| Pigs are more similar to humans regarding their genomic, metabolic physiology and body size. The investigation on porcine embryonic stem cell could be helpful for therapy and research in human disease. Establishment of porcine haploid parthenogenetic embryonic stem cell could make the clone of oocyte come true, then make an approach in therapy for human sterility and yeld disease resulted from gamets ungenesis. It also could become a tool for research on genomic imprinting and improvement of animals cloning efficiency. Establishment of embryonic stem cell from porcine IVF embryos could be used not only for investigation on cells transfer to recipients with organ pathological changes and damnification but also for medicine screening. It also could offer target cells for producing gene knockout animals.In this study, the effects of different basic medium, addition of small moleculars, LIF and bFGF and different feeder cells on the establishment of porcine haploid parthenogenetic embryonic stem cells and IVF embryonic stem cells were investigated. This study consisted of four parts.1. We compared different methods for activating porcine oocyte, in order to obtain high efficient method to produce porcine haploid parthenogenetic embyos.2. The method which could result in high percentage haploid parthenogenetic embyos was used to produce parthenogenetic embyos, and these embryos were used for culture of haploid parthenogenetic embryonic stem cell.3. The IVF embryos were produced with conventional method and used for culture of porcine embryonic stem cell.4. The development rate and quality of parthenogenetic and IVF blastocysts were compared, in order to elucidate the potential reasons resulted in different competences of these two kinds of embryos to attach the dish and outgrowth cell clone. The results were as follows:1. The different physical and chemical methods were applied to activate MII porcine oocytes. In experiment 1, electrical activation, a combination of electrical activation and cytochalasin B (CB), and ethanol activation at different concentrations and durations were used to activate porcine oocytes. In experiment 2, electrical activation followed by MG-132 or Thimerosal/DTT treatments were investigated for their effects on porcine oocytes activation. The results of experiment 1 indicated that the development rates of blastocysts in CB group (27.34%) were significantly higher than that of electrical activation alone (16.92%) (P<0.05), and both of them were significantly higher than that of all ethanol groups (P<0.05). In all ethanol groups, 9% ethanol treatment for 11min got the highest blastocyst rate (10.20%). The results of experiment 2 showed that the rate of blastocysts formation in MG-132 group (24.69%) was not significantly different with that of CB group (27.50%), but they were significantly higher than that of Thimerosal/DTT (14.10%) and electrical activation (17.5%) groups (P< 0.05). The ploidy analysis of blastocysts derived from different groups indicated that the percentages of haploid blastocysts in electrical activation,9% ethanol and Thimerosal/DTT group were 41.7%,40% and 35.3% respectively, they were significantly higher than that of CB (0) and MG-132 (6.7%) groups (P<0.05).Considering both blastocysts formation rate (out of oocytes) and haploid rate (out of total blastocysts), the final haploid blastocyst percentages (out of oocytes) in electrical activation,9% ethanol, Thimerosal/DTT and MG-132 groups were 7.3%(17.5%*41.7%),4.1%(10.2%*40%),5.0%(14.1%*35.3%) and 1.7% (24.69%*6.7%), respectively. The electrical activation might be the best activation way to obtain haploid embryos.2. The parthenogenetic embryos were removed of zona pellucida and cultured in different media with different feeder cells. The attachment rates of embryos cultured in mediumâ… (DMEM+bFGF+LIF+nucleoside mix+FBS) with MEF as feeder cells were 0. The attachment rate of embryos cultured in mediumâ…¡(bafflo rat liver cell conditioned TCM-199) without feeder cells was 21.7%, but the attached cells differentiated apparently and they did not form clone. Adding LIF in this medium reduced the attachment rate to 9.3%, and no clone was formed. Embryos did not attach when cultured in this medium with MEF as feeder cells. When the embryos were cultured in mediumâ…¢(DMEM/F12+Neurobasal medium+N2B27+ PD0325901+CHIR99021) with MEF or L-cell as feeder cells, the attachment rates were 0. When the mixation of 1/2MEF+1/2L-cell was used as feeder cells, attachment rate was 23.1%. The attached cells did not form clone, though they did not differentiate. The attachment rate of embryos in mediumâ…£(DMEM/F12 +KSR+bFGF) with MEF as feeder cells was 0. The attachment rate of embryos in mediumâ…¤(DMEM+HAM'S-F10+NUTRIENT MIX+LIF+bFGF+KSR+FBS) with MEF as feeder cells was 10%, but the attached cells did not form clone.3. Four kinds of different media (â… ,â…¡,â…¢andâ…£mentioned above) were used to culture IVF embryos. The attachment rate of IVF embryos cultured in mediumâ… with MEF as feeder cells were 0. The attachment rate of IVF embryos cultured in BRL conditioned medium without feeder cells was 37.5%, but the attached cells differentiated apparently and they did not form clone. The attachment rate of embryos cultured in mediumâ…¢with 1/2MEF+1/2L-cell as feeder cells was 27.8%, the attached cells did not form clone. The IVF embryos were cultured in mediumâ…£with MEF or STO as feeder cells. The attachment rate of embryos cultured were 22% and 25% respectively. The rate of embryos formed primary clone was 63.6% and 75.0% respectively. Three attached embryos in MEF group passaged for 3 times. One attached embryo in STO group passaged for 1 time. The cells attached on both MEF and STO were detected positive by AKP staining. The attached cells on MEF were detected positive by Oct-4 and SSEA-1 staining.The results shown that the attached cells were porcine embryonic stem cell-like cells.4. The parthenogenetic and IVF embryos were compared for their development rates, quality and immuno fluorescence staining of blastocyst. The blastocyst formation percentages were not significantly different between parthenogenetic and IVF embryos, they were 28.7% and 25.9% respectively. The cell number of blastocysts did not show significant difference either, they were 51 and 49.Both parthenogenetic and IVF embryos shown positive Oct-4 staining.The results shown that neither poor quality nor the lack of pluripotence could result in the poor attachment and outgrowth competence of parthenogenetic embryos. |
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