Animal Reproduction (AR)
https://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

Downloads: 0
Views: 488

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

References

Benson JF, Sikich JA, Riley SPD. Survival and competing mortality risks of mountain lions in a major metropolitan area. Biol Conserv. 2020;241:108294. http://dx.doi.org/10.1016/j.biocon.2019.108294.

Borges AA, Pereira AF. Potential role of intraspecific and interspecific cloning in the conservation of wild mammals. Zygote. 2019;27(3):111-7. http://dx.doi.org/10.1017/S0967199419000170. PMid:31182179.

Campbell KH, Loi P, Otaegui PJ, Wilmut I. Cell cycle co-ordination in embryo cloning by nuclear transfer. Rev Reprod. 1996;1(1):40-6. http://dx.doi.org/10.1530/ror.0.0010040. PMid:9414437.

Coller HA. What’s taking so long? S-phase entry from quiescence versus proliferation. Nat Rev Mol Cell Biol. 2007;8(8):667-70. http://dx.doi.org/10.1038/nrm2223. PMid:17637736.

Curto M, Cole BK, Lallemand D, Liu C-H, McClatchey AI. Contact-dependent inhibition of EGFR signaling by Nf2/Merlin. J Cell Biol. 2007;177(5):893-903. http://dx.doi.org/10.1083/jcb.200703010. PMid:17548515.

Echeverry DM, Asenjo PA, Rojas DM, Aguilera CJ, Rodríguez-Álvarez L, Castro FO. Characterization of mesenchymal stem cells derived from adipose tissue of a cougar (Puma concolor). Anim Reprod. 2020;17(2):e20190109. http://dx.doi.org/10.1590/1984-3143-ar2019-0109. PMid:32714450.

Gómez MC, Jenkins JA, Giraldo A, Harris RF, King A, Dresser BL, Pope CE. Nuclear transfer of synchronized African wild cat somatic cells into enucleated domestic cat oocytes. Biol Reprod. 2003;69(3):1032-41. http://dx.doi.org/10.1095/biolreprod.102.014449. PMid:12773426.

Gómez MC, Pope CE, Giraldo A, Lyons LA, Harris RF, King AL, Cole A, Godke RA, Dresser BL. Birth of African wildcat cloned kittens born from domestic cats. Cloning Stem Cells. 2004;6(3):247-58. http://dx.doi.org/10.1089/clo.2004.6.247. PMid:15671671.

Gómez MC, Pope CE, Kutner RH, Ricks DM, Lyons LA, Ruhe M, Dumas C, Lyons J, López M, Dresser BL, Reiser J. Nuclear transfer of sand cat cells into enucleated domestic cat oocytes is affected by cryopreservation of donor cells. Cloning Stem Cells. 2008;10(4):469-84. http://dx.doi.org/10.1089/clo.2008.0021. PMid:18795868.

Gómez NA, Ramírez MM, Ruiz-Cortés ZT. Primary fibroblast cell cycle synchronization and effects on handmade cloned (HMC) bovine embryos. Ciênc Anim Bras. 2018;19(0):e48555. http://dx.doi.org/10.1590/1809-6891v19e-48555.

Han Z-M, Chen D-Y, Li J-S, Sun Q-Y, Wang P-Y, Du J, Zhang H-M. Flow cytometric cell-cycle analysis of cultured fibroblasts from the giant panda, Ailuropoda melanoleuca. L Cell Biol Int. 2003;27(4):349-53. http://dx.doi.org/10.1016/S1065-6995(02)00353-0. PMid:12788051.

Hashem A, Bhandari DP, Kang SK, Lee BC, Suk HW. Cell cycle analysis of in vitro cultured goral (Naemorhedus caudatus) adult skin fibroblasts. Cell Biol Int. 2006;30(9):698-703. http://dx.doi.org/10.1016/j.cellbi.2006.04.008. PMid:16793292.

Jun S-M, Park M, Lee JY, Jung S, Lee JE, Shim SH, Song H, Lee DR. Single cell‐derived clonally expanded mesenchymal progenitor cells from somatic cell nuclear transfer‐derived pluripotent stem cells ameliorate the endometrial function in the uterus of a murine model with Asherman’s syndrome. Cell Prolif. 2019;52(3):e12597. http://dx.doi.org/10.1111/cpr.12597. PMid:30896075.

Karandikar H, Serota MW, Sherman WC, Green JR, Verta G, Kremen C, Middleton AD. Dietary patterns of a versatile large carnivore, the puma (Puma concolor). Ecol Evol. 2022;12(6):e9002. http://dx.doi.org/10.1002/ece3.9002. PMid:35784054.

Kim MK, Jang G, Oh HJ, Yuda F, Kim HJ, Hwang WS, Hossein MS, Kim JJ, Shin NS, Kang SK, Lee BC. Endangered wolves cloned from adult somatic cells. Cloning Stem Cells. 2007;9(1):130-7. http://dx.doi.org/10.1089/clo.2006.0034. PMid:17386020.

Kues WA, Anger M, Carnwath JW, Paul D, Motlik J, Niemann H. Cell cycle synchronization of porcine fetal fibroblasts: effects of serum deprivation and reversible cell cycle inhibitors. Biol Reprod. 2000;62(2):412-9. http://dx.doi.org/10.1095/biolreprod62.2.412. PMid:10642581.

LaBarge LR, Evans MJ, Miller JRB, Cannataro G, Hunt C, Elbroch LM. Pumas Puma concolor as ecological brokers: a review of their biotic relationships. Mammal Rev. 2022;52(3):360-76. http://dx.doi.org/10.1111/mam.12281.

Lanza RP, Cibelli JB, Diaz F, Moraes CT, Farin PW, Farin CE, Hammer CJ, West MD, Damiani P. Cloning of an endangered species (Bos gaurus) using interspecies nuclear transfer. Cloning. 2000;2(2):79-90. http://dx.doi.org/10.1089/152045500436104. PMid:16218862.

Leite M, Quinta-Costa M, Leite PS, Guimarães JE. Critical evaluation of techniques to detect and measure cell death-study in a model of UV radiation of the leukaemic cell line HL60. Anal Cell Pathol. 1999;19(3-4):139-51. http://dx.doi.org/10.1155/1999/176515. PMid:10866276.

Lira GPO, Borges AA, Nascimento MB, Aquino LVC, Moura LFMP, Silva HVR, Ribeiro LR, Silva AR, Pereira AF. Morphological, ultrastructural, and immunocytochemical characterization and assessment of puma (Puma concolor Linnaeus, 1771) cell lines after extended culture and cryopreservation. Biopreserv Biobank. 2022;20(6):557-66. http://dx.doi.org/10.1089/bio.2021.0117. PMid:35049356.

Lira GPO, Borges AA, Nascimento MB, Aquino LVC, Moura LFMP, Silva HVR, Ribeiro LR, Oliveira MF, Pereira AF. Effects of somatic tissue cryopreservation on puma (Puma concolor L, 1771) tissue integrity and cell preservation after in vitro culture. Cryobiology. 2021;101:52-60. http://dx.doi.org/10.1016/j.cryobiol.2021.06.003. PMid:34118241.

Loi P, Ptak G, Barboni B, Fulka J Jr, Cappai P, Clinton M. Genetic rescue of an endangered mammal by cross-species nuclear transfer using post‐mortem somatic cells. Nat Biotechnol. 2001;19(10):962-4. http://dx.doi.org/10.1038/nbt1001-962. PMid:11581663.

Machado LC, Roballo KCS, Cury FS, Ambrósio CE. Female reproductive system morphology of crab‐eating fox (Cerdocyon thous) and cryopreservation of genetic material for animal germplasm bank enrichment. Anat Histol Embryol. 2017;46(6):539-46. http://dx.doi.org/10.1111/ahe.12306. PMid:28913836.

Młodawska W, Mrowiec P, Bochenek M, Wnęk K, Kochan J, Nowak A, Niżański W, Prochowska S, Pałys M. Effect of serum starvation and contact inhibition on dermal fibroblast cell cycle synchronization in two species of wild felids and domestic cat. Ann Anim Sci. 2022;22(4):1245-55. http://dx.doi.org/10.2478/aoas-2022-0042.

Moro LN, Hiriart MI, Buemo C, Jarazo J, Sestelo A, Veraguas D, Rodriguez-Alvarez L, Salamone DF. Cheetah interspecific SCNT followed by embryo aggregation improves in vitro development but not pluripotent gene expression. Reproduction. 2015;150(1):1-10. http://dx.doi.org/10.1530/REP-15-0048. PMid:25820926.

Moulavi F, Hosseini SM, Tanhaie-Vash N, Ostadhosseini S, Hosseini SH, Hajinasrollah M, Asghari MH, Gourabi H, Shahverdi A, Vosough AD, Nasr-Esfahani MH. Interspecies somatic cell nuclear transfer in Asiatic cheetah using nuclei derived from post-mortem frozen tissue in absence of cryo-protectant and in vitro matured domestic cat oocytes. Theriogenology. 2017;90:197-203. http://dx.doi.org/10.1016/j.theriogenology.2016.11.023. PMid:28166968.

Nguyen VK, Somfai T, Salamone D, Huong VTT, Nguyen HLT, Huu QX, Hoang AT, Phan HT, Pham YKT, Pham LD. Optimization of donor cell cycle synchrony, maturation media and embryo culture system for somatic cell nuclear transfer in the critically endangered Vietnamese Ỉ pig. Theriogenology. 2021;166:21-8. http://dx.doi.org/10.1016/j.theriogenology.2021.02.008. PMid:33667861.

Nielsen C, Thompson D, Kelly M, Lopez-Gonzalez CA. Puma concolor. The IUCN Red List of Threatened Species. 2015:e.T18868A97216466. Online. http://dx.doi.org/10.2305/IUCN.UK.2015-4.RLTS.T18868A50663436.en.

Pereira AF, Borges AA, Santos MVO, Lira GPO. Use of cloning by nuclear transfer in the conservation and multiplication of wild mammals. Rev Bras Reprod Anim. 2019;43(2):242-7.

Praxedes ÉA, Borges AA, Santos MVO, Pereira AF. Use of somatic cell banks in the conservation of wild felids. Zoo Biol. 2018;37(4):258-63. http://dx.doi.org/10.1002/zoo.21416. PMid:29722907.

Praxedes ÉA, Oliveira LRM, Silva MB, Borges AA, Santos MVO, Silva HVR, Oliveira MF, Silva AR, Pereira AF. Effects of cryopreservation techniques on the preservation of ear skin - an alternative approach to conservation of jaguar, Panthera onca (Linnaeus, 1758). Cryobiology. 2019;88:15-22. http://dx.doi.org/10.1016/j.cryobiol.2019.04.007. PMid:31029688.

Ripple WJ, Estes JA, Beschta RL, Wilmers CC, Ritchie EG, Hebblewhite M, Berger J, Elmhagen B, Letnic M, Nelson MP, Schmitz OJ, Smith DW, Wallach AD, Wirsing AJ. Status and ecological effects of the world’s largest carnivores. Science. 2014;343(6167):1241484. http://dx.doi.org/10.1126/science.1241484. PMid:24408439.

Song J, Hua S, Song K, Zhang Y. Culture, characteristics, and chromosome complement of Siberian tiger fibroblasts for nuclear transfer. In Vitro Cell Dev Biol Anim. 2007;43(7):203-9. http://dx.doi.org/10.1007/s11626-007-9043-3. PMid:17763919.

Veraguas D, Gallegos PF, Castro FO, Rodriguez-Alvarez L. Cell cycle synchronization and analysis of apoptosis-related gene in skin fibroblasts from domestic cat (Felis silvestris catus) and kodkod (Leopardus guigna). Reprod Domest Anim. 2017;52(5):881-9. http://dx.doi.org/10.1111/rda.12994. PMid:28544240.

Verma R, Holland MK, Temple-Smith P, Verma PJ. Inducing pluripotency in somatic cells from the snow leopard (Panthera uncia), an endangered felid. Theriogenology. 2012;77(1):220-8.e-2. http://dx.doi.org/10.1016/j.theriogenology.2011.09.022. PMid:22079579.

Wittayarat M, Thongphakdee A, Saikhun K, Chatdarong K, Otoi T, Techakumphu M. Cell cycle synchronization of skin fibroblast cells in four species of family Felidae. Reprod Domest Anim. 2013;48(2):305-10. http://dx.doi.org/10.1111/j.1439-0531.2012.02149.x. PMid:22834557.

Wolf C, Ripple WJ. Range contractions of the world’s large carnivores. R Soc Open Sci. 2017;4(7):170052. http://dx.doi.org/10.1098/rsos.170052. PMid:28791136.

Yang S, Hwang S, Jang J, Kim M, Gwak J, Jeong SM. PGC1α is required for the induction of contact inhibition by suppressing ROS. Biochem Biophys Res Commun. 2018;501(3):739-44. http://dx.doi.org/10.1016/j.bbrc.2018.05.059. PMid:29753744.
 


Submitted date:
02/01/2023

Accepted date:
03/22/2023

643ed2e7a953956be802d733 animreprod Articles
Links & Downloads

Anim Reprod

Share this page
Page Sections