RESUMO
Chromosome segregation is initiated by cohesin degradation, which is driven by anaphase-promoting complex/cyclosome (APC/C). Chromosome cohesin is removed by activated separase, with the degradation of securin and cyclinB1. Dynamin-related protein 1 (DRP1), a component of the mitochondrial fission machinery, is related to cyclin dynamics in mitosis progression. Here, we show that DRP1 is recruited to the kinetochore by centromeric Centromere protein F (CENP-F) after nuclear envelope breakdown in mouse oocytes. Loss of DRP1 during prometaphase leads to premature cohesin degradation and chromosome segregation. Importantly, acute DRP1 depletion activates separase by initiating cyclinB1 and securin degradation during the metaphase-to-anaphase transition. Finally, we demonstrate that DRP1 is bound to APC2 to restrain the E3 ligase activity of APC/C. In conclusion, DRP1 is a CENP-F-dependent atypical spindle assembly checkpoint (SAC) protein that modulates metaphase-to-anaphase transition by controlling APC/C activity during meiosis I in oocytes.
Assuntos
Segregação de Cromossomos , Meiose , Animais , Camundongos , Ciclossomo-Complexo Promotor de Anáfase/metabolismo , Proteínas de Ciclo Celular/metabolismo , Dinaminas/metabolismo , Cinetocoros/metabolismo , Oócitos/metabolismo , Securina/genética , Securina/metabolismo , Separase/metabolismoRESUMO
As a major protein of the polyhedral coat of coated pits and vesicles, clathrin molecules have been shown to play a stabilization role for kinetochore fibers of the mitotic spindle by acting as inter-microtubule bridges. Clathrin heavy chain 1 (CLTC), the basic subunit of the clathrin coat, plays vital roles in both spindle assembly and chromosome congression during somatic-cell mitosis. However, its function in oocyte meiotic maturation and early embryo development in mammals, especially in domesticated animals, has not been fully investigated. In this study, the expression profiles and functional roles of CLTC in sheep oocytes were investigated. Our results showed that the expression of CLTC was maintained at a high level from the germinal vesicle (GV) stage to metaphase II stage and that CLTC was distributed diffusely in the cytoplasm of cells at interphase, from the GV stage to the blastocyst stage. After GV breakdown (GVBD), CLTC co-localized with beta-tubulin during metaphase. Oocyte treatments with taxol, nocodazole, or cold did not affect CLTC expression levels but led to disorders of its distribution. Functional impairment of CLTC by specific morpholino injections in GV-stage oocytes led to disruptions in spindle assembly and chromosomal alignment, accompanied by impaired first polar body (PB1) emissions. In addition, knockdown of CLTC before parthenogenetic activation disrupted spindle formation and impaired early embryo development. Taken together, the results demonstrate that CLTC plays a vital role in sheep oocyte maturation via the regulation of spindle dynamics and an essential role during early embryo development.
RESUMO
As a posttranscriptional regulatory factor, microRNA (miRNA) plays an important role in the formation of myotubes. However, little is known about the mechanism of miRNA regulating myotube morphogenesis. Here, we aimed to characterize the function of miR-455-5p in myotube morphogenesis by inducing differentiation in C2C12 myoblasts containing murine Mylip fragments with the miR-455-5p target sequence. We found that miR-455-5p overexpression promoted the differentiation and hypertrophy of myotubes, while miR-455-5p inhibition led to the failure of myotube differentiation and formation of short myotubes. Furthermore, we demonstrated that miR-455-5p directly targeted the Mylip 3'-untranslated region, which plays a key role in monitoring myotube morphogenesis. Interestingly, the expression and function of Mylip were opposite to those of miR-455-5p during myogenesis. Our data uncovered novel miR-455-5p targets and established a functional link between Mylip and myotube morphogenesis. Understanding the involvement of Mylip in myotube morphogenesis provides insight into the function of the gene regulatory network.
Assuntos
Diferenciação Celular/fisiologia , MicroRNAs/metabolismo , Mioblastos/citologia , Mioblastos/metabolismo , Animais , Diferenciação Celular/genética , Proliferação de Células/genética , Proliferação de Células/fisiologia , Humanos , MicroRNAs/genética , Desenvolvimento Muscular/genética , Desenvolvimento Muscular/fisiologiaRESUMO
Reproductive dysfunction associated with obesity is increasing among women of childbearing age. Emerging evidence indicates that maternal obesity impairs embryo development and offspring health, and these defects are linked to oxidative stress in the ovary and in oocytes. Phycocyanin (PC) is a biliprotein from Spirulina platensis that possesses antioxidant, anti-inflammatory, and radical-scavenging properties. Our previous studies have shown that PC can reduce reactive oxygen species (ROS) accumulation in oocytes in D-gal-induced aging mice. Here, at the Institute of Cancer Research (ICR) mice fed a high-fat diet (HFD) to model obesity were used to test the effect of PC on reversing the fertility decline caused by obesity. We observed a significant increase in litter size and offspring survival rates after PC administration to obese mice. Further, we found that PC not only ameliorated the level of ovarian antioxidant enzymes, but also reduced the occurrence of follicular atresia in obese female mice. In addition, the abnormal morphology of the spindle-chromosome complex (SCC), and the abnormal mitochondrial distribution pattern in oocytes both recovered. The obesity-related accumulation of ROS, increased number of early apoptotic cells, and the abnormal expression of H3K9me3 in oocytes were all partially reversed after PC administration. In summary, this is the first demonstration that PC can improve fertility by partially increasing ovarian and oocyte quality in obese female mice and provides a new strategy for clinically treating obesity-related infertility in females.
RESUMO
Biomedical named entity recognition (Bio-NER) is the prerequisite for mining knowledge from biomedical texts. The state-of-the-art models for Bio-NER are mostly based on bidirectional long short-term memory (BiLSTM) and bidirectional encoder representations from transformers (BERT) models. However, both BiLSTM and BERT models are extremely computationally intensive. To this end, this paper proposes a temporal convolutional network (TCN) with a conditional random field (TCN-CRF) layer for Bio-NER. The model uses TCN to extract features, which are then decoded by the CRF to obtain the final result. We improve the original TCN model by fusing the features extracted by convolution kernel with different sizes to enhance the performance of Bio-NER. We compared our model with five deep learning models on the GENIA and CoNLL-2003 datasets. The experimental results show that our model can achieve comparative performance with much less training time. The implemented code has been made available to the research community.
Assuntos
AlgoritmosRESUMO
As an effective feed additive in the livestock industry, olaquindox (OLA) has been widely used in domestic animal production. However, it is unclear whether OLA has negative effects on mammalian oocyte quality and fetal development. In this study, toxic effects of OLA were tested by intragastric gavage ICR mice with water, low-dose OLA (5 mg/kg/day), or high-dose OLA (60 mg/kg/day) for continuous 45 days. Results showed that high-dose OLA gavage severely affected the offspring birth and growth. Significantly, high-dose OLA impaired oocyte maturation and early embryo development, indicated by the decreased percentage of germinal vesicle breakdown, first polar body extrusion and blastocyst formation. Meanwhile, oxidative stress levels were increased in oocytes or ovaries, indexed by the increased levels of ROS, MDA, H2O2, NO, and decreased levels of GSH, SOD, CAT, GSH-Px and GSH-Rd. Furthermore, aberrant mitochondria distribution, defective spindle assembly, abnormal H3K4me2/H3K9me3 levels, increased DNA double-strand breaks and early apoptosis rate, were observed after high-dose OLA gavage. Taken together, our results for the first time illustrated that high-dose OLA gavage led to sub-fertility of females, which means that restricted utilization of OLA as feed additive should be considered.
Assuntos
Desenvolvimento Embrionário/efeitos dos fármacos , Aditivos Alimentares/toxicidade , Oócitos/efeitos dos fármacos , Oogênese/efeitos dos fármacos , Quinoxalinas/toxicidade , Administração Oral , Animais , Apoptose/efeitos dos fármacos , Relação Dose-Resposta a Droga , Feminino , Peróxido de Hidrogênio/metabolismo , Camundongos , Camundongos Endogâmicos ICR , Mitocôndrias/efeitos dos fármacos , Oócitos/metabolismo , Oócitos/patologia , Ovário/efeitos dos fármacos , Ovário/metabolismo , Oxirredução , Estresse Oxidativo/efeitos dos fármacosRESUMO
In contrast to the homeokinesis of mitosis, asymmetric division of cytoplasm is the conspicuous feature of meiosis in mammalian oocytes. Protein regulator of cytokinesis 1 (PRC1) is an important regulator during mitotic spindle assembly and cytoplasmic division, but its functions in oocyte meiosis and early embryo development have not been fully elucidated. In this study, we detected PRC1 expression and localization and revealed a nuclear, spindle midzone-related dynamic pattern throughout meiotic and mitotic progressions. Treatment of oocytes with the reagents taxol or nocodazole disturbed the distribution of PRC1 in metaphase II oocytes. Further, PRC1 depletion led to failure of first polar body (PB1) extrusion and spindle migration, aneuploidy and defective kinetochore-microtubule attachment and spindle assembly. Overexpression of PRC1 resulted in PB1 extrusion failure, aneuploidy and serious defects of spindle assembly. To investigate PRC1 function in early embryos, we injected Prc1 morpholino into zygotes and 2-cell stage embryos. Depletion of PRC1 in zygotes impaired 4-cell, morula and blastocyst formation. Loss of PRC1 in single or double blastomeres in 2-cell stage embryos significantly impaired cell division, indicating its indispensable role in early embryo development. Co-immunoprecipitation showed that PRC1 interacts with polo-like kinase 1 (PLK1), and functional knockdown and rescue experiments demonstrated that PRC1 recruits PLK1 to the spindle midzone to regulate cytoplasmic division during meiosis. Finally, kinesin family member 4 knockdown downregulates PRC1 expression and leads to PRC1 localization failure. Taken together, our data suggest PRC1 plays an important role during oocyte maturation and early embryonic development by regulating chromosome dynamics and cytoplasmic division.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Cromossomos , Citoplasma/fisiologia , Desenvolvimento Embrionário , Meiose , Oócitos/fisiologia , Fuso Acromático/fisiologia , Animais , Proteínas de Ciclo Celular/genética , Feminino , Cinesinas/genética , Cinesinas/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos ICR , Oócitos/citologia , Oogênese , Gravidez , Espermatozoides/citologia , Espermatozoides/fisiologia , Zigoto/citologia , Zigoto/fisiologiaRESUMO
Hatching out from the zona pellucida (ZP) is a crucial step for blastocyst implantation and development. However, it is still unknown whether the location of the hatching site relative to the inner cell mass (ICM) affects embryo implantation and foetal development. Here, we classified hatching blastocysts into three categories, 0° ≤ θ ≤ 30°, 30° < θ ≤ 60°, and 60° < θ ≤ 90°, in which θ is determined based on the relative position of the hatching site to the arc midpoint of the ICM. Non-surgical embryo transfer (NSET) devices were employed to evaluate blastocyst implantation and embryo development. Of 1,827 hatching blastocysts, 43.84%, 30.60%, and 21.67% were categorized as 30° < θ ≤ 60°, 0° ≤ θ ≤ 30°, and 60° < θ ≤ 90°, respectively. Embryos with different hatching sites showed no distinct differences in blastocyst implantation; surrogate female pregnancy; embryo development to term; litter size, or offspring survival, gender, or body weight. Our results indicate that mouse blastocyst hatching site is not randomly distributed. Embryo implantation and development are not correlated with the blastocyst hatching site in mice. Thus, assessment of the blastocyst hatching site should not be recommended to evaluate mouse blastocyst implantation and developmental potential.
Assuntos
Implantação do Embrião , Desenvolvimento Fetal , Zona Pelúcida/fisiologia , Animais , Células Cultivadas , Feminino , Tamanho da Ninhada de Vivíparos , Masculino , Camundongos , GravidezRESUMO
For sexual reproduction, oocytes are mammalian female germ cells that provide the majority of maternal genetic material for early stage embryo production and development. Early stage embryos begin the process of multicellular organism formation through cell differentiation. Studies on mammalian female germ cells (oocytes) not only reveal its unique physiological characteristics, but also help understand the mechanism involved in cell differentiation of other cell types. However, because it is difficult to culture in vitro, our understanding of the function of oocytes and early stage embryos remains very limited. Gene editing or manipulation is one of the most commonly used method, which is also useful in the field of gametes study. In this review, we summarized the principles, advantages and disadvantages of techniques, which include conditional knockout, RNA interference, Morpholino, Trim-Away and antibody-mediated inhibition of protein function, currently used for gene manipulation in oocytes and early stage embryos. We also discuss the issues the investigators need to consider. Finally, we highlight the future directions for gene manipulation or editing in female germ cells and early stage embryos.
Assuntos
Edição de Genes , Células Germinativas , Oócitos , Animais , Diferenciação Celular , Embrião de Mamíferos , Feminino , Técnicas de Inativação de Genes , Mamíferos , Interferência de RNARESUMO
Phosphodiesterase (PDE)-mediated reduction of cyclic adenosine monophosphate (cAMP) activity can initiate germinal vesicle (GV) breakdown in mammalian oocytes. It is crucial to maintain oocytes at the GV stage for a long period to analyze meiotic resumption in vitro. Meiotic resumption can be reversibly inhibited in isolated oocytes by cAMP modulator forskolin, cAMP analog dibutyryl cAMP (dbcAMP), or PDE inhibitors, milrinone (Mil), Cilostazol (CLZ), and 3-isobutyl-1-methylxanthine (IBMX). However, these chemicals negatively affect oocyte development and maturation when used independently. Here, we used ICR mice to develop a model that could maintain GV-stage arrest with minimal toxic effects on subsequent oocyte and embryonic development. We identified optimal concentrations of forskolin, dbcAMP, Mil, CLZ, IBMX, and their combinations for inhibiting oocyte meiotic resumption. Adverse effects were assessed according to subsequent development potential, including meiotic resumption after washout, first polar body extrusion, early apoptosis, double-strand DNA breaks, mitochondrial distribution, adenosine triphosphate levels, and embryonic development. Incubation with a combination of 50.0 µM dbcAMP and 10.0 µM IBMX efficiently inhibited meiotic resumption in GV-stage oocytes, with low toxicity on subsequent oocyte maturation and embryonic development. This work proposes a novel method with reduced toxicity to effectively arrest and maintain mouse oocytes at the GV stage.
Assuntos
1-Metil-3-Isobutilxantina/farmacologia , Bucladesina/farmacologia , Núcleo Celular/metabolismo , Meiose/efeitos dos fármacos , Oócitos/metabolismo , Inibidores de Fosfodiesterase/farmacologia , Animais , Apoptose/efeitos dos fármacos , Núcleo Celular/efeitos dos fármacos , Células Cultivadas , Colforsina/farmacologia , AMP Cíclico/metabolismo , Quebras de DNA/efeitos dos fármacos , Desenvolvimento Embrionário/efeitos dos fármacos , Feminino , Camundongos , Camundongos Endogâmicos ICR , Oócitos/efeitos dos fármacosRESUMO
Aneuploidy caused by abnormal chromosome segregation during early embryo development leads to embryonic death or congenital malformation. Centromere protein F (CENPF) is a member of centromere protein family that regulates chromosome segregation during mitosis. However, its necessity in early embryo development has not been fully investigated. In this study, expression and function of CENPF was investigated in mouse early embryogenesis. Detection of CENPF expression and localization revealed a cytoplasm, spindle and nuclear membrane related dynamic pattern throughout mitotic progression. Farnesyltransferase inhibitor (FTI) was employed to inhibit CENPF farnesylation in zygotes. The results showed that CENPF degradation was inhibited and its specific localization on nuclear membranes in morula and blastocyst vanished after FTI treatment. Also, CAAX motif mutation leads to failure of CENPF-C630 localization in morula and blastocyst. These results indicate that farnesylation plays a key role during CENPF degradation and localization in early embryos. To further assess CENPF function in parthenogenetic or fertilized embryos development, morpholino (MO) and Trim-Away were used to disturb CENPF function. CENPF knockdown in Metaphase II (MII) oocytes, zygotes or embryos with MO approach resulted in failure to develop into morulae and blastocysts, revealing its indispensable role in both parthenogenetic and fertilized embryos. Disturbing of CENPF with Trim-Away approach in zygotes resulted in impaired development of 2-cell and 4-cell, but did not affect the morula and blastocyst formation because of the recovered expression of CENPF. Taken together, our data suggest CENPF plays an important role during early embryonic development in mice. Abbreviation: CENPF: centromere protein F; MO: morpholino; FTI: Farnesyltransferase inhibitor; CENPE: centromere protein E; IVF: in vitro fertilization; MII: metaphase II; SAC: spindle assembly checkpoint; Mad1: mitotic arrest deficient 1; BUB1: budding uninhibited by benzimidazole 1; BUBR1: BUB1 mitotic checkpoint serine/threonine kinase B; Cdc20: cell division cycle 20.
Assuntos
Blastocisto/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Embrião de Mamíferos/metabolismo , Desenvolvimento Embrionário/genética , Proteínas dos Microfilamentos/metabolismo , Oócitos/metabolismo , Zigoto/metabolismo , Animais , Centrômero/metabolismo , Proteínas Cromossômicas não Histona/química , Proteínas Cromossômicas não Histona/genética , Embrião de Mamíferos/embriologia , Farnesiltranstransferase/antagonistas & inibidores , Feminino , Técnicas de Silenciamento de Genes , Metáfase/genética , Camundongos , Camundongos Endogâmicos ICR , Proteínas dos Microfilamentos/química , Proteínas dos Microfilamentos/genética , Morfolinos/genética , Morfolinos/farmacologia , Mórula/metabolismo , Oócitos/crescimento & desenvolvimento , Partenogênese/genética , Piperidinas/farmacologia , Gravidez , Prenilação , Piridinas/farmacologia , Zigoto/crescimento & desenvolvimentoRESUMO
Sodium nitrite (NaNO2) is an industrial chemical that is frequently used as a food additive to prevent botulism and enhance glossiness, such as curing meat. In addition, in some regions, water source NaNO2 concentrations exceed standard regulatory levels. Whether the excessive intake of NaNO2 has toxic effects on female fertility and fetal development remain unknown. In this study, we administered ICR mice control saline, low-dose NaNO2 (60â¯mg/kg/day), or high-dose NaNO2 (120â¯mg/kg/day) by intragastric gavage for 21 days. We then assessed oocyte morphology, spindle-chromosome dynamics, mitochondrial distribution, ATP content, apoptotic cell numbers, DNA damage levels, histone modifications, reactive oxygen species (ROS) levels, and offspring survival. Results showed that NaNO2 treatment decreased oocyte number, impaired polar body extrusion, and increased zona pellucida thickness in oocytes. Furthermore, NaNO2 disrupted MII spindle integrity, caused abnormal mitochondrial distribution, decreased ATP content, and increased levels of ROS and H3K4me2. Moreover, the number of oocytes in early stages of apoptosis and with levels of DNA damage increased in NaNO2-treated mice along with decreased offspring numbers and survival rates. We demonstrated the negative effects of NaNO2 on female reproductive abilities in mice.
Assuntos
Aditivos Alimentares/toxicidade , Reprodução/efeitos dos fármacos , Nitrito de Sódio/toxicidade , Trifosfato de Adenosina/metabolismo , Animais , Catalase/metabolismo , Dano ao DNA , Feminino , Coração/efeitos dos fármacos , Coração/crescimento & desenvolvimento , Histonas/metabolismo , Fígado/efeitos dos fármacos , Fígado/crescimento & desenvolvimento , Camundongos Endogâmicos ICR , Mitocôndrias/efeitos dos fármacos , Oócitos/efeitos dos fármacos , Oócitos/metabolismo , Tamanho do Órgão/efeitos dos fármacos , Ovário/efeitos dos fármacos , Ovário/crescimento & desenvolvimento , Espécies Reativas de Oxigênio/metabolismo , Superóxido Dismutase/metabolismoRESUMO
Paracrine factors such as glial cell line-derived neurotrophic factor (GDNF), which was originally derived from the supernatants of a rat glioma cell line, play pivotal roles in oocyte maturation and early embryo development in mammals, such as mice, rats, pigs, sheep, and even humans. However, whether GDNF facilitates in vitro oocyte maturation or early embryo development in bovines is not yet known. We show for the first time that GDNF and its receptor, GDNF family receptor alpha-1 (GFRA1), are presented in ovarian follicles at different stages as well as during oocyte maturation and early embryo development. Immunostaining results revealed the subcellular localizations of GDNF and GFRA1 in oocytes throughout follicle development, first in germinal vesicles and during blastocyst embryo stages. The ability of exogenously applied GDNF to promote oocyte maturation and early embryo development was evaluated in culture, where we found that an optimal concentration of 50â¯ng/mL promotes the maturation of cumulus-oocyte complexes and the nuclei of denuded oocytes as well as the development of embryos after IVF. To further investigate the potential mechanism by which GDNF promotes oocyte maturation, bovine oocytes were treated with morpholinos targeting Gfra1. The suppression of GFRA1 presence blocked endogenous and exogenous GDNF functions, indicating that the effects of GDNF that are essential and beneficial for bovine oocyte maturation and early embryo development occur through this receptor. Furthermore, we show that supplementation with GDNF improves the efficiency of bovine IVF embryo production.
Assuntos
Bovinos/embriologia , Técnicas de Cultura Embrionária , Fator Neurotrófico Derivado de Linhagem de Célula Glial/farmacologia , Técnicas de Maturação in Vitro de Oócitos/veterinária , Oócitos/fisiologia , Animais , Linhagem Celular , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Receptores de Fator Neurotrófico Derivado de Linhagem de Célula Glial/genética , Receptores de Fator Neurotrófico Derivado de Linhagem de Célula Glial/metabolismo , Folículo Ovariano/metabolismoRESUMO
Mammalian oocyte meiotic maturation is the precondition of early embryo development. Lots of microtubules (MT)-associated proteins participate in oocyte maturation process. Cytoskeleton-associated protein 5 (CKAP5) is a member of the XMAP215 family that regulates microtubule dynamics during mitosis. However, its role in meiosis has not been fully studied. Here, we investigated the function of CKAP5 in mouse oocyte meiotic maturation and early embryo development. Western blot showed that CKAP5 expression increased from GVBD, maintaining at high level at metaphase, and decreased after late 1-cell stage. Confocal microscopy showed there is no specific accumulation of CKAP5 at interphase (GV, PN or 2-cell stage). However, once cells enter into meiotic or mitotic division, CKAP5 was localized at the whole spindle apparatus. Treatment of oocytes with the tubulin-disturbing reagents nocodazole (induces MTs depolymerization) or taxol (prevents MTs depolymerization) did not affect CKAP5 expression but led to a rearrangement of CKAP5. Further, knock-down of CKAP5 resulted in a failure of first polar body extrusion, serious defects in spindle assembly, and failure of chromosome alignment. Loss of CKAP5 also decreased early embryo development potential. Furthermore, co-immunoprecipitation showed that CKAP5 bound to clathrin heavy chain 1 (CLTC). Taken together, our results demonstrate that CKAP5 is important in oocyte maturation and early embryo development, and CKAP5 might work together with CLTC in mouse oocyte maturation.
Assuntos
Cadeias Pesadas de Clatrina/metabolismo , Desenvolvimento Embrionário/fisiologia , Proteínas Associadas aos Microtúbulos/metabolismo , Oócitos/metabolismo , Fuso Acromático/metabolismo , Animais , Western Blotting , Feminino , Imunofluorescência , Imunoprecipitação , Meiose/fisiologia , Camundongos , Microscopia ConfocalRESUMO
Kinesin family member 1B (KIF1B) is an important microtubule-dependent monomeric motor in mammals, although little is known about its role in meiosis. We profiled KIF1B expression and localization during oocyte maturation and early embryonic development in mice, revealing a dynamic pattern throughout meiotic progression. Depletion or inhibition of KIF1B leads to abnormal polar body extrusion, disordered spindle dynamics, defects in chromosome congression, increased aneuploidy, and impaired embryonic development. Further, KIF1B depletion affects the distribution of mitochondria and abundance of ATP. Taken together, our study demonstrates that mouse KIF1B is important for spindle assembly, chromosome congression, and mitochondrial distribution during oocyte maturation and early embryonic development. Mol. Reprod. Dev. 83: 1027-1040, 2016 © 2016 Wiley Periodicals, Inc.
Assuntos
Embrião de Mamíferos/embriologia , Desenvolvimento Embrionário/fisiologia , Cinesinas/metabolismo , Meiose/fisiologia , Trifosfato de Adenosina/genética , Trifosfato de Adenosina/metabolismo , Animais , Cromossomos de Mamíferos/genética , Cromossomos de Mamíferos/metabolismo , Embrião de Mamíferos/citologia , Feminino , Cinesinas/genética , Masculino , Camundongos , Mitocôndrias/genética , Mitocôndrias/metabolismo , Oócitos , Corpos Polares/metabolismo , Fuso Acromático/genética , Fuso Acromático/metabolismoRESUMO
Women over 35 have higher rates of infertility, largely due to deterioration of oocyte quality characterized by fragmentation, abnormal meiotic spindle-chromosome complexes, and oxidative stress. C-phycocyanin (PC) is a biliprotein enriched in Spirulina platensis that is known to possess antioxidant, anti-inflammatory, and radical-scavenging properties. D-galactose-induced aging acceleration in mice has been extensively used to study aging mechanisms and for pharmaceutical screening. In this study, adult female B6D2F/1 mice injected with D-galactose were used as a model to test the age-reversing effects of PC on degenerated reproductive ability. Our results show that PC can prevent oocyte fragmentation and aneuploidy by maintaining cytoskeletal integrity. Moreover, PC can reverse the expression of antioxidant genes, increase superoxide dismutase (SOD) activity and decrease methane dicarboxylic aldehyde (MDA) content, and normalize mitochondria distribution. PC exerts its benefit by inhibiting reactive oxygen species (ROS) production, which decreases apoptosis. Finally, we observe a significant increase in litter size after PC administration to D-galactose-induced aging mice. Our study demonstrates for the first time that D-galactose-induced impaired female reproductive capability can be partially rescued by the antioxidant effects of PC.
Assuntos
Fertilidade/efeitos dos fármacos , Ficocianina/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Envelhecimento/efeitos dos fármacos , Envelhecimento/metabolismo , Animais , Apoptose/efeitos dos fármacos , Cromossomos/efeitos dos fármacos , Feminino , Galactose/administração & dosagem , Galactose/toxicidade , Humanos , Masculino , Camundongos , Oócitos/citologia , Oócitos/efeitos dos fármacos , Gravidez , Distribuição Aleatória , Fuso Acromático/efeitos dos fármacosRESUMO
Cumulus cells are a group of closely associated granulosa cells that surround and nourish oocytes. Previous studies have shown that cumulus cells contribute to oocyte maturation and fertilization through gap junction communication. However, it is not known how this gap junction signaling affects in vivo versus in vitro maturation of oocytes, and their subsequent fertilization and embryonic development following insemination. Therefore, in our study, we performed mouse oocyte maturation and insemination using in vivo- or in vitro-matured oocyte-cumulus complexes (OCCs, which retain gap junctions between the cumulus cells and the oocytes), in vitro-matured, denuded oocytes co-cultured with cumulus cells (DCs, which lack gap junctions between the cumulus cells and the oocytes), and in vitro-matured, denuded oocytes without cumulus cells (DOs). Using these models, we were able to analyze the effects of gap junction signaling on oocyte maturation, fertilization, and early embryo development. We found that gap junctions were necessary for both in vivo and in vitro oocyte maturation. In addition, for oocytes matured in vivo, the presence of cumulus cells during insemination improved fertilization and blastocyst formation, and this improvement was strengthened by gap junctions. Moreover, for oocytes matured in vitro, the presence of cumulus cells during insemination improved fertilization, but not blastocyst formation, and this improvement was independent of gap junctions. Our results demonstrate, for the first time, that the beneficial effect of gap junction signaling from cumulus cells depends on oocyte maturation and fertilization methods.
RESUMO
The method of vitrification has been widely used for cryopreservation. However, the effectiveness of this method for mammalian oocytes could be improved by optimizing each step of the process. In the present study, we tested the effects of varying several key parameters to determine the most effective protocol for mouse oocyte vitrification. We found that cryoprotectant containing ethylene glycol and dimethylsulfoxide plus 20% fetal calf serum produced the highest rates of oocyte survival, fertilization, and blastocyst formation. The duration and temperature of oocyte exposure to vitrification and thawing solutions influenced survival rate. The presence of cumulus cells surrounding oocytes and the incubation of thawed oocytes in Toyoda-Yokoyama-Hosoki medium also increased oocyte survival. Open pulled straw and nylon loop methods were more effective than the mini-drop method. Finally, the combination of these improved methods resulted in better spindle morphology when compared to the unimproved methods. These results demonstrate that the outcomes of mouse oocyte vitrification can be improved by a suitable combination of cryopreservation methods, which could be applied to future clinical research with human oocytes.
Assuntos
Sobrevivência Celular/efeitos dos fármacos , Criopreservação/métodos , Crioprotetores/farmacologia , Oócitos/citologia , Animais , Blastocisto/efeitos dos fármacos , Blastocisto/fisiologia , Sobrevivência Celular/fisiologia , Dimetil Sulfóxido/farmacologia , Desenvolvimento Embrionário/efeitos dos fármacos , Desenvolvimento Embrionário/fisiologia , Etilenoglicol/farmacologia , Feminino , Fertilização/efeitos dos fármacos , Fertilização/fisiologia , Fertilização in vitro/efeitos dos fármacos , Fertilização in vitro/métodos , Masculino , Camundongos , VitrificaçãoRESUMO
To improve the outcome of assisted reproductive technology (ART) for patients with ovulation problems, it is necessary to retrieve and select germinal vesicle (GV) stage oocytes with high developmental potential. Oocytes with high developmental potential are characterized by their ability to undergo proper maturation, fertilization, and embryo development. In this study, we analyzed morphological traits of GV stage mouse oocytes, including cumulus cell layer thickness, zona pellucida thickness, and perivitelline space width. Then, we assessed the corresponding developmental potential of each of these oocytes and found that it varies across the range measured for each morphological trait. Furthermore, by manipulating these morphological traits in vitro, we were able to determine the influence of morphological variation on oocyte developmental potential. Manually altering the thickness of the cumulus layer showed strong effects on the fertilization and embryo development potentials of oocytes, whereas manipulation of zona pellucida thickness effected the oocyte maturation potential. Our results provide a systematic detailed method for selecting GV stage oocytes based on a morphological assessment approach that would benefit for several downstream ART applications.
Assuntos
Células do Cúmulo/citologia , Oócitos/citologia , Zona Pelúcida , Animais , Células do Cúmulo/metabolismo , Feminino , Camundongos , Oócitos/metabolismoRESUMO
Casein kinase I alpha (CK1α) is a member of serine/threonine protein kinase, generally present in all eukaryotes. In mammals, CK1α regulates the transition from interphase to metaphase in mitosis. However, little is known about its role in meiosis. Here we examined Ck1α mRNA and protein expression, as well as its subcellular localization in mouse oocytes from germinal vesicle to the late 1-cell stage. Our results showed that the expression level of CK1α was increased in metaphase. Immunostaining results showed that CK1α colocalized with condensed chromosomes during oocyte meiotic maturation and early embryo development. We used the loss-of-function approach by employing CK1α specific morpholino injection to block the function of CK1α. This functional blocking leads to failure of polar body 1 (PB1) extrusion, chromosome misalignment and MII plate incrassation. We further found that D4476, a specific and efficient CK1 inhibitor, decreased the rate of PB1 extrusion. Moreover, D4476 resulted in giant polar body extrusion, oocyte pro-MI arrest, chromosome congression failure and impairment of embryo developmental potential. In addition, we employed pyrvinium pamoate (PP), an allosteric activator of CK1α, to enhance CK1α activity in oocytes. Supplementation of PP induced oocyte meiotic maturation failure, severe congression abnormalities and misalignment of chromosomes. Taken together, our study for the first time demonstrates that CK1α is required for chromosome alignment and segregation during oocyte meiotic maturation and early embryo development.