Steroids in equine testes: the identification of endogenous 19-hydroxy and 19-nor neutral steroids by gas chromatography–mass spectrometry

Abstract

ABSTRACT After homogenization of testicular tissue from stallions aged 1, 2 and 5 years, the unconjugated and conjugated steroids were isolated by a combined solvent–solid extraction procedure. The conjugates were further separated into glucuronides and sulphates by chromatography using Sephadex LH-20. After enzyme hydrolysis and solvolysis of the respective conjugate classes, the three extracts, unconjugated steroids, aglycones and solvolysed sulphates, were purified by chromatography using Kieselgel 60H columns. Five fractions were resolved from each extract; an aliquot of each fraction was derivatized to form the methoxime-trimethylsilyl ethers and the steroids were identified by combined gas chromatography–mass spectrometry. The results have shown that in stallion testes (1) steroidogenesis proceeds by both the 4-ene and the 5-ene pathways, (2) age-linked changes occur in both unconjugated and sulphoconjugated steroid fractions and (3) 19-hydroxy androgens and the 19-nor (C18) neutral steroids (19-norandrostenedione and 19-nortestosterone) are detected only in the unconjugated fraction whereas oestrone, the isomers of oestradiol and of 5(10)-oestrene-3,17-diol are the only steroids detected in the sulphoconjugate fraction. It is suggested that the unconjugated 19-oxygenated androgens present in stallion testes are converted to 19-nor neutral steroids by a reverse aldol reaction and a mechanism showing the putative intermediates in their formation is illustrated. Journal of Endocrinology (1989) 120, 223–229