Immunohistochemistry and in situ hybridization of P-45017 alpha (17 alpha-hydroxylase/17,20-lyase).

Abstract

Cytochrome P45017~ catalyzes both 17a-hydro~~btion and 17,204de-chain cleavage in steroidogenesis and lies at a key branch point in the pathways of steroid hormone biosynthesis. To obtain information on the precise localization of P45017a in swine testis, ovary, and adrenal, we undertook the simultaneous detection Of P45017a mRNA and prottin by combining immunohistochemistry with in situ hybridization. In situ hybridization was performed on 4% paraformaldehyde-bed, paraf6n-embedded sections by employing either a 39-base oligomer or a cDNA insert (1.7 -) of porcine testis U5017a as DNA probe. Immunohistochemi d study was performed by employing anti-P45017a. Hybridization signals were obtained in Leydig cells of the testis, theca intern? of the Cnvian follicle, and zona fasciculata reticularis cells of the adrenal cortex. Oligonucleotide probing yielded lower background signal than the DNA probe. No specific signals were obtained in seminiferous tubules of the testis, medulla, and zona glomerulosa of the adrenal, and in membrana granulasa and interstitial cells of the ovary. Hybridization signals were obtained in the cells where immunoreactivity of the enzyme was observed by immunohistochemistry, except for some Leydig cells of the testis and theca interna cells of the ovary in which only immunoreactivity but not hybridization signal was observed. The present study provided detailed information about the precise cellular localization of P45017a expression at both the protein and mRNA levels in swine adrenal glands and gonads. IntroductionIn the steroid biosynthetic pathways of testes, ovaries, and adrenals, 17a-hydroxylase catalyzes the conversion of pregnenolone to 17-hydroxypregnenolone and that of progesterone to 17-hydroxyprogesterone. 17-Hydroxypregnenolone is then converted by 17,20-lyase to dehydroepiandrosterone. Although 17a-hydroxylase and 17,20-lyase activities are two distinct biochemical reactions, studies in guinea pig (1) and in swine adrenals (2) and testis (3) have shown that both activities reside in a single specific microsomal cytochrome P-450 (P-45017a). Therefore, P-45017~ lies at a very important branch point in the entire system of steroid hormone biosynthesis, i.e., bemen glucocorticoid and mineralocorticoid biosynthesis and between glucocorticoid and androgen biosynthesis. P-45017a is expressed in the testis, ovary, and adrenal gland, but it is also important to know its precise localization for a better understanding of steroid metabolism. For this purpose, techniques directed towards illustrating the enzymes specifically involved in steroidogenesis, especially immunolocalization ofsteroidogenic enzymes, are at present considered as a better method than conventional morphological examination, including electron microscopy and immunohistochemistry of steroid hormones themselves (4-10). Recently, cDNA encoding swine testis P-45017a has been characterized and oligonucleotide probes suitable for in situ hybridization were synthesized (11). In this study, we have further pursued localization...

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 the testis, P450 c17 immunoreactivity was only detected in the Leydig cells, confirming previous light microscope studies performed in the rat and guinea pig (LeGoascogne et al 1991, Suzuki et al 1992. Strong immunolabeling was only observed in the ER.…”

“…At the light microscopic level, immunoreactive P450 c17 was only found in theca interna of rat ovaries (LeGoascogne et al 1991). In the bovine ovary, both theca interna and interstitial cells were shown to express P450 c17 mRNA and the immunoreactive protein (Suzuki et al 1992). In the human ovary, immunoreactive P450 c17 was confined to theca interna cells and a few luteinized theca cells in corpora lutea (Sasano et al 1989).…”

Immunolocalization and Biochemical Determination of Cytochrome P450C17 in Adrenals of Hamsters Treated with ACTH

“…In agreement with our data, Suzuki et al (1992) obtained hybridization signals in the ZF cells of swine adrenals by immunocytochemistry, employing an anti-P450C17, although such signals were also found in the ZR. The same authors also found co-localization of P450C17 protein and mRNA in the same tissue section.…”

17.ALPHA.-Hydroxylase Deficiency Accompanied by Adrenal Myelolipoma.

“…The zona glomerulosa is primarily controlled by angiotensin II to secrete aldosterone, while the zona fasciculata are controlled by ACTHto secrete cortisol, DOCand corticosterone (1 , 9). The mammalian adrenal zona glomerulosa does not express 17oc-hydroxylase cytochrome P450c17, but its expression is high in the zona fasciculata ( 10). Therefore the zona fasciculata may produce more DOCand corticosterone than aldosterone, which is produced in zona glomerulosa in the patients with 17oc-hydroxylase deficiency.…”