Lack of Ovarian Responsiveness to Gonadotropic Hormones in Infantile Rats Sterilized with Busulfan*

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Abstract
Ovarian responsiveness to FSH and LH was examined in infantile rats treated in utero with busulfan (1,4-butanediol dimethanesulfate), a cytotoxic drug which has been shown to cause selective attrition of germ cells in the rat fetus. Pregnant Sprague-Dawley rats were injected ip on day 13 of gestation with busulfan (10 mg/kg BW) suspended in seasame oil or with sesame oil alone (control). Pups were killed on day 6, 8, 10, 12, or 14 postnatally, and trunk blood was collected. The ovaries were removed and either fixed for light microscopy or assessed for responsiveness to FSH and LH by their ability to produce net accumulations of cAMP and gonadal steroids in short term incubations. Ovaries, in which the germ cells were successfully destroyed, consisted of anastomotic cords of the intraovarian rete system surrounded by undifferentiated stromal tissue. A variable number of oocytes usually survived the busulfan treatment and were situated within the cords in irregularly defined follicles. Few treated oocytes proceeded to organize antral follicles by 14 days postnatally, but these follicles showed signs of normal theca and interstitial cell investment. A challenge of FSH or LH in vitro failed to stimulate net accumulations of cAMP, progesterone, androstenedione, or estradiol from treated ovaries whereas these responses were significantly stimulated in controls. Detectable levels of cAMP and steroids were, however, present in incubations of busulfan-treated ovaries on days 12 and 14, and these are likely attributable to the activity of antral follicles that survived the effects of busulfan. From day 8 to 12 plasma gonadotropin levels in treated animals rose significantly above those of controls suggesting that normal ovarian steroidogenesis is also suppressed in treated animals in vivo. Although direct effects of busulfan on somatic cells cannot be dismissed, these results suggest that the presence of germ cells is a prerequisite for the normal development of steroidogenic function in the rat ovary.