The Fungal Sexual Pheromone Sirenin Activates the Human CatSper Channel Complex

Abstract

The basal fungus Allomyces macrogynus (A. macrogynus) produces motile male gametes displaying well-studied chemotaxis toward their female counterparts. This chemotaxis is driven by sirenin, a sexual pheromone released by the female gametes. The pheromone evokes a large calcium influx in the motile gametes, which could proceed through the cation channel of sperm (CatSper) complex. Herein, we report the total synthesis of sirenin in 10 steps and 8% overall yield and show that the synthetic pheromone activates the CatSper channel complex, indicated by a concentration-dependent increase in intracellular calcium in human sperm. Sirenin activation of the CatSper channel was confirmed using whole-cell patch clamp electrophysiology with human sperm. Based on this proficient synthetic route and confirmed activation of CatSper, analogues of sirenin can be designed as blockers of the CatSper channel that could provide male contraceptive agents.

Figure 1

Structure of sirenin.

Scheme 1. NaH-Mediated Cyclopropanation, Identification of Side Products, and Their Conversion to 3

Scheme 2. Side Chain Extension

Scheme 3. Alternate Strategy for Side Chain Extension and Completion of the Sirenin Synthesis

Scheme 4. Synthesis of Ester Epoxides 17–19 and Cyclopropanation Optimization

Scheme 5. Completion of the Sirenin Synthesis

Figure 2

Sirenin activates CatSper in human sperm measured by calcium fluorescence. (A) Raw FLIPR traces showing increases in [Ca²⁺]_i elicited by 3 μM progesterone (Prog; red), 3 μM PGE₁ (blue), and increasing concentrations of sirenin (black) compared to the low pH/low K⁺ buffer (green) control. The sirenin (S) dose response increases from 10 nM to 100 μM by half-log concentrations. Cells were treated with compounds at 150 s (**). (B) Concentration-dependent increases in [Ca]²⁺_i elicited by sirenin (black, EC₅₀ = 2.9 ± 0.7 μM), progesterone (red, EC₅₀ = 7.7 ± 0.9 nM), and PGE₁ (blue, EC₅₀ = 4.2 ± 0.7 nM). (C) Sirenin elicits the same level of calcium influx as two endogenous activators of the CatSper channel, progesterone and PGE₁. Human sperm were treated with 30 μM sirenin or 1 μM progesterone or 1 μM PGE₁ (black), and the rise in [Ca²⁺]_i was measured. Mibefradil (gray bar; 30 μM) reduced the calcium influx for all three compounds. Pretreatment with 30 μM mibefradil decreased the sirenin-induced rise in [Ca²⁺]_i by 55%. Calcium fluorescence is expressed as the percent RFU produced by a saturating concentration of progesterone (3 μM). EC₅₀ values determined using Prism v6.05.

Figure 3

Sirenin increases intracellular calcium in human sperm through activation of the CatSper channel. (A) Representative monovalent I_CatSper whole-cell recordings from human spermatozoa using divalent free bath solution (DVF) in the absence (control; blue) or presence of test compound. Currents were elicited in response to indicated voltage ramp. Left panel, 50 μM sirenin (S; green) and 50 μM sirenin (S) with 30 μM mibefradil (M; purple). Right panel, 1 μM progesterone (P; red). Baseline indicates recordings performed in HS bath solution. (B) Averaged fold amplitude change of I_CatSper recorded from human spermatozoa in the presence of indicated test compound. Potentiation was determined by dividing current amplitudes of I_CatSper at −80 mV (negative, inward current) and +80 mV (positive, outward current) by the amplitude of I_CatSper in the absence of the corresponding compound from the same cell. (C) Averaged current density of I_CatSper recorded from human spermatozoa in the presence of indicated test compound. Where appropriate, data are represented as mean ± SEM with n indicating the number of individual cells recorded.