165 Effect of Dimethyl Sulfoxide Supplementation During In Vitro Maturation on the Genetic Expression Pattern of Bovine Blastocyst

Abstract

Supplementation of bovine oocytes with 0.5% (v/v) dimethylsulfoxide (DMSO) during in vitro maturation (IVM) results in increased blastocysts rates (Ynsaurralde et al. 2016 Reprod. Fertil. Dev. 29, 201-202). Recently, an important role of DMSO in stem cell differentiation has been observed, attributed to modulation of gene expression. However, the effect of DMSO suplementation during in vitro maturation on gene expression profiles and embryo quality have not been evaluated so far. Thus, we examinated the effect of DMSO during IVM on the expression of some key genes (Sox2, Oct4, and Cdx2) and on the degree of DNA fragmentation at the blastocyst stage. To this aim, cumulus–oocyte complexes collected from slaughterhouse ovaries were matured in TCM-199 containing 10% fetal bovine serum, 10 µg mL⁻¹ FSH, 0.3 mM sodium pyruvate, 100 mM cysteamine, and 2% antibiotic-antimycotic for 24 h, at 6.5% CO₂ in humidified air and 38.5°C. Maturation media was supplemented with 0, 0.5, or 0.75% (v/v) DMSO. In vitro fertilization (IVF) was performed with 16 × 10⁶ spermatozoa per mL for 5 h. Afterwards, presumptive zygotes were cultured in SOF for 7 days at 38.5°C and 5% O₂. Three pools of 5 blastocysts were analysed for each treatment. Gene expression analysis was performed by real-time qPCR and DNA fragmentation of blastocysts was measured by TUNEL assay (n = 8, 7, and 14 blastocysts analysed for 0, 0.5, and 0.75% v/v DMSO, respectively). The results were statistically analysed using ANOVA with a completely randomised model by InfoStat software Version 1.1 (https://www.infostat.com.ar/). The pluripotency marker genes Sox2 and Oct4 were up-regulated in blastocysts only when the oocytes were matured in 0.75% DMSO, whereas the trophoblastic marker Cdx2 showed no differences among treatments. No differences were detected in the number of TUNEL-positive cells among treatments: 10/65 (15%) in 0%, 19/110 (18%) in 0.5%, and 18/98 (20%) in 0.75% (v/v) DMSO. In conclusion, supplementation with 0.5% (v/v) DMSO, as previously published, increases the production of blastocysts without disrupting the expression pattern of the evaluated genes.