Unknown,Transcriptomics,Genomics,Proteomics

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Transcriptome analysis of Bovine granulosa cells of preovulatory follicles at 30, 60, 90 and 120 days postpartum

ABSTRACT: Cows in Negative Energy Balance (NEB) may preferentially divert nutrients away from reproduction, thereby experiencing a period of anovulatory anestrus, delayed ovulation of large follicles and a condition of impaired fertility. To better understand the changes occurring in these large follicles as a function of time post-partum granulosa cells of preovulatory follicles have been collected at different times: 30, 60, 90 and 120 days after calving . An analysis of the transcriptome was performed using a global bovine oligo-array microarray to map the differences in genes expression and cellular functions that occur in the follicular microenvironment during the progressive recovery from NEB condition in dairy cow Four time points experiment: 30, 60, 90 and 120 days. Granulosa cells from the 30, 60 and 90 days compare to the 120 days (reference). Biological replicates: 3 from each time point. One replicate per array.

ORGANISM(S): Bos taurus

SUBMITTER: Isabelle Dufort

PROVIDER: E-GEOD-50854 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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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:The fertility of dairy cows is challenged during early lactation and better nutritional strategies need to be developed to address this issue. Combined supplementation of folic acid and vitamin B12 improves energy metabolism in the dairy cow during early lactation. Therefore, the present study was undertaken to explore the effects of this supplement on gene expression in granulosa cells from the dominant follicle during the postpartum period. Multiparous Holstein cows received weekly intramuscular injection of 320 mg folic acid and 10 mg vitamin B12 (treated group) beginning 24 (SD 4) d before calving until 56 d after calving, whereas the control group received saline. The urea plasma concentration was significantly decreased during the pre-calving period, and the concentration of both folate and vitamin B12 were increased in treated animals. Milk production and dry matter intake were not significantly different between the two groups. Plasma concentrations of folates and vitamin B12 were increased in vitamin-treated animals. Daily dry matter intake was not significantly different between the 2 groups before (13.5 kg SE 0.5) and after (23.6 kg SE 0.9) calving. Average energy-corrected milk tended to be greater in vitamin-treated cows, 39.7 (SE 1.4) and 38.1 (SE 1.3) kg/d for treated and control cows, respectively. After calving, average plasma concentration of BHBA tended to be lower in cows injected with the vitamin supplement, 0.47 (SE 0.04) vs. 0.55 (SE 0.03) for treated and control cows, respectively. The ovarian follicle ? 12 mm in diameter was collected by ovarian pick-up after estrus synchronization. Recovered follicular fluid volumes were greater in the vitamin-treated group. A microarray platform was used to investigate the impact of treatment on gene expression of granulosa cells. Lower expression of genes involved in the cell cycle and higher expression of genes associated with granulosa cell differentiation prior to ovulation were observed. Selected candidate genes were analyzed by reverse transcription quantitative polymerase chain reaction. Although the effects of intramuscular injections of folic acid and vitamin B12 on lactational performance and metabolic status of animals were limited, Ingenuity Pathway Analysis of gene expression in granulosa cells suggests a stimulation of cell differentiation in vitamin-treated cows, which may be the result of an increase in LH secretion. Two conditions experiment (Control and Treated). Granulosa cells from the 66h post second PGF2alpha injection. Biological replicates: 3 from each time point. Two technical replicates for each comparison (dye-swap).

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.

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Transcriptome analysis of Bovine granulosa cells of preovulatory follicles at 30, 60, 90 and 120 days postpartum

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