RESUMO
Several equids have gone extinct and many extant equids are currently considered vulnerable to critically endangered. This work aimed to evaluate whether domestic horse oocytes support preimplantation development of zebra embryos obtained by intracytoplasmic sperm injection (ICSI, zebroid) and cloning, and to study the Hippo signaling pathway during the lineage specification of trophectoderm cells and inner cell mass cells. We first showed that zebra and horse sperm cells induce porcine oocyte activation and recruit maternal SMARCA4 during pronuclear formation. SMARCA4 recruitment showed to be independent of the genetic background of the injected sperm. No differences were found in blastocyst rate of ICSI hybrid (zebra spermatozoon into horse egg) embryos relative to the homospecific horse control group. Interestingly, zebra cloned blastocyst rate was significantly higher at day 8. Moreover, most ICSI and cloned horse and zebra blastocysts showed a similar expression pattern of SOX2 and nuclear YAP1 with the majority of the nuclei positive for YAP1, and most SOX2+ nuclei negative for YAP1. Here we demonstrated that horse oocytes support zebra preimplantation development of both, ICSI and cloned embryos, without compromising development to blastocyst, blastocyst cell number neither the expression of SOX2 and YAP1. Our results support the use of domestic horse oocytes as a model to study in vitro zebra embryos on behalf of preservation of valuable genetic.
Assuntos
Desenvolvimento Embrionário , Equidae/embriologia , Equidae/genética , Cavalos/fisiologia , Oócitos/fisiologia , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Núcleo Celular/fisiologia , Clonagem de Organismos/veterinária , Citoplasma/fisiologia , Técnicas de Cultura Embrionária/veterinária , Desenvolvimento Embrionário/genética , Desenvolvimento Embrionário/fisiologia , Espécies em Perigo de Extinção , Equidae/metabolismo , Feminino , Perfilação da Expressão Gênica , Cavalos/genética , Técnicas In Vitro , Masculino , Técnicas de Transferência Nuclear/veterinária , Fatores de Transcrição SOXB1/genética , Injeções de Esperma Intracitoplásmicas/veterinária , Sus scrofaRESUMO
Motility acquisition during sperm maturation and passage through the epididymis is closely related to mitochondrial function and appears to occur in parallel with cytoplasmic droplet (CD) migration. However, such mechanism remains unclear in dogs. Thus, the aim of this study was to characterize the influence of sperm CD in the mitochondrial functionality during epididymal sperm maturation in dogs. Twenty-one adult dogs were submitted to elective bilateral orchiectomy. Testicles were stored for 18-24h at 5°C and epididymal sperm samples were then collected from different segments of the epididymis (caput, corpus and cauda). Samples were evaluated for computer-assisted motility analysis (CASA), presence of CD (eosin/nigrosin stain), ultrastructural CD analysis and sperm mitochondrial activity (3,3' diaminobenzidine technique) and membrane potential (JC-1 probe). Samples collected from the corpus epididymis showed higher motility and mitochondrial activity in comparison to the caput sperm. Moreover, corpus sperm had lower percentage of proximal droplets compared to caput samples, while mitochondrial membrane potential remained unchanged. Cauda samples showed higher motility, mitochondrial activity and potential, however, lower presence of sperm droplets (proximal and distal). In conclusion, the CD is essential for epididymal sperm maturation in dogs, showing important functions along the transit in the epididymis. In the corpus segment, the migration of the CD along the sperm midpiece provides a high mitochondrial activity and the onset of sperm motility. On the other hand, sperm from cauda epididymis lack CD but suffered lipid membrane changes which allow a maximum mitochondrial membrane potential and motility.
Assuntos
Citoplasma/fisiologia , Cães/fisiologia , Maturação do Esperma/fisiologia , Espermatozoides/fisiologia , Animais , Masculino , Potencial da Membrana Mitocondrial/fisiologia , Análise do Sêmen , Espermatozoides/ultraestruturaRESUMO
We investigated whether supplementing the medium used to transport bovine oocytes with different macromolecules [foetal calf serum (FCS) or bovine serum albumin (BSA)] or a mixture of antioxidants (cysteine, cysteamine and catalase) affects their nuclear and cytoplasmic maturation and thereby affects their subsequent embryonic development and cryotolerance. Oocytes were transported for 6 hr in a portable incubator and then subjected to standard in vitro maturation (IVM) for 18 hr. The oocytes in the control groups were cultured (standard IVM) for 24 hr in medium containing 10% FCS (Control FCS) or 10% FCS and the antioxidant mixture (Control FCS+Antiox). The intracellular concentrations of reactive oxygen species (ROS) at the end of IVM period were lower in the oocytes subjected to simulated transport in the presence of a macromolecular supplement or the antioxidant mixture than that of the control group (FCS: 0.62 and BSA: 0.66 vs. Control FCS: 1.00, p < .05; and Transp: 0.58 and Transp Antiox: 0.70 vs. Control FCS: 1.00, p < .05). After IVM, the mitochondrial membrane potentials of the transported oocytes were lower than those of the non-transported oocytes (FCS: 0.41 and BSA: 0.57 vs. Control FCS: 1.00, p < .05; and Transp: 0.48 and Transp Antiox: 0.51 vs. Control FCS: 1.00 and Control Antiox: 0.84, p < .05). The blastocyst formation rates (36.9% average) and the re-expansion rates of vitrified-warmed blastocysts (53%, average) were unaffected (p > .05) by the treatments. In conclusion, supplementing the medium in which bovine oocytes are transported with antioxidants or different macromolecules did not affect their in vitro production of embryos or their cryotolerance.
Assuntos
Antioxidantes/farmacologia , Catalase/farmacologia , Cisteamina/farmacologia , Cisteína/farmacologia , Citoplasma/fisiologia , Técnicas de Cultura Embrionária/veterinária , Técnicas de Maturação in Vitro de Oócitos/veterinária , Animais , Bovinos , Meios de Cultura , Técnicas de Maturação in Vitro de Oócitos/métodos , Potencial da Membrana Mitocondrial , Oócitos , Espécies Reativas de OxigênioRESUMO
Members of the Cyperaceae family exhibit an asymmetric microsporogenesis that results in the degeneration of three out of four meiotic products. Efforts have been made previously to describe the resulting structure, named the pseudomonad, but mechanisms concerning the establishment of cell domains, nuclear development, and programmed cell death are largely unknown. Using the Rhynchospora genus as a model, evidence for cell asymmetry, cytoplasmic isolation, and programmed cell death was obtained by a combination of electron microscopic, cytochemical, immunocytochemical, in situ hybridization, and flow cytometric methods. Degenerative cells were identified at the abaxial region, with the cytoskeleton marking their delimitation from the functional domain after meiosis. After attempting to initiate cell division with an unreplicated genome and abnormal spindle assembly, these cells exhibited a gradual process of cytoplasmic contraction associated with hypermethylation of cytosines and differential loss of DNA. These results indicate that the asymmetric tetrad establishes a functional cell, where one nucleus is preferentially selected to survive. Degenerative haploid cells are then eliminated in a multistep process associated with mitotic disorder, non-random elimination of repetitive DNA, vacuolar cell death, and DNA fragmentation.
Assuntos
Morte Celular/fisiologia , Cyperaceae/fisiologia , Gametogênese Vegetal/fisiologia , Divisão Celular/fisiologia , Cyperaceae/ultraestrutura , Citoplasma/fisiologia , Citoesqueleto/fisiologia , Hibridização In Situ , Meiose/fisiologia , Microscopia EletrônicaRESUMO
Adequate dietary intake of manganese (Mn) is required for normal reproductive performance in cattle. This study was carried out to investigate the effect of Mn during in vitro maturation of bovine cumulus-oocyte complexes (COC) on apoptosis of cumulus cells, cumulus expansion, and superoxide dismutase (SOD) activity in the COC. The role of cumulus cells on Mn transport and subsequent embryo development was also evaluated. Early apoptosis decreased in cumulus cells matured with Mn compared with medium alone. Cumulus expansion did not show differences in COC matured with or without Mn supplementation. SOD activity was higher in COC matured with 6 ng/ml Mn than with 0 ng/ml Mn. Cleavage rates were higher in COC and denuded oocytes co-cultured with cumulus cells, either with or without Mn added to in vitro maturation (IVM) medium. Regardless of the presence of cumulus cells during IVM, the blastocyst rates were higher when 6 ng/ml Mn was supplemented into IVM medium compared with growth in medium alone. Blastocyst quality was enhanced when COC were matured in medium with Mn supplementation. The results of the present study indicated that Mn supplementation to IVM medium enhanced the 'health' of COC, and improved subsequent embryo development and embryo quality.
Assuntos
Blastocisto/citologia , Células do Cúmulo/citologia , Técnicas de Maturação in Vitro de Oócitos/métodos , Manganês/farmacologia , Oócitos/fisiologia , Animais , Apoptose/efeitos dos fármacos , Blastocisto/fisiologia , Bovinos , Meios de Cultura/química , Meios de Cultura/farmacologia , Células do Cúmulo/efeitos dos fármacos , Citoplasma/efeitos dos fármacos , Citoplasma/fisiologia , Relação Dose-Resposta a Droga , Feminino , Fertilização in vitro , Manganês/administração & dosagem , Oócitos/efeitos dos fármacos , Superóxido Dismutase/metabolismoRESUMO
During maturation sperm cells acquire their fertilizing ability; however, this event also produces reactive oxygen species and as such local antioxidant protection is required. Thirteen epididymis from dogs were used, and sperm samples were collected from different segments of the epididymis (i.e. caput, corpus and cauda). The samples were evaluated for motility and vigor, permeability of plasma membrane, presence of cytoplasmic droplet, acrosome integrity and the activity of the antioxidant enzymes glutathione peroxidase (GPx) and superoxide dismutase (SOD). Samples collected from the cauda showed higher motility, vigor and lower permeability of plasma and acrosomal membranes in relation to the corpus and caput, indicating different levels of maturity. The enzyme activity remained unchanged in the samples. A significant positive correlation was observed in the epididymal caput, between distal cytoplasmic droplet and SOD, and a negative correlation, in the epididymal cauda, between proximal cytoplasmic droplet and GPx, indicating the physiological need of the specific antioxidants in these segments.
Assuntos
Citoplasma/fisiologia , Cães , Oxidantes/metabolismo , Maturação do Esperma , Espermatozoides/metabolismo , Acrossomo/metabolismo , Animais , Antioxidantes/metabolismo , Glutationa Peroxidase/metabolismo , Masculino , Oxirredução , Motilidade dos Espermatozoides/fisiologia , Superóxido Dismutase/metabolismoRESUMO
Cell-cell fusion in sexually reproducing organisms is a mechanism to merge gamete genomes and, in multicellular organisms, it is a strategy to sculpt organs, such as muscle, bone, and placenta. Moreover, this mechanism has been implicated in pathological conditions, such as infection and cancer. Studies of genetic model organisms have uncovered a unifying principle: cell fusion is a genetically programmed process. This process can be divided in three stages: competence (cell induction and differentiation); commitment (cell determination, migration, and adhesion); and cell fusion (membrane merging and cytoplasmic mixing). Recent work has led to the discovery of fusogens, which are cell fusion proteins that are necessary and sufficient to fuse cell membranes. Two unrelated families of fusogens have been discovered, one in mouse placenta and one in Caenorhabditis elegans (syncytins and F proteins, respectively). Current research aims to identify new fusogens and determine the mechanisms by which they merge membranes.
Assuntos
Fusão Celular , Animais , Caenorhabditis elegans/fisiologia , Diferenciação Celular/fisiologia , Membrana Celular/fisiologia , Citoplasma/fisiologia , Feminino , Fertilização/genética , Fertilização/fisiologia , Regulação da Expressão Gênica no Desenvolvimento , Células Germinativas/fisiologia , Humanos , Macrófagos/fisiologia , Fusão de Membrana/genética , Fusão de Membrana/fisiologia , Camundongos , Mioblastos/fisiologia , Neurospora crassa/fisiologia , Placenta/fisiologia , Plantas/metabolismo , Gravidez , Saccharomyces cerevisiae/fisiologiaRESUMO
The selection of competent oocytes for in vitro maturation is still a major problem during bovine in vitro embryo production. Markers for in vitro cytoplasmic maturation, based on the organization of cortical granule and mitochondria, are lacking. We examined the pre-selection of immature bovine oocytes by brilliant cresyl blue stain (BCB test) based on glucose-6-phosphate dehydrogenase (G6PDH) activity during oocyte development. Oocytes were recovered from ovarian follicles exposed to 26 µM BCB stain and classified according to the aspect of their cytoplasm: BCB(+) (oocytes with blue cytoplasm) and BCB(-) (unstained cytoplasm) and then in vitro matured into a conventional in vitro maturation (IVM) medium and standard procedure. In Experiment 1, nuclear maturation was determined by polar body identification, while cytoplasmic maturation was based on cortical granule (CG) migration (peripheral) and mitochondria distribution (central). Evidence of polar body, cortical granule migration and of centrally located mitochondria was significantly (p < 0.05) higher in BCB(+) oocytes than in BCB(-) (polar body present: 65% vs 20%; peripheral CG: 72% vs. 14%; and central mitochondria: 85% vs. 19%, respectively). In Experiment 2, the efficiency pre-selection of bovine oocytes by BCB on embryo development in vitro was assessed. Cleavage rates were similar (75%) among control, BCB(+) and BCB(-) groups, while blastocyst rates on D7 were (p < 0.05) higher (35%) in BCB(+) vs BCB(-) (10%) or control (28%). We showed that the BCB test is efficient to identify competent immature bovine oocytes to undergo synchronous nuclear and cytoplasmic in vitro maturation thus yielding higher in vitro embryo development to blastocyst stage.
Assuntos
Blastocisto/fisiologia , Oócitos/citologia , Oócitos/fisiologia , Animais , Bovinos , Células Cultivadas , Citoplasma/fisiologia , Desenvolvimento Embrionário , Feminino , Fertilização in vitro , Glucosefosfato Desidrogenase/metabolismo , Técnicas de Maturação in Vitro de Oócitos , Meiose , Mitocôndrias/fisiologia , Folículo Ovariano/citologia , Oxazinas/análise , Coloração e Rotulagem/métodosRESUMO
BACKGROUND: This study evaluated whether there is a relationship between the zona pellucida birefringence (ZP-BF) intensity and the nuclear (NM) and cytoplasmic (CM) in vitro maturation of human oocytes from stimulated cycles. RESULTS: The ZP-BF was evaluated under an inverted microscope with a polarizing optical system and was scored as high/positive (when the ZP image presented a uniform and intense birefringence) or low/negative (when the image presented moderate and heterogeneous birefringence). CM was analyzed by evaluating the distribution of cortical granules (CGs) throughout the ooplasm by immunofluorescence staining. CM was classified as: complete, when CG was localized in the periphery; incomplete, when oocytes presented a cluster of CGs in the center; or in transition, when oocytes had both in clusters throughout cytoplasm and distributed in a layer in the cytoplasm periphery Nuclear maturation: From a total of 83 germinal vesicle (GV) stage oocytes, 58 of oocytes (69.9%) reached NM at the metaphase II stage. From these 58 oocytes matured in vitro, the high/positively scoring ZP-BF was presented in 82.7% of oocytes at the GV stage, in 75.8% of oocytes when at the metaphase I, and in 82.7% when oocytes reached MII. No relationship was observed between NM and ZP-BF positive/negative scores (P = 0.55). These variables had a low Pearson's correlation coefficient (r = 0.081). Cytoplasmic maturation: A total of 85 in vitro-matured MII oocytes were fixed for CM evaluation. Forty-nine oocytes of them (57.6%) showed the complete CM, 30 (61.2%) presented a high/positively scoring ZP-BF and 19 (38.8%) had a low/negatively scoring ZP-BF. From 36 oocytes (42.3%) with incomplete CM, 18 (50%) presented a high/positively scoring ZPBF and 18 (50%) had a low/negatively scoring ZP-BF. No relationship was observed between CM and ZP-BF positive/negative scores (P = 0.42). These variables had a low Pearson's correlation coefficient (r = 0.11). CONCLUSIONS: The current study demonstrated an absence of relationship between ZP-BF high/positive or low/negative score and nuclear and cytoplasmic in vitro maturation of oocytes from stimulation cycles.
Assuntos
Oócitos/fisiologia , Oogênese/fisiologia , Indução da Ovulação , Zona Pelúcida/fisiologia , Birrefringência , Núcleo Celular/fisiologia , Células Cultivadas , Citoplasma/fisiologia , Feminino , Humanos , Oócitos/citologia , Controle de Qualidade , Projetos de PesquisaRESUMO
The objective was to study the effect of a defined culture system, on nuclear and cytoplasmic maturation of bovine oocytes, using the two-step procedure of IVM to detect possible inhibition and subsequent resumption of meiosis arrest. In the first step, called the prematuration period (PMP), COCs were cultured in T1-non-defined medium (NDM), or T2-defined medium (DM), both for 24 h. In step 2, called the resumption period (RP), COCs were cultured in: NDM (T1); DM + NDM (T3); or DM+DM (T4) for 24 h in each medium. The NDM was composed of TCM-199 supplemented with FCS and FSH. The DM was composed of alpha-MEM supplemented with PVA, insulin, IGF-1, androstenedione, nonessential amino acids, transferrin, and sodium selenium. Oocytes from T2 had a lower (P < 0.05) rate of nuclear maturation (19.8%) than T1 oocytes (83.2%). Also, T2 COCs appeared to be in the process of cytoplasmic maturation, according to the distribution of organelles assessed by transmission electron microscopy (MET). These COCs had characteristics previously described as mature: erect microvilli on the plasmembrane, presence of cortical/evenly distributed mitochondria throughout the ooplasm, and presence of 50% aligned/cluster cortical granules. Immature characteristics such as small PvS, compact cumulus cells, and presence of 50% cortical granule clusters were also observed. The T1 COCs had only characteristics of maturation (P < 0.05). In step 2 (RP), meiosis arrest induced by DM was resumed after an additional 24 h of culture in NDM (T3) with 79.2% mature COCs, whereas in T4, meiosis arrest was maintained, resulting in almost 70% immature COCs (P < 0.05). At the end of RP, T3 COCs had the mature characteristics of mitochondria spread throughout the cytoplasm (P < 0.05), cumulus expansion, and alignment of cortical granules, whereas the T4 group had both immature and mature characteristics. We inferred that DM can be used in lieu of meiosis inhibitors and furthermore, it can provide extra time to study nuclear and cytoplasmic maturation synchrony of IVM.
Assuntos
Bovinos , Técnicas de Cultura de Células , Meios de Cultura/química , Meiose/efeitos dos fármacos , Oócitos/efeitos dos fármacos , Animais , Meios de Cultura/farmacologia , Citoplasma/fisiologia , Estradiol/metabolismo , Feminino , Oócitos/citologia , Oócitos/crescimento & desenvolvimento , Álcool de Polivinil/farmacologia , Fatores de TempoRESUMO
The aim of this study was: (1) to monitor the nucleolar material distribution using cytological and cytochemical techniques and ultrastructural analysis; and (2) to compare the nucleolar material distribution with the formation of the chromatoid body (CB) in the germ epithelium of Tilapia rendalli. Nucleolar fragmentation occurred during the leptotene of prophase I and nucleolus reorganization occurred in the early spermatid nucleus. The area of the early spermatid nucleolus was significantly smaller than that of the spermatogonia nucleolus. Ultrastructural analysis showed an accumulation of nuages, which form the CB, before nucleolar fragmentation in the spermatogonia cytoplasm. The CB was observed in association with mitochondrial clusters in the cytoplasm of primary spermatocytes, as well as in those of initial and later spermatids. In conclusion, the nucleolus seems to be related to CB formation during spermatogenesis of T. rendalli, because at the moment of nucleolus fragmentation in the primary spermatocytes, the CB reaches its largest area and it is able to complete important functions during spermatogenesis. The reorganized nucleolus of the initial spermatids has a lower area due several factors, one of which is the probable migration of nucleolar fragments from the nucleus to the cytoplasm, therefore playing a role in CB formation.
Assuntos
Nucléolo Celular/ultraestrutura , Espermátides/ultraestrutura , Espermatogênese/fisiologia , Espermatogônias/ultraestrutura , Testículo/ultraestrutura , Tilápia/anatomia & histologia , Animais , Nucléolo Celular/fisiologia , Cromatina/fisiologia , Cromatina/ultraestrutura , Citoplasma/fisiologia , Citoplasma/ultraestrutura , Masculino , Meiose/fisiologia , Microscopia Eletrônica de Transmissão , Organelas/fisiologia , Organelas/ultraestrutura , Prófase/fisiologia , Especificidade da Espécie , Espermátides/fisiologia , Espermatogônias/fisiologia , Testículo/fisiologia , Tilápia/fisiologiaRESUMO
The endocannabinoid, N-arachidonoylethanolamine (anandamide; AEA) is known to interact with voltage-gated K(+) (Kv) channels in a cannabinoid receptor-independent manner. AEA modulates the functional properties of Kv channels, converting channels with slowly inactivating current into apparent fast inactivation. In this study, we characterize the mechanism of action and binding site for AEA on Kv1.5 channels expressed on HEK-293 cells using the patch-clamp techniques. AEA exhibited high-potency block (IC(50) approximately 200 nM) from the cytoplasmic membrane surface, consistent with open-channel block. Alanine-scanning mutagenesis revealed that AEA interacts with two crucial beta-branching amino acids, Val505 and Ile508 within the S6 domain. Both residues face toward the central cavity and constitute a motif that forms a hydrophobic ring around the ion conduction pathway. This hydrophobic ring motif may be a critical determinant of cannabinoid receptor-independent AEA modulation in other K(+) channel families.
Assuntos
Ácidos Araquidônicos/farmacologia , Canabinoides/farmacologia , Canal de Potássio Kv1.5/genética , Alcamidas Poli-Insaturadas/farmacologia , Sequência de Aminoácidos , Sítios de Ligação , Linhagem Celular , Membrana Celular/efeitos dos fármacos , Membrana Celular/fisiologia , Clonagem Molecular , Citoplasma/efeitos dos fármacos , Citoplasma/fisiologia , Endocanabinoides , Humanos , Rim , Canal de Potássio Kv1.5/antagonistas & inibidores , Canal de Potássio Kv1.5/fisiologia , Mutagênese Sítio-Dirigida , Plasmídeos , Reação em Cadeia da PolimeraseRESUMO
BACKGROUND: Previous studies have suggested that the addition of heparin to a preservation solution attenuated the autonomic dysfunction observed in rat jejunum and in addition that hypothermic hyperbaric oxygenation may play a role as a preservation technique. However, these studies did not address the lesion indices of the autonomic enteric neurons. We sought to investigate whether the autonomic enteric neurons are injured during cold ischemic preservation and whether administration of heparin or hyperbaric oxygenation prevents this lesion. METHODS: Jejunal segments (2 cm; n = 20) of Wistar rats (12-16 weeks old) were maintained in lactated Ringer's solution without or with heparin (H- and H+, respectively) at 4 degrees C under normobaric conditions. Other jejunal segments (n = 10) were maintained at 4 degrees C in H- under hyperbaric oxygenation conditions (HBO). After preservation for 12 hours, H-, H+, and HBO preparations fixed in 10% formaldehyde were stained with hematoxylin and eosin. The lesion indices were expressed as the mean number of affected neurons (karyorhexis, nuclear dislocation, cytoplasmic vacuolisation) per 100 neurons present in intramural ganglia. Statistical analysis was performed using the Mann-Whitney test (P < .05). RESULTS: The histologic studies showed that enteric autonomic neurons were damaged in H- jejunal segments. The lesion indices observed were: karyorhexis 90/100, nuclear dislocation 85/100, and cytoplasmic vacuolization 82/100. The autonomic neurons in H+ and HBO segments seemed to be normal and significantly well-preserved (P < .001). CONCLUSION: Hypothermic hyperbaric oxygenation and heparin prevented lesions in cold ischemic preservation of enteric autonomic neurons.
Assuntos
Heparina/uso terapêutico , Oxigenoterapia Hiperbárica , Neurônios/fisiologia , Preservação de Órgãos/métodos , Traumatismo por Reperfusão/prevenção & controle , Animais , Sistema Nervoso Autônomo/efeitos dos fármacos , Sistema Nervoso Autônomo/fisiologia , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/fisiologia , Citoplasma/efeitos dos fármacos , Citoplasma/fisiologia , Jejuno/efeitos dos fármacos , Jejuno/inervação , Masculino , Neurônios/citologia , Neurônios/efeitos dos fármacos , Ratos , Ratos Wistar , Vacúolos/efeitos dos fármacos , Vacúolos/fisiologiaRESUMO
Oocyte maturation is a long process during which oocytes acquire their intrinsic ability to support the subsequent stages of development in a stepwise manner, ultimately reaching activation of the embryonic genome. This process involves complex and distinct, although linked, events of nuclear and cytoplasmic maturation. Nuclear maturation mainly involves chromosomal segregation, whereas cytoplasmic maturation involves organelle reorganization and storage of mRNAs, proteins and transcription factors that act in the overall maturation process, fertilization and early embryogenesis. Thus, for didactic purposes, we subdivided cytoplasmic maturation into: (1) organelle redistribution, (2) cytoskeleton dynamics, and (3) molecular maturation. Ultrastructural analysis has shown that mitochondria, ribosomes, endoplasmic reticulum, cortical granules and the Golgi complex assume different positions during the transition from the germinal vesicle stage to metaphase II. The cytoskeletal microfilaments and microtubules present in the cytoplasm promote these movements and act on chromosome segregation. Molecular maturation consists of transcription, storage and processing of maternal mRNA, which is stored in a stable, inactive form until translational recruitment. Polyadenylation is the main mechanism that initiates protein translation and consists of the addition of adenosine residues to the 3' terminal portion of mRNA. Cell cycle regulators, proteins, cytoplasmic maturation markers and components of the enzymatic antioxidant system are mainly transcribed during this stage. Thus, the objective of this review is to focus on the cytoplasmic maturation process by analyzing the modifications in this compartment during the acquisition of meiotic competence for development.
Assuntos
Bovinos , Citoplasma/química , Citoplasma/fisiologia , Oócitos/ultraestrutura , Animais , Citoesqueleto/fisiologia , Citoesqueleto/ultraestrutura , Feminino , Meiose , Oócitos/crescimento & desenvolvimento , Oócitos/metabolismo , Organelas/fisiologia , Organelas/ultraestrutura , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
Prolonged epididymal sperm storage in vespertilionid and rhinolophid bats, provides an interesting experimental model for the study of spermatozoa epididymal maturation. We examined the presence of the cytoplasmic droplet, and the sequential induction of capacitation and the acrosome reaction in spermatozoa obtained from different epididymal regions (caput, corpus, cauda) throughout the annual reproductive cycle of Corynorhinus mexicanus (C. mexicanus). This is a vespertilionid bat that stores spermatozoa in the epididymis for several months after testes regression. The number of sperm recovered from the different epididymal regions indicate that epididymal transit in C. mexicanus is rapid. The persistence of a high percentage of sperm cells with cytoplasmic droplet in cauda epididymis was observed in addition to a low index of capacitation and acrosome reaction in sperm cells obtained from the corpus epididymis. There was a significant increase in the percentage of capacitated and acrosome reacted spermatozoa during the storage of sperm cells in the cauda epididymis and the percentage of capacitated spermatozoa was consistently, and significantly, higher (p < 0.05) in cauda compared to the corpus epididymis at all studied dates. The process of epididymal maturation in C. mexicanus is completed in the caudal region of this organ encompassing a significant period. Our results also indicate that in C. mexicanus, and in other vespertilionid and rhinolophid bats that show the same temporal asynchrony in the function of male reproductive organs, the final phases of epididymal maturation and storage are, apparently, independent of testicular function.
Assuntos
Quirópteros/fisiologia , Epididimo/fisiologia , Maturação do Esperma/fisiologia , Espermatozoides/fisiologia , Acrossomo/fisiologia , Animais , Citoplasma/fisiologia , Epididimo/citologia , Masculino , Modelos Animais , Capacitação Espermática/fisiologia , Espermatozoides/citologiaRESUMO
Nitric oxide (NO) is a highly reactive free radical involved in intra- and intercellular signaling in various stages of reproduction. The objective of the present study was to evaluate the effect of the addition of sodium nitroprusside (SNP), a NO donor, on nuclear and cytoplasmic in vitro maturation of bovine oocytes. Analysis of variance was conducted and the means were compared by t test at a level of 5%. Low (10(-7) and 10(-9)M) and intermediate (10(-5)M) concentrations of SNP had no significant effect on nuclear maturation, however, when a greater concentration of SNP (10(-3)M) was added, oocytes remained in metaphase I (MI) after 24 h culture (P<0.05) and did not show cumulus expansion. To evaluate if this effect was reversible and if a retardation or inhibition had occurred in the progression from MI to MII, oocytes were cultured in presence of 10(-3)M of SNP for 24 h followed by culture for an additional 24 h in medium with or without SNP. After 48 h, the oocytes remained in MI even when the medium was changed at 24 h with or without SNP. The kinetics of nuclear maturation was assessed to evaluate if there had been or not a retardation in the progression of meiosis with the concentration of 10(-3)M SNP. This concentration delayed germinal vesicle breakdown (VGBD) at 8 h of culture (P<0.05), and at 12 h there was no significant difference between the control and the treated group. The concentrations that did not induce alterations in nuclear maturation were evaluated for cytoplasmic maturation. The concentration of 10(-5)M improved the percentage of peripheral cortical granules (P<0.05), and significantly increased the percentage of blastocysts. These results demonstrate that SNP at greater concentrations (10(-3)M) has a cytotoxic effect, but at intermediate (10(-5)M) concentrations it increases blastocyst rates. NO exhibits a dual effect on bovine oocytes, inhibits (10(-3)M of SNP) nuclear and cytoplasmic maturation or stimulates (10(-5)M of SNP) cytoplasmic maturation, depending on concentration in the culture medium.
Assuntos
Bovinos/fisiologia , Doadores de Óxido Nítrico/farmacologia , Nitroprussiato/farmacologia , Oócitos/efeitos dos fármacos , Oócitos/crescimento & desenvolvimento , Animais , Blastocisto/efeitos dos fármacos , Morte Celular/efeitos dos fármacos , Núcleo Celular/fisiologia , Células Cultivadas , Citoplasma/fisiologia , Relação Dose-Resposta a Droga , Feminino , Fertilização in vitro/veterinária , Meiose/efeitos dos fármacos , Nitroprussiato/administração & dosagem , Oócitos/ultraestrutura , Folículo Ovariano/fisiologia , Fatores de TempoRESUMO
The aim of this work is to determine when and how ooplasmic segregation is initiated in the zebrafish egg. To this end, the organization of the ooplasm and vitelloplasm were examined in oocytes and eggs shortly after activation. Ooplasmic segregation, initiated in the stage V oocyte, led to the formation of ooplasmic domains rich in organelles, and ribonucleoproteins. A linear array of closely arranged peripheral yolk globules separated an outer domain of ectoplasm from an inner domain of interconnected endoplasmic lacunae. The structure of this yolk array and the distribution of microinjected labeled tracers suggests that it may provide a barrier limiting ooplasm transit. Loosely arranged yolk globules at the animal hemisphere allow wide connections between the endoplasm and a preblastodisc domain. Activation caused further segregation of ooplasm, reorganization of endoplasmic lacunae, and blastodisc growth. The presence of an endoplasmic cytoskeleton suggests that these changes may be driven by microtubules and microfilaments.
Assuntos
Fase de Clivagem do Zigoto/citologia , Citoplasma/ultraestrutura , Oócitos/citologia , Óvulo/citologia , Peixe-Zebra/embriologia , Animais , Fase de Clivagem do Zigoto/ultraestrutura , Citoplasma/fisiologia , Gema de Ovo/citologia , Gema de Ovo/fisiologia , Gema de Ovo/ultraestrutura , Microtúbulos/ultraestrutura , Oócitos/química , Oócitos/ultraestrutura , Organelas/ultraestrutura , Óvulo/química , Óvulo/ultraestrutura , Ribonucleoproteínas/análiseRESUMO
Although progesterone is the established maturation inducer in amphibians, Bufo arenarum oocytes obtained during the reproductive period (spring-summer) resume meiosis with no need of an exogenous hormonal stimulus if deprived of their enveloping follicle cells, a phenomenon called spontaneous maturation. In this species it is possible to obtain oocytes competent and incompetent to undergo spontaneous maturation according to the seasonal period in which animals are captured. Reinitiation of meiosis is regulated by maturation promoting factor (MPF), a complex of the cyclin-dependent kinase p34cdc2 and cyclin B. Although the function and molecule of MPF are common among species, the formation and activation mechanisms of MPF differ according to species. This study was undertaken to evaluate the presence of pre-MPF in Bufo arenarum oocytes incompetent to mature spontaneously and the effect of the injection of mature cytoplasm or germinal vesicle contents on the resumption of meiosis. The results of our treatment of Bufo arenarum immature oocytes incompetent to mature spontaneously with sodium metavanadate (NaVO3) and dexamethasone (DEX) indicates that these oocytes have a pre-MPF, which activates and induces germinal vesicle breakdown (GVBD) by dephosphorylation on Thr-14/Tyr-15 by cdc25 phosphatase and without cyclin B synthesis. The injection of cytoplasm containing active MPF is sufficient to activate an amplification loop that requires the activation of cdc25 and protein kinase C, the decrease in cAMP levels, and is independent of protein synthesis. However, the injection of germinal vesicle content also induces GVBD in the immature receptor oocyte, a process dependent on protein synthesis but not on cdc25 phosphatase or PKC activity.
Assuntos
Bufo arenarum/crescimento & desenvolvimento , Fator Promotor de Maturação/fisiologia , Oócitos/crescimento & desenvolvimento , Animais , Bufo arenarum/fisiologia , AMP Cíclico/metabolismo , Cicloeximida/farmacologia , Citoplasma/fisiologia , Citoplasma/transplante , Dexametasona/farmacologia , Feminino , Técnicas In Vitro , Fator Promotor de Maturação/química , Meiose/efeitos dos fármacos , Meiose/fisiologia , Oócitos/citologia , Oócitos/efeitos dos fármacos , Oócitos/fisiologia , Proteína Quinase C/metabolismo , Inibidores da Síntese de Proteínas/farmacologia , Estações do Ano , Vanadatos/farmacologia , Fosfatases cdc25/metabolismoRESUMO
In this present study was observed that the spermatids underwent morphological differentiation and modifications, which primarily comprised nuclear elongation, during the process of spermiogenesis in the domestic duck. The acrosome was formed and the flagellum developed concomitantly with nuclear modifications. Thus, various modifications could be observed during this process, especially changes in the distribution of cytoplasmic organelles. Long cisternae of the rough endoplasmic reticulum present in the spermatid cytoplasm dissociated into vesicles and the distal centriole initiated the development of the flagellum in the cellular portion opposite to the acrosome. The ultrastructure of the spermatids of the domestic duck did not show the characteristic development of pre-acrosomal granules, but the acrosomal granule could be directly visualized in this species.
Assuntos
Citoplasma/fisiologia , Patos/fisiologia , Espermátides/ultraestrutura , Espermatogênese/fisiologia , Animais , Citoplasma/ultraestrutura , Masculino , Microscopia Eletrônica/veterinária , Espermátides/fisiologiaRESUMO
The aim of the present study was analyze, by histological and morphometrical studies, mandibular glands of Melipona bicolor queens collected from monogynic and polygynic colonies and compare their level of development. The results showed that the glands ofphysogastric queens from monogynic colony present a higher level of activity in relation to the queens of polygynic colonies; this is explained by the fact that just a unique queen controls the monogynic colony. In the polygynic colonies, the queens may divide such control to each other.