Differential role of bovine serum albumin and HCO3- in the regulation of GSK3 alpha during mouse sperm capacitation.

To become fertile, mammalian sperm are required to undergo capacitation in the female tract or in vitro in defined media containing ions (e.g. HCO3 -, Ca2+, Na+, and Cl-), energy sources (e.g. glucose, pyruvate) and serum albumin (e.g. bovine serum albumin (BSA)). These different molecules initiate sequential and concomitant signaling pathways, leading to capacitation. Physiologically, capacitation induces changes in the sperm motility pattern (e.g. hyperactivation) and prepares sperm for the acrosomal reaction (AR), two events required for fertilization. Molecularly, HCO3 - activates the atypical adenylyl cyclase Adcy10 (aka sAC), increasing cAMP and downstream cAMP-dependent pathways. BSA, on the other hand, induces sperm cholesterol release as well as other signaling pathways. How these signaling events, occurring in different sperm compartments and with different kinetics, coordinate among themselves is not well established. Regarding the AR, recent work has proposed a role for glycogen synthase kinases (GSK3α and GSK3ß). GSK3α and GSK3ß are inactivated by phosphorylation of residues Ser21 and Ser9, respectively, in their N-terminal domain. Here, we present evidence that GSK3α (but not GSK3ß) is present in the anterior head and that it is regulated during capacitation. Interestingly, BSA and HCO3 - regulate GSK3α in opposite directions. While BSA induces a fast GSK3α Ser21 phosphorylation, HCO3 - and cAMP-dependent pathways dephosphorylate this residue. We also show that the HCO3--induced Ser21 dephosphorylation is mediated by hyperpolarization of the sperm plasma membrane potential (Em) and by intracellular pH alkalinization. Previous reports indicate that GSK3 kinases mediate the progesterone-induced AR. Here, we show that GSK3 inhibition also blocks the Ca2+ ionophore ionomycin-induced AR, suggesting a role for GSK3 kinases downstream of the increase in intracellular Ca2+ needed for this exocytotic event. Altogether, our data indicate a temporal and biphasic GSK3α regulation with opposite actions of BSA and HCO3 -. Our results also suggest that this regulation is needed to orchestrate the AR during sperm capacitation.

Discovery and Characterization of Multiple Classes of Human CatSper Blockers.

The cation channel of sperm (CatSper) is a validated target for nonhormonal male contraception, but it lacks selective blockers, hindering studies to establish its role in both motility and capacitation. Via an innovative calcium uptake assay utilizing human sperm we discovered novel inhibitors of CatSper function from a high-throughput screening campaign of 72,000 compounds. Preliminary SAR was established for seven hit series. HTS hits or their more potent analogs blocked potassium-induced depolarization and noncompetitively inhibited progesterone-induced CatSper activation. CatSper channel blockade was confirmed by patch clamp electrophysiology and these compounds inhibited progesterone- and prostaglandin E1-induced hyperactivated sperm motility. One of the hit compounds is a potent CatSper inhibitor with high selectivity for CatSper over hCav1.2, hNav1.5, moderate selectivity over hSlo3 and hERG, and low cytotoxicity and is therefore the most promising inhibitor identified in this study. These new CatSper blockers serve as useful starting points for chemical probe development and drug discovery efforts.

Progesterone and estrogen regulate NALCN expression in human myometrial smooth muscle cells.

During pregnancy, the uterus transitions from a quiescent state to an excitable, highly contractile state to deliver the fetus. Two important contributors essential for this transition are hormones and ion channels, both of which modulate myometrial smooth muscle cell (MSMC) excitability. Recently, the sodium (Na+) leak channel, nonselective (NALCN), was shown to contribute to a Na+ leak current in human MSMCs, and mice lacking NALCN in the uterus had dysfunctional labor. Microarray data suggested that the proquiescent hormone progesterone (P4) and the procontractile hormone estrogen (E2) regulated this channel. Here, we sought to determine whether P4 and E2 directly regulate NALCN. In human MSMCs, we found that NALCN mRNA expression decreased by 2.3-fold in the presence of E2 and increased by 5.6-fold in the presence of P4. Similarly, E2 treatment decreased, and P4 treatment restored NALCN protein expression. Additionally, E2 significantly inhibited, and P4 significantly enhanced an NALCN-dependent leak current in MSMCs. Finally, we identified estrogen response and progesterone response elements (EREs and PREs) in the NALCN promoter. With the use of luciferase assays, we showed that the PREs, but not the ERE, contributed to regulation of NALCN expression. Our findings reveal a new mechanism by which NALCN is regulated in the myometrium and suggest a novel role for NALCN in pregnancy.

CatSper channels are regulated by protein kinase A.

Mammalian sperm must undergo capacitation as a preparation for entering into hyperactivated motility, undergoing the acrosome reaction, and acquiring fertilizing ability. One of the initial capacitation events occurs when sperm encounter an elevated HCO3- concentration. This anion activates the atypical adenylyl cyclase Adcy10, increases intracellular cAMP, and stimulates protein kinase A (PKA). Moreover, an increase in intracellular Ca2+ concentration ([Ca2+] i ) is essential for sperm capacitation. Although a cross-talk between cAMP-dependent pathways and Ca2+ clearly plays an essential role in sperm capacitation, the connection between these signaling events is incompletely understood. Here, using three different approaches, we found that CatSper, the main sperm Ca2+ channel characterized to date, is up-regulated by a cAMP-dependent activation of PKA in mouse sperm. First, HCO3- and the PKA-activating permeable compound 8-Br-cAMP induced an increase in [Ca2+] i , which was blocked by the PKA peptide inhibitor PKI, and H89, another PKA inhibitor, also abrogated the 8-Br-cAMP response. Second, HCO3- increased the membrane depolarization induced upon divalent cation removal by promoting influx of monovalent cations through CatSper channels, which was inhibited by PKI, H89, and the CatSper blocker HC-056456. Third, electrophysiological patch clamp, whole-cell recordings revealed that CatSper activity is up-regulated by HCO3- and by direct cAMP injection through the patch-clamp pipette. The activation by HCO3- and cAMP was also blocked by PKI, H89, Rp-cAMPS, and HC-056456, and electrophysiological recordings in sperm from CatSper-KO mice confirmed CatSper's role in these activation modes. Our results strongly suggest that PKA-dependent phosphorylation regulates [Ca2+] i homeostasis by activating CatSper channel complexes.

CFTR/ENaC-dependent regulation of membrane potential during human sperm capacitation is initiated by bicarbonate uptake through NBC.

To fertilize an egg, sperm must reside in the female reproductive tract to undergo several maturational changes that are collectively referred to as capacitation. From a molecular point of view, the HCO3--dependent activation of the atypical soluble adenylyl cyclase (ADCY10) is one of the first events that occurs during capacitation and leads to the subsequent cAMP-dependent activation of protein kinase A (PKA). Capacitation is also accompanied by hyperpolarization of the sperm plasma membrane. We previously reported that PKA activation is necessary for CFTR (cystic fibrosis transmembrane conductance regulator channel) activity and for the modulation of membrane potential (Em). However, the main HCO3- transporters involved in the initial transport and the PKA-dependent Em changes are not well known nor characterized. Here, we analyzed how the activity of CFTR regulates Em during capacitation and examined its relationship with an electrogenic Na+/HCO3- cotransporter (NBC) and epithelial Na+ channels (ENaCs). We observed that inhibition of both CFTR and NBC decreased HCO3- influx, resulting in lower PKA activity, and that events downstream of the cAMP activation of PKA are essential for the regulation of Em. Addition of a permeable cAMP analog partially rescued the inhibitory effects caused by these inhibitors. HCO3- also produced a rapid membrane hyperpolarization mediated by ENaC channels, which contribute to the regulation of Em during capacitation. Altogether, we demonstrate for the first time, that NBC cotransporters and ENaC channels are essential in the CFTR-dependent activation of the cAMP/PKA signaling pathway and Em regulation during human sperm capacitation.

Src Kinase Is the Connecting Player between Protein Kinase A (PKA) Activation and Hyperpolarization through SLO3 Potassium Channel Regulation in Mouse Sperm.

Plasma membrane hyperpolarization is crucial for mammalian sperm to acquire acrosomal responsiveness during capacitation. Among the signaling events leading to mammalian sperm capacitation, the immediate activation of protein kinase A plays a pivotal role, promoting the subsequent stimulation of protein tyrosine phosphorylation that associates with fertilizing capacity. We have shown previously that mice deficient in the tyrosine kinase cSrc are infertile and exhibit improper cauda epididymis development. It is therefore not clear whether lack of sperm functionality is due to problems in epididymal maturation or to the absence of cSrc in sperm. To further address this problem, we investigated the kinetics of cSrc activation using anti-Tyr(P)-416-cSrc antibodies that only recognize active cSrc. Our results provide evidence that cSrc is activated downstream of PKA and that inhibition of its activity blocks the capacitation-induced hyperpolarization of the sperm plasma membrane without blocking the increase in tyrosine phosphorylation that accompanies capacitation. In addition, we show that cSrc inhibition also blocks the agonist-induced acrosome reaction and that this inhibition is overcome by pharmacological hyperpolarization. Considering that capacitation-induced hyperpolarization is mediated by SLO3, we evaluated the action of cSrc inhibitors on the heterologously expressed SLO3 channel. Our results indicate that, similar to SLO1 K(+) channels, cSrc blockers significantly decreased SLO3-mediated currents. Together, these results are consistent with findings showing that hyperpolarization of the sperm plasma membrane is necessary and sufficient to prepare the sperm for the acrosome reaction and suggest that changes in sperm membrane potential are mediated by cSrc activation.

Flow cytometry analysis reveals that only a subpopulation of mouse sperm undergoes hyperpolarization during capacitation.

To gain fertilizing capacity, mammalian sperm should reside in the female tract for a period of time. The physiological changes that render the sperm able to fertilize are known as capacitation. Capacitation is associated with an increase in intracellular pH, an increase in intracellular calcium, and phosphorylation of different proteins. This process is also accompanied by the hyperpolarization of the sperm plasma membrane potential (Em). In the present work, we used flow cytometry to analyze changes in sperm Em during capacitation in individual cells. Our results indicate that a subpopulation of hyperpolarized mouse sperm can be clearly distinguished by sperm flow cytometry analysis. Using sperm bearing green fluorescent protein in their acrosomes, we found that this hyperpolarized subpopulation is composed of sperm with intact acrosomes. In addition, we show that the capacitation-associated hyperpolarization is blocked by high extracellular K(+), by PKA inhibitors, and by SLO3 inhibitors in CD1 mouse sperm, and undetectable in Slo3 knockout mouse sperm. On the other hand, in sperm incubated in conditions that do not support capacitation, sperm membrane hyperpolarization can be induced by amiloride, high extracellular NaHCO3, and cAMP agonists. Altogether, our observations are consistent with a model in which sperm Em hyperpolarization is downstream of a cAMP-dependent pathway and is mediated by the activation of SLO3 K(+) channels.

Ion permeabilities in mouse sperm reveal an external trigger for SLO3-dependent hyperpolarization.

Unlike most cells of the body which function in an ionic environment controlled within narrow limits, spermatozoa must function in a less controlled external environment. In order to better understand how sperm control their membrane potential in different ionic conditions, we measured mouse sperm membrane potentials under a variety of conditions and at different external K(+) concentrations, both before and after capacitation. Experiments were undertaken using both wild-type, and mutant mouse sperm from the knock-out strain of the sperm-specific, pH-sensitive, SLO3 K(+) channel. Membrane voltage data were fit to the Goldman-Hodgkin-Katz equation. Our study revealed a significant membrane permeability to both K(+) and Cl(-) before capacitation, as well as Na(+). The permeability to both K(+) and Cl(-) has the effect of preventing large changes in membrane potential when the extracellular concentration of either ion is changed. Such a mechanism may protect against undesired shifts in membrane potential in changing ionic environments. We found that a significant portion of resting membrane potassium permeability in wild-type sperm was contributed by SLO3 K(+) channels. We also found that further activation of SLO3 channels was the essential mechanism producing membrane hyperpolarization under two separate conditions, 1) elevation of external pH prior to capacitation and 2) capacitating conditions. Both conditions produced a significant membrane hyperpolarization in wild-type which was absent in SLO3 mutant sperm. Hyperpolarization in both conditions may result from activation of SLO3 channels by raising intracellular pH; however, demonstrating that SLO3-dependent hyperpolarization is achieved by an alkaline environment alone shows that SLO3 channel activation might occur independently of other events associated with capacitation. For example sperm may undergo stages of membrane hyperpolarization when reaching alkaline regions of the female genital tract. Significantly, other events associated with sperm capacitation, occur in SLO3 mutant sperm and thus proceed independently of hyperpolarization.

Bovine and mouse SLO3 K+ channels: evolutionary divergence points to an RCK1 region of critical function.

The slo3 gene encodes a K(+) channel found only in mammalian testis. This is in contrast to slo1, which is expressed in many tissues. Genes pertaining to male reproduction, especially those involved in sperm production, evolve morphologically and functionally much faster than their nonsexual counterparts. A comparison of SLO3 channel amino acid sequences from several species revealed a high degree of structural divergence relative to their SLO1 channel paralogues. To reveal any biophysical differences accompanying this rapid structural divergence, we analyzed the functional properties of SLO3 channels from two species, bovine and mouse. We observed several functional differences including voltage range of activation, kinetics, and pH sensitivity. Although SLO3 channel proteins from these two species lack conservation in many structural regions, we found that the first two of these three functional differences map to the same loop structure in their RCK1 (regulator of K(+) conductance 1) domain, which links the intermediate RCK1 subdomain to the C-terminal subdomain. We found that small structural changes in this region produce major changes in the voltage range of activation and kinetics. This rapidly evolving loop peptide shows the greatest length and sequence polymorphisms within RCK1 domains from many different species. In SLO3 channels this region may permit evolutionary changes that tune the gating properties in different species.

Mouse sperm K+ currents stimulated by pH and cAMP possibly coded by Slo3 channels.

Slo3 channels belong to the high conductance Slo K+ channel family. They are activated by voltage and intracellular alkalinization, and have a K+/Na+ permeability ratio (PK/PNa) of only approximately 5. Slo3 channels have only been found in mammalian sperm. Here we show that Slo3 channels expressed in Xenopus oocytes are also stimulated by elevated cAMP levels through PKA dependent phosphorylation. Capacitation, a maturational process required by mammalian sperm to enable them to fertilize eggs, involves intracellular alkalinization and an increase in cAMP. Our mouse sperm patch clamp recordings have revealed a K+ current that is time and voltage dependent, is activated by intracellular alkalinization, has a PK/PNa > or = 5, is weakly blocked by TEA and is very sensitive to Ba2+. This current is also stimulated by cAMP. All of these properties match those displayed by heterologously expressed Slo3 channels, suggesting that the native current we observe in sperm is indeed carried by Slo3 channels.

Critical residues of the Caenorhabditis elegans unc-2 voltage-gated calcium channel that affect behavioral and physiological properties.

The Caenorhabditis elegans unc-2 gene encodes a voltage-gated calcium channel alpha1 subunit structurally related to mammalian dihydropyridine-insensitive high-threshold channels. In the present paper we describe the characterization of seven alleles of unc-2. Using an unc-2 promoter-tagged green fluorescent protein construct, we show that unc-2 is primarily expressed in motor neurons, several subsets of sensory neurons, and the HSN and VC neurons that control egg laying. Examination of behavioral phenotypes, including defecation, thrashing, and sensitivities to aldicarb and nicotine suggests that UNC-2 acts presynaptically to mediate both cholinergic and GABAergic neurotransmission. Sequence analysis of the unc-2 alleles shows that e55, ra605, ra606, ra609, and ra610 all are predicted to prematurely terminate and greatly reduce or eliminate unc-2 function. In contrast, the ra612 and ra614 alleles are missense mutations resulting in the substitution of highly conserved residues in the C terminus and the domain IVS4-IVS5 linker, respectively. Heterologous expression of a rat brain P/Q-type channel containing the ra612 mutation shows that the glycine to arginine substitution affects a variety of channel characteristics, including the voltage dependence of activation, steady-state inactivation, as well as channel kinetics. Overall, our findings suggest that UNC-2 plays a pivotal role in mediating a number of physiological processes in the nematode and also defines a number of critical residues important for calcium channel function in vivo.