656 



PHYSIOLOGY OF GONADS 



fromTPNHtoDPN: 



TPXH + DPN^ -> DPNH + TPN^ 



The transhydrogenation system can be 

 coupled to glucose 6-phosphate dehydro- 

 genase as well as to isocitric dehydrogenase 

 (Talalay and Williams-Ashman, 1958; Vil- 

 lee and Hagerman, 1958) and presumably 

 can be coupled to any TPNH-generating 

 system. 



If the estrogen-stimulable transhydrogen- 

 ation reaction were readily reversible, an 

 enzyme such as lactic dehydrogenase which 

 requires DPN should be stimulated by 

 estrogen if supplied with substrate amounts 

 of TPN, catalytic amounts of DPN, and 

 a preparation from the placenta containing 

 the transhydrogenase. Experiments to test 

 this prediction were made using lactic de- 

 hydrogenase and alcohol dehydrogenase of 

 both yeast and liver (Villee, 1958a). It was 

 not possible to demonstrate an estrogen 

 stimulation of either enzyme system in 

 either the forward or the reverse direction. 

 The stimulation of the lactic dehydrogen- 

 ase-DPN oxidase system of the rat uterus 

 by estrogens administered in vivo reported 

 by Bever, Velardo and Hisaw (1956) 

 might be explained by the stimulation of 

 a transhydrogenase, but it has not yet been 

 possible to demonstrate a coupling of this 

 transhydrogenase and lactic dehydrogenase. 



The stimulating effect of a number of 

 steroids has been tested with a system in 

 which the transhydrogenation reaction is 

 coupled to isocitric dehydrogenase (Villee 

 and Gordon, 1956; Hollander, Nolan and 

 Hollander, 1958). Estrone, equilin, equi- 

 lenin, and 6-ketoestradiol have activities 

 essentially the same as that of estradiol- 

 17 j3. Samples of 1 -methyl estrone and 2- 

 methoxy-estrone had one-half the activitj'' 

 of estradiol. Estriol is only weakly estro- 

 genic in this system; 33 fig. estriol are less 

 active than 0.1 fig. estradiol- 17/3 (Villee, 

 1957a). The activities of estriol and 16- 

 epiestriol are similar, whereas 16-oxoestra- 

 diol is more active than either, with about 

 10 per cent as much activitv as csti'adiol- 

 17/3. 



Certain analogues of stilbestrol have been 

 shown to be anti-estrogens in vivo. When 

 applied topically to the vagina of the rat, 

 they prevent the cornification normally in- 



duced by the administration of estrogen 

 (Barany, Morsing, Muller, Stallberg, and 

 Stenhagen, 1955). One of these, 1,3-di-p- 

 hydroxyphenylpropane, was found to be 

 strongly anti-estrogenic in the placental 

 system in vitro: it prevented the accelera- 

 tion of the transhydrogenase-isocitric de- 

 hydrogenase system normally produced by 

 estradiol- 17/3 (Villee and Hagerman, 1957). 

 The inhibitory power declines as the length 

 of the carbon chain connecting the two 

 phenolic rings is increased and 1 , 10-di-p- 

 hydroxyphenyldecane had no inhibitory ac- 

 tion. Similar inhibitions of the estradiol- 

 sensitive system were observed with stil- 

 bestrol, estradiol-17a, and a smaller anti- 

 estrogenic effect was found with estriol 

 (Villee, 1957a). The inhibition induced 

 by these compounds can be overcome by 

 adding increased amounts of estradiol-17^. 

 When stilbestrol is added alone at low con- 

 centration, 10~' M, it has a stimulatory ef- 

 fect equal to that of estradiol-17^ (Glass, 

 Loring, Spencer and Villee, 1961). 



The quantitative relations between the 

 amounts of stimulator and inhibitor suggest 

 that this inhibition is a competitive one. It 

 was postulated that this phenomenon in- 

 volves a competition between the steroids 

 for specific binding sites on the estrogen- 

 sensitive enzyme (Villee, 1957b; Hagerman 

 and Villee, 1957). When added alone, estriol 

 and stilbestrol are estrogenic and increase 

 the rate of the estrogen-sensitive enzyme. 

 In the presence of both estradiol and estriol, 

 the total enzyme activity observed is the 

 sum of that due to the enzyme combined 

 with a potent activator, estradiol- 17^, and 

 that due to the enzyme combined with a 

 weak activator, estriol. When the concen- 

 tration of estriol is increased, some of the 

 estradiol is displaced from the enzyme and 

 the total activity of the enzyme system is 

 decreased. 



Two hypotheses have been proposed for 

 the mechanism of action of estrogens on the 

 enzyme system of the placenta. One states 

 that the estrogen combines with an inactive 

 form of the enzyme and converts it to an 

 active form (Hagerman and Villee, 1957). 

 When this theory was formulated the evi- 

 dence indicated that the estrogen acted on 

 a specific DPN-linked isocitric dehydrogen- 

 ase. The theory is equally applicable if the 



