Controlled release of sex steroids through osmotic pump alters brain GnRH1 and catecholaminergic system dimorphically in the catfish, Clarias gariepinus.

The present study aimed to evaluate osmotic pump-mediated controlled release of estrogen in males and androgen in females to analyze the impact on gonadotropin-releasing hormone (GnRH1), catecholamines (CAs) and other associated genes in the catfish, Clarias gariepinus. During pre-spawning phase, catfish were separately implanted osmotic pumps loaded with 17ß-estradiol (E2) in males and 17α-methyltestosterone (MT) in females at a dose of 10 µg/100 µl or saline (100 µl) controls into both sexes to release for 21 days and all fishes were maintained as per the duration. Further, GnRH1 expression levels were analysed in the discrete regions of brain after E2 and MT treatments in male and female catfish, respectively using qPCR which revealed that GnRH1 expression was significantly higher in E2 treated male as compared to the control. On the other hand, GnRH1 expression was lower in MT treated female when compared to the control in the discrete regions of brain. In addition, certain brain and monoaminergic system related genes showed a differential response. Catfish GnRH1 could be localized in preoptic area-hypothalamus (POA-HYP) that correlated with the expression profile in the discrete regions of catfish brain. Serum levels of sex steroids in the treated male fish indicated that the treatment of E2 could maintain and impart feminization effect even in the presence of endogenous androgen during gonadal recrudescence while such an effect was not seen in females with androgen treatment. Measurement of CAs, L-3,4-dihydroxyphenylalanine, dopamine and norepinephrine levels in the male and female brain after the controlled release of E2 and MT, respectively confirmed the modulation of neurotransmitters in the E2treated male than MT treated female fish. These results collectively suggest the severity of estrogenic over androgenic compounds to alter reproductive status even at a minimal dose by targeting CAs and GnRH1 at the level of brain of catfish. This study provides insights into the reproductive toxicity of sex steroid analogues at the level of brain GnRH1 and CA-ergic system in addition to serum T, 11-KT and E2 levels during gonadal recrudescence, which is a crucial period of gametogenesis preceding spawning.

Production of recombinant Japanese eel gonadotropins by baculovirus in silkworm larvae.

Recombinant follicle-stimulating hormone (reFSH) and luteinizing hormone (reLH) of the Japanese eel Anguilla japonica were produced by baculovirus in silkworm Bombyx mori larvae. cDNAs encoding Japanese eel gonadotropin subunits (i.e., FSH beta, LH beta, and common alpha) were introduced into the baculovirus, which was infected into silkworm larvae after propagation of the recombinant virus in B. mori culture cells. A 100ml solution of pooled hemolymph from silkworm larvae containing reFSH or reLH were obtained from approximately 250 infected larvae. Ten milliliters of hemolymph were applied to Ni-affinity choromatography, and 5.6 and 3.5mg of partially purified reFSH and reLH were obtained, respectively. Using Western blot analysis concentrations of reFSH and reLH in the original hemolymph was estimated to be 2.2 and 1.1mg/ml, respectively. Biological activities of reFSH and reLH were assessed in vitro and in vivo. Purified reFSH and reLH induced eel oocyte maturation in vitro, and administration of hemolymph containing reFSH or reLH induced spermatogenesis in vivo in sexually immature Japanese eel. The present study indicates that a baculovirus-silkworm system could produce large amounts of biologically active recombinant fish gonadotropins for use in investigations in reproductive endocrinology and/or aquaculture of fish.