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After ovulation, metaphase II oocytes undergo a time-dependent deterioration in vivo or in vitro, which is referred to as postovulatory oocyte aging, a process during which a series of deleterious molecular and cellular changes occur. In this study, we found that short-term injection of resveratrol (3,5,4'-trihydroxystilbene) effectively ameliorated oxidative stress-induced damage in postovulatory oocyte aging of middle-aged mice in vivo. Resveratrol induced changes that delayed the aging-induced oocyte deterioration including the elevated expression of the anti-aging molecule Sirtuin 1 (SIRT1); it reduced intracellular reactive oxygen species (ROS) level, and improved mitochondria function. In addition, these beneficial changes may also help to prevent apoptosis. Taken together, our data suggest that resveratrol can effectively protect against postovulatory oocyte aging in vivo primarily by preventing ROS production.

Oocyte is arrested at metaphase of the second meiosis until fertilization switching on [Ca2+]i oscillations. Oocyte activation inefficiency is the most challenging problem for failed fertilization ...

Increasing evidences indicate that chronic diseases in offspring may be the result of ancestral environmental exposures. Exposures to environmental compounds in windows of epigenetic susceptibility have been shown to promote epigenetic alterations that can be inherited between generations. DNA methylation, histone modifications, and noncoding RNAs are sound mechanistic candidates for the delivery of environmental information from gametes to zygotes. This review focuses mainly on paternal exposures and assesses the risk of epigenetic alterations in the development of diseases, providing insights into relationships between aberrant sperm epigenetic patterns and offspring health. Elucidation of the mechanisms underlying environmental epigenetic information that survive from epigenetic reprogramming and its transmission to future generations may hold a great promise for providing therapeutic targets for epigenetic diseases associated with environmental exposures.

LSM family member 14 (LSM14) belongs to the RNA-associated protein (RAP) family that is widely expressed in different species, and whose functions include associating and storing mRNAs. In the present study, we found that LSM14b was essential for oocyte meiotic maturation. Lack of LSM14b caused oocyte meiotic arrest at metaphase, and misalignment of chromosomes, as well as abnormal spindle assembly checkpoint (SAC) and maturation promoting factor (MPF) activation. Cyclin B1 and Cdc20 mRNAs, whose contents changed with LSM14b expression, were likely direct targets of LSM14b. We conclude that LSM14b, by functioning as a container of mRNAs, controls protein expression, and thus regulates the oocyte meiotic maturation process.

Geminin plays a critical role in cell cycle regulation by regulating DNA replication and serves as a transcriptional molecular switch that directs cell fate decisions. Spermatogonia lacking Geminin disappear during the initial wave of mitotic proliferation, while geminin is not required for meiotic progression of spermatocytes. It is unclear whether geminin plays a role in pre-meiotic DNA replication in later-stage spermatogonia and their subsequent differentiation. Here, we selectively disrupted Geminin in the male germline using the Stra8-Cre/loxP conditional knockout system. Geminin-deficient mice showed atrophic testes and infertility, concomitant with impaired spermatogenesis and reduced sperm motility. The number of undifferentiated spermatogonia and spermatocytes was significantly reduced; the pachytene stage was impaired most severely. Expression of cell proliferation-associated genes was reduced in Gmnnfl/Δ; Stra8-Cre testes compared to in controls. Increased DNA damage, decreased Cdt1, and increased phosphorylation of Chk1/Chk2 were observed in Geminin-deficient germ cells. These results suggest that geminin plays important roles in pre-meiotic DNA replication and subsequent spermatogenesis.

Diabetes affects oocyte nuclear and cytoplasmic quality. In this study, we generated a type 1 diabetes (T1D) mouse model by STZ injection to study the effects of T1D on zona pellucida and genomic DNA methylation of oocytes and granulosa cells. T1D mice showed fewer ovulated oocytes, reduced ovarian reserve, disrupted estrus cycle, and significantly ruptured zona pellucida in 2-cell in vivo embryos compared to controls. Notably, diabetic oocytes displayed thinner zona pellucida and treatment of oocytes with high concentration glucose reduced the zona pellucida thickness. Differential methylation genes in oocytes and granulosa cells were analyzed by methylation sequencing. These genes were significantly enriched in GO terms by GO analysis, and these GO terms were involved in multiple aspects of growth and development. Most notably, the abnormal methylation genes in oocytes may be related to oocyte zona pellucida changes in diabetic mice. These findings provide novel basic data for further understanding and elucidating dysgenesis and epigenetic changes in type 1 diabetes mellitus.

Polychlorinated biphenyls (PCBs) are a class of organic pollutants that have been widely found in the environment. The chemical 2,3′,4,4′5-pentachlorobiphenyl (PCB118) is an important dioxin-like PCB compound with strong toxicity. PCB118 can accumulate in adipose tissue, serum and milk in mammals, and it is highly enriched in the follicular fluid. In this study, pregnant mice were exposed to 0, 20 and 100 μg/kg/day of PCB118 during pregnancy at the fetal primordial germ cell migration stage. The methylation patterns of the imprinted genes H19, Snrpn, Peg3 and Igf2r as well as the expression levels of Dnmt1, 3a, 3b and 3l, Uhrf1, Tet2 and Tet3 in fully grown germinal vesicle oocytes were measured in offspring. The rates of in vitro maturation, in vitro fertilization, oocyte spindle and chromosomal abnormalities were also calculated. The results showed that prenatal exposure to PCB118 altered the DNA methylation status of differentially methylated regions in some imprinted genes, and the expression levels of Dnmt1, 3a, and 3l, Uhrf1 and Tet3 were also changed. In addition, PCB118 disturbed the maturation process of progeny mouse oocytes in a dose-dependent manner. Therefore, attention should be paid to the potential impacts of PCB118-contaminated dietary intake during pregnancy on the offspring’s reproductive health.

Mitochondrial DNA (mtDNA) is important for oxidative phosphorylation; dysfunctions can play a role in many mitochondrial diseases and can also affect the aging of cells and individuals. DNA methylation is an important epigenetic modification that plays a critical role in regulating gene expression. While recent studies have revealed the existence of mtDNA methylation there are still controversies about mtDNA methylation due to the special structure of mtDNA. Mitochondria and DNA methylation are both essential for regulating oocyte maturation and early embryo development, but whether mtDNA methylation changes during this process is unknown. By employing bisulfite sequencing, we found that in the process of mouse oocyte maturation, postovulatory oocyte aging, and early embryo development, all analyzed mitochondrial genes, including 16S-CpGI, DCR, ND6, 12S, and ATP8, lacked 5'mC. Thus, mtDNA methylation does not occur in the oocyte and early embryo.

Cell division cycle protein CDC6 is essential for the initiation of DNA replication. CDC6 was recently shown to inhibit the microtubule-organizing activity of the centrosome. Here, we show that CDC6 is localized to the spindle from Pro-MI to MII stages of oocytes, and it plays important roles at two critical steps of oocyte meiotic maturation. CDC6 depletion facilitated the G2/M transition (GV breakdown, GVBD) through regulation of Cdh1 and cyclin B1 expression and CDK1 phosphorylation in a GVBD-inhibiting culture system containing milrinone. Furthermore, GVBD was significantly decreased after knockdown of cyclin B1 in CDC6-depleted oocytes, indicating that the effect of CDC6 loss on GVBD stimulation was mediated, at least in part, by raising cyclin B1. Knockdown of CDC6 also caused abnormal localization of γ-tubulin, resulting in defective spindles, misaligned chromosomes, cyclin B1 accumulation and spindle assembly checkpoint (SAC) activation, leading to significant Pro-MI/MI arrest and PB1 extrusion failure. These phenotypes were also confirmed by time-lapse live cell imaging analysis. The results indicate that CDC6 is indispensable for maintaining G2 arrest of meiosis and functions in G2/M checkpoint regulation in mouse oocytes. Moreover, CDC6 is also a key player regulating meiotic spindle assembly and metaphase-to-anaphase transition in meiotic oocytes.Summary statementWe show that CDC6 is indispensable for maintaining G2 arrest of mouse oocytes. Moreover, CDC6 is also a key player regulating meiotic spindle assembly and metaphase-to-anaphase transition in meiotic oocytes.

// Teng Zhang 1, 2, * , Yang Zhou 2, 4, * , Hong-Hui Wang 2 , Tie-Gang Meng 2, 4 , Lei Guo 2 , Xue-Shan Ma 2 , Wei Shen 1 , Heide Schatten 3 , Qing-Yuan Sun 1, 2, 4 1 Institute of Reproductive Sciences, College of Animal Science and Technology, Qingdao Agricultural University, Qingdao, China 2 State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing, China 3 Department of Veterinary Pathobiology, University of Missouri, Columbia, MO, USA 4 University of the Chinese Academy of Sciences, Beijing, China * These authors contributed equally to this work Correspondence to: Qing-Yuan Sun, email: sunqy@ioz.ac.cn Keywords: Spc24, meiosis, oocyte, kinetochore-microtubule attachment, aneuploidy Received: July 10, 2016 Accepted: September 29, 2016 Published: October 04, 2016 ABSTRACT Mammalian oocytes are particularly error prone in chromosome segregation during two successive meiotic divisions. The proper kinetochore-microtubule attachment is a prerequisite for faithful chromosome segregation during meiosis. Here, we report that Spc24 localizes at the kinetochores during mouse oocyte meiosis. Depletion of Spc24 using specific siRNA injection caused defective kinetochore-microtubule attachments and chromosome misalignment, and accelerated the first meiosis by abrogating the kinetochore recruitment of spindle assembly checkpoint protein Mad2, leading to a high incidence of aneuploidy. Thus, Spc24 plays an important role in genomic stability maintenance during oocyte meiotic maturation.

Geminin is an important regulator of DNA replication and cell differentiation, but its role in female reproduction remains uncertain. Maternal geminin does not regulate oocyte meiotic maturation but does control accurate DNA replication. Geminin deletion in oocytes results in impaired embryo development and reduced fertility., Geminin controls proper centrosome duplication, cell division, and differentiation. We investigated the function of geminin in oogenesis, fertilization, and early embryo development by deleting the geminin gene in oocytes from the primordial follicle stage. Oocyte-specific disruption of geminin results in low fertility in mice. Even though there was no evident anomaly of oogenesis, oocyte meiotic maturation, natural ovulation, or fertilization, early embryo development and implantation were impaired. The fertilized eggs derived from mutant mice showed developmental delay, and many were blocked at the late zygote stage. Cdt1 protein was decreased, whereas Chk1 and H2AX phosphorylation was increased, in fertilized eggs after geminin depletion. Our results suggest that disruption of maternal geminin may decrease Cdt1 expression and cause DNA rereplication, which then activates the cell cycle checkpoint and DNA damage repair and thus impairs early embryo development.

The quality of metaphase II oocytes will undergo a time-dependent deterioration following ovulation as the result of the oocyte aging process. In this study, we determined that the expression of sirtuin family members (SIRT1, 2, 3) was dramatically reduced in mouse oocytes aged in vivo or in vitro. Increased intracellular ROS was observed when SIRT1, 2, 3 activity was inhibited. Increased frequency of spindle defects and disturbed distribution of mitochondria were also observed in MII oocytes aged in vitro after treatment with Nicotinamide (NAM), indicating that inhibition of SIRT1, 2, 3 may accelerate postovulatory oocyte aging. Interestingly, when MII oocytes were exposed to caffeine, the decline of SIRT1, 2, 3 mRNA levels was delayed and the aging-associated defective phenotypes could be improved. The results suggest that the SIRT1, 2, 3 pathway may play a potential protective role against postovulatory oocyte aging by controlling ROS generation.

Study question What is the impact of glucocorticoid (GC) on female reproduction? Summary answer Corticosterone (CORT) exposure causes little damage to oocyte quality or developmental competence but has an adverse effect on the uterus, which causes decreased implantation, embryo death and subsequent infertility. What is known already Chronic treatment with high GC doses is effective in controlling most allergic diseases but may lead to metabolic disorders such as obesity that are closely related with reproductive function. Study design, size, duration Hypercortisolism was induced in a female mouse model by supplementing the drinking water with 100 μg/ml of CORT. Controls received vehicle (1% v/v ethanol) only. After 4 weeks treatment mice were either mated or killed in estrus for hormone and organ measurements. In the first experiment, treatment with CORT or control continued during pregnancy but in the second CORT treatment was stopped after mating. To identify the effects of GC exposure on the uterus, blastocysts were generated by IVF of oocytes from CORT and control mice and replaced into recipients receiving the opposite treatment. Participants/materials, setting, methods The effects of hypercortisolism on female mice were first characterized by living body fat content, body weight, food intake, hormone and biochemical measurements, a glucose tolerance test and an insulin resistance test. Fertility was determined with or without CORT-treatment during pregnancy. Oocyte quality was assessed by oocyte maturation, mitochondrial distribution, reactive oxygen species production, mitochondrial DNA mutations and morphology of blastocysts produced in vivo or in vitro. Blastocyst cross-transfer was done to evaluate the causes of embryonic development failure. Fetus development and uterus morphology evaluation as well as culture of oocytes in vitro with gradient concentrations of CORT were also carried out. Main results and the role of chance In the hypercortisolism female mouse model, body weight and food intake were much higher than in the control, and corticosterone, estradiol, cholesterol (CHO) and triglycerides (TG) in the plasma of CORT-treated mice was significantly increased. The hypercortisolism female mice were infertile when CORT-treatment was sustained during pregnancy but fertile if CORT-treatment was stopped after mating. The rate of successful implantation in hypercortisolism mice with sustained CORT-treatment during pregnancy was significantly lower than in the control, and the implanted embryos could not develop beyond 13.5 dpc. Blastocyst cross-transfer showed that blastocysts from CORT-treated mice could develop to term in the uterus of control mice, but blastocysts from control mice failed to develop to term when they were transferred into CORT-treated mice, providing evidence that the infertility was mainly caused by an altered uterine environment. CORT administration did not affect oocyte maturation, mitochondrial distribution, ROS production and blastocyst morphology, but increased mitochondrial DNA mutations. Culture of oocytes in vitro with gradient concentrations of CORT showed that only very high concentrations of CORT caused damage to oocyte developmental competence. Large scale data NA. Limitations, reasons for caution The mouse model has the advantages of a consistent genetic and physiological background and openness to experimental manipulation over clinical studies but may not represent the human situation. Wider implications of the findings Our findings show that special care should be taken when administering CORT during pregnancy, and provide important information concerning female reproduction when treating patients by subjecting them to chronic GC exposure. Study funding/competing interest(s) This study was supported by the National Key R&D Program of China (Nos. 2016YFA0100400 and 2017YFC1000600) and the National Natural Science Foundation of China (31472055). The authors have no conflicts of interest.

Background Diabetes induces many complications including reduced fertility and low oocyte quality, but whether it causes increased mtDNA mutations is unknown. Methods We generated a T2D mouse model by using high-fat-diet (HFD) and Streptozotocin (STZ) injection. We examined mtDNA mutations in oocytes of diabetic mice by high-throughput sequencing techniques. Results T2D mice showed glucose intolerance, insulin resistance, low fecundity compared to the control group. T2D oocytes showed increased mtDNA mutation sites and mutation numbers compared to the control counterparts. mtDNA mutation examination in F1 mice showed that the mitochondrial bottleneck could eliminate mtDNA mutations. Conclusions T2D mice have increased mtDNA mutation sites and mtDNA mutation numbers in oocytes compared to the counterparts, while these adverse effects can be eliminated by the bottleneck effect in their offspring. This is the first study using a small number of oocytes to examine mtDNA mutations in diabetic mothers and offspring. Electronic supplementary material The online version of this article (10.1186/s12958-018-0423-1) contains supplementary material, which is available to authorized users.

Despite significant progress in early detection and improved treatment modalities prostate cancer remains the second leading cause of cancer death in American men which results in about 30,000 deaths per year in the USA. An aggressive phenotype leading to 2.58% risk of dying from prostate cancer still exists and immunotherapy has offered new possibilities to treat metastatic prostate cancer that cannot be treated by other modalities. Cancer immunotherapy is a rapidly growing field of research aimed at identifying biomarkers in immunodiagnosis and to develop new therapies by enabling the immune system to detect and destroy cancer cells. Immunotherapy falls into three different broad categories which are checkpoint inhibitors, cytokines, and vaccine immunotherapy. While immunotherapy to treat prostate cancer is still limited progress has been made; for treatment of advanced prostate cancer sipuleucel-T has been administered to patients in personalized doses to destroy prostate cancer cells which is promising and invites further research to determine immunotherapies for advanced prostate cancer. Antibody-based targeted immunotherapy and dendritic-cell-based vaccination are among the therapies that are currently being evaluated as promising approaches to treat prostate cancer. Combination immunotherapies include prostate cancer vaccines and radiotherapy for castration resistant prostate cancer. Microbial vectors for prostate cancer immunotherapy have been developed and bacterial strains have been engineered to express cancer-specific antigens, cytokines, and prodrug-converting cytokines. These approaches are addressed in the present review.

This chapter provides a brief overview of prostate cancer statistics, grading, diagnosis and treatment strategies that are discussed in more detail in the subsequent chapters of this book and the companion book titled "Clinical Molecular and Diagnostic Imaging of Prostate Cancer and Treatment Strategies". It also points to websites that provide additional useful information for patients affected by prostate cancer and for students and teachers to obtain practical and updated information on research, new diagnostic modalities and new therapies including new updated clinical trials. Three sections are focused on overview of prostate cancer statistics; overview of detection, diagnosis, stages and grading of prostate cancer; and treatment possibilities and options.

The significant role of centrosomes in cancer cell proliferation has been well recognized (reviewed in Schatten H, Histochem Cell Biol 129:667-86 (2008); Schatten H, Sun Q-Y, Microsc Microanal 17(4):506-512 (2011); Schatten H, Sun Q-Y, Reprod Fertil Dev. https://doi.org/10.1071/RD14493 (2015a); Schatten H, Sun Q-Y, Centrosome-microtubule interactions in health, disease, and disorders. In: Schatten H (ed) The cytoskeleton in health and disease. Springer Science+Business Media, New York (2015b)) and new research has generated new interest and new insights into centrosomes as potential targets for cancer-specific therapies. The centrosome is a key organelle serving multiple functions through its primary functions as microtubule organizing center (MTOC) that is also an important communication center for processes involved in cellular regulation; transport to and away from centrosome-organized microtubules along microtubules is essential for cellular activities including signal transduction and metabolic activities. New research on cancer cell centrosomes has generated new insights into centrosome dysfunctions in cancer cells in which centrosome phosphorylation, balance of centrosomal proteins, centrosome regulation and duplication are impaired. Among the hallmarks of cancer cells are multipolar spindles or abnormal bipolar spindles that are formed as a result of centrosome protein expression imbalances, abnormalities in centrosome structure and abnormalities in clustering of centrosomal components that are critical for bipolar mitotic apparatus formation. Centrosome abnormalities in cancer cells can be the result of multiple factors including environmental influences and toxicants that can affect centrosome functions by inducing centrosome pathologies leading to abnormal cancer cell proliferation. These topics are addressed in this review with focus on prostate-specific therapy strategies to target centrosome abnormalities. We will also address loss of cell polarity in cancer cells in which centrosome dysfunctions play a role as well as the loss of primary cilia in prostate cancer development and progression.

N6-methyladenosine (m6A) is the most prevalent and reversible internal modification of mammalian messenger and noncoding RNAs mediated by specific m6A writer, reader, and eraser proteins. As an m6A...

Premature ovarian failure (POF), a major cause of female infertility, is a complex disorder, but the molecular mechanisms underlying the disorder are only poorly understood. Here we report that protein kinase CK2 contributes to maintaining follicular survival through PI3K/AKT pathway and DNA damage response pathway. Targeted deletion of CK2β in mouse oocytes from the primordial follicle stage resulted in female infertility, which was attributed to POF incurring by massive follicle atresia. Downregulated PI3K/AKT signaling was found after CK2β deletion, indicated by reduced level of phosphorylated AKT (S473, T308, and S129) and altered AKT targets related to cell survival. Further studies discovered that CK2β-deficient oocytes showed enhanced γH2AX signals, indicative of accumulative unrepaired DSBs, which activated CHK2-dependant p53 and p63 signaling. The suppressed PI3K/AKT signaling and failed DNA damage response signaling probably contribute to large-scale oocyte loss and eventually POF. Our findings provide important new clues for elucidating the mechanisms underlying follicle atresia and POF.

N6-methyladenosine (m6A) is the most prevalent and reversible internal modification of mammalian messenger and noncoding RNAs mediated by specific m6A writer, reader, and eraser proteins. As an m6A writer, the METTL3–METTL14-WTAP complex dynamically regulates m6A modification and plays important roles in diverse biological processes. However, our knowledge about the complete functions of this RNA methyltransferase complex, the contributions of each component to the methylation and their impacts on different biological pathways, are still very limited. Here, by employing both in vivo and in vitro models, we report that METTL14 was indispensable for post-implantation embryonic development by facilitating the conversion from naïve to primed state of the epiblast. Depletion of Mettl14 lead to conspicuous embryonic growth retardation from E6.5 mainly as a result of resistance to differentiation, which further lead to embryonic lethality early in gestation. Our data highlight the critical function of METTL14, as an m6A modification regulator, in orchestrating early mouse embryogenesis.

Despite significant progress in early detection and improved treatment modalities prostate cancer remains the second leading cause of cancer death in American men which results in about 30,000 deaths per year in the USA. An aggressive phenotype leading to 2.58% risk of dying from prostate cancer still exists and immunotherapy has offered new possibilities to treat metastatic prostate cancer that cannot be treated by other modalities. Cancer immunotherapy is a rapidly growing field of research aimed at identifying biomarkers in immunodiagnosis and to develop new therapies by enabling the immune system to detect and destroy cancer cells. Immunotherapy falls into three different broad categories which are checkpoint inhibitors, cytokines, and vaccine immunotherapy. While immunotherapy to treat prostate cancer is still limited progress has been made; for treatment of advanced prostate cancer sipuleucel-T has been administered to patients in personalized doses to destroy prostate cancer cells which is promising and invites further research to determine immunotherapies for advanced prostate cancer. Antibody-based targeted immunotherapy and dendritic-cell-based vaccination are among the therapies that are currently being evaluated as promising approaches to treat prostate cancer. Combination immunotherapies include prostate cancer vaccines and radiotherapy for castration resistant prostate cancer. Microbial vectors for prostate cancer immunotherapy have been developed and bacterial strains have been engineered to express cancer-specific antigens, cytokines, and prodrug-converting cytokines. These approaches are addressed in the present review.

This chapter provides a brief overview of prostate cancer statistics, grading, diagnosis and treatment strategies that are discussed in more detail in the subsequent chapters of this book and the companion book titled "Clinical Molecular and Diagnostic Imaging of Prostate Cancer and Treatment Strategies". It also points to websites that provide additional useful information for patients affected by prostate cancer and for students and teachers to obtain practical and updated information on research, new diagnostic modalities and new therapies including new updated clinical trials. Three sections are focused on overview of prostate cancer statistics; overview of detection, diagnosis, stages and grading of prostate cancer; and treatment possibilities and options.

The significant role of centrosomes in cancer cell proliferation has been well recognized (reviewed in Schatten H, Histochem Cell Biol 129:667-86 (2008); Schatten H, Sun Q-Y, Microsc Microanal 17(4):506-512 (2011); Schatten H, Sun Q-Y, Reprod Fertil Dev. https://doi.org/10.1071/RD14493 (2015a); Schatten H, Sun Q-Y, Centrosome-microtubule interactions in health, disease, and disorders. In: Schatten H (ed) The cytoskeleton in health and disease. Springer Science+Business Media, New York (2015b)) and new research has generated new interest and new insights into centrosomes as potential targets for cancer-specific therapies. The centrosome is a key organelle serving multiple functions through its primary functions as microtubule organizing center (MTOC) that is also an important communication center for processes involved in cellular regulation; transport to and away from centrosome-organized microtubules along microtubules is essential for cellular activities including signal transduction and metabolic activities. New research on cancer cell centrosomes has generated new insights into centrosome dysfunctions in cancer cells in which centrosome phosphorylation, balance of centrosomal proteins, centrosome regulation and duplication are impaired. Among the hallmarks of cancer cells are multipolar spindles or abnormal bipolar spindles that are formed as a result of centrosome protein expression imbalances, abnormalities in centrosome structure and abnormalities in clustering of centrosomal components that are critical for bipolar mitotic apparatus formation. Centrosome abnormalities in cancer cells can be the result of multiple factors including environmental influences and toxicants that can affect centrosome functions by inducing centrosome pathologies leading to abnormal cancer cell proliferation. These topics are addressed in this review with focus on prostate-specific therapy strategies to target centrosome abnormalities. We will also address loss of cell polarity in cancer cells in which centrosome dysfunctions play a role as well as the loss of primary cilia in prostate cancer development and progression.

In early embryo development, a series of cell divisions take place that partition a large single cell, the fertilized oocyte, to become a complex multicellular organism, the mammalian embryo.

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.

The process of follicular development involves communications between oocyte and surrounding granulosa cells. FURIN is a member of the family of proprotein convertases that is involved in the activation of a large number of zymogens and proproteins by cleavage at its recognition motif. To investigate the functions of FURIN in female fertility, furinflox/flox (furfl/fl) mice were crossed with Zp3-Cre mice and Gdf9-Cre, respectively, to achieve oocyte-specific disruption of FURIN. Here we report for the first time that FURIN is dispensable for primordial follicle maintenance and activation but important for early secondary follicular development, as ablation of FURIN in oocytes caused failure of follicle development beyond the type 4 and/or 5a follicles in mutant mice, resulting in increased number of early secondary follicles and the severely decreased number of mature follicles, thus anovulation and infertility. We also found that the developmental arrest of early secondary follicles might be rooted in the loss of the mature form of ADAMTS1 (85-kDa prodomain truncated) and compromised proliferation of granulosa cells in mutant mice. Taken together, our data highlight the importance of FURIN in follicle development beyond the early secondary follicle stage and indicate that compromised FURIN function leads to follicular dysplasia and female infertility in mice.

Oxidative stress is known as a major contributing factor involved in oocyte aging, which negatively affects oocyte quality and development after fertilization. Melatonin is an effective free radical scavenger and its metabolites AFMK and AMK are powerful detoxifiers that eliminate free radicals. In this study, we used porcine oocytes to test the hypothesis that melatonin could scavenge free radicals produced during oocyte aging, thereby maintaining oocyte quality. We compared reactive oxygen species levels, apoptosis levels, mitochondrial membrane potential ratios, total glutathione contents and expression levels in fresh, aged and melatonin-treated aged porcine oocytes and observed the percentage of blastocyst formation following parthenogenetic activation. We found that melatonin could effectively maintain the morphology of oocytes observed in control oocytes, alleviate oxidative stress, markedly decrease early apoptosis levels, retard the decline of mitochondrial membrane potential and significantly promote subsequent embryonic development in oocytes aged for 24 hr in vitro. These results strongly suggest that melatonin can prevent postovulatory oocyte aging and promote subsequent embryonic development in the pig, which might find practical applications to control oocyte aging in other mammalian species including humans to maintain the quality of human oocytes when performing clinical assisted reproductive technology.

In mammalian cells, 5-methylcytosine (5-meC) can be transformed into 5-hydroxymethylcytosine (5-hmC) by the methylcytosine dioxygenase TET proteins (TET1, TET2 and TET3). Thymine DNA glycosylase (TDG), a downstream enzyme of TET proteins, not only functions in base excision repair, but also acts as a key enzyme that participates in active DNA demethylation. Here we microinjected exogenous TDG-mCherry mRNAs into germinal vesicle (GV) stage mouse oocytes, and found that initially TDG-mCherry localized in the nucleus. Just before GV breakdown (GVBD), TDG-mCherry was released from the nucleus into the cytoplasm. In contrast with TDG, another active DNA demethylation-associated enzyme, activation-induced cytidine deaminase (AID) became localized in the cytoplasm of GV oocytes, but entered the nucleus of oocytes just before GVBD. However, both TDG and AID could enter the G0 stage nuclei of cumulus cells injected into the ooplasm. To analyze the effects of TDG on oocyte maturation, we over-expressed TDG-mCherry in GV oocytes, and found that the rates of both GVBD and polar body extrusion rate were significantly decreased. When the TDG over-expressed oocytes were blocked at the GV stage, the oocyte chromatin became decondensed, and the histone 3 trimethyl lysine 9 (H3K9me3) and H3K9me2 levels were decreased. We also found that TDG could reduce the 5-meC level of oocyte genomic DNA. All these results indicate that aberrant TDG expression causes epigenetic modifications and meiotic cell cycle arrest of mouse oocytes.

In female mice, despite the presence of slight DNA double-strand breaks (DSBs), fully grown oocytes are able to undergo meiosis resumption as indicated by germinal vesicle breakdown (GVBD); however, severe DNA DSBs do reduce and delay entry into M phase through activation of the DNA damage checkpoint. But little is known about the effect of severe DNA DSBs on the spindle assembly checkpoint (SAC) during oocyte maturation. We showed that nearly no first polar body (PB1) was extruded at 12 h of in vitro maturation (IVM) in severe DNA DSBs oocytes, and the limited number of oocytes with PB1 were actually at telophase. However, about 60% of the severe DNA DSBs oocytes which underwent GVBD at 2 h of IVM released a PB1 at 18 h of IVM and these oocytes did reach the second metaphase (MII) stage. Chromosome spread at MI and MII stages showed that chromosomes fragmented after GVBD in severe DNA DSBs oocytes. The delayed PB1 extrusion was due to the disrupted attachment of microtubules to kinetochores and activation of the SAC. At the same time, misaligned chromosome fragments became obvious at the first metaphase (MI) in severe DNA DSBs oocytes. These data implied that the inactivation of SAC during the metaphase-anaphase transition of first meiosis was independent of chromosome integrity. Next, we induced DNA DSBs in vivo, and found that the number of superovulated oocytes per mouse was significantly reduced; moreover, this treatment increased the percentage of apoptotic oocytes. These results suggest that DNA DSBs oocytes undergo apoptosis in vivo.

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.

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 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.

Kif2a is a member of the Kinesin-13 microtubule depolymerases. Here, we report the expression, subcellular localization and functions of Kif2a during mouse oocyte meiotic maturation. Immunoblotting analysis showed that Kif2a was gradually increased form GV to the M I stages, and then decreased slightly at the M II stage. Confocal microscopy identified that Kif2a localized to the meiotic spindle, especially concentrated at the spindle poles and inner centromeres in metaphase and translocated to the midbody at telophase. Kif2a depletion by siRNA microinjection generated severely defective spindles and misaligned chromosomes, reduced microtubule depolymerization, which led to significant pro-M I/M Iarrest and failure of first polar body (PB1) extrusion. Kif2a-depleted oocytes were also defective in spindle pole localization of γ-tubulin and showed spindle assembly checkpoint (SAC) protein Bub3 at the kinetochores even after 10 hr extended culture. These results demonstrate that Kif2a may act as a microtubule depolymerase, regulating microtubule dynamics, spindle assembly and chromosome congression, and thus cell cycle progression during mouse oocyte meiotic maturation.

DNA double-strand breaks are caused by both intracellular physiological processes and environmental stress. In this study, we used laser microbeam cut (abbreviated microcut or cut), which allows specific DNA damage in the pronucleus of a fertilized egg and in individual blastomere(s) of an early embryo, to investigate the response of early embryos to DNA double-strand breaks. Line type γH2AX foci were detected in the cut region, while Chk2 phosphorylation staining was observed in the whole nuclear region of the cut pronuclei or blastomeres. Zygotes with cut male or female pronucleus showed poor developmental capability: the percentage of cleavage embryos was significantly decreased, and the embryos failed to complete further development to blastocysts. The cut blastomeres in 2-cell, 4-cell, and 8-cell embryos ceased cleavage, and they failed to incorporate into compacted morulae, but instead underwent apoptosis and cell death at the blastocyst stage; the uncut part of embryos could develop to blastocysts, with a reduced percentage or decreased cell number. When both blastomeres of the 2-cell embryos were cut by laser microbeam, cell death occurred 24 h earlier, suggesting important functions of the uncut blastomere in delaying cell death of the cut blastomere. Taken together, we conclude that microbeam-induced DNA damage in early embryos causes compromised development, and that embryos may have their own mechanisms to exclude DNA-damaged blastomeres from participating in further development.

During mouse antral follicle development, the oocyte chromatin gradually transforms from a less condensed state with no Hoechst-positive rim surrounding the nucleolus (NSN) to a fully condensed chromatin state with a Hoechst-positive rim surrounding the nucleolus (SN). Compared with SN oocytes, NSN oocytes display a higher gene transcription activity and a lower rate of meiosis resumption (G2/M transition), and they are mostly arrested at the two-cell stage after in vitro fertilization. To explore the differences between NSN and SN oocytes, and the maternal factors required for oocyte developmental competence, we compared the whole-transcriptome profiles between NSN and SN oocytes. First, we found that the NSN and SN oocytes were different in their metabolic pathways. In the phosphatidylinositol signaling pathway, the SN oocytes tend to produce diacylglycerol, whereas the NSN oocytes tend to produce phosphatidylinositol (3,4,5)-trisphosphate. For energy production, the SN oocytes and NSN oocytes differed in the gluconeogenesis and in the synthesis processes. Second, we also found that the key genes associated with oocyte meiosis and/or preimplantation embryo development were differently expressed in the NSN and SN oocytes. Our results illustrate that during the NSN-SN transition, the oocytes change their metabolic activities and accumulate maternal factors for further oocyte maturation and post-fertilization embryo development.

Both endogenous and exogenous factors can induce DNA double-strand breaks (DSBs) in oocytes, which is a potential risk for human-assisted reproductive technology as well as animal nuclear transfer. Here we used bleomycin (BLM) and laser micro-beam dissection (LMD) to induce DNA DSBs in germinal vesicle (GV) stage oocytes and compared the germinal vesicle breakdown (GVBD) rates and first polar body extrusion (PBE) rates between DNA DSB oocytes and untreated oocytes. Employing live cell imaging and immunofluorescence labeling, we observed the dynamics of DNA fragments during oocyte maturation. We also determined the cyclin B1 expression pattern in oocytes to analyze spindle assembly checkpoint (SAC) activity in DNA DSB oocytes. We used parthenogenetic activation to determine if the DNA DSB oocytes could be activated. As a result, we found that the BLM- or LMD-induced DSB oocytes showed lower GVBD rates and took a longer time to undergo GVBD compared with untreated oocytes. PBE was also delayed in DSB oocytes, but once GVBD had occurred, PBE was not affected, even in oocytes with severe DSBs. Compared with control oocytes, the DSB oocytes showed higher SAC activity, as indicated by less Ccnb1-GFP degradation during metaphase I to anaphase I transition. Parthenogenetic activation could activate the metaphase to interphase transition in the DNA DSB mature oocytes, but many oocytes contained multiple pronuclei or numerous micronuclei. These data suggest that DNA damage inhibits or delays the G2/M transition, but once GVBD occurs, DNA-damaged oocytes can complete chromosome separation and polar body extrusion even under a higher SAC activity, causing the formation of numerous micronuclei in early embryos.

Oocyte-specific deletion of Cdc42 has little effect on meiotic spindle organization and migration to the cortex but inhibits polar body emission, although homologous chromosome segregation occurs. The failure of cytokinesis is due to loss of polarized Arp2/3 accumulation and actin cap formation, and thus the defective contract ring., Mammalian oocyte maturation is distinguished by highly asymmetric meiotic divisions during which a haploid female gamete is produced and almost all the cytoplasm is maintained in the egg for embryo development. Actin-dependent meiosis I spindle positioning to the cortex induces the formation of a polarized actin cap and oocyte polarity, and it determines asymmetric divisions resulting in two polar bodies. Here we investigate the functions of Cdc42 in oocyte meiotic maturation by oocyte-specific deletion of Cdc42 through Cre-loxP conditional knockout technology. We find that Cdc42 deletion causes female infertility in mice. Cdc42 deletion has little effect on meiotic spindle organization and migration to the cortex but inhibits polar body emission, although homologous chromosome segregation occurs. The failure of cytokinesis is due to the loss of polarized Arp2/3 accumulation and actin cap formation; thus the defective contract ring. In addition, we correlate active Cdc42 dynamics with its function during polar body emission and find a relationship between Cdc42 and polarity, as well as polar body emission, in mouse oocytes.

Liver Kinase b1 (LKB1/STK11)is a tumor suppressor responsible for the Peutz-Jeghers syndrome, an autosomal-dominant, cancer-prone disorder in which patients develop neoplasms in several organs, including the oviduct, ovary, and cervix. Besides, the C allele of a SNP in the Lkb1 gene impedes the likelihood of ovulation in polycystic ovary syndrome (PCOS) in women treated with metformin, a known LKB1-AMPK activator. It is very likely that LKB1 plays roles in female fertility. To identify the physiological functions of LKB1 in the mouse ovary, we selectively disrupted LKB1 in oocytes by the Cre-LoxP conditional knockout system and found that Lkb1fl/fl; Gdf9-Cre mice were severely subfertile with significantly enlarged ovaries compared to Lkb1fl/fl mice. Interestingly, without Lkb1 expression in oocytes from the primordial follicle stage, the entire primordial follicle pool was activated but failed to mature and ovulate, subsequently causing premature ovarian failure (POF). Further investigation demonstrated that elevated mTOR signaling regulated by an AKT-independent LKB1-AMPK pathway was responsible for the excessive follicle activation and growth. Our findings reveal the role of LKB1 as an indispensable gatekeeper for the primordial follicle pool, offer new functional understanding for the tumor suppressor genes in reproductive organs, and might also provide valuable information for understanding POF and infertility.

Spindle sizes are different in diverse species and cell types. In frogs, the meiotic spindle size is positively correlated with the egg cell volume. Across species, relatively small mouse oocytes (70–80 μm) have a relatively large spindle while larger pig oocytes (about 120 μm) have a considerably smaller spindle. In this study we investigated whether species-specific oocyte spindle size was determined by cytoplasmic or nuclear factors. By exchanging the germinal vesicle between mouse and pig oocytes, we obtained two kinds of reconstructed oocytes: one with mouse ooplasm and pig GV (mCy-pGV oocyte) and the other with pig ooplasm and mouse GV (pCy-mGV oocyte). We show that the MII spindle size of the mCy-pGV oocyte is similar to that of the mouse meiotic spindle and significantly larger than that of the pig meiotic spindle. The timing of oocyte maturation also followed that of the species from which the oocyte cytoplasm arose, although some impact of the origin of the GV was observed. These data suggest that spindle size and the timing of meiotic progression are governed by cytoplasmic components rather than cytoplasmic volume and GV materials.