Polycystic ovarian syndrome is accompanied by repression of gene signatures associated with biosynthesis and metabolism of steroids, cholesterol and lipids
© Salilew-Wondim et al.; licensee BioMed Central. 2015
Received: 19 December 2014
Accepted: 30 March 2015
Published: 13 April 2015
Polycystic ovarian syndrome (PCOS) is a spectrum of heterogeneous disorders of reproduction and metabolism in women with potential systemic sequel such as diabetes and obesity. Although, PCOS is believed to be caused by genetic abnormalities, the genetic background that can be associated with PCOS phenotypes remains unclear due to the complexity of the trait. In this study, we used a rat model which exhibits reproductive and metabolic abnormalities similar to the human PCOS to unravel the molecular mechanisms underlining this complex syndrome.
Female Sprague–Dawley rats were randomly assigned to DHT and control (CTL) groups. Rats in the DHT group were implanted with a silicone capsule continuous-releasing 83 μg 5α-dihydrotestosterone (DHT) per day for 12 weeks to mimic the hyperandrogenic state in women with PCOS. The animals were euthanized at 15 weeks of age and the pairs of ovaries were excised and the ovarian cortex tissues were used for gene expression analysis. Total RNA was from the ovarian cortex was amplified, labeled and hybridized to the Affymetrix GeneChip® Rat Genome 230 2.0 Array. A linear model system for microarray data analysis was used to identify genes affected in DHT treated rat ovaries and the molecular pathway of those genes were analyzed using the Database for Annotation, Visualization and Integrated Discovery (DAVID) analysis tool.
A total of 573 gene transcripts, including CPA1, CDH1, INSL3, AMH, ALDH1B1, INHBA, CYP17A1, RBP4, GAS6, GAS7 and GATA4, were activated while 430 others including HSD17B7, HSD3B6, STAR, HMGCS1, HMGCR, CYP51, CYP11A1 and CYP19A1 were repressed in DHT-treated ovaries. Functional annotation of the dysregulated genes revealed that biosynthesis and metabolism of steroids, cholesterol and lipids to be the most top functions enriched by the repressed genes. However, cell differentiation/proliferation, transcriptional regulation, neurogenesis, cell adhesion and blood vessel development processes were enriched by activated genes.
The dysregulation of genes associated with biosynthesis and metabolism of steroids, cholesterol and lipids, cell differentiation/proliferation in DHT- treated ovaries could be a molecular clue for abnormal steroidogenesis, estrous cycle irregularity, abnormal folliculogenesis, anovulation and lipid metabolism in PCOS patients.
The ovary is a key organ in the female reproductive system and its malfunction due to endocrine abnormalities could result in female infertility. Polycystic ovarian syndrome (PCOS) is one of the common hormonal and metabolic disorders in women of reproductive age . However, due to its heterogeneity and complexity, universally accepted clinical definition of PCOS remains ambiguous [2,3]. Indeed, the presence of polycystic ovarian morphology is one of the common phenomena that can occur in the majority of PCOS patients. About 95% of women with PCOS at their early follicular phase could have polycystic ovaries and reduced level of follicle stimulating hormone  which may lead to antral follicle growth arrest and increased luteinizing hormone level . In addition, PCOS is also associated with hyperandrogenism, menstrual dysfunction, oligo-ovulation and insulin resistance . In this context, PCOS is considered as a complex androgen excess accompanied by different degrees of gonadotropic and metabolic dysregulation controlled by multiple gene interaction and environmental factors . However, to what extent this trait is transmitted to the next generation and the intrinsic molecular factors underlining the occurrence of PCOS is unclear.
Although, the genetic basis of abnormal follicular development, anovulation, metabolic disorder and other heterogenous clinical abnormalities of PCOS patients seems to require detailed investigation, it is suggested that daughters from women exhibiting a characteristics of PCOS could have a higher chance of acquiring hyperandrogenism and other PCOS phenotypes . Moreover, single nucleotide polymorphism in thyroid adenoma associated (THADA), DENN/MADD domain containing 1A (DENND1A), interleukin 6 (IL6) and adiponectin genes has been suggested to be the genetic causes of PCOS [9-11]. In addition, in vitro studies also showed altered expression of CYP11A and CYP17 genes in theca cell derived from PCOS woman . Furthermore, changes in the granulosa and theca cell gene expression have been reported in women with PCOS [13-15]. Although these association studies were performed using the samples of PCOS patients, the majority of gene expression studies were based on the cell culture models which may not necessarily represent and describe the biological and molecular networks governing its complex phenotype. Indeed, this can in part be due to the availability and accessibility of the human sample or small sample size of the study populations  and the complexity of the trait between individuals. However, to addresse the clinical heterogeneity of PCOS, animal models have been described to be the best option to investigate the pathophysiologic mechanisms associated with the etiology of PCOS [16-21]. Therefore, to uncover the broad basis of molecular mechanisms associated with physiological and anatomical changes induced by PCOS, we generated a rat PCOS model that exhibit both polycystic ovaries (PCO) and metabolic abnormalities by implanting silastic capsules containing 5α-dihydrotestosterone (DHT) into their ovary in similar way as previously described by others . Using this rat PCOS model that exhibits both polycystic ovaries (PCO) and metabolic abnormalities, we have previously demonstrated altered expression of 89 miRNAs following chronic androgen treatment . However, the genes that are activated or repressed as well as their molecular functions, gene networks and molecular pathways associated with PCOS phenotypes, remained unclear. Therefore, this study was conducted to gain insight into the genes that are associated with follicular arrest, abnormal steroid and metabolite biosynthesis and metabolism, insulin resistance and ovarian dysfunction.
Materials and methods
Details of the materials and methods used in the present study have been described in our previous publication . Briefly, female Sprague–Dawley rats were randomly assigned to DHT and control (CTL) groups. Rats in the DHT group were implanted with a silicone capsule continuous-releasing 83 μg 5α-dihydrotestosterone (DHT) per day for 12 weeks to mimic the hyperandrogenic state in women with PCOS, whose plasma DHT levels are approximately 1.7-fold higher than those of healthy control and those in CTL group received empty capsule . The animals were euthanized at 15 weeks of age, ovaries were excised and extraneous tissues carefully removed. Corpus luteum (CL) was present in most of control rat ovaries while none or very few CL were observed in DHT-treated rat ovaries. Ovarian cortex tissues were snap-frozen in liquid nitrogen and stored at −80°C for further analysis. The PCOS phenotypic characteristics of DHT-treated rats have been described .
Gene expression analysis using GeneChip@rat genome array
Total RNAs were isolated from 3 independent DHT and CTL rat ovaries using miRNeasy mini kit (Qiagen, Hilden, Germany). Genomic DNA contamination was removed from the RNA samples using TURBO DNA-free™ kit (Ambion, Foster City, CA). The concentration of the RNA was analyzed using the Nanodrop 8000 Spectrophotometer (Thermo Fisher Scientific Inc, DE, USA). The RNA integrity and quality was evaluated using Agilent 2100 Bioanalyzer with RNA 6000 Nano LabChip® Kit (Agilent Technologies Inc, CA, USA).
250 ng of total RNAs from DHT-treated or CTL rat groups in four replicates was amplified and labeled as per the GeneChip®3’ IVT Express Kit (Affymetrix, CA, USA). Eukaryotic poly-A RNA control kit (Affymetrix, CA, USA) was used as a SPIKE-IN control to monitor the entire target labeling process. Following amplification, the biotin labeled amplified RNA (aRNA) was purified and fragmented. The distribution of aRNA fragments were evaluated using Agilent 2100 bioanalyzer with RNA 6000 Nano LabChip® Kit (Agilent Technologies Inc, CA, USA).
Sample hybridization, array washing, staining and scanning
Prior to hybridization, the fragmented and biotin labelled cRNA from each rat ovary was mixed with control oligonucleotide B2 (3 nM), 20× eukaryotic hybridization controls (bioB, bioC, bioD, cre) (Affymetrix, CA, USA), 2× hybridization mix and DMSO. The hybridization cocktail were then incubated at 99°C (5 min) and subsequently at 45°C (5 min). Each sample was then transferred to independent GeneChip® Rat Genome 230 2.0 Array chip. Three biological replicates and one technical replicate (pool of three biological replicates) were hybridized for each rat group for 16 h. The array slides were washed and stained using the Fluidics Station 450/250 (Affymetrix, CA, USA), according to the GeneChip® expression user manual (P/N 702232 Rev. 3). Arrays were scanned with the GeneChip™ 3000 laser confocal slide scanner (Affymetrix, CA, USA) integrated with GeneChip® Operating System (GCOS).
Array data analysis and visualization
The array data was normalized by integrating the bioconductor packages (http://bioconductor.org) in R environment (www.r-project.org), using GC robust multi-array average analysis . Briefly, the cell intensity (CELL) files were imported into R software after loading bioconductor packages (http://bioconductor.org) that suit to the Rat GeneChip affymetrix array. The normalized data and the CELL files are stored in the Gene Expression Omnibus (GEO; http://www.ncbi.nlm.nih.gov/geo/, series entry number GSM1437398). The linear models for microarray data analysis system (LIMMA)  and the Benjamini–Hochberg procedure of false discovery rate adjustment  were employed to discriminate the gene expression profile between the samples. The differentially expressed genes were tested for their gene ontology (GO) terms for over- or under-representation, using a classical hypergeometric test . The molecular pathway enriched by differentially expressed genes were obtained from the Kyoto Encyclopedia of Genes and Genomes (KEGG) and Panther pathway data bases, using The Database for Annotation, Visualization and Integrated Discovery (DAVID) analysis tool . The heatmaps and clustering of differentially expressed genes were constructed using Bioconductor (http://www.bioconductor.org) in R software environment http://www.r-project.org/ and/or PermutMatrix .
Validation of differentially expressed genes
Gene specific primers used for validation of differentially expressed genes
Gene bank acc. No
Primer 5’ to 3’
Ovarian polycystic syndrome is associated with dysregulation of gene expression
Gene transcripts repressed in DHT-treated rats
Functional classification of the genes repressed in ovaries of DHT-treated rats
Gene transcripts activated in ovaries of DHT-treated rats
Molecular functions activated in ovaries of DHT-treated rats
List of molecular functions containing genes with increased level of mRNA in DHT treated compared to CTL rat groups
Zinc ion binding
NR2F2,MAP3K1,PTGR1,CPA1,VEZF1,ACY3,SLC39A8,CPA2,RBM5,ZFP278,ZNF574, ZEB2, JARID1A, PHC1, ZCCHC11, KLF11, ZFPM2, OSR2, MSL2L1, TRIM37, PAN3, ZC3H11A, RGNEF, CRYZ, SIVA1, ZFP26, TRIM35, ZFP61, FOXP2, DTX3, ZNF292, GATA4, KLF15, RNF138, MMP23
Transcription regulator activity
NR2F2, CITED2, CRYM, ARNT2, NFIA, NR3C2, BMP2, SMARCD3, RPL7, MYCN, ZFP278, ZEB2, KLF11, ZFPM2, ARX, TLE1, FOXP2, ZNF292, GATA4, NR0B1,RBPJ, MEIS1, HOXD9, CDH1, FOXO1,TWIST, KLF15
INSL3, INHA, JAK2, AKAP9, CXCL12, INHBB, ARNT2, INHBA, PENK, BMP2,SMARCD3, SEMA3C, EPHA4,SEMA6A, SIVA1, NR0B1, GAS6, ANGPTL1, MDK,STC1, IRS3
Protein dimerization activity
INHA, INHBB, GHR, ARNT2, NR3C2, SHMT1, INHBA, BMP2, MYH6, MYH7, RPL7, PON3, GUCY1A3, FOXP2, NR0B1, MEIS1, ROBO2
Growth factor activity
INHA, JAK2, INHBB, INHBA, BMP2, GAS6, MDK
Carboxylic acid transmembrane transporter activity
SLCO1A4, SLC13A5, SLC1A3, SLC7A5, SLC7A8
Growth factor binding
FGFR2, IGFBP6, CRIM1,HTRA3, CYR61
Insulin-like growth factor binding
IGFBP6, CRIM1, HTRA3, CYR61
Protein phosphatase binding
GHR, PHACTR1, JUP, CDH1
Extracellular matrix binding
VTN, RPSA, CYR61
L-amino acid transmembrane transporter activity
Semaphorin receptor binding
Protein tyrosine phosphatase-like protein binding
Actin-dependent atpase activity
Glycine hydroxyl methyltransferase activity
Peptide antigen binding
Molecular pathways activated or repressed in DHT-treated rats
List of molecular pathways containing genes with activated (↑) and repressed (↓) level of mRNA in DHT treated compared to CTL rat ovary groups
Biosynthesis of steroids
↓ CYP51, ↓HMGCR, ↓SQLE, ↓HSD17B7, ↓FDFT1, ↓LSS, MVD, ↓FDPS, ↓IDI1, ↓SC5DL, ↓EBP, ↓SC4MOL, ↓TM7SF2, ↓DHCR24, ↓NSDHL
TGF-beta signaling pathway
↑INHBB, ↑INHBA, ↑AMH, ↑BMP2, ↑FOXO1, ↑FOXP2, ↓FKBP1A,↑ INHA, ↑ BAMBI, ↑CITED1, ↓CITED2
Leukocyte transendothelial migration
↑CXCL12, ↓THY1, ↓BCAR1, ↓PECAM1,↓ CXCR4, ↑CLDN11, ↑MYL9, ↓SIPA1
↓MVD, ↓HMGCR, ↓HMGCS1, ↓FDPS, ↓IDI1, ↓FDFT1
↓ME1, ↓DLAT, ↑ALDH1B1, ↓ME2, ↓ACAT2, ↓ACSS2, ↑ME3
↓PLA2G1B, ↑PLA2G2A, ↓GPD1, ↓CDS1,↓ PCYT2, ↑ETNK2, ↑CRLS1
Complement and coagulation cascades
↓A2M, ↓C2, ↓PLAU, ↑TFPI, ↑MASP1,↓ CFB, ↓C1QA
↓HK1, ↓HK2, ↓DLAT, ↑FBP2, ↑ALDH1B1,↓ACSS2
Adipocytokine signaling pathway
↑JAK2, ↓NFKBIB, ↑IRS3, ↓ACSL3, ↓ADIPOR, ↑STK11
↓HMGCS1, ↓AACS, ↑ALDH1B1, ↓ACAT2, ↑ACSM5
↓SQLE, ↓FDFT1, ↓FDPS, ↓IDI1
Ether lipid metabolism
↓PLA2G1B, ↑PLA2G2A, ↑PAFAH1B3, ↑ENPP6
Citrate cycle (TCA cycle)
↓ACLY, ↓IDH1, ↓DLAT, ↓DLST
Androgen and estrogen metabolism
↑SRD5A1, ↓ HSD17B1, ↓HSD17B7, ↓HSD3B6
↓HSD3B6, ↓CYP11A1, ↓NSDHL, ↓CYP19A1
↓GPX1, ↓IDH1, ↓GSTT1, ↑GPX7
↑ALDH1B1, ↓DLST, ↓ACAT2, ↑MGC109340
Fatty acid metabolism
↑ACSL3, ↓ACAA2, ↑ALDH1B1, ↓ACAT2
Valine, leucine and isoleucine degradation
↓HMGCS1, ↓ACAA2, ↑ALDH1B1, ↓ACAT2
Validation of microarray data using real time quantitative PCR (qPCR)
The array and qPCR results for selected differentially expressed genes
Myosin, heavy chain 6, cardiac muscle, alpha
14 0 (↑)
Plasminogen activator, urokinase
St3 beta-galactoside alpha-2,3-sialyltransferase 6
Steroidogenic acute regulatory protein
Hydroxysteroid (17-beta) dehydrogenase 7
Oxidative stress induced growth inhibitor 1
Using a rat PCOS model, we have previously reported altered ovarian expression pattern of 83 miRNAs following DHT treatment . In this study, we investigated the gene expression profile of the same ovarian samples and identified 573 activated and 430 repressed gene transcripts in DHT-treated rats, suggesting the presence of transcriptome profile dysregulation due to hyperandrogenism. In addition, the cellular localization of the products of the activated or repressed gene showed 180 of the dysregulated ones were present in the nucleus while the majority were localized in the cytoplasm (Additional file 3: Figure S1), suggesting a possible dysregulation of genes function associated with specific ovarian subcellular localization.
The number of dysregulated ovarian genes in the DHT-treated rats appeared consistent with an earlier report by human ovarian cDNA microarray  in which the number of up-regulated genes (n = 88) was relatively higher than the down-regulated ones (n = 31) in ovaries of PCOS women compared to the non-PCOS subjects. However in another study where the GeneChips HG_U133A and HG_U133B arrays from Affymetrix used, majority of the dysregulated genes in PCOS were found to be down-regulated . Although the reasons for these apparent discrepancies are not clear, the possibility that this could be due to the differences in tissue sampling, the microarray platform used and the statistical analysis cannot be excluded. To evaluate whether altered ovarian genes in DHT-treated rat resemble those of PCOS women, we merged our data with the supplemental data previously reported  (http://press.endocrine.org/doi/suppl/10.1210/me.2004-0074/suppl_file/suppltable1part1me_04_0074.xls) and noted that 160 dysregulated genes were observed in ovaries of both DHT-rats and PCOS women (Additional file 4: Table S3).
In the current study, we have described the key biological processes, molecular functions and pathways affected by dysregulated genes in DHT-treated rats. While the text mining approach would shed light on the relevance of the dysregulated genes with respect to the PCOS phenotype, the present global approach involving pathway analysis and molecular functions provided a more comprehensive understanding of the potential genetic mechanism underling PCOS phenotypes. Our findings also demonstrated ovarian cell type-specific changes in expression of genes involved in granulosa cell proliferation and progesterone biosynthesis [AMH, RBP4 and cytochrome P450s (CYP51, CYP19A1, CYP11A1 and CYP17A1) and dysregulation of the genes associated with cholesterol biosynthesis and metabolism (acetyl-CoA acetyltransferases enzymes, ACTAs and HMGCR) in DHT-treated ovaries. These findings are consistent with the dependence of granulosa cell progesterone biosynthesis on the de novo cholesterol synthesis . More importantly, the action of HMGCR is believed to be the rate limiting step in cholesterol biosynthesis [33,34].
Women with PCOS may have higher abdominal body fat distribution, due to hyperandrogenism and insulin resistance . This phenomenon may be attributed by increased level of lactate, long-chain fatty acids and triglyceride [38-40]. We have previously demonstrated that the rats treated with DHT exhibited higher body weight compared to control . Although data regarding the total fat content of the ovary is lacking, examination into the gene set enrichment analysis revealed 42 genes associated with lipid metabolism and biosynthesis were repressed in DHT-treated rats. Among those, 26 genes including acyl-CoA synthetase and fatty acid synthase are known to be involved in dual roles of lipid synthesis and metabolism, while 16 other genes were related only to lipid metabolism. The dysregulation of genes involving de novo lipid synthesis and metabolism in the DHT group may result in the accumulation of lipid precursors or lack of essential fatty acids which are required for normal ovarian physiology.
One of the characteristics of PCOS is the presence of atretic follicles or premature growth arrest without atresia [41,42]. These phenotypic manifestations could be due to defects in steroid biosynthesis and energy metabolism. In line with this notion, excess androgen, luteinizing hormone and insulin are associated with the recruitment of several but small preovulatory follicles . Indeed, cell cycle progression and proliferation is thought to be controlled by several regulators . In the current study, a total of 45 genes that associated with cell proliferation and differentiation, including AMH and BMP2, were activated in ovaries of DHT-treated rats (Figure 6). AMH inhibits primordial follicle recruitment and decreases the sensitivity of growing follicles to FSH . AMH nulls and heterozygous mice exhibited early depletion of primordial follicles . Similarly, it is possible that BMP2 gene activated in DHT-treated ovaries could participate in the regulation of folliculogenesis and luteinization by modulating gonadotropin receptor expression . Activation of these genes may induce small follicle growth but dominant follicle growth arrest in DHT-treated rats, although this possibility needs further investigation.
In addition, genes involved in glycolysis/gluconeogenesis are also dysregulated by DHT treatment. It is known that hexokinases (HK1/2) convert glucose to glucose 6-phosphate  while pyruvate dehydrogenase complex component x (PDHX) catalyzes the conversion of pyruvate to acetyl coenzyme A [48,49]. In our study, ovarian HK1, HK2, PDHX and ACSS2 were repressed but FBP2 and ALDH1B1 were activated in DHT-treated rats (Table 3), further complicating the metabolic disorders in those groups. This finding is consistent with earlier report indicating the down-regulation of several genes regulating glucose synthesis and consumption in PCOS patients . In addition, down-regulation of the oxidative reductase gene (Figure 3E) and those of citrate acid cycle pathway (Table 3) adds further evidence for dysregulated energy metabolism in DHT-treated ovaries.
In conclusion, we have provided detailed evidence for transcriptome profile changes in a chronically androgenized PCOS rat model. Our data suggest biosynthesis and metabolism of cholesterol, sterols/steroids, lipids and oxidation/reduction are key molecular functions associated with repressed gene expression in DHT-treated rats. On the other hand, cell differentiation/proliferation, transcriptional regulation, neurogenesis, cell adhesion and blood vessel development were enriched by activated genes in this animal model. It is therefore conceivable that these molecular functional alterations could be a molecular clue for abnormal steroidogenesis, estrous cycle irregularity, abnormal folliculogenesis, anovulation, and disorders in carbohydrate regulation and lipid metabolism occurring in PCOS patients. This study contributes significantly to our understanding of the ovarian transcriptome profile and associated molecular functional alterations in DHT-treated rats, and provides the basis for future in-depth functional and mechanistic studies that to shed light on the pathophysiologic significance of the current findings in PCOS.
This work was supported by grants from the Canadian Institutes of Health Research (MOP-119381) and the World Class University (WCU) program through the Ministry of Education, Science and Technology and funded by the National Research Foundation of Korea (R31-10056) and research training awards [CIHR-REDIH Doctoral Scholarship and CIHR-QTNPR Doctoral Scholarship (QW)].
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