Search

Increasing the efficiency of farm animal reproduction is necessary to reduce the environmental impact of food production systems. One approach is to increase the number of healthy eggs (oocytes) produced per female for fertilization, thus it is important to understand factors that decrease oocyte health. One paracrine factor that decreases ovarian follicle growth is fibroblast growth factor 18 (FGF18) secreted by cells in the theca layer of the ovarian follicle, however the factors that regulate FGF18 secretion are unknown. In this study we hypothesized that FGF18 secretion is controled by intrafollicular factors and is linked to fertility, which we tested by using cell culture and sheep genetic models in vivo. Separation of theca cell populations revealed that FGF18 messenger RNA (mRNA) is located mainly in thecal endothelial rather than endocrine cells, and immunohistochemistry localized FGF18 protein to microvessels in the theca layer in situ. Culture of ovine theca-derived endothelial cells was used to demonstrate stimulation of FGF18 mRNA and protein abundance by bone morphogenetic protein 4 (BMP4), a growth factor derived from theca endocrine cells. Taking advantage of a sheep genetic model, we demonstrate reduced ovarian and peripheral FGF18 concentrations in the hyperprolific Booroola ewe harboring the FecB

In this study, we aimed to determine the origin of the difference, in terms of anti-Müllerian hormone production, existing between the bovine and porcine ovaries. We first confirmed by quantitative real-time-Polymerase-Chain Reaction, ELISA assay and immunohistochemistry that anti-Müllerian hormone mRNA and protein production are very low in porcine ovarian growing follicles compared to bovine ones. We then have transfected porcine and bovine granulosa cells with vectors containing the luciferase gene driven by the porcine or the bovine anti-Müllerian hormone promoter. These transfection experiments showed that the porcine anti-Müllerian hormone promoter is less active and less responsive to bone morphogenetic protein stimulations than the bovine promoter in both porcine and bovine cells. Moreover, bovine but not porcine granulosa cells were responsive to bone morphogenetic protein stimulation after transfection of a plasmidic construction including a strong response element to the bone morphogenetic proteins (12 repetitions of the GCCG sequence) upstream of the luciferase reporter gene. We also showed that SMAD6, an inhibitor of the SMAD1-5-8 pathway, is strongly expressed in porcine compared to the bovine granulosa cells. Overall, these results suggest that the low expression of anti-Müllerian hormone in porcine growing follicles is due to both a lack of activity/sensitivity of the porcine anti-Müllerian hormone promoter, and to the lack of responsiveness of porcine granulosa cells to bone morphogenetic protein signaling, potentially due to an overexpression of SMAD6 compared to bovine granulosa cells. We propose that the low levels of anti-Müllerian hormone in the pig would explain the poly-ovulatory phenotype in this species.

Breast Cancer Anti-estrogen Resistance 4 (BCAR4) was previously characterised in bovine species as a gene preferentially expressed in oocytes, whose inhibition is detrimental to in vitro embryo development. But its role in oogenesis, folliculogenesis and globally fertility in vivo remains unknown. Because the gene is not conserved in mice, rabbits were chosen for investigation of BCAR4 expression and function in vivo. BCAR4 displayed preferential expression in the ovary compared to somatic organs, and within the ovarian follicle in the oocyte compared to somatic cells. The transcript was detected in follicles as early as the preantral stage. Abundance decreased throughout embryo development until the blastocyst stage. A lineage of genome-edited rabbits was produced; BCAR4 expression was abolished in follicles from homozygous animals. Females of wild-type, heterozygous and homozygous genotypes were examined for ovarian physiology and reproductive parameters. Follicle growth and the number of ovulations in response to hormonal stimulation were not significantly different between genotypes. Following insemination, homozygous females displayed a significantly lower delivery rate than their heterozygous counterparts (22 ± 7% vs 71 ± 11% (mean ± SEM)), while prolificacy was 1.8 ± 0.7 vs 6.0 ± 1.4 kittens per insemination. In conclusion, BCAR4 is not essential for follicular growth and ovulation but it contributes to optimal fertility in rabbits.

International audience; This prospective study aims to analyze the serum metabolome of female goat kids just before the first breeding in order to characterize the changes in metabolites during sexual maturation. A better knowledge of these changes could help optimizing the time for first breeding and improving the fertilization rates at first insemination. Weekly blood sampling was performed on twenty 6- to 7-month-old female goats from Alpine breed born in February during the 5 weeks preceding their first contact with bucks in September. Progesterone assays and metabolome analysis using 1H Nuclear Magnetic Resonance Spectroscopy were performed on the serum samples. No spontaneous ovulatory cycle was observed before breeding based on progesterone assays. All female goats had reached the pubertal stage of maturity at breeding since all got pregnant. Metabolome analysis allowed the identification of 109 spectral bins in sera. When the metabolic profiles were compared between the 5 weeks preceding the first contact with bucks, 6 metabolites were highlighted that could differentiate between week 5 and week 1, 2 and 3: glucose, citrate, creatinine, 3-hydroxy-isobutyrate and two unidentified bins. Citrate and 3-hydroxy-isobutyrate are of particular interest because their level significantly varied during sexual maturation, with a significant decrease of 3-hydroxy-isobutyrate at week 5 and a significant increase of citrate at week 4 and 5. However, the lack of variability in the fertility results did not allow us to conclude on a potential link between these metabolomics profile and reproduction performances. Further studies are necessary to ascertain the reliability of these metabolites as biomarkers of sexual precocity.

PURPOSE: Vitrification is a well-accepted fertility preservation procedure for cryopreservation of oocytes and embryos but little is known regarding ovarian tissue, for which slow freezing is the current convention. The aim of the present study was to assess the efficiency of non-equilibrium vitrification compared to conventional slow freezing for ovarian cortex cryopreservation. METHODS: Using prepubertal sheep ovaries, the capacity of the tissue to sustain folliculogenesis following cryopreservation and in vitro culture was evaluated. Ovarian cortex fragments were cultured in wells for 9 days, immediately or after cryopreservation by conventional slow freezing or non-equilibrium vitrification in straws. During culture, follicular populations within cortex were evaluated by histology and immunohistochemistry for PCNA and TUNEL. Steroidogenic activity of the tissue was monitored by assay for progesterone and estradiol in spent media. RESULTS: No significant differences in follicle morphology, PCNA, or TUNEL labeling were observed between cryopreservation methods at the initiation of culture. Similar decreases in the proportion of primordial follicle population, and increases in the proportion of growing follicles, were observed following culture of fresh or cryopreserved ovarian tissue regardless of cryopreservation method. At the end of culture, PCNA and TUNEL-positive follicles were not statistically altered by slow freezing or vitrification in comparison to fresh cultured fragments. CONCLUSIONS: Overall, for both cryopreservation methods, the cryopreserved tissue showed equal capacity to fresh tissue for supporting basal folliculogenesis in vitro. Taken together, these data confirm that both non-equilibrium vitrification and slow-freezing methods are both efficient for the cryopreservation of sheep ovarian cortex fragments.

International audience; In mammalian ovaries, the theca layers of growing follicles are critical for maintaining their structural integrity and supporting androgen synthesis. Through combining the postnatal monitoring of ovaries by abdominal magnetic resonance imaging, endocrine profiling, hormonal analysis of the follicular fluid of growing follicles, and transcriptomic analysis of follicular theca cells, we provide evidence that the exposure of ovine fetuses to testosterone excess activates postnatal follicular growth and strongly affects the functions of follicular theca in adulthood. Prenatal exposure to testosterone impaired androgen synthesis in the small antral follicles of adults and affected the expression in their theca cells of a wide array of genes encoding extracellular matrix components, their membrane receptors, and signaling pathways. Most expression changes were uncorrelated with the concentrations of gonadotropins, steroids, and anti-Müllerian hormone in the recent hormonal environment of theca cells, suggesting that these changes rather result from the long-term developmental effects of testosterone on theca cell precursors in fetal ovaries. Disruptions of the extracellular matrix structure and signaling in the follicular theca and ovarian cortex can explain the acceleration of follicle growth through altering the stiffness of ovarian tissue. We propose that these mechanisms participate in the etiology of the polycystic ovarian syndrome, a major reproductive pathology in woman.

Simple Summary Changes in anti-Müllerian hormone (AMH) levels, a fertility marker in dairy cows, during the peripartum period associated with reproductive recovery have not been fully evaluated. We investigated the relationship between changes in AMH concentration and inflammation markers in serum during the peripartum period in dairy cows. We found a relationship between changes in AMH concentration, especially reflected in the AMH ratio during the perinatal period, and the systemic inflammation status of dairy cows. Excessive inflammation during the early postpartum period may decrease AMH levels and subsequently affect the reproductive prognosis of postpartum cows. Elucidating the mechanism of perinatal AMH changes and the beneficial effects of AMH may improve reproductive efficacy in the dairy industry. Abstract The relationships between changes in anti-Müllerian hormone (AMH) concentration and various traits, including milk somatic cell counts (SCC), were evaluated. Blood samples were collected from 43 Holstein cows 14 days before (D-14) and 10 (D10) and 28 days after (D28) parturition, and vaginal discharge score (VDS) and polymorphonuclear leukocyte (PMNL) percentages were assessed in endometrial samples at D28. Cows were separated into four quartiles (Q1–Q4) based on changes in AMH concentration during the peripartum period (AMH ratio: D28/D-14). Correlations between AMH ratio and each parameter were evaluated and classified into high-AMH (Q4, 1.83 ± 0.12, n = 11) and low-AMH (Q1, 0.83 ± 0.05, n = 11) groups. The AMH ratio was positively correlated with magnesium and non-esterified fatty acids levels, and the albumin/globulin ratio at D10 and D28, but negatively correlated with serum amyloid A (SAA) at D10. SAA and γ-globulin levels were significantly higher in the low-AMH group at D28. There was no significant difference in VDS, PMNL percentage, and milk SCC between the two groups. The decreasing AMH ratio from the prepartum to the postpartum period corresponds to high inflammation biomarker levels. Whether it subsequently affects the reproductive prognosis of postpartum cows needs further investigations.

Purpose A protective effect of anti-Müllerian hormone (AMH) on follicle atresia was recently demonstrated using long-term treatments, but this effect has never been supported by mechanistic studies. This work aimed to gain an insight into the mechanism of action of AMH on follicle atresia and on how this could account for the increased follicle pool observed in women with polycystic ovary syndrome (PCOS). Methods In vivo and in vitro experiments were performed to study the effects of AMH on follicle atresia and on the proliferation and apoptosis of granulosa cells (GCs). RNA-sequencing was carried out to identify new AMH target genes in GCs. The expression of some of these genes in GCs from control and PCOS women was compared using microfluidic real time quantitative RT-PCR. Results A short-term AMH treatment prevented follicle atresia in prepubertal mice. Consistent with this result, AMH inhibited apoptosis and promoted proliferation of different models of GCs. Moreover, integrative biology analyses of 965 AMH target genes identified in 1 of these GC models, confirmed that AMH had initiated a gene expression program favoring cell survival and proliferation. Finally, on 43 genes selected among the most up- and down-regulated AMH targets, 8 were up-regulated in GCs isolated from PCOS women, of which 5 are involved in cell survival. Main conclusions Our results provide for the first time cellular and molecular evidence that AMH protects follicles from atresia by controlling GC survival and suggest that AMH could participate in the increased follicle pool of PCOS patients.

International audience; The reproductive neuroendocrine axis, or hypothalamo-pituitary-gonadal (HPG) axis, is a paragon of complex biological system involving numerous cell types, spread over several anatomical levels communicating through entangled endocrine feedback loops. The HPG axis exhibits remarkable dynamic behaviors on multiple time and space scales, which are an inexhaustible source of studies for mathematical and computational biology. In this review, we will describe a variety of modeling approaches of the HPG axis from a cellular endocrinology viewpoint. We will in particular investigate the questions raised by some of the most striking features of the HPG axis: (i) the pulsatile secretion of hypothalamic and pituitary hormones, and its counterpart, the cell signaling induced by frequency-encoded hormonal signals, and (ii) the dual, gametogenic and glandular function of the gonads, which relies on the tight control of the somatic cell populations ensuring the proper maturation and timely release of the germ cells.

International audience; We evaluated the relationship between plasma anti-Müllerian hormone (AMH) concentrations in Japanese Black (JB) heifers at 7-10 months of age and the number of embryos recovered after superovulation treatment in selected ovum pick-up donors, concomitantly with changes in their AMH concentrations before and after parturition. Plasma AMH concentrations in heifers were positively correlated with the total number of follicles (r = 0.647, P < 0.01) and embryos (r = 0.681, P < 0.01) recovered from the animals postpartum, when selected as donor cows, but did not correlate with the total number of transferable embryos. No difference was observed between the plasma AMH concentration at the heifer period and the postpartum period. Additionally, serum AMH concentrations of heifers weakly correlated with the number of follicles and embryos recovered by virgin flush after superovulation treatment at 13-15 months of age. Therefore, a single blood AMH concentration measurement may accelerate intensive JB cattle breeding.

From fetal life until senescence, the ovary is an extremely active tissue undergoing continuous structural and functional changes. These ever-changing events are best summarized by a quotation attributed to Plato when describing motion in space and time—‘nothing ever is but is always becoming…’. With respect to the ovary, these changes include, at the beginning, the processes of follicular formation and thereafter those of follicular growth and atresia, steroidogenesis, oocyte maturation, and decisions relating to the number of mature oocytes that are ovulated for fertilization and the role of the corpus luteum. The aims of this review are to offer some examples of these complex and hitherto unknown processes. The ones herein have been elucidated from studies undertaken in vitro or from normal in vivo events, natural genetic mutations or after experimental inactivation of gene function. Specifically, this review offers insights concerning the initiation of follicular growth, pathologies relating to poly-ovular follicles, the consequences of premature loss of germ cells or oocytes loss, the roles of AMH (anti-Müllerian hormone) and BMP (bone morphogenetic protein) genes in regulating follicular growth and ovulation rate together with species differences in maintaining luteal function during pregnancy. Collectively, the evidence suggests that the oocyte is a key organizer of normal ovarian function. It has been shown to influence the phenotype of the adjacent somatic cells, the growth and maturation of the follicle, and to determine the ovulation rate. When germ cells or oocytes are lost prematurely, the ovary becomes disorganized and a wide range of pathologies may arise.

International audience; The primary aim of this study was to develop a FSH enzyme immunoassay (EIA) for the bovine species. The newly developed EIA was validated for FSH determination in bovine plasma by comparison with an existing bovine FSH radioimmunoassay. The EIA detected bovine FSH with a high sensitivity (0.1 ng/ml). Cross-reactivity of the EIA was 0.01% with bovine LH, 51% with ovine FSH

This review focuses on the role of the dialog between the oocyte and its companion somatic cells in driving folliculogenesis from the primordial to the preovulatory follicle stage. Mouse and sheep genetic models have brought complementary evidence of these cell interactions and their consequences for ovarian function. In mouse, the deletion of genes encoding connexins has shown that functional gap junction channels between oocytes and granulosa cells and between granulosa cells themselves maintain the follicle in a functionally integrated state. Targeted deletions in oocytes or granulosa cells have revealed the cell- and stage-specific role of ubiquist factors belonging to the phosphatidylinositol 3 kinase signaling pathway in primordial follicle activation, oocyte growth and follicle survival. Various models of transgenic mice and sheep carrying natural loss-of-function mutations associated with sterility have established that the oocyte-derived factors, bone morphogenetic protein (BMP) 15 and growth differentiation factor 9 orchestrate follicle development, support cumulus metabolism and maturation and participate in oocyte meiosis arrest. Unexpectedly in sheep, mutations resulting in the attenuation of BMP signaling lead to enhanced ovulation rate, likely resulting from a lowered follicular atresia rate and the enhancement of FSH-regulated follicular maturation. Both the activation level of BMP signaling and an adequate equilibrium between BMP15 and growth differentiation factor 9 determine follicle survival, maturation, and development toward ovulation. The physiological approaches which were implemented on genetic animal models during the last 20 years have opened up new perspectives for female fertility by identifying the main signaling pathways of the oocyte-somatic cell dialog.

Anti-Müllerian hormone (AMH) is produced by the granulosa cells (GCs) of growing follicles and inhibits follicular development. This study aimed to investigate the regulation of the AMH-specific receptor gene (AMHR2) expression in GCs by bone morphogenetic protein 15 (BMP15), BMP4 and growth differentiation factor 9 (GDF9). Their effects on AMHR2 and AMH mRNAs were studied in luteinized human GCs (hGCs) and in ovine GCs (oGCs) from small antral follicles. The effects of BMPs on human AMHR2 and AMH promoter reporter activities were analyzed in transfected oGCs. The in vivo effect of BMP15 on GCs AMHR2 and AMH expression was investigated by using Lacaune and Rasa Aragonesa hyperprolific ewes carrying loss-of-function mutations in BMP15. mRNAs were quantified by real-time RT-PCR. Promoter reporter constructs activities were quantified by the measurement of their luciferase activity. BMP15 and BMP4 enhanced AMHR2 and AMH expression in hGCs and in oGCs whereas GDF9 had no effect. In oGCs, GDF9 increased BMP15 effect on AMH expression. Consistent with these results, BMP15 and BMP4, but not GDF9, enhanced AMHR2 promoter activity in oGCs, whereas GDF9 increased BMP15 effect on AMH promoter activity. Moreover, oGCs from both BMP15 mutant ewes had reduced AMHR2 mRNA levels but unchanged AMH expression compared to wild-type ewes. Altogether, these results suggest that the mechanisms of action of BMP15 on AMHR2 and AMH expression are different, and that by stimulating AMHR2 and AMH expression in GCs BMP15 enhances AMH inhibitory actions in GCs.

In this review, we present multi-scale mathematical models of ovarian follicular development that are based on the embedding of physiological mechanisms into the cell scale. During basal follicular development, follicular growth operates through an increase in the oocyte size concomitant with the proliferation of its surrounding granulosa cells. We have developed a spatio-temporal model of follicular morphogenesis explaining how the interactions between the oocyte and granulosa cells need to be properly balanced to shape the follicle. During terminal follicular development, the ovulatory follicle is selected amongst a cohort of simultaneously growing follicles. To address this process of follicle selection, we have developed a model giving a continuous and deterministic description of follicle development, adapted to high numbers of cells and based on the dynamical and hormonally regulated repartition of granulosa cells into different cell states, namely proliferation, differentiation and apoptosis. This model takes into account the hormonal feedback loop involving the growing ovarian follicles and the pituitary gland, and enables the exploration of mechanisms regulating the number of ovulations at each ovarian cycle. Both models are useful for addressing ovarian physio-pathological situations. Moreover, they can be proposed as generic modelling environments to study various developmental processes and cell interaction mechanisms.

International audience; Plasma anti-Müllerian hormone (AMH) concentrations have been recently found to be predictive of the number of embryos recovered after FSH superovulatory treatment in the cow. However, the sensitivity of the Active Müllerian-inhibiting substance/AMH ELISA (ref. 10–14400; DSL-Beckman-Coulter) used to make these measurements in bovine plasma samples is low because it was developed to measure human AMH levels. To overcome this limitation, we developed an immunoassay specific for the bovine (B), ovine (O), and caprine (C) species, the bovine-ovine-caprine (BOC) ELISA. For this purpose, we produced recombinant bovine AMH for standardization, and we used monoclonal antibodies raised against bovine AMH, previously prepared by our laboratory. We evaluated the precision, accuracy, specificity, limit of detection, and functional sensitivity of the assay. The intra-assay coefficient of variation ranged between 3.4% and 11.3% for AMH concentrations between 23.68 and 1.74 ng/mL, and the interassay coefficient of variation ranged between 4.8% and 20.5% for concentrations between 25.53 and 1.42 ng/mL, respectively. The assay displayed a good linearity, had a detection limit of 0.4 ng/mL and a functional sensitivity of 1.4 ng/mL. It also cross-reacted with ovine and caprine AMHs. Both the mean and median AMH levels measured in 40 cow plasma samples using the BOC ELISA were approximately 44 fold higher than the mean and median AMH levels measured with the Active Müllerian-inhibiting substance/AMH ELISA. Moreover, a higher correlation was observed between the average number of embryos recovered from each cow after superovulatory treatment and AMH concentrations measured with the BOC ELISA. This BOC ELISA provides a very efficient tool for evaluating the ovarian follicular reserve of cows and predicting their embryo production capacity.

International audience; Primordial follicles embedded in the ovarian cortex are the source of developing follicles. Follicle growth activa- tion and development up to the small antral follicle stage are controlled by cell interactions. The sheep ovary offers an appropriate non-rodent model to study these interac- tions, thanks to the development of in vitro and in vivo experimental approaches, ex vivo molecular analyses and in silico mathematical modeling. Each primordial follicle, composed of an oocyte surrounded by a single layer of quiescent granulosa cells, relies on nutrients and growth factors supplied by the surrounding stroma of connective tissue. In vitro cultures of ovarian cortex have shown that primordial follicles are activated by the lifting of mech- anisms maintaining quiescence, some of them involving AMH secreted by already growing follicles. Afterwards, follicle development is supported by a finely tuned molec- ular dialog between the growing oocyte and proliferating granulosa cells. The isolation of preantral follicles and their development in vitro as individual follicles perturb this dialog, leading to an acceleration of follicular matu- ration. In vivo, mutations in the oocyte factors BMP15, GDF9 or their receptor BMPR1B also impair this dia- log, leading to an imbalance between oocyte growth and cell proliferation, which can be reproduced by models for cell dynamics. During the growth of preantral follicles, the recruitment and differentiation of theca cells from the ovarian stroma provide them with a structural and vascularized support. In vivo exposure of sheep fetal ovaries to testosterone imprints the stroma cells so that the expression of genes involved in extracellular matrix organization and cell-cell adhesion is affected in theca at adulthood; this leads to a lower ovarian tissue rigid- ity that can account for the accelerated follicle growth observed in androgenized ewes. A better knowledge of cell interactions during early follicular development will help to improve the biotechnology methods of fertility preservation.

Notice à reprendre pas de clé UT au 14 Janvier 2018; International audience; Primordial follicles embedded in the ovarian cortex are the source of developing follicles. Follicle growth activationand development up to the small antral follicle stage are controlled by cell interactions. The sheep ovary offersan appropriate non-rodent model to study these interactions, thanks to the development of in vitro and in vivoexperimental approaches, ex vivo molecular analyses and in silico mathematical modeling. Each primordial follicle, composed of an oocyte surrounded by a single layer of quiescent granulosa cells, relies on nutrients and growth factors supplied by the surrounding stroma of connective tissue. In vitro cultures of ovarian cortex have shown that primordial follicles are activated by the lifting of mechanisms maintaining quiescence, some of them involving AMH secreted by already growing follicles. Afterwards, follicle development is supported by a finely tuned molecular dialog between the growing oocyte and proliferating granulosa cells. The isolation of preantral follicles and their development in vitro as individual follicles perturb this dialog, leading to an acceleration of follicular maturation. In vivo, mutations in the oocyte factors BMP15, GDF9 or their receptor BMPR1B also impair this dialog, leading to an imbalance between oocyte growth and cell proliferation, which can be reproduced by models for cell dynamics. During the growth of preantral follicles, the recruitment and differentiation of theca cells from the ovarian stroma provide them with a structural and vascularized support. In vivo exposure of sheep fetalovaries to testosterone imprints the stroma cells so that the expression of genes involved in extracellular matrixorganization and cell-cell adhesion is affected in theca at adulthood; this leads to a lower ovarian tissue rigiditythat can account for the accelerated follicle growth observed in androgenized ewes. A better knowledge ofcell interactions during early follicular development will help to improve the biotechnology methods of fertilitypreservation.

International audience; Primary ovarian insufficiency (POI) affects similar to 1% of women before 40 years of age. The recent leap in genetic knowledge obtained by next generation sequencing (NGS) together with animal models has further elucidated its molecular pathogenesis, identifying novel genes/pathways. Mutations of > 60 genes emphasize high genetic heterogeneity. Genome-wide association studies have revealed a shared genetic background between POI and reproductive aging. NGS will provide a genetic diagnosis leading to genetic/therapeutic counseling: first, defects in meiosis or DNA repair genes may predispose to tumors; and second, specific gene defects may predict the risk of rapid loss of a persistent ovarian reserve, an important determinant in fertility preservation. Indeed, a recent innovative treatment of POI by in vitro activation of dormant follicles proved to be successful.

International audience; Context: Anti-Mullerian hormone (AMH) and AMH type II receptor (AMHR2) are overexpressed in granulosa cells (GCs) from women with polycystic ovary syndrome (PCOS), the most common cause of female infertility. Objective: The aim of the study was to compare the regulation of the AMH/AMHR2 system by 5adihydrotestosterone (5a-DHT) and estradiol (E2) in GCs from control subjects and women with PCOS. Design, Setting, Patients: Experiments were performed on follicular fluids (FF) and GCs from women undergoing in vitro fertilization. Main Outcome Measures: FF steroid levels were measured by mass spectrometry, and messenger RNA (mRNA) accumulation was quantified by reverse transcription real-time polymerase chain reaction. Results: Total testosterone (T), free T, and 5 alpha-DHT FF levels were significantly higher (P < 0.001) in women with PCOS than in controls. However, E2 and sex hormone-binding globulin concentrations were comparable between the two groups. In GCs from control women, the AMH and AMHR2 expression were not affected by 5a-DHT treatment, whereas AMH mRNA levels were upregulated by 5a-DHT in GCs from patients with PCOS (2.3-fold, P < 0.01) overexpressing the androgen receptor (1.4-fold, P < 0.05). E2 downregulated the AMH and AMHR2 expression in GCs from control women (1.4-fold, P < 0.001 and 1.8-fold, P < 0.01, respectively) but had no effect on these genes in GCs from women with PCOS. This differential effect of E2 was associated with a higher estrogen receptor 1 expression in GCs from women with PCOS (1.9-fold, P < 0.05). Conclusions: In GCs from women with PCOS, the regulation of AMH and AMHR2 expression is altered in a way that promotes the overexpression of the AMH/AMHR2 system, and could contribute to the follicular arrest observed in these patients.

In this study, we systematically compared the morphological, functional and molecular characteristics of granulosa cells and oocytes obtained by a three-dimensionalin vitromodel of ovine ovarian follicular growth with those of follicles recoveredin vivo. Preantral follicles of 200 µm diameter were recovered and cultured up to 950 µm over a 20-day period. Compared within vivofollicles, thein vitroculture conditions maintained follicle survival, with no difference in the rate of atresia. However, thein vitroconditions induced a slight decrease in oocyte growth rate, delayed antrum formation and increased granulosa cell proliferation rate, accompanied by an increase and decrease inCCND2andCDKN1AmRNA expression respectively. These changes were associated with advanced granulosa cell differentiation in early antral follicles larger than 400 µm diameter, regardless of the presence or absence of FSH, as indicated by an increase in estradiol secretion, together with decreased AMH secretion and expression, as well as increased expression ofGJA1,CYP19A1,ESR1,ESR2,FSHR,INHA,INHBA,INHBBandFST. There was a decrease in the expression of oocyte-specific molecular markersGJA4,KIT,ZP3,WEE2andBMP15 in vitrocompared to thatin vivo. Moreover, a higher percentage of the oocytes recovered from cultured follicles 550 to 950 µm in diameter was able to reach the metaphase II meiosis stage. Overall, thisin vitromodel of ovarian follicle development is characterized by accelerated follicular maturation, associated with improved developmental competence of the oocyte, compared to follicles recoveredin vivo.

Ovarian folliculogenesis corresponds to the development of follicles leading to either ovulation or degeneration, this latter process being called atresia. Even if atresia involves apoptosis, its mechanism is not well-understood. The objective of this project was to analyse global gene expression in pig granulosa cells of ovarian follicles during atresia. The transcriptome analysis was performed using 9216 cDNAs microarray to identify gene networks and candidate genes involved in pig ovarian follicular atresia. One thousand six hundred and eighty four significantly regulated genes were differentially regulated between small healthy follicles and small atretic follicles. Among them, two hundred and eighty seven genes had a fold-change higher than 2 between the two follicle groups. Eleven genes (DKK3, GADD45A, CAMTA2, CCDC80, DAPK2, ECSIT, MSMB, NUPR1, RUNX2, SAMD4A, and ZNF628) having a fold-change higher than 5 between groups could likely serve as markers of follicular atresia. Moreover, automatic confrontation of deregulated genes with literature data enlightened 93 genes as regulatory candidates of pig granulosa cell atresia. Among these genes known to be inhibitors of apoptosis, stimulators of apoptosis or tumor suppressors INHBB, HNF4, CLU, different interleukins (IL5, IL24), TNF-associated receptor (TNFR1), and cytochrome-c oxidase (COX) were suggested as playing an important role in porcine atresia. Present study also enlists key upstream regulators in follicle atresia based on our results and on a literature review. The novel gene candidates and gene networks identified in the current study lead to a better understanding of the molecular regulation of ovarian follicular atresia.

In this study, we systematically compared the morphological, functional and molecular characteristics of granulosa cells and oocytes obtained by a three-dimensional

Recently, one Quantitative Trait Locus (QTL) of female fertility located on Bos Taurus chromosome 3 (BTA3), QTL-F-Fert-BTA3, has been identified in Holstein breed. It is implied in the success rate after the first AI (AI1) in cow. The failure of pregnancy can be due to several factors involved in the different steps of the reproductive process. The aim of our study was to finely phenotype heifers and primiparous cows selected for their haplotype at the QTL-F-Fert-BTA3. We specifically studied the ovarian follicular dynamic and several fertility parameters. Females carrying the favourable haplotype "fertil+" or unfavourable haplotype "fertil-" were monitored by transrectal ultrasonography during their cycle before the first AI (AI1). Follicular dynamic was similar between the two groups. However, the length of the estrus cycle was shorter in heifers than in primiparous cows and two-wave cycles were shorter than three-wave cycles, regardless of the age and the haplotype. The concentration of plasma anti-Mullerian hormone was correlated with the number of small antral follicles. It was higher in heifers than in primiparous cows, independently of their haplotype. The success rate at the AI1 was significantly higher in "fertil+" than in "fertil-" primiparous cows, 35 d after the AI1 (70% vs 39%). In both haplotypes, pregnancy failure occurred mainly before 21 d after AI1. The commencement of luteal activity after calving was significantly earlier in "fertil+" than in "fertil-" primiparous cows. Calving-AI1 and calving-calving intervals were similar between "fertil+" and "fertil-" primiparous cows. Taken together, "fertil+" and "fertil-" primiparous cows present a difference in the success rate after AI1 that is not explained by variations of ovarian dynamics. (C) 2011 Elsevier Inc. All rights reserved.

Anti-Mullerian hormone (AMH) is an endocrine marker that can help predict superovulatory responses to treatments administered to cows for embryo production. However, the optimal time of the estrous cycle at which a blood test should be performed for a highly reliable prognosis has not yet been established. Moreover, little is known about the regulation of AMH production. To answer these questions, a study was designed to investigate the regulation of AMH production in cows selected for their high or low ovulatory responses to superovulation. At the granulosa cell level, AMH production was inhibited by follicle-stimulating hormone but enhanced by bone morphogenetic proteins. At the follicular level, the expression of AMH within the follicle was dependent on the stage of follicular development. At the ovarian level, the size of the pool of small antral growing follicles determined ovarian AMH production. At the endocrine level, AMH followed a specific dynamic profile during the estrous cycle, which occurred independently of the follicular waves of terminal follicular development. Cows selected for their high or low responses to superovulation did not differ in the regulation of AMH production, but cows with higher responses had higher plasma AMH concentrations throughout the cycle. The optimal period of the estrous cycle at which to measure AMH concentrations with the aim of selecting the best cows for embryo production was found to be at estrus and after Day 12 of the cycle. Based on this multiscale study, we propose a model that integrates the different regulatory levels of AMH production.

Ovarian primordial follicles present original features of quiescence and long survival. Follicular growth is triggered by withdrawal of inhibitory mechanisms maintaining the quiescence and supported by a finely tuned molecular dialogue between the oocyte and its surrounding granulosa cells. The reserve of primordial follicles is oversized and functionally heterogeneous. Different spatiotemporal components can account for this heterogeneity.

Dietary supplementation with propylene glycol (PG) increases in vitro production of high-quality embryos in feed-restricted heifers. The aim of the present study was to evaluate the effects of PG in feed-restricted heifers on follicular fluid insulin and insulin-like growth factor (IGF) 1 concentrations, expression of IGF system genes in oocytes and cumulus cells and the expression of selected genes in blastocysts. Feed-restricted (R) heifers were drenched with water or PG during induced oestrous cycles (400 mL of PG or water/drench, daily drenching at 1600 hours for the first 9 days of the oestrous cycle). Ovum pick-up (OPU) was performed after superovulation to produce in vitro embryos and without superovulation to recover oocytes, cumulus cells and follicular fluid. OPU was also performed in a control group (not feed restricted and no drenching). Follicular fluid IGF1 concentrations were reduced by R, and PG restored IGF1 concentrations to those seen in the control group. In cumulus cells, expression of IGF1, IGF1 receptor (IGF1R) and IGF binding protein 4 (IGFBP4) was decreased in the R group, and fully (IGF1 and IGF1R) or partially (IGFBP4) restored to control levels by PG. Blastocyst perilipin 2 (PLIN2; also known as adipophilin), Bcl-2-associated X protein (BAX), SCL2A1 (facilitated glucose/fructose transporter GLUT1), aquaporin 3 (AQP3), DNA (cytosine-5)-methyltransferase 3A (DNMT3A) and heat shock 70-kDa protein 9 (HSPA9B) expression were decreased in R heifers; PG restored the expression of the last four genes to control levels. In conclusion, these results suggest that, during follicular growth, PG exerts epigenetic regulatory effects on gene expression in blastocyst stage embryos.

Cette revue présente l’état des connaissances sur la folliculogenèse et l’ovulation chez les mammifères. La folliculogenèse basale est une phase de croissance folliculaire lente, au cours de laquelle l’ovocyte acquiert la compétence méiotique. La folliculogenèse terminale est une phase de développement rapide, au cours de laquelle le follicule ovulatoire est sélectionné et termine sa maturation, tandis que l’ovocyte acquiert la compétence au développement. La revue décrit les différents changements fonctionnels qui s’opèrent dans le follicule et l’ovocyte au cours de ces deux phases, ainsi que les mécanismes qui les régulent et qui déterminent le quota ovulatoire caractéristique de chaque espèce ou race. Les facteurs-clés identifiés de la folliculogenèse sont les BMP, le KITLG et l’AMH pour le démarrage de croissance folliculaire, l’IGF et l’insuline pour la transition entre folliculogenèse basale et terminale, la FSH pour le début de la folliculogenèse terminale et la sélection des follicules ovulatoires, et la LH pour le développement folliculaire final, la maturation ovocytaire et l’ovulation. L’ovulation est déclenchée par une décharge préovulatoire de LH qui induit la maturation du complexe cumulus-ovocyte dans le follicule ovulatoire, suivie de la rupture de la paroi folliculaire et de la formation du corps jaune. La folliculogenèse ovarienne est contrôlée par des facteurs externes, tels que la photopériode et la nutrition, et c’est essentiellement le système hypothalamo-hypophysaire qui intègre les signaux endocriniens et environnementaux. Des modifications de l’apport alimentaire, relayées par des variations de signaux hormonaux et métaboliques, modulent l’activité de l’axe hypothalamo-hypophyso-ovarien à ses différents étages., This review is a state of the art on follicullogenesis and ovulation in mammals. Basal folliculogenesis is a phase of slow follicular growth, during which the oocyte acquires meiotic competence. Terminal folliculogenesis is a phase of rapid development, during which the ovulatory follicle is selected and completes its maturation while the oocyte acquires developmental competence. This review describes the different functional changes occurring within follicles and oocytes throughout these two phases, the underlying regulatory mechanisms and the mechanisms that determine natural ovulation rate in different species and breeds. Known key factors for folliculogenesis are BNTP, KITLG and AMH for the initiation of follicular growth, IGF and insulin for transition between basal and terminal folliculogenesis, FSH for the outset of terminal folliculogenesis and the selection of ovulatory follicles, and LH for final follicular development, oocyte maturation and ovulation. Ovulation is triggered by a preovulatory LH surge that induces maturation of the cumulus oocyte complex within the ovulatory follicle, rupture of the follicular wall and corpus luteum formation. Ovarian folliculogenesis is controlled by external factors such as photoperiod and nutrition, and the hypothalamo-pituitary system integrates endocrine and environmental signals. Nutritional supply, relayed by hormonal and metabolic signals, can modulate both the hypothalamo-pituitary and ovarian activity.

STUDY HYPOTHESIS Is the c-Jun-N-terminal kinase (JNK) pathway implicated in primordial follicle activation? STUDY FINDING Culture of ovine ovarian cortex in the presence of two different c-Jun phosphorylation inhibitors impeded pre-antral follicle activation. WHAT IS KNOWN ALREADY Despite its importance for fertility preservation therapies, the mechanisms of primordial follicle activation are poorly understood. Amongst different signalling pathways potentially involved, the JNK pathway has been previously shown to be essential for cell cycle progression and pre-antral follicle development in mice. STUDY DESIGN, SAMPLES/MATERIALS, METHODS Ovine ovarian cortex pieces were cultured with varying concentrations of SP600125, JNK inhibitor VIII or anti-Mullerian hormone (AMH) in the presence of FSH for 9 days. Follicular morphometry and immunohistochemistry for proliferating cell nuclear antigen (PCNA), apoptosis and follicle activation (Foxo3a) were assessed. MAIN RESULTS AND THE ROLE OF CHANCE Inhibition of primordial follicle activation occurred in the presence of SP600125, JNK inhibitor VIII and AMH when compared with controls (all P < 0.05) after 2 days of culture. However, only in the highest concentrations used was the inhibition of activation associated with induction of follicular apoptosis (P < 0.05). In growing follicles, PCNA antigen expression was reduced when the JNK inhibitors or AMH were used (P < 0.05 versus control), indicating reduced proliferation of the somatic compartment. LIMITATIONS, REASONS FOR CAUTION Although we evaluated the effects of inhibition of c-Jun phosphorylation on primordial follicle development, we did not determine the cellular targets and mechanism of action of the inhibitors. WIDER IMPLICATIONS OF THE FINDINGS These results are the first to implicate the JNK pathway in primordial follicle activation and could have significant consequences for the successful development of fertility preservation strategies and our understanding of primordial follicle activation. LARGE SCALE DATA n/a. STUDY FUNDING AND COMPETING INTERESTS Dr Michael J. Bertoldo and the laboratories involved in the present study were supported by a grant from 'Region Centre' (CRYOVAIRE, Grant number #320000268). There are no conflicts of interest to declare.

We investigated the effects of in vivo exposure to low zearalenone levels on the anti-Müllerian hormone endocrine levels and the reproductive performance of cattle. Urine and blood samples and reproductive records were collected from two Japanese Black breeding female cattle herds with dietary zearalenone contamination below the threshold levels (

Remerciements : Unité Expérimentale du Domaine du Merle de l'UMR0868 SELMET Systèmes d'Elevage Méditerranéens et Tropicaux, SupAgro Montpellier; In the ovary, anti-Müllerian hormone (AMH) is produced by the granulosa cells of growing follicles and can modulate the recruitment of primordial follicles and the follicle stimulating hormone (FSH)-dependent development of follicles. However, the regulation of its production remains poorly understood. Recently, a stimulating effect of the bone morphogenetic proteins (BMPs) on AMH production by granulosa cells has been shown in vitro, but the molecular mechanisms implicated in this regulation and its physiological importance in ovarian function have not yet been established. In the hyperprolific Booroola ewes carrying the FecB(B) partial loss-of-function mutation in the gene encoding the FecB/BMPR1B receptor, the granulosa cells of antral follicles expressed and secreted low AMH amounts, resulting in low AMH concentrations in blood, despite high numbers of AMH-secreting follicles in ovaries. The presence of the FecB(B) mutation impaired the granulosa cell response to the stimulating action of BMP4 on AMH production, indicating a crucial role of the BMPR1B receptor in AMH regulation. In ovine granulosa cells, BMP4 enhanced the transcriptional activity of the human AMH promoter and this action depended on the presence of SMAD1, acting on a promoter sequence located between -423 and -202 bp upstream of the AMH transcription start site. SMAD1 and SF1 acted in concert to mediate BMP4 action on the AMH promoter. Among the two SF1 binding sites present on the AMH promoter, the most proximal site, located at -92 bp upstream of the AMH transcription start site, was found to be critical for ensuring the response of the AMH promoter to BMP4. In conclusion, AMH could mediate the actions of BMPs in regulating follicular development and contributing to the determination of ovulation numbers. A molecular model of regulation of the AMH promoter transactivation by BMP signaling is proposed.

The hyperprolificacy phenotype of Booroola ewes is due to the presence of the FecB(B) allele at the FecB locus, recently identified as a single amino acid substitution (Q249R) in the bone morphogenetic protein (BMP) type-IB receptor (BMPR1B), and is associated with a more precocious differentiation of ovarian granulosa cells (GCs). To evaluate the consequences of the Booroola mutation on BMPR1B functions, the action of ligands of the transforming growth factor-beta (TGFbeta)/BMP family that act through (growth and differentiation factor-5, BMP-4) or independently of (activin A, TGFbeta-1) BMPR1B were studied on primary cultures of GCs from homozygous FecB(+) and FecB(B) ewes. All the tested TGFbeta/BMP family ligands inhibited progesterone secretion by FecB(+) GCs. Those inhibitory effects were lower for GCs from preovulatory (5-7 mm diameter) than from small antral follicles (1-3 mm diameter). The presence of the Booroola mutation was associated with a 3- to 4-fold (P

The biological meaning of folliculogenesis is to free fertilisable oocytes at the time of ovulation. We approached the study of the control of follicular development at the level of follicular granulosa cells, on the experimental as well as mathematical modelling grounds. We built a mathematical model allowing for the processes of proliferation, differentiation and apoptosis. State variables correspond to the numbers of cells undergoing these different processes, while control variables correspond to the cellular transition rates. The model results raised the notion of proliferative resources, which leads to consider the optimal management of these resources and has motivated the settling of an experiment investigating the changes in the growth fraction within the granulosa throughout terminal development. We are now investigating the way gonadotrophins, and especially FSH, operate on granulosa cells, in order to account for the hormonal control of the divergent commitment of granulosa cells towards either proliferation, differentiation or apoptosis. We are thus focusing on the dynamics of cAMP production, which appears to be a keypoint in FSH signal transduction.

The first ovulation induced by male effect in sheep during seasonal anoestrus usually results in the development of a short cycle that can be avoided by progesterone priming before ram introduction. In elucidating the involvement of the hypothalamic–pituitary–gonadal axis in the occurrence of short cycles, the effects of progesterone and the time of anoestrus on the development of male-induced preovulatory follicles were investigated in anoestrous ewes using morphological, endocrine and molecular approaches. Ewes were primed with progesterone for 2 (CIDR2) or 12 days (CIDR12) and untreated ewes used as controls during early (April) and late (June) anoestrus. The duration of follicular growth and the lifespan of the male-induced preovulatory follicles were prolonged by ∼1.6 days in CIDR12 ewes compared with the controls. These changes were accompanied by a delay in the preovulatory LH and FSH surges and ovulation. Intra-follicular oestradiol concentration and mRNA levels ofLHCGRandSTARin the granulosa and theca cells of the preovulatory follicles were higher in CIDR12 ewes than the control ewes. The expression of mRNA levels ofCYP11A1andCYP17A1also increased in theca cells of CIDR12 ewes. CIDR2 ewes gave intermediate results. Moreover, ewes ovulated earlier in June than in April, without changes in the duration of follicular growth, but these effects were unrelated to the lifespan of corpus luteum. Our results give the first evidence supporting the positive effect of progesterone priming on the completion of growth and maturation of preovulatory follicles induced by male effect in seasonal anoestrous ewes, thereby preventing short cycles.

The growing follicles develop from a reserve of primordial follicles constituted early in life. From this pre-established reserve, a second ovarian reserve is formed, which consists of gonadotropin-responsive small antral growing follicles and is a dynamic reserve for ovulation. Its size, evaluated by direct antral follicular count or endocrine markers, determines the success of assisted reproductive technologies in humans and embryo production biotechnologies in animals. Strong evidence indicates that these two reserves are functionally related. The size of both reserves appears to be highly variable between individuals of similar age, but the equilibrium size of the dynamic reserve in adults seems to be specific to each individual. The dynamics of both follicular reserves appears to result from the fine tuning of regulations involving two main pathways, the phosphatase and tensin homolog (PTEN)/phosphatidylinositol-3 kinase (PI3K)/3-phosphoinositide-dependent protein kinase-1 (PDPK1)/v-akt murine thymoma viral oncogene homolog 1 (AKT1) and the bone morphogenetic protein (BMP)/anti-Mullerian hormone (AMH)/SMAD signaling pathways. Mutations in genes encoding the ligands, receptors, or signaling effectors of these pathways can accelerate or modulate the exhaustion rate of the ovarian reserves, causing premature ovarian insufficiency (POI) or increase in reproductive longevity, respectively. With female aging, the decline in primordial follicle numbers parallels the decrease in the size of the dynamic reserve of small antral follicles and the deterioration of oocyte quality. Recent progress in our knowledge of signaling pathways and their environmental and hormonal control during adult and fetal life opens new perspectives to improve the management of the ovarian reserves.

This study was designed to determine the effects of dietary propylene glycol (PG; Propypact®, DIFAGRI, France) on blood metabolites, metabolic and reproductive hormones and follicular growth in 10 dairy heifers. Treatments consisted of (1) 1.1 kg of sugar beet pulp (Control), (2) 150 g PG (PG150) and (3) 300 g PG (PG300). Each heifer received the three treatments in different randomized orders. A standard hay/concentrate diet, formulated to allow a daily liveweight gain of 900 g/day, was given at 8:00 and the dietary treatments were given at 16:00 from Days 1 to 13 of the oestrous cycle following induced oestrus (Day 0). Oestrus induction treatment consisted of a subcutaneous 3 mg norgestomet implant inserted for 9 days combined with GnRH treatment (i.m.) at implant insertion. Two days before implant removal, 500 µg cloprostenol was administered i.m. Blood samples were collected by jugular venipuncture every 2 h for 24 h on Days 0 and 13 to measure plasma insulin, glucose, β-hydroxybutyrate (BHB) and urea concentrations. Blood samples were also collected to measure insulin-like growth factor-1 (IGF-1), oestradiol, progesterone concentrations on Days 2, 6, 9 and 12 and AMH (Anti-Müllerian hormone) on Days 0, 2, 6, 9 and 12. On Days 2, 6, 9 and 12 ovarian follicular growth was evaluated; the total number of follicles and their diameters were recorded and classed (2-3 mm, 4-7 mm, and >8 mm). Results were analysed by repeated-measures ANOVA. There were no treatment, day and interaction effects on average urea concentrations while there were some differences between Days 0 and 13 for insulin, glucose and BHB. Insulin and glucose concentrations were higher on Day 13 compared to Day 0 and the opposite was observed for BHB. There were treatment, time and interaction effects on glucose and BHB concentrations measured over 24 h on Day 13; glucose concentrations were higher (P

Rapid genetic improvement in cattle requires the production of high numbers of embryos of excellent quality. Increasing circulating insulin and/or glucose concentrations improves ovarian follicular growth, which may improve the response to superovulation. The measurement of anti-Müllerian hormone (AMH) can help predict an animal’s response to superovulation treatment. The aim of the present study was to investigate whether increasing circulating insulin concentrations, through propylene glycol (PG) drenches, could improve in vitro embryo production in oestrus-synchronised superovulated heifers with different AMH profiles. Holstein heifers were grouped according to pre-experimental AMH concentrations as low (L) or high (H). The PG drench increased circulating insulin and glucose concentrations and reduced β-hydroxybutyrate and urea concentrations compared with the control group. AMH was a good predictor of follicle and oocyte numbers at ovum pick-up (OPU), and of oocyte and embryo quality (AMH H > AMH L). PG in the AMH H group increased the number of follicles and blastocyst quality above that in the control group, but did not improve these parameters in the AMH L group. These results indicate that short-term oral PG supplementation modifies an animal’s metabolic milieu and is effective in improving in vitro embryo production, after superovulation–OPU, more markedly in heifers with high rather than low AMH concentrations.