Efeito da adição de lectinas recombinantes de Bauhinia forficata sobre o estresse oxidativo na maturação in vitro de oócitos bovinos

Abstract

The similarity in several aspects in folliculogenesis with monovulatory females makes in vitro maturation (IVM) of bovine oocytes important tool for study to female reproductive. This reproductive biotechnology is widely used for screening of molecules and serves as a model for toxicological investigation in process reproductive for humans. The lectin of Bauhinia forficata (nBfL) is a protein able to bind reversibly to N-D-acetylgalactosamine, performing several functions and one of them is the antiproliferative activity in tumor cells, but its effects have not yet been evaluated in female gametes. The objective of the present study was to determine addition effect of B. forficata recombinants lectins in the medium of maturation in vitro of bovine oocytes in expression of genes related to oxidative stress pathways. To get the proteins, the gene for this recombinant lectin (rBfL) and its truncated isoform (rtBfL) were cloned and expressed in Escherichia coli (E.Coli). The oocytes obtained through follicular puncture were incubated in IVM medium for 24 hours containing concentrations of 10 µg/mL, 50 µg/mL and 100 µg/mL of nBfL, rBfL and rtBfL, and a no treated group as a control. The expansion of the cumulus oophorus cells, the meiotic maturation stage, the nitric oxide (NO) levels in the maturation medium were evaluated. In the groups treated with the concentration of 100 µg / mL, the gene expression of genes involved in oxidative stress SOD2, CAT, GPX-1, GSR, NOS2 and apoptosis BAX, CASP3 were evaluated. The expansion of the cumulus oophorus cells of trated oocytes, as well as the nuclear maturation rate, was not statistically different from the control group, though rBfL increased NO levels in all treatments in relation to the control group. The rtBfL increased the expression of the SOD2, GSR and NOS2 genes and all the tested lectins increased the expression of the CASP3 gene compared to the control group. These findings indicate that the tested concentrations of the B. forficata recombinants lectins probably influence on expression of oxidative stress genes and increase the expression of the apoptotic gene CASP3 during in vitro maturation of bovine oocytes.