Tetradecanoyl phorbol acetate suppresses follicle-stimulating hormone-induced synthesis of the cholesterol side-chain cleavage enzyme complex in rat ovarian granulosa cells.

W H Trzeciak,T Duda,M R Waterman,E R Simpson

doi:10.1016/s0021-9258(18)48166-5

Abstract

The effect of tetradecanoylphorbol acetate (TPA) on follicle-stimulating hormone (FSH)-induced synthesis of the cholesterol side-chain cleavage (SCC) enzyme complex was studied in rat ovarian granulosa cells cultured for 48 h in serum-free medium. Cell proteins were radiolabeled with [35S]methionine, followed by immunoprecipitation of cholesterol side-chain cleavage cytochrome P-450 (P-450SCC) as well as the iron-sulfur protein adrenodoxin. Polyacrylamide gel electrophoresis and fluorography of the immunoprecipitates showed that TPA, when added in combination with FSH (50 ng/ml) or dibutyryl cAMP (Bt2cAMP; 1 mM), suppressed the stimulatory effects of these compounds on the synthesis of the SCC components in a concentration-dependent fashion. The effect of TPA was accompanied by decreased progesterone formation and decreased cAMP accumulation. The structural analog of TPA, phorbol-4 alpha-didecanoate, which does not activate protein kinase C (Ca2+/phospholipid-dependent enzyme), had no effect on the FSH- or Bt2cAMP-stimulated synthesis of SCC and progesterone or on cAMP formation. In addition to inhibiting the synthesis of these proteins, TPA greatly reduced the FSH- and Bt2cAMP-induced increase in levels of mRNA encoding the precursor form of P-450SCC. It is concluded that the effect of the phorbol ester TPA to inhibit FSH-stimulated progesterone formation in cultured ovarian granulosa cells of the rat involves decreased synthesis of the components of the SCC enzyme complex due to reduced levels of mRNA encoding the precursor forms of these proteins. The results are indicative that TPA not only inhibits FSH-mediated stimulation of cAMP formation but also may block cAMP-mediated induction of SCC synthesis. It is postulated that the effects of TPA may reflect the physiological role of protein kinase C in the regulation of ovarian steroidogenesis.

Highlights

The effectof tetradecanoylphorbol acetate (TPAo) n protein kinase C, reduces the requirement of the enzyme for follicle-stimulating hormone (FSH)-induced synthesis of the cholesterol side-chain cleavage (SCC) enzyme complex was studiedin rat ovarian granulosa cells cultured for 48 h in serum-freemedium
With FSH (50 ng/ml) or dibutyryl cAMP (BtzcAMP; Protein kinaseC is present in steroid hormone-synthesizing lmM), suppressed the stimulatory effectosf these com- tissues, including adrenal cortex, testis, and ovary pounds on the synthesis of the SCC components in a [12], and acute and chronic actoifopnhsorbol esters on steroid concentration-dependent fashion
Effect of TPA on FSH-stimulated CAMPand Progesterone Formation-Treatment of cultured granulosa cells with TPA alone for 48 h caused no significant change in the accumulapregnenolone, and 0.3% with 20a-dihydroprogesterone

Summary

Introduction

The effectof tetradecanoylphorbol acetate (TPAo) n protein kinase C, reduces the requirement of the enzyme for follicle-stimulating hormone (FSH)-induced synthesis of the cholesterol side-chain cleavage (SCC) enzyme complex was studiedin rat ovarian granulosa cells cultured for 48 h in serum-freemedium. It is concluded that the hasbeenpostulatedthattheinhibitory effect of TPA on effect of the phorbol ester TPA to inhibit FSH-stimu- gonadotropin-stimulatedsteroidhormoneformationmight lated progesterone formation in cultured ovarian grraens-ult from inhibitionof synthesis of the enzymes involvedin ulosa cells of the rat involves decreased synthesis of the components of the SCC enzyme complex due to reduced levelsof mRNA encoding the precursor forms of these proteins.

Results

Conclusion