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High-throughput analysis of ovarian granulosa cell transcriptome.

ABSTRACT: The quality of follicular oocytes depends on interactions with surrounding granulosa cells. Development of molecular techniques and methods enables better understanding of processes underlying mammalian reproduction on cellular level. The success in reproductive biology and medicine in different species depends on reliable assessment of oocyte and embryo viability which presently mainly bases on embryo morphology. Although successful pregnancies have been achieved using this approach, its precision still should be improved and completed with other, more objective, and accurate assessment strategies. Global profiling of gene expression in follicular cumulus cells using microarrays is continuously leading to the establishment of new biomarkers which can be used to select oocytes with highest developmental potential. Even more potential applications and greater precision could be achieved using next generation sequencing (NGS) of granulosa and cumulus cell RNA (RNA-seq). However, due to the high cost, this method is not used as frequently as microarrays at the moment. In any case, high-throughput technologies offer the possibilities and advantages in ovarian somatic cell analysis on scale that has not been noted so far. The aim of this work is to present current directions and examples of global molecular profiling of granulosa cells and underline its impact on reproductive biology and medicine.

SUBMITTER: Chronowska E

PROVIDER: S-EPMC3966335 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

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High-throughput analysis of ovarian granulosa cell transcriptome.

Chronowska Ewa E

BioMed research international 20140310

The quality of follicular oocytes depends on interactions with surrounding granulosa cells. Development of molecular techniques and methods enables better understanding of processes underlying mammalian reproduction on cellular level. The success in reproductive biology and medicine in different species depends on reliable assessment of oocyte and embryo viability which presently mainly bases on embryo morphology. Although successful pregnancies have been achieved using this approach, its precis ...[more]

PMID: 24711992

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Project description:Polycystic ovary syndrome (PCOS) is one of the most common endocrine disorders in women of reproductive age, which is characterized by ovulatory dysfunction, clinical and/or biochemical androgen excess, polycystic ovaries on ultrasound and genetic heterogeneity. It was well-accepted that many lncRNAs and mRNAs were associated with PCOS, however, remain unclear. Therefore, the purpose of our study was to examine different expression profiles of lncRNAs and mRNAs in ovarian granulosa cells (GCs) in PCOS and Controls, and identify the correlation between lncRNAs, mRNAs and clinical parameters. Sixty five PCOS patients and 65 Controls were enrolled in this study and adopted standard long agonist protocols or GnRH antagonist protocols. Then 6 GCs samples in each group were subjected to high-thoughput sequencing and the remaining samples were used for the further verification by quantitative real-time PCR (qRT-PCR). Gene Oncology (GO), Kyoto Encyclopedia Genes and Genomes (KEGG) enrichment analysis were performed. We predicted the relationship between lncRNAs and mRNAs by Cytoscape software. According to the expression level of lncRNAs, mRNAs and the clinical parameters, we also explored their relationship and evaluate their predictive values for embryos quality and PCOS. We identified 1,049 differential expressed lncRNAs and 3,246 mRNAs (fold-change ≥2, p-value < 0.05). Seven lncRNAs (NONHSAT101926.2, NONHSAT136825.2, NONHSAT227177.1, NONHSAT010538.2, NONHSAT191377.1, NONHSAT230904.1, ENST00000607307) and 3 mRNAs (EREG, ENTPD6, YAP1) were validated consistent with sequence profile. Seven lncRNAs were related to hormone level and follicle counts, 3 mRNAs had connections with lipid metabolism. The area under curve (AUC) of 7 lncRNAs were valuable in distinguishing patients with PCOS from Controls. The AUC of NONHSAT230904.1 and NONHSAT227177.1 were 0.6807 and 0.6410, respectively, for distinguishing whether the rate of high-quality embryos exceeds 50%. Our study showed that the GCs lncRNAs and mRNAs were involved in the occurrence and development of PCOS, which contribute to clarify the pathogenesis mechanism of PCOS.

Project description:BackgroundInfertility is a global reproductive-health problem, and diminished ovarian reserve (DOR) is one of the common causes of female infertility. Long noncoding RNAs (lncRNAs) are crucial regulators of numerous physiological and pathological processes in humans. However, whether lncRNAs are involved in the development of DOR remains to be elucidated.MethodsOvarian granulosa cells (OGCs) extracted from infertile women with DOR and from women with normal ovarian reserve (NOR) were subjected to high-throughput sequencing. Comprehensive bioinformatics analysis was conducted to identify the differential expression of messenger RNAs (mRNAs) and lncRNAs. Sequencing results were validated by the selection of lncRNAs and mRNAs using real-time reverse transcription-quantitative polymerase chain reaction (RT-qPCR).ResultsCompared with the NOR group, a total of 244 lncRNAs were upregulated (53 known and 191 novel), and 222 lncRNAs were downregulated (36 known and 186 novel) in the DOR group. Similarly, 457 mRNAs had differential expression between the two groups. Of these, 169 were upregulated and 288 were downregulated. Bioinformatics analysis revealed that the differentially expressed genes of mRNA and lncRNAs were considerably enriched in "cell adhesion and apoptosis", "steroid biosynthesis", and "immune system". A co-expression network comprising lncRNAs and their predicted target genes revealed the possible involvement of the "thyroid hormone signaling pathway" and "protein binding, digestion and absorption" in DOR pathogenesis. The expression of SLC16A10 was positively regulated by multiple lncRNAs. After RT-qPCR validation of seven differentially expressed lncRNAs and mRNAs, respectively, the expression of lncRNA NEAT1, GNG12, ZEB2-AS1, and mRNA FN1, HAS3, RGS4, SUOX were in accordance with RNA-sequencing.ConclusionsWe presented the first data showing that the expression profiles of lncRNA and mRNA in OGCs between NOR and DOR patients using RNA sequencing. The lncRNAs and mRNAs that we identified may serve as novel diagnostic biomarkers for patients with DOR.

Project description:BackgroundThe major function of the ovary is to produce oocytes for fertilisation. Oocytes mature in follicles surrounded by nurturing granulosa cells and all are enclosed by a basal lamina. During growth, granulosa cells replicate and a large fluid-filled cavity (the antrum) develops in the centre. Only follicles that have enlarged to over 10 mm can ovulate in cows. In mammals, the number of primordial follicles far exceeds the numbers that ever ovulate and atresia or regression of follicles is a mechanism to regulate the number of oocytes ovulated and to contribute to the timing of ovulation. To better understand the molecular basis of follicular atresia, we undertook transcriptome profiling of granulosa cells from healthy (n = 10) and atretic (n = 5) bovine follicles at early antral stages (< 5 mm).ResultsPrincipal Component Analysis (PCA) and hierarchical classification of the signal intensity plots for the arrays showed primary clustering into two groups, healthy and atretic. These analyses and size-frequency plots of coefficients of variation of signal intensities revealed that the healthy follicles were more heterogeneous. Examining the differentially-expressed genes the most significantly affected functions in atretic follicles were cell death, organ development, tissue development and embryonic development. The overall processes influenced by transcription factor gene TP53 were predicted to be activated, whereas those of MYC were inhibited on the basis of known interactions with the genes in our dataset. The top ranked canonical pathway contained signalling molecules common to various inflammatory/fibrotic pathways such as the transforming growth factor-β and tumour necrosis factor-α pathways. The two most significant networks also reflect this pattern of tissue remodelling/fibrosis gene expression. These networks also contain molecules which are present in the canonical pathways of hepatic fibrosis/hepatic stellate cell activation and transforming growth factor-β signalling and were up regulated.ConclusionsSmall healthy antral follicles, which have a number of growth outcomes, exhibit greater variability in gene expression, particularly in genes associated with cell division and other growth-related functions. Atresia, on the other hand, not only involves cell death but clearly is an active process similar to wound healing.

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High-throughput analysis of ovarian granulosa cell transcriptome.

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High-throughput analysis of ovarian granulosa cell transcriptome.

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