CRISP1 as a novel CatSper regulator that modulates sperm motility and orientation during fertilization.

Ca(2+)-dependent mechanisms are critical for successful completion of fertilization. Here, we demonstrate that CRISP1, a sperm protein involved in mammalian fertilization, is also present in the female gamete and capable of modulating key sperm Ca(2+) channels. Specifically, we show that CRISP1 is expressed by the cumulus cells that surround the egg and that fertilization of cumulus-oocyte complexes from CRISP1 knockout females is impaired because of a failure of sperm to penetrate the cumulus. We provide evidence that CRISP1 stimulates sperm orientation by modulating sperm hyperactivation, a vigorous motility required for penetration of the egg vestments. Moreover, patch clamping of sperm revealed that CRISP1 has the ability to regulate CatSper, the principal sperm Ca(2+) channel involved in hyperactivation and essential for fertility. Given the critical role of Ca(2+) for sperm motility, we propose a novel CRISP1-mediated fine-tuning mechanism to regulate sperm hyperactivation and orientation for successful penetration of the cumulus during fertilization.

Electrophysiological evidence for the presence of cystic fibrosis transmembrane conductance regulator (CFTR) in mouse sperm.

Mammalian sperm must undergo a maturational process, named capacitation, in the female reproductive tract to fertilize the egg. Sperm capacitation is regulated by a cAMP/protein kinase A (PKA) pathway and involves increases in intracellular Ca(2+), pH, Cl(-), protein tyrosine phosphorylation, and in mouse and some other mammals a membrane potential hyperpolarization. The cystic fibrosis transmembrane conductance regulator (CFTR), a Cl(-) channel modulated by cAMP/PKA and ATP, was detected in mammalian sperm and proposed to modulate capacitation. Our whole-cell patch-clamp recordings from testicular mouse sperm now reveal a Cl(-) selective component to membrane current that is ATP-dependent, stimulated by cAMP, cGMP, and genistein (a CFTR agonist, at low concentrations), and inhibited by DPC and CFTR(inh) -172, two well-known CFTR antagonists. Furthermore, the Cl(-) current component activated by cAMP and inhibited by CFTR(inh) -172 is absent in recordings on testicular sperm from mice possessing the CFTR ΔF508 loss-of-function mutation, indicating that CFTR is responsible for this component. A Cl(-) selective like current component displaying CFTR characteristics was also found in wild type epididymal sperm bearing the cytoplasmatic droplet. Capacitated sperm treated with CFTR(inh) -172 undergo a shape change, suggesting that CFTR is involved in cell volume regulation. These findings indicate that functional CFTR channels are present in mouse sperm and their biophysical properties are consistent with their proposed participation in capacitation.

Cysteine-rich secretory protein 4 is an inhibitor of transient receptor potential M8 with a role in establishing sperm function.

The cysteine-rich secretory proteins (CRISPs) are a group of four proteins in the mouse that are expressed abundantly in the male reproductive tract, and to a lesser extent in other tissues. Analysis of reptile CRISPs and mouse CRISP2 has shown that CRISPs can regulate cellular homeostasis via ion channels. With the exception of the ability of CRISP2 to regulate ryanodine receptors, the in vivo targets of mammalian CRISPs function are unknown. In this study, we have characterized the ion channel regulatory activity of epididymal CRISP4 using electrophysiology, cell assays, and mouse models. Through patch-clamping of testicular sperm, the CRISP4 CRISP domain was shown to inhibit the transient receptor potential (TRP) ion channel TRPM8. These data were confirmed using a stably transfected CHO cell line. TRPM8 is a major cold receptor in the body, but is found in other tissues, including the testis and on the tail and head of mouse and human sperm. Functional assays using sperm from wild-type mice showed that TRPM8 activation significantly reduced the number of sperm undergoing the progesterone-induced acrosome reaction following capacitation, and that this response was reversed by the coaddition of CRISP4. In accordance, sperm from Crisp4 null mice had a compromised ability to undergo to the progesterone-induced acrosome reaction. Collectively, these data identify CRISP4 as an endogenous regulator of TRPM8 with a role in normal sperm function.

TRPM8 in mouse sperm detects temperature changes and may influence the acrosome reaction.

Changes in the concentration of intracellular Ca(2+) ([Ca(2+) ]i) trigger and/or regulate principal sperm functions during fertilization, such as motility, capacitation, and the acrosome reaction (AR). Members of the large TRP channel family participate in a variety of Ca(2+) -dependent cell signaling processes. The eight TRPM channel members constitute one of the seven groups belonging to this family. Here we document using RT-PCR experiments the presence of Trpm2, 4, 7, and 8 in mouse spermatogenic cells. Trpm8 transcription is up-regulated after day 30. The localization of TRPM8 protein in mouse sperm was confirmed by immunocytochemistry and Western blots. Patch clamp recordings in testicular mouse sperm revealed TRPM8 agonist (menthol and icilin) activated currents sensitive to TRPM8 inhibitors N-(4-t-Butylphenyl)-4-(3-Chloropyridin-2-yl)tetrahydropyrazine-1(2H)-carboxamide (BCTC) and capsazepine. These findings are consistent with the presence of functional TRPM8 in mouse sperm. Furthermore, menthol induced a [Ca(2+) ]i increase and the AR in these cells, that were inhibited by capsazepine (20 µM) and BCTC (1.6 µM). Notably, the progesterone and zona pellucida-induced AR was significantly (>40%) inhibited by BCTC and capsazepine, suggesting the possible participation of TRPM8 channels in this reaction. TRPM family members present in sperm could be involved in other important signaling events, such as thermotaxis, chemotaxis, and mechanosensory transduction.

The SLO3 sperm-specific potassium channel plays a vital role in male fertility.

Here we show a unique example of male infertility conferred by a gene knockout of the sperm-specific, pH-dependent SLO3 potassium channel. In striking contrast to wild-type sperm which undergo membrane hyperpolarization during capacitation, we found that SLO3 mutant sperm undergo membrane depolarization. Several defects in SLO3 mutant sperm are evident under capacitating conditions, including impaired motility, a bent "hairpin" shape, and failure to undergo the acrosome reaction (AR). The failure of AR is rescued by valinomycin which hyperpolarizes mutant sperm. Thus SLO3 is the principal potassium channel responsible for capacitation-induced hyperpolarization, and membrane hyperpolarization is crucial to the AR.

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.