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The impact of superovulation administration at different estrous cycle stages in mice on the maintenance of imprinted genes, H19 and Snrpn
|關鍵字:||人工生殖技術;ART;超級排卵;甲基化差異區域;銘印;Superovulation;DMR;Imprinting||出版社:||動物科學系所||引用:||林俊廷。2007。小鼠動情週期觀察及陰道抹片檢查。實驗動物飼養管理實習，第七章。屏東科技大學，台灣。 Abdalla, H., Y. Yoshizawa, and S. Hochi. 2009. Active demethylation of paternal genome in mammalian zygotes. J. Reprod. Dev. 55:356-360. Adams, R. L., R. H. Burdon, and J. Fulton. 1983. Methylation of satellite DNA. Biochem. Biophys. Res. Commun. 113:695-702. Anckaert, E., T. Adriaenssens, S. Romero, and J. Smitz. 2009. Ammonium accumulation and use of mineral oil overlay do not alter imprinting establishment at three key imprinted genes in mouse oocytes grown and matured in a long-term follicle culture. Biol Reprod. 2009 . 81:666-673. Anckaert, E., T. Adriaenssens, S. Romero , S. Dremier , and J. Smitz. 2009. Unaltered imprinting establishment of key imprinted genes in mouse oocytes after in vitro follicle culture under variable follicle-stimulating hormone exposure. Int. J. Dev. Biol. 53:541-548. Arnaud, P. 2010. 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人工生殖技術（assisted reproductive technology, ART）已常態性應用於治療不孕症、動物研究以及家畜生產，但有研究指出，人工生殖技術可能會增加胎兒發育遲緩、早產、體重過輕以及罹患基因銘印相關疾病之風險，而目前研究多指向於進行人工生殖技術中，超級排卵（superovulation）處理為造成銘印基因甲基化混亂之可能原因，但機制尚未明朗。銘印基因為只表現父方或母方同源染色體上之基因，其主要調控方式為於銘印基因啟動子進行甲基化程度，此區域稱為甲基化差異區域（Differentially Methylated Region, DMR）。本研究之目的為探討小鼠於不同動情週期期別，包含發情前期、發情期、發情後期以及間期施以超級排卵，對銘印基因H19與Snrpn啟動子甲基化狀態之影響。母鼠以陰道抹片確認其動情週期期別後，施以超級排卵，收集第二次減數分裂中期（metaphase II, MII）之卵母細胞，一部分進行孤雌激活（pathenogenetically activated, PA）並培養至囊胚期（blastocyst stage），而有些小鼠經超級排卵處理後，配種並收集原核期（pronuclear stage）受精卵培養至囊胚（fertilized blastocyst, FB）階段再進行分析。增殖之H19與Snrpn片段經亞硫酸鈉處理後，進行定序並分析其甲基化狀態與銘印基因CpG island之關聯性。卵母細胞分析結果顯示，母方銘印並未受到超級排卵處理影響；FB組之囊胚顯示具有異常甲基化銘印位點，而於發情後期施以超級排卵之PA組囊胚，其Snrpn銘印區域甲基化有顯著之缺失。本研究結果顯示於受精後，超級排卵可能造成母系產物異常導致銘印基因混亂，但其機制有待更進一步之研究。
Assisted reproductive technology (ART) is routinely applied to the treatments of subfertility in couples, researches in animal models, as well as production of livestocks. However, accumulating evidences indicate that the generations derived from ART may suffer the increased risk of intrauterine growth retardation, premature birth, low birth weight, or genomic imprinting disorders. Imprinted genes show predominant or exclusive transcription from one parental allele only. Methylation of the DNA in regions close to the imprinted genes promoter is thought to play a key role in regulating imprinted gene expression and loss of methylation in these differentially methylated regions (DMR) is associated with loss of imprinting. Therefore, the aim of this study was to investigate the effects of administration of superovulation at different estrous cycle stages, including proestrus, estrus, metestrus and diestrus, on the methylation status of the imprinted genes H19 and Snrpn DMR. Female mice were superovulated according to the vaginal smears. Metaphase II (MII) oocytes were collected. Some of the MII ooctyes were pathenogenetically activated and cultured to the blastocyst stage (PA). Additionally, fertilized pronuclear stage embryos were also collected and cultured to the blastocyst stage (FB). The methylation status of DMR on H19 and Snrpn were analyzed by cloning and sequencing following DNA bisulfite treatment. The results from the analysis of oocytes demonstrated that the maternal imprinting acquisition was not affected by superovulation in both H19 and Snrpn. However, the imprinting patterns in the FB blastocysts showed the aberrant DNA methylation in the imprinting loci, and the PA blastocysts showed the significant differences in the imprinting loss of Snrpn at the metestrus group. Analysis of imprinting CpG islands indicated the correlation among the methylation sites. These results imply that the imprinting disorder caused by superovulation may change the maternal-inherited gene products required for the imprinting maintenance after fertilization.
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