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.     

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.     

Chemically and mechanically induced membrane fusion: non-activating methods for nuclear transfer in mature human oocytes

Most current studies of nuclear transfer in mammalian oocytes have used electrofusion to incorporate donor cell nuclei into enucleated oocyte cytoplasts. However, the application of electrofusion to human oocytes is hampered by the relative ease with which this procedure induces oocyte activation. Here we tested a previously described chemical fusion technique and an original mechanical fusion procedure in this application. Enucleated metaphase II oocytes were first agglutinated with karyoplasts originating from other metaphase II oocytes and then induced to fuse with the use of polyethylene glycol or by micromanipulation with an intracytoplasmic sperm injection (ICSI) micropipette. Both techniques yielded a high frequency of fusion and did not cause oocyte activation. Moreover, the reconstructed oocytes were easily activated by subsequent treatment with ionophore A23187 and 6-dimethylaminopurine. These techniques may be used in attempts to alleviate female infertility due to insufficiency of ooplasmic factors by nuclear transfer from patients' oocytes to enucleated donor oocyte cytoplasts. For eventual future use in human cloning, they would ensure prolonged exposure of transferred nuclei to metaphase promoting factor, which appears to be required for optimal nuclear reprogramming.     

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.     

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.     

A novel method for chromosome analysis of human sperm using enucleated mouse oocytes

BACKGROUND: Mouse oocytes can be used in conjunction with intracytoplasmic sperm injection (ICSI) as a technique to permit chromosomal analysis of human sperm. However, chromosomes derived both from the human sperm and the mouse oocyte appear simultaneously following ICSI. The present study focused on evaluating whether or not previously enucleated mouse oocytes are usable for the analysis of human sperm chromosomes. METHODS: The metaphase chromosome-spindle complex was removed from a mouse oocyte. Human sperm from a donor with proven fertility were injected into mouse enucleated oocytes or intact oocytes. The presence of pronuclei in the oocytes was confirmed approximately 7-11 h after ICSI, and the oocytes were then fixed so that the nuclei could be observed as chromosome samples at 15-16 h after ICSI. RESULTS: The formation rate of one pronucleus in enucleated oocytes after ICSI was 93.9% (186/198) while that of two pronuclei in intact oocytes after ICSI was 85.4% (88/103). The appearance rate of metaphase chromosomes of human sperm in the enucleated oocytes, 89.4% (160/179), was significantly higher than that in intact oocytes, 78.7% (74/94) (P = 0.017). CONCLUSIONS: An efficient ICSI method using enucleated mouse oocytes was established to allow the visualization of the human sperm chromosome complement without the risk of confusion with mouse oocyte chromosomes.     

Microtubular spindle dynamics and chromosome complements from somatic cell nuclei haploidization in mature mouse oocytes and developmental potential of the derived embryos

BACKGROUND: The aim of this study was to investigate haploidization of somatic cell nuclei in non-enucleated mature oocytes regarding spindle formation, chromosomes and developmental potential. METHODS: Mouse cumulus cells were injected into metaphase II oocytes. Some injected oocytes were examined for morphological changes of chromosomes and the spindle immediately, and at 30 min, 1 h or 2 h after the injections. The remaining oocytes were activated by Sr(2+) after various incubation periods and observed for formation of a second polar body and pseudo-polar body. Cytogenetic analysis was performed for some of the resulting zygotes. The progress to blastocysts in vitro and the possibility of conception in vivo were assessed. RESULTS: Immediately after injection, the cumulus cell nucleus was still in interphase without spindle formation. The occurrence of premature chromosome condensation (PCC) and spindle formation increased as the incubation time increased. The percentages of activated oocytes increased with the incubation time after nuclear transfer, but the difference was not significant between 1 (58%) and 2 h (62%). The incidence of chromosomal aberrations was high for the derived embryos. Development in vitro was poor, and no procreation of pups occurred after transfer of the 324 embryos. CONCLUSIONS: The PCC and spindle formation induced by cumulus cell nuclei in mature oocytes was time dependent, as was the chance for successful activation. The chromosomal abnormalities from segregation errors presented one obvious cause, apart from the potential epigenetic defects, of developmental failure of the semi-cloned embryos.     

Cytoplasmic fragmentation in activated eggs occurs in the cytokinetic phase of the cell cycle, in lieu of normal cytokinesis, and in response to cytoskeletal disorder

The timing of cytoplasmic fragmentation in relation to the cell cycle was studied in mature oocytes and early cleavage stages using mouse oocytes and embryos as experimental models. The central approach was to remove the nuclear apparatus, in whole or in part, from non-activated and activated oocytes and early embryos, and follow their response during subsequent culture in vitro. Oocytes arrested in metaphase of the second meiotic division did not fragment following complete removal of the meiotic apparatus, provided they were not subsequently activated. Exposure of spindle-chromosome-complex-depleted oocytes to activation conditions immediately after enucleation led to fragmentation, although not until control embryos entered first mitosis. Delaying activation until 24 h post-enucleation led to earlier fragmentation. Enucleation of normally fertilized or artificially activated oocytes after emission of the second polar body also led to fragmentation coinciding with the first mitosis in nucleated control embryos. However, if artificially activated oocytes were prevented from completing second meiosis, by exposure to cytochalasin, and then enucleated, this universal wave of fragmentation was preceded in some cytoplasts by limited fragmentation after just a few hours in culture, and coinciding with completion of meiosis II in nucleated oocytes. Fragmentation also occurred in the second mitotic cell cycle, but it was limited to blastomeres of fertilized oocytes that were enucleated in late interphase. These results indicate that fragmentation in oocytes and early embryos, though seemingly uncoordinated, is a precisely timed event that occurs only in mitotically active cells, during the cytokinetic phase of the cell cycle, in lieu of normal cytokinesis, and in response to altered cytoskeletal organization.     

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.     

Chromosome number and development of artificial mouse oocytes and zygotes

BACKGROUND: Infertility due to the absence of gametes is one of the last frontiers in reproductive medicine. Sperm or oocyte donation is currently the only treatment option but this approach lacks the genetic contribution of both partners. Artificial production of gametes through haploidization may offer an alternative strategy. The aim of this study was to evaluate the efficiency of producing artificial oocytes and zygotes with correct chromosome number. METHODS AND RESULTS: Somatic cumulus cell nuclei were injected into non-enucleated oocytes to produce artificial zygotes and into enucleated mature mouse oocytes to produce artificial oocytes. The expected chromosome number of artificial zygotes and oocytes is 40 and 20 chromosomes respectively. Fertilization and developmental potential of artificial zygotes and oocytes inseminated by IVF or ICSI were investigated. The expected chromosome numbers were found in 12% of artificial zygotes and 15% of artificial oocytes. Varying the time interval between injection of the somatic nucleus and activation (3, 5, 8 h) tended to increase the efficiency up to 18 and 23% for zygotes and oocytes respectively. Two-cell formation rates were 90% for artificial zygotes and 37% for artificial oocytes after IVF and 53% for artificial oocytes after ICSI. Blastocyst formation rates were 15, 8 and 9% respectively. CONCLUSIONS: Chromosome number analysis shows that the efficiency of obtaining artificial zygotes and oocytes with correct chromosome number was low and that developmental potential was severely hampered. These observations question the possibility of obtaining chromosomally normal embryos from artificial oocytes or zygotes.     

Karyotyping of human oocytes by chromosomal analysis of the second polar bodies

This paper describes a method for obtaining metaphase chromosomes from human second polar bodies. The second polar body nucleus was injected into the cytoplasm of an enucleated oocyte, which is activated shortly after injection. When the polar body nucleus is transformed into a haploid pronucleus, treatment with okadaic acid was used to induce premature chromosome condensation. A total of 25 analysable chromosome plates were obtained from 38 polar bodies karyotyped using this technique. Whole chromosome painting was used to detect second polar bodies (and respectively, oocytes) with unbalanced translocations. In combination with the first polar body analysis, this technique may be useful in preimplantation genetic diagnosis for patients carrying maternal translocations.     

Maturation in vitro of immature human oocytes for clinical use

Human oocyte maturation is considered as the reinitiation andcompletion of the first meiotic division from the germinal vesiclestage (prophase I) to metaphase II, and the accompanying cytoplasmicmaturation for fertilization and early embryonic development.Immature human oocytes obtained from patients undergoing gynaecologicalsurgery, or ovulation induction or having polycystic ovary syndrome(PCOS) can be matured and fertilized in vitro. To date, 80%of immature oocytes matured to metaphase II when cultured inmaturation medium supplemented with gonadotrophins and 85% ofmatured oocytes fertilized and cleaved in vitro. Following transferof these embryos, pregnancies and live births have been achieved.However, the capacity for oocyte maturation was different whenthe immature oocytes were retrieved from PCOS patients and whenthe oocytes were cryopreserved at germinal vesicle stage.     

Distinct microtubule and chromatin characteristics of human oocytes after failed in-vivo and in-vitro meiotic maturation

BACKGROUND: While a complete failure of meiotic maturation following hCG administration is rare during IVF cycles, cases arise in which patients repeatedly display a high incidence of failure to complete maturation to metaphase II (MII) in vivo. For the immature oocytes of such patients, our objectives were (i) to ask whether progression to MII could be supported in vitro, and (ii) to define their microtubule/chromatin properties following in-vitro maturation (IVM). Together, these studies were aimed at augmenting our understanding of factors underlying meiotic arrest in the human. METHODS: Cases are presented here for two patients (A and B) producing oocytes that recurrently showed the inability to mature to metaphase II in vivo. Following IVM attempts, chromatin and microtubule characteristics were identified in those oocytes that remained arrested during meiosis I. RESULTS: In patient A, meiotically arrested oocytes exhibited clear defects in spindle and chromatin arrangements. In contrast, the majority of oocytes from patient B displayed normal MI and MII spindles with aligned chromosomes, although some oocytes exhibited indications for possible defects in cell cycle control. CONCLUSIONS: Together, these analyses illustrate two cases with oocytes exhibiting a common gross defect, that is meiotic maturation arrest, but revealing different aetiologies or manifestations as evidenced by the presence or absence of abnormal spindle/chromatin organization. This work reinforces the existence of intrinsic defects in oocytes of some patients, the molecular and cellular bases of which merit further investigation.     

Effect of recombinant human gonadotrophins on human, bovine and murine oocyte meiosis, fertilization and embryonic development in vitro

The response of murine, bovine and human oocytes to pure recombinant preparations of human follicle stimulating hormone (rFSH) and luteinizing hormone (rLH) for meiotic maturation and subsequent developmental competence in vitro were examined in the present experiments. Maturation of immature bovine oocytes to the metaphase II stage was significantly increased by the addition of 1 IU/ml of rFSH in combination with either 1 IU/ml rLH or 10 IU/ml rLH. Similarly, embryonic development to the blastocyst stage was improved in bovine oocytes treated with a 1:10 combination of rFSH:rLH. However, no significant difference was observed in the number of inner cell mass or trophectoderm cells of the resulting blastocysts. Although the increased maturation to metaphase II was not significant, human embryonic developmental competence was improved by maturing oocytes in the presence of a 1:10 ratio of rFSH:rLH as only those oocytes exposed to a 1:10 ratio of rFSH: rLH during maturation showed normal cleavage patterns beyond day 2. In addition, 1 IU/ml rFSH and 1 IU/ml rLH increased the expression of oocyte proteins in human oocytes. The inclusion of recombinant gonadotrophins, either singly or in combination, had no significant effect on the maturation, fertilization or embryonic development of in-vitro matured mouse oocytes. These data provide support for the responsiveness of human and bovine oocytes to gonadotrophins in vitro and the need to consider variations in the relative concentrations for optimization of oocyte developmental competence.     

Relationship between meiotic spindle location with regard to the polar body position and oocyte developmental potential after ICSI

BACKGROUND: The recent development of a computer-assisted polarization microscopy system (Polscope) with which the meiotic spindle can be visualized in living oocytes on the basis of its birefringence permits analysis of the meiotic spindles of oocytes subjected to ICSI. Previous studies have shown that the meiotic spindle is not always aligned with the first polar body (PB) in metaphase II human oocytes prepared for ICSI. In the present study, the relationship between the degree of meiotic spindle deviation from the first PB location and ICSI outcome was analysed. METHODS: Oocytes were divided into four groups according to the angle of meiotic spindle deviation from the PB position. The angle of deviation was 0-5 degrees, 6-45 degrees, 46-90 degrees and >90 degrees for groups I to IV respectively. RESULTS: The rates of normal [2 pronuclei (PN)] and abnormal (1PN or >2PN) fertilization did not differ between groups I, II and III. However, the rate of normal fertilization was lower among oocytes in which the meiotic spindle deviation angle was >90 degrees; this led to an increased proportion of tripronucleated zygotes that failed to extrude the second PB. When embryos developed from normally fertilized oocytes were evaluated on day 3 after ICSI, no relationship was found between the angle of meiotic spindle deviation and embryo quality. The meiotic spindle was not detected in only 9% of oocytes, and these showed a higher incidence of fertilization and cleavage abnormalities than did oocytes in which the spindle was detected. When oocytes at metaphase I after cumulus oophorus and corona radiata removal were matured in vitro, the meiotic spindle was detected in 53.8% of those that reached metaphase II. In these in-vitro-matured oocytes the meiotic spindle was always aligned with the first PB, suggesting that misalignment seen in those oocytes matured in vivo resulted from PB displacement during manipulations for cumulus and corona removal. CONCLUSION: High degrees of misalignment between the meiotic spindle and the first PB predict an increased risk of fertilization abnormalities. However, when normal fertilization had occurred, the cleavage potential of embryos developing from such oocytes was not impaired. These findings facilitate the selection of oocytes for ICSI in situations when the creation of supernumerary embryos is to be avoided.     

Morphological analysis of resumption of meiosis observed in oocytes of mouse vesicular follicles

Resumption of meiosis observed in vesicular follicles of immature 32-34 day-old mice was classified morphologically, and the morphological mechanism to induce resumption of meiosis was examined. Resumption of meiosis was observed in 22.1% of vesicular follicles, and the dominant stage of the meiotic division was the first or second metaphase. Cumulus-enclosed and cumulus-free oocytes were separated from ovaries by dissection of ovaries. All of the cumulus-enclosed oocytes contained germinal vesicle, however 57.9% of cumulus-free oocytes did not, suggesting that the disappearance of cumulus cells around oocytes precedes the induction of resumption of meiosis. About 80-90% of the oocytes with germinal vesicle formed first polar body after 15 hrs of incubation. After incubation of oocytes showing resumption of meiosis, but without the first polar body, 36% of the oocytes extruded the first polar body indicating that resumption of meiosis is associated with progression to the second metaphase. Morphological characteristics of the surrounding structures of oocytes showing resumption of meiosis was classified into three categories; extensive proliferation of granulosa cells, degeneration of cumulus cells and their disappearance around oocytes, and detachment of cumulus cell-oocyte complexes from granulosa cell layer. It was suggested that cell-to-cell interaction among cumulus cells, oocytes and granulosa cells was impaired at the time of resumption of meiosis. Granulosa cells secrete an inhibitor of the resumption of meiosis. The resumption of meiosis after the surge of luteinizing hormone follows cumulus dispersion which make oocytes independent from the influence of granulosa cells by the disruption of communication.(ABSTRACT TRUNCATED AT 250 WORDS)     

Embryo development after successful somatic cell nuclear transfer to in vitro matured human germinal vesicle oocytes

BACKGROUND: Somatic cell nuclear transfer (SCNT) involves the transfer of somatic cell nuclei into enucleated oocytes. Because human in vivo matured oocytes are scarcely available, we investigated whether in vitro matured (IVM) germinal vesicle (GV) oocytes could also support preimplantation development of human cloned embryos. METHODS: Three groups were used for SCNT: in vitro matured GV oocytes (IVM oocytes), 'in vivo' matured oocytes (in vivo oocytes) and 'failed fertilized' oocytes after routine-ICSI (FF oocytes). After removal of the chromosome-spindle complex, cumulus cell nuclei were injected, and oocytes were artificially activated and cultured. RESULTS: In total 61, 54 and 45 metaphase II oocytes were used for SCNT in the three groups, respectively. Survival and pronuclear rates were 59, 78 and 58% and 61, 64 and 50%, respectively. Of the 22 activated IVM oocytes, 13 cleaved to the 2-cell stage, whereby 2 morulae were formed. For the in vivo oocytes, 17 of 27 activated oocytes cleaved to the 2-cell stage and 1 morula was observed. Cleavage to the 2-cell stage in the group of FF oocytes was compromised. CONCLUSIONS: To our knowledge, this is the first report describing development of cloned human embryos using IVM oocytes and non-autologous transfer using a conventional method of SCNT.     

Cytoskeletal organization in fresh, aged and spontaneously activated human oocytes

The cytoskeleton of the human oocyte (microtobules and actlnfilaments) has been examined using fluorescence microscopy.In unfertilized oocytes in metaphase of the second meiotic division,microtubules were found exclusively within the spindle whichwas located at the periphery of the cell and oriented radially,with its long axis perpendicular to the surface membrane. Thespindle was anastral and slightly pointed at each pole, thechromosomes being arranged on a metaphase plate at the equator.When treated with taxol, the oocyte spindle became astral andmicrotubules appeared in the cortex of the oocyte in the formof small strands or bundles. Polymerized actin was found tobe present in a dense filamentous layer throughout the cortexof the unfertilized oocyte. Aged unfertilized oocytes displayedan increased inddence of disrupted or abnonnal cytoskeletalorganization. In parthenogenetically activated oocytes in anaphaseand telophase, microtubules were again found predominantly inthe spindle but in addition, cortical strands or bundles ofmicrotubules were often present. Oocytes in late telophase sometimesshowed the presence of a concentrated ring of actin in the deavagefurrow between the oocyte and the second polar body. Activatedoocytes in early interphase contained a dense cortical meshof microtubules and a midhody remnant between the oocyte andthe polar body. The cytoskeletal organizations of mouse andhuman oocytes are compared.     

Mouse spermatid nuclei can support full term development after premature chromosome condensation within mature oocytes.

The nucleus of round spermatids, the earliest haploid male germ cells, can participate in the formation of normal zygotes when incorporated into activated oocytes. In this study, we injected mouse round spermatids into homologous mature oocytes that were kept arrested at metaphase II to induce premature chromosome condensation (PCC) of the spermatid nuclei. After full condensation of the spermatid chromosomes, the oocytes were activated by Sr2+-containing medium, into which cytochalasin B was added to prevent extrusion of the segregated female and male chromosomes as polar bodies. Out of 142 oocytes examined, 104 (73%) formed two male (pseudo)pronuclei and two female pronuclei. To restore the diploid state of these zygotes, one of the female pronuclei was removed. When cultured in vitro for 72 hours, all (n = 37) of the constructed embryos developed to the morula/blastocyst stage. When 2-cell embryos and morulae/blastocysts were transferred into pseudopregnant females, 14 (13/96) and 24% (9/37), respectively, developed into term offspring. This study indicates that the spermatid chromosomes, which had undergone PCC, moved safely to opposite poles after oocyte activation. Since round spermatids contain no (in the mouse) or little (in patients with spermatogenic failure) oocyte-activating factor, this method may be used to rescue oocytes that fail to be activated at the time of spermatid injection.     

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.