[show abstract][hide abstract] ABSTRACT: 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.
PLoS ONE 01/2013; 8(4):e62919. · 3.73 Impact Factor
[show abstract][hide abstract] ABSTRACT: 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.
[show abstract][hide abstract] ABSTRACT: Objectives. To analyze the impact of oocyte denudation and microinjection timings on intracytoplasmic sperm injection (ICSI) outcomes. Study Design. We included ICSI cycles with the following parameters: rank 1 or 2, female age <36 years, male factor infertility, long protocol using GnRH agonist and rFSH for ovarian stimulation, and use of freshly ejaculated sperm (n = 110). Several ICSI parameters were analyzed according to the time between oocyte retrieval and denudation (T(1)) and the time between denudation and ICSI (T(2)) using a statistical logistic regression analysis. Results. Neither T(1) nor T(2) had a significant influence on the Metaphase II (MII) rate but the fertilisation rate (FR) showed a significant improvement when T(1) was longer (optimal results at T(1) = 3 hours) while FR significantly decreased with the increase of T(2). Optimal implantation (IR) and pregnancy (PR) rates were obtained when T(1) was around 2 hours. Conclusion. Incubation of oocytes around 2 hours between retrieval and denudation may not increase MII rate but appears to lead to the optimal combination of FR and IR.
Obstetrics and Gynecology International 01/2012; 2012:403531.
[show abstract][hide abstract] ABSTRACT: X-chromosome inactivation (XCI) in female mammals allows dosage compensation for X-linked gene products between the sexes. The developmental regulation of this process has been extensively investigated in mice, where the X chromosome of paternal origin (Xp) is silenced during early embryogenesis owing to imprinted expression of the regulatory RNA, Xist (X-inactive specific transcript). Paternal XCI is reversed in the inner cell mass of the blastocyst and random XCI subsequently occurs in epiblast cells. Here we show that other eutherian mammals have very different strategies for initiating XCI. In rabbits and humans, the Xist homologue is not subject to imprinting and XCI begins later than in mice. Furthermore, Xist is upregulated on both X chromosomes in a high proportion of rabbit and human embryo cells, even in the inner cell mass. In rabbits, this triggers XCI on both X chromosomes in some cells. In humans, chromosome-wide XCI has not initiated even by the blastocyst stage, despite the upregulation of XIST. The choice of which X chromosome will finally become inactive thus occurs downstream of Xist upregulation in both rabbits and humans, unlike in mice. Our study demonstrates the remarkable diversity in XCI regulation and highlights differences between mammals in their requirement for dosage compensation during early embryogenesis.
[show abstract][hide abstract] ABSTRACT: Both female mice deficient in CD9 tetraspanin- and oocyte-specific glycosyl-phosphatidylinositol-anchored family proteins showed severely reduced fertility due to the failure of sperm-egg fusion. This raises the question of a link between these two groups of proteins at the oocyte membrane. We propose two hypotheses to explain why the absence of one of these proteins from the oocyte membrane results in the same phenotype. The first hypothesis envisages different levels of control by these molecules of the common induced signaling cascade. The second relies on the known involvement of these molecules in the overall organization of the plasma membrane. Their disappearance could thus prevent sperm-egg fusion either by disruption of the signaling cascade and/or by an important disorganization of the oocyte membrane. In this review, describing their structural and functional characteristics, and using published results on the oocyte, we try to analyze how these two protein families could interact.
[show abstract][hide abstract] ABSTRACT: Oocyte integrins have been described as essential for fertilization. But this concept has been challenged by deletion experiments. Recently, we have shown that sperm integrin alpha6beta1 plays a determinant role in mouse gamete interaction. In this study, we demonstrate the presence of alphavbeta3 integrin by Western blot and immunofluorescence on the sperm membrane. Oocytes and/or sperm preincubations with anti-alphav or anti-beta3 antibodies were performed before in vitro fertilization on cumulus-intact and zona-free egg assays. We observed inhibitory effects on the fusion process mostly by means of sperm function. An antibody directed against vitronectin inhibited gametes fusion, whereas the presence of exogenous vitronectin increased its efficiency. We suggest that vitronectin (on multimeric forms) can play a first nonspecific link corresponding to loosely bound spermatozoa to oocyte and that this link could be mediated by means of oocyte proteoglycans or integrins, and sperm alphavbeta3 integrin.
[show abstract][hide abstract] ABSTRACT: Assisted Reproductive Technologies (ART) are increasingly used in humans; however, their impact is now questioned. At blastocyst stage, the trophectoderm is directly in contact with an artificial medium environment, which can impact placental development. This study was designed to carry out an in-depth analysis of the placental transcriptome after ART in mice.
Blastocysts were transferred either (1) after in vivo fertilization and development (control group) or (2) after in vitro fertilization and embryo culture. Placentas were then analyzed at E10.5. Six percent of transcripts were altered at the two-fold threshold in placentas of manipulated embryos, 2/3 of transcripts being down-regulated. Strikingly, the X-chromosome harbors 11% of altered genes, 2/3 being induced. Imprinted genes were modified similarly to the X. Promoter composition analysis indicates that FOXA transcription factors may be involved in the transcriptional deregulations.
For the first time, our study shows that in vitro fertilization associated with embryo culture strongly modify the placental expression profile, long after embryo manipulations, meaning that the stress of artificial environment is memorized after implantation. Expression of X and imprinted genes is also greatly modulated probably to adapt to adverse conditions. Our results highlight the importance of studying human placentas from ART.
PLoS ONE 01/2010; 5(2):e9218. · 3.73 Impact Factor
[show abstract][hide abstract] ABSTRACT: To compare the effects of 2 different media on embryo morphology and development at days 2/3.
Six hundred seventy-six attempts from 512 couples were included in this prospective auto-controlled study. Sibling oocytes of all couples undergoing an IVF (n = 286) or ICSI (n = 390) attempt were randomly assigned to either GIII series (Vitrolife) or ISM (Medicult) media. Primary end points were fertilization and embryo morphology rates.
Fertilization rates in GIII series and ISM (IVF: 59.9 vs 62.0% and ICSI: 65.7 vs 66.8%) respectively were not different. GIII series showed an increase, compared to ISM, of early cleavage rate, (IVF: 25.8 vs 16.2% (p = 0.005); ICSI: 40.8 vs 25.5% (p < 0.0001), and good embryo morphology rate at day 2 [IVF: 64.6 vs 57.3% (p = 0.01); ICSI: 74.2 vs 69.4 (p = 0.03)] and at day 3 [IVF: 57.5 vs 49.0% (p = 0.02); ICSI: 67.2 vs 61.6% (p = 0.01)].
Embryo morphology at days 2/3 was significantly enhanced when the embryos were cultured in GIII series.
Journal of Assisted Reproduction and Genetics 11/2009; 26(11-12):575-81. · 1.82 Impact Factor
[show abstract][hide abstract] ABSTRACT: Endocervical and endometrial damage observed after different procedures of embryo transfer (ET) were investigated using diagnostic hysteroscopy.
Prospective, descriptive and comparative study, in an Infertility centre, University Hospital. Hundred consecutive infertile patients with a normal uterine cavity, included in an IVF program, were enrolled between May 1st, 2006 and April 30th, 2007. All the patients had a diagnostic hysteroscopy immediately after trial ET using soft ET catheters: (i) IVF Sydney Set (Cook, Limerick, Ireland) (n=27), (ii) Elliocath (Ellios, Paris, France) (n=34), (iii) Frydman classic 4.5 (CCD, Paris, France) (n=19), and rigid ET catheters: Memory Frydman 4.5 (CCD, Paris, France) (n=20). All the procedures were recorded and blindly reviewed. Data were analyzed using a Kruskal-Wallis test for age and severity of endometrial lesions, or Fisher's exact test for binary criteria.
Endocervical lesions were more frequently encountered in the soft (63%) and rigid (85%) Frydman's catheter groups compared to other groups (Elliocath: 29%, IVF Sydney Set: 26%; p<0.0001). Presence of blood on the catheter, and endometrial lesions were significantly less frequent in soft catheter groups compared to the rigid catheter group (p<0.0001). Severe endometrial lesions were less frequently observed when soft catheters were used (85%, 53%, 32%, 11% for Memory Frydman, Frydman classic, Elliocath and IVF Sydney Set, respectively; p<0.0001). The presence of blood on the catheter signed severe endometrial lesions.
All ET catheters can lead to endocervical and endometrial damage. Severe endometrial lesions were less frequent when soft catheters were used.
European journal of obstetrics, gynecology, and reproductive biology 09/2009; 147(2):183-6. · 1.97 Impact Factor
[show abstract][hide abstract] ABSTRACT: We aimed to evaluate and compare the embryo quality at early cleavage stages using different oils overlaying media to culture human embryos during IVF/ICSI treatments.
A total of 500 IVF/ICSI treatments from 500 women were analyzed in a prospective randomized study. Oocytes/embryos were treated into microdroplets of appropriate media overlaid with (i) Mineral Oil (CryoBioSystem, L'Aigle, France) (group 1, n=129), (ii) Liquid Paraffin (Medicult, Lyon, France) (group 2, n=126), (iii) Nidoil (Nidacon International, Guthenburg, Sweden) (group 3, n=126) and (iv) Ovoil (Vitrolife, Kungsbacka, Sweden) (group 4, n=119). Comparisons between groups were done using two by two post hoc tests, with 5% significance. The primary endpoint was the embryo quality, defined as good or top quality when embryos were with (i) less than 20% of fragmentation and (ii) 3-5/4 cells at day 2 or 6-10/8 cells at day 3, respectively.
At day 2, the embryo quality was similar in all groups. However, the mean number of top quality embryos at day 3 was statistically higher into the group 4 (1.4+/-1.8) compared to the group 1 (0.9+/-1.0; p=0.03) and 2 (0.8+/-1.3; p=0.05). Furthermore, a significant increase of the mean number of good quality embryos was observed at day 3 into the group 4 (2.6+/-2.6) compared to the group 1 (1.6+/-1.6; p=0.02).
The embryo quality could be modified according to commercial oils used to overlay culture media.
European journal of obstetrics, gynecology, and reproductive biology 08/2009; 147(1):52-6. · 1.97 Impact Factor
[show abstract][hide abstract] ABSTRACT: To evaluate the effects of cyclic QDE peptide (cQDE), derived from sperm fertilin beta (ADAM2), in mouse in vitro fertilization (IVF) and its harmlessness on pups after embryo transfer.
Prospective in vivo and in vitro study in mice.
Murine model in an academic research environment in France.
Normal B6CBF1 mice.
Indirect immunofluorescence was used to evaluate cQDE binding to oolemma. The IVF assays were run in presence of the species-specific tripeptide (cQDE at 100 microM), and embryos were transferred in pseudopregnant mice. Controls were obtained by natural mating and IVF in regular medium followed by embryo transfer.
Evaluation of fertilization, litter size, pup fertility and health.
Biotinylated cQDE peptide binds to oocyte plasma membrane and increases gamete fusion in cumulus-intact IVF assay. Litter size as well as pup development after embryo transfer showed no statistically significant differences compared with controls. Pups born from embryos that were incubated with the peptide were as healthy and normally fertile as control mice over at least three generations.
Cyclic QDE is harmless and improves mouse IVF pregnancy rates. A randomized prospective clinical trial to evaluate the beneficial effect of cyclic FEE on human IVF is required before its clinical use.
Fertility and sterility 09/2008; 91(5 Suppl):2110-5. · 3.97 Impact Factor
[show abstract][hide abstract] ABSTRACT: To examine the relationship between seminal polymorphonuclear (PMN) granulocytes and sperm functions (particularly sperm motility) in infertile men.
A prospective clinical study.
Assisted fertilization program in an academic research environment in France.
Infertile men (n = 138) evaluated and classified as follows: group A with no detectable PMN in semen, group B with less than 0.5 x 10(6)/mL, group C with 0.5-1 x 10(6)/mL, and group D > 1 x 10(6)/mL.
Seminal PMN, elastase concentrations, and sperm characteristics were analyzed. Seminal biochemical markers (free L-carnitine, citrate, zinc, acid phosphatase, and fructose) were measured.
Relation between seminal markers and sperm motility.
Grade "b" motility and epididymal carnitine were statistically significantly increased in group C. Acid phosphatase was the only seminal marker to be statistically significantly impaired in group D. Elastase was proportional to the degree of leukocytospermia.
These results suggest that sperm PMN originate from two regions:  the epididymis, where in small numbers and activated they may play a favorable role in sperm quality, and the  prostate, where their presence in large numbers, reflecting prostatitis, induces decreased secretion, especially of acid phosphatase.
Fertility and sterility 05/2008; 90(6):2257-63. · 3.97 Impact Factor
[show abstract][hide abstract] ABSTRACT: Several families of molecules are implicated in the membrane fusion process between sperm and oocyte. Among these, CD9 tetraspanin, a membrane-organizing molecule, plays a crucial role, since the fertilizing ability of CD9-/- oocytes is dramatically impaired. CD9 controls alpha6-beta1 integrin relocation involved in the membrane reorganization that occurs on oocyte fertilization but is not expressed on sperm. We report here that, together with several other proteins, the CD9 tetraspanin is transferred from the oocyte to the fertilizing spermatozoa present in the perivitelline space before fertilization. Transfer of CD9 from oocyte to sperm from CD9-/- male mice still occurs. CD9 acquisition by sperm results from a transfer of membrane fragments from the plasma membrane of the oocyte, in a process similar to trogocytosis, the recently described mechanism of intercellular exchange of membrane patches. Acquisition of CD9 by the sperm may be crucial for the membrane reorganization in sperm required for fusion with the oocyte, a process that is similar to the role CD9 plays in oocyte membrane reorganization.
The FASEB Journal 12/2007; 21(13):3446-9. · 5.70 Impact Factor
[show abstract][hide abstract] ABSTRACT: Based on inhibition tests, the alpha6beta1 integrin was suggested to be a sperm receptor, but further experiments using gene deletion techniques have shown that neither oocyte alpha6, nor beta1 integrin subunits were essential for mouse fertilization.
Using Western blot analysis and immunofluorescence, we showed that the mouse sperm expresses the alpha6beta1 integrin. As for oocyte, binding of GoH3 anti-alpha6 antibody to sperm induces a specific inhibition of sperm fertilizing ability. Comparing zona-intact and zona-free eggs in fusion tests, we showed that the removal of the zona pellucida by acid treatment bypasses fertilizing oocyte alpha6beta1 integrin's function in the adhesion/fusion process.
These findings show that alpha6beta1 integrin is expressed by both gametes and is functional in their membranes interaction. These results and previous reports, about fertilization of alpha6 or beta1 integrin subunits deleted oocytes by wild type sperm, suggest that the presence of alpha6beta1 integrin on one of the two gamete membranes can rescue the fertilization process. This hypothesis is further supported by the exchange of membrane fragments occurring between gametes prior to fusion that we recently reported.
[show abstract][hide abstract] ABSTRACT: A retrospective study was performed to determine the differences in embryo survival and frozen-thawed embryo transfers outcome between cryopreservation performed on day 3 versus day 2. We conclude that freezing supernumerary embryos on day 3 provides similar thawing survival parameters, better implantation, pregnancy, and live-birth rates compared with day 2 cryopreservation.
Fertility and sterility 12/2006; 86(5):1537-40. · 3.97 Impact Factor
[show abstract][hide abstract] ABSTRACT: Fertilization includes a series of cellular interactions culminating with the fusion of gamete membranes, creating a zygote. Two ADAM proteins present on sperm, fertilin beta and cyritestin, drew much attention. However, gene deletion in mice showed that fusion can happen in their absence. The presence of the integrin alpha6beta1 on egg, a putative fertilin beta receptor, is also dispensable. In contrast, sperm lacking Izumo, a molecule with a single Ig domain, are unable to fuse. On the egg side, a role for GPI-anchored molecules has been shown, and in mice lacking both tetraspanins CD9 and CD81 fertilization is completely blocked.
Seminars in Cell and Developmental Biology 05/2006; 17(2):254-63. · 6.20 Impact Factor
[show abstract][hide abstract] ABSTRACT: The process of gamete fusion has been largely studied in the mouse and has revealed the crucial role of the tetraspanin CD9. By contrast, human gamete fusion remains largely unknown. We now show that an anti-alpha 6 integrin mAb (GoH3) strongly inhibited human sperm-egg fusion in human zona-free eggs. Furthermore, a mAb directed against CD151, a tetraspanin known to associate with alpha 6 beta1, partially inhibited sperm-egg fusion. By contrast, the addition of an anti-CD9 mAb to zona free eggs had no effect. The integrin alpha 6 beta1, CD151 and CD9 tetraspanins were evenly distributed on human zona-intact oocytes. On zona-free eggs, the integrin alpha 6 beta1 and tetraspanin CD151 patched and co-localized but the tetraspanin CD9 remained unchanged. CD9 mAb prevented alpha 6 beta1 integrin clustering and gamete fusion when added prior to, but not after, zona removal. Antibody-mediated aggregation of integrin alpha 6 beta1 yielded patches that were bigger and more heterogeneous in mouse oocytes lacking CD9. Moreover, a strong labelling of alpha 6 beta1 could be observed at the sperm entry point. Altogether, these data show that CD9 controls the redistribution of some membrane proteins including the alpha 6 beta1 integrin into clusters that may be necessary for gamete fusion.
[show abstract][hide abstract] ABSTRACT: In somatic cells, the tetraspanins CD81 and CD9 associate with each other, with additional tetraspanins and with non-tetraspanin molecules to form proteolipidic complexes. Here we show that CD81 is expressed on the surface of oocytes where it associates with tetraspanin-enriched membrane structures. A major CD9 and CD81 partner, CD9P-1, is also expressed by oocytes. Deletion of CD81 gene in mice results in a 40% reduction of female fertility. In vitro insemination indicated that this infertility is due to a deficiency of oocytes to fuse with sperm. While the fertility of CD9-/- mice is severely but not completely impaired, double knock-out CD9-/- CD81-/- mice were completely infertile indicating that CD9 and CD81 play complementary roles in sperm-egg fusion. Finally, a fraction of CD9 was transferred from CD81-/- oocytes to sperm present in the perivitelline space indicating that the defect of fusion of CD81-/- oocytes does not result from an impaired initial gamete interaction.