Pregnenolone metabolites in rat testis: endogenous concentrations, and intracellular distribution in whole testes during incubation in vitro

The intracellular movements of pregnenolone in rat testes were investigated. Whole testes were incubated in the presence or absence of pregnenolone (2.5mM) in the medium for 120 min (in some studies 30, 60, and 90 min). The testes were homogenised, subcellular fractions prepared and analysed in quadruplicate for steroid content by gas chromatography-mass spectrometry with selected ion monitoring. Quantification of pregnenolone and 11 of its metabolites, obtained from non-incubated whole testes, provided values for endogenous amounts. Pregnenolone was the only steroid of quantitative importance found initially in the mitochondrial fraction but was subsequently found in the microsomal fraction, where metabolism occurred. Identification and quantification of metabolites indicated that both classical pathways for testosterone production were operating, with the 4-en-3-oxosteroid pathway predominating. By 120 min, virtually all pregnenolone metabolites, including pregnenolone itself, were found in the cytosol, consistent with an overall movement from mitochondria to endoplasmic reticulum to cytosol.