RESUMO
The timely degradation of proteins that regulate the cell cycle is essential for oocyte maturation. Oocytes are equipped to degrade proteins via the ubiquitin-proteasome system. In meiosis, anaphase promoting complex/cyclosome (APC/C), an E3 ubiquitin-ligase, is responsible for the degradation of proteins. Ubiquitin-conjugating enzyme E2 S (UBE2S), an E2 ubiquitin-conjugating enzyme, delivers ubiquitin to APC/C. APC/C has been extensively studied, but the functions of UBE2S in oocyte maturation and mouse fertility are not clear. In this study, we used Ube2s knockout mice to explore the role of UBE2S in mouse oocytes. Ube2s-deleted oocytes were characterized by meiosis I arrest with normal spindle assembly and spindle assembly checkpoint dynamics. However, the absence of UBE2S affected the activity of APC/C. Cyclin B1 and securin are two substrates of APC/C, and their levels were consistently high, resulting in the failure of homologous chromosome separation. Unexpectedly, the oocytes arrested in meiosis I could be fertilized and the embryos could become implanted normally, but died before embryonic day 10.5. In conclusion, our findings reveal an indispensable regulatory role of UBE2S in mouse oocyte meiosis and female fertility.
Assuntos
Pontos de Checagem da Fase M do Ciclo Celular , Meiose , Animais , Feminino , Camundongos , Ciclossomo-Complexo Promotor de Anáfase/genética , Ciclossomo-Complexo Promotor de Anáfase/metabolismo , Oócitos/metabolismo , Ubiquitinas/metabolismoRESUMO
Miro1, a mitochondrial Rho GTPase1, is a kind of mitochondrial outer membrane protein involved in the regulation of mitochondrial anterograde transport and its subcellular distribution. Mitochondria influence reproductive processes of mammals in some aspects. Mitochondria are important for oocyte maturation, fertilization and embryonic development. The purpose of this study was to evaluate whether Miro1 regulates mouse oocyte maturation by altering mitochondrial homeostasis. We showed that Miro1 was expressed in mouse oocyte at different maturation stages. Miro1 mainly distributed in the cytoplasm and around the spindle during oocyte maturation. Small interference RNA-mediated Miro1 depletion caused significantly abnormal distribution of mitochondria and endoplasmic reticulum as well as mitochondrial dysfunction, resulting in severely impaired germinal vesicle breakdown (GVBD) of mouse oocytes. For those oocytes which went through GVBD in the Miro1-depleted group, part of them were inhibited in meiotic prophase I stage with abnormal chromosome arrangement and scattered spindle length. Our results suggest that Miro1 is essential for maintaining the maturation potential of mouse oocyte.
Assuntos
Meiose , Mitocôndrias , Oócitos , Proteínas rho de Ligação ao GTP , Animais , Feminino , Camundongos , Gravidez , Homeostase , Mitocôndrias/fisiologia , Oócitos/fisiologia , Oogênese , Proteínas rho de Ligação ao GTP/fisiologiaRESUMO
Hematopoiesis is a complex, orderly and conserved developmental process, coordinated by multiple factors including transcription factors and signaling pathways. Dysregulation of any of these factors may cause developmental or functional defects in the blood system, leading to the pathogenesis of blood diseases. Zebrafish hematopoiesis and the underlying molecular mechanisms are highly conserved with those in mammals. The use of zebrafish to recapitulate abnormal changes in pathogenic factors can build models of related blood diseases, thus providing powerful tools for exploring the molecular mechanisms of pathogenesis and progression, visualization of tumorigenesis and high-throughput chemical screening. In this review, we summarize the zebrafish models of blood diseases and their applications. These disease models not only help to improve our understanding of the pathophysiology of the blood system and the molecular mechanisms on pathogeneses of blood diseases, but also provide new ideas for the treatment of clinically relevant hematological malignancies.
Assuntos
Doenças Hematológicas , Peixe-Zebra , Animais , Modelos Animais de Doenças , Doenças Hematológicas/genética , Neoplasias Hematológicas/fisiopatologia , Hematopoese/genéticaRESUMO
Fangchinoline (FA) is an alkaloid derived from the traditional Chinese medicine Fangji. Numerous studies have shown that FA has a toxic effect on various cancer cells, but little is known about its toxic effects on germ cells, especially oocytes. In this study, we investigated the effects of FA on mouse oocyte maturation and its potential mechanisms. Our results showed that FA did not affect meiosis resumption but inhibited the first polar body extrusion. This inhibition is not due to abnormalities at the organelle level, such as chromosomes and mitochondrial, which was proved by detection of DNA damage and reactive oxygen species. Further studies revealed that FA arrested the oocyte at the metaphase I stage, and this arrest was not caused by abnormal kinetochore-microtubule attachment or spindle assembly checkpoint activation. Instead, FA inhibits the activity of anaphase-promoting complexes (APC/C), as evidenced by the inhibition of CCNB1 degeneration. The decreased activity of APC/C may be due to a reduction in CDC25B activity as indicated by the high phosphorylation level of CDC25B (Ser323). This may further enhance Maturation-Promoting Factor (MPF) activity, which plays a critical role in meiosis. In conclusion, our study suggests that the metaphase I arrest caused by FA may be due to abnormalities in MPF and APC/C activity.
Assuntos
Benzilisoquinolinas , Fator Promotor de Maturação , Meiose , Mesotelina , Oócitos , Animais , Meiose/efeitos dos fármacos , Oócitos/efeitos dos fármacos , Feminino , Benzilisoquinolinas/farmacologia , Fator Promotor de Maturação/metabolismo , Camundongos , Fosfatases cdc25/metabolismo , Fosfatases cdc25/genética , Ciclossomo-Complexo Promotor de Anáfase/metabolismo , Camundongos Endogâmicos ICR , Espécies Reativas de Oxigênio/metabolismo , Dano ao DNA/efeitos dos fármacos , Ciclina B1/metabolismo , Ciclina B1/genéticaRESUMO
Environmental factors such as diet and lifestyle can influence the health of both mothers and offspring. However, its transgenerational transmission and underlying mechanisms remain largely unknown. Here, using a maternal lactation-period low-protein diet (LPD) mouse model, we show that maternal LPD during lactation causes decreased survival and stunted growth, significantly reduces ovulation and litter size, and alters the gut microbiome in the female LPD-F1 offspring. The transcriptome of LPD-F1 metaphase II (MII) oocytes shows that differentially expressed genes are enriched in female pregnancy and multiple metabolic processes. Moreover, maternal LPD causes early stunted growth and impairs metabolic health, which is transmitted over two generations. The methylome alteration of LPD-F1 oocytes can be partly transmitted to the F2 oocytes. Together, our results reveal that LPD during lactation transgenerationally affects offspring health, probably via oocyte epigenetic changes.
Assuntos
Dieta com Restrição de Proteínas , Lactação , Animais , Feminino , Lactação/genética , Dieta com Restrição de Proteínas/efeitos adversos , Camundongos , Gravidez , Oócitos/metabolismo , Microbioma Gastrointestinal , Epigênese Genética , Fenômenos Fisiológicos da Nutrição Materna , Transcriptoma/genética , Masculino , Metilação de DNA , Efeitos Tardios da Exposição Pré-Natal/genéticaRESUMO
The anaphase promoting complex/cyclosome (APC/C) and its cofactors CDH1 and CDC20 regulate the accumulation/degradation of CCNB1 during mouse oocyte meiotic maturation. Generally, the CCNB1 degradation mediated by APC/CCDC20 activity is essential for the transition from metaphase to anaphase. Here, by using siRNA and mRNA microinjection, as well as time-lapse live imaging, we showed that Septin 9, which mediates the binding of septins to microtubules, is critical for oocyte meiotic cell cycle progression. The oocytes were arrested at the MI stage and the connection between chromosome kinetochores and spindle microtubules was disrupted after Septin 9 depletion. As it is well known that spindle assembly checkpoint (SAC) is an important regulator of the MI-AI transition, we thus detected the SAC activity and the expression of CDC20 and CCNB1 which were the downstream proteins of SAC during this critical period. The signals of Mad1 and BubR1 still remained on the kinetochores of chromosomes in Septin 9 siRNA oocytes at 9.5 h of in vitro culture when most control oocytes entered anaphase I. The expression of CCNB1 did not decrease and the expression of CDC20 did not increase at 9.5 h in Septin 9 siRNA oocytes. Microinjection of mRNA encoding Septin 9 or CDC20 could partially rescue MI arrest caused by Septin 9 siRNA. These results suggest that Septin 9 is required for meiotic MI-AI transition by regulating the kinetochore-microtubule connection and SAC protein localization on kinetochores, whose effects are transmitted to APC/CCDC20 activity and CCNB1 degradation in mouse oocytes.
Assuntos
Ciclo Celular , Oócitos , Septinas , Animais , Camundongos , Anáfase , Ciclossomo-Complexo Promotor de Anáfase/genética , Ciclossomo-Complexo Promotor de Anáfase/metabolismo , Proteínas Cdc20/genética , Proteínas Cdc20/metabolismo , Proteínas de Ciclo Celular/metabolismo , Metáfase , Oócitos/citologia , Oócitos/metabolismo , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/metabolismo , Septinas/genética , Septinas/metabolismoRESUMO
Maternal ageing is one of the major causes of reduced ovarian reserve and low oocyte quality in elderly women. Decreased oocyte quality is the main cause of age-related infertility. Mitochondria are multifunctional energy stations that determine the oocyte quality. The mitochondria in aged oocytes display functional impairments with mtDNA damage, which leads to reduced competence and developmental potential of oocytes. To improve oocyte quality, mitochondrial supplementation is carried out as a potential therapeutic approach. However, the selection of suitable cells as the source of mitochondria remains controversial. We cultivated endometrial mesenchymal stem cells (EnMSCs) from aged mice and extracted mitochondria from EnMSCs. To improve the quality of oocytes, GV oocytes were supplemented with mitochondria via microinjection. And MII oocytes from aged mice were fertilized by intracytoplasmic sperm injection (ICSI), combining EnMSCs' mitochondrial microinjection. In this study, we found that the mitochondria derived from EnMSCs could significantly improve the quality of aged oocytes. Supplementation with EnMSC mitochondria significantly increased the blastocyst ratio of MII oocytes from aged mice after ICSI. We also found that the birth rate of mitochondria-injected ageing oocytes was significantly increased after embryo transplantation. Our study demonstrates that supplementation with EnMSC-derived mitochondria can improve the quality of oocytes and promote embryo development in ageing mice, which might provide a prospective strategy for clinical treatment.
Assuntos
Oócitos , Sêmen , Masculino , Feminino , Animais , Camundongos , Oócitos/metabolismo , Mitocôndrias , Fertilização , Suplementos NutricionaisRESUMO
The zona pellucida (ZP) is an extracellular glycoprotein matrix surrounding mammalian oocytes. Recently, numerous mutations in genes encoding ZP proteins have been shown to be possibly related to oocyte abnormality and female infertility; few reports have confirmed the functions of these mutations in living animal models. Here, we identified a novel heterozygous missense mutation (NM_001376231.1:c.1616C>T, p.Thr539Met) in ZP2 from a primary infertile female. We showed that the mutation reduced ZP2 expression and impeded ZP2 secretion in cell lines. Furthermore, we constructed the mouse model with the mutation (Zp2T541M) using CRISPR-Cas9. Zp2WT/T541M female mice had normal fertility though generated oocytes with the thin ZP, whereas Zp2T541M female mice were completely infertile due to degeneration of oocytes without ZP. Additionally, ZP deletion impaired folliculogenesis and caused female infertility in Zp2T541M mice. Our study not only expands the spectrum of ZP2 mutation sites but also, more importantly, increases the understanding of pathogenic mechanisms of ZP2 mutations.
RESUMO
Sperm-induced Ca2+ rise is critical for driving oocyte activation and subsequent embryonic development, but little is known about how lasting Ca2+ oscillations are regulated. Here it is shown that NLRP14, a maternal effect factor, is essential for keeping Ca2+ oscillations and early embryonic development. Few embryos lacking maternal NLRP14 can develop beyond the 2-cell stage. The impaired developmental potential of Nlrp14-deficient oocytes is mainly caused by disrupted cytoplasmic function and calcium homeostasis due to altered mitochondrial distribution, morphology, and activity since the calcium oscillations and development of Nlrp14-deficient oocytes can be rescued by substitution of whole cytoplasm by spindle transfer. Proteomics analysis reveal that cytoplasmic UHRF1 (ubiquitin-like, containing PHD and RING finger domains 1) is significantly decreased in Nlrp14-deficient oocytes, and Uhrf1-deficient oocytes also show disrupted calcium homeostasis and developmental arrest. Strikingly, it is found that the mitochondrial Na+ /Ca2+ exchanger (NCLX) encoded by Slc8b1 is significantly decreased in the Nlrp14mNull oocyte. Mechanistically, NLRP14 interacts with the NCLX intrinsically disordered regions (IDRs) domain and maintain its stability by regulating the K27-linked ubiquitination. Thus, the study reveals NLRP14 as a crucial player in calcium homeostasis that is important for early embryonic development.
Assuntos
Cálcio , Nucleosídeo-Trifosfatase , Sêmen , Humanos , Masculino , Cálcio/metabolismo , Homeostase/fisiologia , Oócitos/metabolismo , Sêmen/metabolismo , Trocador de Sódio e Cálcio/genética , Trocador de Sódio e Cálcio/metabolismo , Ubiquitinação , Animais , Camundongos , Nucleosídeo-Trifosfatase/metabolismoRESUMO
Gaucher disease (GD) is an autosomal recessive lysosomal storage disorder caused by mutations in the GBA1 gene, which produces the glucocerebrosidase (GCase) protein. There are more than 500 mutations reported in GBA1, among which L444P (p.Leu444Pro) and F213I (p.Phe213Ile) are the most common in the Chinese population, while the function of F213I mutation remains elusive. This study aims to establish the GD mouse model of partially humanized Gba1 gene with F213I mutation. In vitro GCase activity assays showed that the product of partially humanized Gba1 gene, in which the mouse exons 5-7 were replace by the corresponding human exons, displayed similar activity with the wild-type mouse Gba1, while the F213I mutation in the humanized Gba1 led to significant decrease in enzyme activity. ES cell targeting was used to establish the mice expressing the partially humanized Gba1-F213I. Gba1 F213I/+ mice did not show obviously abnormal phenotypes, but homozygous Gba1 F213I/F213I mice died within 24 h after birth, whose epidermal stratum corneum were abnormal from the wild-type. The GCase activity in Gba1 F213I/F213I mice greatly decreased. In conclusion, our results showed that the partially humanized GD mouse model with the F213I mutation was developed and homozygous F213I mutation is lethal for newborn mice.
RESUMO
The developmental potential of oocytes decreases with time after ovulation in vivo or in vitro. Epitalon is a synthetic short peptide made of four amino acids (alanine, glutamic acid, aspartic acid, and glycine), based on a natural peptide called epithalamion extracted from the pineal gland. It is a potent antioxidant, comparable to melatonin, that may confer longevity benefits. The current study aims to test the protective effects of Epitalon on the quality of post-ovulatory aging oocytes. Epitalon at 0.1mM was added to the culture medium, and the quality of oocytes was evaluated at 6h, 12h, and 24h of culture. We found that 0.1mM Epitalon reduced intracellular reactive oxygen species. Epitalon treatment significantly decreased frequency of spindle defects and abnormal distribution of cortical granules during aging for 12h and 24h, while increased mitochondrial membrane potential and DNA copy number of mitochondria, thus decreasing apoptosis of oocytes by 24h of in vitro aging. Our results suggest that Epitalon can delay the aging process of oocytes in vitro via modulating mitochondrial activity and ROS levels.
Assuntos
Oligopeptídeos , Oócitos , Envelhecimento , Animais , Feminino , Camundongos , Oócitos/metabolismo , Ovulação , Espécies Reativas de Oxigênio/metabolismoRESUMO
Esophageal cancer (ESCC) is one of the most common causes of cancer-associated mortality in China. The present investigation reveals that non-coding RNAs (ncRNAs), including long ncRNAs (lncRNAs), exert a significant effect on the initiation, development and metastasis of malignant tumors, including ESCC. However, to the best of our knowledge, the function of non-protein-coding genes that host small nucleolar RNAs has not been investigated in cancer, particularly in ESCC. The expression of small nucleolar host gene 6 (SNHG6) in 70 ESCC tissues and paired adjacent tissues was measured by reverse transcription quantitative polymerase chain reaction. Analysis demonstrated that SNHG6 expression was significantly increased in ESCC tissues, and associated with tumor size (P=0.040) and Tumor-Node-Metastasis stage (P<0.01). Knockdown of SNHG6 may inhibit proliferative and colony-forming abilities, and induce apoptosis, in ESCC cells. To the best of our knowledge, the data from the present study indicated for the first time that SNHG6 was upregulated in ESCC tissues and cell lines. This novel lncRNA may exert a marked effect on the generation and progression of ESCC, potentially providing a novel perspective on ESCC diagnosis and management.
RESUMO
BACKGROUND: Nowadays, despite great progress in cancer research, the detailed mechanisms of colorectal cancer (CRC) are still poorly understood. Circular RNAs (circRNAs), a new star of the non-coding RNA network, have been identified as critical regulators in various cancers, including CRC. METHODS AND RESULTS: In this study, by using unsupervised hierarchical clustering analysis, a novel dysregulated circRNA, hsa_circ_0000069, was found. The expression of hsa_circ_0000069 was measured in 30 paired CRC tissues and adjacent noncancerous tissues using quantitative polymerase chain reaction. A high expression of hsa_circ_0000069 was observed in CRC tissues and correlated with patients' age and tumor, node, metastasis (TNM) stage (P<0.05). Furthermore, by using specifically designed siRNAs in CRC cells, a functional analysis was performed which revealed that hsa_circ_0000069 knockdown could notably inhibit cell proliferation, migration, and invasion, and induce G0/G1 phase arrest of cell cycle in vitro. CONCLUSION: This study's findings are the first to demonstrate that hsa_circ_0000069, an important regulator in cancer progression, could be a promising target in the diagnosis and therapy in colorectal cancer.
RESUMO
Recent findings indicate that long noncoding RNAs (lncRNAs) were dysregulated in many kinds of tumors including esophageal squamous cell carcinoma (ESCC). LncRNA AFAP1-AS1 was found to be upregulated in hepatocellular carcinoma (HCC), lung cancer, colorectal cancer, esophageal adenocarcinoma (EAC), pancreatic ductal adenocarcinoma, and nasopharyngeal carcinoma, while its clinical value and potential function in ESCC are still unknown. Expression of AFAP1-AS1 was measured in 65 ESCC tissues and corresponding noncancerous tissues by quantitative real-time polymerase chain reaction, which revealed that AFAP1-AS1 expression was markedly elevated in ESCC tissues and significantly associated with advanced TNM stage (P = 0.004) and larger tumor size (P = 0.040). Moreover, by knocking down AFAP1-AS1 expression in ESCC cells, the proliferation and colony-forming ability were inhibited and cell apoptosis was induced. Our data indicated the first time that AFAP1-AS1, a novel oncogene, was remarkably upregulated and played a critical role in the progression of ESCC.