* Animal Reproduction and Biotechnology Laboratory, Department of Biomedical Sciences, Colorado State University, Fort Collins, Colorado 80523-1683; Division of Reproductive Biology and Behavior, Oregon Regional Primate Research Center, Beaverton, Oregon; and
Reproductive Toxicology Division, National Health and Environmental Effects Research Laboratories, USEPA, Research Triangle Park, North Carolina
Received January 22, 2004; accepted March 16, 2004
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ABSTRACT |
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Key Words: dibromoacetic acid; ovary; follicles; rabbit; reproduction.
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INTRODUCTION |
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Despite indications of important reproductive consequences of DBA exposure in males, information on reproductive sequelae following DBA exposure in females is limited. Available data on the effects of DBA in females have linked exposure (90 and 270 mg/kg/day via po gavage for 14 days) to changes in estrous cyclicity and alteration of steroid production by rat preovulatory follicles in vitro (Balchak et al., 2000; Goldman and Murr, 2002
). More recently, DBA has been suggested to suppress estradiol catabolism resulting in elevated circulating concentrations (Goldman and Murr, 2003
). DBA has also been shown to cause dysmorphogenesis in whole embryo culture (Hunter et al., 1996
), but exposure to 62.5, 125, or 250 mg/kg DBA per day during the first eight days of pregnancy did not alter number of implantation sites, number of offspring per litter, or pup weight (Cummings and Hedge, 1998
). Nonetheless, exposure to toxicants during the critical periods of oogenesis and follicular formation could have profound effects on ovarian function and fertility in the adult. Therefore, the overall goal of the present study was to test the hypothesis that chronic exposure of females to DBA would alter gametogenesis.
In all female mammals, the period of germ cell proliferation and follicular formation is strictly limited to fetal or neonatal life (Monniaux et al., 1997). The pool of primordial follicles that is formed during this period comprises the sole source of female gametes and when depleted, reproductive senescence occurs. In smaller mammals (mouse, rat, rabbit), follicular formation does not begin until the perinatal period and is completed within one to two weeks of birth (Mauléon, 1969
; Peters, 1978
; Peters et al., 1965
). In humans, formation of the primordial follicle pool is completed prenatally (Mauléon, 1969
). DBA exposure throughout gestation and lactation did not decrease primordial follicle populations in rats exposed via drinking water to 250 and 650 ppm DBA (22.4 to 132 mg/kg/day; Christian et al., 2002
). In this study (Christian et al., 2002
), total number of primordial follicles counted was very low and information on follicular classification, section thickness, and number of sections counted was not provided. In addition, follicular populations at later stages of development were not assessed. Thus, to our knowledge, follicular populations following DBA exposure have not been thoroughly assessed and any latent effects of DBA on ovarian function and fertility are unknown. Therefore, the objective of the present study was to examine the effects of chronic, relatively low dose exposures to DBA on folliculogenesis in prepubertal and adult female rabbits. In addition, endocrine parameters were examined to determine if the hypothalamic-pituitary-gonadal axis was affected.
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MATERIALS AND METHODS |
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After acclimatization for 45 weeks, 61 rabbit does were artificially inseminated using procedures previously described (Veeramachaneni et al., 2001). Each rabbit doe was given an im injection of 10 µg GnRH (Calbiochem, La Jolla, CA) to induce ovulation and inseminated with 20 million spermatozoa, pooled from semen collected from 11 bucks to increase biological heterogeneity of experimental subjects. Rabbits were palpated for pregnancy 14 days after insemination and resulting pregnant does (n = 45) were randomly assigned to treatment groups for appropriate exposures. On gestation day 28, pregnant does were provided with nesting boxes.
Dosing. Four groups (n = minimum of 10/dose group) of pregnant rabbits were exposed daily to 0 (deionized water; control), 1, 5, or 50 mg DBA/kg body weight via drinking water. Amber, borosilicate glass water bottles fitted with a fluorocarbon septum containing a stainless steel sipper tube, equipped with balls to minimize water dripping were used. Bottle systems were steam-cleaned twice weekly. Individual water bottles were filled daily.
DBA (CAS# 631-64-1, lot# 03807JS, purity 97 %) was obtained from Aldrich Chemical (Milwaukee, WI). To minimize loss due to volatility, stock solutions were prepared twice weekly by adding DBA to deionized water and bringing the pH to 6.87.4 using 1N NaOH and refrigerated. Dosing solutions were prepared daily. DBA concentrations in dosing solutions were computed as follows. Water consumption during a 24-h period was recorded: damsdaily from gestation day 15 (when dosing was begun) until 6 weeks postpartum (when pups were weaned); offspringweekly twice from 612 weeks of age and weekly once from 1224 weeks of age. Body weights were recorded weekly. Dosing solutions were prepared based on the average body weight and average daily water consumption (for each treatment group) during the previous week such that the animals received 1, 5, or 50 mg DBA/kg body weight/day. This strategy ensured continuous delivery of doses very close to those intended.
Dosing of dams began on gestation day 15 and continued through parturition and weaning at 6 weeks postpartum. After weaning, rabbit pups were housed individually and DBA treatments continued until necropsy at 12 weeks (prepuberty; n = 6/dose group) or 24 weeks (postpuberty; n = 10/dose group). Thus, the offspring (experimental units) were exposed to DBA in utero beginning from gestation day 15, throughout nursing via dam's milk, and then in drinking water. Rabbits in each dose group represented at least six different litters.
Tissue collection and processing. During the morning hours of the day before termination of the experiments (at 12 and 24 weeks), a GnRH challenge test was performed to determine the ovulatory response as well as the response of anterior pituitary gland to hypothalamic stimuli. After taking a blood sample by jugular venipuncture, 10 µg GnRH was injected im. Two additional blood samples were collected via jugular venipuncture 30 and 120 min later. Serum was separated and samples stored at 20°C until assayed for gonadotropins.
Twenty to 24 h after GnRH injection, rabbits were euthanized via CO2 asphyxiation and visceral and reproductive organs examined. Ano-genital distance, weights of liver, kidneys, and ovaries were measured. Number of ovulation sites was recorded. Left ovaries were fixed in Bouin's solution for 2436 h, dehydrated in graded series of alcohol, and embedded in paraffin. Ovaries were serially sectioned at a thickness of 6 µm and stained with hematoxylin and eosin. Beginning at a section that contained some medullary tissue, every twelfth section (i.e., at intervals of 72 µm) was evaluated at 200x magnification using a Nikon Eclipse 800 microscope; five sections/rabbit were evaluated for morphometry as described below.
Classification of follicles. All healthy follicles present in sections were categorized into one of five specific developmental stages (Fig. 1). A morphological classification scheme developed by Lundy et al. (1999) and used in our laboratory for morphometry of ovine ovaries (Bodensteiner et al., 2000
) was used. Criteria used to classify rabbit ovarian follicles included the shape (squamous vs. cuboidal) and number of layers of granulosa cells. Primordial follicles were characterized by a single layer of squamous granulosa cells with < 10 cells surrounding the oocyte; primary follicles had 1 but less than 2 complete layers of cuboidal granulosa cells; small preantral follicles had 2 to < 4 layers of cuboidal granulosa cells; large preantral follicles had 4 to 5 layers of cuboidal granulosa cells; and small antral follicles with >5 layers of granulosa cells and an antrum.
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Endocrine analyses. Serum concentrations of FSH and LH were determined by validated radioimmunoassays (Pau et al., 1986) using homologous rabbit reagents provided by Dr. A. Parlow and the Pituitary Hormone Program, National Institutes of Health. The final dilutions of antisera for FSH and LH were 1:72,000 and 1:2,160,000, respectively. For a given hormone, all samples were assayed in a single assay and the intra-assay coefficients of variation were 11.4% for FSH and 4.9% for LH.
Statistical analyses. Differences in parameters were analyzed by one-way ANOVA using the GLM procedure of SAS. If a significant difference (p < 0.05) was indicated, Fisher's protected LSD was used as an indicator of differences among means. Box plots were used to evaluate the variability and distribution of follicular populations (Statview, version 5.0, SAS Institute Inc., Cary, NC).
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RESULTS |
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DBA had no effect on growth rate or total body weight in either age group (Table 2).
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Endocrine Axis and Ovulatory Response
In both prepubertal and adult animals, serum FSH peaked at 30 min and declined by 120 min post-GnRH. In prepubertal animals, baseline concentrations of FSH in the 5 mg/kg dose group were higher than the 1 mg/kg dose group (p < 0.05), but in adult animals FSH concentrations were similar at all time points (Table 3). Serum LH peaked at 30 min post-GnRH in all prepubertal animals, with concentrations in the 1 mg/kg dose group higher than control at this time point (p < 0.05). In adult animals, baseline concentrations of LH were higher in the 5 mg/kg dose group and they remained high even at 120 min post-GnRH (p < 0.05; Table 3). DBA had no effect on the total number of ovulation sites for either age group (Table 4).
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In prepubertal animals, DBA caused a reduction in number of primordial follicles (p < 0.05) and total healthy follicles (p < 0.05) at the 50 mg/kg dose level and decreased numbers of primary (p < 0.05) and small preantral (p < 0.05) follicles at the 1 mg/kg dose level. DBA exposure also increased atretic follicles and follicular remnants in the 1 mg/kg dose group (p < 0.05). When the primary statistical model compared control and DBA treatment with all three dose groups pooled, numbers of primordial, primary, and small preantral follicles were decreased (p < 0.05, p < 0.05, and p = 0.07; respectively), but follicular populations at later stages of development were similar. With pooled data, DBA exposure also decreased total number of healthy follicles (p < 0.05) and increased numbers of follicular remnants (p = 0.08).
In adult animals, total number of healthy follicles was significantly decreased following DBA exposure in the 5 mg/kg treatment group (p < 0.05). In addition, number of primordial follicles was lower in both the 5 (p < 0.01) and 50 (p = 0.1) mg/kg groups as average (±SE) counts were 7.8 ± 0.8, 7.2 ± 0.9, 4.1 ± 0.6, and 5.9 ± 0.6, in the 0, 1, 5, and 50 mg/kg groups, respectively (Fig. 2). Eight animals in the 5 mg/kg group and seven in the 50 mg/kg group had fewer primordial follicles than the control median value (907). Follicular populations at all other stages of development, including small antral follicles, were similar to control, and there was no difference in number of atretic follicles, follicular remnants, or necrotic oocytes. However, although not quantified by morphometric means, it was apparent that aggregation of secondary interstitial cells was more prevalent in DBA-treated groups (Fig. 3). Increased secondary interstitial cell masses reflect increased follicular atresia that had occurred since it is well known that theca cells of atretic follicles become secondary interstitial cells and persist throughout life (Gore-Langton and Armstrong, 1988). This could have contributed to increased ovarian weight in spite of fewer follicles. As in prepubertal rabbits, when data were pooled across all DBA dose groups of adults, number of primordial follicles and total number of healthy follicles were lower (p < 0.05).
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DISCUSSION |
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Decreases in primordial follicle populations in the present study suggest that the process of follicular formation and establishment of the primordial follicle pool may be affected by DBA exposure. Although the underlying mechanism is not known, it is possible that DBA alters expression of oocyte- and granulosa cell-specific growth factors involved in these processes. For example, the oocyte-specific receptor c-kit and its ligand, stem cell factor, have been shown to be necessary for normal primordial germ cell migration and proliferation in mice (Besmer et al., 1993). It has been suggested that stem cell factor may act as an inhibitor of apoptosis in oocytes and granulosa cells during follicular formation in sheep (Tisdall et al., 1997
). Similarly, members of the transforming growth factor-ß superfamily, such as growth and differentiation factor-9, bone morphogenic protein-15, and anti-Müllerian hormone (also called Müllerian inhibitory substance), are required for normal follicular development (Dong et al., 1996
; Durlinger et al., 1999
; Juengel et al., 2002
) and growth and differentiation factor-9 may be necessary for thecal cell recruitment (Elvin et al., 1999
). Absence of Fig-
, an oocyte-specific transcription factor, prevents normal granulosa cell-oocyte interactions during the process of follicular formation in mice, possibly through an effect on basal lamina synthesis (Soyal et al., 2000
). Thus, expression of oocyte- and granulosa cell-specific growth factors must be precisely coordinated for normal follicular formation and disruption of these events could affect future fertility by decreasing the primordial follicle pool.
Disruption of primordial germ cell migration and granulosa cell differentiation have been suggested as mechanisms of toxicity for a variety of xenobiotics (Chen et al., 1981; Mackenzie and Angevine, 1981
; Tam and Snow, 1981
; Wide, 1985
). However, results from a recent study conducted in our laboratory (Weber, 2002
) showed no decrease in primordial follicle populations in mice exposed to a DBA regimen similar to that in the present study with rabbits. Similarly, follicular populations in neonatal rats exposed to 1, 5, and 50 mg DBA/kg (Bodensteiner and Frederick, 2003
) and average number of primordial follicles in adult rats exposed to 4.4 to 132 mg DBA/kg (Christian et al., 2002
) did not differ from controls. The discrepancy between rabbits and rodents may be due to a number of factors including sensitivity of each species and differences among species in the rate at which preantral follicles are recruited into the growing pool or the timing of follicular formation during fetal and neonatal development. For example, entry of oocytes into meiotic prophase occurs on gestation day 13 in mice, gestation day 18 in rats, and on postnatal day 2 in rabbits (Peters et al., 1965
). Thus, beginning DBA exposure on gestation day 15 fails to target the period of meiotic prophase in mice, but not in rats and rabbits. Species differences in reproductive parameters may also be reflective of longer exposure periods in rabbits as compared to mice and rats. For example, the interval between entry of oocytes into meiotic prophase and subsequent primordial follicle formation is eight days in the mouse, five days in the rat, and 12 days in the rabbit (Peters et al., 1965
). The longer period of prepubertal development in the rabbit is more analogous to that of humans, relative to lifespan, and, therefore, rabbit may be a more relevant model to delineate effects of toxic agents on human reproductive health (Veeramachaneni, 2002
; Veeramachaneni et al., 2001
).
Whereas chronic exposure to DBA in adult female rabbits was associated with a decrease in the number of primordial follicles, DBA did not appear to have an effect on later stages of follicular development or ovulation. Furthermore, gonadotropin surges occurred in all animals following exogenous GnRH administration. Similarly, recent data from rats (Christian et al., 2001, 2002
) and rabbits (Christian et al., 2001
) has indicated that chronic DBA exposure does not alter estrous cyclicity or mating performance. These observations indicate that the early stages of follicular development in the rabbit are vulnerable to DBA. Although the rabbit may be a more useful model for toxicological assessment than rodents, the mechanism of action of DBA within the ovary remains unknown. Thus, further studies are needed to determine the precise role of DBA in ovarian follicular toxicity in this species.
Traditionally, qualitative measures of female reproductive capability, such as fertility and abnormal clinical symptoms, have been used to assess toxicity. Recently, however, differential follicle counts have been found to be sensitive, quantifiable endpoints of ovarian injury (Bolon et al., 1997; Yu et al., 1999
). Given the fact that small follicles are numerous, relatively unaffected by hormonal fluctuations, and rarely undergo spontaneous atresia, counting preantral follicular populations can provide a direct estimate of the ovarian functional reserve after exposure to female reproductive toxicants (Bolon et al., 1997
). In addition, the total number of follicles counted from serially sectioned ovaries does not differ from the number obtained when random sections are used (Bucci et al., 1997
; Smith et al., 1991
). Thus, sampling populations of preantral follicles provides a good indication of ovarian injury and may be a more sensitive endpoint of female reproductive toxicity than traditional methods of assessment.
Estimates of exposures to haloacetic acids have detected concentrations of DBA from 0.9 to 1.5 µg/l in drinking water, but higher DBA values ranging from 7.8 to 19 µg/l have been reported (Jacangelo et al., 1989; Krasner et al., 1989
). Thus, the 1 mg/kg dose in the present study was approximately 1000-fold higher than levels typically found in public water supplies. Assuming a person weighing 60 kg consumes 2 liters of water a day containing 20 µg DBA/l, a value typical of drinking water in areas having elevated bromide concentrations (Krasner et al., 1989
), this individual will have a margin of exposure of 1500 at a no-observed-adverse-effect-level of 1 mg DBA/kg (as in this study). The calculation is as follows: (1 mg/kg)/[2 l (0.02 mg/l)/60 kg] = 1500. Humans are exposed to mixtures of disinfection byproducts, however, and additive effects of these compounds have been suggested (Richard and Hunter, 1996
; Teuschler et al., 2000
). Using municipal water concentrations as an indication of exposure can be misleading due to the amounts and ways in which people use water (Weisel et al., 1999
). This is especially true for disinfection byproducts, like DBA, whose primary route of exposure is ingestion. Estimates of DBA exposure are further confounded by possible bioaccumulatory effects and formation of DBA during metabolism of other substances (Kennedy et al., 1993
). Thus, understanding mechanism of action, synergism, and additivity for this class of chemicals is essential for risk assessment and further research is warranted.
In summary, chronic exposure to DBA decreased the number of primordial follicles in adult female rabbits, but did not affect patterns of antral follicular growth or the ovulatory response. While it is clear that primordial follicle pool is diminished by DBA, the long-term reproductive consequences of a decrease in primordial follicles remain to be determined. It seems likely that the number of follicles entering the later stages of development will be diminished at some point leading to early onset of reproductive senescence. Future studies need to be done to evaluate the consequence of similar exposure later in the reproductive life of the rabbit. Moreover, since it is now obvious that different responses occur in rodents and rabbits, comparable studies need to be conducted in other species with appreciably longer periods of reproductive development, e.g., primate, and over longer periods of exposure.
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ACKNOWLEDGMENTS |
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NOTES |
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1 Present address: Biology Department, St. Lawrence University, Canton, NY 13617.
2 Present address: Department of Medical Education and Public Health, University of Wyoming, Laramie, WY 82071.
3 To whom correspondence should be addressed. Fax: (970) 491-3557. E-mail: rao{at}colostate.edu.
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