The ovarian hormone progesterone classically binds to a nuclear receptor, initiating gene transcription. But how does it stimulate the transcriptionally inactive human spermatozoon in preparation for fertilization? This question has long both fascinated and frustrated reproductive biologists. In this issue, Strünker and colleagues1 and Lishko et al.2 provide an unexpected answer: progesterone activates a sperm-specific calcium ion (Ca2+) channel called CatSper.
For a sperm to reach the egg, it must penetrate the cumulus oophorus, a thick layer around the egg composed of granulosa cells embedded in a gelatinous matrix. These cells actively synthesize progesterone, such that its concentration within the cumulus is in the micromolar range. It was first reported more than 20 years ago3 that progesterone, even at concentrations well below those present in the cumulus, induces immediate influx into human sperm of Ca2+ — a factor central to regulation of sperm function4,5. Progesterone is therefore believed to have a crucial role during the events leading to fertilization6.
Sperm cells respond to progesterone within less than a second, which is characteristic of classical signalling pathways that involve cell-surface receptors3,6. Such non-nuclear actions of steroid hormones are quite common. In fact, progesterone and its related hormones are considered to have two distinct modes of action: through intracellular nuclear receptors, which regulate transcription; and through non-genomic receptors, probably at the plasma membrane, which regulate ion channels, G-protein-coupled receptors and signalling pathways mediated by kinase enzymes7. However, the mechanism of progesterone-induced Ca2+ influx in sperm has resisted all attempts at characterization, with even the type of 'receptor', let alone the nature of the Ca2+-influx pathway, remaining a mystery. This has been particularly frustrating because the phenomenon is probably of considerable clinical significance: in human sperm, failure of progesterone-activated Ca2+ influx is correlated with reduced fertility6.
The solution to this mystery follows directly from two crucial advances in the field. First, in 2001 two groups8,9 reported the discovery of the Ca2+-permeable cation channel (CatSper), which is expressed only in the plasma membrane of a domain in the sperm tail called the principal piece. Sperm from genetically manipulated mice that cannot express CatSper have impaired motility and, crucially, cannot display hyperactivation — an extravagant, highly asymmetric form of flagellar beating that is regulated by Ca2+ and is essential for fertilization. CatSper-deficient male mice are infertile.
The second, more recent, advance was the development of a method for applying to sperm the technique of whole-cell patch clamping, which records ionic currents across the entire plasma membrane of a cell. Using this technique, researchers showed that increased alkalinity of the sperm cytoplasm strongly activates CatSper channels, promoting Ca2+ flux into the cell. Strünker et al. (page 382) and Lishko and colleagues (page 387) now use this powerful technique to elucidate the mechanism by which progesterone induces rapid Ca2+ influx into human sperm.
Progesterone-induced membrane currents have identical characteristics to those carried by CatSper. For instance, the biophysical aspects of the currents are indistinguishable, with both progesterone and increased intracellular pH stimulating CatSper by shifting its voltage sensitivity so that it opens at lower voltages (Fig. 1). What's more, pharmacological manipulation has the same effects both on CatSper currents activated by increasing intracellular pH and on those stimulated by progesterone; applied together, progesterone and increased alkalinity act synergistically1,2.
The effect of progesterone on CatSper is not simply a nonspecific effect of steroid hormones: another steroid hormone, oestradiol, has no effect on this channel2. However, several prostaglandins — non-protein mediators that increase intracellular Ca2+ concentration in human sperm — have strikingly similar effects to progesterone. Moreover, Strünker and colleagues' measurements of intracellular Ca2+ concentration in progesterone-stimulated sperm showed that compounds that block CatSper currents also reduce the progesterone-induced rise in Ca2+ concentration, and that stringent buffering of this ion in the external medium abolishes the response to progesterone1. Intriguingly, the efficacy of progesterone is increased by in vitro manipulations designed to induce sperm capacitation (a crucial maturation process that naturally occurs in the female reproductive tract before fertilization).
The non-genomic action of progesterone is much more potent in human sperm than in mouse sperm2. But why? Lishko and colleagues show that, at the intracellular pH of 7.0 (a value within the physiological range), mouse spermatozoa show notable CatSper currents, whereas human sperm show a much smaller current. On applying progesterone to human sperm, the current increases to a level closely resembling that in mouse sperm, but in mouse sperm stimulation with this hormone leads to no further increase in current2. It seems, therefore, that in human sperm, progesterone induces a modulation of CatSper function that in mouse sperm is constitutive (at least under the conditions used in these experiments). This is potentially a crucial species difference in sperm regulation within the female reproductive tract.
The two papers also present a much clearer idea of how progesterone exerts its effect by modulating CatSper. Lishko et al.2 could record progesterone-induced currents even in isolated sperm tails, which precludes indirect effects of progesterone exerted through receptors on the sperm head. Furthermore, Strünker et al.1 provide compelling evidence that progesterone does not stimulate synthesis of the signalling molecule cyclic AMP, and they couldn't detect any effects of manipulating cAMP levels on Ca2+ influx through the sperm membrane. These observations rule out involvement of the cAMP–protein kinase A signalling cascade in the progesterone–CatSper response.
The new data also suggest that progesterone directly activates CatSper, by binding either to the channel itself or to an associated subunit(s). Whether CatSper activation is the only effect of progesterone on Ca2+-signalling in human sperm remains to be seen. Several previous studies have attempted to identify progesterone receptors6. Both novel receptors and truncated versions of the classical (nuclear-type) receptors (some of these apparently localized to the sperm head) were proposed to mediate the effects of this hormone. Although such receptors almost certainly do not contribute to the modulation of CatSper reported here, it is noteworthy that completely blocking CatSper currents inhibits — but does not abolish — the effect of progesterone on intracellular Ca2+ levels1,10.
Mobilization of intracellular Ca2+ stores, leading to complex Ca2+ signalling, occurs in progesterone-stimulated human sperm5. Is this purely a downstream effect of CatSper activation or does progesterone activate a separate pathway? Are store-controlled Ca2+ channels involved? These two studies1,2 provide exciting insights, and there is more to come.
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Diagnosis and Prognosis of Male Infertility in Mammal: The Focusing of Tyrosine Phosphorylation and Phosphotyrosine Proteins
Journal of Proteome Research (2014)