Abstract:

OBJECTIVE: To establish mouse preantral follicles 3-dimensions in vitro growth (3D-IVG) system and an efficient in vitro fertilization system. METHODS：Preantral follicles (PFs) of the 12 d old Kunming mice were isolated. 150 μm PFs were selected from each ovary. Then，PFs were cultured in 3D-IVG system for about 6 d. Ovulation of surviving follicles was induced by HCG and EGF. 36 h later，the mucificated cumulus-oocyte-complexes (COCs) and the matured oocytes were collected. Germinal vesicle breakdown (GVBD)，germinal vesicle (GV) oocytes and the matured oocytes were calculated under the stereomicroscope. Chromosomal abnormalities of matured oocytes including numerical and structural aberrations were examined and recorded by C-band strains compared with that of matured oocytes in vitro convention (IVC)．We put sperms and mature COCs together for 24-48 h，then， observed the zygote formation. RESULTS：In 3D-IVG system，the survival rate of the follicles was 82.5% and the mean diameter of follicles increased rapidly. After 6 d of culture ，the survival rate was 91.7％，the ovulation rate induced by hormone was 82.6％，6.1％ oocytes arrested at GV stage，45.4％ oocytes showed GVBD and 48.5％ oocytes emitted the first polar body．After 6 d 3D-IVG culture，the development rate (48.5%) of PFs was strikingly lower than in vivo oocytes superovulation (IVC，82.9％) (P<0.05). However，the chromosomal abnormalities of 3D-IVG have no significant increase comparing with that of oocytes in vitro maturation (IVM) and oocytes superovulation in vivo. We observed that those matured oocytes cultured from 3D-IVG system could get fertilized，from which we can draw a conclusion that they obtained the ability of developing to embryos. CONLUSION：We established and optimized mouse preantral follicles 3D-IVG system，which may provide a new method for reproductive toxicology and embryo engineering．

Key words: mice, preantral follicles, 3-dimensions in vitro growth system