No abstract

Preimplantation embryos from a variety of mammalian species contrast markedly in their response to culture in vitro. Murine preimplantation embryos display a wider tolerance than other mammalian species to culture environments, and this has contributed to the development of several effective defined culture media. Embryo coculture on somatic cells remains the most effective method of supporting reasonable rates of bovine preimplantation development in vitro. The patterns of gene expression for several antioxidant enzymes during preimplantation murine and bovine development were examined by use of the reverse transcription-polymerase chain reaction technique to determine whether the differential developmental capacity of mammalian preimplantation embryos in culture may reflect variations in the patterns of expression for a series of antioxidant enzymes. Transcripts for catalase, CuZn-containing superoxide dismutase (CuZn-SOD), Mn-SOD, glutathione peroxidase (GPX), and glutamylcysteine synthetase (GCS) were detected in mouse embryos at all stages of development regardless of in vivo or in vitro development. Preimplantation cow embryos produced by in vitro procedures expressed mRNAs for catalase, CuZn-SOD and GPX, whereas transcripts for Mn-SOD were not detected at any stage. GCS transcripts, although present in stages up to the morula, were not detected in cow blastocysts. Analysis of antioxidant gene expression in both bovine primary oviductal cell monolayer cultures and nonattached, ciliated oviductal cell vesicle cultures revealed a constitutive pattern of expression of all five enzymes for the 8-day culture interval. These experiments suggest that differences in gene expression may contribute to the variation in the ability of embryos to develop in vitro with respect to levels of oxygen and dependence on coculture.

The reverse transcription-polymerase chain reaction (RT-PCR) was used to determine the patterns of expression for several growth factor ligand and receptor genes during ovine preimplantation development. Transcripts for insulin-like growth factor (IGF)-I, IGF-II, and the receptors for insulin and IGF-I were detected throughout ovine preimplantation development from the 1-cell to the blastocyst stage. Transforming growth factor alpha (TGF alpha) transcripts were also detected throughout ovine preimplantation development. The mRNAs encoding basic fibroblast growth factor (bFGF) were detected in all stages of the ovine preimplantation embryo, although the relative abundance of this transcript consistently decreased from the 1-cell to the blastocyst stage, suggesting that it may represent a maternal transcript in early sheep embryos. Transcripts encoding ovine trophoblast protein (oTP) were detected only within blastocyst-stage embryos. Primary ovine oviduct cell cultures express the transcripts for IGF-II, IGF-I, TGF alpha, bFGF, TGF beta 1, and the receptors for insulin and IGF-I, suggesting that paracrine growth factor circuits may exist between the oviduct epithelium and the early ovine embryo. Transcripts for insulin, epidermal growth factor (EGF), and nerve growth factor (NGF) were not detected in any stage of the ovine preimplantation embryo or within the oviduct cell preparations. The expression of growth factor transcripts very early in mammalian development would predict that these molecules fulfil a necessary role(s) in supporting the progression of early embryos through the preimplantation interval. Our future efforts will be directed to understanding the nature of these putative regulatory pathways.

No abstract

The chemokine GRO-alpha (CXCL1) has been found to mediate the proliferation of glia progenitor cells during neural development. As malignant gliomas are thought to arise from glia progenitors or their differentiated counterparts, astrocytes or oligodendrocytes, we have investigated whether GRO-alpha regulates the tumor characteristics of glioma cells. We found first that resected glioma specimens were strongly immunoreactive for GRO-alpha expression in cells with the morphology of tumor cells. In culture, the U251 glioma line transfected to overexpress GRO-alpha had elevated levels of motility and invasiveness. GRO-alpha transfectants increased their expression of several proteins associated with migratory behavior, including matrix metalloproteinase-2, beta1-integrin and SPARC. The implantation of GRO-alpha glioma clones into the brain of nude mice caused the early demise of mice and this was associated with the formation of larger intracerebral tumors when compared with mice implanted with vector control lines. These results implicate GRO-alpha in gliomas and suggest that the dysregulation of a glia proliferative factor contributes to tumorigenesis. Targeting GRO-alpha may be a useful therapeutic tool to control brain tumor biology.