In vivo and in vitro effects of prochloraz and nonylphenol ethoxylates on trout spermatogenesis

We investigated the effects of in vivo exposure to non-lethal concentration s of two chemicals commonly discharged into the aquatic environment, prochl oraz and nonylphenol diethoxylate (NP2EO - Igepal((R)) 210), on the develop ment of spermatogenesis in trout. The in vitro effects on basal and insulin -like growth factor-1 (IGF-I) stimulated DNA synthesis by early germ cells were also studied. In vivo, rainbow trout were exposed for 2 or 3 weeks to waterborne prochloraz (21 and 175 nmol/l) and/or NP2EO (68-970 nmol/l) rene wed continuously, or periodically. Only the highest concentrations of NP2EO (225-970 nmol/l) induced a significant increase in blood plasma vitellogen in in juvenile or maturing male trout. When prepubertal fish were exposed f or 15 days to prochloraz, the spermatogenetic process was significantly inh ibited as shown by the stage of gonadal development reached 3 weeks after e xposure. This effect was, to a great extent, reversible within 9 weeks post -exposure. When fish in the initial stage of spermatogenesis were exposed f or 21-27 days to 580 nmol/l NP2EO, a 20-40% reduction of the gonadosomatic index was observed 4.5 weeks post-exposure, and the spermatogenetic process was partly inhibited. In vitro, testicular cells obtained at different sta ges of spermatogenesis were cultured for 4.5 days in the presence or not of the tested molecules and with IGF-I or not. H-3-thymidine (H-3-Tdr) incorp oration was measured according to Loir (Mol. Reprod. Dev. 53 (1999) 424) an d I-125-IGF-I specific binding was determined according to Le Gac et al. (M ol. Reprod. Dev. 44 (1996) 35). Irrespective of the spermatogenetic stage, basal H-3-Tdr incorporation was decreased by prochloraz concentrations grea ter than or equal to 10 mu mol/l. The presence of IGF-I (10-100 ng/ml) stim ulated H-3-Tdr incorporation; this response to IGF-I began to decrease at 2 5-50 mu mol/l prochloraz. Tn parallel, a dose-dependent increase of IGF-I s pecific binding was induced by prochloraz 1-100 mu mol/l. Similarly, basal and IGF-I-stimulated H-3-Tdr incorporation was decreased by nonylphenol pol yethoxylate (NpnEO; starting at 10 mu mol/l), NP2EO and NP (30 mu mol/l); a dose-dependent increase of IGF-I specific binding was also induced by NP a nd NPnEO. While 1-100 nmol/l 17 beta -estradiol had no effect in our in vit ro system, Triton((R)) X-100 acted as NPnEO on H-3-Tdr incorporation. Besid e their known endocrine disrupting effects on sex steroid production or act ion, these lipophilic molecules could act on germ cells by disrupting cell membrane receptivity to peptide hormones like growth factors. (C) 2001 Else vier Science B.V. All rights reserved.