Zhao-Jia Ge

Unique insights into maternal mitochondrial inheritance in mice

In animals, mtDNA is always transmitted through the female and this is termed “maternal inheritance.” Recently, autophagy was reported to be involved in maternal inheritance by elimination of paternal mitochondria and mtDNA in Caenorhabditis elegans; moreover, by immunofluorescence, P62 and LC3 proteins were also found to colocalize to sperm mitochondria after fertilization in mice. Thus, it has been speculated that autophagy may be an evolutionary conserved mechanism for paternal mitochondrial elimination. However, by using two transgenic mouse strains, one bearing GFP-labeled autophagosomes and the other bearing red fluorescent protein-labeled mitochondria, we demonstrated that autophagy did not participate in the postfertilization elimination of sperm mitochondria in mice. Although P62 and LC3 proteins congregated to sperm mitochondria immediately after fertilization, sperm mitochondria were not engulfed and ultimately degraded in lysosomes until P62 and LC3 proteins disengaged from sperm mitochondria. Instead, sperm mitochondria unevenly distributed in blastomeres during cleavage and persisted in several cells until the morula stages. Furthermore, by using single sperm mtDNA PCR, we observed that most motile sperm that had reached the oviduct for fertilization had eliminated their mtDNA, leaving only vacuolar mitochondria. However, if sperm with remaining mtDNA entered the zygote, mtDNA was not eliminated and could be detected in newborn mice. Based on these results, we conclude that, in mice, maternal inheritance of mtDNA is not an active process of sperm mitochondrial and mtDNA elimination achieved through autophagy in early embryos, but may be a passive process as a result of prefertilization sperm mtDNA elimination and uneven mitochondrial distribution in embryos.

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

CRL4 complex regulates mammalian oocyte survival and reprogramming by activation of TET proteins.

Ubiquitin Fertility Insurance The female mammals reproductive lifespan is determined by a pool of ovarian primordial follicles that are generated early in life. Yu et al. (p. 1518) found that in mice, the ubiquitin E3 ligase complex CRL4 is essential for oocyte survival within primordial follicles and for development after fertilization. CRL4 binds to and activates an adaptor protein that mediates ubiquitination, but if any component is deleted, the genes required for oocyte maintenance and early embryo development are silenced and the female mice become infertile. Continuing female fertility in mammals requires the function of a ubiquitin ligase complex in oocytes. The duration of a woman’s reproductive period is determined by the size and persistence of a dormant oocyte pool. Specific oocyte genes are essential for follicle maintenance and female fertility. The mechanisms that regulate the expression of these genes are poorly understood. We found that a cullin-ring finger ligase-4 (CRL4) complex was crucial in this process. Oocyte-specific deletion of the CRL4 linker protein DDB1 or its substrate adaptor VPRBP (also known as DCAF1) caused rapid oocyte loss, premature ovarian insufficiency, and silencing of fertility maintaining genes. CRL4VPRBP activates the TET methylcytosine dioxygenases, which are involved in female germ cell development and zygote genome reprogramming. Hence, CRL4VPRBP ubiquitin ligase is a guardian of female reproductive life in germ cells and a maternal reprogramming factor after fertilization.

Oocyte ageing and epigenetics.

It has become a current social trend for women to delay childbearing. However, the quality of oocytes from older females is compromised and the pregnancy rate of older women is lower. With the increased rate of delayed childbearing, it is becoming more and more crucial to understand the mechanisms underlying the compromised quality of oocytes from older women, including mitochondrial dysfunctions, aneuploidy and epigenetic changes. Establishing proper epigenetic modifications during oogenesis and early embryo development is an important aspect in reproduction. The reprogramming process may be influenced by external and internal factors that result in improper epigenetic changes in germ cells. Furthermore, germ cell epigenetic changes might be inherited by the next generations. In this review, we briefly summarise the effects of ageing on oocyte quality. We focus on discussing the relationship between ageing and epigenetic modifications, highlighting the epigenetic changes in oocytes from advanced-age females and in post-ovulatory aged oocytes as well as the possible underlying mechanisms.

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.

Maternal obesity and diabetes may cause DNA methylation alteration in the spermatozoa of offspring in mice

BackgroundThe adverse effects on offspring of diabetic and/or obese mothers can be passed to the next generation. However, the mechanisms behind this are still unclear. Epigenetics may play a key role during this process.MethodsTo confirm the hypothesis, we investigated the DNA methylation of several imprinted genes in spermatozoa of offspring from diabetic and/or obese mothers utilizing streptozotocin (STZ)- and high-fat-diet (HFD)-induced mouse models.ResultsWe found that the DNA methylation of Peg3 was significantly increased in spermatozoa of offspring of obese mothers compared to that in spermatozoa of offspring of normal mothers. The DNA methylation of H19 was significantly higher in spermatozoa of offspring of diabetic mothers than that in spermatozoa of offspring of non-diabetic mothers.ConclusionsThese results indicate that pre-gestational diabetes and/or obesity can alter DNA methylation in offspring spermatozoa.

Overexpression of SETβ, a protein localizing to centromeres, causes precocious separation of chromatids during the first meiosis of mouse oocytes.

Summary Chromosome segregation in mammalian oocyte meiosis is an error-prone process, and any mistake in this process may result in aneuploidy, which is the main cause of infertility, abortion and many genetic diseases. It is now well known that shugoshin and protein phosphatase 2A (PP2A) play important roles in the protection of centromeric cohesion during the first meiosis. PP2A can antagonize the phosphorylation of rec8, a member of the cohesin complex, at the centromeres and thus prevent cleavage of rec8 and so maintain the cohesion of chromatids. SET&bgr; is a protein that physically interacts with shugoshin and inhibits PP2A activity. We thus hypothesized that SET&bgr; might regulate cohesion protection and chromosome segregation during oocyte meiotic maturation. Here we report for the first time the expression, subcellular localization and functions of SET&bgr; during mouse oocyte meiosis. Immunoblotting analysis showed that the expression level of SET&bgr; was stable from the germinal vesicle stage to the MII stage of oocyte meiosis. Immunofluorescence analysis showed SET&bgr; accumulation in the nucleus at the germinal vesicle stage, whereas it was targeted mainly to the inner centromere area and faintly localized to the interchromatid axes from germinal vesicle breakdown to MI stages. At the MII stage, SET&bgr; still localized to the inner centromere area, but could relocalize to kinetochores in a process perhaps dependent on the tension on the centromeres. SET&bgr; partly colocalized with PP2A at the inner centromere area. Overexpression of SET&bgr; in mouse oocytes caused precocious separation of sister chromatids, but depletion of SET&bgr; by RNAi showed little effects on the meiotic maturation process. Taken together, our results suggest that SET&bgr;, even though it localizes to centromeres, might not be essential for chromosome separation during mouse oocyte meiotic maturation, although its forced overexpression causes premature chromatid separation.

Maternal Diabetes Mellitus and the Origin of Non-Communicable Diseases in Offspring: The Role of Epigenetics

ABSTRACT Offspring of diabetic mothers are susceptible to the onset of metabolic syndromes, such as type 2 diabetes and obesity at adulthood, and this trend can be inherited between generations. Genetics cannot fully explain how the noncommunicable disease in offspring of diabetic mothers is caused and inherited by the next generations. Many studies have confirmed that epigenetics may be crucial for the detrimental effects on offspring exposed to the hyperglycemic environment. Although the adverse effects on epigenetics in offspring of diabetic mothers may be the result of the poor intrauterine environment, epigenetic modifications in oocytes of diabetic mothers are also affected. Therefore, the present review is focused on the epigenetic alterations in oocytes and embryos of diabetic mothers. Furthermore, we also discuss initial mechanistic insight on maternal diabetes mellitus causing alterations of epigenetic modifications.

Maternal diabetes causes abnormal dynamic changes of endoplasmic reticulum during mouse oocyte maturation and early embryo development

BackgroundThe adverse effects of maternal diabetes on oocyte maturation and embryo development have been reported.MethodsIn this study, we used time-lapse live cell imaging confocal microscopy to investigate the dynamic changes of ER and the effects of diabetes on the ER’s structural dynamics during oocyte maturation, fertilization and early embryo development.ResultsWe report that the ER first became remodeled into a dense ring around the developing MI spindle, and then surrounded the spindle during migration to the cortex. ER reorganization during mouse early embryo development was characterized by striking localization around the pronuclei in the equatorial section, in addition to larger areas of fluorescence deeper within the cytoplasm. In contrast, in diabetic mice, the ER displayed a significantly higher percentage of homogeneous distribution patterns throughout the entire ooplasm during oocyte maturation and early embryo development. In addition, a higher frequency of large ER aggregations was detected in GV oocytes and two cell embryos from diabetic mice.ConclusionsThese results suggest that the diabetic condition adversely affects the ER distribution pattern during mouse oocyte maturation and early embryo development.

Effect of postovulatory oocyte aging on DNA methylation imprinting acquisition in offspring oocytes

OBJECTIVE To investigate whether postovulatory aging of oocytes in the mother affects DNA methylation acquisition of imprinted genes in oocytes from the offspring. DESIGN Randomized research experimental study. SETTING Academic basic research laboratory. ANIMAL(S) Mice. INTERVENTION(S) Fresh oocytes and aged oocytes from mothers were artificially inseminated, and oocytes were collected from the resultant offspring. MAIN OUTCOME MEASURE(S) Methylation status was evaluated at differentially methylated regions (DMRs) in oocytes of maternally imprinted genes Peg3, Snrpn, and Peg1 and paternally imprinted gene H19. RESULT(S) Our results showed that methylation patterns at DMRs of Peg3, Snrpn, Peg1, and H19 in oocytes from aged-oocyte offspring were mainly normal, with only a small number of oocytes showing aberrant methylation in the DMR of Peg3. CONCLUSION(S) Postovulatory oocyte aging causes a decline in reproductive outcomes but does not evidently lead to defects in DNA methylation imprinting acquisition in the oocytes from viable offspring.