摘要
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。Abstract
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.Category
Chapter 1: Literature Review 1
Introduction 2
Part I. The factors involving in the successful pregnancy 4
1. Reproductive cyclicity (estrous cycle) 4
2. Fertilization and cleavage 7
3. Embryo implantation 8
4. Endometrium 9
Part II. Biomarkers related to endometrial receptivity and embryo implantation 9
1. Steroidal hormone 10
2. Adhesion molecule and embryo implantation 10
Part III. MicroRNAs and embryo implantation 17
1. History of microRNAs 17
2. Biogenesis of microRNAs 18
3. MicroRNAs and embryo implantation 19
Chapter 2: Study 1-Let-7-mediated Suppression of Mucin 1 Expression in Mouse Uterus During Embryo Implantation 20
1. Abstract 21
2. Introduction 22
3. Materials and Methods 24
3.1. Animals 24
3.2. Isolation of mouse endometrial epithelial cells 24
3.3. Western blotting 24
3.4. Real time reverse transcription-polymerase chain reaction (Real-time PCR) 25
3.5. Transfection and Luciferase Assay 26
3.6. In vitro embryo adhesion assay 27
3.7. Statistical analysis 27
4. Results 28
4.1.Expression of Muc1protein in mouse endometrial epithelial cells during early pregnancy 28
4.2.Expression of let-7a and let-7b in mouse endometrial epithelial cells during early pregnancy 29
4.3. Let-7a and let-7b directly target the 3'UTR of Muc1 30
4.4. Suppression of Muc1 by overexpression of let-7a orlet-7b 32
4.5. Reduction of embryo adhesion on the endometria by let-7a and let-7b 33
5. Discussion 34
Chapter 3: Study 2-Estrous Cycle Determines the Pregnancy Performances in Superovulated Mice 37
1. Abstract 38
2. Introduction 39
3. Materials and Methods 41
3.1. Animals 41
3.2. Determining the stage of estrous cycle and superovulation 41
3.3. Histological study of ovaries 41
3.4. Immunohistochemistry of uterine horns 42
3.5. Investigation of let-7a and let-7b miRNA expressions on endometrial epithelial cells by real-time PCR 42
3.6. Statistical analysis 43
4. Results 44
4.1. Pregnancy performances of superovulated mice was determined by the stage of estrous cycle 44
4.2. The effect of PMSG on ovarian follicle development 47
4.3. Abnormal expression of Muc1 in endometrial epithelial cells of mice superovulated at metestrus and diestrus 49
4.4. Abnormal expression of let-7a and let-7b miRNA in endometrial epithelial cells of superovulated mice at metestrus and diestrus 51
5. Discussion 52
Chapter 4: Study 3-Proteomic Analysis of Differentially Expressed Proteins in Mouse Endometrial Epithelial cells During Early Pregnancy 55
1. Abstract 56
2. Introduction 57
3. Materials and Methods 58
3.1 Animals 58
3.2 Sample collection 58
3.3 Two dimensional gel electrophoresis (2-DE) 58
3.4 Staining and imaging of the 2-DE gels 59
3.5 Analysis of differential protein expression 59
3.6 Protein identification by MALDI-TOF/MS and MALDI- TOF/TOF 60
3.7 Bioinformation analysis 60
3.8 Statistical analysis 60
4. Results 61
4.1 Proteomic analysis of protein expression in mouse endometrial epithelial cells during early pregnancy 61
4.2 Quantitative changes of protein expression in mouse endometrial epithelia during early pregnancy 62
4.3 Classification of the differentially expressed proteins 69
5. Disscussion 71
Chapter 5: Conclusion 73
Appendix 76
1. Western blotting 77
2. Immunohistochemistry (IHC) 81
3. Clone MUC1 3'UTR construct vector 82
References 8
