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Regulation of the Sperm Calcium Channel Catsper by Endogenous Steroids and Plant Triterpenoids

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Regulation of the Sperm Calcium Channel Catsper by Endogenous Steroids and Plant Triterpenoids Regulation of the sperm calcium channel CatSper by endogenous steroids and plant triterpenoids Nadja Mannowetza, Melissa R. Millera, and Polina V. Lishkoa,1 aDepartment of Molecular and Cell Biology, University of California, Berkeley, CA 94720 Edited by David E. Clapham, Howard Hughes Medical Institute, Boston Children’s Hospital, Boston, MA, and approved April 20, 2017 (received for review January 10, 2017) The calcium channel of sperm (CatSper) is essential for sperm CatSper in a manner similar to P4 and explore the possibility of hyperactivated motility and fertility. The steroid hormone pro- PregS binding to the same sperm receptor as progesterone. gesterone activates CatSper of human sperm via binding to the As spermatozoa travel through the male and female repro- serine hydrolase ABHD2. However, steroid specificity of ABHD2 ductive tract, they are exposed to a variety of steroid hormones, has not been evaluated. Here, we explored whether steroid such as testosterone and estrogen. The rising levels of hydro- hormones to which human spermatozoa are exposed in the male cortisone (HC) in the body as a result of stress can impact fer- and female genital tract influence CatSper activation via modula- tility (16) by interfering with spermatogenesis and/or sperm tion of ABHD2. The results show that testosterone, estrogen, and functions. Therefore, we have also explored what influence tes- hydrocortisone did not alter basal CatSper currents, whereas the tosterone, estrogen, and HC have on CatSper activation. The neurosteroid pregnenolone sulfate exerted similar effects as pro- structural precursor of all steroid hormones in animals is the gesterone, likely binding to the same site. However, physiological triterpenoid lanosterol, which is formed upon cyclization of concentrations of testosterone and hydrocortisone inhibited CatSper the linear triterpene squalene. Structurally related plant tri- activation by progesterone. Additionally, testosterone antago- terpenoids have been shown to exhibit antifertility properties in nized the effect of pregnenolone sulfate. We have also explored mice and rats. The most extensively studied plant triterpenoid is whether steroid-like molecules, such as the plant triterpenoids lupeol, which is found in mangoes, grapes, and olives. It has been pristimerin and lupeol, affect sperm fertility. Interestingly, both shown to possess antifertility properties when orally adminis- PHYSIOLOGY compounds competed with progesterone and pregnenolone sul- tered to rats, resulting in a significantly reduced number of fate and significantly reduced CatSper activation by either steroid. motile sperm (17), although the underlying mechanisms are not Furthermore, pristimerin and lupeol considerably diminished hyper- understood. A plausible explanation is that lupeol blocks CatSper, activation of capacitated spermatozoa. These results indicate that which is indispensable for sperm motility (4). Another plant tri- (i) pregnenolone sulfate together with progesterone are the main terpenoid, pristimerin, which is found in Tripterygium wilfordii (also steroids that activate CatSper and (ii) pristimerin and lupeol can act known as “Thunder God Vine”)andCelastrus regelii,actsasa as contraceptive compounds by averting sperm hyperactivation, monoacylglycerol lipase (MAGL) inhibitor. It inhibits the hydro- thus preventing fertilization. lysis of 2-arachidonoylglyceol (2-AG) to arachidonic acid (AA) and glycerol (18). In sperm, 2-AG acts as an endogenous CatSper in- CatSper | steroids | lupeol | triterpenoids | pristimerin hibitor, and 2-AG degradation is mediated by the α/β hydrolase domain-containing protein 2 (ABHD2) in a P4-dependent man- teroid hormones control fundamental organism functions ner (7). Because it is likely that pristimerin, lupeol, as well as Ssuch as development, metabolism, inflammation, ion ho- meostasis, and reproduction. According to the conventional Significance model, steroid hormones signal through a corresponding geno- mic receptor, which upon binding to a steroid hormone initiates The calcium channel of sperm—CatSper—is vital for male fer- the translocation of the hormone–receptor complex to the nu- tility. CatSper is activated by the hormone progesterone, cleus. There it acts as a transcription factor by altering gene but its pharmacological profile is not well studied. By exploring expression, and such a process can take hours (1). However, steroid selectivity of CatSper activation, we found one addi- there is another, much faster pathway that is initiated by steroid tional agonist—pregnenolone sulfate—and the two plant-derived hormone binding to its membrane receptor on the extracellular inhibitors pristimerin and lupeol. By averting sperm hyper- side of the cell. The latter signaling involves second messengers activation, both inhibitors can prevent fertilization, thus acting and signal-transduction cascades, which often result in the acti- as contraceptive agents. Additionally, by exploring CatSper reg- vation of ion channels (1). The female hormone progesterone ulation by endogenous steroids, we explain why CatSper is silent (P4) activates the principal calcium channel of sperm (CatSper) within the male reproductive tract and is only activated in close (2–5) and blocks the potassium channel KSper (6) via these proximity to the egg. Interestingly, both testosterone and hy- nongenomic pathways (7). The modulation of ion channel drocortisone antagonize the action of progesterone at physio- functions by steroid hormones has been reported in the heart (8, logical concentrations, which may explain why elevated levels of 9), neurons (10–12), smooth muscle (13), and pancreatic beta these steroids in the female organism affect fertility. cells (14). In marine invertebrates, the sulfated steroid choles- tane acts as a chemoattractant for sea squirt sperm, which also Author contributions: N.M. and P.V.L. designed research; N.M., M.R.M., and P.V.L. per- formed research; N.M. contributed new reagents/analytic tools; M.R.M. helped with pilot happens via the nongenomic pathway (15). The latter represents experiments; P.V.L. led the research study; N.M. and P.V.L. analyzed data; and N.M. and an unconventional chemoattractant for ascidian sperm, as such P.V.L. wrote the paper. factors are usually proteins or peptides and not steroids. In hu- Conflict of interest statement: P.V.L. and N.M. are inventors on a patent application filed mans, pregnenolone sulfate (PregS), a sulfated steroid hormone by University of California, Berkeley related to the work presented in this paper. similar in structure to P4, is a nongenomic activator of the This article is a PNAS Direct Submission. transient receptor potential cation channel subfamily M member 3 Freely available online through the PNAS open access option. (TRPM3) channel in pancreatic beta cells (14). However, the 1To whom correspondence should be addressed. Email: [email protected]. precise mechanism of this activation is unknown. Here, we This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. report that PregS also activates the sperm calcium channel 1073/pnas.1700367114/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1700367114 PNAS Early Edition | 1of6 Downloaded by guest on October 1, 2021 endogenous steroids can interfere with ABHD2 activity based on E2, or HC). Testosterone, E2, or HC did not alter basal CatSper their structure similarities, we have explored the ability of these activity, whereas P4, as expected, caused a sevenfold potentiation triterpenoids and steroids to prevent CatSper activation and of inward CatSper currents (Fig. 1 A–D and Table S1). However, fertilization. coadministration of testosterone and P4 to spermatozoa, which were preexposed to testosterone, completely prevented ICatSper Results potentiation by P4 (Fig. 1 A and B and Table S1). E2 and HC Testosterone, 17 beta-Estradiol, HC, and PregS Exert Distinct Effects partially reduced CatSper activation by P4 to 3.6- and 4.1-fold, on CatSper. Progesterone (P4) and its closest analog 17-OH-P4 when applied simultaneously with P4 (Fig. 1 C and D and Table have been shown to trigger calcium influx into the sperm fla- S1). To explore the efficiency of P4 inhibition by either testos- gellum (19) via activation of the sperm-specific calcium channel terone, E2, or HC, we have determined the IC50 values for each CatSper (3, 5). However, CatSper specificity toward other ste- of them by measuring CatSper activation by 1 μM of P4 in the roid hormones has not been evaluated. To reveal CatSper reg- presence of various concentrations of the corresponding steroid. ulation by endogenous steroids and steroid-like molecules, we Human spermatozoa were first preexposed to corresponding have analyzed inward monovalent currents through CatSper concentrations of either testosterone, E2, or HC. Subsequently, (ICatSper) of human sperm. Cells were first stimulated with either cells were exposed to both the individual steroid concentration μ 1 M testosterone, 17 beta-estradiol (E2), HC, or PregS alone. from the first stimulus plus 1 μM P4. The respective IC50 values Subsequently, each preexposed cell was stimulated with 1 μM were 429 ± 73 nM (testosterone; Fig. 1 F and I and Table S1), P4 in combination with the corresponding steroid (testosterone, 833 ± 280 nM (E2; Fig. 1 G and I and Table S1), and 153 ± 90 nM A BDC E progesterone (P4) testosterone (T) 17beta-estradiol (E2) hydrocortisone (HC) pregnenolone sulfate (PregS) +80 mV 0 mV -80 mV µ µ 1 µM P4 DVF 1 M E2 +
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