Disruption of the principal, progesterone-activated sperm Ca2+ channel in a CatSper2-deficient infertile patient.

The female steroid hormone progesterone regulates ovulation and supports pregnancy, but also controls human sperm function within the female reproductive tract. Progesterone causes elevation of sperm intracellular Ca(2+) leading to sperm hyperactivation, acrosome reaction, and perhaps chemotaxis toward the egg. Although it has been suggested that progesterone-dependent Ca(2+) influx into human spermatozoa is primarily mediated by cationic channel of sperm (CatSper), the principal flagellar Ca(2+) channel of sperm, conclusive loss-of-function genetic evidence for activation of CatSper by progesterone has yet to be provided. Moreover, it is not clear whether the responsiveness of CatSper to progesterone is an innate property of human spermatozoa or is acquired as the result of exposure to the seminal plasma. Here, by recording ionic currents from spermatozoa of an infertile CatSper-deficient patient, we demonstrate that CatSper is indeed the principal Ca(2+) channel of human spermatozoa, and that it is strongly potentiated by progesterone. In addition, by recording CatSper currents from human epididymal and testicular spermatozoa, we show that CatSper sensitivity to progesterone arises early in sperm development and increases gradually to a peak when spermatozoa are ejaculated. These results unambiguously establish an important role of CatSper channel in human sperm nongenomic progesterone signaling and demonstrate that the molecular mechanism responsible for activation of CatSper by progesterone arises early in sperm development concurrently with the CatSper channel itself.

Polymorphisms of human placental alkaline phosphatase are associated with in vitro fertilization success and recurrent pregnancy loss.

Fertility is a quantitative, complex character governed by a considerable number of genes. Despite clinical and scientific advances, several cases of human infertility remain unexplained. In the present study, using a positional cloning approach in a mouse model of interspecific recombinant lines, a candidate gene, ALPP, encoding the placental alkaline phosphatase, was identified as being potentially involved in recurrent spontaneous abortion. We then analyzed patients for detecting putative associations between ALPP polymorphisms, in vitro fertilization failures, and miscarriages. ALPP was sequenced in 100 controls and 100 patients affected by recurrent spontaneous abortion, from the same ethnic background. The frequency of several alleles and allelic combinations were different between recurrent spontaneous abortion and control women. One polymorphism induced a coding substitution (Ile89Leu) that was associated with a decreased risk of abortion and in vitro fertilization failure. Thereafter, the population was increased by the analysis of 92 additional controls and 612 additional patients for the coding polymorphism Ile89Leu. We finally show, by functional analysis, that the 89Leu placental alkaline phosphatase has an enhanced alkaline phosphatase activity. This study suggests that ALPP genotyping could be a strong predictor of implantation success.

Moonlighting proteins in sperm-egg interactions.

Sperm-egg interaction is a highly species-specific step during the fertilization process. The first steps consist of recognition between proteins on the sperm head and zona pellucida (ZP) glycoproteins, the acellular coat that protects the oocyte. We aimed to determine which sperm head proteins interact with ZP2, ZP3 and ZP4 in humans. Two approaches were combined to identify these proteins: immunoblotting human spermatozoa targeted by antisperm antibodies (ASAs) from infertile men and far-Western blotting of human sperm proteins overlaid by each of the human recombinant ZP (hrZP) proteins. We used a proteomic approach with 2D electrophoretic separation of sperm protein revealed using either ASAs eluted from infertile patients or recombinant human ZP glycoproteins expressed in Chinese-hamster ovary (CHO) cells. Only spots highlighted by both methods were analysed by MALDI-MS/MS for identification. We identified proteins already described in human spermatozoa, but implicated in different metabolic pathways such as glycolytic enzymes [phosphokinase type 3 (PK3), enolase 1 (ENO1), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), aldolase A (ALDOA) and triose phosphate isomerase (TPI)], detoxification enzymes [GST Mu (GSTM) and phospholipid hydroperoxide glutathione peroxidase (PHGPx) 4], ion channels [voltage-dependent anion channel 2 (VDAC2)] or structural proteins (outer dense fibre 2). Several proteins were localized on the sperm head by indirect immunofluorescence, and their interaction with ZP proteins was confirmed by co-precipitation experiments. These results confirm the complexity of the sperm-ZP recognition process in humans with the implication of different proteins interacting with the main three ZP glycoproteins. The multiple roles of these proteins suggest that they are multifaceted or moonlighting proteins.

The ciliary pocket: an endocytic membrane domain at the base of primary and motile cilia.

Cilia and flagella are eukaryotic organelles involved in multiple cellular functions. The primary cilium is generally non motile and found in numerous vertebrate cell types where it controls key signalling pathways. Despite a common architecture, ultrastructural data suggest some differences in their organisation. Here, we report the first detailed characterisation of the ciliary pocket, a depression of the plasma membrane in which the primary cilium is rooted. This structure is found at low frequency in kidney epithelial cells (IMCD3) but is associated with virtually all primary cilia in retinal pigment epithelial cells (RPE1). Transmission and scanning electron microscopy, immunofluorescence analysis and videomicroscopy revealed that the ciliary pocket establishes closed links with the actin-based cytoskeleton and that it is enriched in active and dynamic clathrin-coated pits. The existence of the ciliary pocket was confirmed in mouse tissues bearing primary cilia (cumulus), as well as motile cilia and flagella (ependymal cells and spermatids). The ciliary pocket shares striking morphological and functional similarities with the flagellar pocket of Trypanosomatids, a trafficking-specialised membrane domain at the base of the flagellum. Our data therefore highlight the conserved role of membrane trafficking in the vicinity of cilia.

Membrane transfer from oocyte to sperm occurs in two CD9-independent ways that do not supply the fertilising ability of Cd9-deleted oocytes.

Spermatozoa undergo regulation of their functions along their lifespan through exchanges via vesicles or interactions with epithelial cells, in the epididymis, in the seminal fluid and in the female genital tract. Two different ways of oocyte membrane transfer to spermatozoa have been described: trogocytosis and exosomes. We here report an analysis of in vitro exchanges between the membranes of unfertilised oocytes and capacitated spermatozoa. We showed that optimum conditions are fulfilled when unfertilised oocytes interact with acrosome-reacted spermatozoa, a scenario mimicking the events occurring when the fertilising spermatozoon is inside the perivitelline space. Although CD9 tetraspanin is an essential molecule for fertilisation, exosome and trogocytosis transfer persists in Cd9-null oocytes in spite of their dramatic fusion failure. These exchanges are CD9 tetraspanin independent. We also confirm that mice sperm express CD9 tetraspanin and that when Cd9-null oocytes were inseminated with sperm covered with oocyte membrane materials, including CD9 tetraspanin, no rescue of the oocytes' fertilisability could be obtained. Thus, the existence of two ways of exchange between gametes during fertilisation suggests that these events could be of a physiological importance in this process.

Interspecific resources: a major tool for quantitative trait locus cloning and speciation research.

Positional cloning of the quantitative trait locus (QTL) still encounters numerous difficulties, which explains why thousands of QTL have been mapped, while only a few have been identified at the molecular level. Here, we focus on a specific mapping tool that exists in plant and animal model species: interspecific recombinant congenic strains (IRCSs) or interspecific nearly isogenic lines (NILs). Such panels exhibit a much higher sequence diversity than intraspecific sets, thus enhancing the contrasts between phenotypes. In animals, it allows statistical significance to be reached even when using a limited number of individuals. Therefore, we argue that interspecific resources may constitute a major genetic tool for positional cloning and for understanding some bases of speciation mechanisms.

From ultrastructural flagellar sperm defects to the health of babies conceived by ICSI.

The objective of this retrospective study was to describe a population of patients displaying impaired sperm motility due to ultrastructural flagellar defects and to analyse the intracytoplasmic sperm injection (ICSI) results and neonatal outcomes in this population. The fertilization rate, embryo quality, clinical pregnancy rate, implantation rate, birth rate and perinatal health of babies were determined. Patients (n = 20) were divided into seven categories according to ultrastructural flagellar abnormalities. The type of flagellar abnormality significantly affected the fertilization rate (P <0.025). Two types of flagellar abnormalities showed slower early embryo cleavage kinetics (P <0.001) when axonemal central structures and periaxonemmal columns were abnormal or absent. Of 53 ICSI attempts, 14 resulted in clinical pregnancies (26.4% per cycle) after fresh and frozen embryo transfer. Three (21.4%) of these pregnancies ended in miscarriages and, in the remaining, 12 infants were born (7.2% of transferred embryos). The outcomes differed according to the ultrastructural defect. This study demonstrates that a high proportion of patients could father a child (45.0%). However, flagellar abnormalities appear to influence ICSI results and fetal development.

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.

In vitro spermicidal activity of peptides from amphibian skin: dermaseptin S4 and derivatives.

Sexually transmitted infections and unplanned pregnancies present a great risk to the reproductive health of women. Therefore, female-controlled vaginal products directed toward disease prevention and contraception are needed urgently. In the present study, efforts were made to evaluate the contraceptive potency of dermaseptin DS4, an antimicrobial peptide derived from frog skin. To assess the structure-activity relationship between the native DS4 and its derivatives, a set of chemically modified peptides was synthesized and evaluated. Normal human semen samples were used to detect the spermicidal activity of the new compounds. HeLa cultures were used to determine the safety of compounds toward their toxicity. Fluorescent-binding assays were performed to evaluate the rapidity and the irreversibility of the sperm-immobilizing activity of peptides. All DS4 derivatives elicited concentration-dependent spermicidal activity at microgram concentrations (EC(100) values: 25 microg/ml-l mg/ml). The order was K4S4=S4a>S4>K4S4(1-16)a>S4(6-28). In cytotoxicity assay, some compounds were found to be significantly safer than nonoxynol-9, the most widely used spermicide, and their activity was not accompanied by total loss of plasma membrane integrity as detected by fluorescent microscopy. Our data also show that increasing the number of positive charges of the peptide resulted in a reduced cytotoxicity without affecting the spermicidal effect. This study indicates that dermaseptins are spermicidal molecules that deserve to be tested as topical contraceptive with useful activities that can add to their prophylaxis, safety, and effectiveness.

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

BACKGROUND: The 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 unfertilized oocytes. RESULTS: The hZP of fertilised oocytes retained their ability to bind sperm (albeit less strongly than that from unfertilized oocytes), to induce an intraspermatic calcium influx through voltage-dependent channels similar to that observed with hZP from unfertilized oocytes and to promote the acrosome reaction at a rate similar to that induced by the ZP of unfertilized oocytes (61.6 +/- 6.2% vs 60.7 +/- 9.1% respectively). Conversely, the rate of hZP penetrated by sperm was much lower for fertilised than for unfertilized 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. CONCLUSION: The 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.

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-ß-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.

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.

Fidgetin-like1 is a strong candidate for a dynamic impairment of male meiosis leading to reduced testis weight in mice.

BACKGROUND: In a previous work, using an interspecific recombinant congenic mouse model, we reported a genomic region of 23 Mb on mouse chromosome 11 implicated in testis weight decrease and moderate teratozoospermia (∼20-30%), a Quantitative Trait Locus (QTL) called Ltw1. The objective of the present study is to identify the gene underlying this phenotype. RESULTS: In the present study, we refined the QTL position to a 5 Mb fragment encompassing only 11 genes. We showed that the low testis weight phenotype was due to kinetic alterations occurring during the first wave of the spermatogenesis where we could point out to an abnormal lengthening of spermatocyte prophase. We identify Fidgetin-like 1 (Fignl1) as the gene underlying the phenotype, since if fulfilled both the physiological and molecular characteristics required. Indeed, amongst the 11 positional candidates it is the only gene that is expressed during meiosis at the spermatocyte stage, and that presents with non-synonymous coding variations differentiating the two mouse strains at the origin of the cross. CONCLUSIONS: This work prompted us to propose Fignl1 as a novel actor in mammal's male meiosis dynamics which has fundamental interest. Besides, this gene is a new potential candidate for human infertilities caused by teratozoospermia and blockades of spermatogenesis. In addition this study demonstrates that interspecific models may be useful for understanding complex quantitative traits.

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.

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.

[Molecules involved in sperm-zona pellucida interaction in mammals. Role in human fertility]. / Les partenaires moléculaires impliqués dans l'interaction entre spermatozoïdes et zone pellucide chez les mammifères. Conséquences pour la fertilité humaine.

Fertilization in mammals requires an initial interaction of sperm with the oocyte envelope, the zona pellucida (ZP), before it reaches the oocyte. ZP is a highly glycosylated structure, composed of three (mouse) or four (rabbit, boar, bovine, humans...) glycoproteins. The presence of ZP around the oocyte does not allow heterospecific fertilization. This barrier is principally due to the presence of species-specific glycosylations on ZP proteins. Sperm bind ZP by means of membrane receptors which recognize carbohydrate moieties on ZP glycoproteins according to a well-precised sequential process. Upon initial attachment, spermatozoa bind ZP3/ZP4 which induces the sperm acrosome exocytosis followed by a secondary binding of acrosome reacted spermatozoa to ZP2 and by ZP penetration. The sperm receptors are adhesive proteins or integral plasma membrane proteins linked to intraspermatic signalling pathways activating the acrosome reaction. Over the last twenty years, numerous studies have been carried out to identify sperm receptors to ZP in several species, but the data in humans are still incomplete. Work initiated in our research group has identified several proteins interacting with recombinant human ZP2, ZP3 and ZP4, among which are glycolytic enzymes. These enzymes are involved in the gamete interaction by means of their affinity to sugars and not by their catalytic properties. From a clinical point of view, an observed lack or weak expression of some sperm receptors to ZP3 in cases of idiopathic infertility associated with in vitro fertilization failure suggests that knowing the molecular mechanism driving the gamete recognition can be important at the diagnostic level. Furthermore, it has been shown that proteins that mediate gamete recognition diverge rapidly, as a result of positive darwinian selection. A sexual conflict can drive co-evolution of reproductive molecules in both sexes resulting in reproductive isolation and species emergence.

Gene expression regulation in the context of mouse interspecific mosaic genomes.

BACKGROUND: Accumulating evidence points to the mosaic nature of the mouse genome. However, little is known about the way the introgressed segments are regulated within the context of the recipient genetic background. To address this question, we have screened the testis transcriptome of interspecific recombinant congenic mouse strains (IRCSs) containing segments of Mus spretus origin at a homozygous state in a Mus musculus background. RESULTS: Most genes (75%) were not transcriptionally modified either in the IRCSs or in the parent M. spretus mice, compared to M. musculus. The expression levels of most of the remaining transcripts were 'dictated' by either M. musculus transcription factors ('trans-driven'; 20%), or M. spretus cis-acting elements ('cis-driven'; 4%). Finally, 1% of transcripts were dysregulated following a cis-trans mismatch. We observed a higher sequence divergence between M. spretus and M. musculus promoters of strongly dysregulated genes than in promoters of similarly expressed genes. CONCLUSION: Our study indicates that it is possible to classify the molecular events leading to expressional alterations when a homozygous graft of foreign genome segments is made in an interspecific host genome. The inadequacy of transcription factors of this host genome to recognize the foreign targets was clearly the major path leading to dysregulation.

Expression, localization and functions in acrosome reaction and sperm motility of Ca(V)3.1 and Ca(V)3.2 channels in sperm cells: an evaluation from Ca(V)3.1 and Ca(V)3.2 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 Ca(V)3.1 or Ca(V)3.2 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 Ca(V)3.2 deficient mice. This current is thus only due to Ca(V)3.2 channels. We showed that Ca(V)3.2 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 Ca(V)3.2 null sperm in comparison to control cells, demonstrating that Ca(V)3.2 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 Ca(V)3.1 channels in sperm. Despite the Ca(V)3.2 channels contribution in KCl-induced calcium influx, the reproduction parameters remained intact in Ca(V)3.2 deficient mice. These data demonstrate that in sperm, besides Ca(V)3.2 channels, other types of VDCC are activated during the voltage-dependent calcium influx of AR, these channels likely belonging to high-voltage activated Ca(2+) channels family. The conclusion is that voltage-dependent calcium influx during AR is due to the opening of redundant families of calcium channels.

The human "prion-like" protein Doppel is expressed in both Sertoli cells and spermatozoa.

The prion-like Doppel protein (Dpl) has many biochemical and structural properties in common with the cellular prion protein (PrP(c)), and the physiological role of neither protein is known. Experimental data suggest either direct or indirect interaction between the two proteins. In this study, we investigated the expression pattern and biochemical characteristics of Dpl in human tissues and in Chinese hamster ovary cells transfected with wild-type or variant human Dpl gene constructs. Human Dpl appears to be a glycosylphosphatidylinositol-anchored glycoprotein with N- and O-linked sugars. It was found on Sertoli cells in the testis, on the flagella of epididymal and mature spermatozoa, and in seminal plasma. Dpl coexists only with N-terminally truncated isoforms of PrP(c) on mature spermatozoa. The localization of human Dpl on both Sertoli cells (somatic cells) and spermatozoa (germinal cells) strongly suggests that this protein may play a major role in human male fertility. Finally, our data indicate that spermatozoa are thus an interesting model for studies of the potential interaction between Dpl and PrP(c).