Immunohistochemistry and in situ hybridization of P-45017 alpha (17 alpha-hydroxylase/17,20-lyase).
Cytochrome P-45017 alpha catalyzes both 17 alpha-hydroxylation and 17,20-side-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 P-45017 alpha in swine testis, ovary, and adrenal, we undertook the simultaneous detection of P-45017 alpha mRNA and protein by combining immunohistochemistry with in situ hybridization. In situ hybridization was performed on 4% paraformaldehyde-fixed, paraffin-embedded sections by employing either a 39-base oligomer or a cDNA insert (1.7 KB) of porcine testis P-45017 alpha as DNA probe. Immunohistochemical study was performed by employing anti-P-45017 alpha. Hybridization signals were obtained in Leydig cells of the testis, theca interna of the ovarian follicle, and zona fasciculata reticularis cells of the adrenal cortex. Oligonucleotide probing yielded lower background signal than the cDNA probe. No specific signals were obtained in seminiferous tubules of the testis, medulla, and zona glomerulosa of the adrenal, and in membrana granulosa 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 P-45017 alpha expression at both the protein and mRNA levels in swine adrenal glands and gonads. This approach of simultaneous immunohistochemistry and in situ hybridization analysis of steroidogenic enzymes can be applied in the future to tissues exhibiting abnormal steroid metabolism and should contribute to a better understanding of steroidogenesis.
Suzuki
T
T
Department of Pathology, Tohoku University School of Medicine, Sendai, Japan.
Sasano
H
H
Sawai
T
T
Mason
J I
JI
Nagura
H
H
D000311:Adrenal Glands / Q000201:enzymology; D000446:Aldehyde-Lyases / Q000032:analysis* / Q000235:genetics; D000818:Animals; D001483:Base Sequence; D003577:Cytochrome P-450 Enzyme System / Q000032:analysis* / Q000235:genetics; D005260:Female; D008297:Male; D008969:Molecular Sequence Data; D010053:Ovary / Q000201:enzymology; D012333:RNA, Messenger / Q000032:analysis; D013254:Steroid 17-alpha-Hydroxylase / Q000032:analysis* / Q000235:genetics; D013256:Steroids / Q000096:biosynthesis*; D013552:Swine; D013737:Testis / Q000201:enzymology
D012333:RNA, Messenger; D013256:Steroids; D003577:Cytochrome P-450 Enzyme System; D013254:Steroid 17-alpha-Hydroxylase; D000446:Aldehyde-Lyases