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research-article |
Department of Epidemiology and Preventive Medicine,3 University of Maryland School of Medicine, Baltimore, Maryland 21201
Department of Veterinary Biosciences,4 University of Illinois Urbana-Champaign, Illinois 61802
ABSTRACT
The aryl hydrocarbon receptor (AHR) is a known transcription factor. Although studies indicate that Ahr-deficient (AhRKO) mice have defects in female reproduction, only a few studies have examined the role of AHR in the ovary. Previous studies have suggested, without directly testing, that AhRKO mice have slower follicular growth than wild-type (WT) mice. Therefore, the first objective of the present study was to examine whether AhRKO follicles grow slower than WT follicles and if so, to determine whether the mechanism by which Ahr affects follicular growth is through effects on antrum size, granulosa cell proliferation, and regulators of cell cycle progression. Since estradiol (E2) is critical for the normal growth of ovarian follicles, the second objective of the present study was to determine the role of Ahr in regulating E2 production and responsiveness. The third objective of the present study was to determine whether E2 replacement restores follicular growth of AhRKO follicles to WT levels in vitro. We found that AhRKO follicles grew slower than WT follicles in vitro. While AhRKO and WT follicles had similar antrum sizes, AhRKO follicles showed decreased granulosa cell proliferation and reduced mRNA and protein levels of cell cycle regulators, as compared to WT follicles. Furthermore, the AhRKO mice had lower serum and follicle-produced E2 levels and showed decreased Esr1 and Esr2 mRNA levels compared to WT mice. Finally, E2 treatment of AhRKO follicles restored follicular growth to WT levels in vitro. Collectively, these findings suggest that the AHR affects follicular growth via mechanisms that involve E2 regulation and responsiveness.
estrdiol, estradiol receptor, follicle, ovary, toxicology
The aryl hydrocarbon receptor (AHR) is a basic helix-loop-helix (bHLH) and Per-Arnt-Sim (PAS)-containing transcription factor that regulates the expression of genes in a ligand-dependent manner [1, 2]. The major known role of the AHR is to mediate the toxic effects of various environmental contaminants, such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and 7,12-dimethylbenz[a]anthracene (DMBA) [1–4]. Upon binding of a ligand to AHR, a cascade of events occurs that often leads to the induction of various metabolizing enzymes and some toxic effects [1–6].
In addition to its role in mediating toxic responses, studies using Ahr-deficient (AhRKO) mice have provided evidence for an endogenous role of AHR. These mice have decreased liver size, hepatic fibrosis, decreased expression of various xenobiotic-metabolizing enzymes, and immune system impairment [6, 7]. AhRKO mice have also been shown to have defects in the female reproductive system [8]. Specifically, AhRKO mice have reduced fertility, small litters, difficulty maintaining conceptuses, difficulty surviving pregnancy and lactation, and difficulty rearing pups to weaning [8]. Collectively, these data suggest that AHR can mediate biological responses in nonreproductive and reproductive tissues.
Benedict et al. [9, 10] have examined whether the reduced fertility in AhRKO mice is partly due to ovarian defects and have shown that AhRKO ovaries have significantly fewer preantral and antral follicles compared to wild-type (WT) ovaries [9]. Later work by Benedict et al. [10] has shown that the decrease in the number of antral follicles in AhRKO mice compared to WT mice is not due to follicle death (atresia). Instead, they have hypothesized that the decreased number of antral follicles is due to an impaired ability of AhRKO follicles to grow to the antral stage [10]. This hypothesis is supported by their data indicating that by Postnatal Day (PD) 53, almost twice as many follicles reached the antral stage in WT ovaries as in AhRKO ovaries in vivo [10].
While the in vivo data obtained by Benedict et al. [10] provide some support for the hypothesis that Ahr regulates follicle growth, it is difficult to measure accurately the growth of individual mouse ovarian follicles over time in vivo. Instead, investigators count the relative numbers of follicles at each stage of development at selected time-points. Thus, the first objective of the present study was to test directly using an in vitro system whether AhRKO follicles grow slower than WT follicles. The in vitro system provides a means to measure the growth of individual follicles over time [11–14].
The second objective of the present study was to examine the mechanism by which Ahr affects follicular growth. One possible mechanism by which follicle growth may be altered in AhRKO mice is through reduced granulosa cell proliferation. To test this possibility, granulosa cell proliferation and the levels of mRNA and protein of factors required for the cell cycle progression that drives normal cell proliferation (cyclin-dependent kinase 4 [Cdk4, CDK4] and cyclin D2 [Ccnd2, CCND2]) were compared in AhRKO and WT follicles.
Alternatively, it is possible that the mechanism by which Ahr regulates follicle growth involves Ahr regulation of estradiol (E2) levels. This possibility is supported by several lines of evidence, which indicate that E2 is required for the normal growth of ovarian follicles and for granulosa cell proliferation [15–18]. For example, studies using aromatase-deficient (ArKO) mice have shown that ArKO mice lack the ability to produce estrogen and are infertile due to impaired folliculogenesis and inability to ovulate [18]. To test whether the mechanism by which Ahr regulates follicular growth involves Ahr regulation of E2 levels, we examined the levels of E2 in AhRKO and WT mice, as well as the levels of E2 produced by AhRKO and WT follicles. Furthermore, it is possible that deleting Ahr alters the mRNA or protein levels of estrogen receptors (Esrs, ESRs), thereby causing reduced responsiveness of AhRKO follicles to E2, a scenario that could slow follicular growth. The possibility that AhRKO mice may have decreased expression of Esrs is supported by evidence of Ahr-Esr cross-talk in a variety of cells and tissues [19–21]. To test whether this is the case, the levels of Esr1, Esr2, ESR1, and ESR2 were compared in AhRKO and WT follicles. Lastly, we investigated whether administering E2 to AhRKO follicles restores AhRKO follicle growth to WT levels.
AhRKO mice were originally generated as described by Schmidt et al. [22], and breeding pairs were generously provided by Dr. Christopher A. Bradfield (McArdle Laboratory for Cancer Research) and Dr. Richard E. Peterson (University of Wisconsin). The colony of AhRKO and WT mice used in these experiments was housed in individual clear plastic cages and maintained on a 12L:12D cycle (lights on at 0600 h) 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 University of Maryland and the University of Illinois approved all protocols involving mice.
The screening protocol used in the present study was adopted from a previous study [9]. Briefly, ear tissue punches were lysed in a proteinase K buffer (7.5 µl dH20 and 1.5 µl proteinase K) at room temperature (RT) for 30 min, followed by incubation at 100°C for 3 min. The lysates were subjected to PCR assays using the primers described previously [9]. The PCR products were sized by agarose gel electrophoresis. The WT (Ahr+/+) mice were identified by the presence of a 670-bp product. Homozygous AhRKO (Ahr–/–) mice were identified by the presence of a 580-bp product. Heterozygous (Ahr+/–) mice were identified by the presence of both the 580-bp and 670-bp products. Only homozygous AhRKO and WT mice were used in each experiment.
Measurement of Follicular Growth and Granulosa Cell Proliferation In Vitro
An established follicle culture system was used to measure follicle growth and granulosa cell proliferation over time [11–14]. During culture, the follicles attach themselves to the bottom of the plate, and the granulosa cells proliferate to form a dome-shaped cover over the follicles, resulting in an increase in follicular size. Measurements are taken on two perpendicular axes and averaged to get the final measure, which indicates total follicle growth resulting from granulosa cell proliferation [11–14].
Specifically, AhRKO and WT ovaries were collected on PD 30–35. Antral follicles of 300–400 µm were mechanically isolated from the ovaries using fine watchmaker forceps under a dissecting microscope. Upon isolation, at least ten follicles per mouse were placed into individual wells of a 96-well culture plate with 
-Minimal Essential Medium (-MEM) that was supplemented 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 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). Incubation conditions were 37°C in 95% air and 5% CO2. To maintain culture viability, the medium was replaced with fresh medium after 72 h and 120 h of culture. Growth/proliferation was examined by measuring follicle diameter on two perpendicular axes daily for 168 h. Follicle growth and change over time were calculated to compare the growth rates of the AhRKO and WT follicles. Samples of the culture supernatants were collected and stored at –20°C for E2 measurements, as described below.
Measurement of Granulosa Cell Proliferation In Vivo
Measurements of granulosa cell proliferation in vivo were performed by immunostaining for proliferating cell nuclear antigen (PCNA), as previously described [23–29]. PCNA was selected as the proliferation marker for these studies as it has been used successfully and reliably in ovarian tissues by several investigators [27–29]. Briefly, ovaries were removed from AhRKO and WT mice, fixed in Bouin solution, dehydrated in gradient alcohols, cleared in xylene, and embedded in paraffin. Sections were cut at 5 µm and subjected to staining for PCNA using a commercially available monoclonal antibody (1:100 dilution; Oncogene Research Products, Boston, MA). The biotinylated streptavidin Histomouse-SP system (Zymed Laboratories, San Francisco, CA) with background blocking was used with 3-amino-9-ethyl carbazole chromogenic visualization and Mayer hematoxylin counterstaining. In all experiments, negative controls (no primary antibody) were run in parallel.
The levels of PCNA staining in AhRKO and WT ovaries were quantified using the methods of Masters et al. [30], with minor modifications. Specifically, images from stained sections obtained from at least four different animals per genotype and age were digitally captured using a Leica DFC 290 camera and analyzed using the ImageJ software (http://rsb.info.nih.gov/nih-image/). Digital images were initially converted to 8-bit grayscale images and then converted to pseudocolored images. Colors were based on relative stain intensity, as defined digitally. Areas with no staining appeared black, while areas with the most intense staining appeared deep orange to red. The PCNA labeling index, expressed as a percentage of positively stained area per follicle, was determined for all follicles in each section (7–20 follicles per section from at least four separate animals per genotype). Values for PCNA labeling indices were generated by dividing the PCNA positively stained area per follicle by the total area per follicle and multiplying the values by 100. All ovaries were analyzed without knowledge of genotype, to avoid bias.
Measurement of Antrum Size In Vivo
To determine whether follicle growth in vivo was also occurring via changes in antral size, AhRKO and WT follicles were collected on PD 54 and histologically processed, as described by Benedict et al. [9]. Antrum size was calculated digitally using the IP Lab software for looking at the ratio of antrum size versus the size of the follicle. A boundary was drawn around the antral space and a secondary boundary was drawn around the follicle itself. The IP Lab software then calculated area that the antral space covered in the whole follicle.
RNA Isolation and Real-Time PCR
The levels of Cdk4, Ccnd2, Esr1, and Esr2 mRNA expression in AhRKO and WT follicles were compared using Real-time PCR, as described by Tomic et al. [23]. Antral follicles were collected from AhRKO and WT ovaries on PD 32–35 and immediately stored at –70°C until RNA extraction. Total RNA was extracted from the follicles using the RNeasy Mini Kit (Qiagen, Valencia, CA) according to the manufacturers protocol. RNA (0.5–1.0 µg) was then reverse-transcribed using the Omniscript reverse transcriptase kit (Qiagen) with random primers. Real-time PCR was conducted using the Opticon PCR machine (MJ Research) and the accompanying software. The Opticon machine quantifies the amount of PCR product generated by measuring the amount of dye (SYBR Green) that fluoresces when bound to double-stranded DNA. A standard curve was generated from five serial dilutions of purified PCR product. For each primer sequence described below, a melting curve was performed. Real-time PCR amplification of the individual genes was performed using 3 µl of cDNA from AhRKO and WT mice, respectively. The following specific primer sequences were used: for Cdk4, forward 5'-TGGCTGCCACTCGATATGAAC-3' and reverse 5'-CCTCAGGTCCTGGTCTATATG-3' [31]; for Ccnd2, forward 5'-AGCTGTCCCTGATCCGCAAG-3' and reverse 5'-GTCAACATCCCGCACGTCTG-3' [32]; for Esr1, forward 5'-AATTCTGACAATCGACGCCAG-3' and reverse 5'-GTGCTTCAACATTCTCCCTCCTC-3' [33]; and for Esr2, forward 5'-CTTGGTCACGTACCCCTTAC-3' and reverse 5'-GTATCGCGTCACTTTCCTTT-3' [33]. Primers specific for mouse ß-actin were used as an internal control as previously described [33].
Measurements of ESR1, ESR2, CDK4, and CCND2 Protein Levels
Antral follicles from AhRKO and WT mice were collected on PDs 30–35 and immediately snap-frozen. Each frozen sample was homogenized in lysis buffer (20 mM Tris-HCl [pH 8.0], 1% NP40, 140 mM NaCl, 2 mM EDTA, 10 mM NaF, 10% glycerol) that contained a protease inhibitor cocktail tablet (Roche Diagnostics, Mannheim, Germany). After homogenization, the amount of protein in each sample was evaluated using a bicinchoninic acid (BCA) protein assay kit (Pierce, Rockford, IL). Protein lysates (15 µg/lane for ESR1 and ESR2, and 10 µg/lane for CDK4 and CCND2) were subjected to Western blot analysis using anti-ESR1 (1:100; Santa Cruz Biotechnology, Santa Cruz, CA), anti-ESR2 (1 µg; Abcam, Cambridge, MA), CDK4 (1:2000; Abcam), and anti-CCND2 (1:2000; Abcam) polyclonal antibodies. For ESR1 and ESR2, a horseradish peroxidase (HRP)-conjugated anti-mouse antibody (1:100; Santa Cruz Biotechnology) was used as the secondary antibody. For CDK4 and CCND2, a HRP-conjugated rabbit polyclonal mouse IgG antibody (1:2000; Abcam) was used as the secondary antibody. Immune complexes were visualized using an enhanced chemiluminescence (ECL) detection kit (Cell Signaling Technologies, Beverly, MA). To ensure that proteins were loaded in equal amounts in each lane, the blots were stripped using the ImmunoPure elution buffer (Pierce). For ESR1 and ESR2, the blots were then incubated with ß-actin (1:300 dilution; Santa Cruz Biotechnology) followed by a HRP-conjugated anti-mouse polyclonal antibody (1:100; Santa Cruz Biotechnology). For CDK4 and CCND2, the blots were incubated with -tubulin (1:5000; Abcam) followed by goat polyclonal anti-rabbit IgG (1:2000; Abcam). It was necessary to use -tubulin instead of ß-actin for the CDK4 and CCND2 blots because the bands of interest were similar in size to ß-actin. Scanning densitometry using the ImageJ software was used to compare the protein levels in the AhRKO and WT samples.
Measurements of Hormone Levels
To measure serum E2 levels, blood samples were collected from AhRKO and WT mice on PDs 30 and 90 on the same day of the estrus cycle (estrus) to minimize natural fluctuations in hormone levels. The samples were centrifuged at 15 000 rpm for 15 min to isolate serum and then subjected to an ELISA (Diagnostic System Laboratories, Webster, TX). To measure E2 in supernatant samples from the follicle cultures, samples from 72 h and 168 h were removed from the cultures described above and subjected to ELISAs. All samples were run in duplicate in a single assay. The minimum detection limit, as stated in the instructions of the kit, was 7 pg/ml. The average intraassay coefficient of variation was 4.2% and the average interassay coefficient of variation was 8.2%. Culture supernatant samples at 168 h were normalized for follicle size, to control for differences in follicle size between the AhRKO and WT follicles.
AhRKO and WT mice ovaries were collected on PDs 30–35. Antral follicles of 
300–400 µm were mechanically isolated from the ovaries and cultured as described above. WT follicles were treated with dimethylsulfoxide (DMSO) as a vehicle control. AhRKO follicles were treated with DMSO, 1 nM E2, 10 nM E2 or 15 nM E2 (Sigma-Aldrich, St. Louis, MO). Growth of follicles due to granulosa cell proliferation was examined by measuring follicle diameter daily for 168 h on two perpendicular axes [11–14]. Follicle growth and percentage change over time were calculated to compare the growth rates of the AhRKO and WT follicles.
The data were analyzed using the SPSS statistical software (SPSS, Chicago, IL). Independent sample t-tests were used to compare mean differences between the AhRKO and WT samples. ANOVA and the Tukey post-hoc test were used for multiple comparisons between treatment groups. The data are presented as means ± standard error of the mean (SEM). A P value less than or equal to 0.05 was considered statistically significant.
Effect of Ahr Deletion on Follicle Growth and Granulosa Cell Proliferation In Vitro
Using the follicle culture system, the effect of Ahr deletion on follicle growth was evaluated for 168 h to compare the growth of AhRKO follicles and WT follicles due to granulosa cell proliferation. At the beginning of culture, there were no differences between the diameters of the AhRKO follicles and WT follicles (AhRKO = 367 ± 9 µm; WT = 353 ± 10 µm; n = 3 separate cultures, each with at least 10 follicles per genotype; P = 0.53) (Fig. 1A). After 168 h, the AhRKO follicles had significantly smaller follicle diameters than the WT follicles (AhRKO = 489 ± 17 µm; WT = 615 ± 17 µm; n = 3 mice per genotype; P 
0.001). Furthermore, the total change in follicle diameter of the AhRKO follicles was significantly less than that of the WT follicles (AhRKO = 72 ± 11 µm; WT = 136 ± 12 µm; n = 3 separate cultures, each with at least 10 follicles per genotype; P 0.001) (Fig. 1B).
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Effect of Ahr Deletion on Follicle Cell Proliferation In Vivo
To determine whether Ahr deletion affected the proliferation of follicular cells in vivo, the levels of PCNA staining were compared for the AhRKO and WT ovaries. WT follicles contained more PCNA-stained area than AhRKO follicles at all time-points (Fig. 2). On PD 30, WT follicles contained approximately 15% more PCNA-stained area than AhRKO follicles (Fig. 2A–D; WT = 55.9 ± 3.0% positive area, AhRKO = 40.9 ± 3.9% positive area; n = 7–20 follicles per section from at least four animals per genotype; P 0.002). On PD 54, the WT follicles contained approximately 50% more PCNA-stained area than the AhRKO follicles (WT = 77.6 ± 2.5% positive area, AhRKO = 26.6 ± 6.6% positive area; n = 7–20 follicles per section from at least four animals per genotype; P 0.0001) (Fig. 2, E–H). On PD 90, the WT follicles contained approximately 16% more PCNA-stained area than the AhRKO follicles (WT = 49.5 ± 3.6% positive area, AhRKO = 33.2 ± 3.3% positive area; n = 7–20 follicles per section from at least four animals per genotype; P 0.001) (Fig. 2, I–L).
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To investigate further whether Ahr deletion affects granulosa cell proliferation, the mRNA expression and protein levels of factors that control cell cycle progression (Ccnd2, Cdk4, CCND2, and CDK2) were examined in isolated AhRKO and WT follicles. AhRKO follicles had decreased mRNA expression of Ccnd2 compared to WT follicles (AhRKO = 0.78 ± 0.04 genomic equivalents (ge), WT = 1.69 ± 0.18 ge; n = 3 mice per genotype and at least 10 follicles per mouse; P 0.008) (Fig. 3A). The AhRKO follicles also had decreased CCND2 levels compared to the WT follicles (AhRKO = 0.35 ± 0.05 densitometric units, WT = 0.68 ± 0.09 densitometric units; n = 3–4 mice per genotype and at least 10 follicles per mouse; P 0.03) (Fig. 3B). Similarly, the AhRKO follicles had decreased mRNA expression of Cdk4 compared to the WT follicles (AhRKO = 0.84 ± 0.03 ge; WT = 1.25 ± 0.09 ge; n = 3 mice per genotype and at least 10 follicles per mouse; P 0.012) (Fig. 3A). The AhRKO follicles also had decreased CDK4 levels compared to the WT follicles (AhRKO = 0.58 ± 0.04 densitometric units, WT = 0.79 ± 0.04 densitometric units; n = 3–4 mice per genotype and at least 10 follicles per mouse; P 0.03) (Fig. 3B).
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Effect of Ahr Deletion on Antrum Size In Vivo
To determine if differences in follicle growth in AhRKO ovaries compared to WT ovaries in vivo were also due to differences in antrum size, the sizes of the antral spaces in AhRKO and WT ovaries were compared. The percentage of antrum/follicle area for the antral follicles was 13.9 ± 1.4% in the AhRKO ovaries and 15.5 ± 1.6% in the WT ovaries (n = 3 ovaries per genotype and at least five follicles per ovary; P = 0.97). Thus, there were no significant differences in the sizes of the antral spaces between AhRKO and WT follicles.
Effect of Ahr Deletion on E2 Pathways
Since AhRKO follicles had slower follicle growth compared to WT follicles and since E2 is an important regulator of follicle growth, the serum levels of E2 were compared in AhRKO and WT mice (Fig. 4A). On PD 30, more than a 2-fold decrease in E2 levels was observed in AhRKO serum samples compared to WT samples (AhRKO = 40 ± 19 pg/ml; WT = 110 ± 14 pg/ml; n = 3–6 mice per genotype; P 0.03). On PD 90, the AhRKO serum samples had on average almost 2-fold less E2 than the WT serum samples (AhRKO = 23 ± 4 pg/ml; WT = 42 ± 3 pg/ml; n = 3–4 mice per genotype; P 0.014).
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The E2 levels were also compared in supernatant samples from AhRKO and WT follicle cultures (Fig. 4B). In the 72-h samples, there were no differences between the E2 levels in the AhRKO and WT samples (AhRKO = 280 ± 68 pg/ml; WT = 387 ± 75 pg/ml; n = 3 mice per genotype, with at least 7–8 samples per mouse; P = 0.37). However, in the 168-h samples, more than a 2-fold decrease in E2 levels was observed in the AhRKO samples compared to the WT samples even after normalizing for differences in follicle size (AhRKO = 971 ± 315 pg/ml; WT = 2463 ± 508 pg/ml; P 0.02).
Effect of Ahr Deletion on Estrogen Receptor Levels
The levels of Esr1 and Esr2 mRNA were compared in AhRKO and WT follicles (Fig. 5A). The AhRKO follicles had significantly lower levels of Esr1 mRNA compared to the WT follicles (AhRKO = 0.83 ± 0.05 ge; WT = 1.33 ± 0.17 ge; n = 3 mice per genotype and at least five samples per mouse; P 0.05). AhRKO follicles also had significantly lower levels of Esr2 mRNA than WT follicles (AhRKO = 0.80 ± 0.04 ge; WT = 1.43 ± 0.16 ge; n = 3 mice per genotype and at least 10 samples per mouse; P 0.02).
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The levels of ESR1 and ESR2 proteins were also compared in AhRKO and WT follicles (Fig. 5B). No differences were observed in ESR1 protein levels between AhRKO and WT follicles (WT = 1.04 ± 0.05 densitometric units, AhRKO = 0.91 ± 0.06 densitometric units; n = 3; P = 0.2). Similarly, no differences were observed in ESR2 protein levels between AhRKO and WT follicles (WT = 0.89 ± 0.02 densitometric units, AhRKO = 0.90 ± 0.13 densitometric units; n = 3; P = 0.8).
Effect of E2 Replacement on AhRKO Follicle Growth In Vitro
E2 was administered to AhRKO follicles, and follicle growth due to granulosa cell proliferation was compared in the AhRKO and WT follicles (Fig. 6). At the beginning of culture, there were no differences between the follicle diameters of AhRKO follicles (regardless of treatment) and WT follicles treated with DMSO (AhRKO DMSO = 368 ± 8 µm; AhRKO 1 nM E2 = 363 ± 9 µm; AhRKO 10 nM E2 = 372 ± 8 µm, AhRKO 15 nM E2 = 371 ± 9 µm; WT DMSO = 376 ± 9 µm; n = 3 WT mice and n = 6 AhRKO mice, with at least eight follicles per treatment group; P = 0.87). However, after 168 h of culture, AhRKO follicles treated with DMSO or 1 nM E2 had significantly smaller follicle diameters than WT follicles treated with DMSO (AhRKO DMSO = 468 ± 6 µm; AhRKO 1nM E2 = 477 ± 5 µm; WT DMSO = 580 ± 9 µm; n = 3 for WT and n = 6 for AhRKO, with at least eight follicles per treatment group; P 0.001). AhRKO follicles treated with 10 nM E2 had similar follicle diameters to WT follicles treated with DMSO (AhRKO 10 nM E2 = 556 ± 10 µm; WT DMSO = 580 ± 9 µm; n = 3 for WT and n = 6 for AhRKO, with at least eight follicles per treatment group; P = 0.16). Similarly, no difference was observed in the follicle diameters of 15 nM E2-treated AhRKO follicles and WT follicles treated with DMSO (AhRKO 15 nM E2 = 564 ± 7 µm; WT DMSO = 580 ± 9 µm; n = 3 for WT and n = 6 for AhRKO, with at least eight follicles per treatment group; P = 0.55).
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Previous studies have indicated that deletion of the Ahr gene adversely affects fertility and ovarian follicle numbers in mice [8, 9]. Furthermore, studies by Benedict et al. [10] have suggested, but did not directly test, that follicular growth in AhRKO mice compared to WT mice is altered due to slower follicle growth to the antral stage in vivo. Therefore, an objective of the present study was to investigate the mechanism by which Ahr deletion affects follicular growth. To test directly whether AhRKO follicles grow slower than WT follicles, we utilized an in vitro system to compare the growth patterns of AhRKO and WT follicles. Our results indicate that AhRKO follicles grow slower than WT follicles in vitro. Specifically, we noticed that by 168 h in culture, AhRKO follicles had significantly smaller follicle diameters than WT follicles. These data suggest that the Ahr plays an important role in regulating follicle growth in the mouse ovary. During the culture procedure, the follicles attached themselves to the bottom of the plate, and the granulosa cells proliferated to form a dome-shaped cover over the follicles, which resulted in an increase in follicular size [11–14]. Thus, the data also indicate that the slow growth of AhRKO follicles is due to reduced granulosa cell proliferation in AhRKO follicles compared to WT follicles in vitro. These findings are consistent with the recent work of Bussman et al. [34], which indicates that AHR ligands modulate the proliferation of rat granulosa cells.
We further investigated whether Ahr regulates granulosa cell proliferation in vivo. Our results indicate that AhRKO follicles have less granulosa cell proliferation than WT follicles, as determined by PCNA immunohistochemistry. It is likely that the decreased granulosa cell proliferation leads to slow follicular growth in vivo, since studies conducted by Goldenberg et al. [35] have indicated that increases in follicle size during folliculogenesis are directly due to granulosa cell proliferation. Our present studies also show that AhRKO follicles have significantly lower levels of Ccnd2, Cdk4, CCND2, and CDK4 compared to WT follicles. CCND2, via binding to CDK4, is involved in the regulation of cell cycle progression from the G1-to-S phase of the cell cycle [36]. Therefore, these data may explain the reduced granulosa cell proliferation seen in AhRKO follicles, as AHR has been shown to facilitate G1 cell cycle progression independently of ligand activation [37]. These data may also explain the slow follicular growth seen in AhRKO follicles, as both Ccnd2 and Cdk4 have been shown to regulate granulosa cell proliferation in the mouse ovary [23, 38]. Furthermore, previous studies have suggested a link between AHR and Ccnd2 and/or Cdk4 in nonovarian tissues. Specifically, studies have shown that the AHR ligand TCDD affects cell proliferation in liver cells, and that the changes in proliferation are correlated with changes in the expression of Ccnd2 and/or Cdk4 [39–40]. Similarly, TCDD modulates the proliferation and Cdk4 levels of endocervical cells [41]. Studies have also shown that the AHR ligand methylcholanthrene affects the proliferation of Leydig TM3 cells through a pathway that includes cyclins [42]. Similarly, methylcholanthrene affects the proliferation of MCF-7 breast cancer cells through a cyclin pathway [43]. Interestingly, although AHR or AHR ligands inhibit the proliferation of liver, breast, and endocervical cells, Ahr stimulates the proliferation of follicles (present study) and developing thymus cells [44]. The reasons for the tissue-specific differences in the role of Ahr in proliferation are unclear but may be related to cell-specific metabolic enzymes, receptors or growth factors.
We considered the possibility that the differences in follicle growth observed for the AhRKO ovaries in vivo could stem from differences in vascularization, which would cause differences in the amounts of fluid and thus, the sizes of the antral follicles in AhRKO mice compared to WT littermates. Therefore, we measured the size of antral space in each follicle and estimated the percentage antral space area per follicle. Our results indicate that WT and AhRKO follicles of similar sizes have antral spaces of similar size. These data suggest that if follicles grow to the antral stage in AhRKO mice, they appear to have a normal-sized antrum. Thus, our data showing that AhRKO follicles grow slower than WT follicles are probably not due to differences in antrum size. Collectively, our in vitro and in vivo data suggest that Ahr affects follicle growth via mechanisms that involve the regulation of granulosa cell proliferation.
Studies by Richards et al. [45] have shown that E2 is responsible for the stimulation of granulosa cell proliferation in follicles. Thus, we examined the levels of E2 in AhRKO and WT mice in vivo and in vitro. We show that AhRKO mice have significantly lower levels of serum E2 compared to WT mice. Since antral follicles secrete E2 [46], the lower levels of E2 in the serum may reflect the fact that AhRKO mice have reduced numbers of antral follicles [9]. Therefore, it was important to determine whether AhRKO follicles produce less E2 compared to WT follicles. To account for differences in follicle size, E2 production was measured in AhRKO and WT follicles with follicle diameters of similar sizes (72 h). No differences were observed in E2 production at this time-point. At 168 h, AhRKO follicles produced significantly lower levels of E2 compared to WT follicles, even after adjustment for differences in follicle size. Thus, these data suggest that the decreased levels of serum E2 in AhRKO mice may be due to lower numbers of antral follicles in AhRKO ovaries, as well as decreased production of E2 by AhRKO follicles. Collectively, these data suggest that Ahr regulates follicular growth via mechanisms that involve alterations in E2 production.
Since the actions of E2 are mediated via binding to its receptors, ESR1 and ESR2 [47], we examined the possibility that AhRKO follicles have reduced responsiveness to E2 via altered Esr or ESR levels. Our results show that AhRKO follicles express lower Esr1 and Esr2 mRNA levels compared to WT follicles. As Esr2 is expressed in the granulosa cells [47], the decreased mRNA levels of Esr2 are not surprising, since AhRKO follicles have decreased granulosa cell proliferation. The decreased expression of Esr1 mRNA in AhRKO follicles suggests that the thecal cells may also be affected, as Esr1 is expressed predominately in the thecal and interstitial cells [47]. Furthermore, since previous studies have shown that there is cross-talk between Ahr and Esr1 and Esr2 in nonovarian tissues [19–21], it is possible that the decreased mRNA expression of Esrs in AhRKO ovaries compared to WT ovaries is due to an ability of Ahr to regulate Esrs mRNA expression.
Our data also indicate that while Ahr deletion decreases the mRNA expression of Esr1 and Esr2, it does not affect the protein levels of ESR1 and ESR2. The reasons for this are unknown. It is possible that Ahr regulates the transcription of Esr1 and Esr2 through direct or indirect mechanisms, without affecting translation processes. It is also possible that the reduced levels of Esr1 and Esr2 transcription are compensated by increased protein stability or reduced protein degradation. The possibility that Ahr regulates transcription is supported by studies indicating that the agonist-activated AHR associates with Esr1 and Esr2, that this association leads to the activation of transcription and estrogenic effects [48], and that the AHR ligand TCDD suppresses Esr1 transcription in the ovary and uterus [49]. The possibility that Ahr deletion affects ESR protein degradation is supported by the findings of Wormke et al. [50], which indicate that AHR mediates the degradation of ESR1 in the rodent uterus and mammary gland through the activation of proteasomes [50].
E2 replacement studies were conducted to test whether AhRKO follicle growth could be restored to control (WT follicles treated with DMSO) levels in vitro. Our results indicate that DMSO and 1 nM E2-treated AhRKO follicles are not able to grow to WT levels. However, the 10 nM E2- and 15 nM E2-treated AhRKO follicles were able to grow to WT levels. These data suggest that E2 treatment can restore follicular growth of AhRKO follicles to WT levels in a dose-dependent manner in vitro.
The present study has further elucidated the physiological role of AHR in the ovary. Specifically, we have shown that Ahr deletion results in impaired follicle growth through the regulation of granulosa cell proliferation, cell cycle progression, E2 production, and E2 responsiveness. The finding that E2 treatment could restore the growth of AhRKO follicles suggests that the slower follicular growth in AhRKO mice is due, in part, to decreased E2 levels. One way to explain these findings is that in the absence of the Ahr, antral follicles produce abnormally low levels of E2, thereby altering E2 responsive genes (Ccnd2, Cdk4, Esr1, and Esr2). Alterations in these genes in turn cause the follicles to have reduced responsiveness to E2, which could ultimately slow the growth of ovarian follicles.
The reduced E2 levels further suggest that Ahr is involved in regulating E2 biosynthesis in the ovary. This is supported by studies that show that Ahr is expressed in steroid-producing organs, such as the ovary, adrenal glands, and testes [1–6, 51]. A link between AHR and steroid hormone synthesis has already been shown in the testis [52–53]. Furthermore, AHR has been shown to control steroid hormone production in adrenal cells by altering Star gene expression [51]. Therefore, it is possible that Ahr also regulates steroid production in the ovary. Future studies will test whether Ahr deletion affects E2 biosynthesis in the ovary.
ACKNOWLEDGMENTS
The authors thank Janice Babus for her wonderful technical assistance. The authors also thank Bethany Crean and Frank Bartol for their extremely helpful advice on PCNA quantification.
FOOTNOTES
1Supported by NIH HD 047275 and NIH MARC Predoctoral Fellowship F31 GM072195-01. 
Correspondence: 2Jodi Anne Flaws, University of Illinois, Department of Veterinary Biosciences, 3223 VMBS Building, 2001 S. Lincoln Ave, Urbana, IL 61802. FAX: 217 244 1652; e-mail: jflaws{at}uiuc.edu
Received: 25 September 2006.
First decision: 11 October 2006.
Accepted: 27 February 2007.
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