174 Follicular fluid extracellular vesicles obtained from Holstein cows kept under thermoneutral or heat stress conditions modify gene expression of in vitro-matured oocytes

Abstract

Heat stress (HS) has a massive impact on bovine reproduction. In cows, some of these deleterious effects involve follicular development and oocyte quality. Extracellular vesicles (EV) secreted by granulosa cells play a critical role in the intrafollicular environment by directly influencing cumulus cells and oocyte functions. The objective of this study was to investigate the effect of follicular fluid EV obtained from Holstein cows kept under thermoneutral (TN) or HS conditions, on in vitro bovine oocyte maturation. Non-lactating Holstein cows were synchronized with the Ovsynch protocol and also received an intravaginal progesterone device. From ovulation day (Day 1), cows were randomly assigned to TN (26°C, 73% humidity; n = 12) or HS (36°C, 70% humidity; n = 12) environments. On Day 9, 2 follicles (F1 and F2) were individually aspirated and all remained follicles were ablated. Then, on Day 14, newly formed F1 and F2 were also aspirated. Follicular fluid from all follicles from each treatment was pooled and EV were obtained according to Silveira et al. (2017 PLoS One 12, 1-25) and diluted in PBS (100 μL of PBS per mL of follicular fluid centrifuged). Pools of 20 cumulus-oocyte complexes (COC) grade 1 or 2 (Stojkovic et al. Biol Reprod 2001 64, 904-992], predominantly from Bos indicus, were submitted to the following treatment groups: Control (n = 4): matured in 90 μL of TCM-199 with Eagles’ salts, glutamine, NaHCO₃, pyruvate, amikacin, and FSH (base medium); TN (n = 4): matured in 81 μL of base medium + 9 μL of TN EV suspension; and HS (n = 4): matured in 81 μL + 9 μL of HS EV suspension. All treatments were carried out at 38.5°C for 24 h in a humid atmosphere with 5% CO₂. After 24 h of maturation, COC were evaluated for meiotic progression (Hoechst 33342 stain), DNA integrity (TUNEL stain), and expression of genes related to oocyte quality (TaqMan assay, Applied Biosystems/Thermo Fisher Scientific, Waltham, MA, USA). Results were analysed using ANOVA followed by Tukey post hoc test (P < 0.05). When the experimental groups were compared with the control group, there was no treatment effect on meiotic progression, DNA integrity, or gene expression of cumulus cells. In the oocytes of the TN group, the genes HSF1, IGFBP2, BMP15, GDF9, CDCA8, HAS2, RPL15, STAT3,and PFKP were less expressed. We concluded that oocytes matured in the presence of EV from follicular fluid of cows kept under TN conditions had lesser expression of genes related to oocyte quality.

This study was supported by FAPESP (Grant #2012/18297-7) and CAPES Foundation of Brazil.