Transformations of C21 and C19 steroids in porcine ovarian preparations and the chromatographic separation of the metabolites.
The separation of several C21 steroids, such as 17-hydroxyprogesterone, and androgens, such as testosterone, from the non-polar steroids like 16-androstenes has been achieved on one thin-layer chromatographic plate using a two-dimensional technique. This method has been further developed to include a separation of some oestrogens from C19 steroids. The thin-layer chromatographic development was then utilised to separate the metabolites of porcine ovarian incubations. Homogenised preparations of corpora lutea (5-14 days post ovulation) were incubated separately with [4-14C] testosterone, [4-14C] progesterone and [4-14C] pregnenolone, using NADPH as cofactor. After two-dimensional thin-layer chromatography the metabolites were identified by establishing their radiochemical purity either by repeated thin-layer chromatography or by gas-fraction collection. Pregnenolone was converted to small yields of 4,16-androstadien-3-one (0.13-0.28%) and 5 alpha-androst-16-en-3-one (less than 0.1%) and also to 17-hydroxypregnenolone and 17-hydroxyprogesterone (0.8 and 0.37% respectively). Increased activity of 5-ene-3 beta-hydroxysteroid dehydrogenase/4,5-isomerase was shown by the high yields (73-83%) of progesterone obtained from pregnenolone. This was associated with decreased C-17.20 lyase activity as reflected in the relatively small amounts of 4-androstenedione obtained from both pregnenolone and progesterone. None of the well-known oestrogens or 16-androstenes was formed from progesterone or testosterone, but the latter was converted into 4-androstenedione, in small yield. Both pregnenolone and progesterone gave rise to a metabolite in 1-2% yield (of similar polarity to the 16-androstenes but separated from them by thin-layer chromatography on AgNO3-impregnated plates) which has been tentatively identified as 5 alpha-pregnane-3,20-dione. Incubations of porcine follicular fluid and tissue with labelled pregnenolone or progesterone did not result in the biosynthesis of labelled 16-androstenes.
D000728:Androgens / Q000096:biosynthesis / Q000302:isolation & purification; D000818:Animals; D002855:Chromatography, Thin Layer; D003338:Corpus Luteum / Q000378:metabolism; D005260:Female; D006080:Ovarian Follicle / Q000378:metabolism; D010053:Ovary / Q000378:metabolism*; D011284:Pregnenolone / Q000378:metabolism*; D011374:Progesterone / Q000378:metabolism*; D011372:Progestins / Q000096:biosynthesis / Q000302:isolation & purification; D013552:Swine; D013739:Testosterone / Q000378:metabolism*
D000728:Androgens; D011372:Progestins; D013739:Testosterone; D011374:Progesterone; D011284:Pregnenolone