Development of a long-term serum-free culture system for immature granulosa cells from diethylstilboestrol-treated prepubertal rabbits: influence of androstenedione and fibronectin on FSH-induced cytodifferentiation

Abstract

Granulosa cells from diethylstilboestrol-treated prepubertal rabbits were cultured for 6 days in M199 with FSH (1-100 ng ml(-1)) in uncoated or fibronectin-coated plates with or without androstenedione to define the time course profile of oestradiol and progesterone secretion, and the possible modulator role of androstenedione and fibronectin during FSH-induced rabbit granulosa cell differentiation. Every 48 h, cultures were photographed and samples of medium were collected and assayed by ELISA for oestradiol and progesterone. FSH increased oestradiol secretion in a dose-dependent manner. Androstenedione augmented FSH-stimulated oestradiol secretion, and led to a decrease in secretion of oestradiol with time in culture. FSH stimulated progesterone secretion in a dose-dependent manner. This was increased by androstenedione with 10 ng FSH ml(-1) (0-96 h) and 1 ng FSH ml(-1) (96-144 h). FSH-stimulated (100 ng ml(-1)) progesterone secretion decreased at 48-96 h. Fibronectin prevented this decrease, without affecting oestradiol or progesterone secretion at other time points. FSH caused cell reaggregation at 48 h. In conclusion, this serum-free culture system is appropriate for the study of mechanisms of rabbit granulosa cell differentiation. FSH induced cytodifferentiation and reaggregation of granulosa cells. Androstenedione appeared to act synergistically with FSH to promote steroidogenesis. Fibronectin sustained progesterone secretion during differentiation.

Effects of Basal Media and Supplements on Diethylstilbestrol‐Treated Immature Mouse Primary Granulosa Cell Growth and Regulation of Steroidogenesis In Vitro

“…As similar results were obtained in the present study, we choose oestrogen‐treated immature mouse as our experimental animal model. In addition, compared with daily injection for 3 days (Picazo et al. 2000; Vitale et al.…”

“…1982; Sirotkin 2010), sheep (McNatty et al. 2010), rabbit (Picazo et al. 2000) and bovine (Maillard et al.…”

“…Therefore, various serum‐free systems were developed in culturing GCs in an attempt to allow expression of hormonal effects and differentiation (Erickson 1983; Langhout et al. 1991; Picazo et al. 2000), propagate without inducing progesterone production (Broussard et al.…”

“…As an initial step towards developing the in vitro primary GCs culture system, better source of primary GCs was important. Diethylstilbestrol (DES), the analogue of oestrogen, was used to generate large amount of homogeneous GCs in vivo (Picazo et al. 2000; Vitale et al.…”