| Trophoblasts (TR)are specialized cells of the placenta and play an important role in embryo implantation. The development and differentiation of TR cells is regulated by a variety of molecules and pathways, which is essential to reveal the mechanism involved in the embryonic development. The in vitro culture of trophoblasts provided an important tool to investigate the mechanisms of implantation, and the best model for placenta development and differentiation is trophoblast stem cells (TSC), as they are precursor cells of TR lineage. However, TSC were established only in a few species, including mice, porcine TSC has not been reported. Several researches reported that Pig TR cells were derived from in vivo preimplantation embryos, but the describtion without comprehensive. Researches showed that ROCKs (Rho-associated coiled coil-protein kinases), as a downstream effector of Rho, participated in migration, proliferation and apoptosis of trophoblast cells. Therefore, it is likely that ROCKs has a role in the function and differentiation of TR cells. In the present study, the differences of pig in vitro fertilized (IVF) and parthenogenetically activated (PA) blastocysts were compared, and the two type blastocysts were cultured in medium supplemented with KnockOut serum replacement (KOSR) and basic fibroblast growth factor (bFGF) on STO feeder layers. The derived pIVFTR and pPATR cells were characterized by alkaline phosphatase assay, TSC, TR and pluripotent related genes or proteins detection, analysis of chromosome number and differentiation ability. The difference of pIVFTR and pPATR cells was investagated. We also analyzed the effects of ROCK inhibiter Y-27632 on pig TR cells. The main results are as below:1 The preparation of pig PA and IVF embryo and the quality comparison of blastocystsThe quality of pig PA and IVF blastocysts was analyzed before they used for the derivation of trophoblast cells. The results showed that the development of pig IVF and PA blastocysts was synchronous, with the formation of 4 cells on 2d,6-8 cells on 3d and expanded blastocysts on 6d. The ratio of cleavage and blastocyst was no significant differenc. But the average diameter of IVF blastocysts significantly greater than the PA blastocyst (p< 0.01), and cells number of whole IVF blastocysts (Total) and inner cell mass (ICM) was significantly more than the PA blastocyst (p< 0.05). The ICM/Total ratio was higher in IVF blastocysts and the TE (trophectoderm)/Total ratio was higher in PA blastocyst. The proteins OCT4, CDX2 and SOX2 were expressed in both IVF and PA blastocyst and no obvious difference. These results demonstrated that the quality of IVF blastocysts was better than that of PA blastocysts, and the essential difference of blastocysts cell numbers was in ICM. Since PA blastocysts had no difference with IVF blastocysts in morphology, development rate and the expression ofkey pluripotent factors, high quality PAblastocysts canbeused to culture trophoblast cells as IVF blastocysts.2 The culture and identification of trophoblast cells from pig PA and IVFThe zona pellucida of PA and IVF blastocysts were removed and cultured in the system of KOSR/bFGF medium and STO feeder layers to derive TR cell. The pPATR and pIVFTR cells can be cultured in vitro for a long-term. The TR cells were epithelium-like and contained abundant lip id droplets. TR related genes (TEAD4, CDX2,ELF5,HAND1, CDH3, GATA3,ETS2,MASH2,PAG,KRT18and GCM1)and telomerase activity related genes (TERC and TERF2) were detected in pig TR cells, but the expression level of some genes (CDX2,ELF5,KRT18,HAND1, GCM1 and PAG) was different between pPATR and pIVFTR cells. Imprinted genes IGF2, PEG1 and PEG10, expressed lower in pPATR than in pIVFTR cells. Alkaline phosphatase and anti-KRT7, KRT18, CDX2 and SSEA1 antibodies were positive stained in pPATR and pIVFTR cells. The karyo type of pPATR were tetraploid or aneuploid and pIVFTR were normal diploid. In addition, pPATR and pIVFTR had the ability to differentiate into mature cells, when the cells cultured in the differentiation media.3 The effect of ROCK inhibitor Y-27632 on porcine trophoblast cellsThe growth of TR cells cultured as single cells was improved by ROCK inhibitor Y-27632. Growth curve and average cell colony area curve showed the TR growth were significantly improved by Y-27632, but the growth of control cells (pig fetal fibroblast) was supressed. Furthermore, we found the expression of ROCK1 and ROCK2 were suppressed, the trophoblast related genes were up-regulated, and CDX2 protein was remarkly increased in Y-27632 treated TRpig cells.Taken together, the pig PA and IVF blastocysts both can be used to derive the TR cells; pig TR can be obtained using KOSR/bFGF medium and STO feeder layer, and these cells had the typical TR features and some features of TSC; ROCK inhibitor Y-27632 has an effect of promoting pig TR growth and stimulateing trophoblast gene expression. These results could be a reference for exploring the better culture conditions of pig TSC or TR cells and provided an alternative tool for the reseash on placenta development and differentiation. |
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