Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

Granulosa cell differentiation in vivo and developmental competence of oocytes in the bovine model

ABSTRACT: Transcriptional profiling of in vivo granulosa cells collected at 20h, 44h, 68h, and 92h of coasting in lactating cow (breed Holstein) 4 conditions experiment, 3 biological replicates, one repliacte per array, dye swap experiment, two colors, 20h vs 44h, 44h vs 68h, 68h vs 92h, 20h vs 68h, 20h vs 92h, 44h vs 92h

ORGANISM(S): Bos taurus

SUBMITTER: Anne-Laure Nivet

PROVIDER: E-GEOD-40916 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

Changes in granulosa cells' gene expression associated with increased oocyte competence in bovine.

Nivet Anne-Laure AL Vigneault Christian C Blondin Patrick P Sirard Marc-André MA

Reproduction (Cambridge, England) 20130521 6

One of the challenges in mammalian reproduction is to understand the basic physiology of oocyte quality. It is believed that the follicle status is linked to developmental competence of the enclosed oocyte. To explore the link between follicles and competence in cows, previous research at our laboratory has developed an ovarian stimulation protocol that increases and then decreases oocyte quality according to the timing of oocyte recovery post-FSH withdrawal (coasting). Using this protocol, we h ...[more]

PMID: 23564726

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Project description:The main objective of superovulatory treatment is to achieve multiple ovulations and produce multiple embryos. The technique is intended to rescue a cohort of follicles within a follicular wave that would otherwise regress. Superstimulory treatments change the intra-follicular and systemic hormonal milieu, but it is not clear how and to what extent treatment alters gene expression of follicular cells. In this study, a genome-wide bovine oligo-microarray was used to compare the gene expression of granulosa cells from post-LH preovulatory dominant follicle with those from follicles of the same status after superstimulatory treatment. Cows were allocated randomly to two groups (superstimulation group and control group, n=6 cows per group). A new follicular wave was induced by transvaginal ultrasound-guided ablation of follicles ?5 mm in diameter, and a progesterone-releasing device (CIDR) was placed in vagina. The superstimulation group was given eight doses of 25 mg FSH im at 12-h intervals starting from the day of wave emergence (Day 0), whereas the control group was not given any FSH treatment. Both groups were given prostaglandin F2? im twice, 12 h apart, on Day 3 and the CIDR was removed at the second injection; 25 mg pLH was given im 24 h after CIDR removal, and cows were ovariectomized 24 h later. Granulosa cells were collected for RNA extraction, amplification and microarray hybridization. To translate microarray results to a physiological context, a list of differentially expressed transcripts were biologically annotated. A total of 190 genes were down-regulated and 280 genes were up-regulated in superstimulated group when compared with the reference (non-superstimulated control) group. straight comparison of superstimulation group (the treatment; group 1) versus non-superstimulated ( the reference; group 4) using 3 different aninals (biological replicates) in each group and performed dye swap. For example on array 1: group1 cow 1 versus group 4 cow 1 (cy3 vs cy5) and on array 4 is the dey swap group 4 cow 1 versus group 1 cow 1 (cy3 vs cy5).

Project description:Background The domestic pig is an important livestock species for meat production worldwide and is becoming an established biomedical research model. As a result, there is a strong interest in the factors that affect the efficient production of viable embryos and offspring in this species using either in vivo or in vitro production methods. A limited understanding of the molecular mechanisms involved in this critical physiological process has inhibited our ability to fully elucidate these factors. The use of next generation deep sequencing and microarray technology are powerful tools for delineation of molecular pathways during early embryonic development of mammals. Here, we report on the assessment of a porcine-embryo-specific microarray platform created from a large expressed sequence tag (EST) analysis generated by Roche/454 next-generation sequencing of cDNAs constructed from critical stages of in vivo or in vitro porcine preimplantation embryos. Results Two cDNA libraries constructed from in vitro and in vivo produced preimplantation porcine embryos were normalized and sequenced using the 454 Titanium pyrosequencing technology. Treatment of cDNA libraries with BAL 31 nuclease digestion resulted in a 2 fold improvement of sequencing quality compared with untreated libraries. Over one million high quality EST sequences were obtained from this process and used to create an augmented porcine genome catalogue. Using the resulting dataset the EMbryogene Porcine Version 1 (EMPV1) microarray was developed and is composed of 43,795 probes printed onto a 4 × 44 K Agilent array. Based on the initial probe sequences annotation, the EMPV1 featured 17,409 protein-coding, 473 pseudogenes, 46 retrotransposed, 2,359 non-coding RNA (snRNA, snoRNA, etc.), 4,121 splice variants in 2,862 genes and a total of 12,324 Novel Transcript Regions (NTR). After re-annotation, the total unique genes increased from 11,961 to 16,281 and 1.9% of them belonged to a large olfactory receptor (OR) gene family. Quality control of EMPV1 was performed using porcine cumulus–oocyte complexes (COC) as well as early developmental stages of embryos. This revealed an even distribution of ten clusters of spike-in control spots and array to array (dye-swap) correction was 0.97. Further bioinformatics analysis revealed that our microarray probes hybridized with more developmental related transcripts from embryonic labelled targets when compared to COC. Conclusions Using next-generation deep sequencing we have produced a large EST dataset to provide the selection of probe sequences for the development of the EMPV1 microarray platform. The quality of this embryo- specific array was confirmed with the high level of reproducibility using current Agilent microarray technology. Despite the current limitations for full NTR annotation, due to the incomplete porcine genome sequencing project, a significant number of NTR were annotated using Version 10 of porcine genome and human RefSeq RNA database to enrich the orthologous genes with unique gene symbol (GS) for Gene Ontology (GO) search. GO terms confirmed that many are related relevant developmental processes. With more than an estimated 20 thousands unique genes represented on the EMPV1, this platform will provide the foundation for future research into the in vivo and in vitro factors that affect the viability of the porcine embryos, as well as the effects of these factors on the live offspring that result from these embryos. Two biological samples.

Dataset Information

Granulosa cell differentiation in vivo and developmental competence of oocytes in the bovine model

Publications

Changes in granulosa cells' gene expression associated with increased oocyte competence in bovine.

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