Bisphenol A (BPA) and Bisphenol S (BPS) exposure disrupt ovarian function and granulosa cell (GC) steroidogenesis. Extracellular vesicles (EVs) and their miRNA cargo, as mediators of cellular response to environmental stimuli, might be involved in fertility and folliculogenesis. This study explored modulation of microRNA (miRNA) expression after 48 h BPA or BPS exposure (10 µM) in ovine primary GC and EVs from corresponding conditioned medium (CM EVs). Small RNA sequencing of control (0 h) and 48 h treated GC, CM EVs allowed identification of 526 ovine miRNAs, including 128 new sequences. BPA did not alter miRNA expression in GC, while BPS decreased cellular oar-24b miRNA. In contrast, BPA modified expression of 4 miRNAs in CM EVs, including 3 new sequences, and 2 miRNAs were modified by BPS. Both compounds reduced expression of a sequence homologous to miR-1306. Further studies are required to decipher their roles in bisphenol toxicity in GC.

The size of the ovarian reserve, defined by the pool of primordial follicles, is established during fetal life in women and sheep. The prenatal period is a critical window of sensitivity, especially for ovarian development. Maternal metabolic status and nutrition during gestation can influence fetal programming, and affect the establishment of ovarian reserve. In addition, the developing fetus is particularly sensitive to endocrine disruptors. Bisphenol A (BPA), a plasticizer widely used in food packaging, has deleterious effects on fetal folliculogenesis. BPA has been banned from the food industry and has been replaced mainly by bisphenol S (BPS), whose effects on ovarian development remain poorly characterized. The objective of this study was to determine the effects of in utero exposure to BPS (50 µg/kg/day) on ovarian development in female fetuses, and lambs, using histology and/or magnetic resonance imaging (MRI). In addition, pregnant ewes exhibited contrasted metabolic status (lean versus fat), allowing assessment of the interaction between maternal nutrition and BPS exposure. This study was divided into two experiments. In the first, pregnant ewes received daily subcutaneous injection of BPS for 3 months, and fetal ovarian development was analyzed at 130 days of gestation. In the second, pregnant ewes were exposed daily to BPS through food for 3 months and female lambs were then monitored up to 4-month-old (pre-puberty). In offspring of fat mothers, the body weight of the fetuses increased but this increase was not observed anymore in lambs. Plasma anti-Mullerian hormone (AMH) concentrations decreased in 1-month-old lambs and the antral follicle number increased in both fetuses and 4-month-old lambs from well-fed mothers. In addition, a significant interaction between maternal metabolic status and gestational BPS exposure was reported for the numbers of primordial, intermediate and total follicles of fetuses, with a similar trend for pre-antral follicles. Such significant interaction was also reported for the number of intermediate follicles in 4-month-old lambs, with a similar tendency being reported for AMH concentration. In conclusion, maternal nutrition and gestational exposure to BPS had deleterious consequences on the body growth and ovarian follicular development in the offspring. In addition, BPS effects differ according to the maternal metabolic status. Thus, the maternal environment affected folliculogenesis in both fetuses and lambs. Further research is needed to determine whether these alterations have long-term deleterious effects on their reproductive function in adulthood.

Most in vitro models of oviduct epithelial cells (OEC) used thus far to gain insights into embryo-maternal communication induce cell dedifferentiation or are technically challenging. Moreover, although the presence of developing embryos has been shown to alter gene expression in OEC, the effect of embryos on OEC physiology remains largely unknown. Here, we propose a model based on bovine oviduct epithelial spheroids (OES) with specific shape and diameter (100-200 mu m) criteria. The aims of this study were to i) determine the appropriate culture conditions of bovine OES cultured in suspension by evaluating their morphology, total cell number, viability, and activity of ciliated cells; ii) monitor gene expression in OES at the time of their formation (day 0) and over the 10 days of culture; and iii) test whether the vicinity of developing embryos affects OES quality criteria. On day 10, the proportions of vesicle-shaped OES (V-OES) were higher in M199/500 (500 mu l of HEPESbuffered TCM-199) and synthetic oviduct fluid (SOF)/25 (25-mu L droplet of SOF medium under mineral oil) than in M199/25 (25-mu L droplet of M199 under mineral oil). The proportion of viable cells in V-OES was not affected by culture conditions and remained high (>80%) through day 10. The total number of cells per V-OES decreased over time except in SOF/25, while the proportions of ciliated cells increased over time in M199/500 but decreased in M199/25 and SOF/25. The movement amplitude of OES in suspension decreased over time under all culture conditions. Moreover, the gene expression of ANXA1, ESR1, HSPA8, and HSPA1A in OES remained stable during culture, while that of PGR and OVGP1 decreased from day 0 to day 10. Last, the co-culture of developing embryos with OES in SOF/25 increased the rates of blastocysts on days 7 and 8 compared to embryos cultured alone, and increased the proportion of V-OES compared to OES cultured alone. In conclusion, M199/ 500 and SOF/25 provided the optimal conditions for the long-time culture of OES. The supporting effect of OES on embryo development and of developing embryos on OES morphology was evidenced for the first time. Altogether, these results point OES as an easy-to-use, standardizable, and physiological model to study embryo-maternal interactions in cattle.

Bisphenol S (BPS) is the main substitute for bisphenol A (BPA), a well-known plasticiser and endocrine disruptor. BPS disrupts ovarian function in several species. Moreover, a few studies have reported that the effects of BPS might be modulated by the metabolic status, and none have characterised the granulosa cell (GC) proteome after chronic BPS exposure. This study aimed to decipher the mechanisms of action of chronic BPS exposure on the proteome of ewe GCs while considering the interaction between a deliberate contrasted metabolism and reproductive function. Forty ewes were split into two groups with contrasted diets: restricted (R, n = 20) and well-fed (WF, n = 20). The R and WF ewes were subdivided according to the dose of BPS administered through the diet (0–50 µg/kg/day), forming four groups: R0, R50, WF0 and WF50. After 3-month BPS daily exposure, GCs were recovered during the pre-ovulatory stage and proteins were analysed by nano-liquid chromatography coupled with tandem mass spectrometry. Chronic exposure to BPS affected the GC proteome differently according to the ewe metabolic status. Fifty-nine out of 958 quantified proteins were differentially abundant between groups and are mainly involved in carbohydrate and lipid pathways. Unsupervised hierarchical clustering of differentially abundant proteins (DAPs) identified four clusters of 34, 6, 5 and 14 proteins according to the BPS exposure and diet interaction. Pairwise comparisons between groups also revealed a strong effect of BPS exposure and diet interaction. Functional analysis of DAPs highlighted that BPS upregulated β-glucuronidase (GUSB; p = 0.002), a protein especially able to deconjugate bisphenol glucuronides (BP-g). Moreover, among unexposed ewes, GUSB was detected only in well-fed ewes. Conjugation of glucuronides inhibits the oestrogenic activity of bisphenols. Upregulation of GUSB in ewes dosed with BPS would prolong the oestrogenic effects of BPS by deconjugating BPS-g into free BPS. In addition, literature has reported an up-regulation of GUSB in people suffering from obesity. Therefore, people suffering from obesity could be subjected to prolonged and aggravated exposure to BPS. These data highlighted the deleterious effects of BPS and its interaction with metabolic status.

Abstract 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 ERβ). 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.

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 (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 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 mu g/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.

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.

Reproduction, Fertility and Development is an international journal publishing original research , review and comment in the fields of reproduction and developmental biology in humans, domestic animals and wildlife

Protein palmitoylation is a reversible post-translational modification by fatty acids (FA), mainly a palmitate (C16:0). Palmitoylation allows protein shuttling between the plasma membrane and cytosol to regulate protein stability, sorting and signaling activity and its deficiency leads to diseases. We aimed to characterize the palmitoyl-proteome of ovarian follicular cells and molecular machinery regulating protein palmitoylation within the follicle. For the first time, 84 palmitoylated proteins were identified from bovine granulosa cells (GC), cumulus cells (CC) and oocytes by acyl-biotin exchange proteomics. Of these, 32 were transmembrane proteins and 27 proteins were detected in bovine follicular fluid extracellular vesicles (ffEVs). Expression of palmitoylation and depalmitoylation enzymes as palmitoyltransferases (ZDHHCs), acylthioesterases (LYPLA1 and LYPLA2) and palmitoylthioesterases (PPT1 and PPT2) were analysed using transcriptome and proteome data in oocytes, CC and GC. By immunofluorescence, ZDHHC16, PPT1, PPT2 and LYPLA2 proteins were localized in GC, CC and oocyte. In oocyte and CC, abundance of palmitoylation-related enzymes significantly varied during oocyte maturation. These variations and the involvement of identified palmitoyl-proteins in oxidation-reduction processes, energy metabolism, protein localization, vesicle-mediated transport, response to stress, G-protein mediated and other signaling pathways suggests that protein palmitoylation may play important roles in oocyte maturation and ffEV-mediated communications within the follicle.