Lack of ovarian responsiveness to gonadotropic hormones in infantile rats sterilized with busulfan.

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 sesame oil or with sesame oil alone (control). Pups were killed on days 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.

Interactions of a phosphodiesterase inhibitor, 3-isobutyl-1-methyl xanthine, with prostaglandin E2, follicle-stimulating hormone, luteinizing hormone, and dibutyryl cyclic 3',5'-adenosine monophosphate (cAMP) in cAMP and steroid production by neonatal rat ovaries in vitro.

The development of responsiveness to prostaglandin E2 (PGE2), FSH, LH, and [Bu]2cAMP was examined in whole ovaries isolated from neonatal Sprague-Dawley rats on days 0 (birth), 2, 4, or 6 postpartum. Pairs of ovaries were incubated with these stimuli in the absence or presence of 3-isobutyl-1-methyl xanthine (MIX), a potent phosphodiesterase inhibitor, and accumulations in the medium of cAMP, androstenedione, and estradiol were measured. PGE2 stimulated marked cAMP accumulation on day 0 whereas similar responses to FSH and LH did not develop until days 2 and 4, respectively. No cAMP accumulation was detectable in the absence of MIX. Ovaries gradually acquired the ability to produce both cAMP and steroids in response to FSH and LH over the first postnatal week. No steroid accumulation was measurable in incubations conducted on days 0 or 2; however, steroidogenesis was stimulable in day-4 ovaries by (Bu)2cAMP. PGE2, FSH, and LH also stimulated steroid accumulation on day 4, but only when MIX was present in the incubation, suggesting that high levels of endogenous cAMP can also lead to steroid production. By day 6, all stimuli elicited steroid accumulation in a dose-dependent fashion. MIX potentiated the responses to lower doses of these stimuli but not to the higher doses at this age. In the absence of MIX, LH was approximately 100 times more potent than FSH in stimulating steroid production; however, the two gonadotropins were nearly equipotent in this regard when MIX was present in the incubation. These results support the notion that a cAMP-sensitive steroidogenic apparatus is present in the rat ovary as early as the fourth day postpartum. Because of the marked effects of MIX on gonadotropin-induced steroidogenesis, it may be that modulation of phosphodiesterase activity is one way by which steroidogenesis is regulated in the neonatal rat ovary.

PGE2 and dibutyryl-cAMP enhance the response of rat granulosa cells to FSH.

Granulosa cells isolated from immature Sprague-Dawley rat ovaries produce progesterone (31.7 pg/micrograms cell protein) in response to an acute FSH stimulus (5 micrograms/ml NIH-FSH-S11, 2 H). After culture for 48 h in the absence of hormones (control culture), progesterone production by the granulosa cells in response to FSH is significantly reduced (2.9 pg/micrograms cell protein). Cells cultured with prostaglandin E2 (PGE2, 1 microgram/ml) or dibutyryl-cAMP (dbcAMP, 1 mM) exhibited a discernibly greater steroidogenic response to FSH (12.5 and 53.4 pg/microgram cell protein, respectively) than that of control cultures. Therefore the presence of PGE2 or dbcAMP in the culture medium helps to maintain the steroidogenic capacity of granulosa cells in culture. It is probable that this capacity is maintained at a locus distal to the production of cAMP by FSH. Paradoxically, granulosa cells cultured with PGE2 produce less cAMP in response to FSH stimulation than cells in control cultures (15.9 vs. 250.3 fm/micrograms cell protein). This may be due to a suppressive effect of prior exposure to PGE2 on the subsequent activity of adenylate cyclase when the FSH is introduced and a concomitant elevation of phosphodiesterase activity.