Communication between female tract and sperm: Saying NO* when you mean yes.

Signaling through [Ca(2+)](i) is central to regulation of sperm activity and is likely to be the mechanism that transduces signals from the female reproductive tract to regulate sperm motility. In a recent paper1 we showed that exposure of sperm to nitric oxide mobilizes stored Ca(2+) in human sperm, an effect that occurs through nitrosylation of protein thiols. Not only did we find that NO* production by cells of the human female tract would be sufficient to elicit this effect, but progesterone, which is also present in the female tract and is synthesized by the oocyte vestments, acted synergistically with NO* to mobilize Ca(2+) and enhance flagellar beating. Here we argue that a Ca(2+) store at the junction of the sperm head and flagellum is subject to regulation by both progesterone and NO* and that ryanodine receptors at the store may be the point at which coincidence detection and synergistic interaction occurs.

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

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.

Human spermatozoa contain multiple targets for protein S-nitrosylation: an alternative mechanism of the modulation of sperm function by nitric oxide?

Nitric oxide (NO) enhances human sperm motility and capacitation associated with increased protein phosphorylation. NO activates soluble guanylyl cyclase, but can also modify protein function covalently via S-nitrosylation of cysteine. Remarkably, this mechanism remains unexplored in sperm although they depend on post-translational protein modification to achieve changes in function required for fertilisation. Our objective was to identify targets for S-nitrosylation in human sperm. Spermatozoa were incubated with NO donors and S-nitrosylated proteins were identified using the biotin switch assay and a proteomic approach using MS/MS. 240 S-nitrosylated proteins were detected in sperm incubated with S-nitroso-glutathione. Minimal levels were observed in glutathione or untreated samples. Proteins identified consistently based on multiple peptides included established targets for S-nitrosylation in other cells e.g. tubulin, GST and HSPs but also novel targets including A-kinase anchoring protein (AKAP) types 3 and 4, voltage-dependent anion-selective channel protein 3 and semenogelin 1 and 2. In situ localisation revealed S-nitrosylated targets on the postacrosomal region of the head and throughout the flagellum. Potential targets for S-nitrosylation in human sperm include physiologically significant proteins not previously reported in other cells. Their identification will provide novel insight into the mechanism of action of NO in spermatozoa.

Characterization of cyclic adenine dinucleotide phosphate ribose levels in human spermatozoa.

OBJECTIVE: To determine the presence of the Ca2+-releasing pyridine nucleotide derivative, cyclic adenine dinucleotide phosphate ribose (cADPR), in human spermatozoa and to investigate its role in progesterone-induced Ca2+ oscillations in spermatozoa. DESIGN: Biochemical investigation on human spermatozoa from healthy volunteers. SETTING: Healthy volunteers in an academic research environment. PATIENT(S): Ten volunteers. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): The cADPR levels. RESULT(S): Human spermatozoa contain micromolar concentrations of cADPR that do not change significantly during sperm capacitation. An active synthetic machinery for cADPR is present in human spermatozoa, whereas degradation activity is minimal. Although progesterone-induced Ca2+ oscillations are dependent on the ryanodine receptor, they are unaffected by cADPR antagonists. CONCLUSION(S): It appears that cADPR does not to play a role in Ca2+ oscillations in spermatozoa, but the presence of high concentrations of cADPR suggests that, instead, it may be introduced into the egg at fertilization and play a role in the Ca2+ transient immediately following sperm-egg fusion.