Grant-in-Aid for Scientific Research (B)
|Allocation Type||Single-year Grants |
Applied veterinary science
|Research Institution||Osaka Prefecture University |
INABA Toshio Osaka Prefecture University, Graduate School of Life and Environmental Sciences, Professor, 生命環境科学研究科, 教授 (00137241)
SUGIURA Kikuya Osaka Prefecture University, Graduate Sch. of Life and Environ.Sci., Associate Prof., 生命環境科学研究科, 助教授 (30171143)
KUMAGAI Daijiro Osaka Prefecture University, Graduate Sch. of Life and Environ.Sci., Research Assoc., 生命環境科学研究科, 助手 (70316016)
TANI Hiroyuki Osaka Prefecture University, Graduate Sch. of Life and Environ.Sci., Research Assoc., 生命環境科学研究科, 助手 (00305658)
TORII Ryuzo Siga University of Medical Science, Research Center for Animal Life Science, Professor, 動物生命科学研究センター, 教授 (50106647)
|Project Period (FY)
2002 – 2005
Completed (Fiscal Year 2005)
|Budget Amount *help
¥14,800,000 (Direct Cost: ¥14,800,000)
Fiscal Year 2005: ¥2,200,000 (Direct Cost: ¥2,200,000)
Fiscal Year 2004: ¥2,200,000 (Direct Cost: ¥2,200,000)
Fiscal Year 2003: ¥4,500,000 (Direct Cost: ¥4,500,000)
Fiscal Year 2002: ¥5,900,000 (Direct Cost: ¥5,900,000)
|Keywords||dog / embryonic stem cell / oocyte / in vitro maturation / in vitro fertilization / embryonic fibroblast cell / estrogen / estrus induction / 上皮成長因子 / 精子 / EGF / MHC欠損マウス / 胚性幹細胞 / 卵丘細胞 / エストロジェンレセプター遺伝子 / 性腺刺激ホルモン放出ホルモン / アロマターゼ遺伝子|
Embryonic stem (ES) cells are pluripotent cells with the capacity to generate any type of cell. The objective of this study was to establish ES cell lines from the dog for regenerative veterinary medicine, and the following results were obtained.
1.The rise in the levels of hypothalamic P450 aromatase mRNA, hypothalamic and pituitary estrogen receptor α and β mRNAs, and plasma estradiol-17β was associated with termination of anestrus in the bitch.
2.The intranasal administration of a depot-injectable form of the potent GnRH agonist leuprolide acetate was a useful method for controlling the induction of estrus in anestrous bitches that appears to cause the animals little stress, and a combination of leuprolide acetate and oral estradiol-17β might be more effective in inducing estrus.
3.Although the in vitro maturation rate of canine MI oocytes cultured with estrogen and epidermal growth factor was higher than for oocytes cultured in culture media only, the rate that reached the MII stage w
as not different in both groups.
4.Co-culture of embryonic fibroblast cells enhanced nuclear and cytoplasmic maturation of canine oocytes, and co-culture with canine embryonic fibroblasts developed the embryo up to the 16-cell stage, and with mouse embryonic fibroblasts up to morula stage.
5.Primary canine ES-like cell colonies were formed in 67.9% of inner cell masses from hatched blastocysts, which was significantly higher than from blastocysts (25.6%) and d morulae stage (0%).
6.Canine ES-like cells had typical stem cell-like morphology, expressed specific markers such as alkaline phosphatase activity, stage specific embryonic antigen-1 and Oct-4, and formed embryoid bodies (EBs) in a suspension culture ; extended culture of EBs resulted in the formation of cystic EBs. When the simple Ms were cultured in tissue culture plates, they differentiated into several types of cells including neuron-like, epithelium-like, fibroblast-like, melanocyte-like and myocardium-like cells.
7.Dissociation of canine ES-like cell colonies with collagenase resulted in immediate differentiation, but with mechanical disaggregation these cells remain undifferentiated, and two ES-like cell lines passed 8 passages. Less