183 GENE EXPRESSION OF IN VITRO-MATURATED OOCYTES CAN BE MODULATED BY FOLLICLE EXOSOMES FROM COWS KEPT UNDER THERMONEUTRAL OR HEAT STRESS CONDITIONS
Authors: Dalanezi F. M. , Destro F. C. , Ferrazza R. A. , Mogollon García H. D. , Franchi F. F. , Fontes P. K. , Castilho A. C. S. , Sartori R. , Ferreira J. C. P. 
Published: December 2016

There are several intrafollicular agents that have the ability to interfere with the metabolism and development of the oocyte, among these we highlight the exosomes (EXO). Thus, the aim of this study was to evaluate the capacity of EXO extracted from the follicular fluid of cows kept under thermoneutral or heat stress conditions to modulate oocyte maturation in vitro. Twenty-four Holstein cows were subjected to the following treatments for 14 days: heat stress (HS; n = 12), 38°C, 60% RH, temperature-humidity index = 88; and thermo-neutral (TN; n = 12), 24°C, 60% RH, temperature-humidity index = 71. Cows had their follicles aspirated when their diameter reached 9 to 12 mm; all follicles with this diameter were aspirated. All follicular fluid aspirated from cows subjected to HS or TN was pooled forming the groups (HS and TN). The EXO were obtained by ultracentrifugation of follicular fluid (120,000 × g for 70 min at 4°C, twice) and had their presence confirmed by transmission electron microscopy. Bos indicus cumulus-oocyte complexes (COC) collected from ovaries obtained in commercial slaughterhouse, were pooled in groups of 20 COC and randomly subjected to 1 of the following treatments: Control, matured in standard medium (TCM 199, supplemented with Earle’s salts, glutamine, NaHCO3, pyruvate, FSH, and amikacin); HS-EXO, matured in standard medium added with 10 µL of a solution of follicular EXO from HS cows; and TN-EXO, matured in standard medium added with 10 µL of a solution of follicular EXO from TN cows. The procedures were repeated 4 times, always with 20 COC per treatment in each replica. After 22 h of maturation, COC were recovered and the expression of genes related to apoptosis protection (BCL2), cell viability (STAT3), cell maintenance (RPL15), oocyte competence (BMP15), oxidative stress (CPT1B), cumulus cell expansion (HAS2), cell cycle (CDCA8), and heat stress protection (HSF1) were assessed. Oocyte genes were differentially expressed according to the source of EXO. Groups were statistically analysed using ANOVA and Tukey tests. All genes, except CPT1B, showed lower expression in TN-EXO oocytes when compared with control and HS-EXO (P < 0.05). CPT1B showed a higher expression in HS-EXO oocytes (P < 0.05). The results showed that the addition of EXO from exogenous follicles can modulate the expression of oocytes genes related to cell viability and survival. The lower expression of these genes in TN-EXO suggested that the EXO obtained in TN conditions attenuate several genes related to the oocytes maturation and viability. Surprisingly, the control oocytes showed a similar gene expression pattern of the HS-EXO. In conclusion, EXO derived from follicular fluid of cows submitted to TN or HS conditions can modulate the gene expression of oocytes matured in vitro. These results open new perspectives for the use of theses EXO as a tool to increase the efficiency of in vitro oocyte maturation.
Financial support from FAPESP #12/18297–7.