[Effects of assisted-electroacupuncture on recovery of fast tracking anesthesia in mPCNL].

OBJECTIVE: To compare the effects on anesthesia recovery between assisted-electroacupuncture fast tracking anesthesia and simple fast tracking anesthesia in patients with minim ally invasive percutaneous nephrolithotomy (mPCNL). METHODS: Eighty cases of mPCNL were selected and randomly divided into a treatment group and a control group. Fentanyl (1-2 microg/kg), sevoflurane (8%) and rocuronium (0.5 mg/kg) were applied to perform anesthesia induction in both groups, and endotracheal inhalation of sevoflurane and intravenous pump injection of remifentanil were adopted to main anesthesia status during the operation. 20 min before anesthesia induction, bilateral Neiguan (PC 6), Neimadian, Hegu (LI 4), Yangxi (LI 5), Zhongji (CV 3), Qichong (ST 30), Zuwuli (LR 10) were selected and punctured in the treatment group, and elecctroacupuncture was given after arrival of qi until 30 min after the wake-up from anesthesia and withdrawal of endotracheal tube. The dosage for anesthesia maintenance, recovery time of awareness, extubation time, incidences of nausea, vomiting and chill and irritation of urethral catheters were observed and recorded. RESULTS: (1) The dosages of remifentanil and sevoflurane in the treatment group during the operation were obviously less than those in the control group [remifentanil: (5. 27 +/-1.23) micro g/kg h vs (7.35+/-1.70) micro g/kg . h; sevoflurane: (1.12+/-0.43) vol% vs (2.35+/-0.87) vol% , both P<0. 001]. (2) The recovery time of awareness and extubation time in the treatment group were significantly earlier than those in the control group [recovery time of awareness: (5.65 +/- 2.34) min vs (8. 87 +/- 6. 84) min, P<0. 01; extubation time : (7. 23+/-4. 35) min vs (10. 62+/-8. 16) min, P<0. 05]. (3) The incidences of nausea, vomiting and chill in the treatment group were significantly less than those in the control group (all P<0. 05). (4) The irritation of urethral catheters on urethra in the treatment group was significantly less than that in the control group (P<0. 001). CONCLUSION: The assisted-electroacupuncture anesthesia could reduce the dosage of remifentanil and sevoflurane in mPCNL fast tracking anesthesia in urinary surgery, reduce the incidences of nausea, vomiting, chill and irritation of urethral catheters during recovery stage, and prompt recovery of mPCNL patients.

Number of blastomeres and distribution of microvilli in cloned mouse embryos during compaction.

The events resulting in compaction have an important influence on the processes related to blastocyst formation. To analyse the quality of the embryos obtained by somatic cell nuclear transfer (SCNT) in aspects different from previous studies, not only the number of blastomeres of cloned embryos during the initiation of compaction, but also the distribution of microvilli in cloned, normal, parthenogenetic, and tetraploid embryos before and after compaction was preliminarily investigated in mouse. Our results showed that during compaction the number of blastomeres in SCNT embryos was fewer than that in intracytoplasmic sperm injection (ICSI) embryos and, before compaction, there was a uniform distribution of microvilli over the blastomere surface, but microvilli became restricted to an apical region after compaction in the four types of embryos. We also reported here that the time course of compaction in SCNT embryos was about 3 h delayed compared with that in ICSI embryos, while there was no significant difference between SCNT and ICSI embryos when developed to the 4-cell stage. We concluded that: (i) the cleavage of blastomeres in cloned embryos was slow at least before compaction; (ii) the distribution of microvilli in cloned, normal, parthenogenetic, and tetraploid embryos was coherent before and after compaction; and (iii) the initiation of compaction in SCNT embryos was delayed compared with that of ICSI embryos.

[Regulation of compaction initiation in mouse embryo].

Developmental events in preimplantation mouse embryos include the first cleavage, the activation of the embryonic genome, the compaction of the blastomeres to form morula (MO), and the formation of the blastocyst (BL). Compaction, the first cell differentiation event in mammalian development, occurs at the late eight-cell stage in the mouse and may be described in terms of some types of morphological change, which involve reorganization within a cell and intercellular reorganization. Surface microvilli became restricted to a few basal sites and to an externally facing (apical) pole. Prior to compaction, the blastomeres are spherical and lack specialized intercellular junctions. During compaction, the cells were flattened against one another, thus maximizing intercellular contact and obscuring intercellular boundaries. It is believed that the events of compaction have an important influence on the processes involved in blastocyst formation, namely the initiation of inner cell mass and trophectoderm differentiation. The inner cell mass will form the future embryo proper, whereas the trophectoderm cells will form only extraembryonic tissues. Compaction is initiated by E-cadherin mediated cell adhesion, which is regulated post-translationally via protein kinase C. With E-cadherin knock-out, maternal E-cadherin is able to mediate the compaction process at the morula stage. Initial adhesion is mediated by homophilic interactions between E-cadherin extracellular domains.In this review, we attempted to describe this process in detail.

[Dynamic changes of gamma-tubulin in preimplantation development of parthenogenetic mouse embryos.].

Tubulin is the major protein of microtubule. alpha- and beta- tubulins form heterodimers, while gamma-tubulin regulates microtubule organization. The present study aimed to observe the dynamic changes of gamma-tubulin in preimplantation development of parthenogenetic mouse embryos. Immunofluorescence and laser confocal microscopy were used to detect the location of gamma-tubulin in preimplantation parthenogenetic embryos activated by SrCl2. The oocytes were collected at 13-14 h after hCG injection, and then activated with 10 mmol/L SrCl2 in Ca(2+)-free CZB medium with 5 mmol/L cytochalasin B (CB), fixed at 1 h intervals until 6 h after activation. The results showed that spindle was paralleled with the cell membrane all the time, when the meiosis of MII mouse oocytes resumed. The rotation of spindle was inhibited, but karyokinesis was not influenced. At 0 h after activation, i.e. at metaphase, gamma-tubulin was distributed mainly on the two poles of spindle. At 1 h after activation, i.e. at anaphase, following the separation of chromosomes, gamma-tubulin was transformed from dense to disperse. At 2 h after activation, gamma-tubulin was localized between the segregated sister chromatids at telophase. However, at 3-6 h after activation, gamma-tubulin concentrated around the two female pronuclei during their formation and juxtaposition. Moreover, another group of MII oocytes were activated for 6 h and cultured in droplets of KSOM medium under mineral oil in 5% CO2 in air at 37 degrees C to permit parthenogenetic development. The embryos were collected and fixed at 3 h, 14 h, 16 h, and 18 h of culture. At 3 h after culture, i.e. at mitotic interphase, it was shown that amorphous gamma-tubulin distributed around the nuclei of early parthenogenetic embryos. At 24 h after culture, i.e. at prometaphase, gamma-tubulin migrated along the spindle microtubule to the two poles. Our results showed that gamma-tubulin had similar location patterns at metaphase, anaphase and telophase in meiosis and mitosis. It was concluded that gamma-tubulin assembly in parthenogenetically activated oocytes facilitated the formation of negative pole cap and the stabilization of microtubule, thus promoting the spindle formation at meiosis and mitosis. The relocation of gamma-tubulin at anaphase and telophase might be induced by the event of segregation of homologous chromosome being pulled away by the spindle. gamma-tubulin might contribute to the migration and juxtaposition of the two female pronuclei as well.

[Location and role of protein kinase Cα in parthenogenetic and tetraploid preimplantation embryonic development in mouse].

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.].

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.