Search

Recently, Gray and Ostby (Toxicol. Appl. Pharmacol. 133, 285-294, 1995) reported that in utero and lactational TCDD exposure causes striking abnormalities in the rat female reproductive system, including reduced fecundity and vaginal threads. The mechanism by which TCDD induces such abnormalities is unknown. Thus, we sought to determine: (1) whether TCDD reduced fecundity by destroying ovarian follicles and (2) whether the vaginal threads resulted from a TCDD-induced developmental defect during embryogenesis or abnormal vaginal opening at puberty. Pregnant Holtzman rats were treated with 1.0 microgram TCDD/kg or vehicle by a single oral dose on gestation day (GD) 11, 15, or 18. Female offspring were monitored for vaginal opening and terminated on postnatal days 2, 21, and 42. The reproductive tract was removed and evaluated for structural abnormalities. The number of primordial follicles also was determined for each ovary. TCDD exposure on GD 11, 15, or 18 did not change the day of vaginal opening, affect ovarian morphology, or reduce the number of primordial follicles. However, this exposure induced the cleft clitoris and vaginal thread originally described by Gray and Ostby (1995) in approximately 55-96% and 36-44% of the litters in our study, respectively. Histologically the thread presented as a thick cord of mesenchyme surrounded by epithelial cells. This defect was clearly visible in histological sections at birth and was noted in the closed vaginas of prepubertal animals. These data suggest that in utero and lactational exposure to TCDD does not reduce the size of the primordial follicle pool; however, it induces developmental abnormalities in the vaginal canal.

Previous studies have determined that 4-vinylcyclohexene diepoxide (VCD) causes specific destruction of oocytes contained in small pre-antral (primordial and primary) ovarian follicles of Fischer 344 rats following 30 days of daily dosing with VCD. The purposes of this study were to identify the type of VCD-induced cell death occurring in small pre-antral follicles and to determine the earliest time following the onset of dosing when evidence of follicular destruction could first be detected. A significant decrease in the number of oocytes contained in small pre-antral follicles in ovaries of rats after 15 days of daily dosing (ip) with VCD (80 mg/kg) had been observed in preliminary experiments. Therefore, a study was conducted to determine the time of the onset of this follicular destruction by examination of follicular DNA integrity. Female Fischer 344 rats were dosed daily (80 mg/kg, i.p.) for 6, 8, 10, 12, or 14 days, and ovaries were removed 1, 4, or 24 hr after the final dose. Small pre-antral follicles (25-100 microns) were isolated by gentle dissociation of ovaries with collagenase, and follicles were sorted with micropipets. Genomic DNA was isolated from follicles and radiolabeled with [32P]dideoxy ATP, and the degree of fragmentation quantified by agarose gel electrophoresis and autoradiography. Degradation of DNA was evaluated by 32P content in low-molecular-weight fragments ( < 4 kilobase pairs). Degradation of DNA was not observed in follicles collected 24 hr after the final dose on any day. However, a random pattern of DNA degradation was observed, and was significantly greater (p < 0.05) compared with controls, when follicles were collected 4 hr following VCD administration on Days 10 and 12, but not on Days 6 or 8, of dosing. Although not significant, there was also evidence of DNA degradation in dosed animals on Day 14. Histological evaluation of small pre-antral follicles in ovarian sections during the early stages of VCD-induced DNA degradation (Day 10; 4 hr) demonstrated margination of chromatin along the nuclear membrane in oocytes and disruptions in focal contact between granulosa cells and oocytes, both features indicative of apoptosis. Furthermore, there was no sign of ruptured membranes in granulosa cells or oocytes or of an inflammatory response, characteristics of necrosis (pathological cell death). Whereas biochemical and morphological evidence of follicular destruction was seen 4 hr after dosing on Day 10, numbers of oocyte-containing primordial and primary follicles in VCD-treated animals were not different from controls at that time. These results demonstrate that the initial evidence of impending destruction of small pre-antral follicles is first consistently visualized following 10 days of daily dosing with VCD, although a measurable reduction in oocyte numbers has not yet occurred. Despite the fact that internucleosomal cleavage of genomic DNA was not observed, morphological evaluations support that granulosa cells and oocytes in primordial and primary follicles are destroyed via the induction of apoptosis.

4-Vinylcyclohexene diepoxide (1-epoxyethyl-3,4-epoxycyclohexane, VCD), an industrial chemical, is a potential health hazard because it destroys oocytes in small preantral follicles in rats. We proposed that VCD destroys oocytes in these follicles because of their reduced capacity to detoxify VCD (convert VCD to tetrol, 4-(1,2-dihydroxy)ethyl-1,2-dihydroxycyclohexane). Ovaries, livers, and adrenal glands were collected from immature and mature Fischer 344 rats. Tissues were dissociated and ovarian tissue was separated into distinct follicular fractions. Tissues were incubated with [14C]VCD and media were assayed for [14C]tetrol by HPLC. In immature rats, conversion of VCD to tetrol in large preantral follicles and hepatocytes was 1.5-fold greater than in small preantral follicles and 4-fold greater than in ovarian interstitial cells (p < 0.05). In adults, conversion of VCD to tetrol in large preantral follicles and hepatocytes was, respectively, 3- and 10-fold greater than in small preantral follicles and interstitial cells (p < 0.05). Compared with immature rats, all tissues from adult rats converted more VCD to tetrol (p < 0.05). These data demonstrate that interstitial cells and small preantral follicles from adult and immature rats have a reduced capacity to convert VCD to tetrol compared to large preantral follicles and liver cells. This may explain their increased susceptibility to VCD-induced ovotoxicity. Furthermore, adult rats may be less susceptible to VCD-induced ovotoxicity than immature rats.