Catherine Serres

The acrosome reaction in human spermatozoa

During gamete interaction, sperm acrosome reaction (AR) induced by oocyte investment is a prerequisite event for the spermatozoa to pass through the zona pellucida (ZP), fuse with and penetrate the oocyte. Progesterone (P4), secreted by cumulus cells, is an important cofactor for the occurrence of this exocytosis event. The AR results from the fusion between outer acrosomal and plasma membranes, leading to inner acrosomal membrane exposure. Binding of agonists, P4 or ZP3 glycoprotein, to plasma membrane sperm receptors activates intraspermatic signals and enzymatic pathways involved in the AR. Among the proteins or glycoproteins described as potential sperm receptors for ZP, Gi/Go protein‐coupled and tyrosine kinase receptors have been described. Sperm receptors for P4 are poorly characterized, except a putative GABAA‐like receptor. ZP‐ and P4‐promoted AR is mediated by an obligatory intracellular calcium increase, appearing first at the acrosome equatorial segment and spreading throughout the head. The plasma membrane channels involved in calcium entry are operated by a plasma membrane depolarization and protein phosphorylations mediated by protein kinase C and tyrosine kinase protein. Part of the calcium increase could also be due to intracellular store release through IP3‐ and nucleotide (cAMP)‐gated channels. Besides adenylate cyclase and phospholipase C activations, intracellular calcium increase also stimulates PLA2 activity and actin depolymerization, leading to membrane fusion. Evaluation of AR by staining or fluorescent probes can be useful to predict fertilization success and to direct the therapeutic strategy in male infertility.

Genetic male infertility and mutation of CATSPER ion channels

A clinically significant proportion of couples experience difficulty in conceiving a child. In about half of these cases male infertility is the cause and often genetic factors are involved. Despite advances in clinical diagnostics ∼50% of male infertility cases remain idiopathic. Based on this, further analysis of infertile males is required to identify new genetic factors involved in male infertility. This review focuses on cation channel of sperm (CATSPER)-related male infertility. It is based on PubMed literature searches using the keywords ‘CATSPER’, ‘male infertility’, ‘male contraception’, ‘immunocontraception’ and ‘pharmacologic contraception’ (publication dates from January 1979 to December 2009). Previously, contiguous gene deletions including the CATSPER2 gene implicated the sperm-specific CATSPER channel in syndromic male infertility (SMI). Recently, we identified insertion mutations of the CATSPER1 gene in families with recessively inherited nonsyndromic male infertility (NSMI). The CATSPER channel therefore represents a novel human male fertility factor. In this review we summarize the genetic and clinical data showing the role of CATSPER mutation in human forms of NSMI and SMI. In addition, we discuss clinical management and therapeutic options for these patients. Finally, we describe how the CATSPER channel could be used as a target for development of a male contraceptive.

Expression, localization and functions in acrosome reaction and sperm motility of CaV3.1 and CaV3.2 channels in sperm cells: An evaluation from CaV3.1 and CaV3.2 deficient mice

In spermatozoa, voltage‐dependent calcium channels (VDCC) have been involved in different cellular functions like acrosome reaction (AR) and sperm motility. Multiple types of VDCC are present and their relative contribution is still a matter of debate. Based mostly on pharmacological studies, low‐voltage‐activated calcium channels (LVA‐CC), responsible of the inward current in spermatocytes, were described as essential for AR in sperm. The development of CaV3.1 or CaV3.2 null mice provided the opportunity to evaluate the involvement of such LVA‐CC in AR and sperm motility, independently of pharmacological tools. The inward current was fully abolished in spermatogenic cells from CaV3.2 deficient mice. This current is thus only due to CaV3.2 channels. We showed that CaV3.2 channels were maintained in sperm by Western‐blot and immunohistochemistry experiments. Calcium imaging experiments revealed that calcium influx in response to KCl was reduced in CaV3.2 null sperm in comparison to control cells, demonstrating that CaV3.2 channels were functional. On the other hand, no difference was noticed in calcium signaling induced by zona pellucida. Moreover, neither biochemical nor functional experiments, suggested the presence of CaV3.1 channels in sperm. Despite the CaV3.2 channels contribution in KCl‐induced calcium influx, the reproduction parameters remained intact in CaV3.2 deficient mice. These data demonstrate that in sperm, besides CaV3.2 channels, other types of VDCC are activated during the voltage‐dependent calcium influx of AR, these channels likely belonging to high‐voltage activated Ca2+ channels family. The conclusion is that voltage‐dependent calcium influx during AR is due to the opening of redundant families of calcium channels. J. Cell. Physiol. 212:753–763, 2007.

Centimorgan-range one-step mapping of fertility traits using interspecific recombinant congenic mice.

In mammals, male fertility is a quantitative feature determined by numerous genes. Until now, several wide chromosomal regions involved in fertility have been defined by genetic mapping approaches; unfortunately, the underlying genes are very difficult to identify. Here, 53 interspecific recombinant congenic mouse strains (IRCSs) bearing 1–2% SEG/Pas (Mus spretus) genomic fragments disseminated in a C57Bl/6J (Mus domesticus) background were used to systematically analyze male fertility parameters. One of the most prominent advantages of this model is the possibility of analyzing stable phenotypes in living animals. Here, we demonstrate the possibility in one-step fine mapping for several fertility traits. Focusing on strains harboring a unique spretus fragment, we could unambiguously localize two testis and one prostate weight-regulating QTL (Ltw1, Ltw2, and Lpw1), four QTL controlling the sperm nucleus shape (Sh1, Sh2, Sh3, and Sh4), and one QTL influencing sperm survival (Dss1). In several cases, the spretus DNA fragment was small enough to propose sound candidates. For instance, Spata1, Capza, and Tuba7 are very strong candidates for influencing the shape of the sperm head. Identifying new genes implied in mammalian fertility pathways is a necessary prerequisite for clarifying their molecular grounds and for proposing diagnostic tools for masculine infertilities.

Induction of a Sodium Ion Influx by Progesterone in Human Spermatozoa1

Abstract In human spermatozoa, progesterone (P4) induces a depolarization of the plasma membrane, a rapid calcium (Ca2+) influx, and a chloride efflux. The sodium ion (Na+) was partly responsible for the P4-induced depolarizing effect but was not required for calcium influx. We used fluorescent probes for spectrofluorometry to investigate whether P4 induced a Na+ influx and whether voltage-operated channels were involved in Na+ and/or Ca2+ entries. We found that 10 μM P4 significantly increased intracellular Na+ concentration from 17.8 ± 2.0 mM to 27.2 ± 1.6 mM (P < 0.001). Prior incubation of spermatozoa with 10 μM flunarizine, a Na+ and Ca2+ voltage-dependent channel blocker, inhibited the sodium influx induced by 10 μM P4 by 84.6 ± 15.4%. The Ca2+ influx induced by 10 μM P4 was also significantly inhibited in a Na+-containing medium by 10 μM flunarizine or 10 μM pimozide (P < 0.01). In contrast, flunarizine had no inhibitory effect on the Ca2+ influx induced by 10 μM P4 in spermatozoa incubated in Na+-depleted medium. The P4-promoted acrosome reaction (AR) was significantly higher when spermatozoa were incubated in Na+-containing medium as compared to Na+-depleted medium. These data demonstrate that P4 stimulates a Na+ influx that could be involved in the AR completion. They also suggest that voltage-dependent Na+ and Ca2+ channels are implicated in P4-mediated signaling pathway in human spermatozoa.

Spatio-Developmental Distribution of the Prion-Like Protein Doppel in Mammalian Testis: A Comparative Analysis Focusing on Its Presence in the Acrosome of Spermatids

Abstract The first prion-like protein doppel, officially designed as prion protein dublet, does not seem to be needed for prion disease progression, whereas its physiological function seems to be related to male fertility. Its expression is primarily detected in the male genital tract, and Prnd-inactivated male mice are sterile. We investigated the location of Doppel in the testis of various species of mammal to determine its physiological function. Doppel is expressed early during ontogenesis, and is found in both germ cells and Sertoli cells in mice, rats, boars, and humans. Doppel is permanently expressed in the Sertoli cells but at different levels according to species. Its expression in testicular germ cells was primarily detected in spermatids, with a transient presence in the acrosome. These data suggest that Doppel may play a physiological role in acrosome biogenesis and may be of use in studies of patients suffering from idiopathic infertility.

Cholesterol Depletion Disorganizes Oocyte Membrane Rafts Altering Mouse Fertilization

Drastic membrane reorganization occurs when mammalian sperm binds to and fuses with the oocyte membrane. Two oocyte protein families are essential for fertilization, tetraspanins and glycosylphosphatidylinositol-anchored proteins. The firsts are associated to tetraspanin-enriched microdomains and the seconds to lipid rafts. Here we report membrane raft involvement in mouse fertilization assessed by cholesterol modulation using methyl-β-cyclodextrin. Cholesterol removal induced: (1) a decrease of the fertilization rate and index; and (2) a delay in the extrusion of the second polar body. Cholesterol repletion recovered the fertilization ability of cholesterol-depleted oocytes, indicating reversibility of these effects. In vivo time-lapse analyses using fluorescent cholesterol permitted to identify the time-point at which the probe is mainly located at the plasma membrane enabling the estimation of the extent of the cholesterol depletion. We confirmed that the mouse oocyte is rich in rafts according to the presence of the raft marker lipid, ganglioside GM1 on the membrane of living oocytes and we identified the coexistence of two types of microdomains, planar rafts and caveolae-like structures, by terms of two differential rafts markers, flotillin-2 and caveolin-1, respectively. Moreover, this is the first report that shows characteristic caveolae-like invaginations in the mouse oocyte identified by electron microscopy. Raft disruption by cholesterol depletion disturbed the subcellular localization of the signal molecule c-Src and the inhibition of Src kinase proteins prevented second polar body extrusion, consistent with a role of Src-related kinases in fertilization via signaling complexes. Our data highlight the functional importance of intact membrane rafts for mouse fertilization and its dependence on cholesterol.

Identification of Quantitative Trait Loci responsible for embryonic lethality in mice assessed by ultrasonography.

Recurrent Spontaneous Abortion (RSA) is a frequent pathology affecting 1 to 5% of couples. In approximately 50 % of cases, the aetiology is unknown suggesting a subtle interaction between genetic and environmental factors. Previous attempts to describe genetic factors using the candidate gene approach have been relatively unsuccessful due to the physiological, cellular and genetic complexity of mammalian reproduction. Indeed, fertility can be considered as a quantitative feature resulting from the interaction of genetic, epigenetic and environmental factors. Herein, we identified Quantitative Trait Loci (QTL) associated with diverse embryonic lethality phenotypes and the subsequent embryonic resorption in 39 inter-specific recombinant congenic mice strains, using in vivo ultrasound bio-microscopy. The short chromosomal intervals related to the phenotypes will facilitate the study of a restricted number of candidate genes which are potentially dysregulated in patients affected by RSA.

Zona pellucida from fertilised human oocytes induces a voltage-dependent calcium influx and the acrosome reaction in spermatozoa, but cannot be penetrated by sperm

BackgroundThe functions of three zona glycoproteins, ZP1, ZP2 and ZP3 during the sperm-zona pellucida (ZP) interaction are now well established in mice. The expression of an additional zona glycoprotein, ZPB/4, in humans, led us to reconsider the classical mouse model of gamete interaction. We investigated the various functions of human ZP (hZP) during the interaction of spermatozoa with fertilised and unfertilised oocytes.ResultsThe hZP of fertilised oocytes retained their ability to bind sperm (albeit less strongly than that from unfertilised oocytes), to induce an intraspermatic calcium influx through voltage-dependent channels similar to that observed with hZP from unfertilised oocytes and to promote the acrosome reaction at a rate similar to that induced by the ZP of unfertilised oocytes (61.6 ± 6.2% vs60.7 ± 9.1% respectively). Conversely, the rate of hZP penetrated by sperm was much lower for fertilised than for unfertilised oocytes (19% vs 57% respectively, p < 0.01). We investigated the status of ZP2 in the oocytes used in the functional tests, and demonstrated that sperm binding and acrosome reaction induction, but not ZP penetration, occurred whether or not ZP2 was cleaved.ConclusionThe change in ZP function induced by fertilisation could be different in human and mouse species. Our results suggest a zona blocking to polyspermy based at the sperm penetration level in humans.

Refined Mapping of a Quantitative Trait Locus on Chromosome 1 Responsible for Mouse Embryonic Death

Recurrent spontaneous abortion (RSA) is defined as the loss of three or more consecutive pregnancies during the first trimester of embryonic intrauterine development. This kind of human infertility is frequent among the general population since it affects 1 to 5% of women. In half of the cases the etiology remains unelucidated. In the present study, we used interspecific recombinant congenic mouse strains (IRCS) in the aim to identify genes responsible for embryonic lethality. Applying a cartographic approach using a genotype/phenotype association, we identified a minimal QTL region, of about 6 Mb on chromosome 1, responsible for a high rate of embryonic death (∼30%). Genetic analysis suggests that the observed phenotype is linked to uterine dysfunction. Transcriptomic analysis of the uterine tissue revealed a preferential deregulation of genes of this region compared to the rest of the genome. Some genes from the QTL region are associated with VEGF signaling, mTOR signaling and ubiquitine/proteasome-protein degradation pathways. This work may contribute to elucidate the molecular basis of a multifactorial and complex human disorder as RSA.