Hiroyuki Oshima

Studies of the human testis: IV. Testicular 20α-hydroxysteroid dehydrogenase and steroid 17α-hydroxylase

Abstract Homogenates of frozen human testicular tissues were fractionated into soluble (105000 × g supernatant) and microsomal fractions 10000 × g-105000 × g precipitate). Soluble fraction contained NADPH-dependent 20α-hydroxysteroid dehydrogenase which catalyzed reduction of the 20-ketone group of progesterone and 17α-hydroxy progesterone as well as pregnenolone. The Michaelis constants (Km) were 2.0 · 10−6 M for progesterone 5.0 · 10−5 M for 17α-hydroxy progesterone and 7.0 · 10−6 M for pregnenolone. Microsomal fraction contained NADPH-dependent steroid 17α-hydroxylase (steroid, NADPH: oxygen oxidoreductase, 17α-hydroxylating, EC 1.14.99.9) which catalyzed 17α-hydroxylation of progesterone and pregnenolone. Km values were 2.3 · 10−6 M for progesterone and 2.5 · 10−6 M for pregnenolone. Both progesterone and pregnenolone served as competitive substrates for the enzyme. The inhibitor constants (Ki) were 8.0 · 10−7 M for pregnenolone and 3.2 · 10−6 M for progesterone. 20α-Dihydropregnenolone as well as 20α-dihydroprogesterone inhibited activity of human testicular steroid 17α-hydroxylase. 17α,20α-Dihydroxy-4-pregnen-3-one at the same concentration, however, showed no effect. 20α-Dihydropregnenolone functioned as a competitive inhibitor of steroid 17α-hydroxylase for either pregnenolone or progesterone. Ki values were 1.0 · 10−6 M for 17α-hydroxylation of pregnenolone and 1.5 · 10−6 M for progesterone. Since formation of these 20-ketone reduced steroids was mediated by 20α-hydroxysteroid dehydrogenase, it is postulated that the role of 20α-hydroxysteroid dehydrogenase in regulating androgen formation in the human testis is by competing with steroid 17α-hydroxylase to utilize pregnenolone and progesterone and by inhibiting steroid 17α-hydroxylase by its reaction products, 20α-dihydropregnenolone and 20α-dihydroprogesterone.

Studies of the human testis. XVI. Evaluation of multiple indexes of testicular function in relation to advanced age, idiopathic oligospermia, or varicocele *

Testicular function was evaluated by morphologic and biochemical methods in 11 infertile men with idiopathic oligospermia, 12 men with varicocele, and 14 elderly men. The results indicate the presence of two separate subgroups with idiopathic oligospermia with distinct endocrine and morphologic parameters and suggest the involvement of different pathogenetic factors for testicular disorders among these four groups. Biochemical indexes provide further confirmation of the validity of the Leydig cell cluster index (number of Leydig cell clusters per tubular section) as a useful evaluation guide. The results support the importance of combined studies of circulating hormones and testicular histologic features in providing insight into spermatogenic disorders. Recognition of etiologic diversity is an essential basis for further efforts to delineate and apply current or new hormonal techniques for therapy.

Studies of the human testis. XX—effect of NADH on 17α-hydroxypregnene C17–20 lyase activity

A study of the cofactor requirements of C17-20 lyase was carried out using human testis tissue obtained at the time of orchiectomy for treatment of prostatic carcinoma. Washed microsomal fractions were prepared from frozen human testes using a KCl containing buffer. The preparation revealed dose-dependent activity of C17-20 lyase in the presence of either NADPH or commercial or purified NADH. The Km of NADH for the enzyme was of the order of 10(-3) M and the Km of NADPH was determined as 1.6 X 10(-5) M. NADH also provided synergistic enhancement of NADPH-mediated lyase activity, and decreased the Km of NADPH for the lyase but did not change the Vmax of NADPH-mediated lyase activity. Carbon monoxide inhibited both NADH and NADPH-mediated lyase activities indicating that both activities are catalyzed by cytochrome P-450. Cations including Ca2+, Mg2+ and Mn2+ were found to inhibit the NADPH-mediated lyase activity but enhanced the lyase activity in the presence of NADH. The results indicate both the presence of NADH-mediated C17-20 lyase activity and the synergistic effect of NADH on NADPH-mediated lyase activity in the human testis.