The sperm-specific Ca2+ channel CatSper (cation channel of sperm) controls the intracellular Ca2+ concentration and, thereby, the swimming behavior of sperm. Human CatSper is activated by progesterone (1, 2), an oviductal hormone, which stimulates Ca2+ influx and motility responses. By patch-clamp recording from human sperm, Mannowetz et al.(3) studied the action of the steroids pregnenolone sulfate (pregS), testosterone, hydrocortisone, and 17-β-estradiol (estradiol) as well as that of the plant triterpenoids pristimerin and lupeol on CatSper currents. Here, we report data that contradict most of these results. We agree with Mannowetz et al.(3) that pregS activates CatSper, and that pregS and progesterone use the same binding site. We also observe that pregS enhances CatSper currents (Fig. 1 E and G); moreover, we show that pregS evokes a rapid Ca2+ influx (Fig. 1 A, C, and D) and that human sperm (from a patient with deafness-infertility syndrome) that lack CatSper do not respond to pregS (Fig. 1B). Using Ca2+ fluorimetry, we and others showed by cross-desensitization experiments that progesterone and its derivatives (eg, 17-OH-progesterone) employ the same binding site to activate CatSper (1, 4). Similarly, sequential application of progesterone and pregS (and vice versa) leads to cross-desensitization (Fig. 1 J and K), confirming that both steroids act via the same binding site. However, we disagree with the claim of Mannowetz et al.(3) that, by contrast to pregS, the steroids testosterone, hydrocortisone, and estradiol represent true antagonists that abolish CatSper activation by progesterone but themselves do not activate CatSper. Our data lead to entirely different conclusions. Testosterone, hydrocortisone, and estradiol all enhance CatSper currents (Fig. 1 F and G) and stimulate Ca2+ influx via CatSper (Fig. 1 A and B), although with different potency (Fig. 1C) and efficacy (Fig. 1D). Furthermore, these steroids do not antagonize progesterone-induced

CatSper currents (Fig. 1 F and H), and simultaneous application of the steroids with progesterone does not antagonize progesterone-induced Ca2+ influx (Fig. 1I). Thus, testosterone, hydrocortisone, and estradiol, in fact, are agonists that activate CatSper rather than antagonists. Cross-desensitization experiments demonstrate that all these steroids use the same binding site to activate CatSper: Sequential application of progesterone and testosterone, hydrocortisone, or estradiol (and vice versa) leads to cross-desensitization (Fig. 1 L and M). Finally, Mannowetz et al.(3) claim that pristimerin and lupeol antagonize CatSper activation by progesterone. We fail to reproduce this result: Neither simultaneous application of these triterpenoids with progesterone nor preincubation affects progesteroneinduced Ca2+ influx (Fig. 2 A and B), and progesteroneinduced membrane currents were similar in the absence and presence of the triterpenoids (Fig. 2 C–E). Thus, pristimerin and lupeol do not antagonize CatSper activation by progesterone. Of note, at≥ 3 μM, pristimerin alone, but not lupeol, evoked a sizeable Ca2+ increase (Fig. 2B).