Radioimmunoassay of prolactin in plasma of bullfrog tadpoles

Antiserum to prolactin (PRL) of bullfrog (f), Rana catesbeiana, was produced by immunizing rabbits with highly purified fPRL obtained from adenohypophyses of adult bullfrogs. In Ouchterlony's agar double diffusion test, a single precipitin line was produced between the fPRL antiserum and fPRL or the bullfrog pituitary extract. No precipitin line was produced between the antiserum and ovine PRL, bovine PRL, ovine growth hormone (GH) or fGH. The acetone-dried powder of bullfrog pituitary glands which had been incubated with the fPRL antiserum had much less potency in promoting collagen synthesis of the tail fin of bullfrog tadpoles than that incubated with normal rabbit serum. Histological studies on bullfrog adenohypophyses revealed that the cells that immunologically reacted with the antiserum to fPRL were erythrosinophilic. The antiserum to fPRL was used to develop a radioimmunoassay (RIA) in which fPRL and ¹²⁵I-fPRL were employed as the standard of the radioligand, respectively. Several dilutions of plasma of both adult and larval bullfrogs yielded dose-response curves which were parallel to the standard curve. Bovine PRL, ovine PRL, ovine GH and fGH did not react in this assay. Plasma from hypophysectomized bullfrogs had no detectable immunoreactive prolactin. Pituitary homogenates of Rana catesbeiana and Bufo bufo japonicus gave inhibition curves which were parallel to the standard. Pituitary homogenates of Xenopus laevis and Hynobius tokyoensis gave inhibition curves which did not parallel the standard. RIA of plasma PRL in larval bullfrogs of various developmental stages were performed. Average concentrations of prolactin during premetamorphosis (st. X), prometamorphosis (st. XVI-XIX) and at early climax stage (st. XX-XXII) were 18, 21-25 and 27-35 ng/ml, respectively. At advanced climax stage PRL levels were quite high. Average values at stages XXIII, XXIV and XXV were 98, 169 and 116 ng/ml, respectively. The significance of PRL levels in relation to metamorphosis is discussed.