Influência da interleucina 1 beta (IL-1 β) e do fator de necrose tumoral alfa (TNF-α) sobre o crescimento e maturação in vitro de oócitos bovinos provenientes de folículos antrais pequenos

Abstract

The aim of this study was to verify the effect of Interleukin 1 beta (IL-1β) and tumor necrosis factor alpha (TNF-α) during the in vitro growth of bovine oocytes from small antral follicles (1-3 mm). Therefore, the recovered COCs were cultured in TCM-199 alone or supplemented with IL-1β (10 ng/ml), TNF-α (10 ng/ml) or both IL-1β and TNF-α. After 48 hours of culture, oocyte diameter was measured and the COCs were then pre-matured (pre-MIV) for 20 hours in TCM-199 medium supplemented with 10 μM cilostamide. After pre-maturation period, half of the COCs were submetted to matureted in vitro (IVM) for 22 hours, while the other half was used to verify the meiotic progression by staining of Hoechst 33342. In addition, the mitochondrial distribution of oocytes was e valuated after culture in the different treatments, as well as oocytes from follicles grown in vivo (diameters ≤ 3 mm or > 5 mm). The expression of mRNA for GDF-9, c-Mos, Cyclin-1β and H1foo was also analyzed after in vitro pre-maturation. For statistical analysis, paired t-test was used to compare oocyte diameters before and after culture. ANOVA one way analyzed oocyte growth, while the Kruskal-Wallis test compared levels of mesenteric RNAs to gene expression. The Chi-square test was used to evaluate the meiotic progression of oocytes. The results demonstrated that after 48 hours there was a significant increase in oocyte diameter in all treatments. However, oocytes cultured in the presence of TNF-α or IL-1β and TNF-α showed larger diameter when compared to those cultured in the control medium. Regarding the mitochondrial distribution, a heterogeneous (pericortical) distribution was observed in oocytes from follicles grown in vivo (> 5 mm), as well as after pre-IVM in TCM-199 alone or supplemented with TNF-α. However, after IVM, mitochondria had a heterogeneous distribution only in TCM-199. Regarding gene expression, TNF-α, IL-1β or both did not influence the expression of GDF-9, c-Mos, Cyclin-1β and H1foo. In conclusion, TNF-α or both TNF-α and IL-1β favored oocyte growth during the pre-maturation period. However, these substances did not induce effects during maturation of oocytes from small antral follicles