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1.
为探讨卵母细胞减数分裂异常及其与年龄相关变化之间的关系,对不同年龄段昆明白小鼠卵母细胞进行了生发泡(GV)移植研究。应用显微操作和电融合技术,将6~8周龄小鼠GV期卵母细胞分别与6月龄9、月龄和12月龄小鼠GV期卵母细胞进行GV互换,所形成的6种GV-胞质体复合体的融合率(89.7%~95.6%)和6种重组卵母细胞的成熟率(83.5%~88.2%)并不因小鼠年龄的改变而有所变化。成熟的6种重组卵母细胞经体外受精后,形成原核期胚和2-细胞期胚的比率(分别为80.0%~87.3%和42.7%~50.9%)并不因不同年龄小鼠卵母细胞GV互换所带来的细胞质或细胞核的改变而受到影响。  相似文献   

2.
生发泡(germinal vesicle,GV)移植到去核的GV期卵母细胞后,获得重构卵,重构卵在体外能成熟,受精和进行胚胎发育。GV移植到去核的第二次减数分裂中期(metaphase Ⅱ,MII)卵母细胞后,重构卵能发生GV破裂,但难以排出第一极体。GV移植后,通过连续核移植,重构合子具有发育到终期的能力。GV移植为研究卵母细胞的发育提供了一种重要工具。  相似文献   

3.
为研究不同年龄来源的细胞质或细胞核对卵母细胞成熟、钙振荡及核型的影响,在6~8周龄(6W)小鼠与9月龄(9M)和12月龄(12M)小鼠卵母细胞之间进行了生发泡(GV)互换。通过显微操作和电融合获得了5组重组卵母细胞。重组卵母细胞和对照卵母细胞经Sr2 诱导后呈现相似的钙震荡模式。6WGV-6W胞质体组、6WGV-9M胞质体组和6WGV-12M胞质体组成熟卵母细胞染色单体提早分离的比率与6~8周龄对照组比较无差异(P>0.05),但显著低于12月龄对照组(P<0.01)。而9MGV-6W胞质体组和12MGV-6W胞质体组成熟卵母细胞染色单体提早分离的比率则明显增加。这些结果表明由年轻与老龄小鼠之间GV互换所重组的卵母细胞能够正常成熟和产生钙震荡,与衰老相关的减数分裂异常似乎归因于细胞核或染色体而不是细胞质。  相似文献   

4.
本研究探讨卵丘细胞对猪GV期卵母细胞玻璃化冷冻效果的影响。根据卵丘细胞层数将体外收集的猪卵母细胞分成对照组、A组(3层以上卵丘细胞)、B组(2~3层以上卵丘细胞)、C组(裸卵),研究卵母细胞玻璃化冷冻后的存活率、成熟率和孤雌激活后发育潜能的变化。结果表明:玻璃化冷冻后B组和A组的卵母细胞存活率显著高于C组(p0.01);冻融后的卵母细胞在成熟率方面的表现为:A、B、C三组的成熟率分别为22.17%、27.2%和18.15%,其中B组的成熟率显著高于C组(p0.05),但均明显低于对照组(p0.01)。通过进一步的孤雌激活发现,冻融后卵母细胞的卵裂率、4~8细胞卵裂率和囊胚率在3组中均无显著性差异(p0.05),均显著低于对照组(p0.01),但B组与其中两组相比具有上升趋势。综上所述,玻璃化冷冻时保留部分卵丘细胞可以降低冷冻对猪GV期卵母细胞造成的损伤。  相似文献   

5.
灵长类卵母细胞细胞质成熟调控研究较为滞后,使得体外成熟的灵长类卵母细胞的胚胎发育潜能十发低下。提高其潜能对治疗人类不育症有重要的应用价值,并可推动灵长类胚胎发育的研究。基于猕猴卵母细胞质成熟调控研究,讨论了无血清成熟培养基中雌激素和孕激素、能量物质、氨基酸对卵母细胞质成熟的影响,以及动物年龄和生殖周期与发育潜能的关系。从分子水平进一步解释这些因素的作用,阐明卵母细胞质成熟的机理将是今后工作的方向。  相似文献   

6.
研究以银鲫为材料, 根据银鲫(Carassius auratus gibelio)卵母细胞生发泡(Germinal vesicle, GV)边移程度及剥离GV中减数分裂前期染色体的凝集状态, 将银鲫Ⅳ时相的卵母细胞分为GV0、GV1、GV2和GV3四个时期; 并进一步比较了分别处于这4个时期银鲫卵母细胞体外诱导培养的成熟率、卵裂率和孵化率。结果表明, GV1期之后的卵母细胞均可有效进行体外诱导成熟, 可正常受精发育, 由于GV1期卵母细胞有较长时间用于显微操作, 因此GV1期卵母细胞被选为进行体外诱导的最早时期的卵母细胞。以GV1期卵母细胞为研究材料, 摸索了银鲫卵母细胞体外诱导成熟的适宜条件: 取GV1期的Ⅳ时相卵母细胞, 放置于pH 8.5、加有1 μg/mL孕酮激素(17α, 20β-dihydroxy-4-pregnen-3-one, DHP)的格氏平衡盐溶液(Gey’s balanced salt solution, GBSS)中, 在23℃培养箱中体外诱导12h后, 将滤泡膜剥离后再进行人工体外授精, 其所获胚胎的孵化率可达55.5%。此外, 将体外转录合成的带GFP标签的h2af1o mRNA注射到GV1期卵母细胞, 发现经显微操作和体外诱导后不仅可以通过GFP绿色荧光信号活体观察GVBD、受精、卵裂和早期胚胎发育的全过程, 而且诱导成熟的卵子仍可正常受精和胚胎发育。研究建立的银鲫卵母细胞体外诱导成熟技术为银鲫和其他鱼类卵母细胞发育过程研究及其相关基因和细胞显微操作提供了技术平台。  相似文献   

7.
为研究玻璃化冷冻后猪卵母细胞纺锤体、染色体和微丝的变化,从屠宰猪卵巢表面直径2—5 mm卵泡中采集未成熟(GV)期卵母细胞,由GV期卵母细胞经成熟培养获得体外成熟(MⅡ)期卵母细胞。GV期和MⅡ期卵母细胞各分为3组:对照组、冷冻保护剂处理组和玻璃化冷冻组。MⅡ期卵母细胞经分组处理后直接用于激光扫描共聚焦显微镜(LSCM)观察样本;而GV期卵母细胞处理后先经44 h成熟培养,再用作LSCM观察样本。供试卵母细胞经固定、免疫荧光染色后,于LSCM下观察。结果表明,冷冻保护剂处理组GV期卵母细胞经成熟培养后,其纺锤体结构、染色体排列与微丝分布正常率分别为42.9%、89.6%和28.6%;玻璃化冷冻组此3项指标的正常率分别为10.1%、36.4%和16.9%,两组间差异显著(P<0.05);除冷冻保护剂处理组染色体正常率与对照组无较大差异外,两试验组的其他指标均明显低于对照组(分别为79.5%、93.1%和72.3%,P<0.05)。MⅡ期卵母细胞冷冻保护剂处理组的纺锤体结构、染色体排列与微丝分布正常率分别为34.4%、61.3%和47.9%,而冷冻组分别为12.9%、56.7%和37.2%,两组均显著低于对照组(分别为78.3%、90.1%和72.8%,P<0.05)。结果表明,猪GV期和MⅡ期卵母细胞经冷冻保护剂处理或玻璃化冷冻保存后,均造成了纺锤体、染色体和微丝不可逆的损伤,这可能是影响卵母细胞成熟、受精与发育的重要原因。  相似文献   

8.
为了提高猪体细胞核移植重构胚发育潜力,本研究对体外成熟28 h、32 h、36 h、40 h、44 h、48 h、52 h和56 h的猪卵母细胞分别进行去核构建重构胚.研究结果表明,成熟44 h的卵母细胞核移植后有较高的融合率(58.99%)、卵裂率(67.52%)和囊胚率(22.78%),而成熟48 h的卵母细胞则分别为56.51%、65.73%和15.96%;且卵龄为44 h的卵母细胞核移植后分裂率与囊胚率显著高于卵龄为40 h、36 h、32 h、28 h的卵母细胞的分裂率与囊胚率(P<0.05).卵龄为48 h的卵母细胞融合率高于卵龄为52 h卵母细胞的融合率(P<0.05).同时我们还探讨了不同去核方法(盲吸法、Hochest33342染色法和Spindle-view system)对猪体细胞核移植重构胚发育能力的影响.研究结果发现,盲吸法、Hoechest33342染色法和Spindle-view system法的去核率分别达到76.33%,100.00%和98.40%.Hoechest染色法去核率显著高于盲吸法的去核率(P>0.05),而与Spindle-view法去核率没有差异(P>0.05).三种方法在融合率和囊胚率方面差异不显著(P>0.05),但Hoechest染色法的分裂率较低,差异显著(P<0.05).进一步的研究表明,细胞质内注射进行核移植构建重构胚的分裂率和囊胚率分别为68.13%和6.44%;透明带下注射法则为60.37%和8.08%,两者差异不显著(P<0.05);两者均可运用于猪体细胞的核移植,这为建立有效的猪体细胞核移植体系提供了参考.  相似文献   

9.
绵羊卵泡成分对卵母细胞体外减数分裂调控的研究   总被引:1,自引:0,他引:1  
哺乳动物卵巢中的卵母细胞一直处于减数分裂的停滞状态,卵泡内各成分被认为是产生抑制因子的主要来源。本研究以绵羊卵泡各成分为研究对象,用共培养的方法对卵丘细胞、颗粒细胞、膜细胞在卵母细胞体外减数分裂过程中的作用加以探讨。结果表明:1.卵泡整体及卵泡分泌物在体外可以有效地维持减数分裂停滞,经过24h培养,这两个处理组中,处于GV期的卵母细胞分别为69.6%和49.1%。经抑制处理后的卵母细胞脱离抑制环境后可以继发成熟,MⅡ比率可达88.9%。去掉卵丘细胞的裸卵其减数分裂过程不能被卵泡分泌物有效抑制,24h培养后其GV期比例为17.8%。以上结果说明卵泡中的抑制因子主要是通过卵丘细胞束发挥其调控作用的。2.用颗粒细胞与卵母细胞共培养,结果发现具有颗粒细胞卵丘细胞缝隙连接的卵母细胞(COCGs)在培养24小时后47.4%达到MⅡ,与在不具有细胞连接的总浮颗粒细胞中共培养的卵母细胞之间存在无显差异,无论是紧密连接的颗粒细胞层还是悬浮在培养液中的颗粒细胞都不能有效抑制生发泡破裂(GVBD)的发生,只能将卵母细胞抑制在MⅡ以前的各个时期。以上结果说明颗粒细胞在体外分泌抑制图子的活力大大下降。3.卵泡膜细胞具有分泌抑制成熟分裂因子的能力,与膜细胞层共培养的卵母细胞在8h和24h时,其GV期的比例为34.4%和32.7%,显高于没有膜细胞层的对照组(4.5%和1.1%)。综上所述,绵羊卵泡中的抑制因子不仅来自于颗粒细胞,而且膜细胞也参与了成熟分裂的抑制,这些细胞在体外仍具有分泌抑制因子的能力,只是与体内分泌能力有所不同。  相似文献   

10.
为研究玻璃化冷冻后猪卵母细胞纺锤体、染色体和微丝的变化,从屠宰猪卵巢表面直径2—5mm卵泡中采集未成熟(GV)期卵母细胞,由GV期卵母细胞经成熟培养获得体外成熟(MⅡ)期卵母细胞。GV期和MⅡ期卵母细胞各分为3组对照组、冷冻保护剂处理组和玻璃化冷冻组。MⅡ期卵母细胞经分组处理后直接用于激光扫描共聚焦显微镜(LSCM)观察样本;而GV期卵母细胞处理后先经44h成熟培养,再用作LSCM观察样本。供试卵母细胞经固定、免疫荧光染色后,于LSCM下观察。结果表明,冷冻保护剂处理组GV期卵母细胞经成熟培养后,其纺锤体结构、染色体排列与微丝分布正常率分别为42.9%、89.6%和28.6%;玻璃化冷冻组此3项指标的正常率分别为10.1%、36.4%和16.9%,两组间差异显著(P<0.05);除冷冻保护剂处理组染色体正常率与对照组无较大差异外,两试验组的其他指标均明显低于对照组(分别为79.5%、93.1%和72.3%,P<0.05)。MⅡ期卵母细胞冷冻保护剂处理组的纺锤体结构、染色体排列与微丝分布正常率分别为34.4%、61.3%和47.9%,而冷冻组分别为12.9%、56.7%和37.2%,两组均显著低于对照组(分别为78.3%、90.1%和72.8%,P<0.05)。结果表明,猪GV期和MⅡ期卵母细胞经冷冻保护剂处理或玻璃化冷冻保存后,均造成了纺锤体、染色体和微丝不可逆的损伤,这可能是影响卵母细胞成熟、受精与发育的重要原因。  相似文献   

11.
Abnormal oocyte spindle due to the improper function of ooplasm is associated with female infertility of advanced maternal age. A possible way to overcome this problem is to transfer an oocyte germinal vesicle (GV) which contains genetic materials of a patient with a history of poor embryo development to the cytoplast from a donor oocyte. Here we demonstrate that GV transfer is feasible using a rabbit model. When the GVs were transferred to auto- or hetero-cytoplasts of GV stage oocytes, around 80% of the reconstructed oocytes could mature in vitro and 7.1-9.4% of the oocytes developed to blastocyst stage after intracytoplasmic sperm injection (ICSI). Transfer of 93 fertilized eggs reconstructed via GV transfer into six recipients resulted in two live offspring. Results of this experiment indicate that GV transfer can potentially become a new approach in treatment of infertility because of advanced maternal age.  相似文献   

12.
13.
In our study, we have examined the pattern of global histone modification changes in somatic cell nuclei after their transfer into mouse oocytes at different stages of maturation or after their parthenogenetic activation. While germinal vesicle (GV) staged immature oocytes are strongly labeled with anti-acetylated histone H3 and H4 antibodies, the signal is absent in both metaphase I and metaphase II oocytes (MI, MII). In contrast, the oocytes of all maturation stages show a presence of trimethylated H3/K4 in their chromatin. When somatic cells were fused to intact or enucleated GV oocytes, both the GV and the somatic cell nucleus showed a very strong signal for all the antibodies used. On the other hand, when somatic cells nuclei that are AcH3 and AcH4 positive before fusion are introduced into either intact or enucleated MI or MII oocytes, their acetylation signal decreased rapidly and was totally absent after a prolonged culture. This was not the case when anti-trimethyl H3/K4 antibody was used. The somatic cell chromatin showed only a slight decrease in the intensity of labeling after its transfer into MI or MII oocytes. This decrease was, however, evident only after a prolonged culture. These results suggest not only a relatively higher stability of the methylation modification but also some difference between the oocyte and somatic chromatin. The ability to deacetylate the chromatin of transferred somatic nuclei disappears rapidly after the oocyte activation. Our results indicate that at least some reprogramming activity appears in the oocyte cytoplasm almost immediately after GV breakdown (GVBD), and that this activity rapidly disappears after the oocyte activation.  相似文献   

14.
Protein kinase C (PKC) is a family of Ser/Thr protein kinases that can be activated by Ca2+, phospholipid and diacylglycerol. There is evidence that PKC plays key roles in the meiotic maturation and activation of mammalian oocytes. The present study aimed to monitor the effect of age, germinal vesicle (GV) transfer and modified nucleoplasmic ratio on the subcellular distribution profile of PKCα, an important isozyme of PKC, in mouse oocytes undergoing meiotic maturation and following egg activation. Germinal vesicle oocytes were collected from 6-8-week-old and 12-month-old mice. Germinal vesicle-reconstructed oocytes and GV oocytes with one-half or one-third of the original oocyte volume were created using micromanipulation and electrofusion. The subcellular localization of PKCα was detected by immunocytochemistry and laser confocal microscopy. Our study showed that PKCα had a similar location pattern in oocytes and early embryos from young and old mice. PKCα was localized evenly in ooplasm, with weak staining in GV at the GV stage, and present in the entire meiosis II (MII) spindle at the MII stage. In pronuclear and 2-cell embryos, PKCα was concentrated in the nucleus except for the nucleolus. After the GV oocytes were reconstructed, the resultant MII oocytes and embryos showed a similar distribution of PKCα between reconstructed and unreconstructed controls. After one-half or two-thirds of the cytoplasm was removed from the GV oocytes, PKCα still had a similar location pattern in MII oocytes and early embryos from the GV oocytes with modified nucleoplasmic ratio. Our study showed that age, GV transfer and modified nucleocytoplasmic ratio does not affect distribution of PKCα during mouse oocyte maturation, activation, and early embryonic mitosis.  相似文献   

15.
In mammals, oocyte acquires a series of competencies sequentially during folliculogenesis that play critical roles at fertilization and early stages of embryonic development. In mouse, chromatin in germinal vesicle (GV) undergoes dynamic changes during oocyte growth and its progressive condensation has been related to the achievement of developmental potential. Cumulus cells are essential for the acquisition of meiotic competence and play a role in chromatin remodeling during oocyte growth. This study is aimed to characterize the chromatin configuration of growing and fully grown bovine oocytes, the status of communications between oocyte and cumulus cells and oocyte developmental potential. Following nuclear staining, we identified four discrete stages of GV, characterized by an increase of chromatin condensation. GV0 stage represented 82% of growing oocytes and it was absent in fully grown oocytes. GV1, GV2, and GV3 represented, respectively, 24, 31, and 45% of fully grown oocytes. Our data indicated a moderate but significant increase in oocyte diameter between GV0 and GV3 stage. By dye coupling assay the 98% of GV0 oocytes showed fully open communications while the number of oocytes with functionally closed communications with cumulus cells was significantly higher in GV3 group than GV1 and GV2. However, GV0 oocytes were unable to progress through metaphase II while GV2 and GV3 showed the highest developmental capability. We conclude that in bovine, the progressive chromatin condensation is related to the sequential achievement of meiotic and embryonic developmental competencies during oocyte growth and differentiation. Moreover, gap-junction-mediated communications between oocyte and cumulus cells could be implicated in modulating the chromatin remodeling process.  相似文献   

16.

Background

Oocytes are the female gametes which establish the program of life after fertilization. Interactions between oocyte and the surrounding cumulus cells at germinal vesicle (GV) stage are considered essential for proper maturation or ‘programming’ of oocytes, which is crucial for normal fertilization and embryonic development. However, despite its importance, little is known about the molecular events and pathways involved in this bidirectional communication.

Methodology/Principal Findings

We used differential detergent fractionation multidimensional protein identification technology (DDF-Mud PIT) on bovine GV oocyte and cumulus cells and identified 811 and 1247 proteins in GV oocyte and cumulus cells, respectively; 371 proteins were significantly differentially expressed between each cell type. Systems biology modeling, which included Gene Ontology (GO) and canonical genetic pathway analysis, showed that cumulus cells have higher expression of proteins involved in cell communication, generation of precursor metabolites and energy, as well as transport than GV oocytes. Our data also suggests a hypothesis that oocytes may depend on the presence of cumulus cells to generate specific cellular signals to coordinate their growth and maturation.

Conclusions/Significance

Systems biology modeling of bovine oocytes and cumulus cells in the context of GO and protein interaction networks identified the signaling pathways associated with the proteins involved in cell-to-cell signaling biological process that may have implications in oocyte competence and maturation. This first comprehensive systems biology modeling of bovine oocytes and cumulus cell proteomes not only provides a foundation for signaling and cell physiology at the GV stage of oocyte development, but are also valuable for comparative studies of other stages of oocyte development at the molecular level.  相似文献   

17.
Selective enucleation (SE) was applied to germinal vesicle (GV) oocytes by removing the chromatin attached to nuclear envelope, and leaving the liquid contents of GV in the cytoplast. However, after reconstruction with 1/8 blastomeres or fetal fibroblasts (FFs) neither the maturation efficiency nor the frequency of normal (asymmetric) division was improved as compared with completely enucleated (CE) oocytes. Chromosomal aberrations introduced with somatic nuclei were not rescued in SE oocytes either. On the other hand, timing of maturation division in SE GV oocytes, but not in CE GV oocytes, reconstructed with GV-karyoplasts was like in the control. After maturation and fertilization in vitro, SE oocytes reconstructed with 1/8 blastomeres developed nucleolated donor pronuclei, contrary to CE oocytes. The latter could be rescued with nucleoli-containing nucleus, but not anucleolate nucleus, from a 1/2 blastomere. SE oocytes reconstructed with FFs contained nucleolated pronuclei upon activation, unlike CE GV oocytes. These experiments show that the ooplast nucleolar material and/or embryonic nucleolus are indispensable for pronuclei formation. SE oocytes reconstructed with 1/8 blastomeres or FFs failed to cleave after activation or in vitro fertilization. Control GV oocytes enucleolated before fertilization seized cleavage at the 6-cell stage, as oppose to intact GV oocytes, which in 50.9% yielded morulae/blastocysts. These results suggest that ooplast nucleolar material is essential for the cleavage divisions. Activation of cumulus-enclosed SE GV oocytes matured in hormone-supplemented medium and fused to 1/2 blastomere-karyoplasts, yielded morulae, and blastocysts in 45.5% and 23.4% of reconstructed oocytes, respectively.  相似文献   

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