Alteration in ovarian gene expression in response to 2,3,7,8-tetrachlorodibenzo-<em>p</em>-dioxin: reduction of cyclooxygenase-2 in the blockage of ovulation.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a reproductive toxicant and endocrine disrupter that is known to block ovulation. This study was designed to investigate alterations in relevant ovarian genes that may be involved in the blockage of ovulation by TCDD in immature intact rats primed with equine chorionic gonadotropin (eCG). In this ovulation model, rats were given either 32 microg/kg TCDD or corn oil by gavage on 25 days of age. The next day, eCG (5 IU) was injected subcutaneously (s.c.) to stimulate follicular development. Ovulation occurs 72 h after administration of eCG in controls of this model. TCDD blocked ovulation at the expected time and also reduced both ovarian and body weights. At 72 h after eCG (the morning after expected ovulation), TCDD did not alter significantly serum concentrations of progesterone (P4) and androstenedione (A4). However, estradiol (E2) was significantly higher at 72 h after eCG in TCDD-treated rats when compared with controls. Western blots revealed that ovarian CYP1A1 was induced by TCDD. In addition, the aryl hydrocarbon receptor (AhR) and AhR nuclear translocator (ARNT) were down- and up-regulated by TCDD, respectively, indicating that AhR-mediated signal transduction was altered in the ovary. Ovarian estrogen receptor (ER)alpha, ER beta and progesterone receptor (PR) were not altered significantly by TCDD, but ovarian glucocorticoid receptor (GR) was increased at 24h after TCDD and decreased at 72 h after eCG when compared with controls. TCDD induced the early appearance of ovarian plasminogen activator inhibitor type-1 (PAI-1), plasminogen activator inhibitor type-2 (PAI-2), urokinase plasminogen activator (uPA), and tissue plasminogen activator (tPA) at 24h after dosing when compared with controls. On the morning after ovulation (72 h after eCG), no significant differences between control and TCDD-treated rats were observed except that TCDD had still increased tPA and decreased PAI-2 when compared with controls. Interestingly, ovarian COX-2 was induced on the morning after ovulation (72 h after eCG) in controls, but was greatly inhibited in TCDD-treated rats at that time. On the other hand, COX-1 was constitutively expressed throughout the ovulatory period and remained unaffected by TCDD. Immunolocalization of COX-2 in the ovary revealed that TCDD inhibited COX-2 expression in the granulosa cell layer when assessed in the morning of expected ovulation. In conclusion, AhR signaling is activated in the ovary by TCDD and inhibition of COX-2 appeared to be a critical step in the TCDD blockage of ovulation because blockage or reduction of COX-2 expression is well known to be associated with failure of ovulation

2,3,7,8-Tetrachlorodibenzo-<em>p</em>-dioxin decreases responsiveness of the hypothalamus to estradiol as a feedback inducer of preovulatory gonadotropin secretion in the immature gonadotropin- primed rat.

Sprague-Dawley rats (23-day-old) were dosed with TCDD (32 mug/kg) in corn oil or vehicle alone. Equine chorionic gonadotropin (eCG) was injected (5 IU, sc) 24 h later to induce follicular development. Another 24 h later, half of TCDD- or corn oil-treated rats were injected (sc) with 17 beta -estradiol-cypionate (ECP, at 0.004 to 0.5 mg/kg), Blood and ovaries were collected on expected proestrous (preovulatory period) at 51, 54, and 58 h after eCG injection as well as in the morning after ovulation (72 h after eCG), Serum concentrations of 17 beta -estradiol (E), progesterone (P), luteinizing hormone (LH), and follicle-stimulating hormone (FSH) were determined by radioimmunoassay, The number of ova shed was measured at 72 h after injection of eCG by irrigating ova from oviducts, During the preovulatory period (similar to 58 h after eCG injection), a circulating level of 70-100 pg E/ml coincided with LH and FSH surges and later normal ovulation of 10 to 12 ova/rat was observed in controls. However, the same concentration of E was not associated with LH and FSH surges in rats treated with TCDD (32 mug/kg), resulting in reduced ovarian weight gain and reduction of ovulation by 70 to 80% (2-3 ova/rat), Blockage of the gonadotropin surge, reduced ovarian weight gain, and ovulation were all reversed completely by the lowest effective dose of ECP (0.1 mg/kg), At 72 h after eCG, serum P secretion was reduced and serum E levels were significantly increased compared to those of corn oil-treated controls. ECP alone had no effect on serum P levels at any time point, but in rats treated with TCDD and ECP, both the reduction of P (at 58 and 72 h) and the increase in E secretion (72 h) were completely reversed. Further studies confirmed that restoration by ECP of gonadotropin surges and associated ovulation could not be attained until circulating levels of E rose sufficiently high to trigger the LH and FSH surges. The new action threshold of E for inducing gonadotropin surges in rats treated with TCDD (32 mug/kg) was determined to be eight- to 10-fold higher than that in controls. Thus, it is apparent that TCDD decreased the responsiveness of the hypothalamus to E as a feedback inducer of preovulatory gonadotropin secretion

Calcitonin Directly Inhibits Luteinizing Hormone-Stimulated Progesterone Production in Granulosa Cells of the Largest Follicle of Hen

The present study was performed to demonstrate the presence of receptors for avian calcitonin (cCT) in ovarian granulosa cells of the largest follicle of laying hens and to determine the effect of cCT on LH-stimulated P4 production. The cCT binding in the granulosa cells of the largest follicle was determined using a [125I]CT binding assay. The binding properties in the granulosa cells satisfied the criteria of a receptor-ligand interaction in terms of specificity, saturation, high affinity and limited capacity. During incubation of ovarian granulosa cells in vitro, cCT alone did not affect the P4 production alone, but cCT decreased the amount of LH-stimulated P4 production and the sensitivity to LH. The present study suggests that cCT may act directly on the granulosa cell of the largest follicle via its receptor binding and inhibit P4 production in response to LH in hens