Soluble adenylyl cyclase inhibition prevents human sperm functions essential for fertilization

Abstract

Soluble adenylyl cyclase (sAC: ADCY10) has been genetically confirmed to be essential for male fertility in mice and humans. In mice, ex vivo studies of dormant, caudal epididymal sperm demonstrated that sAC is required for initiating capacitation and activating motility. We now use an improved sAC inhibitor, TDI-10229, for a comprehensive analysis of sAC function in mouse and human sperm. In contrast to caudal epididymal mouse sperm, human sperm are collected post-ejaculation, after sAC activity has already been stimulated. In addition to preventing the capacitation-induced stimulation of sAC and protein kinase A activities, tyrosine phosphorylation, alkalinization, beat frequency and acrosome reaction in dormant mouse sperm, sAC inhibitors interrupt each of these capacitation-induced changes in ejaculated human sperm. Furthermore, we show for the first time that sAC is required during acrosomal exocytosis in mouse and human sperm. These data define sAC inhibitors as candidates for non-hormonal, on-demand contraceptives suitable for delivery via intravaginal devices in women.

Keywords: acrosome reaction; capacitation; contraception; cyclic AMP; sperm motility.

Figure 1.

TDI-10229 is more potent than LRE1 and does not inhibit transmembrane adenylyl cyclases. (a) Molecular structure of TDI-10229. (b) Concentration-response curves of LRE1 (blue) and TDI-10229 (grey) on purified recombinant human soluble adenylyl cyclase (sAC) protein in the presence of 1 mM ATP, 2 mM Ca²⁺, 4 mM Mg²⁺, and 40 mM ${\text{HCO}}_3^{-}$, normalized to the respective dimethylsulfoxide (DMSO)-treated control; mean ± SEM (n = 5). (c) Concentration-response curves of LRE1 and TDI-10229 on sAC-overexpressing 4/4 cells. Cellular accumulation of cAMP measured in cells treated with 500 μM 3-isobutyl-1-methylxanthine (IBMX) for 5 min, normalized to the respective DMSO-treated control; mean ± SEM (n = 5). (d) Adenylyl cyclase (AC) activities of HEK 293 cell lysates overexpressing each of the indicated transmembrane adenylyl cyclase (tmACs) activated by 50 μM forskolin or 100 μM GTPyS in the absence or presence of 10 μM TDI-10229, normalized to the respective unstimulated DMSO-treated control; mean ± SEM (n = 3 with individual replicates indicated by symbols). (e) Concentration-response of TDI-10229 on cellular accumulation of cAMP in forskolin-stimulated sAC knockout mouse embryonic fibroblasts (KO MEFs), normalized to unstimulated DMSO control; mean ± SEM (n = 8). Differences between conditions were analyzed using (d) two-tailed, unpaired t-test.

Figure 2.

sAC inhibition by TDI-10229 prevents capacitation-induced cAMP increase and protein kinase A activation in mouse and human sperm. (a, c) Intracellular cAMP levels in mouse and human sperm detected at different time points during capacitation after incubation in (a) Toyoda Yokoyama Hoshi (TYH) medium with 25 mM ${\text{HCO}}_3^{-}$ and 3 mg/ml bovine serum albumin (BSA) (0–90 min) or in (c) human tubal fluid (HTF) with 25 mM ${\text{HCO}}_3^{-}$ and 3 μl/ml human serum albumin (HSA) (0–120 min), time-course in non-capacitated sperm is shown as a control; mean ± SEM (n ≥ 5). (b, d) Intracellular cAMP levels in (b) mouse (at 10 min) and (d) human sperm (at 40 min) in non-capacitating or capacitating media in the absence or presence of 5 μM TDI-10229; mean + SEM (n ≥ 4 with individual replicates indicated by symbols). (e, g) Protein kinase A activity levels in (e) mouse and (g) human sperm detected at different time points during capacitation after incubation in non-capacitating or capacitating media (mouse: 0–90 min, human: 0–120 min); mean ± SEM (n ≥ 3). (f, h) Protein kinase A activity levels in (f) mouse (at 45 min) and (h) human sperm (at 60 min) in non-capacitating or capacitating media in the absence or presence of 5 μM TDI-10229; mean + SEM (n ≥ 3 with individual replicates indicated by symbols). (i, k) Phosphorylation of PKA substrates of non-capacitated and capacitated (i) mouse and (k) human sperm in the absence or presence of different concentrations of TDI-10229, rescued with 5 mM dibutyryl cAMP (db-cAMP)/500 μM IBMX, shown are representative Western blots. Respective uncropped Western blot images are shown as Supplementary Fig. S4a and b. (j, l) Quantitation of PKA substrate phosphorylation Western blots of (j) mouse and (l) human sperm, normalized to DMSO-treated non-capacitated control; mean ± SEM (n ≥ 6 with individual replicates indicated by symbols). Differences between conditions were analyzed using one-way ANOVA compared to the first time point (a, b), first time-point of non-capacitated control (e, f), DMSO-treated non-capacitated control (c, d, g, h), or DMSO-treated capacitated control (j, l), *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Figure 3.

sAC inhibition by TDI-10229 prevents capacitation in mouse and human sperm. (a, b) Intracellular pH of non-capacitated and capacitated (a) mouse and (b) human sperm in the absence or presence of 5 μM TDI-10229; mean ± SEM (n = 5 with individual replicates indicated by symbols). (c, d) Phosphorylation of tyrosine residue Western blots of non-capacitated and capacitated (c) mouse and (d) human sperm in the absence or presence of different concentrations of TDI-10229, rescued with 5 mM db-cAMP/500 μM IBMX, shown are representative Western blots. Respective uncropped Western blot images are shown as Supplementary Fig. S4c, d. (e, f) Quantitation of tyrosine phosphorylation (pY) Western blots of (e) mouse and (f) human sperm, normalized to DMSO-treated non-capacitated control; mean ± SEM (n ≥ 6 with individual replicates indicated by symbols). Differences between conditions were analyzed using one-way ANOVA compared to DMSO-treated non-capacitated control (a, b) or DMSO-treated capacitated control (e, f), *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Figure 4.

Characterization of mouse sperm beating pattern in the absence or after inhibition of sAC. (a) Flagellar waveform of wild-type sperm in the absence or presence of 5 μM TDI-10229 and sAC KO sperm before and after stimulation with 25 mM NaHCO₃ or 5 mM db-cAMP/500 μM IBMX. Superimposed color-coded frames taken every 5 ms, illustrating one flagellar beat cycle; scale bar: 30 µm. (b, c) (b) Mean amplitude of the curvature angle and (c) flagellar beat frequency along the flagellum of wild-type sperm in the absence or presence of 5 μM TDI-10229 and sAC KO sperm. Solid lines indicate the time-averaged values, dotted lines the SEM, n = 3, ≥50 individual sperm from three different mice.

Figure 5.

sAC inhibition by TDI-10229 prevents bicarbonate-induced changes in the flagellar beating pattern of human sperm. (a) Flagellar waveform of human sperm incubated in the presence of 3 μl/ml human serum albumin in the absence or presence of 0.2 μM TDI-10229 before and after stimulation with 25 mM NaHCO₃ or 5 mM db-cAMP/500 μM IBMX. Superimposed color-coded frames taken every 5 ms, illustrating one flagellar beat cycle; scale bar: 15 µm. (b, c) (b) Mean flagellar beat frequency and (c) mean amplitude of the curvature angle along the flagellum of mouse sperm in the absence or presence of 5 μM TDI-10229 before and after stimulation with 25 mM NaHCO₃ or 5 mM db-cAMP/500 μM IBMX. Solid lines indicate the time-averaged values, dotted lines the SEM, n = 3, ≥50 individual sperm from three different donors.

Figure 6.

TDI-10229 blocks acrosome reaction post capacitation and fertilization of mouse sperm in vitro. (a) Acrosome reaction in wild-type mouse sperm evoked by 50 isolated zonae pellucidae after incubation for 90 min in capacitating media in the absence or presence of 5 µM TDI-10229, rescued with 5 mM db-cAMP/500 μM IBMX. For the striped bars, TDI-10229 was added concomitantly with zonae pellucidae; mean ± SEM (n ≥ 3). (b) Acrosome reaction in human sperm evoked by 10 μM progesterone after incubation for 180 min in capacitating media in the absence or presence of 1 µM TDI-10229, rescued with 5 mM db-cAMP/500 μM IBMX. For the striped bars, TDI-10229 was added concomitantly with progesterone; mean ± SEM (n ≥ 4 with individual replicates indicated by symbols). (c, d) Rate of fertilization of oocytes after incubation of (c) C57Bl/6 and (d) CD1 mouse oocytes with capacitated C57Bl/6 and CD1 sperm, respectively in the absence or presence of 5 or 50 μM TDI-10229; mean ± SEM (n = 5), numbers indicate the total number of oocytes from three independent experiments, with individual replicates indicated by symbols. Differences between conditions were analyzed using one-way ANOVA compared to respective DMSO-treated control, *P < 0.05, **P < 0.01, ****P < 0.0001.