RESUMO
Although fusion of nucleoli was observed during pronuclear development of zygotes and the behavior of nucleoli in pronuclei has been suggested as an indicator of embryonic developmental potential, the mechanism for nucleolar fusion is unclear. Although both cytoskeleton and the nucleolus are important cellular entities, there are no special reports on the relationship between the two. Role of cytoskeleton in regulating fusion of nucleoli was studied using the activated mouse oocyte model. Mouse oocytes were cultured for 6 h in activating medium (Ca²âº-free CZB medium containing 10 mM SrCl2) supplemented with or without inhibitors for cytoskeleton or protein synthesis before pronuclear formation, nucleolar fusion, and the activity of maturation-promoting factor (MPF) were examined. Whereas treatment with microfilament inhibitor cytochalasin D or B or intermediate filament inhibitor acrylamide suppressed nucleolar fusion efficiently, treatment with microtubule inhibitor demecolcine or nocodazole or protein synthesis inhibitor cycloheximide had no effect. The cytochalasin D- or acrylamide-sensitive temporal window coincided well with the reported temporal window for nucleolar fusion in activated oocytes. Whereas a continuous incubation with demecolcine prevented pronuclear formation, pronuclei formed normally when demecolcine was excluded during the first hour of activation treatment when the MPF activity dropped dramatically. The results suggest that 1) microfilaments and intermediate filaments but not microtubules support nucleolar fusion, 2) proteins required for nucleolar fusion including microfilaments and intermediate filaments are not de novo synthesized, and 3) microtubule disruption prevents pronuclear formation by activating MPF.
Assuntos
Nucléolo Celular/metabolismo , Citoesqueleto/metabolismo , Fator Promotor de Maturação/metabolismo , Oócitos/citologia , Oogênese , Citoesqueleto de Actina/efeitos dos fármacos , Citoesqueleto de Actina/metabolismo , Animais , Nucléolo Celular/efeitos dos fármacos , Citoesqueleto/efeitos dos fármacos , Ectogênese/efeitos dos fármacos , Técnicas de Cultura Embrionária , Feminino , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Técnicas de Maturação in Vitro de Oócitos , Filamentos Intermediários/efeitos dos fármacos , Filamentos Intermediários/metabolismo , Masculino , Fator Promotor de Maturação/antagonistas & inibidores , Fusão de Membrana/efeitos dos fármacos , Mesotelina , Camundongos Endogâmicos , Microtúbulos/efeitos dos fármacos , Microtúbulos/metabolismo , Oócitos/efeitos dos fármacos , Oócitos/metabolismo , Oogênese/efeitos dos fármacos , Inibidores da Síntese de Proteínas/farmacologia , Estrôncio/farmacologia , Moduladores de Tubulina/farmacologiaRESUMO
In this study, using a mouse model, we tested the hypothesis that restraint stress would impair the developmental potential of oocytes by causing oxidative stress and that antioxidant supplementation could overcome the adverse effect of stress-induced oxidative stress. Female mice were subjected to restraint stress for 24âh starting 24âh after equine chorionic gonadotropin injection. At the end of stress exposure, mice were either killed to recover oocytes for in vitro maturation (IVM) or injected with human chorionic gonadotropin and caged with male mice to observe in vivo development. The effect of antioxidants was tested in vitro by adding them to IVM medium or in vivo by maternal injection immediately before restraint stress exposure. Assays carried out to determine total oxidant and antioxidant status, oxidative stress index, and reactive oxygen species (ROS) and glutathione levels indicated that restraint stress increased oxidative stress in mouse serum, ovaries, and oocytes. Whereas the percentage of blastocysts and number of cells per blastocyst decreased significantly in oocytes from restraint-stressed mice, addition of antioxidants to IVM medium significantly improved their blastocyst development. Supplementation of cystine and cysteamine to IVM medium reduced ROS levels and aneuploidy while increasing glutathione synthesis and improving pre- and postimplantation development of oocytes from restraint-stressed mice. Furthermore, injection of the antioxidant epigallocatechin gallate into restraint-stressed mice significantly improved the blastocyst formation and postimplantation development of their oocytes. In conclusion, restraint stress at the oocyte prematuration stage impaired the developmental potential of oocytes by increasing oxidative stress and addition of antioxidants to IVM medium or maternal antioxidant injection overcame the detrimental effect of stress-induced oxidative stress. The data reported herein are helpful when making attempts to increase the chances of a successful outcome in human IVF, because restraint was applied at a stage similar to the FSH stimulation period in a human IVF program.
Assuntos
Antioxidantes/administração & dosagem , Citoproteção/efeitos dos fármacos , Oócitos/efeitos dos fármacos , Estresse Oxidativo/fisiologia , Estresse Psicológico/metabolismo , Animais , Células Cultivadas , Cisteamina/administração & dosagem , Cistina/administração & dosagem , Suplementos Nutricionais , Embrião de Mamíferos , Desenvolvimento Embrionário/efeitos dos fármacos , Feminino , Masculino , Camundongos , Oócitos/fisiologia , Gravidez , Restrição Física/psicologiaRESUMO
Studies in both humans and animals suggest detrimental effects of psychological stress on reproduction. Although our recent study shows that maternal-restraint stress diminishes oocyte developmental potential, the mechanism behind this effect is unknown. This prompted us to study the potential role of maternal-restraint stress in the genesis of aneuploidy during meiosis I. At 24 h after equine chorionic gonadotropin injection, mice were subjected to restraint stress for 24 h. After the restraint, some mice were killed to recover immature oocytes for in vitro maturation, while others were injected with human chorionic gonadotropin to recover in vivo matured oocytes. Analysis on chromosome complements of both mature oocytes and parthenotes confirmed that maternal restraint increased aneuploidy in both in vivo and in vitro matured oocytes and that the percentage of aneuploid oocytes were three times higher in the earlier matured oocytes than in the later matured ones. Further observations indicated that maternal restraint 1) impaired metaphase I (MI) spindle assembly while inhibiting MAPK activities, 2) accelerated progression of anaphase I while down-regulating the expression of spindle assembly checkpoint (SAC) proteins, and 3) induced intraoocyte oxidative stress. The following possible model was proposed to explain the results. Maternal-restraint stress increased oocyte aneuploidy by impairing MI spindle assembly and decreasing the SAC. Whereas abnormal spindles would affect centromere attachments, a reduction in SAC would accelerate the anaphase I progression. Failure of centromere attachment, together with the hastened anaphase, would result in nondisjunction of the unattached chromosomes. Furthermore, maternal-restraint stress might also impair spindle assembly and SAC function by inducing intraoocyte oxidative stress, which would then reduce MAPK activity, a critical regulator of microtubule assembly and the establishment and maintenance of the SAC during oocyte maturation.
Assuntos
Aneuploidia , Pontos de Checagem da Fase M do Ciclo Celular/fisiologia , Metáfase/fisiologia , Oócitos/citologia , Fuso Acromático/fisiologia , Estresse Psicológico/fisiopatologia , Anáfase/fisiologia , Animais , Centrômero/fisiologia , Gonadotropina Coriônica/farmacologia , Feminino , Técnicas In Vitro , Camundongos , Camundongos Endogâmicos , Quinases de Proteína Quinase Ativadas por Mitógeno/fisiologia , Modelos Animais , Oócitos/efeitos dos fármacos , Estresse Oxidativo/fisiologia , Restrição FísicaRESUMO
Fusion of nucleoli or nucleolus precursor bodies (NPBs) has been observed during somatic cell interphase and pronuclear development of human zygotes; however, the underlying mechanism is unknown. NPB fusion and its regulation by mitogen-activated protein kinase (MAPK) and maturation-promoting factor (MPF) were studied in activated mouse oocytes. Small NPBs appeared about 4 h after ethanol activation, and took about 1.5 h to fuse into a large NPB, which persisted for about 10 h before disappearance. Analysis of the temporal windows for kinase action indicated that a high MAPK activity during the first 2 h and a low MPF activity during the first 3-4 h after activation were essential for subsequent NPB fusion. A preactivation decline in MAPK activity was associated with decreased NPB fusion following activation of aged oocytes. While MAPK inactivation by regulator U0126 prevented NPB fusion in oocytes activated by ethanol or 5 min Sr2+ treatments, it had no effect on oocytes fertilized or activated by 6 h Sr2+ treatment. In most cases, while rates of pronuclear formation did not differ, rates of NPB fusion differed significantly between different treatments. Our results suggest that: (i) the MAPK and MPF activities at the initial stage of activation regulate NPB fusion after pronuclear formation; (ii) pronuclear assembly and NPB fusion are two separable events that might be controlled by different mechanisms; and (iii) high MAPK activity and low MPF activity at the initial stage of activation is essential for NPB fusion when only one calcium rise is induced by ethanol, while inhibition of MAPK activity does not affect NPB fusion when the repetitive intracellular Ca2+ rises are induced after fertilization.
Assuntos
Nucléolo Celular/metabolismo , Fator Promotor de Maturação/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Oócitos/metabolismo , Animais , Butadienos/farmacologia , Nucléolo Celular/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Feminino , Mesotelina , Camundongos , Nitrilas/farmacologia , Oócitos/efeitos dos fármacosRESUMO
In this study, microRNA (miRNA) profiles in postovulatory aging mouse oocytes were analyzed by microarray screening and RT-qPCR. Hierarchical cluster analysis on the microarray data and KEGG pathway enrichment analysis on the mRNAs targeted by differentially expressed (DE) miRNAs between two adjacent egg-ages suggest that while only a mild alteration in miRNA expression occurred from 13 to 18 h, a great change took place from 18 to 24 h post hCG injection. Theoretical exploration on functions of the predicted target genes suggest that KEGG pathways enriched by 13-18 h DE miRNAs are correlated with early events of oocyte aging while pathways most enriched by 18-24 h or 24-30 h DE miRNAs are correlated with the late symptoms of aged oocytes. Experimental verification on functions of the key proteins predicted by the KEGG analysis and injection of miR-98 mimics or inhibitors further confirmed that miRNAs played stimulatory/inhibitory roles in postovulatory oocyte aging. In conclusion, marked changes in miRNA expression are associated with significant alterations in function and morphology of postovulatory aging oocytes.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento/genética , MicroRNAs/biossíntese , Oócitos/fisiologia , Animais , Cálcio/metabolismo , Caspase 3/biossíntese , Caspase 3/genética , Biologia Computacional , Feminino , Perfilação da Expressão Gênica , Camundongos , MicroRNAs/antagonistas & inibidores , MicroRNAs/genética , Análise em Microsséries , Oócitos/metabolismo , Ovulação , Reação em Cadeia da Polimerase , Transdução de Sinais/genéticaRESUMO
The developmental capacity of in vitro-matured (IVM) oocytes is markedly lower than that of their in vivo-matured (IVO) counterparts, suggesting the need for optimization of IVM protocols in different species. There are few studies on IVM of rat oocytes, and there are even fewer attempts to improve ooplasmic maturation compared to those reported in other species. Furthermore, rat oocytes are well known to undergo spontaneous activation (SA) after leaving the oviduct; however, whether IVM rat oocytes have lower SA rates than IVO oocytes and can potentially be used for nuclear transfer is unknown. In this study, we investigated the effects of maturation protocols on cytoplasmic maturation of IVM rat oocytes and observed the possibility to reduce SA by using IVM rat oocytes. Ooplasmic maturation was assessed using multiple markers, including pre- and postimplantation development, meiotic progression, CG redistribution, redox state, and the expression of developmental potential- and apoptosis-related genes. The results showed that the best protocol consisting of modified Tissue Culture Medium-199 (TCM-199) supplemented with cysteamine/cystine and the cumulus cell monolayer dramatically improved the developmental competence of rat oocytes and supported both pre- and postimplantation development and other ooplasmic maturation makers to levels similar to that observed in ovulated oocytes. Rates of SA were significantly lower in IVM oocytes than in IVO oocytes when observed at the same intervals after nuclear maturation. In conclusion, we have optimized protocols for IVM of rat oocytes that sustain ooplasmic maturation to a level similar to ovulated oocytes. The results suggest that IVM rat oocytes might be used to reduce SA for rat cloning.
Assuntos
Desenvolvimento Embrionário , Técnicas de Maturação in Vitro de Oócitos/métodos , Oócitos/citologia , Oócitos/fisiologia , Ovulação/fisiologia , Animais , Calibragem , Células Cultivadas , Feminino , Fertilização in vitro , Técnicas de Maturação in Vitro de Oócitos/normas , Gravidez , Ratos , Ratos Sprague-DawleyRESUMO
AIM: Although previous studies found that 1-time acute stress applied during follicle maturation impaired oocyte competence, it is unknown whether repeated chronic stress, which is known to cause animal behavioral adaptation, would damage oocytes when applied during follicle growth. METHODS AND RESULTS: In this study, female mice were exposed to repeated restraint stress (RRS) or unpredictable stress (UPS) for different days before equine chorionic gonadotropin injection to initiate oocyte prematuration development and to observe effects of different stressors on oocytes in the growing follicles. The results showed that although oocyte pre- and postimplantation development was unaffected when mice were exposed to RRS or UPS once a day for 4 days, development was impaired when mice were exposed to RRS for 8 or more days or to UPS twice a day for 4 days (4 × 2). The 4 × 2 UPS caused more oxidative stress in oocytes and severer apoptosis in antral follicles than did the 4-day RRS. The RRS mice were stressed consistently from days 1 to 23 of restraint, and the stress that a mouse had 4 × 2 UPS was severer than that from 4-day RRS. CONCLUSION: The results suggest that (1) the degree that a stress damages oocytes is the product of duration × severity of the stress; (2) RRS impaired oocyte developmental potential through cumulative effects on growing follicles; and (3) preantral follicles were not as sensitive to stress as antral follicles were.
Assuntos
Oócitos/fisiologia , Folículo Ovariano/crescimento & desenvolvimento , Folículo Ovariano/fisiopatologia , Estresse Psicológico/fisiopatologia , Animais , Ansiedade/etiologia , Ansiedade/fisiopatologia , Apoptose , Peso Corporal , Feminino , Glucocorticoides/sangue , Glutationa/metabolismo , Hidrocortisona/sangue , Camundongos , Oócitos/metabolismo , Restrição Física , Estresse Psicológico/complicaçõesRESUMO
Efforts to improve the quality of in vitro matured oocytes by blocking germinal vesicle breakdown (GVBD) and allowing more time for ooplasmic maturation have achieved little due to a lack of knowledge on the molecular events during GVBD blocking. Such knowledge is also important for studies aimed at regulating gene expression in maturing oocytes prior to GVBD. We studied species difference and signaling pathways leading to the carrying-over effect of GVBD blocking on post-blocking meiotic progression (PBMP). Overall, GVBD-blocking with roscovitine decelerated PBMP of mouse oocytes but accelerated that of pig oocytes. During blocking culture, whereas cyclin B of pig oocytes increased continuously, that of mouse oocytes declined first and then increased slowly. In both species, (a) whereas active CDC2A showed a dynamics similar to cyclin B, inactive CDC2A decreased continuously; (b) when oocytes were blocked in blocking medium containing cycloheximide, PBMP was decelerated significantly while cyclin B and active CDC2A decreasing to the lowest level; (c) whereas sodium vanadate in blocking medium reduced PBMP, epidermal growth factor (EGF) in blocking medium accelerated PBMP significantly with no effect on cyclin B levels. In conclusion, the EGF signaling cascade accelerated PBMP by promoting the pre-MPF (M-phase-promoting factor) to MPF conversion during GVBD blocking with roscovitine. The significant difference in PBMP observed between mouse and pig oocytes was caused by species difference in cyclin B dynamics during blocking culture as no species difference was observed in either pre-MPF to MPF conversion or the EGF signaling activity.
Assuntos
Fator Promotor de Maturação/metabolismo , Meiose , Oócitos/fisiologia , Precursores de Proteínas/metabolismo , Transdução de Sinais , Animais , Proteínas de Ciclo Celular/metabolismo , Células Cultivadas , Feminino , Mesotelina , Camundongos , Purinas/farmacologia , Roscovitina , Especificidade da Espécie , Sus scrofaRESUMO
Although oocytes from prepubertal animals are found less competent than oocytes from adults, the underlying mechanisms are poorly understood. Using the mouse oocyte model, this paper has tested the hypothesis that the developmental potential of prepubertal oocytes is compromised due mainly to their impaired potential for glutathione synthesis. Oocytes from prepubertal and adult mice, primed with or without eCG, were matured in vitro and assessed for glutathione synthesis potential, oxidative stress, Ca(2+) reserves, fertilization and in vitro development potential. In unprimed mice, abilities for glutathione synthesis, activation, male pronuclear formation, blastocyst formation, cortical granule migration and polyspermic block were all compromised significantly in prepubertal compared to adult oocytes. Cysteamine and cystine supplementation to maturation medium significantly promoted oocyte glutathione synthesis and blastocyst development but difference due to maternal age remained. Whereas reactive oxygen species (ROS) levels increased, Ca(2+) storage decreased significantly in prepubertal oocytes. Levels of both catalytic and modifier subunits of the γ-glutamylcysteine ligase were significantly lower in prepubertal than in adult oocytes. Maternal eCG priming improved all the parameters and eliminated the age difference. Together, the results have confirmed our hypothesis by showing that prepubertal oocytes have a decreased ability to synthesize glutathione leading to an impaired potential to reduce ROS and to form male pronuclei and blastocysts. The resulting oxidative stress decreases the intracellular Ca(2+) store resulting in impaired activation at fertilization, and damages the microfilament network, which affects cortical granule redistribution leading to polyspermy.
Assuntos
Blastocisto/metabolismo , Glutationa/biossíntese , Oócitos/metabolismo , Maturidade Sexual/fisiologia , Fatores Etários , Animais , Blastocisto/citologia , Blastocisto/efeitos dos fármacos , Cálcio/metabolismo , Células Cultivadas , Gonadotropina Coriônica/farmacologia , Meios de Cultura , Cisteamina/metabolismo , Cisteamina/farmacologia , Cistina/metabolismo , Cistina/farmacologia , Combinação de Medicamentos , Técnicas de Cultura Embrionária , Embrião de Mamíferos , Desenvolvimento Embrionário , Feminino , Fertilização in vitro , Gonadotropinas Equinas/farmacologia , Camundongos , Oócitos/citologia , Oócitos/efeitos dos fármacos , Oócitos/crescimento & desenvolvimento , Estresse Oxidativo , Espécies Reativas de Oxigênio/metabolismoRESUMO
Rat oocytes are well known to undergo spontaneous activation (SA) after leaving the oviduct, but the SA is abortive with oocytes being arrested in metaphase III (MIII) instead of forming pronuclei. This study was designed to investigate the mechanism causing SA and MIII arrest. Whereas few oocytes collected from SD rats at 13 h after hCG injection that showed 100% of mitogen-activated protein kinase (MAPK) activities activated spontaneously, all oocytes recovered 19 h post hCG with MAPK decreased to below 75% underwent SA during in vitro culture. During SA, MAPK first declined to below 45% and then increased again to 80%; the maturation-promoting factor (MPF) activity fluctuated similarly but always began to change ahead of the MAPK activity. In SA oocytes with 75% of MAPK activities, microtubules were disturbed with irregularly pulled chromosomes dispersed over the spindle and the spindle assembly checkpoint (SAC) was activated. When MAPK decreased to 45%, the spindle disintegrated and chromosomes surrounded by microtubules were scattered in the ooplasm. SA oocytes entered MIII and formed several spindle-like structures by 6 h of culture when the MAPK activity re-increased to above 80%. While SA oocytes showed one Ca(2+) rise, Sr(2+)-activated oocytes showed several. Together, the results suggested that SA stimuli triggered SA in rat oocytes by inducing a premature MAPK inactivation, which led to disturbance of spindle microtubules. The microtubule disturbance impaired pulling of chromosomes to the spindle poles, caused spindle disintegration and activated SAC. The increased SAC activity reactivated MPF and thus MAPK, leading to MIII arrest.
Assuntos
Sistema de Sinalização das MAP Quinases , Fator Promotor de Maturação/metabolismo , Oócitos/citologia , Fuso Acromático/metabolismo , Animais , Núcleo Celular/metabolismo , Gonadotropina Coriônica/metabolismo , Feminino , Pontos de Checagem da Fase M do Ciclo Celular , Metáfase , Microscopia Confocal/métodos , Fosforilação , Proteínas Serina-Treonina Quinases/metabolismo , Ratos , Ratos Sprague-Dawley , Proteína Smad2/metabolismo , Fatores de TempoRESUMO
The removal of chromosomes from recipient oocytes is one of the key steps in nuclear transfer cloning. Although microtubule interrupters have been successfully used for oocyte enucleation, their potential side effect on oocyte developmental potential should be considered, and less harmful drugs should be explored for chemical-assisted enucleation. Based on our previous findings that any maturation promoting factor-activating agent induces ooplasmic protrusion without disrupting microtubules, we have studied the feasibility to use caffeine or MG132 for chemical-assisted enucleation. Experiments using goat oocytes showed that treatments for 30 min with 1-mM caffeine or 5-µM MG132-induced ooplasmic protrusions in about 85% of the oocytes, a percentage similar to that achieved with optimal demecolcine treatment. Rates of enucleation, cell fusion and in vitro blastulation were similar among caffeine, MG132, and demecolcine enucleation but significantly higher than blind aspiration. Furthermore, neither rates of pregnancy on days 90 and 120 nor the general rate of live births/embryos transferred differed significantly (p > 0.05) between caffeine and demecolcine enucleation. Although oocytes treated with caffeine did not retract protrusions until 2 h, many oocytes treated with MG132 withdrew protrusions as early as 0.5 h after treatment. The optimal treatment to induce ooplasmic protrusion in 75% pig oocytes was 8-mM caffeine for 60 min. Mouse oocytes responded poorly to demecolcine or caffeine with less than 40% forming inconspicuous protrusions following optimal treatments. It is concluded that caffeine can be used for enucleation of goat and pig oocytes with similar results as demecolcine, and live kids were born after caffeine-assisted enucleation.