A mRNA landscape of bovine embryos after standard and MAPK-inhibited culture conditions: a comparative analysis

Background

Genes and signalling pathways involved in pluripotency have been studied extensively in mouse and human pre-implantation embryos and embryonic stem (ES) cells. The unsuccessful attempts to generate ES cell lines from other species including cattle suggests that other genes and pathways are involved in maintaining pluripotency in these species. To investigate which genes are involved in bovine pluripotency, expression profiles were generated from morula, blastocyst, trophectoderm and inner cell mass (ICM) samples using microarray analysis. As MAPK inhibition can increase the NANOG/GATA6 ratio in the inner cell mass, additionally blastocysts were cultured in the presence of a MAPK inhibitor and changes in gene expression in the inner cell mass were analysed.

Results

Between morula and blastocyst 3,774 genes were differentially expressed and the largest differences were found in blastocyst up-regulated genes. Gene ontology (GO) analysis shows lipid metabolic process as the term most enriched with genes expressed at higher levels in blastocysts. Genes with higher expression levels in morulae were enriched in the RNA processing GO term. Of the 497 differentially expressed genes comparing ICM and TE, the expression of NANOG, SOX2 and POU5F1 was increased in the ICM confirming their evolutionary preserved role in pluripotency. Several genes implicated to be involved in differentiation or fate determination were also expressed at higher levels in the ICM. Genes expressed at higher levels in the ICM were enriched in the RNA splicing and regulation of gene expression GO term. Although NANOG expression was elevated upon MAPK inhibition, SOX2 and POU5F1 expression showed little increase. Expression of other genes in the MAPK pathway including DUSP4 and SPRY4, or influenced by MAPK inhibition such as IFNT, was down-regulated.

Conclusion

The data obtained from the microarray studies provide further insight in gene expression during bovine embryonic development. They show an expression profile in pluripotent cells that indicates a pluripotent, epiblast-like state. The inability to culture ICM cells as stem cells in the presence of an inhibitor of MAPK activity together with the reported data indicates that MAPK inhibition alone is not sufficient to maintain a pluripotent character in bovine cells.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1448-x) contains supplementary material, which is available to authorized users.     

The single copy gene coding for human α1 (IV) procollagen is located at the terminal end of the long arm of chromosome 13

Summary Using dual-laser sorted chromosomes and spot-blot analysis, we have previously assigned genomic DNA sequences coding for human 1(IV) procollagen to chromosome 13 (Pihlajaniemi et al. 1985). By in situ hybridization to normal chromosomes and chromosomes with 13q deletions, we now report the localization of this gene to the terminal end of the long arm of chromosome 13. In addition, Southern and slot blot hybridization analysis clearly show that these genomic sequences are present only once per haploid genome.     

Expression of decorin by sprouting bovine aortic endothelial cells exhibiting angiogenesis in vitro.

In our recent studies, we have demonstrated that monolayer cultures of bovine aortic endothelial (BAE) cells that do not express type I collagen also fail to express and synthesize decorin, a small chondroitin/dermatan sulfate proteoglycan that interacts with type I collagen and regulates collagen fibrillogenesis in vitro. However, BAE cells exhibiting a spontaneous sprouting phenotype and a predisposition toward the formation of cords and tube-like structures (an in vitro model for angiogenesis) initiate the synthesis of type I collagen during their morphological transition from a polygonal monolayer to an angiogenic phenotype. In the present study, we examined whether BAE cells also initiate the synthesis of the proteoglycan decorin during this morphological transition. We show by Northern blot analysis and by immunochemical methods that BAE cell cultures containing sprouting cells and cords, but not monolayer cultures of these cells, express and synthesize decorin (M(r) approximately 100,000). We also show that type I collagen expression by BAE cell cultures is initiated concomitantly. However, the localization of decorin and type I collagen in cord and tube-forming BAE cell cultures is not completely identical. Type I collagen is detected only in sprouting BAE cells and in endothelial cords, whereas decorin is also apparent in BAE cells surrounding the cords and tubes. Our results indicate that the synthesis of decorin as well as type I collagen is associated with endothelial cord and tube formation in vitro.     

Repair of Chromosome Ends after Telomere Loss in Saccharomyces

Removal of a telomere from yeast chromosome VII in a strain having two copies of this chromosome often results in its loss. Here we show that there are three pathways that can stabilize this broken chromosome: homologous recombination, nonhomologous end joining, and de novo telomere addition. Both in a wild-type and a recombination deficient rad52 strain, most stabilization events were due to homologous recombination, whereas nonhomologous end joining was exceptionally rare. De novo telomere addition was relatively rare, stabilizing <0.1% of broken chromosomes. Telomere addition took place at a very limited number of sites on chromosome VII, most occurring close to a 35-base pair stretch of telomere-like DNA that is normally approximately 50 kb from the left telomere of chromosome VII. In the absence of the Pif1p DNA helicase, telomere addition events were much more frequent and were not concentrated near the 35-base pair tract of telomere-like DNA. We propose that internal tracts of telomere-like sequence recruit telomerase by binding its anchor site and that Pif1p inhibits telomerase by dissociating DNA primer-telomerase RNA interactions. These data also show that telomeric DNA is essential for the stable maintenance of linear chromosomes in yeast.     

Freshwater crayfish invasions in South Africa: past,present and potential future

Freshwater crayfish invasions have been studied around the world, but less so in Africa, a continent devoid of native freshwater crayfish. The present study reviews historical and current information on alien freshwater crayfish species introduced into South Africa and aims to indicate which areas are at risk from invasion. As is the case elsewhere, South Africans have shown a keen interest in both farming and keeping freshwater crayfish as pets, which has resulted in Cherax cainii, Cherax destructor, Cherax quadricarinatus and Procambarus clarkii being introduced to the country. There is evidence of successful establishment in the wild for C. quadricarinatus and P. clarkii in different parts of the country. Species distribution models suggest that the eastern part of the country and parts of the Eastern and Western Cape are at higher risk of invasion. At present, illegal translocations represent the most likely pathway of crayfish spread in South Africa. A continued risk of invasion by freshwater crayfish species in South Africa is highlighted, which reinforces the need for more research, as well as for strong mitigation measures, such as stronger policing of existing regulations, management or eradication where feasible and public education.     

祁连山大野口流域青海云杉种群数量动态

种群数量动态揭示了种群的结构特征及其潜在的驱动机制,有助于预测种群未来的动态,进而为森林生态系统的保护与恢复提供理论依据。本研究基于10.2 hm²青海云杉动态监测样地数据,以种群径级结构代替年龄结构,编制静态生命表,绘制径级结构图、存活曲线、死亡率曲线、消失率曲线和4个生存分析函数曲线,分析青海云杉种群数量特征,并利用种群数量动态变化指数和时间序列模型对种群数量动态进行预测。结果表明：（1）青海云杉种群的年龄结构近似于倒\"J\"型,幼苗和小树储量丰富;（2）种群存活曲线趋近于Deevey-Ⅱ型,为稳定型种群,死亡率曲线和消失率曲线变化趋势基本一致,均在第2、8龄级出现高峰期;（3）生存率曲线呈下降趋势,累计死亡率曲线呈上升趋势,死亡密度曲线缓慢下降,而危险率曲线逐渐上升,该种群具有：前期减少、中期稳定、后期衰退的生长特点;（4）种群数量变化动态指数V_pi>0,表明该种群属于增长型种群,V_pi''>0且趋近于0,则表明该种群趋近于稳定型;（5）时间序列预测分析表明,在未来2、4、6、8个龄级时间后,种群呈稳定增长趋势。研究显示,祁连山大野口流域青海云杉种群为稳定增长型种群,只要未来不遭受强烈干扰,种群数量会保持逐渐增长。针对该种群幼龄个体在前期的更新过程死亡率较高情况,建议在今后的经营管理中应重点加强对第1、2龄级植株生存环境的保护和改善,提高幼苗和小树的存活率。