Mechanisms Underlying Diethylstilbestrol And 17α-ethynylestradiol-induced Inhibition Of Spermatogenesis In Fish

To investigate the influences of EDC on hypothalamus-pituitary-gonadal axis, firstly, adult male zebrafish(Danio rerio) were exposed to DES(0.1, 1 and 10 μg/L) for 20 days. The histological results demonstrated that the DES exposure led to severe impacts on zebrafish spermatogenesis. To further elucidate the mechanisms underlying, c DNAs of vasa and dmc1 were cloned and their expression patterns were analyzed at the tissue and cellular levels. Results showed that vasa was expressed exclusively in spermatogonia and primary spermatocytes of testis, and dmc1 was specifically expressed in spermatocytes of testis. Semi-quantitative RT-PCR result revealed that DES dramatically suppressed the expressions of dmc1(while not vasa) in a doseand time-dependent manner. Moreover, the expression of dmrt1(the male sex determining gene) and P450 11β(the key enzyme responsible for 11-KT synthesis) were also suppressed by DES exposure, but cyp19a(the key enzyme responsible for estrogen synthesis) was up-regulated. Given the roles of these genes in meiosis and spermatogenesis, we speculated that DES might induce the male germ cell apoptosis and meiosis arrest in zebrafish by suppressing the expression of dmrt1, P450 11β and dmc1.Meanwhile, full length c DNA sequences of two subunits of gonadotropin hormone(FSHβ and LHβ) were cloned from the pituitary of Pelteobagrus fulvidraco by RACE. Subsequently, tissue and seasonal expression patterns were investigated. Further, the effects of 17α-Ethynylestradiol(EE2) on the expression regulations of three gonadotropin subunits in male Pelteobagrus fulvidraco were investigated. Results demonstrated that the FSHβ c DNA sequences was 528 bp in length including an open reading frame(ORF) of 399 bp, encoding a precursor protein of 132 amino acids(aa), while LHβ c DNA sequences was 870 bp in length including an ORF of 417 bp, encoding a precursor protein of 138 aa. Sequence analyses showed that, FSHβ contained a predicted signal peptide of 17 aa, and 2 N-glycosylation sites and 13 cysteine residues, while LHβ contained a predicted signal peptide of 18 aa, 1 N-glycosylation site and 12 cysteine residues. Phylogenetic analysis showed that FSHβ and LHβ shared close relationship with those of other fishes of Siluriformes. Tissue expression analysis suggested that all of the gonadotropin subunits were expressed specifically in pituitary. Seasonal expression pattern analysis showed that, in both sexes, Gt Hα and LHβ m RNA peaked in May and decreased gradually thereafter, and the expression of FSHβ also peaked in May in the female, while remained unchanged in male. Semi-quantitative RT-PCR results demonstrated that the expressions of three gonadotropin subunits were dramatically suppressed by EE2. Thus, we speculated that, EE2 probably induce inhibited spermatogenesis, maturation of sperm and spermiation by suppressing expression of FSHβ and LHβ in pituitary of male Pelteobagrus fulvidraco, and further impaired testicular development and reproduction.