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Insulin signaling in bovine and canine corpus luteum under the influence of high and low 17²-estradiol concentrations

Grant number: 14/00739-9
Support type:Research Projects - Thematic Grants
Duration: December 01, 2014 - November 30, 2019
Field of knowledge:Agronomical Sciences - Veterinary Medicine - Animal Reproduction
Principal Investigator:Paula de Carvalho Papa Keohane
Grantee:Paula de Carvalho Papa Keohane
Home Institution: Faculdade de Medicina Veterinária e Zootecnia (FMVZ). Universidade de São Paulo (USP). São Paulo , SP, Brazil
Co-Principal Investigators:Maria Denise Lopes
Assoc. researchers:Ana Augusta Pagnano Derussi ; Bruno Moura Monteiro ; Ines Cristina Giometti Ceda ; Liza Margareth Medeiros de Carvalho Sousa ; Mario Binelli ; Mariusz Kowalewski ; Roberto Sartori Filho ; Ubiratan Fabres Machado
Associated scholarship(s):18/09579-5 - Insulin signaling in bovine and canine corpus luteum under the influence of high and low 17b-estradiol concentrations, BP.IC
16/22589-4 - Cortisol influence on lutein cells response to insulin stimulation in bitches, BP.MS
16/16648-8 - Cell death type evaluation of canine luteal cells in the cyclical and gestational diestrus, BP.IC
15/02551-0 - Morphological, molecular and functional aspects of the corpus luteum of cows treated with equine chorionic gonadotropin (ECG) before or after ovulation, BP.PD

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 (exp.) 1, 16 cows (Bos taurus) will be synchronized and treated with propileneglycol to increase plasma insulin from 72 h prior to LH administration to induce ovulation. In exp. 2, Bos taurus heifers will be treated with 400 IU eCG at the third day after ovulation. In exp. 1 cows will undergo follicular aspiration for granulosa cells collection and in a second round, follicular aspiration will not occur and D6, D10 and D14 CL will be biopsied. This luteal biopsy procedure will also be conducted in exp. 2. 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 exp. 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 exp. 4, 24 bitches will be neutered in the diestrous phase (days 10-60 after ovulation) and in exp. 5, 18 bitches will be neutered at early, mid and late pregnancy. Their CLs 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 (exp. 6) and canine (exp. 7) luteal cells under different concentrations of E2. The functional role of selected genes will be assessed 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 aims, besides unraveling the role of insulin in CL function, to revisit the mechanisms of CL regulation in these two species, since the RNA-seq experiments open new prospects for the global understanding of this phenomenon. (AU)