Nucleoli from growing oocytes inhibit the maturation of enucleolated, full-grown oocytes in the pig

In mammals, the nucleolus of full‐grown oocyte is essential for embryonic development but not for oocyte maturation. In our study, the role of the growing oocyte nucleolus in oocyte maturation was examined by nucleolus removal and/or transfer into previously enucleolated, growing (around 100 µm in diameter) or full‐grown (120 µm) pig oocytes. In the first experiment, the nucleoli were aspirated from growing oocytes whose nucleoli had been compacted by actinomycin D treatment, and the enucleolated oocytes were matured in vitro. Most of non‐treated or actinomycin D‐treated oocytes did not undergo germinal vesicle breakdown (GVBD; 13% and 12%, respectively). However, the GVBD rate of enucleolated, growing oocytes significantly increased to 46%. The low GVBD rate of enucleolated, growing oocytes was restored again by the re‐injection of nucleoli from growing oocytes (23%), but not when nucleoli from full‐grown oocytes were re‐injected into enucleolated, growing oocytes (49%). When enucleolated, full‐grown oocytes were injected with nucleoli from growing or full‐grown oocytes, the nucleolus in the germinal vesicle was reassembled (73% and 60%, respectively). After maturation, the enucleolated, full‐grown oocytes injected with nucleoli from full‐grown oocytes matured to metaphase II (56%), whereas injection with growing‐oocyte nucleoli reduced this maturation to 21%. These results suggest that the growing‐oocyte nucleolus is involved in the oocyte's meiotic arrest, and that the full‐grown oocyte nucleolus has lost the ability. Mol. Reprod. Dev. 78:426–435, 2011. © 2011 Wiley‐Liss, Inc.     

Immunocytochemical localization of actin in the nucleolus of rat oocytes

Summary— In order to determine the localization of actin, growing and fully grown rat oocytes were immunocytochemically examined using a post-embedding ultrastructural protein-A gold technique. In quiescent oocytes, the nucleoplasm showed slightly lower levels of actin signal when compared to the surrounding cytoplasm. The highest levels of labeling were found on nucleoli showing a reticular type morphology. In oocytes at the diakinesis stage in which nucleolar compaction had occurred, the levels of labeling increased by 5–6 times those found in quiescent oocytes. Except for conspicuous accumulation of actin under the plasma membrane, compact nucleoli had significantly higher levels of labeling when compared with those found on the general cytoplasm, while the nucleoplasm with homogeneously dispersed chromatin showed significantly lower levels of associated actin signal than the general cytoplasm. In oocytes at metaphase I, the cytoplasmic region had comparable or lower levels of labeling than the cytoplasm of oocytes at diakinesis. The meiotic spindle embedded in material with medium electron density showed a similar level of labeling as the surrounding cytoplasm. On the other hand, significantly higher levels of associated actin were observed on the chromosomes of metaphase I. The actin signals were dispersed over the chromosomes and not concentrated on a specific region. These results suggest that nuclear actin may be involved in the process of chromosome construction and also the formation of the compacted structure of the nucleolus.     

Localization and function of tyrosine-phosphorylated protein in pig oocytes

Several proteins with phosphorylated tyrosine residues have been shown to be closely involved in the control of meiotic nuclear division. We identified a 42-kD protein in pig oocytes, using a polyclonal antibody to a synthetic phosphotyrosine construct that increases significantly in amount after 12 hr of maturation culture, and is discretely localized to condensing and condensed chromosomes. However, since microinjection of the antibody into oocytes blocks spindle formation, the role of this protein appears to be at that stage rather than directly in chromosome condensation. Specificity of action of the 42-kD protein indicates that it may be a phosphorylation-dependent component necessary for successful spindle assembly. © 1996 Wiley-Liss, Inc.     

Activation of protein kinase C suppresses fragmentation of pig oocytes aged in vitro

When cultured for an extended time, pig oocytes that matured in vitro to the stage of metaphase II undergo the complex process designated as ageing. Under our conditions, some pig oocytes aged 3 days remained at the stage of metaphase II (22%), but others underwent spontaneous parthenogenetic activation (45%), and still others perished through fragmentation (28%) or lysis (5%). Activation of protein kinases C (PKCs) using phorbol-12-myristate-13-acetate (PMA) protects oocytes from fragmentation. None of the oocytes were fragmented after 3 days of aging in 50 nM of PMA. A similar effect (8% of fragmented oocytes) was observed after a 3-day treatment of aging oocytes with 100 μM of 1-stearoyl-2arachidonoyl-sn-glycerol (STEAR). PMA and STEAR activate both calcium-dependent and calcium-independent PKCs. This combined effect on PKCs seems to be essential for the protection of oocytes from fragmentation. Neither the specific activator of calcium-dependent PKCs 1-oleoyl-2-acetyl-sn-glycerol (OLE) nor the specific activator of calcium-independent PKCs dipalmitoyl-l-α-phosphatidylinositol-3,4,5-triphosphate heptaammonium salt (DIPALM) suppressed the fragmentation of aging pig oocytes. Twenty-one percentage of oocytes fragmented when aged for 3 days in 10 μM OLE and 26% of aged oocytes fragmented in 100 nM of DIPALM. However, fragmentation was significantly suppressed to 7% when the oocytes were exposed to the combination of both 10 μM OLE and 100 nM DIPALM. Aging pig oocytes cultured for 1 day with PMA maintained a high capability of being parthenogenetically activated (86% of activated oocytes), using calcium ionophore with 6-dimethylaminopurine. Ageing oocytes treated with PMA also had high capability of cleavage (82%) after their artificial parthenogenetic activation. However, their ability to develop to the stage of blastocyst (12%) was suppressed when compared with oocytes activated immediately after their maturation (29%).     

Ultrastructural changes in hamster spermatogenic cell nuclei after incorporation into homologous oocytes by electrofusion

Isolated hamster spermatogenic cells at various stages of spermatogenesis were examined by thin‐sectioning techniques after electrofusion with activated homologous oocytes. The nuclei and attached organelles of the cells remained almost unchanged within the ooplasm three to five hours after the fusion pulse, by which time the oocytes had developed to the pronuclear stage. Only the spherical nuclei of primary spermatocytes and early spermatids in the Golgi and cap phases underwent modifications in their fine structures. They gained the morphological characteristics of well‐developed mammalian pronuclei; e.g., electron‐dense round nucleolus‐like bodies and blebbing of the nuclear envelope. In contrast, the elongated nuclei of later spermatids in the acrosome and maturation phases retained their original features, except that their acrosomes were deformed. Thus, ooplasm‐mediated transformation within activated oocytes at the pronuclear stage occurred only in nuclei containing dispersed chromatin and having nuclear pores in the envelope. Mol. Reprod. Dev. 52:66–73, 1999. © 1999 Wiley‐Liss, Inc.     

The cortical reaction in pig oocytes during in vivo and in vitro fertilization

The structure of pig oocytes after in vivo and in vitro fertilization and following treatment with the ionophore A23187 with differing levels of calcium are described, with particular reference to the cortical granules. Fertilization in vivo and in vitro resulted in cortical granule exocytosis. Sperm penetration in vivo was more rapid than in vitro and resulted in the dispersal of the cortical granules' contents in the perivitelline space following exocytosis. The contents of the granules remained adjacent to the plasmalemma after in vitro fertilization and suboolemmar vesicles were less numerous than after in vivo fertilization. High calcium levels were necessary to induce the dispersal of the cortical granule contents following treatment with ionophore. The observations are discussed regarding their relevance to the blockage to polyspermy.     

Utility of ultrasound stimulation for activation of pig oocytes matured in vitro

The present study was carried out to examine the development of pig oocytes after exposing to ultrasound under various conditions. When oocytes were exposed to ultrasound in the sorbitol medium, the blastocyst formation rate was significantly (P < 0.01) higher than that of oocytes exposed in HEPES-TLP-PVA. Optison, an echo-contrast microbubble, prevented the development into blastocysts of oocytes exposed to ultrasound in the sorbitol medium (P < 0.01). The mean number of cells in the blastocysts developed from oocytes exposed to ultrasound with 10% duty cycle was significantly (P < 0.05) higher than that obtained by using ultrasound with 50% duty cycle. The blastocyst formation rate of oocytes exposed to ultrasound for 30 sec was significantly (P < 0.05) higher than that exposed for 10 sec. There were no significant differences in the rates of oocytes developed to the blastocyst stage and the mean numbers of cells in the blastocysts among different intensities of ultrasound. The pronuclear formation and second polar body extrusion rates of oocytes exposed to ultrasound did not differ from eclectically activated oocytes. Although there was no significant difference in the blastocyst formation rates between different activation methods, the mean number of cells in the blastocysts developed from oocytes activated by exposing to ultrasound was significantly (P < 0.05) higher than that obtained by applying electric pulses. The results of the present study showed that ultrasound stimulation can induce the nuclear activation and parthenogenetic development of pig oocytes matured in vitro.     

Ultrastructural changes in the spermatid nucleolus during spermiogenesis in the guinea pig

Morphological changes in the ring-shaped nucleoli of spermatids were observed by electron microscopy during spermiogenesis in the guinea pig. In the early acrosomal phase each nucelolus was composed of a bundule of fibers about 25 nm in diameter and 250 nm in length. In cross section the fibers were seen to be closely packed in hexagonal fashion. From the early maturation phase through maturation of the spermatids the fibers of the nuceloli appeared to be replaced by a fine filamentous structure. As the condensation of chromatin proceeded, the filamentous structure decreased in density, and finally the space of the nucleolus was replaced by a nucleolar vacuole.     

人-山羊异种核移植胚胎发育的初步研究

以体外分离培养的人胚胎成纤维细胞为核供体,经血清饥饿培养后,通过显微操作技术移入山羊去核卵母细胞中,采用化学方法激活重组胚.通过体外培养观察,2-细胞胚胎发育率可达51.33%,4-细胞发育率为31.42%,但发育至桑椹胚阶段的胚胎数目大大减少,仅为9.73%.虽然目前尚未能获得异种核移植囊胚,但实验结果说明山羊成熟卵母细胞可以支持人体细胞核完成重编程,人-山羊异种体细胞核移植重组胚可在体外完成其早期发育.     

Asynchronous cytoplast and karyoplast transplantation reveals that the cytoplasm determines the developmental fate of the nucleus in mouse oocytes

The relationship between nucleus and cytoplasm can be well revealed by nuclear transplantation. Here, we have investigated the behavior changes of the reconstructed oocytes after transferring the karyoplasts from mouse GV, MI, and MII oocytes into the cytoplasts at the different developmental stages. When the GV cytoplast was used as recipient and MI or MII karyoplast was used as donor (MI-GV pair and MII-GV pair), the reconstructed pairs extruded a polar body after electrofusion and culture. Both the cytoplasm and the polar body had a metaphase spindle in the MI-GV pair, while only a clutch of condensed chromatin was observed in the cytoplasm and polar body of the MII-GV pair. When the MI cytoplast was used as recipient and GV or MII karyoplast was used as donor (GV-MI pair and MII-MI pair), the reconstructed pairs also extruded a polar body. Each had one spindle and a group of metaphase chromosomes in the cytoplasm and polar body, respectively. When the MII cytoplast was used as recipient and GV or MI karyoplast was used as donor (GV-MII pair and MI-MII pair), the reconstructed pairs were activated, became parthenogenetic embryos and even developed to hatching blastocysts after electrofusion. The result from immunoblotting showed that MAP kinase activity was high in the MI and MII cytoplasts, while not detected in GV cytoplast. The results demonstrate that the cytoplasmic environment determines the behavior of asynchronous donors.     

Nuclear envelope breakdown in starfish oocytes proceeds by partial NPC disassembly followed by a rapidly spreading fenestration of nuclear membranes

Breakdown of the nuclear envelope (NE) was analyzed in live starfish oocytes using a size series of fluorescently labeled dextrans, membrane dyes, and GFP-tagged proteins of the nuclear pore complex (NPC) and the nuclear lamina. Permeabilization of the nucleus occurred in two sequential phases. In phase I the NE became increasingly permeable for molecules up to approximately 40 nm in diameter, concurrent with a loss of peripheral nuclear pore components over a time course of 10 min. The NE remained intact on the ultrastructural level during this time. In phase II the NE was completely permeabilized within 35 s. This rapid permeabilization spread as a wave from one epicenter on the animal half across the nuclear surface and allowed free diffusion of particles up to approximately 100 nm in diameter into the nucleus. While the lamina and nuclear membranes appeared intact at the light microscopic level, a fenestration of the NE was clearly visible by electron microscopy in phase II. We conclude that NE breakdown in starfish oocytes is triggered by slow sequential disassembly of the NPCs followed by a rapidly spreading fenestration of the NE caused by the removal of nuclear pores from nuclear membranes still attached to the lamina.     

Nuclear ultrastructure in bovine oocytes after inhibition of meiosis by chemical and biological inhibitors

Various components of the ovarian follicle as well as different chemicals can suppress the resumption of meiosis in cumulus-oocyte complexes (COCs). In this study the nuclear ultrastructure of bovine COCs was assessed after 8 h of meiotic inhibition with 50 microM roscovitine (ROSC), 50 microM butyrolactone (BL-I), 2 mM 6-DMAP, 2 microM cycloheximide (CX), or a theca cell monolayer (TC). COCs were recovered according to standard in vitro methods, cultured in a simple and defined medium, and processed for transmission electron microscopy. Control COCs were processed before onset of culture and multiple oocytes were evaluated for each treatment. In all groups, the oocyte nucleus presented a dense fibrillar nucleolus consisting of a fibrillar sphere with a fibrillar center. In TC and 6-DMAP inhibited COCs condensed chromatin adhered to the nucleolus while in all other groups the perinuclear chromatin was separated from the nucleolus. In ROSC inhibited COCs, the nuclear envelope presented only slight small amplitude undulation. The BL-I-inhibited COCs presented an intermediate level of low amplitude undulation of the NE. In CX, 6-DMAP, and TC inhibited COCs the nuclear envelope presented extensively low amplitude undulations. In ROSC inhibited COCs, electron-dense granules formed ring-shaped structures. In some of the BL-I inhibited COCs multiple stellate crystal-like structures were found, and in these COCs the nuclear envelope and the perinuclear cisternae appeared less distinct than in the other BL-I inhibited COCs. In 6-DMAP inhibited COCs interchromatin-like granule clusters were present. In conclusion, the oocyte nuclei in all COCs presented a dense fibrillar nucleolus resembling that in control COCs. However, variations were observed in 1) the nuclear envelope morphology; 2) the chromatin location in relation to the nucleolus; and 3) the presence of different populations of intranuclear granules. Although all treatments inhibited oocyte nucleus breakdown, the mechanisms underlying these effects are different and require further characterization. Mol. Reprod. Dev. 59: 459-467, 2001.     

Maintenance of meiotic arrest in bovine oocytes in vitro using butyrolactone I: effects on oocyte ultrastructure and nucleolus function

In this study, we have shown that butyrolactone I (BL-I), a potent inhibitor of cyclin-dependent kinases, affects oocyte cytoplasmic morphology and nuclear function in terms of nucleolar ultrastructure and immunocytochemistry. Bovine oocytes were recovered from three classes of follicle size: 1.5-2, 2-3, and 3-6 mm. The oocytes were incubated for 40 hr with BL-I, and subsequently processed for transmission electron microscopy or immunocytochemistry. A control group of oocytes were processed immediately upon recovery (0 hr). In general, incubation with BL-I for 40 hr disrupted contact between cells of the cumulus oophorous and the oocyte, caused degeneration of the cortical granules and the peripheral migration of all cytoplasmic organelles. At the level of the nucleus, it induced convolution of the nuclear membrane and caused acceleration of nucleolar compaction in oocytes from follicles < 3 mm and fragmentation of nucleoli, particularly evidenced by immunocytochemistry, in oocytes from follicles > 3 mm. Furthermore, the effects appear to be more profound in fully-grown oocytes.     

Ultrastructural cytochemistry of the nucleus and nucleolus in growing rabbit oocytes

Summary— Ultrastructural changes of the germinal vesicle during the growth of rabbit oocytes were studied by means of light and electron microscopy, ³H-uridine autoradiography, Ag-NOR staining and E-PTA staining. Particular interest was paid to the nucleologenesis and condensation of chromatin. In contrast to other mammalian species, chromosome condensation in rabbit oocytes occurred concomitantly with rRNA synthesis-dependent nucleolar compaction and preceded nuclear envelope breakdown and resumption of meiosis.     

猪体细胞核移植重构胚的体外发育(英文)

以卵丘细胞为核供体细胞组成重构胚 ,卵裂率达到 5 6.7% ,发育至桑椹胚率达到1 1 .7% ,囊胚率为 6.7% ,显著高于成纤维细胞重构胚 (P <0 .0 5 )。本文还研究了卵母细胞的采集方法、激活程序和卵龄对卵丘细胞核移植重构胚体外发育的影响。以血清饥饿法将卵丘细胞诱导至G0 G1 期 ,抽吸法 解剖法采集卵母细胞 ,体外培养 3 3～ 44h ,将卵丘细胞放至去核卵母细胞的卵周隙中 ,重构胚以钙离子载体A2 3 81 7或电脉冲结合 6 DMAP激活处理 ,体外培养 6d。研究表明 ,卵母细胞采集方法、激活液中细胞松弛素 (CB)、激活程序并不影响重构胚的发育 (以卵龄 44h的卵母细胞为受体 ) ;而以电脉冲结合 6 DMAP激活处理能提高重构胚发育能力 (以卵龄 3 3h的卵母细胞为受体 ) (P <0 .0 5 )。本研究显示 ,以电脉冲结合 6 DMAP激活卵丘细胞重构胚 ,体外能发育至囊胚