Action of zona pellucida glycoproteins in mouse and human sperm

For fertilization, sperm have to penetrate the oocyte’s vestment, the zona pellucida (ZP). In mouse and human, the ZP consists of three or four different ZP glycoproteins, respectively. Binding of sperm to ZP glycoproteins evokes sperm hyperactivation and acrosome reaction, which enables sperm to penetrate the ZP. However, the ZP-induced signaling pathways underlying these behavioral responses are ill-defined. Therefore, in my thesis I investigated ZP signaling in mouse and human sperm. In both species, mixing with ZP glycoproteins evoked rapid changes in intracellular pH (pHi) and intracellular Ca2+ concentration ([Ca2+]i). I was able to confirm that the sperm-specific Ca2+ channel CatSper mediates the ZP-evoked Ca2+ influx in mouse and human sperm. However, my experiments demonstrate that the molecular mechanism underlying CatSper activation by ZPs are distinctively different in mouse and human. In human sperm, CatSper activation does not require pHi alkalization. Here, human ZP glycoproteins directly activate CatSper. However, in mouse sperm, the alkalization is essential for the ZP-evoked Ca2+ influx via CatSper. Moreover, my experiments reveal that ZP-induced alkalization is mediated by different proteins in mouse and human. In mouse sperm, ZP-evoked alkalization requires extracellular Na+; however, the prominent candidate to control ZP-evoked alkalization, the sperm-specific Na+/H+ exchanger, is not fulfilling this function. My results rather demonstrate that another protein of the Na+/H+ exchanger family, the sodium-proton antiporter 1 (NHA1), controls ZP-evoked alkalization. In NHA1 KO mice, the ZP-evoked increase in pHi and [Ca2+]i was strongly attenuated. The mechanism underlying NHA1 activation remains, however, elusive. ZP-evoked alkalization was suppressed under depolarized membrane potentials, indicating that a polarized membrane potential is crucial for NHA1 activation. In human sperm, a polarized membrane potential was also required for ZP-evoked pHi signaling, however, ZP-evoked alkalization is, in contrast to mouse sperm, not mediated by a Na+/H+ exchange. My work provides important new insights into the molecular mechanism underlying the action of ZP glycoproteins in mouse and human sperm and reveals fundamental differences between the two species, questioning the mouse as appropriate model system to study fertilization in human

Capacitation induces changes in metabolic pathways supporting motility of epididymal and ejaculated sperm

Mammalian sperm require sufficient energy to support motility and capacitation for successful fertilization. Previous studies cataloging the changes to metabolism in sperm explored ejaculated human sperm or dormant mouse sperm surgically extracted from the cauda epididymis. Due to the differences in methods of collection, it remains unclear whether any observed differences between mouse and human sperm represent species differences or reflect the distinct maturation states of the sperm under study. Here we compare the metabolic changes during capacitation of epididymal versus ejaculated mouse sperm and relate these changes to ejaculated human sperm. Using extracellular flux analysis and targeted metabolic profiling, we show that capacitation-induced changes lead to increased flux through both glycolysis and oxidative phosphorylation in mouse and human sperm. Ejaculation leads to greater flexibility in the ability to use different carbon sources. While epididymal sperm are dependent upon glucose, ejaculated mouse and human sperm gain the ability to also leverage non-glycolytic energy sources such as pyruvate and citrate

Mouse sperm energy restriction and recovery (SER) revealed novel metabolic pathways

Mammalian sperm must undergo capacitation to become fertilization-competent. While working on mice, we recently developed a new methodology for treating sperm in vitro, which results in higher rates of fertilization and embryo development after in vitro fertilization. Sperm incubated in media devoid of nutrients lose motility, although they remain viable. Upon re-adding energy substrates, sperm resume motility and become capacitated with improved functionality. Here, we explore how sperm energy restriction and recovery (SER) treatment affects sperm metabolism and capacitation-associated signaling. Using extracellular flux analysis and metabolite profiling and tracing via nuclear magnetic resonance (NMR) and mass spectrometry (MS), we found that the levels of many metabolites were altered during the starvation phase of SER. Of particular interest, two metabolites, AMP and L-carnitine, were significantly increased in energy-restricted sperm. Upon re-addition of glucose and initiation of capacitation, most metabolite levels recovered and closely mimic the levels observed in capacitating sperm that have not undergone starvation. In both control and SER-treated sperm, incubation under capacitating conditions upregulated glycolysis and oxidative phosphorylation. However, ATP levels were diminished, presumably reflecting the increased energy consumption during capacitation. Flux data following the fate of 13C glucose indicate that, similar to other cells with high glucose consumption rates, pyruvate is converted into 13C-lactate and, with lower efficiency, into 13C-acetate, which are then released into the incubation media. Furthermore, our metabolic flux data show that exogenously supplied glucose is converted into citrate, providing evidence that in sperm cells, as in somatic cells, glycolytic products can be converted into Krebs cycle metabolites

Optimization of lead compounds into on-demand, non-hormonal contraceptives: leveraging a public-private drug discovery institute collaboration

Efforts to develop new male or female non-hormonal, orally available contraceptives assume that to be effective and safe, targets must be (1) essential for fertility; (2) amenable to targeting by small-molecule inhibitors; and (3) restricted to the germline. In this perspective, we question the third assumption and propose that despite its wide expression, soluble adenylyl cyclase (sAC: ADCY10), which is essential for male fertility, is a valid target. We hypothesize that an acute-acting sAC inhibitor may provide orally available, on-demand, non-hormonal contraception for men without adverse, mechanism-based effects. To test this concept, we describe a collaboration between academia and the unique capabilities of a public-private drug discovery institut

Targeted inactivation of the mouse epididymal beta-defensin 41 alters sperm flagellar beat pattern and zona pellucida binding

During epididymal maturation, sperm acquire the ability to swim progressively by interacting with proteins secreted by the epididymal epithelium. Beta-defensin proteins, expressed in the epididymis, continue to regulate sperm motility during capacitation and hyperactivation in the female reproductive tract. We characterized the mouse beta-defensin 41 (DEFB41), by generating a mouse model with iCre recombinase inserted into the first exon of the gene. The homozygous Defb41(iCre/iCre) knock-in mice lacked Defb41 expression and displayed iCre recombinase activity in the principal cells of the proximal epididymis. Heterozygous Defb41(iCre/+) mice can be used to generate epididymis specific conditional knock-out mouse models. Homozygous Defb41(iCre/iCre) sperm displayed a defect in sperm motility with the flagella primarily bending in the pro-hook conformation while capacitated wild-type sperm more often displayed the anti-hook conformation. This led to a reduced straight line motility of Defb41(iCre/liCre) sperm and weaker binding to the oocyte. Thus, DEFB41 is required for proper sperm maturation. (C) 2016 Elsevier Ireland Ltd. All rights reserved.Peer reviewe

Soluble adenylyl cyclase inhibition prevents human sperm functions essential for fertilization

Soluble adenylyl cyclase (sAC: ADCY10) has been genetically confirmed to be essential for male fertility in mice and humans. In mice, ex vivo studies of dormant, caudal epididymal sperm demonstrated that sAC is required for initiating capacitation and activating motility. We now use an improved sAC inhibitor, TDI-10229, for a comprehensive analysis of sAC function in mouse and human sperm. In contrast to caudal epididymal mouse sperm, human sperm are collected post-ejaculation, after sAC activity has already been stimulated. In addition to preventing the capacitation-induced stimulation of sAC and protein kinase A activities, tyrosine phosphorylation, alkaliniza- tion, beat frequency and acrosome reaction in dormant mouse sperm, sAC inhibitors interrupt each of these capacitation-induced changes in ejaculated human sperm. Furthermore, we show for the first time that sAC is required during acrosomal exocytosis in mouse and hu- man sperm. These data define sAC inhibitors as candidates for non-hormonal, on-demand contraceptives suitable for delivery via intravagi- nal devices in women.Fil: Balbach, Melanie. Cornell University; Estados UnidosFil: Ghanem, Lubna. Cornell University; Estados UnidosFil: Rhossetti, Thomas. Cornell University; Estados UnidosFil: Kaur, Navpreet. Cornell University; Estados UnidosFil: Ritagliati, Carla. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; ArgentinaFil: Ferreira, Jacob. Cornell University; Estados UnidosFil: Krapf, Dario. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; ArgentinaFil: Puga Molina, Lis del Carmen. Washington University in St. Louis; Estados UnidosFil: Santi, Celia. Washington University in St. Louis; Estados UnidosFil: Hansen, Jan. Universitat Bonn; AlemaniaFil: Wachten, Dagmar. Universitat Bonn; AlemaniaFil: Fushimi, Makoto. Cornell University; Estados UnidosFil: Meinke, Peter. Cornell University; Estados UnidosFil: Buck, Jochen. Cornell University; Estados UnidosFil: Levin, Lonny. Cornell University; Estados Unido

A novel cross-species inhibitor to study the function of CatSper Ca 2 + channels in sperm

Background and PurposeSperm 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 ApproachWe 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 ResultsRU1968 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 ImplicationsWe propose RU1968 as a novel tool to elucidate the function of CatSper channels in sperm across species