The acrosome reaction is a secretory event that must be completed by the sperm of many animal species prior to fusion with eggs. In mammals, exocytosis in triggered by ZP3, a glycoprotein component of the egg pellucida, following gamete contact. ZP3 promotes a sustained influx of Ca 2+ into sperm that is necessary for the acrosome reaction. Here, we discuss the mechanism by which ZP3 generates Ca 2+ entry, as well as the upstream events leading to this influx and downstream processes that couple it with exocytosis.

Although substantial evidence exists that sperm ATP production via glycolysis is required for mammalian sperm function and male fertility, conflicting reports involving multiple species have appeared regarding the ability of individual glycolytic or mitochondrial substrates to support the physiological changes that occur during capacitation. Several mouse models with defects in the signaling pathways required for capacitation exhibit reductions in sperm ATP levels, suggesting regulatory interactions between sperm metabolism and signal transduction cascades. To better understand these interactions, we conducted quantitative studies of mouse sperm throughout a 2-h in vitro capacitation period and compared the effects of single substrates assayed under identical conditions. Multiple glycolytic and nonglycolytic substrates maintained sperm ATP levels and comparable percentages of motility, but only glucose and mannose supported hyperactivation. These monosaccharides and fructose supported the full pattern of tyrosine phosphorylation, whereas nonglycolytic substrates supported at least partial tyrosine phosphorylation. Inhibition of glycolysis impaired motility in the presence of glucose, fructose, or pyruvate but not in the presence of hydroxybutyrate. Addition of an uncoupler of oxidative phosphorylation reduced motility with pyruvate or hydroxybutyrate as substrates but unexpectedly stimulated hyperactivation with fructose. Investigating differences between glucose and fructose in more detail, we demonstrated that hyperactivation results from the active metabolism of glucose. Differences between glucose and fructose appeared to be downstream of changes in intracellular pH, which rose to comparable levels during incubation with either substrate. Sperm redox pathways were differentially affected, with higher levels of associated metabolites and reactive oxygen species generated during incubations with fructose than during incubations with glucose.

STUDY QUESTIONAre significant abnormalities of CatSper function present in IVF patients with normal sperm concentration and motility and if so what is their functional significance for fertilization success?SUMMARY ANSWERSperm with a near absence of CatSper current failed to respond to activation of CatSper by progesterone and there was fertilization failure at IVF.WHAT IS KNOWN ALREADYIn human spermatozoa, Ca2+ influx induced by progesterone is mediated by CatSper, a sperm-specific Ca2+ channel. A suboptimal Ca2+ influx is significantly associated with, and more prevalent in, men with abnormal semen parameters, and is associated with reduced fertilizing capacity. However, abnormalities in CatSper current can only be assessed directly using electrophysiology. There is only one report of a CatSper-deficient man who showed no progesterone potentiated CatSper current. A CatSper 2 genetic abnormality was present but there was no information on the [Ca2+]i response to CatSper activation by progesterone. Additionally, the semen samples had indicating significant abnormalities (oligoasthenoteratozoospermia) multiple suboptimal functional responses in the spermatozoon. As such it cannot be concluded that impaired CatSper function alone causes infertility or that CatSper blockade is a potential safe target for contraception.STUDY DESIGN, SIZE, DURATIONSpermatozoa were obtained from donors and subfertile IVF patients attending a hospital assisted reproductive techniques clinic between January 2013 and December 2014. In total 134 IVF patients, 28 normozoospermic donors and 10 patients recalled due to a history of failed/low fertilization at IVF took part in the study.PARTICIPANTS/MATERIALS, SETTING, METHODSSamples were primarily screened using the Ca2+ influx induced by progesterone and, if cell number was sufficient, samples were also assessed by hyperactivation and penetration into viscous media. A defective Ca2+ response to progesterone was defined using the 99% confidence interval from the distribution of response amplitudes in normozoospermic donors. Samples showing a defective Ca2+ response were further examined in order to characterize the potential CatSper abnormalities. In men where there was a consistent and robust failure of calcium signalling, a direct assessment of CatSper function was performed using electrophysiology (patch clamping), and a blood sample was obtained for genetic analysis.MAIN RESULTS AND THE ROLE OF CHANCEA total of 101/102 (99%) IVF patients and 22/23 (96%) donors exhibited a normal Ca2+ response. The mean (±SD) normalized peak response did not differ between donors and IVF patients (2.57 ± 0.68 [n = 34 ejaculates from 23 different donors] versus 2.66 ± 0.68 [n = 102 IVF patients], P = 0.63). In recall patients, 9/10 (90%) showed a normal Ca2+ response. Three men were initially identified with a defective Ca2+ influx. However, only one (Patient 1) had a defective response in repeat semen samples. Electrophysiology experiments on sperm from Patient 1 showed a near absence of CatSper current an...

The TRPC cation channel family has been implicated in receptor- or phospholipase C (PLC)-mediated Ca2+ entry into animal cells. These channels are present in mammalian sperm and are assigned a role in ZP3-evoked Ca2+ influx that drives acrosome reactions. However, the mechanisms controlling channel activity and coupling Ca2+ entry through these channels to cellular responses are not well understood. A yeast two-hybrid screen was carried out to identify TRPC-interacting proteins that would be candidate regulators or effectors. We identified a novel protein, enkurin, that is expressed at high levels in the testis and vomeronasal organ and at lower levels in selected other tissues. Enkurin interacts with several TRPC proteins (TRPC1, TRPC2, TRPC5, but not TRPC3) and colocalizes with these channels in sperm. Three protein-protein interaction domains were identified in enkurin: a C-terminal region is essential for channel interaction; an IQ motif binds the Ca2+ sensor, calmodulin, in a Ca2+-dependent manner; and a proline-rich N-terminal region contains predicted ligand sequences for SH3 domain proteins, including the SH3 domain of the p85 regulatory subunit of 1-phosphatidylinositol-3-kinase. We suggest that enkurin is an adaptor that functions to localize a Ca2+ sensitive signal transduction machinery in sperm to a Ca2+-permeable ion channel.

STUDY QUESTIONAre significant abnormalities in outward (K+) conductance and resting membrane potential (Vm) present in the spermatozoa of patients undertaking IVF and ICSI and if so, what is their functional effect on fertilization success?SUMMARY ANSWERNegligible outward conductance (≈5% of patients) or an enhanced inward conductance (≈4% of patients), both of which caused depolarization of Vm, were associated with a low rate of fertilization following IVF.WHAT IS KNOWN ALREADYSperm-specific potassium channel knockout mice are infertile with defects in sperm function, suggesting that these channels are essential for fertility. These observations suggest that malfunction of K+ channels in human spermatozoa might contribute significantly to the occurrence of subfertility in men. However, remarkably little is known of the nature of K+ channels in human spermatozoa or the incidence and functional consequences of K+ channel defects.STUDY DESIGN, SIZE AND DURATIONSpermatozoa were obtained from healthy volunteer research donors and subfertile IVF and ICSI patients attending a hospital assisted reproductive techniques clinic between May 2013 and December 2015. In total, 40 IVF patients, 41 ICSI patients and 26 normozoospermic donors took part in the study.PARTICIPANTS/MATERIALS, SETTING, METHODSSamples were examined using electrophysiology (whole-cell patch clamping). Where abnormal electrophysiological characteristics were identified, spermatozoa were further examined for Ca2+ influx induced by progesterone and penetration into viscous media if sufficient sample was available. Full exome sequencing was performed to specifically evaluate potassium calcium-activated channel subfamily M α 1 (KCNMA1), potassium calcium-activated channel subfamily U member 1 (KCNU1) and leucine-rich repeat containing 52 (LRRC52) genes and others associated with K+ signalling. In IVF patients, comparison with fertilization rates was done to assess the functional significance of the electrophysiological abnormalities.MAIN RESULTS AND THE ROLE OF CHANCEPatch clamp electrophysiology was used to assess outward (K+) conductance and resting membrane potential (Vm) and signalling/motility assays were used to assess functional characteristics of sperm from IVF and ICSI patient samples. The mean Vm and outward membrane conductance in sperm from IVF and ICSI patients were not significantly different from those of control (donor) sperm prepared under the same conditions, but variation between individuals was significantly greater (P< 0.02) with a large number of outliers (>25%). In particular, in ≈10% of patients (7/81), we observed either a negligible outward conductance (4 patients) or an enhanced inward current (3 patients), both of which caused depolarization of Vm. Analysis of clinical data from the IVF patients showed significant association of depolarized Vm (≥0 mV) with low fertilization rate (P= 0.012). Spermatozoa with electrophysiological abnormities (conductance and Vm) responded normally to progesterone with elevation of [Ca2+]i and penetr...

Pkdrej, a member of the polycystin-1 gene family, is expressed only in the male germ line. Male mice that are homozygous for a targeted mutation in the Pkdrej allele (Pkdrej tm/tm ) are fertile in unrestricted mating trials, but exhibit lower reproductive success when competing with wild-type males in sequential mating trials and in artificial insemination of mixed-sperm populations. Following mating, sperm from Pkdrej tm/tm mice require >2 h longer than those of wild-type males to be detected within the egg/cumulus complex in the oviduct. Sperm from mice of both genotypes are able to capacitate in vitro. However, one of the component processes of capacitation, the ability to undergo a zona pellucidaevoked acrosome reaction, develops more slowly in sperm from Pkdrej tm/tm animals than in sperm from wild-type males. In contrast, a second component process of capacitation, the transition to hyperactivated flagellar motility, develops with a similar time course in both genotypes. These two behavioral consequences of capacitation, exocytotic competence and altered motility, are therefore differentially regulated. These data suggest that Pkdrej controls the timing of fertilization in vivo through effects on sperm transport and exocytotic competence and is a factor in postcopulatory sexual selection.capacitation ͉ evolution ͉ fertilization ͉ polycystin ͉ sexual selection

One often-noted feature of homeobox genes is the conservation of the homeodomain among orthologous genes from distantly related species. This sequence conservation is presumed to reflect functional conservation, which indeed has been demonstrated in several cases. We analyzed the evolution of an orphan homeobox gene, Pem, which is expressed preferentially in male and female reproductive tissue. Sequence analysis of 12 species of mice and rats indicated that the Pem gene has evolved at a remarkably high rate. The most rapidly evolving region of the Pem protein is the amino portion of the homeodomain, including the flexible N-terminal arm, helices I and II, and the linker regions between the helices. In contrast, the third helix, which is known to mediate base-specific DNA contacts in other homeodomains, is conserved in the Pem protein. Analysis of the ratio of nonsynonymous and synonymous codon substitution rates within the Pem homeodomain suggested that its divergence was driven by adaptive selection. The rate of nonsynonymous substitutions in Pem was higher than that of the sex-determination gene Sry, which also appears to have undergone directional selection over a short evolutionary period. Despite the rapid evolution of the Pem gene, we detected no Pem polymorphisms and observed no variation in the homeobox sequence among closely related Mus species. This suggests that purifying episodes followed phases in which selection pressure drove the rapid divergence of this locus. We propose that transcription factors that function in reproductive events can be subject to rapid adaptive selection.

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