Unknown,Transcriptomics,Genomics,Proteomics

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The Analysis of Genes that Influence Sheep Follicular Development by Different Nutrition Levels during the Luteal Phase using Expression Profiling

ABSTRACT: Purpose: Nutrition is an important factor that regulates reproductive performance of sheep and affects follicle development. However, the correlation between nutrition and follicle development is poorly understood at molecular level. In order to study its possible molecular mechanisms, we performed expression profiling of granulosa cells isolated from sheep that were fed with different nutrition levels during luteal phase. Methods: To do this, ewes received a maintenance diet (M) and their estrus was synchronized by intravaginal progestogen sponges for 12 days. Ewes were randomly divided into the short-term dietary restricted group (R; 0.5ÃM) and nutrient-supplemented group (S; 1.5ÃM). RNA samples were extracted from granulosa cells. Transcriptome libraries from each group were constructed by Illumina Sequencing. Results: In 18,468 detected genes, 170 genes were significantly differentially expressed; of which, 140 genes were up-regulated and 30 genes were down-regulated in group S relative to group R. These genes could be candidates regulating follicular development in sheep. GO, KEGG and Clustering analyses were performed. Conclusions: These data in our study are abundant genomic recourse to expand our understanding of the molecular and cellular events underlying follicle development. Sixteen multiparous Hu sheep (3-4 years; 42.2 Â± 1.2 kg) were randomly assigned to two groups: the short-term nutrient-supplemented group (S; n=8), and the dietary restricted group (R; n=8).Expression profiling of granulosa cells were performed by Illumina sequencing platform (HiSeq 2000).

ORGANISM(S): Ovis aries

SUBMITTER: Feifei Luo

PROVIDER: E-GEOD-76232 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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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:The molecular mechanisms that regulate the pivotal transformation processes observed in the follicular wall following the pre-ovulatory LH-surge, are still not established, particularly for cells of the thecal layer. To elucidate thecal and granulosa cell type-specific biological functions and signaling pathways, large dominant bovine follicles were collected before and 21 hrs after an exogenous GnRH induced LH surge. Because LH receptor density varies within the granulosa cell populations, antral granulosa (aGC; those aspirated by follicular puncture) and membrane associated granulosa (mGC; those scraped from the follicular wall) were compared to thecal cell expression profiles determined by mRNA microarrays. Thecal cell gene expression was less affected in the peri-ovulatory follicle when compared to granulosa cells, as evidenced by only 2% versus 25% of the ~11,000 genes expressed changing in response to the LH surge, respectively. The majority of the 203 LH-regulated thecal genes were also LH regulated in granulosa cells, leaving a total of 58 genes as LH-regulated theca cell specific genes. Most of the 58 genes (i.e., 74%) thecal specific genes including several known thecal markers (CYP17A1, NR5A1) were downregulated, while most genes identified are new to theca. Many of the newly identified upregulated thecal genes (e.g., PTX3, RND3, PPP4R4) were also upregulated in granulosa. Minimal expression differences were observed between aGC and mGC, however, transcripts encoding extracellular proteins (NID2) and matrix modulators (ADAMTS1, SASH1) predominated these differences. We also identified large numbers of unknown LH-regulated granulosa cell genes and discuss their putative roles in ovarian function. The single dominant ovarian follicle was collected from each cow before the LH surge or 22 hours after GnRH (used to induce LH surge). RNA was extracted from three independent cells within each follicle and there were hybridized on Affymetrix microarrays.

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The Analysis of Genes that Influence Sheep Follicular Development by Different Nutrition Levels during the Luteal Phase using Expression Profiling

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