Pharmacological Inactivation of CatSper Blocks Sperm Fertilizing Ability Independently of the Capacitation Status of the Cells: Implications for Non-hormonal Contraception

Abstract

Cation channel of sperm (CatSper), the main sperm-specific Ca2+ channel, plays a key role in mammalian fertilization, and it is essential for male fertility, becoming an attractive target for contraception. Based on this, in the present work, we investigated the effects of CatSper inactivation on in vitro and in vivo sperm fertilizing ability and the mechanisms underlying such effects. Exposure of cauda epididymal mouse sperm to different concentrations (1–20 μM) of the potent CatSper inhibitor HC-056456 (HC) during in vitro capacitation showed no effects on sperm viability but significantly affected Ca2+ entry into the cells, progressive motility, protein tyrosine phosphorylation, induced acrosome reaction, and hyperactivation, as well as the sperm’s ability to in vitro fertilize cumulus oocyte complexes and zona-free eggs. Whereas the presence of HC during gamete coincubation did not affect in vitro fertilization, exposure of either non-capacitating or already capacitated sperm to HC prior to gamete coincubation severely reduced fertilization, indicating that sperm function is affected by HC when the cells are incubated with the drug before sperm–egg interaction. Of note, insemination of HC-treated sperm into the uterus significantly or completely reduced the percentage of oviductal fertilized eggs showing, for the first time, the effects of a CatSper inhibitor on in vivo fertilization. These observations, together with the finding that HC affects sperm fertilizing ability independently of the sperm capacitation status, provide further insights on how CatSper regulates sperm function and represent a solid proof of concept for developing a male/female non-hormonal contraceptive based on the pharmacological blockage of CatSper activity.

Steroidal Antagonists of Progesterone- and Prostaglandin E1-Induced Activation of the Cation Channel of Sperm

“…The most studied tool compounds to explore CatSper physiology and pharmacology are the T-type CCBs mibefradil (Strunker et al, 2011) and NNC55-0396 ( Lishko et al, 2011 ). These and other reported CatSper inhibitors, including HC-056456 ( Curci et al, 2021 ), MDL12330A ( Brenker et al, 2012 ) and RU1968 ( Rennhack et al, 2018 ), also inhibit the sperm-specific K + channel Slo3 with similar potencies, although RU1968 appears to be the most selective for CatSper over Slo3 ( Rennhack et al, 2018 ). In addition, the two structurally related T-type CCBs produce an anomalous calcium influx at elevated concentrations (Strunker et al, 2011), are not selective for CatSper over Ca v 3.1, Ca v 3.2, and Ca v 3.3 T-type calcium channels ( Martin et al, 2000 ) and display cytotoxic effects in sperm ( Tamburrino et al, 2014 ), fibroblasts (unpublished observations), and peripheral blood mononuclear cells ( Lijnen et al, 1999 ).…”

“…Indeed, in certain infertility cases, sperm from otherwise healthy men failed to respond to a PROG stimulus ( Smith et al, 2013 ). This observation validated the hypothesis that compounds able to prevent PROG-induced influx of Ca 2+ by CatSper could serve as contraceptives, which is strengthened by the finding that the CatSper inhibitor HC-056456 greatly reduced both in vitro and in vivo fertilization in mice ( Curci et al, 2021 ).…”

Capacitation-Induced Mitochondrial Activity Is Required for Sperm Fertilizing Ability in Mice by Modulating Hyperactivation

“…Intrauterine insemination assays were performed as previously described ( Curci et al, 2021 ). Briefly, female mice were superovulated by an injection of eCG, followed by hCG 46 h later.…”

High-throughput screening method for discovering CatSper inhibitors using membrane depolarization caused by external calcium chelation and fluorescent cell barcoding

“…This is not observed when CatSper is absent (CatSper1 KO), in which a population of sperm with high DiSC 3 (5) fluorescence remained detectable after EGTA addition ( Figures 2A–C ). Similarly, pharmacological inhibition of CatSper using HC-056456 or RU 1968 ( Rennhack et al, 2018 ; Curci et al, 2021 ) (two well characterized CatSper inhibitors), also prevented the EGTA-induced depolarization ( Figures 2D–F ). This pharmacological approach was also tested using two different mouse strains, C57BL/6 and hybrid F1 (BALB/c female × C57BL/6 male), and no relevant differences were found ( Supplementary Figure S2 ).…”