Nakajima Noriaki, Inomata Tomo, Ito Junya, Kashiwazaki Naomi
Laboratory of Animal Reproduction, Graduate School of Veterinary Science, Azabu University, Sagamihara, Kanagawa, Japan.
Cloning Stem Cells. 2008 Dec;10(4):461-8. doi: 10.1089/clo.2008.0038.
In several mammalian species including rats, successfully cloned animals have been generated using somatic cell nuclear transfer (SCNT). However, in the case of rats, additional treatment with MG132, a proteasome inhibitor, before enucleation of oocytes seems to be required for successful cloning because ovulated rat oocytes are spontaneously activated, and hence, their suppression is the key to successful cloning. A previous study on rats demonstrated that matured oocytes potentially possess lower cytostatic factor (CSF) activity compared to mouse oocytes, resulting in a low incidence of premature chromosome condensation in the reconstructed embryos after SCNT. It is known that mice having more than two pronuclei are generally observed in nuclear-transferred oocytes after induction of premature chromosome condensation, which implies successful reprogramming. This leads us to the hypothesis that MG132 treatment affects not only the inhibition of spontaneous activation but also the reprogramming and developmental ability of reconstructed rat embryos. If so, prolonged MG132 treatment during and/or after SCNT may further improve the survivability. However, the effect of MG132 treatment on reconstructed embryos after SCNT has been very limited in rats and other species. We show here that prolonged MG132 treatment during and after SCNT improves survival and the number of pronuclei in reconstructed rat embryos after activation. These reconstructed embryos treated before, during, and after SCNT showed significantly higher p34(cdc2) kinase activity involving CSF activity compared to that of the control embryos. On the other hand, p34(cdc2) kinase activity was not recovered in nuclear-transferred oocytes without MG132, which suggested that the enucleation had detrimental effects on the development of reconstructed oocytes. Taken together, MG132 treatment during SCNT increases survival and pronuclear numbers in reconstructed rat embryos via maintenance of high CSF activity. The data suggest that MG132 treatment is indispensable for at least rat SCNT.
在包括大鼠在内的几种哺乳动物中，已经通过体细胞核移植（SCNT）成功克隆出了动物。然而，就大鼠而言，由于排卵后的大鼠卵母细胞会自发激活，因此在卵母细胞去核前用蛋白酶体抑制剂MG132进行额外处理似乎是成功克隆所必需的，所以抑制这种自发激活是成功克隆的关键。先前一项针对大鼠的研究表明，与小鼠卵母细胞相比，成熟卵母细胞潜在地具有较低的细胞静止因子（CSF）活性，这导致在SCNT后重构胚胎中过早染色体凝聚的发生率较低。众所周知，在诱导过早染色体凝聚后，核移植卵母细胞中通常会观察到有两个以上原核的小鼠，这意味着重编程成功。这使我们提出一个假设，即MG132处理不仅影响对自发激活的抑制，还影响重构大鼠胚胎的重编程和发育能力。如果是这样，在SCNT期间和/或之后延长MG132处理时间可能会进一步提高存活率。然而，在大鼠和其他物种中，MG132处理对SCNT后重构胚胎的影响非常有限。我们在此表明，在SCNT期间和之后延长MG132处理时间可提高激活后重构大鼠胚胎的存活率和原核数量。与对照胚胎相比，在SCNT之前、期间和之后接受处理的这些重构胚胎显示出涉及CSF活性的p34（cdc2）激酶活性显著更高。另一方面，在没有MG132的核移植卵母细胞中，p34（cdc2）激酶活性没有恢复，这表明去核对重构卵母细胞的发育有不利影响。综上所述，SCNT期间的MG132处理通过维持高CSF活性提高了重构大鼠胚胎的存活率和原核数量。数据表明，MG132处理至少对大鼠SCNT是必不可少的。
