The sperm-specific K+ channel Slo3 is inhibited by albumin and steroids contained in reproductive fluids

Abstract

To locate and fertilize the egg, sperm probe the varying microenvironment prevailing at different stages during their journey across the female genital tract. To this end, they are equipped with a unique repertoire of mostly sperm-specific proteins. In particular, the flagellar Ca²⁺ channel CatSper has come into focus as a polymodal sensor used by human sperm to register ligands released into the female genital tract. Here, we provide the first comprehensive study on the pharmacology of the sperm-specific human Slo3 channel, shedding light on its modulation by reproductive fluids and their constituents. We show that seminal fluid and contained prostaglandins and Zn²⁺ do not affect the channel, whereas human Slo3 is inhibited in a non-genomic fashion by diverse steroids as well as by albumin, which are released into the oviduct along with the egg. This indicates that not only CatSper but also Slo3 harbours promiscuous ligand-binding sites that can accommodate structurally diverse molecules, suggesting that Slo3 is involved in chemosensory signalling in human sperm.

Keywords: follicular fluid; human sperm; ion channel; reproductive tract; sperm signalling.

FIGURE 1

The action of seminal fluid, prostaglandins, and Zn²⁺ on human Slo3 (A) Membrane K⁺ currents recorded from CHO cells co-expressing human Slo3 and LRRC52 in the whole-cell configuration before and after perfusion with dilute (%) solutions of seminal fluid (SF). (B) Relative current amplitude (mean ± SD) at +100 mV in the presence of a given dilution of seminal fluid relative to that under control conditions (set to 1) (n ≥ 3). Grey dots indicate individual values. (C) Slo3 currents before (control, black) and after perfusion with 1 mM Zn²⁺ (red). (D) Current amplitudes (mean ± SD) at +100 mV in the presence of 1 mM Zn²⁺ relative to that under control conditions (set to 1) (n = 4). Red dots indicate individual recordings. (E) Slo3 currents before (black) and after perfusion with prostaglandin E1 or E2 at a concentration of 50 μM (red). (F) Relative current amplitude (mean ± SD) at +100 mV in the presence of 50 µM PGE1/2 (n = 3). Red dots indicate individual recordings.

FIGURE 2

Human Slo3 is inhibited by follicular fluid (A) Slo3 currents before and after perfusion with dilute (%) solutions of follicular fluid (FF). (B) Current amplitudes (mean ± SD) at +100 mV in the presence of a given dilution of follicular fluid relative to that under control conditions (set to 1) (n ≥ 3). Grey dots indicate individual values. The continuous red line represents a fit of the Hill equation, yielding the dose-response relationship (ID₅₀ = 5.7 ± 0.5%, Standard error of the fit). (C) Slo3 currents recorded from human sperm at +100 mV before and after perfusion with FF. (D) Current amplitudes (mean ± SD) in the presence of FF relative to that under control conditions (set to 1) (n = 8). (E) Slo3 currents recorded from CHO cells in the presence of 1 mM intracellular Ca²⁺ and pH 8.0 before and after perfusion with FF. (F) Current amplitudes (mean ± SD) in the presence of FF relative to that under control conditions (set to 1) (n = 4). *p < 0.05, **p < 0.01, ****p < 0.0001.

FIGURE 3

The action of steroids contained in reproductive fluids on human Slo3 (A) Slo3 currents before (black) and after perfusion with a given steroid (50 μM, red). (17-OH-Prog. = 17α-Hydroxyprogesterone, 17-OH-Preg. = 17α-Hydroxypregnenolone, DHEA = Dehydroepiandrosterone, Testo. = Testosterone). Vertical and horizontal scale bars represent pA and ms, respectively. Top right: Voltage protocol used for all recordings. (B) Current amplitude (mean ± SD) at +100 mV in the presence of 50 µM of the respective steroid relative to that under control conditions (set to 1) (n ≥ 3). Red dots indicate individual values; ****p < 0.0001.

FIGURE 4

Dose-response relationship for the steroid inhibition of human Slo3 (A) Slo3 currents before and after perfusion with different concentrations of a given steroid. (B) Current amplitudes (mean ± SD) at +100 mV in the presence of different concentrations of the respective steroid relative to that under control conditions (set to 1) (n ≥ 3). Continuous lines represent fits of the Hill equation to yield the dose-response relationships (n ≥ 3). (C) Experimentally determined dose-response relationship of the Slo3 inhibition by follicular fluid shown in Figure 2B (FF, continuous line) and modelled dose-response relationship (dotted line) assuming that its action exclusively rests on the contained steroids (see explanation in the text).

FIGURE 5

Human Slo3 is inhibited by charcoal-stripped follicular fluid (A) Slo3 currents before and after perfusion with dilute (%) solutions of charcoal-stripped follicular fluid (sFF). (B) Current amplitudes (mean ± SD) at +100 mV in the presence of a given dilution of sFF relative to that under control conditions (set to 1) (n ≥ 3). Grey dots indicate individual values. The continuous green line represents a fit of the Hill equation, yielding the dose-response relationship (ID₅₀ = 5.7 ± 1.3%, Standard error of the fit). For comparison, the dose-response relation for FF from Figure 2B is shown in red. Additionally, the predicted dose-response relations based on the steroid concentrations determined in FF (dotted red) and sFF (dotted green) are shown.

FIGURE 6

Human serum albumin inhibits human Slo3 (A) Slo3 currents before and after perfusion with different concentrations of albumin. (B) Current amplitudes (mean ± SD) at +100 mV in the presence of given concentration of albumin relative to that under control conditions (set to 1) (n = 4). The continuous line represents a fit of the Hill equation to yield the dose-response relationship. Grey dots indicate individual recordings. (C) Comparison of measured (continous red line) and predicted dose-response relations for FF based on the contained concentrations of steroids (dotted red line), HSA (dotted brown line), or both (dotted orange line). (D) Slo3 currents recorded from human sperm at +100 mV before and after perfusion with albumin. (E) Current amplitudes (mean ± SD) in the presence of albumin relative to that under control conditions (set to 1) (n = 4). (F) Slo3 currents recorded from CHO cells in the presence of 1 mM intracellular Ca²⁺ and pH 8.0 before and after perfusion with albumin. (G) Current amplitudes (mean ± SD) in the presence of albumin relative to that under control conditions (set to 1) (n = 6); ****p < 0.0001.

FIGURE 7

The action of albumin on Slo3 channels is transient (A) Time course of the inhibition of Slo3 channels recorded from CHO cells after perfusion with albumin. (B) Time constant of the relieve of inhibition in the presence of albumin.