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Biology of Reproduction 66, 1621-1626 (2002)
© 2002 Society for the Study of Reproduction, Inc.

Regular Article

In Utero and Lactational Exposure to 2,3,7,8-Tetrachlorodibenzo-p-Dioxin and 2,3,4,7,8-Pentachlorodibenzofuran Reduces Growth and Disrupts Reproductive Parameters in Female Rats1

Travis B. Salisburya, and Jennifer L. Marcinkiewicz2,a

a Department of Biological Sciences, Kent State University, Kent, Ohio 44242


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) and 2,3,4,7,8-pentachlorodibenzofuran (PCDF) are widespread environmental pollutants. TCDD is well known for its adverse effects on female reproduction when administered acutely to immature or adult rats. It is also known that fetal/neonatal exposure to this compound alters reproductive parameters. It is unknown whether exposure to PCDF causes similar adverse effects in offspring. The objectives of the study were to investigate the effects of in utero and lactational (IUL) exposure to TCDD and PCDF on subsequent growth, estrous cycles, and ovulation. Additionally a gonadotropin-primed immature rat model was used to investigate possible direct effects on the ovary after IUL exposure to TCDD (2.5 µg/kg) by evaluating 1) ovarian morphometrics and 2) serum estradiol concentrations. Body weights were reduced in animals with IUL exposure to TCDD and PCDF relative to those in controls at 10 days of age (P < 0.05 for each), and this difference was maintained until termination of the experiment at 125–165 days of age (P < 0.05). Exposure to TCDD or PCDF also disrupted regular estrous cycles and inhibited ovulation rate. On Day 23 (before eCG stimulation), ovaries from animals exposed to TCDD contained the same number of primordial, primary, secondary, preantral, and antral follicles as ovaries from control animals. On Day 25 (48 h after eCG stimulation), ovaries from TCDD-exposed rats had significantly fewer large preovulatory follicles when compared with ovaries from controls. The numbers of smaller follicles (both antral and small antral) were not different. Serum estradiol was significantly lower in TCDD-exposed animals 48 h after eCG stimulation.

follicle, follicular development, ovary, ovulatory cycle, toxicology


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Polychlorinated dibenzo-p-dioxins (PCDDs) and dibenzofurans (PCDFs) are environmental contaminants that accumulate in the environment because of their lipophilic properties and stability [1]. PCDDs and PCDFs are introduced into the environment in a variety of ways such as insecticide and herbicide use, the manufacture of paper pulp [1], and municipal waste incineration [2]. PCDDs and PCDFs have been found in trace amounts in wildlife species [3] and within human tissue and milk [2, 4, 5]. The dioxin congener 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is the most toxic and well-studied compound among the PCDDs [6] and has been shown to be a potent disrupter of developmental and reproductive systems in numerous species (reviewed in [7]). In contrast, there have been few studies examining the effects that 2,3,4,7,8-pentachlorodibenzofuran (PCDF) may have on reproduction and development. PCDF is considered the most active of the polychlorinated dibenzofurans, exhibiting the highest binding affinity for the aromatic hydrocarbon receptor (AHR) [6], and was therefore chosen for study in the present investigation. The actions of TCDD and PCDF are mediated through the AHR, a ligand-induced transcription factor. After binding, the activated ligand-receptor complex migrates from the cytoplasm to the nucleus and binds to specific regions on the DNA resulting in either gene expression or modulation of existing gene expression [8]. The AHR has been found in numerous tissues and species, including rat [9] and primate ovarian tissue [10]. Additionally, in mice, the AHR has been shown to be expressed in oocytes and granulosa cells of follicles in all stages of development [11].

After a single maternal administration, TCDD is transferred to offspring across the placenta and through the milk [12, 13]; therefore, this model has been termed in utero and lactational (IUL) exposure. Gray and Ostby [14] studied the effects of TCDD (1.0 µg/kg) on offspring of pregnant Long Evans Hooded and Holtzman rats given TCDD on Gestational Days (GDs) 8 and 15. The investigators found that TCDD caused malformations of external genitalia, characterized by partial clefting of the phallus and a persistent thread of tissue extending across the vaginal opening. Additionally, TCDD delayed puberty and altered estrous cyclicity in exposed offspring, inducing constant estrus at 125 days of age. The fertility rate was also compromised: strikingly, only 17% (GD 15) and 19% (GD 8) of exposed offspring were able to produce a fifth litter, versus 51% of control animals. Terminal ovarian weight (GD 8 and 15) and body weight (GD 15) were also reduced in exposed animals. In other studies, prenatal exposure to TCDD (1.0 µg/kg) on GD 15 reduced serum estradiol (E2) concentrations and altered estrogen receptor mRNA levels in the hypothalamus, pituitary, ovary, and uterus in female Holtzman pups on postnatal Day 21 [15].

In addition to IUL exposure to TCDD, acute administration of TCDD has also been shown to disrupt reproductive parameters in rats. Administering a single dose of TCDD (10 µg/kg) to adult Sprague Dawley rats disrupts estrous cyclicity and reduces ovulation rate [16]. Several investigators have demonstrated similar effects of TCDD (reviewed in [17]) and PCDF [18] when these compounds are administered to immature rats and followed by eCG treatment, demonstrating that these compounds reduce eCG-stimulated ovarian weight gain, alter serum levels of gonadotropins (LH and FSH), and reduce ovulation number.

The objectives of the present investigation were to evaluate the IUL effects of both TCDD and PCDF on growth, estrous cyclicity, and ovulation in female pups. The effects of IUL exposure to TCDD on eCG-stimulated follicular growth and serum E2 concentration were also evaluated.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Female Sprague Dawley rats (Hilltop, Scottsdale, PA) were obtained on Gestational Day 12. Rats were kept in constant L:D (on 0730 h and off 1930 h) and temperature (22°C) conditions and provided food and water ad libitum. On the morning of GD 15, pregnant rats were treated with a single oral dose, via gavage, of TCDD (1.0 µg/kg, n = 4; 2.5 µg/kg, n = 3), PCDF (1.0 µg/kg, n = 4; 10.0 µg/kg, n = 3), or vehicle (acetone:corn oil, 19:1, n = 5). The total volume administered ranged from 0.5–1.4 ml, depending on dose and body weight of dams. Control rats received a weight-matched volume of vehicle. The doses used, as well as the method and timing of administration, were based on those used in previous studies involving IUL exposure of rats to TCDD [14, 15]. Although the compounds are administered only once, it has previously been demonstrated that the maternal half-life is approximately 21 days, and there is significant transfer of TCDD to fetuses and neonates through both the placenta and the milk after this exposure paradigm [13]. On Postnatal Day 1, litter size was adjusted to 13 pups per litter to allow for similar lactational exposure between litters. TCDD and PCDF (98% purity) were purchased from Cambridge Isotope Laboratories (Woburn, MA). Acetone was purchased from Sigma (St. Louis, MO). Corn oil (Mazola) was purchased from a local grocery store. All studies were conducted with the approval of the Kent State University Animal Care and Use Committee and in accordance with the Guide for Care and Use of Laboratory Animals set forth by the Society for the Study of Reproduction. All materials contaminated with TCDD and PCDF were treated as hazardous waste. These materials included rat carcasses, blood and tissues, soiled bedding, cages, and disposable laboratory supplies. Investigators wore respirators, gloves, goggles, and laboratory coats when handling the powdered material and subsequent solutions. The disposal of contaminated materials was monitored by the university safety office.

On Postnatal Days 45–66, estrous cycles were monitored daily between 0900 and 1000 h. Estrous cycle stage was evaluated by inspection of cells obtained by vaginal lavage with 0.9% NaCl as previously described [19]. Diestrus and metestrus were collectively termed "diestrus." Pups from each group were weighed on Postnatal Days 10, 15, and 20. A final weight was obtained from rats at the time that ovulation was studied.

Ovulation was evaluated on the first morning of estrus (Days 125–165). A terminal weight was obtained, and the animals were decapitated after anesthesia with Metafane (Mallinckrodt Veterinary, Inc., Mundelein, IL). Ovaries with accompanying oviducts were isolated, and eggs were counted after the oviducts were flushed with hyaluronidase (10 µg/kg in 0.9% NaCl) using a 30-gauge needle. Eggs were counted under a dissecting microscope.

To evaluate follicle growth, a gonadotropin-primed immature rat model was used. On Postnatal Day 23, control rats and rats from dams treated with TCDD (2.5 µg/kg) on GD 15 were injected s.c. with eCG (10 IU in 0.1 ml of 0.9% saline; Sigma). Ovaries were collected on Day 23 (before eCG stimulation) and on Day 25 (48 h after eCG stimulation) for histologic analysis. Serum was collected on Day 23 (before eCG stimulation) and Day 25 (48 h after eCG stimulation) to evaluate serum E2 levels. Serum E2 concentrations were measured using an ultrasensitive E2 RIA kit (DSL-4800; Diagnostic Systems Laboratories Inc, Webster, TX).

Morphometric analysis was used to evaluate follicle numbers. Ovaries were fixed in Bouin solution (VWR, Bridgeport, NJ) for 48 h and processed through graded alcohols and Hemo-D (xylene substitute) (Fisher, Pittsburgh, PA) using standard protocols [20] and embedded in paraffin blocks. Eight-micron sections were sliced throughout the entire ovary, and every sixth section was stained with hematoxylin. Follicles were counted and classified as primordial, primary, secondary, or preantral. Primordial follicles were characterized as an oocyte surrounded by a single layer of flattened granulosa cells. Primary follicles were characterized as having a single layer of cuboidal granulosa cells. Secondary follicles contained two layers of granulosa cells, and preantral follicles contained numerous layers of granulosa cells but lacked an antrum. Follicles containing an antrum were characterized based on the greatest cross-sectional area of each follicle using the equation for an ellipse, {pi}(A)(B), in which A and B are one half the greatest length and width of the follicle, respectively. Antral follicles were placed into the following groups <50 000, 50 000–75 000, 75 000–100 000, and >100 000 µm2, a classification scheme similar to that used in previous studies [21]. Because small follicle numbers did not differ (primordial, primary, and secondary) on Day 23, and because follicles at this stage are relatively unresponsive to gonadotropins, only preantral and antral follicles were counted 48 h after eCG stimulation.

Values in all figures are given as mean ± SEM. Statistical analysis was performed by one-way ANOVA followed by the Fisher least significant difference test. Results were considered significantly different when P < 0.05.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Body Weight

Female pups exposed to TCDD and PCDF had significantly lower weights at several time points (Fig. 1, A–D). At 10 days of age, pups exposed to TCDD (maternal dose, 1 µg/kg) and to PCDF (maternal dose, 10 µg/kg) weighed less than controls (P < 0.05, Fig. 1A). Similarly, at 15 days, pups exposed to TCDD (maternal dose, 1 and 2.5 µg/kg) and PCDF (maternal dose, 10 µg/kg) had significantly lower body weights than controls (P < 0.05, Fig. 1B). At 20 days, pups exposed to TCDD (maternal dose, 1 and 2.5 µg/kg) and PCDF (maternal dose, 10 µg/kg) also had lower weights when compared with controls (P < 0.05, Fig. 1C). Terminal body weights, taken at the time rats were killed for ovulation evaluation, showed a significant 10%–15% reduction in body weight in animals exposed to TCDD (maternal dose, 1 and 2.5 µg/kg) and to PCDF (maternal dose, 10 µg/kg). Although terminal weights were obtained at different ages for individual rats, this difference in age does not explain the reduction in weight because the average age at termination of treated rats (137 days for 1 µg/kg TCDD; 148 days for 2.5 µg/kg TCDD; 140 days for 1 µg/kg PCDF; 140 days for 10 µg/kg PCDF) was greater than the age of control rats (129 days). The inhibitory effect of these compounds on body weight may be slightly underestimated, given the small difference in the mean age of rats at necropsy. Exposed rats were older because their disrupted estrous cycles (longer cycles with missed estrus days) made it more difficult to obtain rats in estrus (discussed subsequently).



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  		FIG. 1. Mean body weights of rats. A) Day 10 body weights of rats from dams treated with TCDD (1.0 µg/kg, n = 4; 2.5 µg/kg, n = 19), PCDF (1.0 µg/kg, n = 21, 10.0 µg/kg, n = 19), and vehicle (n = 18). B) Day 15 body weights of rats from dams treated with TCDD (1.0 µg/kg, n = 10; 2.5 µg/kg, n = 6), PCDF (1.0 µg/kg, n = 18; 10.0 µg/kg, n = 15), and vehicle (n = 13). C) Day 20 body weights of rats from dams treated with TCDD (1.0 µg/kg, n = 11; 2.5 µg/kg, n = 9), PCDF (1.0 µg/kg, n = 18; 10.0 µg/kg, n = 14), and vehicle (n = 8). D) Terminal body weights of rats from dams treated with TCDD (1.0 µg/kg, n = 6; 2.5 µg/kg, n = 6), PCDF (1.0 µg/kg, n = 18; 10.0 µg/kg, n = 5), and vehicle (n = 6). * Significant difference from the vehicle control group (P < 0.05)

Estrous Cycles

As indicated in Figure 2, IUL exposure to TCDD at maternal doses of 1.0 and 2.5 µg/kg significantly reduced the number of days spent in estrus (P < 0.05). Additionally, PCDF (maternal dose, 1.0 and 10.0 µg/kg) also reduced the total number of days spent in estrus. The decrease in estrus was accompanied by a comparable increase in diestrus.



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  		FIG. 2. The mean ± SEM percentage of the total number of days in estrus of rats from dams treated with vehicle (n = 8), TCDD (1.0 µg/kg, n = 6; 2.5 µg/kg, n = 6), and PCDF (1.0 µg/kg, n = 18; 10.0 µg/kg, n = 5). * Significant difference from the controls (P < 0.05)

Ovulation Rate

Rats with IUL exposure to TCDD at a maternal dose of 2.5 µg/kg had a reduced ovulation rate compared with controls (Fig. 3A). The ovulation rate in rats with exposure to the lower dose of TCDD was not significantly different from that in controls. Both doses of PCDF significantly reduced ovulation in exposed rats when compared with controls (P < 0.05). In all exposed groups, there were animals that failed to ovulate, compared with the control group in which all animals ovulated. The mean numbers of ova per rat were still significantly reduced in animals with PCDF exposure (maternal dose, 10 µg/kg) vs. control animals when nonovulating rats were excluded from the analysis (Fig. 3B). The group exposed to PCDF (maternal dose, 10 µg/kg) not only had the lowest ovulation rate, but also exhibited the greatest number of abnormal estrous cycles.



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  		FIG. 3. The mean ± SEM number of ova retrieved from rats within individual groups (ages, 125–165 days). Rats from dams treated with vehicle (n = 6) TCDD (1.0 µg/kg, n = 6; 2.5 µg/kg, n = 6), or PCDF (1.0 µg/kg, n = 18; 10.0 µg/kg, n = 5). A) Number of ova collected per total number of rats. B) Number of ova collected from ovulating rats (nonovulating rats were excluded). Numbers in parentheses above bars are the number of rats ovulating per total number of rats. * Significant difference from the controls (P < 0.05)

Ovarian Histology

Exposure to TCDD (maternal dose, 2.5 µg/kg) did not significantly alter the number of primordial, primary, secondary, or preantral follicles (Fig. 4A) in immature rat ovaries on Day 23. Additionally, the number of antral follicles of all sizes was not significantly different when compared with that in controls (Fig. 4B). In contrast, 48 h after eCG stimulation (Day 25), animals exposed to TCDD (maternal dose, 2.5 µg/kg) had significantly fewer large preovulatory follicles when compared with controls (Fig. 4D). The number of preantral and antral follicles in other smaller-sized classes of follicles was not significantly different when compared with that in controls (Fig. 4, C and D). Accompanying the reduction in number of large antral follicles in TCDD-exposed animals was an apparent increase in atresia characterized by numerous pyknotic nuclei in granulosa cells. Only healthy follicles were included in the analysis of follicle number.



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  		FIG. 4. Follicle numbers (n = 5 rats per group). A) The mean number of primordial, primary, and secondary follicles of Day 23 rats from pregnant dams treated with vehicle or TCDD (2.5 µg/kg). B) The mean number of preantral and antral follicles of Day 23 rats from pregnant dams treated with vehicle or TCDD (2.5 µg/kg). C) The mean number of preantral follicles and antral follicles <50 000 µm2 of Day 25 (48 h after eCG stimulation) rats from pregnant dams treated with vehicle or TCDD (2.5 µg/kg). D) The mean number of antral follicles of Day 25 (48 h after eCG stimulation) rats from pregnant dams treated with vehicle or TCDD (2.5 µg/kg). * Significant difference from the controls (P < 0.05)

Estradiol

Serum E2 levels were significantly lower on Day 25 in animals exposed to TCDD (maternal dose, 2.5 µg/kg) (n = 5) when compared with controls (n = 8) (Fig. 5). Serum E2 levels were below assay sensitivity (1.4 pg/ml) on Day 23 in both control and exposed animals (Fig. 5).



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  		FIG. 5. Mean serum E2 levels on Days 23 and 25 of rats from pregnant dams treated with vehicle (n = 5) or TCDD (2.5 µg/kg, n = 8). On Day 23, serum E2 levels were undetectable (detection limit of the kit, ~1.4 pg/ml). * Significant difference from the vehicle controls (P < 0.05)


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The significant findings of this study were that IUL exposure to both TCDD and PCDF induce long-term disturbances in growth, estrous cyclicity, and ovulation in female pups. Additionally, TCDD (2.5 µg/kg) appeared to reduce eCG-stimulated follicle growth and E2 production. The present study adds to previous investigations that have clearly demonstrated that IUL exposure to low levels of TCDD can induce long-term disruptions in reproduction and development (reviewed in [7]). Administration of TCDD at varied doses and times of exposure can bring about similar disruptions in reproductive parameters. For example previous investigations have demonstrated reductions in ovulation using doses of TCDD ranging from 4 to 32 µg/kg in rats exposed as adults and as juveniles (reviewed in [17]). The present investigation demonstrated that prenatal and lactational exposure to TCDD also reduces ovulation rate into adulthood. It seems reasonable to speculate that the different modes of TCDD exposure between the present investigation and previous studies may act to disrupt ovulation and other end points in different ways.

Findings of the present study demonstrate that IUL exposure to TCDD and PCDF reduced body weight at 10, 15, and 20 days of age and at termination at 125–165 days of age. Our finding that IUL exposure to TCDD significantly reduced body weight is in agreement with the findings of previous investigations [14]. This study also demonstrates that PCDF and TCDD have similar effects on growth. Previous investigators have provided a possible explanation for reductions in weight by demonstrating that IUL exposure to TCDD significantly reduces liver insulinlike growth factor I mRNA (by 33%) in 21-day-old rats, despite slightly elevated levels of serum growth hormone [22]. TCDD has also been shown to induce goiters and to decrease serum thyroxine levels in exposed rats [23], suggesting that alterations in thyroid hormones may also contribute to changes in growth and/or reproductive end points.

The present study demonstrated that IUL exposure to TCDD or PCDF disrupted estrous cyclicity between 45 and 66 days of age. Cycles were characterized by prolonged periods of diestrus and a decrease in the number of estrus periods. TCDD exposure, occurring either gestationally and lactationally [14] or by direct administration to immature or cycling rats [16], has been shown previously to disrupt estrous cycles. For instance, TCDD induces immediate alterations in estrous cycles in cycling rats that are treated with a single oral dose of TCDD (10 µg/kg) [16]. Alterations include prolonged periods of diestrus and a reduction in the time spent in estrus and proestrus. Previous studies have also demonstrated that TCDD induces constant estrus prematurely in adult rats that were exposed to TCDD on GD 8 [14]. Estrous cyclicity is a reflection of the cyclic nature of ovarian steroid hormone production controlled by the hypothalamopituitary-ovary axis. It is possible that TCDD and PCDF disrupted estrous cycles by decreasing E2 production. This hypothesis is supported by previous studies demonstrating that IUL exposure to TCDD significantly reduces serum E2 concentrations in rats (21 days old) [15]. IUL exposure to TCDD decreases the number of growing follicles, thereby eliminating a significant source of E2 [21]. Additionally, the present investigation demonstrated that IUL exposure to TCDD and PCDF reduces the number of large follicles and serum E2 concentrations 48 h after eCG stimulation.

Another significant finding of this study is that IUL exposure to PCDF or TCDD significantly reduces ovulation rate in 125- to 165-day-old rats. Previous studies have shown that acute exposure of mature rats to TCDD or PCDF administered as a single oral dose results in an immediate reduction in ovulation. Acute exposure to TCDD (32 µg/kg) and PCDF significantly reduces ovulation in gonadotropin-primed immature rats (reviewed in [17]). Additionally, Petroff et al. [24] demonstrated that ovulation is reduced in rats after intrabursal injection of TCDD. TCDD may act to reduce ovulation at least in part by exerting a direct effect on ovarian function. This hypothesis is supported by previous investigations that showed that acute exposure to TCDD reduces ovulation in hypophysectomized rats treated with eCG and exogenous LH [25]. Thus, TCDD may act to disrupt the processes that lead to rupture of the preovulatory follicle. Histologic evaluation of ovaries obtained from rats treated acutely with TCDD at doses of 32 µg/kg [26] and 10 µg/kg [25] shows an increase in the number of oocytes trapped within preovulatory follicles and a concomitant decrease in the number of corpora lutea in TCDD-exposed rats. TCDD might interfere with ovulation by decreasing levels of plasminogen activator [25, 27], a factor implicated in follicular rupture [28, 29].

In addition to directly disrupting ovarian function, TCDD may act to alter the hormonal system that regulates ovulation. A study by Li et el. [25] demonstrated that TCDD may block the LH surge that is essential to induce ovulation. Recent investigations have provided insight into the mechanism by which direct exposure to TCDD (32 µg/kg) acts to reduce the LH surge and, as a consequence, ovulation [30, 31]. Administration of exogenous GnRH to immature gonadotropin-primed rats treated acutely with TCDD (32 µg/kg) partially restores ovulation. Additionally, exogenous GnRH reverses TCDD-induced reductions in FSH and LH, further supporting the idea that acute exposure to TCDD acts to impair hypothalamic function [30]. Gao et al. [31] also showed that acute exposure to TCDD decreases the responsiveness of the hypothalamus to circulating levels of E2 in treated animals. Administration of synthetic estrogen was able to stimulate the necessary FSH and LH surges and restore ovulation in treated rats. Investigations using the IUL exposure model have demonstrated that TCDD significantly reduces mRNA for the estrogen receptor (ER) in the pituitary and that ER DNA-binding activity is also significantly reduced in hypothalamic extracts [15]. Therefore, decreased E2 concentrations or hypothalamic responsiveness to E2 may account for LH surge deficits and impaired ovulation.

Ovulation rate may have been reduced because of the decreased number of large preovulatory follicles demonstrated in the present study and others [21]. In the present study, IUL exposure to TCDD significantly reduced serum E2 levels and numbers of large preovulatory follicles, 48 h after eCG stimulation. These data suggest that TCDD decreases the growth of gonadotropin-responsive follicles into large preovulatory follicles even in the presence of exogenous gonadotropins. One mechanism for this targeted disruption could be a reduction in responsiveness of the developing follicle to gonadotropins. The reduction in follicular growth could also be due to a reduction in E2 production in treated animals because E2 acts in a synergistic fashion with gonadotropins to facilitate follicular growth and responsiveness by up-regulating its own receptors and FSH and LH receptors (reviewed in [32]). Addition of TCDD (3.1 nM) to cultured granulosa cells reduced the expression and activity of specific steroidogenic enzymes (P450 side-chain cleavage and aromatase) and reduced E2 production [33]. The reduction in E2 production after exposure to TCDD may also be a consequence of a reduction in the number of large follicles. Previous investigations have demonstrated that rat granulosa cells cotreated with TCDD have significantly fewer FSH-stimulated FSH receptors [34]. Previous studies [33] have also suggested that TCDD may affect steroidogenesis. Currently, it is unknown if IUL exposure to TCDD reduces E2 levels by acting on specific steps in the steroidogenic pathway.

In conclusion, this study demonstrates that prenatal and lactational exposure to TCDD and PCDF disrupts reproductive physiology, specifically estrous cyclicity and ovulation, in female offspring. Additionally, IUL exposure to TCDD and PCDF reduces weight gain into adulthood. Results from this investigation also suggest that PCDFs induce disruptions in growth, estrous cyclicity, and ovulation that are similar to those induced by PCDDs. Furthermore, the results of this study support the hypothesis that TCDD-induced disruptions in female reproduction may in part be due to a direct effect on the ovary, since TCDD reduced follicular growth even in the presence of exogenous gonadotropins. Future studies are clearly needed to elucidate the mechanism by which TCDD and PCDF induce long-term disruptions in female reproduction after IUL exposure.


   ACKNOWLEDGMENTS
 
The authors thank Dr. Walter Horne for his assistance in animal procedures.


   FOOTNOTES
 
First decision: 30 August 2001.

1 This research was supported by a Research Challenge Grant from the Ohio Board of Regents. Back

2 Correspondence. FAX: 330 672 3713; jmarcink{at}kent.edu Back

Accepted: December 19, 2001.

Received: July 16, 2001.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Poland A, Knutson JC. 2,3,7,8-Tetrachlorodibenzo-p-dioxin and related halogenated aromatic hydrocarbons: examination of the mechanism of toxicity. Annu Rev Pharmacol Toxicol 1982; 22:517-554[CrossRef][Medline]
  2. Schecter A, Papke O, Ball M, Lis A, Brandt-Rauf P. Dioxin concentrations in the blood of workers at municipal waste incinerators. Occup Environ Med 1995; 52:385-387[Abstract]
  3. Giesy JP, Kannan K, Kubitz JA, Williams LL, Zabik MJ. Polychlorinated dibenzo-p-dioxins (PCDDs) and dibenzofurans (PCDFs) in muscle and eggs of salmonid fishes from the Great Lakes. Arch Environ Contam Toxicol 1999; 36:432-446[CrossRef][Medline]
  4. Vartiainen T, Jaakkola J, Saarikoski S, Tuomisto J. Birth weight and sex of children and the correlation to the body burden of PCDDs/PCDFs and PCBs of the mother. Environ Health Perspect 1998; 106::61-66[Medline]
  5. Nakagawa R, Hirakawa H, Matsueda T, Nagayama J. Maternal body burden of organochloride pesticides and dioxins. J AOAC Int 1999; 82:716-724[Medline]
  6. Bandiera S, Sawyer T, Romkes M. Polychlorinated dibenzofurans (PCDF): effects of structure on binding to the 2,3,7,8-TCDD cytosolic receptor protein, AHH induction and toxicity. Toxicology 1984; 32::131-144[CrossRef][Medline]
  7. Birnbaum LS. Developmental effects of dioxins and related endocrine disrupting chemicals. Toxicol Lett 1995; 82:743-750
  8. Hankinson O. The aryl hydrocarbon receptor complex. Annu Rev Pharmacol Toxicol 1995; 35:307-340[CrossRef][Medline]
  9. Chaffin CL, Hutz RJ. Regulation of the aromatic hydrocarbon receptor (AHR) by in utero and lactational exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). J Reprod Dev 1997; 43:47-51[CrossRef]
  10. Chaffin CL, Heimler I, Rawlins RG, Wimpee BA, Sommer C, Hutz R J. Estrogen receptor and the aromatic hydrocarbon receptor in the primate ovary. Endocrine 1996; 13:315-321
  11. Robles R, Morita Y, Mann KK, Perez GI, Yang S, Matikainen T, Sherr DH, Tilly JL. The aryl hydrocarbon receptor, a basic helix-loop-helix transcription factor of the PAS gene family, is required for normal ovarian germ cell dynamics in the mouse. Endocrinology 2000; 141::450-453[Abstract/Free Full Text]
  12. Hurst CH, Abbot BD, DeVito MJ, Birnbaum LS. 2,3,7,8-Tetrachlorodibenzo-p-dioxin in pregnant Long Evans rats: disposition to maternal and embryo/fetal tissues. Toxicol Sci 1998; 45:129-136[Abstract/Free Full Text]
  13. Li X, Weber LW, Rozman KK. Toxicokinetics of 2,3,7,8-tetrachlorodibenzo-p-dioxin in female Sprague-Dawley rats including placental and lactational transfer to the fetus and the neonate. Fundam Appl Toxicol 1995; 27:70-76[CrossRef][Medline]
  14. Gray LE, Ostby JS. In utero 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) alters reproductive morphology and function in female rat offspring. Toxicol Appl Pharmacol 1995; 133:285-294[CrossRef][Medline]
  15. Chaffin CL, Peterson RE, Hutz RJ. In utero and lactational exposure of female Holtzman rats to 2,3,7,8-tetrachlorodibenzo-p-dioxin: modulation of the estrogen signal. Biol Reprod 1996; 55:62-67[Abstract]
  16. Li X, Johnson DC, Rozman KK. Effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on estrous cyclicity and ovulation in female Sprague-Dawley rats. Toxicol Lett 1995; 78:219-222[CrossRef][Medline]
  17. Petroff BK, Roby KF, Gao X, Son D, Williams S, Johnson D, Rozman KK, Terranova PF. A review of the mechanisms controlling ovulation with implications for the anovulatory effects of polychlorinated dibenzo-p-dioxins in rodents. Toxicology 2001; 158:91-107[CrossRef][Medline]
  18. Gao X, Terranova PF, Rozman KK. Effects of polychlorinated dibenzofurans, biphenyls, and their mixture with dibenzo-p-dioxins on ovulation in the gonadotropin-primed immature rat: support for the toxic equivalency concept. Toxicol Appl Pharmacol 2000; 163:115-124[CrossRef][Medline]
  19. Allen E. The oestrous cycle in the mouse. Am J Anat 1922; 30:297-371[CrossRef]
  20. Presnell JK, Schreibman MR. Humason's Animal Tissue Techniques, 5th ed. Baltimore: The Johns Hopkins University Press; 1997
  21. Heimler I, Trewin AL, Chaffin CL, Rawlins RG, Hutz RJ. Modulation of ovarian follicle maturation and the effects on apoptotic cell death in Holtzman rats exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in utero and lactationally. Reprod Toxicol 1998; 12:69-73[CrossRef][Medline]
  22. Chaffin CL, Brogan RS, Peterson RE, Hutz RJ, Wehreberg WB. Modulation of growth axis gene expression by in utero and lactational exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in the weanling Holtzman rat. Endocrine 1996; 5:129-134
  23. Bastomsky CH. Enhanced thyroxine metabolism and high uptake goiters in rats after a single dose of 2,3,7,8-tetrachlorodibenzo-p-dioxin. Endocrinology 1977; 101:292-296[Abstract]
  24. Petroff BK, Gao X, Rozman KK, Terranova PF. Interaction of estradiol and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in an ovulation model: evidence for systemic potentiation and local ovarian effects. Reprod Toxicol 2000; 14:247-255[CrossRef][Medline]
  25. Li X, Johnson DC, Rozman KK. Reproductive effects of 2,3,7,8,-tetrachlorodibenzo-p-dioxin (TCDD) in female rats: ovulation, hormonal regulation, and possible mechanisms. Toxicol Appl Pharmacol 1995; 133:321-327[CrossRef][Medline]
  26. Gao X, Son D, Terranova PF, Rozman KK. Toxic equivalency factors of polychlorinated dibenzo-p-dioxins in an ovulation model: validation of the toxic equivalency concept for one aspect of endocrine disruption. Toxicol Appl Pharmacol 1999; 157:107-116[CrossRef][Medline]
  27. Gierthy JF, Lincoln DW, Gillespie MB, Seeger JI, Martinez HL, Dickerman HW, Kumar SA. Suppression of estrogen-related extracellular tissue plasminogen activator activity of MCF-7 cells by 2,3,7,8-tetrachlorodibenzo-p-dioxin. Cancer Res 1987; 47:6198-6203[Abstract/Free Full Text]
  28. Beers WH. Follicular plasminogen and plasminogen activator and the effect of plasmin on ovarian follicle wall. Cell 1975; 6:379-386[CrossRef][Medline]
  29. Beers WH, Strickland S, Reich E. Ovarian plasminogen activator: relationship to ovulation and hormonal regulation. Cell 1975; 6:387-394[CrossRef][Medline]
  30. Gao X, Petroff BK, Rozman KK, Terranova PF. Gonadotropin-releasing hormone (GnRH) partially reverses the inhibitory effect of 2,3,7,8-tetrachlorodibenzo-p-dioxin on ovulation in the immature gonadotropin-treated rat. Toxicology 2000; 147:15-22[CrossRef][Medline]
  31. Gao X, Mizuyachi K, Terranova PF, Rozman KK. 2,3,7,8-Tetrachlorodibenzo-p-dioxin decreases responsiveness of the hypothalamus to estradiol as a feedback inducer of preovulatory gonadotropin secretion in the immature gonadotropin-primed rat. Toxicol Appl Pharmacol 2001; 170:181-190[CrossRef][Medline]
  32. Hirshfield AN. Development of follicles in the mammalian ovary. Int Rev Cytol 1991; 124:43-99[Medline]
  33. Dasmahapatra AK, Wimpee BA, Trewin AL, Wimpee CF, Ghoria JK, Hutz RJ. Demonstration of 2,3,7,8-tetrachlorodibenzo-p-dioxin attenuation of P450 steroidogenic enzyme in rat granulosa cell in vitro by competitive reverse transcriptase-polymerase chain reaction assay. Mol Cell Endocrinol 2000; 1:5-18
  34. Hirakawa T, Minegishi T, Abe K, Kishi H, Inoue K, Ibuki Y, Miyamoto K. Effect of 2,3,7,8-tetrachlorodibenzo-p-dioxin on the expression of follicle-stimulating hormone receptors during cell differentiation in cultured granulosa cells. Endocrinology 2000; 141:1470-1476[Abstract/Free Full Text]



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