RESUMO
Precise regulation of kinetochore-microtubule attachments is essential for successful chromosome segregation. Central to this regulation is Aurora B kinase, which phosphorylates kinetochore substrates to promote microtubule turnover. A critical target of Aurora B is the N-terminal "tail" domain of Hec1, which is a component of the NDC80 complex, a force-transducing link between kinetochores and microtubules. Although Aurora B is regarded as the "master regulator" of kinetochore-microtubule attachment, other mitotic kinases likely contribute to Hec1 phosphorylation. In this study, we demonstrate that Aurora A kinase regulates kinetochore-microtubule dynamics of metaphase chromosomes, and we identify Hec1 S69, a previously uncharacterized phosphorylation target site in the Hec1 tail, as a critical Aurora A substrate for this regulation. Additionally, we demonstrate that Aurora A kinase associates with inner centromere protein (INCENP) during mitosis and that INCENP is competent to drive accumulation of the kinase to the centromere region of mitotic chromosomes. These findings reveal that both Aurora A and B contribute to kinetochore-microtubule attachment dynamics, and they uncover an unexpected role for Aurora A in late mitosis.
Assuntos
Aurora Quinase A/metabolismo , Aurora Quinase B/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Segregação de Cromossomos/fisiologia , Cinetocoros/metabolismo , Microtúbulos/metabolismo , Proteínas Nucleares/metabolismo , Animais , Linhagem Celular Tumoral , Centrômero/metabolismo , Proteínas do Citoesqueleto , Células HeLa , Humanos , Metáfase/fisiologia , Fosforilação , Potoroidae , Ligação Proteica/fisiologia , Fuso Acromático/metabolismoRESUMO
This cohort study evaluated whether rescue spontaneous maturation (RSM) could be a valid method to increase the number of embryos available for transfer and whether transfers with RSM-derived embryos would contribute to clinical outcomes of poor-responder patients in ovarian stimulation cycles. The study included 440 patients undergoing intracytoplasmic sperm injection cycles in which fewer than five metaphase II (MII) oocytes and at least one immature oocyte were retrieved after follicle aspiration. Patients were allocated into two groups based on the injected oocytes' nuclear maturation status: MII group (n=330), in which only embryos derived from MII oocytes were transferred, and RSM group (n=110), in which at least one embryo derived from an RSM oocyte was transferred. No differences between the MII and RSM groups were observed for pregnancy (16.7% versus 16.5%) or miscarriage (25.5% versus 29.4%) rates, respectively. The RSM group had a higher number of transferred embryos (1.87+/-1.24 versus 2.35+/-1.22; P<0.001), a lower embryo transfer cancellation rate (14.5% versus 6.36%; P=0.025) and lower implantation rate (15.4+/-31.5% versus 10.5+/-22.3%; not significant). These findings suggest that RSM did not contribute to the outcomes in poor-responder cycles.
Assuntos
Metáfase/fisiologia , Oócitos/fisiologia , Indução da Ovulação/métodos , Adulto , Estudos de Coortes , Implantação do Embrião , Transferência Embrionária , Feminino , Fertilização/fisiologia , Humanos , Gravidez , Taxa de Gravidez , Injeções de Esperma Intracitoplásmicas/métodosRESUMO
A maturação in vitro (MIV) de oócitos caninos tem sido objeto de inúmeros estudos focados no estabelecimento de um protocolo capaz de elevar os índices de maturação à metáfase II (MII), atualmente considerados baixos. Dentre as inúmeras variáveis atualmente pesquisadas, estão aspectos relacionados às doadoras (idade, raça, fase estral) e às condições de cultivo, como composição ideal do meio de maturação, adição de diversas fontes proteicas, hormônios, fatores de crescimento e agentes antioxidantes, sendo que os resultados atuais mostram-se ainda pouco eficientes em elevar consideravelmente as taxas de MII. Tal fato pode justificar-se nas diversas características peculiares da biologia reprodutiva desta espécie. Desta maneira, a presente revisão tem como escopo apresentar os aspectos gerais destas características, bem como uma compilação dos aspectos atualmente estudados para a maturação oocitária canina.(AU)
The canine oocyte in vitro maturation (IVM) has been the subject of several studies focused on the establishment of a protocol able to increase the maturation rates to metaphase II (MII), currently considered low. Among the many variables currently researched are aspects related to the donors (age, breed, estrous stage) and to the culture conditions, as the ideal composition of the maturation medium, the addition of several protein resources, hormones, growth factors and anti oxidation factors, being that the current results still present little efficiency in increasing considerably the MII rates. Such fact may be justified in the many peculiar characteristics of the reproductive biology of this specie. Therefore, the current review is aiming to present the general aspects of these characteristics, as well as a compilation of the aspects currently studied for canine oocyte maturation.
Assuntos
Animais , Feminino , Adulto , Cães , Transferência Embrionária/métodos , /tendências , Transferência Embrionária/veterinária , Oócitos/transplante , Metáfase/fisiologiaRESUMO
A maturação in vitro (MIV) de oócitos caninos tem sido objeto de inúmeros estudos focados no estabelecimento de um protocolo capaz de elevar os índices de maturação à metáfase II (MII), atualmente considerados baixos. Dentre as inúmeras variáveis atualmente pesquisadas, estão aspectos relacionados às doadoras (idade, raça, fase estral) e às condições de cultivo, como composição ideal do meio de maturação, adição de diversas fontes proteicas, hormônios, fatores de crescimento e agentes antioxidantes, sendo que os resultados atuais mostram-se ainda pouco eficientes em elevar consideravelmente as taxas de MII. Tal fato pode justificar-se nas diversas características peculiares da biologia reprodutiva desta espécie. Desta maneira, a presente revisão tem como escopo apresentar os aspectos gerais destas características, bem como uma compilação dos aspectos atualmente estudados para a maturação oocitária canina.
The canine oocyte in vitro maturation (IVM) has been the subject of several studies focused on the establishment of a protocol able to increase the maturation rates to metaphase II (MII), currently considered low. Among the many variables currently researched are aspects related to the donors (age, breed, estrous stage) and to the culture conditions, as the ideal composition of the maturation medium, the addition of several protein resources, hormones, growth factors and anti oxidation factors, being that the current results still present little efficiency in increasing considerably the MII rates. Such fact may be justified in the many peculiar characteristics of the reproductive biology of this specie. Therefore, the current review is aiming to present the general aspects of these characteristics, as well as a compilation of the aspects currently studied for canine oocyte maturation.
Assuntos
Feminino , Animais , Adulto , Cães , Metáfase/fisiologia , Oócitos/transplante , Transferência Embrionária/métodos , Transferência Embrionária/veterináriaRESUMO
Porcine immature oocyte quality (i.e., that of live oocytes at the germinal vesicle stage) was evaluated according to features of the surrounding cumulus, aiming to establish maturational competence of different subpopulations of such cumulus-oocyte complexes. Six subpopulations were identified: A1 (with a dense cumulus), A2 (with a translucent cumulus), B1 (with the corona radiata), B2 (partly naked oocytes), C (naked oocytes), D (with a dark cumulus). The percent incidence of live oocyte in these subpopulations changed significantly as related to cumulus features, however the occurrence of oocytes in the germinal vesicle stage was lower in class D only. Similar metaphase II rates achieved in A1, A2, B1 and B2 classes after in vitro maturation suggest that the nucleus may in fact mature in vitro, in spite of the different accompanying cumulus features which are typical of these classes. In contrast, a higher cytoplasmic maturation rate obtained in class A may indicate a stronger dependence of this variable upon cumulus features than that shown by nuclear maturation. When different types of cumulus expansion after in vitro maturation were considered (i.e., fully expanded cumulus, partly expanded cumulus, and partly naked oocyte), no differences were found in the percent of oocytes reaching metaphase II or cytoplasmic maturation. It is concluded that morphological features of the collected porcine cumulus-oocyte complexes (rather than cumulus behavior during culture) may be useful for selection of potentially competent oocytes for in vitro fertilization and embryo production.
Assuntos
Animais , Feminino , Fertilização in vitro , Folículo Ovariano/citologia , Metáfase/fisiologia , Oócitos/citologia , Suínos/fisiologiaRESUMO
Porcine immature oocyte quality (i.e., that of live oocytes at the germinal vesicle stage) was evaluated according to features of the surrounding cumulus, aiming to establish maturational competence of different subpopulations of such cumulus-oocyte complexes. Six subpopulations were identified: A1 (with a dense cumulus), A2 (with a translucent cumulus), B1 (with the corona radiata), B2 (partly naked oocytes), C (naked oocytes), D (with a dark cumulus). The percent incidence of live oocyte in these subpopulations changed significantly as related to cumulus features, however the occurrence of oocytes in the germinal vesicle stage was lower in class D only. Similar metaphase II rates achieved in A1, A2, B1 and B2 classes after in vitro maturation suggest that the nucleus may in fact mature in vitro, in spite of the different accompanying cumulus features which are typical of these classes. In contrast, a higher cytoplasmic maturation rate obtained in class A may indicate a stronger dependence of this variable upon cumulus features than that shown by nuclear maturation. When different types of cumulus expansion after in vitro maturation were considered (i.e., fully expanded cumulus, partly expanded cumulus, and partly naked oocyte), no differences were found in the percent of oocytes reaching metaphase II or cytoplasmic maturation. It is concluded that morphological features of the collected porcine cumulus-oocyte complexes (rather than cumulus behavior during culture) may be useful for selection of potentially competent oocytes for in vitro fertilization and embryo production.(AU)
Assuntos
Animais , Feminino , Fertilização in vitro , Metáfase/fisiologia , Oócitos/citologia , Folículo Ovariano/citologia , Suínos/fisiologiaRESUMO
The lifespan of intestinal epithelial cells is predetermined by the process of cell proliferation that occurs constantly in the crypt. The control of this process involves some endogenous factors, such as hormones, as well as exogenous factors, like food and natural light variations. These last two exogenous factors seem to be the major modulators of the cell proliferation process. Fasting treatment was conducted to assess the role of food and its effect on the metaphase index (MI) of the intestinal epithelium at different times and periods (light and dark) of the day. The effects of short- (5 hr) and long-term (25 hr) fasting on the MI in the jejunal epithelium of young rats were investigated at 09:00 h, 15:00 hr, 21:00 hr, and 02:00 hr using the arrested metaphases method. The present study demonstrates that 5 hr and 25 hr of fasting treatment decrease the MI at 09:00 hr. It was observed from MI analysis that there is an interaction between the fed/fasted status of the animal and the different times of the day. This result suggests that during the transition from youth to adulthood, the control of MI by the light/dark cycle seems to be more pronounced as compared with control by food intake at some periods of the day, although at other times food had a greater impact on the MI.
Assuntos
Animais Lactentes/fisiologia , Ritmo Circadiano/fisiologia , Jejum/fisiologia , Mucosa Intestinal/crescimento & desenvolvimento , Jejuno/crescimento & desenvolvimento , Desmame , Envelhecimento/fisiologia , Animais , Animais Recém-Nascidos , Animais Lactentes/anatomia & histologia , Proliferação de Células , Escuridão , Digestão/fisiologia , Digestão/efeitos da radiação , Feminino , Privação de Alimentos/fisiologia , Mucosa Intestinal/citologia , Jejuno/citologia , Luz , Masculino , Metáfase/fisiologia , Estimulação Luminosa , Ratos , Ratos Wistar , Fatores de TempoRESUMO
This study aimed to evaluate the effect of demecolcine, a microtubule-depolymerizing agent, on microtubule kinetics; to determine the best concentration of demecolcine as a chemically assisted enucleation agent in metaphase I (MI) and metaphase II (MII) bovine oocytes, and to evaluate the embryonic development after nuclear transfer (NT) using chemically assisted enucleation of recipient oocytes. Oocytes in vitro matured for 12 h (MI) and 21 h (MII) were exposed to several concentrations of demecolcine and evaluated for enucleation or membrane protrusion formation. Demecolcine concentration of 0.05 microg/mL produced the highest rates of enucleation in group MI (15.2%) and protrusion formation in group MII (55.1%), and was employed in the following experiments. Demecolcine effect was seen as early as 0.5 h after treatment, with a significant increase in the frequency of oocytes with complete microtubule depletion in MI (58.9%) and MII (21.8%) compared to initial averages at 0 h (27.4% and 1.9%, respectively). Microtubule repolymerization was observed when MII-treated oocytes were cultured in demecolcine-free medium for 6 h (42.4% oocytes with two evident sets of microtubules). Chemically assisted enucleated oocytes were used as recipient cytoplasts in NT procedures to assess embryonic development. For NT, 219 of 515 oocytes (42.5%) formed protrusions and were enucleated, and reconstructed, resulting in 58 nuclear-transferred one-cell embryos. Cleavage (84.5%) and blastocyst development (27.6%) rates were assessed. In conclusion, demecolcine can be used at lower concentrations than routinely employed, and the chemically assisted enucleation technique was proven to be highly efficient allowing embryonic development in bovine.
Assuntos
Clonagem de Organismos/métodos , Demecolcina/farmacologia , Microtúbulos/efeitos dos fármacos , Técnicas de Transferência Nuclear/veterinária , Oócitos/efeitos dos fármacos , Moduladores de Tubulina/farmacologia , Animais , Bovinos , Membrana Celular/efeitos dos fármacos , Membrana Celular/fisiologia , Clonagem de Organismos/veterinária , Feminino , Metáfase/efeitos dos fármacos , Metáfase/fisiologia , Microtúbulos/fisiologia , Oócitos/citologia , Oócitos/fisiologiaRESUMO
Porcine immature oocyte quality (i.e., that of live oocytes at the germinal vesicle stage) was evaluated according to features of the surrounding cumulus, aiming to establish maturational competence of different subpopulations of such cumulus-oocyte complexes. Six subpopulations were identified: A1 (with a dense cumulus), A2 (with a translucent cumulus), B1 (with the corona radiata), B2 (partly naked oocytes), C (naked oocytes), D (with a dark cumulus). The percent incidence of live oocyte in these subpopulations changed significantly as related to cumulus features, however the occurrence of oocytes in the germinal vesicle stage was lower in class D only. Similar metaphase II rates achieved in A1, A2, B1 and B2 classes after in vitro maturation suggest that the nucleus may in fact mature in vitro, in spite of the different accompanying cumulus features which are typical of these classes. In contrast, a higher cytoplasmic maturation rate obtained in class A may indicate a stronger dependence of this variable upon cumulus features than that shown by nuclear maturation. When different types of cumulus expansion after in vitro maturation were considered (i.e., fully expanded cumulus, partly expanded cumulus, and partly naked oocyte), no differences were found in the percent of oocytes reaching metaphase II or cytoplasmic maturation. It is concluded that morphological features of the collected porcine cumulus-oocyte complexes (rather than cumulus behavior during culture) may be useful for selection of potentially competent oocytes for in vitro fertilization and embryo production.
Assuntos
Células do Cúmulo/citologia , Oócitos/citologia , Folículo Ovariano/citologia , Suínos/fisiologia , Animais , Feminino , Fertilização in vitro , Metáfase/fisiologiaRESUMO
In the hexaploid (2n = 6x = 54) accession B176 of Brachiaria brizantha, one cytological characteristic differentiated it from the other accessions previously analyzed with the same ploidy level. Nearly 40% of meiocytes displayed the chromosome set arranged at two metaphase plates at the poles of the cell, close to the membrane. In these cells, both metaphase plates were arranged in an angle to form a typical tripolar spindle. Therefore, cells did not show normal chromosome segregation at anaphase I. Only nine univalent chromosomes migrated from each plate to the opposite pole with the remainder staying immobile on the plate. As a result of such spindle orientation and chromosome behavior, trinucleate telophases I were recorded. After telophase, cytokinesis eliminated the small nuclei into a microcyte. The second division proceeded normally, with the presence of microcytes in all phases. The origin of such an abnormality was explained on the hexaploid level of the accession which could have resulted by chromosome doubling of a triploid derived from species that did not display the same behavior for spindle organization. The high percentage of meiotic abnormalities recorded in this accession compromises fertility and renders it inadequate for the breeding program.