Steve Publicover

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 Ca2+ 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 [Ca2+]i. The effects of NO, GSNO and dithiothreitol on sperm protein S-nitrosylation, assessed using the biotin switch method, closely paralleled their actions on [Ca2+]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 Ca2+ in human sperm. Pre-treatment of sperm with NO greatly enhanced the effect of progesterone on [Ca2+]i, resulting in a prolonged increase in flagellar excursion. We conclude that NO regulates mobilisation of stored Ca2+ 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.

Patch clamp studies of human sperm under physiological ionic conditions reveal three functionally and pharmacologically distinct cation channels

Whilst fertilizing capacity depends upon a K+ conductance (GK) that allows the spermatozoon membrane potential (Vm) to be held at a negative value, the characteristics of this conductance in human sperm are virtually unknown. We therefore studied the biophysical/pharmacological properties of the K+ conductance in spermatozoa from normal donors held under voltage/current clamp in the whole cell recording configuration. Our standard recording conditions were designed to maintain quasi-physiological, Na+, K+ and Cl− gradients. Experiments that explored the effects of ionic substitution/ion channel blockers upon membrane current/potential showed that resting Vm was dependent upon a hyperpolarizing K+ current that flowed via channels that displayed only weak voltage dependence and limited (∼7-fold) K+ versus Na+ selectivity. This conductance was blocked by quinidine (0.3 mM), bupivacaine (3 mM) and clofilium (50 µM), NNC55-0396 (2 µM) and mibefradil (30 µM), but not by 4-aminopyridine (2 mM, 4-AP). Progesterone had no effect upon the hyperpolarizing K+ current. Repolarization after a test depolarization consistently evoked a transient inward ‘tail current’ (ITail) that flowed via a second population of ion channels with poor (∼3-fold) K+ versus Na+ selectivity. The activity of these channels was increased by quinidine, 4-AP and progesterone. Vm in human sperm is therefore dependent upon a hyperpolarizing K+ current that flows via channels that most closely resemble those encoded by Slo3. Although 0.5 µM progesterone had no effect upon these channels, this hormone did activate the pharmacologically distinct channels that mediate ITail. In conclusion, this study reveals three functionally and pharmacologically distinct cation channels: Ik, ITail, ICatSper.

Sperm motility: things are moving in the lab!

Sperm motility is one of the three fundamental tenets of semen analysis providing diagnostic and prognostic information for both natural and assisted reproduction technology (ART) conception. The landmark papers of John MacLeod and Ruth Gold demonstrated clear differences between subfertile and fertile men in sperm motility (MacLeod and Gold, 1951) which have been confirmed by a plethora of subsequent studies (Barratt et al., 2011). The WHO new reference values for motility, as well as sperm concentration (Cooper et al., 2010), are remarkably similar to those proposed by MacLeod and Gold in 1951 (32% motile cells as a 5% centile of a fertile population), suggesting that these observations are robust. The primary conclusion from all these studies is that although there is no magic number or threshold for in vivo fertility, at the lower ends of the motility spectrum, there are significantly higher chances of subfertility (Fig. 1). This overwhelming body of clinical information ignited a series of studies developing new methods to obtain sperm kinematic data. Initial information was very encouraging, e.g. using the biological endpoints of penetration of cervical mucus (Mortimer et al., 1986). With the advent of computer-assisted semen analysis (CASA) machines, making kinematics more widely available, there was an explosion in studies reporting the relationship of quantitative motility and kinematic parameters with conception in vivo (Barratt et al., 1992), in donor insemination (Irvine and Aitken, 1986), in IUI (Bollendorf et al., 1996), IVF (Liu et al., 1991) and even ICSI (Van den Bergh et al., 1998). However, in the last 15 years, there have been a paucity of further studies perhaps due to the limitations in CASA technology but more likely due to the perceived lack of clinical need following the development of ICSI. Another clinically relevant aspect of sperm motility research that has apparently been shelved in the archives is the potential for in vitro enhancement of motility using a wide variety of drugs that target phosphodiesterases (PDEs) and increase [cAMP]. Simplistically, increasing the number of functional cells at the site of fertilization would appear to be a rational objective (Fig. 1). Historically, the most widely used drug was pentoxifylline where results were encouraging in both IVF and IUI (Yovich et al., 1990; Stone et al., 1999). However, the lack of recent research is particularly noticeable in the context of the overwhelming progress on specific (third generation) PDE inhibitors. While targeting of PDE5 is not likely to be very useful (Lefievre et al., 2000), there are a number of new-generation PDE inhibitors available that may positively influence sperm function. It is of course not just PDEs that require investigation and the modification of other enzymes, e.g. kinases and phosphatases, will provide a wealth of interesting data. In contrast to the above, which could be described as sleeping giants of ‘clinical andrology’, there has been breathtaking progress in the understanding of sperm motility in the research laboratory, largely brought on by developments in technology: patch clamping, imaging and the rudimentary synergy of mathematics/physics with sperm biology (systems biology of sperm). A primary example is the role of Ca2+ in regulating the activity of the flagellum. Central to this has been the discovery of CatSper channels and elucidation of their role(s) in sperm function. In mice, the main source of Ca2+ entry for hyperactivation appears to be via pH-dependent CatSper channels in the plasma membrane of the flagellar principal piece. These channels are essential for male fertility as sperm from CatSper null mice are motile but unable to hyperactivate, rendering them unable to migrate to or within the oviduct and unable to fertilize oocytes even by IVF (Ren et al., 2001; Carlson et al., 2003; Quill et al., 2003). CatSper channels are also present in the flagellum of human

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

Signaling through [Ca2+]i is central to regulation of sperm activity and is likely to be the mechanism by which signal from the female tract regulate motility of sperm. In a recent paper1 we showed that exposure of sperm to nitric oxide mobilizes stored Ca2+ 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 Ca2+ and enhance flagellar beating. Here we argue that a Ca2+ 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.