PLA2 may be regulated by PKC because progesterone- or ZP-induced release of arachidonic acid was blocked by the PKC inhibitors staurosporine or chelerythrine chloride. PLA2 could also be regulated by the cAMP-PKA pathway; inclusion of the PKA inhibitor 14-22 amide or H-89 led to a reduction in arachidonic acid release or exocytosis after progesterone or ZP. Taken together, these results suggest that PLA2 plays an essential role in progesterone or ZP-stimulated exocytosis with progesterone priming ZP action.

Involvement of CFTR in uterine bicarbonate secretion and the fertilizing capacity of sperm.

Cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP-activated chloride channel expressed in a wide variety of epithelial cells, mutations of which are responsible for the hallmark defective chloride secretion observed in cystic fibrosis (CF). Although CFTR has been implicated in bicarbonate secretion, its ability to directly mediate bicarbonate secretion of any physiological significance has not been shown. We demonstrate here that endometrial epithelial cells possess a CFTR-mediated bicarbonate transport mechanism. Co-culture of sperm with endometrial cells treated with antisense oligonucleotide against CFTR, or with bicarbonate secretion-defective CF epithelial cells, resulted in lower sperm capacitation and egg-fertilizing ability. These results are consistent with a critical role of CFTR in controlling uterine bicarbonate secretion and the fertilizing capacity of sperm, providing a link between defective CFTR and lower female fertility in CF.

Effects of demethylzeylasteral and celastrol on spermatogenic cell Ca2+ channels and progesterone-induced sperm acrosome reaction.

The male antifertility effect of a water-chloroform extract of Tripterygium wilfordii Hook. f. (GTW) and several monomers isolated from GTW has attracted worldwide interest. In the present study, the effects of two isolated monomers from GTW, demethylzeylasteral and celastrol, on the Ca(2+) channels in mouse spermatogenic cells and on the sperm acrosome reaction were investigated by whole-cell patch-clamp recording and chlortetracycline staining methods, respectively. The results showed that demethylzeylasteral concentration-dependently and in a partially reversible manner inhibited the Ca(2+) current in spermatogenic cells with an IC(50) of 8.8 microg/ml. Celastrol decreased the Ca(2+) current in the cells time-dependently and irreversibly. The changes in the activation and inactivation time constants of Ca(2+) currents after application of these two compounds were also examined. Demethylzeylasteral increased both activation and inactivation time constants of Ca(2+) currents, and celastrol had no significant effect on them. The two compounds also inhibited significantly the sperm acrosome reaction initiated by progesterone. These data suggest that inhibition of Ca(2+) currents could be responsible for the antifertility activity of these compounds.