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In mammals, oocytes are arrested at G2/prophase for a long time, which is called germinal vesicle (GV) arrest. After puberty, fully-grown oocytes are stimulated by a gonadotropin surge to resume meiosis as indicated by GV breakdown (GVBD). CCNB1 is accumulated to a threshold level to trigger the activation of maturation promoting factor (MPF), inducing the G2/M transition. It is generally recognized that the anaphase-promoting complex/cyclosome (APC/C) and its cofactor CDH1 (also known as FZR1) regulates the accumulation/degradation of CCNB1. Here, by using small interfering RNA (siRNA) and messenger RNA (mRNA) microinjection, immunofluorescence and confocal microscopy, immunoprecipitation, time-lapse live imaging, and immunoblotting analysis, we showed that Septin 4 regulates the G2/M transition by regulating the accumulation of CCNB1 via APC/CCDC20 . Depletion of Septin 4 caused GV arrest by reducing CCNB1 accumulation. Unexpectedly, the expression level of CDC20 was higher in Septin 4 siRNA-injected oocytes than in control oocytes, but there was no significant change in the expression level of CDH1. Importantly, the reduced GVBD after Septin 4 depletion could be rescued not only by over-expressing CCNB1 but also could be partially rescued by depleting CDC20. Taken together, our results demonstrate that Septin 4 may play a critical role in meiotic G2/M transition by indirect regulation of CCNB1 stabilization in mouse oocytes.

Parathyroid hormone-related protein (PTHrP), the main cause of humoral hypercalcemia in malignancies, promotes cell proliferation and delays terminal cell maturation during embryonic development. Our previous study reported that PTHrP plays important roles in blastocyst formation, pluripotency gene expression, and histone acetylation during mouse preimplantation embryonic development. In this study, we further investigated the mechanism of preimplantation embryonic development regulated by PTHrP. Our results showed that Pthrp depletion decreased both the developmental rate of embryos at the cleavage stage and the cell number of morula-stage embryos. Pthrp-depleted embryos had significantly decreased levels of cyclin D1, phospho (p)-AKT (Thr308) and E2F1. However, Pthrp depletion did not cause significant changes in CDK4, β-catenin or RUNX2 expression. In addition, our results indicated that Pthrp depletion promoted HDAC4 translocation from the cytoplasm to the nucleus in cleavage-stage embryos by stimulating the activity of protein phosphatase 2A (PP2A), which resulted in dephosphorylation of HDAC4. Taken together, these results suggest that PTHrP regulates cleavage division progression and blastocyst formation through the AKT/cyclin D1 pathway and that PTHrP modulates histone acetylation patterns through nuclear translocation of HDAC4 via PP2A-dependent HDAC4 dephosphorylation during preimplantation embryonic development in mice.

The formation of zygote is the beginning of mammalian life, and dynamic epigenetic modifications are essential for mammalian normal development. H3K27 di-methylation (H3K27me2) and H3K27 tri-methylation (H3K27me3) are marks of facultative heterochromatin which maintains transcriptional repression established during early development in many eukaryotes. However, the mechanism underlying establishment and regulation of epigenetic asymmetry in the zygote remains obscure. Here we show that maternal EZH2 is required for the establishment of H3K27me3 in mouse zygotes. However, combined immunostaining with ULI-NChIP-seq (ultra-low-input micrococcal nuclease-based native ChIP-seq) shows that EZH1 could partially safeguard the role of EZH2 in the formation of H3K27me2. Meanwhile, we identify that EHMT1 is involved in the establishment of H3K27me2, and that H3K27me2 might be an essential prerequisite for the following de novo H3K27me3 modification on the male pronucleus. In this work, we clarify the establishment and regulatory mechanisms of H3K27me2 and H3K27me3 in mouse zygotes., Dynamic arrangement of epigenetic modifications such as repressive H3K27 methylation is essential for zygote development. Here the authors show that establishment of genome-wide H3K27me3 in zygotes requires EZH2, that EZH1 partially compensates for EZH2 loss, and that EHMT1 is involved in H3K27me2 establishment.

Mammalian cyclin A1 is prominently expressed in testis and essential for meiosis in the male mouse, however, it shows weak expression in ovary, especially during oocyte maturation. To understand why cyclin A1 behaves in this way in the oocyte, we investigated the effect of cyclin A1 overexpression on mouse oocyte meiotic maturation. Our results revealed that cyclin A1 overexpression triggered meiotic resumption even in the presence of germinal vesicle breakdown inhibitor, milrinone. Nevertheless, the cyclin A1-overexpressed oocytes failed to extrude the first polar body but were completely arrested at metaphase I. Consequently, cyclin A1 overexpression destroyed the spindle morphology and chromosome alignment by inducing premature separation of chromosomes and sister chromatids. Therefore, cyclin A1 overexpression will prevent oocyte maturation although it can promote meiotic resumption. All these results show that decreased expression of cyclin A1 in oocytes may have an evolutional significance to keep long-lasting prophase arrest and orderly chromosome separation during oocyte meiotic maturation.

Protein phosphatase 6 (PP6) is a member of the PP2A-like subfamily, which plays a critical role in many fundamental cellular processes. We recently reported that PP6 is essential for female fertility. Here, we report that PP6 is involved in meiotic recombination and that germ cell-specific deletion of PP6 by Stra8-Cre causes defective spermatogenesis. The PP6-deficient spermatocytes were arrested at the pachytene stage and defects in DSB repair and crossover formation were observed, indicating that PP6 facilitated meiotic double-stranded breaks (DSB) repair. Further investigations revealed that depletion of PP6 in the germ cells affected chromatin relaxation, which was dependent on MAPK pathway activity, consequently preventing programmed DSB repair factors from being recruited to proper positions on the chromatin. Taken together, our results demonstrate that PP6 has an important role in meiotic recombination and male fertility.

The quality of oocytes is a vital factor for embryo development. Meiotic progression through metaphase I usually takes a relatively long time to ensure correct chromosome separation, a process that is critical for determining oocyte quality. Here, we report that cell division cycle 5-like (Cdc5L) plays a critical role in regulating metaphase-to-anaphase I transition during mouse oocyte meiotic maturation. Knockdown of Cdc5L by small interfering RNA injection did not affect spindle assembly but caused metaphase I arrest and subsequent reduced first polar body extrusion due to insufficient anaphase-promoting complex/cyclosome activity. We further showed that Cdc5L could also directly interact with securin, and Cdc5L knockdown led to a continuous high expression level of securin, causing severely compromised meiotic progression. The metaphase-to-anaphase I arrest caused by Cdc5L knockdown could be rescued by knockdown of endogenous securin. In summary, we reveal a novel role for Cdc5L in regulating mouse oocyte meiotic progression by interacting with securin.

NEK5, a member of never in mitosis-gene A-related protein kinase, is involved in the regulation of centrosome integrity and centrosome cohesion at mitosis in somatic cells. In this study, we investigated the expression and function of NEK5 during mouse oocyte maturation and preimplantation embryonic development. The results showed that NEK5 was expressed from germinal vesicle (GV) to metaphase II (MII) stages during oocyte maturation with the highest level of expression at the GV stage. It was shown that NEK5 localized in the cytoplasm of oocytes at GV stage, concentrated around chromosomes at germinal vesicle breakdown (GVBD) stage, and localized to the entire spindle at prometaphase I, MI and MII stages. The small interfering RNA-mediated depletion of Nek5 significantly increased the phosphorylation level of cyclin-dependent kinase 1 in oocytes, resulting in a decrease of maturation-promoting factor activity, and severely impaired GVBD. The failure of meiotic resumption caused by Nek5 depletion could be rescued by the depletion of Wee1B. We found that Nek5 depletion did not affect CDC25B translocation into the GV. We also found that NEK5 was expressed from 1-cell to blastocyst stages with the highest expression at the blastocyst stage, and Nek5 depletion severely impaired preimplantation embryonic development. This study demonstrated for the first time that NEK5 plays important roles during meiotic G2/M transition and preimplantation embryonic development.

Family with sequence similarity 46, member C (FAM46C) is a highly conserved non-canonical RNA polyadenylation polymerase that is abundantly expressed in human and mouse testes and is frequently mutated in patients with multiple myeloma. However, its physiological role remains largely unknown. In this study, we found that FAM46C is specifically localized to the manchette of spermatids in mouse testes, a transient microtubule-based structure mainly involved in nuclear shaping and intra-flagellar protein traffic. Gene knockout of FAM46C in mice resulted in male sterility, characterized by the production of headless spermatozoa in testes. Sperm heads were intermittently found in the epididymides of FAM46C knockout mice, but their fertilization ability was severely compromised based on the results of intracytoplasmic sperm injection assays. Interestingly, our RNA-sequencing analyses of FAM46C knockout testes revealed that mRNA levels of only nine genes were significantly altered compared to wild-type ones (q < 0.05). When considering alternate activities for FAM46C, in vitro assays demonstrated that FAM46C does not exhibit protein kinase or AMPylation activity against general substrates. Together, our data show that FAM46C in spermatids is a novel component in fastening the sperm head and flagellum.

AIM To understand the agreement, precision, and accuracy between other estimated glomerular filtration rate (eGFR) equations and the Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) creatinine-cystatin C equation (EPI_Cr_CysC). MATERIALS AND METHODS We conducted a cross-sectional study of 1,913 CKD patients. The eGFRs were calculated separately by creatinine clearance rate and Cockcroft-Gault equation corrected for standard body surface area (Ccr_BSA and eCcr_BSA); CKD-EPI creatinine equation (EPI_Cr); CKD-EPI cystatin C equation (EPI_CysC); EPI_Cr_CysC equation; Modification of Diet in Renal Disease (MDRD) Study equation with standardized serum creatinine; and full-age spectrum creatinine equation (FAS). The EPI_Cr_CysC equation was used as the reference. RESULTS When compared with the EPI_Cr_CysC equation, the EPI_Cr equation achieved the highest agreement in eGFRs (Lin's concordance correlation coefficient = 0.936, 95% confidence interval (CI) = 0.930, 0.941). eCcr_BSA and EPI_Cr equations achieved the first and second highest percentage agreement in the accurate classification of CKD stage (72.55 vs. 71.25%). The MDRD equation had minimal bias and was closely followed by the EPI_Cr equation (median difference = -1.3, 95% CI = -2.0, -0.8 vs. median difference = 2.5, 95% CI = 1.7, 3.3 mL/min/1.73m2). The EPI_CysC and EPI_Cr equations achieved the first and second highest precision (interquartile range (IQR) of the difference = 12.2, 95% CI = 11.6, 12.9 vs. IQR of the difference = 15.5, 95% CI = 14.7, 16.3 mL/min/1.73m2). The EPI_Cr and MDRD equations performed similarly and both had the highest accuracy at 30% (1 - P30 = 18.6, 95% CI = 16.9, 20.4 vs. 1 - P30 = 18.6, 95% CI = 16.8, 20.3%). CONCLUSION For assessment of renal function, the EPI_Cr equation performed the best and remained an acceptable alternative to the EPI_Cr_CysC equation in the absence of cystatin C. .

PKCβI, a member of the classical protein kinase C family, plays key roles in regulating cell cycle transition. Here, we report the expression, localization and functions of PKCβI in mouse oocyte meiotic maturation. PKCβI and p-PKCβI (phosphor-PKCβI) were expressed from germinal vesicle (GV) stage to metaphase II (MII) stage. Confocal microscopy revealed that PKCβI was localized in the GV and evenly distributed in the cytoplasm after GV breakdown (GVBD), and it was concentrated at the midbody at telophase in meiotic oocytes. While, p-PKCβI was concentrated at the spindle poles at the metaphase stages and associated with midbody at telophase. Depletion of PKCβI by specific siRNA injection resulted in defective spindles, accompanied with spindle assembly checkpoint activation, metaphase I arrest and failure of first polar body (PB1) extrusion. Live cell imaging analysis also revealed that knockdown of PKCβI resulted in abnormal spindles, misaligned chromosomes, and meiotic arrest of oocytes arrest at the Pro-MI/MI stage. PKCβI depletion did not affect the G2/M transition, but its overexpression delayed the G2/M transition through regulating Cyclin B1 level and Cdc2 activity. Our findings reveal that PKCβI is a critical regulator of meiotic cell cycle progression in oocytes. Abbreviations: PKC, protein kinase C; COC, cumulus-oocyte complexes; GV, germinal vesicle; GVBD, germinal vesicle breakdown; Pro-MI, first pro-metaphase; MI, first metaphase; Tel I, telophase I; MII, second metaphase; PB1, first polar body; SAC, spindle assembly checkpoint.

There are two important events in oocyte meiotic maturation, the G2/M transition and metaphase I progression. Thousands of proteins participate in regulating oocyte maturation, which highlights the importance of the ubiquitin proteasome system (UPS) in regulating protein synthesis and degradation. Skp1–Cullin–F-box (SCF) complexes, as the best characterized ubiquitin E3 ligases in the UPS, specifically recognize their substrates. F-box proteins, as the variable adaptors of SCF, can bind substrates specifically. Little is known about the functions of the F-box proteins in oocyte maturation. In this study, we found that depletion of FBXO34, an F-box protein, led to failure of oocyte meiotic resumption due to a low activity of MPF, and this phenotype could be rescued by exogenous overexpression of CCNB1. Strikingly, overexpression of FBXO34 promoted germinal vesicle breakdown (GVBD), but caused continuous activation of spindle assembly checkpoint (SAC) and MI arrest of oocytes. Here, we demonstrated that FBXO34 regulated both the G2/M transition and anaphase entry in meiotic oocytes.

RNA-binding proteins (RBPs) have essential functions during germline and early embryo development. However, current methods are unable to identify the in vivo targets of a RBP in these low-abundance cells. Here, by coupling RBP-mediated reverse transcription termination with linear amplification of complementary DNA ends and sequencing, we present the LACE-seq method for identifying RBP-regulated RNA networks at or near the single-oocyte level. We determined the binding sites and regulatory mechanisms for several RBPs, including Argonaute 2 (Ago2), Mili, Ddx4 and Ptbp1, in mature mouse oocytes. Unexpectedly, transcriptomics and proteomics analysis of Ago2-/- oocytes revealed that Ago2 interacts with endogenous small interfering RNAs (endo-siRNAs) to repress mRNA translation globally. Furthermore, the Ago2 and endo-siRNA complexes fine-tune the transcriptome by slicing long terminal repeat retrotransposon-derived chimeric transcripts. The precise mapping of RBP-binding sites in low-input cells opens the door to studying the roles of RBPs in embryonic development and reproductive diseases.

Cadmium (Cd) is a bioaccumulative heavy metal element with potential toxicity on the female reproductive system, but the exact molecular mechanisms have not yet been clearly defined. In this study, female mice were exposed to 0.5 mg/kg/day of CdCl2 for 60 consecutive days. We found that chronic Cd exposure significantly decreased the fecundity of female mice by affecting oocyte meiotic progression as indicated by disrupted spindle assembly, chromosome alignment and kinetochore-microtubule attachments, consequently resulting in aneuploid oocytes. Further studies showed that the periodic fluctuations of MPF activity and cyclin B1 expression were disturbed in Cd-exposed oocytes probably by affecting the spindle assembly checkpoint protein Bub3. In addition, Cd exposure induced oxidative stress as indicated by an increased level of reactive oxygen species and apoptosis in oocytes, leading to oocyte quality deterioration. Taken together, these data suggest that Cd exposure causes disrupted molecular events of meiotic progression and deterioration of oocyte quality via oxidative stress, leading to decrease of female fertility.

// Lei Guo 1, * , Shi-Bin Chao 2, 3, * , Lu Xiao 4 , Zhen-Bo Wang 2, 5 , Tie-Gang Meng 2, 4 , Yuan-Yuan Li 2 , Zhi-Ming Han 2 , Ying-Chun Ouyang 2 , Yi Hou 2 , Qing-Yuan Sun 2, 5 and Xiang-Hong Ou 1 1 Center for Reproductive Medicine, Guangdong Second Provincial General Hospital, Guangzhou 510317, PR China 2 State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, PR China 3 The ART Center, Jiujiang Maternal and Child Health Care Hospital, Jiangxi 332000, PR China 4 Department of Obstetrics and Gynecology, Nanfang Hospital, Southern Medical University, Guangzhou 510515, PR China 5 College of Life Sciences, University of Chinese Academy of Sciences, Beijing 100049, PR China * These authors have contributed equally to this work Correspondence to: Xiang-Hong Ou, email: ouxianghong2003@163.com Qing-Yuan Sun, email: sunqy@ioz.ac.cn Keywords: sperm RNA, RNase, intracytoplasmic sperm injection, blastocyst formation, offspring Received: March 28, 2017 Accepted: May 22, 2017 Published: June 27, 2017 ABSTRACT Recently, numerous studies have reported that the mature sperm contains both coding and non-coding RNAs and the sperm delivers some RNAs to the oocyte at fertilization. However, the functions of the RNAs carried to the oocyte by sperm at fertilization in embryonic development remains a mystery. In this study, the mature spermatozoa were treated with lysolecithin, pronase and RNases (RNase A and RNase H) to remove the sperm-carried RNAs, and then injected into normal mature oocyte. The results showed that after the treatment, the content of the sperm RNAs was decreased by about 90%. The blastocyst formation rate and the live birth rate of the embryos from intracytoplasmic sperm injection (ICSI) using the treated sperm were significantly decreased ( P 0.05), but the body weight of F1 mice from sperm-treated group was lower than that in control group after two weeks of birth ( P

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.

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.

Chromosome segregation is driven by separase, activity of which is inhibited by binding to securin and cyclin B1/CDK1. In meiosis, premature separase activity will induce aneuploidy or abolish chromosome segregation owing to the untimely destruction of cohesin. Recently, we have proved that cyclin B2 can compensate for cyclin B1 in CDK1 activation for the oocyte meiosis G2/M transition. In the present study, we identify an interaction between cyclin B2/CDK1 and separase in mouse oocytes. We find that cyclin B2 degradation is required for separase activation during the metaphase I-anaphase I transition because the presence of stable cyclin B2 leads to failure of homologous chromosome separation and to metaphase I arrest, especially in the simultaneous absence of securin and cyclin B1. Moreover, non-phosphorylatable separase rescues the separation of homologous chromosomes in stable cyclin B2-arrested cyclin B1-null oocytes. Our results indicate that cyclin B2/CDK1 is also responsible for separase inhibition via inhibitory phosphorylation to regulate chromosome separation in oocyte meiosis, which may not occur in other cell types.

Background Systemic lupus erythematosus (SLE) is a multisystem autoimmune disease that commonly affects the kidneys. Research into markers that can predict the prognosis of tubulointerstitial lupus nephritis (LN) has been impeded by the lack of well-designed studies. Methods In this study, we selected and merged 3 sets of renal biopsy tubulointerstitial data from GSE32591, GSE69438, and GSE127797, including 95 LN and 15 living healthy donors. CIBERSORTx was utilized for differentially infiltrating immune cell (DIIC) analysis. Weighted Gene Co-Expression network analysis (WGCNA) was employed to explore differentially expressed gene (DEG) related modules. Combined WGCNA hub genes and protein-protein interaction (PPI) validation was used for immune marker identification. Lastly, unsupervised clustering was carried out to validate the correlation between these markers and clinical characteristics. Results Our findings unveiled TYROBP, C1QB, LAPTM5, CTSS, PTPRC as the 5 immune markers, which were negatively correlated with glomerular filtration rate (GFR). Specifically, the expression levels of TYROBP and C1QB were significantly different between proliferative LN (PLN) and membranous LN (MLN). Unsupervised clustering could aggregate LN by these immune marker expression spectrums. Conclusions This study is the first to identify infiltrating immune cells and associated molecular patterns in the tubulointerstitium of LN by utilizing bioinformatics methods. These findings contribute to a better understanding of the mechanisms behind LN, and promote more precise diagnosis.

Nek11, a member of the never in mitosis gene A (NIMA) family, is activated in somatic cells associated with G1/S or G2/M arrest. However, its function in meiosis is unknown. In this research, the expression, localization and functions of NEK11 in the mouse oocyte meiotic maturation were examined. Western blotting indicated that NEK11S was the major NEK11 protein in mouse oocyte. MYC-tagged Nek11 mRNA microinjection and immunofluorescent staining showed that NEK11 was localized to the meiotic spindles at MI and MII stage. Knockdown of Nek11 by microinjection of siRNA did not affect germinal vesicle breakdown (GVBD) and the first polar body extrusion, but caused formation of 2-cell-like eggs. These results demonstrate that Nek11 regulates asymmetric cell division during oocyte meiotic maturation.

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...

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.

Zygotic chromatin undergoes extensive reprogramming immediately after fertilization. It is generally accepted that maternal factors control this process. However, little is known about the underlying mechanisms. Here we report that maternal RAD9A, a key protein in DNA damage response pathway, is involved in post-zygotic embryo development, via a mouse model with conditional depletion of Rad9a alleles in oocytes of primordial follicles. Post-zygotic losses originate from delayed zygotic chromatin decondensation after depletion of maternal RAD9A. Pronucleus formation and DNA replication of most mutant zygotes are therefore deferred, which subsequently trigger the G2/M checkpoint and arrest development of most mutant zygotes. Delayed zygotic chromatin decondensation could also lead to increased reabsorption of post-implantation mutant embryos. In addition, our data indicate that delayed zygotic chromatin decondensation may be attributed to deferred epigenetic modification of histone in paternal chromatin after fertilization, as fertilization and resumption of secondary meiosis in mutant oocytes were both normal. More interestingly, most mutant oocytes could not support development beyond one-cell stage after parthenogenetic activation. Therefore, RAD9A may also play an important role in maternal chromatin reprogramming. In summary, our data reveal an important role of RAD9A in zygotic chromatin reprogramming and female fertility.

SUN (Sad1 and UNC84 domain containing)-domain proteins are reported to reside on the nuclear membrane playing distinct roles in nuclear dynamics. SUN5 is a new member of the SUN family, with little knowledge regarding its function. Here, we generated Sun5−/− mice and found that male mice were infertile. Most Sun5-null spermatozoa displayed a globozoospermia-like phenotype but they were actually acephalic spermatozoa. Additional studies revealed that SUN5 was located in the neck of the spermatozoa, anchoring sperm head to the tail, and without functional SUN5 the sperm head to tail coupling apparatus was detached from nucleus during spermatid elongation. Finally, we found that healthy heterozygous offspring could be obtained via intracytoplasmic injection of Sun5-mutated sperm heads for both male mice and patients. Our studies reveal the essential role of SUN5 in anchoring sperm head to the tail and provide a promising way to treat this kind of acephalic spermatozoa-associated male infertility.

Elder women suffer from low or loss of fertility because of decreasing oocyte quality as maternal aging. As energy resource, mitochondria play pivotal roles in oocyte development, determining oocyte quality. With advanced maternal age, increased dysfunctions emerge in oocyte mitochondria, which decrease oocyte quality and its developmental potential. Mitochondria supplement as a possible strategy for improving egg quality has been in debate due to ethnic problems. Heterogeneity is an intractable problem even transfer of germinal vesicle, spindle, pronuclei or polar body is employed. We proposed that the autologous adipose tissue-derived stem cell (ADSC) mitochondria could improve the fertility in aged mice. We found that autologous ADSC mitochondria could promote oocyte quality, embryo development and fertility in aged mice, which may provide a promising strategy for treatment of low fertility or infertility in elder women.

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.

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.

Aim Accumulating evidence showed that transient receptor potential channels play an important role in the regulation of cardiomyocyte differentiation. The vanilloid receptor 1 (VR1) is a member of the transient receptor channel super family and is expressed in cardiomyocytes. However, its function in cardiomyocytes remains unclear. Methods Herein, the aim of this study was to investigate the functional role of VR1 in the cardiomyocyte differentiation of bone marrow-derived mesenchymal stem cells (BMSCs), and to elucidate the potential molecular mechanisms. Results Immunofluorescence assay showed that cardiomyocyte marker cardiac troponin T (cTnT) was found significantly elevated in differentiated BMSCs induced by 5-azacytidine compared with control. Similarly, VR1 expression was also found significantly increased in induced BMSCs differentiation. Additionally, we examined the role of VR1 in BMSC differentiation processes through VR1 siRNAs. We found that the expression of cardiomyocyte marker genes, such as alpha-myosin heavy chain (α-MHC), α-cardiac actin and Nkx2.5 (cardiac-specific transcription factor) was significantly decreased when VR1 was silenced. Furthermore, we found that inhibition of VR1 expression is associated with downregulation of Wnt/β-catenin signaling. Conclusions To summarize, our data demonstrate important role of VR1 in BMSCs differentiation into cardiomyocytes in conjunction of Wnt/β-catenin signaling. This article is protected by copyright. All rights reserved.

Background: Cardiovascular calcifications can be prevented by vitamin K and are accelerated by vitamin K antagonists. These effects are believed to be mainly mediated by the vitamin K-dependent matrix Gla protein. Another vitamin K-dependent protein, Gas6, is also expressed in vascular smooth muscle cells (VSMC). In vitro Gas6 expression was shown to be regulated in VSMC calcification and apoptotic processes. Methods: We investigated the role of Gas6 in vitro using VSMC cultures and in vivo in young and old Gas6-deficient (Gas6(-/-)) and wildtype (WT) mice. In addition, Gas6(-/-) and WT mice were challenged by (a) warfarin administration, (b) uninephrectomy (UniNX) plus high phosphate diet, or (c) UniNX plus high phosphate plus electrocautery of the residual kidney. Results: In vitro VSMC from WT and Gas6(-/-) mice exposed to warfarin showed increased apoptosis and calcified similarly. In vivo, aortic, cardiac and renal calcium content in all groups was similar, except for a lower cardiac calcium content in Gas6(-/-) mice (group a). Von Kossa staining revealed small vascular calcifications in both WT and Gas6(-/-) mice (groups a-c). In aging, non-manipulated mice, no significant differences in vascular calcification were identified between Gas6(-/-) and WT mice. Gas6(-/-) mice exhibited no upregulation of matrix Gla protein in any group. Cardiac output was similar in all treatment groups. Conclusions: Taken together, in our study Gas6 fails to aggravate calcification against the previous assumption.

Septin 7 is a conserved GTP-binding protein. In this study, we examined the localization and functions of Septin 7 during mouse oocyte meiotic maturation. Immunofluorescent analysis showed that intrinsic Septin 7 localized to the spindles from the pro-MI stage to the MII stage. Knockdown of Septin 7 by siRNA microinjection caused abnormal spindles and affected extrusion of the first polar body. Septin 7 mRNA tagged with myc was injected into GV stage oocytes to overexpress Septin 7. Overexpressed Myc-Septin 7 localized to the spindle and beneath the plasma membrane displaying long filaments. Fluorescence intensity of spindle α-tubulin in myc-Septin 7-injected oocytes was weaker than that of the control group, demonstrating that Septin 7 may influence recruitment of α-tubulin to spindles. MII oocytes injected with myc-Septin 7 exhibited abnormal chromosome alignment, and parthenogenetic activation failed to allow extrusion of the second polar body, suggesting that overexpression of Septin 7 may affect extrusion of the polar body by disturbing the alignment of chromosomes and regulating α-tubulin recruitment to spindles. In summary, Septin 7 may regulate meiotic cell cycle progression by affecting microtubule cytoskeletal dynamics in mouse oocytes.

Checkpoint kinase 1 (Chk1) plays key roles in all currently defined cell cycle checkpoints, but its functions in mouse oocyte meiosis remain unclear. In this study, we report the expression, localization and functions of Chk1 in mouse oocyte meiosis. Chk1 was expressed from germinal vesicle (GV) to metaphase II (MII) stages and localized to the spindle from pro-metaphase I (pro-MI) to MII stages in mouse oocytes. Chk1 depletion facilitated the G 2/M transition while Chk1 overexpression inhibited the G 2/M transition as indicated by germinal vesicle breakdown (GVBD), through regulation of Cdh1 and Cyclin B1. Chk1 depletion did not affect meiotic cell cycle progression after GVBD, but its overexpression after GVBD activated the spindle assembly checkpoint and prevented homologous chromosome segregation, thus arresting oocytes at pro-MI or metaphase I (MI) stages. These results suggest that Chk1 is indispensable for prophase I arrest and functions in G 2/M checkpoint regulation in meiotic oocytes. Moreover, Chk1 overexpression affects meiotic spindle assembly checkpoint regulation and thus chromosome segregation.

Rab family members play important roles in membrane trafficking, cell growth, and differentiation. Almost all components of the cell endomembrane system, the nucleus, and the plasma membrane are closely related to RAB proteins. In this study, we investigated the distribution and functions of three members of the Rab family, Rab3A, Rab27A, and Rab35, in mouse oocyte meiotic maturation and activation. The three Rab family members showed different localization patterns in oocytes. Microinjection of siRNA, antibody injection, or inhibitor treatment showed that (1) Rab3A regulates peripheral spindle and cortical granule (CG) migration, polarity establishment, and asymmetric division; (2) Rab27A regulates CG exocytosis following MII-stage oocyte activation; and (3) Rab35 plays an important role in spindle organization and morphology maintenance, and thus meiotic nuclear maturation. These results show that Rab proteins play important roles in mouse oocyte meiotic maturation and activation and that different members exert different distinct functions.

STUDY QUESTION: There is an unexplored physiological role of N-WASP (neural Wiskott-Aldrich syndrome protein) in oocyte maturation that prevents completion of second meiosis. SUMMARY ANSWER: In mice, N-WASP deletion did not affect oocyte polarity and asymmetric meiotic division in first meiosis, but did impair midbody formation and second meiosis completion. WHAT IS KNOWN ALREADY: N-WASP regulates actin dynamics and participates in various cell activities through the RHO-GTPase-Arp2/3 (actin-related protein 2/3 complex) pathway, and specifically the Cdc42 (cell division cycle 42)-N-WASP-Arp2/3 pathway. Differences in the functions of Cdc42 have been obtained from in vitro compared to in vivo studies. STUDY DESIGN, SAMPLES/MATERIALS, METHODS: By conditional knockout of N-WASP in mouse oocytes, we analyzed its in vivo functions by employing a variety of different methods including oocyte culture, immunofluorescent staining and live oocyte imaging. Each experiment was repeated at least three times, and data were analyzed by paired-samples t-test. MAIN RESULTS AND THE ROLE OF CHANCE: Oocyte-specific deletion of N-WASP did not affect the process of oocyte maturation including spindle formation, spindle migration, polarity establishment and maintenance, and homologous chromosome or sister chromatid segregation, but caused failure of cytokinesis completion during second meiosis (P

Dynein light chain, Tctex-type 3 (DYNLT3), is a member of the cytoplasmic dynein DYNLT light chain family and has been reported to have a potential role in chromosome congression in human mitosis. However, its role in mammalian meiosis is unclear. In this study, we examined its localization, expression, and functions in mouse oocyte meiosis. Immunofluorescent staining showed that DYNLT3 was restricted to the germinal vesicle and associated with kinetochores at the germinal vesicle breakdown stage, metaphase I and metaphase II. The expression level of DYNLT3 was similar at all meiotic stages. Depletion of DYNLT3 by antibody injection resulted in chromosome misalignment and decrease of the polar body extrusion rate. We further found that DYNLT3-depleted oocytes displayed kinetochore-microtubule detachments. Chromosome-spread experiments showed that depletion of DYNLT3 inhibited the metaphase-anaphase transition by preventing homologous chromosome segregation in meiosis I. Our data suggest that DYNLT3 is required for chromosome alignment and homologous chromosome segregation during mouse oocyte meiosis.

The bipolar spindle is a complex molecular machinery that drives chromosome congression and segregation. During meiosis in the mouse multiple microtubule organizing centers aggregate to form a bipolar intermediate followed by elongation and establishment of the barrel-shaped acentriolar meiotic spindle. Previous studies have shown that septin1 is localized to spindle poles in mitosis, suggesting its possible involvement in spindle assembly. We, therefore, asked whether perturbation of septin1 will impair the process of spindle assembly and investigated localization and function during mouse oocyte meiotic maturation. Septin1 was localized to the spindle at metaphase and at the midbody during cytokinesis. Disruption of septin1 function using siRNA caused a decrease in PBE and extensive spindle defects. Moreover, the process of chromosome congression was impaired. However, septin1 depletion did not cause aneuploidy in oocyte with an extruded polar body. Taken together, our results show that septin1 is a key player in spindle assembly and chromosome congression in mouse meiosis. Developmental Dynamics 240:2281–2289, 2011. © 2011 Wiley-Liss, Inc.

P38αMAPK (p38α) is usually activated in response to various stresses and plays a role in the inhibition of cell proliferation and tumor progression, but little is known about its roles in meiotic spindle assembly. In this study, we characterized the dynamic localization of p38α and explored its function in mouse oocyte meiotic maturation. P38α specifically colocalized with γ-tubulin and Plk1 at the center of MTOCs and spindle poles. Depletion of p38α by specific morpholino injection resulted in severely defective spindles and misaligned chromosomes probably via MK2 dephosphorylation. Notably, depletion of p38α led to significant spindle pole defects, spindle elongation, non-tethered kinetochore microtubules and increased microtubule tension. The disruption of spindle stability was coupled with decreased γ-tubulin and Plk1 at MTOCs. Overexpression of Eg5, a conserved motor protein, also caused spindle elongation and its morpholino injection almost completely rescued spindle elongation caused by p38α depletion. In addition, p38α-depletion decreased BubR1 and interfered with spindle assembly checkpoint (SAC), which resulted in aneuploid oocytes. Together, these data indicate that p38α is an important component of MTOCs, which regulates spindle assembly and spindle length, as well as stabilizes the spindle and spindle poles. Perturbed SAC and abnormal microtubule tension may be responsible for the misaligned chromosomes and high aneuploidy in p38α-depleted mouse oocytes.

Sumoylation is an important post-translational modification in which SUMO (small ubiquitin-related modifier) proteins are bonded covalently to their substrates. Studies on the roles of sumoylation in cell cycle regulation have been emerging in both mitosis from yeast to mammals and meiosis in budding yeast, but the functions of sumoylation in mammalian meiosis, especially in oocyte meiotic maturation are not well known. Here, we examined the localization and expression of SUMO-1 and SUMO-2/3, the two basic proteins in the sumoylation pathway and investigated their roles through over-expression of Senp2 during mouse oocyte maturation. Immunofluorescent staining revealed differential patterns of SUMO-1 and SUMO-2/3 localization: SUMO-1 was localized to the spindle poles in prometaphase I, MI and MII stages, around the separating homologues in anaphase I and telophase I stages of first meiosis, while SUMO-2/3 was mainly concentrated near centromeres during mouse oocyte maturation. Immunoblot analysis uncovered the different expression profiles of SUMO-1 and SUMO-2/3 modified proteins during mouse oocyte maturation. Over-expression of Senp2, a SUMO-specific isopeptidase, caused changes of SUMO-modified proteins and led to defects in MII spindle organization in mature eggs. These results suggest that the SUMO pathway may play an indispensable role during mouse oocyte meiotic maturation.

This work describes a new approach to the construction of an amperometric biosensor for hydrogen peroxide detection based on in situ electrosynthesized gold/polyaniline core–shell nanocomposites on conducting ITO electrode. The immobilization of the enzyme, horseradish peroxidase (HRP), on the polyaniline nanofilm was carried out by electrostatic attachment approach. Results showed that the immobilized HRP exhibited enhanced performance toward the reduction of H 2 O 2 , in comparison with other bulk polyaniline (PANI)-based H 2 O 2 biosensor and metallic nanoparticles incorporated PANI systems. The resulting biosensor shows a fast amperometric response ( 2 O 2 . A linear range from 0.2 to 80 μM for the detection of H 2 O 2 was observed with a sensitivity of 20.5 μA/mM and a detection limit of 0.16 μM at a signal-to-noise ratio of 3. Moreover, the biosensor has a good reproducibility, and long-term stability.

The biological effects of estrogens are largely mediated through estrogen receptors (ERs), which belong to the nuclear receptor gene family of transcription factors. ER-alpha36 has been recently identified as a new variant of ERalpha, but its expression and roles in female reproduction system remain unknown. Immunocytochemistry and confocal microscopy were employed to observe ER-alpha36 distribution in mouse ovary during postnatal development and in oocyte during meiotic maturation. ER-alpha36 was consistently present in the nuclei of oocytes regardless of follicular growth stage and mouse age until germinal vesicle breakdown (GVBD). Its immunosignal was smeared in granulosa cells. However, the ER-alpha36 signal is up-regulated and found in cytoplasm with little or no nuclear staining during corpus luteum development. ER-alpha36 was also found in theca cells. We showed by Western blot that ER-alpha36 was expressed in mouse oocytes at various maturation stages. When the function of nuclear ER-alpha36 was blocked by microinjecting anti-ER-alpha36 specific antibody into the germinal vesicle (GV) of mouse oocytes, the first polar body emission occurred earlier in a higher proportion of oocytes compared to the control. These results suggest that ER-alpha36 may play critical roles in mouse ovarian folliculogenesis and oocyte development.

BRCA1 as a tumor suppressor has been widely investigated in mitosis, but its functions in meiosis are unclear. In the present study, we examined the expression, localization, and function of BRCA1 during mouse oocyte meiotic maturation. We found that expression level of BRCA1 was increased progressively from germinal vesicle to metaphase I stage, and then remained stable until metaphase II stage. Immunofluorescent analysis showed that BRCA1 was localized to the spindle poles at metaphase I and metaphase II stages, colocalizing with centrosomal protein gamma-tubulin. Taxol treatment resulted in the presence of BRCA1 onto the spindle microtubule fibers, whereas nocodazole treatment induced the localization of BRCA1 onto the chromosomes. Depletion of BRCA1 by both antibody injection and siRNA injection caused severely impaired spindles and misaligned chromosomes. Furthermore, BRCA1-depleted oocytes could not arrest at the metaphase I in the presence of low-dose nocodazole, suggesting that the spindle checkpoint is defective. Also, in BRCA1-depleted oocytes, gamma-tubulin dissociated from spindle poles and MAD2L1 failed to rebind to the kinetochores when exposed to nocodazole at metaphase I stage. Collectively, these data indicate that BRCA1 regulates not only meiotic spindle assembly, but also spindle assembly checkpoint, implying a link between BRCA1 deficiency and aneuploid embryos.

Spindle movement, including spindle migration during first meiosis and spindle rotation during second meiosis, is essential for asymmetric divisions in mouse oocytes. Previous studies by others and us have shown that microfilaments are required for both spindle migration and rotation. In the present study, we aimed to further investigate the mechanism controlling spindle movement during mouse oocyte meiosis. By employing drug treatment and immunofluorescence microscopy, we showed that dynamic microtubule assembly was involved in both spindle migration and rotation. Furthermore, we found that the calcium/CaM/CaMKII pathway was important for regulating spindle rotation.

To investigate the influence of histone deacetylases on nuclear reprogramming after nuclear transfer, we treated the cloned embryos with a histone deacetylase inhibitor, Trichostatin A (TSA). In the present study, global changes in acetylation of histone H3-lysine 14, histone H4-lysine 12, and histone H4-lysine 5 were studied in rabbit in vivo fertilized embryos, somatic cell nuclear transfer (SCNT) embryos, and TSA-treated SCNT embryos. From the pronuclear to the morula stage, the deacetylation-reacetylation changes in acetylation of histone H3-lysine 14 and histone H4-lysine 12 occurred in both fertilized embryos and TSA-treated cloned embryos; however, the distribution pattern in untreated cloned embryos failed to display such changes. More interesting, the signal of acetylation of histone H4-lysine 12 in cloned embryos was detected in both the inner cell mass and the trophectoderm, whereas TSA-treated cloned embryos showed the same staining pattern as fertilized embryos and the staining was limited to the inner cell mass. The histone acetylation pattern of TSA-treated SCNT embryos appeared to be more similar to that of normal embryos, indicating that TSA could improve nuclear reprogramming after nuclear transfer.

The behavior of mitochondria during early oogenesis remains largely unknown in zebrafish. We used three mitochondrial probes (Mito Tracker Red CMXRos, Mito Tracker Green FM, and JC-1) to stain early zebrafish oocyte mitochondria, and confocal microscopy to analyze mitochondrial aggregation and distribution. By using fluorescence recovery after photobleaching (FRAP), we traced mitochondrial movement. The microtubule assembly inhibitor nocodazole and microfilament inhibitor cytochalasin B (CB) were used to analyze the role of microtubules and microfilaments on mitochondrial movement. By using the dual emission probe, JC-1, and oxidative phosphorylation uncoupler, carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP), we determined the distribution of active and inactive (low-active) mitochondria. Green/red fluorescence ratios of different sublocations in different oocyte groups stained by JC-1 were detected in merged (green and red) images. Our results showed that mitochondria exhibited a unique distribution pattern in early zebrafish oocytes. They tended to aggregate into large clusters in early stage I oocytes, but in a threadlike state in latter stage I oocytes. We detected a lower density mitochondrial area and a higher density mitochondrial area on opposite sides of the germinal vesicle. The green/red fluorescence ratios in different sublocations in normal oocytes were about 1:1. This implies that active mitochondria were distributed in all sublocations. FCCP treatment caused significant increases in the ratios. CB and nocodazole treatment caused an increase of the ratios in clusters and mitochondrial cloud, but not in dispersed areas. Mitochondria in different sublocations underwent fast dynamic movement. Inhibition or disruption of microtubules or microfilaments resulted in even faster mitochondrial free movement.

The interspecies somatic cell nuclear transfer (iSCNT) technique for therapeutic cloning gives great promise for treatment of many human diseases. However, the incomplete nuclear reprogramming and the low blastocyst rate of iSCNT are still big problems. Herein, we observed the effect of TSA on the development of rabbit-rabbit intraspecies and rabbit-human interspecies cloned embryos. After treatment with TSA for 6 hr during activation, we found that the blastocyst rate of rabbit-rabbit cloned embryos was more than two times higher than that of untreated embryos; however, the blastocyst rate of TSA-treated rabbit-human interspecies cloned embryos decreased. We also found evident time-dependent histone deacetylation-reacetylation changes in rabbit-rabbit cloned embryos, but not in rabbit-human cloned embryos from fusion to 6 hr after activation. Our results suggest that TSA-treatment does not improve blastocyst development of rabbit-human iSCNT embryos and that abnormal histone deacetylation-reacetylation changes in iSCNT embryos may account for their poor blastocyst development.

Because some animals and human beings potentially engage in sexual activity at any day of the menstrual cycle, this may cause fertilization of postovulatory aged oocytes, which result in decreased potential of embryo development and longevity of offspring. To investigate the involvement of histone acetylation in the function of postovulatory aging, we examined the changes of histone acetylation by immunostaining with specific antibodies against various acetylated lysines on histones H3 and H4. We found that the acetylation levels of lysine 14 on histone H3 and lysines 8 and 12 on histone H4 in mouse oocytes were gradually increased during in vivo and in vitro postovulatory aging. Furthermore, the acetylation levels on these sites were markedly decreased or increased when the process of postovulatory aging was artificially delayed or accelerated, respectively. These results indicated that the gradual acetylation on some lysines of histones H3 and H4 is one of the phenomena in the process of postovulatory aging. Moreover, raising the level of histone acetylation by trichostatin A can accelerate the progression of postovulatory aging, suggesting that alteration of the acetylation on histones H3 and H4 can affect the progression of postovulatory aging in mouse oocytes.

The objective was to investigate, using a mouse model, the effects of caffeine on the number of ovulated oocytes, the rate of oocyte maturation, the susceptibility of oocytes to activating stimuli, spindle morphology, and distribution of cortical granules (CGs). Mice were given caffeine (150 mg/kg body weight ip) at various times relative to hCG (-2, 0, and +2h); in an in vitro study, 1, 5 or 10 mM caffeine was added to the maturation culture. Caffeine had no effect on the quality of oocytes in vivo maturation, but caffeine was detrimental to the quality of oocytes matured in vitro. Further studies are needed to determine caffeine concentration in follicles relative to that in culture medium.

We recently reported that MEK1/2 plays an important role in microtubule organization and spindle pole tethering in mouse oocytes, but how the intracellular transport of this protein is regulated remains unknown. In the present study, we investigated the mechanisms of poleward MEK1/2 transport during the prometaphase I/metaphase I transition and MEK1/2 release from the spindle poles during the metaphase I/anaphase I transition in mouse oocytes. Firstly, we found that p-MEK1/2 was colocalized with dynactin at the spindle poles. Inhibition of the cytoplasmic dynein/dynactin complex by antibody microinjection blocked polar accumulation of p-MEK1/2 and caused obvious spindle abnormalities. Moreover, coimmunoprecipitation of p-MEK1/2 and dynein or dynactin from mouse oocyte extracts confirmed their association at metaphase I. Secondly, disruption of microtubules by nocodazole resulted in the failure of poleward p-MEK1/2 transport. Whereas, when the nocodazole-treated oocytes were recovered in fresh culture medium, the spindle reformed and p-MEK1/2 relocalized to the spindle poles. Finally, we examined the mechanism of p-MEK1/2 release from the spindle poles. In control oocytes, polar p-MEK1/2 was gradually released during metaphase I/anaphase I transition. By contrast, in the presence of nondegradable cyclin B (Delta90), p-MEK1/2 still remained at the spindle poles at anaphase I. Our results indicate that poleward MEK1/2 transport is a cytoplasmic dynein/dynactin-mediated and spindle microtubule-dependent intracellular movement, and that its subsequent anaphase release from spindle poles is dependent on cyclin B degradation.

Cyclin B3 is a relatively new member of the cyclin family whose functions are little known. We found that depletion of cyclin B3 inhibited metaphase-anaphase transition as indicated by a well-sustained MI spindle and cyclin B1 expression in meiotic oocytes after extended culture. This effect was independent of spindle assembly checkpoint activity, since both Bub3 and BubR1 signals were not observed at kinetochores in MI-arrested cells. The metaphase I arrest was not rescued by either Mad2 knockdown or cdc20 overexpression, but it was rescued by securin RNAi. We conclude that cyclin B3 controls the metaphase-anaphase transition by activating APC/Ccdc20 in meiotic oocytes, a process that does not rely on SAC activity.