Central role of soluble adenylyl cyclase and cAMP in sperm physiology.

Cyclic adenosine 3',5'-monophosphate (cAMP), the first second messenger to be described, plays a central role in cell signaling in a wide variety of cell types. Over the last decades, a wide body of literature addressed the different roles of cAMP in cell physiology, mainly in response to neurotransmitters and hormones. cAMP is synthesized by a wide variety of adenylyl cyclases that can generally be grouped in two types: transmembrane adenylyl cyclase and soluble adenylyl cyclases. In particular, several aspects of sperm physiology are regulated by cAMP produced by a single atypical adenylyl cyclase (Adcy10, aka sAC, SACY). The signature that identifies sAC among other ACs, is their direct stimulation by bicarbonate. The essential nature of cAMP in sperm function has been demonstrated using gain of function as well as loss of function approaches. This review unifies state of the art knowledge of the role of cAMP and those enzymes involved in cAMP signaling pathways required for the acquisition of fertilizing capacity of mammalian sperm. This article is part of a Special Issue entitled: The role of soluble adenylyl cyclase in health and disease.

Compartmentalization of distinct cAMP signaling pathways in mammalian sperm.

Fertilization competence is acquired in the female tract in a process known as capacitation. Capacitation is needed for the activation of motility (e.g. hyperactivation) and to prepare the sperm for an exocytotic process known as acrosome reaction. Although the HCO3(-)-dependent soluble adenylyl cyclase Adcy10 plays a role in motility, less is known about the source of cAMP in the sperm head. Transmembrane adenylyl cyclases (tmACs) are another possible source of cAMP. These enzymes are regulated by stimulatory heterotrimeric Gs proteins; however, the presence of Gs or tmACs in mammalian sperm has been controversial. In this study, we used Western blotting and cholera toxin-dependent ADP-ribosylation to show the Gs presence in the sperm head. Also, we showed that forskolin, a tmAC-specific activator, induces cAMP accumulation in sperm from both WT and Adcy10-null mice. This increase is blocked by the tmAC inhibitor SQ22536 but not by the Adcy10 inhibitor KH7. Although Gs immunoreactivity and tmAC activity are detected in the sperm head, PKA is only found in the tail, where Adcy10 was previously shown to reside. Consistent with an acrosomal localization, Gs reactivity is lost in acrosome-reacted sperm, and forskolin is able to increase intracellular Ca(2+) and induce the acrosome reaction. Altogether, these data suggest that cAMP pathways are compartmentalized in sperm, with Gs and tmAC in the head and Adcy10 and PKA in the flagellum.

The use of alternative polyadenylation sites renders integrin ß1 (Itgb1) mRNA isoforms with differential stability during mammary gland development.

Integrins are heterodimeric cell-surface adhesion receptors that play a critical role in tissue development. Characterization of the full-length mRNA encoding the ß1 subunit (Itgb1) revealed an alternative functional cleavage and polyadenylation site that yields a new Itgb1 mRNA isoform 578 bp shorter than that previously reported. Using a variety of experimental and bioinformatic approaches, we found that the two Itgb1 isoforms are expressed at different levels in a variety of mouse tissues, including the mammary gland, where they are differentially regulated at successive developmental stages. The longer mRNA species is prevelant during lactation, whereas the shorter is induced after weaning. In 3D cultures, where expression of integrin ß1 protein is required for normal formation of acini, experimental blockade of the longer isoform induced enhanced expression of the shorter species which allowed normal morphological mammary differentiation. The short isoform lacks AU-rich motifs and miRNA target sequences that are potentially implicated in the regulation of mRNA stability and translation efficiency. We further determined that the AU-binding protein HuR appears to selectively stabilize the longer isoform in the mammary gland. In summary, the results of the present study identify a new regulatory instance involved in the fine-tuning of Itgb1 expression during mammary gland development and function.

cSrc is necessary for epididymal development and is incorporated into sperm during epididymal transit.

Changes that occur to mammalian sperm upon epididymal transit and maturation render these cells capable of moving progressively and capacitating. Signaling events leading to mammalian sperm capacitation depend on the modulation of proteins by phosphorylation and dephosphorylation cascades. Recent experiments have demonstrated that the Src family of kinases plays an important role in the regulation of these events. However, sperm from cSrc null mice display normal tyrosine phosphorylation associated with capacitation. We report here that, despite normal phosphorylation, sperm from cSrc null mice display a severe reduction in forward motility, and are unable to fertilize in vitro. Histological analysis of seminiferous tubules in the testes, caput and corpus epididymis do not reveal obvious defects. However, the cauda epididymis is significantly smaller, and expression of key transport proteins in the epithelial cells lining this region is reduced in cSrc null mice compared to wild type littermates. Although previously, we and others have shown the presence of cSrc in mature sperm from cauda epididymis, a closer evaluation indicates that this tyrosine kinase is not present in sperm from the caput epididymis, suggesting that this protein is acquired by sperm later during epididymal maturation. Consistent with this observation, cSrc is enriched in vesicles released by the epididymal epithelium known as epididymosomes. Altogether, these observations indicate that cSrc is essential for cauda epididymal development and suggest an essential role of this kinase in epididymal sperm maturation involving cSrc extracellular trafficking.

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.

Participation of the Cl-/HCO(3)- exchangers SLC26A3 and SLC26A6, the Cl- channel CFTR, and the regulatory factor SLC9A3R1 in mouse sperm capacitation.

Sperm capacitation is required for fertilization and involves several ion permeability changes. Although Cl(-) and HCO(3)(-) are essential for capacitation, the molecular entities responsible for their transport are not fully known. During mouse sperm capacitation, the intracellular concentration of Cl(-) ([Cl(-)](i)) increases and membrane potential (Em) hyperpolarizes. As in noncapacitated sperm, the Cl(-) equilibrium potential appears to be close to the cell resting Em, opening of Cl(-) channels could not support the [Cl(-)](i) increase observed during capacitation. Alternatively, the [Cl(-)](i) increase might be mediated by anion exchangers. Among them, SLC26A3 and SLC26A6 are good candidates, since, in several cell types, they increase [Cl(-)](i) and interact with cystic fibrosis transmembrane conductance regulator (CFTR), a Cl(-) channel present in mouse and human sperm. This interaction is known to be mediated and probably regulated by the Na(+)/H(+) regulatory factor-1 (official symbol, SLC9A3R1). Our RT-PCR, immunocytochemistry, Western blot, and immunoprecipitation data indicate that SLC26A3, SLC26A6, and SLC9A3R1 are expressed in mouse sperm, localize to the midpiece, and interact between each other and with CFTR. Moreover, we present evidence indicating that CFTR and SLC26A3 are involved in the [Cl(-)](i) increase induced by db-cAMP in noncapacitated sperm. Furthermore, we found that inhibitors of SLC26A3 (Tenidap and 5099) interfere with the Em changes that accompany capacitation. Together, these findings indicate that a CFTR/SLC26A3 functional interaction is important for mouse sperm capacitation.

Replacement of calcium for strontium in hamster sperm incubation media: effect on sperm function.

Calcium (Ca(2+)) is an absolute requirement for a decisive sperm function event: the acrosome reaction (AR). Physiologically, sperm capacitation is a prerequisite for this specialized exocytosis and both events are intimately related. In an effort to separate capacitation from AR, we have been using a modified sperm incubation medium where Ca(2+) is replaced by Strontium (Sr(2+)). The aim of this report is to analyze with more detail the difference between sperm incubated with Ca(2+) or Sr(2+) in several events. We found that sperm undergo the capacitation-related changes in the chlortetracycline (CTC) pattern and tyrosine phosphorylation, and also bind to the zona pellucida (ZP) when using Sr(2+)-instead of Ca(2+)-containing media. However, the spontaneous AR typical of hamster sperm does not take place in Sr(2+)-medium, even if sperm are previously capacitated with Ca(2+). Nevertheless, Sr(2+) was able to sustain AR when cells were treated with thapsigargin or depolarized with K(+) in Na(+)-depleted medium. Considering that the absence of Na(+) increased spontaneous AR in Sr(2+)-medium, we tested whether Na(+)-transport systems could be involved in the inability of Sr(2+)-incubated sperm to undergo AR. We found that when sperm incubated in Sr(2+)-medium are treated with amiloride to inhibit epithelial Na(+) channel (ENaC), they are able to undergo spontaneous AR. The same result was obtained when analyzing AR on the ZP. On the contrary, addition of ouabain (a Na(+)/K(+)-ATPase inhibitor) or DIDS (a Na(+)/HCO3(-) co-transporter inhibitor) showed no effect. These results suggest that, differing from what happens in Ca(2+)-incubated sperm, cells incubated in Sr(2+)-modified medium would have an active ENaC.

Inhibition of Ser/Thr phosphatases induces capacitation-associated signaling in the presence of Src kinase inhibitors.

Signaling events leading to mammalian sperm capacitation rely on activation/deactivation of proteins by phosphorylation. This cascade includes soluble adenylyl cyclase, an atypical bicarbonate-stimulated adenylyl cyclase, and is mediated by protein kinase A and the subsequent stimulation of protein tyrosine phosphorylation. Recently, it has been proposed that the capacitation-associated increase in tyrosine phosphorylation is governed by Src tyrosine kinase activity. This conclusion was based mostly on the observation that Src is present in sperm and that the Src kinase family inhibitor SU6656 blocked the capacitation-associated increase in tyrosine phosphorylation. Results in the present manuscript confirmed these observations and provided evidence that these inhibitors were also able to inhibit protein kinase A phosphorylation, sperm motility, and in vitro fertilization. However, the block of capacitation-associated parameters was overcome when sperm were incubated in the presence of Ser/Thr phosphatase inhibitors such as okadaic acid and calyculin-A at concentrations reported to affect only PP2A. Altogether, these data indicate that Src is not directly involved in the observed increase in tyrosine phosphorylation. More importantly, this work presents strong evidence that capacitation is regulated by two parallel pathways. One of them requiring activation of protein kinase A and the second one involving inactivation of Ser/Thr phosphatases.

Activity of the Na,K-ATPase alpha4 isoform is important for membrane potential, intracellular Ca2+, and pH to maintain motility in rat spermatozoa.

While the function of the ubiquitous Na,K-ATPase alpha1 subunit has been well documented, the role of the sperm-specific alpha4 isoform of this ion transporter is less known. We have explored the importance of alpha4 in rat sperm physiology by taking advantage of the high sensitivity of this isoform for the inhibitor ouabain. Using concentrations that selectively block alpha4 activity, we found ouabain to reduce not only sperm total motility, but also multiple parameters of sperm movement, including progressive motility, straight line, curvilinear, and average path velocities, lateral head displacement, beat cross frequency, and linearity. According to a direct role of alpha4 in Na(+) transport, ouabain inhibition of alpha4 increased [Na(+)](i) in the male gametes. In addition, interference of alpha4 activity with ouabain produced cell membrane depolarization, diminished pH, and increased [Ca(2)(+)](i) in spermatozoa. Inhibition of alpha4 was sufficient to cause all these effects and additional blockage of alpha1, the other Na,K-ATPase alpha isoform expressed in sperm, and higher doses of ouabain did not result in further changes in the cell parameters studied. These results show that alpha4 is the Na,K-ATPase isoform primarily involved in controlling the transmembrane Na(+) gradient in sperm, and that alpha4 activity is necessary for maintaining membrane potential, [Ca(2)(+)](i), and [H(+)](i) in the cells. The high dependence of sperm motility on membrane excitability, [Ca(2)(+)](i), and acid-base balance suggests that their regulation is the mechanism by which alpha4 maintains motility of the male gametes.

Identification of proteins undergoing tyrosine phosphorylation during mouse sperm capacitation.

Mammalian sperm are not able to fertilize immediately upon ejaculation; they become fertilization-competent after undergoing changes in the female reproductive tract collectively termed capacitation. Although it has been established that capacitation is associated with an increase in tyrosine phosphorylation, little is known about the role of this event in sperm function. In this work we used a combination of two dimensional gel electrophoresis and mass spectrometry to identify proteins that undergo tyrosine phosphorylation during capacitation. Some of the identified proteins are the mouse orthologues of human sperm proteins known to undergo tyrosine phosphorylation. Among them we identified VDAC, tubulin, PDH E1 beta chain, glutathione S-transferase, NADH dehydrogenase (ubiquinone) Fe-S protein 6, acrosin binding protein precursor (sp32), proteasome subunit alpha type 6b and cytochrome b-c1 complex. In addition to previously described proteins, we identified two testis-specific aldolases as substrates for tyrosine phosphorylation. Genomic and EST analyses suggest that these aldolases are retroposons expressed exclusively in the testis, as has been reported elsewhere. Because of the importance of glycolysis for sperm function, we hypothesize that tyrosine phosphorylation of these proteins can play a role in the regulation of glycolysis during capacitation. However, neither the Km nor the Vmax of aldolase changed as a function of capacitation when its enzymatic activity was assayed in vitro, suggesting other levels of regulation for aldolase function.

Role of Hedgehog Signaling in Breast Cancer: Pathogenesis and Therapeutics.

Breast cancer (BC) is the leading cause of cancer-related mortality in women, only followed by lung cancer. Given the importance of BC in public health, it is essential to identify biomarkers to predict prognosis, predetermine drug resistance and provide treatment guidelines that include personalized targeted therapies. The Hedgehog (Hh) signaling pathway plays an essential role in embryonic development, tissue regeneration, and stem cell renewal. Several lines of evidence endorse the important role of canonical and non-canonical Hh signaling in BC. In this comprehensive review we discuss the role of Hh signaling in breast development and homeostasis and its contribution to tumorigenesis and progression of different subtypes of BC. We also examine the efficacy of agents targeting different components of the Hh pathway both in preclinical models and in clinical trials. The contribution of the Hh pathway in BC tumorigenesis and progression, its prognostic role, and its value as a therapeutic target vary according to the molecular, clinical, and histopathological characteristics of the BC patients. The evidence presented here highlights the relevance of the Hh signaling in BC, and suggest that this pathway is key for BC progression and metastasis.

Characterization of a P-Rex1 gene signature in breast cancer cells.

The Rac nucleotide Exchange Factor (Rac-GEF) P-Rex1 is highly expressed in breast cancer, specifically in the luminal subtype, and is an essential mediator of actin cytoskeleton reorganization and cell migratory responses induced by stimulation of ErbB and other tyrosine-kinase receptors. Heregulin (HRG), a growth factor highly expressed in mammary tumors, causes the activation of P-Rex1 and Rac1 in breast cancer cells via ErbB3, leading to a motile response. Since there is limited information about P-Rex1 downstream effectors, we carried out a microarray analysis to identify genes regulated by this Rac-GEF after stimulation of ErbB3 with HRG. In T-47D breast cancer cells, HRG treatment caused major changes in gene expression, including genes associated with motility, adhesion, invasiveness and metastasis. Silencing P-Rex1 expression from T-47D cells using RNAi altered the induction and repression of a subset of HRG-regulated genes, among them genes associated with extracellular matrix organization, migration, and chemotaxis. HRG induction of MMP10 (matrix metalloproteinase 10) was found to be highly sensitive both to P-Rex1 depletion and inhibition of Rac1 function by the GTPase Activating Protein (GAP) ß2-chimaerin, suggesting the dependence of the P-Rex1/Rac1 pathway for the induction of genes critical for breast cancer invasiveness. Notably, there is a significant association in the expression of P-Rex1 and MMP10 in human luminal breast cancer, and their co-expression is indicative of poor prognosis.

Rac signaling in breast cancer: a tale of GEFs and GAPs.

Rac GTPases, small G-proteins widely implicated in tumorigenesis and metastasis, transduce signals from tyrosine-kinase, G-protein-coupled receptors (GPCRs), and integrins, and control a number of essential cellular functions including motility, adhesion, and proliferation. Deregulation of Rac signaling in cancer is generally a consequence of enhanced upstream inputs from tyrosine-kinase receptors, PI3K or Guanine nucleotide Exchange Factors (GEFs), or reduced Rac inactivation by GTPase Activating Proteins (GAPs). In breast cancer cells Rac1 is a downstream effector of ErbB receptors and mediates migratory responses by ErbB1/EGFR ligands such as EGF or TGFα and ErbB3 ligands such as heregulins. Recent advances in the field led to the identification of the Rac-GEF P-Rex1 as an essential mediator of Rac1 responses in breast cancer cells. P-Rex1 is activated by the PI3K product PIP3 and Gßγ subunits, and integrates signals from ErbB receptors and GPCRs. Most notably, P-Rex1 is highly overexpressed in human luminal breast tumors, particularly those expressing ErbB2 and estrogen receptor (ER). The P-Rex1/Rac signaling pathway may represent an attractive target for breast cancer therapy.

Chloride Is essential for capacitation and for the capacitation-associated increase in tyrosine phosphorylation.

After epididymal maturation, sperm capacitation, which encompasses a complex series of molecular events, endows the sperm with the ability to fertilize an egg. This process can be mimicked in vitro in defined media, the composition of which is based on the electrolyte concentration of the oviductal fluid. It is well established that capacitation requires Na(+), HCO(3)(-), Ca(2+), and a cholesterol acceptor; however, little is known about the function of Cl(-) during this important process. To determine whether Cl(-), in addition to maintaining osmolarity, actively participates in signaling pathways that regulate capacitation, Cl(-) was replaced by either methanesulfonate or gluconate two nonpermeable anions. The absence of Cl(-) did not affect sperm viability, but capacitation-associated processes such as the increase in tyrosine phosphorylation, the increase in cAMP levels, hyperactivation, the zona pellucidae-induced acrosome reaction, and most importantly, fertilization were abolished or significantly reduced. Interestingly, the addition of cyclic AMP agonists to sperm incubated in Cl(-)-free medium rescued the increase in tyrosine phosphorylation and hyperactivation suggesting that Cl(-) acts upstream of the cAMP/protein kinase A signaling pathway. To investigate Cl(-) transport, sperm incubated in complete capacitation medium were exposed to a battery of anion transport inhibitors. Among them, bumetanide and furosemide, two blockers of Na(+)/K(+)/Cl(-) cotransporters (NKCC), inhibited all capacitation-associated events, suggesting that these transporters may mediate Cl(-) movements in sperm. Consistent with these results, Western blots using anti-NKCC1 antibodies showed the presence of this cotransporter in mature sperm.

Signalling pathways involved in sperm capacitation.

After ejaculation, mammalian sperm have not yet acquired full fertilising capacity. They will require a finite period of residence in the female reproductive tract before they become fertilisation competent. The molecular, biochemical, and physiological changes that occur to sperm while in the female tract are collectively referred to as capacitation. During capacitation, changes in membrane properties, enzyme activities, and motility render spermatozoa responsive to stimuli that induce the acrosome reaction and prepare spermatozoa for penetration of the egg investments prior to fertilisation. These changes are facilitated by the activation of cell signalling cascades in the female reproductive tract in vivo or in defined media in vitro. The purposes of this review are to consider some recent contributions towards our understanding of capacitation, to summarise open questions in this field, and to discuss future avenues of research.

Involvement of cystic fibrosis transmembrane conductance regulator in mouse sperm capacitation.

Mammalian sperm acquire fertilizing ability in the female tract during a process known as capacitation. In mouse sperm, this process is associated with increases in protein tyrosine phosphorylation, membrane potential hyperpolarization, increase in intracellular pH and Ca2+, and hyperactivated motility. The molecular mechanisms involved in these changes are not fully known. Present evidence suggests that in mouse sperm the capacitation-associated membrane hyperpolarization is regulated by a cAMP/protein kinase A-dependent pathway involving activation of inwardly rectifying K+ channels and inhibition of epithelial sodium channels (ENaCs). The cystic fibrosis transmembrane conductance regulator (CFTR) is a Cl- channel that controls the activity of several transport proteins, including ENaCs. Here we explored whether CFTR is involved in the regulation of ENaC inhibition in sperm and therefore is essential for the capacitation-associated hyperpolarization. Using reverse transcription-PCR, Western blot, and immunocytochemistry, we document the presence of CFTR in mouse and human sperm. Interestingly, the addition of a CFTR inhibitor (diphenylamine-2-carboxylic acid; 250 microM) inhibited the capacitation-associated hyperpolarization, prevented ENaC closure, and decreased the zona pellucida-induced acrosome reaction without affecting the increase in tyrosine phosphorylation. Incubation of sperm in Cl- -free medium also eliminated the capacitation-associated hyperpolarization. On the other hand, a CFTR activator (genistein; 5-10 microM) promoted hyperpolarization in mouse sperm incubated under conditions that do not support capacitation. The addition of dibutyryl cyclic AMP to noncapacitated mouse sperm elevated intracellular Cl-. These results suggest that cAMP-dependent Cl- fluxes through CFTR are involved in the regulation of ENaC during capacitation and thus contribute to the observed hyperpolarization associated with this process.

Sperm N-acetylglucosaminidase is involved in primary binding to the zona pellucida.

The glycosidase-recognizing N-acetylglucosamine terminal residue, N-acetylglucosaminidase (NAG), has been repetitively implicated in fertilization. Nevertheless, its role in the multiple steps comprising this process is a matter of debate because it has been involved in zona pellucida (ZP) binding and penetration and polyspermy block. In this study, the involvement of NAG during sperm interaction with the ZP was analysed. Soluble ZP was able to inhibit sperm NAG activity, suggesting that it can be recognized as a ligand by this enzyme. Sperm-ZP binding assays were carried out under conditions where acrosome reaction (AR) could not take place (salt-stored oocytes and a modified medium where Ca(2+) was replaced by Sr(2+)). Different NAG-specific reagents-an inhibitor (2-acetamido-2-deoxy-D-glucono-1,5-lactone), a substrate (p-nitrophenyl-N-acetylglucosaminide) and an anti-NAG antibody-were able to impair sperm binding to the ZP when present during these assays. The lactone was also able to inhibit oocyte penetration during IVF assays, although not when present after primary binding had taken place. This result was not related to the interference of lactone with AR or zona penetrability. Exogenous NAG also inhibited sperm-oocyte interaction when present during binding and IVF assays or used for oocyte pre-incubation. These results suggest the participation of NAG in sperm primary binding to the ZP.

Sodium and epithelial sodium channels participate in the regulation of the capacitation-associated hyperpolarization in mouse sperm.

In a process called capacitation, mammalian sperm gain the ability to fertilize after residing in the female tract. During capacitation the mouse sperm plasma membrane potential (E(m)) hyperpolarizes. However, the mechanisms that regulate sperm E(m) are not well understood. Here we show that sperm hyperpolarize when external Na(+) is replaced by N-methyl-glucamine. Readdition of external Na(+) restores a more depolarized E(m) by a process that is inhibited by amiloride or by its more potent derivative 5-(N-ethyl-N-isopropyl)-amiloride hydrochloride. These findings indicate that under resting conditions an electrogenic Na(+) transporter, possibly involving an amiloride sensitive Na(+) channel, may contribute to the sperm resting E(m). Consistent with this proposal, patch clamp recordings from spermatogenic cells reveal an amiloride-sensitive inward Na(+) current whose characteristics match those of the epithelial Na(+) channel (ENaC) family of epithelial Na(+) channels. Indeed, ENaC-alpha and -delta mRNAs were detected by reverse transcription-PCR in extracts of isolated elongated spermatids, and ENaC-alpha and -delta proteins were found on immunoblots of sperm membrane preparations. Immunostaining indicated localization of ENaC-alpha to the flagellar midpiece and of ENaC-delta to the acrosome. Incubations known to produce capacitation in vitro or induction of capacitation by cell-permeant cAMP analogs decreased the depolarizing response to the addition of external Na(+). These results suggest that increases in cAMP content occurring during capacitation may inhibit ENaCs to produce a required hyperpolarization of the sperm membrane.

Analysis of the participation of N-acetylglucosamine in the different steps of sperm-zona pellucida interaction in hamster.

Glycoproteins and lectin-like proteins mediate sperm-zona pellucida interaction. The present study analysed the participation of carbohydrates in the different stages of sperm interaction with the zona pellucida in hamster, by determining the effects of different monosaccharides. N-acetylglucosamine (GlcNAc, 1 mM) reduced sperm ability to bind to the zona pellucida. Surprisingly, spontaneous acrosome reaction (AR) was also inhibited by this sugar. In order to analyse the effect of GlcNAc on sperm-zona pellucida binding, independent of its effect on the AR, strontium (Sr) was used as a calcium (Ca) replacement in the sperm capacitation and co-incubation medium. Sr seemed to be able to replace Ca for sperm capacitation, at least when measured as the ability to bind to the zona pellucida, and undergo AR when Ca is provided. Moreover, sperm-zona pellucida binding could also take place in a Sr-modified medium. When binding assays were carried out in the Sr medium, GlcNAc also produced an inhibitory effect. This could be reproduced when sperm, but not oocytes, were pre-incubated with the monosaccharide. IVF assays were also carried out to analyse the participation of GlcNAc in the different steps of sperm-oocyte interaction. Taken together, the results support the involvement of the GlcNAc residues of the zona pellucida in the early steps of the interaction with sperm.