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

The biosynthesis of steroid hormones in endocrine steroid-secreting glands results from a series of successive steps involving both cytochrome P450 en zymes, which are mixed-function oxidases, and steroid dehydrogenases. So fa r, the subcellular distribution of steroidogenic enzymes has been mostly st udied following subcellular fractionation, performed in placenta and adrena l cortex. In order to deter-mine in situ the intracellular distribution of some steroidogenic enzymes, we have investigated the ultrastructural locali zation of the three key enzymes: P450 side chain cleavage (scc) which conve rts cholesterol to pregnenolone; 3 beta -hydroxysteroid dehydrogenase (3 be ta -HSD) which catalyzes the conversion of 3 beta -hydroxy-5-ene steroids t o 3-oxo-4-ene steroids (progesterone and androstenedione); and P450(c17) wh ich is responsible for the transformation of C-21 into C-19 steroids (dehyd roepiandrosterone and androstenedione). Immunogold labeling was used to loc alize the enzymes in rat adrenal cortex and gonads. The tissues were fixed in 1% glutaraldehyde and 3% paraformaldehyde and included in LR gold resin. In the adrenal cortex, both P450(scc) and 3 beta -HSD immunoreactivities w ere detected in the reticular, fascicular and glomerular zones. P450(scc) w as exclusively found in large mitochondria. In contrast, 3 beta -HSD antige nic sites were mostly observed in the endoplasmic reticulum (ER) with some gold particles overlying crista and outer membranes of the mitochondria. P4 50(c17) could not be detected in adrenocortical cells. In the testis, the t hree enzymes were only found in Leydig cells. Immunolabeling for P450(scc) and 3 beta -HSD was restricted to mitochondria, while P450(c17) immunoreact ivity was exclusively observed in ER. In the ovary, P450(scc) and 3 beta -H SD immunoreactivities were found in granulosa, theca interna and corpus lut eum cells. The subcellular localization of the two enzymes was very similar to that observed in adrenocortical cells. P450(c17) could also be detected in theca interna cells of large developing and mature follicles. As observ ed in Levdig cells, P450(c17) immunolabeling could only be found in the ER. These results indicate that in different endocrine steroid-secreting cells P450(scc), 3 beta -HSD and P450(c17) have the same association with cytopl asmic organelles (with the exception of 3 beta -HSD in Leydig cells), sugge sting similar intracellular pathways for biosynthesis of steroid hormones.