Enrique O. Hernández-González

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 (Em) hyperpolarizes. However, the mechanisms that regulate sperm Em 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 Em 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 Em. 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-α and -δ mRNAs were detected by reverse transcription-PCR in extracts of isolated elongated spermatids, and ENaC-α and -δ proteins were found on immunoblots of sperm membrane preparations. Immunostaining indicated localization of ENaC-α to the flagellar midpiece and of ENaC-δ 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.

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 μm) 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 μm) 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.

TRPM8, a Versatile Channel in Human Sperm

Background The transient receptor potential channel (TRP) family includes more than 30 proteins; they participate in various Ca2+ dependent processes. TRPs are functionally diverse involving thermal, chemical and mechanical transducers which modulate the concentration of intracellular Ca2+ ([Ca2+]i). Ca2+ triggers and/or regulates principal sperm functions during fertilization such as motility, capacitation and the acrosome reaction. Nevertheless, the presence of the TRPM subfamily in sperm has not been explored. Principal Findings Here we document with RT-PCR, western blot and immunocitochemistry analysis the presence of TRPM8 in human sperm. We also examined the participation of this channel in sperm function using specific agonists (menthol and temperature) and antagonists (BCTC and capsazepine). Computer-aided sperm analysis revealed that menthol did not significantly alter human sperm motility. In contrast, menthol induced the acrosome reaction in human sperm. This induction was inhibited about 70% by capsazepine (20 µM) and 80% by BCTC (1.6 µM). Activation of TRPM8 either by temperature or menthol induced [Ca2+]i increases in human sperm measured by fluorescence in populations or individual sperm cells, effect that was also inhibited by capsazepine (20 µM) and BCTC (1.6 µM). However, the progesterone and ZP3-induced acrosome reaction was not inhibited by capsazepine or BCTC, suggesting that TRPM8 activation triggers this process by a different signaling pathway. Conclusions This is the first report dealing with the presence of a thermo sensitive channel (TRPM8) in human sperm. This channel could be involved in cell signaling events such as thermotaxis or chemotaxis.

Participation of the Cl−/HCO3− Exchangers SLC26A3 and SLC26A6, the Cl− Channel CFTR, and the Regulatory Factor SLC9A3R1 in Mouse Sperm Capacitation

ABSTRACT Sperm capacitation is required for fertilization and involves several ion permeability changes. Although Cl− and HCO3− 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.

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+, \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{HCO}_{3}^{-}\) \end{document}, Ca2+, 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.

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

Background: Sperm capacitation, a process associated with phosphorylation and membrane potential changes, is required for acrosome reaction and fertilization. Results: Inducing hyperpolarization in non-capacitated sperm does not result in protein tyrosine phosphorylation but allows physiologically-induced [Ca2+]i increases and acrosome reaction. Conclusion: Sperm hyperpolarization appears to be necessary and sufficient for acrosome reaction. Significance: Advancing our understanding of capacitation, the acrosome reaction and fertilization. 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 (pHi) and Ca2+ ([Ca2+]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 [Ca2+]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 [Ca2+]i. Altogether, these results suggest that sperm hyperpolarization by itself is key to enabling mice sperm to undergo the acrosome reaction.

Involvement of an F-actin skeleton on the acrosome reaction in guinea pig spermatozoa

The acrosome reaction (AR) is a regulated exocytotic process. In several cell types, an actin network situated under the plasma membrane (PM) acts as a physical barrier to prevent this exocytosis. In seeking a function for a cortical skeleton in guinea pig spermatozoa, the PM and the outer acrosomal membrane (OAM) were investigated for the presence of F-actin and spectrin, proteins generally found in cell cortical skeletons. Both membrane types were visualized in whole-mount preparations by electron microscopy. PM proteins gave positive reaction to the Na(+),K(+)-ATPase antibody and the OAM proteins did not react to the antibody. Furthermore, a Triton X-100-resistant skeleton was obtained from both membrane types. Using gold immunoelectron microscopy, F-actin was visualized in the PM and in the OAM skeletons, while spectrin was only detected in the PM skeleton. The presence of an F-actin cortical skeleton in the sperm PM suggests that F-actin may be involved in the AR. The significantly higher number of AR elicited by cytochalasin D (Cyt-D) treatment(P<0.005) and data showing a significant (P>0.03) decrease in F-actin relative concentration in capacitating spermatozoa, agree with this suggestion. Furthermore, the proposal is strengthened by the fact that stabilization of F-actin by phalloidin (Ph) significantly (P>0.01) diminished AR induced by Ca(2+) in a streptolysin O (SLO)-permeabilized sperm model.

Presence, processing, and localization of mouse ADAM15 during sperm maturation and the role of its disintegrin domain during sperm-egg binding.

Successful fertilization requires gametes to complete several stages, beginning with maturation and transport along the male and female reproductive tracts and ending with the interaction between the sperm and the egg. This last step involves sperm-egg adhesion and membrane fusion. ADAMs (disintegrin and metalloprotease domain proteins) are a family of membrane-anchored glycoproteins that are thought to play diverse roles in cell-cell adhesion through their interaction with integrins. This study analyzes the presence, location, processing, and possible role of ADAM15 in mouse sperm. The presence of ADAM15 in mouse spermatozoa was detected by Western blotting, which revealed that ADAM15 is post-translationally processed, during epididymal sperm maturation and the acrosome reaction. The 35 kDa antigen present in the acrosome-reacted sperm is the last proteolytic product of the 110/75 kDa ADAM15 found in non-capacitated sperm. This 35 kDa protein contains the disintegrin domain. By indirect immunofluorescence, ADAM15 was identified in the acrosomal region and along the flagellum of mouse spermatozoa. In acrosome-reacted sperm, ADAM15 was lost from the acrosomal region, but remained diffusely distributed throughout the head and flagellum. Furthermore, the ADAM15 disintegrin domain (RPPTDDCDLPEF) partially inhibited fusion and almost completely inhibited sperm-oolemma adhesion. In conclusion, our data indicate that ADAM15 is present in the testis and in spermatozoa from the caput, corpus, and cauda epididymis, as well as in non-capacitated and acrosome-reacted gametes. Results also indicate that ADAM15 is processed during epididymal maturation and acrosome reaction and that it may play a role during sperm-egg binding.

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.

Absence of Dp71 in mdx3cv mouse spermatozoa alters flagellar morphology and the distribution of ion channels and nNOS.

In muscle, the absence of dystrophin alters the dystrophin-associated protein complex (DAPC), which is involved in the clustering and anchoring of signaling proteins and ion and water channels. Here we show that mice spermatozoa express only dystrophin Dp71 and utrophin Up71. The purpose of this study was to explore the effect of the absence of Dp71 on the morphology and membrane distribution of members of the DAPC, ion channels and signaling proteins of spermatozoa obtained from dystrophic mutant mdx3cv mice. Our work indicates that although the absence of Dp71 results in a dramatic decrease in β-dystroglycan, it induces membrane redistribution and an increase in the total level of α-syntrophin, voltage-dependent Na+ (μ1) and K+ (Kv1.1) channels and neural nitric oxide synthase (nNOS). The short utrophin (Up71) was upregulated and redistributed in the spermatozoa of mdx3cv mice. A significant increase in abnormal flagella morphology was observed in the absence of Dp71, which was partially corrected when the plasma membrane was eliminated by detergent treatment. Our observations point to a new phenotype associated with the absence of Dp71. Abnormal flagellar structure and altered distribution of ion channels and signaling proteins may be responsible for the fertility problems of mdx3cv mice.