Full confluency, serum starvation, and roscovitine for inducing arrest in the G0/G1 phase of the cell cycle in puma skin-derived fibroblast lines.

IF 1.6 4区 农林科学 Q2 AGRICULTURE, DAIRY & ANIMAL SCIENCE Animal Reproduction Pub Date : 2023-01-01 DOI:10.1590/1984-3143-AR2023-0017
Luanna Lorenna Vieira Rodrigues, Yasmin Beatriz França Moura, João Vitor da Silva Viana, Lhara Ricarliany Medeiros de Oliveira, Érika Almeida Praxedes, José de Brito Vieira, Sarah Leyenne Alves Sales, Herlon Victor Rodrigues Silva, Maria Claudia Dos Santos Luciano, Claudia Pessoa, Alexsandra Fernandes Pereira
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Abstract

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 G0/G1 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 G0/G1 (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 G0/G1 (P 0.05). In summary, full confluency induces puma fibroblast cell cycle synchronization at the G0/G1 stage without affecting cell viability. These outcomes may be valuable for planning donor cells for somatic cell nuclear transfer in pumas.

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在美洲狮皮肤源性成纤维细胞系中,完全融合、血清饥饿和罗斯维汀诱导细胞周期G0/G1期阻滞
美洲狮种群数量在不断减少,通过体细胞核移植克隆可以保护该物种。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融合(~100%)、血清饥饿(0.5%血清)和罗斯科维汀(15µM)处理对美洲狮皮肤源性成纤维细胞G0/G1期细胞周期同步的影响。此外,我们使用显微镜工具评估了这些同步方法对形态学、活力和凋亡水平的影响。结果表明,融合培养24 h(84.0%)、48 h(84.6%)、72 h(84.2%)和血清饥饿培养96 h(85.4%),细胞停留在G0/G1期的细胞比例(P 0.05)显著高于未进行细胞周期同步的细胞(73.9%)。然而,虽然血清饥饿降低了活细胞百分比,但在全融合和罗斯科维汀处理中没有观察到差异(p0.05)。罗斯科维汀作用12 h(78.6%)和24 h(82.1%)不能同步G0/G1细胞(P < 0.05)。综上所述,在G0/G1期,完全融合诱导成纤维美洲狮细胞周期同步,而不影响细胞活力。这些结果可能对规划供细胞用于美洲狮体细胞核移植有价值。
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来源期刊
Animal Reproduction
Animal Reproduction AGRICULTURE, DAIRY & ANIMAL SCIENCE-
CiteScore
2.30
自引率
11.80%
发文量
49
审稿时长
70 days
期刊介绍: Animal Reproduction (AR) publishes original scientific papers and invited literature reviews, in the form of Basic Research, Biotechnology, Applied Research and Review Articles, with the goal of contributing to a better understanding of phenomena related to animal reproduction. The scope of the journal applies to students, researchers and practitioners in the fields of veterinary, biology and animal science, also being of interest to practitioners of human medicine. Animal Reproduction Journal is the official organ of the Brazilian College of Animal Reproduction in Brazil.
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