Abstract
The incidence of infertility among individuals of reproductive age has been growing due to genetic and environmental factors, and considerable research efforts are focused on solving this issue. Ovarian development is an overly complex process in the body, involving the interaction between primordial germ cells and gonad somatic cells. However, follicles located in the center of the in vitro ovary are poorly formed owing to ovarian complexity, nutrient deficiency, and signaling deficiency. In the present study, we optimized methods for dissociating gonads and culture conditions for the in vitro generation of miniaturized ovaries. The gonads from embryos were dissociated into cell masses and cultured on a Transwell-COL membrane for 3-5 weeks. Approximately 12 follicles were present per in vitro ovary. We observed that miniaturized ovaries successfully matured to MII oocytes in vitro from 150 to 100 µm gonad masses. This method will be useful for investigating follicle development and oocyte production.