RESUMO
Fully grown oocytes remain transcriptionally quiescent, yet many maternal mRNAs are synthesized and retained in growing oocytes. We now know that maternal mRNAs are stored in a structure called the mitochondria-associated ribonucleoprotein domain (MARDO). However, the components and functions of MARDO remain elusive. Here, we found that LSM14B knockout prevents the proper storage and timely clearance of mRNAs (including Cyclin B1, Btg4 and other mRNAs that are translationally activated during meiotic maturation), specifically by disrupting MARDO assembly during oocyte growth and meiotic maturation. With decreased levels of storage and clearance, the LSM14B knockout oocytes failed to enter meiosis II, ultimately resulting in female infertility. Our results demonstrate the function of LSM14B in MARDO assembly, and couple the MARDO with mRNA clearance and oocyte meiotic maturation.
Assuntos
Oogênese , RNA Mensageiro Estocado , Feminino , Humanos , Meiose/genética , Oócitos/fisiologia , Oogênese/genética , RNA Mensageiro/genética , RNA Mensageiro Estocado/genética , Camundongos Endogâmicos C57BL , Masculino , Animais , CamundongosRESUMO
Maternal-to-zygotic transition (MZT) is the first and key step in the control of animal development and intimately related to changes in chromatin structure and histone modifications. H2AK119ub1, an important epigenetic modification in regulating chromatin configuration and function, is primarily catalyzed by PRC1 and contributes to resistance to transcriptional reprogramming in mouse embryos. In this study, the genome-wide dynamic distribution of H2AK119ub1 during MZT in mice was investigated using chromosome immunoprecipitation and sequencing. The results indicated that H2AK119ub1 accumulated in fully grown oocytes and was enriched at the TSSs of maternal genes, but was promptly declined after meiotic resumption at genome-wide including the TSSs of early zygotic genes, by a previously unidentified mechanism. Genetic evidences indicated that ubiquitin-specific peptidase 16 (USP16) is the major deubiquitinase for H2AK119ub1 in mouse oocytes. Conditional knockout of Usp16 in oocytes did not impair their survival, growth, or meiotic maturation. However, oocytes lacking USP16 have defects when undergoing zygotic genome activation or gaining developmental competence after fertilization, potentially associated with high levels of maternal H2AK119ub1 deposition on the zygotic genomes. Taken together, H2AK119ub1 level is declined during oocyte maturation by an USP16-dependent mechanism, which ensures zygotic genome reprogramming and transcriptional activation of essential early zygotic genes.
Assuntos
Histonas , Lisina , Animais , Cromatina/genética , Regulação da Expressão Gênica no Desenvolvimento , Histonas/genética , Histonas/metabolismo , Lisina/metabolismo , Camundongos , Oócitos/metabolismo , Oogênese/genética , Ubiquitina Tiolesterase/genética , Ubiquitina Tiolesterase/metabolismo , ZigotoRESUMO
Post-transcriptional RNA modifications critically regulate various biological processes. N4-acetylcytidine (ac4C) is an epi-transcriptome, which is highly conserved in all species. However, the in vivo physiological functions and regulatory mechanisms of ac4C remain poorly understood, particularly in mammals. In this study, we demonstrate that the only known ac4C writer, N-acetyltransferase 10 (NAT10), plays an essential role in male reproduction. We identified the occurrence of ac4C in the mRNAs of mouse tissues and showed that ac4C undergoes dynamic changes during spermatogenesis. Germ cell-specific ablation of Nat10 severely inhibits meiotic entry and leads to defects in homologous chromosome synapsis, meiotic recombination and repair of DNA double-strand breaks during meiosis. Transcriptomic profiling revealed dysregulation of functional genes in meiotic prophase I after Nat10 deletion. These findings highlight the crucial physiological functions of ac4C modifications in male spermatogenesis and expand our understanding of its role in the regulation of specific physiological processes in vivo.
Assuntos
Citidina , Meiose , Masculino , Camundongos , Animais , Meiose/genética , Citidina/genética , Pareamento Cromossômico , Células Germinativas , MamíferosRESUMO
Zygotic cleavage failure (ZCF) is a unique early embryonic phenotype resulting in female infertility and recurrent failure of in vitro fertilization (IVF) and/or intracytoplasmic sperm injection (ICSI). With this phenotype, morphologically normal oocytes can be retrieved and successfully fertilized, but they fail to undergo cleavage. Until now, whether this phenotype has a Mendelian inheritance pattern and which underlying genetic factors play a role in its development remained to be elucidated. B cell translocation gene 4 (BTG4) is a key adaptor of the CCR4-NOT deadenylase complex, which is involved in maternal mRNA decay in mice, but no human diseases caused by mutations in BTG4 have previously been reported. Here, we identified four homozygous mutations in BTG4 (GenBank: NM_017589.4) that are responsible for the phenotype of ZCF, and we found they followed a recessive inheritance pattern. Three of them-c.73C>T (p.Gln25Ter), c.1A>G (p.?), and c.475_478del (p.Ile159LeufsTer15)-resulted in complete loss of full-length BTG4 protein. For c.166G>A (p.Ala56Thr), although the protein level and distribution of mutant BTG4 was not altered in zygotes from affected individuals or in HeLa cells, the interaction between BTG4 and CNOT7 was abolished. In vivo studies further demonstrated that the process of maternal mRNA decay was disrupted in the zygotes of the affected individuals, which provides a mechanistic explanation for the phenotype of ZCF. Thus, we provide evidence that ZCF is a Mendelian phenotype resulting from mutations in BTG4. These findings contribute to our understanding of the role of BTG4 in human early embryonic development and provide a genetic marker for female infertility.
Assuntos
Proteínas de Ciclo Celular/genética , Infertilidade Feminina/genética , Mutação/genética , Zigoto/patologia , Animais , Linhagem Celular Tumoral , Desenvolvimento Embrionário/genética , Exorribonucleases/genética , Feminino , Células HeLa , Homozigoto , Humanos , Infertilidade Feminina/patologia , Camundongos , Fenótipo , Estabilidade de RNA/genéticaRESUMO
The most significant feature of meiosis is the recombination process during prophase I. CXXC finger protein 1 (CXXC1) binds to CpG islands and mediates the deposition of H3K4me3 by the SETD1 complex. CXXC1 is also predicted to recruit H3K4me3-marked regions to the chromosome axis for the generation of double-strand breaks (DSBs) in the prophase of meiosis. Therefore, we deleted Cxxc1 before the onset of meiosis with Stra8-Cre The conditional knockout mice were completely sterile with spermatogenesis arrested at MII. Knockout of Cxxc1 led to a decrease in the H3K4me3 level from the pachytene to the MII stage and caused transcriptional disorder. Many spermatogenesis pathway genes were expressed early leading to abnormal acrosome formation in arrested MII cells. In meiotic prophase, deletion of Cxxc1 caused delayed DSB repair and improper crossover formation in cells at the pachytene stage, and more than half of the diplotene cells exhibited precocious homologous chromosome segregation in both male and female meiosis. Cxxc1 deletion also led to a significant decrease of H3K4me3 enrichment at DMC1-binding sites, which might compromise DSB generation. Taken together, our results show that CXXC1 is essential for proper meiotic crossover formation in mice and suggest that CXXC1-mediated H3K4me3 plays an essential role in meiotic prophase of spermatogenesis and oogenesis.
Assuntos
Troca Genética/fisiologia , Histona-Lisina N-Metiltransferase/metabolismo , Histonas/metabolismo , Meiose/fisiologia , Transativadores/fisiologia , Animais , Células Cultivadas , Embrião de Mamíferos , Feminino , Masculino , Meiose/genética , Metilação , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Oogênese/genética , Processamento de Proteína Pós-Traducional/genética , Espermatogênese/genética , Transativadores/genéticaRESUMO
Strategies to maximize individual fertility chances are constant requirements of ART. In vitro folliculogenesis may represent a valid option to create a large source of immature ovarian follicles in ART. Efforts are being made to set up mammalian follicle culture protocols with suitable FSH stimuli. In this study, a new type of recombinant FSH (KN015) with a prolonged half-life is proposed as an alternative to canonical FSH. KN015 supports the in vitro development of mouse follicles from primary to preovulatory stage with higher efficiency than canonical FSH and enhanced post-fertilization development rates of the ovulated oocytes. The use of KN015 also allows us to compare the dynamic transcriptome changes in oocytes and granulosa cells at different stages, in vivo and in vitro. In particular, KN015 facilitates mRNA accumulation in growing mouse oocytes and prevents spontaneous luteinization of granulosa cells in vitro. Novel analyses of transcriptome changes in this study reveal that the in vivo oocytes were more efficient than in vitro oocytes in terms of maternal mRNA clearing during meiotic maturation. KN015 promotes the degradation of maternal mRNA during in vitro oocyte maturation, improves cytoplasmic maturation and, therefore, enhances embryonic developmental potential. These findings establish new transcriptome data for oocyte and granulosa cells at the key stages of follicle development, and should help to widen the use of KN015 as a valid and commercially available hormonal support enabling optimized in vitro development of follicles and oocytes.
Assuntos
RNA Mensageiro Estocado , Transcriptoma , Feminino , Camundongos , Animais , RNA Mensageiro Estocado/metabolismo , Oogênese/genética , Oócitos/metabolismo , Células da Granulosa , Hormônio Foliculoestimulante/genética , Hormônio Foliculoestimulante/farmacologia , Hormônio Foliculoestimulante/metabolismo , Meiose , MamíferosRESUMO
With the rapid change of people's lifestyle, more childbearing couples live with irregular schedules (i.e., staying up late) and suffer from decreased fertility and abortion, which can be caused by luteal phase defect (LPD). We used continuous light-exposed mice as a model to observe whether continuous light exposure may affect luteinization and luteal function. We showed that the level of progesterone in serum reduced (p < .001), the number of corpus luteum (CL) decreased (p < .01), and the expressions of luteinization-related genes (Lhcgr, Star, Ptgfr, and Runx2), clock genes (Clock and Per1), and Mt1 were downregulated (p < .05) in the ovaries of mice exposed to continuous light, suggesting that continuous light exposure induces defects in luteinization and luteal functions. Strikingly, injection of melatonin (3 mg/kg) could improve luteal functions in continuous light-exposed mice. Moreover, we found that, after 2 h of hCG injection, the level of pERK1/2 in the ovary decreased in the continuous light group, but increased in the melatonin administration group, suggesting that melatonin can improve LPD caused by continuous light exposure through activating the ERK1/2 pathway. In summary, our data demonstrate that continuous light exposure affects ovary luteinization and luteal function, which can be rescued by melatonin.
Assuntos
Melatonina , Ovário , Feminino , Gravidez , Camundongos , Animais , Ovário/metabolismo , Camundongos Endogâmicos ICR , Melatonina/farmacologia , Melatonina/metabolismo , Corpo Lúteo/metabolismo , Progesterona/metabolismo , LuteinizaçãoRESUMO
BACKGROUND: Recurrent preimplantation embryo developmental arrest (RPEA) is the most common cause of assisted reproductive technology treatment failure associated with identified genetic abnormalities. Variants in known maternal genes can only account for 20%-30% of these cases. The underlying genetic causes for the other affected individuals remain unknown. METHODS: Whole exome sequencing was performed for 100 independent infertile females that experienced RPEA. Functional characterisations of the identified candidate disease-causative variants were validated by Sanger sequencing, bioinformatics and in vitro functional analyses, and single-cell RNA sequencing of zygotes. RESULTS: Biallelic variants in ZFP36L2 were associated with RPEA and the recurrent variant (p.Ser308_Ser310del) prevented maternal mRNA decay in zygotes and HeLa cells. CONCLUSION: These findings emphasise the relevance of the relationship between maternal mRNA decay and human preimplantation embryo development and highlight a novel gene potentially responsible for RPEA, which may facilitate genetic diagnoses.
Assuntos
Infertilidade Feminina , Blastocisto , Feminino , Células HeLa , Humanos , Infertilidade Feminina/diagnóstico , Infertilidade Feminina/genética , Gravidez , RNA Mensageiro Estocado , Fatores de Transcrição/genética , Sequenciamento do ExomaRESUMO
During oogenesis, oocytes gain competence and subsequently undergo meiotic maturation and prepare for embryonic development; trimethylated histone H3 on lysine-4 (H3K4me3) mediates a wide range of nuclear events during these processes. Oocyte-specific knockout of CxxC-finger protein 1 (CXXC1, also known as CFP1) impairs H3K4me3 accumulation and causes changes in chromatin configurations. This study investigated the changes in genomic H3K4me3 landscapes in oocytes with Cxxc1 knockout and the effects on other epigenetic factors such as the DNA methylation, H3K27me3, H2AK119ub1 and H3K36me3. H3K4me3 is overall decreased after knocking out Cxxc1, including both the promoter region and the gene body. CXXC1 and MLL2, which is another histone H3 methyltransferase, have nonoverlapping roles in mediating H3K4 trimethylation during oogenesis. Cxxc1 deletion caused a decrease in DNA methylation levels and affected H3K27me3 and H2AK119ub1 distributions, particularly at regions with high DNA methylation levels. The changes in epigenetic networks implicated by Cxxc1 deletion were correlated with the transcriptional changes in genes in the corresponding genomic regions. This study elucidates the epigenetic changes underlying the phenotypes and molecular defects in oocytes with deleted Cxxc1 and highlights the role of CXXC1 in orchestrating multiple factors that are involved in establishing the appropriate epigenetic states of maternal genome.
Assuntos
Epigênese Genética , Oócitos/metabolismo , Transativadores/fisiologia , Animais , Células Cultivadas , Metilação de DNA , Feminino , Deleção de Genes , Genoma , Código das Histonas , Histonas/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Regiões Promotoras Genéticas , Transativadores/genética , Transcrição GênicaRESUMO
Meiotic resumption-coupled degradation of maternal transcripts occurs during oocyte maturation in the absence of mRNA transcription. The CCR4-NOT complex has been identified as the main eukaryotic mRNA deadenylase. In vivo functional and mechanistic information regarding its multiple subunits remains insufficient. Cnot6l, one of four genes encoding CCR4-NOT catalytic subunits, is preferentially expressed in mouse oocytes. Genetic deletion of Cnot6l impaired deadenylation and degradation of a subset of maternal mRNAs during oocyte maturation. Overtranslation of these undegraded mRNAs caused microtubule-chromosome organization defects, which led to activation of spindle assembly checkpoint and meiotic cell cycle arrest at prometaphase. Consequently, Cnot6l-/- female mice were severely subfertile. The function of CNOT6L in maturing oocytes is mediated by RNA-binding protein ZFP36L2, not maternal-to-zygotic transition licensing factor BTG4, which interacts with catalytic subunits CNOT7 and CNOT8 of CCR4-NOT Thus, recruitment of different adaptors by different catalytic subunits ensures stage-specific degradation of maternal mRNAs by CCR4-NOT This study provides the first direct genetic evidence that CCR4-NOT-dependent and particularly CNOT6L-dependent decay of selective maternal mRNAs is a prerequisite for meiotic maturation of oocytes.
Assuntos
Meiose , Oócitos/metabolismo , Estabilidade de RNA , RNA Mensageiro/metabolismo , Ribonucleases/metabolismo , Animais , Exorribonucleases , Feminino , Deleção de Genes , Camundongos , Camundongos Knockout , Oócitos/citologia , Proteínas/genética , Proteínas/metabolismo , RNA Mensageiro/genética , Proteínas Repressoras , Ribonucleases/genética , Tristetraprolina/genética , Tristetraprolina/metabolismoRESUMO
BACKGROUND: Maternal obesity is a global issue that has devastating effects across the reproductive spectrum such as meiotic defects in oocytes, consequently worsening pregnancy outcomes. Different studies have shown that such types of meiotic defects originated from the oocytes of obese mothers. Thus, there is an urgent need to develop strategies to reduce the incidence of obesity-related oocyte defects that adversely affect pregnancy outcomes. Multiple growth factors have been identified as directly associated with female reproduction; however, the impact of various growth factors on female fertility in response to obesity remains poorly understood. METHODS: The immature GV-stage oocytes from HFD female mice were collected and cultured in vitro in two different groups (HFD oocytes with and without 50 nM IGF2), however; the oocytes from ND mice were used as a positive control. HFD oocytes treated with or without IGF2 were further used to observe the meiotic structure using different analysis including, the spindle and chromosomal analysis, reactive oxygen species levels, mitochondrial functional activities, and early apoptotic index using immunofluorescence. Additionally, the embryonic developmental competency and embryos quality of IGF2-treated zygotes were also determined. RESULTS: In our findings, we observed significantly reduced contents of insulin-like growth factor 2 (IGF2) in the serum and oocytes of obese mice. Our data indicated supplementation of IGF2 in a culture medium improves the blastocyst formation: from 46% in the HFD group to 61% in the HFD + IGF2-treatment group (50 nM IGF2). Moreover, adding IGF2 to the culture medium reduces the reactive oxygen species index and alleviates the frequency of spindle/chromosome defects. We found increased mitochondrial functional activity in oocytes from obese mice after treating the oocytes with IGF2: observed elevated level of adenosine triphosphate, increased mitochondrial distribution, higher mitochondrial membrane potentials, and reduced mitochondrial ultrastructure defects. Furthermore, IGF2 administration also increases the overall protein synthesis and decreases the apoptotic index in oocytes from obese mice. CONCLUSIONS: Collectively, our findings are strongly in favor of adding IGF2 in culture medium to overcome obesity-related meiotic structural-developmental defects by helping ameliorate the known sub-optimal culturing conditions that are currently standard with assisted reproduction technologies.
Assuntos
Desenvolvimento Embrionário , Oócitos , Animais , Feminino , Humanos , Fator de Crescimento Insulin-Like II/genética , Camundongos , Camundongos Obesos , Obesidade/complicações , Oócitos/metabolismo , Gravidez , Espécies Reativas de Oxigênio/metabolismoRESUMO
An important event of the maternal-to-zygotic transition (MZT) in animal embryos is the elimination of a subset of the maternal transcripts that accumulated during oogenesis. In both invertebrates and vertebrates, a maternally encoded mRNA decay pathway (M-decay) acts before zygotic genome activation (ZGA) while a second pathway, which requires zygotic transcription, subsequently clears additional mRNAs (Z-decay). To date the mechanisms that activate the Z-decay pathway in mammalian early embryos have not been investigated. Here, we identify murine maternal transcripts that are degraded after ZGA and show that inhibition of de novo transcription stabilizes these mRNAs in mouse embryos. We show that YAP1-TEAD4 transcription factor-mediated transcription is essential for Z-decay in mouse embryos and that TEAD4-triggered zygotic expression of terminal uridylyltransferases TUT4 and TUT7 and mRNA 3'-oligouridylation direct Z-decay. Components of the M-decay pathway, including BTG4 and the CCR4-NOT deadenylase, continue to function in Z-decay but require reinforcement from the zygotic factors for timely removal of maternal mRNAs. A long 3'-UTR and active translation confer resistance of Z-decay transcripts to M-decay during oocyte meiotic maturation. The Z-decay pathway is required for mouse embryo development beyond the four-cell stage and contributes to the developmental competence of preimplantation embryos.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas de Ciclo Celular/genética , Proteínas de Ligação a DNA/genética , Genoma/genética , Proteínas Musculares/genética , RNA Mensageiro/genética , Fatores de Transcrição/genética , Zigoto/crescimento & desenvolvimento , Animais , Embrião de Mamíferos , Desenvolvimento Embrionário/genética , Feminino , Regulação da Expressão Gênica no Desenvolvimento/genética , Proteínas de Homeodomínio/genética , Camundongos , Nucleotidiltransferases/genética , Oócitos/crescimento & desenvolvimento , Estabilidade de RNA/genética , Receptores CCR4/genética , Fatores de Transcrição de Domínio TEA , Proteínas de Sinalização YAPRESUMO
CxxC-finger protein 1 (CFP1)-mediated trimethylated histone H3 at lysine-4 (H3K4me3) during oocyte development enables the oocyte genome to establish the competence to generate a new organism. Nevertheless, it remains unclear to which extent this epigenetic modification forms an instructive component of ovarian follicle development. We investigated the ovarian functions using an oocyte-specific Cxxc1 knockout mouse model, in which the H3K4me3 accumulation is downregulated in oocytes of developing follicles. CFP1-dependent H3K4 trimethylation in oocytes was necessary to maintain the expression of key paracrine factors and to facilitate the communication between an oocyte and the surrounding granulosa cells. The distinct gene expression patterns in cumulus cells within preovulatory follicles were disrupted by the Cxxc1 deletion in oocytes. Both follicle growth and ovulation were compromised after CFP1 deletion, because Cxxc1 deletion in oocytes indirectly impaired essential signaling pathways in granulosa cells that mediate the functions of follicle-stimulating hormone and luteinizing hormone. Therefore, CFP1-regulated epigenetic modification of the oocyte genome influences the responses of ovarian follicles to gonadotropin in a cell-nonautonomous manner.
Assuntos
Histonas/metabolismo , Oócitos/metabolismo , Folículo Ovariano/metabolismo , Transativadores/metabolismo , Animais , Células do Cúmulo/metabolismo , Feminino , Hormônio Foliculoestimulante/metabolismo , Células da Granulosa/citologia , Células da Granulosa/metabolismo , Hormônio Luteinizante/metabolismo , Metilação , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Folículo Ovariano/crescimento & desenvolvimento , Ovulação , Comunicação Parácrina , Fosfatidilinositol 3-Quinases/química , Fosfatidilinositol 3-Quinases/metabolismo , Transdução de Sinais , Transativadores/deficiência , Transativadores/genéticaRESUMO
Meiotic maturation of mammalian oocytes depends on the temporally and spatially regulated cytoplasmic polyadenylation and translational activation of maternal mRNAs. Cytoplasmic polyadenylation is controlled by cis-elements in the 3'-UTRs of mRNAs including the polyadenylation signal (PAS), which is bound by the cleavage and polyadenylation specificity factor (CPSF) and the cytoplasmic polyadenylation element (CPE), which recruits CPE binding proteins. Using the 3'-UTRs of mouse Cpeb1, Btg4 and Cnot6l mRNAs, we deciphered the combinatorial code that controls developmental stage-specific translation during meiotic maturation: (i) translation of a maternal transcript at the germinal vesicle (GV) stage requires one or more PASs that locate far away from CPEs; (ii) PASs distal and proximal to the 3'-end of the transcripts are equally effective in mediating translation at the GV stage, as long as they are not close to the CPEs; (iii) Both translational repression at the GV stage and activation after germinal vesicle breakdown require at least one CPE adjacent to the PAS; (iv) The numbers and positions of CPEs in relation to PASs within the 3'-UTR of a given transcript determines its repression efficiency in GV oocytes. This study reveals a previously unrecognized non-canonical mechanism by which the proximal PASs mediate 3'-terminal polyadenylation and translation of maternal transcripts.
Assuntos
Proteínas de Ciclo Celular/genética , Oócitos/crescimento & desenvolvimento , Biossíntese de Proteínas , Ribonucleases/genética , Fatores de Transcrição/genética , Fatores de Poliadenilação e Clivagem de mRNA/genética , Regiões 3' não Traduzidas/genética , Animais , Citoplasma/genética , Feminino , Células Germinativas/crescimento & desenvolvimento , Meiose/genética , Camundongos , Oócitos/metabolismo , Oogênese/genética , Poliadenilação/genética , RNA Mensageiro/genéticaRESUMO
Mammalian oocyte maturation depends on the translational activation of stored maternal mRNAs upon meiotic resumption. Cytoplasmic polyadenylation element binding protein 1 (CPEB1) is a key oocyte factor that regulates maternal mRNA translation. However, the signal that triggers CPEB1 activation at the onset of mammalian oocyte maturation is not known. We provide evidence that a mitogen-activated protein kinase (MAPK) cascade couples maternal mRNA translation to meiotic cell cycle progression in mouse oocytes by triggering CPEB1 phosphorylation and degradation. Mutations of the phosphorylation sites or ubiquitin E3 ligase binding sites in CPEB1 have a dominant-negative effect in oocytes, and mimic the phenotype of ERK1/2 knockout, by impairing spindle assembly and mRNA translation. Overexpression of the CPEB1 downstream translation activator DAZL in ERK1/2-deficient oocytes partially rescued the meiotic defects, indicating that ERK1/2 is essential for spindle assembly, metaphase II arrest and maternal-zygotic transition (MZT) primarily by triggering the translation of key maternal mRNAs. Taken together, ERK1/2-mediated CPEB1 phosphorylation/degradation is a major mechanism of maternal mRNA translational activation, and is crucial for mouse oocyte maturation and MZT.
Assuntos
Sistema de Sinalização das MAP Quinases , Oócitos/citologia , Oócitos/metabolismo , RNA Mensageiro Estocado/genética , RNA Mensageiro Estocado/metabolismo , Animais , Ciclo Celular , Proteínas de Ciclo Celular/genética , Feminino , Sistema de Sinalização das MAP Quinases/genética , Sistema de Sinalização das MAP Quinases/fisiologia , Meiose , Camundongos , Camundongos Knockout , Modelos Biológicos , Oogênese , Fosforilação , Estabilidade de RNA , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Suínos , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Ativação Transcricional , Zigoto/citologia , Zigoto/metabolismo , Fatores de Poliadenilação e Clivagem de mRNA/genética , Fatores de Poliadenilação e Clivagem de mRNA/metabolismoRESUMO
In mammals, maternal-to-zygotic transition (MZT), or oocyte-to-embryo transition, begins with oocyte meiotic resumption due to the sequential translational activation and destabilization of dormant maternal transcripts stored in the ooplasm. It then continues with the elimination of maternal transcripts during oocyte maturation and fertilization and ends with the full transcriptional activation of the zygotic genome during embryonic development. A hallmark of MZT in mammals is its reliance on translation and the utilization of stored RNAs and proteins, rather than de novo transcription of genes, to sustain meiotic maturation and early development. Impaired maternal mRNA clearance at the onset of MZT prevents zygotic genome activation and causes early arrest of developing embryos. In this review, we discuss recent advances in our knowledge of the mechanisms whereby mRNA translation and degradation are controlled by cytoplasmic polyadenylation and deadenylation which set up the competence of maturing oocyte to accomplish MZT. The emphasis of this review is on the mouse as a model organism for mammals and BTG4 as a licensing factor of MZT under the translational control of the MAPK cascade.
Assuntos
Desenvolvimento Embrionário/genética , Mamíferos/embriologia , Mamíferos/genética , Biossíntese de Proteínas , Estabilidade de RNA/fisiologia , RNA Mensageiro Estocado/metabolismo , Zigoto/metabolismo , Animais , Morte , Embrião de Mamíferos , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Parto/fisiologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Mensageiro Estocado/genéticaRESUMO
In mammals, oocytes within the primordial follicles require a number of essential factors to maintain their survival. However, the survival factors for activated oocytes have been poorly characterized. Recently we reported that damaged DNA binding protein-1 (DDB1), the linker subunit of the cullin ring-finger ubiquitin E3 ligase-4 (CRL4) complex, and its substrate adaptor, DDB1-CUL4 associated factor-1 (DCAF1), were essential for primordial follicle maintenance. In this study we specifically deleted these in the oocytes of growing follicles, to investigate if DDB1 and DCAF1 were also survival factors for activated oocytes. In the ovaries of Ddb1(fl/fl);Zp3-Cre mice, the primordial follicle pool was intact, but awakened oocytes and growing follicles beyond the primary stage were rapidly depleted. In the ovaries of Dcaf1(fl/fl);Pten(fl/fl);Gdf9-Cre and Ddb1(fl/fl);Pten(fl/fl);Gdf9-Cre mice, global primordial follicle activation was stimulated by enhanced PI3K signaling, but the awakened oocytes were rapidly lost due to no CRL4(DCAF1) activity. These mouse models provided original evidence that CRL4(DCAF1) was essential for maintaining oocyte survival, not only those in dormancy at the primordial follicle stage, but also naturally awakened oocytes and those awakened by hyper-activation of PI3K signaling. Interestingly, the oocyte-specific Ddb1 or Dcaf1 knockout mice had ovulation defects even before oocyte exhaustion. CRL4(DCAF1) within oocytes was required for cumulus expansion and ovulation-related somatic gene expression in a cell non-autonomous manner. Granulosa cells that surrounded these Ddb1 or Dcaf1-deleted oocytes exhibited increased rates of apoptosis and showed poor responses to ovulation signals. These results suggested that CRL4 in oocytes also regulated granulosa cell functions in a cell non-autonomous manner.
Assuntos
Proteínas de Transporte/metabolismo , Folículo Ovariano/metabolismo , Ovulação/fisiologia , Animais , Proteínas de Transporte/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Feminino , Células da Granulosa/metabolismo , Fator 9 de Diferenciação de Crescimento/genética , Fator 9 de Diferenciação de Crescimento/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Oócitos/metabolismo , Folículo Ovariano/fisiologia , Ovulação/genética , PTEN Fosfo-Hidrolase/genética , PTEN Fosfo-Hidrolase/metabolismo , Transdução de SinaisRESUMO
Steel slag and waste clay bricks are two prevalent solid waste materials generated during industrial production. The complex chemical compositions of these materials present challenges to their utilization in conventional alumina silicate ceramics manufacturing. A new type of ceramic tile, which utilizes steel slag and waste clay brick as raw materials, has been successfully developed in order to effectively utilize these solid wastes. The optimal composition of the ceramic material was determined through orthogonal experimentation, during which the effects of the sample molding pressure, the soaking time, and the sintering temperature on the ceramic properties were studied. The results show that the optimal ceramic tile formula was 45% steel slag, 35% waste clay bricks, and 25% talc. The optimal process parameters for this composition included a molding pressure of 25 MPa, a sintering temperature of 1190 °C, and a soaking time of 60 min. The prepared ceramic tile samples had compositions in which solid waste accounted for more than 76% of the total material. Additionally, they possessed a modulus of rupture of more than 73.2 MPa and a corresponding water absorption rate of less than 0.05%.
RESUMO
OBJECTIVE: Serum metabolites from 19 myasthenia gravis (MG) patients and 15 normal controls were analyzed via untargeted metabolomics, including 6 pre/post-treatment paired MG patients, to assess the value of serum metabolites as biomarkers in monitoring MG. METHOD: Differential metabolites between MG patients and normal controls were identified through liquid and gas chromatography-mass spectrometry simultaneously. Principal component analysis and orthogonal partial least squares-discriminant analysis were conducted to identify the differential metabolites. Candidate metabolites and pathways associated with MG were selected through a random forest machine learning model. RESULT: A total of 310 differential metabolites were identified with a threshold of variable projected importance > 1 and P value < 0.05. Among these, 158 metabolites were upregulated and 152 were downregulated. The random forest machine learning model selected 5 metabolites as potential biomarkers associated with MG: lignoceric acid (AUC=0.944), uridine diphosphate-N-acetylglucosamine (AUC=0.951), arachidonic acid (AUC=0.951), beta-glycerophosphoric acid (AUC=0.933), and L-Asparagine (AUC=0.877). Further analysis using 6 paired MG patients pre- and post-immunosuppression treatment revealed 25 upregulated and 6 downregulated metabolites in post-treatment serum, which might be relevant to disease intervention. The significance remains elusive due to the limited number of patients. CONCLUSION: A subset of differential metabolites was identified in the serum of MG patients, some of which changed with immunosuppressive therapy. Small molecule metabolites may serve as valuable biomarkers for disease monitoring in MG.
Assuntos
Biomarcadores , Metabolômica , Miastenia Gravis , Humanos , Miastenia Gravis/sangue , Miastenia Gravis/diagnóstico , Biomarcadores/sangue , Masculino , Feminino , Pessoa de Meia-Idade , Adulto , Idoso , Estudos de Casos e Controles , Adulto JovemRESUMO
To improve the heat extraction efficiency from the wellbore fluids to the stratum in the geothermal well, thermal insulation cement, which contains of waste glass powder as a heat-insulating material, is proposed to apply in geothermal well's middle and upper sections in the paper. Effect of such glass powers on mechanic and thermal property of thermal insulation cement was then investigated. Various tests were carried out to measure compressive strength, thermal conductivity, microstructure porosity etc. parameters of the thermal insulation cement. Results showed that the waste glass powder would enhance its the compressive strength and improve its the thermal insulation performance. Correlation study between contents of the added waste glass powder in geothermal cements and its mechanic and thermal property was conducted. It was found that thermal insulation cement exhibited its optimum performance when the added content of glass powers was 20% in weight. Analysis of the microstructure porosity with SEM found that the pores in thermal insulation cement with added waste glass powders were mostly closed, tiny and even, and therefore contributed to the compressive strength of the thermal insulation cement; such pores would be also beneficial to improving its thermal insulation performance.