Synthesis and Functional Characterization of Novel RU1968-Derived CatSper Inhibitors with Reduced Stereochemical Complexity.

The sperm-specific Ca2+ channel CatSper (cation channel of sperm) controls the intracellular Ca2+ concentration and, thereby, the swimming behavior of sperm from many species. The steroidal ethylenediamine RU1968 (1) represents a well-characterized, potent, and fairly selective cross-species inhibitor of CatSper. Due to its two additional centers of chirality in the amine-bearing side chain, RU1968 is a mixture of diastereomeric pairs of enantiomers and, thus, difficult to synthesize. This has hampered the use of this commercially not available inhibitor as a powerful tool for research. Here, simplifying both structure and synthesis, we introduced novel stereochemically less complex and enantiomerically pure aminomethyl RU1968 analogues lacking the C-21 CH3 moiety. Starting from (+)-estrone, a five-step synthesis was developed comprising a Wittig reaction as the key step, leading to a diastereomerically pure 17ß-configured aldehyde. Subsequent reductive amination yielded diastereomerically and enantiomerically pure amines. Compared to RU1968, the novel ethylenediamine 2d and homologous trimethylenediamine derivative 2e inhibited CatSper with similar and even twofold enhanced potency, respectively. Considering that these aminomethyl analogues are enantiomerically pure and much easier to synthesize than RU1968, we envisage their common use in future studies investigating the physiology of CatSper in sperm.

The Action of Reproductive Fluids and Contained Steroids, Prostaglandins, and Zn2+ on CatSper Ca2+ Channels in Human Sperm.

The sperm-specific Ca2+ channel CatSper registers chemical cues that assist human sperm to fertilize the egg. Prime examples are progesterone and prostaglandin E1 that activate CatSper without involving classical nuclear and G protein-coupled receptors, respectively. Here, we study the action of seminal and follicular fluid as well of the contained individual prostaglandins and steroids on the intracellular Ca2+ concentration of sperm from donors and CATSPER2-deficient patients that lack functional CatSper channels. We show that any of the reproductive steroids and prostaglandins evokes a rapid Ca2+ increase that invariably rests on Ca2+ influx via CatSper. The hormones compete for the same steroid- and prostaglandin-binding site to activate the channel, respectively. Analysis of the hormones' structure-activity relationship highlights their unique pharmacology in sperm and the chemical features determining their effective properties. Finally, we show that Zn2+ suppresses the action of steroids and prostaglandins on CatSper, which might prevent premature prostaglandin activation of CatSper in the ejaculate, aiding sperm to escape from the ejaculate into the female genital tract. Altogether, our findings reinforce that human CatSper serves as a promiscuous chemosensor that enables sperm to probe the varying hormonal microenvironment prevailing at different stages during their journey across the female genital tract.