Abstract
The aim of this project is to identify components of the insulin signaling pathway involved in glucose uptake and steroidogenesis according to 17²-estradiol ( E2 ) cyclical variations in the canine cyclic and gestational corpus luteum (CL) and in the bovine cyclic CL, treated or not with eCG. For that, in Experiment 1, 24 heifers (Bos taurus) will be synchronized and treated with propileneglycol 48 hours prior to LH administration to increase plasma insulin. In experiment 2, the same synchronized heifers will be treated with 400IU eCG at the third day after ovulation. In exp. 1 heifers will undergo follicular aspiration for granulosa cells collection and in both experiments, CL biopsy at days 7 and 12 after the beginning of the cycle. Global gene expression will be determined by RNA sequencing (RNA-seq), from which differentially expressed genes related to the insulin signaling will be selected. Then, the expression of these genes will be validated by qPCR, western blotting and immunohistochemistry. In experiment 3, samples of CL from cyclic cows, treated or not with eCG and previously analyzed by microarray, will be validated for insulin signaling pathway genes as previously described. In experiment 4, 24 bitches will be neutered in the diestrus phase (days 10-60 after ovulation) and in experiment 5, 18 bitches will be neutered at early, mid and late pregnancy. Their CL will be collected and submitted to RNA-seq and subsequent validations. After obtaining the results from global gene analysis, we will evaluate the effects of insulin in cultured bovine (experiment 6) and canine (experiment 7) luteal cells under different concentrations of E2. The functional role of selected genes will be performed by RNAi technique and culture samples will be submitted to RNA-seq and subsequent validations. RNA-seq analyses will be performed by aligning the generated sequences (reads) against bovine and canine genome, respectively, which are converted into transcripts, whose expression level is estimated using the index RPKM (reads/Kb/Million). The sequencing data will be published in the NCBI library (Sequence Read Arquives - SRA). After validations, the obtained results will be tested by GraphPad Prism 5.0 program (GraphPad Software, USA), according to their homogeneity and normality and represented as mean ± SEM. Differences of p d 0.05 will be considered. Thus, this project wants to revisit the concepts of luteal function regulation in these two species, since the RNA-seq experiments open new prospects for the global understanding of this phenomenon.
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