Ghylène Goudet

Effect of Growth Hormone (GH) on In Vitro Nuclear and Cytoplasmic Oocyte Maturation, Cumulus Expansion, Hyaluronan Synthases, and Connexins 32 and 43 Expression, and GH Receptor Messenger RNA Expression in Equine and Porcine Species

Abstract The aim of this study was to investigate the role of growth hormone (GH) on in vitro cumulus expansion and oocyte maturation in equine and porcine cumulus-oocyte complexes (COCs), and to approach its way of action. Equine COCs were cultured in a control medium (TCM199, 5 mg/ml BSA, 1 μg/ml estradiol, and antibiotics) supplemented with either 0.5 μg/ml equine GH or 5 μg/ml equine LH. Porcine COCs were cultured in a basal medium (TCM199 with 570 μM cysteamine) supplemented with 0, 0.1, 0.5, or 1 μg/ml porcine GH or in a control medium (basal medium with 10 ng/ml epidermal growth factor and 400 ng/ml FSH) supplemented with 0 or 0.5 μg/ml porcine GH. After culture, cumulus expansion and nuclear stage were assessed. The cytoplasmic maturation of porcine oocytes was evaluated by in vitro fertilization and development for 7 days. The modifications of the expression of proteins implicated in cumulus expansion were analyzed in equine COCs by SDS-PAGE with antibodies against connexins 32 and 43 and hyaluronan synthases (Has) 1, 2, and 3. The expression of GH receptor mRNA was studied in oocytes and cumulus cells of the two species using reverse transcription-polymerase chain reaction with specific primers. The addition of GH in maturation medium increased cumulus expansion in equine but not porcine COCs. It improved nuclear maturation in equine and porcine, but had no effect on porcine fertilization and embryo development. The GH receptor mRNA was detected in equine and porcine oocytes and cumulus cells. GH did not influence the expression of Has 1, Has 3, and connexin 43 in equine cumulus cells.

Intrafollicular Concentrations of Steroids and Steroidogenic Enzymes in Relation to Follicular Development in the Mare

Abstract The objective of the present study was to determine the changes in follicular fluid steroid concentrations and in granulosa cell steroidogenic enzyme expression during the follicular phase, in relation to follicular size and physiological status in the mare. Follicular fluid and follicular cells were recovered by ultrasound-guided follicular punctures either around the time of emergence of the dominant follicle, at the end of the dominant follicle growth, or at the preovulatory stage, after injection of gonadotropin to induce ovulation. Cellular relative amounts of steroidogenic acute regulatory protein (StAR), P450-side chain cleavage (P450scc), 3β-hydroxysteroid dehydrogenase (3βHSD), 17α-hydroxylase, and aromatase were assessed by semiquantitative Western blot and densitometry. Follicular fluid was assayed for cholesterol concentrations by colorimetric assay and for progesterone, testosterone, and estradiol-17β concentrations by RIA. Intrafollicular concentrations of progesterone and estradiol-17β significantly increased in the dominant follicle during growth. After injection of gonadotropin, follicular maturation was characterized by a decrease in estradiol-17β concentrations and a further increase in progesterone concentrations. Granulosa cells from dominant follicles had increased levels of StAR, P450scc, 3βHSD, and aromatase during growth, but decreased levels during maturation. Levels of StAR, P450scc, 3βHSD, and aromatase, as well as progesterone and estradiol-17β, were lower in granulosa cells from subordinate than from dominant follicles. We did not observe a relationship between the steroidogenic activity of follicles and the capacity of their enclosed oocytes to complete meiosis in vitro.

Cysteamine supplementation of in vitro maturation media: a review.

Under in vitro culture conditions, oxidative modifications of cell components via increased reactive oxygen species (ROS) represent a major culture induced stress. Anti-oxidant systems such as glutathione (GSH) can attenuate the deleterious effects of oxidative stress by scavenging ROS. It has been suggested that GSH content in oocytes may serve as a reservoir protecting the zygote and the early embryos from oxidative damage before genomic activation and de novo GSH synthesis occur. Addition of low molecular weight compounds to culture media, such as cysteamine, can increase GSH levels by increasing cysteine uptake. Quite naturally, effects of supplementation of in vitro maturation (IVM) media with low molecular weight thiols have been studied in various species. This article reviews the use of cysteamine supplementation for IVM, its effects on maturation rates and further embryo development.

Cytoplasmic lipid droplets and mitochondrial distribution in equine oocytes: Implications on oocyte maturation, fertilization and developmental competence after ICSI

Lipid droplets (LDs) and mitochondria in the ooplasm are essential for energy production required for maturation, fertilization and embryo development. This study investigates the correlations between cytoplasmic LDs polar aggregation and: (1) nuclear maturation (Experiment 1); (2) mitochondrial (mt) distribution pattern and localization (Experiment 2); (3) fertilization and embryonic development after intracytoplasmic sperm injection (ICSI; Experiment 3) in equine oocytes recovered from slaughtered mares and matured in vitro. Morphologically normal oocytes were selected after culture and categorized as having polar (P) aggregation or uniform (U) distribution of LDs. In Experiment 1, the maturation rate was significantly higher in P compared with U oocytes (69%, 40/58 vs. 32%, 13/41; P<0.001). In Experiment 2, it was observed that P and U oocytes showed heterogeneous mt distribution at comparable rates (68%, 25/37 vs. 50%, 2/4 for P and U respectively; NS). Moreover, only in 8/25 (32%) of P oocytes, LDs overlapped with mt aggregates in the area containing meiotic spindle. In Experiment 3, normal fertilization (51%, 19/37 vs. 60%, 6/10, for P and U) and cleavage rates (83%, 20/24 vs. 67%, 4/6, for P and U) did not differ between groups, also in oocytes with LDs located nearby the polar body. Overall, P aggregation of LDs was related to cumulus expansion at collection. In conclusion, in equine matured oocytes, P aggregation of LDs is related with cumulus expansion and nuclear maturation. However, it is not related with heterogeneous mt distribution and cannot be considered a predictive indicator for normal fertilization and embryo development.

New Insights into the Mechanisms of Fertilization: Comparison of the Fertilization Steps, Composition, and Structure of the Zona Pellucida Between Horses and Pigs

The mechanism of fertilization remains largely enigmatic in mammals. Most studies exploring the molecular mechanism underlying fertilization have been restricted to a single species, generally the mouse, without a comparative approach. However, the identification of divergences between species could allow us to highlight key components in the mechanism of fertilization. In the pig, in vitro fertilization (IVF) and polyspermy rates are high, and spermatozoa penetrate easily through the zona pellucida (ZP). In contrast, IVF rates are low in the horse, and polyspermy is scarce. Our objective was to develop a comparative strategy between these two divergent models. First, we compared the role of equine and porcine gametes in the following five functions using intraspecific and interspecific IVF: ZP binding, acrosome reaction, penetration through the ZP, gamete fusion, and pronucleus formation. Under in vitro conditions, we showed that the ZP is a determining element in sperm-ZP attachment and penetration, whereas the capacity of the spermatozoa is of less importance. In contrast, the capacity of the spermatozoa is a key component of the acrosome reaction step. Second, we compared the composition and structure of the equine and porcine ZP. We observed differences in the number and localization of the ZP glycoproteins and in the mesh-like structure of the ZP between equine and porcine species. These differences might correlate with the differences in spermatozoal attachment and penetration rates. In conclusion, our comparative approach allows us to identify determining elements in the mechanism of fertilization.

Changes in histone H4 acetylation during in vivo versus in vitro maturation of equine oocytes

Epigenetic modifications are established during gametogenesis and preimplantation embryonic development. Any disturbance of the normal natural environment during these critical phases could cause alterations of the epigenetic signature. Histone acetylation is an important epigenetic modification involved in the regulation of chromatin organization and gene expression. The present study was aimed to determine whether the proper establishment of post-translational histone H4 acetylation at lysine 8 (AcH4K8), 12 (AcH4K12) and 16 (AcH4K16) of equine oocytes is adversely affected during in vitro maturation (IVM) when compared with in vivo matured oocytes collected from naturally cycling mares not undergoing ovarian hyperstimulation. The acetylation patterns were investigated by means of indirect immunofluorescence staining with specific antibodies directed against the acetylated lysine residues. Our results indicate that the acetylation state of H4 is dependent on the chromatin configuration in immature germinal vesicle (GV) stage oocytes and it changes in a residue-specific manner along with the increase of chromatin condensation. In particular, the levels of AcH4K8 and AcH4K12 increased significantly, while AcH4K16 decreased significantly from the fibrillar to the condensed state of chromatin configuration within the GV. Moreover, during meiosis, K8 and K12 were substantially deacetylated without any differences between in vivo and in vitro conditions, while K16 displayed a strong acetylation in oocytes matured in vivo, and in contrast, it was markedly deacetylated following IVM. Although the functional meaning of residue-specific acetylation during oocyte differentiation and meiotic resumption needs further investigation, our results support the hypothesis that IVM conditions can adversely affect oocyte ability to regulate the epigenetic reprogramming, critical for successful meiosis and subsequent embryonic development.

The secretions of oviduct epithelial cells increase the equine in vitro fertilization rate: are osteopontin, atrial natriuretic peptide A and oviductin involved?

BackgroundOviduct epithelial cells (OEC) co-culture promotes in vitro fertilization (IVF) in human, bovine and porcine species, but no data are available from equine species. Yet, despite numerous attempts, equine IVF rates remain low. Our first aim was to verify a beneficial effect of the OEC on equine IVF. In mammals, oviductal proteins have been shown to interact with gametes and play a role in fertilization. Thus, our second aim was to identify the proteins involved in fertilization in the horse.Methods & resultsIn the first experiment, we co-incubated fresh equine spermatozoa treated with calcium ionophore and in vitro matured equine oocytes with or without porcine OEC. We showed that the presence of OEC increases the IVF rates. In the subsequent experiments, we co-incubated equine gametes with OEC and we showed that the IVF rates were not significantly different between 1) gametes co-incubated with equine vs porcine OEC, 2) intact cumulus-oocyte complexes vs denuded oocytes, 3) OEC previously stimulated with human Chorionic Gonadotropin, Luteinizing Hormone and/or oestradiol vs non stimulated OEC, 4) in vivo vs in vitro matured oocytes.In order to identify the proteins responsible for the positive effect of OEC, we first searched for the presence of the genes encoding oviductin, osteopontin and atrial natriuretic peptide A (ANP A) in the equine genome. We showed that the genes coding for osteopontin and ANP A are present. But the one for oviductin either has become a pseudogene during evolution of horse genome or has been not well annotated in horse genome sequence. We then showed that osteopontin and ANP A proteins are present in the equine oviduct using a surface plasmon resonance biosensor, and we analyzed their expression during oestrus cycle by Western blot. Finally, we co-incubated equine gametes with or without purified osteopontin or synthesized ANP A. No significant effect of osteopontin or ANP A was observed, though osteopontin slightly increased the IVF rates.ConclusionOur study shows a beneficial effect of homologous and heterologous oviduct cells on equine IVF rates, though the rates remain low. Furthers studies are necessary to identify the proteins involved. We showed that the surface plasmon resonance technique is efficient and powerful to analyze molecular interactions during fertilization.

Evolution of Genes Involved in Gamete Interaction: Evidence for Positive Selection, Duplications and Losses in Vertebrates

Genes encoding proteins involved in sperm-egg interaction and fertilization exhibit a particularly fast evolution and may participate in prezygotic species isolation [1], [2]. Some of them (ZP3, ADAM1, ADAM2, ACR and CD9) have individually been shown to evolve under positive selection [3], [4], suggesting a role of positive Darwinian selection on sperm-egg interaction. However, the genes involved in this biological function have not been systematically and exhaustively studied with an evolutionary perspective, in particular across vertebrates with internal and external fertilization. Here we show that 33 genes among the 69 that have been experimentally shown to be involved in fertilization in at least one taxon in vertebrates are under positive selection. Moreover, we identified 17 pseudogenes and 39 genes that have at least one duplicate in one species. For 15 genes, we found neither positive selection, nor gene copies or pseudogenes. Genes of teleosts, especially genes involved in sperm-oolemma fusion, appear to be more frequently under positive selection than genes of birds and eutherians. In contrast, pseudogenization, gene loss and gene gain are more frequent in eutherians. Thus, each of the 19 studied vertebrate species exhibits a unique signature characterized by gene gain and loss, as well as position of amino acids under positive selection. Reflecting these clade-specific signatures, teleosts and eutherian mammals are recovered as clades in a parsimony analysis. Interestingly the same analysis places Xenopus apart from teleosts, with which it shares the primitive external fertilization, and locates it along with amniotes (which share internal fertilization), suggesting that external or internal environmental conditions of germ cell interaction may not be the unique factors that drive the evolution of fertilization genes. Our work should improve our understanding of the fertilization process and on the establishment of reproductive barriers, for example by offering new leads for experiments on genes identified as positively selected.

The extracellular calcium-sensing receptor is expressed in the cumulus–oocyte complex in mammals and modulates oocyte meiotic maturation

The extracellular calcium-sensing receptor (CASR) plays an important role in cells involved in calcium (Ca2+) homeostasis by directly sensing changes in the extracellular Ca2+ ion concentration. We previously reported the localization and quantitative expression of CASR protein in human oocytes. In this study, we examined the expression and the functional role of CASR during oocyte meiotic maturation in a large mammal animal model, the horse. As in humans, CASR protein was found to be expressed in equine oocytes and cumulus cells. Western-blot analysis revealed a single 130 kDa band in denuded oocytes and a doublet of 130-120 kDa in cumulus cells. CASR labeling was observed by confocal microscopy in cumulus cells and in oocytes on the plasma membrane and within the cytoplasm at all examined stages of meiosis. Functionally, the CASR allosteric effector NPS R-467, in the presence of 2.92 mM external Ca2+, increased oocyte maturation rate in a dose-dependent manner and its stimulatory effect was attenuated by pre-treatment with the CASR antagonist NPS 2390. NPS R-467 had no effect in suboptimal external Ca2+ (0.5 mM), indicating that it requires higher external Ca2+ to promote oocyte maturation. In oocytes treated with NPS R-467, CASR staining increased at the plasmalemma and was reduced in the cytosol. Moreover, NPS R-467 increased the activity of MAPK, also called ERK, in cumulus cells and oocytes. These results provide evidence of a novel signal transduction pathway modulating oocyte meiotic maturation in mammals in addition to the well-known systemic hormones.

Efficiency of embryonic development after intrafollicular and intraoviductal transfer of in vitro and in vivo matured horse oocytes.

In vivo techniques, such as intraoviductal oocyte transfer (OT) and intrafollicular oocyte transfer (IFOT), can be considered as alternatives to bypass the lack of efficient superovulation treatments and the inadequacy of conventional in vitro fertilization techniques in the horse. We compared embryo production after transfer of in vivo recovered oocytes (1) into a recipients oviduct or (2) into her preovulatory follicle either immediately after ovum pick-up or (3) after in vitro maturation (IVM). Recipients were inseminated with fresh semen of a stallion with a known normal fertility. Ten days after surgery, rates of embryos collected in excess to the number of ovulations were calculated and compared for each group. Embryo collection rates were 32.5% (13 of 40), 5.5% (3 of 55), and 12.8% (6 of 47) for OT, post-IVM IFOT, and immediate IFOT, respectively. Oocyte transfer significantly yielded more embryos than did immediate IFOT and post-IVM IFOT. We also showed that in vitro matured oocytes could successfully be used for IFOT. Our results also suggest that improvement of the IFOT technique could turn it into an inexpensive and easy-to-perform procedure that could be an answer to the inefficiency of superovulation treatments in the mare.