Ultrastructural Changes and Methylation of Human Oocytes Vitrified at the Germinal Vesicle Stage and Matured in vitro after Thawing.

AIM: The study aimed to assess the effect of in vitro vitrification and maturation on the nuclear configuration, cytoplasmic maturation and global DNA methylation pattern of human germinal vesicle (GV) stage oocytes. METHODS: Human oocytes from infertile women were randomly assigned to one of 3 groups: (i) metaphase II (MII) oocytes matured in vivo (vivo-MII, n = 56) as controls; (ii) MII oocytes matured in vitro (vitro-MII, n = 106); and (iii) MII oocytes that were vitrified at the GV stage, warmed and matured in vitro (cryo-MII, n = 122). All MII oocytes were fixed and immunofluorescence staining for spindle, chromosome, mitochondrion and cortical granules (CGs) were performed; examination was done using immunofluorescence laser scanning confocal microscope. The expression of 5-methycytosine in these MII oocytes was also assessed. RESULTS: No significant difference was observed between vitro-MII and cryo-MII groups with respect to oocyte maturation rate (72.4 vs. 78.3%). No significant differences were observed in the distribution of mitochondria, migration of CG and global DNA methylation pattern among the 3 study groups. However, the abnormal configuration of spindle and chromosome was significantly higher in cryo-MII group (78.9 and 84.2%) as compared to that in the vitro-MII (45.0 and 50.0%, p < 0.05) and vivo-MII groups (27.3 and 36.4%, p < 0.05). CONCLUSION: GV oocytes appeared to resist vitrification and retain their potential for maturation to MII stage oocytes after thawing. However, GV oocytes vitrification combined with in vitro maturation could affect the organization of spindle and chromosome.

Abnormality of maternal-to-embryonic transition contributes to MEHP-induced mouse 2-cell block.

Mono (2-ethylhexyl) phthalate (MEHP), an environmental contaminant, is known to cause many serious diseases, especially in reproductive system. However, little is known about the effect of MEHP on preimplantation embryo development. In this study, we found that the development of mouse 2-cell embryo was blocked by 10(-3) M MEHP. A significant increase in the level of reactive oxygen species (ROS) was observed in arrested 2-cell embryo following 10(-3) M MEHP treatment for 24 h. However, antioxidants, catalase (CAT), and superoxide dismutase (SOD), reduced intracellular ROS and protected MEHP-exposed embryos from death but failed to return the arrested embryos. Further experiments demonstrated that the level of apoptosis was not altered in live arrested 2-cell embryo and increased in dead arrested 2-cell embryo after MEHP treatment, which implied that ROS and apoptosis were not related with 2-cell block. During analysis of the indicators of embryonic genome activation (EGA) initiation (Hsc70, MuERV-L, Hsp70.1, eIF-1A, and Zscan4) and maternal-effect genes (OCT4 and SOX2), we found that MEHP treatment could significantly decline Hsc70, MuERV-L mRNA level and SOX2 protein level, and markedly enhance Hsp70.1, eIF-1A, Zscan4 mRNA level, and OCT4 protein level at 2-cell to 4-cell stage. Supplementation of CAT and SOD did not reverse the expression tendency of EGA related genes. Collectively, this study demonstrates for the first time that MEHP-induced 2-cell block is mediated by the failure of EGA onset and maternal-effect genes, not oxidative stress and apoptosis.

Exposure to mono-n-butyl phthalate disrupts the development of preimplantation embryos.

Dibutyl phthalate (DBP), a widely used phthalate, is known to cause many serious diseases, especially in the reproductive system. However, little is known about the effects of its metabolite, mono-n-butyl phthalate (MBP), on preimplantation embryo development. In the present study, we found that treatment of embryos with 10⁻³ M MBP impaired developmental competency, whereas exposure to 10⁻4 M MBP delayed the progression of preimplantation embryos to the blastocyst stage. Furthermore, reactive oxygen species (ROS) levels in embryos were significantly increased following treatment with 10⁻³ M MBP. In addition, 10⁻³ M MBP increased apoptosis via the release of cytochrome c, whereas immunofluorescent analysis revealed that exposure of preimplantation embryos to MBP concentration-dependently (10⁻5, 10⁻4 and 10⁻³ M) decreased DNA methylation. Together, the results indicate a possible relationship between MBP exposure and developmental failure in preimplantation embryos.

MiR-185 is involved in human breast carcinogenesis by targeting Vegfa.

MiR-185 expression has been associated with many cancers. However, the roles of miR-185 in human breast cancer remain elusive. Here, we found that miR-185 expression was decreased in human breast cancer tissues compared with healthy tissue controls. Up-regulation of miR-185 inhibited breast cancer cell proliferation and invasion and vice versa. MiR-185 was shown to bind to the 3'-untranslated region (UTR) of vascular endothelial growth factor a (Vegfa), and a significant inverse correlation was found between miR-185 and Vegfa. Vegfa overexpression partially restored the inhibition of cell proliferation and invasion that was induced by miR-185, and vice versa. Additionally, Vegfa expression was found to be high in human breast cancer tissues. Thus, miR-185-mediated Vegfa targeting may be involved in breast cancer formation.