ETUDE DE L'ACTION DU FACTEUR HYPOTHALAMIQUE LRF (LH-RELEASING FACTOR) CHEZ LE RAT IN VIVO & IN VITRO

Abstract

A highly purified ovine LH-releasing factor (LRF) was obtained by a modification of the method previously described. After the fractionation of a crude hypothalamic extract on a Sephadex G-25 column, the LRF fraction was desalted and partially purified by chromatography on a Dowex 50 × 12 column and on an Amberlite CG 4B column. The last step of this method, chromatography on a CMC column, gave a purification of about 1600 times with respect to the crude extract. The action of this highly purified LRF preparation was studied on rat pituitary glands in vivo and in vitro. The method used in vivo was the evaluation of the LH-releasing effect of LRF in chronically ovariectomized, steroid-blocked rats (Ramirez & McCann 1963 b). A procedure was developed which allows a 4-fold concentration of the plasma LH from these rats, so that it can be assayed by a 4-point assay method. In the in vitro method, the pituitary glands of ovariectomized steroidblocked rats (Schally & Bowers 1964 a) were incubated in a Krebs-Ringer buffer with or without LRF, and the LH released into the medium was assayed using the O.A. A.D. method of Parlow. A dose-response curve was established between the log doses of LRF and the amount of LH released. This method can be used as a sensitive and specific assay for LRF. It was shown that a dose of 1.22 μg of LRF releases approximately 5 μg of LH per mg of pituitary tissue. This is about double of the amount of this hormone originally present in the pituitary glands of these rats (2.7 μg/mg). This leads us to the conclusion that the excess of this hormone was probably synthetized during the process of incubation. The amount of steroids injected as a blocking agents, appears to be very important for both in vivo and in vitro tests.