Action of steroids and plant triterpenoids on CatSper Ca2+ channels in human sperm.

The sperm-specific Ca channel CatSper (cation channel of sperm) controls the intracellular Ca concentration and, thereby, the swimming behavior of sperm. Human CatSper is activated by progesterone (1, 2), an oviductal hormone, which stimulates Ca 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 andG); moreover, we show that pregS evokes a rapid Ca 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 Ca fluorimetry, we and others showed by cross-desensitization experiments that progesterone and its derivatives (e.g., 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 ofMannowetz 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 Ca 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 Ca 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 Ca influx (Fig. 2 A and B), and progesteroneinducedmembrane 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 Ca increase (Fig. 2B). These obvious discrepancies are likely not due to differences in buffer composition, voltage protocols, or drug concentrations. Considering that various steroids coexist in male and female reproductive fluids, it is required to distinguish between agonistic and antagonistic mechanisms of pharmacological action. Future studies need to address the physiological consequences and potential medical application of the promiscuous steroid-binding site controlling CatSper.