Nocodazole sensitivity, age-related aneuploidy, and alterations in the cell cycle during maturation of mouse oocytes

To detect age-related alterations in the formation and function of the spindle apparatus, we examined in vitro maturing oocytes obtained from young (2-4 mo) and aged (greater than 9 mo) diestrous CBA/Ca mice. Observation of cells processed for antitubulin immunofluorescence revealed that oocytes from aged females progress faster through first maturation division than those from young animals. They are also more prone to nondisjunction, as shown by a significantly higher level of aneuploidy in C-banded cells arrested at metaphase II. The ability of oocytes to recover from treatment with a microtubule inhibitor, nocodazole, and the effect of the drug on spindle integrity and chromosome segregation were also studied. In both age groups, treatment of metaphase I oocytes with 10 microM nocodazole caused rapid and complete microtubule depolymerization and chromosome scattering. Upon recovery, oocytes from both age groups were able to reestablish a spindle apparatus, proceed through anaphase, and extrude a first polar body. However, nocodazole treatment led to a dramatic increase of aneuploidy. Unexpectedly, the relative rise in hyperploids was greater in oocytes from young mice than in those from aged mice, so that the absolute percentage of hyperploid metaphase II cells was similar in both age groups after drug treatment. Concomitantly, nocodazole exposure abolished or, at least, diminished intrinsic differences in the cell cycle and anaphase trigger present in the controls (e.g., the earlier onset of chromosome separation in oocytes from aged females). It shortened the period available for spindle formation before chromosome segregation in all oocytes. Therefore, our study implies that temporal differences in the progression of oocytes through maturation, in particular, the shortening of the time available for alignment of bivalents before chromosome separation occurs in oocytes of old females, are mainly responsible for age-related rises in aneuploidy. There is no indication that (1) the spindle apparatus of oocytes from aged mammals is more labile or susceptible to disturbances than the spindle apparatus of oocytes from young individuals or that (2) an increase in the number of univalents makes oocytes from aged mammals particularly prone to nondisjunction.     

Nondisjunction, disturbances in spindle structure, and characteristics of chromosome alignment in maturing oocytes of mice heterozygous for Robertsonian translocations

To correlate the chromosomal constitution of meiotic cells with possible disturbances in spindle function and the etiology of nondisjunction, we examined the spindle apparatus and chromosome behavior in maturing oocytes and analyzed the chromosomal constitution of metaphase II-arrested oocytes of CD/Cremona mice, which are heterozygous for a large number of Robertsonian translocation chromosomes (18 heterobrachial metacentrics in addition to two acrocentric chromosomes 19 and two X chromosomes). Spreading of oocytes during prometaphase 1 revealed that nearly all oocytes of the heterozygotes contained one large ring multivalent, apart from the bivalents of the two acrocentric chromosomes 19 and the X chromosomes, indicating that proper pairing and crossing-over between the homologous chromosome arms of all heterobrachial chromosomes took place during prophase. A large proportion of in vitro-matured oocytes arrested in metaphase II exhibited numerical chromosome aberrations (26.5% hyperploids, 40.8% hypoploids, and 6.1% diploids). In addition, some of the oocytes with euploid chromosome numbers (26.5% of the total examined) appeared to be nullisomic for one chromosome and disomic for another chromosome, so that aneuploidy levels may even be higher than expected on the basis of chromosome counts alone. Although oocytes of the complex heterozygous mice seemed able initially to form a bipolar spindle during first prometaphase, metaphase I spindles were frequently asymmetrical. Chromosomes in the multivalent did not align properly at the equator, centromeres of neighboring chromosomes in the multivalent remained maloriented, and pronounced lagging of chromosomes was observed at telophase I in oocytes obtained from the Robertsonian translocation heterozygotes. Therefore, disturbance in spindle structure and chromosome behavior appear to correlate with the chromosomal constitution in these oocytes and, ultimately, with failures in proper chromosome separation. In particular, reorientation appears to be a rare event, and malorientation of chromosomes may remain uncorrected throughout prometaphase, as we could not find many typical metaphase I stages in heterozygotes. This, in turn, could be the basis for malsegregation at anaphase and may ultimately induce a high rate of nondisjunction and aneuploidy in the oocytes of CD/Cremona mice, leading to total sterility in heterozygous females.     

Alterations to the microtubular cytoskeleton and increased disorder of chromosome alignment in spontaneously ovulated mouse oocytes aged in vivo: an immunofluorescence study

Alterations in the organization of the microtubular cytoskeleton and chromosome alignment were examined by tubulin immunofluorescence and DAPI staining during in vivo ageing of naturally ovulated, metaphase-arrested oocytes of CBA/Ca mice in the fallopian tubes. In oocytes isolated from young mice on the day of oestrus, a few hours after ovulation, when they are still tightly surrounded by cumulus, the anti-tubulin fluorescence is almost exclusively restricted to the metaphase spindle. Only some faintly staining foci are observed in the cytoplasm, which presumably represent cytoplasmic MTOC not involved in spindle formation. The spindle is usually barrel-shaped or slightly pointed at its poles and does not possess astral fibres. In oocytes aged for more than 12 h in the fallopian tubes cytoplasmic asters develop, while microtubules seem to become gradually lost from the spindle, preferentially in its central area near the chromosomes. Astral fibres are observed radiating out from the polar centrosomes into the cytoplasm. In oocytes free of cumulus, and consequently more than 24 h post-ovulation, a pronounced shrinking of the spindle is observed. The mean pole-to-pole distance becomes significantly reduced in postovulatory aged cells. At the same time astral microtubules in the cytoplasm appear to become gradually depolymerized. Age-dependent alterations in the microtubular cytoskeleton do not seem to result from a changed pattern of the post-translational detyrosylation of -tubulin in certain sets of microtubules. In freshly ovulated oocytes chromosomes in most spindles are well ordered and precisely arranged at the equatorial plane. In 11% of the cells only, there was dislocation of one or several of the chromosomes from the spindle equator. By contrast, 61.4% of bipolar spindles of postovulatory aged oocytes have chromosomes displaced from the centre of the spindle towards one of the spindle poles. The implications of the observed alterations in the microtubular cytoskeleton, shrinking of the spindle and increased disorder of chromosome alignment are discussed with regard to predisposition to aneuploidy and reduction of developmental potential of postovulatory aged oocytes.     

Sexual immaturity and maternal age: incidence of aneuploidy and polyploidy in first-cleavage mouse embryos

The influence of maternal age on the incidence of aneuploidy and polyploidy was studied, using C57Bl/6J X CBA/Ca hybrid mice, including immature females, as gamete donors. The age of the females ranged from 3.5-4 wk (immature or prepubertal), to 10-12 wk (young adults), to 24-28 wk (aged females). Ovulation was induced with gonadotrophins, and the differential condensation of paternal and maternal chromosomes was used to elucidate the origin of chromosome abnormalities in first-division metaphase plates. The results indicated a high incidence of aneuploid oocytes in immature and older female mice, as compared to young adult females. Eggs of immature female mice underwent polyspermic fertilization more often than those of young adults and older females, and the production of diploid oocytes was more frequent in immature females than in the other age groups.     

Chromosomal and cytoplasmic context determines predisposition to maternal age-related aneuploidy: brief overview and update on MCAK in mammalian oocytes

It has been known for more than half a century that the risk of conceiving a child with trisomy increases with advanced maternal age. However, the origin of the high susceptibility to nondisjunction of whole chromosomes and precocious separation of sister chromatids, leading to aneuploidy in aged oocytes and embryos derived from them, cannot be traced back to a single disturbance and mechanism. Instead, analysis of recombination patterns of meiotic chromosomes of spread oocytes from embryonal ovary, and of origins and exchange patterns of extra chromosomes in trisomies, as well as morphological and molecular studies of oocytes and somatic cells from young and aged females, show chromosome-specific risk patterns and cellular aberrations related to the chronological age of the female. In addition, analysis of the function of meiotic- and cell-cycle-regulating genes in oogenesis, and the study of the spindle and chromosomal status of maturing oocytes, suggest that several events contribute synergistically to errors in chromosome segregation in aged oocytes in a chromosome-specific fashion. For instance, loss of cohesion may differentially predispose chromosomes with distal or pericentromeric chiasmata to nondisjunction. Studies on expression in young and aged oocytes from human or model organisms, like the mouse, indicate that the presence and functionality/activity of gene products involved in cell-cycle regulation, spindle formation and organelle integrity may be altered in aged oocytes, thus contributing to a high risk of error in chromosome segregation in meiosis I and II. Genes that are often altered in aged mouse oocytes include MCAK (mitotic-centromere-associated protein), a microtubule depolymerase, and AURKB (Aurora kinase B), a protein of the chromosomal passenger complex that has many targets and can also phosphorylate and regulate MCAK localization and activity. Therefore we explored the role of MCAK in maturing mouse oocytes by immunofluorescence, overexpression of a MCAK-EGFP (enhanced green fluorescent protein) fusion protein, knockdown of MCAK by RNAi (RNA interference) and inhibition of AURKB. The observations suggest that MCAK is involved in spindle regulation, chromosome congression and cell-cycle control, and that reductions in mRNA and protein in a context of permissive SAC (spindle assembly checkpoint) predispose to aneuploidy. Failure to recruit MCAK to centromeres and low expression patterns, as well as disturbances in regulation of enzyme localization and activity, e.g. due to alterations in activity of AURKB, may therefore contribute to maternal age-related rises in aneuploidy in mammalian oocytes.     

Intra-oocyte localization of MAD2 and its relationship with kinetochores, microtubules, and chromosomes in rat oocytes during meiosis

The present study was designed to investigate subcellular localization of MAD2 in rat oocytes during meiotic maturation and its relationship with kinetochores, chromosomes, and microtubules. Oocytes at germinal vesicle (GV), prometaphase I (ProM-I), metaphase I (M-I), anaphase I (A-I), telophase I (T-I), and metaphase II (M-II) were fixed and immunostained for MAD2, kinetochores, microtubules and chromosomes. The stained oocytes were examined by confocal microscopy. Some oocytes from GV to M-II stages were treated by a microtubule disassembly drug, nocodazole, or treated by a microtubule stabilizer, Taxol, before examination. Anti-MAD2 antibody was also injected into the oocytes at GV stage and the injected oocytes were cultured for 6 h for examination of chromosome alignment and spindle formation. It was found that MAD2 was at the kinetochores in the oocytes at GV and ProM-I stages. Once the oocytes reached M-I stage in which an intact spindle was formed and all chromosomes were aligned at the equator of the spindle, MAD2 disappeared. However, when oocytes from GV to M-II stages were treated by nocodazole, spindles were destroyed and MAD2 was observed in all treated oocytes. When nocodazole-treated oocytes at M-I and M-II stages were washed and cultured for spindle recovery, it was found that, once the relationship between microtubules and chromosomes was established, MAD2 disappeared in the oocytes even though some chromosomes were not aligned at the equator of the spindle. On the other hand, when oocytes were treated with Taxol, MAD2 localization was not changed and was the same as that in the control. However, immunoblotting of MAD2 indicated that MAD2 was present in the oocytes at all stages; nocodazole and Taxol treatment did not influence the quantity of MAD2 in the cytoplasm. Significantly higher proportions of anti-MAD2 antibody-injected oocytes proceeded to premature A-I stage and more oocytes had misaligned chromosomes in the spindles. The present study indicates that MAD2 is a spindle checkpoint protein in rat oocytes during meiosis. When the spindle was destroyed by nocodazole, MAD2 was reactivated in the oocytes to overlook the attachment between chromosomes and microtubules. However, in this case, MAD2 could not check unaligned chromosomes in the recovered spindles, suggesting that a normal chromosome alignment is maintained only in the oocytes without any microtubule damages during maturation.     

Kinetochore microtubule establishment is defective in oocytes from aged mice

Errors in chromosome segregation in mammalian oocytes increase in number with advancing maternal age, and are a major cause of pregnancy loss. Why chromosome segregation errors are more common in oocytes from older females remains poorly understood. In mitosis, accurate chromosome segregation is enabled by attachment of kinetochores to microtubules from appropriate spindle poles, and erroneous attachments increase the likelihood of mis-segregation. Whether attachment errors are responsible for age-related oocyte aneuploidy is unknown. Here we report that oocytes from naturally aged mice exhibit substantially increased chromosome misalignment, and fewer kinetochore pairs that make stable end-on attachments to the appropriate spindle poles compared with younger oocytes. The profile of mis-attachments exhibited is consistent with the types of chromosome segregation error observed in aged oocytes. Loss of chromosome cohesion, which is a feature of oocytes from older females, causes altered kinetochore geometry in meiosis-I. However, this has only a minor impact upon MT attachment, indicating that cohesion loss is not the primary cause of aneuploidy in meiosis-I. In meiosis-II, on the other hand, age-related cohesion loss plays a direct role in errors, since prematurely individualized sister chromatids misalign and misattach to spindle MTs. Thus, whereas cohesion loss leading to precocious sister chromatid separation is a direct cause of errors in meiosis-II, cohesion loss plays a more minor role in the etiology of aneuploidy in meiosis-I. Our data introduce altered MT-kinetochore interactions as a lesion that explains aneuploidy in meiosis-I in older females.     

Age‐dependent aneuploidy in mammalian oocytes instigated at the second meiotic division

Ageing severely affects the chromosome segregation process in human oocytes resulting in aneuploidy, infertility and developmental disorders. A considerable amount of segregation errors in humans are introduced at the second meiotic division. We have here compared the chromosome segregation process in young adult and aged female mice during the second meiotic division. More than half of the oocytes in aged mice displayed chromosome segregation irregularities at anaphase II, resulting in dramatically increased level of aneuploidy in haploid gametes, from 4% in young adult mice to 30% in aged mice. We find that the post‐metaphase II process that efficiently corrects aberrant kinetochore‐microtubule attachments in oocytes in young adult mice is approximately 10‐fold less efficient in aged mice, in particular affecting chromosomes that show small inter‐centromere distances at the metaphase II stage in aged mice. Our results reveal that post‐metaphase II processes have critical impact on age‐dependent aneuploidy in mammalian eggs.     

Merotelic kinetochore attachment in oocyte meiosis II causes sister chromatids segregation errors in aged mice

Mammalian oocyte chromosomes undergo 2 meiotic divisions to generate haploid gametes. The frequency of chromosome segregation errors during meiosis I increase with age. However, little attention has been paid to the question of how aging affects sister chromatid segregation during oocyte meiosis II. More importantly, how aneuploid metaphase II (MII) oocytes from aged mice evade the spindle assembly checkpoint (SAC) mechanism to complete later meiosis II to form aneuploid embryos remains unknown. Here, we report that MII oocytes from naturally aged mice exhibited substantial errors in chromosome arrangement and configuration compared with young MII oocytes. Interestingly, these errors in aged oocytes had no impact on anaphase II onset and completion as well as 2-cell formation after parthenogenetic activation. Further study found that merotelic kinetochore attachment occurred more frequently and could stabilize the kinetochore-microtubule interaction to ensure SAC inactivation and anaphase II onset in aged MII oocytes. This orientation could persist largely during anaphase II in aged oocytes, leading to severe chromosome lagging and trailing as well as delay of anaphase II completion. Therefore, merotelic kinetochore attachment in oocyte meiosis II exacerbates age-related genetic instability and is a key source of age-dependent embryo aneuploidy and dysplasia.     

Sex-dependent frequency and type of autosomal univalency at the first meiotic metaphase in mouse germ cells

Univalents at the first meiotic metaphase in mouse spermatocytes occur mainly in the XY pair, making it difficult to compare the amounts of univalency in males and females. In this study, the amounts of autosomal univalency in male and female meiosis were compared using the model strain CBA-T6, in which univalency of the small marker autosome pair T6 has been shown to occur very frequently in spermatocytes. Mice from inbred CBA and DBA strains were also analysed. The total frequencies of univalency (sex chromosomes plus autosomes) in metaphase I spermatocytes were 45.6% in CBA, 36.9% in CBA-T6, and 37.3% in DBA males. The aneuploidy in metaphase II spermatocytes ranged from 1.4 to 3% in these strains, which was in agreement with previous findings that most primary spermatocytes with abnormal chromosome configurations are arrested in their development before metaphase II. In the CBA-T6 strain, autosomal univalency at metaphase I mostly involved chromosome pair T6; however, its frequency differed significantly between the sexes, amounting to 18.9% in spermatocytes and 4.3% in oocytes. In the CBA strain, autosomal univalents at metaphase I were seen in 7.7% of the spermatocytes and 1.4% of the oocytes and, in DBA mice, in 4.9% of the spermatocytes and 3.8% of the oocytes. However, in DBA oocytes, when univalency occurred it usually concerned a greater number of bivalents in one cell (range: 2-19 disjoined bivalents), a phenomenon very rare in males of this strain. This study shows that univalent formation differs between the male and female types of meiosis.     

The use of translocation-derived "marker-bivalents" for studying the origin of meiotic instability in female mice

Female mice of two age groups, 3--4 and 11--14 months old, homozygous for the T(1;13)70H reciprocal mouse translocation were used for cytological observations of bivalents (in primary oocytes) and metaphase II chromosomes (in secondary oocytes). Special attention was given to the behavior of the long (131) and short (113) marker chromosomes. In primary oocytes, univalents were considered "true" or "opposite". The aged females showed an eight-folded increase in "true" univalent frequency for chromosomes 113 over the young ones. A nine-fold rise for nondisjunction with regard to this chromosome was observed. For the other chromosomes, these factors were 2 and 1.7, respectively. The absolute levels of nondisjunction remained low at old age (1.42% for chromosome 113, 1.22% for all other chromosomes). The long marker bivalent 131 was used for chiasma counts. No change in chiasma number with age was observed. It is argued that poorer physiological conditions within the maturing oocytes of older females are the major cause for both the increasing frequencies of "true" and "opposite" univalents and the increased incidence for nondisjunction.     

Exposure of mouse oocytes to bisphenol A causes meiotic arrest but not aneuploidy

Mouse oocytes isolated from large antral follicles were exposed to a wide range of concentrations of bisphenol A (BPA) during maturation in vitro (50 ng/ml to 10 microg/ml BPA in medium). Exposure to high concentrations of BPA (10 microg/ml) affected spindle formation, distribution of pericentriolar material and chromosome alignment on the spindle (termed congression failure), and caused a significant meiotic arrest. However, BPA did not increase hyperploidy at meiosis II at any tested concentration. Some but not all meiosis I arrested oocytes had MAD2-positive foci at centromeres of chromosomes in bivalents, suggesting that they had failed to pass the spindle checkpoint control. In a second set of experiments prepubertal mice were exposed sub-chronically for 7 days to low BPA by daily oral administration, followed by in vitro maturation of the denuded oocytes to metaphase II in the absence of BPA, as this treatment protocol was previously reported to induce chromosome congression failure and therefore suspected to cause aneuploidy in oocytes. The sub-chronic exposure subtly affected spindle morphology and oocyte maturation. However, as with the exposure in vitro, there was no evidence that low BPA doses increased hyperploidy at meiosis II. In conclusion, the data suggest that mouse oocytes from mice respond to BPA-induced disturbances in spindle formation by induction of meiotic arrest. This response might result from an effective checkpoint mechanism preventing the occurrence of chromosome malsegregation and aneuploidy. Low chronic BPA exposure in vivo as such does not appear to pose a risk for induction of errors in chromosome segregation at first meiosis in mouse oocytes. Additional factors besides BPA may have caused the high rate of congression failure and the temporary increase in hyperploidy in mouse metaphase II oocytes reported previously.     

MLN8054, a small-molecule inhibitor of Aurora A, causes spindle pole and chromosome congression defects leading to aneuploidy

Aurora A kinase plays an essential role in the proper assembly and function of the mitotic spindle, as its perturbation causes defects in centrosome separation, spindle pole organization, and chromosome congression. Moreover, Aurora A disruption leads to cell death via a mechanism that involves aneuploidy generation. However, the link between the immediate functional consequences of Aurora A inhibition and the development of aneuploidy is not clearly defined. In this study, we delineate the sequence of events that lead to aneuploidy following Aurora A inhibition using MLN8054, a selective Aurora A small-molecule inhibitor. Human tumor cells treated with MLN8054 show a high incidence of abnormal mitotic spindles, often with unseparated centrosomes. Although these spindle defects result in mitotic delays, cells ultimately divide at a frequency near that of untreated cells. We show that many of the spindles in the dividing cells are bipolar, although they lack centrosomes at one or more spindle poles. MLN8054-treated cells frequently show alignment defects during metaphase, lagging chromosomes in anaphase, and chromatin bridges during telophase. Consistent with the chromosome segregation defects, cells treated with MLN8054 develop aneuploidy over time. Taken together, these results suggest that Aurora A inhibition kills tumor cells through the development of deleterious aneuploidy.     

Follicle-stimulating hormone affects metaphase I chromosome alignment and increases aneuploidy in mouse oocytes matured in vitro

Follicle-Stimulating Hormone (FSH) at a wide range of doses is routinely added to culture media during in vitro maturation (IVM) of oocytes, but the effects on oocyte health are unclear. The suggestion that superovulation may cause aneuploidy and fetal abnormalities prompted us to study the potential role of FSH in the genesis of chromosomal abnormalities during meiosis I. Mouse cumulus-oocyte complexes (COCs) isolated from the antral follicles of unprimed, sexually immature B6CBF1 mice were cultured in increasing concentrations of FSH. Following culture, matured oocytes were isolated, spread, stained with DAPI, and the numbers of chromosomes counted. Significantly increased aneuploidy, arising during the first meiotic division, was observed in metaphase II oocytes matured in higher concentrations of FSH (> or =20 ng/ml). The effect of FSH on spindle morphology and chromosome alignment during metaphase I was then explored using immunocytochemistry and three-dimensional reconstruction of confocal sections. High FSH had no effect on gross spindle morphology but did alter chromosome congression during prometaphase and metaphase, with the spread of chromosomes across the spindle at this time being significantly greater in oocytes cultured in 2000 ng/ml compared with 2 ng/ml FSH. Analysis of three-dimensional reconstructions of spindles in oocytes matured in 2000 ng/ml FSH shows that chromosomes are more scattered and farther apart than they are following maturation in 2 ng/ml FSH. These results demonstrate that exposure to high levels of FSH during IVM can accelerate nuclear maturation and induce chromosomal abnormalities and highlights the importance of the judicious use of FSH during IVM.     

Does human oocyte cryopreservation affect equally on embryo chromosome aneuploidy?

Oocytes cryopreservation as an important part of assisted reproductive technologies, which should ensure after warming not only intact oocyte morphological characteristics, but also their genetic apparatus stability. However, the meiotic spindle is very sensitive to the temperature fluctuations that can lead to unequal chromosome segregation during meiosis and as a consequence can cause embryo aneuploidy after oocyte fertilization. The aim of the study was to estimate the oocytes cryopreservation impact on human embryo chromosome aneuploidy. It has been shown that fertilization rate of the cryopreserved oocytes did not differ from fresh ones (83.1% vs 84% respectively). The number of blastocysts obtained from cryopreserved oocytes was less than that obtained from fresh oocytes, however, their morphological characteristics were better if compared the fresh oocytes. Our results showed different cryopreservation impact on aneuploidy rates of certain chromosomes in embryos obtained from cryopreserved oocytes. They had an increased aneuploidy of chromosome 13 and a decreased nondisjunction of chromosome 18 and sex chromosomes.     

Asymmetric division of spindle microtubules and microfilaments during bovine meiosis from metaphase I to metaphase III

The kinetics of spindle and chromosomes during bovine oocyte meiosis from meiosis I to meiosis III is described. The results of this study showed that (1) oocytes began to extrude the first polar body (Pb1) at the early anaphase I stage and the Pb1 totally separated from the mother cell only when oocytes reach the MII stage; (2) the morphology of the spindle changed from barrel-shaped at the metaphase stage to cylinder-shaped at early anaphase, and then to a thin, long triangle-shaped cone at late anaphase and telophase stages; (3) chromosome morphology went from an individual visible stage at metaphase to a less defined chromatin state during anaphase and telophase stages, and then back to visible individual chromosomes at the next metaphase; (4) chromatin that connected with the floor of the cone became the polar bodies and expelled, and almost all of the microtubules (MTs) and microfilaments (MFs) composing the spindles moved towards and contributed to the polar bodies; and (5) the size of the metaphase I (MI) spindle was larger than the metaphase II (MII) and metaphase III (MIII) spindles. The MII spindle, however, is more barrel-shaped than the MI spindle. This study suggests that spindle MTs and MFs during bovine oocyte meiosis are asymmetrically divided into the polar bodies.     

Requirement of functional telomeres for metaphase chromosome alignments and integrity of meiotic spindles

Telomerase deficiency in the mouse eventually leads to loss of telomeric repeats from chromosome ends and to end-to-end chromosome fusions, which result in defects in highly proliferative tissues. We show that telomere dysfunction resulting from telomerase deficiency leads to disruption of functional meiotic spindles and misalignment of chromosomes during meiotic division of oocytes in late-generation (G₄) mice. However, oocytes from first-generation (G₁) mice lacking telomerase showed no appreciable telomere dysfunction and exhibited chromosome alignment at the metaphase plates of meiotic spindles, in a manner similar to that of wild-type mouse oocytes. These findings suggest that telomerase does not directly influence chromosome alignment and spindle integrity. Rather, functional telomeres may be involved in mediating metaphase chromosome alignment and maintaining functional spindles during meiotic division.     

Premiers stades de l'oogenèse de Rhabdophaga saliciperda (Cecidomyiidae,Diptera)

The females of Rhabdophaga saliciperda have in their somatic cells 8 chromosomes and the males 6. The type of sex determination is therefore: X₁X₁X₂X₂—♀; X₁X₂—♂. The cells of the germinal line have 46 chromosomes, but a variation of their number was observed. In the oogonia and spermatogonia the number of heterochromatic chromosomes may exceed the number of E chromosomes, i.e. 8. In the beginning of the growth stage of the oocytes an incorporation of somatic cells was observed. The nuclei of these somatic cells persist in the cytoplasm of the oocytes until the maturation divisions. The possibility of their participation in the reconstruction of the nucleus of the mature egg is envisaged. The metaphase of the I segmentation division has a complex character. During prophase of the first meiotic division the E chromosomes form 4 bunches of 6–8 chromosomes each. Some univalents may also be present. The 8 S chromosomes form 4 regular bivalents. The 4 groups of E chromosomes persist until metaphase I. During metaphase I a phenomenon of expulsion of the majority of E chromosomes from the metaphase spindle was observed. The 4 bivalents remain in the equatorial plain of the spindle with some E Chromosomes. After this expulsion 2 groups of chromosomes are formed. In connection with them 2 spindles develop. An irregular distribution of E chromosomes follows without their division. The bivalents are probably separated in regular manner. These 2 spindles correspond to the I maturation division. The II maturation division was not observed because of lack of respective stages.     

Cellular and morphological traits of oocytes retrieved from aging mice after exogenous ovarian stimulation.

The present study aims to shed light on the origin of abnormal oocytes ovulated by aged females. In order to reach this goal, cellular and morphological traits of ovulated oocytes from hybrid (C57Bl/6JIco female x CBA/JIco male) female mice retrieved after exogenous ovarian stimulation at the age of 12, 40-42, 50-52, or 57-62 wk were analyzed. Aging of female mice was associated with 1) decreased number of ovulated oocytes; 2) increased percentage of cumulus-free oocytes; 3) raised percentage of oocytes with intracellular mitochondrial aggregates; 4) reduced percentage of oocytes displaying a normal distribution of chromosomes in the metaphase-II plate; 5) increased percentage of normal oocytes exhibiting a DNA-containing polar body (PB); 6) higher percentage of oocytes with chromosome scattering; 7) increased percentage of chromosome-scattered oocytes without a DNA-containing PB and with intracytoplasmic mitochondrial aggregates; 8) raised percentage of oocytes exhibiting chromosome decondensation; 9) lower percentage of chromosome-decondensed oocytes lacking both a DNA-containing PB and intracytoplasmic mitochondrial aggregates; 10) increased percentage of abnormal/degenerated oocytes; 11) reduced percentage of abnormal/degenerated oocytes displaying cellular fragmentation; and 12) higher percentage of abnormal/degenerated oocytes with mitochondrial aggregates exhibiting no nuclear/chromosomal DNA fluorescence, cellular fragmentation, milky or dark cytoplasm, or cellular remains enclosed by the zona pellucida. Although several studies suggest aging females may ovulate aged or overripened oocytes, these data support the hypothesis that old females ovulate an increased percentage of atretic/apoptotic oocytes coming from rescued follicles that would have become atretic earlier in life.