The isolation and measurement of luteinizing hormone-releasing hormone (LHRH) from the rat testis

Abstract

Although LHRH has been implicated in the direct control of rat Leydig cell function, LHRH has not been previously detected in the rat testis. An optimized fractionation procedure, which involved acid extraction, bulk fractionation on ODS-silica, extraction in chloroform/ethanol, ether extraction, gel filtration on Sephadex G-15 and RP-HPLC, was employed to isolate LHRH from lyophilized adult rat testes. LHRH activity was assessed by an in vitro LHRH bioassay system employing rat anterior pituitary cells in monolayer culture and several radioimmunoassays for LHRH. LHRH recoveries were monitored by the addition of exogenous LHRH to some samples of lyophilized testes prior to extraction. LHRH bioactivity was non-detectable in the LHRH region of the gel filtration profile of testis extracts in the absence of exogenous LHRH; however, using the most specific LHRH RIA procedure, LHRH immunoactivity which co-chromatographed with LHRH following RP-HPLC was found in all extracts. Based on an average LHRH recovery of 31.0%, as determined from the extracts containing exogenous LHRH, the level of endogenous LHRH immunoactivity in the extracts was determined to be equivalent to 5.6 pg LHRH/g dry weight or 1.0 pg LHRH/testis. These results indicate that the levels of LHRH in the adult rat testis are considerably less than those of the hypothalamus. Based on these findings a simplified fractionation/assay procedure with sufficient sensitivity can be devised for the quantitation of testicular LHRH as a means of clarifying its physiological role in gonadal function.