Molecular Mechanism Underlying the Action of Zona-pellucida Glycoproteins on Mouse Sperm.

Mammalian oocytes are enveloped by the zona pellucida (ZP), an extracellular matrix of glycoproteins. In sperm, stimulation with ZP proteins evokes a rapid Ca2+ influx via the sperm-specific, pH-sensitive Ca2+ channel CatSper. However, the physiological role and molecular mechanisms underlying ZP-dependent activation of CatSper are unknown. Here, we delineate the sequence of ZP-signaling events in mouse sperm. We show that ZP proteins evoke a rapid intracellular pH i increase that rests predominantly on Na+/H+ exchange by NHA1 and requires cAMP synthesis by the soluble adenylyl cyclase sAC as well as a sufficiently negative membrane potential set by the spem-specific K+ channel Slo3. The alkaline-activated CatSper channel translates the ZP-induced pH i increase into a Ca2+ response. Our findings reveal the molecular components underlying ZP action on mouse sperm, opening up new avenues for understanding the basic principles of sperm function and, thereby, mammalian fertilization.

The flagellar protein Enkurin is required for mouse sperm motility and for transport through the female reproductive tract.

Enkurin was identified initially in mouse sperm where it was suggested to act as an intracellular adaptor protein linking membrane calcium influx to intracellular signaling pathways. In order to examine the function of this protein, a targeted mutation was introduced into the mouse Enkurin gene. Males that were homozygous for this mutated allele were subfertile. This was associated with lower rates of sperm transport in the female reproductive tract, including reduced entry into the oviduct and slower migration to the site of fertilization in the distal oviduct, and with poor progressive motility in vitro. Flagella from wild-type animals exhibited symmetrical bending and progressive motility in culture medium, and demembranated flagella exhibited the "curlicue" response to Ca2+ in vitro. In contrast, flagella of mice homozygous for the mutated allele displayed only asymmetric bending, nonprogressive motility, and a loss of Ca2+-responsiveness following demembrantion. We propose that Enkurin is part of a flagellar Ca2+-sensor that regulates bending and that the motility defects following mutation of the locus are the proximate cause of subfertility.

Evolutionary Proteomics Uncovers Ancient Associations of Cilia with Signaling Pathways.

Cilia are organelles specialized for movement and signaling. To infer when during evolution signaling pathways became associated with cilia, we characterized the proteomes of cilia from sea urchins, sea anemones, and choanoflagellates. We identified 437 high-confidence ciliary candidate proteins conserved in mammals and discovered that Hedgehog and G-protein-coupled receptor pathways were linked to cilia before the origin of bilateria and transient receptor potential (TRP) channels before the origin of animals. We demonstrated that candidates not previously implicated in ciliary biology localized to cilia and further investigated ENKUR, a TRP channel-interacting protein identified in the cilia of all three organisms. ENKUR localizes to motile cilia and is required for patterning the left-right axis in vertebrates. Moreover, mutation of ENKUR causes situs inversus in humans. Thus, proteomic profiling of cilia from diverse eukaryotes defines a conserved ciliary proteome, reveals ancient connections to signaling, and uncovers a ciliary protein that underlies development and human disease.

Evolution of the voltage sensor domain of the voltage-sensitive phosphoinositide phosphatase VSP/TPTE suggests a role as a proton channel in eutherian mammals.

The voltage-sensitive phosphoinositide phosphatases provide a mechanism to couple changes in the transmembrane electrical potential to intracellular signal transduction pathways. These proteins share a domain architecture that is conserved in deuterostomes. However, gene duplication events in primates, including humans, give rise to the paralogs TPTE and TPTE2 that retain protein domain organization but, in the case of TPTE, have lost catalytic activity. Here, we present evidence that these human proteins contain a functional voltage sensor, similar to that in nonmammalian orthologs. However, domains of these human proteins can also generate a noninactivating outward current that is not observed in zebra fish or tunicate orthologs. This outward current has the anticipated characteristics of a voltage-sensitive proton current and is due to the appearance of a single histidine residue in the S4 transmembrane segment of the voltage sensor. Histidine is observed at this position only during the eutherian radiation. Domains from both human paralogs generate proton currents. This apparent gain of proton channel function during the evolution of the TPTE protein family may account for the conservation of voltage sensor domains despite the loss of phosphatase activity in some human paralogs.

Mechanisms of sperm-egg interactions: between sugars and broken bonds.

A model of the early events of mammalian fertilization has emerged during the past 30 years. However, studies during the past decade have used newly available mouse models to readdress these processes. Here, we will consider these new data in light of the existing model and point to areas of reconciliation and of controversy.

Shedding light on sperm pHertility.

The acquisition of fertilization capacity by sperm is regulated by intracellular pH (pH(i)), but the transport pathways that regulate pH(i) are not well understood. Lishko et al. (2010) now report that Hv1, the voltage-sensitive proton channel, is present in human sperm and is an important regulator of the functional maturation of sperm.

Regulating the acrosome reaction.

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 Ca2+ into sperm that is necessary for the acrosome reaction. Here, we discuss the mechanism by which ZP3 generates Ca2+ entry, as well as the upstream events leading to this influx and downstream processes that couple it with exocytosis.

A polycystin-1 controls postcopulatory reproductive selection in mice.

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 pellucida-evoked 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.

Phosphoinositide-dependent pathways in mouse sperm are regulated by egg ZP3 and drive the acrosome reaction.

Sperm of many animals must complete an exocytotic event, the acrosome reaction, in order to fuse with eggs. In mammals, acrosome reactions are triggered during sperm contact with the egg extracellular matrix, or zona pellucida, by the matrix glycoprotein ZP3. Here, we show that ZP3 stimulates production of phosphatidylinositol-(3,4,5)-triphosphate in sperm membranes. Phosphatidylinositol-3-kinase antagonists that prevent acrosome reactions and fertilization in vitro, while generation of this phosphoinositide in the absence of ZP3 triggered acrosome reactions. Downstream effectors of phosphatidylinositol-(3,4,5)-triphosphate in sperm include the protein kinases, Akt and PKCzeta. These studies outline a signal transduction pathway that plays an essential role in the early events of mammalian fertilization.

Functional characterization of PKDREJ, a male germ cell-restricted polycystin.

Polycystin-1 regulates a number of cellular processes through the formation of complexes with the polycystin-2 ion channel or with other signal transduction proteins. Polycystin-1 is expressed in many tissues but other members of this gene family are distributed in a more restricted fashion. PKDREJ expression has been detected only in the mammalian testis, where it is restricted to the spermatogenic lineage and retained in mature sperm. However, the functional characteristics of this protein and its role in sperm biology are not well understood. In this study it is shown that PKDREJ can modulate G protein signaling and associates with several members of the polycystin-2 family. These interactions, as well as polycystin-2 association with TRPC channels, are consistent with a role of this protein in the regulation of the acrosome reaction and in other aspects of sperm physiology.

Enkurin is a novel calmodulin and TRPC channel binding protein in sperm.

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.

Bovine sperm capacitation: assessment of phosphodiesterase activity and intracellular alkalinization on capacitation-associated protein tyrosine phosphorylation.

Mammalian sperm capacitation is the obligatory maturational process leading to the development of the fertilization-competent state. Heparin is known to be a unique species-specific inducer of bovine sperm capacitation in vitro and glucose a unique inhibitor of this induction. Heparin-induced capacitation of bovine sperm has been shown to correlate with protein kinase A (PKA)-dependent protein tyrosine phosphorylation driven by an increase in intracellular cAMP. This study examines the possible roles of cyclic nucleotide phosphodiesterase (PDE) activity and intracellular alkalinization on bovine sperm capacitation and the protein tyrosine phosphorylation associated with it. Measurement of whole cell PDE kinetics during capacitation reveals neither a substantial change with heparin nor one with glucose: PDE activity is effectively constitutive in maintaining intracellular cAMP levels during capacitation. In contrast to a transient increase in intracellular pH, a sustained increase in medium pH by switching from 5% CO(2)/95% air incubation to 1% CO(2)/99% air incubation over 4 hr in the absence of heparin resulted in an increase in protein tyrosine phosphorylation and in the extent of induced acrosome reaction comparable to that observed following heparin-induced capacitation in 5% CO(2). These results suggest that increased bicarbonate-dependent adenylyl cyclase activity, driven by alkalinization, increases intracellular cAMP and so increases PKA activity mediating protein tyrosine phosphorylation. Quantitative analysis of the lactic acid production rate by bovine sperm glycolysis accounts fully for intracellular acidification sufficient to offset heparin-induced alkalinization, thus inhibiting capacitation. The mechanism by which heparin uniquely induces intracellular alkalinization in bovine sperm leading to capacitation remains obscure, inviting future investigation.

In the beginning: lessons from fertilization in mice and worms.

Sexual reproduction proceeds by fertilization; formation of new individuals by the union of haploid gametes. Recent reports in Cell and in Developmental Cell may provide new insights as to how this process begins and is regulated.