Nuclear-cytoplasmic interactions in rat oocytes and reconstructed eggs derived by somatic cell nuclear transfer.

摘要

Somatic cell nuclear transfer (SCNT) enables the generation of transgenic animal models from genetically modified cells and it is a potential alternative to the ES cell technology, which is still not established yet in rats. However, there are many problems to resolve to establish stable protocols for SCNT in rats. The objectives of this study are (1) to investigate the possible reasons and mechanism of rat spontaneous oocyte activation, (2) to optimize development of parthenogenic and SCNT-derived oocytes in rats by investigating the patterns of nuclear changes, the changes of MPF and MAP kinase activities, and in vitro embryo development after various activation treatments, and (3) to investigate optimal cell cycle coordination between donor cells and recipient oocytes for rat SCNT and to analyse the expression of genes and proteins involved in the cytoskeleton of in vitro cultured reconstructed eggs.; Soon after exposure to an in vitro environment, ovulated rat oocytes are activated spontaneously; this spontaneous oocyte activation is characterized by resumption of meiotic division followed by the cytoplasmic scattering of chromosomes. Neither in vivo aging in oviducts after ovulation nor hyaluronidase treatment affected spontaneous oocyte activation. L-type calcium channel blocker, IP3R inhibitor and inhibitor of calcium/calmodulin-dependent kinase II (CaMKII) prevented spontaneous oocyte activation in calcium-containing medium. The activity of CaMKII increased at 20 min and remained high for 30 min followed by decreased activity by 60 min after oocyte recovery. Constitutively active CaMKII was localized close to the meiotic spindle after oocyte recovery. Our findings indicate that rat oocytes are very sensitive to extracellular calcium in vitro conditions and CaMKII is one of the upstream signals that activate rat oocytes spontaneously after recovery.; Oocyte activation is an essential step in successful cloning by SCNT. In our study, oocytes were activated by electrical stimulation (EST) alone or in combination with 6-dimethylaminopurine (DMAP), cycloheximide (CHX)/cytochalashin B (CB), and roscovitine (ROS)/CB. All combination groups effectively induced inactivation of MPF activity. The patterns of MAP kinase varied in different treatment groups. DMAP induced faster inactivation of MAP kinase than CHX/CB and ROS/CB treatment groups. CHX/CB-treated oocytes showed synchronous nuclear breakdown and cleavage after activation treatment, whereas DMAP and ROS/CB treated groups showed asynchronous patterns. Although in vitro development to the blastocyst stage was efficient after parthenogenesis, development of SCNT-derived embryos was arrested at 2-cell stage in all regimens examined.; The procedure of micromanipulation, coordination of cell cycle between donor nuclei and recipient oocytes, and artificial oocyte activation are very important steps in the procedure for cloning animals. Metaphase II (MII) stage and pre-activated telophase II (TII) stage oocytes were used as a recipient cytoplasm with GO/G1, M, and S/G2-phases donor cells. Moreover, pronuclear and 2-cell stage blastomeres derived from SCNT were used as donor cells with enucleated zygotic and parthenogenetic ooplasts for serial cloning. No significant difference in cleavage rate was observed among activation groups after SCNT. M-phase donor cells had a significantly higher cleavage rate than G0/G1-phase donor cells with MII oocytes and G2-phase donor cells with TII oocytes. However, no reconstructed embryo was able to develop beyond the 2-cell stage during in vitro culture. Moreover, reconstructed embryos cultured in vivo, i.e. after transfer to the oviduct of surrogate females, were also unable to develop further. To better understand the causes of developmental arrest, reconstructed 2-cell stage embryos were analyzed to examine the distribution of cytoskeletal proteins and transcription of mRNAs. Abnormal microtubule distribution and downregulated expression o