Sperm Aster Research Articles

Water-soluble extracts of the diatom Thalassiosira rotula induce aberrations in embryonic tubulin organisation of the sea urchin Paracentrotus lividus

Eggs and embryos of the sea urchin Paracentrotus lividus were used as a model to study the effect at the cellular level of potential anti-mitotic compounds extracted from the diatom Thalassiosira rotula. Eggs and embryos incubated in a water-soluble diatom extract, corresponding to 5 × 106 and 107 cells ml−1, were totally blocked (i.e. cell division was blocked) at the one-cell stage. At lower concentrations (2.5 and 1.25 × 106 cells ml−1), the first mitotic division was inhibited in 32 ± 26% and 25 ± 3.5% of the zygotes, respectively, demonstrating the dose-dependent effect of diatom extracts on sea urchin development. Immunofluorescence dyes, specific for DNA and α-tubulin subunits, were used to stain nuclei and microtubules in sea urchin embryos during various phases of development. Images with the confocal laser scanning microscope showed that tubulin was not organised in filaments at the sperm aster and cortex levels, and that the pronuclei were not fused in embryos incubated soon after fertilisation with water-soluble diatom extracts corresponding to 107 cells ml−1. At lower diatom-extract concentrations (4 × 106 cells ml −1), fusion of the pronuclei occurred but the mitotic spindle was not formed. Microtubules were clearly de-polymerised and the chromatin appeared globular and compacted at the centre of the cell. A similar structure was observed for sea urchin embryos incubated with 0.1 mM colchicine, a potent anti-mitotic compound. When sea urchin embryos were incubated in water-soluble diatom extracts at different times prior to the first mitotic division, microtubules appeared de-polymerised at each step, from pronuclear fusion to telophase, and cell division was blocked. At the histological level, embryos incubated with 4 × 106 cells ml−1 diatom extract showed nuclear fragmentation without cytokinesis. The possible use of sea urchin embryos as a bioassay to test for other unknown compounds with cytotoxic activity in phytoplankton species is discussed.

Interactions between cytoskeletal components during myoplasm rearrangement in ascidian eggs.

Ooplasmic segregation in ascidian eggs consists of two phases of cytoplasmic movement, the first phase is mediated by the microfilament system and the second is mediated by the microtubule system. Recently, two novel proteins, p58 and myoplasmin-C1, which are localized to the myoplasm, were suggested to have important roles in muscle differentiation. In order to analyze the molecular mechanisms underlying ooplasmic segregation, the interactions between actin, tubulin, p58 and myoplasmin-C1 were examined. During the first segregation, microtubule meshwork in the unfertilized egg disappeared. At the second segregation, a novel structure of the microtubules that extended from the sperm aster and localized in the cortical region of the myoplasm was found. Moreover, uniform distribution of the cortical actin filament was observed at the second segregation. During the course of myoplasm rearrangement, p58 and myoplasmin-C1 are colocalized and can form a molecular complex in vitro. This complex of p58 and myoplasmin-C1 is a good candidate for a cytoskeletal component of the myoplasm, and is likely to be involved in the correct distribution of cytoplasmic determinants.

XMAP230 is required for the organization of cortical microtubules and patterning of the dorsoventral axis in fertilized Xenopus eggs.

The dorsoventral axis of Xenopus embryos is specified by a rotation of the egg cortex relative to the underlying yolky cytoplasm. This cortical rotation, which occurs during the first cell cycle after fertilization, is dependent upon an array of parallel microtubules in the subcortical cytoplasm. We have used confocal immunofluorescent microscopy and microinjection of affinity-purified anti-XMAP230 antibody to address the role of XMAP230, one of three high-molecular-weight microtubule-associated proteins (MAPs) in Xenopus eggs, in the assembly and organization of the cortical microtubule array and specification of the dorsoventral axis. Confocal immunofluorescence microscopy revealed that XMAP230 was associated with cortical microtubules shortly after their appearance in the subcortical cytoplasm. XMAP230 staining became more prominent as microtubules were aligned and bundled during the cortical rotation. Loss of XMAP230 appeared to precede disassembly of cortical microtubules at the end of the first cell cycle. Deeper within the cytoplasm, XMAP230 was associated with microtubules early in the assembly of the sperm aster. However, later in the first cell cycle, XMAP230 was associated with microtubules (MTs) of the first mitotic spindle, spindle asters, and the cortical MTs, but not with microtubule remnants of the sperm aster. Microinjection of anti-XMAP230 antibody locally disrupted the assembly and organization of microtubules in the cortex of activated or fertilized eggs and resulted in defects in the dorsoventral patterning of embryos. These results indicate that the assembly and/or organization of cortical microtubules in fertilized Xenopus eggs and subsequent specification of the dorsoventral axis are dependent upon XMAP230.

Fertilization of porcine oocytes following intracytoplasmic spermatozoon or isolated sperm head injection.

We demonstrated normal fertilization processes (as determined by pronuclear formation, pronuclear apposition and syngamy) in porcine oocytes either following intracytoplasmic spermatozoon (ICSI) or isolated sperm head injection. Microtubule organization and chromatin configuration were investigated in these oocytes during the first cell cycle. Following ICSI, the microtubular aster was organized from the neck of the spermatozoon and filled the whole cytoplasm. These male-derived microtubules appear to move both pronuclei to the center of oocytes. These cytoskeletal changes are analogous to those seen following conventional fertilization. In contrast, following isolated sperm head injection, the sperm aster was not seen. Instead, the microtubule matrix was organized from the cortex and then filled the whole cytoplasm in all cases in normally fertilized oocytes following injection (n=35). This organization is similar to what has been shown in the parthenogenetically activated oocytes. Chromosome analysis revealed that the oocytes injected with isolated sperm heads were fertilized normally. At 7 days following injection, the incidence of blastocoele formation following ICSI (38%) and isolated sperm head injection (22%) was higher than that following sham injection (2%). These results suggested that successful fertilization and preimplantation development occurred in porcine oocytes following either ICSI or isolated sperm head injection. Our results also indicated that fertilization processes can occur by self-assembled microtubules within cytoplasm in the absence of a sperm centrosome.

Gamma-tubulin during the differentiation of spermatozoa in various mammals and man.

The distribution of gamma-tubulin as a marker of microtubule organizing centres (MTOC) was studied during spermiogenesis in rodents and in rabbit, monkey and man. A polyclonal antibody directed against human gamma-tubulin was used both for indirect immunofluorescence (IIF) and post-embedding immunogold procedures. In all species, gamma-tubulin was detected in the proximal and distal centrioles of round spermatids. In elongating spermatids, gamma-tubulin was predominantly found in the pericentriolar material (PCM) of both centrioles and particularly around the adjunct of the proximal centriole. At this level, some labelling was also associated with manchette microtubules, but other parts of the manchette and the nuclear ring were never labelled. We propose a role for distal centriole gamma-tubulin in axoneme nucleation and centriolar adjunct gamma-tubulin in manchette nucleation. The disappearance of gamma-tubulin in mature spermatozoa indicates that sperm aster nucleation should be dependent on oocyte gamma-tubulin. Remnants of gamma-tubulin in some human spermatozoa suggest that paternal gamma-tubulin also could contribute to sperm aster formation.

Caulerpenyne interferes with microtubuledependent events during the first mitotic cycle of sea urchin eggs

Caulerpenyne (Cyn), the major secondary metabolite synthesized by the green alga Caulerpa taxifolia proliferating in the Mediterranean Sea, is a cytotoxic sesquiterpene. As this compound has an antiproliferative potency by inhibiting division of many types of cells, we examined the precise effects of Cyn during the early development of the sea urchin Paracentrotus lividus. Whereas Cyn (60 microM) had no effect on fertilization, it blocked the first cell division in the same manner whether added before or after fertilization, provided the drug was added before or during metaphase. Immunofluorescence localization revealed that Cyn had no effect on the microtubular sperm aster formation, pronuclei migration and fusion, chromosome condensation, nuclear envelope breakdown, and bipolar mitotic spindle assembly. However, mitosis was blocked in a metaphase-like stage at which most chromosomes were aligned at the equatorial plate, while a few of them had not even migrated towards the metaphase plate. When added after the metaphase-anaphase transition, the first division occurred normally but the second division was inhibited with the same phenotype as described above. We previously showed that Cyn did not affect protein synthesis or H1 kinase activation or deactivation (Pesando et al., 1996, Aquat. Toxicol. 35, 139), but that it partially inhibited DNA synthesis. Our results establish that Cyn does not affect the microfilament-dependent processes of fertilization and cytokinesis and allows the beginning of mitosis, but prevents normal DNA replication and results in metaphase-like arrest of sea urchin embryos.

Microtubule Organization and Chromatin Configurations in Hamster Oocytes During Fertilization and Parthenogenetic Activation, and After Insemination With Human Sperm

The cytoskeletal components of hamster oocytes, zygotes, and spontaneously activated parthogenotes were examined after immunocytochemical labeling. Microtubules were found only in the anastral, tangentially arranged second meiotic spindle of unfertilized oocytes. Taxol treatment of unfertilized oocytes greatly augmented astral microtubules in both the metaphase II spindle and the cortex. Disruption of the meiotic spindle microtubules with nocodazole resulted in cortical chromosomal scattering. During hamster sperm incorporation and pronuclear formation, no sperm aster was detected in association with the male DNA. Instead, a large overlapping array of microtubules assembled in the cortex. By mitosis, this interphase array disassembled and an anastral metaphase spindle formed. Microtubule and chromatin configurations were also imaged in hamster oocytes injected with human sperm. Astral microtubules were absent from the sperm centrosome. The implications of these results are discussed in relation to the hamster oocyte penetration assay, a test commonly used by in vitro fertilization clinics to demonstrate the fertilizing ability of human sperm. We conclude that since hamsters and humans follow different methods of centrosome inheritance, maternal and paternal, respectively, the hamster may be an inappropriate model for exploring microtubule and centrosomal defects in humans or for assaying postinsemination forms of human male fertility defects.

The Tomaj mutant alleles of alpha Tubulin67C reveal a requirement for the encoded maternal specific tubulin isoform in the sperm aster, the cleavage spindle apparatus and neurogenesis during embryonic development in Drosophila.

The three dominant TomajD and their eleven revertant (TomajR) alleles have been localized to the alpha Tubulin67C gene of Drosophila melanogaster. Although the meiotic divisions are normally completed in eggs laid by TomajD/+, TomajD/-, TomajR/- females, embryogenesis arrests prior to the gonomeric division. The arrest is caused by: (1) the failure of prominent sperm aster formation; and (2) a consequent lack of female pronuclear migration towards the male pronucleus. Concomitant with the sperm aster defect, the four female meiotic products fuse (tetra-fusion), similar to what is seen in eggs of wild-type virgin females. In eggs of females heterozygous for weaker TomajR alleles, embryogenesis comes to a cessation before or shortly after cortical migration of cleavage nuclei. The apparent source of embryonic defect is the cleavage spindle apparatus. One of the three TomajD alleles is cold-sensitive and its cold-sensitive period coincides with the completion of female meiosis and pronuclear migration. Disorganized central and peripheral nervous systems are also characteristic of embryos derived from the temperature-sensitive TomajD/+ females. The Tomaj mutant phenotypes indicate an involvement of the normal alpha Tubulin67C gene product in: (1) the formation of the sperm aster; (2) cleavage spindle apparatus formation/function; and (3) the differentiation of the embryonic nervous system. The TomajD alleles encode a normal-sized alpha Tubulin67C isotype. Sequence analyses of the TomajD alleles revealed the replacement in different positions of a single negatively charged or neutral amino acid with a positively charged one. These residues presumably identify important functional sites.

The promotory effect of growth hormone on the developmental competence of in vitro matured bovine oocytes is due to improved cytoplasmic maturation.

In a previous study we have shown that the addition of growth hormone (GH) during in vitro maturation accelerates nuclear maturation, induces cumulus expansion, and promotes subsequent cleavage and embryonic development. The aim of this study was to investigate whether the promotory effect of GH on subsequent cleavage and blastocyst formation is due to an improved fertilization and whether this effect is caused by an improved cytoplasmic maturation of the oocyte. Therefore, bovine cumulus oocyte complexes (COCs) were cultured for 22 hours in M199 supplemented with 100 ng/ml bovine GH (NIH-GH-B18). Subsequently the COCs were fertilized in vitro. Cultures without GH served as controls. To verify whether the promoted fertilization is caused by the effect of GH on cumulus expansion or oocyte maturation, cumulus cells were removed from the oocytes after in vitro maturation (IVM) and denuded MII oocytes were selected and fertilized in vitro. Both IVM and in vitro fertilization (IVF) were performed at 39 degrees C in a humidified atmosphere with 5% CO2 in air. At 18 hours after the onset of fertilization, the nuclear stage of the oocytes was assessed using 4,6-diamino-2-phenylindole (DAPI) staining. Oocytes with either an metaphase I (MI) or MII nuclear stage and without penetrated sperm head were considered unfertilized; oocytes with two pronuclei, zygotes, and cleaved embryos were considered normally fertilized; and oocytes with more than two pronuclei were considered polyspermic. To evaluate cytoplasmic maturation, the distribution of cortical granules 22 hours after the onset of IVM, and sperm aster formation 8 hours after the onset of fertilization were assessed. In addition, to assess the sperm-binding capacity, COCs were fertilized in vitro, and 1 hour after the onset of fertilization the number of spermatozoa bound to the oocytes was counted. The addition of GH during IVM significantly (P < 0.001) enhanced the proportion of normal fertilized oocytes. Removal of the cumulus cells prior to fertilization and selection of the MII oocytes did not eliminate the positive effect of GH on fertilization. No effect of GH on the sperm-binding capacity of the oocyte was observed. In addition, GH supplementation during IVM significantly (P < 0.001) enhanced the migration of cortical granules and sperm aster formation. It can be concluded that the promotory effect of GH on the developmental competence of the oocyte is due to a higher fertilization rate as a consequence of an improved cytoplasmic maturation.

Paternal centrosomal dynamics in early human development and infertility.

Our purpose was to demonstrate the dynamics of the human sperm centrosome during fertilization and cleavage. Human gametes, fertilized oocytes, and preimplantation embryos were examined by transmission electron microscopy. The functional sperm centrosome containing a typical centriole (proximal) is inherited at fertilization and forms a sperm monoaster. It then replicates and is perpetuated during cleavage. It organizes the mitotic apparatus at each stage of cleavage up to the hatching blastocyst stage. Bipolar spindles are formed in all monospermic and most dispermic embryos. Occasionally, two sperm asters and tripolar spindles are formed in dispermic embryos. Centrioles are associated with pronuclei and nuclei at interphases when they duplicate and occupy pivotal positions at spindle poles during mitoses. The maternal centrosome is not functional. The human embryo shows paternal centrosome inheritance and perpetuation like most other animals. Inheritance of defective centrosomes may lead to abnormal cleavage and contribute to infertility.

染色体操作によるサケ科魚類の三倍体及び雌性発生二倍体の生物学的特性並びに形質の遺伝に関する研究 ＶＩＩＩ 染色体操作を施したニジマス初期はいの核の行動における細胞学的観察

Heat shock treatment for 20min from 15min after insemination suppressed the formation of the second polar body of the normally developed eggs, and the genetic material of the second polar body was retained in the egg. These processes did not result in the formation of a diploidy female pronucleus, but formed some karyomeres. They were tugged with the female pronucleus toward the male pronucleus already present in the center of the sperm aster. Thus, they adhered, and formed a fertilized nucleic group like a cluster of grapes. The genetic inactivated sperm irradiated with ultraviolet rays (UV-sperm) became swollen and formed a male pronucleus after passing through the micropile. It behaved like normal sperm until the connection with the female pronucleus. The paternal genetic material in gynogenetic embryos disappeared during the anaphase of the first cleavage, caused by the incomplete chromosome formation from the male pronucleus, and the paternal DNA remained near the cleavage plane. In the gynogenetic diploid embryo inseminated with UV-sperm, and polyploidized with heat shock treatment, the above mentioned cytological events caused by dual artificial manipulation occurred in the same egg, i.e., the genetic material formed a triploidy nucleus like a cluster of grapes, and a male pronucleus derived from UV-sperm disappeared during the anaphase of the first cleavage. The developmental rate was higher in triploid embryos polyploidized with heat shock, and lower in the gynogenetic haploid embryo than in the normal diploid embryos. In the gynogenetic diploid embryos, the metaphase of the first cleavage occurred at the same time as normal diploid embryos.

Microtubule organization and chromatin configurations in hamster oocytes during fertilization and parthenogenetic activation, and after insemination with human sperm.

The cytoskeletal components of hamster oocytes, zygotes, and spontaneously activated parthogenotes were examined after immunocytochemical labeling. Microtubules were found only in the anastral, tangentially arranged second meiotic spindle of unfertilized oocytes. Taxol treatment of unfertilized oocytes greatly augmented astral microtubules in both the metaphase II spindle and the cortex. Disruption of the meiotic spindle microtubules with nocodazole resulted in cortical chromosomal scattering. During hamster sperm incorporation and pronuclear formation, no sperm aster was detected in association with the male DNA. Instead, a large overlapping array of microtubules assembled in the cortex. By mitosis, this interphase array disassembled and an anastral metaphase spindle formed. Microtubule and chromatin configurations were also imaged in hamster oocytes injected with human sperm. Astral microtubules were absent from the sperm centrosome. The implications of these results are discussed in relation to the hamster oocyte penetration assay, a test commonly used by in vitro fertilization clinics to demonstrate the fertilizing ability of human sperm. We conclude that since hamsters and humans follow different methods of centrosome inheritance, maternal and paternal, respectively, the hamster may be an inappropriate model for exploring microtubule and centrosomal defects in humans or for assaying postinsemination forms of human male fertility defects.

Recruitment of maternal material during assembly of the zygote centrosome in fertilized sea urchin eggs.

Spindle poles of sea urchin embryos contain centrosomal material derived from maternal as well as paternal sources. To examine how maternal centrosomal material becomes recruited into spindle poles during the first cell cycle, fertilized sea urchin eggs were fixed and labeled with an anti-centrosomal antibody at sequential timepoints after insemination. Immunolabeling patterns demonstrate that the unfertilized egg contains small foci of immunoreactive material dispersed throughout the cytoplasm. Shortly after insemination, the diffuse foci coalesce to form a dense aggregate close to the sperm nucleus. Subsequently, centrosomal material spreads over the surface of the zygote nucleus and becomes partitioned into two masses during spindle pole formation. The involvement of the cytoskeleton in the translocation and targeting of maternal centrosomal material through the first cell cycle was examined by treating eggs with cytoskeletal disrupting agents, a general kinase inhibitor, and by re-inseminating fertilized eggs. These experiments indicate that the initially diffuse centrosomal material is transported centripetally to the sperm nucleus by the sperm aster and the centrosomal material is subsequently sequestered around the zygote nucleus by a microtubule-mediated mechanism. Remarkably, 6-dimethylaminopurine treatment shifted the targeting of maternal centrosomal material from the sperm nucleus to the female pronucleus; upon recovery, some of these zygotes formed spindle poles that flanked only the maternal chromosomes.

Assembly of the zygotic centrosome in the fertilized Drosophila egg

Zygotic centrosome assembly in fertilized Drosophila eggs was analyzed with the aid of an antiserum Rb188, previously shown to be specific for CP190, a 190 kDa centrosome-associated protein (Whitfield et al. (1988) J. Cell Sci. 89, 467–480; Whitfield et al. (1995) J. Cell Sci. 108, 3377–3387). The CP190 protein was detected in two discrete spots, associated with the anterior and posterior ends of the elongating nucleus of Drosophila spermatids. As the spermatids matured, this labelling gradually disappeared and was no longer visible in sperm dissected from spermathecae and ventral receptacles. γ-Tubulin was also found in association with the posterior end of the sperm nucleus during spermiogenesis, but was not detected in mature sperm. This suggests that CP190 and γ-tubulin are not present in detectable quantities in fertilizing sperm. CP190 was not detected in association with the sperm nucleus of newly fertilized eggs removed from the uterus, whereas many CP190-positive particles were associated with microtubules of the sperm aster from anaphase I to anaphase II. These particles disappeared during early telophase II and only one pair of CP190-positive spots remained visible at the microtubule focus of the sperm aster. These spots were associated with one aster through telophase, and then moved away to form two smaller asters from which the first mitotic spindle was organized. Colchicine treatment suggested that at least some CP190 protein is an integral part of the centrosome rather than merely being transported along microtubules. Centrosomal localization of the CP190 antigen was prevented by incubation of the permeabilized zygote in 20 mM EDTA.

The Drosophila kinesin-like protein KLP3A is required for proper behavior of male and female pronuclei at fertilization.

Drosophila melanogaster females homozygous for mutations in the gene encoding the kinesin-like protein KLP3A are sterile (Williams et al., 1995). We have investigated the basis of this sterility. The eggs produced by KLP3A mutant mothers are fertilized by sperm, and female meiosis appears to occur normally. However, the large majority of these embryos arrest their development soon thereafter with a characteristic phenotype. The four nuclei produced by female meiosis associate together in a polar body-like structure, while a bipolar spindle is established around the metaphase-arrested male pronucleus. Thus, the major defect caused by depletion of the KLP3A protein is either in specification of the female pronucleus, or in migration of the male and female pronuclei toward each other. We have also found that the KLP3A protein is located throughout the metaphase spindle during meiosis and the early embryonic mitotic divisions, but later accumulates specifically at the midzone of these same spindles during telophase. The protein is also present on two other microtubule structures: the sperm aster; and the radial, monastral array of microtubules established between the two meiosis II spindles. We discuss these results in light of possible functions of the KLP3A protein in pronuclear specification and migration.

Changes in microtubule structures during the first cell cycle of physiologically polyspermic newt eggs.

The unfertilized egg of the newt, Cynops pyrrhogaster, has a second meiotic spindle at the animal pole and numerous cortical cytasters. After physiologically polyspermic fertilization, all sperm nuclei incorporated into the egg develop sperm asters, and the cortical cytasters change into bundles of cortical microtubules. The size of the sperm asters in the animal hemisphere is approximately 5.6-fold larger than that in the vegetal hemisphere. Only one sperm nucleus moves toward the center of the animal hemisphere to form a zygote nucleus with the egg nucleus. This movement is inhibited by nocodazole, but not by cytochalasin B. The centrosome in the zygote nucleus divides into two parts to form a bipolar spindle for the first cleavage synchronously with the nuclear cycle, but centrosomes of accessory sperm nuclei in the vegetal hemisphere remained to form monopolar interphase asters and subsequently degenerate around the first cleavage stage. The size of sperm asters in monospermically fertilized Xenopus eggs was approximately 37-fold larger than those in Cynops eggs. Since sperm asters that formed in polyspermically fertilized Xenopus eggs exclude each other, the formation of a zygote nucleus is inhibited. Cynops sperm nuclei form larger asters in Xenopus eggs, whereas Xenopus sperm nuclei form smaller asters in Cynops eggs compared with those in homologous eggs. Since there was no significant difference in the concentration of monomeric tubulin between those eggs, the size of sperm asters is probably regulated by a component(s) in egg cytoplasm. Smaller asters in physiologically polyspermic newt eggs might be useful for selecting only one sperm nucleus to move toward the egg nucleus.

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.

Movement of nuclei along microtubules in Xenopus egg extracts

Microtubules are implicated in the movement and positioning of nuclei in many cell types. Nuclei can be moved by forces acting on microtubules nucleated at the spindle pole body, as in fungi [1], or microtubules nucleated at the centrosome, as during migration of the male (sperm) pronucleus towards the centre of the zygote after fertilization [2–4]. The dramatic movements of the female pronucleus towards the male pronucleus potentially involve another mechanism: movement along the microtubules of the sperm aster towards their slower growing, attached or ‘minus’ ends [3,5]. Here, we have reconstituted this last type of nuclear movement in vitro. Synthetic nuclei assembled in cytoplasmic extracts made from interphase Xenopus eggs move along microtubules towards their minus ends. We provide strong experimental evidence that cytoplasmic dynein is the motor for nuclear movement in this in vitro system, and discuss our results in terms of current knowledge of motility of the endoplasmic reticulum.

The implications of a paternally derived centrosome during human fertilization: consequences for reproduction and the treatment of male factor infertility.

Successful fertilization in humans follows a complex series of events, including the completion of meiotic maturation of the oocyte with the extrusion of the second polar body, the decondensation of the sperm nucleus and the maternal chromosomes into male and female pronuclei, the restoration of the sperm centrosome, and the nucleation of microtubule-mediated motility necessary to bring the male and female pronuclei into close apposition. These events occur after both fertilization in vitro and after intracytoplasmic sperm injection (ICSI), a new technique which is currently being applied in many clinics to overcome severe male infertility. Defects in any of the events leading to fertilization can be lethal to the zygote and may prove to be causes of infertility. Imaging of inseminated human and rhesus oocytes using immunohistochemical techniques reveals several phases at which fertilization arrests. Oocytes from some infertile patients failed to complete fertilization due to failure of the sperm aster microtubules in uniting the sperm and egg nuclei. The rate of sperm aster formation, size, and organization during fertilization has been used as a measurement of bovine sperm quality. The development of an assay using Xenopus laevis oocyte extract can also be used to test sperm from various species for their ability to form esters and perform other centrosomal functions in vitro, as well as another indicator of sperm quality. Semen from men with questionable fertility was found to contain sperm which are generally incapable of producing sperm asters. In addition, the activity of centrosomal proteins such as gamma-tubulin and centrin have been detected in mammalian eggs and sperm. The levels of gamma-tubulin increase markedly after exposure to X. laevis egg extract. Defects in either male or female nucleus decondensation also resulted in the arrest of fertilization and was found to occur in both inseminated human oocytes and in rhesus oocytes fertilized by ICSI. These discoveries on the molecular basis of infertility in humans have important implications for infertility diagnosis and managing reproduction.

The human sperm centrosome is responsible for normal syngamy and early embryonic development.

As early as 1887, it was postulated that the mature oocyte possesses all of the elements necessary for embryonic development with the exception of an active division centre, and that the spermatozoon contains such a centre, but lacks the substrate in which to operate. This division centre is called the centrosome. The precise definition of this structure is still a subject for debate. It consists of two centrioles in a perpendicular arrangement and pericentriolar material, and is considered to be responsible for nucleation of microtubules and the formation of the mitotic spindle. There is a paternal pattern of inheritance of the centrosome in humans; thus, human oocytes lack centrioles but the spermatozoa carry two. At gamete fusion the sperm tail is incorporated into the ooplasm, and the centriolar region forms the sperm aster while the sperm head is decondensing; this aster acts to guide the female pronucleus towards the male pronucleus. The centriole duplicates during the pronuclear stage, and at syngamy centrioles are found at opposite poles of the first cleavage. The centrosome has several implications for human infertility. It is possible that immotile or nonprogressively motile spermatozoa may possess centriolar abnormalities or an absence of centrioles. Similarly, antisperm antibodies against centrioles may be responsible for mitotic arrest. One way of solving this problem would be the use of donor centrosomes. To this end, we have assessed the ability of embryos injected with physically separated sperm segments (head only, head and tail separated or isolated tail) to develop normally. Fluorescent in situ hybridization revealed an almost universal mosaicism in these embryos, suggesting that physical disruption of the spermatozoa compromises the ability of the centrosome to function in the zygote. Thus far, centrosome donation with centriole-carrier flagellae obtained by this dissection method does not appear to be feasible.