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Cell death type evaluation of canine luteal cells in the cyclical and gestational diestrus

Grant number: 16/16648-8
Support type:Scholarships in Brazil - Scientific Initiation
Effective date (Start): December 01, 2016
Effective date (End): November 30, 2017
Field of knowledge:Agronomical Sciences - Veterinary Medicine - Animal Reproduction
Principal researcher:Maria Denise Lopes
Grantee:Ana Carolina Tancredo das Neves
Home Institution: Faculdade de Medicina Veterinária e Zootecnia (FMVZ). Universidade Estadual Paulista (UNESP). Campus de Botucatu. Botucatu , SP, Brazil
Associated research grant:14/00739-9 - Insulin signaling in bovine and canine corpus luteum under the influence of high and low 17²-estradiol concentrations, AP.TEM

Abstract

The goal of this project is to characterize the mechanism of action of insulin for glucose uptake and steroidogenesis according to the cyclical variations of 17²-estradiol (E2) in the cyclic and gestational corpus luteum (CL) in the canine species and cyclic CL in bovine, and in this species resulting or not of treatment with eCG. For this purpose, in the experiment (exp.) 1 will be used 16 cows (Bos taurus taurus), which will be synchronized and half of them treated with propylene glycol for increasing circulating insulin. In the exp. 2, Bos taurus heifers will be synchronized and treated with 400 IU of eCG, 3 days after ovulation. In exp. 1, after supplementation with propylene glycol on strategic days (before ovulation or during the luteal development), the corpora luta will be assessed by ultrasound and subsequently biopsied at D7 and D14 of the estrous cycle, respectively. The luteal biopsy procedure will also be conducted in exp. 2. The global gene expression will be determined by RNA sequencing (RNA-seq), from which will be selected genes differentially expressed related to the insulin signaling pathways, in order to be validated by qPCR, western blotting and immunohistochemistry. In exp. 3, CL samples from cyclic cows, treated or not with eCG and previously analyzed by microarray, will be validated for genes related to insulin signaling in the manner already described above. In exp. 4, 24 bitches will be castrated in the diestrus phase (days 10 to 60 after the ovulation) and in exp. 5, 18 pregnant bitches will be castrated on the stages: initial, intermediate and late pregnancy. The CLs will be collected and also submited to RNA-seq and validations. After obtaining the results from the global gene analysis, we will assess in vitro the effects of insulin in bovine luteal cells (exp. 6) and canine (exp.7) cultivated and subjected to different concentrations of E2. The verification of the functional role of selected genes will be performed by RNAi technique and the cultivation samples will also be submitted to RNA-seq and subsequent validations. The RNA-seq analysis will be performed by aligning the generated sequences (reads) against bovine and canine genomes, respectively, which will be converted into transcripts, whose expression level will be estimated through the RPKM index (reads/Kb/Million). The sequencing data will be published on the Library of NCBI (Sequence Read Arquives - SRA). After validations, the obtained results will be tested using GraphPad Prism 5.0 software (GraphPad Software, USA), according to their normality and homogeneity, and presented as mean ± SEM. Differences of p d 0.05 will be considered significant. Thus, the present study aims, in addition to unveil the role of insulin in the CL function, develop a review of the CL regulatory mechanisms in these two addressed species, due to the fact that the RNA-seq experiments will open new prospects for global understanding of this phenomenon. (AU)

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