Development of real-time RT-PCR assays for eel gonadotropins and their application to the comparison of in vivo and in vitro effects of sex steroids

Gonadotropins, luteinizing hormone (LH) and follicle-stimulating hormone (FSH), are key factors in the brain-pituitary-gonad axis and understanding their regulation remains essential for future management of eel reproduction. In this regard, we developed quantitative real-time RT-PCR (qrtRT-PCR) assays for the expression of European eel LHb, FSHb and GPa subunits, using the Light Cycler system. The qrtRT-PCR was adapted to permit detection of the three gonadotropin subunit mRNAs in individual pituitaries and in dispersed pituitary cells. The validated assays were applied to investigate the effects of sex steroids (estrogens and androgens) on gonadotropin subunit expression, in vivo in steroid-injected eels, and in vitro by steroid treatments of primary cultures of eel pituitary cells. In vivo, a stimulation of LHb mRNA was observed after estradiol (E2) treatments, while testosterone (T) or the non-aromatizable androgen dihydrotestosterone (DHT) had no effect. Concerning FSHb expression, slight but non-significant decreases were observed after both E2 and androgen treatments. Different results were obtained in vitro: E2 induced an increase in FSHb mRNA levels but had no effect on LHb expression. In contrast, androgens (T and DHT) stimulated LHb expression while no significant variation was observed on FSHb mRNA levels following androgen treatment. Concerning the GPa mRNA, no significant effect of sexual steroids was observed in vivo or in vitro. This demonstrated specific direct actions of steroids on gonadotropin subunit expression. The differences observed between in vivo and in vitro experiments may be explained by the involvement of cerebral control, including GnRH and dopamine neurons, and their specific regulation by sex steroids. The data indicate that sex steroid feedbacks on gonadotropins are exerted via multiple pathways, indirectly at the brain level and directly on pituitary gonadotrope cells.