CAMP-dependent and tissue-specific expression of genes encoding steroidogenic enzymes in bovine luteal and granulosa cells in primary culture

Abstract

Steroidogenic enzymes are differentially expressed throughout the ovarian cycle. The complex pattern of cell-specific up- and down-regulation accounts, at least in part, for the cyclic production of estrogens, androgens and progesterone. The gonadotropins follicle-stimulating hormone and luteinizing hormone are the main regulators of ovarian steroid hormone production and act primarily via the cAMP second-messenger system. Previous studies have identified cAMP-responsive sequences (CRS) in a number of genes encoding steroidogenic enzymes. In the present study we attempted to compare the cAMP responsiveness of some of these sequences with each other and with the classical cAMP-responsive element (CRE), as identified in the somatostatin gene. In addition, we were interested to determine whether or not the information for tissue-specific expression is contained by these sequences. Using transient transfection of reporter gene constructs, comprizing the CRS of bCYP11A, bCYP17, hCYP21B and bovine adrenodoxin, we investigated cAMP-dependent and tissue-specific expression in primary cultures of bovine luteal and granulosa cells. Treatment of transfected luteal cells with forskolin markedly increased the expression of all but the CYP17-specific reporter gene constructs. A similar pattern of forskolin responsiveness was observed when these reporter gene constructs were transfected in bovine granulosa cells in primary culture. Furthermore, when a reporter gene construct containing the classical CRE genomic was transfected in bovine luteal cells, its expression was also highly stimulated upon treatment with forskolin. Thus, the classical cAMP/CRE system appears to be functional in these cells. Northern blot analysis of primary cultures of bovine luteal and granulosa cells revealed that bCYP21B and bCYP21B are not expressed in control and forskolin-treated cultures. In order to explain why the CYP21B-specific reporter gene construct is highly expressed in forskolin-treated cells, whereas the endogenous gene is not, we examined the regulation of expression of a set of chimeric DNA constructs containing increasing deletions of the hCYP21B 5' flanking region. These experiments indicated that the control of tissue-specific expression of this gene may lie within the region −612 to −250 bp. In conclusion, the data presented in this study suggest that cAMP-dependent regulation of genes encoding steroidogenic enzymes is mediated by different molecular mechanisms, distinct from the classical cAMP/CRE system. In addition, we provide evidence that the information for tissue-specific expression may be located within (CYP17) or outside (CYP21B) the cAMP-responsive sequences.