Immunocytochemical studies of aromatase in early and full-term human placental tissues: comparison with biochemical assays.
An immunocytochemical method for visualizing the aromatase P450 enzyme with a specific monoclonal antibody has been developed for use with unfixed, frozen tissue sections. We compared both monoclonal and polyclonal aromatase-specific antibodies and found that placental aromatase was consistently and exclusively located in the syncytiotrophoblast layer of chorionic villi. The monoclonal antibody had the highest affinity, with negligible associated background stain. Fixation was found to impair stain reaction. Examination of first trimester and term placentae revealed identical immunostaining patterns of similar intensity in 9 of 10 samples. The immunostain reactions of first trimester and term placentae were compared with their respective microsomal aromatase activity, determined simultaneously by both indirect radiometric tritiated water (3H2O) assay, and direct product isolation by HPLC, using [1, 2, 6, 7(-3)H] androstenedione as substrate. The two assays were found to be comparable for enzyme activity estimates of term placental specimens. However, when first trimester specimens were analyzed, the direct-product measurements were significantly larger than the corresponding 3H2O assay results. Nonetheless, biochemical aromatase activity was found to correlate positively with immunostain reaction. Although 17 beta-hydroxysteroid dehydrogenase activity was not directly measured, differences in the estradiol:estrone product ratio (2.49 +/- 0.68 first trimester vs. 0.89 +/- 0.15 term) suggest differential control of this enzyme at the two stages of pregnancy. One first trimester specimen with an atypical, patchy immunostain distribution also had extremely low aromatase activity. The results indicate that both antibodies recognize functional aromatase enzyme and suggest that immunocytochemical detection is a sensitive, qualitative technique for investigating this important steroidogenic enzyme.
Inkster
S E
SE
Department of Pharmacology and Experimental Therapeutics, School of Medicine, University of Maryland, Baltimore 21201.
Brodie
A M
AM
D000911:Antibodies, Monoclonal / Q000276:immunology; D001141:Aromatase / Q000032:analysis / Q000276:immunology / Q000378:metabolism*; D005260:Female; D006801:Humans; D007124:Immunoenzyme Techniques; D007150:Immunohistochemistry; D010920:Placenta / Q000032:analysis / Q000166:cytology / Q000201:enzymology*; D011247:Pregnancy; D011261:Pregnancy Trimester, First / Q000378:metabolism*; D011263:Pregnancy Trimester, Third / Q000378:metabolism*
D000911:Antibodies, Monoclonal; D001141:Aromatase