女性年龄对卵母细胞纺锤体和染色体构型的影响

目的探讨女性年龄对卵母细胞纺锤体和染色体构型的影响。方法取试管婴儿术后卵龄1天的未受精卵母细胞,采用免疫细胞化学和激光共聚焦术检测卵母细胞纺锤体和染色体构型。结果25~29岁女性卵母细胞Ⅰ级纺锤体和染色体比率分别为33%和31%,显著高于30~34岁(P<0.05)和35~40岁女性(P<0.01);而25~29岁女性卵母细胞的Ⅲ级纺锤体和染色体率均为54%,明显低于30~34岁(P<0.01)和35~40岁女性(P<0.05)。结论卵母细胞纺锤体和染色体构型异常率与女性年龄存在明显的相关性。     

Cell division patterns and chromosomal segregation defects in oral cancer stem cells

Oral squamous cell carcinoma (OSCC) is a serious public health problem caused primarily by smoking and alcohol consumption or human papillomavirus. The cancer stem cell (CSC) theory posits that CSCs show unique characteristics, including self‐renewal and therapeutic resistance. Examining biomarkers and other features of CSCs is critical to better understanding their biology. To this end, the results show that cellular SOX2 immunostaining correlates with other CSC biomarkers in OSCC cell lines and marks the rare CSC population. To assess whether CSC division patterns are symmetrical, resulting in two CSC, or asymmetrical, leading to one CSC and one cancer cell, cell size and fluorescence intensity of mitotic cells stained with SOX2 were analyzed. Asymmetrical SOX2 distribution in ≈25% of the mitoses analyzed was detected. Chromosomal instability, some of which is caused by chromosome segregation defects (CSDs), is a feature of cancer cells that leads to altered gene copy numbers. We compare chromosomal instability (as measured by CSDs) between CSCs (SOX2+) and non‐CSCs (SOX2−) from the same OSCC cell lines. CSDs were more common in non‐CSCs (SOX2−) than CSCs (SOX2+) and in symmetrical CSC (SOX2+) mitotic pairs than asymmetrical CSC (SOX2+/SOX2−) mitotic pairs. CSCs showed fewer and different types of CSDs after ionizing radiation treatment than non‐CSCs. Overall, these data are the first to demonstrate both symmetrical and asymmetrical cell divisions with CSDs in OSCC CSC. Further, the results suggest that CSCs may undergo altered behavior, including therapeutic resistance as a result of chromosomal instability due to chromosome segregation defects. © 2016 Wiley Periodicals, Inc.     

Variation of mouse oocyte sensitivity to griseofulvin-induced aneuploidy and meiotic delay during the first meiotic division

The effects of varying the time of chemical treatment on the induction of areuploidy and meiotic delay in metaphase II (Mll) oocytes were studied by administering 1,500 mg/kg griseofulvin (GF) at 0, 2, 4, 6, or 8 hr after on injection of human chorionic gonadotrophin (HCG). The results show that the oocytes have a different sensitivity to GF-induced aneuploidy and meiotic delay during the course of meiotic maturation. Although not restricted to a particular period of meiotic maturation, the frequency of aneuploidy was highest (P < 0.05) when GF was given at 2, 4, or 6 hr after HCG. The maximum frequency of hyperploidy (42.4%) occurred at the 4-hr treatment time. Also, GF treatment resulted in the induction of meiotic delay as demonstrated by ovulated metaphase I (Ml) and polyploid Mll oocytes. The meiotic delay data depict a period of relative resistance between two periods of sensitivity in that the percentages of ovulated Ml oocytes were 53.3, 21.3, 3.5, 6.7, and 25.7 when GF was given at 0, 2, 4, 6, and 8 hr after HCG, respectively. Also, at these treatment times the percentages of polyploid oocytes were 0.6, 1.7, 7.7, 20.1, and 15.4, respectively. Therefore, the oocytes seem to be more sensitive to GF-induced meiotic delay during the periods preceding and following meiotic spindle assembly. In conclusion, the results demonstrate that the time of chemical treatment influences the frequency of aneuploidy and the degree of meiotic delay. Also, the results emphasize that to thoroughly characterize the aneugenic potential of a specific chemical several treatment times may be needed. © 1994 Wiley-Liss, Inc.     

NO调节在小鼠生发泡期卵母细胞体外成熟中的作用

目的观察不同浓度硝普钠(SNP)、左旋硝基精氨酸甲基酯(L-NAME)对小鼠生发泡(GV)期卵母细胞体外成熟的影响。方法给小鼠腹腔注射PMSG 8IU,取其双侧卵巢,用卵母细胞体外培养法,分为L-NAME组、SNP+dbc AMP+L-NAME组、P+dbc AMP+SNP+L-NAME组,在倒置生物显微镜下观察卵母细胞生发泡破裂(GVBD)率及第一极体(PB1)排出率。结果 SNP+dbc AMP组的GVBD率与PB1率分别为(52.5±0.4与32.5±0.1)显著高于dbc AMP组的(28.0±0.2与11.1±0.7)(P0.05)。P+dbc AMP+SNP+L-NAME组的GVBD率与PB1率分别为(62.8±0.3与20±1.4)显著低于P+dbc AMP+SNP组的(90.1±1.3与66.6±0.9),GVBD率与PB1率显著性降低(P0.05)。结论 L-NAME与二丁酰环腺苷酸(dbc AMP)抑制小鼠卵母细胞成熟时,小剂量NO呈现促进作用,这种促进作用在孕酮(P)存在时进一步加强。     

排卵后老化对卵母细胞姐妹染色单体平衡性早分离的影响

目的探讨排卵后老化对卵母细胞姐妹染色单体平衡性早分离（balanced predivision，BP）的影响。方法分别采用荧光原位杂交和免疫细胞化学方法检测排卵后不同培养时间下小鼠成熟卵母细胞16号染色体的核型及纺锤体和染色体构象的变化。结果新鲜小鼠卵母细胞中BP的发生率为7％，培养24h、48h和72h后，卵母细胞BP发生率分别为32％、53％和6l％，明显高于新鲜卵母细胞（P〈0．01）；新鲜小鼠卵母细胞的纺锤体和染色体异常比率为9％，明显低于培养24h、48h和72h后卵母细胞的纺锤体和染色体异常率（P〈0．01）。结论BP可发生于卵母细胞排卵后老化过程中，其发生可能与纺锤体和染色体构象改变有关。     

Study of two markers of apoptosis and meiotic segregation in ejaculated sperm of chromosomal translocation carrier patients

BACKGROUND: To try to explain the infertility of chromosomal translocation carrier patients, we compared the expression of two markers of apoptosis in the sperm of patients and of fertile donors, and we studied the meiotic segregation in the ejaculated sperm of these translocation carriers. METHODS: Twenty semen samples of translocation carriers, [reciprocal (n=14) and Robertsonian translocations (n=6)], were compared with the semen samples of donors (n=20). Different tests were applied: annexin V binding assay; terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labelling (TUNEL); and fluorescence in situ hybridization (FISH). RESULTS: The annexin V binding assay in sperm of patients with chromosomal translocation (n=17) showed a significantly increased proportion of sperm with externalized phosphatidylserine (PS) than in the control group (n=20, P相似文献   

Nucleocytoplasmic ratio of fully grown germinal vesicle oocytes is essential for mouse meiotic chromosome segregation and alignment, spindle shape and early embryonic development

BACKGROUND: This study examined the effect of nucleocytoplasmic ratio of fully grown germinal vesicle (GV) oocytes on meiotic chromosome segregation and alignment, spindle shape, Ca(2+) oscillations and capacity of early embryonic development in mouse. METHODS: GV oocytes with reduced volume (equal to 1/5 to 4/5 of an intact oocyte) were made by micromanipulation to remove different amounts of cytoplasm, and then matured and fertilized in vitro. RESULTS: When >1/2 of GV oocyte cytoplasm was removed, the time-course of GV breakdown (GVBD) was delayed and oocyte maturation rate decreased significantly. Abnormal chromosome segregation rate increased if >1/2 of the cytoplasm was removed from the oocyte. Length and structure of meiotic spindle and chromosome alignment were also impaired by the reduction of cytoplasmic volume. Once matured in vitro, the oocytes could undergo Sr(2+)-induced Ca(2+) oscillations and form pronuclei in a manner independent of nucleocytoplasmic ratio, but their ability to develop to 2-cell embryos was affected if >1/2 of their cytoplasm was removed from the GV oocytes. CONCLUSIONS: These results suggest that nucleocytoplasmic ratio is essential for normal meiotic chromosome segregation, spindle formation and chromosome alignment over the metaphase spindle, and development to 2-cell stage, for which 1/2 of the volume of the GV oocyte appears to be a threshold.     

Variation of mouse oocyte sensitivity to griseofulvin-induced aneuploidy during the second meiotic division

The effects of griseofulvin (GF) treatment during the secondmeiotic division of oogenesis were investigated by cytogeneticanalysis of mouse one-cell (1-CI) zygotes. After determiningthe duration of fertilization and the second meiotic division,1500 mg/kg GF were administered to superovulated mice at 10,12 or 14 h after human chorionic gonadotrophin (HCG) injection.The results showed that GF reduced the frequencies of fertilizedoocytes (P<0.01) and of 1-CI zygotes that reached the firstmitotic metaphase stage (P< 0.001). Aneuploidy was inducedregardless of the moment of treatment, but the various treatmenttimes were associated with statistically different (P < 0.05)levels of hyperdiploidy. The maximum frequency of hyperdiploidy(39.6%) occurred when GF was given 10 h after HCG. Polyploid1-CI zygotes were significantly induced only at the 10 and 12h treatment times and their level never exceeded 1.8% of fertilizedoocytes. GF also induced abnormalities of chromosome condensationalong with centromeric chromosome associations. These resultssupport the conclusion that GF treatment during the second meioticdivision induced aneuploidy, polyploidy, and reduced rates offertilization and zygotic development. Also, the time of chemicaltreatment influenced the frequencies of these effects. ¹To whom correspondence should be addressed at present address: Biology and Biotechnology Research Program, Lawrence Livermore National Laboratory, L-452, PO Box 808, Livermore, CA 94550, USA     

Vitrification of in vitro matured oocytes: effects on meiotic spindle configuration and mitochondrial function

Background: In the assisted reproductive technique, cryopreserving in vitro-matured oocytes is a new strategy to extend the pool of total oocytes. However, oocyte cryopreservation technique is still unsatisfied. So the assessment of cyro-damage on meiotic spindle and mitochondrial function is necessary to evaluate and refine the current protocols. Material and Methods: The immature oocytes were donated from women undergoing ICSI cycles. Cytoskeleton was assessed by α-tubulin and mitochondria through the fluorescent ΔΨm reporter JC-1. Results: Relative inner membrane potential in MII oocytes from vIVM group sharply decreased, compared with the control (n=30) (1.397 vs. 1.019, P<0.05). 45.2% defective spindles were observed in fIVM group, compared with 48.0% in vIVM group (P>0.05). Oocytes in fIVM (35.5%, 11/31) and vIVM (40.0%, 10/25) displayed abnormal chromosome (P>0.05). Conclusion: In vitro maturation (IVM) has an adverse effect on the organization of spindle and chromosome, and no significantly effect on spindle and chromosome was discovered after vitrification-thaw cycle, while there was obvious damage of oocyte mitochondrial function of in vitro-matured oocyte detected after warming, which may be the reason of the low following developmental potential.     

Fertilization and early embryology: Influence of maternal age on meiotic spindle assembly oocytes from naturally cycling women

To examine the effects of maternal ageing on the meiotic apparatus,we obtained oocytes from naturally cyding women in two age groups,including younger (aged 20–25 years) and older (aged 40–45years) women. Using high- resolution confocal microscopy weobtained a detailed picture of the meiotic spindle and chromosomeplacement during various phases of meiosls. Our data revealedthat the meiotic spindle in older women is frequently abnormal,both with regard to chromosome alignment and the micro- tubulematrix that comprise the meiotic spindle. The spindle in 79%of the oocytes from the older group exhibited abnormal tubulinplacement and one or more chromosomes were displaced from themetaphase plate during the second meiotic division. In contrast,only 17% of the oocytes from the younger age group exhibitedaneuploid conditions. The majority of eggs from this group possesseda well ordered, meiotlc spindle containing chromosomes thatwere fully aligned within a distinct metaphase plate in thespindle. Chromosome management during meiosis is directed bymicrotubule assembly within the spindle. These data suggestthat the regulatory mechanisms responsible for assembly of themeiotic spindle are significantly altered in older women, leadingto the high prevalence of aneuploidy.     

Influence of follicular fluid meiosis-activating sterol on aneuploidy rate and precocious chromatid segregation in aged mouse oocytes

BACKGROUND: Follicular fluid meiosis-activating sterol (FF-MAS) protects young oocytes from precocious chromatid separation (predivision). Reduced expression of cohesion and checkpoint proteins and predivision has been hypothesized to occur in age-related aneuploidy in oocytes. METHODS: To know whether FF-MAS also protects aged oocytes from predivision and from age-related non-disjunction, we analysed chromosome constitution in mouse oocytes matured spontaneously with or without 10 microM FF-MAS and in hypoxanthine (HX)-arrested young and aged oocytes induced to resume maturation by FF-MAS. Messenger RNA for checkpoint protein MAD2 and cohesion protein SMC1beta was compared between oocytes matured with or without FF-MAS. RESULTS: Aged oocytes possessed many bivalents with single distal chiasma at meiosis I. Predivision was especially high in aged oocytes cultured sub-optimally to metaphase II in alpha-minimum essential medium (alpha-MEM). FF-MAS reduced predivision significantly (P < 0.001) but neither reduced non-disjunction nor induced aneuploidy in aged oocytes. Polyploidy was high in FF-MAS-stimulated maturation, in particular in the aged oocytes (P > 0.001). Relative levels of Smc1beta mRNA appeared increased by maturation in FF-MAS, and mitochondrial clustering was restored. CONCLUSIONS: Sister chromatids of aged oocytes appear to be highly susceptible to precocious chromatid separation, especially when maturation is under sub-optimal conditions, e.g. in the absence of cumulus and FF-MAS. This may relate to some loss of chromatid cohesion during ageing. FF-MAS protects aged oocytes from predivision during maturation, possibly by supporting Smc1beta expression, thus reducing risks of meiotic errors, but it cannot prevent age-related non-disjunction. Aged oocytes appear prone to loss of co-ordination between nuclear maturation and cytokinesis suggesting age-related relaxed cell cycle control.     

The influence of postovulatory ageing on the retardation of mouse oocyte maturation and chromosome segregation induced by vinblastine

Certain compounds can induce ovulated metaphase I (MI) oocytes.To study if these MI oocytes can overcome this blockage, ICRmice were given human chorionic gonadotrophin and 0.6 mg/kgvinblastine sulfate (VBS). Their ovulated oocytes were collectedat 17, 19, 21, 23 and 25 h later. The results showed that thefrequencies of MI oocytes decreased, the proportions of diploidmetaphase II (MII) oocytes increased and the frequencies ofhyperploid MII oocytes did not significantly differ (P>0.05)among the five harvest or postovulatory times. We also foundthat the proportions of MII oocytes exhibiting premature anaphaseII and premature centromere separation increased with postovulatoryageing, and that these frequencies were consistently higherin controls than in the VBS groups. These findings indicatethat some of the oocytes blocked in MI can overcome this inhibitionand produce primarily diploid MII oocytes. The ultimate fateof ovulated MI and diploid oocytes on aneuploid production hingesupon the formation of a functioning meiotic spindle, which isaffected by both dosage and the specific chemical. ¹To whom correspondence should be addressed     

Cryopreservation of mouse and human oocytes using 1, 2-propanediol and the configuration of the meiotic spindle

Human and mouse oocytes were cryopreserved by a slow freeze,rapid thaw method, using propanediol (PROH) as the cryoprotectant.A simulated cryopreservation was also included in the studyto detect the level of damage attributable to the PROH alone.Comparison of the mouse and human oocytes cryopreserved by thesame method showed opposing results, with a poor morphologicalsurvival rate of 4% observed for mouse oocytes and a subsequentnormal fertilization rate of 0%. In 171 cryopreserved humanoocytes a higher survival rate of 64% was achieved, and thisshowed more similarity to the mouse pronuclear oocytes survivalof 53%. A comparison of human oocytes, cryopreserved withinthe cumulus and denuded of cumulus and corona prior to cryopreservation,demonstrated a higher survival rate in the denuded oocytes of69% compared to 48%. A delay prior to cryopreservation of 1or 2 days had no effect on the immediate survival of oocytes,but culture for a further 24 h after thawing reduced survival,with the day 1 oocytes exhibiting the most dramatic reductionin survival (28%). Using a lectin binding method, abundant corticalgranules were observed in all cryopreserved oocytes analysed.The meiotic spindle and chromosomes were examined in cryopreservedoocytes using fluorescence microscopy and 60% of the survivingoocytes had a normal spindle and chromosome configuration.     

The incidence of chromosomal abnormalities in frozen-thawed mouse oocytes after in-vitro fertilization.

Cryopreservation of mouse oocytes induced a high rate of atresia. Frozen oocytes observed immediately after thawing did not exhibit any alteration in the frequency of chromosomal abnormalities, aneuploidy or polyploidy. After in-vitro fertilization attempts, the cleavage rate of frozen-thawed mouse oocytes was decreased. Cytogenetical observations of inseminated eggs also confirmed this decrease in fertilization rate. First and second cleavages were delayed compared to fresh controls but subsequent development to the 4-cell stage was not altered. Freeze-thawing increased the incidence of chromosomal abnormalities in inseminated oocytes but this only concerned the frequency of triploidy and not monosomic or trisomic aneuploidy. The increase in triploidy seemed to be largely due to the presence of digynic embryos. Second polar body retention seemed to be mainly responsible for this high rate of polyploidy.     

Effects of low O2 and ageing on spindles and chromosomes in mouse oocytes from pre-antral follicle culture

To assess their quality, spindles were analysed in mouse oocytes from pre-antral follicle culture. High or low oxygen tension was present during the last 16 or 20 h post human chorionic gonadotrophin (HCG)/epidermal growth factor (EGF) addition. Most oocytes from pre-antral follicle culture possessed typical anastral spindles with flat poles resembling those of ovulated, in-vivo-matured oocytes of sexually mature mice, while denuded oocytes in-vitro matured to metaphase II (MII) formed significantly longer, slender spindles with pointed, narrow poles. Spindles in oocytes from follicle culture were only slightly shorter and less compact at the equator as compared with those of oocytes matured in vivo. Chromosomes were well aligned at the equator in MII oocytes obtained from follicle culture with high oxygen. Maturation rate was significantly reduced by lowering oxygen tension to 5% O2. Prolonged culture and the presence of only 5% O2 dramatically increased the percentage of MII oocytes with unaligned chromosomes. These observations indicate that sufficient oxygen supply and time of retrieval after initiation of resumption of maturation by HCG as well as the microenvironment and cell-cell interactions between oocytes and their somatic compartment are critical in affecting the oocyte's capacity to mature to MII, to form a functional spindle, and to align chromosomes correctly.     

Sensitivity of mouse oocytes to nicotine-induced perturbations during oocyte meiotic maturation and aneuploidy in vivo and in vitro

Oocyte meiosis is sensitive to endogenous and exogenous perturbations that upset the temporal sequence of biochemical reactions during oocyte maturation (OM) and predispose oocytes to aneuploidy. Nicotine is an alkaloid that has been reported to disrupt the rate of OM, reduce ovulation and fertilization rates, and increase diploidy. The objective of this study was to test the hypothesis that nicotine perturbs the rate of OM and induces aneuploidy in mouse oocytes in vivo and in vitro. Female mice were given 7.5 IU pregnant mare's serum and either 0, 5.0, 7.5, or 10 mg/kg nicotine in vivo at -3, 0, and +3 h relative to a 5 IU injection of HCG. Oocytes were also cultured in vitro in the presence of 0, 1.0, 5.0, or 10.0 mmol/l nicotine. In vivo, significant (P < 0.05) differences in the proportions of oocytes with premature centromere separation and premature anaphase were found at 10.0 mg/kg nicotine suggesting that the rate of OM was advanced. Also, at this dose the proportion of ovulated oocytes was reduced by approximately 50% relative to controls. In vitro, only non-significant differences were found among the parameters measured. Although nicotine reduced the ovulation rate and perturbed the rate of OM in vivo, these data show that the rate of aneuploidy was not significantly elevated.     

Knockdown of the cAMP-dependent protein kinase (PKA) Type Ialpha regulatory subunit in mouse oocytes disrupts meiotic arrest and results in meiotic spindle defects.

In mammalian oocytes, cyclic AMP-dependent protein kinase (PKA) is responsible for maintaining meiotic arrest. We examined the role of the predominant regulatory subunit, RIalpha in regulating PKA activity during mouse oocyte maturation by knocking down the protein levels using an RNA interference approach. In oocytes in which RIalpha protein was reduced to non-detectable levels, compensatory decreases were also observed in the RIIalpha and catalytic (Calpha) subunit levels. These oocytes resumed meiosis, despite culture under conditions that maintain elevated intracellular cAMP levels, suggesting that the remaining Calpha was not sufficient to maintain meiotic arrest. The resulting eggs, however, displayed meiotic spindle abnormalities and abnormal cleavage planes leading to extrusion of large polar bodies. These results demonstrate that RIalpha is required for regulating PKA activity in maturing oocytes and that compensatory upregulation of RII does not occur. Furthermore, we implicate PKA as a modulator of spindle morphology and function during meiosis.