Effects of angiotensin II, atrial natriuretic peptide and endothelin-1 on proliferation and steroidogenic output of bovine granulosa cells cultured in a chemically defined system.

The role of local factors in the modulation of granulosa cell (GC) proliferation and differentiation is well described in the literature. The present work used a long-term bovine GC culture, in chemically defined medium without gonadotropins, to study the effects of angiotensin II (Ang II), atrial natriuretic peptide (ANP) and endothelin-1 (EDN1) on the steroidogenesis and cellular proliferation. Small follicles (3-5mm in diameter) from ovaries obtained in the slaughterhouse were selected according to their vascularization and follicular fluid color in order to isolate GC. Granulosa cells were plated at a density of 5×10(4)cells/well in supplemented alpha-MEM containing 3 levels (0, 10(-8)M and 10(-7)M) of Ang II, ANP, and EDN1 for up to 96h. Proliferation was evaluated by tritiated thymidine incorporation. The results showed that Ang II, ANP, and EDN1 modulate the steroidogenic output and proliferation index of GCs depending on the dose and time of culture. The selected vasoactive peptides increased androstenedione (A4) consumption in parallel with increased estradiol (E2). Although the peptides also promoted a significant increase in pregnenolone (P5) and progesterone (P4) production, the E2:P4 ratio was maintained at a high at most of the tested doses. Taken together, our in vitro data suggest that these vasoactive factors may have a direct effect on physiological follicular deviation, favoring dominance of the selected follicle.