Development and use of an in vitro technique to investigate the effect of pharmaceutical agents on female germ cell development

Abstract

With meiosis spanning from embryonic development to the end of reproductive life in females, scientists have faced considerable limitations in studying female meiosis and the effects of toxicants on the developing oocyte. Over the last half century, various culture methods have been developed with the aim of studying the mechanisms of early ovary development, as well as for use in reproductive toxicology. However, very few of the established embryonic ovary culture systems have been used to investigate potential reproductive toxicants on the embryonic ovary, in particular when compared with the vast number of in vitro reproductive toxicity studies on the post-natal ovary. Here, a novel test compound, a topoisomerase II inhibitor: AstraZeneca Test Compound (AZTC), was used to investigate the efficacy and validity of ovarian culture methods when compared with in vivo reprotoxicity studies. AZTC was selected due to preliminary in vivo studies demonstrating its detrimental effects on spermatogenesis in male rats. AZTC targets bacterial type II topoisomerases that might have mammalian homologues involved in meiosis. Topoisomerase-II α was expressed within the female germ cells pre-natally, but became localised to the granulosa and stroma cells post-natally. This occurred both in vivo and in vitro. Ovaries from female rats exposed pre-natally to AZTC in vivo were analysed histologically and a significant increase in the number of primordial follicles was observed within the ovaries, as well as an increase in the number of unhealthy follicles. A novel mouse embryonic ovary culture system was developed by adapting, improving and bridging existing available culture techniques. The culture system supported growth of pre-meiotic mouse germ cells through prophase I of meiosis, the formation of primordial follicles and initiation of follicle growth. Cultured ovaries contained follicles at stages in comparable ratios to those in vivo and appeared morphologically normal and healthy. The culture also supported meiotic progression of oocytes to the pachytene stage, albeit with a slight delay. AZTC was used to validate the novel embryonic ovary culture by comparing the results with those from the in vivo study, where AZTC exposure had also occurred during embryonic development. Similar results were consistently observed between the in vivo and in vitro studies. In vitro effects of AZTC on the post-natal mouse ovary were also investigated, where neonatal mouse ovaries cultured with AZTC had fewer primordial follicles and more unhealthy follicles than did control ovaries. AZTC therefore demonstrated different effects when exposure occurred pre-natally vs. post-natally. The embryonic ovary culture was then used to examine the effects of another topoisomerase II inhibitor, etoposide, on the pre-natal ovary. Etoposide is a chemotherapy agent and has previously been prescribed to pregnant women. A significant reduction in the size of the follicle pool was observed in exposed cultured embryonic ovaries, where primordial and transitional follicles were targeted. Overall, establishment of post-natal culture systems have become a useful addition to in vivo reproductive toxicology studies. The embryonic ovary culture system developed here could become a valuable and powerful tool to screen potential reproductive toxicants, as well as to study the dynamics and regulation of early ovary development.