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

Abstract

The objectives of this study were to investigate the effects of basal media, supplements, including mixture of 5 mg/ml insulin, 5 mg/ml transferrin and 5 g/ml selenium (ITS), bovine serum albumin (BSA), sodium pyruvate (PNa) and epidermal growth factor (EGF) compared with serum on the proliferation of diethylstilbestrol-treated C57BL/6J mouse primary granulosa cells (GCs) to develop a serum-free culture system and to use this system to evaluate the effects of these supplements on GCs' processes of steroidogenesis and related regulation mechanism. DMEM/F12, McCoy's 5a, RPMI Medium 1640, Medium 199 and a-MEM were chosen as basal media, and McCoy's 5a proliferated the GCs greatly (p < 0.05) with a better attachment and morphology. Granulosa cells showed a better proliferation status in serum-containing media than serum-free media, BSA, ITS, PNa and EGF increased the proliferation of GCs in serum-free system gradually, while the latter three had the same effect on supporting GCs' proliferation with serum. After 2-day culture, the cells cultured remained P450arom positive, which sustained a normal morphology and there was no sign of apoptosis when supplements were added in the serum-free condition. Only ITS induced oestradiol production, while ITS, BSA and PNa enhanced progesterone secretion significantly indicating the promotion of differentiation. And these supplements functioned partly through increasing the transcription level of StAR, 3β-HSD, P450scc or P450arom. In conclusion, McCoy's 5a medium was a better basal medium, and ITS and PNa promoted both the proliferation and differentiation of primary GCs in serum-free culture system, showing a satisfactory state for GCs' physiological study.

CNP/NPR2 signaling maintains oocyte meiotic arrest in early antral follicles and is suppressed by EGFR‐mediated signaling in preovulatory follicles

“…The collected granulosa cells were washed twice, and passed through a 40‐µm cell strainer (BD Falcon, Franklin Lakes, NJ, USA) to remove oocytes. The granulosa cells were cultured in a humidified atmosphere of 5% CO 2 at 37°C in McCoy's 5a medium containing 5% FBS and ITS (10 µg/ml insulin; 5.5 µg/ml transferrin; and 6.7 ng/ml sodium selenite), which is highly effective in the survival and proliferation of primary granulosa cells (Ding et al, 2012). The medium was replaced with McCoy's 5a medium containing ITS without FBS 48 hr later, and the cells were further incubated for 24 hr.…”

Phosphodiesterases in the rat ovary: effect of cAMP in primordial follicles

“…An explanation for the previous reported effects of cAMP on primordial follicle activation could be due to changes in hormone/growth factor secretion from later follicular stages affecting the primordial follicles, as the proportion of secondary follicles was higher in the human study. Another important difference between the two studies is the inclusion of FSH in the culture media in the present study, as additional cAMP may not stimulate the follicle growth further (Ding et al 2012). However, 8-bromocAMP did increase the expression of Inha suggesting that the ovaries were cAMP responsive.…”

Supplemented αMEM/F12‐based medium enables the survival and growth of primary ovarian follicles encapsulated in alginate hydrogels

“…Base medium had a significant influence on follicle survival and growth, which supports the notion that the metabolic environment plays a critical role in early follicle development. Previous reports have investigated various base medium formulations, such as αMEM, DMEM/F12, Waymouth, and McCoy's 5A, on follicle and oocyte development (Ding et al, ; Eppig et al, ; Hirao, ; Nonowaki, ; Picton et al, ; van de Sandt et al, ; Yoshida et al, ). However, results vary due to differences in species, medium supplements, and culture system.…”