Topoisomerase II Is Required for the Proper Separation of Heterochromatic Regions during Drosophila melanogaster Female Meiosis

Heterochromatic homology ensures the segregation of achiasmate chromosomes during meiosis I in Drosophila melanogaster females, perhaps as a consequence of the heterochromatic threads that connect achiasmate homologs during prometaphase I. Here, we ask how these threads, and other possible heterochromatic entanglements, are resolved prior to anaphase I. We show that the knockdown of Topoisomerase II (Top2) by RNAi in the later stages of meiosis results in a specific defect in the separation of heterochromatic regions after spindle assembly. In Top2 RNAi-expressing oocytes, heterochromatic regions of both achiasmate and chiasmate chromosomes often failed to separate during prometaphase I and metaphase I. Heterochromatic regions were stretched into long, abnormal projections with centromeres localizing near the tips of the projections in some oocytes. Despite these anomalies, we observed bipolar spindles in most Top2 RNAi-expressing oocytes, although the obligately achiasmate 4^th chromosomes exhibited a near complete failure to move toward the spindle poles during prometaphase I. Both achiasmate and chiasmate chromosomes displayed defects in biorientation. Given that euchromatic regions separate much earlier in prophase, no defects were expected or observed in the ability of euchromatic regions to separate during late prophase upon knockdown of Top2 at mid-prophase. Finally, embryos from Top2 RNAi-expressing females frequently failed to initiate mitotic divisions. These data suggest both that Topoisomerase II is involved in the resolution of heterochromatic DNA entanglements during meiosis I and that these entanglements must be resolved in order to complete meiosis.     

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.     

Induction of DNA replication in the germinal vesicle of the growing mouse oocyte

Growing mouse oocytes are physiologically arrested in the G2 phase of prophase of the first meiotic division. Growing oocytes were isolated from ovaries of 9- to 12-day-old mice and fused with parthenogenetic one-cell eggs or two-cell embryos derived from fertilized eggs. Resulting hybrids were injected with Dig-11-dUTP and examined for DNA replication using immunofluorescence. Parthenogenetic one-cell eggs fused at telophase II, G1, and middle-to-late S phase, and also S-phase two-cell blastomeres, were able to trigger DNA synthesis in oocyte germinal vesicle (GV) in the majority of hybrids cultured to the end of the first cell cycle. Activation of replication in the GV occurred within 2-3 h after fusion of growing oocytes with S-phase eggs. We show indirectly that the reactivation of replication in GVs was not dependent on the breakdown of the GV envelope. Although GVs had the ability to renew DNA replication after fusion, the G2 blastomere nuclei were incapable of reinitiating DNA replication under the influence of S-phase one-cell eggs. We hypothesize that the nuclei of growing oocytes arrested in meiotic prophase are in a physiological state that is equivalent to replication-competent G1, and not G2, nuclei.     

Origin of the mitotic spindle in onion root cells

Summary Immunofluorescence studies on microtubule arrangement during the transition from prophase to metaphase in onion root cells are presented. The prophase spindle observed at late preprophase and prophase is composed of microtubules converged at two poles near the nuclear envelope; thin bundles of microtubules are tracable along the nuclear envelope. Prior to nuclear envelope breakdown diffuse tubulin staining occurs within the prophase nuclei. During nuclear envelope breakdown the prophase spindle is no longer identifiable and prominent tubulin staining occurs among the prometaphase chromosomes. Patches of condensed tubulin staining are observed in the vicinity of kinetochores. At advanced prometaphase kinetochore bundles of microtubules are present in some kinetochore regions. At metaphase the mitotic spindle is mainly composed of kinetochore bundles of microtubules; pole-to-pole bundles are scarce. Our observations suggest that the prophase spindle is decomposed at the time of nuclear envelope breakdown and that the metaphase spindle is assembled at prometaphase, with the help of kinetochore nucleating action.     

α-endosulfine (ENSA) regulates exit from prophase I arrest in mouse oocytes

Mammalian oocytes in ovarian follicles are arrested in meiosis at prophase I. This arrest is maintained until ovulation, upon which the oocyte exits from this arrest, progresses through meiosis I and to metaphase of meiosis II. The progression from prophase I to metaphase II, known as meiotic maturation, is mediated by signals that coordinate these transitions in the life of the oocyte. ENSA (α-endosulfine) and ARPP19 (cAMP-regulated phosphoprotein-19) have emerged as regulators of M-phase, with function in inhibition of protein phosphatase 2A (PP2A) activity. Inhibition of PP2A maintains the phosphorylated state of CDK1 substrates, thus allowing progression into and/or maintenance of an M-phase state. We show here ENSA in mouse oocytes plays a key role in the progression from prophase I arrest into M-phase of meiosis I. The majority of ENSA-deficient oocytes fail to exit from prophase I arrest. This function of ENSA in oocytes is dependent on PP2A, and specifically on the regulatory subunit PPP2R2D (also known as B55δ). Treatment of ENSA-deficient oocytes with Okadaic acid to inhibit PP2A rescues the defect in meiotic progression, with Okadaic acid-treated, ENSA-deficient oocytes being able to exit from prophase I arrest. Similarly, oocytes deficient in both ENSA and PPP2R2D are able to exit from prophase I arrest to an extent similar to wild-type oocytes. These data are evidence of a role for ENSA in regulating meiotic maturation in mammalian oocytes, and also have potential relevance to human oocyte biology, as mouse and human have genes encoding both Arpp19 and Ensa.     

Chromosome aberrations in metaphase II-oocytes. Stage sensitivity in the mouse oogenesis to amethopterin and cyclophosphamide

The stage sensitivity in oogenesis of C₃H mice was investigated by transplacental treatment of embryonic oogonia and oocytes at meiotic prophase I. After birth the stages of early and late dictyotene as well as the preovulatory and ovulatory phases were treated. Chromosome analysis was performed in unfertilized metaphase II-oocytes after induced ovulation [pregnant mare's serum (PMS) and human chorionic gonadotrophin (HCG)]. As test compounds both the folic acid antagonist amethopterin (M) and the alkylating agent cyclophosphamide (C) were used.Embryonic oogonia as well as the preovulatory phase of oogenesis proved to be most sensitive for the induction of chromosomal aberrations. The investigation with graded doses during the preovulatory stage demonstrated the dose-dependent frequency of the induced types of chromosomal abnormalities.The high sensitivity of these stages where chromosome segregation takes place, e.g. oogonia, preovulatory stage, seems to be related to an additional induction of aneuploidies.     

Comparative analysis of meiotic progression in female mice bearing mutations in genes of the DNA mismatch repair pathway

The DNA mismatch repair (MMR) family functions in a variety of contexts to preserve genome integrity in most eukaryotes. In particular, members of the MMR family are involved in the process of meiotic recombination in germ cells. MMR gene mutations in mice result in meiotic disruption during prophase I, but the extent of this disruption often differs between male and female meiocytes. To address the role of MMR proteins specifically in female meiosis, we explored the progression of oocytes through prophase I and the meiotic divisions in mice harboring deletions in members of the MMR pathway (Mlh1, Mlh3, Exo1, and an ATPase-deficient variant of Mlh1, Mlh1(G67R)). The colocalization of MLH1 and MLH3, key proteins involved in stabilization of nascent crossovers, was dependent on intact heterodimer formation and was highly correlated with the ability of oocytes to progress through to metaphase II. The exception was Exo1(-/-) oocytes, in which normal MLH1/MLH3 localization was observed followed by failure to proceed to metaphase II. All mutant oocytes were able to resume meiosis after dictyate arrest, but they showed a dramatic decline in chiasmata (to less than 25% of normal), accompanied by varied progression through metaphase I. Taken together, these results demonstrate that MMR function is required for the formation and stabilization of crossovers in mammalian oocytes and that, in the absence of a functional MMR system, the failure to maintain chiasmata results in a reduced ability to proceed normally through the first and second meiotic divisions, despite near-normal levels of meiotic resumption after dictyate arrest.     

Congression of achiasmate chromosomes to the metaphase plate in Drosophila melanogaster oocytes

Chiasmata established by recombination are normally sufficient to ensure accurate chromosome segregation during meiosis by physically interlocking homologs until anaphase I. Drosophila melanogaster female meiosis is unusual in that it is both exceptionally tolerant of nonexchange chromosomes and competent in ensuring their proper segregation. As first noted by Puro and Nokkala [Puro, J., Nokkala, S., 1977. Meiotic segregation of chromosomes in Drosophila melanogaster oocytes. A cytological approach. Chromosoma 63, 273-286], nonexchange chromosomes move precociously towards the poles following formation of a bipolar spindle. Indeed, metaphase arrest has been previously defined as the stage at which nonexchange homologs are symmetrically positioned between the main chromosome mass and the poles of the spindle. Here we use studies of both fixed images and living oocytes to show that the stage in which achiasmate chromosomes are separated from the main mass does not in fact define metaphase arrest, but rather is a component of an extended prometaphase. At the end of prometaphase, the nonexchange chromosomes retract into the main chromosome mass, which is tightly repackaged with properly co-oriented centromeres. This repackaged state is the true metaphase arrest configuration in Drosophila female meiosis.     

Role of germinal vesicle on protein synthesis in rat oocyte during in vitro maturation

To investigate the role of the germinal vesicle (GV) on in vitro maturation (IVM) of rat oocytes, we examined protein synthesis during IVM by comparing polypeptide patterns in control and enucleated oocytes using one and two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Separation of polypeptides extracted from the cytoplasm of GV by one-dimensional SDS-PAGE revealed that a 55 kDa polypeptide was present only in the GVs of rat oocytes. At 0, 12, 24, 36, and 44 hr after PMSG injection, prior to the initiation of maturation, enucleated oocytes synthesized the same major polypeptides as cumulus intact (CI) oocytes. During meiotic maturation, no major changes were detected in protein synthesis from prophase (GV stage) to prometaphase I (0–6 hr IVM). However, after entry into prometaphase I (7 hr IVM), striking changes were seen; a 24 kDa polypeptide disappeared and expression of a 34 kDa polypeptide became stronger. This pattern lasted until metaphase II. We detected no major differences in the pattern of protein synthesis between CI and enucleated oocytes using two-dimensional PAGE. These results indicate that protein synthesis in the maturing rat oocyte is controlled by cytoplasmic regulators rather than intrinsic nuclear components. © 1996 Wiley-Liss, Inc.     

Light and electron microscopy of Rat kangaroo cells in mitosis

Rat kangaroo (PtK₂) cells were fixed and embedded in situ. Cells in mitosis were studied with the light microscope and thin sections examined with the electron microscope. Pericentriolar, osmiophilic material, rather than the centrioles, is probably involved in the formation of astral microtubules during prophase. Centriole migration occurs during prophase and early prometaphase. The nuclear envelope ruptures first in the vicinity of the asters. Nuclear pore complexes disintegrate as envelope fragments are dispersed to the periphery of the mitotic spindle. Microtubules invade the nucleus through gaps of the fragmented envelope. The number of microtubules and the degree of spindle organization increase during prometaphase and are maximal at metaphase. At this stage, chromosomes are aligned on the spindle equator, sister kinetochores facing opposite poles. Cytoplasmic organelles are excluded from the spindle. Prominent bundles of kinetochore microtubules converge towards the poles. Spindles in cold-treated cells consist almost exclusively of kinetochore tubules. Separating daughter chromosomes in early anaphase are connected by chromatin strands, possibly reflecting the rupturing of fibrous connections occasionally observed between sister chromatids in prometaphase. Breakdown of the spindle progresses from late anaphase to telophase, except for the stem bodies. Chromosomes decondense to form two masses. Nuclear envelope reconstruction, probably involving endoplasmic reticulum, begins on the lateral faces. Nuclear pores reappear on membrane segments in contact with chromatin. Microtubules are absent from reconstructed daughter nuclei.This report is to a large part based on a dissertation submitted by the author to the Graduate Council of the University of Florida in partial fulfillment of the requirements for the degree of Doctor of Philosophy.     

The mitotic apparatus during unequal microspore division observed by a confocal laser scanning microscope

Summary The structure of the mitotic apparatus during the microspore division ofTradescantia paludosa, which has a distinctively unequal division of large vegetative and small generative cells, was studied using -tubulin immunofluorescence methods and confocal laser scanning microscopy. Mitotic apparatuses began to develop asynchronously during early prophase at the vegetative pole (VP) and during prometaphase at the generative pole (GP). Both, however, reached completion together at the same time during metaphase. At the VP from prophase to prometaphase, microtubules (MTs) did not converge on the pole, and there was a circular area containing only a few MTs. The prophase spindles on the VP side were in the form of domes or cones that lacked the top. In the metaphase, however, the MTs concentrated at the pole to form a representative cone-shaped half-spindle. At the GP from prometaphase to metaphase, the MTs did not concentrate, and a circular area existed that lacked MTs. The half-spindles formed truncated cones. When the phragmoplast developed and curved around the generative nucleus during the telophase. it first grew toward the long axis of the ellipsoidal-shaped microspore; and after it arrived at the inner membrane of the microspore, it again curved past the generative nucleus toward the short axis. In conclusion, it was found that the mitotic apparatus ofT. paludosa microspores with its asynchronous growth and asymmetrical spindle structure and with its three dimensional growth of phragmoplasts had a peculiar developmental manner related to unequal division.     

Ecdysteroids and meiotic reinitiation in Palaemon serratus (Crustacea Decapoda Natantia) and in Locusta migratoria (Insecta orthoptera). A comparative study

Summary

Meiotic reinitiation has been studied in Locusta migratoria and Palaemon serratus in relation to the titre of free ecdysteroids present in the maturing oocyte. In both species meiotic reinitiation is characterized by two meiotic arrests, in prophase I and in metaphase I, and the first meiotic resumption which leads to germinal vesicle breakdown (GVBD) is correlated with increasing titres of ecdysteroids in the oocyte. Meiotic reinitiation has been successfully triggered in the oocytes of both species by incubation with physiological doses of ecdysteroids.     

Localization of anti-topoisomerase IIα antibodies under normal conditions and after artificial chromosome condensation and decondensation

By means of immunofluorescence method, localization of DNA-topoisomerase IIα (Topo IIα) in interphase nuclei and chromosomes at different stages of mitosis was studied in situ under normal conditions and after treatment with condensing and decondensing solutions. In non-isolated mitotic M-HeLa cell chromosomes, Topo IIα was uniformly distributed along chromatids after fixation and permeabilization in situ. After treatment of cells with decondensing solutions (10 mM Tris; 0.1 mM CaCl₂ in 10 mM Tris; 0.3 mM CaCl₂ in 10 mM Tris; 15% DMEM; 75 mM KCl), Topo IIα was evenly distributed along chromatids in prophase, prometaphase and metaphase; its concentration was the highest in the pericentromere region. After treatment of cells with condensing solutions containing 0.7 mM, 1 mM, 2 mM or 3 mM CaCl₂ in 10 mM Tris, Topo IIα was not detected in prophase, metaphase and anaphase. However, in late telophase anti-Topo IIα antibodies were found in reforming nuclei under identical conditions. After sequential treatment with condensing and decondensing solutions, the distribution patterns of Topo IIα in chromosomes were the same as after treatment with only decondensing solutions. In anaphase and telophase, Topo IIα was evenly distributed along chromatids, while in prophase, prometaphase and metaphase it was predominantly localized in the pericentromere region. After the treatment of cells with condensing solutions chromosome staining was not observed, apparently due to “masking” of binding sites for anti-Topo IIα antibodies. Homogenous distribution of Topo IIα along chromatids in non-isolated chromosomes was preserved after the treatment of cells with hypotonic solutions; however, under these conditions Topo IIα concentration was higher in centromeres.     

Involvement of ecdysone in the control of meiotic reinitiation in oocytes of Locusta migratoria (Insecta,orthoptera)

Following their biosynthesis in the follicle cells of vitellogenic ovaries, large amounts of ecdysteroids pass into the oocytes where they accumulate and persist during ovulation and egg-laying. The present paper shows that free ecdysone is unevenly distributed in the oocytes exhibiting the highest concentrations in the region of the posterior pole where the final sequences of nuclear maturation, including germinal vesicle breakdown (GVBD), occur. A correlative study indicates that the concentrations of free ecdysone in this region are particularly high (10 to 20 μM) during two periods of meiotic reinitiation observed in the oocytes: reinitiation I, leading from prophase I to metaphase I with GVBD; and reinitiation II, from metaphase I to the end of meiosis. In vitro incubations of oocytes in meiotic arrest (prophase I) in the presence of exogenous ecdysone demonstrate that complete reinitiation (including GVBD) can be triggered in a dose-dependent manner by this hormone.     

A combination of FSH and dibutyryl cyclic AMP promote growth and acquisition of meiotic competence of oocytes from early porcine antral follicles

Growing porcine oocytes from early antral follicles (1.2-1.5 mm in diameter) do not mature to metaphase II (MII, 4%) under culture conditions which supported maturation (MII, 95%) of fully grown oocytes from large (4-6 mm) antral follicles. We hypothesized that FSH and dbcAMP supported growth and acquisition of meiotic competence. Growing oocytes (113.0 ± 0.4 μm, mean ± SEM) were cultured for 5 d in medium supplemented with 1 mM dbcAMP, 0.01 IU/mL FSH or both; in these media, oocytes reached, 120.5 ± 0.4, 123.5 ± 0.4 and 125.7 ± 0.2 μm, respectively, after 5 d, and then were matured in vitro for 48 h. Oocytes remained enclosed by cumulus cells when cultured with FSH (82%) or both FSH and dbcAMP (80%), but not with dbcAMP alone (0%). Furthermore, oocytes cultured with FSH maintained trans-zonal projections of cumulus cells. Oocytes remained at the GV stage at higher rates when cultured with dbcAMP and FSH (99%), or dbcAMP (97%), than with FSH (64%), or without either (75%). Following in vitro maturation, oocytes reached MII after in vitro growth with dbcAMP (19%), FSH (11%), or both (68%). When oocytes were cultured with both FSH and dbcAMP, activation of Cdc2 and MAP kinases in growing oocytes was similar to fully grown oocytes. In conclusion, growing porcine oocytes grew and acquired meiotic competence in medium supplemented with dbcAMP and FSH; the former maintained oocytes in meiotic arrest, whereas the latter maintained trans-zonal projections of cumulus cells to oocytes during in vitro growth culture.     

Chromatin behaviour under influence of puromycin and 6-DMAP at different stages of mouse oocyte maturation

Preovulatory mouse oocytes were cultured in vitro up to each subsequent stages of maturation: germinal vesicle (GV), germinal vesicle breakdown (GVBD), groups of not yet individualized bivalents, circular bivalents, late prometaphase I, metaphase I, anaphase I and telophase I. The stages were identified in living oocytes by fluorescence microscopy using Hoechst 33342 as a specific vital dye. Oocytes from each stage of development developed in vitro and ovulated metaphase II oocytes were subsequently cultured in the presence of puromycin or 6-dimethylaminopurine (6-DMAP), an inhibitor of protein phosphorylation. The effects on chromatin of these drugs were studied during and at the end of culture by fluorescence and electron microscopy. We found that puromycin and 6-DMAP stop meiosis when applied at all stages of oocyte maturation, except for metaphase II. Oocytes at this stage are activated by puromycin. Reaction of the oocytes to the two drugs is different at GV and at metaphase II. All of the other stages react to the drugs by chromatin compaction, which can be followed by chromatin decondensation to form a nucleus. Our results suggest that late prophase chromatin condensation, bivalent individualization and retention of their individuality, as well as individualization of monovalents from telophase and retention of their individuality at metaphase II, are dependent on protein phosphorylation. The events occurring between metaphase I and telophase I are independent of protein synthesis and phosphorylation. The events occurring between metaphase II and formation of the nucleus are independent of protein synthesis.by U. Scheer     

Behaviour of the X chromosomes during growth and maturation of parthenogenetic eggs of Amphorophora tuberculata (Homoptera,Aphididae), in relation to sex determination

Prophase chromosomes of growing oocytes from thelytokous, viviparous females of Amphorophora tuberculata Brown and Blackman (n=2) were studied using a modified propionic acid squash technique with Feulgen staining. In early prophase, prior to the growth phase of the oocyte, the X chromosomes are partially condensed and looped together so that all four ends appear to be associated. Later in prophase the X chromosomes separate in oocytes destined to be female, but remain associated in presumptive male oocytes. The autosomes condense gradually throughout prophase. The nucleus of the presumptive male oocyte is further characterised by the formation of a spherical Feulgen-positive body, which attains a large size (7 m diameter) in late prophase. At this stage, the X chromosomes are no longer visible as separate entities, and are apparently included in the spherical body. At metaphase this disappears, leaving the X chromosomes still united as a condensed bivalent. The spherical body seems to have nucleolar as well as chromatin constituents; nucleolar organisers are present at the ends of the X chromosomes where it first arises. It may function in maintaining the cohesion between the X chromosomes through prophase, and could also facilitate correct orientation of the X bivalent on the spindle of the maturation division. As sex determination in aphids is controlled by juvenile hormone concentration, it appears that the hormone may interact with the X chromosomes during prophase, bringing about their separation in female oocytes, perhaps by inhibiting the formation of the spherical body.     

Light and electron microscopy of rat kangaroo cells in mitosis

Kinetochores in rat kangaroo (PtK₂) cells in prophase of mitosis are finely fibrillar, globular bodies, 5000–8000 Å in diameter. Sister kinetochores are attached to opposite lateral faces in the primary constriction of chromosomes. No microtubules (MTs) occur in prophase nuclei. During prometaphase the ball-shaped kinetochores differentiate into trilaminar plaques. An outer kinetochore layer, less electron dense than chromatin, appears first in the fibrillar matrix. The inner layer, continuous with, but more electron dense than the chromosome, is formed later. Kinetochore-spindle MT interaction is evident at the very beginning of prometaphase. As a result, kinetochore shape is very variable, but three types of kinetochores can be distinguished by fine structure analysis. A comparison of kinetochore structure and chromosome position in the mitotic spindle yielded clues regarding initial orientation and congression. At the time the nuclear envelope (NE) breaks down chromosomes near asters orient first. Chromosomes approximately equidistant from the two spindle poles amphi-orient immediately. Chromosomes closer to one pole probably achieve mono-orientation first, then amphi-orient and congress. In normal metaphase all the chromosomes lie at or near the spindle equator and kinetochores are structurally uniform. Paraxial and para-equatorial sections revealed that they are trilaminar, roughly circular plaques of 4000–6000 Å diameter. Inner and outer layers are 400 Å, and the electron translucent middle layer which separates them is 270 Å thick. From 16 to 40 MTs are anchored in the outer layer. In cold-treated cells the kinetochores are trilaminar, but in colcemid-treated cells the inner layer is lacking. Both kinetochores and their MTs are disorganized beginning in late anaphase. In telophase the inner layer persists for some time as an electron dense patch apposed to the NE, while the outer layer disintegrates.     

Steroid hormones promote bovine oocyte growth and connection with granulosa cells

Many approaches have been investigated for growing oocytes in vitro in mammals. To support oocyte growth in vitro, the culture systems must meet certain conditions for maintaining connections between oocytes and surrounding granulosa cells. The aims of this study were to determine the effects of combinations of 17β-estradiol (E₂) and androstenedione (A₄) on in vitro growth of bovine oocytes and to determine the number of connections between the oocyte and granulosa cells. Oocyte–granulosa cell complexes (OGCs) collected from early antral follicles (0.4−0.7 mm in diameter) were cultured for 14 days in a medium with different concentrations of E₂ and A₄, either alone or in combinations. We then assessed the number of transzonal projections (TZPs), which extend from granulosa cells through the zona pellucida to the oolemma. During in vitro growth culture, OGC structures were maintained in the medium with steroid hormones. The mean diameter of oocytes grown in the medium with both E₂ and A₄ was increased from 95.8 μm to around 120 μm, larger than oocytes grown without steroid hormones (109.9 μm) and similar in size to in vivo fully grown oocytes (119.4 μm) from 4- to 6-mm antral follicles. In subsequent in vitro maturation culture (22 hours), 30% (12 of 40) and 34% (14 of 41) of oocytes grown with E₂ or A₄ alone, respectively, matured to metaphase II; meanwhile, oocytes grown with a combination of E₂ and A₄ matured to metaphase II at a high rate (58%, 23 of 40). Growing oocytes isolated from early antral follicles had many uniformly distributed TZPs throughout the zona pellucida. After 14 days of culture, there was a significant decrease in the number of TZPs in oocytes grown without steroid hormones, whereas the number of TZPs was maintained in oocytes grown with steroid hormones. In particular, oocytes grown with E₂ alone or with a combination of E₂ and A₄ had numbers of TZPs similar to oocytes before growth culture. In conclusion, a combination of E₂ and A₄ maintained the connections between oocytes and granulosa cells during in vitro growth culture of bovine oocytes for 14 days, resulting in the complete oocyte growth and the acquisition of meiotic competence in more than half the oocytes.     

Microinjected centromere [corrected] kinetochore antibodies interfere with chromosome movement in meiotic and mitotic mouse oocytes [published erratum appears in J Cell Biol 1990 Dec;111(6 Pt 1):following 2800]

Kinetochores may perform several functions at mitosis and meiosis including: (a) directing anaphase chromosome separation, (b) regulating prometaphase alignment of the chromosomes at the spindle equator (congression), and/or (c) capturing and stabilizing microtubules. To explore these functions in vivo, autoimmune sera against the centromere/kinetochore complex are microinjected into mouse oocytes during specific phases of first or second meiosis, or first mitosis. Serum E.K. crossreacts with an 80-kD protein in mouse cells and detects the centromere/kinetochore complex in permeabilized cells or when microinjected into living oocytes. Chromosome separation at anaphase is not blocked when these antibodies are microinjected into unfertilized oocytes naturally arrested at second meiotic metaphase, into eggs at first mitotic metaphase, or into immature oocytes at first meiotic metaphase. Microtubule capture and spindle reformation occur normally in microinjected unfertilized oocytes recovering from cold or microtubule disrupting drugs; the chromosomes segregate correctly after parthenogenetic activation. Prometaphase congression is dramatically influenced when antikinetochore/centromere antibodies are introduced during interphase or in prometaphase-stage meiotic or mitotic eggs. At metaphase, these oocytes have unaligned chromosomes scattered throughout the spindle with several remaining at the poles; anaphase is aberrant and, after division, karyomeres are found in the polar body and oocyte or daughter blastomeres. Neither nonimmune sera, diffuse scleroderma sera, nor sham microinjections affect either meiosis or mitosis. These results suggest that antikinetochore/centromere antibodies produced by CREST patients interfere with chromosome congression at prometaphase in vivo.