RESUMEN
Actin-interacting protein 1 (AIP1), also known as WD repeat-containing protein 1 (WDR1), is ubiquitous in eukaryotic organisms, and it plays critical roles in the dynamic reorganization of the actin cytoskeleton. However, the biological function and mechanism of AIP1 in mammalian oocyte maturation is still largely unclear. In this study, we demonstrated that AIP1 boosts ADF/Cofilin activity in mouse oocytes. AIP1 is primarily distributed around the spindle region during oocyte maturation, and its depletion impairs meiotic spindle migration and asymmetric division. The knockdown of AIP1 resulted in the gathering of a large number of actin-positive patches around the spindle region. This effect was reduced by human AIP1 (hAIP1) or Cofilin (S3A) expression. AIP1 knockdown also reduced the phosphorylation of Cofilin near the spindle, indicating that AIP1 interacts with ADF/Cofilin-decorated actin filaments and enhances filament disassembly. Moreover, the deletion of AIP1 disrupts Cofilin localization in metaphase I (MI) and induces cytokinesis defects in metaphase II (MII). Taken together, our results provide evidence that AIP1 promotes actin dynamics and cytokinesis via Cofilin in the gametes of female mice.
Asunto(s)
Factores Despolimerizantes de la Actina/metabolismo , Citocinesis/fisiología , Metafase/fisiología , Oocitos/metabolismo , Proteínas Activadoras de ras GTPasa/metabolismo , Actinas/metabolismo , Animales , Células Cultivadas , Femenino , Humanos , Ratones , Ratones Endogámicos ICR , Fosforilación/fisiología , Huso Acromático/metabolismoRESUMEN
Homologous recombination (HR) plays a critical role in facilitating replication fork progression when the polymerase complex encounters a blocking DNA lesion, and it also serves as the primary mechanism for error-free DNA repair of double-stranded breaks. DNA repair protein RAD51 homolog 1 (RAD51) plays a central role in HR. However, the role of RAD51 during porcine early embryo development is unknown. In the present study, we examined whether RAD51 is involved in the regulation of early embryonic development of porcine parthenotes. We found that inhibition of RAD51 delayed cleavage and ceased development before the blastocyst stage. Disrupting RAD51 activity with RNAi or an inhibitor induces sustained DNA damage, as demonstrated by the formation of distinct γH2AX foci in nuclei of four-cell embryos. Inhibiting RAD51 triggers a DNA damage checkpoint by activating the ataxia telangiectasia mutated (ATM)-p53-p21 pathway. Furthermore, RAD51 inhibition caused apoptosis, reactive oxygen species accumulation, abnormal mitochondrial distribution and decreased pluripotent gene expression in blastocysts. Thus, our results indicate that RAD51 is required for proper porcine parthenogenetic activation (PA) embryo development.
Asunto(s)
Blastocisto/efectos de los fármacos , Desarrollo Embrionario/efectos de los fármacos , Recombinasa Rad51/antagonistas & inhibidores , Animales , Apoptosis/efectos de los fármacos , Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Blastocisto/metabolismo , Reparación del ADN/efectos de los fármacos , Femenino , Embarazo , Recombinasa Rad51/metabolismo , Transducción de Señal/efectos de los fármacos , Porcinos , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
Fatty acid synthase (FAS) is an important enzyme involved in the de novo synthesis of long-chain fatty acids. During development, the function of FAS in growth is greater than that in energy storage pathways; therefore, we hypothesized that knockout of FAS would affect early embryonic development owing to the induction of endoplasmic reticulum (ER) stress. In the present study, the function of FAS was studied using the CRISPR (clustered regularly interspaced short palindromic repeats)/ CRISPR-associated protein 9 (Cas9) system. Cas9 and single-guide RNA (sgRNA) were injected into parthenotes to decrease the number of FAS-positive embryos. The efficiency of knockout was assayed by DNA sequencing. We found that FAS knockout caused excessive production of reactive oxygen species (ROS). Excess ROS induced ER stress, resulting in activation of the adaptive unfolded protein response (UPR). FAS knockout caused splicing of the X-box binding protein 1 gene (XBP1) and expression of spliced XBP1 mRNA. In addition, FAS knockout caused phosphorylation of PKR-like ER kinase (PERK), and an increase in the mRNA expression of the ER stress-regulated genes, activating transcription factor 4 (ATF4), and C/EBP homologous protein (CHOP). Finally, Ca2+ was released from the ER and taken up by the mitochondria. As the ER stress became intolerable, apoptosis was initiated. These results demonstrate that FAS knockout induced ROS generation, which mediated the activation of UPR via the ER stress, ultimately leading to apoptosis induction.
Asunto(s)
Apoptosis/genética , Estrés del Retículo Endoplásmico/genética , Ácido Graso Sintasas/genética , Proteína 1 de Unión a la X-Box/genética , Factor de Transcripción Activador 4/genética , Animales , Desarrollo Embrionario/genética , Retículo Endoplásmico/genética , Femenino , Técnicas de Inactivación de Genes , Embarazo , Especies Reactivas de Oxígeno/metabolismo , Transducción de Señal/genética , Porcinos , Factor de Transcripción CHOP/genética , Respuesta de Proteína Desplegada/genéticaRESUMEN
Coxsackie virus and adenovirus receptor (CXADR) is a member of the immunoglobulin superfamily as well as a member of the junctional adhesion molecule family of adhesion receptor. In human pre-implantation embryos, CXADR was detected and co-localized with tight junction (TJ) proteins on the membrane of the trophectoderm. However, its physiological roles were not elucidated in terms of blastocyst formation. Here, we reported expression patterns and biological functions of CXADR in porcine pre-implantation embryos. The transcripts of CXADR were detected at all stages of pre-implantation. Particularly, its expression dramatically increased and preferentially localized at the edge of cell-cell contacts, rather than in the nucleus from the eight-cell stage onwards. CXADR expression was knocked down (KD) by microinjecting double-stranded RNA into one-cell parthenotes. The vast majority of CXADR KD embryos failed to develop to the blastocyst stage, and a few developed KD blastocysts did not expand fully. Analysis of adherens junction (AJ)- and TJ-associated genes/proteins using qRT-PCR, immunocytochemistry and assessment of TJ permeability using FITC-dextran uptake assay revealed that the developmental failure and relatively small cavities are attributed to the defects of TJ assembly. In summary, CXADR is necessary for the AJ and TJ assembly/biogenesis during pre-implantation development.
Asunto(s)
Uniones Adherentes/metabolismo , Blastocisto/citología , Blastocisto/metabolismo , Proteína de la Membrana Similar al Receptor de Coxsackie y Adenovirus/metabolismo , Regulación del Desarrollo de la Expresión Génica , Uniones Estrechas/metabolismo , Animales , Western Blotting , Proteína de la Membrana Similar al Receptor de Coxsackie y Adenovirus/genética , Humanos , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , PorcinosRESUMEN
Measurements of the three-dimensional (3D) structure of spermatozoon are crucial for the study of developmental biology and for the evaluation of in vitro fertilization. Here, we present 3D label-free imaging of individual spermatozoon and perform quantitative analysis of bovine, porcine, and mouse spermatozoa morphologies using refractive index tomography. Various morphological and biophysical properties were determined, including the internal structure, volume, surface area, concentration, and dry matter mass of individual spermatozoon. Furthermore, Holstein cows and Korean native cattle spermatozoa were systematically analyzed and revealed significant differences in spermatozoa head length, head width, midpiece length, and tail length between the two breeds. This label-free imaging approach provides a new technique for understanding the physiology of spermatozoa.
Asunto(s)
Imagenología Tridimensional , Espermatozoides/citología , Animales , Bovinos , Masculino , Refractometría , Especificidad de la Especie , Espermatozoides/metabolismoRESUMEN
Autophagy is an essential cellular mechanism that degrades cytoplasmic proteins and organelles to recycle their components; however, the contribution of autophagy during meiosis has not been studied in porcine oocytes maturing in vitro. In this study, we observed that the autophagy-related gene, LC3, was expressed in porcine oocytes during maturation for 44 h in vitro. Knockdown of the autophagy-related gene, BECN1, reduced both BECN1 and LC3 protein expression levels. Moreover, BECN1 knockdown and treatment with the autophagy inhibitor, LY294002, during maturation of porcine oocytes in vitro impaired polar body extrusion, disturbed mitochondrial function, triggered the DNA damage response, and induced early apoptosis in porcine oocytes. Autophagy inhibition during oocyte maturation also impaired the further developmental potential of porcine oocytes. These results indicate that autophagy is required for the in vitro maturation of porcine oocytes.
Asunto(s)
Autofagia , Meiosis , Oocitos/citología , Animales , Apoptosis , Daño del ADN , Femenino , Espacio Intracelular/metabolismo , Potencial de la Membrana Mitocondrial , Especies Reactivas de Oxígeno/metabolismo , PorcinosRESUMEN
Polo-like kinaseâ 1 (PLK1) plays crucial roles in various stages of oocyte maturation. Recently, we reported that the peptidomimetic compound AB103-8, which targets the polo box domain (PBD) of PLK1, affects oocyte meiotic maturation and the resumption of meiosis. However, to overcome the drawbacks of peptidic compounds, we designed and synthesized a series of pyrrole-based small-molecule inhibitors and tested them for their effects on the rates of porcine oocyte maturation. Among them, the macrocyclic compound (E/Z)-3-(2,16-dioxo-19-(4-phenylbutyl)-3,19-diazabicyclo[15.2.1]icosa-1(20),6,17-trien-3-yl)propyl dihydrogen phosphate (4) showed the highest inhibitory activity with enhanced inhibition against embryonic blastocyst formation. Furthermore, the addition of this compound to culture media efficiently blocked the maturation of porcine and mouse oocytes, indicating its ability to penetrate the zona pellucida and cell membrane. We investigated mouse oocytes treated with compound 4, and the resulting impairment of spindle formation confirmed PLK1 inhibition. Finally, molecular modeling studies with PLK1 PBD also confirmed the presence of significant interactions between compound 4 and PLK1 PBD binding pocket residues, including those in the phosphate, tyrosine-rich, and pyrrolidine binding pockets. Collectively, these results suggest that the macrocyclic compound 4 may serve as a promising template for the development of novel contraceptive agents.
Asunto(s)
Proteínas de Ciclo Celular/antagonistas & inhibidores , Compuestos Macrocíclicos/farmacología , Oocitos/efectos de los fármacos , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Proto-Oncogénicas/antagonistas & inhibidores , Pirroles/farmacología , Animales , Compuestos de Azabiciclo/farmacología , Permeabilidad de la Membrana Celular , Compuestos Macrocíclicos/síntesis química , Compuestos Macrocíclicos/metabolismo , Ratones , Simulación del Acoplamiento Molecular , Oligopéptidos/farmacología , Organofosfatos/síntesis química , Organofosfatos/farmacología , Dominios Proteicos , Pirroles/síntesis química , Pirroles/metabolismo , Huso Acromático/efectos de los fármacos , Huso Acromático/fisiología , Porcinos , Zona Pelúcida/efectos de los fármacos , Zona Pelúcida/fisiología , Quinasa Tipo Polo 1RESUMEN
Progesterone (P4), an ovarian steroid hormone, is an important regulator of female reproduction. In this study, we explored the influence of progesterone on porcine oocyte nuclear maturation and cytoplasmic maturation and development in vitro. We found that the presence of P4 during oocyte maturation did not inhibit polar body extrusions but significantly increased glutathione and decreased reactive oxygen species (ROS) levels relative to that in control groups. The incidence of parthenogenetically activated oocytes that could develop to the blastocyst stage was higher (p < 0.05) when oocytes were exposed to P4 as compared to that in the controls. Cell numbers were increased in the P4-treated groups. Further, the P4-specific inhibitor mifepristone (RU486) prevented porcine oocyte maturation, as represented by the reduced incidence (p < 0.05) of oocyte first polar body extrusions. RU486 affected maturation promoting factor (MPF) activity and maternal mRNA polyadenylation status. In general, these data show that P4 influences the cytoplasmic maturation of porcine oocytes, at least partially, by decreasing their polyadenylation, thereby altering maternal gene expression.
RESUMEN
The purpose of this study was to investigate the effect of antifreeze glycoprotein 8 (AFGP8) supplementation during vitrification on the survival, fertilization, and embryonic development of bovine oocytes and the underlying molecular mechanism(s). Survival, fertilization, early embryonic development, apoptosis, DNA double-strand breaks, reactive oxygen species levels, meiotic cytoskeleton assembly, chromosome alignment, and energy status of mitochondria were measured in the present experiments. Compared with that in the nonsupplemented group; survival, monospermy, blastocyst formation rates, and blastomere counts were significantly higher in the AFGP8-supplemented animals. Oocytes of the latter group also presented fewer double-strand breaks and lower cathepsin B and caspase activities. Rates of normal spindle organization and chromosome alignment, actin filament impairment, and mitochondrial distribution were significantly higher in the AFGP8-supplemented group. In addition, intracellular reactive oxygen species levels significantly decreased in the AFGP8-supplemented groups, maintaining a higher ΔΨm than that in the nonsupplemented group. Taken together, these results indicated that supplementation with AFGP8 during vitrification has a protective effect on bovine oocytes against chilling injury.
Asunto(s)
Proteínas Anticongelantes/farmacología , Bovinos/fisiología , Criopreservación/veterinaria , Oocitos/efectos de los fármacos , Oocitos/fisiología , Vitrificación , Animales , Blastocisto , Técnicas de Cultivo de Célula , Técnicas de Cultivo de Embriones , Desarrollo Embrionario , Fertilización In Vitro/veterinariaRESUMEN
In the present study, we investigated the potential role of glucose and pyruvate in the cytoplasmic maturation of porcine oocytes by investigating the effect of glucose and/or pyruvate supplementation, in the presence or absence of 10% porcine follicular fluid (PFF), on meiotic maturation and subsequent embryo development. In the absence of 10% PFF, without exogenous addition of glucose and pyruvate, the medium seemed unable to support maturation. In the presence of 10% PFF, the addition of 5.6 mM glucose and/or 2 mM pyruvate during in vitro maturation of cumulus enclosed oocytes increased MII oocyte and blastocyst rates. In contrast, oocytes denuded of cumulus cells were not able to take full advantage of the glucose in the medium, as only pyruvate was able to increase the MII rate and the subsequent early embryo developmental ability. Treatment of cumulus enclosed oocytes undergoing maturation with 200 µM dehydroepiandrosterone (DHEA), a pentose phosphate pathway inhibitor, or 2 µM iodoacetate (IA), a glycolysis inhibitor, significantly reduced GHS, intra-oocyte ATP, maternal gene expression, and MPF activity levels. DHEA was also able to increase ROS and reduce the levels of NADPH. Moreover, blastocysts of the DHEA- or IA-treated groups presented higher apoptosis rates and markedly lower cell proliferation cell rates than those of the non-treated group. In conclusion, our results suggest that oocytes maturing in the presence of 10% PFF can make full use of energy sources through glucose metabolism only when they are accompanied by cumulus cells, and that pentose phosphate pathway (PPP) and glycolysis promote porcine oocyte cytoplasmic maturation by supplying energy, regulating maternal gene expression, and controlling MPF activity.
Asunto(s)
Regulación de la Expresión Génica/fisiología , Glucosa/metabolismo , Glucólisis/fisiología , Meiosis/fisiología , Oocitos/metabolismo , Animales , Apoptosis/efectos de los fármacos , Apoptosis/fisiología , Células del Cúmulo/citología , Células del Cúmulo/metabolismo , Femenino , Regulación de la Expresión Génica/efectos de los fármacos , Glucólisis/efectos de los fármacos , Ácido Yodoacético/farmacología , Meiosis/efectos de los fármacos , Oocitos/citología , Ácido Pirúvico/metabolismo , Ácido Pirúvico/farmacología , Especies Reactivas de Oxígeno/metabolismo , PorcinosRESUMEN
In a mammalian oocyte, completion of meiosis is suspended until fertilization by a sperm, and the cell cycle is arrested by a biochemical activity called cytostatic factor (CSF). Emi2 is one of the CSFs, and it maintains the protein level of maturation promoting factor (MPF) by inhibiting ubiquitin ligase anaphase promoting complex/cyclosome (APC/C). Degradation of Emi2 via ubiquitin-mediated proteolysis after fertilization requires phosphorylation by Polo-like kinase 1 (Plk1). Therefore, recognition and phosphorylation of Emi2 by Plk1 are crucial steps for cell cycle resumption, but the binding mode of Emi2 and Plk1 is poorly understood. Using biochemical assays and X-ray crystallography, we found that two phosphorylated threonines (Thr(152) and Thr(176)) in Emi2 are each responsible for the recruitment of one Plk1 molecule by binding to its C-terminal polo box domain (PBD). We also found that meiotic maturation and meiosis resumption via parthenogenetic activation were impaired when Emi2 interaction with Plk1-PBD was blocked by a peptidomimetic called 103-8. Because of the inherent promiscuity of kinase inhibitors, our results suggest that targeting PBD of Plk1 may be an effective strategy for the development of novel and specific contraceptive agents that block oocyte maturation and/or fertilization.
Asunto(s)
Proteínas de Ciclo Celular/química , Proteínas F-Box/química , Peptidomiméticos/química , Dominios y Motivos de Interacción de Proteínas , Proteínas Serina-Treonina Quinasas/química , Proteínas Proto-Oncogénicas/química , Animales , Sitios de Unión , Proteínas de Ciclo Celular/metabolismo , Diferenciación Celular/efectos de los fármacos , Proteínas F-Box/metabolismo , Fertilización/efectos de los fármacos , Meiosis/efectos de los fármacos , Mesotelina , Ratones , Modelos Moleculares , Oocitos/citología , Oocitos/efectos de los fármacos , Oocitos/metabolismo , Peptidomiméticos/administración & dosificación , Peptidomiméticos/farmacología , Fosforilación , Unión Proteica , Conformación Proteica , Proteínas Serina-Treonina Quinasas/metabolismo , Proteolisis , Proteínas Proto-Oncogénicas/metabolismo , Huso Acromático/metabolismo , Relación Estructura-Actividad , Xenopus , Quinasa Tipo Polo 1RESUMEN
To determine whether exogenous amino acids affect gene transcription patterns in parthenogenetic porcine embryos, we investigated the effects of amino acid mixtures in culture medium. Parthenogenetic embryos were cultured in PZM3 medium under four experimental conditions: 1) control (no amino acids except L-glutamine and taurine); 2) nonessential amino acids (NEAA); 3) essential amino acids (EAA); and 4) NEAA and EAA. The rate of development of embryos to the four-cell stage was not affected by treatment. However, fewer (P<0.05) embryos cultured with EAA (12.8%) reached the blastocyst stage as compared with the control group (25.6%) and NEAA group (30.3%). Based on these findings, we identified genes with altered expression in parthenogenetic embryos exposed to medium with or without EAAs. The results indicated that EAA influenced gene expression patterns, particularly those of imprinted genes (e.g., H19, IGF2R, PEG1, XIST). However, NEAAs did not affect impaired imprinted gene expressions induced by EAA. The results also showed that mechanistic target of rapamycin (MTOR) mRNA expression was significantly increased by EAA alone as compared with control cultures, and that the combined treatment with NEAA and EAA did not differ significantly from those of control cultures. Our results revealed that gene transcription levels in porcine embryos changed differentially depending on the presence of EAA or NEAA. However, the changes in the H19 mRNA observed in the parthenogenetic blastocysts expression level was not related to the DNA methylation status in the IGF2/H19 domain. The addition of exogenous amino acid mixtures affected not only early embryonic development, but also gene transcription levels, particularly those of imprinted genes. However, this study did not reveal how amino acids affect expression of imprinted genes under the culture conditions used. Further studies are thus required to fully evaluate how amino acids affect transcriptional regulation in porcine embryos.