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H. MIYAMOTO and M. C. CHANG

Summary.

About 64% of 854 superovulated mouse eggs were fertilized in vitro by epididymal spermatozoa and were capable of cleaving in chemically defined media. Approximately 10% of such eggs fertilized in vitro or in vivo could develop to the blastocyst stage in culture. When two-cell eggs fertilized in vitro and in vivo were transferred to the oviducts of pseudopregnant mice, only 13% and 16%, respectively, developed into normal fetuses.

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Y. Hatanaka, T. Nagai, T. Tobita and M. Nakano

Summary. Several hundred fertilized pig eggs were prepared by an in vitro fertilization (IVF) technique in which follicular phase ovarian eggs were matured in vitro to metaphase II before incubation with capacitated epididymal spermatozoa for 12 h at 39°C. Parthenogenetic eggs were also prepared by stimulation of the mature eggs with an electric pulse. The zonae were solubilized with 0·2% pronase/phosphate-buffered saline (PBS) or lactic acid/PBS. The time taken for solubilization was 30–40% shorter than for unfertilized eggs, indicating that zona hardening was induced during fertilization. At the same time, the sperm receptor activity of the zona was reduced. Electrophoretic analyses of zona glycoproteins from the ovarian, mature and fertilized eggs revealed that the amount of 90 kDa proteins decreased substantially during fertilization. This fraction could barely be detected in the zonae from parthenogenetic eggs. However, modification with a fluorescent probe showed that the general architecture of the zona remained unchanged during fertilization. These results suggest that the minor 90 kDa proteins are specifically degraded by the protease(s) released from the oocyte at fertilization, thereby leading to the block to polyspermy.

Keywords: zona pellucida; fertilization; pig; in vitro fertilization

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I. Maudlin and Lynn R. Fraser

Summary. Analysis of the chromosomes of 1-cell mouse embryos fertilized in vitro showed that the incidence of polyploidy is independent of both sperm and egg genotype. No significant differences were detected between groups of eggs from 5 strains of mice fertilized by spermatozoa from a single strain, TO, or between groups of (C57BL/10 × CBA) F1 eggs fertilized by spermatozoa from 2 strains, CBA/H-T6 and TO, although there was significant heterogeneity between individual males in the latter set of experiments. While the majority of polyploids were triploids resulting from dispermic fertilization, some derived from diploid spermatozoa were also found.

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S. Iwasaki, N. Yoshiba, H. Ushijima, S. Watanabe and T. Nakahara

Summary. The morphology and proportion of inner cell mass (ICM) of bovine blastocysts cultured in vitro or in vivo in rabbit oviducts after in-vitro fertilization of in-vitro matured follicular oocytes were compared with those of blastocysts fertilized in vivo by a differential fluorochrome staining technique. The delineation of each ICM cell was improved by the transfer of embryos derived from in-vitro fertilization to a rabbit oviduct although the cell–cell contacts of ICM cells were not as tight as those from in-vivo fertilization. The proportions (15·8 and 14·9%) of ICM in blastocysts cultured in vitro at early and expanded stages were significantly lower than those cultured in rabbit oviducts after in-vitro fertilization and fertilized in vivo. These results show that the transfer of bovine embryos derived from in-vitro fertilization to the rabbit oviduct increased the proliferation of ICM cells to the level of embryos fertilized in vivo although the cell–cell contact of ICM cell is not improved by the process.

Keywords: cattle; blastocysts; inner cell mass; in-vitro fertilization

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Sally Perreault, L. J. D. Zaneveld and B. Jane Rogers

Summary. Sodium aurothiomalate, a low molecular weight inhibitor of hyaluronidase, blocked in-vitro fertilization in hamsters at the level of the zona pellucida: concentrations of 25–250 pg inhibitor/ml prevented fertilization of cumulus-intact and cumulus-denuded eggs. Fertilization of zona-free ova was not affected. In-vivo fertilization was also reduced (from 100% in controls to 37·5%) by 10 mg inhibitor/ml added to an epididymal sperm suspension before artificial insemination into the uterus. These findings suggest that hyaluronidase may play a role in zona penetration or that sodium aurothiomalate blocks sperm penetration of the zona by inhibiting an enzyme(s) other than hyaluronidase.

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Mattan Levi, Bernard Maro and Ruth Shalgi

Fertilization of mammalian oocytes triggers their exit from the second meiotic division metaphase arrest. The extrusion of the second polar body (PBII) that marks the completion of meiosis is followed by the first mitotic cleavage of the zygote. Several lines of evidence in somatic cells imply the involvement of Fyn, an Src family kinase (SFK), in cell cycle control and actin functions. In this study, we demonstrate, using live cell confocal imaging and microinjection of Fyn cRNAs, the recruitment of Fyn to the oocyte's cortical area overlying the chromosomes and its colocalization with filamentous actin (F-actin) during exit from the meiotic metaphase. Fyn concentrated asymmetrically at the cortical site designated for ingression of the PBII cleavage furrow, where F-actin had already been accumulated, and then redispersed throughout the entire cortex only to be recruited again to the cleavage furrow during the first mitotic division. Although microinjection of dominant negative Fyn did not affect initiation of the cleavage furrow, it prolonged the average duration of ingression, decreased the rates of PB extrusion and of the first cleavage, and led to the formation of bigger PBs and longer spindles. Extrusion of the PBII was blocked in oocytes exposed to SU6656, an SFK inhibitor. Our results demonstrate, for the first time, a continuous colocalization of Fyn and F-actin during meiosis and imply a role for the SFKs, in general, and for Fyn, in particular, in regulating pathways that involve actin cytoskeleton, during ingression of the meiotic and mitotic cleavage furrows.

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Dong Zhang, Shen Yin, Man-Xi Jiang, Wei Ma, Yi Hou, Cheng-Guang Liang, Ling-Zhu Yu, Wei-Hua Wang and Qing-Yuan Sun

The present study was designed to investigate the localization and function of cytoplasmic dynein (dynein) during mouse oocyte meiosis and its relationship with two major spindle checkpoint proteins, mitotic arrest-deficient (Mad) 1 and Mad2. Oocytes at various stages during the first meiosis were fixed and immunostained for dynein, Mad1, Mad2, kinetochores, microtubules, and chromosomes. Some oocytes were treated with nocodazole before examination. Anti-dynein antibody was injected into the oocytes at germinal vesicle (GV) stage before the examination of its effects on meiotic progression or Mad1 and Mad2 localization. Results showed that dynein was present in the oocytes at various stages from GV to metaphase II and the locations of Mad1 and Mad2 were associated with dynein’s movement. Both Mad1 and Mad2 had two existing states: one existed in the cytoplasm (cytoplasmic Mad1 or cytoplasmic Mad2), which did not bind to kinetochores, while the other bound to kinetochores (kinetochore Mad1 or kinetochore Mad2). The equilibrium between the two states varied during meiosis and/or in response to the changes of the connection between microtubules and kinetochores. Cytoplasmic Mad1 and Mad2 recruited to chromosomes when the connection between microtubules and chromosomes was destroyed. Inhibition of dynein interferes with cytoplasmic Mad1 and Mad2 transportation from chromosomes to spindle poles, thus inhibits checkpoint silence and delays anaphase onset. These results indicate that dynein may play a role in spindle checkpoint inactivation.

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Josie K Collins and Keith T Jones

DNA damage acquired during meiosis can lead to infertility and miscarriage. Hence, it should be important for an oocyte to be able to detect and respond to such events in order to make a healthy egg. Here, the strategies taken by oocytes during their stages of growth to respond to DNA damaging events are reviewed. In particular, recent evidence of a novel pathway in fully grown oocytes helps prevent the formation of mature eggs with DNA damage. It has been found that fully grown germinal vesicle stage oocytes that have been DNA damaged do not arrest at this point in meiosis, but instead undergo meiotic resumption and stall during the first meiotic division. The Spindle Assembly Checkpoint, which is a well-known mitotic pathway employed by somatic cells to monitor chromosome attachment to spindle microtubules, appears to be utilised by oocytes also to respond to DNA damage. As such maturing oocytes are arrested at metaphase I due to an active Spindle Assembly Checkpoint. This is surprising given this checkpoint has been previously studied in oocytes and considered to be weak and ineffectual because of its poor ability to be activated in response to microtubule attachment errors. Therefore, the involvement of the Spindle Assembly Checkpoint in DNA damage responses of mature oocytes during meiosis I uncovers a novel second function for this ubiquitous cellular checkpoint.

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W Ma, Y Hou, QY Sun, XF Sun and WH Wang

Centromere proteins (CENPs) are required for the attachment of microtubules to chromosomes. However, their structure and mechanism of action are not well understood, especially in mammalian meiosis. The present study was conducted to examine (i). whether a human nuclear centromere autoantibody can be used to localize the CENPs in pig oocytes and (ii). the dynamics of CENPs and their association with microtubules and chromosomes during meiosis in pigs. Oocytes at various stages were double-labelled for CENPs, chromosomes or microtubules and examined by confocal fluorescence microscopy. Quantification of tubulin and CENPs in the oocytes was determined by immunoblotting. CENPs were detected in all oocytes from germinal vesicle (GV) to metaphase II (MII) stages. The changes in the location were associated with chromosome movement and spindle formation. Tubulin was detected in the oocytes from GV to MII stages and no differences in content were observed. Two major CENPs at 80 kDa (CENP-B) and 50 kDa (CENP-D) were also found in the oocytes by the autoantibody and its content was significantly lower in the oocytes at GV stage compared with oocytes at other stages. These results indicate that the autoantibody used in this study can be used to detect CENPs in the kinetochores, and the proteins are expressed in pig oocytes at all stages during meiosis. As the localization of CENPs is associated with spindle formation and chromosome movement, CENPs may participate in cell cycle changes during meiosis in mammals.

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S E Racedo, V Y Rawe and H Niemann

For successful fertilization by the male gamete, oocyte cytoplasmic organelles such as the Golgi apparatus have to undergo specific changes: the entire process is known as cytoplasmic maturation. The goal of this study was to unravel the dynamics of the Golgi apparatus in bovine oocytes at critical stages of in vitro maturation, i.e. germinal vesicle (GV), GV breakdown (GVBD), metaphase I (MI) and metaphase II, and to investigate the role of various molecules critically involved therein. The cytoplasmic distribution of proteins was assessed by immunocytochemistry and laser confocal microscopy. We applied specific inhibitors, including nocodazole to unravel the functional role of the microtubular elements; sodium orthovanadate, which primarily inhibits cytoplasmic dynein ATPase activity; monastrol which inhibits the kinesin EG5; and roscovitine to inhibit the kinase cyclin-dependent kinase 2A (CDC2A). Prior to GVBD, the Golgi apparatus was translocated from the centre of the cytoplasm to the cortical area in the periphery, where it underwent fragmentation. A second translocation was observed between GVBD and MI stages, when the Golgi apparatus was moved from the cortex to the centre of the cytoplasm. Incubation with the specific inhibitors revealed that microtubules played an active role in the final localization at GVBD, while CDC2A was essential for Golgi fragmentation at GVBD stage. This partitioning was a precondition for the second movement. In conclusion, for the first time we show basic mechanisms critically involved in the regulation of the dynamic changes of Golgi apparatus during meiosis of the bovine oocyte.