In vitro culture of bovine steroidogenic luteal cells and non-steroidogenic. effec...
| Grant number: | 13/07439-8 |
| Support type: | Regular Research Grants |
| Duration: | July 01, 2013 - December 31, 2015 |
| Field of knowledge: | Agronomical Sciences - Veterinary Medicine - Animal Reproduction |
| Principal Investigator: | João Carlos Pinheiro Ferreira |
| Grantee: | João Carlos Pinheiro Ferreira |
| Home Institution: | Faculdade de Medicina Veterinária e Zootecnia (FMVZ). Universidade Estadual Paulista (UNESP). Campus de Botucatu. Botucatu , SP, Brazil |
| Assoc. researchers: | Fernanda da Cruz Landim ; Ian Martin |
Abstract
This work aims to perform in vitro culture of bovine luteal cells as an experimental model for the study of physiological processes occurring in corpus luteum (CL), such as luteolysis. 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, PGFS, HPGD, IL-8 and EDN1. Progesterone (P4) will be dosed 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) to observe in vitro induction of luteolysis in these three cell types. 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 5 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. (AU)