LETTER doi:10.1038/nature09767 Progesterone activates the principal Ca21 channel of human sperm Polina V. Lishko1, Inna L. Botchkina1 & Yuriy Kirichok1 Steroid hormone progesterone released by cumulus cells surround- Under normal physiological conditions, mouse and human CatSper ing the egg is a potent stimulator of human spermatozoa. It attracts channels are Ca21 selective, but pass monovalent ions (Cs1 or Na1) spermatozoa towards the egg and helps them penetrate the egg’s pro- under divalent-free conditions12,13. Because monovalent CatSper tective vestments1.ProgesteroneinducesCa21 influx into spermato- currents are significantly larger, we studied CatSper currents under zoa1–3 and triggers multiple Ca21-dependent physiological responses divalent-free conditions. The monovalent human CatSper current essential for successful fertilization, such as sperm hyperactivation, (ICatSper) was overall smaller (Fig. 1a, blue) than mouse ICatSper acrosome reaction and chemotaxis towards the egg4–8.Asanovarian (Fig. 1c, blue), especially at negative membrane potentials (inward cur- hormone, progesterone acts by regulating gene expression through a rent). The virtual absence of human ICatSper at the negative potentials well-characterized progesterone nuclear receptor9. However, the effect normally found across the sperm plasma membrane was puzzling. of progesterone upon transcriptionally silent spermatozoa remains Interestingly, addition of 500 nM progesterone to the bath solution unexplained and is believed to be mediated by a specialized, non- dramatically increased the amplitude of human monovalent ICatSper 5,10 genomic membrane progesterone receptor . The identity of this (Fig. 1a, red). Mouse monovalent ICatSper did not increase after addition non-genomic progesterone receptor and the mechanism by which it of 500 nM progesterone (Fig. 1c, red) or 10 mM progesterone (Sup- causes Ca21 entry remain fundamental unresolved questions in plementary Fig. 1). human reproduction. Here we elucidate the mechanism of the non- Similar results were obtained with divalent ICatSper. At intracellular 21 genomic action of progesterone on human spermatozoa by identify- pH 7.0, human Ba ICatSper was very small but increased dramatically ing the Ca21 channel activated by progesterone. By applying the after addition of 500 nM progesterone (Supplementary Fig. 2a). In 21 patch-clamp technique to mature human spermatozoa, we found that contrast, mouse Ba ICatSper was easily detected at intracellular pH nanomolar concentrations of progesterone dramatically potentiate 7.0 and was not affected by progesterone (Supplementary Fig. 2b). 21 CatSper, a pH-dependent Ca channel of the sperm flagellum. We After potentiation with P, the amplitude of human ICatSper remained demonstrate that human CatSper is synergistically activated by stable. The potentiation was reversible and could be reproduced again elevation of intracellular pH and extracellular progesterone. on the same cell (Supplementary Fig. 3). Another major ovarian steroid Interestingly, human CatSper can be further potentiated by prosta- hormone, oestradiol (17-b-oestradiol)9,10, did not affect human glandins, but apparently through a binding site other than that of CatSper (Supplementary Fig. 4). progesterone. Because our experimental conditions did not support We next conducted a series of experiments to confirm that the second messenger signalling, CatSper or a directly associated protein progesterone-activated current is indeed mediated by CatSper. serves as the elusive non-genomic progesterone receptor of sperm. Triturating human spermatozoa with a micropipette leads to the sepa- Given that the CatSper-associated progesterone receptor is sperm ration of a few spermatozoa into the head and the flagellum (contain- specific and structurally different from the genomic progesterone ing midpiece and principal piece) at the neck region. Patch-clamp receptor, it represents a promising target for the development of a recording from isolated flagella detected monovalent CatSper currents new class of non-hormonal contraceptives. that were strongly potentiated by progesterone and were indistinguish- CatSper is a sperm-specific Ca21 channel located in the principal able from those recorded from whole human spermatozoa (Fig. 1b, piece of the flagellum11,12. Weakly voltage-dependent, pH-sensitive compare with Fig. 1a). Mean amplitudes of currents recorded from CatSper is the only constitutively active Ca21 conductance present in whole human spermatozoa before and after stimulation with proges- mouse and human spermatozoa as recorded using the whole-cell patch- terone also closely matched those recorded from flagella (Fig. 2a), clamp technique12,13.Ca21 influx through CatSper triggers sperm suggesting that similar to CatSper, the progesterone-activated conduc- hyperactivation, a special high-amplitude asymmetrical flagellar beat tance originates from the sperm flagellum. required for penetration through viscous luminal fluids of the female Next, we discovered that human ICatSper is potently blocked by an reproductive tract and protective vestments of the egg14–16. CatSper is inhibitor of T-type voltage-gated Ca21 channels, NNC55-0396 (ref. 21) also ideally positioned to control sperm chemotaxis, because ‘chemo- (NNC, Fig. 2b, left panel), and tested whether NNC also inhibits the tactic turns’ that guide spermatozoa towards the egg depend on asym- progesterone-activated current. Indeed, 2 mM NNC completely inhi- metrical flagellar motion triggered by Ca21 influx into the flagellum4,17. bited the whole-cell current in the presence of progesterone (Fig. 2b, Furthermore, CatSper-mediated Ca21 influx into the flagellum leads to right panel), confirming that the progesterone-activated current is Ca21 elevation even in the sperm head18 (probably by causing Ca21- carried by CatSper. Interestingly, because just 2 mM NNC completely 21 dependent Ca release from the intracellular store located in the sperm suppressed human ICatSper, we estimate that the affinity of NNC to neck19) and thus can contribute to Ca21-dependent acrosome reac- CatSper is at least ten times greater than that to T-type voltage-gated 20 21 21 tion . Because CatSper may cause all three Ca -dependent responses Ca channels (half-maximum inhibitory concentration (IC50) < 7 mM triggered by progesterone (hyperactivation, chemotaxis and acrosome for T-type channels)21. NNC blocked mouse CatSper in a similar range 1 reaction), we tested the hypothesis that progesterone activates CatSper of concentrations (Supplementary Fig. 5). Furthermore, the Cs ICatSper using the recently developed method for patch-clamping completely was blocked by the same concentrations of Ca21 as the progesterone- mature ejaculated human spermatozoa13. activated current, suggesting that the progesterone-activated channels 1Department of Physiology, University of California San Francisco, UCSF Mail Code 2140, Genentech Hall Room N272F, 600 16th Street, San Francisco, California 94158, USA. 17 MARCH 2011 | VOL 471 | NATURE | 387 ©2011 Macmillan Publishers Limited. All rights reserved RESEARCH LETTER a 8), suggesting that the ligand-binding site of the CatSper-associated +80 mV 0 mV progesterone receptor is structurally different from that of the classic nuclear progesterone receptor. –80 mV CatSper activation by intercellular alkalinization can be explained at Human least partially by shifting voltage-activation (G/V) curve to more nega- whole tive, physiologically relevant potentials12. Therefore we tested whether sperm progesterone activates human CatSper through a similar mechanism. Indeed, at intracellular pH 7.4, addition of 500 nM progesterone shifted CatSper half-activation voltage (V1/2) from 185 mV to Baseline Control 152 mV (Fig. 3c, black curves, and Supplementary Fig. 9a). On the other hand, the maximal amplitude of the CatSper tail currents did not 100 pA change significantly after addition of 500 nM progesterone (control 500 nM P maximal tail current was 97 6 3% of that with progesterone (n 5 7); 0.2 s see Supplementary Fig. 9a). These results suggest that progesterone Human activates human CatSper primarily by shifting its voltage dependence b flagellum towards more physiological, negative membrane potentials, while the only single-channel conductance is not significantly affected. Because in vitro capacitation (incubation of spermatozoa under conditions similar to those found in the Fallopian tubes) was shown to increase progesterone-induced Ca21 transients in human spermato- Control Baseline zoa23, we investigated the effect of capacitation on the progesterone- induced shift in the CatSper G/V curve. Capacitation caused a 15 mV negative shift of the CatSper G/V curve, and slightly increased the 500 nM P amplitude of the negative shift induced by progesterone: addition of 500 nM progesterone to capacitated spermatozoa shifted CatSper half- 130 mM + activation voltage (V )from170 mV to 130 mV (Fig. 3c, red curves, Cs 1/2 and Supplementary Fig. 9b). Again, no change in the maximal ampli- c Mouse pH whole tude of the CatSper tail currents was observed after stimulation of 7.35 sperm capacitated spermatozoa with 500 nM progesterone: control maximal tail current was 99 6 4% of that with progesterone (n 5 6, Supplemen- + 140 mM Cs tary Fig. 9b). Thus capacitation enhances the effect of progesterone upon CatSper channel by providing additional negative shift of the CatSper G/V curve. Control Baseline The experiments with CatSper voltage activation also revealed why, in the absence