* Program in Toxicology and
Department of Epidemiology and Preventive Medicine, University of Maryland, 660 West Redwood Street, Howard Hall 133 B, Baltimore, Maryland 21201; and
School of Pharmacy, University of Wisconsin, Madison, Wisconsin 53706
Received February 4, 2000; accepted April 11, 2000
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ABSTRACT |
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Key Words: aryl hydrocarbon receptor (AhR); ovary; mouse; follicle..
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INTRODUCTION |
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AhR agonists can also cause reproductive abnormalities. For example, a prototype compound 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin (TCDD) has been found to have a profound effect on male and female reproductive tract development and function (Malby et al., 1992a,b
,c
). Malby et al. (1992a,b) showed that in utero exposure to TCDD decreases prostate weight and epididymal sperm numbers in male rats. Gray and Ostby (1995) showed that Holtzman rats exposed to TCDD in utero had vaginal clefting in 100% of exposed progeny and vaginal thread formation in 83% of exposed progeny. Flaws et al. (1997) also showed cleft clitoris and vaginal thread formation in 5596% and 3644% of litters, respectively, following 1.0 µg/kg TCDD exposure on gestational days 11, 15, and 18.
In addition to malformations of external genitalia in female rat offspring, TCDD has been shown to directly affect the ovary. Gray and Ostby (1995) demonstrated that exposure to TCDD in utero results in a 23% decrease in ovarian weight. Silbergeld and Mattison (1987) demonstrated that postnatal exposure to TCDD decreases ovarian weight and the number of corpora lutea. Heimler et al. (1998) showed that in utero and lactational exposure to TCDD on gestational day 15 results in a reduction in the number of both pre-antral and antral follicles at PNDs 21/22 in Holtzman rats compared to controls. However, in utero and lactational TCDD exposure is not thought to affect the number of primordial follicles in Holtzman rats (Flaws et al., 1997).
Although the AhR is known to mediate toxic responses to TCDD and other AhR agonists in both non-reproductive and reproductive tissues, little is known about the physiological role of the AhR. To our knowledge, only one study has examined the role of the AhR in the female reproductive system. In this study, Abbott et al. (1999) found that AhR deficient female and male mice (AhRKO) were both fertile, but female AhRKO mice produced fewer total and live pups at birth. Also, both AhRKO dams and pups had a decreased ability to survive lactation compared to wild-type animals. The purpose of the current study was to further explore the role of the AhR in the female reproductive system. Specifically, we tested the hypothesis that the AhR plays a role in the development of the mammalian ovary. To test this hypothesis, we examined whether the AhR is required for the normal morphological appearance of the ovary and the development of germ cells, primordial follicles, primary follicles, and antral follicles.
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MATERIALS AND METHODS |
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Screening/Genotyping of Mice
Individually housed heterozygous females (AhRKO +/), between 90120 days old, were mated with heterozygous males (AhRKO +/). Offspring were genotyped to determine whether they were +/+, +/, or / for the AhR (Schmidt et al., 1996). Ear punch tissue from pups was lyzed in 25 µl of proteinase K buffer (50 mM TrisHCl, 20 mM NaCl, 1 mM EDTA and 1% SDS, pH 8.0) containing 1 µl of 20 mg/ml proteinase K. Digestion was carried out at 65°C for 30 min with a 15-s vortex after the 15- and 30-min incubation times. The lysate was then subjected to the polymerase chain reaction (PCR) using the following primers: (1) Neo F TTG GGT GGA GAG GCT ATT CG, (2) Neo R CCA TTT TCC ACC ATG ATA TTC G, (3) AhRI3F TCT TGG GCT CGA TCT TGT GTC A, and (4) AhR x 2RB TTG ACT TAA TTC CTT CAG CGG. The conditions for PCR were as follows: 94°C, 2-min initial denaturation, 35 cycles of 94°C for 5 s, 60°C for 30 s, and 72°C for 1 min, with a final extension at 72°C for 2 min. PCR products were then subjected to agarose gel electrophoresis; wild-type AhR products were 670 bp and mutant AhR products were 580 bp.
Selection of Time Points for Ovarian Tissue Collection
Ovaries were collected from AhRKO (/) and wild-type mice at several times to determine if the AhR played a role in influencing the appearance and/or number of follicles in the gestational, pubertal, and adult ovary. Ovaries were collected from AhRKO and wild-type mice on gestational day 18 because, at this time, the ovary has a finite pool of naked germ cells that have not yet become surrounded by granulosa cells to form primordial follicles (Hirshfield, 1991; Hogan et al., 1994
). Ovaries were collected on PND 23 because, at this time, some germ cells have become surrounded by a layer of fusiform granulosa cells to form primordial follicles. Single, naked (not surrounded by somatic cells) germ cells still exist in the ovary at this time as well (Hirshfield, 1991
; Hogan et al., 1994
). Ovaries were collected on PND 8 because by that day there is a complete pool of fully formed primordial follicles, and few, if any, naked germ cells exist in the ovary. It is also when some primordial follicles have grown into more mature primary follicles (Hirshfield, 1991
; Hogan et al., 1994
). Ovaries were collected on PND 3235 because that is when the ovary contains follicles in many stages of development (primordial, primary, and antral) and the animal is experiencing sexual maturity (Hirshfield, 1991
; Hogan et al., 1994
). Ovaries were collected on PND 53 because that is when the ovary contains follicles in many stages of growth and wild-type mice are usually considered cycling adults (Hirshfield, 1991
; Hogan et al., 1994
).
Histological Evaluation of Gestational Mouse Ovaries
AhRKO and wild-type control embryos were harvested at gestational day 18 (GD 18) and fixed in Bouin's solution (glacial acetic acid, 3740% formalin, and saturated aqueous picric acid) for 18 h, followed by washes in 70% ethanol. Gestational ovaries then were removed and embedded in a 2% agar pellet to ensure that the integrity of the tissue was not lost during the dehydration and embedding processes. The ovaries then were placed in cassettes, dehydrated through an ethanol series, embedded in Paraplast (VWR Scientific, Baltimore, MD), serially sectioned (8 µm), mounted onto glass slides, and stained with Weigert's hematoxylin-picric acid methyl blue.
Qualitative evaluation of germ cells was made by light microscopy using a Zeiss microscope. Normal germ cells were classified as those that were round in appearance and with an intact nucleus. Abnormal germ cells were classified as those that were misshapen or undergoing apoptosis (i.e., germ cells with condensed, fragmented nuclei or pyknotic bodies).
To quantitatively evaluate the number of germ cells, sections from each ovary were arranged in order and every tenth section with a random start was marked. The number of germ cells present in each marked section then was counted and multiplied by 10 to account for every tenth section being used in the analysis (Flaws et al., 1997; Smith et al., 1991
). All quantitative and qualitative evaluations of germ cells were done without knowledge of genotype.
Histological Evaluation of Postnatal Mouse Ovaries
Ovaries were harvested from AhRKO and wild-type mice on PNDs 23, 8, 3235, and 53 and processed as described above. To qualitatively evaluate the morphological appearance of follicles, each section was examined for healthy and atretic follicles. Healthy follicles were classified as those that contained an intact oocyte, organized granulosa cell layers, and few, if any, apoptotic bodies. Atretic follicles were classified as those that contained a degenerating oocyte, disorganized granulosa cell layers, and/or more than 10% of granulosa cells being pyknotic.
To quantitatively evaluate the number of primordial, primary, pre-antral/antral follicles, slides from each ovary were arranged in order and every tenth section was marked for analysis. Follicles containing an intact oocyte with a nucleolus and a single layer of fusiform granulosa cells were classified as primordial. Follicles consisting of an intact oocyte with visible nucleolus and a single layer of cuboidal granulosa cells were termed primary follicles. Follicles containing an intact oocyte with nucleolus and more than one layer of granulosa or thecal cells were scored as pre-antral/antral. To obtain an estimate of the total number of follicles per ovary, the total number of primordial, primary, and pre-antral/antral follicles present in the marked section was multiplied by 10 to account for every tenth section being used in the analysis (Flaws et al., 1997; Smith et al., 1991
). All ovaries were coded prior to histological evaluation to ensure that all morphological and quantitative evaluation was conducted without knowledge of genotype. This method of follicle counting allows for reasonable estimation and comparison of ovarian follicle numbers between animal groups. However, it may slightly overestimate the actual number of follicles in the ovary.
Statistical Analysis
The mean number of germ cells and follicles was calculated using ovaries from at least 3 different animals per group. Litter independence was maintained during the experiments, and ovaries were used from at least 3 different litters per time point. Differences in germ cell and follicle numbers were evaluated by the Mann-Whitney test, with statistical significance assigned at p 0.05. All results are presented as mean ± SE.
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RESULTS |
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On PNDs 2, 3, 8, 3235, and 53, AhRKO and wild-type mice also appeared similar (Fig. 1). On PND 23, both AhRKO and wild-type ovaries contained many primordial follicles as well as some single, naked germ cells. The primordial follicles were healthy and there appeared to be no evidence of degeneration of germ cells or somatic cells. On PND 8, both AhRKO and wild-type ovaries contained primordial and primary follicles with intact oocytes surrounded by a single somatic cell layer. There were no major signs of follicular degeneration. On PND 3235 and 53, ovaries from both wild-type mice and AhRKO mice contained healthy looking primordial, primary, and antral follicles. In addition, both AhRKO and wild-type ovaries contained atretic follicles.
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DISCUSSION |
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Our data suggest that in the presence of the AhR, the process of surrounding single germ cells by somatic cells to form primordial follicles is slower than in the absence of the AhR. For example, on PND 23, AhRKO mice had significantly more fully formed primordial follicles and fewer naked germ cells compared with wild-type mice. By PND 8, however, AhRKO and wild-type mice had similar numbers of primordial follicles. Thus, it is possible that the AhR affects the rate at which germ cells become surrounded by somatic cells to form primordial follicles. It is unclear how the AhR may regulate primordial follicle formation, largely because little is known about this biologic process in general. To our knowledge, the results of this study are the first to identify a factor involved in the rate at which germ cells are surrounded by somatic cells to form primordial follicles. Other studies have focused on the role of c-kit and its ligand (steel factor) in ovarian development (Horie et al., 1991; Manova et al., 1993
; Pesce et al., 1997
). Although these studies indicate that these factors are important for the migration and proliferation of germ cells, they have not assessed whether c-kit or steel factor alter the rate at which somatic cells surround germ cells to form primordial follicles in postnatal life.
In a recent study, Robles et al. (2000) also demonstrated that AhRKO mice have approximately two-fold more primordial follicles per ovary than wild-type mice on PND 4. These authors proposed that this increased number of primordial follicles might be due to increased numbers of germ cells during gestational life. Although Robles et al. did not report any data on actual germ cell numbers during gestational life, our results indicate that there is no difference in numbers.
Our results also suggest that the AhR may be involved in the regulation of the number of antral follicles in postnatal life. The decreased antral follicle numbers that we observed in AhRKO mice may possibly explain some of the reproductive abnormalities seen in AhRKO mice described by Abbott et al. (1999). Adequate numbers of antral follicles are required to synthesize and secrete reproductive hormones (e.g., estrogen) and to maintain estrous cyclicity and fertility. Perhaps, the reduced number of antral follicles present in AhRKO mice could lead to abnormal hormone levels and disturbed estrous cyclicity in these animals. In turn, the altered hormone levels could affect the fertility of the animals as well as the ability of AhRKO pregnant dams to sustain pregnancies and maintain lactation. It is interesting to note that despite the reduction in antral follicle numbers, AhRKO mice are still fertile (Abbott et al., 1999). However, it is possible that the AhRKO animals may experience early reproductive senescence compared with wild-type animals.
The AhR may regulate antral follicle numbers by affecting the rate of ovarian atresia (i.e., AhRKO may have more ovarian atresia than wild-type mice). In our study, we did not observe any significant difference in the rate of ovarian atresia in AhRKO and wild-type mice at any time point (PNDs 23, 8, 3235, and 53). It is possible, however, that the significant difference in atresia occurs between PNDs 3235 and 53. It also is possible that it is difficult to detect small changes in the rates of atresia using histological methods. Other investigators have also had difficulty detecting changes in the rate of atresia preceding antral follicle loss (Heimler et al., 1998).
In the course of this study, we noticed that some of the ovarian phenotypes in the AhRKO were similar to those reported in TCDD-treated animals (Abbott et al., 1999; Heimler et al., 1998
; Wolf et al., 1999
). The reason for the similarities between AhRKO and TCDD-treated mice is unknown. Perhaps, TCDD down-regulates the expression of the AhR, producing an AhR deficiency. This hypothesis is supported by several studies that indicate that TCDD exposure decreases AhR protein and mRNA levels in the male rat reproductive tract and in the mouse palatal shelf (Abbott et al., 1994
; Roman et al., 1998
; Sommer et al., 1999
). It is also possible that AhRKO mice and TCDD treatment result in some similar reproductive phenotypes because TCDD toxicity is mediated through AhR independent events in some tissues. In studies by Peters et al. (1999) and Mimura et al. (1997), TCDD exposure elicited adverse effects in AhRKO mice such as TCDD induced litter resorptions and reduced fetal weight. In fact, the number of litter resorptions and reduction in fetal weight was more pronounced in the AhRKO mice treated with TCDD than in wild-type mice treated with TCDD. These data suggest that TCDD may work via some AhR independent mechanism and that the toxic effects of TCDD may actually be enhanced in the absence of the AhR.
In conclusion, we found that AhRKO mice had significantly more primordial follicles and fewer single, naked germ cells compared to wild-type controls on PND 23. AhRKO mice also had significantly fewer antral follicles compared to wild-type controls on PND 53. These results indicate that the AhR may have an important role in the rate at which single germ cells are surrounded by somatic cells to form primordial follicles. The AhR may also have a role in the regulation of antral follicles. Future studies should address the mechanism by which the AhR influences the rate of primordial follicle formation and the number of antral follicles. Future studies also should examine why AhRKO- and TCDD-treated mice have similar phenotypes. Specifically, studies need to investigate the effect of TCDD on AhRKO mice to determine if certain signs of TCDD toxicity in the female reproductive system occur via non-AhR-mediated pathways.
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ACKNOWLEDGMENTS |
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NOTES |
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REFERENCES |
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