In Vitro Differentiation of Bovine Theca and Granulosa Cells into Small and Large Luteal-like Cells: Morphological and Functional Characteristics1

Abstract

This study was undertaken to investigate whether bovine granulosa and theca interna cells could be luteinized in vitro into luteal-like cells. Granulosa and theca cells were cultured for 9 days in the presence of forskolin (10 microM), insulin (2 micrograms/ml), insulin-like growth factor I (100 ng/ml), or a combination of these agents. During the first day of culture, granulosa and theca cells secreted estradiol and androstenedione, respectively; progesterone rose only after 3-5 days in culture and reached a maximum on the ninth day of culture. Cells incubated in the presence of forskolin plus insulin exhibited morphological and functional characteristics of luteal cells isolated from the corpus luteum. It was found that cell diameter, basal and stimulated progesterone secretion, and pattern of cell replication for both cell types were comparable to those of luteal cells. Numerous lipid droplets and intensified mitochondrial adrenodoxin staining also indicated active steroidogenesis in luteinized cells. After 9 days in culture, stimulants were withdrawn, and the culture proceeded in basal medium for an additional 5 days; elevated progesterone levels were maintained by luteinized granulosa cells (LGC), whereas in contrast a dramatic drop in progesterone production was observed in luteinized theca cells (LTC). On Day 9, cells were challenged for 3 h with LH (10 ng/ml), forskolin (10 microM), or cholera toxin (100 ng/ml), resulting in a 4-fold increase in progesterone secretion by LTC; the same treatments failed to stimulate progesterone in LGC.(ABSTRACT TRUNCATED AT 250 WORDS)

Transforming growth factor-β1 inhibits luteinization and promotes apoptosis in bovine granulosa cells

“…In vitro, granulosa cells harvested from follicles always spontaneously luteinize (Meidan et al 1990). However, in cultured bovine granulosa cells harvested from small bovine antral follicles, the addition of low dose of FSH maintains follicle characteristics by stimulating E 2 and CYP19A1 for several days (Gutierrez et al 1997, Silva & Price 2002, Sahmi et al 2004) and appears to counteract the process of luteinization by inhibiting STAR mRNA expression (Zheng et al 2008).…”

Effects of gonadotropins and insulin-like growth factors-I and -II on in vitro steroid production by bovine granulosa cells

“…Follicles are highly dependent on gonadotropins to achieve ovulatory size (Fortune and Hilbert 1986). Starting when they are approximately 4 mm diameter, the expression of P450 aromatase increases and follicle growth ceases if FSH is suppressed (Meidan et al 1990;Xu et al 1995a). After reaching approximately 8 mm diameter, bovine follicles express LH receptors on the GC (Xu et al 1995a).…”

In Vitro Differentiation of Bovine Granulosa Cells Obtained from Small Antral Follicles.

“…Based on these data, one could assume that bovine granulosa cells obtained from small antral follicles can differentiate into large luteal-like cells during culture in vitro, and that the cultured granulosa cells can be used as a model of large luteal cells. Meidan et al [8] showed that bovine granulosa cells obtained from preovulatory follicles differentiated into large luteal-like cells during culture in the presence of insulin plus forskolin. In the present study, granulosa cells obtained from small antral follicles could also be luteinized when they were incubated with insulin plus forskolin.…”