The calcium channel of sperm (CatSper) is essential for sperm hyperactivated motility and fertility. The steroid hormone progesterone activates CatSper of human sperm via binding to the serine hydrolase ABHD2. However, steroid specificity of ABHD2 has not been evaluated. Here, we explored whether steroid hormones to which human spermatozoa are exposed in the male and female genital tract influence CatSper activation via modulation of ABHD2. The results show that testosterone, estrogen, and hydrocortisone did not alter basal CatSper currents, whereas the neurosteroid pregnenolone sulfate exerted similar effects as progesterone, likely binding to the same site. However, physiological concentrations of testosterone and hydrocortisone inhibited CatSper activation by progesterone. Additionally, testosterone antagonized the effect of pregnenolone sulfate. We have also explored whether steroid-like molecules, such as the plant triterpenoids pristimerin and lupeol, affect sperm fertility. Interestingly, both compounds competed with progesterone and pregnenolone sulfate and significantly reduced CatSper activation by either steroid. Furthermore, pristimerin and lupeol considerably diminished hyperactivation of capacitated spermatozoa. These results indicate that (i) pregnenolone sulfate together with progesterone are the main steroids that activate CatSper and (ii) pristimerin and lupeol can act as contraceptive compounds by averting sperm hyperactivation, thus preventing fertilization.
Our results demonstrate that testosterone, estrogen, and hydrocortisone alone did not activate CatSper but that they reduce or prevent CatSper activation by progesterone (P4). In the case of testosterone and hydrocortisone, the effects were the strongest with a significant inhibition of P4-mediated CatSper stimulation under physiological concentrations of either steroid. One possibility for this effect could be that testosterone binds with a much higher affinity to ABHD2 than P4, thereby preventing CatSper activation. The blood serum concentration of P4 in men is about 2 nM (22), whereas the minimum concentration needed for CatSper activation is 10 pM, with an EC50 of 7.7 nM (3). Testosterone blocked CatSper activation even in the presence of 1 µM P4 with an IC50 of 429 nM, which is within the physiological range, as testosterone concentrations reach 2 µM in the blood plasma of men (23). CatSper can also be activated by PregS, which can reach high concentrations in male reproductive tissues. However, testosterone also inhibits CatSper response to PregS by shifting its EC50 10-fold to 172 nM. Therefore, even if sperm are exposed to elevated concentrations of PregS, high concentrations of testosterone in the male genital tract prevent premature CatSper activation by P4, or even PregS. It is therefore possible that testosterone acts as an anticapacitation factor by preventing CatSper activation until it is removed in the female reproductive tract, presumably by chelation with albumin.
Resting serum E2 levels in women are about 110 pM, which peak at ovulation to concentrations of around 403 pM (24). Therefore, sperm encounter an E2-enriched milieu in the uterus and the fallopian tube. Our results show that E2 did not alter resting CatSper currents but that it also did not allow full channel activation by P4. We assume that elevated levels of E2 during ovulation render CatSper in its closed state to prevent premature calcium influx and thus sperm activation. However, the significantly weaker IC50 for E2 of 833 nM indicates that E2 acts as a much less potent P4 antagonist. Right after ovulation, E2 levels decrease, whereas P4 concentrations surge to ∼7 nM (24) in the blood. Because cumulus cells surrounding the oocyte also secrete P4 (25⇓⇓–28), sperm travel through a P4 gradient with maximal concentrations in close proximity to the egg. It is therefore possible that P4 outcompetes E2, leading to CatSper activation, as P4 is the natural E2 antagonist (29).
Various conditions, such as stress and elevated levels of glucocorticoids, particularly hydrocortisone, are known to impair male fertility (16) by either inhibiting spermatogenesis (30) or reducing sperm counts and sperm motility (31). Stress and elevated hydrocortisone levels can also affect female fertility. One study shows that in women who underwent in vitro fertilization, baseline urine cortisol levels increased from ∼230 nM to ∼500 nM (32). According to our results, hydrocortisone blocked CatSper activation by P4 with an IC50 of 153 nM. It is therefore possible that elevated levels of stress hormones in the female genital tract impair not only early stages but also late stages of sperm acquisition of their fertilizing potential, thus significantly contributing to infertility.
Another interesting candidate among the hormones tested in this study was the sulfated neurosteroid PregS, as it stimulates CatSper currents via the P4-related pathway. Although significant, the response to PregS was not as pronounced as the response to P4 and the EC50 of PregS was twofold higher than the EC50 of P4 [15 nM vs. 7 nM (3)]. Nevertheless, we show that PregS acted via an ABHD2 mechanism to activate CatSper—the same mechanism of channel activation as demonstrated for P4 (7). These findings identify PregS as the third steroid hormone to exert nongenomic actions on CatSper apart from P4 and its close analog 17-OH-P4 (3, 5, 33) and demonstrate the importance of sulfated steroids to regulate physiological processes in human sperm. Even though the concentrations of PregS in human testes are higher than those of pregnenolone (51 μg/100 g tissue) (34), it is unclear whether elevated concentrations of PregS exist within the entire testis or only in specific domains. Because testosterone inhibits the response to PregS with an IC50 of 172 nM, it will prevent CatSper activation by PregS even if sperm cells are exposed to high PregS concentrations. Interestingly, the plasma concentration of PregS in women is about 14 nM (35). This concentration matches the EC50 we determined for PregS to activate CatSper. It is therefore possible that once testosterone is removed from sperm cells within the female genital tract, both P4 and PregS can bind to ABHD2, resulting in CatSper activation. These two compounds indeed compete for the ABHD2 binding site(s), but further studies are needed to reveal whether PregS and P4 act synergistically or independently to activate CatSper.
Our earlier findings identified the acylglycerol lipase ABHD2 as the P4 binding partner (7). Therefore, we tested whether the monoacylglycerol lipase inhibitor pristimerin, a plant triterpenoid (18), can inhibit both the P4- and the PregS-mediated activation of CatSper of human sperm. Although basal CatSper currents were not affected, both P4 and PregS-mediated CatSper potentiation was significantly reduced. Lupeol, another plant triterpenoid, had similar effects on ICatSper as pristimerin. It is possible that both triterpenoids occupy the steroid binding site of ABHD2, thus preventing CatSper activation by P4 via a competitive antagonist-type mechanism. Both compounds were also able to inhibit sperm hyperactivation and slightly reduced basal motility of capacitated sperm cells, as evident from computer-assisted sperm analyses (CASAs). Interestingly, pristimerin and lupeol had no effect on sperm motility of noncapacitated cells, which indicates their low toxicity effect toward spermatozoa. These results correlate with our electrophysiological data, which showed a significant reduction of ICatSper with pristimerin + P4 and lupeol + P4, respectively. Because CatSper is indispensable for hyperactivated sperm motility (36⇓⇓⇓–40), it is evident that the reduction of sperm hyperactivation by both triterpenoids may significantly impair sperm ability to fertilize an egg.
CASA experiments also revealed that although P4 increased hyperactivated motility, PregS failed to do so. This could be due to the fact that PregS is a charged molecule, which cannot pass the plasma membrane (41). It is therefore possible that the nonpolar P4 can activate additional intracellular pathways, contributing to a more pronounced activation of CatSper. Indeed, for full sperm hyperactivation in vitro, two events must be met at the same time: (i) CatSper must be relieved from inhibition by 2-AG, and (ii) the sperm plasma membrane needs to be depolarized. Because CatSper is a voltage-dependent channel, it requires at least +30 mV for half-activation (3). The latter can be achieved via the P4-mediated inhibition of the potassium channel KSper (6), which creates membrane depolarization required for full CatSper activation. If P4 inhibits KSper from the intracellular side, which PregS fails to do, as it cannot cross plasma membrane, then P4 is able to cause a more pronounced hyperactivation.
In conclusion, our findings show that apart from P4, PregS is another steroid hormone that can activate CatSper via ABHD2 in human spermatozoa, whereas testosterone, E2, and HC may bind to ABHD2 competitively to modulate the response to P4 and PregS. In addition, we describe two plant triterpenoids that can serve as promising candidates for contraception, as they reduce the number of hyperactive spermatozoa, thus preventing sperm from reaching and fertilizing an egg.
Materials and Methods
Progesterone and pristimerin were purchased from Calbiochem (EMD Millipore). MAFP was from Cayman Chemical Company, and NNC 55–0396 was from Tocris. All other compounds were from Sigma Aldrich. Testosterone was purchased in accordance with the controlled substance protocol (CS084484), as a collaborative effort with Yuriy Kirichok (University of California, San Francisco).
Donors and Purification of Human Ejaculated Spermatozoa.
The participation of four healthy human sperm donor volunteers was approved by the Committee on Human Research at the University of California, Berkeley (protocol number 2013–06-5395). All donors provided informed consent. Freshly ejaculated semen samples were obtained by masturbation. Sperm were purified with the swim-up technique (3) using artificial human tubal fluid solution (HTF), containing (in mM) 21 Hepes, 21 lactic acid, 98 NaCl, 4.7 KCl, 3 glucose, 2 CaCl2, 0.3 KH2PO4, 0.3 sodium pyruvate, and 0.2 MgSO4, pH 7.4 (adjusted with NaOH).
All recordings were performed as described in ref. 3. Briefly, gigaohm seals between patch pipette and spermatozoa were formed at the cytoplasmic droplet in high saline (HS) solution containing (in mM) 135 NaCl, 20 Hepes, 10 lactic acid, 5 KCl, 5 glucose, 2 CaCl2, 1 MgSO4, 1 sodium pyruvate, pH 7.4 (adjusted with NaOH), and ∼320 mOsm/L. Transition into whole-cell mode was achieved by applying suction and short voltage pulses. For CatSper recordings, the divalent-free bath solution contained (in mM) 140 Cs-methanesulfonate, 40 Hepes, 1 EDTA, pH 7.4 (adjusted with CsOH), and ∼325 mOsm/L. Pipettes (10–15 MΩ) were filled with 130 mM Cs-methanesulfonate, 70 mM Hepes, 3 mM EGTA, 2 mM EDTA, 0.5 mM Tris·HCl, pH 7.4 (adjusted with CsOH), and ∼335 mOsm/L. Access resistance was 42–60 MΩ. Cells were stimulated every 5 s, and data were sampled at 2–5 kHz and filtered at 1 kHz. All experiments were performed at room temperature and currents elicited by a voltage ramp from –80 mV to 80 mV with a holding potential of 0 mV. Data were analyzed with Clampfit 9.2 and OriginPro 9.0. To build dose–response curves, data were fitted with the Hill-based equation: y = Imin + (Imax – Imin)/(1 + (x/IC50)k), where Imax is close to 100% activation, Imin is close to inactivation, and k is the Hill slope factor.
Purified human spermatozoa were capacitated for 3.5 h at 37 °C and 5% CO2 in capacitation media (HS supplemented with 15 mM NaHCO3 and 5% BSA) as reported in ref. 7. Aliquots of the cell suspension were preincubated for 15 min with 3 μM pristimerin or 3 μM lupeol before exposure to steroids (3 μM progesterone or 3 μM PregS). Sperm motility was analyzed at 37 °C with an HTM-IVOS sperm analysis system (version 12.3, Hamilton Thorne Biosciences). We pipetted 10 μL of sperm suspension into a two-chamber slide (Leja), and sperm movement of a minimum of 300 cells was recorded. Parameters measured were the four motility classes (A–D), average path velocity (VAP, μm/s), straight line velocity (VSL, μm/s), and VCL (μm/s). Measurements on a given day were performed in duplicates and defined as one experiment.
Statistical data were calculated as the mean ± SEM, and n indicates number of individual cells analyzed unless stated otherwise. Statistical significance (unpaired t test) is indicated by *P < 0.05, **P < 0.005, ***P < 0.001, and ****P < 0.0001.