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In Vitro Steroidogenesis by Dissociated Rat Follicles, Primary to Antral, Before and After Injection of Equine Chorionic Gonadotropin¹

^a Department of Molecular and Integrative Physiology and Ralph L. Smith Research Center, University of Kansas Medical Center, Kansas City, Kansas 66160-7401

Prepubertal female rats were injected s.c. with 5.0 IU eCG, and ovaries were collected 24 and 48 h post-eCG, on Day 25, as well as from an untreated group also on Day 25. Large antral follicles were manually dissected, and the ovarian remnants were incubated with collagenase overnight to liberate preantral follicles from adhering stromal cells. The viability of the follicles was established by normal histology and lack of pyknotic granulosa cells (GCs) and by their ability to secrete steroids. After a 1-h baseline incubation, either 10 ng LH or 100 ng FSH was added for an additional hour, and the media—before and after gonadotropin administration—were used to measure progesterone, androstenedione, and estradiol by RIA. A distinct hierarchy existed in steroid synthesis, with the maximal production by the largest (700 µm) antral follicles. The major steroid that had accumulated after addition of LH at 48 h post-eCG was androstenedione (1099 pg/follicle per hour), followed by equal amounts of progesterone (155 pg/follicle per hour) and estradiol (191 pg/follicle per hour). There was a precipitous drop in steroid production by 550-µm and 400-µm antral follicles, especially in estradiol for the latter-sized follicles (0.08 pg/follicle per hour). Preantral follicles also produced progesterone and androstenedione after addition of LH. For example, follicles 222 µm in diameter with 4–5 layers of GCs and well-developed theca responded to LH at 48 h post-eCG by accumulating androstenedione (37 pg/follicle per hour) and progesterone (6 pg/follicle per hour) but negligible estradiol. The smallest follicles secreting steroids, 110–148 µm in diameter, had 2–4 layers of GCs. However, primary follicles (1 layer of GCs and no theca) did not synthesize appreciable amounts of any steroid. Although small preantral follicles were consistently stimulated by LH, FSH was ineffective. This result differs from findings in the hamster showing that intact preantral follicles with 1–4 layers of GCs and no theca respond to FSH by secreting progesterone in vitro (Roy and Greenwald, Biol Reprod 1987; 31:39–46). The technique developed to collect intact rat follicles should be useful for numerous investigations.

¹ Supported by the National Institutes of Health (HD-00596 and HD-02528 [G.S.G.]).

² Correspondence: G. Greenwald, Department of Molecular and Integrative Physiology, University of Kansas Medical Center, 3901 Rainbow Boulevard, Kansas City, KS 66160-7401. FAX: 913 588 7430; ggreenwa{at}kumc.edu

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