RESUMO
Embryological studies in fish species are useful to the understanding of their biology and systematics. The available biological data in Leiarius marmoratus are scarce and additional information about its reproductive biology is needed, mainly because this species has been commercially exploited and used in production of hybrid lineages. In order to evaluate the temporal-morphological embryonic modifications in L. marmoratus, samples of nearly 200 embryos were collected at random at different stages of development, starting from fecundation (time zero). Embryos were fixed in modified Karnovsk's solution and 2.5% glutaraldehyde, processed and analysed under optic and electron microscopy. The incubation period of L. marmoratus was equal to 14.42 h at a mean temperature of 28.3 ± 0.07°C. The following stages of embryonic development were established: zygote, cleavage, gastrula, organogenesis and hatching. These stages were divided into phases, as follows: cleavage - phases of 2, 4, 8, 16, 32 and 64 cells and morula; gastrula - phases of 25, 50, 75 and 90% of epiboly and blastopore closure; and organogenesis - neurula, segmentation and pre-larval phases. The embryogenesis of L. marmoratus was typical of neotropical teleosteans, with peculiarities in species development.
Assuntos
Peixes-Gato/embriologia , Embrião não Mamífero/citologia , Embrião não Mamífero/ultraestrutura , Desenvolvimento Embrionário/fisiologia , Morfogênese/fisiologia , Organogênese/fisiologia , Animais , Blástula/citologia , Blástula/ultraestrutura , Gástrula/citologia , Gástrula/ultraestrutura , Microscopia Eletrônica de Varredura/métodos , Mórula/citologia , Mórula/ultraestrutura , Oócitos/citologia , Oócitos/ultraestrutura , Zigoto/citologia , Zigoto/ultraestruturaRESUMO
We describe irregularly calcified egg and eggshell morphologies for the first time in nests of the broad-snouted caiman, Caiman latirostris. Research is based on detailed descriptions of 270 eggs from a total sample of 46,800 collected between 2005 and 2011 in Santa Fe Province, Argentina, and encompasses animals from both natural habitats and held in captivity. We discuss possible reasons for the occurrence of eggs with different mineralisation patterns in our extensive C. latirostris field sample and its conservation significance; the chemistry of egg laying in amniotes is sensitive to environmental contamination which, in turn, has biological implications. Based on our egg sample, we identify two caiman eggshell abnormalities: (1) regularly calcified eggs with either calcitic nodules or superficial wrinkles at one egg end and (2) irregularly calcified eggs with structural gaps that weaken the shell. Some recently laid clutches we examined included eggs with most of the shell broken and detached from the flexible membrane. Most type 1 regularly calcified eggs lost their initial calcified nodules during incubation, suggesting that these deposits do not affect embryo survival rates. In contrast, irregularly calcified caiman eggs have a mean hatching success rate of 8.9% (range 0-38%) across our sample compared to a mean normal success of 75%. Most irregularly calcified caiman eggs probably die because of infections caused by fungi and bacteria in the organic nest material, although another possible explanation that merits further investigation could be an increase in permeability, leading to embryo dehydration.
Assuntos
Jacarés e Crocodilos/anatomia & histologia , Jacarés e Crocodilos/fisiologia , Calcificação Fisiológica/fisiologia , Zigoto/química , Animais , Argentina , Microscopia Eletrônica de Varredura , Análise de Sobrevida , Zigoto/metabolismo , Zigoto/ultraestruturaRESUMO
This paper presents and discusses archaeological samples of Pediculus humanus capitis nits/eggs in Arica, northern Chile, dating between 2000 B.C. and A.D. 500. Eight samples of nits/eggs taken directly from seven mummified bodies of both the valley and the coast of Arica, were collected and studied. Samples were analysed with scanning electron microscopy (SEM), uncoated, using low and variable pressure modes. The aim was to study the morphology of the nits/eggs, the different degrees of preservation and their research potential. All samples were in good external condition and due to manipulation before SEM analysis, the oldest ones were fractured allowing the observation in situ of the hatching ad portas of an embryo. This inside view of the egg allowed observation and identification of microstructures of the embryo such as abdominal and thoracic spiracles and claws. In the most recent and best preserved samples, external structures characteristic of the egg such as aeropyles and operculum were observed. SEM can contribute significantly to the study of ectoparasites that affected ancient American populations and in this particular case to illustrate the stages and morphology of Andean archaeological specimens of P. humanus capitis.
Assuntos
Fósseis , Pediculus/ultraestrutura , Zigoto/ultraestrutura , Animais , Chile , Humanos , Microscopia Eletrônica de Varredura , Múmias/parasitologia , Pediculus/crescimento & desenvolvimentoRESUMO
Introdução - A malária é uma das principais doenças humanas do mundo e afeta principalmente as populações pobres em regiões tropicais e subtropicais onde as condições ambientais são favoráveis tanto para a proliferação dos agentes etiológicos como dos mosquitos vetores. No Brasil, An. darlingi é considerado vetor primário de plasmódios humanos. Devido à importância médica, esse inseto tem sido objeto de campanhas de controle populacional. No entanto, não foi considerada a ocorrência de microevolução em An. darlingi e consequentemente, a possibilidade de populações diferentes apresentarem características biológicas distintas. Objetivo Verificar a presença de variabilidade genética e morfológica em populações de An. darlingi no Brasil. Metodologia - Foram analisados e comparados 26 atributos da morfologia externa de ovos de nove populações, bem como a morfometria geométrica da forma alar de espécimes de 10 localidades diferentes de Anopheles darlingi. Além disso, foram empregadas sequências do gene mitocondrial citocromo oxidase subunidade I (COI) para analisar a estrutura populacional de An. darlingi. Resultados e Conclusão Apesar dos atributos dos ovos apresentarem variação, somente as amostras de Tocantins e Pará foram diferenciadas das demais populações. As variações nas estruturas externas dos ovos são provavelmente adaptativas, com influência de fatores ambientais, como temperatura, umidade e disponibilidade de alimento para as fêmeas. A comparação da morfometria geométrica da asa demonstrou que existe maior similaridade entre as populações da costa (estados do Espírito Santo , do cerrado (estados de Goiás e Tocantins), do norte do rio Amazonas (estados do Amazonas e Amapá), e do sul do rio Amazonas (estados de Mato Grosso e Pará).
Assuntos
Animais , Anopheles/anatomia & histologia , Anopheles/genética , Insetos Vetores/anatomia & histologia , Insetos Vetores/genética , Brasil , Complexo IV da Cadeia de Transporte de Elétrons/genética , Asas de Animais/anatomia & histologia , Zigoto/ultraestruturaRESUMO
PURPOSE: To evaluate the prognostic significance of triploidy incidence on the outcomes of embryos derived from normally fertilized oocytes from the same cohort. METHODS: This study included 1500 ICSI cycles. Logistic regression models were used to study the influence of abnormal fertilization on the development and clinical outcomes of embryos derived from normally fertilized oocytes from the same cohort RESULTS: We observed a negative influence of the percentages of triploid zygotes on fertilization (75.2% and 56.8%, P < 0.0001), high-quality embryos (58.9% and 48.2%, P = 0.0001), pregnancy (34.1% and 28.2%, P = 0.0540) and implantation rates (20.0% and 13.3%, P = 0.0012). When the 3PN zygote rate was >25%, the percentages of normal fertilization, high-quality embryos and implantation rates were significantly lower than in the control group. CONCLUSIONS: We observed an approximately 50% lower risk of pregnancy and a 3.5-fold higher risk of miscarriage in cycles with a 3PN incidence of >25%.
Assuntos
Injeções de Esperma Intracitoplásmicas , Triploidia , Zigoto/ultraestrutura , Aborto Espontâneo/diagnóstico , Aborto Espontâneo/epidemiologia , Aborto Espontâneo/genética , Adulto , Feminino , Humanos , Incidência , Masculino , Pessoa de Meia-Idade , Gravidez , Resultado da Gravidez , Prognóstico , Estudos RetrospectivosRESUMO
The developmental stages for the embryonic and larval periods of the silver catfish (Rhamdia quelen) kept at different temperatures (21, 24, 27 and 30 degrees C) are described. Fish were analysed under light and scanning electron microscopy. For embryonic development, we described 25 stages, which were grouped into seven periods named zygote, cleavage, blastula, gastrula, segmentation, pharyngula and hatching periods. For larval development, we defined three stages (early, mid, and late larvae). Additionally, the main ontogenetic events during the post-larvae and early juvenile periods were also described. This species presents a well developed lateral line and chemosensory systems that grow up during the larval period, maturing in the post-larvae. All tested temperatures are viable to R. quelen development, but a shorter incubation period was necessary to complete the development at lower temperatures. However, some malformations (heart edema) were verified at 30 degrees C.
Assuntos
Peixes-Gato/embriologia , Animais , Blástula/metabolismo , Blástula/ultraestrutura , Feminino , Peixes/embriologia , Masculino , Organogênese , Temperatura , Zigoto/metabolismo , Zigoto/ultraestruturaRESUMO
The hybrid fish Pseudoplatystoma spp. has been raised on a large scale by several fish farmers, despite the fact that little is known about its biology. This is because it presents a number of zootechnical advantages over the parental species. In order to provide information about the early morphology of this important species, we analyzed the fertilization and embryonic development of the hybrid between spotted females and barred males of sorubim specimens by light microscopy and by scanning (SEM) and transmission electron microscopy (TEM) after induced spawning. Samples were collected at pre-established moments up to larval hatching. Seven distinct stages of hybrid embryonic development were identified: zygote, cleavage, morula, blastula, gastrula, histogenesis and organogenesis, and hatching. Under SEM, we observed spermatozoa at the micropyle entrance, the formation of a fertilization cone in the eggs, the differentiation of cephalic and caudal regions, the neural tube and embryo growth along the cephalo-caudal axis, as well as rudimentary optic vesicle and barbels. Under light microscopy, cytoplasmic movement was apparent with the consequent formation of animal and vegetative poles in eggs, in addition to epiboly movements and a small notochord portion. Under TEM, the oocyte chorion and eggs presented a sieve-like aspect in transversal cuts, coupled with the rupture of cortical alveoli and chorion elevation, thus enlarging the perivitelline space. Several mitochondria in the cortical cytoplasm were detected in both oocytes and eggs. Overall, we observed that the larvae hatched without visible morphological alterations, and seemed to be as viable in captive systems as they are in the natural environment.
Assuntos
Peixes-Gato/embriologia , Desenvolvimento Embrionário , Animais , Blastocisto/ultraestrutura , Blástula/ultraestrutura , Quimera , Córion/embriologia , Córion/ultraestrutura , Citoplasma/ultraestrutura , Feminino , Fertilização , Gástrula/ultraestrutura , Larva/ultraestrutura , Masculino , Mitocôndrias/ultraestrutura , Mórula/ultraestrutura , Oócitos/ultraestrutura , Espermatozoides/ultraestrutura , Zigoto/ultraestruturaRESUMO
The embryogenesis of Brycon cephalus was established in seven stages: zygote, cleavage, blastula, gastrula, segmentation, larval and hatching, in an incubation period of 11 h (26 degrees C). The zygote phase was observed directly after fertilization and egg hydration. Cleavage began at 0.5 h of incubation and extended up to the morula phase (1.5 h; +100 blastomeres). Cleavage was meroblastic and underwent the following division pattern: the first five divisions were vertical and perpendicular to each other, following the model 2 x 2, 4 x 2, 4 x 4 and 4 x 8. The sixth division was horizontal and occurred at 1.25 h after fertilization, giving rise to two cell layers (4 x 8 x 2) with 64 blastomeres. At the blastula stage (1.25-1.5 h), an irregular space between the blastomeres, the blastocoele, could be detected and the periblast structure initiated. The gastrula (1.75-6.0 h) was characterized by the morphogenetic movements of epiboly, convergence and cell involution, and formation of the embryonic axis. The segmentation stage (7-9 h) comprised the development of somites, the notochord, optic, otic and Kupffer's vesicles, neural tube, primitive intestine and ended with the release of the tail. The larval stage (up to 10 h) was characterized by the presence of 30 somites and growth and elongation of the larvae. At the hatching stage, the embryos presented more than 30 somites and exhibited swimming movements and a soft chorion. The blastomeres presented euchromatic nuclei, indicating a high mitotic activity and many yolk globules in the cytoplasm. The periblast was constituted of a layer with several nuclei and many vesicles, which grew during the epiboly movement.
Assuntos
Peixes/embriologia , Animais , Blástula/metabolismo , Blástula/ultraestrutura , Fertilização , Peixes/metabolismo , Gástrula/metabolismo , Gástrula/ultraestrutura , Morfogênese , Zigoto/metabolismo , Zigoto/ultraestruturaRESUMO
In the present work, we analyzed the structure of oocytes and fertilized eggs of the piracanjuba fish (Brycon orbignyanus) under light and scanning electron microscopy. After inducing spawning, samples were collected at the moment of oocyte extrusion, when oocytes and semen were mixed (time 0), as well as at 10, 20 and 30 s after mixing, every minute up to 10 min, and then at 15 and 20 min. The oocytes are spherical, translucent and greenish with a mean diameter of 1.3 +/- 0.11 mm. During the extrusion, cytoplasmic movement was observed in eggs towards the micropyle, characterizing the animal pole. At the moment of fertilization, the cortical cytoplasm showed a higher concentration of cortical alveoli at the animal pole than at the vegetal pole. The cortical alveoli breakdown promoted the elevation of the chorion with a consequent increase in egg diameter (1.95 +/- 0.08 mm). The penetration of the spermatozoon promotes the formation of a fertilization cone of spherical external structure, which obstructs the opening of the micropyle. This structure acts as a main mechanism to avoid polyspermy, intercepting the access of supernumerary spermatozoa. Such studies about the reproductive biology of fish are important to species survival and conservation programmes.
Assuntos
Fertilização/fisiologia , Peixes/fisiologia , Oócitos/ultraestrutura , Zigoto/ultraestrutura , Animais , Citoplasma/ultraestrutura , Desenvolvimento Embrionário/fisiologia , Feminino , Masculino , Microscopia Eletrônica de Varredura , Espermatozoides/ultraestruturaRESUMO
Forensic entomology is the science that studies the role of insects in decomposing corpses and one of the most common uses is to estimate the post-mortem interval (PMI) based on insect activity on a decomposing body. Usually, flies are the first insects to reach a carcass and are able to oviposit on carrion within a few hours after death. Scanning electron microscopy (SEM) gives detailed information about morphological characters helping to identify the immature forms of flies and consequently serves as a tool in crime scene investigations. Sometimes, only eggs and larvae are found in corpses. Some dipteral species are important because their larvae develop in organic matter. The aim of this study is to identify eggs of species of forensic importance, such as Chrysomya megacephala, Chrysomya putoria, Lucilia cuprina, Lucilia eximia and Ophyra aenescens, using scanning electron microscopy (SEM). C. megacephala had no anastomosis or holes at the top of the islands and C. putoria had few anastomoses and no holes, whereas L. eximia and O. aenescens were found to have anastomoses and holes and L. cuprina had only anastomoses. The median area was bifurcated anteriorly in C. megacephala, L. eximia and O. aenescens and rounded in C. putoria and L. cuprina. Also the sculptures observed in the chorionic cells, the length and the way that median area ends up posteriorly are characteristics of great diagnostic value to identify muscoids of forensic importance.
Assuntos
Dípteros/classificação , Dípteros/ultraestrutura , Medicina Legal/métodos , Microscopia Eletrônica de Varredura/métodos , Animais , Entomologia/métodos , Especificidade da Espécie , Zigoto/classificação , Zigoto/ultraestruturaRESUMO
PURPOSE: to determine the relationship between the morphology of the first spindle pole of human oocytes and rates of fertilization, fragmentation and embryo quality in procedures of Intracytoplasmic Sperm Injection (ICSI). METHODS: retrospective study of 582 consecutive ICSI cycles, from July 2003 to July 2005. The morphology of the first spindle pole (SP) was assessed through the analysis of 3,177 oocytes in metaphase II, immediately before the ICSI procedure, always by the same observer. SP has been classified in the following categories: normal size intact, fragmented or augmented SP. Fertilization rate and fragmentation, and the number and rate of good quality embryos in each one of the three groups studied have been evaluated, 48 hours after ICSI (D2). Embryos with four cells, without fragmentation and with symmetric blastomeres in D2 were considered as of good quality. RESULTS: rates of fertilization, fragmentation and of good quality embryo formation, resulting from oocyte insemination, with augmented SP (20.7, 16.7 and 5% respectively) were significantly lower than the ones from intact and normal size SP (70.8, 62.5 and 19%, respectively) or from fragmented SP oocytes (69.7, 60.5 and 17.1%, respectively). CONCLUSIONS: it has been observed that the presence of augmented first spindle pole is related to worse rates of fertilization, fragmentation and bad quality embryo formation. Nevertheless, fragmentation in the first spindle pole of the oocyte does not seem to affect ICSI results.
Assuntos
Fase de Clivagem do Zigoto , Embrião de Mamíferos , Fertilização/fisiologia , Injeções de Esperma Intracitoplásmicas/normas , Zigoto/citologia , Humanos , Estudos Retrospectivos , Fatores de Tempo , Zigoto/ultraestruturaRESUMO
We had previously reported that a cysteine-protease catalyzes the sperm histones (SpH) degradation associated to male chromatin remodeling in sea urchins. We found that this protease selectively degraded the SpH leaving maternal cleavage stage (CS) histone variants unaffected, therefore we named it SpH-protease. It is yet unknown if the SpH-protease catalyzes the SpH degradation while these histones are organized as nucleosomes or if alternatively these histones should be released from DNA before their proteolysis. To investigate this issue we had performed an in vitro assay in which polynucleosomes were exposed to the active purified protease. As shown in this report, we found that sperm histones organized as nucleosomes remains unaffected after their incubation with the protease. In contrast the SpH unbound and free from DNA were readily degraded. Interestingly, we also found that free DNA inhibits SpH proteolysis in a dose-dependent manner, further strengthening the requirement of SpH release from DNA before in order to be degraded by the SpH-protease. In this context, we have also investigated the presence of a sperm-nucleosome disassembly activity (SNDA) after fertilization. We found a SNDA associated to the nuclear extracts from zygotes that were harvested during the time of male chromatin remodeling. This SNDA was undetectable in the nuclear extracts from unfertilized eggs and in zygotes harvested after the fusion of both pronuclei. We postulate that this SNDA is responsible for the SpH release from DNA which is required for their degradation by the cysteine-protease associated to male chromatin remodeling after fertilization.
Assuntos
Montagem e Desmontagem da Cromatina/fisiologia , Histonas/metabolismo , Meiose , Nucleossomos/ultraestrutura , Espermatozoides/fisiologia , Animais , Montagem e Desmontagem da Cromatina/efeitos dos fármacos , Cisteína Endopeptidases/farmacologia , Cisteína Endopeptidases/fisiologia , Feminino , Fertilização , Histonas/efeitos dos fármacos , Masculino , Meiose/fisiologia , Nucleossomos/química , Nucleossomos/efeitos dos fármacos , Ouriços-do-Mar , Espermatozoides/citologia , Espermatozoides/metabolismo , Zigoto/química , Zigoto/ultraestruturaRESUMO
Después de manifestar la relevancia del tema, se exponen algunos textos de la OPS sobre el mismo, se distinguen varios paradigmas sobre el ser humano y las ideas generales del pensamiento complejo que se relacionan con el tema, las pruebas de la existencia del espíritu como elemento diferenciador o unificador en las definiciones de ser humano y persona, y varios argumentos, con los que se puede concluir cuándo comienza a existir el espíritu en el ser humano. También se propone una referencia, para que la toma de decisiones sobre seres humanos sea ética. El artículo refleja algunas ideas expuestas en el libro Ser humano y persona, editado en el año 2003 por la Universidad de La Sabana y el Celam
Assuntos
Humanos , Bioética/educação , Bioética/tendências , Zigoto/crescimento & desenvolvimento , Zigoto/fisiologia , Zigoto/metabolismo , Zigoto/ultraestruturaRESUMO
This pilot project was designed to determine if normal kids could be produced after microinjection in pronuclear embryos and subsequent transfer to recipients in a transgenic goat program in Brazil. Twelve donors of the Saanen breed and 17 recipients of an undefined breed were used. The estrus of both donors and recipients was synchronized by a standard progestagen treatment and superovulation obtained by six pFSH injections. Donors in estrus were mated with fertile Saanen bucks. Zygotes were recovered surgically by flushing oviducts. The recovered zygotes with visible pronuclei were microinjected with 500 to 1000 copies of the human G-CSF gene. Two or four embryos were surgically transferred into the oviducts of recipients. One recipient became pregnant and two kids were born. No transgenic goat was identified after PCR analysis. Even though transgenic goats were not obtained, this experiment establishes the basis of a synchronization and superovulation regimen for use in goats raised in Brazil, for the purpose of collecting and manipulating the pronuclear embryos. This project also showed that microinjected one-cell goat embryos can survive to produce live young following surgical transfer
Assuntos
Animais , Feminino , Gravidez , Animais Geneticamente Modificados/embriologia , Cabras/genética , Transferência Embrionária , Fator Estimulador de Colônias de Granulócitos/genética , Zigoto/ultraestrutura , Brasil , Cabras/embriologia , Microinjeções , Projetos PilotoRESUMO
PURPOSE: To analyze best parameter to select embryos according to sperm origin in ICSI cycles. METHODS: One hundred seventy-two ICSI cycles were divided among three different groups: A (ejaculated spermatozoa from nonmale factor infertility), B (ejaculated spermatozoa from oligospermia), and C (spermatozoa from azoospermia). Embryos were divided on Day 1 into two patterns: S0 (pronuclei (PN) aligned and close with normal arrangement of nucleoli) and S1 (when these characteristics were absent) and also on transfer day according to morphological features. RESULTS: Relationships of PN patterns related to sperm origin were noted. More S0 embryos were detected with better sperm quality. Higher number of good quality embryos was obtained when male factor was absent. Ejaculated and epididymal spermatozoa provide better quality embryos than do testicular spermatozoa. CONCLUSIONS: PN classification associated with transfer day morphology is valuable additional noninvasive criterion for elective embryo transfer, mainly in the cases with severe male factor.
Assuntos
Nucléolo Celular/ultraestrutura , Transferência Embrionária , Injeções de Esperma Intracitoplásmicas/métodos , Zigoto/ultraestrutura , Aborto Espontâneo , Fase de Clivagem do Zigoto , Ejaculação , Implantação do Embrião , Epididimo/fisiologia , Feminino , Humanos , Masculino , Oligospermia , Gravidez , Taxa de Gravidez , Espermatozoides/fisiologia , Testículo/fisiologiaRESUMO
This pilot project was designed to determine if normal kids could be produced after microinjection in pronuclear embryos and subsequent transfer to recipients in a transgenic goat program in Brazil. Twelve donors of the Saanen breed and 17 recipients of an undefined breed were used. The estrus of both donors and recipients was synchronized by a standard progestagen treatment and superovulation obtained by six pFSH injections. Donors in estrus were mated with fertile Saanen bucks. Zygotes were recovered surgically by flushing oviducts. The recovered zygotes with visible pronuclei were microinjected with 500 to 1000 copies of the human G-CSF gene. Two or four embryos were surgically transferred into the oviducts of recipients. One recipient became pregnant and two kids were born. No transgenic goat was identified after PCR analysis. Even though transgenic goats were not obtained, this experiment establishes the basis of a synchronization and superovulation regimen for use in goats raised in Brazil, for the purpose of collecting and manipulating the pronuclear embryos. This project also showed that microinjected one-cell goat embryos can survive to produce live young following surgical transfer.
Assuntos
Animais Geneticamente Modificados/embriologia , Transferência Embrionária , Cabras/genética , Fator Estimulador de Colônias de Granulócitos/genética , Zigoto/ultraestrutura , Animais , Brasil , Feminino , Cabras/embriologia , Microinjeções , Projetos Piloto , GravidezRESUMO
The formation and bipolar translocation of an ectoplasmic cytoskeleton of rings and meridional bands was studied in interphase zygotes of the glossiphoniid leech Theromyzon trizonare. Zygotes consisted of a peripheral organelle-rich ectoplasm and an internal yolk-rich endoplasm. After microinjection of labeled tubulin and/or actin, zygotes were examined by time-lapse video imaging, immunofluorescence and confocal microscopy. The rings and meridional bands were formed by condensation of a network of moving cytasters that represented ectoplasmic secondary centers of microtubule and actin filament nucleation. In some cases the network of cytasters persisted between the rings. The cytoskeleton had an outer actin layer and an inner microtubule layer that merged at the irregularly-shaped boundary zone. Bipolar translocation of the rings, meridional bands, or the network of cytasters led to accumulation of the cytoskeleton at both zygote poles. Translocation of the cytoskeleton was slowed or arrested by microinjected taxol or phalloidin, in a dose-dependent fashion. Results of drug treatment probably indicate differences in the degree and speed at which the cytoskeleton becomes stabilized. Moreover, drugs that selectively stabilized either microtubules or actin filaments stabilized and impaired movement of the entire cytoskeleton. Microtubule poisons and latrunculin-B failed to disrupt the cytoskeleton. It is concluded that the microtubule and actin cytoskeletons are dynamic, presumably cross-linked and resistant to depolymerizing drugs. They probably move along each other by a sliding mechanism that depends on the instability of microtubules and actin filaments.
Assuntos
Citoesqueleto de Actina/metabolismo , Citoesqueleto/metabolismo , Sanguessugas/fisiologia , Microtúbulos/metabolismo , Zigoto/ultraestrutura , Citoesqueleto de Actina/ultraestrutura , Animais , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Colchicina/farmacologia , Citoesqueleto/ultraestrutura , Corantes Fluorescentes/metabolismo , Interfase/fisiologia , Sanguessugas/citologia , Microinjeções , Microscopia de Vídeo , Microtúbulos/ultraestrutura , Paclitaxel/farmacologia , Faloidina/farmacologia , Transporte Proteico/fisiologia , Tiazóis/farmacologia , Tiazolidinas , Fatores de Tempo , Zigoto/fisiologiaRESUMO
We analysed the distribution of beta-tubulins, acetylated alpha-tubulins and chromatin configuration in 113 human zygotes showing abnormal fertilisation, 16-18 h after conventional in vitro fertilisation (IVF) or intracytoplasmic sperm injection (ICSI). After a first characterisation using phase contrast microscopy, immunofluorescence staining was performed in 67 IVF and 46 ICSI zygotes that developed one, three or more pronuclei and/or subnuclei, with or without extrusion of the second polar body. Independently of the number of pronuclei found, beta-tubulins were uniformly distributed throughout the cytoplasm of the abnormal zygotes. We did not observe any kind of microtubule alteration with respect of the ploidy level and/or its origin. The most frequent abnormal fertilisation pattern found after IVF was the presence of three or four pronuclei (74.6%). On the other hand, the presence of one pronucleus (63.0%) was the main pattern found after ICSI. No differences between the two groups were seen in terms of development of subnuclei. Anamolies detected after IVF and ICSI showed different aetiologies such as parthenogenetic activation, gynogenetic or androgenetic development, as well as digynic or diandric fertilisation.
Assuntos
Fertilização in vitro , Genoma Humano , Microtúbulos/ultraestrutura , Cromatina/metabolismo , Feminino , Humanos , Masculino , Microscopia de Fluorescência , Microscopia de Contraste de Fase , Pais , Partenogênese , Ploidias , Injeções de Esperma Intracitoplásmicas , Tubulina (Proteína)/metabolismo , Zigoto/metabolismo , Zigoto/ultraestruturaRESUMO
The organization of the cytoskeleton in the early first interphase zygote and its involvement in organelle redistribution were studied in the glossiphoniid leech Theromyzon trizonare by confocal and electron microscopy, immunofluorescence, and time-lapse video imaging after microinjection of labeled tubulin and/or actin and loading with a mitotracker. The cytoskeleton consists of an inner or endoplasmic and an outer or ectoplasmic domain. The inner domain consists of a monaster whose fibers retract from the zygote periphery by the end of the early first interphase. The outer domain is built upon a network of microtubules and microfilaments cytasters. Short pulses of microinjected labeled actin or tubulin and Taxol treatment demonstrate that cytasters are centers of microtubule and microfilament nucleation. Immunostaining with anti-centrophilin, anti-BX-63, and anti-AH-6 indicates that the network of cytasters includes centrosomal antigens. Cytasters move in an orderly fashion at speeds of 0.5-2 micrometer/min, in an energy-dependent process retarded and finally blocked by the ATP analogue AMP-PNP and high concentrations of Taxol. Colliding cytasters fuse and form larger cytoskeletal nucleation centers. The leech zygote is a highly compartmentalized cell whose cytasters function as articulated components of a very dynamic cytoskeletal system engaged in bulk transportation of organelles during ooplasmic segregation.
Assuntos
Citoesqueleto de Actina/metabolismo , Sanguessugas/citologia , Centro Organizador dos Microtúbulos/metabolismo , Microtúbulos/metabolismo , Zigoto/citologia , Citoesqueleto de Actina/efeitos dos fármacos , Citoesqueleto de Actina/ultraestrutura , Actinas/metabolismo , Adenilil Imidodifosfato/farmacologia , Animais , Transporte Biológico/efeitos dos fármacos , Centrossomo/efeitos dos fármacos , Centrossomo/metabolismo , Centrossomo/ultraestrutura , Imuno-Histoquímica , Interfase/efeitos dos fármacos , Sanguessugas/efeitos dos fármacos , Sanguessugas/metabolismo , Sanguessugas/ultraestrutura , Meiose/efeitos dos fármacos , Microinjeções , Microscopia Eletrônica , Microscopia de Fluorescência , Microscopia de Vídeo , Centro Organizador dos Microtúbulos/efeitos dos fármacos , Centro Organizador dos Microtúbulos/ultraestrutura , Microtúbulos/efeitos dos fármacos , Microtúbulos/ultraestrutura , Movimento/efeitos dos fármacos , Organelas/efeitos dos fármacos , Organelas/metabolismo , Organelas/ultraestrutura , Paclitaxel/farmacologia , Tubulina (Proteína)/metabolismo , Zigoto/efeitos dos fármacos , Zigoto/metabolismo , Zigoto/ultraestruturaRESUMO
To investigate changes in chromatin organization associated with DNA replication during the first stages of development of the sea urchin Tetrapygus niger, we compared micrococcal nuclease (MNase) digestion patterns of chromatin from zygotes harvested during the first S phase and from unfertilized eggs. We observed that the majority of DNA fragments derived from MNase digested zygote nuclei were similar to or smaller than a mononucleosome, while those derived from unfertilized egg nuclei were larger (1,500 to 410 bp). This result indicates that in zygotes, where active DNA replication is occurring, the major chromatin fraction is represented as unfolded nucleosomes. In contrast, in unfertilized eggs chromatin appears to be organized into polynucleosomes. To determine if the unfolded structure of nucleosomes observed during S phase is related to the level of poly (ADP-ribosylation) of cleavage stage (CS) histone variants, zygotes were treated with 20 mM 3-Amino Benzamide (3 ABA) during the interval between 3 and 30 min post-insemination (p.i.). This treatment with 3 ABA decreases the poly (ADP-ribosylation) of CS histone variants and inhibits the first S phase in zygotes [Imschenetzky et al. (1991): J Cell Biochem 46:234-241; Imschenetzky et al. (1993): J Cell Biochem 51:198-205]. When the MNase digested patterns of chromatin from these 3 ABA treated and control zygotes were compared, we found that the unfolded structure of the nucleosomes remains unaltered by the inhibition of the poly(ADP-ribose) synthetase with 3 ABA. This result indicates that the unfolded nucleosomal structure, particular to the chromatin of S phase zygotes, is not contemporaneous to DNA replication and is independent of the normal level of poly(ADP-ribosylation) of CS histone variants.