| There is some evidence that gap junction intercellular communication has been built early before implantation. As the only distinct communication pathway between adjacent cells, connexins exist widely during mammalian preimplantation embryo development. However, the diversity and the temporal and spatial expression of connexins family members make the functional research for a single connexin has been complex and difficult. In this research, RT-PCR, Real-time PCR, immunochemistry and western blot, combined with in vitro fertilization technique, had been employed to detect the diversity of connexins expressed in ovine preimplantation embryos and the expression profiles of connexin43 and connexin45. Second, small molecular fluorescent dye was injected into single blastomere to observe the onset of gap junction intercellular communication. Then, connexin43 and connexin45 dsRNA were injected into zygote to explore the function of these two connexins during ovine preimplantation embryos in vitro. 1 Expression of connexin26, connexin31, connexin32, connexin40, connexin43 and connexin45 mRNA in oocytes and preimplantation embryos by RT-PCRPCR primers of all six connexins were designed according to sequences published on NCBI. Partial cDNA fragment were gained from RT-PCR and confirmed by sequence blast. Then, connexin26, connexin32, connexin43 and connexin45 had been found to express during oocyte maturation and embryos development by RT-PCR, and connexin31 and connexin40 had not been detected.2. The expression profiles of connexin 43 (Cx43) and connexin 45 (Cx45) transcripts and protein.2.1 Detection by Real-time PCRThe results indicated that Cx43 and Cx45 expressed in mature oocytes higher than immature ones. During embryos development, Cx43 transcript was gradually increased from 2-cell to 8-cell stage and maintaining higher mRNA level at morula and blastocyst stages; the expression level of Cx45 gene was not so obvious in each period of embryonic development.2.2 Detection of Cx43 and Cx45 in oocytes and preimplantation embryos by immunochemistry.The results showed that Cx43 and Cx45 proteins existed in immature oocytes, mature oocytes and embryos at all the developmental stages. After antibody staining, green fluorescent that denoted connexins could be observed near the cell membrane, slight fluorescent trace could be found in cell plasma and no trace in cell nucleus. The patterns of expression of Cx43 and Cx45 protein in embryos produced in vivo were similar to the embryos produced in vitro. It seemed that more abundant transcripts of Cx43 and Cx45 had been detected in embryos derived in vivo than that in embryos derived in vitro, and higher transcripts volume in the former always indicated better embryo quality.2.3 Expression of Cx43 detected by westernblot.Three confirmation of Cx43 were detectable by westernblot, containing two phosphorylation forms (PI and P2) and one disphosphorylation form (PO). Total volume of Cx43 expressed higher in mature oocytes compare to immature ones. The abundant of P1 and P2 expressed higher in 2-cell and 8-cell stages. P0 began to increase at morula, then a bit decrease in blastocyst stage. That might be attributed to gap junction gating correlated with some physiological function.3.Observation of gap junction intercellular communication in ovine preimplantation embryos derived in vitro.Lucifer yellow, a small molecular fluorescent dye, was injected into single blastomere of embryos at 4-cell,8-cell and morula stages by microinjection, respectively. The results showed that no dye diffused to adjacent cells could be observed when Lucifer yellow was injected to 4-cell or 8-cell. Then the diffusion phenomenon could be observed at morula stage. It revealed that gap junction intercellular communication began after 8-cell stage.4. Effects of Cx43 and Cx45 on preimplantation development4.1 Effects of dsRNA injection on gene expression of preimplantation embryosCx43 dsRNA, Cx45 dsRNA and water were injected into zygotes, respectively. Control group was cultured without injection. The results detected at blastocyst stage indicated the amounts of Cx43 and Cx45 transcripts had been decreased to 26% and 56% compared to control group, respectively. The abundant of housekeeping gene and relative genes showed no obviously difference among experiment groups, it is indicated that the synthetic dsRNA we used were specific and efficient. The protein levels were estimated by westernblot and immunochemistry. Westernblot result showed that protein level of Cx43 dsRNA injected group decreased visible compared to water and control groups, and results of immunochemistry showed that the fluorescent intensity of both dsRNA injected groups displayed not as brightness as water and control groups after staining.4.2 Effects of Cx43 and Cx45 dsRNA injection on cleavage rates of preimplantation embryos.The cleavage rates of embryos were statistical at 48 hours after culture in vitro. Clevage rates of embryos were 83.7%,84.5%,81.5%; 2-cell rates were 5.6%,4.7%, 4.6%; 4-cell rates were 14.6%,10.8%,10.3% and 8-cell rates were 64.5%,68.9% and 66.6% from the Cx43 dsRNA injected group, water group and control group, respectively. The statistical datas of Cx45 were showed as Cx43 were 81.7%,76.9%, 77.5%; 7.6%,3.6%,3.8%; 13.9%,11.6%,7.2% and 60.3%,61.8%,66.5%, respectively. Except 2-cell rates of Cx45 dsRNA injected group were significantly higher than that of water and control groups (P<0.01) and 4-cell rates of Cx45 dsRNA injected group and water group were significantly higher than that of control group (P<0.01), there were no significantly differences between the rest groups (P>0.05). However, microinjection of Cx43 or Cx45 dsRNA could not affect the total cleavage rates (P>0.05).4.3 Effects of Cx43 and Cx45 dsRNA injection on development rates and embryo qualities of preimplantation embryos.The in vitro development competence of 4-and 8-cell stages embryos was assessed at day 8 after fertilization, and the hatched rate was assessed at day 8-10. The results indicated that blastocyst and hatched blastocyst rates of 4-and 8-cell stages embryos were 20.3%,21.7%and 34.5%and 19.2%,37.5%and 41.3%from the Cx43 dsRNA group, water group and control group, respectively; there was no significant difference of blastocyst rates in groups(P>0.05), but hatched blastocyst rate in control and water group were significantly higher than Cx43 deRNA group (P<0.01). Blastocyst rates and hatched blastocyst rates of Cx45 groups were 20.8%, 19.4%,32.5%and 18.5%,25.0%,34.9%, respectively; there were no significant difference between every two groups (P>0.05). Cell numbers and means of dead cell rates of blastocyst were 74,76,83 and 24.6%,15.4%,18.2%of Cx43 groups and 79, 71,72 and 21.1%,17.9%,15.7%, respectively; no significant difference existed in every two groups (P>0.05). The results showed that Cx43 could affect the quality but did not affect the developmental rate of ovine embryos derived in vitro; Cx45 did not affect both the quality and the developmental rates of ovine embryos derived in vitro.
|
PDF Full Text Request