Establishment Of In-vitro Rat Preantral Follicle Three Dimensional Culture Systems And Application Of It In The Female Reproductive Toxicity Studies

Evaluation of the femal reproductive function by in vivo studies is currently carried out on the basis of parameters related to fertility outcome, these parameters do not allow elucidation the site of action and the mechanism of toxic damage to the ovary. Since in-vivo animal studies are less sensitivity, require longer testing cycle and more test samples than in-vitro bioassay, so it is necessary to develop in-vitro bioassay for testing femal reproductive toxicity. As yet, no adequate in-vitro models have been validated to identify potentially hazardous products with regard to the reproductive system, including ovarian follicle, oocyte, oogenesis which is the key to evaluate femal reproductive toxicity. In rescent years, in-vitro follicle culture systems have been developed with the aim of growing immature ooeytes from early follicle stages to fertilizable oocytes. They are also often used as a tool to study the process of folliculogenesis and oogenesis in-vitro. In-depth study of the culture system demonstrates that the whole in-vitro process mimics normal(in-vivo) physiology from the point of view of morphogenesis, differentiation, hormone production. Ovarian follicle function can be analyzed simultaneously on folliculogenesis, hormonogenesis, and oogenesis on variant developmental stages. These systems have been applied succeed to reproductive biology and physiology studies for the folliculogenesis and follicle regulation.Our department of Wan Xuying lead in establishing a rat preantral follicles in vitro2D culture models, but the development trend of the future in vitro toxicity testing methods is the development of methods based on three-dimensional culture models. Access to foreign literature found that rat preantral follicles in vitro three-dimensional culture model is rare, I only saw two foreign short-term training, And have not been reported in the literature for any species follicles in vitro three-dimensional culture in our countryOn basis of the mouse follicle in-vitro three-dimensional culture system described according to literatures, in-vitro rat preantral follicle three-dimensional culture systems were developed in this study and had never been reported before. The aim of this study is to develop rat preantral follicle three-dimensional culture in-vitro assay in reproductive toxicology testing to provide information regarding the potential mechanism of action and the identification of chemicals which have female reproductive toxicity.Establishment of in-vitro rat preantral follicle three dimensional culture systems Objective: To establish an in-vitro system to support the growth of rat preantral follicles and produce mature oocytes. Method:preantral follicles (140-170μm) were mechanically isolated from12-14-day-old female rat and cultured for10days in a three-dimensional(3D) alginate and two dimensional (2D) flat. Through the morphologic of follicle development, steroid hormone production, ovulation rate and oocyte maturation rate to evaluate the3D culture effect, and compare the results with the2D. Result: During the3D culture, follicles has maintained a three-dimentional structure. But in the2D culture system, After two days culture, theca cells began to adherent growth, in the following culture, theca cells spread to surrounding and granulose cells break the basement membrane in the most of follicles. In the3D culture system, The rates of survival, antrum, the realeased of COCs and PB were higher than the2D culture system,they were95.7%vs90.7%(P<0.05),72.1%vs33.6%(P<0.05),79.3%vs39.8%(P<0.05),57.1%vs31.9%(P<0.05). In the two culture systems, from preantral follicle to oocyte maturation and the formation of first polar body, the morphological observation showed that development characteristics of rat preantral in-vitro were similar to those in-vivo. Secretion patterns of E2and P were consistent with the observed changes in follicle morphology and differentiation. Conclusion(s):This study successfully developed in-vitro rat preantral follicle three dimensional culture system,BPA inhibit the in-vitro growth of rat follicular and oocyte maturationObjective: rat follicles in vitro long-term three-dimensional culture as a novel approach for the first time to explore the impact of BPA on the in vitro growth of rat follicular and oocyte maturation; preliminary exploration the feasibility of the system for reproductive toxicity study. Method:preantral follicles (140-170μm) were mechanically isolated from12-14-day-old female rat, The follicles were randomly divided into three treatment groups (50~150μM) and a control group, and then cultured for10days in the three-dimensional alginate. Through the morphologic of follicle development, steroid hormone production, ovulation rate and oocyte maturation rate to evaluate the result. Result: The vast majority follicles of control group have experienced the preantral follicles, antral follicles and mature follicle stage among the10days of continuous culture. Compared with the control group, with doses increasing, the rates of follicle survival, cavity formation, COCs discharge and the PB were decreased,100μM/ml and over dose difference was statistically significant compared with the control group (P <0.05). Theca cells and granulosa cell proliferation was inhibited in treatment group,100μM and over dose difference was statistically significant compared with the control group(P <0.05). With the doses increasing, the secretion of estradiol and progesterone were decreased, The difference was statistically significant compared with the control group (P <0.05). Conclusion(s):In this study, BPA inhibition of follicle growth and oocyte maturation; This study preliminary validation instructions rat follicles in vitro long-term three-dimensional culture model suitable for toxicity studies.The above results show that the three-dimensional culture system established in this study can support the growth of rat preantral follicles and produce mature oocytes, and the rate of follicle survival, cavity formation, COCs discharge, and PB were higher than the two dimensional culture system; The study of female reproductive toxicants bisphenol-A have shown that the system is suitable for toxicological studies. Rat follicles in vitro long-term three-dimensional culture system is a potential new method which not only can rapid evaluation ovarian poison but also can study female reproductive toxic mechanism.