Graduate Student University of Maryland Front Royal, Virginia, United States
Abstract Authors: Batsheva Marks1,2; Jennifer Nagashima2; Carol Keefer1; Nucharin Songsasen2
1. Department of Animal and Avian Sciences, University of Maryland, College Park, United States
2. Center for Species Survival, Smithsonian National Zoo and Conservation Biology Institute, Front Royal, United States
Abstract Text: Ovarian stromal cells act as crucial support and regulators for in vivo folliculogenesis; however, less is known about their effect on in vitro grown follicles. The objective of this study was to investigate the impact of ovarian stromal cell co-culture or conditioned medium (CM) on survival and development of cat pre-, early, and antral follicles in vitro. Ovaries were obtained from cats older than 6 months (n = 3), then enzymatically digested to release stromal cells. The ovarian stromal cells were allowed to grow to confluency in a T75 flask, before being cryopreserved for long term storage in liquid nitrogen. Cells were thawed one week prior to follicular culture onset, and passaged once before CM collection. CM was subsequently removed 24 - 48 hours after feeding, and stored at -80°C until used. Ovarian follicles were mechanically isolated from cats older than 6 six months (n = 23 cats, 155 follicles), encapsulated in 0.5% alginate hydrogel, and cultured for 13 days in Endothelial Cell Growth Medium. The isolated follicles were then divided into five treatment groups (control, ovarian stromal cell co-culture, 20% CM, 50% CM, and 100% CM), and classified based on initial diameter as preantral (224.4 ± 4.7 μm), early antral (394.8 ± 7.4 μm), or antral (592.2 ± 18.8 μm). The survival and growth of isolated follicles were then evaluated on Day 0, 4, 6, 8, 11 and 13. At the culture period endpoint, follicles were assessed via RTPCR for expression of CYP19A, FSHR, and GDP9 to further quantify development. Statistical analysis was done in R software. Follicles in 100% CM had higher survival up to Day 11 of culture as compared to other treatment groups (Cox proportional hazards model, p ≤ 0.01). Initial stage also influenced survival, with antral follicle survival significantly lower than that of pre- and early antral follicles (p ≤ 0.0001). However, no differences in growth were detected across the treatment groups, nor across initial size classifications (Kruskal-Wallis test, p > 0.05). Post culture RTPCR analysis of the three selected genes showed CYP19A was upregulated in 50% CM follicles compared to the control (ANOVA, p ≤ 0.05). However, there were no differences in CYP19A expression between the control and other treatment groups, or in GDF9 and FSHR expression among culture groups (p > 0.05). In summary, the findings demonstrated that conditioned medium collected from primary culture of ovarian stromal cells improves in vitro survival and modulates CYP19A expression of isolated cat follicles. Further research to identify paracrine factors present in conditioned medium will elucidate the roles of ovarian stromal cells pertaining to follicle survival during in vitro folliculogenesis.
