Prevention of maternal aging-associated oocyte aneuploidy and meiotic spindle defects in mice by dietary and genetic strategies

Increased meiotic spindle abnormalities and aneuploidy in oocytes of women of advanced maternal ages lead to elevated rates of infertility, miscarriage, and trisomic conceptions. Despite the significance of the problem, strategies to sustain oocyte quality with age have remained elusive. Here we report that adult female mice maintained under 40% caloric restriction (CR) did not exhibit aging-related increases in oocyte aneuploidy, chromosomal misalignment on the metaphase plate, meiotic spindle abnormalities, or mitochondrial dysfunction (aggregation, impaired ATP production), all of which occurred in oocytes of age-matched ad libitum-fed controls. The effects of CR on oocyte quality in aging females were reproduced by deletion of the metabolic regulator, peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α). Thus, CR during adulthood or loss of PGC-1α function maintains female germline chromosomal stability and its proper segregation during meiosis, such that ovulated oocytes of aged female mice previously maintained on CR or lacking PGC-1α are comparable to those of young females during prime reproductive life.     

Heterozygosity for a Bub1 mutation causes female-specific germ cell aneuploidy in mice

Aneuploidy, the most common chromosomal abnormality at birth and the main ascertained cause of pregnancy loss in humans, originates primarily from chromosome segregation errors during oogenesis. Here, we report that heterozygosity for a mutation in the mitotic checkpoint kinase gene, Bub1, induces aneuploidy in female germ cells of mice and that the effect increases with advancing maternal age. Analysis of Bub1 heterozygous oocytes showed that aneuploidy occurred primarily during the first meiotic division and involved premature sister chromatid separation. Furthermore, aneuploidy was inherited in zygotes and resulted in the loss of embryos after implantation. The incidence of aneuploidy in zygotes was sufficient to explain the reduced litter size in matings with Bub1 heterozygous females. No effects were seen in germ cells from heterozygous males. These findings show that Bub1 dysfunction is linked to inherited aneuploidy in female germ cells and may contribute to the maternal age-related increase in aneuploidy and pregnancy loss.     

Melatonin protects against defects induced by deoxynivalenol during mouse oocyte maturation

Deoxynivalenol (DON) is one of the most prevalent fusarium mycotoxins in feedstuff and food. DON causes detrimental effects on human and animal reproductive systems by inducing oxidative stress and apoptosis. However, melatonin is a multifunctional endogenous hormone that plays crucial roles in the development of animal germ cells and embryos as a robust deoxidizer. In this study, we explored the effects of melatonin on the DON exposure mouse oocytes. Our in vitro and in vivo results showed that DON adversely affected mouse oocyte maturation and early embryo cleavage, while melatonin administration ameliorated the toxic effects of DON. DON exposure disrupted the meiotic spindle formation and kinetochore‐microtubule attachment, which induced aneuploidy in oocytes. This might be through DON effects on the acetylated tubulin level. Moreover, we found that DON exposure caused the alteration of DNA and histone methylation level, which might affect early embryo cleavage. The toxic effects of DON on oocytes might be through its induction of oxidative stress‐mediated early apoptosis, while the treatment with melatonin significantly ameliorated these phenotypes in DON‐exposed mouse oocytes. Collectively, our results indicated the protection effects of melatonin against defects induced by DON during mouse oocyte meiotic maturation.     

Aging and aneuploidy in human oocytes and follicular cells

The incidence of chromosomal aneuploidy in the oocyte and surrounding follicular cells in women of different ages was investigated. Oocytes and granulosa cells derived from ovarian specimens from a random population of 289 adult patients ages 16 to 76 years undergoing gynecologic surgery for nonovarian pathology were cultured for short periods, and cytogenetic preparations were scored for chromosome number and morphology. Of 91 oocytes harvested at second meiotic metaphase, five oocytes revealed a chromosomal abnormality. Cytogenetic analysis of the granulosa cells revealed aneuploid cells as well as complements with structural changes. A significant difference between the percentage of granulosa cell aneuploidy in the 25 to 34 years group and all other age groups was found, suggesting a decrease of the tendency for granulosa cells to exhibit aneuploidy in older age groups. Previous reports of aneuploidy in human ovary and other somatic cell types suggest that follicular cells behave differently in their response to aging. There also would appear to be a difference between the chromosomal response of oocytes and granulosa cells to aging.     

Premature ovarian aging in mice deficient for Gpr3

After becoming competent for resuming meiosis, fully developed mammalian oocytes are maintained arrested in prophase I until ovulation is triggered by the luteotropin surge. Meiotic pause has been shown to depend critically on maintenance of cAMP level in the oocyte and was recently attributed to the constitutive Gs (the heterotrimeric GTP-binding protein that activates adenylyl cyclase) signaling activity of the G protein-coupled receptor GPR3. Here we show that mice deficient for Gpr3 are unexpectedly fertile but display progressive reduction in litter size despite stable age-independent alteration of meiotic pause. Detailed analysis of the phenotype confirms premature resumption of meiosis, in vivo, in about one-third of antral follicles from Gpr3-/- females, independently of their age. In contrast, in aging mice, absence of GPR3 leads to severe reduction of fertility, which manifests by production of an increasing number of nondeveloping early embryos upon spontaneous ovulation and massive amounts of fragmented oocytes after superovulation. Severe worsening of the phenotype in older animals points to an additional role of GPR3 related to protection (or rescue) of oocytes from aging. Gpr3-defective mice may constitute a relevant model of premature ovarian failure due to early oocyte aging.     

Chromosomal abnormalities in the first and second polar body

Aneuploidy free oocytes may be pre-selected by testing the first and second polar bodies removed from oocytes following their maturation and fertilization. We present here our experience on the application of the method in IVF cycles from patients of advanced maternal age. Overall, 5590 oocytes were obtained from 917 cycles and tested by polar body sampling and fluorescent in situ hybridization (FISH) analysis using specific probes for chromosomes 13,16,18,21 and 22. FISH results were available in 4599 (82.2%) of 5590 oocytes studied, from which 2077(45.2%) were with aneuploidies. Thirty six point one percent of aneuploidies were of the first meiotic origin, and 29.3% of the second meiotic origin. Most errors in the first meiotic division were represented by chromatid errors. The transfer of embryos deriving from 2014 of 2520 aneuploidy free oocytes in 821 treatment cycles resulted in 182 (22.2%) clinical pregnancies and 140 healthy children born after confirmation of the polar body diagnosis. Polar body testing of oocytes provides an approach for pre-selection of aneuploidy free embryos, improving pregnancy rate in IVF patents of advanced maternal age.     

Melatonin protects against maternal obesity‐associated oxidative stress and meiotic defects in oocytes via the SIRT3‐SOD2‐dependent pathway

Maternal obesity in humans is associated with poor outcomes across the reproductive spectrum. Emerging evidence indicates that these defects are likely attributed to factors within the oocyte. Although various molecules and pathways may contribute to impaired oocyte quality, prevention of fertility issues associated with maternal obesity is a challenge. Using mice fed a high‐fat diet (HFD) as an obesity model, we document spindle disorganization, chromosome misalignment, and elevated reactive oxygen species (ROS) levels in oocytes from obese mice. Oral administration of melatonin to HFD mice not only reduces ROS generation, but also prevents spindle/chromosome anomalies in oocytes, consequently promoting the developmental potential of early embryos. Consistent with this finding, we find that melatonin supplement during in vitro maturation also markedly attenuates oxidative stress and meiotic defects in HFD oocytes. Finally, by performing morpholino knockdown and acetylation‐mimetic mutant overexpression assays, we reveal that melatonin ameliorates maternal obesity‐induced defective phenotypes in oocytes through the SIRT3‐SOD2‐dependent mechanism. In sum, our data uncover the marked beneficial effects of melatonin on oocyte quality from obese females; this opens a new area for optimizing culture system as well as fertility management.     

An inhibitory influence of porcine vasoactive intestinal peptide upon mouse oocyte meiosis in culture

The germinal vesicle (GV) of follicle-enclosed oocytes in mice remains arrested at the dictyate state of meiosis. Upon releasing the oocytes from the follicles, the meiotic process resumes, leading to dissolution of the GV, suggesting that factors in the follicular constituents sustain the meiotic arrest of oocytes. Vasoactive intestinal peptide (VIP) was demonstrated in the ovary and found to have a wide range of biological effects. In this study, the possibility of VIP to be a factor which induces meiosis-arrest of oocytes was evaluated. Porcine VIP inhibited resumption of meiosis of cumulus-enclosed mouse oocytes in vitro. Germinal vesicle breakdown (GVBD) was prevented in more than 50% of the oocytes treated by VIP at a concentration of 30 microM. The inhibiting effect of VIP was dose-dependent and reversible. Spontaneous resumption of meiosis of isolated oocytes in vitro was reported to be inhibited by dibutyryl cAMP (dbcAMP) added to the medium, indicating that meiotic arrest can be sustained by maintaining intra-oocyte cAMP above a critical level. It was further reported that follicular fluid contains substances that maintain meiotic arrest in association with exogenous dbcAMP. In the present study, the blocking activity of dbcAMP for the spontaneous resumption of meiosis was not potentiated by the addition of VIP in the medium. The present results suggest that VIP may play a role in the regulation of resumption of oocyte meiosis, and that VIP is not a substance which maintains meiotic arrest in association with cAMP.     

LHP1, the Arabidopsis homologue of HETEROCHROMATIN PROTEIN1, is required for epigenetic silencing of FLC

Vernalization is the acceleration of flowering by prolonged cold that aligns the onset of reproductive development with spring conditions. A key step of vernalization in Arabidopsis is the epigenetic silencing of FLOWERING LOCUS C (FLC), which encodes a repressor of flowering. The vernalization-induced epigenetic silencing of FLC is associated with histone deacetylation and H3K27me2 and H3K9me2 methylation mediated by VRN/VIN proteins. We have analyzed whether different histone methyltransferases and the chromodomain protein LIKE HETEROCHROMATIN PROTEIN (LHP)1 might play a role in vernalization. No single loss-of-function mutation in the histone methyltransferases studied disrupted the vernalization response; however, lhp1 mutants revealed a role for LHP1 in maintaining epigenetic silencing of FLC. Like LHP1, VRN1 functions in both flowering-time control and vernalization. We explored the localization of VRN1 and found it to be associated generally with Arabidopsis chromosomes but not the heterochromatic chromocenters. This association did not depend on vernalization or VRN2 function and was maintained during mitosis but was lost in meiotic chromosomes, suggesting that VRN1 may contribute to chromatin silencing that is not meiotically stable.     

Specific changes in the pattern of protein synthesis during meiotic maturation of mammalian oocytes in vitro.

High resolution two-dimensional electrophoresis has been used to examine the pattern of protein synthesis during meiotic maturation of mouse oocytes in vitro. Fluorograms of [35S]methionine-labeled oocyte proteins have revealed that meiotic progression from dictyate to metaphase II (meiotic maturation) is accompanied by marked changes in the pattern of proteins synthesized by oocytes. Virtually all of the changes observed take place subsequent to the breakdown of the oocyte's germinal vesicle, but are not dependent upon the occurrence of other morphological events, such as spindle formation or polar body emission. These changes in protein synthesis do not take place in oocytes that fail to undergo breakdown of germinal vesicles spontaneously or in oocytes arrested at the germinal vesicle stage by dibutyryl 3':5'-cyclic AMP. These data suggest that mixing of the oocyte's nucleoplasm and cytoplasm may trigger many of the changes in protein synthesis that accompany meiotic maturation of mouse oocytes in vitro.     

Induction of germinal vesicle breakdown in Xenopus laevis oocytes: synergistic action of progesterone and insulin

Progesterone is the hormone that reinitiates the meiotic division of amphibian oocytes and insulin and insulin-like growth factors are also active on defolliculated oocytes in vitro. We have studied Xenopus laevis oocytes (stage 5-6) of different hormonal sensitivities, obtained from unstimulated and from human chorionic gonadotrophin-stimulated females. Some oocytes from unstimulated females were also precultured with a subthreshold level of progesterone. A dose-dependent potentiating effect of the action of progesterone was observed with insulin, and this was particularly remarkable in low-sensitivity oocytes. Since in the presence of insulin, the optimally effective concentration of progesterone was much reduced (as an example from 1 mumol/1 to 50 nmol/l), it is suggested that an insulin-like growth factor may play a physiological role in the reinitiation of meiosis in ovaries.     

Transmission of Y chromosomes from XY female mice was made possible by the replacement of cytoplasm during oocyte maturation

The B6.Y^TIR sex-reversed female mouse is anatomically normal at young ages but fails to produce offspring. We have previously shown that its oocytes go through the meiotic cell cycle up to the second metaphase; however, the meiotic spindle is not properly organized, the second meiotic division goes awry after activation or fertilization, and none of the oocytes initiate embryonic development. In the present study, we transferred the nuclei of GV-stage oocytes from XY females into the enucleated GV-stage oocytes from (B6.DBA)F1.XX females. The resultant reconstructed oocytes properly assembled second meiotic spindles after in vitro maturation and produced healthy offspring after in vitro fertilization. Some male pups inherited maternal Y chromosomes. We conclude that the cytoplasm of the XY oocyte is insufficient to support spindle formation at the second metaphase whereas its replacement with the cytoplasmic material from an XX oocyte allows normal development.     

Alterations of epigenetics and microRNA in hepatocellular carcinoma

Studies have shown that alterations of epigenetics and microRNA (miRNA) play critical roles in the initiation and progression of hepatocellular carcinoma (HCC). Epigenetic silencing of tumor suppressor genes in HCC is generally mediated by DNA hypermethylation of CpG island promoters and histone modifications such as histone deacetylation, methylation of histone H3 lysine 9 (H3K9) and tri‐methylation of H3K27. Chromatin‐modifying drugs such as DNA methylation inhibitors and histone deacetylase inhibitors have shown clinical promise for cancer therapy. miRNA are small non‐coding RNA that regulate expression of various target genes. Specific miRNA are aberrantly expressed and play roles as tumor suppressors or oncogenes during hepatocarcinogenesis. We and other groups have demonstrated that important tumor suppressor miRNA are silenced by epigenetic alterations, resulting in activation of target oncogenes in human malignancies including HCC. Restoring the expression of tumor suppressor miRNA by inhibitors of DNA methylation and histone deacetylase may be a promising therapeutic strategy for HCC.     

Coupling of histone deacetylation to NAD breakdown by the yeast silencing protein Sir2: Evidence for acetyl transfer from substrate to an NAD breakdown product

The Saccharomyces cerevisiae silencing protein Sir2 is the founding member of a universally conserved family of proteins that have been shown to possess NAD-dependent histone deacetylation and ADP-ribosylation activities. Here we show that histone deacetylation by Sir2 is coupled to cleavage of the high-energy bond that links the ADP-ribose moiety of NAD to nicotinamide. Analysis of the NAD cleavage products revealed the presence of nicotinamide, ADP-ribose, and a third product that appeared to be related to ADP-ribose. With the use of label transfer experiments, we show that the acetyl group in the histone substrate is transferred to this NAD breakdown product during deacetylation, forming a product that we conclude to be O-acetyl-ADP-ribose. Detection of this species strongly argues for obligate coupling of histone deacetylation to NAD breakdown by Sir2. We propose reaction mechanisms that could account for this coupling via acetyl-ADP-ribose formation. The unprecedented coupling of amide bond cleavage to cleavage of a high-energy bond raises the possibility that NAD breakdown by Sir2 plays an important role in silencing that is independent of its requirement for deacetylation.     

Age-related decline in fertility.

A progressive decline in fecundity with advancing age is a reality, attributed primarily to the detrimental impact of various aging processes on female gametes. Despite medical advances that have dramatically prolonged the female life span, declining numbers and deteriorating quality of oocytes, and an increasing incidence of meiotic errors and aneuploidy of gametes and embryos, reduce clinical pregnancy rates and escalate pregnancy wastage. Increased fetal aneuploidies in ongoing pregnancies and an increased predisposition to obstetric morbidities further contribute to the diminishing reproductive successes associated with advancing age. The age of male partners, despite the decline in semen parameters and sexual performance with aging, does not appear to have a major impact on the eventual fertility of the aging couple. The contributions of age-related impaired sexuality and ejaculatory problems, although slight albeit significant, to declining fertility in the aging should be appreciated in appropriate cases. With the realization of the age-related detriment on fertility potential and the limitations of available therapeutic interventions, management of subfecundity in women beyond their mid-30s should be approached aggressively. Success of ovulation induction with clomiphine citrate or gonadotropins is marginal in women aged older than 40 years; a case can be made to proceed directly with ART in women in this age group, especially when there is coexisting male factor or pelvic disease. Except for the use of donor oocytes, the outcome of various therapeutic interventions to optimize reproductive performance in women aged older than 44 years remains dismal. A broader application of PGD techniques may contribute to improved live birth rates in reproductively aging women. The greater likelihood of obstetric complications in pregnancies resulting from donor oocytes and an increased prevalence of age-related medical problems complicating pregnancy should prompt a thorough medical evaluation before proceeding with ART.     

Modification of hematopoietic stem cell fate by 5aza 2'deoxycytidine and trichostatin A

Efforts to change the fate of human hematopoietic stem cells (HSCs) and progenitor cells (HPCs) in vitro have met with limited success. We hypothesized that previously utilized in vitro conditions might result in silencing of genes required for the maintenance of primitive HSCs/HPCs. DNA methylation and histone deacetylation are components of an epigenetic program that regulates gene expression. Using pharmacologic agents in vitro that might possibly interfere with DNA methylation and histone deacetylation, we attempted to maintain and expand cells with phenotypic and functional characteristics of primitive HSCs/HPCs. Human marrow CD34(+) cells were exposed to a cytokine cocktail favoring differentiation in combination with 5aza 2'deoxycytidine (5azaD) and trichostatin A (TSA), resulting in a significant expansion of a subset of CD34(+) cells that possessed phenotypic properties as well as the proliferative potential characteristic of primitive HSCs/HPCs. In addition, 5azaD- and TSA-pretreated cells but not the CD34(+) cells exposed to cytokines alone retained the ability to repopulate immunodeficient mice. Our findings demonstrate that 5azaD and TSA can be used to alter the fate of primitive HSCs/HPCs during in vitro culture.     

Effects of cryopreservation on meiotic spindles of oocytes and its dynamics after thawing: clinical implications in oocyte freezing--a review article

Embryo freezing has been a successful practice, but oocyte cryopreservation formerly achieved poorer results. This was mainly due to low rates of survival, fertilization, and development. The major dissimilarities for oocytes to embryos are the character of the plasma membrane, the presence of cortical granules, at the metaphase of meiosis II with the spindle system. In addition, the oocytes must be fertilized by sperm at the appropriate time. To improve the survival rate, a refined slow freezing method with increased sucrose concentration would dehydrate oocytes more sufficiently. Vitrification is another approach to prevent ice crystal formation. Intracytoplasmic sperm injection is used to overcome possible zona hardening from the release of cortical granules. The microtubules of meiotic spindles are vulnerable to the thermal changes and would depolymerize. Cryopreserved oocytes exhibited serious disturbances of the microtubules immediately after thawing. Fertilization of oocytes with disorganized spindles could lead to chromosomal aneuploidy, digyny, and arrest of cleavage. After incubation, the microtubules would repolymerize in a time-dependent way. Normal fertilization and development of cryopreserved oocytes improved after appropriate incubation and timing of insemination, compatible with recovery of the spindles. With the improvement of survival, fertilization, and cleavage, oocyte cryopreservation would gain an imperative role.