# Ovarian Stromal Cell-Conditioned Media, but Not Co-Culture, Improves Survival in Feline Follicles

## TL;DR

Using media from ovarian stromal cells improves survival of cat follicles in the lab, but co-culture does not.

## Contribution

Shows that conditioned media from ovarian stromal cells enhances follicle survival in vitro, but co-culture does not.

## Key findings

- Follicles cultured in 100% conditioned media had higher survival rates.
- CYP19A expression was upregulated in follicles treated with 50% conditioned media.
- Oocyte maturation rates were not significantly different across groups.

## Abstract

Preserving the genetic diversity of wildlife populations is a high priority for endangered species conservation efforts. One way to preserve genes from an animal that dies prematurely is rescuing immature gametes and cryopreserving or growing them in the lab. This study investigated the effect of co-incubating ovarian cells with ovarian follicles in the domestic cat (a model for endangered felids). We found that conditioned media collected from ovarian cell cultures significantly improved follicle survival, although there were no differences in the expression level of developmental markers between follicles cultured with conditioned media and the controls. Furthermore, oocyte maturation rates after culturing follicles were not significantly different among groups. Altogether, this study is an important step toward optimizing a laboratory system for maturing female gametes.

Ovarian stromal cells play roles in in vivo folliculogenesis; however, little is known about their effect on in vitro cultured follicles. This study investigated the impact of ovarian stromal cell co-culture or conditioned medium (CM) on the survival and development of domestic cat follicles in vitro. Preantral (n = 148 follicles), early antral (n = 92), and antral (n = 22) stage cat follicles were divided into five groups (control, ovarian stromal cell co-culture, 20% CM, 50% CM, and 100% CM), cultured for 13 days, and evaluated for survival, growth, and the mRNA expression of CYP19A, GDF9, and FSHR. Additional follicles (n = 199) were isolated, divided into three groups (control, co-culture, and 100% CM), cultured for 10 days, and oocytes were subjected to in vitro maturation (IVM). More follicles (p ≤ 0.01) cultured in 100% CM survived until day 11 of culture than other groups. Antral follicle survival was significantly lower than pre- or early antral (p ≤ 0.0001). However, no differences (p > 0.05) in growth were detected across the treatments. CYP19A expression was upregulated (p ≤ 0.001) in the 50% CM-treated follicles. Furthermore, no differences (p > 0.05) were found in IVM rates between cultures. In summary, the findings demonstrate that conditioned medium collected from primary cultures of ovarian stromal cells improves in vitro survival of isolated cat follicles.