Reproduction Abstracts

Introduction: In vitro cultures are a widely used tool to study ovary development and assess reproductive toxicology of chemicals. However, establishing a culture system whereby mouse ovaries can be cultured from a pre-meiotic stage to a mature oocyte has proved challenging. We have developed a novel culture system that spans meiotic entry to meiotic arrest, germ cell nest break-down, follicle formation, and initiation of follicle growth.

Methods: E13.5 mouse ovaries were cultured for 12 days on an agar block. Follicle health and numbers was analysed histologically, and oocyte meiotic chromosome spreads were analysed to determine meiotic progression of cultured oocytes.

Results: Cultured ovaries contained follicles at stages in comparable ratios to those in P4 in vivo ovaries (77.1% primordial, 17.0% transitional, and 5.6% primary in cultured ovaries vs 88, 7.8, and 2.8% respectively, in vivo). Ovaries and follicles appeared morphologically normal and healthy; with only a slight, non-significant increase in unhealthy follicles in the cultured ovaries compared with P4 ovaries (3.6% increase, P=0.38). Furthermore, 23% of cultured oocytes had reached pachytene after 6 days in culture, which, compared with the 50% pachytene nuclei in an in vivo E18 ovary, demonstrates that the culture system supports development of oocytes pachytene, albeit with a slight delay.

Conclusion: Our results demonstrate that our novel culture system supports growth of pre-meiotic mouse germ cells through prophase I of meiosis to meiotic arrest, the formation of primordial follicles and initiation of follicle growth.