Gene expression of equine oocytes maintained in meiotic blockade

Abstract

To improve quality, immature horse oocytes may undergo a pre-maturation (PM) period to promote synchronization of nuclear and cytoplasmic maturation. PM can be performed using meiosis blockers, such as Butyrolactone I, or by keeping oocytes at room temperature (Choi et al. Theriogenology 2006;66(4):955-963; Martino et al. Reproductive Biology and Endocrinology.2014;12(1):99-110). In this experiment the gene expression profile in COCs maintained in a commercial embryo medium (Botuembryo) and in DMEM/F12 supplemented with Butyrolactone was studied. Equine COCs were retrieved by follicular curettage from slaughterhouse ovaries and divided into 3 groups (n = 74): Control - COCs were destined to IVM during 24hs in DMEM/F12 with 10% FCS and antibiotics; Botuembryo - COCs underwent PM for 20 hours at room temperature in Botuembryo (21 to 25°C) and then 24hs of IVM at the same conditions as Control group, and Butyrolactone - COCs stayed in PM in DMEM/F12 with 10% FCS and 50 μM Butyrolactone for 20hs in a controlled atmosphere incubator at 38,5°C, and then were destined to IVM to an extra 24hs. Four replicates were used. Timepoint 1 (M1) in controls corresponds to the moment just after collection and in Botuembryo and Butyrolactone groups, M1 corresponds to the end of PM. Timepoint 2 (M2) corresponds to the end of IVM in all groups. Oocytes were denuded of cumulus cells (CCs) and both were stored in separate vials at -80°C. Analysis of mRNA expression was done by RT-qPCR with β-actin (ACTB) as reference gene. Ampiregulin (AREG), Connexin 43 (Cx43), Hyalurone Synthetase 2 (HAS2), FSH Receptor (FSHR) and LH Receptor (LHR) were studied in COCs whereas Bone Morphogenetic Protein 15(BMP15), Connexin 37 (Cx37), Growth and Differentiation Factor 9 (GDF9) and Matrix Metalloproteinase 2 (MMP2) were analyzed in oocytes. Data for each timepoint were tested by ANOVA followed by group comparison with the Tukey Kramer test (JMP software). Atypical gene expression (outliers) was identified by the generalized ESD test (GraphPad PRISM) and excluded. Data are presented as mean ± SEM, with P<0.05 being significant. The results (Table 1) indicate a failure in blocking molecular maturation in the Butyrolactone group, with an increase in the abundance of AREG and HAS2 in M1 compared to M2. On the other hand, the Botuembryo group was superior in M2 in relation to the abundance of BMP15, and GDF9, indicating better quality oocytes. More studies need to be performed to confirm the superiority of Botuembryo in maintaining meiotic arrest during PM. Acknowledgement: FAPESP (process 2020/01646-5); CNPq; CAPES; Omics Animal Biotechnology; Frigofava.