Physiological Role of the Aryl Hydrocarbon Receptor in Mouse Ovary Development

Jamie C. Benedict*, Tien-Min Lin{dagger}, I. K. Loeffler{dagger}, Richard E. Peterson{dagger} and Jodi A. Flaws*,{ddagger},1

* Program in Toxicology and {ddagger} Department of Epidemiology and Preventive Medicine, University of Maryland, 660 West Redwood Street, Howard Hall 133 B, Baltimore, Maryland 21201; and {dagger} School of Pharmacy, University of Wisconsin, Madison, Wisconsin 53706

Received February 4, 2000; accepted April 11, 2000


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The aryl hydrocarbon receptor (AhR) regulates the toxicity of environmental contaminants such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). As the physiological role of the AhR in the ovary is unknown, the purpose of this study was to test the hypothesis that the AhR regulates the appearance and numbers of ovarian follicles. Ovaries were harvested from AhR-deficient (AhRKO) and wild-type mice on gestational day 18 (GD 18) and postnatal days (PND) 2–3, 8, 32–35, and 53. Complete serial sections of ovaries were evaluated histologically for the presence of germ cells and follicles. On GD 18, there was no difference in the number of germ cells per ovary between AhRKO and wild-type fetuses. However, by PND 2–3, AhRKO mice had significantly more fully formed primordial follicles (AhRKO = 38,440 ± 3632 versus wild-type = 21,120 ± 2688) and fewer single germ cells than wild-type mice (AhRKO = 12,696 ± 1192 vs. wild-type = 18,160 ± 720). On PND 8 and 32–35, there was no difference in the number of follicles between AhRKO and wild-type mice but by PND 53, AhRKO mice had significantly fewer antral follicles than wild-type (AhRKO = 3416 ± 480 vs. wild-type = 6776 ± 1024). Taken together, these results suggest that the AhR may play a role in the formation of primordial follicles and the regulation of antral follicle numbers.

Key Words: aryl hydrocarbon receptor (AhR); ovary; mouse; follicle..


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that mediates the toxicity of various structurally related environmental contaminants such as the polyhalogenated dibenzo-p-dioxins, dibenzofurans, and biphenyls (Burbach et al., 1992Go; Ema et al., 1992Go). When such environmental contaminants activate the AhR, a signaling cascade of events follows, which can lead to a variety of pathologic responses including thymic involution, tumor promotion, wasting, and carcinogenesis (Fujii-Kuriyama et al., 1994Go).

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., 1992aGo,bGo,cGo). 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 55–96% and 36–44% 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., 1997Go).

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.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Animals and Treatment
AhRKO mice were originally generated as described by Schmidt et al. (1996) and generously provided by Dr. Christopher A. Bradfield, and a colony of these mice and wild-type mice was established in the Wisconsin School of Pharmacy Animal Facility. Dams were kept individually in clear plastic cages and maintained on a 0600–1800 light:dark cycle in a temperature-controlled room (24 ± 1°C) with 35 ± 4% relative humidity. The mice were provided feed (Purina 5015) and tap water ad libitum. The Institutional Animal Use and Care Committees at the Unversity of Wisconsin and the University of Maryland approved all protocols involving mice.

Screening/Genotyping of Mice
Individually housed heterozygous females (AhRKO +/–), between 90–120 days old, were mated with heterozygous males (AhRKO +/–). Offspring were genotyped to determine whether they were +/+, +/–, or –/– for the AhR (Schmidt et al., 1996Go). Ear punch tissue from pups was lyzed in 25 µl of proteinase K buffer (50 mM Tris–HCl, 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, 1991Go; Hogan et al., 1994Go). Ovaries were collected on PND 2–3 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, 1991Go; Hogan et al., 1994Go). 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, 1991Go; Hogan et al., 1994Go). Ovaries were collected on PND 32–35 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, 1991Go; Hogan et al., 1994Go). 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, 1991Go; Hogan et al., 1994Go).

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, 37–40% 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., 1997Go; Smith et al., 1991Go). 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 2–3, 8, 32–35, 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., 1997Go; Smith et al., 1991Go). 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.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Effect of AhR on Ovarian Morphology
On GD 18, there were no gross differences in the morphological appearance of ovaries from AhRKO and wild-type mice. Ovaries from both AhRKO and wild-type mice consisted of single, naked germ cells and somatic cells, but no fully formed follicles.

On PNDs 2, 3, 8, 32–35, and 53, AhRKO and wild-type mice also appeared similar (Fig. 1Go). On PND 2–3, 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 32–35 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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FIG. 1. Effect of the aryl hydrocarbon receptor (AhR) on postnatal ovaries. Ovaries were collected on PNDs 2–3, 8, 32–35, and 53 from AhRKO and wild-type mice, and complete serial sections were prepared for histological examination. Each section was evaluated for morphological appearance of germ cells, primordial follicles, primary follicles, and antral follicles. (A) and (C) show wild-type ovary collected on PNDs 2–3 and 53 respectively. (B) and (D) show AhRKO ovary collected on PNDs 2–3 and 53, respectively. In all panels: gc, single, naked germ cell; p, primordial follicle; Pr, primary follicle; PA, pre-antral follicle; A, antral follicle. Original magnification in (A) and (B) x40; (C) and (D) x10.

 
Effect of the AhR on Number of Germ Cells
On GD 18, AhRKO and wild-type ovaries had similar numbers of germ cells (Fig. 2Go). AhRKO mice had 31,376 ± 1712 germ cells and wild-type mice had 33,616 ± 5104 germ cells (n = 3 for each group, p = 0.83). However, by PND 2–3, the number of naked germ cells in AhRKO ovaries was reduced to 70% of wild-type (Fig. 2, Gon = 3 for wild-type, n = 6 for AhRKO, p <= 0.04).



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FIG. 2. Quantitative evaluation of germ cell numbers. Ovaries were collected from wild-type and AhRKO mice on GD 18 and PND 2–3, and complete serial sections were prepared for evaluation of germ cell numbers. Every tenth section was counted to estimate the total number of germ cells per ovary. Each bar represents the mean ± SE (n = 3 for each group at GD 18 and n = 3 for wild-type, n = 6 for AhRKO at PND 2–3; *p <= 0.04).

 
Effect of the AhR on the Number of Primordial Follicles
On PND 2–3, AhRKO ovaries had almost two times more primordial follicles than the wild-type ovaries (Figs. 1 and 3GoGo). AhRKO ovaries contained 38,440 ± 3632 primordial follicles, whereas wild-type ovaries only contained 21,120 ± 2688 primordial follicles (n = 4 for wild-type, n = 7 for AhRKO, p = 0.008). On PNDs 8, 32–35, and 53, AhRKO and wild-type ovaries contained similar numbers of primordial follicles (n = 8 for wild-type, n = 9 for AhRKO, p = 0.25). By PND 32–35, AhRKO mice had 9352 ± 1680 primordial follicles and wild-type mice had 9816 ± 1744 primordial follicles (n = 6 for wild-type, n = 9 for AhRKO, p = 0.64). By PND 53, AhRKO mice had 9664 ± 1648 primordial follicles and wild-type mice had 12,168 ± 1784 primordial follicles (n = 5 for wild-type, n = 7 for AhRKO, p = 0.22).



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FIG. 3. Quantitative evaluation of primordial follicle numbers. Ovaries were collected from wild-type and AhRKO mice on PNDs 2–3, 8, 32–35, and 53 for evaluation of primordial follicle numbers per ovary. Each bar represents the mean ± SE (n >= 4 in each group at each time point; *p <= 0.05).

 
Effect of the AhR on the Number of Primary Follicles
On PNDs 2–3, 8, 32–35, and 53, AhRKO and wild-type ovaries had similar numbers of primary follicles (Fig. 4Go). On PND 2–3, AhRKO ovaries contained 1,016 ± 256 primary follicles and wild-type ovaries contained 776 ± 176 primary follicles (n = 4 for wild-type, n = 7 for AhRKO, p = 0.70). By PND 8, AhRKO mice had 3840 ± 496 primary follicles and wild-type mice had 3968 ± 368 primary follicles (n = 8 for wild-type, n = 9 for AhRKO, p = 0.81). By PND 32–35, AhRKO mice had 1952 ± 184 primary follicles and wild-type mice had 1744 ± 256 primary follicles (n = 6 for wild-type, n = 9 for AhRKO, p = 0.56). Finally, by PND 53, AhRKO mice had 1952 ± 368 primary follicles and wild-type mice had 2416 ± 208 primary follicles (n = 5 for wild-type, n = 7 for AhRKO, p = 0.29).



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FIG. 4. Quantitative evaluation of primary follicle numbers. Ovaries were collected from wild-type and AhRKO mice on PNDs 2–3, 8, 32–35, and 53 for evaluation of primary follicle numbers per ovary. Each bar represents the mean ± SE (n >= 4 in each group at each time point).

 
Effect of the AhR on the Number of Pre-Antral/Antral Follicles
On PND 2–3, both AhRKO and wild-type mice lacked pre-antral/antral follicles. By PND 8, there were antral follicles present in the ovaries of AhRKO and wild-type mice (Fig. 5Go). However, there was no significant difference in the number of these follicles between AhRKO and wild-type mice (n = 8 for wild-type, n = 9 for AhRKO, p = 0.63). On PND 32–35, AhRKO ovaries still had similar numbers of pre-antral/antral follicles as wild-type ovaries (Fig. 5Go; n = 6 for wild-type, n = 9 for AhRKO, p = 0.81). Approximately, 34% of antral follicles were atretic in both AhRKO and wild-type ovaries at this time (p = 0.64). By PND 53, however, AhRKO ovaries had approximately 54% fewer pre-antral/antral follicles compared to wild-type ovaries (Figs. 1 and 5GoGo; n = 5 for wild-type, n = 7 for AhRKO, p = 0.03). Approximately 31% of antral follicles were atretic in AhRKO ovaries and 21% of antral follicles were atretic in wild-type ovaries (p = 0.29).



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FIG. 5. Quantitative evaluation of pre-antral/antral follicle numbers. Ovaries were collected from wild-type and AhRKO mice on PNDs 8, 32–35, and 53 for evaluation of pre-antral/antral follicle numbers per ovary. Each bar represents the mean ± SE (n >= 4 in each group at each time point; *p <= 0.05).

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The physiological role of the AhR in normal ovarian development is unknown. The transgenic model used in this study allows for investigation of ovarian follicle development in the absence of the AhR. Our results indicate that the AhR may have different roles in the ovary in neonatal and postnatal life. Specifically, the AhR may have a role in the rate of formation of primordial follicles during neonatal life and a role in the regulation of antral follicle numbers during postnatal life.

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 2–3, 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., 1991Go; Manova et al., 1993Go; Pesce et al., 1997Go). 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., 1999Go). 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 2–3, 8, 32–35, and 53). It is possible, however, that the significant difference in atresia occurs between PNDs 32–35 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., 1998Go).

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., 1999Go; Heimler et al., 1998Go; Wolf et al., 1999Go). 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., 1994Go; Roman et al., 1998Go; Sommer et al., 1999Go). 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 2–3. 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.


    ACKNOWLEDGMENTS
 
The authors gratefully acknowledge Janice K. Babus for her assistance with histology. The work presented here was supported by the Veteran's Administration (J. A. F.), the Heinz Family Foundation (J. A. F.), and NIH grant ES01332 (R. E. P.).


    NOTES
 
1 To whom correspondence should be addressed. Fax: (410) 706-1503. E-mail: jflaws{at}som.umaryland.edu. Back


    REFERENCES
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 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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