Emi1 Class of Proteins Regulate Entry into Meiosis and the Meiosis I to Meiosis II Transition in Xenopus Oocytes

Xenopus oocytes are arrested at the G2/prophase boundary of meiosis I and enter meiosis in response to progesterone. A hallmark of meiosis is the absence of DNA replication between the successive cell division phases meiosis I (MI) and meiosis II (MII). After the MI-MII transition, Xenopus eggs are locked in metaphase II by the cytostatic factor (CSF) arrest to prevent parthenogenesis. Early Mitotic Inhibitor 1 (Emi1) maintains CSF arrest by inhibiting the ability of the Anaphase Promoting Complex (APC) to direct the destruction of cyclin B. To investigate whether Emi1 has an earlier role in meiosis, we injected Xenopus oocytes with neutralizing antibodies against Emi1 at G2/prophase and during the MI-MII transition. Progesterone-treated G2/prophase oocytes injected with anti-Emi1 antibody fail to activate Maturation Promoting Factor (MPF), a complex of cdc2/cyclin B, and the MAPK pathway, and do not undergo germinal vesicle breakdown (GVBD). Injection of purified ?90 cyclin B protein or blocking anti-Emi1 antibody with purified Emi1 protein rescues these meiotic processes in Emi1-neutralized oocytes. Acute inhibition of Emi1 in progesterone treated oocytes immediately after GVBD causes rapid loss of cdc2 activity with simultaneous loss of cyclin B levels and inactivation of the MAPK pathway. These oocytes decondense their chromosomes and enter a DNA replication phase instead of progressing to MII. Prior ablation of Cdc20, addition of methyl-ubiquitin, or addition of indestructible ?90 cyclin B rescues the MI-MII transition in Emi1 inhibited oocytes.     

Selective Disruption of Aurora C Kinase Reveals Distinct Functions from Aurora B Kinase during Meiosis in Mouse Oocytes

Aurora B kinase (AURKB) is the catalytic subunit of the chromosomal passenger complex (CPC), an essential regulator of chromosome segregation. In mitosis, the CPC is required to regulate kinetochore microtubule (K-MT) attachments, the spindle assembly checkpoint, and cytokinesis. Germ cells express an AURKB homolog, AURKC, which can also function in the CPC. Separation of AURKB and AURKC function during meiosis in oocytes by conventional approaches has not been successful. Therefore, the meiotic function of AURKC is still not fully understood. Here, we describe an ATP-binding-pocket-AURKC mutant, that when expressed in mouse oocytes specifically perturbs AURKC-CPC and not AURKB-CPC function. Using this mutant we show for the first time that AURKC has functions that do not overlap with AURKB. These functions include regulating localized CPC activity and regulating chromosome alignment and K-MT attachments at metaphase of meiosis I (Met I). We find that AURKC-CPC is not the sole CPC complex that regulates the spindle assembly checkpoint in meiosis, and as a result most AURKC-perturbed oocytes arrest at Met I. A small subset of oocytes do proceed through cytokinesis normally, suggesting that AURKC-CPC is not the sole CPC complex during telophase I. But, the resulting eggs are aneuploid, indicating that AURKC is a critical regulator of meiotic chromosome segregation in female gametes. Taken together, these data suggest that mammalian oocytes contain AURKC to efficiently execute meiosis I and ensure high-quality eggs necessary for sexual reproduction.     

Meiosis in Bovine Oocytes Matured in Vitro and in Vivo

Meiosis in bovine oocytes has; been studied after maturation in vitro or in vivo. Oocytes for in vitro maturation were collected from the ovaries of slaughtered cattle without regard to the phase of the estrous cycle while in vivo maturation was studied in oocytes from gonadotrophin-stimulated heifers at times varying between 6 and 36 h after the beginning of behavioural estrus. Oocytes from slaughtered cattle were classified according to their cumulus complex and ooplasm and were cultured for 6, 12, 18, 24, 36 or 48 h in modified Krebs-Ringer bicarbonate buffer before fixation) for cytogenetic analysis. Oocytes from stimulated heifers were aspirated from follicles or flushed from the oviducts, classified according to cumulus and ooplasm, and fixed within 6 h of collection. Nuclear maturation was more rapid in vitro than in vivo. The largest proportion of oocytes reached maturity (Mil) after 12 to 18 h in culture or 30 to 36 h after the onset of behavioural estrus. Oocytes devoid of cumulus cells or showing signs of vacuolation or degeneration had virtually no capacity for nuclear maturation.     

Cdc20 Is Critical for Meiosis I and Fertility of Female Mice

Chromosome missegregation in germ cells is an important cause of unexplained infertility, miscarriages, and congenital birth defects in humans. However, the molecular defects that lead to production of aneuploid gametes are largely unknown. Cdc20, the activating subunit of the anaphase-promoting complex/cyclosome (APC/C), initiates sister-chromatid separation by ordering the destruction of two key anaphase inhibitors, cyclin B1 and securin, at the transition from metaphase to anaphase. The physiological significance and full repertoire of functions of mammalian Cdc20 are unclear at present, mainly because of the essential nature of this protein in cell cycle progression. To bypass this problem we generated hypomorphic mice that express low amounts of Cdc20. These mice are healthy and have a normal lifespan, but females produce either no or very few offspring, despite normal folliculogenesis and fertilization rates. When mated with wild-type males, hypomorphic females yield nearly normal numbers of fertilized eggs, but as these embryos develop, they become malformed and rarely reach the blastocyst stage. In exploring the underlying mechanism, we uncover that the vast majority of these embryos have abnormal chromosome numbers, primarily due to chromosome lagging and chromosome misalignment during meiosis I in the oocyte. Furthermore, cyclin B1, cyclin A2, and securin are inefficiently degraded in metaphase I; and anaphase I onset is markedly delayed. These results demonstrate that the physiologically effective threshold level of Cdc20 is high for female meiosis I and identify Cdc20 hypomorphism as a mechanism for chromosome missegregation and formation of aneuploid gametes.     

Three Phases of Cortical Maturation during Meiosis Reinitiation in Starfish Oocytes

Oocytes of the starfish, Asterina pectinifera , respond differently to calcium ionophore A23187 depending upon their stage of maturation. Oocytes not-treated with 1-methyladenine (1-MA) formed only a partial fertilization envelope (FE) in response to A23187. Those treated with 1-MA formed no FE if the ionophore was introduced to them before germinal vesicle breakdown (GVBD), in contrast with which they did fully elevate the FE if it was introduced after GVBD. Similar stage-dependent results were obtained if the intracellular concentration of calcium was increased by microinjection of calcium-EGTA buffers. In good accordance with the FE formation, a stage-dependent protease release from oocytes by the ionophore was observed.
It is concluded from these results that, in starfish oocytes, their ability to undergo the exocytosis of cortical granules in response to an increase in intracellular calcium greatly changes along the way of maturation.     

Disruption of Sorting Nexin 5 Causes Respiratory Failure Associated with Undifferentiated Alveolar Epithelial Type I Cells in Mice

Sorting nexin 5 (Snx5) has been posited to regulate the degradation of epidermal growth factor receptor and the retrograde trafficking of cation-independent mannose 6-phosphate receptor/insulin-like growth factor II receptor. Snx5 has also been suggested to interact with Mind bomb-1, an E3 ubiquitin ligase that regulates the activation of Notch signaling. However, the in vivo functions of Snx5 are largely unknown. Here, we report that disruption of the Snx5 gene in mice (Snx5^-/- mice) resulted in partial perinatal lethality; 40% of Snx5^-/- mice died shortly after birth due to cyanosis, reduced air space in the lungs, and respiratory failure. Histological analysis revealed that Snx5^-/- mice exhibited thickened alveolar walls associated with undifferentiated alveolar epithelial type I cells. In contrast, alveolar epithelial type II cells were intact, exhibiting normal surfactant synthesis and secretion. Although the expression levels of surfactant proteins and saturated phosphatidylcholine in the lungs of Snx5^-/- mice were comparable to those of Snx5^+/+ mice, the expression levels of T1α, Aqp5, and Rage, markers for distal alveolar epithelial type I cells, were significantly decreased in Snx5 ^-/- mice. These results demonstrate that Snx5 is necessary for the differentiation of alveolar epithelial type I cells, which may underlie the adaptation to air breathing at birth.     

Mitogen-Activated Protein Kinase-Activated Protein Kinase 2 Deficiency Reduces Insulin Sensitivity in High-Fat Diet-Fed Mice

Adipose tissue inflammation is considered an important contributor to insulin resistance. Mitogen-activated protein kinase-activated protein kinase 2 (MK2) is a major downstream target of p38 MAPK and enhances inflammatory processes. In line with the role of MK2 as contributor to inflammation, MK2^−/− mice are protected against inflammation in different disease models. Therefore, MK2 is considered an attractive therapeutic target for the treatment of chronic inflammatory diseases. This study tested the impact of MK2-deficiency on high-fat diet (HFD)-induced adipose tissue inflammation and insulin resistance. After feeding MK2^−/− and WT control mice a HFD (60% energy from fat) for 24 weeks, body weight was not different between groups. Also, liver weight and the amount of abdominal fat remained unchanged. However, in MK2^−/− mice plasma cholesterol levels were significantly increased. Surprisingly, macrophage infiltration in adipose tissue was not altered. However, adipose tissue macrophages were more skewed to the inflammatory M1 phenotype in MK2^−/− mice. This differerence in macrophage polarization did however not translate in significantly altered expression levels of Mcp-1, Tnfα and Il6. Glucose and insulin tolerance tests demonstrated that MK2^−/− mice had a significantly reduced glucose tolerance and increased insulin resistance. Noteworthy, the expression of the insulin-responsive glucose transporter type 4 (GLUT4) in adipose tissue of MK2^−/− mice was reduced by 55% (p<0.05) and 33% (p<0.05) on the mRNA and protein level, respectively, compared to WT mice. In conclusion, HFD-fed MK2^−/− display decreased glucose tolerance and increased insulin resistance compared to WT controls. Decreased adipose tissue expression of GLUT4 might contribute to this phenotype. The data obtained in this study indicate that clinical use of MK2 inhibitors has to be evaluated with caution, taking potential metabolic adverse effects into account.     

Urea-Induced Meiosis Reinitiation in Oocytes of the Starfish, Marthasterias glacialis

In the absence of hormone stimulation, prophase-blocked oocytes of Marthasterias glacialis have been induced to undergo meiosis reinitiation up to female pronucleus formation by pulse incubation in isoosmotic urea-sea water solutions. Even when this procedure was not effective all along the breeding season, it could trigger full maturation when applied to so-called "incompetent oocytes" that did not complete maturation following microinjection-induced mixing of their nucleoplasm and cytoplasm.
³²P phosphate incorporation into proteins and cell fusion experiments demonstrate that this treatment produces an increased protein phosphorylation which appears tightly associated with the production of M-phase promoting factor (MPF). Instead, when oocytes are maintained in the inducing medium, dephosphorylation soon occurs and MPF is no longer present to support meiosis. Under these conditions, the GV-disrupted oocytes present a permanent nucleolus and do not form a meiotic spindle. The same cytological aspect was also obtained when the oocytes were treated in the presence of 90 μM emetine or 150 μM of the intracellular chelator Quin 2-AM.
These data suggest that urea-induced maturation may involve an intracellular Ca²⁺ shift which would be required to activate both MPF precursor molecules and the resting female centers which stand in the animal cortex outside the nucleus and give rise to the poles of the first maturation spindle. They also show that nuclear disruption alone, without protein phosphorylation, cannot trigger meiosis reinitiation of incompetent oocytes.     

Plasminogen Deficiency Causes Reduced Corticospinal Axonal Plasticity and Functional Recovery after Stroke in Mice

Tissue plasminogen activator (tPA) has been implicated in neurite outgrowth and neurological recovery post stroke. tPA converts the zymogen plasminogen (Plg) into plasmin. In this study, using plasminogen knockout (Plg^-/-) mice and their Plg-native littermates (Plg^+/+), we investigated the role of Plg in axonal remodeling and neurological recovery after stroke. Plg^+/+ and Plg^-/- mice (n = 10/group) were subjected to permanent intraluminal monofilament middle cerebral artery occlusion (MCAo). A foot-fault test and a single pellet reaching test were performed prior to and on day 3 after stroke, and weekly thereafter to monitor functional deficit and recovery. Biotinylated dextran amine (BDA) was injected into the left motor cortex to anterogradely label the corticospinal tract (CST). Animals were euthanized 4 weeks after stroke. Neurite outgrowth was also measured in primary cultured cortical neurons harvested from Plg^+/+ and Plg^-/- embryos. In Plg^+/+ mice, the motor functional deficiency after stroke progressively recovered with time. In contrast, recovery in Plg^-/- mice was significantly impaired compared to Plg^+/+ mice (p<0.01). BDA-positive axonal density of the CST originating from the contralesional cortex in the denervated side of the cervical gray matter was significantly reduced in Plg^-/- mice compared with Plg^+/+ mice (p<0.05). The behavioral outcome was highly correlated with the midline-crossing CST axonal density (R²>0.82, p<0.01). Plg^-/- neurons exhibited significantly reduced neurite outgrowth. Our data suggest that plasminogen-dependent proteolysis has a beneficial effect during neurological recovery after stroke, at least in part, by promoting axonal remodeling in the denervated spinal cord.     

Rictor/mTORC2 Pathway in Oocytes Regulates Folliculogenesis,and Its Inactivation Causes Premature Ovarian Failure

Molecular basis of ovarian folliculogenesis and etiopathogenesis of premature ovarian failure (POF), a common cause of infertility in women, are not fully understood. Mechanistic target of rapamycin complex 2 (mTORC2) is emerging as a central regulator of cell metabolism, proliferation, and survival. However, its role in folliculogenesis and POF has not been reported. Here, we showed that the signaling activity of mTORC2 is inhibited in a 4-vinylcyclohexene diepoxide (VCD)-induced POF mouse model. Notably, mice with oocyte-specific ablation of Rictor, a key component of mTORC2, demonstrated POF phenotypes, including massive follicular death, excessive loss of functional ovarian follicles, abnormal gonadal hormone secretion, and consequently, secondary subfertility in conditional knock-out (cKO) mice. Furthermore, reduced levels of Ser-473-phosphorylated Akt and Ser-253-phosphorylated Foxo3a and elevated pro-apoptotic proteins, Bad, Bax, and cleaved poly ADP-ribose polymerase (PARP), were observed in cKO mice, replicating the signaling alterations in 4-VCD-treated ovaries. These results indicate a critical role of the Rictor/mTORC2/Akt/Foxo3a pro-survival signaling axis in folliculogenesis. Interestingly, loss of maternal Rictor did not cause obvious developmental defects in embryos or placentas from cKO mice, suggesting that maternal Rictor is dispensable for preimplantation embryonic development. Our results collectively indicate key roles of Rictor/mTORC2 in folliculogenesis, follicle survival, and female fertility and support the utility of oocyte-specific Rictor knock-out mice as a novel model for POF.     

Regulation of Separase in Meiosis: Separase is Activated at the Metaphase I-II Transition in Xenopus Oocytes during Meiosis

Separase is a cysteine protease conserved in all eukaryotes and functions to remove the sisterchromatid cohesion in anaphase by cleaving the SCC1 subunit of the cohesin complex. Theregulation of separase activity as the degradation of its inhibitor, securin, and the downregulationof the inhibitory phosphorylation has never been directly investigated in themeiotic cell cycle of vertebrates. In this study, we cloned the full-length gene encodingXenopus separase from an oocyte cDNA library. Purified xSeparase can cleave the human ?-kleisin subunit of cohesin in vitro but cannot bind with hSecurin when these two proteins areco-expressed in 293T cells. Similar to its human counterpart, xSeparase cleaves itself uponactivation but at a single site. The cleavage site is conserved with one of the three selfcleavagesites in hSeparase. Using self-cleavage as a reporter for its activation, wedemonstrated that xSeparase was transiently activated between the two meioses and may beinvolved in the homologous chromosome separation, as observed in other organisms. Takingthe advantage of the inability of xSecurin to interact with hSeparase, we demonstrated that theCSF extract re-inhibit both full-length and auto-cleaved hSeparase, indicating thatphosphorylation inhibition of separase does occur under the physiological condition. Inaddition, we found that the endogenous xSecurin was accumulated in response toprogesterone-induced oocyte maturation, and was degraded at both the anaphase I and II in anAPC/C-dependent manner.     

Deletion of STK40 Protein in Mice Causes Respiratory Failure and Death at Birth

STK40 is a putative serine/threonine kinase and was shown to induce extraembryonic endoderm differentiation from mouse embryonic stem cells. However, little is known about its physiological function in vivo. Here, we generate Stk40 knock-out mice and demonstrate that loss of the Stk40 gene causes neonatal lethality at birth. Further examination reveals that the respiratory distress and atelectasis occur in the homozygous mutants. The maturation of lung and alveolar epithelium is delayed in the mutant, as indicated by narrowed air spaces, thickened interstitial septa, and increased glycogen content in the lungs of Stk40^−/− mice. The reduction in levels of T1-α, SP-B, and SP-C indicates delayed maturation of both type I and type II respiratory epithelial cells in Stk40^−/− lungs. Moreover, Stk40 is found to be most highly expressed in lungs of both fetal and adult mice among all organs tested. Mechanistically, a genome-wide RNA microarray analysis reveals significantly altered expression of multiple genes known to participate in lung development. The expression of some genes involved in lipid metabolism, immune response, and glycogen metabolism is also disrupted in the lung of Stk40^−/− mice. Protein affinity purification identifies RCN2, an activator of ERK/MAPK signaling, as an STK40-associated protein. Consistently, Stk40 deficiency attenuates the ERK/MAPK activation, and inhibition of ERK/MAPK activities reduces surfactant protein gene expression in lung epithelial cells. Collectively, this study uncovers an important role of STK40 for lung maturation and neonatal survival. STK40 may associate with RCN2 to activate ERK/MAPK signaling and control the expression of multiple key regulators of lung development.     

Roles of Rho-associated Kinase in Cytokinesis; Mutations in Rho-associated Kinase Phosphorylation Sites Impair Cytokinetic Segregation of Glial Filaments

Rho-associated kinase (Rho-kinase), which is activated by the small GTPase Rho, regulates formation of stress fibers and focal adhesions, myosin fiber organization, and neurite retraction through the phosphorylation of cytoskeletal proteins, including myosin light chain, the ERM family proteins (ezrin, radixin, and moesin) and adducin. Rho-kinase was found to phosphorylate a type III intermediate filament (IF) protein, glial fibrillary acidic protein (GFAP), exclusively at the cleavage furrow during cytokinesis. In the present study, we examined the roles of Rho-kinase in cytokinesis, in particular organization of glial filaments during cytokinesis. Expression of the dominant-negative form of Rho-kinase inhibited the cytokinesis of Xenopus embryo and mammalian cells, the result being production of multinuclei. We then constructed a series of mutant GFAPs, where Rho-kinase phosphorylation sites were variously mutated, and expressed them in type III IF-negative cells. The mutations induced impaired segregation of glial filament (GFAP filament) into postmitotic daughter cells. As a result, an unusually long bridge-like cytoplasmic structure formed between the unseparated daughter cells. Alteration of other sites, including the cdc2 kinase phosphorylation site, led to no remarkable defect in glial filament separation. These results suggest that Rho-kinase is essential not only for actomyosin regulation but also for segregation of glial filaments into daughter cells which in turn ensures correct cytokinetic processes.     

NOTCH1 Gain of Function in Germ Cells Causes Failure of Spermatogenesis in Male Mice

NOTCH1 is a member of the NOTCH receptor family, a group of single-pass trans-membrane receptors. NOTCH signaling is highly conserved in evolution and mediates communication between adjacent cells. NOTCH receptors have been implicated in cell fate determination, as well as maintenance and differentiation of stem cells. In the mammalian testis expression of NOTCH1 in somatic and germ cells has been demonstrated, however its role in spermatogenesis was not clear. To study the significance of NOTCH1 in germ cells, we applied a cre/loxP approach in mice to induce NOTCH1 gain- or loss-of function specifically in male germ cells. Using a Stra8-icre transgene we produced mice with conditional activation of the NOTCH1 intracellular domain (NICD) in germ cells. Spermatogenesis in these mutants was progressively affected with age, resulting in decreased testis weight and sperm count. Analysis of downstream target genes of NOTCH1 signaling showed an increased expression of Hes5, with a reduction of the spermatogonial differentiation marker, Neurog3 expression in the mutant testis. Apoptosis was significantly increased in mouse germ cells with the corresponding elevation of pro-apoptotic Trp53 and Trp63 genes'' expression. We also showed that the conditional germ cell-specific ablation of Notch1 had no effect on spermatogenesis or male fertility. Our data suggest the importance of NOTCH signaling regulation in male germ cells for their survival and differentiation.     

绵羊卵泡液和次黄嘌呤对卵母细胞体外减数分裂的影响

目的　利用在培养液中添加绵羊卵泡液和次黄嘌呤 ,抑制卵母细胞GVBD发生 ,延长转录活性 ,从而使卵母细胞真正成熟 ,提高胚胎质量及生产效率。方法　利用体外成熟技术对有屠宰采集的绵羊卵母细胞进行培养 ,培养液中添加卵泡液及次黄嘌呤 ,检查成熟效果。结果　将卵母细胞培养在 5 0 %和 10 0 %的卵泡液中 ,2 4h后处于GV期的卵母细胞分别为 19% (8 4 2 )和 33 3% (13 39)。在含有 4mmol L次黄嘌呤的培养液中 ,2 4h后有2 1 6 % (16 74 )的卵母细胞处GV期 ,而对照组中只有 6 % (3 5 0 ) ,经过次黄嘌呤处理的卵母细胞多数都停滞于PⅠ期(44 6 % ,33 74 )。在 4mmol L次黄嘌呤培养液中添加FSH并未使受到抑制的卵母细胞诱导成熟。结论　卵泡液和次黄嘌呤只能在有限的程度上抑制减数分裂的重新启动 ,并对减数分裂的全过程都有影响 ,这种影响程度与抑制因子的浓度相关 ,存在明显的剂量效应。