Ageing-associated aberration in meiosis of oocytes from senescence-accelerated mice

BACKGROUND: The senescence-accelerated mouse (SAM) has been shown to exhibit ageing-associated mitochondrial dysfunction and oxidative stress, and early decline in fertility. METHODS: We compared meiotic progression of germinal vesicle oocytes between young (2-3 months) and old (10-14 months) SAM mice using triple immunostaining and fluorescence microscopy as well as Pol-Scope imaging. RESULTS: At 8-9 h of in-vitro maturation (IVM), most young SAM oocytes (86%, 32/37) were at meiosis I (MI) stage, with chromosomes aligned in the mid-region of MI spindles, whereas disrupted MI spindles and/or chromosome misalignments (45%, 18/40) and a few oocytes (20%, 8/40) with abnormal MII spindles were found in old SAM oocytes. At 15-17 h of IVM, old SAM oocytes, despite errors at MI stage, extruded a first polar body at an incidence of 88% (n = 85), which did not differ from that (92%, n = 106) of young SAM oocytes. However, oocytes from old SAM (64%, 32/50) showed aberrant MII, with chromosome misalignment and dispersal, in contrast to normal MII in most young SAM oocytes (87%, 65/75), showing chromosome alignment at the metaphase plate of MII spindles. Moreover, Pol-Scope imaging non-invasively detected disrupted or absent visible spindles and possibly aberrant chromosome alignment. CONCLUSIONS: Spindle disruption and/or chromosome misalignments at both MI and MII are associated with maternal ageing in the SAM mouse. Our findings also suggest that meiotic division lacks a competent checkpoint for spindle integrity and chromosome alignment during reproductive ageing-associated oocyte senescence.     

Germinal vesicle transfer between fresh and cryopreserved immature mouse oocytes.

BACKGROUND: We assessed the maturational competence and the chromosomal pattern of mouse oocytes reconstructed by germinal vesicle (GV) transfer technique using nuclear and/or cytoplasmic components from cryopreserved GV stage oocytes. METHODS: From 657 GV oocytes (326 fresh and 331 frozen/thawed), four groups of reconstructed oocytes were obtained by micromanipulation and electrofusion: fresh GV-fresh cytoplast (FF), thawed GV-thawed cytoplast (TT), fresh GV-thawed cytoplast (FT), thawed GV-fresh cytoplast (TF). All reconstructed oocytes were cultured in vitro to metaphase II. RESULTS: Survival rate after manipulation and electrofusion, as well as progression to metaphase II, did not differ significantly among the four groups. Comparing reconstructed oocytes with fresh and thawed control pools, the only difference was a slightly but significantly higher maturation rate in the TT pool versus matched controls (P < 0.01). Cytogenetic analysis of 25 reconstructed oocytes showed the expected number of 20 chromosomes in 88% of them. CONCLUSIONS: We conclude that both nuclear and cytoplasmic components derived from cryopreserved immature oocytes are suitable for GV transfer procedure, and generate chromosomally normal oocytes able to progress to metaphase II in vitro. The possibility of using cryostored immature oocytes as a source of nuclei and cytoplasm could help in applying GV transfer procedure, both in research and clinical settings.     

Technical approaches to correction of oocyte aneuploidy

BACKGROUND: This study describes the technical approaches used in treatment of age-related oocyte aneuploidy, the efficiency of each step of nuclear transplantation into mouse and human oocytes, and the ability of germinal vesicle (GV) transplantation to restore artificially induced ooplasmic damage. Finally, it examines the possibility of constructing viable female gametes by transferring diploid somatic cell nuclei into enucleated oocytes. METHODS: GV stage mouse oocytes were collected from unstimulated ovaries, and human GV oocytes were donated from consenting patients undergoing ICSI. Stromal (somatic) cells were isolated from uterine biopsies of consenting patients. Mouse cumulus cells were obtained after ovarian stimulation. GV ooplasts prepared by removing nuclei were transplanted either with GV nuclei or with somatic cells by micromanipulation. Grafted oocytes were electrofused and cultured to allow maturation, following which they were inseminated or analysed cytogenetically. Ooplasmic dysfunction was induced by photosensitization with a mitochondria-specific fluorescent dye. RESULTS: GV transplantation had an overall efficiency of 87 and 73% in the mouse and humans respectively. Maturation rates of 95 (mouse) and 64% (human) following reconstitution were comparable with those in control oocytes, as was the incidence of aneuploidy for five chromosome-specific probes after aneuploidy among the reconstituted oocytes. Photosensitization of oocytes significantly reduced the maturation rate to 4.2%, whereas 61.9% of oocytes matured after transfer of photosensitized GV karyoplasts into healthy ooplasts, with 52% of these mature oocytes being successfully fertilized by ICSI. Enucleated immature oocytes receiving mouse cumulus or human endometrial cell nuclei extruded a polar body in >40% of cases. Five out of seven successfully transferred aged human nuclei exhibited the expected number of signals with five chromosome-specific probes suggesting an appropriate chromosome separation in young ooplasm. CONCLUSIONS: Nuclear transplantation itself does not appear to interfere with chromosome segregation and can possibly rescue oocytes with damaged mitochondria. Finally, immature mouse ooplasm supported separation of somatic chromosomes to expected numbers, implying that haploidization may be occurring. The roles of genetic imprinting and fidelity of chromosome segregation are unknown.     

Reconstruction of mouse oocytes by germinal vesicle transfer: maturity of host oocyte cytoplasm determines meiosis.

We evaluated the maturational competence of mouse oocytes reconstructed by the transfer and electrofusion of germinal vesicles (GV) into anuclear cytoplasts of GV stage oocytes (both auto- and hetero-transfers), metaphase II stage oocytes or zygotes. Following in-vitro culture, the maturation rates of the reconstructed oocytes to metaphase II did not significantly differ between auto- and hetero-transfers (40/70 versus 95/144 respectively); these rates also did not differ from those of control oocytes (57/97) which were matured in vitro without micromanipulation and electrofusion. In contrast, when a GV was transferred into an enucleated metaphase II oocyte or a zygote, only a few reconstructed oocytes underwent germinal vesicle breakdown (5/30 and 2/21 respectively); moreover, none reached metaphase II stage. Cytogenetic and immunofluorescence analyses were conducted on hetero-GV oocytes that extruded a first polar body. Each oocyte showed two groups of chromosomes, one in the cytoplast and one in the polar body, as well as a bipolar spindle with twenty univalent chromosomes. Our findings suggest that oocytes reconstructed by GV transfer into a cytoplast of the same developmental stage mature normally in vitro through metaphase II. Such oocytes may be a useful research model to elucidate the cytoplasmic and nuclear mechanisms regulating meiosis and the relationships between meiotic errors and age-related changes in the oocyte.     

Metaphase II nuclei generated by germinal vesicle transfer in mouse oocytes support embryonic development to term

BACKGROUND: Cytoplasmic defects are thought to cause aneuploidies in oocytes and embryos and oocyte 'reconstruction' by germinal vesicle (GV) transfer may circumvent such defects. In mice 'reconstructed' oocytes undergo meiosis and fertilize normally, but early embryonic development is compromised if their ooplasm matured in vitro. This study employs sequential MII spindle and/or pronucleus (PN) transfer to assess the embryonic potential of MII nuclei that form following GV transfer. METHODS AND RESULTS: Mouse embryos generated by these procedures were transferred to the oviducts of pseudopregnant mice to monitor pregnancy outcome. Following GV transfer, the resultant metaphase II (MII) nuclei were activated either in situ or transferred and activated in ooplasts from in-vivo matured oocytes. When exchanged with the female PN of a fertilized zygote, only the PNs that developed in in-vivo matured ooplasts generated live offspring. Viable offspring also resulted when MII nuclei were transferred to in-vivo matured ooplasts and fertilized by insemination with sperm or by artificial activation and male PN transfer. Significantly, the offspring displayed normal fertility as adults. CONCLUSION: This report of live births following GV transfer in mice illustrates the importance of the maturational history of the ooplasm at PN formation for normal embryonic and fetal development.     

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.     

A reliable technique of nuclear transplantation for immature mammalian oocytes.

Transplanting a germinal vesicle (GV) to another enucleated oocyte provides a possible way to avoid age-related aneuploidy in metaphase II (MII) oocytes from older women. This study was conducted to examine the efficiency of each step of nuclear transplantation as reflected in the survival and maturation capacity of immature mouse oocytes subjected to this procedure. GV stage oocytes were retrieved from unstimulated ovaries. A GV removed with a small amount of cytoplasm (karyoplast) was transferred subzonally into a previously enucleated oocyte, which was then exposed to direct current to promote fusion. Such reconstituted oocytes were placed in culture to allow maturation, and some that had extruded a first polar body were fixed and processed for chromosome analysis. Each step of nuclear transplantation - survival, enucleation, grafting, and reconstitution - was successful in >90%, with the overall efficiency of reconstitution being 80%. The observation of normal karyotypes confirmed that the procedure did not increase chromosomal aneuploidy. An electrolytic medium, revealed to be superior for the reconstitution procedure, also allowed haploidization of the transplanted nucleus. These findings suggest that this technique can be applied to study the effects of a 'younger' woman's ooplasm on the disjunction of an 'older' woman's chromosomes during meiosis I.     

Preliminary findings in germinal vesicle transplantation of immature human oocytes

Transplanting a germinal vesicle (GV) from an aged woman's oocyte into a younger ooplasm has been proposed as a possible way to reduce the incidence of oocyte aneuploidy which is considered to be responsible for age-related infertility. In this study, we have assessed the efficiency of each step involved in nuclear transplantation-specifically cell survival, nuclear-cytoplasmic reconstitution, and the capacity of the reconstituted oocytes for in-vitro maturation. In addition, we have evaluated the fertilizability and karyotypic status of the manipulated oocytes by intracytoplasmic sperm injection (ICSI) and fluorescent in-situ hybridization technique respectively. Nuclear transplantation was accomplished with an overall efficiency of 73%. Due to the limited availability of materials, most nuclear transplantation procedures were performed between sibling oocytes. The maturation rate of 62% following reconstitution was comparable with that of control oocytes, as was the incidence of aneuploidy among the reconstituted oocytes. The ICSI results of the reconstituted oocytes yielded a survival rate of 77%, a fertilization rate of 52%, and a satisfactory early embryonic cleavage. Furthermore, in a limited number of observations where the nucleus of an aged oocyte was transferred into a younger ooplasm, there was an appropriate chromosomal segregation. These findings demonstrate that human oocytes reconstituted with GV nuclei are able to undergo maturation, fertilization, and early embryo cleavage, and maintain a normal ploidy. Although in-vitro maturation seems to be a limiting step, this technique would allow us to investigate further the nuclear-ooplasmic relationship during meiotic maturation.     

Effects of myo-inositol on the in-vitro maturation and subsequent development of mouse oocytes

BACKGROUND: The study aim was to assess whether the incorporation of myo-inositol (MI) into culture medium could improve oocyte maturation in vitro. METHODS AND RESULTS: We performed a controlled prospective study using female ICR strain mice superovulated with pregnant mare's serum gonadotrophins. Cumulus-enclosed germinal vesicle (GV) oocytes were randomly cultured in medium with or without MI supplementation. The kinetics of GV breakdown after 4 h of incubation was significantly higher in oocytes incubated with 30 mmol/l of MI than in controls (P < 0.001). Accordingly, this concentration of MI was used for subsequent experiments. The proportion of metaphase II oocytes achieved after 24 h of culture, their fertilization and cleavage rates were significantly higher in the MI-treated group (P < 0.01, P < 0.05, P < 0.05 respectively). This group also demonstrated significant improvement in postimplantation development after transferring the 2-cell embryos to pseudopregnant mice. Confocal microscopy revealed spontaneous intracellular Ca(2+) oscillations within competent GV oocytes and treatment with MI caused an earlier onset of these Ca(2+) signals. CONCLUSIONS: Our results suggest that MI may affect meiotic progression of mouse GV oocytes possibly by enhancing the intracellular Ca(2+) oscillations. Supplementation of MI in culture medium may be useful for human oocyte maturation.     

A confocal microscopy analysis of the spindle and chromosome configurations of human oocytes cryopreserved at the germinal vesicle and metaphase II stage

BACKGROUND: Routine oocyte cryopreservation remains an elusive technique in the wide range of assisted reproductive technologies available. This study examines the effect of a cryopreservation protocol on the spindle and chromosome configurations of human oocytes cryopreserved at the germinal vesicle (GV) and metaphase II (MII) stage. METHODS: GV oocytes were randomly assigned to one of three groups: (i) control oocytes matured in vitro to MII stage (n = 156); (ii) oocytes cryopreserved at the GV stage and then matured in vitro (n = 90); (iii) oocytes cryopreserved at the MII stage (n = 147). Following cryopreservation and in-vitro maturation, immunostaining of tubulin and chromatin was performed, before visualization using confocal microscopy. RESULTS: A statistically significant increase was observed in the survival rate in group 2 (73.3%, 66/90) compared to group 3 (55.7%, 82/147) (P < 0.007). Exposure of oocytes to the cryoprotective solutions without freezing had no effect on the structure of their second meiotic spindle. However, statistically significant differences were observed on both spindle and chromosome configurations of oocytes from group 2 (5.2 and 5.2% respectively) and group 3 (16.2 and 18.8% respectively) compared with group 1 oocytes (71.6 and 82.0% respectively) (P < 0.001 in all cases). CONCLUSIONS: The protocol followed results in high rates of survival and potential for in-vitro maturation, but has a deleterious effect on the organization of the meiotic spindle of human oocytes cryopreserved at both the GV and MII stages.     

Ovary and ovulation: DNA fragmentation of oocytes in aged mice

To investigate whether female fertility decreases with age dueto poor oocyte quality, we examined the presence of DNA fragmentationin ovulated oocytes from young, mature and aged mice. Oocytesfrom three age groups of female mice (7–8, 20–24and 40–48 weeks) were retrieved from the ovlducts 15 hafter human chorionic gonadotrophin (HCG) injection. Oocytesfrom each mouse were incubated in a CO₂ Incubator for 0–60h in human tubal fluid (HTF). After incubation, each oocytewas stained with the terminal deoxynucleotidyl transferase-mediateddUDP nick-end labelling (TUNEL) method. The rate of DNA fragmentation(interpreted as apoptotic changes) was significantly higherfor oocytes from aged mice, and the fertilization rate was significantlylower, compared with oocytes from young and mature mice. Ourresults suggest that DNA fragmentation of oocytes might be oneof the reasons for poor oocyte quality and lower fertility inthe aged group.     

A successful model to assess embryo development after transplantation of prophase nuclei

BACKGROUND: Germinal vesicle transplantation (GVT) provides a means of investigating interactions between karyoplasts and cytoplasts isolated from different cells. Technically, GVT can be accomplished with a high degree of efficiency without compromising the maturation of either the human or mouse oocyte nucleus. Although maturation, fertilization and preimplantation development have been established using GVT, full-term development has been reported only after supplementation with fresh mature ooplasm. In this study, we assess the ability of immature oocytes collected from gonadotrophin-primed ovaries to mature in vitro after GVT and develop to full-term. METHODS: GV oocytes were retrieved from either non-stimulated or pregnant mare's serum gonadotrophin (PMSG)-primed female mice. Microsurgically isolated GV karyoplasts were transplanted into previously enucleated oocytes. Oocytes successfully reconstituted by electrofusion were cultured for 14 h to allow nuclear maturation. Metaphase II oocytes were subjected to Piezo-ICSI, and those fertilized normally were cultured to the blastocyst stage. Some such embryos were transferred to pseudopregnant female mice to examine their potential for normal development. Cumulus-denuded non-manipulated oocytes that were matured in vitro served as controls. RESULTS: The reconstitution and maturation rates were comparable in oocytes isolated from PMSG-primed and from unstimulated ovaries. The rate of normal fertilization in oocytes from primed ovaries was significantly higher than that of their non-primed counterparts (63.5 versus 39.6%; P < 0.01). This difference was also confirmed in terms of blastocyst development (31.8 versus 7.9%; P < 0.01). Of a total of 70 embryos transferred to the oviduct of five recipient mice, 21.4% developed to normal live offspring. All developed as normal adults and proved to be fertile. The live birth rate was comparable to that obtained using non-manipulated control oocytes (22.3%). CONCLUSIONS: Higher rates of fertilization and blastocyst formation were obtained after GVT of mouse oocytes isolated from PMSG-primed ovaries compared with their non-primed counterparts. These represent the first mouse offspring derived from in vitro matured, cumulus-denuded oocytes treated by allo-GVT and fertilized by ICSI. Thus, GVT appears not to impair oocyte maturation, fertilization and pre- and post-implantation development and, after gonadotrophin priming, allows generation of healthy mouse offspring without mature ooplasm supplementation.     

Nuclear competence for maturation and pronuclear formation in mouse oocytes

BACKGROUND: In response to gonadotrophins, a fully grown mouse oocyte matures to the metaphase of the second meiotic division and becomes competent for the development of female and male pronuclei after fertilization. The present study was carried out to clarify when during the growth period an oocyte nucleus acquires the ability to promote pronuclei formation after fertilization. METHODS: Fully grown germinal vesicle (GV) oocytes were enucleated and fused with nuclei from growing oocytes from 1-20 day old mice by standard nuclear transfer technique. The reconstructed oocytes were matured and fertilized in vitro, and pronuclear formation was assessed. RESULTS: The oocytes whose nuclei were exchanged for those of the non-growing-stage oocytes matured to the metaphase of the second meiotic stage, but no normal female pronuclei were formed. Female pronuclei first formed in 27% of the oocytes reconstituted with the nuclei of oocytes from 8 day old pups after fertilization. Recondensed sperm chromatin was detected in 27% of the oocytes reconstructed with oocyte nuclei from 8 day old mice, and a male pronucleus was first formed in 6% of the oocytes that had been reconstructed with the nuclei of oocytes from 15 day old mice. The sizes of the female and male pronuclei increased with oocyte donor age, and reached normal size when the oocytes from 15 and 20 day old mice respectively were used. An electron microscopic study using oocytes that had received the oocyte nuclei of 8 day old mice confirmed these results. CONCLUSION: The factors required for pronuclear formation are derived from fully grown GV oocytes, and the transformation from decondensed sperm chromatin to a recondensed male pronucleus is governed by GV-derived factors.     

影响高龄小鼠卵母细胞质量的超微结构因素

目的 探讨高龄小鼠卵母细胞线粒体、皮质颗粒、微丝、纺锤体及染色体形态和排布的变化规律。 方法 将雌性昆明小鼠分为年轻组（4～8周）和高龄组（48～52周）。腹腔内注射孕马血清促性腺激素（PMSG）10 IU/只行促排卵,48 h后腹腔内注射人绒毛膜促性腺激素（hCG）10 IU/只,14 h后收集输卵管的卵冠丘复合物(OCCC)。脱去颗粒细胞,获取成熟卵母细胞（MⅡ期）经2%多聚甲醛固定。抗小鼠α-tubulin单克隆抗体染色卵母细胞纺锤体的微管,罗丹明标记的鬼笔环肽染色微丝,FITC结合的小扁豆凝集素染色皮质颗粒,Mito Tracker Green FM染色线粒体。激光扫描共焦显微镜下观察。 结果 共获取小鼠卵母细胞372枚,其中年轻组179枚,高龄组193枚。高龄组小鼠卵母细胞纺锤体异常率（71.78% ±1.27%）、染色体排列异常率（65.16% ±1.52%）明显高于年轻组（18.93% ±1.27%,32.24% ±1.10%）（P<0.01,P<0.01）;高龄组小鼠卵母细胞线粒体的荧光强度（28.09±6.62）明显低于年轻组（45.07 ±5.90）（P<0.01）;高龄组小鼠成熟皮质颗粒（Ⅲ级皮质颗粒）（51.00%±100%）明显低于年轻组（94.04% ±0.71%）（P<0.01）。 结论 高龄小鼠卵母细胞中各主要细胞器、细胞骨架及染色体排列异常率明显增加,可能是导致高龄受孕率低的重要原因。     

Meiotic and mitotic nondisjunction: lessons from preimplantation genetic diagnosis

Direct testing of the outcome of the first and second meiotic divisions has become possible with the introduction of preimplantation genetic diagnosis (PGD) for aneuploidies. Testing of oocytes by fluorescent in situ hybridization (FISH) analysis of the first and second polar bodies showed that more than half of oocytes from the IVF patients aged 35 years and older had chromosomal abnormalities, which originated from errors in meiosis I or meiosis II, or both: 41.9% of oocytes were aneuploid after meiosis I and 37.3% aneuploid after meiosis II, with 29.1% of these oocytes having both meiosis I and meiosis II errors. As a result, one third of oocytes detected as normal after meiosis I contained the meiosis II errors, and two thirds of those with meiosis II errors were already abnormal following meiosis I. Although the rates of chromosomal abnormalities deriving from meiosis I and II were comparable, meiosis I errors predominantly resulted in extra chromosome (chromatid) material in oocytes, in contrast to a random distribution of extra and missing chromatids after meiosis II. The majority of meiosis I abnormalities were represented by chromatid errors, which seem to be the major source of chromosomal abnormalities in the resulting embryos. Approximately one third of aneuploid oocytes deriving from sequential errors in the first and second meiotic divisions resulted in a balanced karyotype, representing a possible phenomenon of "aneuploidy rescue" during the second meiotic division. However, the majority of the embryos resulting from such oocytes appeared to be abnormal for the same or different chromosome(s), or were mosaic, suggesting a possible predisposition of the resulting embryos to further mitotic errors. Although the origin of a high frequency of mosaicism at the cleavage stage is not sufficiently understood, the mosaic embryos may originate from the chromosomally abnormal oocytes, as a result of a "trisomy rescue" mechanism during the first mitotic divisions, which renders polar body FISH analysis to have important clinical value for reliable pre-selection of aneuploidy-free embryos for transfer.     

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.     

Intracytoplasmic sperm injection in the mouse

Intracytoplasmic sperm injection (ICSI) into mouse oocytes involvesa very low survival rate. This study was designed to determinewhy ICSI frequently fails in mice. Metaphase II oocytes wereobtained from superovulated 4–6 week old F₁ hybrid mice.Spermatozoa were retrieved from the epididymis of 12–14week old F₁ hybrid mice. The spiked microinjection pipette usedto inject a spermatozoon into the ooplasm had outer and innerdiameters of 10 and 8 µm respectively. The oocytes usedin the first part of the study were not activated (group 1).Some oocytes were incubated with calcium ionophore for 5 min(group 2). The injected oocytes were evaluated 6, 20, 48 and72 h after injection. A total of 143 eggs in each group underwentICSI. In group 1, sperm heads escaped into the perivitellinespace. In all, 63 (47%) of the remaining oocytes were damagedduring the injection or had degenerated by the first evaluation.The survival rate was 53%, but fertilization did not occur.In group 2, 31 oocytes (22%) were damaged during microinjectionor soon degenerated. Two oocytes underwent accidental subzonalinsemination. Six oocytes were fertilized (4.2%) among the 78%of survivors. After injection, the sperm tail was found in thecytoplasm (27 and 31% in groups 1 and 2 respectively), the perivitellinespace (45% in both groups) or protruding through the zona pellucida(28 and 23% respectively). More oocytes degenerated when thetail remained in the cytoplasm, i.e. 78% in group 1 and 36%in group 2.     

Nuclear and cytoplasmic maturation of in vitro matured human oocytes after temporary nuclear arrest by phosphodiesterase 3-inhibitor

BACKGROUND: The use of hormones for controlled ovarian stimulation results in follicular heterogeneity, with oocytes at diverse stages of nuclear and cytoplasmic development. This study evaluated the impact of temporary nuclear arrest by a specific phosphodiesterase 3-inhibitor (PDE3-I), cilostamide, on nuclear and cytoplasmic maturation of cumulus-free germinal vesicle (GV) human oocytes from controlled ovarian stimulated cycles. METHODS: GV oocytes (n = 234) were cultured in: (i) medium without the inhibitor (control); (ii) medium supplemented with 1 microM cilostamide and (iii) medium supplemented with 10 microM cilostamide. Oocytes in groups (ii) and (iii) were exposed to cilostamide for 24 h. The PDE3-I was subsequently removed by transfer of oocytes to fresh in vitro maturation (IVM) medium and the reversibility of GV arrest was assessed during IVM culture for maximum 48 h. RESULTS: Cilostamide (1 and 10 microM) could maintain >80% of the oocytes at the GV stage, without affecting subsequent maturation to metaphase II. Oocytes exposed to 1 microM cilostamide were more likely to have normal bipolar spindles with aligned chromosomes than control oocytes (P < 0.05). When GV chromatin configurations before and after arrest were compared, a significantly higher proportion of oocytes had acquired a nucleolus completely surrounded by a rim of highly condensed chromatin (P < 0.05). CONCLUSIONS: Temporary nuclear arrest of human GV oocytes with PDE3-I proved to be beneficial for obtaining normal spindle and chromosome configurations after IVM. It resulted also in synchronization within the population of GV oocytes.     

Rescue ICSI of unfertilized oocytes after IVF

BACKGROUND: Failed fertilization after IVF occurs in 10-20% of cycles. Conflicting results of rescue fertilization by ICSI have been reported. We therefore compared the success rate in terms of fertilization and pregnancy of cycles in which rescue ICSI was performed with those from a matched control group of primarily ICSI cycles. METHODS: Unfertilized oocytes from IVF cycles with total fertilization failure where at least four metaphase II oocytes were available were treated by ICSI (group I; n = 120). A matched control group was established with patients undergoing ICSI during the same period (group II; n = 280). RESULTS: Both fertilization rate and the proportion of embryos with four blastomeres on day 2 after ICSI were significantly higher in the control group (P < 0.05). Embryo quality, however, was comparable in both groups. The pregnancy rate in the control group was 25.3% whereas in group I with rescue ICSI, no pregnancy was obtained. CONCLUSIONS: Although unfertilized oocytes after IVF can be fertilized by ICSI, the developmental potential of the ensuing embryos is very poor. Therefore, rescue ICSI after total failure of fertilization is not recommended.     

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.