Mobilisation of Ca2+ stores and flagellar regulation in human sperm by S-nitrosylation: a role for NO synthesised in the female reproductive tract

Abstract

Generation of NO by nitric oxide synthase (NOS) is implicated in gamete interaction and fertilisation. Exposure of human spermatozoa to NO donors caused mobilisation of stored Ca(2+) by a mechanism that did not require activation of guanylate cyclase but was mimicked by S-nitroso-glutathione (GSNO; an S-nitrosylating agent). Application of dithiothreitol, to reduce protein -SNO groups, rapidly reversed the actions of NO and GSNO on [Ca(2+)](i). The effects of NO, GSNO and dithiothreitol on sperm protein S-nitrosylation, assessed using the biotin switch method, closely paralleled their actions on [Ca(2+)](i). Immunofluorescent staining revealed constitutive and inducible NOS in human oviduct and cumulus (the cellular layer investing the oocyte). 4,5-diaminofluorescein (DAF) staining demonstrated production of NO by these tissues. Incubation of human sperm with oviduct explants induced sperm protein S-nitrosylation resembling that induced by NO donors and GSNO. Progesterone (a product of cumulus cells) also mobilises stored Ca(2+) in human sperm. Pre-treatment of sperm with NO greatly enhanced the effect of progesterone on [Ca(2+)](i), resulting in a prolonged increase in flagellar excursion. We conclude that NO regulates mobilisation of stored Ca(2+) in human sperm by protein S-nitrosylation, that this action is synergistic with that of progesterone and that this synergism is potentially highly significant in gamete interactions leading to fertilisation.

Figures

Fig. 1

Expression of eNOS in human oviductal and cumulus cells. A, C and E show staining of human oviductal (ampullary) primary culture (A), human cumulus (C) and human granulosa cell line (COV 434; E) for eNOS. B, D and F show corresponding phase images of these samples.

Fig. 2

NO mobilises stored Ca2+ in sperm. (A) Spermine NONOate causes a slowly-developing rise in [Ca2+]i in human sperm. Responses of 4 separate cells are shown. Red trace shows example of cell generating [Ca2+]i oscillations. (B) In low-Ca2+ medium ([Ca2+]o≤5 μM) the response to NONOate was similar, but oscillations were rarely seen. Responses of 7 cells shown. (C) Pseudocolour image series showing NONOate-induced rise in [Ca2+]i in the sperm neck/midpiece. Numbers show minutes since application of 100 μM spermine NONOate. (D) Mean normalised increase in fluorescence 10 minutes after application of 100 μM spermine NONOate to cells bathed in sEBSS (271 cells; 3 experiments) and low-Ca2+ sEBSS (214 cells; 3 experiments). (E) A rapid decrease in [Ca2+]i followed washout of NONOate, followed by slow recovery. Upon re-introduction of NONOate many cells generated oscillations in the neck/midpiece region. Responses of 5 individual cells shown. Lower panel shows pseudocolour images series of a single [Ca2+]i oscillation (numbers show time in seconds).

Fig. 3

Mobilisation of stored Ca2+ by NO does not involve cGMP. (A) 100 μM 8-bromo cGMP causes rapid elevation of [Ca2+]i in human sperm. Responses of 6 cells shown. (B) Response to 8-bromo cGMP is greatly reduced and slowed in cells exposed to cGMP in low-Ca2+ saline. Responses of 5 cells shown. (C) Ca2+-dependence of the response to 100 μM 8-bromo cGMP. Light grey bars show responses of cells bathed in sEBSS (72 cells; 2 experiments), dark grey bars show cells bathed in low-Ca2+ sEBSS (122 cells; 3 experiments). (D) Pre-treatment with the sGC inhibitor ODQ (10 μM; white bar) does not inhibit the increase in [Ca2+]i induced by exposure to 100 μM spermine NONOate (arrow). Responses of 7 cells are shown. (E) Mean normalised increase in fluorescence 10 minutes after application of 100 μM spermine NONOate under control conditions (208 cells; 3 experiments) and after pre-treatment with 10 μM ODQ (267 cells; 3 experiments). Pre-treatment did not modify the amplitude of the response.

Fig. 4

NO and protein S-nitrosylation in sperm. (A) 100 μM GSNO, a nitrosylating agent, causes a rise in [Ca2+]i similar to that seen with NONOate but onset of the effect is more rapid. Responses of 6 cells shown. (B) 100 μM GSH rapidly reverses the action of 100 μM GSNO on sperm [Ca2+]i. Responses of 5 cells shown. (C) GSNO causes rapid S-nitrosylation of sperm proteins: Lane 1 shows background levels in cells processed immediately for assay (indicated by *). Lane 2 shows that, after 60 minutes of incubation of the cells in sEBSS, this level does not change. Lanes 3, 4, 5, 6 and 7 show increased S-nitrosylation in cells processed for assay immediately upon exposure to 50 μM GSNO (*) and those incubated with GSNO for 5, 10, 30 and 60 minutes respectively. S-nitrosylation reaches near steady-state levels in the sample processed immediately (approximately 5 minutes for preliminary centrifugation; see methods). (D) S-nitrosylation of sperm proteins is rapidly reversible. Left panel shows S-nitrosylated proteins in untreated cells incubated for 10 minutes (lane 1), cells exposed to GSNO and cys-SNO (lanes 2 and 4) and cells exposed to GSH and exhausted cys-NO (lanes 3 and 5; controls). Right panel shows same treatments but cells were washed in PBS immediately before processing for the assay. S-nitrosylation caused by GSNO and CSNO is rapidly reversed upon removal of the agent.

Fig. 5

Thiol reducing agents reverse NO effects. (A) DTT rapidly reverses nitrosylation of sperm proteins. Lane 1 shows endogenous S-nitrosylation in cells incubated in sEBSS for 60 minutes. Lanes 2 and 3 show cells incubated in the presence of 1 mM GSH (control) and 100 μM GSNO. Lane 4 shows cells incubated as for lane 3 but 1 mM DTT was added to the incubation 5 minutes before processing for the assay. (B) DTT reverses the action of 100 μM spermine NONOate. Upon application of 1 mM DTT, the increase in fluorescence induced by spermine NONOate is rapidly reduced or completely reversed. Responses of 5 separate cells shown. (C) DTT induced-decrease in fluorescence is correlated with the preceding NONOate-stimulated increase in fluorescence. Scattergram shows data from a single experiment, representative of 5 repeats. R2=0.33. (D) Action of DTT is not due to e−-dependent mitochondrial Ca2+ accumulation. After application of 100 μM spermine NONOate to mobilise Ca2+, the cells were exposed to 10 μM CCCP to collapse the mitochondrial inner membrane potential. The effect of subsequent application of 1 mM DTT resembled that seen in cells with functioning mitochondria. Responses of 5 cells shown.

Fig. 6

NO production by female tract cells induces S-nitrosylation in human sperm. S-nitrosylated proteins were identified using fluorescently-tagged methanethiosulfonate, as described in the text. Negligible levels of labelling were present in controls but treatment with 100 μM spermine NONOate or GSNO caused clear labelling, particularly at the back of the sperm head. Incubation of sperm with primary cultures derived from endometrial of tubal explants (ampulla and isthmus) induced levels of S-nitrosyaltion at least as great as those seen with NONOate.

Fig. 7

Pre-treatment with 100 μM spermine NONOate potentiates responses of sperm to 3 μM progesterone. (A) When sperm were exposed to 3 μM progesterone after pre-treatment with spermine NONOate (100 μM for 10 minutes), the initial [Ca2+]i transient was enlarged (in some cells) and significantly prolonged compared to that seen in control cells (insert: 3 single cell responses, scales as for main plot). Responses of 8 cells shown. (B) Co-stimulation with spermine NONOate increases the proportion of cells in which a prolonged [Ca2+]i transient occurs in response to stimulation with 3 μM progesterone. Data are plotted as percentage of cells in each class (defined by [Ca2+]i transient duration). Control cells (black bars; n=27) were from the same sample as cells exposed to NO before and during progesterone stimulation (grey bars; n=69) and cells in which NO was washed off as progesterone was applied (white bars; 44 cells). (C) Progesterone (3 μM) causes a brief increase in flagellar displacement. Red line and shading show the mean ± 2 s.d. of frame-to-frame midpiece displacement during the control period. Graph shows response of 1 cell (representative of > 100 cells in 2 experiments). (D) Pre-treatment with spermine NONOate (100 μM) prolonged and intensified the effect of progesterone on flagellar activity. Red line and shading show the mean ± 2 s.d. of frame-to-frame midpiece displacement during the control period. The graph shows response of 1 sperm cell (representative of > 100 cells in 2 experiments).