Some Characteristics and Optimum Incubation Conditions ofin VitroProgesterone Synthesis by Bovine Corpora Lutea

Abstract

In view of the widespread use of in vitro incubations of corpus luteum slices for a variety of purposes, experiments were conducted to establish optimum incubation conditions and to determine some of the sources of error variation observed in this system. A 2-hr. incubation period was sufficient to detect an LH (luteinizing hormone) induced stimulation of progesterone synthesis; longer incubation times yielded higher levels of synthesis but did not increase the precision of the assay. The pH optimum for the incubation system was between 7.15 and 7.55. Addition of 1 [mu]mole NADPH2 resulted in a consistent stimulation of progesterone synthesis, even in those groups already maximally stimulated with LH, suggesting that 2 different mechanisms are operative for the 2 stimulating agents. NADPH2 did not change the regression coefficient or the precision of the assay. Addition of nicotinamide (SO m[image]) resulted in higher rates of progesterone synthesis, presumably by maintaining the integrity of the pyridine nucleotides. This effect was most pronounced in groups of highest LH stimulation having the greatest rate of steroid synthesis. Addition of nicotinamide to the incubation buffer resulted in a slightly better index of precision. An attempt was made to substitute cholesterol for progesterone as the final parameter measured, thus conducting a cholesterol depletion rather than a progesterone accumulation assay. However, no depletion of total cholesterol was observed upon stimulation with LH in vitro. Luteinizing hormone in vitro increased the incorporation of mevalonate-2-Hc into progesterone. However, the increase was independent of LH concentration, indicating that while mevalonate serves as substrate during LH induced steroidogenesis, it does so in an indirect fashion upon depletion of immediate precursors for progesterone synthesis. Cows were injected with HCG [human chorionic gonadotropin] on days 2 through 10 of the estrous cycle in order to increase the weight of the corpora lutea and hence the tissue available for the assay. This treatment yielded corpora which although greatly increased in weight, were less responsive to LH in vitro. An analysis of the methodology experiments indicated that the observed error variance was due to variation in initial progesterone contents, variation in pool size of immediate precursors for steroid hormone formation, and possibly to variation in the amount of enzyme per unit tissue weight. No end-product inhibition of progesterone could be established.