APCFZR1 prevents nondisjunction in mouse oocytes by controlling meiotic spindle assembly timing

FZR1 is an anaphase-promoting complex (APC) activator best known for its role in the mitotic cell cycle at M-phase exit, in G1, and in maintaining genome integrity. Previous studies also established that it prevents meiotic resumption, equivalent to the G2/M transition. Here we report that mouse oocytes lacking FZR1 undergo passage through meiosis I that is accelerated by ∼1 h, and this is due to an earlier onset of spindle assembly checkpoint (SAC) satisfaction and APC^CDC20 activity. However, loss of FZR1 did not compromise SAC functionality; instead, earlier SAC satisfaction was achieved because the bipolar meiotic spindle was assembled more quickly in the absence of FZR1. This novel regulation of spindle assembly by FZR1 led to premature bivalent attachment to microtubules and loss of kinetochore-bound MAD2. Bivalents, however, were observed to congress poorly, leading to nondisjunction rates of 25%. We conclude that in mouse oocytes FZR1 controls the timing of assembly of the bipolar spindle and in so doing the timing of SAC satisfaction and APC^CDC20 activity. This study implicates FZR1 as a major regulator of prometaphase whose activity helps to prevent chromosome nondisjunction.     

Trichlorfon exposure, spindle aberrations and nondisjunction in mammalian oocytes

Consumption of trichlorfon-poisoned fish by women in a small Hungarian village has been associated with trisomy resulting from an error of meiosis II in oogenesis. We therefore examined mouse oocytes exposed for 3 h during fertilization to 50 μg/ml trichlorfon. Spindle morphology was not visibly altered by the pesticide. Chromosomes segregated normally at anaphase II with no induction of aneuploidy. However, formation of a spindle was disturbed in many oocytes resuming meiosis I in the presence of trichlorfon. In spite of the spindle aberrations and the failure of bivalents to align properly at the equator, oocytes did not become meiotically arrested but progressed to metaphase II. At this stage, spindles were highly abnormal, and chromosomes were often totally unaligned, unattached or dispersed on the elongated and disorganized spindle. By causing spindle aberrations and influencing chromosome congression, trichlorfon appears, therefore, to predispose mammalian oocytes to random chromosome segregation, especially when they undergo a first division and develop to metaphase II during exposure. This is the first case in which environmentally induced human trisomy can be correlated with spindle aberrations induced by chemical exposure. Our observations suggest that oocytes may not possess a checkpoint sensing displacement of chromosomes from the equator at meiosis I and may therefore be prone to nondisjunction. Received: 2 August 1998; in revised form: 12 September 1998 / Accepted: 13 September 1998     

Rotation of meiotic spindle is controlled by microfilaments in mouse oocytes

The completion of meiosis requires the spatial and temporal coordination of cytokinesis and karyokinesis. During meiotic maturation, many events, such as formation, location, and rotation of the meiotic spindle as well as chromosomal movement, polar body extrusion, and pronuclear migration, are dependent on regulation of the cytoskeleton system. To study functions of microfilaments in meiosis, we induced metaphase II (MII) mouse oocytes to resume meiosis by in vitro fertilization or parthenogenetic activation, and we treated such oocytes with cytochalasin B (CB). The changes of the meiotic spindle, as visualized in preparations stained for beta-tubulin and chromatin, were observed by fluorescent confocal microscopy. The meiotic spindle of MII oocytes was observed to be parallel to the plasmalemma. After meiosis had resumed, the spindle rotated to the vertical position so that the second polar body could be extruded into the perivitelline space. When meiosis resumed and oocytes were treated with 10 micro g/ml of CB, the spindle rotation was inhibited. Consequently, the oocyte formed an extra pronucleus instead of extruding a second polar body. These results indicate that spindle rotation is essential for polar body extrusion; it is the microfilaments that play a crucial role in regulating rotation of the meiotic spindle.     

Preferential nondisjunction of specific bivalents in oocytes from Djungarian hamsters (Phodopus sungorus) following colchicine treatment

In an attempt to study the mechanisms leading to nondisjunction during meiosis I, Djungarian hamster females were treated with colchicine (3 mg/kg), which binds specifically to tubulin. The number of ovulated oocytes per female was significantly reduced following colchicine treatment (8.2 +/- 5.3, compared to 10.6 +/- 5.9 in controls receiving saline solution only). Application of colchicine rather late during oocyte maturation (ie, 5.5 h after injection of human chorionic gonadotrophin) caused a significant increase in the number of ovulated diploid (34.5%) and hyperhaploid (11.7%) oocytes, compared to the frequencies observed in the saline-treated controls (0.8% and 3.5%, respectively). Specific bivalents (viz, the large meta- and submetacentric chromosomes of groups A, B, and C) were preferentially involved in colchicine-induced nondisjunction. The same pattern of chromosomal malsegregation was previously observed in oocytes from this hamster species following hypergonadotrophic stimulation. Preferential involvement of bivalents in the process of nondisjunction, whether induced by colchicine or hypergonadotrophic stimulation, is explained by an interference with microtubular function affecting those bivalents that are the last to segregate.     

Porcine oocytes are most vulnerable to the mycotoxin deoxynivalenol during formation of the meiotic spindle

Deoxynivalenol (DON, vomitoxin) is a secondary metabolite and mycotoxin produced by Fusarium species that occurs with a high prevalence in cereals and grains intended for human and animal consumption. Pigs are considered to be the most sensitive animal species and exposure to DON results in reduced feed intake, reduced performance and cause alterations in the expression of markers of inflammation and cell cycle regulation. The objective of this study was to determine how DON possibly affects the oocyte developmental potential in vitro at concentrations which correspond to those observed in practice. To evaluate DON toxicity during specific stages of oocyte meiosis, cumulus-oocyte complexes were exposed to 0.02, 0.2, or 2 μM DON. Exposure to the highest DON concentration inhibited cumulus expansion and induced cumulus cell death. After exposure for 42 h, DON at all concentrations reduced Metaphase II formation and led to malformations of the meiotic spindle. Despite spindle malformations, exposure to different concentrations of DON did not lead to increased percentages of blastomeres with abnormal ploidy in embryos. Spindle malformation occurred by DON exposure during formation of meiotic spindles at Metaphase I and II, but embryo development was also reduced when oocytes were exposed to DON during Prophase I. Together, these results indicate that exposure to DON via contaminated food or feed can affect oocyte developmental competence by interfering directly with microtubule dynamics during meiosis, and by disturbing oocyte cytoplasmic maturation through other as yet undetermined mechanisms.     

Intranuclear membranes and the formation of the first meiotic spindle in Xenos peckii (Acroschismus wheeleri) oocytes

The ultrastructure of spindle formation during the first meiotic division in oocytes of the Strepsipteran insect Xenos peckii Kirby (Acroschismus wheeleri Pierce) was examined in serial thick (0.25- micron) and thin sections. During late prophase the nuclear envelope became extremely convoluted and fenestrated. At this time vesicular and tubular membrane elements permeated the nucleoplasm and formed a thin fusiform sheath, 5-7 micron in length, around each of the randomly oriented and condensing tetrads. These membrane elements appeared to arise from the nuclear envelope and/or in association with annulate lamellae in the nuclear region. All of the individual tetrads and their associated fusiform sheaths became aligned within the nucleus subsequent to the breakdown of the nuclear envelope. Microtubules (MTs) were found associated with membranes of the meiotic apparatus only after the nuclear envelope had broken down. Kinetochores, with associated MTs, were first recognizable as electron-opaque patches on the chromosomes at this time. The fully formed metaphase arrested Xenos oocyte meiotic apparatus contained an abundance of membranes and had diffuse poles that lacked distinct polar MT organizing centers. From these observations we conclude that the apparent individual chromosomal spindles--seen in the light microscope to form around each Xenos tetrad during "intranuclear prometaphase" (Hughes-Schrader, S., 1924, J. Morphol. 39:157-197)--actually form during late prophase, lack MTs, and are therefore not complete miniature bipolar spindles, as had been commonly assumed. Thus, the unique mode of spindle formation in Xenos oocytes cannot be used to support the hypothesis that chromosomes (kinetochores) induce the polymerization of their associated MTs. Our observation that MTs appeared in association with and parallel to tubular membrane components of the Xenos meiotic apparatus after these membranes became oriented with respect to the tetrads, is consistent with the notion that membranes associated with the spindle determine the orientation of spindle MTs and also play a part in regulating their formation.     

The meiotic stage of nondisjunction in trisomy 21: Determination by using DNA polymorphisms

We have studied DNA polymorphisms at loci in the pericentromeric region on the long arm of chromosome 21 in 200 families with trisomy 21, in order to determine the meiotic origin of nondisjunction. Maintenance of heterozygosity for parental markers in the individual with trisomy 21 was interpreted as resulting from a meiosis I error, while reduction to homozygosity was attributed to a meiosis II error. Nondisjunction was paternal in 9 cases and was maternal in 188 cases, as reported earlier. Among the 188 maternal cases, nondisjunction occurred in meiosis I in 128 cases and in meiosis II in 38 cases; in 22 cases the DNA markers used were uninformative. Therefore meiosis I was responsible for 77.1% and meiosis II for 22.9% of maternal nondisjunction. Among the 9 paternal nondisjunction cases the error occurred in meiosis I in 2 cases (22.2%) and in meiosis II in 7 (77.8%) cases. Since there was no significant difference in the distribution of maternal ages between maternal I error versus maternal II error, it is unlikely that an error at a particular of maternal ages between maternal I error versus maternal II error, it is unlikely that an error at a particular meiotic stage contributes significantly to the increasing incidence of Down syndrome with advancing maternal age. Although the DNA polymorphisms used were at loci which map close to the centromere, it is likely that rare errors in meiotic-origin assignments may have occurred because of a small number of crossovers between the markers and the centromere.(ABSTRACT TRUNCATED AT 250 WORDS)     

Increased spindle resistance to antimicrotubule agents in women of high risk for meiotic nondisjunction

Summary Spindle sensitivity of phytohemagglutinin (PHA)-stimulated lymphocytes to three antimicrotubule drugs was compared in two groups of women who differ in their predisposition to meiotic aneuploidy: young women of low-risk age (ranging from 22 to 34 years) and middle-aged women of high-risk age (ranging from 40 to 52 years). Numerical sensitivity values for the antimicrotubule drugs, colchicine, podophyllotoxin, and vinblastine were obtained for each woman by recording the percentage of fully arrested metaphases out of the total metaphase cell population, i.e., cells exhibiting short, thick, and condensed chromosomes with sister chromatids clearly separated at their distal parts. Sensitivity increased linearly with increasing drug concentrations and was highly correlated with youth: its rate was significantly higher for women of the low-risk group. In addition, dividing lymphocytes of young mothers (26–33 years old) of Down syndrome children revealed significantly lower sensitivity to colchicine and podophyllotoxin than those of all young women of the low-risk group and similar sensitivity to that of the middle-aged women, i.e., the high-risk age group. The data are consistent with the theory that factors involved in meiotic nondisjunction may be concurrently operating in somatic cells. These factors presumably shift the equilibrium between tubulin and microtubules towards microtubules stabilization and thereby affect some of their functions.     

The meiotic stage of preovulatory oocytes in mares

Confusion exists as to whether the oocytes of the domestic horse are ovulated at the first meiotic metaphase (MI) or the second (MII). In this study eight oocytes were collected from the preovulatory follicles of 16 mares 36 h after human chorionic gonadotropin CG treatment. Six of the eight oocytes were judged to be at MII by the presence of the first polar body and this judgement was confirmed by semithin sectioning in one. Of the two that had no polar body, one was found to be at MII after fixation for chromosomal analysis and the meiotic stage of the other remained undetermined. Since all seven oocytes yielding conclusive evidence were at MII, it was concluded that horse oocytes, like those of most mammals studied, are ovulated after completion of the first meiotic division and formation of the first polar body.     

Identification of microtubule-associated proteins in the meiotic spindle of surf clam oocytes

Meiotic spindles isolated from surf clam oocytes to morphological purity are biochemically complex, consisting of many polypeptides. These proteins fall into two classes: (a) polypeptides that are apparently cytoplasmic proteins and are not specifically associated with the spindle; and (b) polypeptides that are specifically associated with the spindle. A subset of the spindle-associated proteins, including a 250,000 mol wt component, remain with spindle tubulin through cycles of cold depolymerization and warm polymerization, showing that they are microtubule-associated proteins.     

Meiotic nondisjunction in oocytes from aged Djungarian hamsters correlates with an alteration in meiosis rate but not in univalent formation

Summary The effect of maternal ageing on the meiotic rate, on chiasma and univalent frequency as well as on heteroploidy in secondary oocytes from Djungarian hamsters was exammed. The frequency of hyperhaploid oocytes increased from 0.6% in young (8–14 weeks) to 2.8% in middle-aged (26–46 weeks) and reached 3.6% in the oldest females (49–75 weeks). On the basis of malsegregated bivalents per oocyte, nondisjunction occurred most often in the middle-aged group (5.42x10^-2 bivalents per oocyte). Hereby, the large meta- and submetacentric A-D chromosomes were preferentially involved. Furthermore, the pattern of nondisjunction was not different from that expected on the basis of chromosome length or induced by colchicine. The large A-D chromosomes did not show any alteration in chiasma or univalent frequency. Terminalized chiasmata were only detected in the E group and univalents increased slightly, but not significantly in the small chromosomes (G group). At higher ages, both chromosome group were not preferentially involved in nondisjunction. Presegregation slightly increased with age and affected more or less all bivalents, whereas the incidence of diploidy significantly decreased. With respect to the rate of meiosis in oocytes from aged females, the resumption was delayed at metaphase I. Our data suggest that failures in the control of oocyte proliferation are involved in nondisjunction rather than the production-line. Furthermore, a model is proposed to explain nondisjunction of specific bivalents at certain maternal ages.     

The chromosomal basis of meiotic acentrosomal spindle assembly and function in oocytes

Several aspects of meiosis are impacted by the absence of centrosomes in oocytes. Here, we review four aspects of meiosis I that are significantly affected by the absence of centrosomes in oocyte spindles. One, microtubules tend to assemble around the chromosomes. Two, the organization of these microtubules into a bipolar spindle is directed by the chromosomes. Three, chromosome bi-orientation and attachment to microtubules from the correct pole require modification of the mechanisms used in mitotic cells. Four, chromosome movement to the poles at anaphase cannot rely on polar anchoring of spindle microtubules by centrosomes. Overall, the chromosomes are more active participants during acentrosomal spindle assembly in oocytes, compared to mitotic and male meiotic divisions where centrosomes are present. The chromosomes are endowed with information that can direct the meiotic divisions and dictate their own behavior in oocytes. Processes beyond those known from mitosis appear to be required for their bi-orientation at meiosis I. As mitosis occurs without centrosomes in many systems other than oocytes, including all plants, the concepts discussed here may not be limited to oocytes. The study of meiosis in oocytes has revealed mechanisms that are operating in mitosis and will probably continue to do so.     

The kinesinlike protein Subito contributes to central spindle assembly and organization of the meiotic spindle in Drosophila oocytes

In the oocytes of many species, bipolar spindles form in the absence of centrosomes. Drosophila melanogaster oocyte chromosomes have a major role in nucleating microtubules, which precedes the bundling and assembly of these microtubules into a bipolar spindle. Here we present evidence that a region similar to the anaphase central spindle functions to organize acentrosomal spindles. Subito mutants are characterized by the formation of tripolar or monopolar spindles and nondisjunction of homologous chromosomes at meiosis I. Subito encodes a kinesinlike protein and associates with the meiotic central spindle, consistent with its classification in the Kinesin 6/MKLP1 family. This class of proteins is known to be required for cytokinesis, but our results suggest a new function in spindle formation. The meiotic central spindle appears during prometaphase and includes passenger complex proteins such as AurB and Incenp. Unlike mitotic cells, the passenger proteins do not associate with centromeres before anaphase. In the absence of Subito, central spindle formation is defective and AurB and Incenp fail to properly localize. We propose that Subito is required for establishing and/or maintaining the central spindle in Drosophila oocytes, and this substitutes for the role of centrosomes in organizing the bipolar spindle.     

Maternal ageing and nondisjunction: A comparative study of two chromosomal techniques on the formation of univalents in first meiotic metaphase oocytes of the mouse

The incidence of univalents was compared between slides prepared according to two clearly different chromosomal methods, i.e. Tarkowski's method and ours, in order to examine whether a univalent pair could be formed artifactually at the first meiotic metaphase (MI). The oocytes used were obtained from young (2–3 months) and old (12–15 months) age groups of both C57BL/6 and dd mice. In Tarkowski's method only a single fixative was used, while in our method three different fixatives were used successively in order to fix oocytes without their being ruptured. Despiralized, fuzzy and loosely associated chromatids were seen frequently in the slides prepared by Tarkowski's method, while such features were seen less frequently in the slides prepared by our method. The incidence of oocytes with univalents in the slides made by Tarkowski's method was much higher than in those made by ours in both age and strain groups (P<0.05–0.001). Thus, it was confirmed that the so-called univalents could be produced artifactually. The results did not support the production line hypothesis of Henderson and Edwards (1968) which was based on their observation of an increased incidence of univalents in MI oocytes from aged female mice.     

2-methoxyestradiol induces spindle aberrations, chromosome congression failure, and nondisjunction in mouse oocytes

2-Methoxyestradiol (2-ME) is a metabolite of 17beta-estradiol and a natural component of follicular fluid. Local concentrations of 2-ME may be increased by exposure to environmental pollutants that activate the expression of enzymes in the metabolic pathway from 17beta-estradiol to 2-ME. It has been suspected that this may have adverse effects on spindle formation in maturing oocytes, which would affect embryo quality. To study the dose-response patterns, we exposed denuded mouse oocytes to 2-ME during in vitro maturation. Meiotic progression, spindle morphology, centrosome integrity, and chromosome congression were examined by immunofluorescence and noninvasive polarizing microscopy (PolScope). Chromosomal constituents were assessed after spreading and C-banding. 2-ME sustained MAD2L1 expression at the centromeres and increased the number of meiosis I-blocked oocytes in a dose-dependent manner. 2-ME also caused dramatic dose-dependent increases in the hyperploidy of metaphase II oocytes. Some of these meiosis II oocytes contained anaphase I-like chromosomes, which suggests that high concentrations of the catecholestradiol interfere with the physical separation of chromosomes. Noninvasive PolScope analysis and tubulin immunofluorescence revealed that perturbations in spindle organization, which resulted in severe disturbances of the chromosome alignment at the spindle equator (congression failure), were caused by 2-ME at meiosis I and II. Pericentrin-positive centrosomes failed to align at the spindle poles, and multipolar spindles and prominent arrays of cytoplasmic microtubule asters were induced in 2-ME-exposed metaphase II oocytes. In conclusion, a micromolar level of 2-ME is aneugenic for mammalian oocytes. Therefore, exposure to 2-ME and conditions that increase the intrinsic local concentration of 2-ME in the ovary may affect fertility and increase risks for chromosomal aberrations in the oocyte and embryo.     

Astrin regulates meiotic spindle organization,spindle pole tethering and cell cycle progression in mouse oocytes

Astrin has been described as a microtubule and kinetochore protein required for the maintenance of sister chromatid cohesion and centrosome integrity in human mitosis. However, its role in mammalian oocyte meiosis is unclear. In this study, we find that Astrin is mainly associated with the meiotic spindle microtubules and concentrated on spindle poles at metaphase I and metaphase II stages. Taxol treatment and immunoprecipitation show that Astrin may interact with the centrosomal proteins Aurora-A or Plk1 to regulate microtubule organization and spindle pole integrity. Loss-of-function of Astrin by RNAi and overexpression of Tof the coiled-coil domain results in spindle disorganization, chromosome misalignment and meiosis progression arrestT. Thr24, Ser66 or Ser447 may be the potential phosphorylated sites of Astrin by Plk1, as site-directed mutation of these sites causes oocyte meiotic arrest at HTmetaphaseTH I with highly disordered spindles and disorganized chromosomes, although mutant Astrin localizes to the spindle apparatus. Taken together, these data strongly suggest that Astrin is critical for meiotic spindle assembly and maturation in mouse oocytes.     

BRCA1 is required for meiotic spindle assembly and spindle assembly checkpoint activation in mouse oocytes

BRCA1 as a tumor suppressor has been widely investigated in mitosis, but its functions in meiosis are unclear. In the present study, we examined the expression, localization, and function of BRCA1 during mouse oocyte meiotic maturation. We found that expression level of BRCA1 was increased progressively from germinal vesicle to metaphase I stage, and then remained stable until metaphase II stage. Immunofluorescent analysis showed that BRCA1 was localized to the spindle poles at metaphase I and metaphase II stages, colocalizing with centrosomal protein gamma-tubulin. Taxol treatment resulted in the presence of BRCA1 onto the spindle microtubule fibers, whereas nocodazole treatment induced the localization of BRCA1 onto the chromosomes. Depletion of BRCA1 by both antibody injection and siRNA injection caused severely impaired spindles and misaligned chromosomes. Furthermore, BRCA1-depleted oocytes could not arrest at the metaphase I in the presence of low-dose nocodazole, suggesting that the spindle checkpoint is defective. Also, in BRCA1-depleted oocytes, gamma-tubulin dissociated from spindle poles and MAD2L1 failed to rebind to the kinetochores when exposed to nocodazole at metaphase I stage. Collectively, these data indicate that BRCA1 regulates not only meiotic spindle assembly, but also spindle assembly checkpoint, implying a link between BRCA1 deficiency and aneuploid embryos.     

Nuclear and cytoplasmic maturation of sow oocytes are not synchronized by specific meiotic inhibition with roscovitine during in vitro maturation

The effect of roscovitine exposure prior to IVM was studied on cumulus expansion, on changes of cumulus-oocyte contacts and on nuclear and cytoplasmic maturation of sow oocytes. It was hypothesized that delayed nuclear maturation and prolonged contact with cumulus cells allows prolonged cytoplasmic differentiation and therefore improves oocyte developmental potential. Cumulus-oocyte complexes (COCs) were exposed for 22 h or 44 h to 0, 25 or 50 microM of roscovitine and subsequently cultured for 22, 29 or 44 h without roscovitine. COCs were examined for cumulus expansion and oocytes for nuclear status and dynamics of transzonal microfilaments. Oocyte developmental potential was assessed by blastocyst formation after IVF. Fifty muM of roscovitine inhibited cumulus expansion for the first 22 h of culture, and maintained oocytes in meiotic arrest for 44 h. Roscovitine treatment during 22 h prior to culture for 44 h without roscovitine did not increase embryo development, but oocytes cultured for 66 h without roscovitine had reduced blastocyst formation. Oocytes cultured for 29 h after roscovitine exposure showed reduced blastocyst rates compared with their counterparts cultured for 44 h. Roscovitine treatment during 44 h prior to culture for 22 h or 44 h without roscovitine reduced embryo development. Transzonal microfilaments were reduced after culture with roscovitine, and disappeared during culture without roscovitine. It is concluded that prolonged contact with cumulus cells does not improve oocyte developmental potential. Furthermore, it is suggested that nuclear and cytoplasmic maturation in vitro cannot be seen as two independent processes.     

Formin-2, polyploidy,hypofertility and positioning of the meiotic spindle in mouse oocytes

Successful reproduction in mammals requires a competent egg, which is formed during meiosis through two assymetrical cell divisions. Here, we show that a recently identified formin homology (FH) gene, formin-2 (Fmn2), is a maternal-effect gene that is expressed in oocytes and is required for progression through metaphase of meiosis I. Fmn2(-/-) oocytes cannot correctly position the metaphase spindle during meiosis I and form the first polar body. We demonstrate that Fmn2 is required for microtubule-independent chromatin positioning during metaphase I. Fertilization of Fmn2(-/-) oocytes results in polyploid embryo formation, recurrent pregnancy loss and sub-fertility in Fmn2(-/-) females. Injection of Fmn2 mRNA into Fmn2-deficient oocytes rescues the metaphase I block. Given that errors in meiotic maturation result in severe birth defects and are the most common cause of chromosomal aneuploidy and pregnancy loss in humans, studies of Fmn2 may provide a better understanding of infertility and birth defects.