Location and role of protein kinase Cα in parthenogenetic and tetraploid preimplantation embryonic development in mouse / 生理学报

Protein kinase C (PKC) is a critical molecule in cellular signal transduction in mammals. It is involved in many biological processes in embryonic development, including nuclear remodeling, cell cycle adjustment and cellular polarity regulation. The present study aimed to observe the location of PKCα, an important isozyme of PKC, in fertilized, parthenogenetic and tetraploid preimplantation embryos, and compare the expression of PKCα during embryonic compaction in Kunming mice. The location of PKCα was detected by immunochemistry and laser confocal microscopy. Western blot was performed to quantify PKCα expression during embryonic compaction in the three kinds of embryos. In the experiment, fertilized embryos were flushed from oviduct or uterus at 45, 52, 69, 76 and 93 h after injection of human chorionic gonadotrophin (hCG); parthenogenetic embryos were collected by SrCl2 activation of oocytes for 6 h; and tetraploid embryos were produced by electrofusion of 2-cell embryos. Embryos were fixed at different developmental stages for immunofluorescent staining. 8-cell/4-cell embryos and morula were lysed for Western blot. The results showed that PKCα had similar location pattern in different embryos. It was distributed mainly in the nuclear aggregating around chromatin at different developmental stages. However, PKCα expressed strongly in the interphase than in mitotic blastomere. Before embryonic compaction, PKCα was localized at the blastomere boundary. At late blastocyst stage of fertilized embryos, PKCα was localized only in the polar trophoblast, but not in other trophoblast. At late stage of pathenogenetic blastocyst, there was no clear PKCα signal in the polar trophoblast. Tetraploid embryos had larger blastomere than other embryos and compacted after 4-cell stage, but not after 8-cell stage. Meanwhile, there was PKCα signal at the blastomere boundary at 4-cell stage. Our results showed that the expression of PKCα lasted through all the preimplantation stage. Although there were different expression levels among different stages, the expression increased around embryonic compaction. Quantification of expression of PKCα by Western blot demonstrated that the expression increased after compaction, indicating that the compaction was possibly dependent on the relocation of PKCα. Moreover, it was shown that the second relocation of PKCα occurred during the blastocyst formation. PKCα had different expression patterns in the three kinds of preimplantation embryos. However, the effects of PKCα on embryonic development started in early stage. There must be a necessary connection between PKCα relocation and cell adhesion starting at embryonic compaction.

Dynamic changes of microtubule in parthenogenetic and in vitro fertilized preimplantation embryos in mouse / 生理学报

In this study we detected dynamic changes and function of beta-tubulin, a subtype of microtubule, during the first cleavage period in mouse parthenogenetic and in vitro fertilized embryos. Firstly, we compared the developmental potential of in vitro fertilized, parthenogenetic, and in vivo fertilized embryos in culture. Then, the dynamic changes of beta-tubulin and nucleus in parthenogenetic and in vitro fertilized preimplantation embryos were detected by immunofluorescence and confocal microscopy to analyze the role of microtubules in meiotic division and embryonic development. The results indicated that the development rate of in vivo fertilized embryos was significantly higher than that of in vitro fertilized or parthenogenetic embryos (P<0.05). However, there was no significant difference in developmental potential between in vitro fertilized and parthenogenetic embryos. During in vitro fertilization, oocyte was activated when sperm entered it. Oocyte resumed the second meiotic division. Condensed maternal chromosomes aligning at the equator of the spindle were pulled to the spindle poles by kinetochore microtubules in anaphase. Furthermore, in telophase, there were microtubules between the two sets of decondensed maternal chromosomes. One set formed the second polar body (Pb(2)), which was extruded to the perivitelline space. The other set formed female pronucleus. Meanwhile, 5-8 h after fertilization, sperm chromatin condensed and decondensed to form male pronucleus. Microtubule composed mesosome and cytaster remodeling around male and female pronuclei to form long microtubules, which pull the pronuclei to get close. During 4-6 h parthenogenetic activation, SrCl(2) activated oocytes to resume meiosis. As a consequence, sister chromatids were pulled to spindle poles. Cytochalasin B, which was applied in the medium, inhibited the extrusion of Pb(2). Two haploid pronuclei in the cytoplasm were connected by microtubules. Compared with that in in vitro fertilization, oocyte is easier to be activated in parthenogenetic activation. Chemical activation is more efficient than sperm penetration in in vitro fertilization as indicated by earlier and better remodeling of the microtubules.