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.

Abstract STUDY QUESTION Can pharma drug discovery approaches be utilized to transform investigation into novel therapeutics for male infertility? SUMMARY ANSWER High-throughput screening (HTS) is a viable approach to much-needed drug discovery for male factor infertility. WHAT IS KNOWN ALREADY There is both huge demand and a genuine clinical need for new treatment options for infertile men. However, the time, effort and resources required for drug discovery are currently exorbitant, due to the unique challenges of the cellular, physical and functional properties of human spermatozoa and a lack of appropriate assay platform. STUDY DESIGN, SIZE, DURATION Spermatozoa were obtained from healthy volunteer research donors and subfertile patients undergoing IVF/ICSI at a hospital-assisted reproductive techniques clinic between January 2012 and November 2016. PARTICIPANTS/MATERIALS, SETTING, METHODS A HTS assay was developed and validated using intracellular calcium ([Ca2+]i) as a surrogate for motility in human spermatozoa. Calcium fluorescence was detected using a Flexstation microplate reader (384-well platform) and compared with responses evoked by progesterone, a compound known to modify a number of biologically relevant behaviours in human spermatozoa. Hit compounds identified following single point drug screen (10 μM) of an ion channel-focussed library assembled by the University of Dundee Drug Discovery Unit were rescreened to ensure potency using standard 10 point half-logarithm concentration curves, and tested for purity and integrity using liquid chromatography and mass spectrometry. Hit compounds were grouped by structure activity relationships and five representative compounds then further investigated for direct effects on spermatozoa, using computer-assisted sperm assessment, sperm penetration assay and whole-cell patch clamping. MAIN RESULTS AND THE ROLE OF CHANCE Of the 3242 ion channel library ligands screened, 384 compounds (11.8%) elicited a statistically significant increase in calcium fluorescence, with greater than 3× median absolute deviation above the baseline. Seventy-four compounds eliciting ≥50% increase in fluorescence in the primary screen were rescreened and evaluated further, resulting in 48 hit compounds that produced a concentration-dependent increase in [Ca2+]i. Sperm penetration studies confirmed in vitro exposure to two hit compounds (A and B) resulted in significant improvement in functional motility in spermatozoa from healthy volunteer donors (A: 1 cm penetration index 2.54, 2 cm penetration index 2.49; P < 0.005 and B: 1 cm penetration index 2.1, 2 cm penetration index 2.6; P < 0.005), but crucially, also in patient samples from those undergoing fertility treatment (A: 1 cm penetration index 2.4; P = 0.009, 2 cm penetration index 3.6; P = 0.02 and B: 1 cm penetration index 2.2; P = 0.0004, 2 cm penetration index 3.6; P = 0.002). This was primarily as a result of direct or indirect CatSper channel action, supported by evidence from electrophysiology studies of individual sperm. LIMITATIONS, REASONS FOR CAUTION Increase and fluxes in [Ca2+]i are fundamental to the regulation of sperm motility and function, including acrosome reaction. The use of calcium signalling as a surrogate for sperm motility is acknowledged as a potential limitation in this study. WIDER IMPLICATIONS OF THE FINDINGS We conclude that HTS can robustly, efficiently, identify novel compounds that increase [Ca2+]i in human spermatozoa and functionally modify motility, and propose its use as a cornerstone to build and transform much-needed drug discovery for male infertility. STUDY FUNDING/COMPETING INTEREST(S) The majority of the data were obtained using funding from TENOVUS Scotland and Chief Scientist Office NRS Fellowship. Additional funding was provided by NHS Tayside, MRC project grants (MR/K013343/1, MR/012492/1) and University of Abertay. The authors declare that there is no conflict of interest. TRAIL REGISTRATION NUMBER N/A.

STUDY QUESTION Are 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 ANSWER Negligible 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 ALREADY Sperm-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 DURATION Spermatozoa 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, METHODS Samples 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 CHANCE Patch 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 penetration of viscous medium, indicating retention of cation channel of sperm (CatSper) channel function. LIMITATIONS, REASONS FOR CAUTION For practical, technical, ethical and logistical reasons, we could not obtain sufficient additional semen samples from men with conductance abnormalities to establish the cause of the conductance defects. Full exome sequencing was only available in two men with conductance defects. WIDER IMPLICATIONS OF THE FINDINGS These data add significantly to the understanding of the role of ion channels in human sperm function and its impact on male fertility. Impaired potassium channel conductance (Gm) and/or Vm regulation is both common and complex in human spermatozoa and importantly is associated with impaired fertilization capacity when the Vm of cells is completely depolarized. STUDY FUNDING/COMPETING INTEREST(S) The majority of the data were obtained using funding from MRC project grants (#MR/K013343/1, MR/012492/1). Additional funding was provided by NHS Tayside, TENOVUS, Chief Scientist Office NRS Fellowship and University of Abertay. The authors declare that there is no conflict of interest. TRIAL REGISTRATION NUMBER Not applicable.

STUDY QUESTION: Are 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 ANSWER: Sperm 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 ALREADY: In 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, DURATION: Spermatozoa 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, METHODS: Samples 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 CHANCE: A 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 and exon screening demonstrated no mutations in the coding regions of the CatSper complex. There was no increase in penetration of viscous media when the spermatozoa were stimulated with progesterone and importantly there was failed fertilization at IVF.LIMITATIONS, REASONS FOR CAUTION: A key limitation relates to working with a specific functional parameter (Ca2+ influx induced by progesterone) in fresh sperm samples from donors and patients that have limited viability. Therefore, for practical, technical and logistical reasons, some men (similar to 22% of IVF patients) could not be screened. As such the incidence of significant Ca2+ abnormalities induced by progesterone may be higher than the similar to 1% observed here. Additionally, we used a strict definition of a defective Ca2+ influx such that only substantial abnormalities were selected for further study. Furthermore, electrophysiology was only performed on one patient with a robust and repeatable defective calcium response. This man had negligible CatSper current but more subtle abnormalities (e.g. currents present but significantly smaller) may have been present in men with either normal or below normal Ca2+ influx.WIDER IMPLICATIONS OF THE FINDINGS: These data add significantly to the understanding of the role of CatSper in human sperm function and its impact on male fertility. Remarkably, these findings provide the first direct evidence that CatSper is a suitable and specific target for human male contraception.

PKDREJ is a testis-specific protein thought to be located on the sperm surface. Functional studies in the mouse revealed that loss of PKDREJ has effects on sperm transport and the ability to undergo an induced acrosome reaction. Thus, PKDREJ has been considered a potential target of post-copulatory sexual selection in the form of sperm competition. Proteins involved in reproductive processes often show accelerated evolution. In many cases, this rapid divergence is promoted by positive selection which may be driven, at least in part, by post-copulatory sexual selection. We analysed the evolution of the PKDREJ protein in primates and rodents and assessed whether PKDREJ divergence is associated with testes mass relative to body mass, which is a reliable proxy of sperm competition levels. Evidence of an association between the evolutionary rate of the PKDREJ gene and testes mass relative to body mass was not found in primates. Among rodents, evidence of positive selection was detected in the Pkdrej gene in the family Cricetidae but not in Muridae. We then assessed whether Pkdrej divergence is associated with episodes of sperm competition in these families. We detected a positive significant correlation between the evolutionary rates of Pkdrej and testes mass relative to body mass in cricetids. These findings constitute the first evidence of post-copulatory sexual selection influencing the evolution of a protein that participates in the mechanisms regulating sperm transport and the acrosome reaction, strongly suggesting that positive selection may act on these fertilization steps, leading to advantages in situations of sperm competition.

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.

Molecular Reproduction and DevelopmentVolume 79, Issue 5 p. Fm i-Fm i EditorialFree Access Coup d'oeil of sperm Harvey M. Florman, Harvey M. Florman Department of Cell Biology, University of Massachusetts Medical School, Worcester MA 01655Search for more papers by this authorWenlei Cao, Wenlei Cao Department of Cell Biology, University of Massachusetts Medical School, Worcester MA 01655Search for more papers by this authorMelissa K. Jungnickel, Melissa K. Jungnickel Department of Cell Biology, University of Massachusetts Medical School, Worcester MA 01655Search for more papers by this authorKeith A. Sutton, Keith A. Sutton Department of Cell Biology, University of Massachusetts Medical School, Worcester MA 01655Search for more papers by this authorPei-Shiue Tsai, Pei-Shiue Tsai Department of Cell Biology, University of Massachusetts Medical School, Worcester MA 01655Search for more papers by this author Harvey M. Florman, Harvey M. Florman Department of Cell Biology, University of Massachusetts Medical School, Worcester MA 01655Search for more papers by this authorWenlei Cao, Wenlei Cao Department of Cell Biology, University of Massachusetts Medical School, Worcester MA 01655Search for more papers by this authorMelissa K. Jungnickel, Melissa K. Jungnickel Department of Cell Biology, University of Massachusetts Medical School, Worcester MA 01655Search for more papers by this authorKeith A. Sutton, Keith A. Sutton Department of Cell Biology, University of Massachusetts Medical School, Worcester MA 01655Search for more papers by this authorPei-Shiue Tsai, Pei-Shiue Tsai Department of Cell Biology, University of Massachusetts Medical School, Worcester MA 01655Search for more papers by this author First published: 16 April 2012 https://doi.org/10.1002/mrd.22039AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onEmailFacebookTwitterLinkedInRedditWechat No abstract is available for this article. Volume79, Issue5May 2012Pages Fm i-Fm i RelatedInformation

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.

Sage advice, particularly if you are a hermaphrodite organism seeking to take advantage of sexual reproduction. Promotion of out-breeding requires that male gametes do not fertilize the eggs being released from the same organism, or only do so at very low efficiency. This problem impacts a variety of organisms from flowering plants (Rea and Nasrallah, 2008) to ascidians, the sea squirts. Indeed the basic process is equally fundamental to the vertebrate immune response (Burnet, 1971). Interestingly the receptor/ligand pairs that mediate gamete self/non-self discrimination (allorecognition) are not phylogenetically conserved, indicating that these systems are a striking example of convergent evolution. Ciona intestinalis (photo by Lixy Yamada) In the ascidian Ciona intestinalis it has long been known that the key allorecognition event occurs at the level of the egg's vitelline coat (Morgan, 1923). In the current issue, Akira Yamaguchi and colleagues from Hitoshi Sawada's laboratory move us a significant step closer to understanding how the gametes of C. intestinalis manage this task. Previous work from this group had identified two highly polymorphic, unlinked loci: themis A and B, which each encode two products, a sperm membrane receptor (s-themis) and an egg coat ligand (v-themis). Should the sperm s-themis receptors bind to v-themis ligand encoded by the same allele, “self” is signaled and fertilization is prevented (Harada et al., 2008). While this elegant system explains Morgan's observations on gamete compatibility, which gave rise to the haploid sperm hypothesis, it is not complete. After all it is the failure to recognize self, for example, no binding between s-themis on the sperm head and v-themis in the vitelline coat, that is the signal for fertilization. Clearly there must be additional receptor–ligand pairs involved in sperm–egg interactions. A clue comes from other studies by the same group using a second ascidian species, Halocynthia roretzi. This work implicated a second pair of proteins: HrVC70, an EGF repeat protein in the vitelline coat, and HrUrabin, a GPI linked CRISP protein on the sperm plasma membrane (Urayama et al., 2008). Blockade of the HrUrabin/HrVC70 interaction blocks fertilization in this species. Is a similar interaction occurring in C. intestinalis? In the current article, the authors clone CiUrabin, a protein that interacts with the C. intestinalis ortholog of HrVC70, CiVC57. Despite low levels of amino acid identity, both CiUrabin and HrUrabin are members of the CRISP family of proteins and have a similar domain structure, have key conserved cysteine residues, and appear to be attached to the sperm membrane by GPI linkages. This allows the proposal of an elegant two-step model to allorecognition: (1) initial binding of sperm of C. intestinalis to the vitelline coat via CiUrabin/CiVC57 is followed by (2) an allorecognition event mediated by the themis proteins. If there is no recognition of self then fertilization proceeds, but if s-themis A and B bind to their allelic v-themis then a signaling cascade triggers the shedding of the sperm from the vitelline coat and self-fertilization is prevented. This model raises a number of questions. One is the nature of the signaling cascade derived from s-themis receptors. This signaling event must be rapid enough to prevent sperm from penetrating the vitelline coat, but it requires that both s-themis proteins (A and B) be bound to prevent fertilization. How does the spermatozoon manage this task? While both s-themis proteins are members of the polycystin family, only s-themis-B is structurally complete with 11 transmembrane domains. The s-themis A protein is truncated, suggesting that two distinct signaling cascades are activated and must cooperate to block fertilization. A second question is the diversity in the molecules ascidians use to mediate gamete recognition, and how this relates to the allorecognition event. In H. roretzi HrVC70 is highly polymorphic and has been proposed to play an indirect role in gamete allorecognition as it shows a higher binding affinity towards non-self sperm (Sawada et al., 2004). It is unknown if a similar phenomenon occurs in C. intestinalis. Divergence of the gamete allorecognition system within ascidians may not be that surprising given that in the colonial form of Botryllus schlosseri, the allorecognition molecules that govern colony fusion versus rejection are distinct from both the themis and HrVC70/CiVC57/Urabin proteins (McKitrick et al., 2011). The lack of primary sequence conservation between the HrUrabin and CiUrabin proteins may also hint at selective pressures driving their divergence. Analysis of the allelic structures of CiVC57 and also determining the presence or absence of themis loci within H. roretzi will be essential. It may well be that there are many paths to self-knowledge.

In mammals the number of sperm introduced into the female tract is orders of magnitude greater than the number of eggs available for fertilization. By the time spermatozoa reach the site of fertilization the ratio of sperm to egg has fallen due to regulation of sperm transport by the female tract. Male mice homozygous for a targeted mutation in pkdrej (pkdrej(tm/tm)) show normal fertility in unrestricted matings but sperm from these animals compete poorly with those of wild type males in vivo. Pkdrej is a member of the polycystin-1 family of genes which encode large 11 pass transmembrane proteins. Mutations in another member of the family, Pkd1, underly the majority of cases of autosomal dominant polycystic kidney disease. Unlike other family members pkdrej expression is limited to the male germline in both mouse and human. In vitro analysis showed that while motility and other aspects of the capacitation program are normal, there is a delay in the acquisition of the potential to undergo a zona pellucida induced acrosome reaction. To examine the basis for the under performance of sperm from these mice, we observed the migration of sperm within the oviducts of hormonally superovulated females mated at the time of ovulation. Using trans-illumination of oviducts to observe the behavior of sperm within, we found apparent differences between the behavior of sperm from wild type males and pkdrej(tm/tm) males. At 1.5 h after mating, sperm from pkdrej(tm/tm) mice showed reduced colonization of the extramural uterotubal junction and lower isthmus, which comprise the site of the sperm storage reservoir. The mutant sperm in this region were mostly hyperactivated and free swimming; whereas, most sperm from wild type males were bound by their heads to the oviductal epithelium. A few wild type sperm were seen beyond the reservoir in the upper isthmus and ampulla (oviducts from 6 mated females), but no sperm from pkdrej(tm/tm) males were found beyond the region of the resevoir (oviducts from 5 mated females). These observations indicate that sperm from pkdrej(tm/tm) males are defective in colonization of the storage reservoir and subsequent movement up the oviduct, which could explain the reduced competitiveness of the mutant males with wild type males for fertilization of oocytes. Supported by NIH 1R03HD062471-01 (S. Suarez), 5R03HD060034-01 (K. Sutton) (poster)

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.

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.

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.

It is a curious and intriguing situation that mammalian sperm are introduced into the female reproductive tract in an infertile state and must be educated there before they are able to fertilize oocytes. The recognition that mammalian sperm must first be switched into a competent state, or capacitated, was exploited in the development of in vitro fertilization methods and has proved essential for the dependent technologies of clinical assisted reproduction and infertility treatments. Yet many aspects of the mechanisms of capacitation remain unclear (for recent reviews, see refs. 1 and 2). In a recent issue of PNAS, Xu et al. (3) advanced our understanding of capacitation by showing that CFTR, the cystic fibrosis transmembrane regulator, plays an essential role in some aspects of this process.