Angiotensin II Interferes with Steroidogenesis in Porcine Granulosa Cells1

The elements for the synthesis and activity of the vasopressor angiotensin II (AII) are present in the mammalian ovary. In the present investigation, the effects of AII were determined on three parameters of steroidogenic function in porcine granulosa cells in vitro: the accumulation of progesterone, the cellular content of the enzyme 3β-hydroxysteroid dehydrogenase Δ5−4 isomerase (3β-HSD), and the accumulation of mRNA for 3β-HSD. Cells were incubated with LH (200 ng/ml) in the presence or absence of AII (10−7 M) or phorbol 12-myristate 13-acetate (PMA, 10−7 M); doses of AII from 10−10 to 10−6 M in the presence or absence of LH; the AII reactor antagonist saralasin (10−6 M) in the presence of AII or in combination of AII and LH; and AII in the presence or absence of (Bu)2 cAMP. The results demonstrate that LH increased progesterone, 3β-HSD message, and 3β-HSD content. Both PMA and AII interfered with the LH-induced progesterone accumulation, reducing the response by 50% or more. All also abrogated the LH-induced increases in 3β-HSD mRNA and 3β-HSD enzyme content in porcine granulosa cells. The AII inhibition was dose-dependent. The AII receptor antagonist saralasin blocked the inhibitory effects of AII on LH-induced steroidogenic events. AII interfered with the (Bu)2 cAMP induction of steroidogenesis and 3β-HSD mRNA and enzyme accumulation when (Bu)2 cAMP was present at a concentration of 30 μM. Pretreatment of cell cultures with PMA for 24 h to downregulate protein kinase C (PKC) reduced basal and LH-stimulated progesterone as well as 3β-HSD and mRNA accumulation. Comparison across the PMA down-regulated cultures demonstrated that LH mildly stimulated progesterone accumulation, but increased 3β-HSD mRNA accumulation 4-fold relative to the PMA down-regulated control. All had no effect on LH-stimulated 33βHSD mRNA accumulation, further suggesting that All acts through the PKC pathway. The results indicate that the mechanism by which All inhibits steroidogenesis includes inhibition of the transcription of the 3β-HSD gene or alteration of 3β-HSD mRNA stability. All functions through its receptor to express this inhibition, which includes some interference with cellular function beyond the generation of cAMR All may be a paracrine agent associated with the limitation of follicular development in the pig ovary.