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research-article |
Center for Reproductive Sciences3 and Departments of Internal Medicine4 and Molecular and Integrative Physiology,5 University of Kansas Medical Center, Kansas City, Kansas 66160
Department of Animal Physiology,6 University of Warmia and Mazury in Olsztyn, Olsztyn 10-718, Poland
ABSTRACT
The aryl hydrocarbon receptor (AHR) mediates the effects of many endocrine disruptors and contributes to the loss of fertility in polluted environments. While previous work has focused on mechanisms of short-term endocrine disruption and ovotoxicity in response to AHR ligands, we have shown recently that chronic exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) induces premature reproductive senescence in female rats without depletion of ovarian follicular reserves. In the current study, premature reproductive senescence was induced using a range of low-dose exposure to TCDD (0, 1, 5, 50, and 200 ng kg–1 wk–1) beginning in utero and continuing until the transition to reproductive senescence. Doses of 50 and 200 ng TCDD kg–1 wk–1 delayed the age at vaginal opening and accelerated the loss of normal reproductive cyclicity with age without depletion of follicular reserves. Serum estradiol concentrations were decreased in a dose-dependent fashion (
5 ng kg–1 wk–1) across the estrous cycle in perisenescent rats still displaying normal cyclic vaginal cytology. Serum FSH, LH, and progesterone profiles were unchanged by TCDD. The loss of reproductive cyclicity following chronic exposure to TCDD was not accompanied by decreased responsiveness to GnRH. Ovarian endocrine disruption is the predominant functional change preceding the premature reproductive senescence induced by chronic exposure to low doses of the AHR-specific ligand TCDD.
aging, aryl hydrocarbon receptor, estradiol, menopause, ovary, pituitary, rat, reproduction, toxicology
The aryl hydrocarbon receptor (AHR) is conserved across phyla, and AHR knockout mice display a number of defects, suggesting that in addition to transducing xenobiotic signals, the AHR is involved in the regulation of fundamental physiological processes [1–4]. TCDD (2,3,7,8-tetrachloro-dibenzo-p-dioxin) is the most potent toxicant of the AHR ligands due to its accumulation in the environment, resistance to breakdown, and impact on multiple organ systems. Short-term exposure to high doses of TCDD and similar ligands induces endocrine disruption, endometriosis, teratogenesis, and abortion [5–7]; alters sexual behavior; decreases spermatogenesis; and diminishes fertility [8]. However, few studies have addressed the impact of realistic, chronic activation of the AHR pathway on aging of the reproductive system.
Current demographic and economic trends, including an aging of the population seeking pregnancy, rising body fat content, and progressive industrialization, all increase exposure to lipophilic pollutants such as TCDD and the subsequent risk of chronic reproductive sequelae [5, 9]. Our group recently monitored reproductive function in female rats exposed chronically to TCDD across their reproductive lifespan, revealing a dose-dependent acceleration of reproductive senescence [10]. The present study was carried out to evaluate the sensitivity of the aging reproductive system to AHR activation and to determine the mechanisms underlying premature reproductive senescence in a simulation of polluted environments.
Adult pregnant Sprague Dawley dams (n = 15; Charles River Laboratories) were purchased and housed under a 12L:12D photoperiod (0600 h–1800 h) and controlled temperature (23°C ± 2°C) and humidity. Food (Purina Rat Chow; Ralston Purina Co.) and water were provided ad libitum. TCDD (CAS 1746-01-6; MW: 321.9; purity > 99%) was obtained from Cambridge Isotope Laboratories, Inc (Lenexa, KS). All procedures were approved by the University of Kansas Medical Center Institutional Animal Care and Use Committee.
Pregnant dams were dosed with corn oil vehicle or TCDD (0, 1, 5, 50, or 200 ng kg–1 wk–1 by gavage) on Days 14 and 21 of gestation and Days 7 and 14 postnatally to expose pups gestationally and latationally. Female pups (n = 10 per dose) were weaned on postnatal Day 21 and given these same doses weekly across their reproductive lifespan until killing near the transition to reproductive senescence. The doses of TCDD were chosen to approximate a daily exposure of 150 to 30 000 pg kg–1 wk–1 or a total cumulative exposure of roughly 50 to 10 000 ng kg–1 across the normal reproductive lifespan. The lowest dose was based upon the estimated cumulative exposure of women in the general population of the United States to dioxins across their reproductive life span (50 years x 365 days–1 year x 3 pg kg–1 day–1 average exposure 
55 ng kg–1 cumulative dose for women [11] vs. 52 weeks x 1 ng kg–1 wk–1 52 ng kg–1 cumulative dose for rats). The higher doses mimic exposure of high-risk populations to dioxinlike compounds [12] and have been well characterized for other endpoints in previous chronic studies [13].
Female pups were monitored daily for vaginal opening beginning at weaning on Day 21 of age. Beginning at vaginal opening, reproductive cycles were monitored by vaginal cytology for 7–10 days each month until the transition to reproductive senescence, as defined by the occurrence of two consecutive prolonged (6–9 days) cycles [14, 15].
In order to assess early changes in the reproductive endocrinology with chronic AHR ligand exposure, normally cycling rats (250–300 g body weight) accustomed to handling were cannulated and serial blood samples collected when the transition to reproductive senescence was first evident in the highest-dose group (9 months). Rats (n = 6 to 8 per group, 36 total) were cannulated with a sterile silicon jugular cannula on the morning of proestrus under anesthesia using ketamine (80 mg/kg) and xylazine (8 mg/kg). Cannulation and anesthesia were scheduled to avoid effects on the daily neural signal for the gonadotropin surge in the rat [16, 17]. Blood (300 µl per sample) was collected at 0900, 1700, and 2200 h on proestrus and at 0900 and 1700 h on estrus and diestrus and replaced with equal volumes of normal saline. This sampling technique and schedule has been used successfully in past studies of female reproductive endocrinology in rats [18–20]. Cannula patency was maintained during this infrequent sampling with Burr solution (25% glycerol in normal saline, heparin 25 IU/ml) equivalent to the residual volume of the jugular cannula.
In a separate experiment to test the impact of TCDD on the responsiveness of pituitary glands of rats exposed to TCDD, aged rats (11 months old, n = 6 per group) from vehicle control and 200 ng kg–1 wk–1 TCDD-treated groups were ovariectomized and injected with GnRH (1 µg per rat s.c.; Sigma Chemical, St. Louis, MO) 48 h later. Blood samples were collected at ovariectomy (OVX), at GnRH injection, and at 1 h after treatment with GnRH. All serum samples were separated by centrifugation at 1700 x g at 4°C for 15 min and were kept in a freezer at –80°C for determination of FSH and LH.
Assay for 17ß-estradiol, Progesterone, FSH, and LH
Serum hormone concentrations of FSH and LH were measured by the Ligand Assay and Analysis Core in the Center for Research in Reproduction at the University of Virginia (Charlottesville, VA) [10, 21]. FSH was measured by RIA, and LH was measured by a modified two-site sandwich ELISA with assay sensitivities of 0.07 ng/ml and 1.5 ng/ml and intraassay coefficients of variance of 4.7% and 4.6%, respectively. Serum E2 and P4 were measured by ELISA kits (E2, P4; Estradiol EIA, DSL-10–4300; Progesterone EIA, DSL-10–3900; Diagnostic Systems Laboratories Inc., Webster, TX). The assay sensitivity was 7 pg/ml for E2 and 0.13 ng/ml for P4. The intra-assay coefficients of variation for E2 and P4 were 4.8% and 3.4%.
Evaluation of Follicular Reserves
All rats were killed at 11 mo, and ovaries from the control and 200 ng kg–1 groups were fixed in 4% paraformaldehyde, dehydrated through graded ethanols, embedded in paraffin, and sectioned (8-µm thickness) for histological analysis. Follicles were classified with one layer of granulosa cells, two to five layers of granulosa cells, as well as antral follicles [10, 22], and counted every fifth section, and a total of 15 sections from the midsagittal region of each ovary were used. Follicles were enumerated only in the section through the nucleolus to avoid overcounting.
All data were presented as mean ± SEM. The effect of TCDD on the onset of puberty was analyzed using a one-way analysis of variance (ANOVA). Serum concentrations of E2, P4, FSH, and LH were compared using two-way ANOVA with TCDD and time of sampling as main effects. Similarly, ANOVA with OVX, GnRH, and TCDD as main effects followed by Fisher PLSD test was used to assess the differences in responsiveness of the pituitary to GnRH. Follicular class and number were analyzed by Student t-test. Percentages of reproductive cyclicity with aging among groups were tested by chi-square analysis. A value of P < 0.05 was considered significant.
Effect of TCDD on Vaginal Opening and Vaginal Cytology
Vaginal opening was slightly but significantly delayed by a dose of 200 ng kg–1 wk–1 TCDD compared with the control (Fig. 1). There also was a tendency (P 
0.10) toward delayed vaginal opening at the 50 ng kg–1 wk–1 dose, but this was not significant. The transition to reproductive senescence was accelerated by TCDD in a dose-responsive manner (Fig. 2). Beginning at age 9 mo, normal cyclicity was decreased in the 50 and 200 ng kg–1 wk–1 groups. In contrast, the majority of rats in the control, 1, and 5 ng kg–1 wk–1 TCDD groups maintained normal cyclicity throughout the experiment, although there was a transient decrease in normal cyclicity at 8 mo in the 5 ng kg–1 wk–1 group as well.
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No differences in number or size distribution of ovarian follicles were found between the control and 200 ng kg–1 wk–1 TCDD groups (Fig. 3).
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Secretion of Steroid Hormones and Gonadotropins Across the Estrous Cycle
TCDD treatment decreased serum E2 concentrations at all time points across the estrous cycle in a dose-dependent manner with a lowest observable effect at 5 ng kg-1 wk–1 (Fig. 4A). Serum concentrations of P4, FSH, and LH across the estrous cycle were unaffected by chronic exposure to TCDD, although peak LH concentrations of TCDD-treated groups tended to be higher on proestrus than the control group (P = 0.10; Figure 4D).
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Responsiveness of Pituitary Gland to GnRH
GnRH treatment significantly increased serum FSH and LH concentrations in both the control and 200 ng kg–1 wk–1 groups by 1 h after injection (Fig. 5). TCDD treatment was associated with greater concentrations of LH at 1 h after GnRH than in controls. There were no significant changes in FSH concentrations between the control and 200 ng kg–1 wk–1 groups, and FSH concentrations were similarly increased by OVX in both groups.
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Delayed puberty, a dose-dependent loss of reproductive cyclicity with age, decreased serum E2 concentrations across the estrous cycle, and unchanged follicular reserves, together with an unimpaired sensitivity of pituitary gland to GnRH were the salient effects of chronic exposure to TCDD in the current study. This study is in agreement with our earlier work concerning the impact of lifetime exposure to AHR ligands on the loss of reproductive function with age [10]. Taken together, these data suggest that chronic exposure to TCDD hastens reproductive aging in females predominantly via local ovarian endocrine disruption without depletion of follicular reserves.
We found that chronic exposure of 200 ng kg–1 wk–1 delayed vaginal opening significantly, as has been reported elsewhere [10, 23, 24], but the mechanisms underlying delayed puberty in rats exposed to TCDD are not yet clear. Chronic AHR activation led to a gradual prevalence of abnormal reproductive cycles in the current study, indicating a premature transition to reproductive senescence. This is in agreement with previous work using equal or higher doses of TCDD [10, 23, 25]. In the present study, chronic exposure to TCDD did not alter ovarian follicular reserves. Flaws et al. found that a single dose of 1 µg kg–1 wk–1 TCDD on gestational Day 11, 15, or 18 did not change numbers of primordial follicles in offspring [26]. In another study, numbers of primordial, primary, secondary, preantral, and antral follicles were not altered by an intrauterine and lactational exposure of 2.5 µg kg–1 wk–1 TCDD, but an inhibitory effect of TCDD on ovulation was observed [27]. Overall, premature reproductive senescence by chronic low-dose TCDD exposure does not involve depletion of follicular reserves.
In the current study, the predominant endocrine change preceding accelerated reproductive senescence due to TCDD was reduced serum E2 concentrations even at exposures as low as 5 ng kg–1 wk–1. This is in agreement with previous experiments in zebrafish [28], rodents [29, 30], nonhuman primates [31], and in vitro cell cultures [32, 33]. Most recently, a negative association between body burdens of AHR ligands and serum estradiol was found in pregnant women [34]. In particular, the endocrine changes seen here are in close agreement with the observations of Hutz and colleagues following a single intrauterine exposure to 1 µg TCDD kg–1 in rats [30]. Nonetheless, some workers showed decreased serum concentrations of P4 without changes in serum E2 levels [35, 36], or no acute effect of TCDD on either ovarian steroid [37]. We previously found no effect of high-dose acute exposure to TCDD on estradiol catabolism in rats [38], suggesting that TCDD exposure decreases serum E2 through effects on estrogen biosynthesis. This may involve altered gonadotropin receptor expression or activation [39] or alterations in steroidogenic enzymes [32, 40].
Previously, we found that high doses of TCDD treatment reduced FSH and LH secretion at ovulation but induced a premature surge, suggesting adverse effects of TCDD on hypothalamus or pituitary gland [8, 41, 42]. Interestingly, in the current study chronic exposure of TCDD did not significantly alter the patterns of serum FSH or LH across the estrous cycle compared with controls. These results imply an unimpeded function of hypothalamus and pituitary gland in females exposed chronically to our highest dose of TCDD, and this is further verified by the equal or greater responsiveness of TCDD-treated, ovariectomized females to GnRH. Indeed, TCDD-treated rats often remained cyclic with normal gonadotropin profiles despite suppressed E2, again failing to support any direct inhibitory effect of TCDD on the hypothalamo-hypophyseal axis.
In summary, chronic environmentally relevant exposure to TCDD beginning in utero accelerated the transition to reproductive senescence, as indicated by increasing abnormal reproductive cyclicity with age in female rats. This premature loss of cyclicity was triggered neither by depletion of ovary follicular reserves, nor by altered responsiveness of pituitary gland to GnRH. It is more likely that TCDD exerts direct effects on ovarian function. The mechanisms of premature reproductive senescence during chronic exposure to AHR ligands appear distinct from those following acute high-dose exposures.
ACKNOWLEDGMENTS
We thank Dr. Renata Ciereszko for critical reading of the manuscript.
FOOTNOTES
1Supported by the United States National Institutes of Health (NIH/NIEHS ES012916, to B.K.P.), the NIH Center for Reproductive Sciences, the University of Kansas Medical Center, and the University of Virginia NIH Center for Research in Reproduction Ligand Analysis Core. 
Correspondence: 2Brian K. Petroff, Center for Reproductive Sciences, Department of Internal Medicine, University of Kansas Medical Center, 3901 Rainbow Blvd., Kansas City, KS 66160. FAX: 913 588 7180; e-mail: bpetroff{at}kumc.edu
Received: 18 May 2006.
First decision: 10 June 2006.
Accepted: 10 October 2006.
REFERENCES
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