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Growth factors and ovarian function in cattle. The IGF system and the oestrogen receptor

Final Activity Report Summary - OESTROGEN RECEPTOR (Growth factors and ovarian function in cattle. The IGF system and the oestrogen receptor)

The project objective was to investigate the control of oestrogen receptor function in granulosa cells by growth factors, through ligand-independent mechanisms.

Follicle growth and development is controlled by multiple factors affecting granulosa and theca cell proliferation and antrum formation. Oestrogen receptors are expressed in the granulosa cells of all species in which they have been investigated including cattle. Granulosa cell oestrogen receptors (ER) are functional, as indicated by the stimulatory effect of administered oestradiol in various species on cell growth and viability, as well as by the maintenance of Follicle-stimulating hormone (FSH) and Luteinising hormone (LH) receptor levels and induction of the Insulin-like growth factor I (IGF-I) synthesis.

The development of primordial follicles into primary follicles, which is independent of pituitary gonadotrophins, was suggested to involve growth factors produced within the ovary itself. Among these paracrine factors were the growth factors IGF-I and -II, Epidermal growth factor (EGF), Fibroblast growth factor (FGF) and Transforming growth factor beta (TGF-ß). ERs were subject to ligand-independent control by those growth factors. The effects of the growth factors on follicle development were possibly modulated via phosphorylation of the ERs through activation of the Mitogen-activated protein (MAP) kinase.

We wished to use an oestrogen responsive reporter gene construct to test this hypothesis, introducing the construct into granulosa cells by a procedure of transient transfection. Granulosa cell phenotype was maintained after transfection and short-term culture and no cytotoxic effect was observed. The effects on the oestrogen receptor function of different factors involved in responses to diet were then tested. Insulin, IGF-I, IGF-II and EGF, in addition to 17-oestradiol and androstenedione were used, either alone or in combination, in experiments of bovine granulosa cell transfection to check the activation of the oestrogen receptor. Other effects, such as cell association in culture, expression of connexin 43 as a marker of gap-junctions measured by immunofluorescence, aromatase expression measured by immunocytochemistry, oestradiol and progesterone production in culture medium were also assessed.

In general, IGF-I, IGF-II and EGF stimulated the cell activity of bovine granulosa cells. IGF-I showed the highest level of cellular stimulation and activated the ligand-independent activity of the oestrogen receptor. It stimulated the expression of gap junction proteins and the formation of cellular clumping, reduced granulosa cell death and loss of oestrogen receptor after short-term culture; however oestradiol production by granulosa cells was not increased by the presence of IGF-I. The effect was independent of the parity of the cow or size of follicle, with highest responses occurring in medium-sized follicles from cows. The inhibition of the MAP-kinase pathway partially modified the main effects that IGF-I produced on granulosa cell metabolism. The oestrogen receptor was detectable via a binding assay, which showed that levels of the receptor were higher in mural granulosa cells from large than in those from medium-sized follicles, although this did not apply to cumulus cells. IGF-I increased oestrogen receptor binding, but only in the presence of oestradiol.

Our results suggested that short-term changes in the plane of nutrition with increased concentrations of IGF-I could lead to an increase in fertility through, among others, a general effect on ovarian cell activity.