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The Journal of Clinical Endocrinology & Metabolism Vol. 92, No. 2 705-713
Copyright © 2007 by The Endocrine Society

Molecular Abnormalities in Oocytes from Women with Polycystic Ovary Syndrome Revealed by Microarray Analysis

Jennifer R. Wood, Daniel A. Dumesic1, David H. Abbott and Jerome F. Strauss, III1

Department of Animal Science (J.R.W.), University of Nebraska, Lincoln, Nebraska 68583; the National Primate Research Center (D.A.D., D.H.A.) and the Department of OB/GYN (D.H.A.), University of Wisconsin, Madison, Wisconsin 53715; the Mayo Clinic (D.A.D.), Rochester, Minnesota 55905; Reproductive Medicine and Infertility Associates (D.A.D.), Woodbury, Minnesota 55125; and the Department of OB/GYN (J.F.S.), Virginia Commonwealth University, Richmond, Virginia 23298

Address all correspondence and requests for reprints to: Jennifer R. Wood, Department of Animal Science, University of Nebraska-Lincoln, 3800 Fair Street, Lincoln, Nebraska 68583-0908. E-mail: jwood5{at}unl.edu.


   Abstract
Top
 Abstract
Introduction
 Subjects and Methods
 Results
 Discussion
 References
 
Context: Polycystic ovary syndrome (PCOS), the most common cause of anovulatory infertility, is characterized by increased ovarian androgen production and arrested follicle development and is frequently associated with insulin resistance. These PCOS phenotypes are associated with exaggerated ovarian responsiveness to FSH and increased pregnancy loss.

Objective: The objective of this study was to examine whether the perturbations in follicle growth and the intrafollicular environment affect gene expression and ultimately development of the PCOS oocyte.

Design: Oocyte cDNA was subjected to microarray and PCR analysis.

Setting: This study was conducted at a university laboratory.

Patients: The study comprised 10 normal ovulatory women and nine women with PCOS.

Intervention: The intervention was GnRH analog/recombinant human FSH therapy for in vitro fertilization.

Main Outcome Measure: The main outcome measure was mRNA abundance of oocyte-expressed genes.

Results: Cluster analysis revealed differences in global gene expression profiles between normal and PCOS oocytes. Of the 8123 transcripts expressed in the oocytes, 374 genes showed significant differences in mRNA abundance in PCOS oocytes. Annotation of the data demonstrated that a subset of these genes was associated with chromosome alignment and segregation during mitosis and/or meiosis. Furthermore, 68 of the differentially expressed genes contained putative androgen receptor and/or peroxisome proliferating receptor {gamma} binding sites.

Conclusions: These analyses demonstrated that normal and PCOS oocytes that are morphologically indistinguishable and of high quality exhibit different gene expression profiles. Promoter analysis suggests that androgens and other activators of nuclear receptors may play a role in differential gene expression in the PCOS oocyte. Likewise, annotation of the differentially expressed genes suggests that defects in meiosis or early embryonic development may contribute to reduced developmental competency of PCOS oocytes.


   Introduction
Top
 Abstract
 Introduction
Subjects and Methods
 Results
 Discussion
 References
 
POLYCYSTIC OVARY SYNDROME (PCOS) is a common endocrine and metabolic disorder that is characterized by increased circulating androgen levels, anovulatory infertility, and frequently, insulin resistance and hyperinsulinemia (1, 2, 3). Despite overcoming anovulation with pharmacological agents or by lifestyle intervention, some, but not all, women with PCOS are at increased risk for pregnancy loss (4, 5) possibly from prolonged folliculogenesis or a suboptimal intrauterine environment resulting from the endocrinopathy or ovulation induction itself (6, 7). Furthermore, enhanced theca cell androgen biosynthesis (8) with exaggerated granulosa cell responsiveness to FSH (9) alter the intrafollicular microenvironment in patients with PCOS undergoing in vitro fertilization (IVF), which may increase the risks of implantation failure (10), although the underlying molecular defect(s) remain unknown.

The female germ cell is unique compared with somatic cells. Specifically, transcription of oocyte-expressed genes is regulated by alternate promoters, the half-life (t1/2) of individual mRNAs is long (up to 28 d), transcription and translation are uncoupled, and the meiotic cell cycle starts and stops several times during the oocyte lifespan (11, 12, 13). Transcription is high during the growth phase of the oocyte and is negligible after meiotic resumption. As a result of uncoupling of transcription and translation and long transcript t1/2s, the oocyte cytoplasm contains an abundance of maternal mRNAs, which are used for the completion of the meiotic cell cycle, the first embryonic mitotic cell cycle, and activation of the embryonic genome (13, 14, 15). Thus, although the complement of mRNAs in a mature, MII-arrested oocyte represents the accumulation of transcripts during the growth phase, disruption of gene transcription and/or mRNA stability could negatively impact oocyte growth, oocyte maturation, and/or early embryonic development.

Previous microarray analyses have demonstrated that whole ovaries and isolated theca cells from normal (NL) women and women with PCOS have unique gene expression profiles (16, 17, 18, 19, 20). PCOS follicles have elevated androgen levels before and after GnRH analog/recombinant human FSH (rhFSH) therapy for IVF (21, 22) and contain excess amounts of insulin that are positively correlated with adiposity (23). Taken together, these studies suggest that the altered PCOS hormonal milieu and/or intrinsic abnormalities might alter gene expression in the PCOS oocyte, which could impact oocyte developmental competence. To this end, we have compared the gene expression profiles of NL and PCOS oocytes using Affymetrix microarray chips.


   Subjects and Methods
Top
 Abstract
 Introduction
 Subjects and Methods
Results
 Discussion
 References
 
The study was approved by the Mayo Institutional Review Board before sample collection, and all women signed informed consent forms before participating in the study. Study subjects included 10 nonhirsute ovulatory NL women and nine women with PCOS undergoing gonadotropin therapy for IVF who were willing to donate one healthy oocyte for research, provided a sufficient number of oocytes were available for their own use (22). The indication for IVF gonadotropin therapy was nonovarian (male or tubal factor infertility) in the NL group and failure to conceive by ovulation induction or treatment of male factor infertility in the PCOS group.

The general inclusion criteria have been previously described (22). All NL women had regular menstrual cycles occurring every 21 to 35 d, luteal serum progesterone values indicative of ovulation, absence of hirsutism (30), and normal ovarian morphology as assessed by ultrasound scanning. All patients with PCOS had intermenstrual intervals longer than 35 d, hirsutism, and/or elevated total or free serum testosterone levels (31) and at least one ovary with polycystic morphology (24). All patients with PCOS fulfilled the 1990 National Institutes of Health and the revised 2003 Rotterdam consensus diagnostic criteria for PCOS (24).

Baseline blood sampling

Serum hormone determinations and a 75-g 2-h oral glucose tolerance test were performed in patients with PCOS during a period of amenorrhea and in NL women between cycle d 5 through 10 of the menstrual cycle preceding IVF. Age, body mass index, and serum FSH, dehydroepiandrosterone sulfate as well as glucose levels were similar between female groups (supplemental Table 1, published as supplemental data on The Endocrine Society’s Journals Online web site at http://jcem.endojournals.org). Serum LH, free testosterone, and androstenedione concentrations were higher in patients with PCOS than NL women, as were fasting and 2-h postprandial serum insulin levels.

Ovarian stimulation and oocyte retrieval

Ovarian stimulation and oocyte retrieval protocols were carried out as previously described (22). Briefly, pituitary down-regulation was started with midluteal phase GnRH analog, leuprolide acetate (Lupron; TAP Pharmaceuticals, Deerfield, IL). Once adequate pituitary down-regulation was confirmed [absence of ovarian cysts ≥18 mm in diameter and serum estradiol (E2) levels of <50 pg/ml], rhFSH (Gonal-F; Serono Laboratories, Norwell, MA) therapy was administered. Human chorionic gonadotropin (hCG; 10,000 IU, Profasi; Serono Laboratories) was given im when two or more follicles were at least 18 mm in diameter and the serum E2 levels were at least 300 pg/ml per dominant follicle. The amount of rhFSH administered and the duration of rhFSH treatment were similar in NL women and patients with PCOS, causing comparable maximum serum E2 levels on the day of hCG administration (supplemental Table 2).

Oocyte retrieval was performed 36 h after hCG administration. Follicular fluid was aspirated from the first follicle of each ovary, which was selected by size (at least 15 mm in diameter) and accessibility. One healthy, metaphase II oocyte (i.e. one polar body in the perivitelline space and no visible nuclear structure in the cytoplasm) was collected at random from either of these two follicles, placed immediately in TRIzol (Sigma, St. Louis, MO), and stored at –80 C. Total number of oocytes retrieved and average diameter of follicles containing donated oocytes were similar in patients with PCOS vs. NL women (supplemental Table 2). Follicles of patients with PCOS had elevated E2 levels despite diminished FSH availability and increased testosterone concentrations with normal amounts of progesterone, bioactive LH, and insulin (supplemental Table 1).

RNA isolation and amplification

Total RNA was isolated from each oocyte and subjected to three rounds of linear amplification with the Ovation Biotin RNA Amplification and Labeling System (NuGen Technologies, San Carlos, CA). Briefly, double-stranded cDNA was generated using a proprietary, chimeric RNA/DNA primer resulting in a unique RNA/DNA heteroduplex at one end of the DNA. The double-stranded cDNA was then subjected to three rounds of linear amplification using primers complementary to the RNA/DNA heteroduplex. The amplified cDNA was fragmented and labeled with biotin (supplemental Table 3). RNA from the GeneChip Eukaryotic Poly-A RNA Control Kit (Affymetrix, Santa Clara, CA), which contains mRNAs from Bacillus subtilis genes (lys, phe, thr, and dap) were amplified and labeled under the same conditions as positive controls.

Microarray hybridization

Affymetrix GeneChip Human Genome U133 Plus 2.0 microarray chips (Affymetrix) were hybridized at the University of Pennsylvania Microarray Core Facility as previously described (19, 20). Briefly, the linear-amplified, biotin-labeled cDNA from six NL (N1–N6) and six PCOS (P1–P6) oocytes (supplemental Table 2) was hybridized to individual Affymetrix U133 chips. The fluorescence intensity of each chip was normalized to a trimmed mean signal of 150. Each transcript on the U133 chip was defined as present or absent in each oocyte sample using the Affymetrix Microarray Suite 5.0. The raw gene expression profile data has been deposited in the NCBI Gene Expression Omnibus (www.ncbi.nlm.nih.gov/geo) as series GSE5850.

Gene expression analysis

GeneSpring 7.0 (Agilent Technologies Inc., Palo Alto, CA) was used to compare the microarray data of each sample within and between each experimental group as previously described (19, 20). The fluorescence intensity of each transcript in NL and PCOS oocytes was normalized to the median fluorescence intensity of each transcript in NL oocytes. Each transcript was identified as expressed in the NL or PCOS oocytes if it was called present in at least four of the six samples in each group (supplemental Table 4). In parallel analyses, the data from each transcript was subjected to parametric, one-way ANOVA, which was corrected using the cross-gene error model (Agilent Technologies) and to parametric, one-way ANOVA with variance averaging (NIA; http://lgsun.grc.nia.nih.gov/ANOVA/). A transcript was identified as differentially expressed in PCOS oocytes if it was called present and showed a P value less than 0.05 by both statistical analyses. The lists of expressed and differentially expressed transcripts were annotated using EASE (25) and the Affymetrix (www.affymetrix.com/analysis/index.affx), NCBI (www.ncbi.nlm.nih.gov/), and UCSC genome browser (http://genome.ucsc.edu/cgi-bin/hgGateway?db=hg10) databases.

Semiquantitative PCR (S-QPRC) and quantitative PCR (QPCR)

Approximately 1 ng of linear-amplified, biotin-labeled cDNA from four NL (N7–N10) and three PCOS oocytes (P7–P9) was combined with primers for BUB3, BMPR1A, or growth differentiation factor 9 (GDF9) (supplemental Table 5), and the PCR was carried out in the presence of 32P-labeled dCTP. The PCR products from each sample were resolved on 10% Tris-Boric Acid-EDTA (TBE) polyacrylamide gels and detected using phosphor screens. Similarly, approximately 1 ng of cDNA from N7–N10 and P7–P9 were combined with primers for CDC2-related protein kinase 7 (CRK7), MATER, synaptonemal complex protein (SYCP)2, SYCP3, or TAX1BP1 and SYBR-green master mix (Applied Biosystems) and real-time, QPCR was carried out. The relative abundance of each gene in each sample was determined (19, 20). The relative abundance of each transcript was normalized to the concentration of input cDNA as determined by the Agilent 6000 Pico Assay (Agilent Technologies).

Multiple Em for Motif Elicitation (MEME), Motif Alignment and Search Tools (MAST), and JASPAR analyses

To define common sequence motifs in the promoter region of the transcripts that were differentially expressed in the PCOS oocytes, the alignment tools MEME and MAST were used (26, 27) (http://meme.sdsc.edu/meme/intro.html). The promoter sequences (1000 bps 5' of the predicted transcriptional start-site) of the Atrx, Bub3, Cep70, Diaph2, Ect2, Eme1, Eml1, Fgfr1op2, Fmn2, Nbn1, Nek2, Nek4, Pcm1, Spast, and Tacc1 genes were obtained using the human genome browser (http://genome.ucsc.edu/cgi-bin/hgGateway?db=hg10). The sequences were compared and aligned based on position-dependent letter-probability matrices using MEME. Common sequence motifs 1, 2, and 3 were identified with log likelihood ratios of 369, 346, and 29 and E-values of 19e-52, 2.4e-46, and 5.0e-15, respectively. A MAST search was subsequently carried out using the promoter sequences (1000 bps 5' of the predicted transcriptional start site) for the 294 known genes and full-length cDNAs that were differentially expressed in the PCOS oocytes and the position-specific scoring matrix for each of the three sequence motifs. The MAST search identified 68 independent promoter sequences, which contained one or more of the sequence motifs with E-values less than or equal to 9.5e-10.

To define putative transcription factor binding sites within the three common sequence motifs, the position-specific probability matrices generated for each motif by MEME was compared with the JASPAR collection of position weight matrices for transcription factor DNA-binding preferences (28, 29).


   Results
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
Discussion
 References
 
Gene expression profiles of NL and PCOS oocytes

To define and compare NL and PCOS oocyte transcriptomes, total RNA from MII-arrested, nondeselected oocytes were isolated and subjected to three rounds of linear amplification (supplemental Table 3). This is an accurate and reproducible method to detect and compare mRNA abundance in human, bovine, and mouse oocytes and embryos (32, 33, 34, 35).

To analyze global gene expression in individual oocytes, linear-amplified cDNA from six NL (N1–N6) and six PCOS (P1–P6) oocytes was hybridized to individual Affymetrix Human Genome U133 Plus 2.0 Array chips. Of the 50,000 transcripts interrogated, 8123 were detected as present in four of six NL or PCOS oocytes (supplemental Table 4). Furthermore, 428 known, oocyte-expressed mRNAs were detected by microarray hybridization, including bone morphogenic protein 15 (BMP15), GDF9, Fig{alpha}, Kit, maternal antigen that embryos require (Mater/NALP5), SYCP2, and SYCP3 (35, 36, 37, 38, 39) (supplemental Table 6) indicating that the Affymetrix microarray platform reliably detected oocyte-expressed genes.

Hierarchical clustering and principal component analyses (Fig. 1Go, A and B, respectively) demonstrated that the gene expression profiles of PCOS and NL oocytes are distinctly different and showed that the PCOS oocytes had greater transcriptome homology than the NL oocytes, suggesting surprising similarity in perturbed transcription between PCOS oocytes. When the fluorescent intensity of each expressed transcript in each sample was compared with the median fluorescence intensity of each transcript in the six NL oocytes, individual transcripts with increased (red) and decreased (blue) mRNA abundance in individual PCOS oocytes were identified (Fig. 1CGo). Furthermore, 374 genes exhibited statistically significant increased or decreased mRNA abundance in PCOS oocytes (Fig. 1DGo and supplemental Table 7). Interestingly, 80% of the differentially expressed genes had increased mRNA abundance in the PCOS oocytes. The 374 genes were annotated and represented several functional categories, including signal transduction (51), cellular metabolism (103), DNA transcription (44), RNA processing (18), cellular architecture (24), and novel genes and expressed sequence tags (134).


Figure 1
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  		FIG. 1. Distinct differences identified in the global gene expression profiles of NL and PCOS oocytes. A, Hierarchical cluster analysis was carried out using GeneSpring 7.0 and the microarray hybridization data for the six NL (N1–N6) and six PCOS (P1–P6) oocytes. B, Principal component analysis (PCA) was carried out using GeneSpring 7.0. The gene expression profile for each transcript from oocytes N1–N6 and P1–P6 was represented by two PCA components and the results plotted on a two-dimensional graph. C, For each PCOS and NL oocyte sample, the fluorescence intensity of the 8123 transcripts, which were called present on the microarray hybridization, were compared with the median fluorescence intensity of the transcript in the six NL oocyte samples. Increased (red), decreased (blue), and unchanged (yellow) mRNA levels for each transcript are indicated for each sample. D, For each of the 8123 expressed transcripts, the mean log fluorescence intensity for the six NL oocytes (x-axis) is plotted against the mean log fluorescence intensity for the six PCOS oocytes (y-axis). Transcripts with a statistically significant increased (red) or decreased (green) mRNA abundance were determined using the Student’s t test corrected in parallel with the cross-gene error model and variance averaging (P value < 0.05 using both correction algorithms).

 
Validation of the microarray data using S-QPCR and real-time QPCR provided independent confirmation that the microarray analysis identified good candidate genes, which have altered expression in the PCOS oocyte. Specifically, S-QPCR and QPCR showed similar levels of GDF9, SYCP2, and SYCP3 in seven independent oocyte samples (N7–N10 and P7–P9) (Fig. 2Go). Conversely, mRNA abundance of budding uninhibited by benzimidazoles 3 (BUB3), bone morphogenetic protein receptor 1A (BMPR1A), CRK7, maternal antigen that embryos require (Mater/NALP5), and T-cell leukemia virus type 1-binding protein (TAX1BP1) showed a similar trend of increased or decreased (CRK7) mRNA levels in the three PCOS oocyte samples (Fig. 2Go). Despite the lack of statistical confirmation of differential mRNA abundance, these results were remarkable given that independent oocyte cDNA samples were used to obtain the microarray and the PCR data.


Figure 2
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  		FIG. 2. S-QPCR and QPCR validation of differentially expressed mRNAs identified by microarray analysis. S-QPCR (A) and real-time QPCR (B) were carried out using primers for the oocyte-expressed mRNAs GDF9, BUB3, BMPR1A (A), and SYCP2, SYCP3, CRK7, MATER (NALP5), TAX1BP1 (B). Primers were combined with linear-amplified cDNA from four NL (N7–N10) and three PCOS (P7–P9) oocytes, which were not represented on the microarray analysis. Cycle conditions and primer concentrations were determined empirically for each PCR. S-QPCRs were resolved on 10% TBE polyacrylamide gels and the resulting gene-specific band detected using a phosphorimager. QPCRs were normalized to input cDNA, which was determined using the Agilent 6000 Pico Assay.

 
Differential expression of maternal-effect and cell-cycle regulation genes in PCOS oocytes

Annotation of the transcripts with altered mRNA abundance in the PCOS oocytes revealed two interesting groups of genes, maternal-effect genes and genes involved in the meiotic/mitotic cell cycle. Maternal-effect genes produce mRNA and/or protein during oogenesis that is required for development before and after activation of the zygotic genome (40). Although only seven mammalian maternal-effect genes (Mater, Hsf1, Dnmt1, Zar1, Npm2, Stella, Fmn2, and Bnc1) have been identified to date, three exhibited increased mRNA abundance in PCOS oocytes. Specifically, the microarray analysis demonstrated that the transcript levels of Mater/NALP5, which is required for progression of development past the two-cell stage (41) are increased approximately 3-fold in PCOS oocytes (supplemental Table 7). Likewise, QPCR showed consistently increased levels of Mater/NALP5 in PCOS compared with NL oocytes (Fig. 2BGo). In addition to Mater/NALP5, basonuclin (BNC1), and formin 2 (FMN2), which regulate transcription of rRNA during oogenesis and spindle dynamics during meiosis, respectively (42, 43), had 4-fold increased mRNA levels in the PCOS oocytes (supplemental Table 7). Although most studies have examined loss-of-function mutations for these maternal-effect genes, these microarray data suggest that increased expression of Mater/Nalp5, Bnc1,and/or Fmn2 may negatively impact early embryonic development.

Meiosis represents a major developmental process in oocyte maturation. Defects in meiosis result in the formation of aneuploid/polyploid oocytes and are a major contributing factor to embryonic loss (44, 45). Annotation of the array data revealed that several genes that are involved in the meiotic and/or mitotic cell cycle in yeast or mammals have altered mRNA abundance in PCOS oocytes (Fig. 3Go). Specifically, eight genes involved in chromosome alignment (Atrx, Eme1, Nbn) (46, 47, 48), spindle dynamics (Ect2, Eml1, Diaph2, Fmn2) (42, 49, 50, 51), and cell-cycle checkpoint (Bub3) (52) exhibit altered mRNA abundance in the PCOS oocytes. Although the meiotic cell cycle generally takes place in the absence of functional centrosomes (53), seven centrosome-associated genes (Cep70, Fgfr1op2, Nek2, Nek4, Pcm1, Spast, Tacc1) (54, 55, 56, 57, 58) have increased mRNA abundance in the PCOS oocytes (Fig. 3Go). The expression of these collective genes, which are generally not a part of the human oocyte transcriptome, suggests that this pathway is disrupted in PCOS oocytes and thereby may also contribute to abnormalities in early embryonic development.


Figure 3
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  		FIG. 3. Fifteen genes involved in the meiotic/mitotic cell cycle pathway exhibited altered mRNA abundance in PCOS oocytes. Schematic representation of the chromosomes (orange), spindle microtubules (blue), and centrioles/centrosomes (pink/yellow) at the mitotic division (adapted from Ref. 58 ). Genes that were differentially expressed in the PCOS oocytes, which are involved in spindle dynamics, homologous recombination/chromosome alignment, cell-cycle checkpoint, and centrosome function, are indicated.

 
Promoter analysis reveals common sequence motifs in differentially expressed PCOS genes

Given that the meiotic cell cycle is exquisitely regulated in the mammalian oocyte, altered mRNA abundance of ATRX, BUB3, CEP70, DIAPH2, ECT2, EME1, EML1, FMN2, FGFR1OP2, NBN, NEK2, NEK4, PCM1, SPAST, and TACC1 suggested that transcription of these genes may be commonly regulated. To determine if these 15 genes contained common putative transcription factor binding sites, 1000 bases of their proximal promoters were compared. The promoter sequences were analyzed using MEME, which uses position-dependent letter-probability matrices to align common sequences within multiple samples (26). Three common sequence motifs were defined in the promoters of Eme1, Nek2, Tacc1, Nbn, Nek4, Spast, Diaph2, and Fmn2 (Fig. 4Go, A and B). Subsequently, these common sequence motifs were compared with matrix-based transcription factor binding profiles using the JASPAR collection of position weight matrices for transcription factor DNA-binding preferences (28, 29). The JASPAR analysis revealed that each of the sequence motifs has at least 70% identity with the androgen receptor (AR), peroxisome proliferating receptor {gamma} (PPAR{gamma}), and/or PPAR{gamma}-retinoid X receptor (RXR) binding sites (Fig. 4CGo). Although our microarray analysis did not detect mRNAs for these nuclear receptors, previous studies have demonstrated AR, PPAR{gamma}, and RXR expression in mammalian oocytes (39, 59, 60) suggesting that nuclear receptor mRNA levels in a single oocyte were below the sensitivity of our array analysis.


Figure 4
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  		FIG. 4. Three common sequence motifs, which contain putative nuclear-receptor binding sites, were identified in genes differentially expressed in PCOS oocytes. A, MEME identified three nonoverlapping, common sequence motifs (blue, yellow, and pink squares) within the proximal promoter (1000 base pairs) of the EME1, NEK2, TACC1, NBN, NEK4, SPAST, DIAPH2, and FMN2 genes. B, The multilevel consensus sequences for motif 1 (blue), 2 (yellow), and 3 (pink) are shown. C, The position-specific probability matrix for each motif was compared with the JASPAR collection of position weight matrices for transcription factor DNA-binding preferences. This comparison revealed that each motif contained a putative binding site for the androgen receptor, PPAR{gamma}, and/or PPAR{gamma}-RXR{alpha}. The percent identity of each motif with each transcription factor binding site is indicated.

 
Given that intrafollicular androgen and insulin levels are higher in the patients with PCOS (supplemental Table 1 and Ref. 22), the promoter regions of all the full-length cDNAs (i.e. excluding the 80 expressed sequence tags), which were differentially expressed in the PCOS oocytes (supplemental Table 6), were analyzed using the MAST, which searches for known motifs within a sequence using the parameters generated by MEME (26, 27). When the promoter sequences of the 294 differentially expressed known genes and full-length cDNAs were searched for the three common sequence motifs identified by MEME, MAST identified at least one of the sequence motifs in 68 of the differentially expressed genes (supplemental Table 8). Taken together, these data indicated that the altered hormonal environment likely contributes to altered gene expression in the PCOS oocyte, including increased expression of genes, which play a role in the meiotic cell cycle.


   Discussion
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
References
 
It is clear from differences in IVF success rates and in reproductive capacity among women that not all MII-arrested oocytes are created equal. Indeed, the microarray data indicate that MII oocytes that are morphologically of high quality and thus indistinguishable in gross appearance (61) from NL and PCOS ovaries have distinctly different transcriptomes. The limitations on sample size in this study make it difficult to determine if these differences in gene expression are the result of the altered endocrine microenvironment of the oocyte, altered function of the supporting somatic cells of the follicle (i.e. granulosa or theca cell function), or an intrinsic defect in the PCOS oocyte. Although outside the scope of this study, future experiments will be required to determine the mechanism of altered mRNA abundance in the PCOS oocyte and the ramification of altered mRNA levels on oocyte competency for fertilization or embryonic development. Despite these limitations, the current study highlights altered expression of mitotic cell cycle and maternal effect genes in the PCOS oocyte. Furthermore, promoter analysis of the differentially expressed genes suggests that androgens and other activators of nuclear receptors may regulate gene expression in the oocyte.

Knockout mouse models and in vitro assays demonstrate a role for nuclear receptors including AR and PPAR{gamma} in normal follicular development and ovulation (62, 63, 64). Although most studies have examined the function of these nuclear receptors in the somatic cells of the follicle, recent studies in Xenopus and mouse oocytes demonstrate that nongenomic signaling through the classic AR promotes meiotic resumption and oocyte maturation (59, 65, 66). The current microarray analysis identified putative binding sites for AR, PPAR{gamma}, and/or PPAR{gamma}-RXR within the proximal promoter of several genes differentially expressed in the PCOS oocytes suggesting that these nuclear receptors also promote transcriptional activation in the oocyte. Interestingly, Zhang et al. (67) have also shown that insulin signaling phosphorylates PPAR{gamma} resulting in its activation in the absence of endogenous ligand. Given that genes involved in spindle dynamics and centrosome function contained these putative nuclear receptor binding sites (Fig. 4Go) and that intrafollicular testosterone levels and circulating insulin concentrations are increased in our patients with PCOS undergoing IVF (supplemental Table 1), the collective data suggest that the altered endocrine and metabolic environment surrounding the PCOS oocyte may negatively affect the meiotic cell cycle of the oocyte and/or the initial mitotic cell cycle of the one-cell embryo.

The microarray data also demonstrated that overall mRNA abundance is higher in PCOS compared with NL oocytes. Previous array analysis of NL and PCOS theca cells demonstrated that the differentially expressed genes were represented by nearly equivalent numbers of transcripts with increased or decreased mRNA abundance (19, 20). Conversely, 80% of the genes differentially expressed in PCOS oocytes had increased mRNA abundance, including the maternal effect genes Mater/Nalp5, Bnc1, and Fmn2. In the mouse, there is a dramatic change in the gene expression profile at the two-cell stage, which presumably marks the maternal-to-zygotic genome transition (68). Several groups have postulated that the timely degradation of maternal RNAs at this transition is critical for appropriate embryonic development (69, 70, 71). Furthermore, Giraldez et al. (72) have demonstrated in zebrafish that inhibition of maternal transcript degradation leads to neural tube defects and have suggested that the lack of maternal transcript clearance results in mixed developmental states that is detrimental to the normal progression of embryogenesis. Given that the PCOS oocytes have increased levels of mRNA, the maternal-to-zygotic transition may be disrupted, which could contribute to developmental failures.

In conclusion, our studies suggest for the first time that there are molecular abnormalities in PCOS oocytes, which could account, in part, for the reduced fecundity that characterizes PCOS. The discovery that a number of the genes whose transcript abundance is altered in the PCOS oocyte contain putative nuclear receptor binding sites suggests that pharmacological or lifestyle interventions might correct the molecular defects in the PCOS oocyte and improve pregnancy outcome. In addition to PCOS, moreover, other insulin-resistant states such as obesity and type II diabetes and/or environmental factors that could alter the intrafollicular hormone environment might modulate transcriptional events in the oocyte and reduce its competency for embryonic development.


   Acknowledgments
 
We thank Rebekah R. Herrman for technical assistance during this study.


   Footnotes
 
This work was supported by the National Institutes of Health (NIH) Grants U01 HD044650, U54 HD34449, and R01 RR 013635; Mayo Clinical Research Grant 2123-01; Mayo Grant M01-RR-00585; P51 RR000167 from the National Center for Research Resources (NCRR), a component of NIH, to the Wisconsin National Primate Research Center, University of Wisconsin–Madison; and a contribution of the University of Nebraska Agricultural Research Division, supported in part by funds provided through the Hatch Act. This work was also partially supported by the NIH as part of the National Institute of Child Health and Human Development National Cooperative Program on Female Health and Egg Quality under cooperative agreement U01 HD044650 and by Serono Pharmaceuticals. This research was conducted in part at a facility constructed with support from Research Facilities Improvement Program Grants RR15459-01 and RR020141-01. This publication’s contents are solely the responsibility of the authors and do not necessarily represent the official views of NCRR or NIH. Likewise, mention of a trade name, proprietary products, or company name is for presentation clarity and does not imply endorsement by the authors of the University of Nebraska.

Disclosure Statement: J.R.W. and D.H.A. have nothing to declare. D.A.D. has received grant support from Serono Pharmaceuticals. J.F.S. consults for Burroughs Wellcome Fund, Berlex Foundation, TAP Pharmaceuticals, Serono Pharmaceuticals, and Johnson & Johnson and has equity interests in FemmePharma, Matrix Pharma, and EndoCeutics.

First Published Online December 5, 2006

1 D.A.D. and J.F.S. contributed equally to the manuscript. Back

Abbreviations: AR, Androgen receptor; BNC1, basonuclin; CRK7, CDC2-related protein kinase 7; E2, estradiol; FMN2, formin 2; GDF, growth differentiation factor; hCG, human chorionic gonadotropin; IVF, in vitro fertilization; MAST, Motif Alignment and Search Tools; MEME, Multiple Em for Motif Elicitation; NL, normal; PCOS, polycystic ovary syndrome; PPAR, peroxisome proliferating receptor; QPCR, quantitative PCR; rhFSH, recombinant human FSH; RXR, retinoid X receptor; S-QPCR, semi-QPCR; SYCP, synaptonemal complex protein.

Received September 28, 2006.

Accepted November 28, 2006.


   References
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
 References
 

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