Bioscientifica Proceedings

Summary. To examine the regulation of P-450_scc expression at the molecular level, a transfection protocol specific for bovine luteal cell cultures was developed. Among several commonly used transfection methods, electroporation yielded highest transfection efficiencies. Transfection of primary cultures of bovine luteal cells with chimaeric DNA constructs containing increasing deletions of the 5'-flanking region of P-450_scc fused to the chloramphenicol acetyl transferase (CAT) reporter gene allowed two conclusions. Firstly, sequences of the P-450_scc 5'-flanking region were capable of conferring basal expression and cAMP responsiveness to the CAT reporter gene and secondly, the region — 186 to —100 bp appears to be required for these two types of regulation of gene expression. On the other hand, 5'-flanking regions of P-450_17α were not capable of conferring any regulation of gene expression to the CAT reporter gene in these cells. Thus, the physiologically observed regulation of the endogenous cytochromes P-450_scc and P-450_17α is closely reflected by CAT reporter gene expression. These experiments will allow investigation of the molecular mechanisms underlying the regulation of the genes encoding steroidogenic enzymes throughout the ovarian cycle.

Keywords: gene expression; corpus luteum, cytochrome P-450; transfection; luteal cell cultures

© 1991 Journals of Reproduction & Fertility Ltd