Molecular mechanism underlying the action of zona-pellucida glycoproteins on mouse sperm

Mammalian oocytes are enveloped by the zona pellucida (ZP), an extracellular matrix of glycoproteins. In sperm, stimulation with ZP proteins evokes a rapid Ca2+ influx via the sperm-specific, pH-sensitive Ca2+ channel CatSper. However, the physiological role and molecular mechanisms underlying ZP-dependent activation of CatSper are unknown. Here, we delineate the sequence of ZP-signaling events in mouse sperm. We show that ZP proteins evoke a rapid intracellular pH i increase that rests predominantly on Na+/H+ exchange by NHA1 and requires cAMP synthesis by the soluble adenylyl cyclase sAC as well as a sufficiently negative membrane potential set by the spem-specific K+ channel Slo3. The alkaline-activated CatSper channel translates the ZP-induced pH i increase into a Ca2+ response. Our findings reveal the molecular components underlying ZP action on mouse sperm, opening up new avenues for understanding the basic principles of sperm function and, thereby, mammalian fertilization

Synergistic activation of CatSper Ca2+ channels in human sperm by oviductal ligands and endocrine disrupting chemicals

Does the chemosensory activation of CatSper Ca2+ channels in human sperm give rise to additive, sub-additive or even synergistic actions among agonists? SUMMARY ANSWER: We show that oviductal ligands and endocrine disrupting chemicals (EDCs) activate human CatSper highly synergistically. WHAT IS KNOWN ALREADY: In human sperm, the sperm-specific CatSper channel controls the intracellular Ca2+ concentration and, thereby, several crucial stages toward fertilization. CatSper is activated by oviductal ligands and structurally diverse EDCs. The chemicals mimic the action of the physiological ligands, which might interfere with the precisely coordinated sequence of events underlying fertilization. STUDY DESIGN, SIZE, DURATION: For both oviductal ligands and EDCs, we examined in quantitative terms whether stimulation of human sperm in vitro with mixtures results in additive, sub-additive or synergistic actions. PARTICIPANTS/MATERIALS, SETTING, METHODS: We studied activation of CatSper in sperm of healthy volunteers, using kinetic Ca2+ fluorimetry and patch-clamp recordings. The combined action of progesterone and prostaglandins and of the EDCs benzylidene camphor sulfonic acid (BCSA) and α-Zearalenol was evaluated by curve-shift analysis, curvilinear isobolographic analysis and the combination-index method. MAIN RESULTS AND THE ROLE OF CHANCE: Analysis of the action of progesterone/prostaglandin and BCSA/α-Zearalenol mixtures in human sperm by fluorimetry revealed that the oviductal ligands and EDCs both evoke Ca2+ influx via CatSper in a highly synergistic fashion. Patch-clamp recordings of CatSper currents in human sperm corroborated the synergistic ligand-activation of the channel. LIMITATIONS, REASONS FOR CAUTION: This is an in vitro study. Future studies have to assess the physiological relevance in vivo. WIDER IMPLICATIONS OF THE FINDINGS: These findings indicate that the fertilization process is orchestrated by multiple oviductal CatSper agonists that act in concert to control the behavior of sperm. Moreover, our results substantiate the concerns regarding the negative impact of EDCs on male reproductive health. So far, safety thresholds like the "No Observed Adverse Effect Level (NOAEL)" or "No Observed Effect Concentration (NOEC)" are set for individual EDCs. Our finding that EDCs act synergistically in human sperm challenges the validity of this procedure. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by the German Research Foundation (SFB 645; CRU326), the Cells-in-Motion (CiM) Cluster of Excellence, Münster, (FF-2016-17), the 'Innovative Medical Research' of the University of Münster Medical School (BR121507), an EDMaRC research grant from the Kirsten and Freddy Johansen's Foundation, and the Innovation Fund Denmark (InnovationsFonden; 14-2013-4). The authors have no competing financial interests

A novel cross-species inhibitor to study the function of CatSper Ca2+ channels in sperm

BACKGROUND AND PURPOSE Sperm from many species share the sperm-specific Ca2+ channel CatSper that controls the intracellular Ca2+ concentration and, thereby, the swimming behaviour. A growing body of evidence suggests that the mechanisms controlling the activity of CatSper and its role during fertilization differ among species. A lack of suitable pharmacological tools has hampered the elucidation of the function of CatSper. Known inhibitors of CatSper exhibit considerable side effects and also inhibit Slo3, the principal K+ channel of mammalian sperm. The compound RU1968 was reported to suppress Ca2+ signaling in human sperm by an unknown mechanism. Here, we examined the action of RU1968 on CatSper in sperm from humans, mice, and sea urchins. EXPERIMENTAL APPROACH We resynthesized RU1968 and studied its action on sperm from humans, mice, and the sea urchin Arbacia punctulata by Ca2+ fluorimetry, single-cell Ca2+ imaging, electrophysiology, opto-chemistry, and motility analysis. KEY RESULTS RU1968 inhibited CatSper in sperm from invertebrates and mammals. The compound lacked toxic side effects in human sperm, did not affect mouse Slo3, and inhibited human Slo3 with about 15-fold lower potency than CatSper. Moreover, in human sperm, RU1968 mimicked CatSper dysfunction and suppressed motility responses evoked by progesterone, an oviductal steroid known to activate CatSper. Finally, RU1968 abolished CatSper-mediated chemotactic navigation in sea urchin sperm. CONCLUSION AND IMPLICATIONS We propose RU1968 as a novel tool to elucidate the function of CatSper channels in sperm across species