The effects of TETs on DNA methylation and hydroxymethylation of mouse oocytes after vitrification and warming.

Oocyte vitrification has extensively been applied in the field of embryo engineering and in the preservation of genetic resources of fine livestock. Following our previous work in oocyte vitrification and the level change of DNA methylation, here we further explored the dynamic change of three active demethylation proteins: Ten-Eleven-Translocation 1/2/3(TET1/2/3), 5-methylcytosine (5 mC) and 5-hydroxymethycytosine (5hmC) after vitrification and warming. In order to observe the active demethylation in vitrified oocytes, two small molecular regulators, i.e. Vitamin C (VC) and dimethyloxaloylglycine (DMOG) were used to adjust activity and level of the TET 3 protein. The results showed that the levels of 5 mC and 5hmC were significantly decreased after 2 h of vitrification (P < 0.01). Moreover, the level of TET3 protein was significantly increased after 2 h warming (P < 0.01). And the relative gene expression of TET2/3 did not change in the first 2 h, but significantly increased after 2 h (P < 0.01). When VC was added to vitrification and recovery medium, it could not significantly improve the level of TET3 gene expression, and affect 5 mC and 5hmC expression (P > 0.05). When the DMOG was added to the solutions of vitrification, the level of 5hmC showed significantly increase (P < 0.01). In conclusion, the oocyte vitrification procedure reduced DNA methylation and hydroxymethylation in MII oocytes, but adding VC and DMOG to vitrification medium can prevent the reduction of DNA hydroxymethylation by increasing activity of TET3 methylation protein after vitrification and warming.

Evidence that downregulation of Wilms' tumor 1 (WT1) is involved in cortical stromal cell differentiation into theca cells in adult bovine ovaries.

Bovine theca cells are thought to differentiate from cortical stromal cells, and ovary-derived Wilms' tumor 1+ (WT1+ ) cells are the primary source of mouse theca cells. However, it is not known whether the differentiation of cortical stromal cells is regulated by WT1. Here, we identified WT1 in the cortical stroma and theca layer of the bovine ovary and analyzed the theca cell functional markers in cortical stromal cells and theca cells; in addition, we determined the effects of this gene on the secretion of androstenedione and progesterone by cortical stromal cells and the responsiveness of cortical stromal cells to luteinizing hormone (LH) in vitro. We used quantitative reverse-transcription polymerase chain reaction (RT-qPCR), western blot analysis, and immunohistochemistry to discover that the cortical stroma had higher WT1 expression than the theca layer. We used RT-qPCR and ELISA analyses to determine that the cortical stromal cells had lower levels of androstenedione and progesterone secretion and LHR messenger RNA expression than the levels of the theca cells. In cultured bovine cortical stromal cells, we found that WT1 downregulation increased androstenedione and progesterone secretion but had no effect on the LH responsiveness. Notably, the increase in androstenedione and progesterone secretion was associated with an increase in 3-ß-hydroxysteroid dehydrogenase expression. In conclusion, the results suggest that WT1 is involved in the differentiation of cortical stromal cells into cells with characteristics similar to theca cells of antral follicles in adult bovine ovaries.

Effect of oocyte vitrification on DNA damage in metaphase II oocytes and the resulting preimplantation embryos.

As an assisted reproduction technology, vitrification has been widely used for oocyte and embryo cryopreservation. Many studies have indicated that vitrification affects ultrastructure, gene expression, and epigenetic status. However, it is still controversial whether oocyte vitrification could induce DNA damage in metaphase II (MII) oocytes and the resulting early embryos. This study determined whether mouse oocytes vitrification induce DNA damage in MII oocytes and the resulting preimplantation embryos, and causes for vitrification-induced DNA damage. The effects of oocyte vitrification on reactive oxygen species (ROS) levels, γ-H2AX accumulation, apoptosis, early embryonic development, and the expression of DNA damage-related genes in early embryos derived by in vitro fertilization were examined. The results indicated that vitrification significantly increased the number of γ-H2AX foci in zygotes and two-cell embryos. Trp53bp1 was upregulated in zygotes, two-cell embryos and four-cell embryos in the vitrified group, and Brca1 was increased in two-cell embryos after vitrification. Vitrification also increased the ROS levels in MII oocytes, zygotes, and two-cell embryos and the apoptotic rate in blastocysts. Resveratrol (3,5,4'-trihydroxystilbene) treatment decreased the ROS levels and the accumulation of γ-H2AX foci in zygotes and two-cell embryos and the apoptotic rate in blastocysts after vitrification. Overall, vitrification-induced abnormal ROS generation, γ-H2AX accumulation, an increase in the apoptotic rate and the disruption of early embryonic development. Resveratrol treatment could decrease ROS levels, γ-H2AX accumulation, and the apoptotic rate and improve early embryonic development. Vitrification-associated γ-H2AX accumulation is at least partially due to abnormal ROS generation.

Resveratrol improved the developmental potential of oocytes after vitrification by modifying the epigenetics.

Resveratrol (Res) has been reported to be able to improve oocyte vitrification because of its antioxidative properties. The objective of this study was to further assess the positive effect of Res addition on the developmental potential of vitrified mouse oocytes from the perspective of epigenetic alterations. First, 2 µM Res was chosen as the optimal concentration on the basis of its effects on survival and its antioxidative properties. We found that Res addition significantly promoted fertilization (63.8% vs. 42.9%) and blastocyst formation (68.3% vs. 50.2%) after oocyte vitrification. The quality of the derived blastocysts was also higher after Res treatment. Regarding epigenetic aspects, the expression of the important deacetylase SIRT1 was found to decrease significantly upon vitrification, but it was rescued by Res. The abnormal levels of H3K9 acetylation and DNA methylation in vitrified oocytes were restored by Res addition. Moreover, the expression of several imprinted genes was affected by oocyte vitrification. Among them, abnormal Gtl2 and Peg3 expression levels were restored by Res addition. Therefore, the methylation of their imprinted control regions (ICRs) was examined. Surprisingly, the abnormal patterns of Gtl2 and Peg3 methylation in blastocysts developed from vitrified oocytes were both restored by Res addition. Finally, the full-term embryonic development showed that the birth rate was improved significantly by Res addition (56.2% vs. 38.1%). Collectively, Res was beneficial for the pre- and postimplantation embryonic development. Except for the antioxidative activity, Res also played a role in the correction of some abnormal epigenetic modifications caused by oocyte vitrification.

Confirmation of the abnormal lipid metabolism as a risk factor for the disease of leukoaraiosis.

Our purpose is to screen out medical history indicators and test indicators linked to lipid metabolism which is closely correlated to leukoaraiosis (LA), and to build assistant diagnosis model based on support vector machine (SVM), which provided theoretical evidence for genesis and development of LA. One thousand LA patients who underwent magnetic resonance imaging (MRI) examination in Imaging Department was retrospectively analyzed and divided into LA group and non-LA group in accordance with examination results. Detailed clinical statistics of the two groups were collected, including test indicators related to lipid metabolism, such as total cholesterol (TC), triglyceride (TG), low density lipoprotein (LDL), high density lipoprotein (HDL), medical history indicators, age, sex, diabetes, hypertension, hyperlipidemia, history of intracranial infection, history of cerebral hemorrhage, cerebral infarction, lacunar infarction and relevant biochemical indexes. The study shows that patients' incidence of LA was 31.10%; in accordance with Logistic analysis, the incidence of LA is significantly correlated to factors like age, hypertension, history of cerebral hemorrhage, cerebral infarction, lacunar infarction and triglyceride elevation; two SVMs, one including all variables and the other containing all screened variables were successfully established, and the former's accuracy, specificity and sensitivity respectively were 85.0%, 85.0% and 85.0% while the latter's 90.0%, 100.0% and 80.0%. Test indicators and medical history indicators of lipid metabolism correlated to LA were screened out successfully. Meanwhile, an effective SVM model also was built successfully, which is able to predict LA relatively accurately and can be used as assistant diagnostic tool for clinicians.

An archean impact layer from the Pilbara and Kaapvaal cratons.

The Barberton greenstone belt of South Africa and the eastern Pilbara block of Western Australia provide information about Earth's surface environments between 3.2 and 3.5 billion years ago, including evidence for four large bolide impacts that likely created large craters, deformed the target rocks, and altered the environment. We have obtained identical single-zircon uranium-lead ages of 3470 +/- 2 million years ago for the oldest impact events from each craton. These deposits represent a single global fallout layer that is associated with sedimentation by an impact-generated tsunami and in Western Australia is represented by a major erosional unconformity.