Yanchang Wei

Paternally induced transgenerational inheritance of susceptibility to diabetes in mammals

Significance Increasing evidence suggests that certain acquired traits can be transmitted to the next generation. However, controversy over the inheritance of acquired traits remains, as the exact molecular and mechanistic basis for these observations remains largely unclear. In this study, using a nongenetic prediabetes mouse model, we have shown that environmentally induced epigenetic alterations in sperm can be inherited to the next generation. Paternal prediabetic conditions affect epigenetic marks in offspring and can be inherited for several generations. This finding provides a molecular basis for the inheritance of acquired traits and may have implications in explaining the prevalence of obesity, type 2 diabetes, and other chronic metabolic diseases. The global prevalence of prediabetes and type 2 diabetes (T2D) is increasing, and it is contributing to the susceptibility to diabetes and its related epidemic in offspring. Although the impacts of paternal impaired fasting blood glucose and glucose intolerance on the metabolism of offspring have been well established, the exact molecular and mechanistic basis that mediates these impacts remains largely unclear. Here we show that paternal prediabetes increases the susceptibility to diabetes in offspring through gametic epigenetic alterations. In our findings, paternal prediabetes led to glucose intolerance and insulin resistance in offspring. Relative to controls, offspring of prediabetic fathers exhibited altered gene expression patterns in the pancreatic islets, with down-regulation of several genes involved in glucose metabolism and insulin signaling pathways. Epigenomic profiling of offspring pancreatic islets revealed numerous changes in cytosine methylation depending on paternal prediabetes, including reproducible changes in methylation over several insulin signaling genes. Paternal prediabetes altered overall methylome patterns in sperm, with a large portion of differentially methylated genes overlapping with that of pancreatic islets in offspring. Our study uniquely revealed that prediabetes can be inherited transgenerationally through the mammalian germ line by an epigenetic mechanism.

DNA Methylation in Oocytes and Liver of Female Mice and Their Offspring: Effects of High-Fat-Diet–Induced Obesity

Background: Maternal obesity has adverse effects on oocyte quality, embryo development, and the health of the offspring. Objectives: To understand the underlying mechanisms responsible for the negative effects of maternal obesity, we investigated the DNA methylation status of several imprinted genes and metabolism-related genes. Methods: Using a high-fat-diet (HFD)-induced mouse model of obesity, we analyzed the DNA methylation of several imprinted genes and metabolism-related genes in oocytes from control and obese dams and in oocytes and liver from their offspring. Analysis was performed using combined bisulfite restriction analysis (COBRA) and bisulfite sequencing. Results: DNA methylation of imprinted genes in oocytes was not altered in either obese dams or their offspring; however, DNA methylation of metabolism-related genes was changed. In oocytes of obese mice, the DNA methylation level of the leptin (Lep) promoter was significantly increased and that of the Ppar-α promoter was reduced. Increased methylation of Lep and decreased methylation of Ppar-α was also observed in the liver of female offspring from dams fed the high-fat diet (OHFD). mRNA expression of Lep and Ppar-α was also significantly altered in the liver of these OHFD. In OHFD oocytes, the DNA methylation level of Ppar-α promoter was increased. Conclusions: Our results indicate that DNA methylation patterns of several metabolism-related genes are changed not only in oocytes of obese mice but also in oocytes and liver of their offspring. These data may contribute to the understanding of adverse effects of maternal obesity on reproduction and health of the offspring. Citation: Ge ZJ, Luo SM, Lin F, Liang QX, Huang L, Wei YC, Hou Y, Han ZM, Schatten H, Sun QY. 2014. DNA methylation in oocytes and liver of female mice and their offspring: effects of high-fat-diet–induced obesity. Environ Health Perspect 122:159–164; http://dx.doi.org/10.1289/ehp.1307047

The root of reduced fertility in aged women and possible therapentic options: Current status and future perspects

It is well known that maternal ageing not only causes increased spontaneous abortion and reduced fertility, but it is also a high genetic disease risk. Although assisted reproductive technologies (ARTs) have been widely used to treat infertility, the overall success is still low. The main reasons for age-related changes include reduced follicle number, compromised oocyte quality especially aneuploidy, altered reproductive endocrinology, and increased reproductive tract defect. Various approaches for improving or treating infertility in aged women including controlled ovarian hyperstimulation with intrauterine insemination (IUI), IVF/ICSI-ET, ovarian reserve testing, preimplantation genetic diagnosis and screening (PGD/PGS), oocyte selection and donation, oocyte and ovary tissue cryopreservation before ageing, miscarriage prevention, and caloric restriction are summarized in this review. Future potential reproductive techniques for infertile older women including oocyte and zygote micromanipulations, derivation of oocytes from germ stem cells, ES cells, and iPS cells, as well as through bone marrow transplantation are discussed.

Aberrant expression and methylation status of putatively imprinted genes in placenta of cloned piglets.

Unlike embryos derived from fertilization, most cloned embryos die during postimplantation development, and those that survive to term are frequently defective. Many of the observed defects involve placenta. Abnormal placentation has been described in several cloned species. Imprinted genes are important regulators of placenta growth, and may be subjected to faulty reprogramming during somatic cell nuclear transfer. We aimed to determine the expression levels and methylation patterns of imprinted genes in placentas of live cloned piglets and dead ones. Quantitative real-time reverse transcriptase-polymerase chain reaction (RT-PCR) analysis showed that the expression of all four imprinted genes (IGF2, H19, PEG3, and GRB10) was significantly reduced in placentas of dead clones compared with placentas of live cloned piglets and controls (p < 0.05). In contrast, both live and dead cloned piglets exhibited steady-state mRNA levels for these genes within the control range (p > 0.05). Transcript levels for these genes in live clones rarely differed from those of controls in both piglets and placentas. Examination of the methylation status of DMR2 of IGF2 and CTCF3 of H19 genes revealed that both genes exhibited significant high methylation levels in placentas of dead clones compared with placentas of live clones and controls. In contrast, both genes showed a normal differential methylation pattern in live cloned piglets and their placentas compared with controls. Importantly, dead cloned piglets also showed a normal pattern. Our results suggest that abnormal expression of imprinted genes in placenta may contribute to the development failure in pig somatic cell nuclear transfer (SCNT), which may be caused by abnormal methylation patterns in differentially methylated regions (DMRs) of imprinted genes as a result of incomplete reprogramming during SCNT.

Spindle Assembly Checkpoint Regulates Mitotic Cell Cycle Progression during Preimplantation Embryo Development

Errors in chromosome segregation or distribution may result in aneuploid embryo formation, which causes implantation failure, spontaneous abortion, genetic diseases, or embryo death. Embryonic aneuploidy occurs when chromosome aberrations are present in gametes or early embryos. To date, it is still unclear whether the spindle assembly checkpoint (SAC) is required for the regulation of mitotic cell cycle progression to ensure mitotic fidelity during preimplantation development. In this study, using overexpression and RNA interference (RNAi) approaches, we analyzed the role of SAC components (Bub3, BubR1 and Mad2) in mouse preimplantation embryos. Our data showed that overexpressed SAC components inhibited metaphase-anaphase transition by preventing sister chromatid segregation. Deletion of SAC components by RNAi accelerated the metaphase-anaphase transition during the first cleavage and caused micronuclei formation, chromosome misalignment and aneuploidy, which caused decreased implantation and delayed development. Furthermore, in the presence of the spindle-depolymerizing drug nocodazole, SAC depleted embryos failed to arrest at metaphase. Our results suggest that SAC is essential for the regulation of mitotic cell cycle progression in cleavage stage mouse embryos.

Maternal Diabetes Causes Alterations of DNA Methylation Statuses of Some Imprinted Genes in Murine Oocytes

ABSTRACT Maternal diabetes has adverse effects not only on oocyte quality but also on embryo development. However, it is still unknown whether the DNA imprinting in oocytes is altered by diabetes. By using streptozotocin (STZ)-induced and nonobese diabetic (NOD) mouse models we investigated the effect of maternal diabetes on DNA methylation of imprinted genes in oocytes. Mice which were judged as being diabetic 4 days after STZ injection were used for experiments. In superovulated oocytes of diabetic mice, the methylation pattern of Peg3 differential methylation regions (DMR) was affected in a time-dependent manner, and evident demethylation was observed on Day 35 after STZ injection. The expression level of DNA methyltransferases (DNMTs) was also decreased in a time-dependent manner in diabetic oocytes. However, the methylation patterns of H19 and Snrpn DMRs were not significantly altered by maternal diabetes, although there were some changes in Snrpn. In NOD mice, the methylation pattern of Peg3 was similar to that of STZ-induced mice. Embryo development was adversely affected by maternal diabetes; however, no evident imprinting abnormality was observed in oocytes from female offspring derived from a diabetic mother. These results indicate that maternal diabetes has adverse effects on DNA methylation of maternally imprinted gene Peg3 in oocytes of a diabetic female in a time-dependent manner, but methylation in offsprings oocytes is normal.

Unfaithful Maintenance of Methylation Imprints Due to Loss of Maternal Nuclear Dnmt1 during Somatic Cell Nuclear Transfer

The low success rate of somatic cell nuclear transfer (SCNT) in mammalian cloning is largely due to imprinting problems. However, little is known about the mechanisms of reprogramming imprinted genes during SCNT. Parental origin-specific DNA methylation regulates the monoallelic expression of imprinted genes. In natural fertilization, methylation imprints are established in the parental germline and maintained throughout embryonic development. However, it is unclear whether methylation imprints are protected from global changes of DNA methylation in cloned preimplantation embryos. Here, we demonstrate that cloned porcine preimplantation embryos exhibit demethylation at differentially methylated regions (DMRs) of imprinted genes; in particular, demethylation occurs during the first two cell cycles. By RNAi-mediated knockdown, we found that Dnmt1 is required for the maintenance of methylation imprints in porcine preimplantation embryos. However, no clear signals were detected in the nuclei of oocytes and preimplantation embryos by immunofluorescence. Thus, Dnmt1 is present at very low levels in the nuclei of porcine oocytes and preimplantation embryos and maintains methylation imprints. We further showed that methylation imprints were rescued in nonenucleated metaphase II (MII) oocytes. Our results indicate that loss of Dnmt1 in the maternal nucleus during SCNT significantly contributes to the unfaithful maintenance of methylation imprints in cloned embryos.

GM130, a cis-Golgi protein, regulates meiotic spindle assembly and asymmetric division in mouse oocyte

GM130, a cis-Golgi protein, plays key roles in various mitotic events, but its function in mammalian oocyte meiosis remains unknown. In this study, we found that GM130 was localized to the spindle poles at both metaphase I and metaphase II stages and associated with the midbody at telophase I stage. The association of GM130 with spindle poles was further confirmed by its colocalization with the centrosome-associated proteins, MEK1/2. By nocodazole treatment, we clarified that GM130 localization was consistently dependent on spindle assembly. Then we investigated the possible function of GM130 by specific morpholino microinjection. This treatment caused abnormal spindle formation, and decreased first polar body extrusion. Our results showed that knockdown of GM130 impaired the localization of MTOCs proteins γ-tubulin and Plk1. Using live cell imaging we observed that depletion of GM130 affected spindle migration and resulted in elongated spindle and large polar body extrusion. We further found that depletion of GM130 blocked p-MEK1/2 accumulation at the spindle poles. And, it was shown that GM130 detached from the spindle poles in oocytes treated with MEK specific inhibitor U0126. Taken together, our results suggested that GM130 regulates microtubule organization and might cooperate with the MAPK pathway to play roles in spindle organization, migration and asymmetric division during mouse oocyte maturation.

The G Protein Coupled Receptor 3 Is Involved in cAMP and cGMP Signaling and Maintenance of Meiotic Arrest in Porcine Oocytes

The arrest of meiotic prophase in mammalian oocytes within fully grown follicles is dependent on cyclic adenosine monophosphate (cAMP) regulation. A large part of cAMP is produced by the Gs-linked G-protein-coupled receptor (GPR) pathway. In the present study, we examined whether GPR3 is involved in the maintenance of meiotic arrest in porcine oocytes. Expression and distribution of GPR3 were examined by western blot and immunofluorescence microscopy, respectively. The results showed that GPR3 was expressed at various stages during porcine oocyte maturation. At the germinal vesicle (GV) stage, GPR3 displayed a maximal expression level, and its expression remained stable from pro-metaphase I (MI) to metaphase II (MII). Immunofluorescence staining showed that GPR3 was mainly distributed at the nuclear envelope during the GV stage and localized to the plasma membrane at pro-MI, MI and MII stages. RNA interference (RNAi) was used to knock down the GPR3 expression within oocytes. Injection of small interfering double-stranded RNA (siRNA) targeting GPR3 stimulated meiotic resumption of oocytes. On the other hand, overexpression of GPR3 inhibited meiotic maturation of porcine oocytes, which was caused by increase of cGMP and cAMP levels and inhibition of cyclin B accumulation. Furthermore, incubation of porcine oocytes with the GPR3 ligand sphingosylphosphorylcholine (SPC) inhibited oocyte maturation. We propose that GPR3 is required for maintenance of meiotic arrest in porcine oocytes through pathways involved in the regulation of cAMP and cGMP.

Short-term Preservation of Porcine Oocytes in Ambient Temperature: Novel Approaches

The objective of this study was to evaluate the feasibility of preserving porcine oocytes without freezing. To optimize preservation conditions, porcine cumulus-oocyte complexes (COCs) were preserved in TCM-199, porcine follicular fluid (pFF) and FCS at different temperatures (4°C, 20°C, 25°C, 27.5°C, 30°C and 38.5°C) for 1 day, 2 days or 3 days. After preservation, oocyte morphology, germinal vesicle (GV) rate, actin cytoskeleton organization, cortical granule distribution, mitochondrial translocation and intracellular glutathione level were evaluated. Oocyte maturation was indicated by first polar body emission and spindle morphology after in vitro culture. Strikingly, when COCs were stored at 27.5°C for 3 days in pFF or FCS, more than 60% oocytes were still arrested at the GV stage and more than 50% oocytes matured into MII stages after culture. Almost 80% oocytes showed normal actin organization and cortical granule relocation to the cortex, and approximately 50% oocytes showed diffused mitochondria distribution patterns and normal spindle configurations. While stored in TCM-199, all these criteria decreased significantly. Glutathione (GSH) level in the pFF or FCS group was higher than in the TCM-199 group, but lower than in the non-preserved control group. The preserved oocytes could be fertilized and developed to blastocysts (about 10%) with normal cell number, which is clear evidence for their retaining the developmental potentiality after 3d preservation. Thus, we have developed a simple method for preserving immature pig oocytes at an ambient temperature for several days without evident damage of cytoplasm and keeping oocyte developmental competence.