1.Progress with research on the permeability characteristics of reproductive cell membranes.
Zheng ZHOU ; Guangming CHEN ; Shaozhi ZHANG
Journal of Biomedical Engineering 2012;29(2):383-386
The successful cryopreservation of reproductive cells has important practical significance in many fields. In order to improve the recovery rate and viability of cryopreserved cells, it is necessary to study the permeability characteristics of cell membrane to both water and cryoprotectant. In this paper we review the studies on membrane permeability of animal reproductive cell for the recent years. We firstly list the typical permeability data of spermatozoa and oocyte membrane for water and cryoprotectant. We then analyze the effects of these characteristics on the design of cryopreservation protocol. We also introduce the latest experimental methods to measure the cell membrane permeability.
Animals
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Cell Membrane Permeability
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physiology
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Cryopreservation
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methods
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Female
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Humans
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Male
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Oocytes
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cytology
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Spermatozoa
;
cytology
2.Tail-suspended model simulating mouse oocytes maturation inhibited with microgravity.
Changli WU ; Li LI ; Hengxi WEI ; Zhenfang WU ; Qingyan JIANG ; Shouquan ZHANG
Journal of Biomedical Engineering 2012;29(4):687-696
We studied the effects of simulated microgravity on mouse oocytes maturation, and analyzed whether the tail-suspended model can be applied to investigate simulated microgravity effects on reproductive processes in female mice. Mouse oocytes were cultured in vitro with microgravity simulated by a rotating wall vessel bioreactor and by tail-suspended model, and the maturation rate of the mouse oocytes in the two models were examined in vivo. The maturation rate of mouse oocytes cultured in simulated microgravity was 8.93%, and that was 72.33% in 1g gravity. In ratio, oocyte maturation rate had no significant difference between the rotational group and control group. Microgravity simulated by the tail-suspended model inhibited mouse oocytes maturation and increased the rate of oocytes abnormity. The maturation rate of tail-suspended mouse oocytes was 14.54%, which was significantly lower than that of control group. Tail-suspended model should be an ideal model to investigate simulated microgravity effects on reproductive processes of female mice.
Animals
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Cells, Cultured
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Female
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Hindlimb Suspension
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Mice
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Oocytes
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cytology
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physiology
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Oogenesis
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physiology
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Weightlessness Simulation
3.Thickness of cumulus cell layer is a significant factor in meiotic competence of buffalo oocytes.
Hassan M WARRIACH ; Kazim R CHOHAN
Journal of Veterinary Science 2004;5(3):247-251
This study evaluated the meiotic competence of buffalo oocytes with different layers of cumulus cells. A total of 588 oocytes were collected from 775 ovaries averaging 0.78 oocytes per ovary. Oocytes with homogenous cytoplasm (n = 441) were selected for in vitro maturation (IVM) and divided into four groups based on their cumulus morphology: a) oocytes with > or == 3 layers of cumulus cells, b) 1-2 layers of cumulus cells and oocytes with partial remnants or no cumulus cells to be cocultured c) with or d) without cumulus cells. Oocytes in all four groups were matured in 100 microL drop of TCM-199 supplemented with 10microgram/mL follicle stimulating hormone (FSH), 10microgram/mL luteinizing hormone (LH), 1.5microgram/mL estradiol, 75microgram/mL streptomycin, 100 IU/mL penicillin, 10 mM Hepes and 10% FBS at 39degrees C and 5% CO2 for 24 hours. After IVM, cumulus cells were removed from oocytes using 3 mg/mL hyaluronidase, fixed in 3% glutaraldehyde, stained with DAPI and evaluated for meiotic competence. The oocytes with > or ==3 layers of cumulus cells showed higher maturation rates (p <0.05: 64.5%) than oocytes with partial or no cumulus cells (8.6%) and oocytes co-cultured with cumulus cells (34.5%) but did not differ from oocytes having 1-2 layers of cumulus cells (51.4%). The degeneration rates were higher (p < 0.05) for oocytes with partial or no cumulus cells (51%) than rest of the groups (range: 13.8% to 17.4%). These results suggest that buffalo oocytes with intact layers of cumulus cells show better IVM rates than oocytes without cumulus cells and the co-culture of poor quality oocytes with cumulus cells improves their meiotic competence.
Animals
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Buffaloes/*physiology
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Female
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Fluorescent Dyes/chemistry
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Indoles/chemistry
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Meiosis/*physiology
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Microscopy, Fluorescence/veterinary
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Oocytes/cytology/growth&development/*physiology
4.Effect of vitrification at the germinal vesicle stage on the global methylation status in mouse oocytes subsequently matured in vitro.
Jie YAN ; Lu ZHANG ; Tianren WANG ; Rong LI ; Ping LIU ; Liying YAN ; Jie QIAO
Chinese Medical Journal 2014;127(23):4019-4024
BACKGROUNDIt is still unclear whether the vitrification procedure itself is associated with the incidence of abnormal DNA methylation during oocytes vitrification. The purpose of this study was to evaluate the epigenetic profile of mouse oocytes, which went through vitrification either at a mature stage or at an immature stage following in vitro maturation (IVM) by analyzing the global DNA methylation.
METHODSMetaphase II (M II) stage and germinal vesicle (GV) stage oocytes were collected from adult female mice and were vitrified respectively. The M II oocytes were assessed for cryo-survival and global DNA methylation. The GV oocytes were assessed for cryo-survival and only the surviving GV oocytes were cultured in vitro for subsequent assessment of global DNA methylation in mature oocytes. In vivo matured fresh M II oocytes without undergoing vitrification were used as control. The level of global DNA methylation in the M II oocytes was then examined by immunofluorescence using an anti-5-methylcytosine (anti-5-MeC) monoclonal antibody and fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse IgG under a laser scanning confocal microscope.
RESULTSIn terms of the effect of vitrification on global DNA methylation status in matured oocytes, in the M II-v group, all the examined oocytes (90/90) were found with hypermethylation, including 63.3% (57/90) of them displaying DNA methylation of a very high level, 25.6% (23/90) with a high level, and 11.1% (10/90) with an intermediate level, whereas in the GV-v group, all the matured oocytes (129/129) were also examined with hypermethylation, including 67.4% (87/129) of them displaying DNA methylation of a very high level, 23.3% (30/129) with a high level, and 9.3% (12/129) with an intermediate level. Statistically, it was similar between both groups, which were similar to the control: 68.6% (83/121) of fresh M II oocytes displayed DNA methylation of a very high level, 21.5% (26/121) with a high level, and 9.9%(12/121) with an intermediate level (P > 0.05). In terms of the effect of IVM on global DNA methylation status in matured oocytes, in the in vivo matured oocytes group, all oocytes examined (94/94) were found with hypermethylation, including 80.9% (76/94) displaying DNA methylation of a very high level and 19.1% (18/94) with a high level, whereas in the in vitro matured oocytes group, all oocytes examined (69/69) were also found with hypermethylation: 85.2% (56/69) of them displayed with DNA methylation of very high level, 11.9% (11/69) with high level, and 2% (2/69) with intermediate level. This result was similar to that in in vivo matured fresh M II oocytes (P > 0.05).
CONCLUSIONThe vitrification procedure at GV stage does not induce widespread alteration of global DNA methylation status of mouse oocytes subsequently matured in vitro.
Animals ; DNA Methylation ; physiology ; Female ; Fertilization in Vitro ; Mice ; Microscopy, Confocal ; Oocytes ; cytology ; metabolism ; Vitrification
5.Dynamic changes of gamma-tubulin in preimplantation development of parthenogenetic mouse embryos..
Qing-Hua ZHANG ; Zhi-Yan SHAN ; Na GUAN ; Yan-Ning XU ; Jing-Ling SHEN ; Shu-Qi ZHONG ; Lei LEI
Acta Physiologica Sinica 2008;60(6):777-782
Tubulin is the major protein of microtubule. alpha- and beta- tubulins form heterodimers, while gamma-tubulin regulates microtubule organization. The present study aimed to observe the dynamic changes of gamma-tubulin in preimplantation development of parthenogenetic mouse embryos. Immunofluorescence and laser confocal microscopy were used to detect the location of gamma-tubulin in preimplantation parthenogenetic embryos activated by SrCl2. The oocytes were collected at 13-14 h after hCG injection, and then activated with 10 mmol/L SrCl2 in Ca(2+)-free CZB medium with 5 mmol/L cytochalasin B (CB), fixed at 1 h intervals until 6 h after activation. The results showed that spindle was paralleled with the cell membrane all the time, when the meiosis of MII mouse oocytes resumed. The rotation of spindle was inhibited, but karyokinesis was not influenced. At 0 h after activation, i.e. at metaphase, gamma-tubulin was distributed mainly on the two poles of spindle. At 1 h after activation, i.e. at anaphase, following the separation of chromosomes, gamma-tubulin was transformed from dense to disperse. At 2 h after activation, gamma-tubulin was localized between the segregated sister chromatids at telophase. However, at 3-6 h after activation, gamma-tubulin concentrated around the two female pronuclei during their formation and juxtaposition. Moreover, another group of MII oocytes were activated for 6 h and cultured in droplets of KSOM medium under mineral oil in 5% CO2 in air at 37 degrees C to permit parthenogenetic development. The embryos were collected and fixed at 3 h, 14 h, 16 h, and 18 h of culture. At 3 h after culture, i.e. at mitotic interphase, it was shown that amorphous gamma-tubulin distributed around the nuclei of early parthenogenetic embryos. At 24 h after culture, i.e. at prometaphase, gamma-tubulin migrated along the spindle microtubule to the two poles. Our results showed that gamma-tubulin had similar location patterns at metaphase, anaphase and telophase in meiosis and mitosis. It was concluded that gamma-tubulin assembly in parthenogenetically activated oocytes facilitated the formation of negative pole cap and the stabilization of microtubule, thus promoting the spindle formation at meiosis and mitosis. The relocation of gamma-tubulin at anaphase and telophase might be induced by the event of segregation of homologous chromosome being pulled away by the spindle. gamma-tubulin might contribute to the migration and juxtaposition of the two female pronuclei as well.
Animals
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Embryo, Mammalian
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Embryonic Development
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Female
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Meiosis
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Mice
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Mitosis
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Oocytes
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cytology
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Parthenogenesis
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Spindle Apparatus
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physiology
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Tubulin
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physiology
6.Program optimization for bovine somatic cells nuclear transfer.
Anmin LEI ; Xiaoling MA ; Zhimin GAO ; Yongce HU ; Jinqiang SUI ; Weiwei HUANG ; Linsen ZAN ; Zhongying DOU
Chinese Journal of Biotechnology 2009;25(9):1424-1432
To optimize program of bovine somatic nuclear transfer, we used two different enucleation procedures (by Spindle-view system & Hoechst 33342 staining), two different procedures to introduce donor nuclei (by ooplasm microinjection & electrofusion), and three different group electrofusion parameters (group 1: 1.9 kV/cm, 10 micros, two; group 2: 1.5 kV/cm, 25 micros, two; group 3: 0.6 kV/cm, 100 micros, one) to reconstruct bovine cloned embryos. The cleavation rates and blastocyst development rates of cloned embryos were used to assess the efficiency of different operational procedure. Finally, the best combination of operational procedure, that the spindle-viewer system was used for oocytes enucleating, and donor cell was electrofused into ooplasm by electrical pulse (1.9 kV/cm, 10 micros, two) to reconstruct bovine cloned embryos. Then the excellent blastocysts were transferred to fosters for producing cloned cattle 80 high-quality cloned blastocysts were transferred into 33 fosters, two cloned calves were produced. According to the results, the optimized program could be used to produce cloned cattle.
Animals
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Cattle
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Cell Nucleus
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physiology
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Cloning, Organism
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veterinary
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Embryo Transfer
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methods
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Embryo, Mammalian
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cytology
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physiology
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Female
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Microinjections
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Nuclear Transfer Techniques
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veterinary
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Oocytes
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cytology
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physiology
7.Construction of human-bovine interspecies embryos and investigation of interspecies embryonic mitochondrial source.
Lu YANG ; Dong ZHANG ; Yongsheng WANG ; Daquan SUN ; Yong ZHANG
Chinese Journal of Biotechnology 2008;24(7):1210-1215
UNLABELLEDObtaining human blastocysts is a prerequisite for cell replacement therapy using embryonic stem cells. We established an interspecies somatic cell nuclear transfer (iSCNT) technique for producing blastocysts without sacrificing human oocytes. Human foetal fibroblasts were used as donor cells injected into the enucleated bovine oocytes in nuclear transfer, whereas bovine foetal fibroblasts were used to produce intraspecies embryos. We also examined the fate of human and bovine mitochondrial DNA (mtDNA) during preimplantation development after nuclear transfer by PCR. PCR analysis for the detection of human and bovine mtDNA was done at the 2,8-morula, and blastocyst stages of the embryos.
RESULT2.8% interspecies embryos developed to blastocysts after cultured in an SOF medium, while blastocyst rate of intraspecies embryos were 10.1%. Both human and bovine mtDNAs existed until the morula stage, whereas only the bovine mtDNA was found at the blastocyst stage. These results indicated that interspecies cloning without using human oocytes could generate human blastocysts. Because of the incoordination between bovine mtDNA and human nuclear gene, developmental rate of interspecies embryos was significantly lower than intraspecie. Whether the embryonic stem cell could be used for cell replacement therapy need further research.
Animals ; Blastocyst ; cytology ; physiology ; Cattle ; Cloning, Organism ; DNA, Mitochondrial ; genetics ; Embryonic Development ; physiology ; Female ; Fibroblasts ; physiology ; Humans ; Nuclear Transfer Techniques ; Oocytes ; physiology ; Species Specificity
8.Soluble Human Leukocyte Antigen G Level in Fluid from Single Dominant Follicle and the Association with Oocyte Competence.
Byung Chul JEE ; Chang Suk SUH ; Seok Hyun KIM ; Shin Yong MOON
Yonsei Medical Journal 2011;52(6):967-971
PURPOSE: To investigate the direct relationship between the follicular fluid (FF) level of soluble human leukocyte antigen G (HLA-G) and fertilizability of the corresponding oocyte as well as the morphological quality of the corresponding embryo. MATERIALS AND METHODS: Sixty-three patients were stimulated with recombinant FSH combined with gonadotropin-releasing hormone (GnRH) agonist long (n=5) or antagonist protocol (n=58) for standard in vitro fertilization (IVF). At the time oocyte retrieval, follicular fluid was obtained from single dominant follicle in 63 patients, and the level of soluble HLA-G was measured by sandwich enzyme-liked immunosorbent assay (ELISA). Normal fertilization and individual embryo quality were evaluated, and were graded to four categories by morphological criteria (the embryo with symmetrical blastomeres and no fragmentation were assigned as grade A). Good-quality embryo was defined as those with grade A or B. RESULTS: Soluble HLA-G was not detected in 15 FF samples. In the group with positive FF soluble HLA-G (sHLA-G) (n=48), high levels of sHLA-G (>117.758 U/mL) could predict the failure of fertilization with statistical significance {area under the curve (AUC) 0.676, 95% confidence interval (CI) 0.525-0.804}. However, the FF sHLA-G level was not related with the formation of good-quality embryo. CONCLUSION: High level of FF sHLA-G could predict the fertilization failure of the corresponding oocyte, but was not related with the formation of good-quality embryo.
Adult
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Enzyme-Linked Immunosorbent Assay
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Female
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Follicular Fluid/*metabolism
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HLA-G Antigens/*metabolism
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Humans
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Oocytes/*cytology/physiology
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Ovarian Follicle/*cytology/physiology
9.In vitro maturation--n vitro fertilization--embryo transfer--frozen-thawed of human oocytes.
Yi-min ZHU ; Ying-hui YE ; Hui-juan GAO ; Chen-ming XU ; Yu-li QIAN ; Fan JIN ; He-feng HUANG
Journal of Zhejiang University. Medical sciences 2007;36(5):443-448
OBJECTIVETo investigate the influence of different cycles, ovarian follicle size and IVM culture media on the number of retrieved immature oocytes, maturation rate, fertilization rate, embryo quality and implantation rate, pregnancy rate, delivery rate, survival and development of frozen-thawed embryos from IVM.
METHODSThe oocytes were obtained by follicular aspiration from 19 women undergoing oocyte retrieval for in vitro maturation due to the possible risk of ovarian hyperstimulation in IVF-ET program. One patient was in natural cycle, four patients were in ovulation induction cycles with gonadotropine and fourteen patients is controlled ovarian stimulated cycles. All the oocytes retrieved from follicles with 10.0 - 13.5 mm in maximumdiameter were allowed to culture in medium M-199 (TCM 199) or HTF supplemented with other substance.
RESULTWhen there were nonuniform diameters of follicles and the diameter of largest oocyte exceeded 12 mm, the retrieval rate of oocytes, fertilization rate, and the number of high-quality embryos decreased. The high-quality embryos formation rate was higher for the oocytes cultured in TCM 199 medium than in HTF medium (P<0.01). After being frozen-thawed, the IVM embryos could achieve the same outcome when compared with the conventional IVF treatment. In addition, the offspring were healthy.
CONCLUSIONWhen the nonuniform diameters of follicles and the diameter of largest oocyte exceeds 12 mm,the retrieval rate of oocytes, fertilization rate, and the number of high-quality embryos decreased. TCM199-based medium is better to improve the developmental potential and implantation rate of embryos derived from in vitro matured oocytes. After being frozen-thawed, the IVM embryos could achieve the same outcome when compared with the conventional IVF treatment. In addition, the offspring are healthy.
Adult ; Cryopreservation ; methods ; Embryo Transfer ; Embryo, Mammalian ; cytology ; physiology ; Female ; Fertilization in Vitro ; methods ; Humans ; Infertility, Female ; therapy ; Oocytes ; cytology ; physiology ; Pregnancy ; Pregnancy Outcome ; Pregnancy Rate
10.Influence of postovulatory ageing on balanced predivision of sister chromatid in mouse oocytes.
Wen CHEN ; Qun LIU ; Zhou LI ; Xin-rong WANG ; Gui-jin ZHU
Chinese Journal of Medical Genetics 2006;23(3):256-259
OBJECTIVETo study the impact of postovulatory ageing to balanced predivision of oocyte sister chromatid.
METHODSThe mouse oocytes were cultured 0-72 h. Then chromosome 16 was detected by fluorescence in situ hybridization (FISH). The oocyte spindle and chromosome configuration were examined by immunocytochemistry.
RESULTSFor freshly ovulated mouse oocyte, the balanced predivision of sister chromatid occurred only at 7%. However, for oocytes cultured for 24 h, 48 h and 72 h in vitro, the balanced predivision of sister chromatid occurred up to at 32%, 51% or 62% respectively (P< 0.01). The abnormal cell spindle and chromosome configuration occurred at 9% of freshly ovulated oocytes, but it increased to 63%, 83% and 98% when the oocytes were cultured in vitro for 24 h, 48 h or 72 h respectively (P< 0.01).
CONCLUSIONThe occurrence of balanced predivision of oocyte sister chromatid may result during postovulatory ageing, and may be related to change of oocyte spindle and chromosome configuration.
Aging ; physiology ; Animals ; Cells, Cultured ; Chromatids ; genetics ; Chromosomes, Mammalian ; genetics ; Female ; Immunohistochemistry ; In Situ Hybridization ; Mice ; Oocytes ; cytology ; metabolism