Nadine Gérard

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.

In vivo effect of interleukin-1beta and interleukin-1RA on oocyte cytoplasmic maturation, ovulation, and early embryonic development in the mare

A growing body of evidence suggests that the interleukin-1 system is involved in periovulatory events. Previous work from our lab demonstrated that in the mare, interleukin-1beta (IL-1beta) increases the ovulatory rate of metaphase II oocytes. The present study was conducted to analyze in vivo the effect of IL-1 on oocyte cytoplasmic maturation, ovulation and pregnancy rate. In the present work, IL-1beta (experiment 1, n = 13; experiment 2, n = 25) and interleukin-1RA (IL-1RA; experiment 1, n = 25) were injected intrafollicularly by using the transvaginal ultrasound-guided injection method. Injections were performed on cyclic mares when the diameter of the growing dominant follicle reached 30–34 mm. In experiment 1, mares were inseminated the day of the treatment and all the other day until ovulation. The time of ovulation was determined and a pregnancy diagnosis was performed 14 days after ovulation of the injected follicle. In experiment 2, the cumulus-oocyte complex from each injected follicle was collected by transvaginal ultrasound-guided aspiration 38 h after the intrafollicular injection. Oocyte nuclear stage and oocyte cytoplasmic maturation were assessed by analyzing chromatin configuration, cortical granules migration and mitochondria distribution under a confocal microscope. The results from experiment 1 confirm that an intrafollicular injection of 1 microgram IL-1beta induces ovulation in the mare whereas IL-1RA has no effect at the dose used in the present study. Furthemore, we demonstrated, that in our experimental conditions, IL-1beta and IL-1RA induced a decrease in embryo development. Experiment 2 leads us to observe that IL-1beta is unable to induce cortical granules migration and remodelling of mitochondria, that commonly occurs during oocyte maturation, whereas it acts on nuclear maturation. This result may explain the decrease in embryo development we observed after IL-1beta intrafollicular injection. In conclusion, the present study tends to demonstrate that IL-1beta plays a role in the ovulatory process and may acts on oocyte maturation in the mare, but additional factors are required to complete equine oocyte cytoplasmic maturation to allow embryo development.

Steroid hormones content and proteomic analysis of canine follicular fluid during the preovulatory period

BackgroundFollicular fluid contains substances involved in follicle activity, cell differentiation and oocyte maturation. Studies of its components may contribute to better understanding of the mechanisms underlying follicular development and oocyte quality. The canine species is characterized by several ovarian activity features that are not extensively described such as preovulatory luteinization, oocyte ovulated at the GV stage (prophase 1) and poly-oocytic follicles. In this study, we examined the hypothesis that the preovulatory LH surge is associated with changes in steroid and protein content of canine follicular fluid prior to ovulation.MethodsFollicular fluid samples were collected from canine ovaries during the preovulatory phase, before (pre-LH; n = 16 bitches) and after (post-LH; n = 16) the LH surge. Blood was simultaneously collected. Steroids were assayed by radioimmunoassay and proteomic analyses were carried out by 2D-PAGE and mass spectrometry.ResultsThe concentrations of 17beta-estradiol and progesterone at the pre-LH stage were 737.2 +/- 43.5 ng/ml and 2630.1 +/- 287.2 ng/ml in follicular fluid vs. 53 +/- 4.1 pg/ml and 3.9 +/- 0.3 ng/ml in plasma, respectively. At that stage, significant positive correlations between follicular size and intra-follicular steroid concentrations were recorded. After the LH peak, the intrafollicular concentration of 17beta-estradiol decreased significantly (48.3 +/- 4.4 ng/ml; p < 0.001), whereas that of progesterone increased (11690.2 +/- 693.6 ng/ml; p < 0.001). Plasmatic concentration of 17beta-estradiol was not modified (49 +/- 9.6 pg/ml) after the LH peak, but that of progesterone significantly increased (9.8 +/- 0.63 ng/ml).Proteomic analysis of canine follicular fluid identified 38 protein spots, corresponding to 21 proteins, some of which are known to play roles in the ovarian physiology. The comparison of 2D-PAGE patterns of follicular fluids from the pre- and post-LH stages demonstrated 3 differentially stained single spot or groups of spots. One of them was identified as complement factor B. A comparison of follicular fluid and plasma protein patterns demonstrated a group of 4 spots that were more concentrated in plasma than in follicular fluid, and a single spot specific to follicular fluid. These proteins were identified as gelsolin and clusterin, respectively.ConclusionOur results provide the first demonstration of size-related changes in the steroid concentrations in canine follicular fluid associated with the LH surge. 2D protein mapping allowed identification of several proteins that may play a role in follicle physiology and ovarian activity at the preovulatory stage. This may help in the future to explain and to better understand the species specificities that are described in dogs.

Proteomes of the Female Genital Tract During the Oestrous Cycle

The female genital tract includes several anatomical regions whose luminal fluids successively interact with gametes and embryos and are involved in the fertilisation and development processes. The luminal fluids from the inner cervix, the uterus and the oviduct were collected along the oestrous cycle at oestrus (Day 0 of the cycle) and during the luteal phase (Day 10) from adult cyclic ewes. The proteomes were assessed by GeLC-MS/MS and quantified by spectral counting. A set of 940 proteins were identified including 291 proteins differentially present along the cycle in one or several regions. The global analysis of the fluid proteomes revealed a general pattern of endocrine regulation of the tract, with the cervix and the oviduct showing an increased differential proteins abundance mainly at oestrus while the uterus showed an increased abundance mainly during the luteal phase. The proteins more abundant at oestrus included several families such as the heat shock proteins (HSP), the mucins, the complement cascade proteins and several redox enzymes. Other proteins known for their interaction with gametes such as oviductin (OVGP), osteopontin, HSPA8, and the spermadhesin AWN were also overexpressed at oestrus. The proteins more abundant during the luteal phase were associated with the immune system such as ceruloplasmin, lactoferrin, DMBT1, or PIGR, and also with tissue remodeling such as galectin 3 binding protein, alkaline phosphatase, CD9, or fibulin. Several proteins differentially abundant between estrus and the luteal phase, such as myosin 9 and fibronectin, were also validated by immunohistochemistry. The potential roles in sperm transit and uterine receptivity of the proteins differentially regulated along the cycle in the female genital tract are discussed.

Differences in the metabolomic signatures of porcine follicular fluid collected from environments associated with good and poor oocyte quality

The microenvironment of the developing follicle is critical to the acquisition of oocyte developmental competence, which is influenced by several factors including follicle size and season. The aim of this study was to characterise the metabolomic signatures of porcine follicular fluid (FF) collected from good and poor follicular environments, using high-resolution proton nuclear magnetic resonance (1H-NMR) spectroscopy. Sow ovaries were collected at slaughter, 4 days after weaning, in summer and winter. The contents of small (3-4  mm) and large (5-8  mm) diameter follicles were aspirated and pooled separately for each ovary pair. Groups classified as summer-small (n=8), summer-large (n=15), winter-small (n=9) and winter-large (n=15) were analysed by 1H-NMR spectroscopy. The concentrations of 11 metabolites differed due to follicle size alone (P<0.05), including glucose, lactate, hypoxanthine and five amino acids. The concentrations of all these metabolites, except for glucose, were lower in large FF compared with small FF. Significant interaction effects of follicle size and season were found for the concentrations of glutamate, glycine, N-acetyl groups and uridine. Succinate was the only metabolite that differed in concentration due to season alone (P<0.05). The FF levels of progesterone, androstenedione and oestradiol were correlated with the concentrations of most of the metabolites examined. The results indicate that there is a distinct shift in follicular glucose metabolism as follicles increase in diameter and suggest that follicular cells may be more vulnerable to oxidative stress during the summer months. Our findings demonstrate the power of 1H-NMR spectroscopy to expand our understanding of the dynamic and complex microenvironment of the developing follicle.

Data for chicken semen proteome and label free quantitative analyses displaying sperm quality biomarkers

Understanding of biology of the avian male gamete is essential to improve the conservation of genetic resources and performances in farming. In this study, the semen proteome of the main domestic avian species (Gallus gallus) and evaluation of the molecular phenotype related to sperm quality were investigated using GeLC–MS/MS approach and label-free quantitative proteomic based on Spectral Counting (SC) and extracted ion chromatograms (XIC) methods. Here we describe in details the peptide/protein inventory of chicken ejaculated spermatozoa (SPZ) and seminal plasma (SP). We also show differential analyses of chicken semen (SPZ and corresponding SP) from 11 males demonstrating different levels of fertilizing capacity and sperm motility. The interpretation and description of these data can be found in a research article published by Labas and colleagues in the Journal of Proteomics in 2014 [1]. This is a new resource for exploring the molecular mechanisms involved in fertilizing capacity and to reveal new sets of fertility biomarkers.

In vivo and in vitro effects of interleukin-1β on equine oocyte maturation and on steroidogenesis and prostaglandin synthesis in granulosa and cumulus cells

We analysed the effect of interleukin-1 on oocyte maturation and on steroid and prostaglandin production by equine granulosa and cumulus cells. In Experiment 1, interleukin-1beta (IL-1beta) was injected into the growing dominant follicle, which was punctured 38 h later. Follicular fluid was assayed for steroids and prostaglandin-F2alpha (PGF2alpha). Granulosa cells were analysed for 3beta-hydroxysteroid dehydrogenase (3beta-HSD), progesterone receptor (PR), cyclooxygenase 1 and 2 (Cox 1 and Cox 2) and steroidogenic acute regulatory protein (StAR) mRNAs. In Experiment 2, cumulus-oocyte complexes (COCs) were collected from slaughterhouse ovaries and cultured in different media: control group (TCM199 + BSA); Group 2 (+ IL-1beta); Group 3 (+ EGF); Group 4 (+ EGF + IL-1beta); and Group 5 (+ EGF + IL-1beta + IL-1RA). Cumulus cells were analysed for 3beta-HSD, PR, Cox 1, Cox 2 and StAR mRNAs. After injections of crude equine gonadotropin (CEG; LH effect) or IL-1beta, progesterone and PGF2alpha levels increased, whereas 17beta-oestradiol decreased. EGF induced an increase in the rate of in vitro maturation (P < 0.05), whereas IL-1beta had a limited effect. IL-1beta significantly decreased the rate of EGF-induced oocyte maturation (P < 0.05). Cox 2 mRNA level increases in granulosa cells after CEG injection (P = 0.07). In cumulus cells, StAR and PR mRNAs were lower in Group 2 and 3beta-HSD mRNA was higher in Groups 4 and 5. These data confirm that IL-1 is involved in equine oocyte in vitro maturation. We demonstrated in vivo that IL-1beta has an effect on steroids and PGF2alpha secretion in the preovulatory follicle.

Reproductive Physiology and Ovarian Folliculogenesis Examined via 1H-NMR Metabolomics Signatures: A Comparative Study of Large and Small Follicles in Three Mammalian Species (Bos taurus, Sus scrofa domesticus and Equus ferus caballus)

The aim of this study was to characterize the composition of follicular fluid (FF) collected from the small and large follicles of three mammalian species, Bos taurus, Sus scrofa domesticus, and Equus ferus caballus, that display distinct ovulatory properties. For each species, five large FF samples and five small FF samples were analyzed using 1H-NMR spectroscopy. The FF metabolic profiles of the three species were very distinct. In cows and mares, the metabolic profiles of large FF and small FF were also very distinct. The concentrations of seventeen identified metabolites differed significantly between the sample groups. In mares, fourteen metabolites were found at much greater concentrations in large FF than in small FF (p<0.05). In cows, four metabolites differed in concentration between the large FF and small FF samples (p<0.05). A common feature of the monovulatory species was that the concentrations of α- and β-glucose were much greater in large FF compared with small FF (p<0.05). Sow FF was characterized by the apparent absence of citrate (detected in cow and mare FF), and the presence of succinate (not detected in cow and mare FF). Another obvious difference between species was the concentration of lactate, which was minimal in mare FF compared with cow and sow FF (p<0.05). The findings provide valuable insights into reproductive physiology broadly, and indicate that the activities of central metabolic enzymes differ enormously between these species. Future investigations into species-specific differences in follicle metabolism would increase our understanding of the processes critical to folliculogenesis and the acquisition of oocyte developmental competence.

Eggshell matrix proteins OC-116, OC-17 and OCX36 in hen's sperm storage tubules

While uterine epithelium secretes eggshell matrix proteins to regulate eggshell structural organization, uterovaginal junction (UVJ) epithelium supports sperm storage in tubules (SST). Here, we examined the presence of OCX36, OC-116 and OC-17 eggshell matrix proteins in SSTs. Two experimental lines of hens displaying either a long (F+ line) or a short (F- line) potential to store sperm were used, before and 24h after insemination. Using immunohistochemistry and western blot, we analyzed the presence of OC-116, OC-17 and OCX36 proteins in the SSTs. Using lectin and calcium staining, we examined the presence in SSTs of Gal/GalNAc (Galactose/N-acetylgalactosamine) and Glc/GlcNAc (Glucose/N-acetylglucosamine) glycans, as well as calcium ions. Our results indicate that in both F+ and F- hens, the eggshell matrix proteins OC-116 and OCX36 were identified in SST cells and lumen, in contact with spermatozoa. The OC-17 protein was found associated with calcium in F+ and F- hens, only in the SST lumen 24h after insemination. Glycans Gal/GalNAc and Glc/GlcNAc were found to be more abundant in the apical cytoplasmic area of the SST cells of F+ hens than in that of F- hens after insemination. This is the first report demonstrating the presence in SSTs of the OC-116, OC-17 and OCX36 eggshell matrix proteins, and their concomitant presence with Gal/GalNAc and Glc/GlcNAc glycans, as well as with calcium. Our results suggest that the OC-116, OC-17 and OCX36 eggshell matrix proteins may be involved in sperm storage.