Miranda Moysés dos Santos, Bressan Fabiana Fernandes, Zecchin Karina Gottardello, Vercesi Anibal Eugênio, Mesquita Ligia Garcia, Merighe Giovana Krempel Fonseca, King William Allan, Ohashi Otávio Mitio, Pimentel José Rodrigo Valim, Perecin Felipe, Meirelles Flávio Vieira
Departamento de Ciências Básicas, FZEA-USP, Pirassununga-SP, Brazil.
Cloning Stem Cells. 2009 Dec;11(4):565-73. doi: 10.1089/clo.2009.0028.
Cell cycle synchronization by serum starvation (SS) induces apoptosis in somatic cells. This side effect of SS is hypothesized to negatively affect the outcome of somatic cell nuclear transfer (SCNT). We determined whether apoptotic fibroblasts affect SCNT yields. Serum-starved, adult, bovine fibroblasts were stained with annexin V-FITC/propidium iodide to allow apoptosis detection by flow cytometry. Positive and negative cells sorted by fluorescence activated cell sorting (FACS) and an unsorted control group were used as nuclear donors for SCNT. Reconstructed embryos were cultured in vitro and transferred to synchronized recipients. Apoptosis had no effect on fusion and cleavage rates; however, it resulted in reductions in blastocyst production and quality measured by apoptotic index. However, reconstructed embryos with apoptotic cells resulted in pregnancy rates similar to that of the control on day 30, and generated one live female calf. In conclusion, we showed that apoptotic cells present in serum-starved cultures negatively affect embryo production after SCNT without compromising full-term development. Further studies will evaluate the ability of the oocyte to reprogram cells in specific phases of apoptosis.
血清饥饿（SS）诱导的细胞周期同步化会导致体细胞凋亡。据推测，SS的这种副作用会对体细胞核移植（SCNT）的结果产生负面影响。我们确定了凋亡的成纤维细胞是否会影响SCNT的成功率。用膜联蛋白V-异硫氰酸荧光素/碘化丙啶对血清饥饿的成年牛成纤维细胞进行染色，以便通过流式细胞术检测细胞凋亡。通过荧光激活细胞分选（FACS）分选的阳性和阴性细胞以及一个未分选的对照组用作SCNT的核供体。重构胚胎在体外培养并移植到同步化的受体中。细胞凋亡对融合率和分裂率没有影响；然而，通过凋亡指数测量，它导致囊胚产量和质量下降。然而，含有凋亡细胞的重构胚胎在第30天时的妊娠率与对照组相似，并产下一头存活的雌性小牛。总之，我们表明血清饥饿培养物中存在的凋亡细胞会对SCNT后的胚胎产生负面影响，但不会影响足月发育。进一步的研究将评估卵母细胞对处于特定凋亡阶段的细胞进行重编程的能力。
