TY - JOUR
AU1 - Sobinoff, Alexander, P.
AU2 - Nixon,, Brett
AU3 - Roman, Shaun, D.
AU4 - McLaughlin, Eileen, A.
AB - Abstract 3-Methylcholanthrene (3MC) is a potent ovotoxicant capable of causing premature ovarian failure through primordial follicle depletion. Despite 3MCs ovotoxicity having been established for 30 years, relatively little information exists on the mechanisms. In this study, we examined the effects of 3MC exposure on the immature ovarian follicle population. Microarray analysis revealed a complex mechanism of 3MC-induced ovotoxicity involving a number of cellular processes associated with xenobiotic metabolism, ovarian cancer, cell cycle progression, and cell death. 3MC exposure was also found to induce developing follicle atresia and aberrant primordial follicle activation via the stimulation of PI3K/Akt and mammalian target of rapamycin (mTOR) signaling pathways. Inhibition of PI3K/Akt signaling resulted in the severe depletion of the primordial follicle pool, with further analysis identifying increased Akt1-stimulated Bad phosphoinhibition in 3MC-treated primordial follicles. Our results suggest that the primordial follicle pool enters a “prosurvival” state upon 3MC exposure and that its depletion is due to a vicious cycle of primordial follicle activation in an attempt to replace developing follicles undergoing follicular atresia. 3-methylcholanthrene, ovary, primordial follicle, cellular survival, PI3K/Akt/mTOR signaling The major function of the mammalian ovary is the development of mature oocytes for ovulation and fertilization. Oocyte maturation occurs in ovarian follicles, which are first assembled within the primitive ovary, when primary oocytes are enveloped by a single layer of flattened pregranulosa cells (McNatty et al., 2000). The resulting structure is known as the primordial follicle, the most immature stage of follicular development. These precious follicles cannot be regenerated postfetal development and represent the total number of germ cells available to the mammalian female throughout her entire reproductive life (Edson et al., 2009). Primordial follicles remain in a quiescent state for many months or years until they are selectively recruited to join the growing population. This event occurs in regular waves and is continuous from birth until the primordial follicle pool becomes depleted, resulting in menopause (McGee and Hsueh, 2000). Overall, < 1% of all recruited follicles will complete maturation and undergo ovulation, with the vast majority being lost to an apoptotic process known as atresia (Hirshfield, 1991). Therefore, the female reproductive life span can be shortened if anything untoward happens to the primordial follicle pool, such as aberrant activation and/or follicular atresia (McLaughlin and McIver, 2009). An increasing trend in the number of women opting to delay childbirth (> 30 years) has led to an increased awareness of the impact of environmental chemicals on reproductive function (Hamilton et al., 2010). Ongoing research has identified a number of ovotoxic xenobiotics, which specifically target ovarian follicles for destruction, leading to perturbed fertility (Mark-Kappeler et al., 2011). Of chief interest are those ovotoxic xenobiotics that specifically target irreplaceable primordial follicles, resulting in premature ovarian failure (POF). Evidence suggests that these ovotoxic chemicals cause POF by inducing proapoptotic signaling events (caspase activation and proapoptotic Bcl2 signaling) resulting in primordial follicle atresia and by stimulating members of the PI3K/Akt and mammalian target of rapamycin (mTOR) signaling pathways, resulting in accelerated primordial follicle activation (Hu et al., 2001; Matikainen et al., 2001; Sobinoff et al., 2012a, 2011). Although patterns are starting to emerge as to how environmental ovotoxicants cause primordial follicle depletion, the mechanisms underlying their ovotoxicity remain largely unknown (Sobinoff et al., 2012a). One group of environmental chemicals, which are notorious reproductive toxicants are the polycyclic aromatic hydrocarbons (PAH). PAH are major constituents of cigarette smoke and are produced via various combustion reactions (including automotive exhaust and charbroiled foods), making these compounds ubiquitous pollutants (Centers for Disease Control and Prevention , 2009). The three PAH, benzo(a)pyrene (BaP), 9:10-dimethyl-1:2-benzanthracene (DMBA), and 3-methylcholanthrene (3MC), are all thought to specifically target immature follicles for destruction, causing POF (Borman et al., 2000; Mattison et al., 1983). Although there are a number of studies characterizing the ovarian response to both BaP and DMBA exposure, relatively little information exists on the mechanisms behind 3MC-induced ovotoxicity (Bhattacharya and Keating, 2011; Neal et al., 2007; Sobinoff et al., 2011, 2012b). Initial studies in rodents suggest that 3MC specifically targets immature primordial follicle oocytes for destruction (Borman et al., 2000; Mattison, 1980). Inhibition of the aryl hydrocarbon receptor by alpha-naphthoflavone nullifies 3MC-induced primordial follicle depletion, revealing its ovotoxicity is dependent upon metabolic bioactivation (Shiromizu and Mattison, 1985). To further characterize the mechanisms behind 3MC-induced ovotoxicity, we examined its effects on the ovarian transcriptome of cultured neonatal mouse ovaries. Microarray analysis revealed 3MC significantly altered the expression of genes involved in cancer, cellular growth and proliferation, and cell death. In contrast to the current literature, histomorphological and immunohistological analysis revealed 3MC specifically targeted developing follicles for destruction, not primordial follicles. Our results suggest that 3MC exposure induces primordial follicle depletion via an overstimulation of primordial follicle activation involving downstream members of the PI3K/Akt and mTOR signaling pathways (McLaughlin and Sobinoff, 2010). However, inhibition of PI3K using the inhibitor LY294002 resulted in the severe depletion of the primordial follicle pool, suggesting that PI3K/Akt signaling prevents 3MC-induced primordial follicle destruction in addition to promoting primordial follicle activation. We also provide evidence of increased prosurvival BAD S136 phosphorylation in 3MC-exposed primordial follicles, indicating the population enters a “prosurvival state” upon exposure to 3MC. MATERIALS AND METHODS Reagents. 3MC (> 95% purity), LY294002 (L9908), rapamycin (R0395), and custom designed primers were purchased from Sigma Chemical Co. (St Louis, MO) and were of molecular biology or research grade. Mouse monoclonal anti-proliferating cell nuclear antigen antibody (anti-PCNA, NA03T) was obtained from Merck KGaA (Darmstadt, Germany). Rabbit polyclonal antiactive caspase 3 antibody (anti-Casp3, ab13847), rabbit polyclonal antiactive caspase 2 antibody (anti-Casp2, ab2251), rabbit monoclonal anti-Akt1 (phospho S473) (anti-pAkt1 [S474], ab81283), rabbit monoclonal anti-Akt1 (phospho T308) (anti-pAkt1 [T308], ab5626) rabbit polyclonal anti-Foxo3a (anti-Foxo3a, ab47409), rabbit polyclonal anti-Tsc2 (phospho S939) (anti-pTsc2 [S939], ab59269), and rabbit polyclonal anti-Bad (phosphor S139) (anti-pBad [S139], ab28824) were obtained from Abcam (Cambridge, MA). Mouse monoclonal antihuman anti-Müllerian hormone (anti-AMH, MCA2246) was obtained from AbD Serotec (Kidlington, U.K.). Rabbit polyclonal anti-Akt (anti-Akt, #9272) was obtained from Cell Signaling Technologies (Beverly, MA). Alexa Fluor 594 goat anti-rabbit IgG (A11012), Alexa Fluor 594 goat anti-mouse IgG (A11005), fetal bovine serum, l-glutamine, and Insulin-Transferrin-Selenium were purchased from the Invitrogen Co. (Carlsbad, CA). l-Ascorbic acid was obtained from MP Biomedicals (Solon, OH). Rabbit polyclonal anti-phospho-mTOR (phosphor S2448) (anti-pmTOR [S2448], 09-213SP), rabbit polyclonal anti-phospho-mTOR (phosphor T2446) (anti-pmTOR [T2446], 09-345SP), and 0.4 μm culture plate inserts were purchased from Millipore (Billerica, MA). All culture dishes and cell culture plates were obtained from Greiner Bio-One (Monroe, NC). Oligo(dT)15 primer, RNasin, deoxynucleotide triphosphates (dNTPs), M-MLV-Reverse Transcriptase, RQ1 DNase, GoTaq Flexi, MgCl2, GoTaq qPCR master mix, and proteinase K were purchased from the Promega Corporation (Madison, WI). Animals. All experimental procedures involving the use of animals were performed with the approval of the University of Newcastle's Animal Care and Ethics Committee (ACEC). Swiss mice were obtained from a breeding colony held at the institute's central animal facility and maintained according to the recommendations prescribed by the ACEC. Mice were housed under a controlled lighting regime (16L:8D) at 21–22°C and supplied with food and water ad libitum. Animal dosing. Female Swiss neonatal mice (day 4; 6–10 animals per treatment group) were weighed and administered (ip) seven daily, consecutive doses of either sesame oil containing vehicle control (< 10 μl/kg/daily dimethyl sulfoxide [DMSO]) or sesame oil containing a low and high dose of 3MC (5 mg and 10 mg/kg/daily). The dosage, routes of administration, and dosing time courses were based on previous studies and were chosen with the intention of inducing ovotoxicity with minimal cytotoxicity (Borman et al., 2000). Animals were observed daily for symptoms of toxicity and mortality. Treated animals were culled by CO2 asphyxiation 24 h after the last injection. Ovarian culture. Ovaries from day 3–4 Swiss neonatal mice were cultured as described previously (Sobinoff et al., 2010). Briefly, Swiss neonates were sacrificed by CO2 inhalation followed by decapitation. Ovaries were excised, trimmed of excess tissue, and placed on culture plate inserts in six-well tissue culture plate wells floating atop 1.5 ml Dulbecco's Modified Eagle's Medium/F12 medium containing 5% (vol/vol) fetal calf serum, 1 mg/ml bovine serum albumin, 50 μg/ml ascorbic acid, 27.5 μg/ml ITS, 2.5mM glutamine, and 5 U/ml penicillin/streptomycin. Media were supplemented with 40 ng/ml basic fibroblast growth factor, 50 ng/ml leukemia inhibitory factor, and 25 ng/ml stem cell factor. A drop of medium was placed over the top of each ovary to prevent drying. Ovaries were cultured for 4 days at 37°C and 5% CO2 in air, with media changes every 2 days. Pilot studies performed in our laboratory identified this time point as a “middle ground” between the initial and end stages of 3MC-induced ovotoxicity (i.e., after exposure and before complete follicular depletion). This would allow us to identify both causative and responsive changes in ovarian gene expression induced by 3MC exposure. Ovaries were treated with vehicle control medium (0.01% DMSO) or 3MC (5μM) ± LY294002 (15μM) or rapamycin (50nM). The 3MC and LY294002/rapamycin culture concentrations were determined by pilot studies performed in our laboratory with the intention of inducing overt ovotoxicity. Histological evaluation of follicles. Following in vitro culture/in vivo dosing, ovaries were placed in Bouin's fixative for 4 h, washed in 70% ethanol, paraffin embedded, and serially sectioned (4 μm thick) throughout the entire ovary, with every fourth slide counterstained with hematoxylin and eosin. Healthy oocyte-containing follicles were then counted in every hematoxylin- and eosin-stained section. Follicles with eosinophilic (pyknotic) oocytes were considered as degenerating or atretic and so were not counted. Primordial follicles were classified as those with a single layer of squamous granulosa cells. Activating follicles were identified as those, which contained one or more cuboidal granulosa cells in a single layer. Primary follicles were classified as those that contained more than four cuboidal granulosa cells in a single layer. Secondary follicles were identified as those with two layers of granulosa cells, and preantral follicles were classified as those with more than two layers of granulosa cells. Both in vitro and in vivo treated ovaries did not contain follicles beyond the preantral stage. Immunohistochemistry. Ovaries for immunohistochemistry were fixed in Bouin's and sectioned 4 μm thick. PCNA, active Casp2, active Casp3, AMH, Akt, pAkt1, pmTOR, pTsc2, pBad, and Foxo3a were stained using the same protocol with the exception of the primary antibody. Slides were deparaffinized in xylene and rehydrated with subsequent washes in ethanol. Antigen retrieval was carried out by microwaving sections for 3 × 3min in Tris buffer (50mM, pH 10.6). Sections were then blocked in 3% bovine serum albumin (BSA)/Tris-buffered saline (TBS) for 1.5 h at room temperature. The following solutions were diluted in TBS containing 1% BSA. Sections were incubated with either anti-PCNA (1:80), anti-Casp2 (1:200), anti-Casp3 (1:200), anti-AMH (1:200), anti-Akt (1:100), anti-pAkt1 (1:100), or anti-Foxo3a (1:200) for 1 h at room temperature. After washing in TBS containing 0.1% Triton X-100, sections were incubated with the appropriate fluorescent conjugated secondary antibodies (Alexa Fluor 594 goat anti-rabbit IgG, Alexa Fluor 594 goat anti-mouse IgG; 1:200 dilution) for 1 h. Slides were then counter stained with 4′-6-diamidino-2-phenylindole (DAPI) for 5 min, mounted in Mowiol, and observed on an Axio Imager A1 fluorescence microscope (Carl Zeiss MicroImaging, Inc., Thornwood, NY) under fluorescence optics and pictures taken using an Olympus DP70 microscope camera (Olympus America, Center Valley, PA). Protein staining was quantified according to Cy5 intensity in primordial follicle oocytes using ImageJ software (NCBI). Negative controls lacking the primary antibody were performed alongside each experiment (Supplementary Data). Terminal deoxynucleotidyl transferase dUTP nick end labeling analysis. Bouin's fixed sections were deparaffinized and rehydrated as mentioned previously. Sections were then boiled in Tris buffer (50mM, pH 10.6) for 20 min and treated with 20 μg/ml Proteinase K for 15 min in a humidified chamber. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) analysis was then performed using an In Situ Cell Death Detection Kit, fluorescein (Roche Diagnostics Pty Ltd; Dee Why, New South Wales, Australia) according to the manufacturer's instructions. Slides were then counter stained with DAPI for 5 min, mounted in Mowiol, and observed using an Axio Imager A1 epifluorescent microscope (Carl Zeiss), and images captured using an Olympus DP70 microscope camera (Olympus). Negative controls lacking the TUNEL enzyme and positive controls pretreated with DNase enzyme were performed alongside each experiment (Supplementary DataSupplementary Data). RNA extraction. Total RNA was isolated from ovaries using two rounds of a modified acid guanidinium thiocyanate-phenol-chloroform protocol (Chomczynski and Sacchi, 1987): Washed cells were resuspended in lysis buffer (4M guanidinium thiocyanate, 25mM sodium citrate, 0.5% sarkosyl, and 0.72% β-mercaptoethanol). RNA was isolated by phenol/chloroform extraction and isopropanol precipitated. Real-time PCR (Quantitative PCR). Reverse transcription was performed with 2 μg of isolated RNA, 500 ng oligo(dT)15 primer, 40 U of RNasin, 0.5mM dNTPs, and 20 U of M-MLV reverse transcriptase. Total RNA was DNase treated prior to reverse transcription to remove genomic DNA. Real-time PCR was performed using SYBR Green GoTaq qPCR master mix according to manufacturer's instructions on an MJ Opticon 2 (MJ Research, Reno, NV). Primer sequences along with annealing temperatures have been supplied as Supplementary Data (Supplementary DataSupplementary Data). Reactions were performed on complementary DNA equivalent to 100 ng of total RNA and carried out for 40 amplification cycles. SYBR Green fluorescence was measured after the extension step at the end of each amplification cycle and quantified using Opticon Monitor Analysis software Version 2.02 (MJ Research). For each sample, a replicate omitting the reverse transcription step was undertaken as a negative control. Reverse transcription reactions were verified by β-actin PCR, performed for each sample in all reactions in triplicate. Real-time data were analyzed using the equation 2−ΔΔC(t), where C(t) is the cycle at which fluorescence was first detected above background fluorescence. Data were normalized to cyclophilin, and are presented as the average of each replicate normalized to an average of the reference genes (± SEM). Microarray analysis. Total RNA (approximately 3 μg) was isolated from 3MC-cultured neonatal ovaries and prepared for microarray analysis at the Australian Genome Research Facility (AGRF) using an Illumina Sentrix Mouse ref8v2 BeadChip. Labeling, hybridizing, washing, and array scanning were performed by the AGRF using the Illumina manual on an Illumina BeadArray Reader, and normalized according to the quantile normalization method using GenomeStudio version 1.6.0 (Illumina, Inc., San Diego, CA). All experiments were performed in triplicate with independently extracted RNAs. Statistically significant genes with more than a 1.5-fold difference in gene expression (p < 0.05) determined through the use of a “volcano plot” were then analyzed using Ingenuity Pathways Analysis (IPA) (Ingenuity Systems, Redwood City, CA) software to identify canonical signaling pathways influenced by 3MC exposure. The data discussed in this publication have been deposited in NCBI's Gene Expression Omnibus and are accessible through GEO Series accession number GSE35836 (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE35836). Statistics. Comparisons between the control and 3MC-treated groups were performed using one-way ANOVA, Student's t-test, and Tukey's honestly significant difference test. The assigned level of significance for all tests was p < 0.05. RESULTS Effects of 3MC Exposure on the Neonatal Ovarian Transcriptome 3MC exposure caused a significant change in ovarian gene expression, altering the expression of 428 genes representing 1.7% of the total number present on the array (Fig. 1A). Though the use of IPA software, these altered genes were identified as components of molecular networks implicated in cell cycle regulation, cancer, cell death, cell to cell signaling, free radical scavenging, and drug metabolism (Fig. 1B). Grouping according to their molecular and cellular function revealed a large proportion of significantly altered genes were also implicated in cellular growth and proliferation, genetic disorder, and reproductive system disease (Table 1; Supplementary Data). These results suggest that 3MC exposure alters a small subset of genes involved in a variety of cellular processes that contribute to its ovotoxicity. TABLE 1 Functional Classification of Genes Upregulated or Downregulated by 3MC Exposure in Cultured Neonatal Ovaries Molecular and cellular function Upregulated Downregulated Cellular growth and proliferation 102 55 Genetic disorder 88 55 Cellular development 86 43 Tissue development 82 51 Reproductive system disease 77 19 Cell death 77 49 Cell to cell signaling and interaction 59 46 Inflammatory response 52 58 Cell cycle 43 18 Cellular function 40 48 Organismal development 58 1 Molecular and cellular function Upregulated Downregulated Cellular growth and proliferation 102 55 Genetic disorder 88 55 Cellular development 86 43 Tissue development 82 51 Reproductive system disease 77 19 Cell death 77 49 Cell to cell signaling and interaction 59 46 Inflammatory response 52 58 Cell cycle 43 18 Cellular function 40 48 Organismal development 58 1 Note. Genes were analyzed using ipa (ingenuity systems) for molecular and cellular functions. Only those genes exhibiting a greater than twofold change in expression were categorized (p < 0.05). Note that some genes are listed in multiple functional groups. Open in new tab TABLE 1 Functional Classification of Genes Upregulated or Downregulated by 3MC Exposure in Cultured Neonatal Ovaries Molecular and cellular function Upregulated Downregulated Cellular growth and proliferation 102 55 Genetic disorder 88 55 Cellular development 86 43 Tissue development 82 51 Reproductive system disease 77 19 Cell death 77 49 Cell to cell signaling and interaction 59 46 Inflammatory response 52 58 Cell cycle 43 18 Cellular function 40 48 Organismal development 58 1 Molecular and cellular function Upregulated Downregulated Cellular growth and proliferation 102 55 Genetic disorder 88 55 Cellular development 86 43 Tissue development 82 51 Reproductive system disease 77 19 Cell death 77 49 Cell to cell signaling and interaction 59 46 Inflammatory response 52 58 Cell cycle 43 18 Cellular function 40 48 Organismal development 58 1 Note. Genes were analyzed using ipa (ingenuity systems) for molecular and cellular functions. Only those genes exhibiting a greater than twofold change in expression were categorized (p < 0.05). Note that some genes are listed in multiple functional groups. Open in new tab FIG. 1. Open in new tabDownload slide Microarray analysis of control-cultured ovaries versus 3MC-cultured ovaries. Ovaries were excised from neonatal mice (4 days old, n = 15) and cultured in xenobiotic-treated medium for 96 h. RNA extracted and subjected to microarray analysis as described in the Materials and Methods section. (A) Summary of microarray results. Total number of genes found on an Illumina Sentrix Mouse ref8v2 BeadChip are presented as nonregulated (black) and regulated (white) genes with a significant change in expression (> 1.5-fold change, p < 0.05). The top bar represents the number of positively regulated genes, and the bottom bar represents the number of negatively regulated genes in xenobiotic-cultured ovaries. (B) Molecular networks of significantly altered genes influenced by 3MC exposure. Significantly altered genes were overlaid onto a global molecular network developed from information contained in the Ingenuity Pathways Knowledge Base (Ingenuity Systems). Networks of significantly altered genes were then algorithmically generated based on their connectivity. The networks are ranked according to their scores, and the five highest-ranking networks of genes are displayed. The numbers of genes in each network are shown in brackets. FIG. 1. Open in new tabDownload slide Microarray analysis of control-cultured ovaries versus 3MC-cultured ovaries. Ovaries were excised from neonatal mice (4 days old, n = 15) and cultured in xenobiotic-treated medium for 96 h. RNA extracted and subjected to microarray analysis as described in the Materials and Methods section. (A) Summary of microarray results. Total number of genes found on an Illumina Sentrix Mouse ref8v2 BeadChip are presented as nonregulated (black) and regulated (white) genes with a significant change in expression (> 1.5-fold change, p < 0.05). The top bar represents the number of positively regulated genes, and the bottom bar represents the number of negatively regulated genes in xenobiotic-cultured ovaries. (B) Molecular networks of significantly altered genes influenced by 3MC exposure. Significantly altered genes were overlaid onto a global molecular network developed from information contained in the Ingenuity Pathways Knowledge Base (Ingenuity Systems). Networks of significantly altered genes were then algorithmically generated based on their connectivity. The networks are ranked according to their scores, and the five highest-ranking networks of genes are displayed. The numbers of genes in each network are shown in brackets. Canonical Pathways Significantly Upregulated by 3MC Exposure To further characterize the mechanisms behind 3MC-induced ovotoxicity, differentially expressed genes were also analyzed for signaling pathways and molecular functions using IPA (Fig. 2; Supplementary DataSupplementary Data). 3MC exposure influenced genes belonging to pathways involved in xenobiotic metabolism (aryl hydrocarbon receptor signaling, xenobiotic metabolism signaling), tumorigenesis (molecular mechanisms of cancer, ovarian cancer signaling), cell death and DNA damage repair (p53 signaling, cell cycle G2/M DNA damage checkpoint regulation, cell cycle G1/S checkpoint regulation), cellular proliferation (cyclins and cell cycle regulation), and folliculogenesis (aryl hydrocarbon receptor signaling, integrin-linked kinase (ILK) signaling, PI3K/Akt signaling). The upregulation of pathways associated with increased proliferation and follicular development is intriguing as it suggests 3MC-induced ovotoxicity is not limited to primordial follicle atresia. FIG. 2. Open in new tabDownload slide Top canonical pathways that were significantly upregulated by 3MC-cultured neonatal ovaries as identified by IPA. The significance of the association between upregulated genes and the canonical pathway was evaluated using a right-tailed Fisher's exact test to calculate a p value determining the probability that the association is explained by chance alone (bars, y-axis). Ratios referring to the proportion of upregulated genes from a pathway related to the total number of molecules that make up that particular pathway are also displayed (line graph, z-axis). FIG. 2. Open in new tabDownload slide Top canonical pathways that were significantly upregulated by 3MC-cultured neonatal ovaries as identified by IPA. The significance of the association between upregulated genes and the canonical pathway was evaluated using a right-tailed Fisher's exact test to calculate a p value determining the probability that the association is explained by chance alone (bars, y-axis). Ratios referring to the proportion of upregulated genes from a pathway related to the total number of molecules that make up that particular pathway are also displayed (line graph, z-axis). Quantitative PCR Validation of Microarray Results Validation of microarray results was performed by examining the levels of expression for different genes using quantitative PCR (QPCR) (Table 2). Similar gene expression patterns were observed for all targets measured by QPCR when compared with the results of the microarray gene expression study. Of these genes, five were associated with xenobiotic detoxification/metabolism (Cyp1b1, Aldh1l2, Aldh4a1, Noq1, and Ugt1a10), three with increased cellular proliferation and follicular development (Ccng1, Ccnd1, and Hspa8), two with cell cycle arrest and apoptosis (Cdkn1a and Aldh1l2), and one with increased cell stress (Hspa8). These results provide insight into ovarian 3MC metabolism and support the notion 3MC exposure is not limited to primordial follicle destruction. TABLE 2 QPCR Validation of Microarray Results for Select Transcripts Upregulated by 3MC-cultured Neonatal Ovaries Gene symbol Gene name Summary of function Fold change Cdkn1a Cyclin-dependent kinase inhibitor 1A Regulator of cell cycle progression at G1 phase; implicated in DNA damage repair (Binková et al., 2000) 8.64 ± 1.9 Ccng1 Cyclin G1 Regulator of cell cycle progression at G2/M phase; implicated in DNA damage repair and apoptosis; associated with granulosa cell proliferation and differentiation (Kimura et al., 2001; Liu et al., 2006) 3.9 ± 0.4 Cyp1b1 Cytochrome P450, family 1, subfamily b, polypeptide 1 Phase I detoxifying enzyme; implicated in the bioactivation of PAH into DNA adduct forming metabolites (Shimada and Fujii-Kuriyama, 2004) 3.78 ± 0.4 Aldh1l2 Aldehyde dehydrogenase 1 family, member L2 Mitochondrial homolog of proapoptotic 10-formyltetrahydrofolate dehydrogenase (Ghose et al., 2009; Krupenko et al., 2010) 3.16 ± 0.2 Nqo1 NAD(P)H-quinone oxidoreductase Phase II detoxifying enzyme; detoxifies quinones into quinols; implicated in 3MC metabolism (Kondraganti et al., 2008; Ross, 2004; ) 2.61 ± 0.2 Aldh4a1 Aldehyde dehydrogenase 4 family, member A1 Mitochondrial matrix protein essential for proline degradation; implicated in p53 mediated protection against oxidative stress (Yoon et al., 2004) 2.45 ± 0.1 Ugt1a10 Uridine diphosphate glycosyltransferase 1 family, polypeptide A10 Phase II detoxifying enzyme; implicated in the detoxification of bioactivated PAH metabolites (Dellinger et al., 2006) 1.87 ± 0.3 Ccnd1 Cyclin D1 Promotes cell cycle progression from G1-S phase; overexpression associated with breast/ovarian cancer (Bali et al., 2004; Robker and Richards, 1998). 1.7 ± 0.4 Hspa8 Heat shock protein 8 Stress related chaperone; expression increases during cell cycle G1 phase; regulates cyclin D1 accumulation and maintains its activity in the presence of inhibitory Cdkn1a (Diehl et al., 2003) 1.51 ± 0.1 Cdkn1a Cyclin-dependent kinase inhibitor 1A Regulator of cell cycle progression at G1 phase; implicated in DNA damage repair (Binková et al., 2000) 8.64 ± 1.9 Gene symbol Gene name Summary of function Fold change Cdkn1a Cyclin-dependent kinase inhibitor 1A Regulator of cell cycle progression at G1 phase; implicated in DNA damage repair (Binková et al., 2000) 8.64 ± 1.9 Ccng1 Cyclin G1 Regulator of cell cycle progression at G2/M phase; implicated in DNA damage repair and apoptosis; associated with granulosa cell proliferation and differentiation (Kimura et al., 2001; Liu et al., 2006) 3.9 ± 0.4 Cyp1b1 Cytochrome P450, family 1, subfamily b, polypeptide 1 Phase I detoxifying enzyme; implicated in the bioactivation of PAH into DNA adduct forming metabolites (Shimada and Fujii-Kuriyama, 2004) 3.78 ± 0.4 Aldh1l2 Aldehyde dehydrogenase 1 family, member L2 Mitochondrial homolog of proapoptotic 10-formyltetrahydrofolate dehydrogenase (Ghose et al., 2009; Krupenko et al., 2010) 3.16 ± 0.2 Nqo1 NAD(P)H-quinone oxidoreductase Phase II detoxifying enzyme; detoxifies quinones into quinols; implicated in 3MC metabolism (Kondraganti et al., 2008; Ross, 2004; ) 2.61 ± 0.2 Aldh4a1 Aldehyde dehydrogenase 4 family, member A1 Mitochondrial matrix protein essential for proline degradation; implicated in p53 mediated protection against oxidative stress (Yoon et al., 2004) 2.45 ± 0.1 Ugt1a10 Uridine diphosphate glycosyltransferase 1 family, polypeptide A10 Phase II detoxifying enzyme; implicated in the detoxification of bioactivated PAH metabolites (Dellinger et al., 2006) 1.87 ± 0.3 Ccnd1 Cyclin D1 Promotes cell cycle progression from G1-S phase; overexpression associated with breast/ovarian cancer (Bali et al., 2004; Robker and Richards, 1998). 1.7 ± 0.4 Hspa8 Heat shock protein 8 Stress related chaperone; expression increases during cell cycle G1 phase; regulates cyclin D1 accumulation and maintains its activity in the presence of inhibitory Cdkn1a (Diehl et al., 2003) 1.51 ± 0.1 Cdkn1a Cyclin-dependent kinase inhibitor 1A Regulator of cell cycle progression at G1 phase; implicated in DNA damage repair (Binková et al., 2000) 8.64 ± 1.9 Note. Total RNA was isolated from xenobiotic cultured ovaries, reverse transcribed, and qPCR performed with primers specific for the complementary DNA of indicated genes as described in the materials and methods section. Genes selected for validation were chosen from those most significantly altered by DMBA exposure as detected via microarray analysis. Preference was given to those genes with the highest changes in gene expression. Fold change (mean ± SE) and summary of function relating to folliculogenesis are included. All fold changes were statistically significant (p < 0.05). Open in new tab TABLE 2 QPCR Validation of Microarray Results for Select Transcripts Upregulated by 3MC-cultured Neonatal Ovaries Gene symbol Gene name Summary of function Fold change Cdkn1a Cyclin-dependent kinase inhibitor 1A Regulator of cell cycle progression at G1 phase; implicated in DNA damage repair (Binková et al., 2000) 8.64 ± 1.9 Ccng1 Cyclin G1 Regulator of cell cycle progression at G2/M phase; implicated in DNA damage repair and apoptosis; associated with granulosa cell proliferation and differentiation (Kimura et al., 2001; Liu et al., 2006) 3.9 ± 0.4 Cyp1b1 Cytochrome P450, family 1, subfamily b, polypeptide 1 Phase I detoxifying enzyme; implicated in the bioactivation of PAH into DNA adduct forming metabolites (Shimada and Fujii-Kuriyama, 2004) 3.78 ± 0.4 Aldh1l2 Aldehyde dehydrogenase 1 family, member L2 Mitochondrial homolog of proapoptotic 10-formyltetrahydrofolate dehydrogenase (Ghose et al., 2009; Krupenko et al., 2010) 3.16 ± 0.2 Nqo1 NAD(P)H-quinone oxidoreductase Phase II detoxifying enzyme; detoxifies quinones into quinols; implicated in 3MC metabolism (Kondraganti et al., 2008; Ross, 2004; ) 2.61 ± 0.2 Aldh4a1 Aldehyde dehydrogenase 4 family, member A1 Mitochondrial matrix protein essential for proline degradation; implicated in p53 mediated protection against oxidative stress (Yoon et al., 2004) 2.45 ± 0.1 Ugt1a10 Uridine diphosphate glycosyltransferase 1 family, polypeptide A10 Phase II detoxifying enzyme; implicated in the detoxification of bioactivated PAH metabolites (Dellinger et al., 2006) 1.87 ± 0.3 Ccnd1 Cyclin D1 Promotes cell cycle progression from G1-S phase; overexpression associated with breast/ovarian cancer (Bali et al., 2004; Robker and Richards, 1998). 1.7 ± 0.4 Hspa8 Heat shock protein 8 Stress related chaperone; expression increases during cell cycle G1 phase; regulates cyclin D1 accumulation and maintains its activity in the presence of inhibitory Cdkn1a (Diehl et al., 2003) 1.51 ± 0.1 Cdkn1a Cyclin-dependent kinase inhibitor 1A Regulator of cell cycle progression at G1 phase; implicated in DNA damage repair (Binková et al., 2000) 8.64 ± 1.9 Gene symbol Gene name Summary of function Fold change Cdkn1a Cyclin-dependent kinase inhibitor 1A Regulator of cell cycle progression at G1 phase; implicated in DNA damage repair (Binková et al., 2000) 8.64 ± 1.9 Ccng1 Cyclin G1 Regulator of cell cycle progression at G2/M phase; implicated in DNA damage repair and apoptosis; associated with granulosa cell proliferation and differentiation (Kimura et al., 2001; Liu et al., 2006) 3.9 ± 0.4 Cyp1b1 Cytochrome P450, family 1, subfamily b, polypeptide 1 Phase I detoxifying enzyme; implicated in the bioactivation of PAH into DNA adduct forming metabolites (Shimada and Fujii-Kuriyama, 2004) 3.78 ± 0.4 Aldh1l2 Aldehyde dehydrogenase 1 family, member L2 Mitochondrial homolog of proapoptotic 10-formyltetrahydrofolate dehydrogenase (Ghose et al., 2009; Krupenko et al., 2010) 3.16 ± 0.2 Nqo1 NAD(P)H-quinone oxidoreductase Phase II detoxifying enzyme; detoxifies quinones into quinols; implicated in 3MC metabolism (Kondraganti et al., 2008; Ross, 2004; ) 2.61 ± 0.2 Aldh4a1 Aldehyde dehydrogenase 4 family, member A1 Mitochondrial matrix protein essential for proline degradation; implicated in p53 mediated protection against oxidative stress (Yoon et al., 2004) 2.45 ± 0.1 Ugt1a10 Uridine diphosphate glycosyltransferase 1 family, polypeptide A10 Phase II detoxifying enzyme; implicated in the detoxification of bioactivated PAH metabolites (Dellinger et al., 2006) 1.87 ± 0.3 Ccnd1 Cyclin D1 Promotes cell cycle progression from G1-S phase; overexpression associated with breast/ovarian cancer (Bali et al., 2004; Robker and Richards, 1998). 1.7 ± 0.4 Hspa8 Heat shock protein 8 Stress related chaperone; expression increases during cell cycle G1 phase; regulates cyclin D1 accumulation and maintains its activity in the presence of inhibitory Cdkn1a (Diehl et al., 2003) 1.51 ± 0.1 Cdkn1a Cyclin-dependent kinase inhibitor 1A Regulator of cell cycle progression at G1 phase; implicated in DNA damage repair (Binková et al., 2000) 8.64 ± 1.9 Note. Total RNA was isolated from xenobiotic cultured ovaries, reverse transcribed, and qPCR performed with primers specific for the complementary DNA of indicated genes as described in the materials and methods section. Genes selected for validation were chosen from those most significantly altered by DMBA exposure as detected via microarray analysis. Preference was given to those genes with the highest changes in gene expression. Fold change (mean ± SE) and summary of function relating to folliculogenesis are included. All fold changes were statistically significant (p < 0.05). Open in new tab 3MC Exposure Induces Developing Follicle Atresia and Primordial Follicle Activation InVitro and InVivo To gain a better understanding of the effects of 3MC exposure on the primordial follicle pool, we cultured neonatal ovaries in the presence of 3MC and probed for markers of follicular atresia and proliferation (Fig. 3). The early markers of apoptosis, Casp2 and Casp3, were both detected in 3MC-treated developing follicle oocytes and granulosa cells but were absent in 3MC-treated primordial follicles. TUNEL, a technique used to detect DNA strand breaks and therefore end stage atresia, was also detected in 3MC-treated developing follicles and absent from treated primordial follicles. These results suggest that 3MC specifically targets developing follicles for destruction, not primordial follicles. PCNA, a marker of cellular proliferation and primordial follicle activation, was detected in the majority of 3MC-treated primordial follicle oocyte and granulosa cells, suggesting increased levels of primordial follicle activation (Fig. 3). Again these results are intriguing as they suggest that 3MC-induced primordial follicle depletion may be due to excessive activation not atresia. FIG. 3. Open in new tabDownload slide 3MC exposure causes immature follicular destruction and primordial follicle activation in vitro. Fluorescent immunohistological and TUNEL staining as visualized via epifluorescent microscopy. Ovaries excised from neonatal mice (4 days old) were cultured in 3MC-treated medium for 96 h and processed for immunohistochemistry and TUNEL analysis as described in the Materials and Methods section. Ovarian sections were probed with antibodies against PCNA, active caspase 2, and active caspase 3 or subjected to TUNEL analysis. The results presented here are representative of n = 3 experiments. The percentage of labeled follicles per section is represented by the following scale present in the top right-hand corner; * ≤ 25%, ** = 25–50%, *** = 51–75%, **** = 76–100%. Thin arrow = primordial follicle highlighted in insert at higher magnification; arrow head = primary follicle; scale bar is equal to 100 μm. FIG. 3. Open in new tabDownload slide 3MC exposure causes immature follicular destruction and primordial follicle activation in vitro. Fluorescent immunohistological and TUNEL staining as visualized via epifluorescent microscopy. Ovaries excised from neonatal mice (4 days old) were cultured in 3MC-treated medium for 96 h and processed for immunohistochemistry and TUNEL analysis as described in the Materials and Methods section. Ovarian sections were probed with antibodies against PCNA, active caspase 2, and active caspase 3 or subjected to TUNEL analysis. The results presented here are representative of n = 3 experiments. The percentage of labeled follicles per section is represented by the following scale present in the top right-hand corner; * ≤ 25%, ** = 25–50%, *** = 51–75%, **** = 76–100%. Thin arrow = primordial follicle highlighted in insert at higher magnification; arrow head = primary follicle; scale bar is equal to 100 μm. To confirm the observed effects of 3MC exposure on folliculogenesis in vivo, we treated PND4 neonatal mice with both low- (5 mg/kg/daily) and high- (10 mg/kg/daily) dose exposures over a period of 7 days and collected their ovaries for analysis. PCNA was detected in large groups of primordial follicles reminiscent of localization patterns observed in vitro in the high dose–treated primordial follicles but not in the low dose– and control-treated primordial follicles (Fig. 4). Histomorphological analysis revealed a slight reduction in primordial follicle composition coupled with a comparable increase in activating and primary follicles (Fig. 5A). There was no difference between the average number of follicles per section between the vehicle control (DMSO) and low dose–treated ovaries. The high dose treatment induced a significant decrease in primordial follicle composition (∼twofold) and a significant increase in activating follicle composition (∼threefold) (Fig. 5B). There was also a significant decrease in the total number of follicles per section between the high-dose and control treatment (69% of the control). These results suggest that considerable 3MC-induced follicular depletion occurs alongside the profound primordial follicle activation observed in vivo. FIG. 4. Open in new tabDownload slide Fluorescent immunolocalization of PCNA protein in low- and high-dose 3MC-treated ovaries in vivo. Neonatal mice (4 days old) were treated with either a low or high dose of 3MC over a 7-day period, culled, and their ovaries extracted and processed of immunohistochemistry as outlined in the Materials and Methods section. The results presented here are representative of n = 3 experiments.The percentage of labeled follicles per section is represented by the following scale present in the top right-hand corner; * ≤ 10%, ** = 10–50%, *** = 51–75%, **** = 76–100%. Thin arrow = primordial follicle highlighted in insert at higher magnification; scale bar is equal to 100 μm. FIG. 4. Open in new tabDownload slide Fluorescent immunolocalization of PCNA protein in low- and high-dose 3MC-treated ovaries in vivo. Neonatal mice (4 days old) were treated with either a low or high dose of 3MC over a 7-day period, culled, and their ovaries extracted and processed of immunohistochemistry as outlined in the Materials and Methods section. The results presented here are representative of n = 3 experiments.The percentage of labeled follicles per section is represented by the following scale present in the top right-hand corner; * ≤ 10%, ** = 10–50%, *** = 51–75%, **** = 76–100%. Thin arrow = primordial follicle highlighted in insert at higher magnification; scale bar is equal to 100 μm. FIG. 5. Open in new tabDownload slide Effect of 3MC exposure on ovarian follicle composition and number in vivo. Neonatal mice (4 days old) were treated with either a low or high dose of 3MC over a 7-day period as described in the Materials and Methods section. Ovarian sections were stained with hematoxylin and eosin, and healthy oocyte-containing follicles were classified and counted under a microscope. (A) Low-dose ovarian follicle composition (left panel) and average number of follicles per counted section (right panel). (B) High-dose ovarian follicle composition (left panel) and average number of follicles per counted section (right panel). Values are mean ± SEM, n = 3–5 ovaries from 3 to 5 mice. The symbol ** represents p < 0.01 in comparison with control values. FIG. 5. Open in new tabDownload slide Effect of 3MC exposure on ovarian follicle composition and number in vivo. Neonatal mice (4 days old) were treated with either a low or high dose of 3MC over a 7-day period as described in the Materials and Methods section. Ovarian sections were stained with hematoxylin and eosin, and healthy oocyte-containing follicles were classified and counted under a microscope. (A) Low-dose ovarian follicle composition (left panel) and average number of follicles per counted section (right panel). (B) High-dose ovarian follicle composition (left panel) and average number of follicles per counted section (right panel). Values are mean ± SEM, n = 3–5 ovaries from 3 to 5 mice. The symbol ** represents p < 0.01 in comparison with control values. 3MC Exposure Increases Primordial Follicle Akt1 Phosphorylation To investigate the mechanisms behind 3MC-induced primordial follicle activation, we probed cultured neonatal ovaries for total Akt protein, pAkt1 (S473), and pAkt1 (T308) (Fig. 6). Total Akt protein was detected in both the oocyte (cytoplasm and nucleus) and granulosa cells of 3MC and control-treated follicles at all stages of folliculogenesis, with no distinguishable differences between the two treatments (Fig. 6A). Both pAkt1 (S473) and pAkt1 (T308) were also detected in the oocyte and granulosa cells at all stages of folliculogenesis (Figs. 6B and C). However, quantitative analysis revealed a significant upregulation in pAkt1 (S473) and pAkt1 (T308) phosphorylation in 3MC-treated primordial follicle oocytes (∼1.5-fold) (Figs. 6B and C). Levels of pAkt1 (S473) and pAkt1 (T308) did not differ between 3MC-treated and control developing follicles (Supplementary Data). These results suggest that 3MC exposure causes increased primordial follicle Akt1 phosphorylation, an event that is crucial for PI3K/Akt-induced follicular activation. FIG. 6. Open in new tabDownload slide Fluorescent immunolocalization of Akt (A), pAkt1 (S473)(B), and pAkt1 (T308)(C) in 3MC-cultured ovaries. Ovaries excised from neonatal mice (4 days old) were cultured in 3MC-treated medium for 96 h and processed for immunohistochemistry as outlined in the Materials and Methods section. Representative image of both control and 3MC-treated ovaries (left panel) and quantification of oocyte nuclear staining (right panel). The results presented here are representative of n = 3 experiments. Arrow = primordial follicle; scale bar is equal to 100 μm. The symbol ** represents p < 0.01 in comparison with control values. FIG. 6. Open in new tabDownload slide Fluorescent immunolocalization of Akt (A), pAkt1 (S473)(B), and pAkt1 (T308)(C) in 3MC-cultured ovaries. Ovaries excised from neonatal mice (4 days old) were cultured in 3MC-treated medium for 96 h and processed for immunohistochemistry as outlined in the Materials and Methods section. Representative image of both control and 3MC-treated ovaries (left panel) and quantification of oocyte nuclear staining (right panel). The results presented here are representative of n = 3 experiments. Arrow = primordial follicle; scale bar is equal to 100 μm. The symbol ** represents p < 0.01 in comparison with control values. 3MC Exposure Upregulates Positive mTOR Signaling in Primordial Follicle Oocytes Given the positive upregulation of Akt1 phosphorylation in 3MC-treated primordial follicles, we next observed the levels of downstream targets pTsc2 (S930), pmTOR (S2448), and pmTOR (T2446) in primordial follicle oocytes (Fig. 7). pTsc2 (S930) phosphorylation was predominantly localized in the oocyte nucleus of developing follicles in control-treated ovaries but was significantly upregulated in 3MC-treated primordial follicles (∼1.8-fold) (Fig. 7A). pmTOR (S2448) phosphorylation was localized to both the oocyte nucleus and cytoplasm and was significantly upregulated in 3MC-treated primordial follicles (∼1.8-fold) (Fig. 7B). pmTOR (T2446) phosphorylation was primarily localized to the nucleus of control-treated primordial and primary follicle oocytes and was severely reduced in 3MC-treated primordial follicles (∼fivefold) (Fig. 7C). These events correspond with the positive upregulation of the mTOR pathway, which also plays an essential role in primordial follicle activation. FIG. 7. Open in new tabDownload slide Fluorescent immunolocalization of pTsc2 (S939)(A), pmTOR (S2448)(B), and pmTOR (T2446)(C) in 3MC-cultured ovaries. Ovaries excised from neonatal mice (4 days old) were cultured in 3MC-treated medium for 96 h and processed for immunohistochemistry as outlined in the Materials and Methods section. Representative image of both control and 3MC-treated ovaries (left panel) and quantification of oocyte nuclear staining (right panel). The results presented here are representative of n = 3 experiments. Arrow = primordial follicle; scale bar is equal to 100 μm. The symbol ** represents p < 0.01 in comparison with control values. FIG. 7. Open in new tabDownload slide Fluorescent immunolocalization of pTsc2 (S939)(A), pmTOR (S2448)(B), and pmTOR (T2446)(C) in 3MC-cultured ovaries. Ovaries excised from neonatal mice (4 days old) were cultured in 3MC-treated medium for 96 h and processed for immunohistochemistry as outlined in the Materials and Methods section. Representative image of both control and 3MC-treated ovaries (left panel) and quantification of oocyte nuclear staining (right panel). The results presented here are representative of n = 3 experiments. Arrow = primordial follicle; scale bar is equal to 100 μm. The symbol ** represents p < 0.01 in comparison with control values. PI3K Inhibition Causes Complete Primordial Follicle Depletion in 3MC-Cultured Neonatal Ovaries To examine the role of PI3K/Akt signaling in 3MC-induced primordial follicle ovotoxicity, neonatal ovaries were cultured in vehicle control and 3MC supplemented medium in the presence of the PI3K inhibitor LY294002 and probed for markers of primordial follicle activation and atresia (Fig. 8). PCNA was detected in both the oocyte and granulosa cells of LY294002-treated ovaries from the primary stage onward and was absent from primordial follicles. Although PCNA was also localized to the oocyte and granulosa cells of developing follicles in 3MC + LY294002 cultured ovaries, almost no primordial follicles were detected, suggesting a complete lack of the primordial follicle pool. This was confirmed via histomorphological analysis, which revealed a 20-fold reduction in primordial follicle number compared with the LY294002 control treatment (Supplementary Data). Casp2, Casp3, and TUNEL were all localized to the majority of primary and secondary follicles present in 3MC + LY294002 cultured ovaries, suggesting follicular atresia. In addition, Casp2 and TUNEL staining were also detected in the sparse primordial follicle population of 3MC + LY294002 cultured ovaries (Fig. 8B). These results suggest that PI3K/Akt signaling is essential for primordial follicle survival when exposed to 3MC but does not play a role in 3MC-induced developing follicle ovotoxicity. Akt1 stimulates phosphorylation of prosurvival pBad on S136. Therefore, we examined 3MC-cultured neonatal ovaries for pBad (136) phosphorylation (Fig. 9). Phosphorylation was significantly increased in 3MC-exposed primordial follicles (∼eightfold), suggesting that these follicles have entered a “prosurvival state.” FIG. 8. Open in new tabDownload slide PI3K inhibition causes complete primordial follicle depletion in 3MC-exposed ovaries in vitro. (A) Fluorescent immunohistological and TUNEL staining as visualized via epifluorescent microscopy. (B) Primordial follicle Casp2 and TUNEL staining in 3MC + LY294002–treated ovaries. Ovaries excised from neonatal mice (4 days old) were cultured in 3MC-treated medium + LY294002 for 96 h and processed for immunohistochemistry and TUNEL analysis as described in the Materials and Methods section. Ovarian sections were probed with antibodies against PCNA, active caspase 2, and active caspase 3 or subjected to TUNEL analysis. The results presented here are representative of n = 3 experiments. The percentage of labeled follicles per section is represented by the following scale present in the top right-hand corner; * ≤ 25%, ** = 25–50%, *** = 51–75%, **** = 76–100%. Thin arrow = primordial follicle highlighted in insert at higher magnification; arrow head = primary follicle; scale bar is equal to 100 μm. FIG. 8. Open in new tabDownload slide PI3K inhibition causes complete primordial follicle depletion in 3MC-exposed ovaries in vitro. (A) Fluorescent immunohistological and TUNEL staining as visualized via epifluorescent microscopy. (B) Primordial follicle Casp2 and TUNEL staining in 3MC + LY294002–treated ovaries. Ovaries excised from neonatal mice (4 days old) were cultured in 3MC-treated medium + LY294002 for 96 h and processed for immunohistochemistry and TUNEL analysis as described in the Materials and Methods section. Ovarian sections were probed with antibodies against PCNA, active caspase 2, and active caspase 3 or subjected to TUNEL analysis. The results presented here are representative of n = 3 experiments. The percentage of labeled follicles per section is represented by the following scale present in the top right-hand corner; * ≤ 25%, ** = 25–50%, *** = 51–75%, **** = 76–100%. Thin arrow = primordial follicle highlighted in insert at higher magnification; arrow head = primary follicle; scale bar is equal to 100 μm. FIG. 9. Open in new tabDownload slide Fluorescent immunolocalization of pBad (S136) in 3MC-cultured ovaries. Ovaries excised from neonatal mice (4 days old) were cultured in 3MC-treated medium for 96 h and processed for immunohistochemistry as outlined in the Materials and Methods section. Representative image of both control and 3MC-treated ovaries (left panel) and quantification of oocyte nuclear staining (right panel). The results presented here are representative of n = 3 experiments. Arrow head = primordial follicle; scale bar is equal to 100 μm. The symbol ** represents p < 0.01 in comparison with control values. FIG. 9. Open in new tabDownload slide Fluorescent immunolocalization of pBad (S136) in 3MC-cultured ovaries. Ovaries excised from neonatal mice (4 days old) were cultured in 3MC-treated medium for 96 h and processed for immunohistochemistry as outlined in the Materials and Methods section. Representative image of both control and 3MC-treated ovaries (left panel) and quantification of oocyte nuclear staining (right panel). The results presented here are representative of n = 3 experiments. Arrow head = primordial follicle; scale bar is equal to 100 μm. The symbol ** represents p < 0.01 in comparison with control values. DISCUSSION The primary focus of this study was to characterize the mechanisms behind 3MC-induced ovotoxicity. Microarray analysis revealed a diverse response in ovarian gene expression to 3MC exposure. 3MC influenced the expression of a number of genes involved in cellular processes consistent with its bioactivation into DNA adduct forming compounds, such as drug metabolism, cell death, genetic disorder, and cancer (Table 1; Fig. 1) (Wood et al., 1978). In addition, 3MC also caused the upregulation of a number of genes involved in cellular processes associated with positive follicular development, such as cellular growth/proliferation, cellular development, and cell cycle regulation. This was surprising as these developmental processes have not been previously associated with 3MC-induced ovotoxicity. Canonical pathway analysis also identified a number of significantly upregulated signaling pathways associated with positive proliferation and folliculogenesis (Fig. 2). These pathways included PI3K/Akt and ILK signaling, both of which are essential for primordial follicle activation/growth and survival (Reddy et al., 2010; Troussard et al., 2003) and Ahr signaling, which plays an important physiological role in the regulation of follicular growth and development (Robles et al., 2000). In the context of its reported ovotoxicity, Ahr signaling is also essential for 3MC bioactivation, with Ahr receptor inhibition nullifying 3MC-induced primordial follicle depletion (Shiromizu and Mattison, 1985). Pathways associated with DNA damage and programmed cell death (p53 signaling, G2/M and G1/S checkpoint control) were also upregulated by 3MC exposure, supporting follicular atresia as a mechanism of its ovotoxicity. Ovarian cancer signaling was also significantly upregulated by 3MC exposure, suggesting that the ovotoxicant may also stimulate ovarian tumorigenesis in addition to causing POF. QPCR analysis on genes upregulated in our microarray data confirmed an increase in nine individual genes associated with a variety of cellular processes (Table 2). One of these genes was cyp1b1, a cytochrome p450 oxidase phase I detoxifying enzyme upregulated by Ahr signaling in response to xenobiotic exposure. Cyp1b1 has been identified as one of the initial enzymes, which results in the bioactivation of a number of PAH into DNA adduct forming metabolites, resulting in toxicity (Shimada and Fujii-Kuriyama, 2004). As inhibition of Ahr nullifies 3MC ovotoxicity, cyp1b1 most likely plays a role in ovarian 3MC bioactivation (Shiromizu and Mattison, 1985). 3MC exposure also upregulated aldh1l2 and aldh4a1, two members of the aldehyde dehydrogenase (ALDH) superfamily of NAD(P)+-dependent phase I aldehyde oxidizers (Marchitti et al., 2008). Aldehydes are highly reactive molecules that are pathogenically produced as a consequence of oxidative stress (Esterbauer et al., 1991). As xenobiotics generate reactive oxygen species as a consequence of their detoxification, the upregulation of these two detoxifying enzymes may be a consequence of 3MC induced oxidative stress (Sobinoff et al., 2012a). Indeed, the ALDH Aldh3a1 contains multiple xenobiotic response elements in its promoter region and is upregulated by 3MC exposure (Reisdorph and Lindahl, 2007). Individually, aldh1l2 is a mitochondrial homolog of proapoptotic 10-formyltetrahydrofolate dehydrogenase, whereas aldh4a1 is a mitochondrial matrix protein implicated in p53 mediated protection against oxidative stress, DNA repair, and cell survival (Ghose et al., 2009; Krupenko et al., 2010; Yoon et al., 2004). In addition to phase I detoxifying enzymes, 3MC exposure also upregulated two phase II enzymes, ugt1a10 and noq1. Ugt1a10 is a uridine diphosphate-glucuronosyltransferase that converts phase I bioactivated xenobiotics into water-soluble metabolites for excretion. Ugt1a10 metabolism results in the detoxification of Cyp1b1 bioactivated metabolites and is upregulated by 3MC exposure (Dellinger et al., 2006; Horio and Horie, 1997). Nqo1 is an antioxidant flavoprotein that detoxifies quinones into quinols and has been previously implicated in 3MC metabolism (Kondraganti et al., 2008; Ross, 2004). In addition, Nqo1 has also been associated with p53-dependent apoptosis (Ross, 2004). The upregulation of these phase I and II genes in response to 3MC exposure provides insight into its ovarian bioactivation into ovotoxic compounds. 3MC exposure also upregulated a number of genes involved in cell cycle regulation. One of these genes was cdkn1a, a well-known regulator of cell cycle progression at G1 phase, involved in p53-induced apoptosis and DNA damage repair (Bartek and Lukas, 2001). The upregulation of this cell cycle checkpoint protein would suggest that 3MC-induced ovotoxicity involves reduced cellular proliferation alongside DNA damage and apoptosis. However, 3MC exposure also upregulated genes associated with positive cell cycle progression, such as the cyclins, ccnd1 and ccng1. Ccnd1 promotes cell cycle progression from G1-S phase and is exclusively expressed in theca cells within the ovary (Robker and Richards, 1998). As theca cells are only present in developing follicles, ccnd1 represents a marker of developing follicle growth and an indicator of increased follicular activation. Intriguingly, ccnd1 binds to cdk4 and promotes cell cycle progression from the G1-S phase, whereas cdkn1a binds to ccnd1/cdk4 complexes to inhibit G1 phase progression (Harris and Levine, 2005). Another gene upregulated by 3MC was hspa8, a heat shock protein responsible for regulating protein function and maturation (Agashe and Hartl, 2000). It is now known that hspa8 expression increases during the G1 phase, where it binds to ccnd1 complexes and prevents the inhibitory effect of cdkn1a, allowing the formation of an active ccnd1/cdk4/cdkn1a complex (Diehl et al., 2003). Therefore, hspa8 upregulation in response to 3MC exposure may prevent cdkn1a repression and allow ccnd1-mediated cell cycle progression. Increased ccnd1 expression has also been associated with an increased incidence in ovarian cancer, providing further support for a hypothesis of 3MC-induced ovarian tumorigenesis (Bali et al., 2004). Ccng1, a regulator of cell cycle progression at the G2/M phase, was also upregulated by 3MC exposure (Kimura et al., 2001). In terms of ovarian function and development, ccng1 is essential for granulosa cell proliferation and differentiation and may therefore represent a marker of primordial follicle activation (Liu et al., 2006). Overall, our microarray results reveal a complex mechanism of 3MC-induced ovotoxicity involving a number of cellular processes. These results are comparable to what we observed in other studies investigating the mechanisms of DMBA- and BaP-induced ovotoxicity and imply a similar mechanism of follicular depletion (Sobinoff et al., 2011, 2012b). However, our results are at odds with previous studies as they suggest that 3MC-induced ovotoxicity is limited to primordial follicle oocyte atresia, a hypothesis based exclusively on histomorphological analysis (Borman et al., 2000; Mattison et al., 1983). Therefore, we reexamined the effects of 3MC exposure on folliculogenesis using markers of follicular activation and atresia. PCNA, a marker of cellular proliferation and primordial follicle activation, was detected in both the oocyte and granulosa cells of primordial follicles in both cultured and in vivo high dose–treated ovaries (Figs. 3 and 4). Conversely, markers of follicular atresia (Casp 2, Casp3, and TUNEL) were all detected in developing follicles but not primordial follicles (Fig. 3). Although these markers of apoptosis do not appear to be involved in physiological primordial follicle atresia, they do participate in stress-induced primordial follicle cell death (Hanoux et al., 2006; Sobinoff et al., 2010; Tingen et al., 2009). Therefore, these results suggest that 3MC does not specifically target primordial follicle oocytes for atresia and instead causes developing follicle atresia and primordial follicle activation. These results coincide with our previous studies, in which we found that ovotoxic xenobiotics cause primordial follicle loss through activation and not atresia (Sobinoff et al., 2010, 2011, 2012b). As primordial follicle activation occurred alongside developing follicle atresia, we hypothesized that 3MC-induced follicular activation was due to a homeostatic mechanism of developing follicle replacement. To support this hypothesis, we probed 3MC-cultured ovaries for AMH, a negative regulator of follicular activation secreted by developing follicles (Supplementary Data) (Reddy et al., 2010). Histomorphological analysis revealed a significant reduction in the number of developing follicles expressing AMH. These results support a homeostatic replacement hypothesis of primordial follicle depletion, whereby 3MC directly targets developing follicles for atresia, resulting aberrant primordial follicle activation due to a decrease in negative signaling (Sobinoff et al., 2012a). Therefore, previous investigations into 3MC ovotoxicity relying solely on histomorphological analysis may have seen no evidence of atresia in the developing follicular pool due to its successful maintenance through primordial follicle activation, with the resulting depletion in the primordial follicle pool being put down to atresia (Borman et al., 2000; Mattison et al., 1983). Histomorphological analysis supports this in vivo, as significant 3MC-induced follicular depletion occurred alongside significant primordial follicle activation, with no apparent change in the developing pools composition (Fig. 5B). To further investigate the mechanisms behind 3MC-induced primordial follicle activation, we probed 3MC-cultured ovaries for members of the PI3K/Akt and mTOR pathways of primordial follicle activation. Akt1 is a serine-threonine protein kinase central to the PI3K signaling pathway and is phosphorylated upon PI3K stimulation (Reddy et al., 2010). We observed increased levels of both pAkt1 (T308) and pAkt1 (S437) in 3MC-treated primordial follicle oocytes (Fig. 6). Akt1 is phosphorylated on its T308 residue by Pdpk1 (3-phosphoinositide-dependent protein kinase), an indirect measurement of PI3K activity and a marker of primordial follicle activation (Reddy et al., 2009). Akt1 is also phosphorylated on its S437 residue by activated mTORC2, an event, which coincides with Akt1 T308 phosphorylation during follicular activation (Polak and Hall, 2006). In addition to Akt1 phosphorylation levels, we also investigated Tsc2 and mTOR phosphorylation (Fig. 7). Tsc2 is a tumor suppressor protein, which forms a heterodimer with Tsc1 to suppress mTOR phospoactivation and therefore prevents the overstimulation of primordial follicle recruitment (Adhikari et al., 2009). Tsc2 is phosphorylated on its S939 residue by pAkt1 as part of the PI3K/Akt signaling cascade, resulting in its sequestration from Tsc1 by 14-3-3 proteins to allow mTOR activation (Cai et al., 2006). Tsc2 was localized to the oocyte of developing follicles in control-treated ovaries but was significantly increased in 3MC-treated primordial follicles, suggesting a promotion of mTOR activation. mTOR itself is a central component in the multimeric kinase mTORC1, a key regulator of primordial follicle activation (Adhikari et al., 2010). Its activity is regulated via a phosphorylation-dependent molecular switch, whereby mTOR S2448 phosphorylation results in activation, and mTOR T2446 phosphorylation causes repression (Cheng et al., 2004). mTOR S2448 phosphorylation was significantly increased in 3MC-treated primordial follicle oocytes, whereas mTOR T2446 phosphorylation was significantly decreased (Fig. 7). This suggests an upregulation of mTOR activation upon 3MC exposure and therefore primordial follicle activation. Overall, these results suggest that 3MC induces primordial follicle depletion via activation through synergistic PI3K/Akt1 and mTOR signaling. To confirm a role for PI3K signaling in 3MC-induced primordial follicle depletion, we exposed neonatal ovaries to both 3MC and the PI3K inhibitor LY294002 (Keating et al., 2011). Immunohistological analysis for markers of follicular activation and follicular atresia revealed a severely reduced population of primordial follicles showing signs of atresia (Casp2, TUNEL) in 3MC + LY294002 cultured ovaries (Fig. 8) (Supplementary Data). These results suggest that the upregulation of the PI3K pathway may play a role in preventing 3MC-induced primordial follicle depletion by promoting cell survival. Indeed, there is extensive documentation detailing PI3K and its downstream targets involvement in the promotion of cell survival by inhibiting apoptosis (Franke et al., 2003). To confirm this hypothesis of PI3K-induced primordial follicle survival, we probed 3MC-exposed neonatal ovaries for pBad S136 phosphorylation. Bad is a member of the BCL-2 family of cell death regulators, which binds to, and inhibits proapoptotic members from the same family (Kim et al., 2006). Upon stimulation of PI3K signaling, activated Akt1 phosphorylates Bad on its S136 residue, resulting in its sequestration from prosurvival factors by 14-3-3 proteins to prevent apoptosis (Blume-Jensen et al., 1998; Masters et al., 2001). pBAD (S136) was significantly increased in 3MC-exposed primordial follicles, suggesting the inhibition of apoptosis via Akt1 and therefore PI3K signaling (Fig. 9). These results are intriguing, as they directly conflict with the current ovarian toxicology literature in suggesting that exposure to 3MC actually activates prosurvival pathways in primordial follicle, not atresia (Borman et al., 2000; Mattison, 1980; Mattison et al., 1983). To confirm a role for mTOR signaling in 3MC-induced primordial follicle depletion, we exposed neonatal ovaries to both 3MC and the mTORC1 complex inhibitor rapamycin (Kim et al., 2002). Immunohistological analysis for markers of follicular activation and follicular atresia suggest that inhibition of mTORC1 does not alter 3MC-induced primordial follicle activation and developing follicle atresia as evidenced by primordial follicle PCNA staining in the oocyte and granulosa cells and markers of follicular atresia (Casp 2, Casp3, and TUNEL) being detected in developing follicles (Supplementary Data). It is now known that mTOR signaling acts synergistically with PI3K/Akt signaling to induce primordial follicle activation and that the inhibition of one pathway is not sufficient to prevent activation (Tingen et al., 2009). Therefore, these results suggest that mTORC1 does not play a role in 3MC-induced primordial follicle survival and act in a synergistic role with PI3K signaling in promoting 3MC-induced primordial follicle growth. In conclusion, this study represents one of the first comprehensive studies into the mechanism of 3MC-induced ovotoxicity. Our results suggest that the ovary directly combats 3MC-induced primordial follicle depletion by promoting cellular survival and that the resulting depletion of the primordial follicle pool occurs due to a homeostatic mechanism of developing follicle replacement in an attempt to replace maturing follicles lost due to 3MC-induced follicular atresia. FUNDING National Health and Medical Research Council (Project grant no. 510735) to E.A.M., S.D.R., and B.N. The authors gratefully acknowledge the financial assistance to E.A.M. by the Australian Research Council, Hunter Medical Research Institute, and the Newcastle Permanent Building Society Charitable Trust. 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TI - Staying Alive: PI3K Pathway Promotes Primordial Follicle Activation and Survival in Response to 3MC-Induced Ovotoxicity
JF - Toxicological Sciences
DO - 10.1093/toxsci/kfs137
DA - 2012-07-01
UR - https://www.deepdyve.com/lp/oxford-university-press/staying-alive-pi3k-pathway-promotes-primordial-follicle-activation-and-1dS96pEHzU
SP - 258
EP - 271
VL - 128
IS - 1
DP - DeepDyve
ER -