RESUMEN
The oocytes of female mammals will undergo aging after ovulation, also known as postovulatory oocyte aging (POA). Until now, the mechanisms of POA have not been fully understood. Although studies have shown that cumulus cells accelerate POA over time, the exact relationship between the two is still unclear. In the study, by employing the methods of mouse cumulus cells and oocytes transcriptome sequencing and experimental verification, we revealed the unique characteristics of cumulus cells and oocytes through ligand-receptor interactions. The results indicate that cumulus cells activated NF-κB signaling in oocytes through the IL1-IL1R1 interaction. Furthermore, it promoted mitochondrial dysfunction, excessive ROS accumulation, and increased early apoptosis, ultimately leading to a decline in the oocyte quality and the appearance of POA. Our results indicate that cumulus cells have a role in accelerating POA, and this result lays a foundation for an in-depth understanding of the molecular mechanism of POA. Moreover, it provides clues for exploring the relationship between cumulus cells and oocytes.
Asunto(s)
Senescencia Celular , Células del Cúmulo , Oocitos , Receptores Tipo I de Interleucina-1 , Animales , Femenino , Ratones , Envejecimiento/metabolismo , Senescencia Celular/fisiología , Células del Cúmulo/metabolismo , Interleucina-1/metabolismo , Mamíferos , Oocitos/metabolismo , Transducción de SeñalRESUMEN
Olaquindox (OLA), a potent antibacterial agent, has been widely used as a feed additive and growth promoter in animal husbandry. Our previous study has shown that OLA administration in female mice could markedly cause sub-fertility. Here we established the model in male mice to investigate the toxic effects of OLA on mammalian spermatozoa quality and fetal development. After continuous 45 days of OLA gavage, the dosage of 60 mg/kg/day (high dose) significantly affected body weight, organ weights and coefficients, and the morphology of the testis seminiferous tubule in male mice. Dosage of 60 mg/kg/day also reduced sperm count, motility, and viability. OLA at both low-dose (5 mg/kg/day) and high-dose induced peroxidation, early apoptosis, and abnormal mitochondrial membrane potential in sperm. Significantly, high-dose OLA impaired in vitro fertilized embryo development, indicated by the decreased percentages of 2-cell and blastocyst formation. Surprisingly, the natural fertility of males was unaffected after OLA gavage, which was indicated by the comparable litter size after mating. However, paternal gavage of OLA significantly decreased the survival rate of the offspring from the age of 4 weeks. In sum, our study showed that OLA gavage in male mice damages sperm quality and offspring survival, illustrating the use of OLA as a feed additive should be strictly restricted.
RESUMEN
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.
Asunto(s)
1-Metil-3-Isobutilxantina/farmacología , Bucladesina/farmacología , Núcleo Celular/metabolismo , Meiosis/efectos de los fármacos , Oocitos/metabolismo , Inhibidores de Fosfodiesterasa/farmacología , Animales , Apoptosis/efectos de los fármacos , Núcleo Celular/efectos de los fármacos , Células Cultivadas , Colforsina/farmacología , AMP Cíclico/metabolismo , Roturas del ADN/efectos de los fármacos , Desarrollo Embrionario/efectos de los fármacos , Femenino , Ratones , Ratones Endogámicos ICR , Oocitos/efectos de los fármacosRESUMEN
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.
Asunto(s)
Desarrollo Embrionario/efectos de los fármacos , Aditivos Alimentarios/toxicidad , Oocitos/efectos de los fármacos , Oogénesis/efectos de los fármacos , Quinoxalinas/toxicidad , Administración Oral , Animales , Apoptosis/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Femenino , Peróxido de Hidrógeno/metabolismo , Ratones , Ratones Endogámicos ICR , Mitocondrias/efectos de los fármacos , Oocitos/metabolismo , Oocitos/patología , Ovario/efectos de los fármacos , Ovario/metabolismo , Oxidación-Reducción , Estrés Oxidativo/efectos de los fármacosRESUMEN
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.
Asunto(s)
Edición Génica , Células Germinativas , Oocitos , Animales , Diferenciación Celular , Embrión de Mamíferos , Femenino , Técnicas de Inactivación de Genes , Mamíferos , Interferencia de ARNRESUMEN
SummaryPropofol is a intravenous anaesthetic most commonly used in ultrasound oocyte retrieval. We studied if the use of propofol had an effect on mouse oocyte maturation, pregnancy, childbirth and progeny and investigated the correlation between propofol side effects and reproductive performance in mice. There was no statistical difference in mating, pregnancy, childbirth, litter size, the number of stillbirths and survival between each group (P>0.05). Propofol also had no effect on polar body extrusion in oocyte maturation as well as on pronucleus formation and, subsequently, early embryo development (P>0.05). An increased concentration of propofol had no effect on this result, although propofol at more than 0.01 mg/ml reduced polar body extrusion. Different concentrations of propofol had no effect on oocyte culture in vitro, pronucleus formation and early embryo development.
Asunto(s)
Anestésicos Intravenosos/farmacología , Desarrollo Embrionario/efectos de los fármacos , Desarrollo Fetal/efectos de los fármacos , Técnicas de Maduración In Vitro de los Oocitos/métodos , Oocitos/crecimiento & desarrollo , Oogénesis/efectos de los fármacos , Propofol/farmacología , Animales , Células Cultivadas , Femenino , Masculino , Ratones , Oocitos/efectos de los fármacos , EmbarazoRESUMEN
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.
Asunto(s)
Embrión de Mamíferos/embriología , Desarrollo Embrionario/fisiología , Cinesinas/metabolismo , Meiosis/fisiología , Adenosina Trifosfato/genética , Adenosina Trifosfato/metabolismo , Animales , Cromosomas de los Mamíferos/genética , Cromosomas de los Mamíferos/metabolismo , Embrión de Mamíferos/citología , Femenino , Cinesinas/genética , Masculino , Ratones , Mitocondrias/genética , Mitocondrias/metabolismo , Oocitos , Cuerpos Polares/metabolismo , Huso Acromático/genética , Huso Acromático/metabolismoRESUMEN
In vitro maturation (IVM) and cryopreservation of goat oocytes are important for establishing a valuable genetic bank for domesticated female animals and improving livestock reproductive efficiency. C-Phycocyanin (PC) is a Spirulina extract with antioxidant, antiinflammatory, and radical scavenging properties. However, whether PC has positive effect on goat oocytes IVM or developmental competence after vitrification is still unknown. In this study, we found that first polar body extrusion (n = 293), cumulus expansion index (n = 269), and parthenogenetic blastocyst formation (n = 281) were facilitated by adding 30 µg/mL PC to the oocyte maturation medium when compared with the control groups and that supplemented with 3, 10, 100 or 300 µg/mL PC (P < 0.05). Although PC supplementation did not affect spindle formation or chromosome alignment (n = 115), it facilitated or improved cortical granules migration (n = 46, P < 0.05), mitochondria distribution (n = 39, P < 0.05), and mitochondrial membrane potential (n = 46, P < 10-4). Meanwhile, supplementation with 30 µg/mL PC in the maturation medium could significantly inhibit the reactive oxygen species accumulation (n = 65, P < 10-4), and cell apoptosis (n = 42, P < 0.05). In addition, PC increased the oocyte mRNA levels of GPX4 (P < 0.01), and decreased the mRNA and protein levels of BAX (P < 0.01). Next, we investigated the effect of PC supplementation in the vitrification solution on oocyte cryopreservation. When compared with the those equilibrate in the vitrification solution without PC, recovered oocytes in the 30 µg/mL PC group showed higher ratios of normal morphology (n = 85, P < 0.05), survival (n = 85, P < 0.05), first polar body extrusion (n = 62, P < 0.05), and parthenogenetic blastocyst formation (n = 107, P < 0.05). Meanwhile, PC supplementation of the vitrification solution increased oocyte mitochondrial membrane potential (n = 53, P < 0.05), decreased the reactive oxygen species accumulation (n = 73, P < 0.05), promoted mitochondria distribution (n = 58, P < 0.05), and inhibited apoptosis (n = 46, P < 10-3). Collectively, our findings suggest that PC improves goat oocyte IVM and vitrification by reducing oxidative stress and early apoptosis, which providing a novel strategy for livestock gamete preservation and utilization.
Asunto(s)
Criopreservación , Cabras , Técnicas de Maduración In Vitro de los Oocitos , Oocitos , Ficocianina , Vitrificación , Animales , Oocitos/efectos de los fármacos , Técnicas de Maduración In Vitro de los Oocitos/veterinaria , Técnicas de Maduración In Vitro de los Oocitos/métodos , Vitrificación/efectos de los fármacos , Criopreservación/veterinaria , Criopreservación/métodos , Ficocianina/farmacología , Femenino , Especies Reactivas de Oxígeno/metabolismo , Potencial de la Membrana Mitocondrial/efectos de los fármacosRESUMEN
PURPOSE: The cryopreservation process damages oocytes and impairs development potential. As a potent antioxidant, C-phycocyanin (PC) regulates reproductive performance. However, its beneficial effects on vitrified human oocytes remain unknown. METHODS: In this study, human GV-stage oocytes obtained from controlled ovarian hyperstimulation (COH) cycles were randomly allocated to three groups: fresh oocyte without freezing (F group), vitrification in medium supplemented with PC (P group), and vitrification in medium without PC as control group (C group). After warming, viable oocytes underwent in vitro maturation. RESULTS: Our results showed that 3 µg/mL PC treatment increased the oocyte maturation rate after cryopreservation. We also found that PC treatment maintains the regular morphological features of oocytes. After PC treatment, confocal fluorescence staining showed a significant increase in the mitochondrial membrane potential of the vitrified oocytes, along with a notable decrease in intracellular reactive oxygen species and the early apoptosis rate. Finally, after in vitro maturation and parthenogenetic activation, vitrified oocytes had a higher potential for cleavage and blastocyst formation after PC treatment. CONCLUSION: Our results suggest that PC improves the developmental potential of cryopreserved human GV-stage oocytes by attenuating oxidative stress and early apoptosis and increasing the mitochondrial membrane potential.
Asunto(s)
Criopreservación , Ficocianina , Humanos , Especies Reactivas de Oxígeno/metabolismo , Ficocianina/farmacología , Criopreservación/métodos , Oocitos , VitrificaciónRESUMEN
Clathrin heavy chain 1 (CLTC) has been considered a "moonlighting protein" which acts in membrane trafficking during interphase and in stabilizing spindle fibers during mitosis. However, its roles in meiosis, especially in mammalian oocyte maturation, remain unclear. This study investigated CLTC expression and function in spindle formation and chromosome congression during mouse oocyte meiotic maturation. Our results showed that the expression level of CLTC increased after germinal vesicle breakdown (GVBD) and peaked in the M phase. Immunostaining results showed CLTC distribution throughout the cytoplasm in a cell cycle-dependent manner. Appearance and disappearance of CLTC along with ß-tubulin (TUBB) could be observed during spindle dynamic changes. To explore the relationship between CLTC and microtubule dynamics, oocytes at metaphase were treated with taxol or nocodazole. CLTC colocalized with TUBB at the enlarged spindle and with cytoplasmic asters after taxol treatment; it disassembled and distributed into the cytoplasm along with TUBB after nocodazole treatment. Disruption of CLTC function using stealth siRNA caused a decreased first polar body extrusion rate and extensive spindle formation and chromosome congression defects. Taken together, these results show that CLTC plays an important role in spindle assembly and chromosome congression through a microtubule correlation mechanism during mouse oocyte maturation.
Asunto(s)
Ciclo Celular , Segregación Cromosómica , Cadenas Pesadas de Clatrina/metabolismo , Oocitos/fisiología , Huso Acromático/metabolismo , Animales , Ratones , Tubulina (Proteína)/metabolismoRESUMEN
In oocytes, the cytoplasmic polyadenylation and maternal mRNAs translation is regulated by cis-elements, including polyadenylation signal (PAS) and cytoplasmic polyadenylation element (CPE) in 3'-UTR. Recent studies illustrate non-canonical polyadenylation mechanisms of translational regulation in mouse oocytes, which is different from that in Xenopus oocytes. However, it is still unclear if this regulation in rodent oocytes functions in the domestic animal oocyte. Here, by using sheep as an animal model, we cloned the 3'-UTRs of Cpeb1 or Btg4 and ligated it into the pRK5-Flag-Gfp vector. Variant numbers and positions of PASs and CPEs within the 3'-UTRs were constructed to detect their effects on translational control. After in vitro-transcription and microinjection into sheep fully grown germinal vesicle stage oocytes, the expression efficiency of mRNAs was detected by the GFP and flag expression. Our results show that: (i) PAS located at the proximal end of 3'-UTR can mediate the translation of the maternal mRNAs, as long as they locate far from CPEs; (ii) The proximal PAS has higher efficiency in regulating transcription than the distal one; (iii) increase of PAS number can promote the translational activity more efficiently; (iv) a single CPE located close to PAS (<50 bp) in 3'-UTRs of Cpeb1 or Btg4 could partially repress translation. In 3'-UTRs of Btg4, two CPEs have a higher inhibitory effect, and three CPEs can completely inhibit mRNA translation. These results confirm the existence of the non-canonical mechanism in domestic animal oocytes.
Asunto(s)
Poliadenilación , Biosíntesis de Proteínas , Animales , Ratones , Ovinos/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Oocitos/metabolismo , Citoplasma/metabolismo , Regiones no Traducidas , Regiones no Traducidas 3'RESUMEN
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.
Asunto(s)
Segregación Cromosómica , Meiosis , Animales , Ratones , Ciclosoma-Complejo Promotor de la Anafase/metabolismo , Proteínas de Ciclo Celular/metabolismo , Dinaminas/metabolismo , Cinetocoros/metabolismo , Oocitos/metabolismo , Securina/genética , Securina/metabolismo , Separasa/metabolismoRESUMEN
For in vitro produced embryos generated from in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI) procedure, the intra- and extra-environmental factors during in vitro culture have significant impact on latter embryo development and fetus growth. Assisted hatching (AH), an effective approach to facilitate hatchability for in vitro generated embryos, is an essential step for successful embryo implantation in the uterus. However, regarding the different AH methods reported in clinical practice, it is still unknown whether zona pellucida (ZP) broken is based on AH applied in diverse stages of embryos affect implantation and fetal development. Here, piezo-mediated AH treatments were classified into four categories: (1) drilling one small hole (SH) with a diameter of 10 µm on ZP (SH); (2) drilling one large hole (LH) with a diameter of 40 µm on ZP (LH); (3) made a small area with diameter of 40-µm thinner on ZP [small area thinner (ST)]; (4) made a large area with a diameter of 80-µm thinner [large area thinner (LT)]. These four AH treatments were applied in different stage embryos including two-cell, four-cell, and morula. The most efficient AH approach was chosen according to the final hatch rate at 120 h after fertilization. We found that the approach of SH applied in morula-stage embryos obtained the highest hatch rate. To further investigate if this treatment has any side effect on later development after embryo transfer, we evaluated embryo implantation, gestational period, litter size, and growth. Our results showed that SH applied in morula-stage embryos could facilitate the implantation process and increase litter size. Meanwhile, this approach had no side effect on birth weight, growth, or gender ratio in the offspring. We conclude that drilling a SH on ZP in morula-stage embryos is an effective and reliable AH approach for in vitro cultured embryos in rodent. And this approach is worth further investigating in human-assisted reproductive technology.
RESUMEN
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.
RESUMEN
Mechanisms providing for temporally complex patterns of maternal mRNA translation after fertilization are poorly understood. We employed bioinformatics analysis to compare populations of mRNAs enriched specifically on polysomes at the metaphase II (MII) stage oocyte and late one-cell stages and a detailed deletion/truncation series to identify elements that regulate translation. We used the Bag4 3' untranslated region (UTR) as a model. Bioinformatics analysis revealed one conserved motif, subsequently confirmed by functional studies to be a key translation repressor element. The deletion/truncation studies revealed additional regulatory motifs, most notably a strong translation activator element of <30 nt. Analysis of mRNA secondary structure suggests that secondary structure plays a key role in translation repression. Additional bioinformatics analysis of the regulated mRNA population revealed a diverse collection of regulatory motifs found in small numbers of mRNAs, highlighting a high degree of sequence diversity and combinatorial complexity in the overall control of the maternal mRNA population. We conclude that translational control after fertilization is driven primarily by negative regulatory mechanisms opposing strong translational activators, with stage-specific release of the inhibitory influences to permit recruitment. The combination of bioinformatics analysis and deletion/truncation studies provides the necessary approach for dissecting postfertilization translation regulatory mechanisms.
Asunto(s)
Embrión de Mamíferos/fisiología , Regulación del Desarrollo de la Expresión Génica , Biosíntesis de Proteínas , ARN Mensajero Almacenado/metabolismo , Secuencias Reguladoras de Ácidos Nucleicos , Regiones no Traducidas 3' , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Biología Computacional , Embrión de Mamíferos/citología , Ratones , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , ARN Mensajero Almacenado/química , ARN Mensajero Almacenado/genéticaRESUMEN
Embryos produced by somatic cell nuclear transfer (SCNT) display low term developmental potential. This is associated with deficiencies in spindle composition prior to activation and at early mitotic divisions, including failure to assemble certain proteins on the spindle. The protein-deficient spindles are accompanied by chromosome congression defects prior to activation and during the first mitotic divisions of the embryo. The molecular basis for these deficiencies and how they might be avoided are unknown. Proteomic analyses of spindles isolated from normal metaphase II (MII) stage oocytes and SCNT constructs, along with a systematic immunofluorescent survey of known spindle-associated proteins were undertaken. This was the first proteomics study of mammalian oocyte spindles. The study revealed four proteins as being deficient in spindles of SCNT embryos in addition to those previously identified; these were clathrin heavy chain (CLTC), aurora B kinase, dynactin 4, and casein kinase 1 alpha. Due to substantial reduction in CLTC abundance after spindle removal, we undertook functional studies to explore the importance of CLTC in oocyte spindle function and in chromosome congression defects of cloned embryos. Using siRNA knockdown, we demonstrated an essential role for CLTC in chromosome congression during oocyte maturation. We also demonstrated rescue of chromosome congression defects in SCNT embryos at the first mitosis using CLTC mRNA injection. These studies are the first to employ proteomics analyses coupled to functional interventions to rescue a specific molecular defect in cloned embryos.
Asunto(s)
Segregación Cromosómica/efectos de los fármacos , Clonación de Organismos , Oocitos/ultraestructura , Proteómica/métodos , Huso Acromático/química , Animales , Aurora Quinasa B , Aurora Quinasas , Quinasa de la Caseína I/deficiencia , Segregación Cromosómica/genética , Cadenas Pesadas de Clatrina/análisis , Cadenas Pesadas de Clatrina/efectos de los fármacos , Complejo Dinactina , Embrión de Mamíferos , Ratones , Ratones Endogámicos , Proteínas Asociadas a Microtúbulos/deficiencia , Oocitos/química , Proteínas Serina-Treonina Quinasas/deficiencia , Proteínas/análisis , ARN Mensajero/administración & dosificación , ARN Mensajero/uso terapéuticoRESUMEN
Mammalian oocytes remain dormant in the diplotene stage of prophase I until the resumption of meiosis characterized by germinal vesicle breakdown (GVBD) following the preovulatory gonadotropin stimulation. Based on genome-wide analysis of peri-ovulatory DNA microarray to identify paracrine hormone-receptor pairs, we found increases in ovarian transcripts for endothelin-1 and endothelin receptor type A (EDNRA) in response to the preovulatory luteinizing hormone (LH)/human chorionic gonadotropin (hCG) stimulation. Immunohistochemical analyses demonstrated localization of EDNRA in granulosa and cumulus cells. In cultured preovulatory follicles, treatment with endothelin-1 promoted oocyte GVBD. The stimulatory effect of endothelin-1 was blocked by cotreatment with antagonists for the type A, but not related type B, receptor. The stimulatory effect of hCG on GVBD was partially blocked by the same antagonist. The endothelin-1 promotion of GVBD was found to be mediated by the MAPK/ERK pathway but not by the inhibitory G protein. Studies using cumulus-oocyte complexes and denuded oocytes demonstrated that the endothelin-1 actions are mediated by cumulus cells. Furthermore, intrabursal administration with endothelin-1 induced oocyte GVBD in preovulatory follicles. Our findings demonstrate a paracrine role of endothelin-1 in the induction of the resumption of meiosis and provide further understanding on the molecular mechanisms underlying the nuclear maturation of oocytes induced by the preovulatory LH surge.
Asunto(s)
Endotelina-1/fisiología , Meiosis , Oocitos/citología , Comunicación Paracrina/fisiología , Animales , Gonadotropina Coriónica/fisiología , Células del Cúmulo , Femenino , Hormona Luteinizante/fisiología , Ratones , Folículo Ovárico , Receptores de Endotelina/metabolismoRESUMEN
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.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Cromosomas , Citoplasma/fisiología , Desarrollo Embrionario , Meiosis , Oocitos/fisiología , Huso Acromático/fisiología , Animales , Proteínas de Ciclo Celular/genética , Femenino , Cinesinas/genética , Cinesinas/metabolismo , Masculino , Ratones , Ratones Endogámicos ICR , Oocitos/citología , Oogénesis , Embarazo , Espermatozoides/citología , Espermatozoides/fisiología , Cigoto/citología , Cigoto/fisiologíaRESUMEN
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.
Asunto(s)
Implantación del Embrión , Desarrollo Fetal , Zona Pelúcida/fisiología , Animales , Células Cultivadas , Femenino , Tamaño de la Camada , Masculino , Ratones , EmbarazoRESUMEN
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.