20 beta-hydroxysteroid dehydrogenase activity in nonflagellated germ cells of rainbow trout testis

Nonflagellated germ cells were isolated from rainbow trout testis to determine their ability to synthesize 17,20 beta-dihydroxy-4-pregnen-3-one (17,20 beta OHP), a progestin involved in the control of the release of sperm. Germ cells were obtained by enzymatic dissociation (collagenase; 3 mg . ml(-1), 4.5 h, 12 degrees C) from testes that were immature and at the beginning of spermatogenesis. Somatic cells were eliminated by adhesion to the culture plates. Dose-related amounts of 17,20 beta OHP were measured by RIA in culture media of germ cells incubated with increasing dosages of 17-hydroxyprogesterone (17OHP; 0.05-10 mu g . ml(-1)) for 20 h at 12 degrees C. Furthermore, H-3-17,20 beta OHP was identified by chromatography and co-crystallization with a reference in incubating cells provided by H-3-17OHP (2.5 and 4 h, 12 degrees C). Other metabolites were detected but not identified. 11-Ketotestosterone (11KT) was either nondetectable by RIA in control cultures or, when detected, was found at very low levels. In no case was 11KT stimulated by addition of 17OHP or gonadotropin II (GtH II; 400 ng . ml(-1)); this indicated the absence of contamination by Leydig cells. Thus, to our knowledge, this report demonstrates steroidogenic activities in nonflagellated germ cells of fish testis for the first time. 20 beta-Hydroxysteroid dehydrogenase (20ssHSD) activity was identified, showing that germ cells are able to synthesize 17,20 beta OHP at an early stage in rainbow trout testis.