Effects Of Bisphenol A On The Primordial Follicle Growth In Vitro Of Neonatal Mice Ovary

BackgroundEndocrine disruptor chemicals (EDCs), an exogenous compounds, could affect human and animal’s endocrine system, and consequently causes adverse health effects of the corresponding organ, or even the progeny. Because of its widespread presence in nature and the adverse impact on health, EDCs has garnered much concern in recent years.Bisphenol A (BPA) is the highest volume endocrine disruptors so far. It present in many common consumer products, including plastic (as a polymer, polycarbonate plastic), polyvinyl chloride (PVC), food packaging, dental sealants, and so on. Humans could absorb BPA through food, air inhalation and skin contact. In China, BPA has been detected in approximately50%of blood samples and90%of urine samples collected from the general population. Similar results have been observed in other countries, including the United States, Germany, and other Asian countries. Furthermore, owing to the widespread use of BPA, all humans, regardless of age, are at high risk for BPA exposure. Though the use of BPA has been conducted severely, the effect of BPA cannot be ignored. In order to prevent the environment-related disease, we should make it clear that what disease BPA induced and how BPA did.The primordial follicle pool governs the onset of female fertility and determines reproductive ages. During the female reproductive lifespan, the primordial follicles in the ovary serve as the source of fertilizable ova. In mice, the primordial follicles are formed just after birth and are later synchronously activated in a wave-like manner. The primordial follicle to primary follicle transition initiates follicle development. Of the remaining follicles in the dormant state, a few follicles undergo atresia. The rate of follicle growth and atresia is of critical importance to maintain the primordial follicle pool. The phosphatidylinositol-3-kinase (PI3K) pathway is a "classic" signaling pathway that is involved in the regulating cell proliferation, migration, and metabolism (reviewed by Cantley). Recently, the PI3K pathway in oocytes has been found to be important for early follicular development since it activates the downstream expression of the serine/threonine protein kinase Akt, which controls follicle and oocyte development. The kit ligand is a well-known molecule that regulates early follicle development by stimulating the PI3K/Akt pathway.Numerous studies have investigated the influence of BPA on human health, especially reproductive health, since the ovary is an active organ that is susceptible to harmful environmental exposure. Previous studies revealed that BPA can suppress ovarian follicle growth by causing perturbations in estrogen metabolism or meiotic division. Recent evidence suggests that BPA perturbs various ovarian functions such as folliculogenesis and follicle formation in vitro. However, few studies have focused on the primordial follicle pool in neonates. Therefore, in this study, we used the mouse neonatal ovarian culture system to investigate the effect of BPA on the primordial follicle pool.Methods1. Establishment of in vitro culture system with neonatal mice ovary and its effect on primordial follicle poolThe whole ovary from the PND-4C57BL/6mice was cultured in vitro. All the results were compared with the ovary in vivo. HE staining was used to observe the pathological change and evaluate the follicle number of in vitro culture system.To evaluate the the growth and apoptosis of follicles, Ki-67and caspase3immunohistochemical analysis were employed.2. Observe the effect of BPA on primordial follicle growthThe ovaries were randomly divided into four groups:the control group (DMSO); the low concentration of bisphenol A group (0.1μM); the moderate concentrations of bisphenol A group (1μM); the high concentration of bisphenol A group (10μM). All the bisphenol A groups were compared with the control group. HE staining was used to observe the pathological change and evaluate the follicle number of in vitro culture system.To evaluate the the growth and apoptosis of follicles, Ki-67and caspase3immunohistochemical analysis were employed.3.Examine the change of PI3K/Akt signaling pathway with BPA exposureThe key molecule of PI3K/Akt pathway was examined in all groups. Realtime-PCR was used to detect the levels of mRNA expression. Protein expression levels were quantified by Western-blot and localized with immunohistochemical analysis. Primary follicles number were counted after Blocking the PI3K/Akt pathway by using PI3K inhibitors.4. Statistical analysisData are expressed as mean±SEM (standard error of the mean). All of the statistical analyses were conducted using GraphPad Prism5.0software. P<0.05was regarded as statistically significant. When the groups of data were not normally distributed, the differences between the groups were compared using a Kruskal-Wallis-test. A one-way analysis of variance (ANOVA) test was used for all other analysis.Results1. primordial follicle growth in vitro was similar with that in vivoThe average diameter of ovary in vitro was slightly less than that in vivo, but the difference was not statistically significant. HE staining showed that there is no obvious change in the pathology of cultured ovary. Primordial follicles number with culture system decreased and primary follicles increased significantly. But the number of primary follicles and secondary follicles was not significantly different. Ki-67and caspase3immunohistochemistry showed that follicles apoptosis increased in vitro, especially the primordial follicles. But there is no statistical significance of growth rate between ovary in vitro and in vivo.2. BPA exposure reduced the primordial follicle pool and increased the primary follicle numberAll the concentrations of bisphenol A groups were compared with the control group. HE staining showed no pathological changes with bisphenol A exposure. The number of primary follicles increased significantly in1μM,10μM bisphenol A groups. The difference between0.1μM bisphenol A group and control group was not statistically significant. The number of secondary follicles and total follicles in all bisphenol A groups showed no significant difference with the control group. Ki-67and caspase3immunohistochemistry showed bisphenol A increased the growth of follicles and reduced the apoptosis.3. BPA promote primordial follicle pool through activating PI3K/Akt signaling pathwayAll the concentrations of bisphenol A groups significantly increased mRNA expression of PI3K and protein expression of PI3K and Akt phosphorylation. Immunohistochemical analysis showed that bisphenol A significantly increased protein expression of PI3K and Akt phosphorylation in follicles. Ovaries co-treated with BPA (all concentrations) and LY294002showed a significant reduction in primary follicle number compared to that in ovaries treated with BPA alone. No difference in primary follicle number was observed between the control group treated with LY294002and the BPA group treated with LY294002.ConclusionThe growth of primordial follicle in vitro was similar with that of in vivo. In vitro culture system with neonatal mice ovary was a good media to explore the mechanism of primordial follicle pool. Bisphenol A (BPA) could significantly reduce the number of primordial follicles in vitro by activation of primordial follicles growth and reduce of primordial follicles apoptosis. BPA increased the expression of PI3K/Akt signaling pathway, thus contributing to the development of primordial follicles. However, how the effects of bisphenol A on the PI3K/Akt pathway requires further exploration and research.