豬出生前胚胎死亡率高達30%~40﹪，許多卵母細胞在發育至囊胚期前即面臨死亡的命運，目前對於排卵前起動豬卵母細胞發育的基因表現情形及所知甚少。本論文的主要目標就是利用抑制扣除雜交(suppression subtraction hybridization, SSH)技術來尋找豬卵母細胞體外培養成熟與濾泡腔（直徑≧ 3 mm）內兩個不同發育階段有差異表現的基因。欲徹底瞭解涉及豬卵母細胞體外培養成熟與胚胎體外生長的機制，獲得基因與其所轉錄的mRNA的資訊是非常重要的。我們取自本地屠宰場豬的卵巢，收集濾泡直徑大於3mm的卵母細胞，卵丘細胞包被完全的卵母細胞放置於成熟培養液中培養44小時後，收集排出第一極體的卵母細胞，去掉卵丘細胞，隨即進行mRNA的萃取，此即為SSH技術中的測試者(tester)。另外，我們取26個去除卵丘細胞的卵，不進行體外培養，直接抽mRNA，此即為SSH技術的驅動者(driver)。
經由正反向扣除的過程，最後獲得豬卵母細胞體外培養成熟與濾泡腔（直徑≧ 3 mm）內兩個不同發育階段有差異表現的基因。我們分析135個選殖DNA序列，每條序列均進行BLAST軟體進行分析比對，結果對應到50個與資料庫高度吻合( E值≦1e-4)的DNA序列。在這50個表現序列標記(expressed sequence tag, EST)中，我們挑選21個利用點漬墨cDNA矩陣 (dot blot cDNA array)技術分析，結果找到幾個有差異表現的基因，像是zona pellucida glycoprotein (ZP1), 1-aminocyclopropane-1-carboxylate synthase (ACS), 和death associated protein 5（DAP 5），而剩餘的部分尚未確認。有關在體內卵母細胞發育所需的基因表現仍然有待進一步的研究、探討。

Prenatal mortality in the swine ranges from 30%~40%. Little is known about genes that involve in the preovulation events that the initiation of swine oocyte development. The main objective of this study was to utilize suppression subtractive hybridization（SSH）to delineate the differential gene expression between in vitro culture mature and antral-follicle oocytes of swine development. The knowledge of genes and their accumulated mRNA is essential to better understand the mechanisms involved in the oocyte maturation and the survival of the in vitro produced embryos. Porcine ovaries obtained from the slaughterhouse were used to collect oocytes from follicle with a diameter ≥ 3 mm. After in vitro mature for two days, oocytes with first polar body were subjected to as the testers and were lysed for mRNA extraction. Pools of 26 denuded oocytes without culture were submitted to suppressive subtraction hybridization (SSH) as the drivers.
Forward and reverse subtractions were performed to identify candidate genes differentially expressed between in vitro culture mature and primordial-follicle oocytes. A total of one hundred and thirty-five differential expressed plasmid clones were sequenced, and each was analyzed by BLAST programs. Of these transcripts, 40 clones were subjected to differential screening by a dot blot cDNA array. We identified three genes like zona pellucida glycoprotein (ZP1), 1-aminocyclopropane-1-carboxylate synthase (ACS), and death associated protein 5（DAP 5）while other numerous clones remain novel. The in vivo functions of the genes remain further investigation.

Chinese Abstract•••••••••••••••••••••••••••••••••••••I
English Abstract••••••••••••••••••••••••••••••••••••II
Thesis••••••••••••••••••••••••••••••••••••••••••••••1
Introduction•••••••••••••••••••••••••••••••••••••••••1
Materials and Methods••••••••••••••••••••••••••••••••5
Results••••••••••••••••••••••••••••••••••••••••••••10
Super SMART cDNA Amplification••••••••••••••••••••••••10
Rsa I Digestion of Synthesized cDNA••••••••••••••••••••10
Analysis of Secondary PCR Products•••••••••••••••••••••10
PCR Analysis of Subtraction Efficiency•••••••••••••••••11
Subtraction cDNA Library Construction••••••••••••••••••11
Identification of Inserted Fragments in Plasmids of Library by PCR•••••••••••••••••••••••••••••••••••••••••••••11
Annotation of BLASTx-nr Results•••••••••••••••••••••••11
Statistics Analysis of Gene Function Classification••••••12
Differential Screening of the Subtracted cDNA Library••••12
Discussion••••••••••••••••••••••••••••••••••••••••••14
Conclusion••••••••••••••••••••••••••••••••••••••••••18
References••••••••••••••••••••••••••••••••••••••••••19
Tables•••••••••••••••••••••••••••••••••••••••••••••21
Figures••••••••••••••••••••••••••••••••••••••••••••30
Appendix•••••••••••••••••••••••••••••••••••••••••••41

Altschul, S., W. Gish, W. Miller, E. Myers and D. Lipman. 1990. Basic local alignment search tool. J. Mol. Biol. 215: 403-410.
Bachvarova, R.F. 1992. A maternal tail of poly (A): the long and the short of it. Cell 69: 895-897.
Dynal, A. S. 1995. Biomagnetic Techniques in Molecular Biology: Technical Handbook. 2nd ed. Oslo, Norway.
Eppig, J. J., R.M. Schultz, M. O'Brien, F. Chesnel. 1994. Relationship between the developmental programs controlling nuclear and cytoplasmic maturation of mouse oocytes. Dev. Biol. 164(1): 1-9.
Fan, H. Y., Q.Y. Sun. 2004. In vitro maturation and fertilization of pig oocytes Page 227-234 in Germ Cell Protocols. Humana Press 999 Riverview Dr. Suite 208 Totowa, NJ 07512 USA
Fellmann, F., J. L. Pretet, and D. Fellmann. 1996. Simplified protocol of solid-phase cDNA libraries for multiple PCR amplification. BioTechniques. 21: 766-770
Gurr, S. J., M. J. McPherson, C. Scollan, H. J. Atkinson, and D. J. Bowles. 1991. Gene expression in nematode-infected plant roots. Mol. Gen. Genet. 226: 361-366.
Leibfried, L. and N. L. First. 1979. Characterization of bovine follicular oocytes and their ability to mature in vitro. J. Anim. Sci. 48: 76-86.
Leibfried-Rutledge, M. L., E. S. Critser, J. J. Parish and N. L. First. 1989. In vitro maturation and fertilization of bovine oocytes. Theriogenology. 31: 61.
Marchal, R., C. Vigneron, C. Perreau, A. Bali-Papp, P. Mermillod. 2002. Effect of follicular size on meiotic and developmental competence of porcine oocytes. Theriogenology. 57(5): 1523-1532.
Motlik, J., N. Crozet, J. Fulka. 1984. Meiotic competence in vitro of pig oocytes isolated from early antral follicles. J. Reprod. Fertil. 72(2): 323-328.
Paynton, B.V., R. Bachvarova. 1994. Polyadenylation and deadenylationof maternal mRNAs during oocyte growth and maturation in the mouse. Mol. Reprod. Dev. 37: 172-178
Plante, L., C. Plante, D. L. Shepherd, W.A. King. 1994. Cleavage and 3Huridine incorporation in bovine embryos of high in vitro developmental potential. Mol. Reprod. Dev. 39: 375-383.
Raineri, I., C. Moroni and H.P. Senn. 1991. Improved efficiency for single-sided PCR by creating a reusable pool of first-strand cDNA coupled to a solid phase. Nucleic Acids Res. 19: 4010.
Tsafriri, A. and C. P. Channing. 1975a. Influence of follicular maturation and culture conditions on the meiosis of pig oocytes in vitro. J. Reprod. Fertil. 43 (1): 149-152.
Tsafriri, A. and C. P. Channing. 1975b. An inhibitory influence of granulose cells and follicular fluid upon porcine oocyte meiosis in vitro. Endocrinology. 96: 922-927.
Verrotti, A.C., S. Strickland. 1997. Oocyte selection of mutations affecting cytoplasmic polyadenylation of maternal mRNAs. Mol. Reprod. Dev. 46: 482-488.
Xie, H., A. Wasserman, Z. Levine, A. Novik, V. Grebinskiy, A. Shoshan, and L. Mintz. 2002. Large-scale protein annotation through gene ontology. Genome Res. 12: 785-794.