Structural changes in oocyte nucleoli ofXenopus laevis during oogenesis and meiotic maturation

Immunoelectron microscopy with anti-nucleolin defined substructures within the multiple nucleoli of biosynthetically active stage II–III oocytes and within the nucleoli of relatively quiescent stage VI oocytes of Xenopus laevis. Dense fibrillar components (DFCs) of nucleoli from stage II–III oocytes consisted of nucleolonemas that radiated from a continuous DFC sheath surrounding fibrillar centers (FCs). Discernible granular regions (GRs) were absent in these same nucleoli. Conversely, stage VI oocyte nucleoli displayed compacted DFCs and prominent GRs. Immunofluorescence microscopy then tracked fibrillarin, nucleolin, and condensed DNA through oogenesis and into progesterone-induced meiotic maturation and nuclear breakdown. In stage II–III oocyte nucleoli, fibrillarin was enriched near the FC-DFC boundaries, while nucleolin was distributed throughout these same DFCs. Both proteins were enriched within the compacted DFCs of stage VI oocyte nucleoli. Staining with (DAPI) 4′,6-diamidino-2-phenylindole showed condensed DNA within nucleolar FCs of both stage II–III and stage VI oocyte. Upon nuclear breakdown, we found fibrillarin and nucleolin in small particles and in the surrounding cytoplasm. Although we saw no trace of fibrillarin or nucleolin in nuclear remnants prepared just minutes later, DAPI-stained particles remained within these preparations, thus suggesting that FCs were at least slow to disassemble. Received: 18 March 1996 / Accepted: 16 April 1996     

Location of DNA within the nucleolus of rat oocytes during the early stages of follicular growth.

We have investigated the DNA distribution within the rat oocyte nucleolus during the early stages of follicular growth by means of the in situ terminal deoxynucleotidyl transferase method. In the fibrillogranular nucleolus, label is visualized on small clumps of peri- and intranucleolar chromatin. Such labeled clumps are frequently observed inside the interstices surrounding the fibrillar centers. Label is also consistently found in the fibrillar centers whereas the dense fibrillar component and the granular component are devoid of gold particles. These results contradict earlier data but conform with other recent immunocytochemical observations, obtained in nucleoli of a variety of somatic cell types, concerning the correlation between structure and function in the nucleolus.     

The effect of okadaic acid on meiotic maturation of canine oocytes of different size

The present study was conducted to determine the effect of okadic acid (OA), a potent inhibitor of seronine/treonine 1 and 2A phosphatase, on meiotic resumption and progression in canine oocytes with different diameters. Cumulus-oocyte complexes were collected from ovaries of bitches at different oestrous phases. In Experiment 1, to determine the optimal concentration of OA (0.5 or 2 μM), the oocytes were pre-incubated for 1, 3, and 20 h in TCM 199 supplemented with 20% SCE and thereafter cultured in the same medium without OA. In Experiment 2, the selected oocytes were divided into three groups according to their diameter: <110 μm, 110-120 μm, >120 μm, and pre-incubated in OA 0.5 μM for 1 h. Oocytes were cultured in vitro as previously described. After 72 h of IVM, in Experiment 1, significantly more oocytes reached MII stage with 0.5 μM for 1 h (30.8% P <0.001%) for oocytes cultured in other OA condition and in control group. In Experiment 2, OA induced a significantly higher incidence of MII oocytes in the 110-120 μm and >120 μm groups (P <0.001) compared to control group, but a significantly higher proportion of the oocytes >120 μm pre-incubated with OA progressed to MII (51.3% P <0.001). In contrast, smaller oocytes (<110) did not develop to MII stage with or without OA. In conclusion, treatment of canine oocytes with 0.5 μM for 1 h, improves meiotic maturation. The culture of fully grown (>120 μm) oocytes with OA at the onset of in vitro maturation can result in a higher frequency of meiotic maturation.     

Oocyte diameter in relation to meiotic competence and sperm penetration

This study was conducted to determine the diameter of canine oocytes that are able to attain full meiotic competence and sperm penetration. Oocytes were collected from ovaries of bitches at various stages of the estrous cycle. Only healthy-looking cumulus-oocyte complexes were used for in vitro maturation, and were divided into four groups based on diameter: <100, 100 to <110, 110 to <120 and >120 microm. Following in vitro maturation or fertilization, oocytes were stained to assess nuclear maturation and penetration rates. The mean oocyte diameter was 108.5 +/- 0.4 microm. The oocytes displayed size-related ability to undergo meiotic maturation. After culture for 72 h, the rates of oocytes that remained at the germinal vesicle stage in the <110 microm groups were significantly higher (P<0.01) than in the > or = 110 microm groups. None of the oocytes <110 microm reached metaphase II (MU), but 4.9 and 21.5% of the oocytes that were greater than 110 and 120 microm, respectively, progressed to MII. After in vitro fertilization for 20 h, 10 to 25% of oocytes were penetrated by spermatozoa, but there were no clear relationships between oocyte diameter and penetration rates of the oocyte by sperm. In the <120 microm groups, sperm penetration was mostly found in oocytes arrested at the germinal vesicle stage. However, a total of eight oocytes > or = 120 microm in diameter were penetrated by spermatozoa, of which five oocytes reached MII. These results suggest that there is a clear relationship between oocyte diameter and meiotic competence, but no relationship between oocyte diameter and sperm penetration. Canine oocytes may have acquired meiotic competence once they reach at a diameter of 120 microm, but the oocytes may allow the entry of spermatozoa into the ooplasm irrespective of oocyte diameter.     

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.     

小鼠卵母细胞体外成熟及受精过程中细胞核的时空变化规律

用电镜方法研究小鼠卵母细胞的发育及受精虽然已有很多报道,但大多数是有关细胞质、尤其是皮质颗粒、高尔基复合体及线粒体的形态及分布变化的。从卵母细胞体外成熟培养、第一次减数分裂恢复到受精后第二次减数分裂完成,细胞核经历了复杂的变化,有关的系统研究却很少。本实验详细地研究了小鼠卵母细胞体外成熟及受精过程中两性生殖细胞内细胞核的时空变化规律。从卵巢中采集生发泡（ＧＶ）期卵母细胞,进行体外成熟培养,经超排获得的成熟卵母细胞去卵丘和透明带后,用于体外受精。于体外成熟培养及受精后的不同时间,用光镜及电镜方法观察细胞核变化及极体排放。结果表明,尽管大多数卵母细胞在体外培养２至４小时生发泡破裂（ＧＶＢＤ）,但有１３．６％在培养８小时后仍处于ＧＶ期（图１）。电镜观察揭示,不发生ＧＶＢＤ的卵母细胞核的核仁由颗粒性纤维成分、空泡及纤维中心组成。有时核仁表面有空泡。只有核仁完全致密化、核仁周围有核仁相随染色质分布时,卵母细胞才获得恢复减数分裂的能力。ＧＶＢＤ发生时,随着核仁相随染色质向核膜侧扩散迁移,核仁越来越小;与此同时,核膜打折,染色质团块中央出现电子致密的芯。核仁的消失早于核膜的破裂,提示核仁成分可能参与核膜打折及破裂,体外培     

Ultrastructural study of the relationships between the various nucleolar components in Ehrlich tumour and HEp-2 cell nucleoli after acetylation

In the present study, we analysed the relationships between various nucleolar components in Ehrlich tumour and HEp-2 cells, using acetylation. Under these conditions, we found contacts between the condensed intranucleolar chromatin and the fibrillar centre, illustrating the continuity between the DNA present inside the fibrillar centre and that of condensed associated chromatin. We also found that although the dense fibrillar component is usually situated at the periphery of the fibrillar centre, it is sometimes found inside the centre. On the other hand, the layer of dense fibrils bordering the fibrillar centre is interrupted by nucleolar interstices. In addition, in HEp-2 cell nucleoli with a reticulated appearance, the numerous small fibrillar centres are bound together by strands of dense fibrillar component. These observations are discussed in terms of relationships between nucleolar ultrastructure and function(s).     

Chromatin organization during mouse oocyte growth

We investigated the changes in the organization of oocyte nuclear chromatin and nucleolar-associated chromatin throughout folliculogenesis. Zona-free oocytes were isolated from ovaries, grouped into seven classes according to size and chromatin organization, and analyzed after staining with Hoechst 33342. We show that oocyte differentiation from the dictyate stage to the conclusion of maturation is associated with either of two chromatin configurations. Initially, all oocytes are in the NSN configuration (nonsurrounded nucleolus oocytes; characterized by a Hoechst positive-chromatin pattern of small clumps forming a network on the nuclear surface, with a nucleolus nonsurrounded by chromatin). While growing, some of these NSN oocytes continue their development in the NSN configuration, whereas others shift (from class IV on) into the SN configuration (surrounded nucleolus oocytes; characterized by a threadlike chromatin organization that may partially surround the nucleolus or project towards the nuclear periphery). The percentage of SN oocytes increases both with increasing size of the oocyte (class I–III, 10–40 μm in diameter: 100% NSN vs. 0% SN; class VII 70–80 μm in diameter: 47.3% NSN vs. 52.3 SN, in 4–6-week-old females), and with aging (class VII: 94.1% NSN vs. 5.9% SN in 2-week-old females; 11.8% NSN vs. 8.2% SN in 56-week-old females). Further, we suggest as a working hypothesis that those oocytes that switch to the SN chromatin organization early in maturation may not be ovulated, even though this particular chromatin structure normally occurs just prior to ovulation. © 1995 Wiley-Liss, Inc.