Steroid production, cell proliferation, and apoptosis in cultured bovine antral and mural granulosa cells: Development of an in vitro model to study estradiol production

Abstract

This study was undertaken to characterize the relationship between changes in steroid production, cell cycle activity (ie, cell proliferation) and apoptosis in antral and mural bovine granulosa cells cultured in vitro. This was done to select conditions promoting optimal estradiol production by bovine granulosa cells cultured in completely defined conditions. In the first experiment, antral granulosa cells were cultured over the entire 4 days of the culture period in the presence of either 0, 2, or 10 ng/ml of FSH (chronic conditions) or were maintained under minimal FSH support (0.5 ng/ml FSH) for the first 3 days of culture and then were challenged over the fourth day of culture with either 0, 2, or 10 ng/ml FSH (challenged conditions). Compared with cells exposed to constant FSH levels (chronic conditions), the FSH‐induced production of estradiol was higher (P < 0.006) and that of progesterone was lower (P < 0.02) over the last 24 h of culture, when antral granulosa cells were maintained under minimal FSH support during the first 3 days of culture (challenged conditions). In the second experiment, dynamics of estradiol and progesterone productions, conversion of [14C]androstenedione into subsequent steroid metabolites, DNA content, cell cycle activity, and apoptosis (as assessed by flow cytometry) of antral and mural granulosa cells over the first 3 days of culture under minimal FSH support and in response to a challenge with FSH during the last 24 h of culture were evaluated. Estradiol production as well as the conversion of androstenedione into testosterone and estradiol were greater (P< 0.01) in antral than in mural granulosa cells cultured under challenged conditions. A higher proportion of mural than antral granulosa cells were in the proliferative state at the end of culture (P < 0.03). This may be related to the decreased ability of mural cells to produce estradiol. FSH suppressed (P < 0.05) the spontaneous onset of apoptosis in both cell types. These results suggest that functional differences between these two cell compartments need to be considered in studying bovine granulosa cells in vitro. Because of their large (400 to 600%) FSH‐induced estradiol production, antral granulosa cells cultured under challenged conditions provide a model that can be used to examine substances for their ability to alter estradiol production and apoptosis in bovine granulosa cells. Mol. Reprod. Dev. 50:170–177, 1998. © 1998 Wiley‐Liss, Inc.

Norepinephrine stimulates progesterone production in highly estrogenic bovine granulosa cells cultured under serum-free, chemically defined conditions

“…In contrast to findings reported from other bovine GC primary cultures [28,29,32] and even buffalo GC serum-free culture [33], P4 was low during all time points evaluated in our cell cultures and a significant increase was observed only after 96h. A low although increasing P4 production is an important feature of the ideal GC culture system that represents physiological events of an E2 active follicle that is growing towards dominance.…”

“…High E2 production by GCs cultured in the presence of bovine serum albumin or other serum replacers has also been described in earlier studies [16,28]. Although attempts to propagate bovine GCs using serum replacers have succeeded in some cases [16,28,29], some of those serum replacers were contaminated with steroids and other interfering factors [19]. Previous experiments from our group suggest that replacement with PVA (and additional defined supplements) allows for greater and earlier E2 production by GCs when compared with bovine serum albumin, a widely used serum replacer [18].…”

“…In the serum-free culture system described here, GCs cultivated under chemically defined medium supplemented with PVA continued to secrete E2 throughout the 144h of culture under gonadotropin-independent conditions. High E2 production by GCs cultured in the presence of bovine serum albumin or other serum replacers has also been described in earlier studies [16,28]. Although attempts to propagate bovine GCs using serum replacers have succeeded in some cases [16,28,29], some of those serum replacers were contaminated with steroids and other interfering factors [19].…”

Effects of FSH and LH on Steroid Production by Buffalo (Bubalus bubalis) Granulosa Cells CulturedIn VitroUnder Serum-Free Conditions

“…1997; Silva and Price 2000) by inclusion of insulin and IGF‐I, both of which have strong mitogenic and steroidogenic effects, whereas in other studies, inclusion of insulin at concentrations ranging from 50 to 1000 ng/ml was found to be unable to stop the reduction in oestradiol production during the course of the culture (Berndtson et al. 1995; Rouillier et al. 1996, 1998; Yang and Rajamahendran 1998).…”

“…Also, the differences in the culture systems employed for the culturing of granulosa cells has to be considered while comparing the results. The culture systems used in nearly all studies contained either insulin at supraphysiological concentrations of 50 (Rouillier et al. 1996, 1997, 1998), 500 (Yang and Rajamahendran 1998) or 1000 ng/ml (Roberts and Echternkamp 1994; Berndtson et al.…”