RESUMO
Protein phosphatase 4 (PPP4) is a protein phosphatase that, although highly expressed in the testis, currently has an unclear physiological role in this tissue. Here, we show that deletion of PPP4 catalytic subunit gene Ppp4c in the mouse causes male-specific infertility. Loss of PPP4C, when assessed by light microscopy, did not obviously affect many aspects of the morphology of spermatogenesis, including acrosome formation, nuclear condensation and elongation, mitochondrial sheaths arrangement and '9 + 2' flagellar structure assembly. However, the PPP4C mutant had sperm tail bending defects (head-bent-back), low sperm count, poor sperm motility and had cytoplasmic remnants attached to the middle piece of the tail. The cytoplasmic remnants were further investigated by transmission electron microscopy to reveal that a defect in cytoplasm removal appeared to play a significant role in the observed spermiogenesis failure and resulting male infertility. A lack of PPP4 during spermatogenesis causes defects that are reminiscent of oligoasthenoteratospermia (OAT), which is a common cause of male infertility in humans. Like the lack of functional PPP4 in the mouse model, OAT is characterized by abnormal sperm morphology, low sperm count and poor sperm motility. Although the causes of OAT are probably heterogeneous, including mutation of various genes and environmentally induced defects, the detailed molecular mechanism(s) has remained unclear. Our discovery that the PPP4C-deficient mouse model shares features with human OAT might offer a useful model for further studies of this currently poorly understood disorder.
Assuntos
Infertilidade Masculina/genética , Fosfoproteínas Fosfatases/deficiência , Cauda do Espermatozoide/patologia , Animais , Feminino , Fertilização , Fertilização in vitro , Infertilidade Masculina/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos ICR , Fosfoproteínas Fosfatases/metabolismo , Contagem de Espermatozoides , Motilidade dos Espermatozoides/genética , Cauda do Espermatozoide/metabolismo , Espermatogênese/genéticaRESUMO
Rab family members play important roles in membrane trafficking, cell growth, and differentiation. Almost all components of the cell endomembrane system, the nucleus, and the plasma membrane are closely related to RAB proteins. In this study, we investigated the distribution and functions of three members of the Rab family, Rab3A, Rab27A, and Rab35, in mouse oocyte meiotic maturation and activation. The three Rab family members showed different localization patterns in oocytes. Microinjection of siRNA, antibody injection, or inhibitor treatment showed that (1) Rab3A regulates peripheral spindle and cortical granule (CG) migration, polarity establishment, and asymmetric division; (2) Rab27A regulates CG exocytosis following MII-stage oocyte activation; and (3) Rab35 plays an important role in spindle organization and morphology maintenance, and thus meiotic nuclear maturation. These results show that Rab proteins play important roles in mouse oocyte meiotic maturation and activation and that different members exert different distinct functions.
Assuntos
Meiose , Oócitos/citologia , Oócitos/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Proteína rab3A de Ligação ao GTP/metabolismo , Animais , Células Cultivadas , Imunofluorescência , Camundongos , Camundongos Endogâmicos ICR , Proteínas rab de Ligação ao GTP/análise , Proteínas rab de Ligação ao GTP/genética , Proteínas rab27 de Ligação ao GTP , Proteína rab3A de Ligação ao GTP/análise , Proteína rab3A de Ligação ao GTP/genéticaRESUMO
DNA damage has recently been shown to inhibit or delay germinal vesicle breakdown (GVBD) in mouse oocytes, but once meiosis resumes, DNA-damaged oocytes are able to extrude the first polar body. In this study, using porcine oocytes, we showed that DNA damage did not affect GVBD, but inhibited the final stages of maturation, as indicated by failure of polar body emission. Unlike mitotic cells in which chromosome mis-segregation causes DNA double-strand breaks, meiotic mouse oocytes did not show increased DNA damage after disruption of chromosome attachment to spindle microtubules. Nocodazole-treated oocytes did not display increased DNA damage signals that were marked by γH2A.X signal strength, but reformed spindles and underwent maturation, although aneuploidy increased after extended nocodazole treatment. By using the mouse for parthenogenetic activation studies, we showed that early cleavage stage embryos derived from parthenogenetic activation of nocodazole-treated oocytes displayed normal activation rate and normal γH2A.X signal strength, indicating that no additional DNA damage occured. Our results suggest that DNA damage inhibits porcine oocyte maturation, while nocodazole-induced dissociation between chromosomes and microtubules does not lead to increased DNA damage either in mouse meiotic oocytes or in porcine oocytes.
Assuntos
Cromossomos/genética , Quebras de DNA de Cadeia Dupla , Microtúbulos/genética , Oócitos/citologia , Fuso Acromático/genética , Aneugênicos/farmacologia , Aneuploidia , Animais , Antibióticos Antineoplásicos/farmacologia , Bleomicina/farmacologia , Feminino , Histonas/metabolismo , Meiose/genética , Camundongos , Camundongos Endogâmicos ICR , Nocodazol/farmacologia , Folículo Ovariano/citologia , Partenogênese/genética , Suínos , Moduladores de Tubulina/farmacologiaRESUMO
BACKGROUND: Strategies to detect the presence of suicidal ideation (SI) or characteristics of ideation that indicate marked suicide risk are critically needed to guide interventions and improve care during care transition periods. Some studies indicate that machine learning can be applied to momentary data to improve classification of SI. This study examined whether the classification accuracy of these models varies as a function of type of training data or characteristics of ideation. METHODS: A total of 257 psychiatric inpatients completed a 3-week battery of ecological momentary assessment and measures of suicide risk factors. The accuracy of machine learning models in classifying the presence, duration, or intensity of ideation was compared across models trained on baseline and/or momentary suicide risk data. Relative feature importance metrics were examined to identify the risk factors that were most important for outcome classification. RESULTS: Models including both baseline and momentary features outperformed models with only one feature type, providing important information in both correctly classifying and differentiating individual characteristics of SI. Models classifying SI presence, duration, and intensity performed similarly. LIMITATIONS: Results of this study may not generalize beyond a high-risk, psychiatric inpatient sample, and additional work is needed to examine temporal ordering of the relationships identified. CONCLUSIONS: Our results support using machine learning approaches for accurate identification of SI characteristics and underscore the importance of understanding the factors that differentiate and drive different characteristics of SI. Expansion of this work can support use of these models to guide intervention strategies.
RESUMO
Taxol blocks the migrations of the sperm and egg nuclei in fertilized eggs and induces asters in unfertilized eggs of the sea urchins Lytechinus variegatus and Arbacia punctulata. Video recordings of eggs inseminated in 10 microM taxol demonstrate that sperm incorporation and sperm tail motility are unaffected, that the sperm aster formed is unusually pronounced, and that the migration of the egg nucleus and pronuclear centration are inhibited. The huge monopolar aster persists for at least 6 h; cleavage attempts and nuclear cycles are observed. Colcemid (10 microM) disassembles both the large taxol-stabilized sperm aster in fertilized eggs and the numerous asters induced in unfertilized eggs. Antitubulin immunofluorescence microscopy demonstrates that in fertilized eggs all microtubules are within the prominent sperm aster. Within 15 min of treatment with 10 microM taxol, unfertilized eggs develop numerous (greater than 25) asters de novo. Transmission electron microscopy of unfertilized eggs reveals the presence of microtubule bundles that do not emanate from centrioles but rather from osmiophilic foci or, at times, the nuclear envelope. Taxol-treated eggs are not activated as judged by the lack of DNA synthesis, nuclear or chromosome cycles, and the cortical reaction. These results indicate that: (a) taxol prevents the normal cycles of microtubule assembly and disassembly observed during development; (b) microtubule disassembly is required for the nuclear movements during fertilization; (c) taxol induces microtubules in unfertilized eggs; and (d) nucleation centers other than centrioles and kinetochores exist within unfertilized eggs; these presumptive microtubule organizing centers appear idle in the presence of the sperm centrioles.
Assuntos
Alcaloides/farmacologia , Fertilização/efeitos dos fármacos , Microtúbulos/fisiologia , Ouriços-do-Mar/efeitos dos fármacos , Animais , Núcleo Celular/fisiologia , Demecolcina/farmacologia , Feminino , Masculino , Microtúbulos/efeitos dos fármacos , Movimento/efeitos dos fármacos , Óvulo/efeitos dos fármacos , Óvulo/ultraestrutura , Paclitaxel , Motilidade dos Espermatozoides/efeitos dos fármacos , Cauda do Espermatozoide/efeitos dos fármacos , Tubulina (Proteína)/metabolismoRESUMO
We have used in situ hybridization and cell fractionation methods to follow the distribution of U1 RNA and immunofluorescence microscopy to follow the distribution of snRNP proteins in oocytes, eggs, and embryos of several sea urchin species. U1 RNA and U1-specific snRNP antigens are concentrated in germinal vesicles of oocytes. Both appear to relocate after oocyte maturation because they are found primarily, if not exclusively, in the cytoplasm of mature unfertilized eggs. This cytoplasmic residence is maintained during early cleavage and U1 RNA is first detectable in nuclei of micromeres at the 16-cell stage. Between morula and gastrula stages the steady-state concentrations of both RNA and antigens gradually increase in nuclei and decrease in cytoplasm. Surprisingly, analysis of the distribution of newly synthesized U1 RNA shows that it does not equilibrate with the maternal pool. Instead new transcripts are confined to nuclei, while cytoplasmic U1 RNAs are of maternal origin. This lack of equilibration and the conversion of maternal U1 RNAs from nuclear species in oocytes to cytoplasmic in embryos suggests that these RNPs (or RNAs) are structurally altered when released to the cytoplasm at oocyte maturation.
Assuntos
Embrião não Mamífero/citologia , RNA Nuclear Pequeno/genética , Ouriços-do-Mar/embriologia , Animais , Divisão Celular , Núcleo Celular/ultraestrutura , Feminino , Imunofluorescência , Hibridização de Ácido Nucleico , RNA Nuclear Pequeno/análise , Ouriços-do-Mar/citologia , Transcrição GênicaRESUMO
This paper explores the mode of action of the tranquillizers chloral hydrate and diazepam during fertilization and mitosis of the first reproductive cell cycles in sea urchin eggs. Most striking effects of these drugs are the alteration of centrosomal material and the abnormal microtubule configurations during exposure and after recovery from the drugs. This finding is utilized to study the mechanisms of centrosome compaction and decompaction and the dynamic configurational changes of centrosomal material and its interactions with microtubules. When 0.1% chloral hydrate or 350-750 microM diazepam is applied at specific phases during the first cell cycle of sea urchin eggs, expanded centrosomal material compacts at distinct regions and super-compacts into dense spheres while microtubules disassemble. When eggs are treated before pronuclear fusion, centrosomal material aggregates around each of the two pronuclei while microtubules disappear. Upon recovery, atypical asters oftentimes with multiple foci are formed from centrosomal material surrounding the pronuclei which indicates that the drugs have affected centrosomal material and prevent it from functioning normally. Electron microscopy and immunofluorescence studies with antibodies that routinely stain centrosomes in sea urchin eggs (4D2; and Ah-6) depict centrosomal material that is altered when compared to control cells. This centrosomal material is not able to reform normal microtubule patterns upon recovery but will form multiple asters around the two pronuclei. When cells are treated with 0.1% chloral hydrate or 350-750 microM diazepam during mitosis, the bipolar centrosomal material becomes compacted and aggregates into multiple dense spheres while spindle and polar microtubules disassemble. With increased incubation time, the smaller dense centrosome particles aggregate into bigger and fewer spheres. Upon recovery, unusual irregular microtubule configurations are formed from centrosomes that have lost their ability to reform normal mitotic figures. These results indicate that chloral hydrate and diazepam affect centrosome structure which results in the inability to reform normal microtubule formations and causes abnormal fertilization and mitosis.
Assuntos
Ciclo Celular/efeitos dos fármacos , Centrossomo/efeitos dos fármacos , Hidrato de Cloral/farmacologia , Diazepam/farmacologia , Fertilização/efeitos dos fármacos , Óvulo/efeitos dos fármacos , Animais , Núcleo Celular/efeitos dos fármacos , Centrossomo/fisiologia , Centrossomo/ultraestrutura , Microtúbulos/efeitos dos fármacos , Mitose/efeitos dos fármacos , Óvulo/citologia , Óvulo/fisiologia , Ouriços-do-MarRESUMO
Griseofulvin (4-6 X10-5 and 1 X 10-4 M) prevents the formation of any microtubule-based structures of sea urchin (Strongylocentrotus purpuratus, Lytechinus variegatus, Arbacia punctulata) eggs at fertilization. Sperm incorporation occurs, though the migrations of the pronuclei, dependent on the formation of the sperm aster, are arrested. Similarly in "streak" and the mitotic apparatus fail to assemble. Cycles of DNA synthesis, chromosome activity, nuclear breakdown and reconstitution, and even cleavage attempts occur on schedule in the absence of any mitotic movements. The action of griseofulvin, unlike that of colchicine, is readily reversible by the removal of the drug. Microtubules are formed, and the chromosome are separated. At 1 X 10-6 M, diminutive microtubule-based structures (e.g. sperm aster, mitotic apparatus) are observed though syngamy and division are arrested. These results demonstrate an independence of the cycle of microtubule-mediated events from other cyclical processes during the first cell cycles.
Assuntos
Fertilização/efeitos dos fármacos , Griseofulvina/farmacologia , Microtúbulos/efeitos dos fármacos , Mitose/efeitos dos fármacos , Ouriços-do-Mar/embriologia , Animais , Ciclo Celular/efeitos dos fármacos , Cromossomos/metabolismo , DNA/biossínteseRESUMO
The regulation of the microtubule-mediated motions within eggs during fertilization was investigated in relation to the shift in intracellular pH (pHi) that occurs during the ionic sequence of egg activation in the sea urchins Lytechinus variegatus and Arbacia punctulata. Microtubule assembly during formation of the sperm aster and mitotic apparatus was detected by anti-tubulin immunofluorescence microscopy, and the microtubule-mediated migrations of the sperm and egg nuclei were studied with time-lapse video differential interference contrast microscopy. Manipulations of intracellular pH were verified by fluorimetric analyses of cytoplasmic fluorescein incorporated as fluorescein diacetate. The ionic sequence of egg activation was manipulated i) to block the pHi shift at fertilization or reduce the pHi of fertilized eggs to unfertilized values, ii) to elevate artificially the pHi of unfertilized eggs to fertilized values, and iii) to elevate artificially or permit the normal pHi shift in fertilized eggs in which the pHi shift at fertilization was previously prevented. Fertilized eggs in which the pHi shift was suppressed did not assemble microtubules or undergo the normal microtubule-mediated motions. In fertilized eggs in which the pHi was reduced to unfertilized levels after the assembly of the sperm aster, no motions were detected. If the intracellular pH was later permitted to rise, normal motile events leading to division and development occurred, delayed by the time during which the pH elevation was blocked. Microtubule-mediated events occurred in eggs in which the intracellular pH was elevated, even in unfertilized eggs in which the pH was artificially increased. These results indicate that the formation and normal functioning of the egg microtubules is initiated, either directly or indirectly, by the shift in intracellular pH that occurs during fertilization.
Assuntos
Fertilização , Concentração de Íons de Hidrogênio , Microtúbulos/ultraestrutura , Óvulo/ultraestrutura , Zigoto/ultraestrutura , Animais , Feminino , Microtúbulos/fisiologia , Óvulo/fisiologia , Ouriços-do-Mar , Tubulina (Proteína)/fisiologia , Zigoto/fisiologiaRESUMO
We investigated the role of androgen-induced oxidative stress in prostate cancer using the androgen-responsive LNCaP human prostate cancer cell line exposed to a 1-nM concentration of the synthetic androgen R1881 (which correlates with serum androgen levels). Such exposure, which decreases growth rate and increases oxidative stress in LNCaP cells, induced statistically significant mitochondrial changes. A 40% increase in 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) reduction, indicative of mitochondrial dehydrogenase activity, occurred 24 hr after androgen treatment. This change preceded 50-110% increases, 40-96 hr after R1881 exposure, in levels of cellular peroxides and hydroxyl radicals as measured by 2'7'-dicholorofluorescin diacetate (DCF) fluorescence. On the basis of electron microscopy measurements, R1881 treatment increased the area fraction of mitochondria per cell by approximately 100% at 72 hr. In agreement, mitochondrial mass at 96 hr, evaluated by the fluorescent dye nonyl acridine orange (NAO), was 80% higher in treated cells. R1881 exposure for 24 hr lowered the activities of electron transport system (ETS) complexes, I, II, and IV by 17-27% and ATP levels by 50%. The ETS inhibitors, rotenone and antimycin A, lowered androgen-induced DCF fluorescence readings to control levels thereby suggesting ETS involvement in androgen-induced oxidant production. Addition of alpha-tocopherol succinate abrogated R1881-induced elevations in MTT reduction. In sum, androgens may, directly or indirectly, contribute to oxidative stress in LNCaP cells by regulating mitochondrial number, activity, and oxidant production by mechanisms that are, at least in part, sensitive to an antioxidant.
Assuntos
Metribolona/farmacologia , Mitocôndrias/efeitos dos fármacos , Estresse Oxidativo , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/ultraestrutura , Congêneres da Testosterona/farmacologia , Vitamina E/análogos & derivados , Antimicina A/farmacologia , Corantes/metabolismo , DNA/análise , Transporte de Elétrons/efeitos dos fármacos , Fluoresceínas/metabolismo , Corantes Fluorescentes/metabolismo , Humanos , Radical Hidroxila/metabolismo , Masculino , Mitocôndrias/metabolismo , Mitocôndrias/ultraestrutura , Peróxidos/metabolismo , Rotenona/farmacologia , Sais de Tetrazólio/metabolismo , Tiazóis/metabolismo , Fatores de Tempo , Tocoferóis , Células Tumorais Cultivadas , Desacopladores/farmacologia , Vitamina E/farmacologiaRESUMO
Centrosomes play crucial roles in the union of sperm and egg nuclei during fertilization and in the equal separation of genomic material during cell division. While many studies in recent years have focused on the molecular composition of centrosomes, this article focuses on the structural behavior of centrosomes and on factors that play a role in centrosome functions under normal, artificially altered, and abnormal conditions. We review here how studies in the classic sea urchin egg model have contributed to our knowledge on the centrosome cycle within the cell cycle, on compaction and decompaction of centrosomal material, and on the contributions of maternal and paternal centrosomes during fertilization. Centrosome material is activated in unfertilized eggs by increasing pH with ammonium and by increasing calcium with the ionophore A23187, which are conditions that are normally induced by sperm. D(2)O and taxol also induce centrosome aggregation in the unfertilized egg. Maternal and paternal centrosome material both contribute to the formation of a functional centrosome but the formation of a bipolar centrosome requires material from the paternal centrosome. Fertilization of taxol-treated eggs reveals that the male centrosome possesses the capability to attract maternal centrosome material. When pronuclear fusion of the male and female pronuclei is inhibited with agents such as the disulfide reducing agent dithiothreitol (DTT) a bipolar mitotic apparatus is formed from the paternal centrosome. Furthermore, one centrosome of the bipolar mitotic apparatus is capable of organizing an additional half spindle that attaches to the female pronucleus indicating a functional and perhaps structural connection between centrosomes and chromatin. Sea urchin eggs are also useful to study centrosome abnormalities and consequences for the cell cycle. While classic studies by Theodor Boveri have shown that dispermic fertilization will result in abnormal cell division because of multiple centrosomes contributed by sperm, abnormal cell division can also be induced by chemical alterations of centrosomes. Compaction and decompaction of centrosome structure is studied using chloral hydrate or the chaotropic agent formamide, which reveals that centrosomes can be chemically altered to produce mono- or multipolar abnormal mitosis and unequal distribution of genomic material upon release from formamide. The patterns of abnormal centrosome reformations after recovery from formamide treatment resemble those seen in cancer cells which argues that structural defects of centrosomes can account for the formation of abnormal mitosis and multipolar cells frequently observed in cancer. In summary, the sea urchin model has been most useful to gain information on the role of centrosomes during fertilization and cell division as well as on adverse conditions that play a role in centrosome dysfunctions and in disease.
Assuntos
Centrossomo/fisiologia , Fertilização/fisiologia , Óvulo/fisiologia , Animais , Calcimicina/farmacologia , Divisão Celular/efeitos dos fármacos , Centrossomo/química , Centrossomo/efeitos dos fármacos , Aberrações Cromossômicas , DNA/análise , Ditiotreitol/farmacologia , Fertilização/efeitos dos fármacos , Formamidas , Concentração de Íons de Hidrogênio , Masculino , Microscopia Eletrônica de Varredura , Microscopia de Fluorescência , Microtúbulos/efeitos dos fármacos , Microtúbulos/ultraestrutura , Óvulo/efeitos dos fármacos , Óvulo/ultraestrutura , Paclitaxel/farmacologia , Compostos de Amônio Quaternário , Ouriços-do-Mar , Espermatozoides/efeitos dos fármacos , Espermatozoides/ultraestruturaRESUMO
The mitogen-activated protein kinase (MAPK) cascade is one of the most important of the intracellular signaling pathways that play a crucial role in cell proliferation, cell differentiation and cell cycle regulation. Since the first report in 1993 of MAPK's involvement in the functional regulation of mammalian oocytes, much work has been done on the role of the MAPK cascade in germ cells in different species of mammals. This review describes the possible involvement of the MOS/MEK/MAPK/RSK cascade in spermatogenesis, sperm function, oocyte meiotic re-initiation, spindle assembly, metaphase II arrest, pronuclear formation and the entry of first mitosis, as well as the cross-talk of this cascade to maturation-promoting factor (MPF) and other signal molecules in mammals.
Assuntos
Proteínas Quinases Ativadas por Mitógeno/metabolismo , Oócitos/fisiologia , Espermatozoides/fisiologia , Animais , Feminino , Fertilização , Humanos , Masculino , Meiose , Mitose , Partenogênese , EspermatogêneseRESUMO
In the present study the effects of two cell-permeant antioxidants, 2(3)-tert-butyl-4-hydroxyanisole (BHA) and nordihydroguaiaretic acid (NDGA), on porcine oocyte meiosis resumption, chromatin behaviour and spindle assembly were investigated. The antioxidants BHA and NDGA stimulated meiosis resumption in a dose-dependent manner in both cumulus-enclosed and denuded porcine oocytes. After in vitro culture for 8 h, few oocytes underwent germinal vesicle breakdown (GVBD) in control groups, whereas GVBD occurred in high percentages of oocytes treated with BHA or NDGA at concentrations that inhibit GVBD in rodent oocytes, although mitogen-activated protein (MAP) kinase was not phosphorylated as revealed by Western immunoblots. Orcein staining and fluorescein isothiocyanate-anti-alpha-tubulin labelling showed that chromosome and spindle formation, respectively, and further meiosis progression were inhibited 20 and 25 h after culture. Instead, chromatin was highly condensed or existed in scattered condensed clusters. Correspondingly, MAP kinase phosphorylation was inhibited by both BHA and NDGA in a dose-dependent manner. The inhibitory effects of BHA on meiosis completion and MAP kinase phosphorylation was reversible. These results suggest that, unlike in rodent oocytes, antioxidants stimulate GVBD in the absence of MAP kinase activation, but inhibit MAP kinase phosphorylation, meiotic apparatus formation and thus the further progression of the meiosis of porcine oocytes.
Assuntos
Antioxidantes/farmacologia , Meiose/efeitos dos fármacos , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Oócitos/citologia , Oócitos/efeitos dos fármacos , Animais , Antioxidantes/administração & dosagem , Hidroxianisol Butilado/administração & dosagem , Hidroxianisol Butilado/farmacologia , Ciclo Celular/efeitos dos fármacos , Cromatina/efeitos dos fármacos , Relação Dose-Resposta a Droga , Feminino , Técnicas In Vitro , Masoprocol/administração & dosagem , Masoprocol/farmacologia , Oócitos/enzimologia , Fosforilação , SuínosRESUMO
Gravity has been a pervasive influence on all living systems and there is convincing evidence to suggest that it alters fertilization and embryogenesis in several developmental systems. Notwithstanding the global importance of gravity on development, it has only been recently possible to begin to design experiments which might directly investigate the specific effects of this vector. The goal of this research program is to explore and understand the effects of gravity on fertilization and early development using sea urchins as a model system. Sea urchin development has several advantages for this project including the feasibility of maintaining and manipulating these cells during spaceflight, the high percentage of normal fertilization and early development, and the abundant knowledge about molecular, biochemical, and cellular events during embryogenesis which permits detailed insights into the mechanism by which gravity might interfere with development. Furthermore, skeletal calcium is deposited into the embryonic spicules within a day of fertilization permitting studies of the effects of gravity on bone calcium deposition.
Assuntos
Fertilização/fisiologia , Ouriços-do-Mar/embriologia , Ouriços-do-Mar/crescimento & desenvolvimento , Voo Espacial/instrumentação , Ausência de Peso , Animais , Divisão Celular , Desenho de Equipamento , Feminino , Sensação Gravitacional , Abrigo para Animais , Masculino , Óvulo , EspermatozoidesRESUMO
The cytoskeleton is a complex network of fibers that is sensitive to environmental factors including microgravity and altered gravitational forces. Cellular functions such as transport of cell organelles depend on cytoskeletal integrity; regulation of cytoskeletal activity plays a role in cell maintenance, cell division, and apoptosis. Here we report cytoskeletal and mitochondria alterations in cultured human lymphocyte (Jurkat) cells after exposure to spaceflight and in insect cells of Drosophila melanogaster (Schneider S-1) after exposure to conditions created by clinostat rotation. Jurkat cells were flown on the space shuttle in Biorack cassettes while Schneider S-1 cells were exposed to altered gravity forces as produced by clinostat rotation. The effects of both treatments were similar in the different cell types. Fifty percent of cells displayed effects on the microtubule network in both cell lines. Under these experimental conditions mitochondria clustering and morphological alterations of mitochondrial cristae was observed to various degrees after 4 and 48 hours of culture. Jurkat cells underwent cell divisions during exposure to spaceflight but a large number of apoptotic cells was also observed. Similar results were obtained in Schneider S-1 cells cultured under clinostat rotation. Both cell lines displayed mitochondria abnormalities and mitochondria clustering toward one side of the cells which is interpreted to be the result of microtubule disruption and failure of mitochondria transport along microtubules. The number of mitochondria was increased in cells exposed to altered gravity while cristae morphology was severely affected indicating altered mitochondria function. These results show that spaceflight as well as altered gravity produced by clinostat rotation affects microtubule and mitochondria organization and results in increases in apoptosis. Grant numbers: NAG 10-0224, NAG2-985.
Assuntos
Apoptose/fisiologia , Citoesqueleto/fisiologia , Mitocôndrias/fisiologia , Voo Espacial , Ausência de Peso , Animais , Antígenos Nucleares , Ciclo Celular , Proteínas de Ciclo Celular , Divisão Celular , Linhagem Celular , Células Cultivadas , Citoesqueleto/ultraestrutura , Drosophila melanogaster , Humanos , Células Jurkat/ultraestrutura , Microscopia Eletrônica , Microtúbulos/fisiologia , Microtúbulos/ultraestrutura , Mitocôndrias/ultraestrutura , Proteínas Associadas à Matriz Nuclear , Proteínas Nucleares , RotaçãoRESUMO
Survivin is the smallest member of the inhibitor of apoptosis protein (IAP) family and acts as a bifunctional protein involved in mitosis regulation and apoptosis inhibition. To identify the physiological role of Survivin in female reproduction, we selectively disrupted Survivin expression in oocytes and granulosa cells (GCs), two major cell types in the ovary, by two different Cre-Loxp conditional knockout systems, and found that both led to defective female fertility. Survivin deletion in oocytes did not affect oocyte growth, viability and ovulation, but caused tetraploid egg production and thus female infertility. Further exploration revealed that Survivin was essential for regulating proper meiotic spindle organization, spindle assembly checkpoint activity, timely metaphase-to-anaphase transition and cytokinesis. Mutant mice with Survivin depleted in GCs showed reduced ovulation and subfertility, caused by defective follicular growth, increased follicular atresia and impaired luteinization. These findings suggest that Survivin has an important role in regulating folliculogenesis and oogenesis in the adult mouse ovary.
Assuntos
Fertilidade , Proteínas Inibidoras de Apoptose/metabolismo , Oócitos/metabolismo , Oogênese , Proteínas Repressoras/metabolismo , Anáfase , Ciclossomo-Complexo Promotor de Anáfase/metabolismo , Animais , Apoptose , Diferenciação Celular , Cromossomos de Mamíferos/metabolismo , Citocinese , Regulação para Baixo , Feminino , Deleção de Genes , Células da Granulosa/metabolismo , Fator 9 de Diferenciação de Crescimento/metabolismo , Integrases/metabolismo , Cinetocoros/metabolismo , Luteinização , Pontos de Checagem da Fase M do Ciclo Celular , Meiose , Camundongos , Oócitos/citologia , Ovulação , SurvivinaRESUMO
The physiological role of cumulus cells (CC) surrounding oocytes is particularly important for normal cytoplasmic maturation of oocytes. However, removal of CC from oocytes is inevitable for some embryo manipulation techniques, such as germinal vesicle (GV) transfer, somatic cell haploidization, and oocyte cryopreservation. The present study was designed to determine an optimal method to culture porcine denuded oocytes (DO). The results indicated CC from cumulus-oocyte complexes at the GV stage (GVCC) or at the metaphase II stage, and mural granulosa cells could not improve the maturation of DO. However, GVCC could enhance the development of matured porcine DO after fertilization; the percentage of blastocysts was increased from 1.1 to 17.2% (P < 0.05), and the relative value of the x-axis and y-axis of spindles was also increased (P < 0.05). Coculture with GVCC had no effect on the distribution of mitochondria and cortical granules. The results contribute to our understanding of the mechanisms by which CC promote oocyte maturation and contribute to optimization of protocols for in vitro maturation of DO.