Vitrification of immature mouse oocytes by the modified-cut standard straw method

Abstract

The feasibility of using the modified-cut standard straw (M-CSS) method for the vitrification of immature mouse oocytes has been tested. The effects of different vitrification methods on oocyte survival, cytoskeletal organization, the distribution of cortical granules (CGs), and apoptosis have also been compared. Immature mouse oocytes were vitrified-thawed using electron microscope grid or M-CSS method, and cultured to meiosis II (MII) stage. Oocyte development, cytoskeletal organization, CG distribution, and the expression of apoptosis-related genes were evaluated. Rates of recovery (91.7 vs. 74.9%) and survival (89.0 vs. 62.6%) were significantly higher in M-CSS group than in EM grid group. The number of oocytes with normal chromosome alignment at the spindle and spindle morphology were similar in both groups. However, the actin cap was significantly degraded in EM grid groups (52.6 vs. 35.1%, respectively). Abnormal release of CGs also frequently occurred in EM grid groups (42.6 vs. 32.7%, respectively). Pro-apoptosis-related gene expression levels of Bax, caspase 3 were expressed lower than control in MII stage oocytes derived from M-CSS group; anti apoptosis-related genes, survivin and heat shock factor-1 (Hsf-1) were slightly increased. However, all genes expression was significantly increased in MII stage oocytes derived from EM grid groups. Vitrification reduces the survival rate of immature mouse oocytes, alters cytoskeletal organization and CG distribution, and promotes apoptosis. However, these effects are less pronounced in vitrified oocytes generated by M-CSS than in those generated by EM grid method. Therefore, the novel M-CSS is a feasible approach for the cryopreservation of immature mouse oocytes.

Keywords: apoptosis-related gene; cortical granule; cytoskeleton; immature mouse oocyte; modified-cut standard straw; vitrification.