Baufeld Anja, Koczan Dirk, Vanselow Jens
Institute of Reproductive Biology, Leibniz Institute for Farm Animal Biology (FBN), Wilhelm-Stahl-Allee 2, 18196, Dummerstorf, Germany.
Institute for Immunology, University of Rostock, 18055, Rostock, Germany.
Reprod Biol Endocrinol. 2017 Jan 5;15(1):3. doi: 10.1186/s12958-016-0221-6.
In previous studies it has been shown that bovine granulosa cells (GC) cultured at a high plating density dramatically change their physiological and molecular characteristics, thus resembling an early stage of luteinization. During the present study, these specific effects on the GC transcriptome were comprehensively analysed to clarify the underlying mechanisms.
GC were cultured in serum free medium with FSH and IGF-1 stimulation at different initial plating density. The estradiol and progesterone production was determined by radioimmunoassays and the gene expression profiles were analysed by mRNA microarray analysis after 9 days. The data were statistically analysed and the abundance of selected, differentially expressed transcripts was re-evaluated by qPCR. Bioinformatic pathway analysis of density affected transcripts was done using Ingenuity Pathway Analysis.
The data showed that at high plating density the expression of 1510 annotated genes, represented by 1575 transcript clusters, showed highly altered expression levels. Nearly two-thirds were up- and one third down-regulated. Within the top up-regulated genes VNN2, RGS2 and PTX3 could be identified, as well as HBA or LOXL2. Down-regulated genes included important key genes of folliculogenesis like CYP19A1 and FSHR. Ingenuity pathway analysis identified "AMPK signaling" as well as "cAMP-mediated signaling" as major pathways affected by the alteration of the expression profile. Main putative upstream regulators were TGFB1 and VEGF, thus indicating a connection with cell differentiation and angiogenesis. A detailed cluster analysis revealed one single cluster that was highly associated with the upstream regulator beta-estradiol. Within this cluster key genes of steroid biosynthesis were not included, but instead, other genes importantly involved in follicular development, like OXT and VEGFA as well as the three most down-regulated genes TXNIP, PAG11 and ARRDC4 were identified.
From these data we hypothesize that high density conditions induce a stage of differentiation in cultured GC that is similar to early post-LH conditions in vivo. Furthermore we hypothesize that specific cell-cell-interactions led to this differentiation including transformations necessary to promote angiogenesis.
在先前的研究中已表明，以高接种密度培养的牛颗粒细胞（GC）会显著改变其生理和分子特征，从而类似于黄体化的早期阶段。在本研究中，对这些对GC转录组的特定影响进行了全面分析，以阐明其潜在机制。
将GC在添加FSH和IGF-1刺激的无血清培养基中以不同的初始接种密度进行培养。通过放射免疫测定法测定雌二醇和孕酮的产生，并在9天后通过mRNA微阵列分析来分析基因表达谱。对数据进行统计学分析，并通过qPCR重新评估选定的差异表达转录本的丰度。使用Ingenuity Pathway Analysis对受密度影响的转录本进行生物信息学通路分析。
数据显示，在高接种密度下，由1575个转录本簇代表的1510个注释基因的表达显示出高度改变的表达水平。近三分之二上调，三分之一下调。在上调的基因中可鉴定出VNN2、RGS2和PTX3，以及HBA或LOXL2。下调的基因包括卵泡发生的重要关键基因，如CYP19A1和FSHR。Ingenuity通路分析确定“AMPK信号传导”以及“cAMP介导的信号传导”是受表达谱改变影响的主要通路。主要的假定上游调节因子是TGFB1和VEGF，因此表明与细胞分化和血管生成有关。详细的聚类分析揭示了一个与上游调节因子β-雌二醇高度相关 的单个簇。在该簇中未包括类固醇生物合成的关键基因，而是鉴定出了其他重要参与卵泡发育的基因，如OXT和VEGFA，以及三个下调程度最高的基因TXNIP、PAG11和ARRDC4。
从这些数据中我们推测，高密度条件会在培养的GC中诱导出一个类似于体内LH后早期条件的分化阶段。此外，我们推测特定的细胞间相互作用导致了这种分化，包括促进血管生成所需的转变。
