CAMP-DEPENDENT AND TISSUE-SPECIFIC EXPRESSION OF GENES ENCODING STEROIDOGENIC ENZYMES IN BOVINE LUTEAL AND GRANULOSA-CELLS IN PRIMARY CULTURE

Steroidogenic enzymes are differentially expressed throughout the ovar ian cycle. The complex pattern of cell-specific up- and down-regulatio n accounts, at least in part, for the cyclic production of estrogens, androgens and progesterone. The gonadotropins follicle-stimulating hor mone and luteinizing hormone are the main regulators of ovarian steroi d hormone production and act primarily via the cAMP second-messenger s ystem. 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 elem ent (CRE), as identified in the somatostatin gene. In addition, we wer e interested to determine whether or not the information for tissue-sp ecific expression is contained by these sequences. Using transient tra nsfection 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 a nd granulosa cells. Treatment of transfected luteal cells with forskol in markedly increase the expression of all but the CYP17-specific repo rter gene constructs. A similar pattern of forskolin responsiveness wa s observed when these reporter gene constructs were transfected in bov ine granulosa cells in primary culture. Furthermore, when a reporter g ene construct containing the classical CRE genomic was transfected in bovine luteal cells, its expression was also highly stimulated upon tr eatment with forskolin. Thus, the classical cAMP/CRE system appears to be functional in these cells. Northern blot analysis of primary cultu res of bovine luteal and granulosa cells revealed that bCYP17 and bCYP 21B are not expressed in control and forskolin-treated cultures. In or der to explain why the CYP21B-specific reporter gene construct is high ly expressed in forskolin-treated cells, whereas the endogenous gene i s not, we examined the regulation of expression of a set of chimeric D NA constructs containing increasing deletions of the hCYP21B 5' flanki ng region. These experiments indicated that the control of tissue-spec ific expression of this gene may lie within the region -612 to -250 bp . In conclusion, the data presented in this study suggest that cAMP-de pendent 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 (C YP21B) the cAMP-responsive sequences.