Sprícigo José Felipe, Morató Roser, Arcarons Núria, Yeste Marc, Dode Margot Alves, López-Bejar Manuel, Mogas Teresa
Facultat de Veterinària, Universitat Autònoma de Barcelona, Cerdanyola del Vallès, Spain; School of Agriculture and Veterinary Medicine, University of Brasilia, Brasília-DF, Brazil.
Facultat de Veterinària, Universitat Autònoma de Barcelona, Cerdanyola del Vallès, Spain.
Theriogenology. 2017 Feb;89:47-57. doi: 10.1016/j.theriogenology.2016.09.035. Epub 2016 Oct 6.
Cryopreservation may lead bovine oocytes to undergo morphological changes and functional damage due to the high-lipid content in the cytoplasm and the formation of reactive oxygen species. Against this background, the present study aimed to improve the cryotolerance and developmental competence of prepubertal calf oocytes by adding L-carnitine (LC) and/or resveratrol (R) to the IVM medium, as the former is involved in lipid metabolism and both are able to scavenge reactive oxygen species. With this purpose, different quality and functional oocyte parameters, such as spindle and chromosome configuration, DNA integrity, caspase activity, and the profile of genes involved in lipid metabolism and oxidative stress were evaluated in IVM bovine oocytes before or after vitrification/warming. Oocytes were matured in the absence (control) or presence of LC (3.03 mM) and/or R (1 μM) and then vitrified/warmed before IVF and embryo culture. All treatment groups (control, LC, R, and LC + R) of nonvitrified IVM oocytes showed similar rates (P > 0.05) of a normal spindle and chromosome configuration to oocytes vitrified/warmed after maturation in the presence of LC + R. When oocytes in all treatment groups were compared before and after vitrification, no significant differences were detected in DNA fragmentation as measured using the TUNEL method. However, the proportion of early apoptotic oocytes increased after vitrification/warming, except when previously matured with R. Vitrified/warmed oocytes matured in the presence of LC did not differ with nonvitrified oocytes in terms of the expression of ACACA, SLC2A1, PLIN2, HSPA1A, GPX1, and SOD1 genes. Similarly, expression of ACACA, SLC2A1, PLIN2, HSPA1A, and SOD1 genes in vitrified/warmed oocytes was similar to that of their fresh counterparts when matured in the presence of R. Finally, while the addition of LC and/or R to IVM medium had no effect on both cleavage and blastocyst rates either in fresh or vitrified oocytes. To conclude, the results of the present study report that the addition of LC and/or R to the IVM medium used for prepubertal bovine oocytes did not increase the embryo development potential of both fresh and vitrified oocytes. However, LC + R supplementation before vitrification decreased spindle damage, R addition-modulated apoptosis, and LC or R addition before vitrification positively affected the gene expression of vitrified/warmed oocytes.
由于细胞质中高脂质含量以及活性氧的形成，冷冻保存可能会导致牛卵母细胞发生形态变化和功能损伤。在此背景下，本研究旨在通过向体外成熟（IVM）培养基中添加左旋肉碱（LC）和/或白藜芦醇（R）来提高青春期前犊牛卵母细胞的抗冻性和发育能力，因为前者参与脂质代谢，且二者都能够清除活性氧。为此，在玻璃化/复温之前或之后，对IVM牛卵母细胞的不同质量和功能的卵母细胞参数进行了评估，如纺锤体和染色体构型、DNA完整性、半胱天冬酶活性以及参与脂质代谢和氧化应激的基因谱。卵母细胞在不存在（对照）或存在LC（3.03 mM）和/或R（1 μM）的情况下成熟，然后在体外受精和胚胎培养之前进行玻璃化/复温。未玻璃化的IVM卵母细胞的所有处理组（对照、LC、R和LC + R）与在LC + R存在下成熟后玻璃化/复温的卵母细胞相比，正常纺锤体和染色体构型的比例相似（P > 0.05）。当比较所有处理组的卵母细胞在玻璃化前后的情况时，使用TUNEL法测量的DNA片段化未检测到显著差异。然而，除了之前用R成熟的情况外，玻璃化/复温后早期凋亡卵母细胞的比例增加。在LC存在下成熟的玻璃化/复温卵母细胞在ACACA、SLC2A1、PLIN2、HSPA1A、GPX1和SOD1基因的表达方面与未玻璃化的卵母细胞没有差异。同样，在R存在下成熟时，玻璃化/复温卵母细胞中ACACA、SLC2A1、PLIN2、HSPA1A和SOD1基因的表达与其新鲜对应物相似。最后，虽然向IVM培养基中添加LC和/或R对新鲜或玻璃化卵母细胞的分裂率和囊胚率均无影响。总之，本研究结果表明，向用于青春期前牛卵母细胞的IVM培养基中添加LC和/或R不会增加新鲜和玻璃化卵母细胞的胚胎发育潜力。然而，玻璃化前补充LC + R可减少纺锤体损伤，添加R可调节凋亡，玻璃化前添加LC或R对玻璃化/复温卵母细胞的基因表达有积极影响。
