In vitro culture of bovine steroidogenic luteal cells and non-steroidogenic. Effect of PGF2a on the expression of genes and proteins associated with steroidogenesis and apoptosis

• Grant number: 13/00992-3
• Support Opportunities: Scholarships in Brazil - Doctorate
• Start date: September 01, 2013
• End date: January 31, 2016
• Field of knowledge: Agronomical Sciences - Veterinary Medicine - Animal Reproduction
• Principal Investigator: João Carlos Pinheiro Ferreira
• Grantee: Flavia Caroline Destro
• Host Institution: Faculdade de Medicina Veterinária e Zootecnia (FMVZ). Universidade Estadual Paulista (UNESP). Campus de Botucatu. Botucatu , SP, Brazil
• Associated scholarship(s): 15/01940-2 - In vitro culture of bovine steroidogenic and non-steroidogenic luteal cells: effect of PGF2a on the expression of genes and proteins associated with steroidogenesis and apoptosis, BE.EP.DR

Abstract

This work aims to perform in vitro culture of bovine luteal cells evaluate the effect of PGF2± on gene expression and protein in substances involved in steroidogenesis (StAR), the production of PGF2± intraluteal (PGTS, PTGFS, HPGD) in the induction of apoptosis (CASP-3, Bax, Bcl2) and genes of immune system that have an increased expression during the process of luteolysis and functional intraluteal that stimulate the synthesis of prostaglandin (IL-8 and FAS) in culture of cells in the corpus luteum. To do so will be conducted three experiments. To achieve this, three experiments will be performed. In Experiment 1 the CLs will be obtained from local slaughterhouses, processed and steroidogenic luteal cells will be cultured for 48 hours to induce in vitro luteolysis of this cell type. This cells will then receive the following treatments: Control Group: 1,0mL of basic culture medium, which will be partially substituted after 48 hours of culture for 1,0mL basic culture medium and then consecutively from 6 to 6 hours; PGF2± Group: 10-6M PGF2± (Sigma® P 0424; KORZEKWA et al., 2008) diluted in 1,0mL basic culture medium. After 48 hours, this medium will be partially substituted by 10-6M PGF2± diluted in 1,0mL basic culture medium from 6 to 6 hours until the completion of 24 hours. Cultured cells will be morphologically evaluated by using Oil Red and Hematoxylin stain, gene (qRT-PCR) and protein expression (Immunofluorescence) will be evaluated for STAR, , PTGS2, PTGFS, HPGD, IL-8, FAS, CASP-3, Bax, Bcl-2 and finally will be dosed the progesterone (P4) of the medium culture by Radioimmunoassay (RIA). In Experiment 2 will be realized co-culture of steroidogenic luteal cells (CLEs), endothelial luteal cells (CLEn) and immune system cells (CSI) in order to observe the action of PGF2± in these cell types in vitro. The CLEs will be cultured again for 48 hours and then CLEn and CSI will be added and pre-incubated for 2 hours (co-culture) and the same treatments and evaluations made in Experiment 1 will be realized. Experiment 3 aims to induce in vitro luteolysis by PGF2±± treatment during different moments. To achieve this, the wells of the dish containing the three cell types (CLEs, CLEn, CSI) will receive 5 doses of PGF2±± or 5 doses of basic culture medium or 3 doses of PGF2±± in different moments (1º, 3º and 5º moment) and the remaining moments (2º and 4º moment) will receive basic culture medium. Morphology evaluation and gene and protein expression will be performed as described in Experiment 1. Data will be tabulated and adequate statistical tests will be performed.

Scientific publications

(References retrieved automatically from Web of Science and SciELO through information on FAPESP grants and their corresponding numbers as mentioned in the publications by the authors)

DESTRO, F. C.; MARTIN, I.; LANDIM-ALVARENGA, F. D. C.; FERREIRA, J. C. P.; PATE, J. L.. Effects of concanavalin A on the progesterone production by bovine steroidogenic luteal cells in vitro. REPRODUCTION IN DOMESTIC ANIMALS, v. 51, n. 5, p. 848-852, OCT 2016. (13/07439-8, 15/01940-2, 13/00992-3)

Academic Publications

(References retrieved automatically from State of São Paulo Research Institutions)

DESTRO, Flavia Caroline. Progesterone production by the steroidogenic luteal cells cultured and co-cultured in vitro in cattle. 2016. Doctoral Thesis - Universidade Estadual Paulista (Unesp). Faculdade de Medicina Veterinária e Zootecnia. Botucatu Botucatu.