Regulation of Glucose-6-Phosphate Dehydrogenase Activity in Uterine Tissue in Organ Culture

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Abstract
An organ culture technique, employing a total defined medium, was developed, in which the levels of glucose-6-phosphate dehydrogenase (G6PD) activity in uterine tissue from estradiol-treated ovariectomized mature rats continue to increase in vitro for 18 h at a rate similar to that seen in vivo. Uterine G6PD levels did not increase in vitro in tissue from estrogen-deprived rats even if estradiol (10-6 M) was added in vitro, but the administration of estradiol (5 .mu.g/rat) in vivo for as little as 2 min permitted G6PD levels to increase by 0.03 units/uterus (from 0.075 to 0.108 units/uterus) after the 18 h incubation. The maximum increase of 0.08 units/uterus (from 0.147 to 0.230 units/uterus) was seen in uteri from rats which were given estradiol 12 h prior to sacrifice. Tissues from animals given estradiol for > 30 h exhibit a net decrease in G6PD levels under the in vitro conditions. The in vitro increase in G6PD activity is inhibited by the addition of either actinomycin D (5 .mu.g/ml), cycloheximide (5 .mu.g/ml), or cordycepin (150 .mu.g/ml), or by the intrauterine injection of actinomycin D (10 .mu.g/rat). Intrauterine administration of cycloheximide (100 .mu.g/rat) inhibited the in vivo increase in enzyme activity; however, the enzyme levels increased after placement of uterine tissues from these animals into organ culture. The removal of cycloheximide, which was added at the beginning of incubation after the 12th h, restores the ability of the tissues to increase the G6PD activity, and this restoration is not blocked by the addition of actinomycin D, suggesting that the mRNA activity for uterine G6PD accumulates during in vitro inhibition of protein synthesis by cycloheximide. The in vitro increase in uterine G6PD is due to an increase in immunologically identifiable G6PD protein and this increase is due, at least in part, to an increase in the de novo synthesis of the enzyme, as measured by the incorporation of [14C]leucine into immunochemically isolated G6PD protein. Once initiated in vivo by estradiol, the uterus may be capable of continuing in vitro those events, including the synthesis of both RNA and protein, which result in an increased rate of synthesis of uterine G6PD.

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