Animal Reproduction (AR)
https://www.animal-reproduction.org/article/doi/10.1590/1984-3143-AR2023-0017
Animal Reproduction (AR)
ORIGINAL ARTICLE

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

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 Neto; 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.

Keywords

felids, cell cycle synchronization, culture to confluence, somatic cell nuclear transfer

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Submitted date:
02/01/2023

Accepted date:
03/22/2023

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