The Effect of Castration and Androgen Replacement on Rat Pituitary Follicle Stimulating Hormone Investigated by Reversed-Phase High Performance Liquid Chromatography

Abstract

Rat follicle stimulating hormone (rFSH) was analysed in extracts of rat anterior pituitary using a newly developed separation method of reversed-phase high performance liquid chromatography (RP-HPLC) and post-column detection of immunoreactivity (rFSH-IR) by radioimmunoassay (RIA) or immunoprecipitation with a specific anti-rFSH antibody. Good separation of rFSH was achieved on a Waters Radial-Peak 8MBC18 cartridge with a linear (60 min) gradient from 10-60% acetonitrile at constant 0.1% trifluoroacetic acid concentration. The elution profiles of radioiodinated purified rFSH [(125I)rFSH] as well as extracts of adult male and female rat pituitaries contained two main immunoreactive components (peaks 1 and 2) which eluted at 16-17 min and 21 min respectively. In the (125I)rFSH elution profile, the peaks of immunobinding corresponded to two major peaks of radioactivity. The male pituitary showed greater binding to anti-rFSH-serum of peak 1 and 2 eluate compared to female pituitary. At day 20 after orchidectomy, the amount of immunoreactive rFSH (rFSH-IR) increased in both peaks (by 30% in peak 1 and 180% in peak 2); however, the rFSH-IR level in peak 2 appeared to be more variable compared with that in peak 1. Subcutaneous implantation of 5 mg or 15 mg of dihydrotestosterone (DHT) at the time of orchidectomy prevented the post-castration elevation of rFSH-IR at 10 and 20 days after surgery. The precise nature and physiological significance of the two elution peaks are not clear at this time; however, only peak 2 showed immunoreactivity with anti-rLH-alpha-subunit antibody (AFP 87713681) and the elution time of peak 2 coincided with that of (125I) rLH-alpha subunit (AFP-7264 B).(ABSTRACT TRUNCATED AT 250 WORDS)