Preparation of Antiserum to Rat Cytochrome P-450 Cholesterol Side Chain Cleavage, and Its Use for Ultrastructural Localization of the Immunoreactive Enzyme by Protein A-Gold Technique*

Abstract

Rabbit antiserum to rat cytochrome P-450 cholesterol side chain cleavage (P-450scc) was produced without a previous biochemical purification of the enzyme. Instead, for immunization we used a single protein band of mol wt 53,000, which was isolated from sodium dodecyl sulfate polyacrylamide gel electrophoresis of rat steroidogenic mitochondrial membranes. The resulting antiserum cross-reacted in a protein-blotting test with affinity purified and biologically active bovine adrenocortical P-450scc enzyme. The antiserum to the rat P-450scc also substantially blocked the conversion of cholesterol to pregnenolone in sonicated steroidogenic mitochondria, suggesting a successful cross-reactivity with the native form of the enzyme, despite the fact that the immunizing antigen was sodium dodecyl sulfate-denatured protein. The antiserum was applied for ultrastructural immunocytochemical visualization of the P-450scc in thin sections of adrenal cortex and immature ovary. Immunoreactive enzyme was identified by the protein-A-gold technique which showed that the gold particles concentrated exclusively in the steroidogenic mitochondria of adrenal zona glomerulosa and fasciculata cells. In the immature ovary, the only zone which was heavily stained with colloidal gold was the population of the interstitial cells. Part of the theca cell population contained P-450scc before PMSG treatment. The granulosa cells were devoided of the enzyme in any follicles before the preovulatory stage. The high resolution of the pAg technique allowed to visualize the localization of the P-450scc antigen in the matrix side of the inner mitochondrial membranes. Moreover, a clear coupling could be demonstrated between the morphological and functional maturation of the steroidogenic mitochondrion in the ovary: from a few lamella cristae devoid of P-450scc in the unstimulated granulosa mitochondria, to numerous tubulovesicular inner membranes, heavily loaded with the enzyme, in the mitochondria of the interstitial cells.

Immunoelectron microscopic localization of three key steroidogenic enzymes (cytochrome P450(scc), 3 beta-hydroxysteroid dehydrogenase and cytochrome P450(c17)) in rat adrenal cortex and gonads

“…In all the reactive cell types, immunolabeling was restricted to mitochondria, with gold particles overlying vesicular structures. These results are in agreement with a previous immunoelectron microscopic study indicating that P450 scc immunoactivity was associated with the matrix side of the inner mitochondrial membranes in rat adrenocortical cells as well in granulosa, theca interna and interstitial cells in the ovary (Farkash et al 1986). They are also in agreement with previous subcellular fractionation studies indicating that in rat and bovine adrenal glands P450 scc activity was associated with mitochondrial fractions (Hanukoglu et al 1981, Kramer et al 1984, Cherradi et al 1994.…”

Parallel early development of zebrafish interrenal glands and pronephros:differential control bywt1andff1b

“…Higher magnification shows that these mitochondria contain tubulo-vesicular cristae (Fig. 3E), which are typical for cells engaged in active steroidogenesis (Farkash et al, 1986). It indicates that these cells have already acquired steroidogenic potential.…”

Immunolocalization of aromatase, 17 -hydroxylase and side-chain-cleavage cytochromes P-450 in the human ovary

“…Farkash et al (1986) recently demonstrated by ultrastructural immunohistochemistry of P450scc in rat ovary that the transition of lamelliform to tubular mitochondrial cristae was associ¬ ated with the expression of P-450scc. The prominent immunoreactivity noted in the present study of P-450scc, which is the mitochondrial cytochrome P-450 and is necessary for progesterone synthesis, in luteinized granulosa cells can also reflect increased progesterone production after ovulation.…”