The effects of prepuberal gonadectomy on adrenal function were studied in both male and female rats. Steroid production was measured in vitro using either adrenal homogenates or slices. Adrenal tissue from castrated animals of either sex produced less steroid when determined by acid fluorescence or ultraviolet absorption. The effect was reversed after replacement with testosterone or estradiol. No difference due to castration was observed when steroid production was measured with blue tetrazolium. Corticosterone added in vitro to adrenal tissue from gonadectomized rats was converted to a fluorescence negative, ultraviolet negative, blue tetrazolium positive metabolite. This conversion was inhibited by gonadal hormone replacement in. vivo. The metabolite was identified by Rf both before and after acetylation on several paper and thin layer chromatographic systems and also by infrared spectroscopy as 3/3,5o:-tetrahydrocorticosterone (compound R). Two additional compounds tentatively identified by chromatography alone were 3a,5a-tetrahydrocorticosterone and 5a-dihydrocorticosterone. Further studies showed that the decreased production of steroids by adrenal homogenates observed after ovariectomy was abolished by preincubation of the tissue as slices. Such preincubation concomitantly reduced the conversion of added corticosterone to compound R. The reaction involves 2 enzymes, 5a-reductase and 3-hydroxysteroid dehydrogenase. Activity of the latter is not increased after castration. The results demonstrate that gonadectomy enhances the capacity of the adrenal gland to convert corticosterone to compound R and other tetrahydro metabolites. The data are consistent with the hypothesis that adrenal 5a-reductase activity is increased after castration and is inhibited by replacement with testosterone or estradiol. These effects may represent a physiologic mechanism for regulation of adrenal steroid secretion. {Endo-crinology 87: 1257{Endo-crinology 87: , 1970 /CONSIDERABLE evidence is available \jk to indicate that pituitary-adrenal function is influenced by the gonadal hormones (2). However, physiologic roles for estradiol and testosterone in the regulation of adrenal function have been difficult to define. The available data seem contradictory since both stimulatory and inhibitory effects have been described for both hormones. Much of the disagreement can be related to wide variations in experimental designs used in different laboratories. For example, dose is a critical factor. The effects of estradiol and testosterone appear to

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Adrenal corticosterone (Cpd B) secretion by the rat is decreased after gonadectomy in either sex and restored by replacement with estradiol or testosterone (1). This response to castration has been shown to be related to enhancement of the capacity of the adrenal gland to convert Cpd B to 3P,5atetrahydrocorticosterone (Cpd R) and other Sa-reduced metabolites (2, 3 ) . The process is likewise reversed by gonadal hormone replacement. The present study demonstrates that the previously observed increment in reduced steroid metabolite formation is explained by increased adrenal 5 a-steroid reductase activity. Some aspects of cofactor and substrate requirements are also presented.Materials and Methods. Male or female rats of the Sherman strain, obtained from Camm Research Institute, Wayne, New Jersey, were used in all experiments. The animads were maintained under standardized conditions of light (14 hr/24 hr) and temperature ( 2 2 3. 0.5") on a diet consisting of Purina Laboratory Chow and water ad libitum. Gonadectomy was performed at 24-28 days of age with no further manipulation (except removal of wound clips). The rats were killed by decapitation 8 weeks later, and adrenal studies were performed. Final body weight exceeded 190 g.Whole adrenal homogenates were prepared in 0.154 M KC1 using glass homogenizers with Teflon pestles. Unless indicated otherwise, they were incubated for 60 rnin at 37" under 95% O2 + 5 % C02 in a reaction mixture containing KCl, 0.154 M ; NaHC03, 20 X M ; Ca2+ 1.25 X 10-3M; glucose-6-phosphate 3.3 X low3 M ; NADP, NADH, 10-3 M ; and 5 to 20 mg of adrenal tissue/ml. Corticosterone or other steroid substrates, 10 or 15 pg in 3 pl of ethanol, was added as indicated, and final volume was 0.5 ml. Incubates were extracted with chloroform and steroid production was measured in aliquots by fluorescence (4) or by ultraviolet (UV) absorption a t 240 mp in methanol. Previous studies showed that the two methods give equivalent results (3). Adrenal 5 a-reductase activity was also assayed in cell fractions according to the method of Tomkins ( 5 ) . Although the technique was described for liver, preliminary studies established that a pH optimum of 6.4 also applied to adrenal tissue. Modifications were trivial and included the substitution of corticosterone as substrate and a final incubation volume of 0.25 ml. Adrenal cell fractions were prepared by homogenization in 0.25 M sucrose supplemented with 0.05 M Tris-maleate buffer, pH 6.4; and 0.02 M nicotinamide. After initial centrifugation for 10 min at 700g, the supernatant was respun for 15 min a t 5OOOg. The sediment (mitochondria1 fraction) was resuspended and washed x 2 with buffer before incubation. The original 5000g supernatant was then centrifuged at 15,OOOg for 15 min. The resulting supernatant was then spun at 105,OOOg for 30 min. The final supernatant (cytosol fraction) was used unchanged. The sediment (microsomal fraction) was resuspended and centrifuged again at 105,OOOg for 30 rnin before use. In some experiments, corticosterone-4-14C, 0.02...