Large intact follicles and granulosa cells were obtained from sheep ovaries at various stages of the oestrous cycle and were cultured in vitro for 7 days. The daily output of steroid hormones into the culture medium was determined using a procedure capable of measuring a wide range of steroids including immunoreactive oestrogen, testosterone, androstenedione, progesterone, 20α-hydroxy-pregn-4-en-3-one, 17α-hydroxyprogesterone, 17α,20α-dihydroxy-pregn-4-en-3-one, pregnenolone, 17α-hydroxypregnenolone and certain hydrogenated metabolites.
Intact follicles explanted from sheep on Day 4 of the cycle produced oestrogen (40 ng/mg follicular tissue per 24 hr) during the first 3 days in culture followed by large amounts of testosterone (100 ng/mg per 24 hr) and finally 17α-hydroxylated progestin. Follicles explanted at midcycle produced constant amounts of oestrogen (30 ng/mg per 24 hr) throughout the culture period; no other steroids were produced in significant quantities. The highest levels of oestrogen (75 ng/mg per 24 hr) were produced by follicles which had been explanted from sheep on Day 14 of the cycle. Oestrogen production declined rapidly in follicles explanted just before oestrus (late Day 15) and was replaced by the production of testosterone and 17α-hydroxylated progestin. The production of oestrogen and testosterone was very low in follicles explanted at oestrus; these follicles produced a transient peak of 17α-hydroxypregnenolone (30 ng/mg per 24 hr) followed by large amounts of progesterone (300 ng/mg per 24 hr) and 20α-hydroxy-pregn-4-en-3-one (250 ng/mg per 24 hr).
Monolayer cultures of granulosa cells produced only progesterone, 20α-hydroxy-pregn-4-en-3-one and pregnenolone, thus implicating the cells of the theca interna as the principal source of oestrogen, androgen and 17α-hydroxylated progestin.
Our findings indicate that the biosynthesis of oestrogen in the cells of the theca interna involve a sequence of steps including pregnenolone → 17α-hydroxypregnenolone→17α-hydroxyprogesterone→ testosterone or androstenedione→ oestrogen. During the transformation of follicles from oestrogen to progesterone secretors, steroid synthetic capacity is transferred from the theca interna to the membrana granulosa. The accumulation first of testosterone and then of 17α-hydroxypregnenolone suggests that the aromatase and then the desmolase systems limit steroid production in the theca interna during the period of transformation.