Microtubule and microfilament organization in maturing human oocytes

Various stages of immature human oocytes were imaged for microtubule, microfilament and chromatin organization. After germinal vesicle breakdown, a small microtubule aster was observed near the condensed chromatin. The asters appeared to elongate and encompass the condensed chromatin. At metaphase I stage, microtubules were detected in the meiotic spindle. The meiotic spindle in metaphase II was a symmetric, barrel-shaped structure containing anastral broad poles, located peripherally and radially oriented. After germinal vesicle breakdown, treatment with taxol induced numerous cytoplasmic foci of microtubules, mainly in the cortex of the oocyte. Microfilaments were observed as a relatively thick uniform area around the cell cortex and were also found near the germinal vesicle position. After germinal vesicle breakdown, the microfilaments were seen in both the cortex and around the female chromatin. In conclusion, this study suggests that both microtubules and microfilaments are closely associated with the reconstruction and proper positioning of chromatin after germinal vesicle breakdown and during meiotic maturation in human oocytes.     

Differential expression and functions of cortical myosin IIA and IIB isotypes during meiotic maturation, fertilization, and mitosis in mouse oocytes and embryos

To explore the role of nonmuscle myosin II isoforms during mouse gametogenesis, fertilization, and early development, localization and microinjection studies were performed using monospecific antibodies to myosin IIA and IIB isotypes. Each myosin II antibody recognizes a 205-kDa protein in oocytes, but not mature sperm. Myosin IIA and IIB demonstrate differential expression during meiotic maturation and following fertilization: only the IIA isoform detects metaphase spindles or accumulates in the mitotic cleavage furrow. In the unfertilized oocyte, both myosin isoforms are polarized in the cortex directly overlying the metaphase-arrested second meiotic spindle. Cortical polarization is altered after spindle disassembly with Colcemid: the scattered meiotic chromosomes initiate myosin IIA and microfilament assemble in the vicinity of each chromosome mass. During sperm incorporation, both myosin II isotypes concentrate in the second polar body cleavage furrow and the sperm incorporation cone. In functional experiments, the microinjection of myosin IIA antibody disrupts meiotic maturation to metaphase II arrest, probably through depletion of spindle-associated myosin IIA protein and antibody binding to chromosome surfaces. Conversely, the microinjection of myosin IIB antibody blocks microfilament-directed chromosome scattering in Colcemid-treated mature oocytes, suggesting a role in mediating chromosome-cortical actomyosin interactions. Neither myosin II antibody, alone or coinjected, blocks second polar body formation, in vitro fertilization, or cytokinesis. Finally, microinjection of a nonphosphorylatable 20-kDa regulatory myosin light chain specifically blocks sperm incorporation cone disassembly and impedes cell cycle progression, suggesting that interference with myosin II phosphorylation influences fertilization. Thus, conventional myosins break cortical symmetry in oocytes by participating in eccentric meiotic spindle positioning, sperm incorporation cone dynamics, and cytokinesis. Although murine sperm do not express myosin II, different myosin II isotypes may have distinct roles during early embryonic development.     

Mitosis in the human embryo: the vital role of the sperm centrosome (centriole)

The pattern of sperm centrosomal (centriolar) inheritance, centrosomal replication and perpetuation during mitosis of the human embryo is reviewed with a series of electron micrographs. Embryonic cleavage involves repeated mitoses, a convenient sequence to study centriolar behaviour during cell division. After the paternal inheritance of centrioles in the human was reported (Sathananthan et al., 1991a), there has been an upsurge of centrosomal research in mammals, which largely follow the human pattern. The human egg has an inactive non-functional centrosome. The paternal centrosome contains a prominent centriole (proximal) associated with pericentriolar material which is transmitted to the embryo at fertilization and persists during sperm incorporation. Centriolar duplication occurs at the pronuclear stage (interphase) and the centrosome initially organizes a sperm aster when male and female pronuclei breakdown (prometaphase). The astral centrosome containing diplosomes (two typical centrioles) splits and relocates at opposite poles of a bipolar spindle to establish bipolarization, a prerequisite to normal cell division. Single or double centrioles occupy pivotal positions on spindle poles and paternal and maternal chromosomes organize on the equator of a metaphase spindle, at syngamy. Bipolarization occurs in all monospermic and in most dispermic ova. Dispermic embryos occasionally form two sperm asters initially and produce tripolar spindles (tripolarization). Anaphase and telophase follows producing two or three cells respectively, completing the first cell cycle. Descendants of the sperm centriole were found at every stage of perimplantation embryo development and were traced from fertilization through cleavage (first four cell cycles) to the morula and hatching blastocyst stage. Centrioles were associated with nuclei at interphase, when they were often replicating and occupied pivotal positions on spindle poles during mitosis. Sperm remnants were associated with centrioles and were found at most stages of cleavage. Centrioles were found in trophoblast, embryoblast and endoderm cells in hatching blastocysts. Pericentriolar, centrosomal material nucleated astral and spindle microtubules. Abnormal nuclear configurations observed in embryos reflect mitotic aberrations. The bovine embryo closely resembles the human embryo in centriolar behaviour during mitosis. It is concluded that the sperm centrosome is the functional active centrosome in humans and is likely the ancestor of centrioles within centrosomes in foetal and adult somatic cells. The role of the sperm centrosome in embryogenesis and male infertility is discussed, since it is of clinical importance in assisted reproduction.     

Effect of ultrasound on the contraction of isolated myocardial cells of adult rats

Microtubules and microfilaments are major cytoskeletal elements in mammalian ova and are important modulators of many fertilization and post-fertilization events. In this study, the integrated distribution of microtubules and microfilaments in pig oocytes were examined under a laser scanning confocal microscope, and the requirements of their assembly during in vitro fertilization and parthenogenesis in in vitro matured pig oocytes were determined. After sperm penetration, an aster of microtubules was produced in the spermatozoon, and this microtubule aster filled the whole cytoplasm during pronuclear movement. During pronuclear formation after activation by insemination, microfilaments became concentrated at the male and female pronuclei and, after electrical stimulation, at the female pronucleus. At metaphase of cleavage, microtubules were detected in the spindle and microfilaments were found mainly in the cortex. At anaphase, microtubule asters assembled at each spindle pole. During cleavage, large asters filled each daughter blastomere and a microfilament-rich cleavage furrow was observed. Cytochalasin B, a microfilament inhibitor, inhibited microfilament polymerization but affected neither pronuclear formation nor movement. However, syngamy and cell division were inhibited in eggs treated with cytochalasin B. Treatment with nocodazole after sperm penetration inhibited microtubule assembly and prevented migration leading to pronuclear union and cell division. These results indicate that microtubule and microfilament assembly in pig oocytes are integrated during fertilization and are required for the union of sperm and egg nuclei and for subsequent cell division.     

Chromatin decondensation, pronucleus formation, metaphase entry and chromosome complements of human spermatozoa after intracytoplasmic sperm injection into hamster oocytes

Obtaining karyotypes from human spermatozoa after microinjection into Syrian golden hamster oocytes is difficult and the hitherto reported results are unsatisfactory. This may be related to the injection and culture technique or to the high susceptibility of the hamster oocytes to undergo parthenogenetic activation or both. Therefore, we investigated the hamster oocyte-human sperm microinjection model using the following two approaches: (i) application of contemporary techniques for injection (touching the sperm tail) and culture (hamster embryo culture medium, HECM-3, 10% CO2) and (ii) omission of Ca2+ from the injection medium. Thus, in the first series of experiments, 252 hamster oocytes were injected with human spermatozoa. Among the 219 (87%) oocytes that survived the injection procedure, the mean percentages of male pronucleus formation [two pronuclei (2PN), two polar bodies (PB)], mitotic metaphase entry and sperm chromosome spreads were 41.4, 27.8 and 18.2% respectively. Analysis of the oocytes which failed to develop the male pronucleus following injection revealed that most of them had developed only the hamster female PN while the sperm nuclei were either intact or swollen (partially decondensed), indicating that failure of oocyte activation was not the likely reason for the failure of male PN formation in these oocytes. In the next series of experiments, sibling oocytes were alternately injected with spermatozoa suspended either in the regular (1.9 mM Ca2+) or Ca2+-free injection medium (experiment set 2, n=278). A significant improvement was noted in the mean percentages of oocytes with 2PN, 2PB, metaphase entry and sperm chromosome spreads in the Ca2+-free group versus the regular group (2PN, 2PB: 51 versus 36.6%, metaphase entry: 36.3 versus 26.9% and sperm chromosome spreads: 28 versus 20.4%; all P < 0.04). Thus, parthenogenetic activation appears to be one of the contributing factors for the failure of male PN formation after heterospecific hamster ICSI. From these experiments it can be concluded that application of the advanced injection and culture techniques and omission of Ca2+ from the injection medium are promising for the routine application of the hamster oocyte microinjection for karyotyping of human spermatozoa with poor fertilizing capacity.     

Anastral meiotic spindle morphogenesis: role of the non-claret disjunctional kinesin-like protein

We have used time-lapse laser scanning confocal microscopy to directly examine microtubule reorganization during meiotic spindle assembly in living Drosophila oocytes. These studies indicate that the bipolarity of the meiosis I spindle is not the result of a duplication and separation of centrosomal microtubule organizing centers (MTOCs). Instead, microtubules first associate with a tight chromatin mass, and then bundle to form a bipolar spindle that lacks asters. Analysis of mutant oocytes indicates that the Non-Claret Disjunctional (NCD) kinesin-like protein is required for normal spindle assembly kinetics and stabilization of the spindle during metaphase arrest. Immunolocalization analyses demonstrate that NCD is associated with spindle microtubules, and that the centrosomal components gamma-tubulin, CP-190, and CP-60 are not concentrated at the meiotic spindle poles. Based on these observations, we propose that microtubule bundling by the NCD kinesin-like protein promotes assembly of a stable bipolar spindle in the absence of typical MTOCs.     

Depletion of glutathione during bovine oocyte maturation reversibly blocks the decondensation of the male pronucleus and pronuclear apposition during fertilization

Oocyte-produced glutathione (the tripeptide gamma-glutamyl-cysteinyl-glycine; GSH) has been implicated in the reduction of disulfide bonds in the sperm nucleus during fertilization and thus in the development of the male pronucleus (PN). In this study, we show that the depletion of endogenous glutathione by 10 mM buthionine sulfoximine (BSO; specific inhibitor of GSH synthesis) during bovine oocyte maturation (24 h in vitro; represents prophase I to metaphase II transition in this species) blocks the formation of a male PN in > 85% of treated oocytes (vs. 6.8% in controls) and prevents the assembly of the sperm aster microtubules in approximately 35%. Consequently, the pronuclear migration and apposition do not occur. Ultrastructural observations suggest that the effect of BSO on pronuclear apposition might be due to incomplete disassembly of the sperm tail connecting piece, which normally leads to the release of the sperm centriole and to the reconstitution of the zygotic centrosome during fertilization. The sperm nucleus decondensation and migration blocks were reversed by the treatment of the GSH-depleted oocytes with 1-10 mM dithiothreitol (a disulfide bond-reducing agent) applied 8 h after insemination: 82% of these oocytes exhibited a normal male PN and pronuclear apposition 20 h after insemination. The pool of glutathione seems to be generated during oocyte maturation since > 80% of oocytes that were matured in the absence of BSO displayed a normal male PN, as apposed to a female PN, when inseminated and cultured in the presence of 10 mM BSO. These data suggest that the reduction of disulfide bonds in the sperm after incorporation is important for the formation of the male PN, as well as for the disassembly of the sperm tail connecting piece and pronuclear apposition. The lack of disulfide-reducing power in the GSH-depleted oocytes can be reversed by treatment with disulfide bond-reducing agents.     

Lack of checkpoint control at the metaphase/anaphase transition: a mechanism of meiotic nondisjunction in mammalian females

A checkpoint mechanism operates at the metaphase/anaphase transition to ensure that a bipolar spindle is formed and that all the chromosomes are aligned at the spindle equator before anaphase is initiated. Since mistakes in the segregation of chromosomes during meiosis have particularly disastrous consequences, it seems likely that the meiotic cell division would be characterized by a stringent metaphase/ anaphase checkpoint. To determine if the presence of an unaligned chromosome activates the checkpoint and delays anaphase onset during mammalian female meiosis, we investigated meiotic cell cycle progression in murine oocytes from XO females and control siblings. Despite the fact that the X chromosome failed to align at metaphase in a significant proportion of cells, we were unable to detect a delay in anaphase onset. Based on studies of cell cycle kinetics, the behavior and segregation of the X chromosome, and the aberrant behavior and segregation of autosomal chromosomes in oocytes from XO females, we conclude that mammalian female meiosis lacks chromosome-mediated checkpoint control. The lack of this control mechanism provides a biological explanation for the high incidence of meiotic nondisjunction in the human female. Furthermore, since available evidence suggests that a stringent checkpoint mechanism operates during male meiosis, the lack of a comparable checkpoint in females provides a reason for the difference in the error rate between oogenesis and spermatogenesis.     

Influence of maternal age on meiotic spindle assembly in oocytes from naturally cycling women

To examine the effects of maternal ageing on the meiotic apparatus, we obtained oocytes from naturally cycling women in two age groups, including younger (aged 20-25 years) and older (aged 40-45 years) women. Using high-resolution confocal microscopy we obtained a detailed picture of the meiotic spindle and chromosome placement during various phases of meiosis. Our data revealed that the meiotic spindle in older women is frequently abnormal, both with regard to chromosome alignment and the microtubule matrix that comprise the meiotic spindle. The spindle in 79% of the oocytes from the older group exhibited abnormal tubulin placement and one or more chromosomes were displaced from the metaphase plate during the second meiotic division. In contrast, only 17% of the oocytes from the younger age group exhibited aneuploid conditions. The majority of eggs from this group possessed a well ordered, meiotic spindle containing chromosomes that were fully aligned within a distinct metaphase plate in the spindle. Chromosome management during meiosis is directed by microtubule assembly within the spindle. These data suggest that the regulatory mechanisms responsible for assembly of the meiotic spindle are significantly altered in older women, leading to the high prevalence of aneuploidy.     

Survival, metabolic activity and conjugative interactions of indigenous and introduced streptomycete strains in soil microcosms

Exocytosis of cortical granules in mouse eggs is required to produce the zona pellucida block to polyspermy. In this study, we examined the role of microfilaments and microtubules in the regulation of cortical granule movement toward the cortex during oocyte maturation and anchoring of cortical granules in the cortex. Fluorescently labeled cortical granules, microfilaments, and microtubules were visualized using laser-scanning confocal microscopy. It was observed that cortical granules migrate to the periphery of the oocyte during oocyte maturation. This movement is blocked by the treatment of oocytes with cytochalasin D, an inhibitor of microfilament polymerization, but not with nocodazole or colchicine, inhibitors of microtubule polymerization. Cortical granules, once anchored at the cortex, remained in the cortex following treatment of metaphase II-arrested eggs with each of these inhibitors; i.e., there was neither inward movement nor precocious exocytosis. Finally, the single cortical granule-free domain that normally becomes localized over the metaphase II spindle was not observed when the chromosomes become scattered following microtubule disruption with nocodazole or colchicine. In these instances a cortical granule-free domain was observed over each individual chromosome, suggesting that the chromosome or chromosome-associated material, and not the spindle, dictates the localization of the cortical granule-free domain.     

A metaphase pause: hormone-induced maturation progresses through a long pause at the first meiotic metaphase in oocytes of the starfish, Pisaster ochraceus

In several species of starfish, it has been reported that the meiotic divisions in fertilized oocytes occur precociously compared to those in unfertilized oocytes. The nature of the 'acceleration' of meiosis was studied using Pisaster ochraceus oocytes. The extent of the acceleration of first polar body formation was found to be completely dependent on the time of fertilization (or artificial activation); fertilization at about 100 min after 1-methyladenine application accelerated meiosis I the most, while earlier or later fertilization resulted in a smaller extent of accelerations of meiosis I. Observation of isolated meiotic spindles and fluorescent visualization of meiotic spindles in whole oocytes showed that progression of meiosis I in Pisasteroocytes pauses transiently at metaphase I for more than 40 min unless they are activated. The activation shortened the duration of metaphase I, which resulted in the acceleration of first polar body formation. A new term 'metaphase pause' is proposed to define this long duration of metaphase I in starfish oocytes.     

Chromosome identification in human oocytes and polar bodies by spectral karyotyping

Sixty unfertilized human oocytes and two fresh polar bodies were karyotyped by spectral karyotyping (SKY). The oocytes were provided by 29 women ranging from 30 to 42 yr of age. The mean hybridization efficiency for oocytes was 95.2% (60/63). Nondisjunction of bivalent chromosomes (13.3%) and predivision of sister chromatids at meiosis I (3.3%) were unequivocally determined by analysis first with SKY and then fluorescence in situ hybridization. Four oocytes (6.7%) were hyperhaploid, six (10.0%) were hypohaploid, one (1.7%) showed balanced predivision, and another (1.7%) was diploid. No specific structural rearrangements were detected. This study demonstrates that the SKY technique can be used successfully as an alternative method of karyotyping second meiotic metaphase chromosomes from human oocytes and polar bodies in appropriate spreads.     

Structural changes in the endoplasmic reticulum of starfish oocytes during meiotic maturation and fertilization

The endoplasmic reticulum (ER) of live starfish oocytes was observed during meiotic maturation and fertilization. The ER was visualized by injection into the cytoplasm of an oil drop saturated with the fluorescent lipophilic dye DiI; DiI spread throughout the oocyte endoplasmic reticulum and the pattern was imaged by confocal microscopy. The ER in the immature (germinal vesicle stage) oocyte was composed of interconnected membrane sheets. In response to 1-methyladenine, the sheets of ER appeared to become associated with the yolk platelets, forming spherical shells. A few of these spherical shells could sometimes be seen in immature oocytes, but their number was much greater in the egg at the first meiotic spindle stage. At about the time that the first polar body formed, the spherical shells disappeared, and the ER returned to a form like that of the immature oocyte. The spherical shells did not reappear during the second meiotic cycle. During maturation, the ER also began to move; the movement was apparent by the time of germinal vesicle breakdown and continued throughout both meiotic cycles and in eggs with second polar bodies. When eggs at the first meiotic spindle stage were fertilized, the form of the ER changed. Within 1 min after sperm addition to the observation chamber, the circular cross sections of the spherical shells of the unfertilized egg ER were no longer distinct. At this point, the form of the ER could not be discerned with the resolution of the light microscope; however, the rate of spreading of DiI from an injected oil drop decreased, providing strong evidence that the ER had become fragmented. The ER remained in this form for several minutes and then gradually, the appearance of the ER and the rate of DiI spreading returned to be like those of the unfertilized egg. Injection of inositol trisphosphate caused a similar change in the ER structure. These results indicate that the ER is a dynamic structure, the form of which changes during oocyte maturation and fertilization.     

Cytogenetic abnormalities of unfertilized oocytes generated from in-vitro fertilization and intracytoplasmic sperm injection: a double-blind study

In the present study we have assessed the cytogenetic abnormalities of unfertilized oocytes from in-vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) programmes during a one year period (July 1995 to July 1996) with the cytogenetic analysis being carried out in a double-blind manner. A total of 88 unfertilized ICSI and 85 unfertilized IVF oocytes were used for the study and of these 51 and 62 oocytes, in each respective group, were suitable for analysis. The haploidy, diploidy and aneuploidy rates between ICSI (62.7, 7.8 and 5.9%) and IVF (61.3, 9.7 and 14.5%) groups were similar. A significant inter-patient variation in the incidence of hypohaploidy was observed within the IVF group. Chromosomal fragmentation or breakage was observed at a similar rate in both groups of unfertilized oocytes (23.5 and 14.5% for ICSI and IVF respectively). A significantly higher proportion of ICSI oocytes contained sperm nuclei (27/51, 52.9%) than did IVF oocytes (20/62, 32.3%, P < 0.01). The distribution and state of sperm head chromatin in relation to oocyte chromosomal complement was studied in both groups. ICSI oocytes contained decondensed or swollen sperm nuclei in association with haploid oocyte chromosomes (12/27, 44.4%) or condensed sperm heads in oocytes showing no chromosomal complements (7/27, 25.9%). In IVF oocytes sperm heads were either arrested in the condensed state (5/20, 25%), metaphase stage (3/20, 15%) or had undergone premature chromosome condensation (PCC; 6/20, 30%) in association with haploid oocyte chromosomes. The incidence of PCC was similar in the two groups. A marked variation in the incidence of total chromosomal abnormality was observed between patients within both ICSI (0-75%) and IVF (0-71%) groups indicating a possible similarity in oocyte quality between the majority of male factor and tubal infertility patients. The type of sperm used in the two fertilization procedures showed an increased incidence of chromosomal breakage with ICSI-MESA (microepididymal sperm aspiration) spermatozoa (4/6, 67%) compared to the ICSI-ejaculated (6/35, 17.1%; P < 0.05), ICSI-testicular biopsy (2/10, 20%) and IVF-normospermic (9/62, 14.5%; P < 0.01) spermatozoa. Chromosomal fragmentation may be associated with the degree of difficulty experienced at sperm injection, especially with sperm retrieved from the reproductive tract. Thus chromosomal fragmentation in ICSI may need further investigation using a larger sample size in order to assess the possible causative factors.     

Provisional bilateral symmetry in Xenopus eggs is established during maturation

Dorsal-ventral patterning in the Xenopus egg becomes established midway through the first cell cycle during a 30 degree rotation of the subcortical yolk mass relative to the egg cortex. This 'rotation of symmetrisation' is microtubule dependent, and its direction is thought to be cued by the usually eccentric sperm centrosome. The fact that parthenogenetically activated eggs also undergo a directed rotation, despite the absence of a sperm centrosome, suggests that an endogenous asymmetry in the unfertilised egg supports the directed polymerisation of microtubules in the vegetal cortex, in the way that an eccentric sperm centrosome would in fertilised eggs. Consistent with this idea, we noticed that the maturation spot is usually located an average of more than 15 degrees from the geometric centre of the pigmented animal hemisphere. In parthenogenetically activated eggs, this eccentric maturation spot can be used to predict the direction of rotation. Although in most fertilised eggs the yolk mass rotates toward the sperm entry point (SEP) meridian, occasionally this relationship is perturbed significantly; in such eggs, the maturation spot is never on the same side of the egg as the SEP. In oocytes tilted 90 degrees from upright during maturation in vitro, the maturation spot developed 15 degrees or more from the centre of the pigmented hemisphere, always displaced towards the point on the equator that was up during maturation. This experimentally demonstrated lability is consistent with an off-axis oocyte orientation during oogenesis determining its eccentric maturation spot position, and, in turn, its endogenous rotational bias.     

Cooperative interactions between the central spindle and the contractile ring during Drosophila cytokinesis

We analyzed male meiosis in mutants of the chickadee (chic) locus, a Drosophila melanogaster gene that encodes profilin, a low molecular weight actin-binding protein that modulates F-actin polymerization. These mutants are severely defective in meiotic cytokinesis. During ana-telophase of both meiotic divisions, they exhibit a central spindle less dense than wild type; certain chic allelic combinations cause almost complete disappearance of the central spindle. Moreover, chic mutant spermatocytes fail to form an actomyosin contractile ring. To further investigate the relationships between the central spindle and the contractile ring, we examined meiosis in the cytokinesis-defective mutants KLP3A and diaphanous and in testes treated with cytochalasin B. In all cases, we found that the central spindle and the contractile ring in meiotic ana-telophases were simultaneously absent. Together, these results suggest a cooperative interaction between elements of the actin-based contractile ring and the central spindle microtubules: When one of these structures is disrupted, the proper assembly of the other is also affected. In addition to effects on the central spindle and the cytokinetic apparatus, we observed another consequence of chic mutations: A large fraction of chic spermatocytes exhibit abnormal positioning and delayed migration of asters to the cell poles. A similar phenotype was seen in testes treated with cytochalasin B and has been noted previously in mutants at the twinstar locus, a gene that encodes a Drosophila member of the cofilin/ADF family of actin-severing proteins. These observations all indicate that proper actin assembly is necessary for centrosome separation and migration.     

Dizygotic twin pregnancy after intracytoplasmic sperm injection of 1 day old unfertilized oocytes

We report on a case where late intracytoplasmic sperm injection (ICSI) on unfertilized oocytes after standard in-vitro fertilization (IVF) cycles resulted in a dizygotic twin pregnancy. Fifteen oocytes were harvested from a patient with a history of salpingotomy. After a single cycle of IVF, only one oocyte showed two pronuclei. Subsequently ICSI was performed on six unfertilized metaphase II oocytes, and three of these oocytes showed two pronuclei. Three fertilized embryos were transferred (two derived from ICSI and one from IVF). A normal twin pregnancy resulted, and after delivery of two healthy boys the twins were confirmed to be dizygotic by DNA analysis of several loci. We conclude that at least one of the embryos was derived from the reinsemination by 'second day ICSI'.     

A defect in synapsis causes male sterility in a T-DNA-tagged Arabidopsis thaliana mutant

Fluorescence microscopy was used to study meiosis in microsporocytes from wild-type Arabidopsis thaliana and a T-DNA-tagged meiotic mutant. Techniques for visualizing chromosomes and beta-tubulin in other plant species were evaluated and modified in order to develop a method for analyzing meiosis in A. thaliana anthers. Like most dicots, A. thaliana microsporocytes undergo simultaneous cytokinesis in which both meiotic divisions are completed prior to cytokinesis. However, two unique events were observed in wild-type A. thaliana that have not been reported in other angiosperms: (1) polarization of the microsporocyte cytoskeleton during prophase I prior to nuclear envelope breakdown, and (2) extensive depolymerization of microtubules just prior to metaphase II. The first observation could have implications regarding a previously uncharacterized mechanism for determining the axis of the metaphase I spindle during microsporogenesis. The second observation is peculiar since microtubules are known to be involved in chromosome alignment in other species; possible explanations will be discussed. A T-DNA-tagged meiotic mutant of A. thaliana (syn1), which had previously been shown to produce abnormal microspores with variable DNA content, was also cytologically characterized. The first observable defect occurs in microsporocytes at telophase I, where some chromosomes are scattered throughout the cytoplasm, usually attached to stray microtubules. Subsequent development stages are affected, leading to complete male sterility. Based on similarities to synaptic mutants that have been described in other species, it is suggested that this mutant is defective in synaptonemal complex formation and/or cohesion between sister chromatids.     

Ultrastructural observations on rabbit, hamster and mouse eggs following electrical stimulation in vitro

The fine structural changes were studied in the plasma membrane, cortical granules (CGs) and meiotic spindle of rabbit, hamster and mouse eggs in response to electrical stimulation. Eggs were collected 16 to 18 hours after HCG injection, and freed from the cumulus oophorus. They were stimulated in vitro by delivering a single monophasic square wave pulse of 150 V for 1 msec. Stimulated and unstimulated eggs were fixed for fine structural observations 1, 30 and 60 minutes after stimulation. Within one minute of stimulation the microvilli of rabbit eggs were long, branching and had bulbous ends. Umbonate protrusions were also present on their surface. By 30 minutes after stimulation the rabbit eggs lacked microvilli and the perivitelline space with detached vesicles. Degenerating changes were readily noticeable in the microvilli of hamster eggs by 60 minutes. Changes were not noted in the microvilli of stimulated mouse eggs. There were markedly fewer CGs in the hamster and mouse, but not rabbit, eggs by 30 minutes after stimulation, and nearly all of them disappeared in the mouse and hamster eggs by 60 minutes. The density of the CGs in the rabbit was not altered. The meiotic spindle of hamster eggs dissipated within one minute of stimulation, however, the microtubules reappeared by 60 minutes after stimulation. Rotation of the meiotic spindle occurred in the mouse and hamster eggs by 30 minutes after stimulation. In some of the mouse eggs the spindle migrated to the center and the chromosomes were in telophase.     

AIR-2: An Aurora/Ipl1-related protein kinase associated with chromosomes and midbody microtubules is required for polar body extrusion and cytokinesis in Caenorhabditis elegans embryos

An emerging family of kinases related to the Drosophila Aurora and budding yeast Ipl1 proteins has been implicated in chromosome segregation and mitotic spindle formation in a number of organisms. Unlike other Aurora/Ipl1-related kinases, the Caenorhabditis elegans orthologue, AIR-2, is associated with meiotic and mitotic chromosomes. AIR-2 is initially localized to the chromosomes of the most mature prophase I-arrested oocyte residing next to the spermatheca. This localization is dependent on the presence of sperm in the spermatheca. After fertilization, AIR-2 remains associated with chromosomes during each meiotic division. However, during both meiotic anaphases, AIR-2 is present between the separating chromosomes. AIR-2 also remains associated with both extruded polar bodies. In the embryo, AIR-2 is found on metaphase chromosomes, moves to midbody microtubules at anaphase, and then persists at the cytokinesis remnant. Disruption of AIR-2 expression by RNA- mediated interference produces entire broods of one-cell embryos that have executed multiple cell cycles in the complete absence of cytokinesis. The embryos accumulate large amounts of DNA and microtubule asters. Polar bodies are not extruded, but remain in the embryo where they continue to replicate. The cytokinesis defect appears to be late in the cell cycle because transient cleavage furrows initiate at the proper location, but regress before the division is complete. Additionally, staining with a marker of midbody microtubules revealed that at least some of the components of the midbody are not well localized in the absence of AIR-2 activity. Our results suggest that during each meiotic and mitotic division, AIR-2 may coordinate the congression of metaphase chromosomes with the subsequent events of polar body extrusion and cytokinesis.