201 The presence of corpus luteum affects gene expression in oocytes and cumulus cells after in vitro maturation in mares

The possibility of obtaining viable individuals from the culture of gametes and embryos under laboratory conditions is presented as an available alternative to solve clinical fertility problems related to mares and horses. However, to reach the full establishment of the technique as an assisted reproduction service for massive and routine use in horses, further studies are necessary to unravel the factors that affect the competence of the complex oocyte clusters. This study aimed to evaluate the effect of the presence and location of the corpus luteum (CL) on mRNA expression of genes associated with cell quality in equine oocytes and cumulus cells subjected to IVM. Pairs of ovaries belonging to each animal were collected in a slaughterhouse; in the laboratory they were evaluated to detect the presence of CL and, accordingly, they were classified into three groups: ovaries with CL (CL+); ovaries without CL, from an animal with CL in the contralateral ovary (CL−); and ovaries from animals without a corpus luteum (NCL). The cumulus–oocyte complexes (COCs) were recovered by curettage and washing the follicular wall with PBS solution supplemented with 1% polyvinyl alcohol (PVA). Groups of 10 COCs recovered from each experimental group (CL+ n = 10 COCs and 8 replicates; CL− n = 10 COCs and 6 replicates; and NCL n = 10 COCs and 5 replicates) were placed in 100 µL of DMEM F-12 medium (supplemented with 20% fetal bovine serum (FBS), cysteine, cystine, melatonin (1 p.m.), gentamicin (50 mg mL⁻¹), FSH, LH, and epidermal growth factor) and matured in an incubator at 38.5°C and 5% CO₂, for 36 h. Subsequently, the cumulus cells were separated from the oocytes by repetitive pipetting. Total RNA from cumulus cells and oocytes was extracted with TRIzol^® reagent according to the manufacturer’s recommendations. Reverse transcription was performed using a commercial kit (ProtoScript^®). The real-time PCR was performed using the kit (Luna^®). The target genes evaluated in both cumulus cells and oocytes were BCL2 associated X (BAX), bone morphogenetic protein 15 (BMP-15), ornithine decarboxylase 1 (ODC1), growth differentiation factor 9 (GDF9), and aurora kinase A (AURKA). Analyzes were performed in SAS version 9.4 using one-way analysis of variance with Tukey post hoc test, and a P-value lower than 0.05 was considered significant. In oocytes, a higher expression for the BMP-15 gene was observed in oocytes from the CL+ group (2.38 ± 0.37 fold), compared with those from the NCL group (P = 0.04). Additionally, in cumulus cells, a higher expression of GDF9 was observed among the CL+ group (2.97 ± 0.48 fold) compared with the NCL group (P = 0.01). For the remaining genes, no differences were observed between groups. In conclusion, the results suggest that the presence of CL increases the mRNA expression of genes related to higher cell quality in oocytes and equine cumulus compared with mares without CL presence in both ovaries.