Ilene R. Cohen

Proliferative Lesions of Ovarian Granulosa Cells and Reversible Hormonal Changes Induced in Rats by a Selective Estrogen Receptor Modulator

This study assessed the effects of raloxifene. a selective estrogen receptor modulator (SERM), on ovarian morphology and circulating hormone levels in rats. Female Fischer-344 rats (65/group) were given dietary raloxifene for 6 months at average daily doses of 0, 15, 75, and 365 mg/kg. Morphologic evaluation of ovaries was conducted on 25 rats/group at the end of the treatment period and from 20 rats per group after 1 and 3 months withdrawal from treatment. Plasma hormone analyses were conducted on 10 rats per group at the end of the treatment period and after each withdrawal period. Treatment with raloxifene for 6 months resulted in disruption of the hypothalamic-pituitary-ovarian axis, manifested by increased plasma concentrations of luteinizing hormone (LH) and estradiol-17beta (E2), and failure of ovulation, manifested by ovarian follicular prominence (retained anovulatory follicles), lack of corpora lutea (CL), and depressed plasma progesterone (P4). Many (56% to 80%) rats in all raloxifene treated groups had focal, minimal to slight hyperplasia of granulosa cells within individual retained follicles. A few treated rats in the mid- and high-dose groups (2 of 25 and 3 of 25, respectively) had more extensive focal proliferation of granulosa cells. These foci were approximately 3 to 6 mm in overall size and were characterized by moderate papillary proliferation of large granulosa cells associated with cystic spaces, often with hemorrhage. In 4 of the 5 rats with this focal cystic granulosa cell hyperplasia, the remainder of the involved ovary and the contralateral ovary were atrophic. After 1 or 3 months of drug withdrawal, most previously treated rats examined had morphologic evidence of ovarian cyclic changes, including developing follicles, various stages of CL, and normal plasma levels of LH, E2, and P4. Continued lack of cyclic changes was limited to 4 of 20 rats from the low-dose group after 1 month of recovery and to 1 low dose rat after 3 months. Intrafollicular granulosa cell hyperplasia was not seen in rats in the reversibility phase. Areas of prior focal cystic granulosa cell hyperplasia were represented by focal sclerosis that included hemorrhage and/or hemosiderin. The foci of sclerosis were associated with cystic spaces after 1 month and were solid after 3 months. A granulosa cell tumor, approximately 12-13 mm diameter, was present in a high-dose rat in the 3-month reversibility group. This tumor effaced 1 ovary and was characterized by proliferative granulosa cells, usually in papillary formations and cords within cystic spaces. This rat had atrophy of the uninvolved ovary, excessive plasma levels of E2 and prolactin, and high P4 levels considering the absence of CL. The results of this study indicate that ovarian granulosa cells in rats are susceptible to proliferative changes when stimulated chronically with excessive trophic hormones. Most of these proliferative changes were reversible upon cessation of the hormonal stimulation. However, the proliferative lesion in one treated rat progressed to apparent autonomous (neoplastic) growth.

The Selective Estrogen Receptor Modulator, Raloxifene: A Segment II/III Delivery Study in Rats

Raloxifene is a nonsteroidal, selective estrogen receptor modulator developed by Eli Lilly and Company as a therapeutic agent for postmenopausal osteoporosis. Raloxifene was administered orally by gavage at doses of 0, 0.1, 1, or 10 mg/kg/d to female CD rats (25/group) on Gestation Day 6 (GD 6) through Postpartum Day 20 (PD 20). Females were allowed to deliver and maintain their progeny until PD 21. All dead pups and pups culled on PD 1 were given internal and external examinations. One pup/sex/litter was assigned to each of the following assessment groups: 1) the primary pair for the F1 generation study, in which survival, growth, development, behavior, indicators of sexual maturation, and reproductive performance were evaluated; 2) terminal necropsy evaluations at PD 21; 3) terminal necropsy evaluations at 60 d of age; and 4) assessments of immune function at 5 to 6 weeks of age. At termination on PD 21, 60, or approximately 140, a necropsy was performed; crown rump and tibia lengths were measured; pituitary weights were taken; and a portion of the anterior pituitary was retained for growth hormone, luteinizing hormone, and prolactin content determinations (control and 10-mg/kg groups only). The remainder of the pituitary and reproductive tissues were retained for histologic evaluations. Dose-related depressions in maternal body weight and food consumption occurred during gestation. Mean gestation length was increased at 1 and 10 mg/kg. Delayed, extended, and/or disrupted parturition occurred in dams given 10 mg/kg, which resulted in a high incidence of maternal morbidity and/or death, increased numbers of dead pups, and the survival of only 66% of live pups to PD 21. Progeny body weights were not decreased at birth, but were depressed progressively in a dose-related manner during the 3-week lactation period. Negative geotaxis and incisor eruption were apparently accelerated in the 1- and 10-mg/kg groups, but eye opening was delayed at 10 mg/kg. Postweaning activity levels, auditory startle, and passive avoidance performance were not affected in the raloxifene groups. Dose-related decreases in spleen cellularity and thymus weights occurred in both sexes, but immune system function, as measured by splenic natural killer cell activity and antibody response to sheep red blood cells, was not affected. Postweaning body weights and growth parameters, as well as pituitary hormone content, were affected in both an age- and sex-specific manner. Preputial separation was not affected, but vaginal patency occurred ca 2 d earlier than controls in females from the 10-mg/kg group. Estrous cycles of the F1 females were not affected during the first two weeks after vaginal opening, but were disrupted at 12 to 14 weeks of age in the 10-mg/kg group. These females showed poorer mating and fertility indices, and litter size was reduced in the two females that were pregnant. Histologically, reproductive organs were not affected in males at any age or in females at PD 21. At PD 60, vaginal mucification occurred in females from the 0.1- and 1-mg/kg groups. At PD 140, the only finding was a high rate of uterine hypoplasia in the 10-mg/kg group, and this finding occurred in the absence of any concomitant ovarian or vaginal changes. These reproductive and developmental findings are consistent with estrogen antagonist activity of raloxifene.

Age-associated alterations in catecholaminergic concentrations, neuronal activity, and α1 receptor densities in female rats

The density of alpha 1-adrenergic receptors and catecholamine concentrations and neuronal activity were assessed in selected estrogen-responsive regions of the hypothalamus and pineal glands. The results of this study demonstrate that the densities of alpha 1-adrenergic receptors and norepinephrine and epinephrine concentrations and activity rate constants are altered in aged rats. The direction and extent of the change depend on the specific brain region and reproductive state of the animals. No widespread decline with increased age was observed in any of the parameters measured.

Androgen Dependent Mammary Gland Virilism in Rats Given the Selective Estrogen Receptor Modulator LY2066948 Hydrochloride

A selective estrogen receptor modulator (SERM) is a nonsteroidal compound with tissue specific estrogen receptor (ER) agonist or antagonist activities. In animals, SERMs may produce morphologic changes in hormonally-sensitive tissues like the mammary gland. Mammary glands from female rats given the SERM LY2066948 hydrochloride (LY2066948) for 1 month at ≥ 175 mg/kg had intralobular ducts and alveoli lined by multiple layers of vacuolated, hypertrophied epithelial cells, resembling in part the morphology of the normal male rat mammary gland. We hypothesized that these SERM-mediated changes represented an androgen-dependent virilism of the female rat mammary gland. To test this hypothesis, the androgen receptor antagonist flutamide was co-administered with LY2066948 (175 mg/kg) to female rats for 1 month. Female rats given SERM alone had hyperandrogenemia and the duct and alveolar changes described here. Flutamide cotreatment did not affect serum androgen levels but completely blocked the SERM-mediated mammary gland change. In the mouse, a species that does not have the sex-specific differences in the mammary gland observed in the rat, SERM treatment resulted in hyperandrogenemia but did not alter mammary gland morphology. These studies demonstrate that LY2066948 produces species-specific, androgen-dependent mammary gland virilism in the female rat.

The reversible effects of raloxifene on luteinizing hormone levels and ovarian morphology in mice.

Raloxifene is a selective estrogen receptor modulator that has estrogen agonist effects on bone and serum lipids and estrogen antagonist effects on breast and uterine tissues. This study assessed the effects of raloxifene hydrochloride (HCl) treatment on circulating luteinizing hormone (LH) levels and ovarian morphology in sexually mature, 15-week-old, female CD-1 mice. Mice were maintained on diets providing average daily doses of 0 or 233 mg/kg raloxifene for 2 weeks (Study 1) or 0, 7.9, or 236 mg/kg raloxifene for 4 weeks (Study 2). At the end of the treatment period, blood samples were collected every 2 hours for 24 h in Study 1 (5 mice per group) and at 10:00 a.m. and 10:00 p.m. in Study 2 (8 mice per group). Serum LH levels were measured by radioimmunoassay. Ovarian histomorphology was evaluated in the 10 mice per group (Study 1) and the 8 mice per group (Study 2). For the reversibility phase (Study 2), mice were fed untreated diets for 3 weeks; serum LH levels and ovarian histomorphology were then assessed. Raloxifene treatment at 233 mg/kg/day for 2 weeks (Study 1) significantly elevated circulating LH levels by 4- to 7-fold compared with control. Raloxifene-treated mice had elevated LH levels sustained over the 24-h sampling period and did not exhibit the preovulatory LH surge evident in some control mice at the 4:00 p.m., 6:00 p.m., and 8:00 p. m. time points. Mice treated with 236 mg/day raloxifene for 4 weeks (Study 2) had elevated LH levels (4.4-fold compared to control), whereas mice exposed to 7.9 mg/kg/day raloxifene had a slight, nonsignificant increase in LH (2-fold compared to control). In both dose groups, LH levels were indistinguishable from controls 3 weeks after raloxifene treatment was discontinued. The ovaries in six of the eight mice treated with 7.9 mg/kg/day raloxifene had dilated and/or anovulatory follicles. One mouse in this group had a single hemorrhagic follicle; however, corpora lutea distribution was normal, indicating that ovulation was occurring. Raloxifene-treated mice in Study 1 and mice treated with a comparable raloxifene dose (236 mg/day) in Study 2 had histomorphological changes in the ovary indicative of arrested follicular maturation, including anovulatory hemorrhagic follicles, some developing follicles, and very few corpora lutea. At the end of the reversibility phase, hemorrhagic follicles were no longer evident and follicular maturation and corpora lutea distribution were normal. Raloxifene treatment in mice produces a dose-dependent, sustained elevation in serum LH levels and is associated with changes in ovarian follicular morphology. These changes are reversible upon discontinuation of raloxifene treatment.

Steroid treatment fails to induce an afternoon luteinizing hormone or prolactin surge in rats exposed to short-term constant light at the time of ovariectomy.

The present study investigated the effects of short-term exposure to constant light, initiated at the time of ovariectomy, on the ability of estradiol (E2) treatment alone or in conjunction with progesterone (P) to induce afternoon surges of LH and prolactin (PRL). Adult Fischer 344 rats, which had been ovariectomized (OVX) and placed into constant light (LL) on day 0, were implanted with Silastic capsules containing E2 on day 7 and an atrial cannula on day 8. On the following day (day 9), hourly blood samples were collected between 12.00 and 20.00 h from LL-exposed animals which had received E2 treatment alone or from LL E2-treated animals which had also received P at 12.20 h. Blood samples from control animals which were OVX and treated with E2, but maintained under a 12-hour light:12-hour dark photoperiod, were also collected. Exposure to 9 days of LL abolished the ability of E2 treatment to induce an afternoon surge of LH or PRL. The addition of P treatment to LL E2-treated animals failed to reinstate an LH or PRL surge. While P treatment in LL E2-treated animals induced a rise in PRL levels, it is unlikely, given the timing, duration, and magnitude of PRL release, that this enhancement was initiated by the same mechanisms which normally generate the afternoon surge. The results from the present study demonstrate that short-term exposure to LL, initiated at the time of OVX, abolishes the E2-induced afternoon surges of LH and PRL.

Lack of effect of hyperprolactinemia on serotonin turnover in ovariectomized and ovariectomized estrogen-treated rats☆

Hyperprolactinemia suppresses luteinizing hormone (LH) and prolactin (PRL) secretion under a variety of experimental conditions. The secretion of both of these hormones is regulated at the hypothalamic level by several neurotransmitters, including serotonin (5-HT). Therefore, we examined the effect of hyperprolactinemia on 5-HT neuronal activity in key hypothalamic areas that are rich in 5-HT terminals and which are known to regulate the release of LH and PRL. Young cycling virgin rats were ovariectomized (day 0). From days 11-16, animals were injected with ovine prolactin (oPRL, 4 mg/kg, s.c.) or vehicle every 8 h. On day 14, one-half of the oPRL- and vehicle-treated rats were implanted with 20-mm long Silastic capsules containing estradiol (180 micrograms/ml). On day 16, animals were killed at 08.00, 12.00 or 18.00 h or treated with pargyline (75 mg/kg) and killed 10 min later. Trunk blood was collected and serum was radioimmunoassayed for LH and endogenous rat PRL (rPRL). Brains were removed, frozen, sectioned and the medial preoptic, suprachiasmatic, and arcuate nuclei, median eminence and globus pallidus were microdissected. Serotonin was measured using high pressure liquid chromatographic methodology. We were unable to detect any feedback effect of hyperprolactinemia on 5-HT turnover in any brain area of ovariectomized or ovariectomized estradiol-treated rats at any time of day that we examined. Several potential reasons for the absence of an effect of hyperprolactinemia on serotonergic function are discussed.

Effects of the serotonin antagonist amesergide on reproduction in female rats

Amesergide, a serotonin (5-HT2) antagonist intended to treat depression, was administered orally to female CD rats (20/group) at doses of 0, 3, 10, or 30 mg/kg to evaluate effects on mating, fertility, litter size, live birth index (100 x total liveborn progeny/litter size), progeny survival, and weight gain of each litter. The treatment period extended from two weeks prior to mating through postpartum day 21 to cover possible effects of estrous cycle, mating, gestation, and postpartum events. Twenty additional female rats were given 30 mg/kg through gestation day 18, after which they received the acacia vehicle (recovery group). All females were allowed to deliver naturally and rear their progeny. On postpartum day 8, progeny in the control, 30 mg/kg and 30 mg/kg recovery groups were removed from dams for 4 h. Progeny were weighed as litters, returned to the dams for a 1-h nursing period, and then weighed again to provide an indication of milk intake. Mating and fertility, using the present study design, were not affected by treatment with amesergide. No effects were observed on litter size, live birth index, or progeny survival. In contrast, treatment with amesergide throughout gestation and lactation produced a significant dose-related depression in progeny body weight gains. However, when treatment was discontinued after day 18 of gestation (30 mg/kg recovery group), progeny body weight gains did not differ from those of the control group.(ABSTRACT TRUNCATED AT 250 WORDS)