Korhonen K, Kananen K, Ketoja E, Matomäki J, Halmekytö M, Peippo J
Biotechnology and Food Research, Animal Genomics, MTT Agrifood Research Finland, Jokioinen, Finland.
Reprod Domest Anim. 2010 Feb;45(1):42-9. doi: 10.1111/j.1439-0531.2008.01203.x. Epub 2008 Dec 30.
Maturation of oocytes and the subsequent outcome of the in vitro production (IVP) are affected by the composition of in vitro maturation (IVM) medium. To determine the use of serum interfering with effects of single molecules, we aimed at developing simplified IVM medium. The experimental IVM media were: (1) M199-medium supplemented with hormones and serum (control), (2) as 1 but serum was substituted with fatty acid-free serum albumin (FAFBSA) and (3) M199-medium without hormonal and serum supplementation (M199). The quality of embryos was assessed on day 7 by morphology and cryotolerance, as well as by Terminal deoxynucleotidyl Transferase Biotin-dUTP Nick End Labeling (TUNEL) and differential staining. Results showed that the nuclear maturation was suppressed in M199 group alone. Embryo cleavage and development rates, and the proportion of quality 1 blastocysts were lower in the FAFBSA and M199 groups compared to the control. Differences in the cell allocation of fresh embryos were observed at the blastocyst stage, but not at the expanded blastocyst stage. The control group blastocysts had larger number of cells allocated to the inner cell mass (ICM), and the FAFBSA group blastocysts larger apoptotic cell proportion compared to the blastocysts derived from other groups. After cryopreservation, the reduction of ICM proportion and increase of apoptotic cell proportion of embryos were equal between the experimental groups. In conclusion, exclusion of serum from the IVM media reduces embryo development and may cause perturbations in blastocyst development. Differences in the cell allocation of blastocysts between IVM media may appear only when the developmental stages are taken into account.
卵母细胞的成熟以及随后的体外生产(IVP)结果会受到体外成熟(IVM)培养基成分的影响。为了确定血清对单分子效应的干扰作用,我们旨在开发简化的IVM培养基。实验性IVM培养基如下:(1)添加激素和血清的M199培养基(对照),(2)与1相同,但血清被无脂肪酸血清白蛋白(FAFBSA)替代,以及(3)不添加激素和血清的M199培养基(M199)。在第7天通过形态学、耐冻性以及末端脱氧核苷酸转移酶生物素-dUTP缺口末端标记(TUNEL)和差异染色来评估胚胎质量。结果显示,仅M199组的核成熟受到抑制。与对照组相比,FAFBSA组和M199组的胚胎分裂和发育率以及1级囊胚的比例较低。在囊胚阶段观察到新鲜胚胎细胞分配存在差异,但在扩张囊胚阶段未观察到。与其他组来源的囊胚相比,对照组囊胚分配到内细胞团(ICM)的细胞数量更多,FAFBSA组囊胚的凋亡细胞比例更高。冷冻保存后,实验组胚胎的ICM比例降低和凋亡细胞比例增加的情况相同。总之,从IVM培养基中排除血清会降低胚胎发育,并可能导致囊胚发育出现扰动。仅在考虑发育阶段时,IVM培养基之间囊胚的细胞分配差异才可能出现。