Development of Cryopresservation technique for ovarian germline stem cells

2007 Fiscal Year Final Research Report Summary

• Project/Area Number: 17580254
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Single-year Grants
• Section: 一般
• Research Field: Basic veterinary science/Basic zootechnical science
• Research Institution: Kyoto University
• Principal Investigator: SUGIMOTO Miki Kyoto University, Graduate School of Agriculture, Assistant Professor (20243074)
• Co-Investigator(Kenkyū-buntansha): MANABE Noboru The University of Tokyo, Graduate School of Agricultural and Life Sciences, Professor (80243070)
• Project Period (FY): 2005 – 2007
• Keywords: Mammals / Female Reproductive Organs / Ovary / Germ cell / Stem cells

Research Abstract

In this study, ovarian germline calls in mammals welt examined. Germline cells in murine ovaries were surveyed by immunohistological techniques. Positive reactions for Vasa homologue (MVH), a germ cell marker, were lend in oocytes and scattered cells on ovarian surface. Incorporation of 5-bromo-2'-deoxyuridine (BrdU) and expression of proliferating cell markers (Ki-67 anf MCM3) were mainly found in granulose cells and were not found in MVH positive cells. To collect ovarian gemiline stem cells, murine ovaries were dispersed and fractionated by sieving, Percoll gradient centrifugation, adhesiveness, and anti-SSEA-1 antibody/ magnetic beads selection. Cell proliferations were hardly observed in MVH positive cells in all fractionation techniches. Cells collected by anti-SSEA-1 antibody/ magnetic beads showed mainly fibroblastic or epithelial morphology and partly aggregated in culture. MVH positive cells were observed but they were negative for cell proliferation. For cryopreservation, ovaries of immature mice were frozen by slow cooling method with dimethyl sulfoxide and trehalose as cryoprogtectants, thawed and then cultured. Damages in MVH positive cells on the surface of ovaries, the candidates fir germline stem cells, were mild, suggesting that these cells can be preserved using the cryopreservation techniques developed for ovarian tissue. Trehalose in high concentration seemed to increase damages in ovarian tissue. Ovaries of neonatal mice were organ cultured in different temperatures. MVH positive calls on the surface of ovaries was small in number and changes were not detected, however distribution of oocytes showed a slight changes by culture temperature. In this study, ovarian germline stem cells were not confeermed, although the results cannot eliminate the possibility of the presence of ovarian germline stem cells completely.