SKF96365 modulates activity of CatSper channels in human sperm.

Exposure of human sperm to progesterone (P4) activates cation channel of sperm (CatSper) channels, inducing an intracellular Ca2+ concentration ([Ca2+]i) transient followed by repetitive [Ca2+]i activity (oscillations), which are believed to be functionally important. We investigated the potential significance of store-operated Ca2+-entry in these oscillations using the inhibitor SKF96365 (30 µM; SKF). Following pre-treatment of human sperm with 3 µM P4, exposure to SKF doubled the proportion of oscillating cells (P = 0.

Complex combined steroid mix of the female tract modulates human sperm.

Human spermatozoa interact with a complex biochemical environment in the female reproductive tract en route to the site of fertilisation. Ovarian follicular fluid contributes to this complex milieu and is known to contain steroids such as progesterone, whose effects on sperm physiology have been widely characterised. We have previously reported that progesterone stimulates intracellular calcium concentration ([Ca]) signalling and acrosome reaction in human spermatozoa.

Cell-penetrating peptides, targeting the regulation of store-operated channels, slow decay of the progesterone-induced [Ca2+]i signal in human sperm.

Previous work has provided evidence for involvement of store-operated channels (SOCs) in [Ca(2+)]i signalling of human sperm, including a contribution to the transient [Ca(2+)]i elevation that occurs upon activation of CatSper, a sperm-specific cation channel localized to the flagellum, by progesterone. To further investigate the potential involvement of SOCs in the generation of [Ca(2+)]i signals in human sperm, we have used cell-penetrating peptides containing the important basic sequence KIKKK, part of the STIM-Orai activating region/CRAC activating domain (SOAR/CAD) of the regulatory protein stromal interaction molecule 1. SOAR/CAD plays a key role in controlling the opening of SOCs, which occurs upon mobilization of stored Ca(2+).

Clinically relevant enhancement of human sperm motility using compounds with reported phosphodiesterase inhibitor activity.

Study Question: Can we identify compound(s) with reported phosphodiesterase inhibitor (PDEI) activity that could be added to human spermatozoa in vitro to enhance their motility without compromising other sperm functions?

Summary Answer: We have identified several compounds that produce robust and effective stimulation of sperm motility and, importantly, have a positive response on patient samples.

What Is Known Already: For >20 years, the use of non-selective PDEIs, such as pentoxifylline, has been known to influence the motility of human spermatozoa; however, conflicting results have been obtained. It is now clear that human sperm express several different phosphodiesterases and these are compartmentalized at different regions of the cells.

2-APB-potentiated channels amplify CatSper-induced Ca(2+) signals in human sperm.

Ca2+i signalling is pivotal to sperm function. Progesterone, the best-characterized agonist of human sperm Ca2+i signalling, stimulates a biphasic [Ca2+]i rise, comprising a transient and subsequent sustained phase. In accordance with recent reports that progesterone directly activates CatSper, the [Ca2+]i transient was detectable in the anterior flagellum (where CatSper is expressed) 1-2 s before responses in the head and neck.

Techniques for imaging Ca2+ signaling in human sperm.

Fluorescence microscopy of cells loaded with fluorescent, Ca(2+)-sensitive dyes is used for measurement of spatial and temporal aspects of Ca(2+) signaling in live cells. Here we describe the method used in our laboratories for loading suspensions of human sperm with Ca(2+)-reporting dyes and measuring the fluorescence signal during physiological stimulation. Motile cells are isolated by direct swim-up and incubated under capacitating conditions for 0-24 h, depending upon the experiment.

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.

Ca2+-stores in sperm: their identities and functions.

Intracellular Ca2+ stores play a central role in the regulation of cellular [Ca2+](i) and the generation of complex [Ca2+] signals such as oscillations and waves. Ca2+ signalling is of particular significance in sperm cells, where it is a central regulator in many key activities (including capacitation, hyperactivation, chemotaxis and acrosome reaction) yet mature sperm lack endoplasmic reticulum and several other organelles that serve as Ca2+ stores in somatic cells. Here, we review i) the evidence for the expression in sperm of the molecular components (pumps and channels) which are functionally significant in the activity of Ca2+ stores of somatic cells and ii) the evidence for the existence of functional Ca2+ stores in sperm.

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).

Mobilisation of stored calcium in the neck region of human sperm--a mechanism for regulation of flagellar activity.

Calcium signalling plays a pivotal role in sperm physiology, being intimately involved in the regulation of acrosome reaction, chemotaxis and hyperactivation. Here we describe briefly the mechanisms of calcium regulation in somatic cells and the ways in which these mechanisms have been adapted to function in mature spermatozoa. We then consider recent data from this and other laboratories on the responses of sperm to three compounds: progesterone and nitric oxide (both products of the cumulus oophorus) and 4-aminopyridine.

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.

Counting sperm does not add up any more: time for a new equation?

Although sperm dysfunction is the single most common cause of infertility, we have poor methods of diagnosis and surprisingly no effective treatment (excluding assisted reproductive technology). In this review, we challenge the usefulness of a basic semen analysis and argue that a new paradigm is required immediately. We discuss the use of at-home screening to potentially improve the diagnosis of the male and to streamline the management of the sub-fertile couple.

Secretory pathway Ca(2+)-ATPase (SPCA1) Ca(2)+ pumps, not SERCAs, regulate complex [Ca(2+)](i) signals in human spermatozoa.

The sarcoplasmic-endoplasmic reticulum Ca(2+)-ATPase (SERCA) inhibitors thapsigargin (0.1-1 microM) and cyclopiazonic acid (10 microM), failed to affect resting [Ca(2+)] in human spermatozoa. Slow progesterone-induced [Ca(2+ i)](i) oscillations in human spermatozoa, which involve cyclic emptying-refilling of an intracellular Ca(2+) store were also insensitive to these inhibitors.

Use of phosphoproteomics to study tyrosine kinase activity in capacitating boar sperm. Kinase activity and capacitation.

It is generally accepted that sperm capacitation is associated with the protein kinase A-mediated appearance of tyrosine phosphoproteins, although the substrates and kinase(s) involved have not been identified. We described a Mr 32,000 tyrosine phosphoprotein, "p32", appearing in porcine sperm coincident with capacitation. We also discovered a tyrosine kinase-like enzyme in boar sperm of Mr 32,000 ("TK-32") with enhanced activity during capacitation.

Implication of cAMP during porcine sperm capacitation and protein tyrosine phosphorylation.

Second messengers are involved in sperm fertilizing potential, as both motility and the acrosome reaction are influenced by cAMP. Moreover, the activity of cyclic nucleotides is implicated in the appearance of tyrosine phosphorylated sperm proteins, which is associated with capacitation in the mammalian spermatozoa. Nevertheless, the involvement of the cAMP/protein kinase A (PK-A) pathway during pig sperm capacitation may be different from that observed in other mammals.

Physiological and proteomic approaches to studying prefertilization events in the human.

This research aims firstly to understand, in cellular and molecular terms, how a mature human spermatozoon is prepared for fertilization, and secondly, to identify what factors are involved in the initial signalling interactions between the egg and spermatozoon. In order to achieve these objectives, a combination of approaches is being used, including single-cell imaging, patch clamping and proteomics. Single-cell imaging reveals hidden complexity and heterogeneity in signalling responses in spermatozoa.

Activation of protein kinase A during human sperm capacitation and acrosome reaction.

Spermatozoa undergo a variety of changes during their life that are prerequisites to their maturation and ability to fertilize eggs. Mammalian sperm capacitation and acrosome reaction are regulated by signal transduction systems involving cyclic adenosine monophosphate (cAMP) as a second messenger. This second messenger acts through the activation of protein kinase A (PKA) and indirectly regulates protein tyrosine phosphorylation.

Presence of cyclic nucleotide phosphodiesterases PDE1A, existing as a stable complex with calmodulin, and PDE3A in human spermatozoa.

Mammalian sperm motility, capacitation, and the acrosome reaction are regulated by signal transduction systems involving cAMP as a second messenger. Levels of cAMP are controlled by two key enzymes, adenylyl cyclase and phosphodiesterases (PDEs), the latter being involved in cAMP degradation. Calmodulin-dependent PDE (PDE1) and cAMP-specific PDE (PDE4) activities were previously identified in spermatozoa via the use of specific inhibitors.

Increase of cAMP upon release from prophase arrest in surf clam oocytes.

Surf clam (Spisula solidissima) oocytes are spawned at the prophase I stage of meiosis, and they remain arrested at this stage until fertilization. Full oocyte meiosis reinitiation, first evidenced by germinal vesicle breakdown (GVBD), may be induced by artificial activators mimicking sperm, such as high K(+) or serotonin. Previous reports indicated that treatments thought to increase the level of oocyte cAMP inhibited sperm- or serotonin-induced, but not KCl-induced, GVBD in clam oocytes.