The histochemical reaction for Δ6-3β-ol-steroid dehydrogenase (3βOHD) and 3β-hydroxysteroid dehydrogenase, using a variety of substrates, was studied in ovaries of rats after spontaneous ovulation had been blocked by sodium pentobarbital for 1, 2 or 3 days. In animals that failed to ovulate, the granulosa of preovulatory type of follicles, adjacent to the theca, had increased enzyme activity after the first day of blockade and remained unchanged thereafter. The reaction of the granulosa cells closer to the antrum remained unchanged, showing either weak or no enzyme activity. This portion of the granulosa disappeared after the second day of blockade. As the blockade of ovulation continued, the size of the preovulatory type of follicles increased. Finally, changes suggesting atresia appeared in the region between the theca and the granulosa of the preovulatory follicles. There was no visible change in enzyme activity in any other ovarian structure. The degree of increase in 3/3OHD activity in the granulosa of the blocked preovulatory type of follicle was comparable for all the substrates that gave a positive reaction in the proestrous ovary. The substrate giving positive reactions were 5-androsten-3β-ol-17-one, 5-androsten-3β, 17α- diol, 5-androsten-3β, 17β-diol, 5-pregnen-3β-ol- 20-one, 5-pregnen-3β, 20α-diol and 5β-androstan- 3β-ol-17-one. No reaction was observed in either the blocked or the normal ovary when 20β3- dihydropregnenolone, 17α-hydroxypregnenolone, 5β-androstan-3α-ol-17-one, DHEA sulfate or pregnenolone sulfate were used as substrates.
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