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.
The metabolism of varying quantities of pregnenolone has been studied in nuclei-free homogenates from Macaca fascicularis testes by using capillary gas chromatography, after derivatization of metabolites as O-methyl oximes/trimethylsilyl ethers. Evidence was obtained indicating that both pathways for testosterone biosynthesis were operating. 5-Androstene-3 beta, 17 beta-diol was formed in especially high quantities. Two 16-androstenes, namely 5,16-androstadien-3 beta-ol and 5 alpha-androst-16-en-3 beta-ol, were also quantitatively important as metabolites. Co-incubation of stored homogenates with relaxin resulted in 80-100% reduction of the formation of all metabolites quantified except for 5 alpha-androst-16-en-3-one, which was stimulated. Freezing the homogenates at -10 degrees C for 3 weeks resulted in marked 4- to 6-fold reduction in the yields of testosterone and of the 5-ene and 4-ene metabolites from pregnenolone.
Using the rapid gas chromatographic steroid profiling technique, a number of metabolites of pregnenolone have been separated and quantified after incubation of this steroid with adult rat and neonatal porcine testicular homogenates. It was shown that the 5-ene-3 beta-hydroxy- and the 4-en-3-oxosteroid pathways for androgen biosynthesis were operating in both species, although the former pathway appeared to be more important in porcine testis. This tissue was characterised by the formation of several odorous, and pheromonal, 16-androstenes, which were quantitatively more important than the androgens. Three non-steroidal anti-inflammatory drugs (NSAIDS) caused dose-related inhibition of androgen and 16-androstene biosynthesis when co-incubated with pregnenolone. The order of potency was flurbiprofen > indomethacin > > > aspirin. The possibility that the NSAIDS may interfere with cytochrome P-450 is discussed, since several steroid-transforming enzymes, known to be dependent on this cytochrome for their activity, were markedly inhibited.
The metabolism of pregnenolone in subcellular fractions of the testes of the macaque (Macaca fascicularis) has been studied using capillary gas chromatography to characterize and quantify the metabolites, after their conversion into the O-methyloxime and/or trimethylsilyl ether derivatives. The microsomal incubations yielded the greatest quantities of metabolites, with lesser amounts in the mitochondrial fraction. The cytosolic fraction contained no significant quantity of metabolites after incubation, except for 5alpha-androst-16-en-3 beta-ol. This, and other odorous androst-16-enes, found in the microsomal fraction, are of particular interest in the context of animal communication because of their possible pheromonal role. Pregnenolone was converted into androst-5-ene-3 beta,17 beta-diol, androst-4-ene-3,17-dione and testosterone, suggesting that both classical pathways for testosterone synthesis were operating. Testosterone was further converted into 5 alpha-reduced androstanediols, especially in the microsomal fraction.
Capillary gas chromatographic 'steroid profiling' has been utilised to separate and quantify the metabolites (derivatized as methyloximes and/or trimethylsilyl ethers) formed from pregnenolone after incubation with rat testicular microsomes. A wide range of steroid metabolites was found, indicating that both the 5-ene and 4-ene pathways of testosterone biosynthesis were operating, as well as 16 alpha-hydroxylation, 20 beta-reduction and the formation of several C19 steroids (the 16-androstenes). At the concentration used, Metyrapone markedly inhibited 16 alpha- and 17-hydroxylation and side-chain cleavage of 17-hydroxylated C21 steroids. 16-Androstene production was also markedly inhibited and the formation of other metabolites was affected to lesser extents. Oxytocin abolished the formation of all C21 and C19 metabolites of pregnenolone.
Twenty authentic steroids, derivatized as O-methyl oximes (MO), trimethylsilyl (TMS) ethers or as MO-TMS ethers have been subjected to capillary gas chromatography using two different columns. Virtually all of the steroid derivatives have been resolved, one difficult pair to separate being 5,16-androstadien-3 beta-ol and 5 alpha-androst-16-en-3 beta-ol on the non-selective phase OV-1. Where syn and anti forms of MO derivatives arose, these were also resolved under the conditions utilised. This technique of 'steroid profiling' has been applied to the separation and quantification of metabolites of pregnenolone which were formed during incubations of the microsomal and cytosolic fractions from rat testes. The majority of the metabolites were found in the microsomal incubation. These compounds included some odorous 16-androstenes as well as other C21 and C19 steroids, the formation of which was consistent with the 5-ene and 4-ene pathways of testosterone biosynthesis being operative. In addition, evidence was obtained for 16 alpha-hydroxylation of C21 steroids. Very much less metabolic activity was found in the cytosolic fraction of rat testes. Metabolic pathways have been proposed which both confirm and extend earlier work. We conclude that the rat testis can only form some of the odorous, possibly pheromonal, 16-androstenes and that these are quantitatively less important than in the porcine testis.