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Cholesterol side chain cleavage cytochrome P450 (P450SCC) and 17 alpha-hydroxylase cytochrome P450 (P450(17) alpha) are key enzymes involved in steroid hormone biosynthesis. The 5'-flanking regions of the corresponding bovine genes have recently been characterized in the adrenal-derived Y1 cell line and specific DNA sequences, required for basal and cAMP-dependent gene expression, were identified. In order to investigate the molecular mechanisms controlling P450SCC and P450(17) alpha gene expression throughout the ovarian cycle, we devised an electroporation protocol to transfect bovine luteal cells in primary culture with specific chimeric DNA constructs. Transfection of such cells with reporter gene constructs containing 186 base pairs (bp) or more of the CYP11A (P450SCC) 5'-regulatory region resulted in cAMP-responsive reporter gene expression. Reporter gene constructs containing 101 bp or less of this regulatory region were expressed neither in the presence nor in the absence of forskolin. Thus, expression of the CYP11A gene in bovine luteal cells in primary culture appears to be controlled by a cis-acting element located between -186 to -101 bp. Transfection of bovine luteal cells in primary culture with reporter gene constructs containing increasing deletions of the bovine CYP17 (P450(17) alpha) 5'-regulatory region clearly demonstrated that none of the sequences were capable of promoting significant reporter gene expression, neither in the presence nor absence of forskolin. Since no 17 alpha-hydroxylase activity and no specific mRNA encoding P450(17) alpha have been detected in the bovine corpus luteum, the failure of expression of the P450(17) alpha reporter gene constructs mimics that of the endogenous gene. These results demonstrate that the regulation of CYP11A gene expression in bovine luteal cells is carried out by the same cis-acting element (-183/-101) which serves this role in the adrenal Y1 cell line. On the other hand, expression of the CYP17 gene in bovine luteal cells appears to be completely repressed, due to the absence of specific positive transcription factor(s) or the presence of negative regulatory factor(s). Thus, transfection experiments using bovine luteal cells in primary culture represent a first step towards the elucidation of molecular mechanisms underlying the regulation of cytochrome P450 gene expression throughout the ovarian cycle.