| Bone morphogenetic protein15(BMP-15), which has the similar biological function asthe Growth differention factor-9(GDF-9), is a member of the transforming differentiationfactor-β (TGF-β) family. Studies have shown that BMP-15specifically expressed in oocyteand it plays a very important role in the growth and development of female germ cells. In thisstudy, BMP-15and GDF-9expression were identified in9porcine tissues and4differentcells. We also constructed the BMP-15and GDF-9reporter plasmid, and then detected theactivity of them via transant transfection in CHO, porcine AFSC and C2C12. Besides that,Reporter gene fragment was injected into GV stage oocyte by micromanipulation to test theexpression of BMP-15in vitro maturation. Because of its tissue specificity, BMP-15isconsidered as a biological marker to make in vitro study of female germ cells more easily.Porcine amniotic fluid stem cells (AFSC) are an Oct4-positive pluripotent stem cells. It hashigh differentiation potential, with a strong proliferation ability and other advantages.Therefore they can be satisfactory biological materials to derive oocyte-like cell (OLC) invitro. By building a stable transfection cell line via pBMP15-EGFP, we got a moniter cell totrace the differentiation process into OLC. Morphology observation, RT-PCR, andimmunofluorescence techniques verified our suspect that BMP-15can be a useful monitor invitro.1. Tissue-specific expression of BMP-15We detected the BMP-15and GDF-9expression in9porcine tissues, including ovary,testis, stomach, liver, muscle, kidney, heart, thymus and lung by RT-PCR method. The resultsshowed that BMP-15was more tissue-specific than GDF-9. It only highly expressed in ovary.RT-PCR detection of BMP-15and GDF-9in porcine oocyte, Chinese hamster ovary cell(CHO), mouse myoblasts cell (C2C12) and porcine AFSC showed that BMP-15only detectedin oocyte and CHO, while GDF-9could also expressed in porcine AFSC. Thus, BMP-15could be a better marker to track the differentiation process of porcine AFSC into OLC. Thenthe immunohistochemistry results illustrated that BMP-15expressed in the maturation porcessof oocyte.2. pBMP15-EGFP, pGDF9-EGFP report vector construction and activity detectionThe cloned BMP-15and GDF-9promoter fragments from porcine genome (2.2kb and 1.7kb) were linked with pEGFP-1vector to construct the pBMP15-EGFP and pGDF9-EGFPreport vectors, repsectively, which were then transfected into CHO, C2C12and porcineAFSC. The results showed that BMP-15promoter have no effect in porcine AFSC, whileGDF-9promoter has the opposite effect. This testified in cell biology that BMP-15is feasibleto be a tracer. To further validate the expression of BMP-15in the process of oocytematuration and parthenogenetic activation, BMP15-EGFP DNA fragment was injected in GVstageoocyte, then observe GFP under fluorescence microscope. The results indicated thatgreen fluorescence positive cells were observed in vitro maturation9h,18h,27h oocytes.However, in2and4-cell stage embryos, the fluorescence intensity decreased significantly,indicating that BMP-15no longer play a major role in the parthenogenetic period.3. BMP-15traces porcine AFSC differentiation into OLCThe pBMP15-EGFP stable transfected porcine AFSC line (BMP15-AFSC) was screenedin this research. Then we specifically induced the cell into OLC. Green fluorescenceexpression and morphological changes were observed by fluorescence microscopy. Theresults showed that in4days differentiation, GFP can be seen in fibroblast-like cells; while in8days, cell became round with green fluorescent protein in; in the12days, most cells areround with high GFP expression. Immunofluorescence results showed that SCP3graduallyappear in the induction process. RT-PCR results showed that12days induced cells expressreproductive marker such as c-MOS, FSHR, p450c17, StAR, ZP3and DAZL. To sum up,these results illustrated that the BMP-15report vector could be used as an oogenesis marker totrack porcine AFSC differentiation into the oocyte. |
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