Chronic low BPS exposure through diet impairs in vitro embryo production parameters according to metabolic status in the ewe.

Bisphenol A (BPA), an endocrine disruptor, has been replaced by structural analogues including bisphenol S (BPS). BPA and BPS exhibited similar effects regarding reproductive functions. Moreover, metabolic status and lipid metabolism are related to female fertility and could worsen BPS effects. The objective was to determine BPS in vivo effects on folliculogenesis and embryo production after chronic exposure through diet, and the influence of metabolic status in adult ewes. Sixty primiparous 2.5 year-old ewes, undergoing a restricted or well fed diet, were exposed to BPS (0, 4 or 50 µg/kg/day) for at least three months. After hormonal oestrus synchronisation and ovarian stimulation, ewes were subjected to ovum pick-up (OPU) procedures to collect immature oocytes, that underwent in vitro maturation, fertilisation and embryo production. Body weight, body condition score and plasma glucose were higher in well-fed compared to restricted ewes, while plasma NEFA was lower during the 4-5 months after the beginning of the diets. Plasma progesterone levels increased on day 5 before OPU session in well-fed compared to restricted ewes. No effect of BPS dose was observed on follicle population, plasma AMH levels and embryo production numbers and rates. However, a significant diet x BPS dose interaction was reported for cleaved embryos, > 4-cell embryos, blastocyst and early blastocyst numbers, and plasma triiodothyronine levels. Our study showed that a contrasted diet did not affect follicle population nor embryo production in adult ewes but could affect the quality and progesterone secretion of the corpus luteum. Chronic low BPS exposure had no effect on follicular population and oocyte competence. Nevertheless, the significant diet x dose interactions observed on embryo production suggest that BPS effect is modulated by metabolic status. Further studies are required to assess the risk of BPS exposure for public reproductive health.

Prenatal programming by testosterone of follicular theca cell functions in ovary.

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.

Bisphenol A and S impaired ovine granulosa cell steroidogenesis.

Bisphenols, plasticisers used in food containers, can transfer to food. Bisphenol A (BPA) has been described as an endocrine disruptor and consequently banned from the food industry in several countries. It was replaced by a structural analogue, Bisphenol S (BPS). BPA action on the steroidogenesis is one of the mechanisms underlying its adverse effects on the efficiency of female reproduction. This study aimed to determine whether BPS is a safe alternative to BPA regarding GC functions. Antral follicles (2-6 mm), of approximatively 1000 adult ewe ovaries, were aspired and GC purified. For 48 h, ovine GC were treated with BPA or BPS (from 1 nM to 200 µM) and the effects on cell viability, proliferation, steroid production, steroidogenic enzyme expression and signalling pathways were investigated. Dosages at and greater than 100 µM BPA and 10 µM BPS decreased progesterone secretion by 39% (P < 0.001) and 22% (P = 0.040), respectively. BPA and BPS 10 µM and previously mentioned concentrations increased oestradiol secretion two-fold (P < 0.001 and P = 0.082, respectively). Only 100 µM BPA induced a decrease (P < 0.001) in gene expression of the enzymes of steroidogenesis involved in the production of progesterone. BPA reduced MAPK3/1 phosphorylation and ESR1 and ESR2 gene expression, effects that were not observed with BPS. BPA and BPS altered steroidogenesis of ovine GC. Thus, BPS does not appear to be a safe alternative for BPA. Further investigations are required to elucidate BPA and BPS mechanisms of action.

Bisphenol S Impaired In Vitro Ovine Early Developmental Oocyte Competence.

INTRODUCTION: Bisphenol A (BPA) is a widespread compound in the plastic industry that is especially used to produce baby bottles, food packaging and metal cans. BPA, an endocrine disruptor, leads to alterations in reproductive function and therefore has been banned from the food industry. Unregulated BPA analogues, particularly Bisphenol S (BPS), have emerged and are now used in the plastic industry. Thus, this study aimed to examine the acute effects of low and environmental doses of BPS on ewe oocyte quality and developmental competence, and its mechanism of action, during in vitro maturation. METHODS: Ewe cumulus-oocyte complexes underwent in vitro maturation in the presence or absence of BPS (1 nM, 10 nM, 100 nM, 1 µM or 10 µM). Oocytes were then subjected to in vitro fertilisation and development. RESULTS: 1 µM BPS induced a 12.7% decrease in the cleavage rate (p = 0.004) and a 42.6% decrease in the blastocyst rate (p = 0.017) compared to control. The blastocyst rate reduction was also observed with 10 nM BPS. Furthermore, 10 µM BPS reduced the oocyte maturation rate, and 1 µM BPS decreased cumulus cell progesterone secretion. PR and AMH gene expression were reduced in cumulus cells. BPS induced a 5-fold increase in MAPK 3/1 activation (p = 0.04). CONCLUSIONS: BPS impaired ewe oocyte developmental competence. The data suggest that BPS might not be a safe BPA analogue. Further studies are required to elucidate its detailed mechanism of action.

Bisphenol S Impaired Human Granulosa Cell Steroidogenesis in Vitro.

Bisphenol S (BPS) is a structural analog of the endocrine disruptor bisphenol A (BPA); it is the main BPA replacement in the plastics industry. Previous studies have shown that BPA and BPS exhibit similar effects on reproduction in fish and rodent species. BPS reportedly alters steroidogenesis in bovine granulosa cells. Luteinised granulosa cells collected from 59 women who were undergoing an in vitro fertilization procedure were cultured for 48 h in the presence or absence of BPS (10 nM, 100 nM, 1 µM, 10 µM or 50 µM). BPS exposure was investigated by assessing follicular fluids from these 59 women for their BPS content. Culture medium, cells, total messenger RNA (mRNA) and total protein extracted from the luteinised granulosa cells were examined for oestradiol and progesterone secretion, cellular proliferation, viability, gene expression, steroidogenic enzyme expression and cell signaling. BPS was measured in follicular fluids using mass spectrometry. Exposure of granulosa cells to 10 or 50 µM BPS for 48 h induced a 16% (p = 0.0059) and 64% (p < 0.0001) decrease, respectively, in progesterone secretion; 50 µM BPS decreased oestradiol secretion by 46% (p < 0.0001). Ten µM BPS also tended to reduce CYP11A1 protein expression by 37% (p = 0.0947) without affecting HSD3B1 and CYP19A1 expression. Fifty µM BPS increased ERRγ expression. Environmental levels of BPS (nanomolar range) did not induce changes in steroidogenesis in human granulosa cells. The effects of BPS were observed after only 48 h of BPS exposure. These acute effects might be similar to chronic effects of physiological BPS levels.

MALDI-TOF Mass Spectrometry Revealed Significant Lipid Variations in Follicular Fluid and Somatic Follicular Cells but Not in Enclosed Oocytes between the Large Dominant and Small Subordinate Follicles in Bovine Ovary.

Lipid metabolism in ovarian follicular cells supports the preparation of an enclosed oocyte to ovulation. We aimed to compare lipid composition of a dominant large follicle (LF) and subordinated small follicles (SFs) within the same ovaries. Mass spectrometry imaging displayed the differences in the distribution of several lipid features between the different follicles. Comparison of lipid fingerprints between LF and SF by Matrix Assisted Laser Desorption/Ionisation Time-Of-Flight (MALDI-TOF) mass spectrometry revealed that in the oocytes, only 8 out of 468 detected lipids (1.7%) significantly changed their abundance (p < 0.05, fold change > 2). In contrast, follicular fluid (FF), granulosa, theca and cumulus cells demonstrated 55.5%, 14.9%, 5.3% and 9.8% of significantly varied features between LF and SF, respectively. In total, 25.2% of differential lipids were identified and indicated potential changes in membrane and signaling lipids. Tremendous changes in FF lipid composition were likely due to the stage specific secretions from somatic follicular cells that was in line with the differences observed from FF extracellular vesicles and gene expression of candidate genes in granulosa and theca cells between LF and SF. In addition, lipid storage in granulosa and theca cells varied in relation to follicular size and atresia. Differences in follicular cells lipid profiles between LF and SF may probably reflect follicle atresia degree and/or accumulation of appropriate lipids for post-ovulation processes as formation of corpus luteum. In contrast, the enclosed oocyte seems to be protected during final follicular growth, likely due in part to significant lipid transformations in surrounding cumulus cells. Therefore, the enclosed oocyte could likely keep lipid building blocks and energy resources to support further maturation and early embryo development.

Docosahexaenoic acid (DHA) effects on proliferation and steroidogenesis of bovine granulosa cells.

BACKGROUND: Docosahexaenoic acid (DHA) is a n-3 polyunsaturated fatty acid (PUFA) belonging to a family of biologically active fatty acids (FA), which are known to have numerous health benefits. N-3 PUFAs affect reproduction in cattle, and notably directly affect follicular cells. In terms of reproduction in cattle, n-3 PUFA-enriched diets lead to increased follicle size or numbers. METHODS: The objective of the present study was to analyze the effects of DHA (1, 10, 20 and 50 µM) on proliferation and steroidogenesis (parametric and/or non parametric (permutational) ANOVA) of bovine granulosa cells in vitro and mechanisms of action through protein expression (Kruskal-Wallis) and signaling pathways (non parametric ANOVA) and to investigate whether DHA could exert part of its action through the free fatty acid receptor 4 (FFAR4). RESULTS: DHA (10 and 50 µM) increased granulosa cell proliferation and DHA 10 µM led to a corresponding increase in proliferating cell nuclear antigen (PCNA) expression level. DHA also increased progesterone secretion at 1, 20 and 50 µM, and estradiol secretion at 1, 10 and 20 µM. Consistent increases in protein levels were also reported for the steroidogenic enzymes, cytochrome P450 family 11 subfamily A member 1 (CYP11A1) and hydroxy-delta-5-steroid dehydrogenase, 3 beta- and steroid delta-isomerase 1 (HSD3B1), and of the cholesterol transporter steroidogenic acute regulatory protein (StAR), which are necessary for production of progesterone or androstenedione. FFAR4 was expressed in all cellular types of bovine ovarian follicles, and in granulosa cells it was localized close to the cellular membrane. TUG-891 treatment (1 and 50 µM), a FFAR4 agonist, increased granulosa cell proliferation and MAPK14 phosphorylation in a similar way to that observed with DHA treatment. However, TUG-891 treatment (1, 10 and 50 µM) showed no effect on progesterone or estradiol secretion. CONCLUSIONS: These data show that DHA stimulated proliferation and steroidogenesis of bovine granulosa cells and led to MAPK14 phosphorylation. FFAR4 involvement in DHA effects requires further investigation, even if our data might suggest FFAR4 role in DHA effects on granulosa cell proliferation. Other mechanisms of DHA action should be investigated as the steroidogenic effects seemed to be independent of FFAR4 activation.

Lipid Identification and Transcriptional Analysis of Controlling Enzymes in Bovine Ovarian Follicle.

Ovarian follicle provides a favorable environment for enclosed oocytes, which acquire their competence in supporting embryo development in tight communications with somatic follicular cells and follicular fluid (FF). Although steroidogenesis in theca (TH) and granulosa cells (GC) is largely studied, and the molecular mechanisms of fatty acid (FA) metabolism in cumulus cells (CC) and oocytes are emerging, little data is available regarding lipid metabolism regulation within ovarian follicles. In this study, we investigated lipid composition and the transcriptional regulation of FA metabolism in 3⁻8 mm ovarian follicles in bovine. Using liquid chromatography and mass spectrometry (MS), 438 and 439 lipids were identified in FF and follicular cells, respectively. From the MALDI-TOF MS lipid fingerprints of FF, TH, GC, CC, and oocytes, and the MS imaging of ovarian sections, we identified 197 peaks and determined more abundant lipids in each compartment. Transcriptomics revealed lipid metabolism-related genes, which were expressed constitutively or more specifically in TH, GC, CC, or oocytes. Coupled with differential lipid composition, these data suggest that the ovarian follicle contains the metabolic machinery that is potentially capable of metabolizing FA from nutrient uptake, degrading and producing lipoproteins, performing de novo lipogenesis, and accumulating lipid reserves, thus assuring oocyte energy supply, membrane synthesis, and lipid-mediated signaling to maintain follicular homeostasis.

Molecular evidence that follicle development is accelerated in vitro compared to in vivo.

In this study, we systematically compared the morphological, functional and molecular characteristics of granulosa cells and oocytes obtained by a three-dimensional in vitro model of ovine ovarian follicular growth with those of follicles recovered in vivo Preantral follicles of 200 µm diameter were recovered and cultured up to 950 µm over a 20-day period. Compared with in vivo follicles, the in vitro culture conditions maintained follicle survival, with no difference in the rate of atresia. However, the in vitro conditions induced a slight decrease in oocyte growth rate, delayed antrum formation and increased granulosa cell proliferation rate, accompanied by an increase and decrease in CCND2 and CDKN1A mRNA 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 of GJA1, CYP19A1, ESR1, ESR2, FSHR, INHA, INHBA, INHBB and FST There was a decrease in the expression of oocyte-specific molecular markers GJA4, KIT, ZP3, WEE2 and BMP15 in vitro compared to that in 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, this in vitro model of ovarian follicle development is characterized by accelerated follicular maturation, associated with improved developmental competence of the oocyte, compared to follicles recovered in vivo.

Visfatin and resistin in gonadotroph cells: expression, regulation of LH secretion and signalling pathways.

Visfatin and resistin appear to interfere with reproduction in the gonads, but their potential action at the hypothalamic-pituitary level is not yet known. The aim of the present study was to investigate the mRNA and protein expression of these adipokines in murine gonadotroph cells and to analyse the effects of different concentrations of recombinant mouse visfatin and resistin (0.01, 0.1, 1 and 10ngmL-1) on LH secretion and signalling pathways in LßT2 cells and/or in primary female mouse pituitary cells. Both visfatin and resistin mRNA and protein were found in vivo in gonadotroph cells. In contrast with resistin, the primary tissue source of visfatin in the mouse was the skeletal muscle, and not adipose tissue. Visfatin and resistin both decreased LH secretion from LßT2 cells after 24h exposure of cells (P<0.03). These results were confirmed for resistin in primary cell culture (P<0.05). Both visfatin (1ngmL-1) and resistin (1ngmL-1) increased AMP-activated protein kinase α phosphorylation in LßT2 cells after 5 or 10min treatment, up to 60min (P<0.04). Extracellular signal-regulated kinase 1/2 phosphorylation was transiently increased only after 5min resistin (1ngmL-1) treatment (P<0.01). In conclusion, visfatin and resistin are expressed in gonadotroph cells and they may affect mouse female fertility by regulating LH secretion at the level of the pituitary.

Effect of a long-chain n-3 polyunsaturated fatty acid-enriched diet on adipose tissue lipid profiles and gene expression in Holstein dairy cows.

The objective of this study was to determine whether fish oil supplement has an effect on adipose tissue lipid profiles and gene expression in postpartum dairy cows. Holstein cows were supplemented with either long-chain n-3 polyunsaturated fatty acid (PUFA; protected fish oil) or control PUFA (n-6; toasted soybeans) for 2mo after calving (n=23 per diet). These cows showed no difference in milk production or metabolic parameters, but exhibited a tendency toward a decrease in early embryo mortality rate after artificial insemination. We hypothesized that, in addition to this effect, modifications in adipose tissue (AT) gene expression and lipid profiles would occur in response to diet. Subcutaneous AT samples were thus collected from the dewlaps of n-3 and n-6 dairy cows at 1mo antepartum, and 1wk, 2mo, and 5mo postpartum for the analysis of lipids and gene expression. Lipid profiles were obtained by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry in both positive and negative modes. We found 37 lipid species in the 200 to 1,200 m/z range, which differed between the n-3 and control groups, suggesting that the n-3 supplement affected the lipid composition through the enrichment of lipids integrating long-chain PUFA from fish oil sources: eicosapentaenoic and docosahexaenoic acid. Moreover, a decrease in triacylglycerolipids was observed in AT of n-3 supplemented cows. The expression of 44 genes involved in fatty acid metabolism and the adipokine system was assessed by real-time reverse-transcription PCR. Hierarchical clustering, according to either postpartum stage or diet, enabled us to group genes exhibiting similar kinetic properties during lactation or by those that varied in similar ways after n-3 supplementation, respectively. Among the genes exhibiting a dietary effect, FABP4, LIPE, CD36, and PLIN1 were overexpressed in n-3 AT samples compared with the control, suggesting an increase in lipolysis due to n-3 supplementation, which was reflected on lipolytic activity at the protein level (i.e., protein expression of fatty acid binding protein 4, phosphorylated perilipin 1, and phosphorylated hormone-sensitive lipase). This increase in lipolysis is relevant to the decrease in triglycerides observed in these samples. Gene expression analyses between n-3 and control AT samples also suggested that the n-3 diet could modulate the secretory functions of AT, possibly by affecting adipokine expression; however, this has to be confirmed at the protein level.

The loss of adipokine genes in the chicken genome and implications for insulin metabolism.

Gene loss is one of the main drivers in the evolution of genomes and species. The demonstration that a gene has been lost by pseudogenization is truly complete when one finds the pseudogene in the orthologous genomic region with respect to active genes in other species. In some cases, the identification of such orthologous loci is not possible because of chromosomal rearrangements or if the gene of interest has not yet been sequenced. This question is particularly important in the case of birds because the genomes of avian species possess only about 15,000 predicted genes, in comparison with 20,000 in mammals. Yet, gene loss raises the question of which functions are affected by the changes in gene counts. We describe a systematic approach that makes it possible to demonstrate gene loss in the chicken genome even if a pseudogene has not been found. By using phylogenetic and synteny analysis in vertebrates, genome-wide comparisons between the chicken genome and expressed sequence tags, RNAseq data analysis, statistical analysis of the chicken genome, and radiation hybrid mapping, we show that resistin, TNFα, and PAI-1 (SERPINE1), three genes encoding adipokines inhibiting insulin sensitivity, have been lost in chicken and zebra finch genomes. Moreover, omentin, a gene encoding an adipokine that enhances insulin sensitivity, has also been lost in the chicken genome. Overall, only one adipokine inhibiting insulin sensitivity and five adipokines enhancing insulin sensitivity are still present in the chicken genome. These genetic differences between mammals and chicken, given the functions of the genes in mammals, would have dramatic consequences on chicken endocrinology, leading to novel equilibriums especially in the regulation of energy metabolism, insulin sensitivity, as well as appetite and reproduction.

Expression of adipokine and lipid metabolism genes in adipose tissue of dairy cows differing in a female fertility quantitative trait locus.

We have previously characterized 2 haplotypes (Fertil+ and Fertil-) of Holstein dairy cows differing in 1 female fertility quantitative trait locus (QTL) located on chromosome 3 (QTL-Fert-F-BTA3) between positions 9.8 and 13.5 cM. This QTL is composed of 124 genes, some of them being involved in metabolism or reproduction. Primiparous Fertil+ and Fertil- cows exhibited 69 and 39% pregnancy rate at first service, respectively. A difference in plasma nonesterified fatty acid concentrations observed between both haplotypes might indicate a difference in adipose tissue mobilization. We compared adipose tissue gene expression in Fertil+ and Fertil- cows during their second lactation, at 2 physiological stages, implying either intense lipid mobilization (1 wk postpartum) or fat storage (5 mo of gestation). We investigated by reverse-transcription quantitative PCR the mRNA gene expression of 5 positional candidate genes located in the QTL-Fert-F-BTA3, as well as 18 other functional candidate genes encoding proteins involved in lipid metabolism and several adipokines. Among them, genes involved in either lipolysis or lipogenesis were chosen as controls because they were previously described in dairy cow adipose tissue. A hierarchical clustering was performed to group genes according to their expression pattern, allowing 2 clusters to be determined. Cluster 1 was composed of genes that were overexpressed during mobilization (ADIPOQ, ADIPOR2, LIPE, FABP4, PLIN1, RARRES, LEPR, and CPT1A) and cluster 2 of genes overexpressed during reconstitution of body reserves (ACACA, FASN, and SCD). Genes belonging to cluster 1 (LIPE, FABP4, PLIN1, and CPT1A) are known to be involved in lipolysis and fatty acid oxidation, and genes belonging to cluster 2 (ACACA, FASN, and SCD) are known to be involved in fatty acid synthesis. The expression of 5 genes from cluster 1 was correlated to plasma nonesterified fatty acid levels and thus to mobilization of body reserves in dairy cows (ADIPOQ, ADIPOR2, LIPE, PLIN1, and FABP4). During the mobilization stage, none of the positional candidate genes belonging to QTL-Fert-F-BTA3 (ADAR, MTX1, SHC1, SPTA1, and PAQR6) showed a difference in expression between the 2 haplotypes. Interestingly, ADIPOQ and ADIPOR2 were the only genes showing a significant mRNA overexpression in Fertil- cows at the mobilization stage. Further studies focusing on plasma adiponectin level and adipokine actions on the ovary are needed to investigate its potential role in dairy cow fertility.

[Adiponectin and resistin: a role in the reproductive functions?]. / Adiponectine et résistine - Un rôle dans les fonctions de reproduction ?

Adipokines are hormones mainly produced by the white adipose tissue, an endocrine organ involved in energy homeostasis. They play an important role in the regulation of lipid and glucose metabolisms, in inflammation and immune disorders. New roles for adipokines have recently emerged in the field of fertility and reproduction. Indeed, adipokines such as adiponectin and resistin are able to regulate the functions of male and female gonads and of the hypothalamic-pituitary axis. For example, they modulate steroidogenesis of gonadic somatic cells, germ cell maturation and secretion of gonadotrope hormones in various species. The reproductive system is tightly coupled with energy balance, and thereby metabolic abnormalities can lead to the development of physiopathological situations such as the polycystic ovary syndrome (PCOS). Obesity and overweight are significantly involved in the declining natural fertility and decrease the effectiveness of treatments. Women with obesity and/or PCOS have abnormal plasma adiponectin and resistin profiles. Thus, these adipokines could be a link between reproduction and energy metabolism and could partly explain some infertility related to obesity or PCOS.

Bisphenol A and bisphenol S both disrupt ovine granulosa cell steroidogenesis but through different molecular pathways.

BACKGROUND: Ovarian granulosa cells (GC) are essential for the development and maturation of a proper oocyte. GC are sensitive to endocrine disruptors, including bisphenol A (BPA) and its analogue bisphenol S (BPS), plasticisers present in everyday consumer products. BPA exhibits greater binding affinity for the membrane oestrogen receptor (GPER) than for the nuclear oestrogen receptors (ERα and ERß). Here, we analysed the effects of BPA and BPS on the steroidogenesis of ovine GC in vitro, as well as their early mechanisms of action, the ovine being a relevant model to study human reproductive impairment. Disruption of GC steroidogenesis might alter oocyte quality and consequently fertility rate. In addition, we compared the effects of a specific GPER agonist (G-1) and antagonist (G-15) to those of BPA and BPS. Ewe GC were cultured with BPA or BPS (10 or 50 µM) or G-1 (1 µM) and/or G-15 (10 µM) for 48 h to study steroidogenesis. RESULTS: Both BPA and BPS (10 µM) altered the secretion of progesterone, however, only BPS (10 µM) affected oestradiol secretion. RNA-seq was performed on GC after 1 h of culture with BPA or BPS (50 µM) or G-1 (10 µM), followed by real-time PCR analyses of differentially expressed genes after 12, 24 and 48 h of culture. The absence of induced GPER target genes showed that BPA and BPS did not activate GPER in GC after 1 h of treatment. These molecules exhibited mainly independent early mechanisms of action. Gene ontology analysis showed that after 1 h of treatment, BPA mainly disrupted the expression of the genes involved in metabolism and transcription, while BPS had a smaller effect and impaired cellular communications. BPA had a transient effect on the expression of CHAC1 (NOTCH signalling and oxidative balance), JUN (linked to MAPK pathway), NR4A1 (oestradiol secretion inhibition), ARRDC4 (endocytose of GPCR) and KLF10 (cell growth, differentiation and apoptosis), while expression changes were maintained over time for the genes LSMEM1 (linked to MAPK pathway), TXNIP (oxidative stress) and LIF (cell cycle regulation) after 12 and 48 h, respectively. CONCLUSION: In conclusion, although they exhibited similar effects, BPA and BPS impaired different molecular pathways in GC in vitro. New investigations will be necessary to follow the temporal changes of these genes over time, as well as the biological processes involved.

Cumulative and potential synergistic effects of seven different bisphenols on human granulosa cells in vitro?

Bisphenol (BP) structural analogues of BPA are widely used. Previous studies showed similar effects of BPA and BPS on reproduction in several species including human. We hypothesised that the similar effects of several bisphenols (BPs) could accumulate in granulosa cells (GCs) and affects steroidogenesis. This study investigated the effects of seven BP analogues and their equimolar cocktail on human granulosa cells (hGC) and assessed BPA, BPS, BPF and BPAF level exposures in the follicular fluid of 277 women undergoing Assisted Reproductive Technology. The hGCs were recovered after women oocyte punctures and treated with the seven BP analogues (BPS, BPA, BPAF, BPF, BPAP, BPE and BPB) or their equimolar cocktail of 7 × 1.43 or 7 × 7.14 µM for each of the seven BPs, the sum of BPs reaching 10 ("∑BPs 10 µM"), or 50 µM ("∑BPs 50 µM"), respectively. Oestradiol and progesterone secretion, cell proliferation, viability and expression of steroidogenic enzymes were investigated. Progesterone secretion was decreased by 6 BPs 10 µM and the cocktail "∑BPs 10 µM", (-17.8 to -41.3%) and by all seven BPs 50 µM and "∑BPs 50 µM" (-21.8 to -84.2%). Oestradiol secretion was decreased only by 50 µM BPAF and BPAP (-37.8% and -44%, respectively), with corresponding decreases in CYP17A1 and CYP19A1 gene expression. Cellular proliferation was decreased after treatment with 50 µM BPAF (-32.2%), BPAP (-29%), BPB (-24%) and the equimolar cocktail "∑BPs 50 µM" (-33.1%). BPB (50 µM) and the cocktail "∑BPs 50 µM" increased HSD3B2 mRNA expression. At least one BP was detected in 64 of 277 (23.1%) women follicular fluids. Similar effects of the seven BPs or their cocktail were observed on progesterone secretion and/or on cell proliferation, suggesting cumulative effects of BPs. Our results highlight the urge to consider all BPs simultaneously and to further investigate the potential additive or synergistic effects of several BPs.

Bisphenol S Impairs Oestradiol Secretion during In Vitro Basal Folliculogenesis in a Mono-Ovulatory Species Model.

Bisphenol S (BPS) affects terminal folliculogenesis by impairing steroidogenesis in granulosa cells from different species. Nevertheless, limited data are available on its effects during basal folliculogenesis. In this study, we evaluate in vitro the effects of a long-term BPS exposure on a model of basal follicular development in a mono-ovulatory species. We cultured ovine preantral follicles (180−240 µm, n = 168) with BPS (0.1 µM (possible human exposure dose) or 10 µM (high dose)) and monitored antrum appearance and follicular survival and growth for 15 days. We measured hormonal secretions (oestradiol (at day 13 [D13]), progesterone and anti-Müllerian hormone [D15]) and expression of key follicular development and redox status genes (D15) in medium and whole follicles, respectively. BPS (0.1 µM) decreased oestradiol secretion compared with the control (−48.8%, p < 0.001), without significantly impairing antrum appearance, follicular survival and growth, anti-Müllerian hormone and progesterone secretion and target gene expression. Thus, BPS could also impair oestradiol secretion during basal folliculogenesis as it is the case during terminal folliculogenesis. It questions the use of BPS as a safe BPA substitute in the human environment. More studies are required to elucidate mechanisms of action of BPS and its effects throughout basal follicular development.

Bisphenol S Alters the Steroidome in the Preovulatory Follicle, Oviduct Fluid and Plasma in Ewes With Contrasted Metabolic Status.

Bisphenol A (BPA), a plasticizer and endocrine disruptor, has been substituted by bisphenol S (BPS), a structural analogue that had already shown adverse effects on granulosa cell steroidogenesis. The objective of this study was to assess the effect of chronic exposure to BPS, a possible endocrine disruptor, on steroid hormones in the ovary, oviduct and plasma using the ewe as a model. Given the interaction between steroidogenesis and the metabolic status, the BPS effect was tested according to two diet groups. Eighty adult ewes were allotted to restricted (R) and well-fed (WF) groups, that were further subdivided into two subgroups. Ewes were exposed to 50 µg BPS/kg/day in their diet (R50 and WF50 groups) or were unexposed controls (R0 and WF0 groups). After at least 3 months of BPS exposure, preovulatory follicular fluid, oviduct fluid and plasma were collected and steroid hormones were analyzed by gas chromatography coupled with tandem mass spectrometry (GC-MS/MS). A deleterious effect of restricted diet on the volume of oviduct fluid and numbers of pre-ovulatory follicles was observed. Exposure to BPS impaired estradiol concentrations in both follicular and oviduct fluids of well-fed ewes and progesterone, estradiol and estrone concentrations in plasma of restricted ewes. In addition, a significant interaction between metabolic status and BPS exposure was observed for seven steroids, including estradiol. In conclusion, BPS acts in ewes as an endocrine disruptor with differential actions according to metabolic status.

Metformin decreases GnRH- and activin-induced gonadotropin secretion in rat pituitary cells: potential involvement of adenosine 5' monophosphate-activated protein kinase (PRKA).

Metformin is an insulin sensitizer molecule used for the treatment of infertility in women with polycystic ovary syndrome and insulin resistance. It modulates the reproductive axis, affecting the release of gonadotropin-releasing hormone (GnRH) and luteinizing hormone (LH). However, metformin's mechanism of action in pituitary gonadotropin-secreting cells remains unclear. Adenosine 5' monophosphate-activated protein kinase (PRKA) is involved in metformin action in various cell types. Here, we investigated the effects of metformin on gonadotropin secretion in response to activin and GnRH in primary rat pituitary cells (PRP), and studied PRKA in rat pituitary. In PRP, metformin (10 mM) reduced LH and follicle-stimulating hormone (FSH) secretion induced by GnRH (10(-8) M, 3 h), FSH secretion, and mRNA FSHbeta subunit expression induced by activin (10(-8) M, 12 or 24 h). The different subunits of PRKA are expressed in pituitary. In particular, PRKAA1 is detected mainly in gonadotrophs and thyrotrophs, is less abundant in lactotrophs and somatotrophs, and is undetectable in corticotrophs. In PRP, metformin increased phosphorylation of both PRKA and acetyl-CoA carboxylase. Metformin decreased activin-induced SMAD2 phosphorylation and GnRH-induced mitogen-activated protein kinase (MAPK) 3/1 (ERK1/2) phosphorylation. The PRKA inhibitor compound C abolished the effects of metformin on gonadotropin release induced by GnRH and on FSH secretion and Fshb mRNA induced by activin. The adenovirus-mediated production of dominant negative PRKA abolished the effects of metformin on the FSHbeta subunit mRNA and SMAD2 phosphorylation induced by activin and on the MAPK3/1 phosphorylation induced by GnRH. Thus, in rat pituitary cells, metformin decreases gonadotropin secretion and MAPK3/1 phosphorylation induced by GnRH and FSH release, FSHbeta subunit expression, and SMAD2 phosphorylation induced by activin through PRKA activation.

Expression and effect of resistin on bovine and rat granulosa cell steroidogenesis and proliferation.

Resistin, initially identified in adipose tissue and macrophages, was implicated in insulin resistance. Recently, its mRNA was found in hypothalamo-pituitary axis and rat testis, leading us to hypothesize that resistin may be expressed in ovary. In this study, we determined in rats and cows 1) the characterization of resistin in ovary by RT-PCR, immunoblotting, and immunohistochemistry and 2) the effects of recombinant resistin (10, 100, 333, and 667 ng/ml) ± IGF1 (76 ng/ml) on steroidogenesis, proliferation, and signaling pathways of granulosa cells (GC) measured by enzyme immunoassay, [(3)H]thymidine incorporation, and immunoblotting respectively. We observed that resistin mRNA and protein were present in several bovine and rat ovarian cells. Nevertheless, only bovine GC abundantly expressed resistin mRNA and protein. Resistin treatment decreased basal but not IGF1-induced progesterone (P<0.05; whatever the dose) and estradiol (P<0.005; for 10 and 333 ng/ml) production by bovine GC. In rats, resistin (10 ng/ml) increased basal and IGF1-induced progesterone secretion (P<0.0001), without effect on estradiol release. We found no effect of resistin on rat GC proliferation. Conversely, in cows, resistin increased basal proliferation (P<0.0001; for 100-667 ng/ml) and decreased IGF1-induced proliferation of GC (P<0.0001; for 10-333 ng/ml) associated with a decrease in cyclin D2 protein level (P<0.0001). Finally, resistin stimulated AKT and p38-MAPK phosphorylation in both species, ERK1/2-MAPK phosphorylation in rats and had the opposite effect on the AMPK pathway (P<0.05). In conclusion, our results show that resistin is expressed in rat and bovine ovaries. Furthermore, it can modulate GC functions in basal state or in response to IGF1 in vitro.