臺灣博碩士論文加值系統

雌性生殖道內具免疫系統來抵禦病菌等外來物的侵入。而精子需具備抵抗雌性生殖道免疫系統攻擊的能力，才能到達位於輸卵管的壺腹進行受精動作。Carcinoembryonic antigen-related cell adhesion molecule 10 （CEACAM10）係成熟公鼠副屬性腺貯精囊液中的蛋白質，在雄性與雌性小鼠的性腺體中，其特別表現於貯精囊中。CEACAM10為分子量36-kDa的分泌性醣蛋白，雖然在體外實驗（in vitro）已證實CEACAM10可結合於精子的表面並促進其活動力，但於雌性生殖系統中所扮演的角色尚待釐清。由公鼠貯精囊液所純化的CEACAM10 與動情期子宮液反應，發現子宮液內特定蛋白質有減少的趨勢。經過LC/MS/MS鑑定，證實該減少的蛋白質為補體complement 3 component（C3）。進一步的實驗發現CEACAM10能造成子宮液內補體C3 β chain的降解，使得補體C3b、iC3b、C3c、C3dg失去完整的結構。在體內實驗（in vivo）著床前期第1天的小鼠子宮液內有CEACAM10的存在，而其子宮液內補體C3 β chain也有減少情形，提示CEACAM10可能由引起C3 β chain的降解而破壞補體C3的結構，使其喪失補體生理活性，藉此抑制補體活化以致提高精子的存活率。人類補體C3與小鼠補體C3在氨基酸序列以及蛋白質序列上有高度的相似性，所以先將人類補體C3與小鼠貯精囊液CEACAM10反應，檢測補體活性的變化。結果顯示：（1）CEACAM10與人類補體C3單獨反應無法造成補體C3降解；（2）加入缺乏人類補體C3血清於CEACAM10與人類補體C3的反應中，可以引起補體C3 β chain降解；（3）在人類補體C3與缺乏人類補體C3血清中，CEACAM10可以抑制羊紅血球溶血。

Innate immunity plays an essential role in the defense of the invaders in the female genital tract. Spermatozoa must therefore evade immunity attack in order to reach the ampulla of oviduct for fertilization. A 36-kDa has been demonstrated carcinoembryonic antigen-related cell adhesion molecule 10（CEACAM10）from adult mouse seminal vesicle secretions. Among the sexual glands of male and female mice, it is exclusively expressed in seminal vesicle. Although, it is able to bind on the entire surface of mouse spermatozoa and enhance sperm motility in vitro, its role in murine female reproductive system has no yet understood. This work aimed to that direction, I incubated purified CEACAM10 with uterine luminal fluid collected from adult female mice during estrus and resolved the protein components by nonreduced SDS-PAGE. A 180-kDa protein was markedly decrease by CEACAM10. This protein was demonstrated to be complement 3 component（C3）based on the peptide sequences determined by LC/MS/MS analysis. Further, I illustrated the degradation of β chain of C3 in the uterine fluid by CEACAM10. As a result, C3b、iC3b、C3c、C3dg became incomplete. In the preimplantation period, CEACAM10 was determined in the uterine fluid and only on day 1. It was accomplished with the degradation of C3 β chain. Taken together, the presence of CEACAM10 in the uterine fluid caused the loss of C3 activity via the degradation of its β chain to prevent the ejaculated spermatozoa in the uterine lumen from the immunity, attacked by the complement system. C3 was highly conserved and I studied the effect of CEACAM10 in the human C3 activity. These results were summarized：（1）Incubation of human C3 and mouse CEACAM10 didn’t cause the C3 degradation；（2）Addition of the human C3 deficient serum to the incubation of human C3 and CEACAM10 caused the human C3 β chain degradation;（3）CEACAM10 was able to inhibit the lysis of sheep blood by human C3 and the C3 deficient serum together.

目錄
中文摘要 I
英文摘要 II
縮寫表 3
第一章 概論 4
小鼠雄性生殖系統 4
小鼠貯精囊的研究 4
小鼠雌性生殖系統 5
小鼠子宮的生理及子宮液的生成 5
補體系統 7
補體系統的活化機制 7
補體的生物活性 9
小鼠C3的構造 10
小鼠子宮內C3的表現情形 11
主要研究方向與實驗背景 12
第二章 材料與方法 13
實驗材料 13
實驗方法 13
一、小鼠子宮液、輸卵管沖洗液與貯精囊液樣品製備 13
1.1 自然週期子宮液之取得 13
1.2著床前期子宮沖洗液之取得 13
1.3著床前期輸卵管沖洗液之取得 14
1.4 貯精囊液的收集 14
二、由貯精囊液純化CEACAM10 的方法 14
2.1陰離子交換樹脂層析法 14
2.2 高效液相層析法 15
三、蛋白質的分析方法 17
3.1 蛋白質定量 17
3.2 SDS-聚丙烯醯胺膠體電泳 17
3.3 Coomassie Blue G-250染色 19
3.4西方墨點法 19
四、補體C3溶血活性分析 21
第三章 實驗結果 25
一、小鼠貯精囊液CEACAM10蛋白質的純化 25
二、小鼠CEACAM10對母鼠子宮液內蛋白質的影響 25
三、小鼠CEACAM10對母鼠子宮液內補體C3的影響 25
四、著床前期母鼠子宮液與輸卵管液內補體C3的變化 26
五、小鼠CEACAM10蛋白質對人類補體C3的影響 27
六、小鼠CEACAM10蛋白質對人類補體C3活化補體傳統路徑的影響 28
第四章 實驗討論 29
一、小鼠CEACAM10對小鼠補體C3與人類補體C3的影響 29
二、小鼠CEACAM10在母鼠生殖系統的生理功能 30
圖次 32
圖一、純化小鼠貯精囊液CEACAM10蛋白的離子交換樹脂層析圖 33
圖二、小鼠子宮液與CEACAM10反應後蛋白質的變化 35
圖三、以質譜分析小鼠子宮液分子量180-kDa位置的蛋白質 37
圖四、免疫偵測小鼠子宮液與CEACAM10反應後補體C3的變化 39
圖五、以質譜分析小鼠子宮液分子量120-kDa位置的蛋白 41
圖六、CEACAM10具有時間遞增性的減少小鼠子宮液內補體C3 43
圖七、CEACAM10具有濃度遞增性的減少小鼠子宮液內補體C3 45
圖八、小鼠著床前期子宮液中補體C3與CEACAM10變化於還原性與非還原性SDS-PAGE的圖形 47
圖九、小鼠著床前期輸卵管液中補體C3與CEACAM10變化於還原性與非還原性SDS-PAGE的圖形 49
圖十、人類補體C3與CEACAM10反應後的變化 51
圖十一、在有缺乏人類補體C3血清的情況下，小鼠CEACAM10具有時間遞增性的減少補體C3 53
圖十二、在有缺乏人類補體C3血清的情況下，小鼠CEACAM10具有濃度遞增性的減少人類補體C3 55
圖十三、人類補體C3活性溶血活性分析法的建立 57
圖十四、小鼠CEACAM10具有時間遞增性的降低人類補體C3溶血反應 59
圖十五、小鼠CEACAM10具有濃度遞增性的降低人類補體C3溶血反應 61
參考文獻 63

參考文獻
1.Chen, Y. H., Pentecost, B. T., McLachlan, J. A. & Teng, C. T. The androgen-dependent mouse seminal vesicle secretory protein IV: characterization and complementary deoxyribonucleic acid cloning. Mol Endocrinol 1, 707-716 (1987).
2.Mills, J. S., Needham, M. & Parker, M. G. A secretory protease inhibitor requires androgens for its expression in male sex accessory tissues but is expressed constitutively in pancreas. Embo J 6, 3711-3717 (1987).
3.Chen, L. Y., Lin, Y. H., Lai, M. L. & Chen, Y. H. Developmental profile of a caltrin-like protease inhibitor, P12, in mouse seminal vesicle and characterization of its binding sites on sperm surface. Biol Reprod 59, 1498-1505 (1998).
4.Luo, C. W., Lin, H. J. & Chen, Y. H. A novel heat-labile phospholipid-binding protein, SVS VII, in mouse seminal vesicle as a sperm motility enhancer. J Biol Chem 276, 6913-6921 (2001).
5.Lin, H. J., Luo, C. W. & Chen, Y. H. Localization of the transglutaminase cross-linking site in SVS III, a novel glycoprotein secreted from mouse seminal vesicle. J Biol Chem 277, 3632-3639 (2002).
6.Walmer, D. K., Wrona, M. A., Hughes, C. L. & Nelson, K. G. Lactoferrin expression in the mouse reproductive tract during the natural estrous cycle: correlation with circulating estradiol and progesterone. Endocrinology 131, 1458-1466 (1992).
7.Aitken, R. J. Changes in the protein content of mouse uterine flushings during normal pregnancy and delayed implantation, and after ovariectomy and oestradiol administration. J Reprod Fertil 50, 29-36 (1977).
8.Teng, C. T. et al. Lactotransferrin gene expression in the mouse uterus and mammary gland. Endocrinology 124, 992-999 (1989).
9.Chu, S. T., Huang, H. L., Chen, J. M. & Chen, Y. H. Demonstration of a glycoprotein derived from the 24p3 gene in mouse uterine luminal fluid. Biochem J 316 ( Pt 2), 545-550 (1996).
10.Huang, H. L., Chu, S. T. & Chen, Y. H. Ovarian steroids regulate 24p3 expression in mouse uterus during the natural estrous cycle and the preimplantation period. J Endocrinol 162, 11-19 (1999).
11.Li, S. H., Huang, H. L. & Chen, Y. H. Ovarian steroid-regulated synthesis and secretion of complement C3 and factor B in mouse endometrium during the natural estrous cycle and pregnancy period. Biol Reprod 66, 322-332 (2002).
12.Lundwall, A., Wetsel, R. A., Domdey, H., Tack, B. F. & Fey, G. H. Structure of murine complement component C3. I. Nucleotide sequence of cloned complementary and genomic DNA coding for the beta chain. J Biol Chem 259, 13851-13856 (1984).
13.Wetsel, R. A. et al. Structure of murine complement component C3. II. Nucleotide sequence of cloned complementary DNA coding for the alpha chain. J Biol Chem 259, 13857-13862 (1984).
14.Domdey, H. et al. Characterization of the mRNA and cloned cDNA specifying the third component of mouse complement. Proc Natl Acad Sci U S A 79, 7619-7623 (1982).
15.Li, S. H., Lee, R. K., Hsiao, Y. L. & Chen, Y. H. Demonstration of a glycoprotein derived from the Ceacam10 gene in mouse seminal vesicle secretions. Biol Reprod 73, 546-553 (2005).
16.Finkenzeller, D. et al. Carcinoembryonic antigen-related cell adhesion molecule 10 expressed specifically early in pregnancy in the decidua is dispensable for normal murine development. Mol Cell Biol 23, 272-279 (2003).
17.Keck, U., Nedellec, P., Beauchemin, N., Thompson, J. & Zimmermann, W. The cea10 gene encodes a secreted member of the murine carcinoembryonic antigen family and is expressed in the placenta, gastrointestinal tract and bone marrow. Eur J Biochem 229, 455-464 (1995).
18.Kent, J. F. & Fife, E. H., Jr. Precise standardization of reagents for complement fixation. Am J Trop Med Hyg 12, 103-116 (1963).
19.Kent, J. F., Otero, A. G. & Harrigan, R. E. Relative specificity of serologic tests for syphilis in Mycobacterium leprae infection. Am J Clin Pathol 27, 539-545 (1957).
20.Jin, M., Larsson, A. & Nilsson, B. O. A functionally active complement system is present in uterine secretion of the mouse prior to implantation. Am J Reprod Immunol 26, 53-57 (1991).
21.Kinoshita, T., Lavoie, S. & Nussenzweig, V. Regulatory proteins for the activated third and fourth components of complement (C3b and C4b) in mice. II. Identification and properties of complement receptor type 1 (CR1). J Immunol 134, 2564-2570 (1985).
22.Davis, A. E., 3rd, Harrison, R. A. & Lachmann, P. J. Physiologic inactivation of fluid phase C3b: isolation and structural analysis of C3c, C3d,g (alpha 2D), and C3g. J Immunol 132, 1960-1966 (1984).
23.Griger, B., Frensdorff, A. & Kraicer, P. F. The soluble proteins of rat seminal vesicle fluid. Some physico-chemical and immunological properties. Immunology 27, 729-738 (1974).
24.Veselsky, L. Immunological properties of seminal vesicle fluid. Arch Androl 7, 1-7 (1981).
25.Robertson, S. A. Seminal plasma and male factor signalling in the female reproductive tract. Cell Tissue Res 322, 43-52 (2005).
26.Harris, C. L., Mizuno, M. & Morgan, B. P. Complement and complement regulators in the male reproductive system. Mol Immunol 43, 57-67 (2006).
27.Lee, Y. L. et al. The embryotrophic activity of oviductal cell-derived complement C3b and iC3b, a novel function of complement protein in reproduction. J Biol Chem 279, 12763-12768 (2004).
28.de Bruijn, M. H. & Fey, G. H. Human complement component C3: cDNA coding sequence and derived primary structure. Proc Natl Acad Sci U S A 82, 708-712 (1985).
29.D''Cruz, O. J., Haas, G. G., Jr., Wang, B. L. & DeBault, L. E. Activation of human complement by IgG antisperm antibody and the demonstration of C3 and C5b-9-mediated immune injury to human sperm. J Immunol 146, 611-620 (1991).
30.Jenne, D. E. & Tschopp, J. Molecular structure and functional characterization of a human complement cytolysis inhibitor found in blood and seminal plasma: identity to sulfated glycoprotein 2, a constituent of rat testis fluid. Proc Natl Acad Sci U S A 86, 7123-7127 (1989).
31.Clark, R. A. & Klebanoff, S. J. Generation of a neutrophil chemotactic agent by spermatozoa: role of complement and regulation by seminal plasma factors. J Immunol 117, 1378-1386 (1976).
32.Shigeoka, A. O., Gobel, R. J., Janatova, J. & Hill, H. R. Neutrophil mobilization induced by complement fragments during experimental group B streptococcal (GBS) infection. Am J Pathol 133, 623-629 (1988).