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|標題:||Analyses of Muc1 and protein expressions in mouse endometrial epithelium during early pregnancy
|關鍵字:||著床;超級排卵;小鼠;Implantation;Superovulation;Mouse||引用:||References Achache, H., and A. Revel. 2006. Endometrial receptivity markers, the journey to successful embryo implantation. Human Reproduction Update 12: 731-746. Adams, A. T. et al. 2014. Two-stage genome-wide methylation profiling in childhood-onset Crohn's Disease implicates epigenetic alterations at the VMP1/MIR21 and HLA loci. Inflamm Bowel Dis 20: 1784-1793. Adams, G. P., M. H. Ratto, W. Huanca, and J. Singh. 2005. Ovulation-inducing factor in the seminal plasma of alpacas and llamas. Biol Reprod 73: 452-457. Afonso, S., L. Romagnano, and B. Babiarz. 1997. The expression and function of cystatin C and cathepsin B and cathepsin L during mouse embryo implantation and placentation. Development 124: 3415-3425. Anderson, T. L., and L. H. Hoffman. 1984. Alterations in epithelial glycocalyx of rabbit uteri during early pseudopregnancy and pregnancy, and following ovariectomy. American Journal of Anatomy 171: 321-334. Aplin, J. 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Mucin 1（Muc1）為膜主體醣蛋白（integral membrane glycoprotein），表現於子宮等不同器官之分泌型或腺狀上皮細胞。子宮上皮細胞表面之Muc1表現消失有助於胎著床。此外，內源性18至24個核苷酸的非蛋白質編譯microRNAs（miRNAs）存在於多種有機物中，並藉由調節轉錄後基因表現，參予許多細胞表現調節過程。Muc1表現於本研究為一檢測指標，用以探討超級排卵（superovulation）時機對小鼠懷孕率之影響。正常情況下，與公鼠自然配種之母鼠其Muc1表現於懷孕第一天時最高，而後表現逐漸降低。然而，以即時聚合酶鏈鎖反應（real-time polymerase chain reaction, real-time PCR）分析發現，let-7a and let-7b miRNA表現則與Muc1相反。Luciferase分析顯示let-7a與let-7b miRNA直接與Muc1之3端UTR結合來調節Muc1表現。超級排卵常應用於增加卵子數目，然而對懷孕可能有不良影響。當不同動情週期（動情前期、動情期、動情後期與動情間期）之小鼠施予超級排卵後，動情前期（59%）與動情期（66%）之懷孕率顯著高於動情後期（23%）（P < 0.05）。動情前期與動情期表現較佳的配種率與懷孕率。我們推測可能與卵巢濾泡發育有關。因此，不同動情週期之小鼠經PMSG處理48小時後收集其卵巢。小鼠於動情前期與動情期施予超級排卵後之卵巢較動情後期與動情間期施打者有較多發育中濾泡與葛拉夫（Graafian）濾泡。相反的，動期後期與動期間期施予超級排卵之小鼠較動情前期與動情期者有較多的黃體（corpus luteum）（P < 0.05）。此外，動情前期與動情期組別之Muc1表現於懷孕第4天時顯著低於懷孕第1天（P < 0.05），然動情後期與動期間期組別並未有顯著差異。let-7a 與let-7b miRNA亦呈相反的表現方式。此外，亦利用二維膠體電泳（two-dimensional polyacrylamide gel electrophoresis，2-DE）分析懷孕第1天與第4天之小鼠子宮內膜上皮細胞之蛋白質表現。共有82個蛋白質點有表現上差異，並鑑定其中的52個蛋白質點。一些蛋白質如Vim、Gstm2、Hsp47、Hspa5（GRP78）與Ctsb可能與懷孕成功與否有關。總結來說，降低Muc1表現對於胚著床是必要的，且Muc1表現可被let-7a and let-7b miRNA調節。Muc1表現未減少，以及較少或較低品質之卵都可能造成懷孕率低落，可能導致不孕。這些發現或許可應用於改進或發展生殖醫療技術以增進懷孕率。
Mucin 1 (Muc1), an transmembrane glycoprotein, is expressed on the apical surface of lumen and glandular epithelial cells in many different organs, including the uterus. The lessening of Muc1 on the surface of uterine epithelial cells is essential for embryo implantation. Also, the endogenous non-protein coding microRNAs (miRNAs) of 18-24 nucleotides exist in various organisms and participate a myriad of cellular processes through regulating gene expressions at post-transcriptional level. In this study, Muc1 expression was an indicator to examine the effects of timing of superovulation on pregnancy rate in mice. In normal situation, the expression of Muc1 in female mice mated with male mice naturally reached the highest level on day 1 of pregnancy and constantly decreased afterwards. However, the expression of let-7a and let-7b miRNA detected by real-time polymerase chain reaction (real-time PCR) showed the reverse patterns compared with Muc1 expression. The direct binding of let-7a and let-7b miRNA to the 3' UTR of Muc1 was shown by luciferase assay, indicating the regulation of Muc1 expression. Superovulation is a common application for increasing the number of oocytes, but it might impair the pregnancy. When superovulation was conducted at the different stage of estrous cycle in mice (i.e. proestrous, estrous, metestrous and diestrous stages), the pregnancy rate was significantly higher at the groups of proestrus (59%) and estrus (66%) than that at the groups of metestrus (23%) (P < 0.05). Proestrus and estrus exhibited the best mating and pregnancy rate. We suspected that this reason might be associated with the development of ovarian follicles. Therefore, mouse ovaries were collected at different stage of estrus cycle after PMSG treatment for 48 h. More number of developing and Graafian follicles were shown in ovarian sections of mice superovulated at proestrous and estrous stages compared with those superovulated at metestrous and diestrous stages. In contrast, the mice superovulated at metestrus and diestrus presented more number of corpus lutea than those superovulated proestrus and estrus (P < 0.05). Additionally, the expression of Muc1 was significantly low at the groups of proestrus and estrus on day 4 of pregnancy compared with the expression on day 1 of pregnancy (P < 0.05), but no significant difference was shown at the groups of metestus and diestrus. The reverse pattern of the expression of let-7a and let-7b miRNA was also presented. Furthermore, the protein expressions of mouse endometrial epithelial cells during day 1 and 4 of pregnancy were also analyzed using two-dimensional polyacrylamide gel electrophoresis (2-DE). Eighty two protein spots were shown differential expression (P < 0.05), and fifty two of them were identified. Some proteins might be associated with successful pregnancy, such as Vim, Gstm2, Hsp47, Hspa5 (GRP78), and Ctsb. In conclusion, the reduction of Muc1 expression is essential for embryo implantation, and the expression is regulated by let-7a and let-7b miRNA. The failure of decrease in Muc1 and less number or low quality of oocytes could result in the low pregnancy rate, which might be associated with infertility. These finding might be applied to improve and develop modern reproductive therapies for higher pregnancy rate.
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