* Program in Toxicology and Department of Epidemiology and Preventive Medicine, and Department of Physiology, University of Maryland, Baltimore, Maryland 21201
1 To whom correspondence should be addressed at Department of Epidemiology and Preventive Medicine, 660 W. Redwood Street, Howard Hall Suite 133, Baltimore, MD 21201. Fax: (410) 706-1503. E-mail: jflaws{at}epi.umaryland.edu.
Received June 14, 2005; accepted August 1, 2005
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ABSTRACT |
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Key Words: methoxychlor; antral follicles; ovary; Bcl-2; Bax.
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INTRODUCTION |
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Females are born with a finite number of primordial follicles (Hirshfield, 1997). Some of these follicles mature to the antral stage and release eggs for fertilization during the reproductive years. The majority of the follicles (greater than 99%), however, undergo atresia (Byskov, 1978
). The process of ovarian atresia is an apoptotic process that is regulated by highly conserved factors, including members of the Bcl-2 family of proteins (Choi et al., 2004
; Gosden and Spears, 1997
; Hsu and Hsueh, 2000
). Two important family members that regulate ovarian atresia are Bcl-2, an anti-apoptotic factor, and Bax, a pro-apoptotic factor (Choi et al., 2004
; Flaws et al., 2001
; Gosden and Spears, 1997
; Hsu et al., 1996
; Hsu and Hsueh, 2000
).
Atresia is a normal process in the ovary that occurs throughout female reproductive life, during all stages of follicular development, and is central to the proper function of the ovary (Gosden and Spears, 1997). Elevating the normal levels of atresia, however, can have a significant impact on the number of follicles available for maturation and fertilization in the adult years. Over time, the ovary becomes devoid of follicles because they are depleted through the processes of ovulation and atresia. Once the ovary is devoid of follicles, the female becomes infertile and is said to have entered reproductive senescence or menopause. Any chemical that increases atresia of follicles could increase the rate at which follicles are depleted from the ovary and result in premature ovarian failure, and early menopause in the case of humans. This is a concern because early menopause has been associated with an increased risk of chronic diseases such as osteoporosis and cardiovascular disease (Jacobsen et al., 1999
; Ohta et al., 1996
; Pouilles et al., 1994
; van der Schouw et al., 1996
).
Methoxychlor (MXC) is a widely used chlorinated organic pesticide that targets the ovary, with outcomes including reduced fertility (Cummings and Gray, 1989; Swartz and Eroschenko, 1998
), ovarian atrophy (Eroschenko et al., 1995
; Okazaki et al., 2001
), persistent estrus (Martinez and Swartz, 1991
; You et al., 2002
), and follicular atresia (Martinez and Swartz, 1991
; Swartz and Corkern, 1992
). Recently, studies have shown that exposure to MXC in vivo results in antral follicle-specific toxicity, characterized by a decreased number of healthy antral follicles, and an increased percentage of antral follicles undergoing atresia versus controls (Borgeest et al., 2002
).
While previous work indicates that MXC increases atresia of antral follicles (Borgeest et al., 2002), it is important to know whether the increased atresia is due to a direct effect of MXC on antral follicles, or an indirect effect caused by toxicity to other components of the ovary or other tissues that contribute to ovarian function (i.e., the hypothalamus or pituitary). To overcome these problems and examine the mechanism of action of MXC in antral follicles, we utilized in vitro culture techniques to directly assess the effects of MXC on isolated ovarian antral follicles. One advantage of this method is that we can directly examine the effect of MXC on antral follicles in a dose and time response manner, without confounding factors characteristic of in vivo toxicity studies. An additional advantage of follicle culture is that metabolism of MXC by the liver is eliminated, allowing us to look at the ovotoxicity of MXC. Furthermore, we can determine the effects of MXC on follicle growth over time, which is difficult to do in vivo. Finally, we can use the in vitro system to examine the mechanism by which MXC damages antral follicles. Specifically, we can determine the role of Bcl-2 family members (anti-apoptotic Bcl-2 and pro-apoptotic Bax) in regulating MXC-induced antral follicle atresia. Therefore, we utilized this system to test the hypothesis that MXC is directly toxic to antral follicles, and that Bcl-2 overexpression, or Bax deletion, protects antral follicles from MXC-induced toxicity.
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MATERIALS AND METHODS |
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Animals.
Female mice were used from breeding colonies currently maintained by our laboratory at the University of Maryland Central Animal Facility. Bcl-2 overexpressing mice were developed by Flaws et al. (2001), and are in an FVB background, along with their wild-type (WT) littermates. Bax-deficient mice were generated by Knudson et al. (1995)
, and are in a C57BL/6 background, along with their WT littermates. All mice were genotyped using polymerase chain reaction (PCR)-based assays that have been previously described (Flaws et al., 2001
; Knudson et al., 1995
). Bcl-2 overexpression in the ovary and antral follicles was confirmed by Western blot analysis (data not shown). Only WT mice and mice with homozygous deletion or overexpression were used in experiments. In addition, female CD-1 mice were obtained from Charles River Laboratories (Wilmington, MA), for quantitative PCR studies that would correlate with previously conducted in vivo studies. We determined that FVB, CD-1, and C57BL/6 mice have similar growth curves and atresia ratings in response to vehicle and MXC treatment in vitro (see figures for growth curves and atresia ratings for FVB and C57BL/6 mice), and respond to MXC toxicity in a similar manner in vivo (Borgeest et al., 2002
, 2004
). Therefore, strain difference was not a limiting factor. Mice from each colony were housed (five animals per cage) at the University of Maryland Central Animal Facility and provided food and water ad libitum. Temperature was maintained at 22 ± 1°C and animals were subjected to 12L:12D cycles. The University of Maryland School of Medicine Institutional Animal Use and Care Committee approved all procedures involving animal care, euthanasia, and tissue collection.
In vitro follicle culture.
Female FVB or C57BL/6 mice were sacrificed on postnatal day (PND) 3035 and their ovaries removed. Antral follicles were isolated mechanically from the ovary based on relative size and cleaned of interstitial tissue using fine watchmaker forceps. Sufficient numbers of antral follicles for experimental significance were isolated from unprimed mouse ovaries; follicles from 24 mice were isolated per day with approximately 2025 antral follicles from each mouse. Upon isolation, follicles were placed individually in wells of a 96-well culture plate with unsupplemented -Minimal Essential Media (
-MEM) prior to treatment. Each experiment contained a minimum of eight follicles per treatment. Supplemented
-MEM was prepared with: 1% ITS (10 ng/ml insulin, 5.5 ng/ml transferrin, 5.5 ng/ml selenium), 100 U/ml penicillin, 100 mg/ml streptomycin, 5 IU/ml human recombinant FSH (Dr. A. F. Parlow, National Hormone and Peptide Program, Harbor-UCLA Medical Center, Torrance, CA), and 5% fetal calf serum (Atlanta Biologicals, Lawrenceville, GA). A dose response regimen of MXC (0.01100 µg/ml) and DMSO controls were individually prepared in supplemented
-MEM, with an equal volume of chemical added for each dose to control for the amount of vehicle in each preparation. For treatment, unsupplemented
-MEM was removed from each well and replaced with 150 µl supplemented
-MEM containing MXC or vehicle. Follicles were then incubated for 096 h at 37°C in 95% air and 5% CO2. DMSO concentrations in all experiments were kept below 0.075%, levels that solubilized MXC in aqueous media without overt changes in growth or atresia. Non-treated (NT) controls were used in each growth experiment to control for culture conditions. Follicles were also determined to be viable for 96 h in culture by trypan blue exclusion experiments (data not shown).
Analysis of follicle growth.
Antral follicles were cultured as described above for 96 h. Follicle growth was examined at 24 h intervals by measuring follicle diameter on two perpendicular axes with an inverted microscope equipped with a calibrated ocular micrometer. Antral follicles were considered as those having diameters of 200 µm or greater (Smitz and Cortvrindt, 2002), which correlates with histological appearance of these follicles. At least three separate culture experiments were performed for each chemical treatment. Follicle diameter measurements were averaged among treatment groups and plotted to compare the effects of chemical treatments on growth over time.
Histological evaluation of atresia.
At the end of each follicle culture, supplemented -MEM was removed from each well and Dietrick's solution was immediately added to fix follicles. Follicles were fixed for at least 24 h in Dietrick's solution and transferred in histology cassettes to 70% ethanol. The tissues were dehydrated, embedded in Paraplast (VWR Scientific, West Chester, PA), serially sectioned (5 µm), mounted on glass slides, and stained with Weigert's hematoxylin and methyl blue:picric acid. Each follicle section was examined for level of atresia as evidenced by the presence of pyknotic bodies and reported at the highest level observed throughout the tissue. Follicles were rated on a scale of 15 for the presence of pyknotic bodies: 1 = healthy, 2 = less than 10% pyknotic bodies (early), 3 = 1030% pyknotic bodies (mid), 4 = greater than 30% pyknotic bodies in an isolated area (late), and 5 = greater than 30% pyknotic bodies over the entire follicle (late and widespread). In addition, follicles were evaluated based on size: small follicles (200349 µm starting diameter) versus large follicles (350 µm and larger starting diameter). This was done to compare follicular atresia in small and large follicles in response to MXC because studies indicate that small and large antral follicles may respond differently to toxicants (Hirshfield, 1988
; Roby, 2001
). At least three separate culture experiments and atresia analyses were performed for each chemical treatment. Ratings were averaged and plotted to compare the effect of chemical treatments on atresia levels.
Quantitative real time polymerase chain reaction (qPCR).
Female CD-1 mouse antral follicles were cultured as described above for 24, 48, 72, or 96 h with MXC (1100 µg/ml) or DMSO. After each time point, follicles were collected and snap-frozen at 80°C for qPCR analysis. Total RNA was extracted from follicles using the RNeasy Mini Kit (Qiagen, Inc., Valencia, CA) according to the manufacturer's protocol. Reverse-transcriptase generation of cDNA was performed with 0.31 µg of total RNA using an Omniscript RT kit (Qiagen, Inc., Valencia, CA) with random primers according to the manufacturer's protocols. qPCR was conducted using an MJ Research Chromo4 Real Time PCR machine (MJ Research, Inc., Waltham, MA) and accompanying software according to the manufacturer's instructions. The Chromo4 quantifies the amount of PCR product generated by measuring a dye (SYBR green) that fluoresces when bound to double-stranded DNA. A standard curve was generated from five serial dilutions of one of the samples, thus allowing analysis of the amount of cDNA in the exponential phase. Primer sequences used were as follows: Bcl-2 (forward) 5'-CTC-GTC-GCT-ACC-GTC-GTG-ACT-TCG-3', (reverse) 5'-CAG-ATG-CCG-GTT-CAG-GTA-CTC-AGTC-3'; Bax (forward) 5'-ACC-AGC-TCT-GAA-CAG-ATC-ATG-3', (reverse) 5'-TGG-TCT-TGG-ATC-CAG-ACA-AG-3'; ß-actin (forward) 5'-GGG-CAC-AGT-GTG-GGT-GAC-3', (reverse) 5'-CTG-GCA-CCA-CAC-CTT-CTAC-3'. qPCR analysis was performed using 3 µl cDNA, forward and reverse primers (5 pmol) for Bcl-2, Bax, or ß-actin, in conjunction with a DyNAmo SYBR Green qPCR kit (Finnzymes, c/o Bio-Rad Laboratories, Hercules, CA). An initial incubation of 95°C for 10 min was followed by denaturing at 94°C for 10 s, annealing at 60°C (Bcl-2 and Bax) or 55°C (ß-actin) for 10 s, and extension at 72°C for 10 s, for 50 cycles (Bcl-2 and Bax) or 39 cycles (ß-actin), followed by final extension at 72°C for 10 min. A melting curve was generated between 5590°C to indicate the generation of a single product. The software also generated a standard curve. ß-Actin was used as an internal standard for each sample. Final values were expressed as genomic equivalents and were calculated as the ratio Bcl-2:ß-actin or Bax:ß-actin. All experiments were performed in triplicate.
Statistical analysis.
All data were analyzed using SPSS statistical software (SPSS, Inc., Chicago, IL). For all comparisons, statistical significance was assigned at p 0.05. For multiple comparisons between DMSO and MXC-treated follicles, and comparisons between genotypes, we used analysis of variance (ANOVA), along with a Dunnett 2-sided post hoc test and multiple regression analysis. At least eight follicles per treatment per experiment were evaluated and the results of at least three separate experiments were combined for data analysis.
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RESULTS |
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DISCUSSION |
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Using the follicle culture assay, we have shown that MXC is directly toxic to antral follicles. Direct exposure of isolated antral follicles to MXC inhibited follicle growth and increased atresia in a dose-dependent manner. These data are consistent with those obtained from in vivo studies indicating that MXC increases atresia of antral follicles (Borgeest et al., 2002). Our results also expand on work previously done in vivo since we were able to show that MXC has a negative effect on antral follicle growth. We have therefore determined that follicle growth is a marker of MXC toxicity that can be directly observed and measured in vitro, and may correlate with the onset of atresia due to chemical exposure.
We have also shown that Bcl-2 and Bax pathways mediate MXC toxicity in the antral follicle. Upon examining mRNA levels of Bcl-2 and Bax from isolated antral follicles exposed to MXC, we found that Bax levels were increased compared to controls as early as 48 h after 100 µg/ml MXC exposure in vitro, and remained high through 96 h. A longer exposure period, however, was necessary for Bcl-2 levels to change, as decreases in Bcl-2 mRNA levels were only observed after 96 h. This expands the findings of previous in vivo studies in our laboratory which indicated that treatment with MXC in vivo (64 mg/kg/day for 20 days) resulted in enhanced immunohistochemical staining for the pro-apoptotic Bax protein compared to controls (Borgeest et al., 2004). Collectively, our data indicate that an apoptotic pathway might be initiated early during exposure, and that the ratio of Bcl-2 to Bax may be altered to drive cells within the follicle toward apoptosis. Cells are protected when Bcl-2 levels are in excess of Bax, leading to the formation of Bcl-2-Bax heterodimers or Bcl-2 homodimers that prevent Bax induction of apoptosis (Basu and Haldar, 1998
). However, apoptosis proceeds when Bax levels are in excess and Bax:Bax homodimers predominate (Basu and Haldar, 1998
). This pattern of expression may explain our findings. Bax levels begin to rise at 48 h, but it is not until 96 h when Bcl-2 levels decline, and Bax levels remain high, that we observe atresia in MXC-treated antral follicles. It is possible that Bcl-2 levels must be high enough prior to 96 h to prevent Bax homodimer formation. These results indicate that Bcl-2 and Bax may work coordinately in antral follicles to mediate MXC toxicity and induce atresia.
Previous studies examining the role of Bcl-2 and Bax in MXC-induced ovarian toxicity in vivo also indicated that ovaries of Bcl-2 overexpressing mice or Bax deficient mice were protected from MXC-induced atresia (Borgeest et al., 2004). To determine whether the protection observed was a function of mechanisms occurring in the antral follicle, or mechanisms occurring in the whole ovary that influence atresia, we investigated the effect of MXC treatment on isolated antral follicles from Bcl-2 OE and BaxKO mice. Our in vitro results indicate that Bcl-2 overexpression and Bax deletion delays MXC-induced growth inhibition in comparison to WT controls, but does not prevent it. In addition, our results indicate that small and large antral follicles were differentially affected by MXC toxicity leading to atresia. Large antral follicles of Bcl-2 OE mice were protected from MXC-induced atresia compared to WT controls, while those from BaxKO mice were not protected from MXC-induced atresia. Furthermore, small antral follicles from both Bcl-2 OE and BaxKO mice were not protected from MXC-induced atresia. Multiple factors may contribute to these results. With regard to growth, it is possible that Bcl-2 and Bax contribute to cellular growth pathways that regulate follicle growth in culture, but that their overexpression or deletion are not sufficient to completely overcome growth inhibition due to MXC. With regard to atresia, we hypothesize that the differences in protection between Bcl-2 OE and BaxKO large antral follicles is due to the fact that our line of BaxKO mice is completely deficient in Bax, when Bel-2 is only elevated in the ovaries of Bcl-2 OE mice (Flaws et al., 2001
; Knudson et al., 1995
). While Bcl-2 overexpression in antral follicles is sufficient to protect large follicles from MXC-induced toxicity, Bax deficiency in the follicle may not be sufficient for protection from in vitro MXC toxicity, and signaling pathways in the whole animal influenced by the deletion of Bax may be necessary to induce the protection we observe upon MXC exposure in vivo. Other investigators have indicated that while Bax mediates granulosa cell apoptosis in atretic follicles, Bax deletion does not prevent the induction of follicle atresia, demonstrating that other factors may be involved to link the two processes (Gosden and Spears, 1997
). It is also possible that other pro-apoptotic Bcl-2 family members, such as Bak, are present and active, and compensate for the loss of Bax to continue to drive the natural process of atresia. Thus, other factors and apoptotic pathways may be involved in MXC-induced atresia and growth inhibition in addition to Bcl-2 and Bax. As for the absence of protection in small antral follicles, we speculate that these smaller follicles may not be able to respond completely to signaling mechanisms altered by overexpression of Bcl-2 or deletion of Bax, as they are less mature than larger antral follicles and remain susceptible to atresia. Another possibility is that in our colony of Bcl-2 OE mice, overexpression of Bcl-2 is driven by the c-kit promoter, which is present in theca cells. It may be that larger follicles have more theca cells, and overexpression of Bcl-2 is at a higher level in these follicles compared to smaller follicles, therefore providing more protection.
The mechanism by which MXC induces changes in Bcl-2 family members is unknown. It is possible that MXC may influence Bcl-2 regulation via the estrogen receptor (ER). Studies have shown that estradiol, a potent ER ligand, blocks injury-induced downregulation of both Bcl-2 and ERß in a parallel manner in the brain, suggesting that ERß may mediate the protective effects of estradiol on Bcl-2-mediated apoptotic pathways (Dubal et al., 1999). Since MXC is an antagonist for ERß, the predominant ER isoform in the ovary, this might provide a mechanism of action linking ER and Bcl-2 regulatory pathways in the ovary. This hypothesis is supported by data from studies on the ovarian surface epithelium, which indicate that MXC regulates growth and apoptosis of the ovarian surface epithelium through ER (Symonds et al., 2005
). Further, other investigators have shown by microarray analysis that MXC regulates the expression of many genes, implicating other pathways of action as well (Larkin et al., 2003
; Terasaka et al., 2004
; Waters et al., 2001
).
In conclusion, these studies have shown that an in vitro ovarian follicle culture model to assess MXC toxicity is able to effectively mimic in vivo models. These studies also expand previous work by indicating that MXC directly inhibits antral follicle growth and increases atresia in part through modulation of Bcl-2- and Bax-mediated apoptotic pathways. Future studies to identify other Bcl-2 family members that mediate MXC-induced apoptosis will be helpful in distinguishing pathways of toxicity and developing ways to prevent atresia and premature ovarian failure caused by environmental chemical exposures.
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ACKNOWLEDGMENTS |
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