一種新的卵母細胞成長因子BMP15, 如同他的同源基因GDF9一樣，是屬於TGF-β superfamily的其中一員。在動物實驗當中，猪卵巢組織裡面所發現的BMP15基因，其表現以及功能性皆尚未被徹底的研究。因此，我們研究：是否BMP15 mRNA與蛋白質在未成熟排卵時期當中，猪卵母細胞與週遭濾泡細胞當中是否會有所改變。另一實驗則是在離體的環境之下將猪卵母細胞進行培養，觀察是否會有BMP15 mRNA與蛋白質的表現量改變。將裸露卵母細胞，卵丘細胞，卵丘與卵母細胞複合體從正在發育以及未排卵前的濾泡當中分離出來，從猪卵母細胞當中萃取Total RNA，並且利用反轉錄聚合酶連鎖反應設計專一性的寡核酸引子完成單股cDNA。第一次觀察到黏液化擴散作用發生的時間點大約是在第18小時，並且在這個時間點我們發現BMP15 mRNA表現量很明顯地增加。當我們將裸露的卵母細胞取代卵丘與卵母細胞複合體進行離體培養，發現BMP15 mRNA隨時間點增加而沒有變化。我們同時也偵測GDF9與BMP15訊息RNA在受精成猪胚胎時期與分化到猪胚胎幹細胞時期的表現。GDF9在發育後期持續會表現，然而BMP15則是沒有表現。當我們偵測BMP15蛋白質隨時間點變化的表現，發現表現量很明顯地增加也是在第18小時的時候。接下來我們將BMP15抗體加入體外成熟培養液當中進行離體培養並且偵測是否有會抑制黏液化擴散作用的發生。我們對於目前的結果猜測能夠促進卵丘細胞從卵母細胞分離擴散，BMP15對於猪卵母細胞發育到成熟是扮演一個關鍵的調節者。

The newly discovered oocyte-derived growth factor BMP15, like GDF9, is a member of the transforming growth factor-β (TGF-β) superfamily. To our knowledge, however, the expression and function of BMP15 in ovarian tissues has not yet been studied in the pig. We asked whether the relative abundance (RA) of BMP15 mRNA changes in oocytes and follicular cells during pre-ovulatory period or culture of cumulus-oocyte-complexs (COCs) in vitro. Denuded oocytes, cumulus cells and COCs were isolated from growing and pre-ovulatory follicles. Total RNA was extracted from the cells, and reverse transcriptase polymerase chain (RT-PCR) was carried out using specific oligo-nucleotide primers. Expansion of COCs was firstly observed at 18 h, when this period we found BMP15 mRNA expression obviously. But when we culture of denuded oocytes instead of COCs, BMP15 mRNA didn’t express throughout the period. We also detected GDF9 and BMP15 mRNA in pig embryonic stage and in differentiation stage of pig stem cell. GDF9 mRNA level continued to express after fertilization, but BMP15 mRNA didn’t appear. We also added BMP15 antibody against Expanding of COCs. The present results support the concept that BMP15 is a key mediator of oocyte-enabled cumulus expansion in pig.

中文摘要…………………………………………………. I
英文摘要………………………………………………….. II
英文縮寫表……………………………………………………….. III
壹、前言 (INTRODUCTION)…………………………………………. 1
貳、文獻探討 (LITERATURE REVIEW)……………………………... 2
1. TGF-

王懷秀 (1998). "受精調控技術" 動物生殖調控, 王建成和張孝榮主編,安徽科學技術出版社: 218-225.
吳信志、鄭三寶、劉炳燦、邱啟明與朱志成。 (1991a). "豬卵母細胞體外成熟及受精後發育的能力: I、卵丘卵母細胞之完整性及添加內泌素於成熟培養液之影響." 中畜會誌 20(4): 455-468.
吳信志、鄭三寶、劉炳燦、邱啟明與朱志成。 (1991b). "豬卵母細胞體外成熟及受精後發育的能力: II、卵巢濾泡之大小和添加濾泡亦於成熟培養液之影響." 中畜會誌 20(4): 469-480.
邱啟明、鄭三寶、楊忠亮、吳信志與朱志成 (1995). "卵丘細胞包被之完整性及培養液中添加之內泌素組合對牛卵母細胞於體外成熟與受精後發育之影響." 中畜會誌 24(4): 457-470.
Anderson, L. D. and T. Hillensjo (1982). "Lack of effect of a gonadotropin-releasing hormone agonist upon meiosis in isolated porcine oocytes." Acta Physiol Scand 114(4): 623-5.
Chang, H., C. W. Brown, et al. (2002). "Genetic analysis of the mammalian transforming growth factor-beta superfamily." Endocr Rev 23(6): 787-823.
Christenson, R. K., J. J. Ford, et al. (1985). "Maturation of ovarian follicles in the prepubertal gilt." J Reprod Fertil Suppl 33: 21-36.
Di Pasquale, E., P. Beck-Peccoz, et al. (2004). "Hypergonadotropic ovarian failure associated with an inherited mutation of human bone morphogenetic protein-15 (BMP15) gene." Am J Hum Genet 75(1): 106-11.
Downs, S. M. and J. J. Eppig (1986). "The role of purines in the maintenance of meiotic arrest in mouse oocytes." Tokai J Exp Clin Med 11(6): 463-9.
Dragovic, R. A., L. J. Ritter, et al. (2005). "Role of oocyte-secreted growth differentiation factor 9 in the regulation of mouse cumulus expansion." Endocrinology.
Duffy, D. M. (2003). "Growth differentiation factor-9 is expressed by the primate follicle throughout the periovulatory interval." Biol Reprod 69(2): 725-32.
Eckery, D. C., L. J. Whale, et al. (2002). "Expression of mRNA encoding growth differentiation factor 9 and bone morphogenetic protein 15 during follicular formation and growth in a marsupial, the brushtail possum (Trichosurus vulpecula)." Mol Cell Endocrinol 192(1-2): 115-26.
Elvin, J. A., A. T. Clark, et al. (1999). "Paracrine actions of growth differentiation factor-9 in the mammalian ovary." Mol Endocrinol 13(6): 1035-48.
Eppig, J. J. and S. M. Downs (1984). "Chemical signals that regulate mammalian oocyte maturation." Biol Reprod 30(1): 1-11.
Findlay, J. K. (1994). "Peripheral and local regulators of folliculogenesis." Reprod Fertil Dev 6(2): 127-39.
Fortune, J. E. (1994). "Ovarian follicular growth and development in mammals." Biol Reprod 50(2): 225-32.
Fujioka, T., Y. Takebayashi, et al. (2000). "Nek2 expression and localization in porcine oocyte during maturation." Biochem Biophys Res Commun 279(3): 799-802.
Gordo, A. C., H. Wu, et al. (2000). "Injection of sperm cytosolic factor into mouse metaphase II oocytes induces different developmental fates according to the frequency of [Ca(2+)](i) oscillations and oocyte age." Biol Reprod 62(5): 1370-9.
Grant, S. A., M. G. Hunter, et al. (1989). "Morphological and biochemical characteristics during ovarian follicular development in the pig." J Reprod Fertil 86(1): 171-83.
Gui, L. M. and I. M. Joyce (2005). "RNA interference evidence that growth differentiation factor-9 mediates oocyte regulation of cumulus expansion in mice." Biol Reprod 72(1): 195-9.
Heikinheimo, O. and W. E. Gibbons (1998). "The molecular mechanisms of oocyte maturation and early embryonic development are unveiling new insights into reproductive medicine." Mol Hum Reprod 4(8): 745-56.
Heldin, C. H., K. Miyazono, et al. (1997). "TGF-beta signalling from cell membrane to nucleus through SMAD proteins." Nature 390(6659): 465-71.
Hensleigh, H. C. and A. G. Hunter (1985). "In vitro maturation of bovine cumulus enclosed primary oocytes and their subsequent in vitro fertilization and cleavage." J Dairy Sci 68(6): 1456-62.
Hunter, M. G. (2000). "Oocyte maturation and ovum quality in pigs." Rev Reprod 5(2): 122-30.
Juengel, J. L., K. J. Bodensteiner, et al. (2004). "Physiology of GDF9 and BMP15 signalling molecules." Anim Reprod Sci 82-83: 447-60.
Juengel, J. L., N. L. Hudson, et al. (2002). "Growth differentiation factor 9 and bone morphogenetic protein 15 are essential for ovarian follicular development in sheep." Biol Reprod 67(6): 1777-89.
Juengel, J. L. and K. P. McNatty (2005). "The role of proteins of the transforming growth factor-beta superfamily in the intraovarian regulation of follicular development." Hum Reprod Update 11(2): 143-60.
Kimura, N., Y. Konno, et al. (2002). "Expression of hyaluronan synthases and CD44 messenger RNAs in porcine cumulus-oocyte complexes during in vitro maturation." Biol Reprod 66(3): 707-17.
Knight, P. G. and C. Glister (2003). "Local roles of TGF-beta superfamily members in the control of ovarian follicle development." Anim Reprod Sci 78(3-4): 165-83.
Laitinen, M., K. Vuojolainen, et al. (1998). "A novel growth differentiation factor-9 (GDF-9) related factor is co-expressed with GDF-9 in mouse oocytes during folliculogenesis." Mech Dev 78(1-2): 135-40.
Liao, W. X., R. K. Moore, et al. (2003). "Effect of intracellular interactions on the processing and secretion of bone morphogenetic protein-15 (BMP-15) and growth and differentiation factor-9. Implication of the aberrant ovarian phenotype of BMP-15 mutant sheep." J Biol Chem 278(6): 3713-9.
Lin, S. Y., J. R. Morrison, et al. (2003). "Regulation of ovarian function by the TGF-beta superfamily and follistatin." Reproduction 126(2): 133-48.
Massague, J. (1990). "The transforming growth factor-beta family." Annu Rev Cell Biol 6: 597-641.
Matzuk, M. M., K. H. Burns, et al. (2002). "Intercellular communication in the mammalian ovary: oocytes carry the conversation." Science 296(5576): 2178-80.
Mazerbourg, S., C. Klein, et al. (2004). "Growth differentiation factor-9 signaling is mediated by the type I receptor, activin receptor-like kinase 5." Mol Endocrinol 18(3): 653-65.
McGaughey, R. W., D. H. Montgomery, et al. (1979). "Germinal vesicle configurations and patterns of polypeptide synthesis of procine oocytes from antral follicles of different size, as related to their competency for spontaneous maturation." J Exp Zool 209(2): 239-54.
McKenzie, L. J., S. A. Pangas, et al. (2004). "Human cumulus granulosa cell gene expression: a predictor of fertilization and embryo selection in women undergoing IVF." Hum Reprod 19(12): 2869-74.
McNatty, K. P., J. L. Juengel, et al. (2005). "Bone morphogenetic protein 15 and growth differentiation factor 9 co-operate to regulate granulosa cell function." Reproduction 129(4): 473-80.
McNatty, K. P., J. L. Juengel, et al. (2003). "Oocyte-derived growth factors and ovulation rate in sheep." Reprod Suppl 61: 339-51.
McNatty, K. P., L. G. Moore, et al. (2004). "The oocyte and its role in regulating ovulation rate: a new paradigm in reproductive biology." Reproduction 128(4): 379-86.
McNatty, K. P., P. Smith, et al. (2005). "Oocyte-expressed genes affecting ovulation rate." Mol Cell Endocrinol 234(1-2): 57-66.
McNeilly, A. S., W. Crow, et al. (1992). "Luteinizing hormone pulses, follicle-stimulating hormone and control of follicle selection in sheep." J Reprod Fertil Suppl 45: 5-19.
McPherron, A. C. and S. J. Lee (1993). "GDF-3 and GDF-9: two new members of the transforming growth factor-beta superfamily containing a novel pattern of cysteines." J Biol Chem 268(5): 3444-9.
Moore, R. K., F. Otsuka, et al. (2003). "Molecular basis of bone morphogenetic protein-15 signaling in granulosa cells." J Biol Chem 278(1): 304-10.
Motlik, J. and J. Fulka (1976). "Breakdown of the germinal vesicle in pig oocytes in vivo and in vitro." J Exp Zool 198(2): 155-62.
Naito, M., F. Yamamura, et al. (1989). "Development, differentiation, and maturation of fetal mouse yolk sac macrophages in cultures." J Leukoc Biol 46(1): 1-10.
Novak, S., F. Paradis, et al. (2004). "Identification of porcine oocyte proteins that are associated with somatic cell nuclei after co-incubation." Biol Reprod 71(4): 1279-89.
Otsuka, F., Z. Yao, et al. (2000). "Bone morphogenetic protein-15. Identification of target cells and biological functions." J Biol Chem 275(50): 39523-8.
Pangas, S. A., C. J. Jorgez, et al. (2004). "Growth differentiation factor 9 regulates expression of the bone morphogenetic protein antagonist gremlin." J Biol Chem 279(31): 32281-6.
Prochazka, R., L. Nemcova, et al. (2004). "Expression of growth differentiation factor 9 messenger RNA in porcine growing and preovulatory ovarian follicles." Biol Reprod 71(4): 1290-5.
Richards, J. S. (1980). "Maturation of ovarian follicles: actions and interactions of pituitary and ovarian hormones on follicular cell differentiation." Physiol Rev 60(1): 51-89.
Richardson, M. C. (1986). "Hormonal control of ovarian luteal cells." Oxf Rev Reprod Biol 8: 321-78.
Salustri, A., A. Camaioni, et al. (1996). "Endocrine and paracrine regulation of cumulus expansion." Zygote 4(4): 313-5.
Shimasaki, S., R. K. Moore, et al. (2004). "The bone morphogenetic protein system in mammalian reproduction." Endocr Rev 25(1): 72-101.
Sidis, Y., T. Fujiwara, et al. (1998). "Characterization of inhibin/activin subunit, activin receptor, and follistatin messenger ribonucleic acid in human and mouse oocytes: evidence for activin's paracrine signaling from granulosa cells to oocytes." Biol Reprod 59(4): 807-12.
Silva, J. R., R. van den Hurk, et al. (2005). "Expression of growth differentiation factor 9 (GDF9), bone morphogenetic protein 15 (BMP15), and BMP receptors in the ovaries of goats." Mol Reprod Dev 70(1): 11-9.
Sirard, M. A. and R. D. Lambert (1986). "Birth of calves after in vitro fertilisation using laparoscopy and rabbit oviduct incubation of zygotes." Vet Rec 119(8): 167-9.
Somfai, T., K. Kikuchi, et al. (2004). "Relationship between the morphological changes of somatic compartment and the kinetics of nuclear and cytoplasmic maturation of oocytes during in vitro maturation of porcine follicular oocytes." Mol Reprod Dev 68(4): 484-91.
Souza, C. J., B. K. Campbell, et al. (2002). "Effect of bone morphogenetic protein 2 (BMP2) on oestradiol and inhibin A production by sheep granulosa cells, and localization of BMP receptors in the ovary by immunohistochemistry." Reproduction 123(3): 363-9.
Su, Y. Q., X. Wu, et al. (2004). "Synergistic roles of BMP15 and GDF9 in the development and function of the oocyte-cumulus cell complex in mice: genetic evidence for an oocyte-granulosa cell regulatory loop." Dev Biol 276(1): 64-73.
ten Dijke, P., K. Miyazono, et al. (2000). "Signaling inputs converge on nuclear effectors in TGF-beta signaling." Trends Biochem Sci 25(2): 64-70.
Tsafriri, A., S. H. Pomerantz, et al. (1976). "Inhibition of oocyte maturation by porcine follicular fluid: partial characterization of the inhibitor." Biol Reprod 14(5): 511-6.
Vanderhyden, B. C., E. A. Macdonald, et al. (2003). "Evaluation of members of the TGFbeta superfamily as candidates for the oocyte factors that control mouse cumulus expansion and steroidogenesis." Reprod Suppl 61: 55-70.
Vitt, U. A., S. Mazerbourg, et al. (2002). "Bone morphogenetic protein receptor type II is a receptor for growth differentiation factor-9." Biol Reprod 67(2): 473-80.
Wilson, T., X. Y. Wu, et al. (2001). "Highly prolific Booroola sheep have a mutation in the intracellular kinase domain of bone morphogenetic protein IB receptor (ALK-6) that is expressed in both oocytes and granulosa cells." Biol Reprod 64(4): 1225-35.
Yan, C., P. Wang, et al. (2001). "Synergistic roles of bone morphogenetic protein 15 and growth differentiation factor 9 in ovarian function." Mol Endocrinol 15(6): 854-66.
Yoshida, M., Y. Ishizaki, et al. (1992). "Effects of pig follicular fluid on maturation of pig oocytes in vitro and on their subsequent fertilizing and developmental capacity in vitro." J Reprod Fertil 95(2): 481-8.