title: Growth of mouse oocytes to maturity from premeiotic germ cells in vitro.
abstract: In the present study, we established an in vitro culture system suitable for generating fertilizable oocytes from premeiotic mouse female germ cells. These results were achieved after first establishing an in vitro culture system allowing immature oocytes from 12-14 day-old mice to reach meiotic maturation through culture onto preantral granulosa cell (PAGC) monolayers in the presence of Activin A (ActA). To generate mature oocytes from premeiotic germ cells, pieces of ovaries from 12.5 days post coitum (dpc) embryos were cultured in medium supplemented with ActA for 28 days and the oocytes formed within the explants were isolated and cocultured onto PAGC monolayers in the presence of ActA for 6-7 days. The oocytes were then subjected to a final meiotic maturation assay to evaluate their capability to undergo germinal vesicle break down (GVBD) and reach the metaphase II (MII) stage. We found that during the first 28 days of culture, a significant number of oocytes within the ovarian explants reached nearly full growth and formed preantral follicle-like structures with the surrounding somatic cells. GSH level and Cx37 expression in the oocytes within the explants were indicative of proper developmental conditions. Moreover, the imprinting of Igf2r and Peg3 genes in these oocytes was correctly established. Further culture onto PAGCs in the presence of ActA allowed about 16% of the oocytes to undergo GVBD, among which 17% reached the MII stage during the final 16-18 hr maturation culture. These MII oocytes showed normal spindle and chromosome assembly and a correct ERK1/2 activity. About 35% of the in vitro matured oocytes were fertilized and 53.44% of them were able to reach the 2-cell stage. Finally, around 7% of the 2-cell embryos developed to the morula/blastocyst stage.
authors: Key Laboratory of Animal Reproduction and Germplasm Enhancement in Universities of Shandong, Qingdao Agricultural University, Qingdao, China.
authors: Zhi-Peng
authors: ZP
authors: Zhang
authors: Gui-Jin
authors: GJ
authors: Liang
authors: Xi-Feng
authors: XF
authors: Zhang
authors: Guo-Liang
authors: GL
authors: Zhang
authors: Hu-He
authors: HH
authors: Chao
authors: Lan
authors: L
authors: Li
authors: Xiao-Feng
authors: XF
authors: Sun
authors: Ling-Jiang
authors: LJ
authors: Min
authors: Qing-Jie
authors: QJ
authors: Pan
authors: Qing-Hua
authors: QH
authors: Shi
authors: Qing-Yuan
authors: QY
authors: Sun
authors: Massimo
authors: M
authors: De Felici
authors: Wei
authors: W
authors: Shen
mesh_terms: D028341:Activins / Q000494:pharmacology; D000818:Animals; D001755:Blastocyst / Q000166:cytology / Q000187:drug effects; D018929:Cell Culture Techniques / Q000379:methods*; D049109:Cell Proliferation / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D018920:Coculture Techniques; D019175:DNA Methylation / Q000187:drug effects; D005260:Female; D006107:Granulosa Cells / Q000166:cytology / Q000187:drug effects; D008540:Meiosis / Q000187:drug effects; D051379:Mice; D009028:Morula / Q000166:cytology / Q000187:drug effects; D009865:Oocytes / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D010053:Ovary / Q000196:embryology
chemical_list: C422405:activin A; D028341:Activins