Cell confluency is as efficient as serum starvation for inducing arrest in the G0/G1 phase of the cell cycle in granulosa and fibroblast cells of cattle

Hayes, Orlando; Ramos, B.; Rodríguez, Lleretny; Aguilar, A.; Badia, Teresita; Castro, FO

doi:10.1016/j.anireprosci.2004.11.011

Cited by 67 publications

(74 citation statements)

References 30 publications

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“…In the present study it is shown that only serum starved and fully confluent states had synchronized more cells at G 0 / G 1 phases which were significantly different than that of cycling state (Table 1). Our results differ with the findings of other authors (Kubota et al, 2000;Kues et al, 2000;Gibbons et al, 2002;Zou et al, 2002;Gomez et al, 2003;Han et al, 2003;Liu et al, 2004;Hayes et al, 2005). Among them, Liu et al (2004) stated that chemical synchronization of the cell cycle stage of rabbit fetal fibroblasts to G 0 /G 1 (25-50%) was less effective as compared to serum deprivation.…”

Section: Discussioncontrasting

confidence: 57%

“…Zou et al (2002) found the frequency of G 0 /G 1 cell cycle stages was 74.2% in SS, 61.6% in cycling and 71.7% in confluent culture conditions in transfected goat fetal fibroblast cells. Hayes et al (2005) reported G 0 /G 1 cell cycle stage frequencies of 69.3% in SS (for 3 days), 40.2% in cycling Molecular Reproduction and Development. DOI 10.1002/mrd and 80.1% in confluent culture conditions in adult fibroblast cells of cattle.…”

Section: Discussionmentioning

confidence: 99%

“…Double staining with propidium iodide (PI) and fluorescein isothiocyanate (FITC) is particularly useful to distinguish G 0 phase from G 1 phase (Boquest et al, 1999). There are many studies which have been done on the synchronization of cell cycles using different chemicals and methods (Blow, 1993;Kubota and Takisawa, 1993;Fenteany et al, 1994;Mayer and Noble, 1994;Vesley et al, 1994;Ferrari et al, 1995;Yan et al, 1995;Cotgreave, 1997;Hitomi and Staney, 1999;Kubota et al, 2000;Kues et al, 2000;Gibbons et al, 2002;Lee and Piedrahita, 2002;Zou et al, 2002;Gomez et al, 2003;Han et al, 2003;Gongalez-Cabrera et al, 2004;Liu et al, 2004;Hayes et al, 2005).…”

Section: Introductionmentioning

confidence: 99%

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Cell cycle analysis and interspecies nuclear transfer of in vitro cultured skin fibroblasts of the Siberian tiger (Panthera tigris Altaica)

Hashem

Bhandari

Kang

et al. 2006

Molecular Reproduction Devel

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The present study was conducted to examine the effect of cell culture conditions, antioxidants, protease inhibitors (PI), and different levels of dimethylsulfoxide (DMSO) for the promotion of synchronization of different cell cycles of Siberian tiger skin fibroblasts. We also compared the ability of somatic cell nuclei of the Siberian tiger in pig cytoplasts and to support early development after reconstruction. Cell cycle synchronization between nuclear donor and recipient cells is considered to be one of the most crucial factors for successful cloning. Five experiments were performed each with a one-way completely randomized design involving three replicates of all treatments. Least significant difference (LSD) was used to determine variation among treatment groups. Experiment I focused in the effects of cycling, serum starved and fully confluent stages of Siberian tiger cells on different cell cycles. In Experiment II, the effects of different antioxidants like b-Mercaptoethanol (b-ME, 10 mM), cysteine (2 mM), and glutathione (2 mM) were examined after cells were fully confluent without serum starvation for 4 hr. In Experiment III, three PI, namely 6-dimethylaminopurine (6-DMAP, 2 mM), cycloheximide (7.5 mg/ml) and cytochalasin B (7.5 mg/ ml) were used in the sane manner as in Experiment II. In Experiment IV, different levels of DMSO at 0%, 0.5%, 1.0%, and 2.5% were tested on different cell cycle stages of Siberian tiger examined by Flowcytometry (FACS). In Experiment I, 67.2% of the Siberian tiger skin fibroblasts reached the G 0 /G 1 stage (2C DNA content) in fully confluent conditions which was more than the cycling (49.8%) and serum starved (SS) medium (65.5%; P < 0.05). Among the chemically treated group, glutathione (72.6%) and cycloheximide (71.3%) had little bit better results for the synchronization of G 0 þ G 1 phases than serum starved and fully confluent. After nuclear transfer we did not see any significant differences on the development of tigerporcine reconstructed embryos at cycling, SS and fully confluent. Data indicate that prolonged culture of cells in the absence of serum as well as using different chemicals for this experiment does not imply a shift in the percentage of cells that enter G 0 /G 1 and that confluency is sufficient to induce quiescence. This finding can be beneficial in nuclear transfer programs in Siberian tiger, because there are negative effects, such as apoptosis associated with serum starvation. Mol. Reprod. Dev. 74: 403-411, 2007. ß

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Section: Discussioncontrasting

confidence: 57%

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Cell cycle analysis and interspecies nuclear transfer of in vitro cultured skin fibroblasts of the Siberian tiger (Panthera tigris Altaica)

Hashem

Bhandari

Kang

et al. 2006

Molecular Reproduction Devel

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“…The overall efficiency of the SCNT procedure was 52.6% which is probably the highest published so far for cloned embryos using HMC [2-4, 7, 8]. In our hands, blastocyst rates of 11% to 20% where historically achieved when using conventional cloning [10][11][12]. For Wagyu cell line, the figures for blastocysts development (65.7%) are to our knowledge the highest ever published for any SCNT experiment reported so far and are similar to those reported for WOW culture of IVP embryos [3].…”

Section: Resultsmentioning

confidence: 52%

High developmental potential in vitro and in vivo of cattle embryos cloned without micromanipulators

Rodríguez

Navarrete

Tovar

et al. 2008

J Assist Reprod Genet

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Purpose In order to simplify cloning, a new method that does not require micromanipulators was used. We aimed to evaluate the developmental potential of two bovine cell lines upon cloning. Materials and methods In vitro matured bovine oocytes, were released from zona pellucida, enucleated, fused to foetal or adult somatic donor cells. The reconstructed embryos were reprogrammed, activated and cultured until blastocyst stage. No micromanipulators were used. Blastocyst rate and quality was scored. Some expanded (d7) blastocysts were transferred to recipient cattle and collected back at d17 to assess elongation. Results High developmental potential in vitro of cloned embryos to expanded (d7) blastocysts was achieved (52.6%). In one cell line, 65.7% of blastocysts was scored. Most blastocysts (87.4%) were graded as excellent. In vivo development to elongation (day-17) in temporary recipient cows also showed a high developmental potential (11/18 transferred blastocysts elongated). Conclusions Hand-made cloning is an efficient alternative for cloning in cattle.

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“…However, serum starvation causes reduced cell survival and increased DNA fragmentation [12] and apoptosis [13] in porcine cells. The confluent cell culture system is as effective for cell cycle synchronization as serum starvation, especially for arresting cells in the G0/G1 phase [14][15][16]. Moreover, SCNT embryos reconstructed with donor cells obtained by confluent cell culture or s e r u m s t a r v a t i o n d o n o t s h o w d i f f e r e n t developmental rates during in vitro development [17].…”

Section: Hida Beef Cattle Research Gifu Prefectural Livestock Researmentioning

confidence: 99%

Effects of Synchronization of Donor Cell Cycle on Embryonic Development and DNA Synthesis in Porcine Nuclear Transfer Embryos

Miyamoto

Hoshino

Minami

et al. 2007

Journal of Reproduction and Development

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Abstract.The relationship between donor cell cycle and the developmental ability of somatic cell nuclear transfer (SCNT) embryos has not fully been elucidated. Donor cells that are usually prepared by serum starvation or confluent-cell culture for SCNT represent a heterogeneous population that includes mainly G0 phase cells, other cells in different phases of the cell cycle and apoptotic cells. In this study, we compared the developmental ability of porcine SCNT embryos reconstructed from G0 phase cells (G0-SCNT embryos) and strictly synchronized-G1 phase cells (G1-SCNT embryos), and examined the developmental rates and timing of first DNA synthesis. The G0 phase cells were synchronized by confluent culture, and the G1 phase cells were prepared from actively dividing M phase cells. The G1-SCNT embryos showed a significantly higher (P<0.05) developmental rate to the blastocyst stage per cleaved embryo (59%) than the G0-SCNT embryos (43%). Moreover, initiation of first DNA synthesis and cleavage occurred significantly earlier in the G1-SCNT embryos than in the G0-SCNT embryos. Delay of initiation of first DNA synthesis in the SCNT embryos by aphidicolin resulted in decreased developmental rates to the blastocyst stage without any effect on cleavage rates. Our data demonstrates that synchronized-G1 phase cells can be used as donor cells for SCNT embryos and that earlier initiation of first DNA synthesis may be important for subsequent development of SCNT embryos. The SCNT system using G1-synchronized cells, in terms of their highly uniform and viable cell states, can be useful for studying the reprogramming processes and embryonic development of SCNT embryos.

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Cell confluency is as efficient as serum starvation for inducing arrest in the G0/G1 phase of the cell cycle in granulosa and fibroblast cells of cattle

Cited by 67 publications

References 30 publications

Cell cycle analysis and interspecies nuclear transfer of in vitro cultured skin fibroblasts of the Siberian tiger (Panthera tigris Altaica)

Cell cycle analysis and interspecies nuclear transfer of in vitro cultured skin fibroblasts of the Siberian tiger (Panthera tigris Altaica)

High developmental potential in vitro and in vivo of cattle embryos cloned without micromanipulators

Effects of Synchronization of Donor Cell Cycle on Embryonic Development and DNA Synthesis in Porcine Nuclear Transfer Embryos

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