Stage‐related production of 21‐hydroxylated progestins by the dogfish (Squalus acanthias) testis

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Abstract
Using shark (Squalus acanthias) testicular microsomes and [3H]progesterone ([3H]P) and [3H]17α-hydroxyprogesterone ([3H]17α-P) and [3H]17α-hydroxyprogesterone ([3H]17α-P) as substrates, two major products of 21-hydroxylase action were identified; respectively, 21-hydroxy-4-pregnen-3-one (11-deoxycorticosterone, DOC) and 17α,21-dihydroxy-4-pregnen-3-one (11-deoxycortisol, S). Additional products of 17α-hydroxylase/C-17,20-lyase action were detected: 17α-hydroxyprogesterone (17αP), testosterone (T), and androstene-dione (AE). When microsomes derived from tissues in premeiotic (PrM), meiotic (M), and postmeiotic (PoM) stages of spermatogenesis were compared, maturation-related increases were observed with both 21-hydroxylase (3- to 20-fold) and 17 α-hydroxylase (2- to 6-fold). With [3H]P as tracer, the half-maximal substrate concentrations (Km = 0.2–1.0 μM) and maximal reaction velocities (Vmax = 9–25 pmol/mg protein/min) were similar for both enzymes when assayed in the same preparation, suggesting they compete for available substrate. Also, the presence of 1- or 10-fold molar excess radioinert DOC reduced conversion of [3H]P to 17α-hydroxylated products. [3H]DOC itself was a substrate of 17α-hydroxylation but not C-17,20-lyase action. Expression of 21-hydroxylase and 17α-hydroxylase activities in cultured spermatocysts (intact germ cell/Sertoli cell units) was confirmed by detection of immunoreactive 17α,20β,21-trihydroxy-4-pregnen-3-one (20β-S), S, 17α,20β-dihydroxy-4-pregnen-3-one (17α,20β-P), DOC, P and T in spent media. 20β-S and S secretion increased and 17α,20β-P decreased progressively with stage of maturation, but DOC was similar in all stages. P secretion was maximal and T lowest in M-stage spermatocysts, but when DOC (0.1 μM) was added to PrM or PoM spermatocysts, T output decreased. Taken together, the data suggest that 21-hydroxylating pathways, via direct and indirect mechanisms, affect accumulation of bioactive steroids (P and T) differentially by stage of spermatogenic development. Whether 21-hydroxylated progestins produced in shark testis have paracrine or endocrine actions in their own right remains to be investigated.