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Microarray analysis of NL and PCOS oocytes

ABSTRACT: Polycystic ovary syndrome (PCOS), the most common cause of anovulatory infertility, is characterized by increased ovarian androgen production, 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. To examine whether the perturbations in follicle growth and the intrafollicular environment affects development of the mature PCOS oocyte, genes that are differentially expressed in PCOS compared to normal oocytes were defined using microarray analysis. This analysis detected approximately 8000 transcripts. Hierarchical clustering and principal component analysis revealed differences in global gene expression profiles between normal and PCOS oocytes. 374 genes had a statistically-significant increase or decrease in mRNA abundance in PCOS oocytes. A subset of these genes was associated with chromosome alignment and segregation during mitosis and/or meiosis, suggesting that increased mRNAs for these proteins may negatively affect oocyte maturation and/or early embryonic development. Of the 374 differentially expressed genes, 68 contained putative androgen receptor, retinoic acid receptor, and/or peroxisome proliferating receptor gamma binding sites, including 9 of the genes involved in chromosome alignment and segregation. These analyses demonstrated that normal and PCOS oocytes that are morphologically indistinguishable and of high quality exhibit different gene expression profiles. Furthermore, altered mRNA levels in the PCOS oocyte may contribute to defects in meiosis and/or mitosis which might impair oocyte competence for early development and therefore contribute to poor pregnancy outcome in PCOS. Keywords: disease state analysis

ORGANISM(S): Homo sapiens

PROVIDER: GSE5850 | GEO | 2007/06/05

SECONDARY ACCESSION(S): PRJNA97255

REPOSITORIES: GEO

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Project description:Polycystic ovary syndrome (PCOS), the most common cause of anovulatory infertility, is characterized by increased ovarian androgen production, 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. To examine whether the perturbations in follicle growth and the intrafollicular environment affects development of the mature PCOS oocyte, genes that are differentially expressed in PCOS compared to normal oocytes were defined using microarray analysis. This analysis detected approximately 8000 transcripts. Hierarchical clustering and principal component analysis revealed differences in global gene expression profiles between normal and PCOS oocytes. 374 genes had a statistically-significant increase or decrease in mRNA abundance in PCOS oocytes. A subset of these genes was associated with chromosome alignment and segregation during mitosis and/or meiosis, suggesting that increased mRNAs for these proteins may negatively affect oocyte maturation and/or early embryonic development. Of the 374 differentially expressed genes, 68 contained putative androgen receptor, retinoic acid receptor, and/or peroxisome proliferating receptor gamma binding sites, including 9 of the genes involved in chromosome alignment and segregation. These analyses demonstrated that normal and PCOS oocytes that are morphologically indistinguishable and of high quality exhibit different gene expression profiles. Furthermore, altered mRNA levels in the PCOS oocyte may contribute to defects in meiosis and/or mitosis which might impair oocyte competence for early development and therefore contribute to poor pregnancy outcome in PCOS. Experiment Overall Design: A single MII oocyte, defined by one polar body in the perivitelline space and no visible nuclear structure in the cytoplasm, was collected from 6 individual NL and 6 individual PCOS ovaries, placed immediately in TRIzol (Sigma, St. Louis MO), and stored at -80 C until further study. 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) per the manufacturerâs instructions. RNA from the GeneChip Eukaryotic Poly-A RNA Control Kit (Affymetrix, Santa Clara CA) was amplified and labeled under the same conditions for a positive control. Affymetrix GeneChip Human Genome U133 Plus 2.0 microarray chips (Affymetrix, Santa Clara, CA) were hybridized at the University of Pennsylvania Microarray Core Facility. Briefly, the linear-amplified, biotin-labled cDNA from 6 NL (N1-N6) and 6 PCOS (P1-P6) oocytes 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.

Project description:Mammalian follicle development is characterised by extensive changes in morphology, endocrine responsiveness, and function, providing the optimum environment for oocyte growth, development, and resumption of meiosis. In cattle, the first signs of transcription activation in the oocyte are observed in the secondary follicle, later than during mouse and human oogenesis. While many studies have generated extensive datasets characterising gene expression in bovine oocytes, they are mostly limited to the analysis of fully grown and matured oocytes. The aim of the present study was to apply single-cell RNA sequencing to interrogate the transcriptome of the growing bovine oocyte from the secondary follicle stage through to the mid-antral follicle stage. Single-cell RNA-seq libraries were generated from oocytes of known diameters (<60 to >120 um), and datasets were binned into non-overlapping size groups for downstream analysis. Combining the results of weighted gene co-expression network and Trendy analyses, and differently expressed genes (DEGs) between size groups, we identified a decrease in oxidative phosphorylation and an increase in maternal -genes and transcription regulators across the bovine oocyte growth phase. In addition, around 5,000 genes did not change in expression, revealing a cohort of stable genes. An interesting switch in gene expression profile was noted in oocytes greater than 100 um in diameter, when the expression of genes related to cytoplasmic activities was replaced by genes related to nuclear activities (e.g., chromosome segregation). The highest number of DEGs were detected in the comparison of oocytes 100-109 versus 110-119 um in diameter, revealing a profound change in the molecular profile of oocytes at the end of their growth phase. The current study provides a unique dataset of the key genes and pathways characteristic of each stage of oocyte development, contributing an important resource for a greater understanding of bovine oogenesis.

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Microarray analysis of NL and PCOS oocytes

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