Rodrigues Luanna Lorenna Vieira, Moura Yasmin Beatriz França, Viana João Vitor da Silva, de Oliveira Lhara Ricarliany Medeiros, Praxedes Érika Almeida, Vieira José de Brito, Sales Sarah Leyenne Alves, Silva Herlon Victor Rodrigues, Luciano Maria Claudia Dos Santos, Pessoa Claudia, Pereira Alexsandra Fernandes
Laboratório de Biotecnologia Animal, Universidade Federal Rural do Semi-Árido, Mossoró, RN, Brasil.
Laboratório de Oncologia Experimental, Universidade Federal do Ceará, Fortaleza, CE, Brasil.
Anim Reprod. 2023 Apr 21;20(1):e20230017. doi: 10.1590/1984-3143-AR2023-0017. eCollection 2023.
The puma population is constantly decreasing, and cloning by somatic cell nuclear transfer can be used to conserve the species. One of the factors determining the success of the development of cloned embryos is the cell cycle stage of the donor cells. We evaluated the effects of full confluency (~100%), serum starvation (0.5% serum), and roscovitine (15 µM) treatments on the cell cycle synchronization in G/G of puma skin-derived fibroblasts by flow cytometric analysis. Also, we assessed the effects of these synchronization methods on morphology, viability, and apoptosis levels using microscopy tools. The results showed that culturing the cells to confluence for 24 h (84.0%), 48 h (84.6%), and 72 h (84.2%) and serum starvation for 96 h (85.4%) yielded a significantly higher percentage of cells arrested in the G/G (P 0.05) phase than cells not subjected to any cell cycle synchronization method (73.9%). Nevertheless, while serum starvation reduced the percentage of viable cells, no difference was observed for the full confluence and roscovitine treatments (P 0.05). Moreover, roscovitine for 12 h (78.6%) and 24 h (82.1%) was unable to synchronize cells in G/G (P 0.05). In summary, full confluency induces puma fibroblast cell cycle synchronization at the G/G stage without affecting cell viability. These outcomes may be valuable for planning donor cells for somatic cell nuclear transfer in pumas.
美洲狮种群数量在持续减少，通过体细胞核移植进行克隆可用于保护该物种。决定克隆胚胎发育成功的因素之一是供体细胞的细胞周期阶段。我们通过流式细胞术分析评估了完全汇合（约100%）、血清饥饿（0.5%血清）和roscovitine（15 μM）处理对美洲狮皮肤来源成纤维细胞在G/G期细胞周期同步化的影响。此外，我们使用显微镜工具评估了这些同步化方法对细胞形态、活力和凋亡水平的影响。结果表明，将细胞培养至汇合24小时（84.0%）、48小时（84.6%）和72小时（84.2%）以及血清饥饿96小时（85.4%）后，与未进行任何细胞周期同步化方法处理的细胞（73.9%）相比，处于G/G期停滞的细胞百分比显著更高（P<0.05）。然而，虽然血清饥饿降低了活细胞百分比，但完全汇合和roscovitine处理未观察到差异（P>0.05）。此外，roscovitine处理12小时（78.6%）和24小时（82.1%）无法使细胞在G/G期同步化（P<0.05）。总之，完全汇合可诱导美洲狮成纤维细胞在G/G期进行细胞周期同步化，且不影响细胞活力。这些结果对于规划美洲狮体细胞核移植的供体细胞可能具有重要价值。
