Supplementing culture medium with the weak acid, 5,5-dimethyl-2,4-oxazolidinedione (DMO) limits the development of aneuploid mouse embryos through a Trp53-dependent mechanism.
Supplementing culture medium with the weak acid, 5,5-dimethyl-2,4-oxazolidinedione (DMO) limits the development of aneuploid mouse embryos through a Trp53-dependent mechanism.
复制标题
在培养基中添加弱酸 5,5-二甲基-2,4-恶唑烷二酮 (DMO) 可通过 Trp53 依赖性机制限制非整倍体小鼠胚胎的发育。
DOI：
10.1007/s10815-023-02788-x
复制
发表时间：
2023
影响因子：
3.1
通讯作者：
Peluso,JohnJ
中科院分区：
文献类型：
--
作者：
Lowther,KatieM;Bartolucci,AlisonF;Massey,RachaelE;Brown,Judy;Peluso,JohnJ
本研究旨在探讨DMO是否通过激活TrP53依赖机制限制非整倍体小鼠胚胎的体外发育。方法用逆转氨酸诱导小鼠卵裂期胚胎非整倍体，或用载体产生对照，然后在添加DMO的培养液中培养，以降低培养液的pH值。用位相显微镜观察胚胎形态。DAPI染色显示固定胚胎的细胞数、有丝分裂形态和凋亡小体。定量聚合酶链式反应(QPCRs)检测TrP53、Oct-4和CDX2基因的表达水平。DepletingTrp53usingTrp53siRNA.ResultsAneuploid-enriched检测Trp53对Oct-4和CDX2表达的影响：晚期囊胚在形态上与对照囊胚没有明显区别，但细胞数量较少，Oct-4和CDX2基因表达水平降低。在8-细胞向囊胚转变的过程中，在培养液中加入1 mM的DMO可以减少非整倍体致富的囊胚的形成，但不能抑制囊胚的发育，并进一步抑制非整倍体致富的胚胎中Oct-4和CDx2mRNAs的表达。在非整倍体致富的胚胎中， >； 的Trp53RNA水平是对照组的两倍，Trp53siRNA的水平降低了一倍，而 和GT； 则增加了一倍。结论这些研究表明，在培养液中加入低剂量的DMO可以抑制形态正常的非整倍体致富集囊胚的发育，导致Trp53mRNA的水平升高，从而抑制Oct-4和CDx2的表达。
PurposeThis study was designed to determine if DMO limits in vitro development of aneuploid-enriched mouse embryos by activating aTrp53-dependent mechanism.MethodsMouse cleavage-stage embryos were treated with reversine to induce aneuploidy or vehicle to generate controls, and then cultured in media supplemented with DMO to reduce the pH of the culture media. Embryo morphology was assessed by phase microscopy. Cell number, mitotic figures, and apoptotic bodies were revealed by staining fixed embryos with DAPI. mRNA levels ofTrp53,Oct-4, andCdx2were monitored by quantitative polymerase chain reactions (qPCRs). The effect ofTrp53on the expression ofOct-4andCdx2was assessed by depletingTrp53usingTrp53siRNA.ResultsAneuploid-enriched late-stage blastocysts were morphologically indistinguishable from control blastocysts but had fewer cells and reduced mRNA levels ofOct-4andCdx2. Adding 1 mM DMO to the culture media during the 8-cell to blastocyst transition reduced the formation of aneuploid-enriched late-stage blastocysts but not control blastocysts and further suppressed the levels ofOct-4andCdx2mRNA.Trp53RNA levels in aneuploid-enriched embryos that were exposed to DMO were > twofold higher than controls, andTrp53siRNA levels reduced the levels ofTrp53and increased levels ofOct-4andCdx2mRNA by > twofold.ConclusionThese studies suggest that the development of morphologically normal aneuploid-enriched mouse blastocysts can be inhibited by adding low amounts of DMO to the culture media, which results in elevated levels ofTrp53mRNA that suppressesOct-4andCdx2expression.