Introduction: Follicle development is complex, and the use of reaggregated ovaries (ROs) allows us to investigate oocyte-somatic cell interactions, since they can be created using different sources of germ and somatic cells. Production of an RO involves the separation of germ and somatic cells using differential plate adhesion, followed by reaggregation into a pellet. The pellet is then transplanted beneath the kidney capsule of an immunocompromised mouse which facilitates follicle development in the ROs however, development cannot be observed. We have developed an in vitro technique that supports RO growth in order to observe follicle development over time.
Methods: This study was approved by the Local Ethical Review Panel (University of Oxford). ROs generated from 4 to 5 neonatal mice (aged P0-P6) were cultured in Waymouth media supplemented with FSH, insulin-transferrin-selenium, ascorbic acid and FBS, for 7 and 14 days. ROs were also transplanted beneath the kidney capsule of an immunocompromised mouse for 21 days. ROs were embedded, sectioned, H&E stained and follicle development assessed.
Results and discussion: ROs cultured for 7 days (n=3) contained primary follicles, whereas after 14 days of culture (n=3), ROs contained primary, secondary and preantral follicles. ROs developed in vivo for 21 days contained primary, secondary, preantral and antral follicles (n=4). The presence of antral follicles in the in vivo developed RO demonstrates the potential for full follicle development in cultured ROs. Although further studies are needed to enable follicles to develop to the later stages in vitro, RO culture provides us with the ability to observe follicle development in real time, which is hugely advantageous to furthering our understanding of follicle function.
This study was partially funded by Nuffield Department Obstetrics and Gynaecology.
11 Jul 2016 - 11 Jul 2016