that pregnant mare serum gonadotrophin (PMS) loses its gonadotrophic activity on incubation with certain quinones. Since it is now well established that oestrogens are converted to 2-hydroxy metabolites by man and other species (Engel, Baggett & Carter, 1957;Kraychy & Gallagher, 1957; King, 1961) and since these might then be further oxidized to quinones, it was decided to investigate the effect of such orthohydroxylated steroids and their oxidation products on the inactivation of PMS.Oestradiol-17\g=b\ and 2-hydroxyoestradiol-17\g=b\ were converted to their o-quinones with mushroom phenolase (Jellinck, 1960;Jellinck & Irwin, 1963). The PMS (150 i.u. in 3\m=.\3 mg.) dissolved in potassium phosphate buffer (0\m=.\067m), pH 7\m=.\0, was incubated in O2 at 37\ s=deg\for 2 hr. with 1 mg. of phenolase (mushroom tyrosinase, grade II, Sigma Chemical Co.) and oestrogen (60 \g=m\g.); total volume, 3 ml. Control tubes were incubated without enzyme or steroid. The protective action of glutathione on PMS inactivation by oxidized oestrogens was also investigated, and in this experiment 1-5 m-moles of glutathione were added at the beginning of the incubation. The bioassay was adapted from that described by Graham et al.
(1960).Female Sprague-Dawley rats (3-5 weeks old) were used; each animal received a single s.c. dose of 0-5 ml. test solution containing 25 i.u. of PMS treated as described above. The animals were killed 3 days later and the ovaries and uteri carefully dissected out and weighed after fixation and partial dehydration in 50% ethanol.The results (Table 1) show that relatively low concentrations of oestrogens (0-07 mï) which themselves had no inhibitory action on PMS, inactivated this gonado¬ trophin after incubation with phenolase. 2-Hydroxyoestradiol was found to be no more active than oestradiol in this system even though it might be expected to be more readily converted to the quinone than the parent oestrogen. The inactivation of PMS could be prevented by glutathione (0-5 him) and did not occur with phenolase alone. It is well established (Mason, 1955) that thiols combine readily with quiñones, and glutathione probably exerts its protective action in this manner. This assumption is supported by biochemical evidence (Jellinck & Lewis, unpublished observations) for conjugation between oestrogen and glutathione under the conditions of the experiment.
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