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Microtubule distribution in somatic cell nuclear transfer bovine embryos following control of nuclear remodeling type
Dae Jin Kwon1, Yu Mi Lee, In Sun Hwang
1College of Veterinary Medicine and Institute of Veterinary Science, Kangwon National University, Chuncheon 200-701, Korea.
Abstract:
This study was conducted to evaluate the microtubule distribution following control of nuclear remodeling by treatment of bovine somatic cell nuclear transfer (SCNT) embryos with caffeine or roscovitine. Bovine somatic cells were fused to enucleated oocytes treated with either 5 mM caffeine or 150 microM roscovitine to control the type of nuclear remodeling. The proportion of embryos that underwent premature chromosome condensation (PCC) was increased by caffeine treatment but was reduced by roscovitine treatment (p < 0.05). The microtubule organization was examined by immunostaining beta- and gamma-tubulins at 15 min, 3 h, and 20 h of fusion using laser scanning confocal microscopy. The gamma-tubulin foci inherited from the donor centrosome were observed in most of the SCNT embryos at 15 min of fusion (91.3%) and most of them did not disappear until 3 h after fusion, regardless of treatment (82.9-87.2%). A significantly high proportion of embryos showing an abnormal chromosome or microtubule distribution was observed in the roscovitinetreated group (40.0%, p < 0.05) compared to the caffeinetreated group (22.1%). In conclusion, PCC is a favorable condition for the normal organization of microtubules, and inhibition of PCC can cause abnormal mitotic division of bovine SCNT embryos by causing microtubule dysfunction.
Insights
Caffeine treatment promotes premature chromosome condensation (PCC) in bovine somatic cell nuclear transfer embryos, aiding normal microtubule organization. Roscovitine inhibits PCC, leading to abnormal chromosome and microtubule distribution during mitotic division.
Area of Science:
- Reproductive Biology
- Cell Biology
- Developmental Biology
Background:
- Somatic cell nuclear transfer (SCNT) is a key technology in assisted reproduction.
- Nuclear remodeling is crucial for successful SCNT embryo development.
- Microtubule organization is essential for accurate chromosome segregation during cell division.
Purpose of the Study:
- To investigate the impact of caffeine and roscovitine on nuclear remodeling and microtubule distribution in bovine SCNT embryos.
- To determine the relationship between premature chromosome condensation (PCC) and microtubule organization in SCNT embryos.
- To evaluate the role of controlled nuclear remodeling in preventing abnormal mitotic division.
Main Methods:
- Bovine somatic cells were fused with enucleated oocytes treated with caffeine or roscovitine.
- Embryos were analyzed for premature chromosome condensation (PCC).
- Microtubule organization was assessed using immunostaining for beta- and gamma-tubulins and confocal microscopy.
Main Results:
- Caffeine treatment increased PCC, while roscovitine treatment decreased it.
- Gamma-tubulin foci from the donor centrosome persisted for at least 3 hours post-fusion in most SCNT embryos.
- Roscovitine treatment resulted in a significantly higher proportion of embryos with abnormal chromosome and microtubule distribution compared to caffeine treatment.
Conclusions:
- Premature chromosome condensation (PCC) is a favorable condition for normal microtubule organization in bovine SCNT embryos.
- Inhibition of PCC by roscovitine can lead to abnormal mitotic division due to microtubule dysfunction.
- Controlling nuclear remodeling through agents like caffeine may improve the developmental potential of SCNT embryos.
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