Effect of Oestrogen Priming on the Pattern of Progesterone Metabolism in the Mouse Uterus

Abstract

Oestrogen treatment in vivo increases the metabolism of progesterone by the rat uterus in vitro (Armstrong & King, 1971;Howard & Wiest, 1972). However, the pattern of metabolites produced is dependent on the concentration of progesterone used in vitro (Saffron, Loeser, Haas & Stavely, 1974). When high concentrations of progesterone were used, oestrogen increased both ring A and C20-ketone metabolism. At low concentrations oestrogen increased C20-ketone metabolism, whereas ring A metabolism was decreased.This present experiment was carried out to see whether oestrogen affected uterine meta¬ bolism of progesterone in vivo in mice (Clark, 1973(Clark, , 1974 in the same way.[1,2,6,7-3H] Progesterone (5 or SOOpg; sp. act. 261 µ ^, Radiochemical Centie, Amersham) was injected intraluminally into both uterine horns of spayed mice 1 or 6 days after oestrogen priming as described by Clark (1973). Five picograms of progesterone may be bound in the uterus but 500 pg would exceed the binding capacity of the uterus (Clark, 1973(Clark, , 1974. On day 6, responsiveness of the uterus to progesterone and oestrogen is qualitatively the same as that of the untreated ovariectomized mouse but the uteri are larger and their responses less variable.Groups of 5 uteri were removed 5 min after injection and immediately extracted three times with 5 ml chloroform:methanol (2:1, v/v). The pooled solvent was evaporated to dryness at 70°C under nitrogen. The residue was dissolved in 10 ml aqueous 70% methanol and stored overnight at -20°C. After centrifugation at 800 g for 10min at 4°C the supernatant was removed and evaporated to dryness. The residue was partitioned between water and ethyl acetate (5 : 10, v/v). The ethyl acetate was removed and an aliquot taken for estimation of total radioactivity. After addition of 50 µg each of unlabelled reference steroids corresponding to compounds previously found in the mouse uterus (5a-pregnane-3a,20a-diol; 20a-hydroxypregn-4-en-3-one; 3a-hydroxy-5a-pregnan-20-one; 5a-pregnane-3,20-dione and progesterone), the progesterone metabolites were separated by thin-layer chromatography (Armstrong & King, 1971). This method separates the above compounds into six discrete spots which were located by exposure to iodine vapour. In this experiment, as previously found by Clark (1974), negligible radioactivity was found in areas outside the spots. Recrystallization to constant specific activity had previously demonstrated that, for mouse uterine metabolism of pro¬ gesterone, radioactivity in each spot is indeed associated with the particular reference com¬ pound (Clark, 1974). Silica gel from reference spots or from intervening areas was sucked into cotton-wool plugged Pasteur pipettes and eluted with 2 2 ml chloroform : methanol (2:1, v/v) into counting vials. Samples were dried overnight: 5 ml toluene containing 0-3 % PPO and 0-1 % POPOP were added to each vial and the radioactivity was counted by liquid scintillation spectrometry. Figure 1 shows that metabolism of progesterone was greatest immediately after oes...

The ovarian-uterine vasculature in relation to unilateral endometrial growth in flying foxes (genus Pteropus, suborder Megachiroptera, order Chiroptera)

“…Like Marshall (1953) (Towers and Martin, 1985). If luteal progesterone were sequestered to, and metabolized to inactive compounds by the endometrium, as in rodents (Clark, 1973(Clark, , 1974(Clark, , 1975 (Walsh et ah, 1979) and women (Bendz et ah, 1982b). Recirculation in this way might be a mechanism to regulate follicle growth, ovulation and luteal function (Bendz et ah, 1982a, b).…”