Follicle-stimulating hormone and its alpha and beta subunits in rainbow trout (Oncorhynchus mykiss): Purification, characterization, development of specific radioimmunoassays, and their seasonal plasma and pituitary concentrations in females

Gonad development in fish, as in mammals, is regulated by two gonadotropins (GTHs), FSH and LH. The function of LH in fish has been clearly establishe d; however, the function(s) of FSH is less certain. The lack of specific an d sensitive assays to quantify FSH and its alpha and beta subunits has hind ered studies to assess physiological function. In this study, gel filtratio n chromatography, ion exchange chromatography, and HPLC were employed to pu rify FSH and its subunits from pituitary glands of rainbow trout (Oncorhync hus mykiss), and the identities of the isolates were confirmed by amino aci d analysis. Polyclonal antibodies were raised against the free GTH alpha2 a nd free FSH beta subunits to develop specific RIAs. The sensitivities of;th e intact FSH, GTH alpha2, and FSH beta assays were 1 ng/ml, 0.2 ng/ml, and 0.1 ng/ml, respectively, and the cross-reaction of these molecules with eac h other and with intact LH in the heterologous assays was < 10.4% throughou t. Pituitary and plasma samples diluted in parallel with the standards in a ll three assays and spiked sample recoveries were > 90% throughout, Measure ment of plasma and pituitary concentrations of intact FSH in female rainbow trout confirmed the established seasonal profiles. Concentrations of free GTH alpha2 subunit were elevated both in the plasma and in the pituitary in females at ovulation (maximum concentrations: 34.93 +/- 6.3 ng/ml in plasm a; 37.63 +/- 5.79 mug/pituitary). In both the plasma and the pituitary, fre e FSH beta subunit was present throughout the reproductive cycle but at ver y low concentrations when compared with both free GTH alpha2 and intact FSH . The presence of free GTH alpha2 subunit in the plasma similarly occurs in mammals, but its functional significance in fish has yet to be established .