Changes in Steroidogenesis in Preovulatory Rat Ovaries Induced to Ovulate with Pregnant Mare Serum Gonadotropin

The pattern of steroids produced by the immature rat ovary is different from that observed in the adult cycling rat. Changes in steroid production were studied several hours before the first ovulation, as it was anticipated that during this transitional period both the immature and mature pattern of steroid formation may coexist in the ovary. Ovarian homogenates (1000 ×g supernatant) obtained from 26-day-old rats treated 52 h previously with 25 IU PMS gonadotropin were incubated in the presence of NADPH with either [¹⁴C]progesterone, 20α- [³H]hydroxy -4 - pregnen - 3 - one (20α-OHP), or [¹⁴C]progesterone and 3α-[₃H]hydroxy- 5α-pregnan-20-one (5α -3α -ol-20-one). The dose of PMS gonadotropin used induces an endogenous LH surge in the afternoon of day 26, which is followed by ovulation. After incubation, radioactive metabolites were isolated, identified, and measured by thin layer and gas-liquid chromatography, with derivative formation and recrystallization to constant specific activity.

5α -Pregnane-3α,20α-diol (5α-3α,20α-diol) and 5aandrostane- 3α,17βdiol (5αA-3α,17β-diol) were found to be the major products formed. 20α -OHP seems to be an intermediate product in the formation of 5αP-3α,20adiol from progesterone because 1) [¹⁴C]20α -OHP was formed with [¹⁴C]progesterone served as substrate and 2) [₃H]20α -OHP was readily converted into [₃H]5αP- 3α,20α-diol by the ovarian supernatant. 5αP-3α,20α-diol was also produced by reduction of 5α -3α-ol-20-one; this was shown by double isotope experiments in which ovarian homogenates were incubated with both [¹⁴C]- progesterone and [₃H]5αP-3α-ol-20-one. The 5α-3α,- 20α-diol isolated was found to be labeled with both isotopes. 5α-3α,20α-Diol was not further metabolized when incubated for 90 min with the ovarian preparation.

In an experiment in which a high concentration of [₃H]5αP-3α -ol-20-one was added concomitantly with [¹⁴C]progesterone, no 14C label was found in 5αP-3α-ol- 20-one isolated at the end of the experiment, while 3α,17α-dihydroxy-5α-pregnan-20-one (5αP-3α,17α-diol- 20-one) was labeled with both isotopes. This indicates the production of 5αP-3α,17α-diol-20-one from progesterone through a 17α-hydroxylated intermediate. As 5α-3α,17β-diol in both double labeling experiments showed a ³H:¹⁴C ratio half of that observed in 5α -3α - 20-one and in 5α-3α,17α-diol-20-one, it was concluded that 5α-3α,17β-diol is also produced by a third route, probably through and ostenedione. Thus, three possible pathways lead to 5α-3α,17β-diol formation in the ovarian preparation under investigation.