Regional Difference in Mechanical Properties of the Cell Surface in Dividing Echinoderm Eggs*

Stiffness of the cell was surface was determined in fertilized sea urchin and starfish eggs by measuring the mechanical resistivity of the cell surface against negative pressure applied to a restricted part with a micropipette in contact with the cell surface at its tip (elastimetry). In both sea urchin and starfish eggs, the stiffness of the cell surface changed almost in parallel between the presumptive furrow and polar surfaces before the onset of the first cleavage, and the stiffness of the furrow surface became larger than that of the polar surface when cleavage started, although temporal changes in the stiffness were different between sea urchin and starfish eggs. The stiffness of the cell surface changed almost in parallel between the surfaces at the equator and at the animal pole in starfish eggs before the onset of polar body formation. The stiffness of the cell surface around the forming polar body increased during the formation of the polar body and remained at a high level after the polar body formation. It seems that the stiffness difference responsible for the formation of the contractile ring develops simultaneously with rather than prior to the formation of the cleavage furrow.     

Plasma Membrane Domains and the Site of Sperm Entrance in Discoglossus pictus (Anura) Eggs

Freeze-fracture quantitative analysis reveals three different plasma membrane (PM) domains in the unfertilized egg of the anuran Discoglossus pictus . One of these is specific to the sperm entrance site (D1). where the plasma membrane shows a larger number of intramembranous particles (IMP) than the rest of the egg surface. Such an increment is due to a markedly higher number of the IMPs anchored to the P-face. The two other domains (D2 and D3) are characterized by a lower IMP density at the P-face with respect to D1. The IMP density decreased within 10 min after fertilization by about 33% in all domains observed, probably due to the insertion of new membrane through exocytosis. The possibility that the IMPs located in D1 may represent putative plasma membrane proteins playing a role in sperm-egg interaction and/or in egg activation is discussed.     

Ultrastructural Study of Asters Induced by Microinjection with Sperm Centriolar Fraction in Sea Urchin Eggs

Multiple asters can be artificially induced in sea urchin fertilized eggs by the microinjection of the centriolar fraction of sperm homogenate. Investigation was continued by the electron microscopy to determine whether the multi-aster formation was due to the centrioles or the contaminants in the injected sperm fraction. Thirty three asters in 3 operated eggs were thoroughly examined, and we confirmed that the presence of centrioles in the central region of 26 asters. We considered that the rest of them might contained the centrioles in the sections lost during the preparation procedures. Fragmented axoneme, the plug of electron dense material, and the centriolar fossa, which were usually accompanied with the isolated centrioles, disappeared from the centrioles in these multiple asters. However, electron dense, amorphous materials were formed associating with the triplet blades and distributed around the centrioles. Many astral microtubules were terminated in these pericentriolar materials. Results obtained suggest that, although the pericentriolar material is acting as the microtubule organizing center, all multiple asters, except those derived from fertilization (2 asters per egg), are most likely induced by the injected centrioles and not by the contaminants.     

Aster Formation in Sea Urchin Eggs Induced by the injection of Enzyme-Treated Sperm Centrioles

To determine the responsible components of isolated sperm centrioles for the aster induction in sea urchin eggs, the sperm centriolar fraction was treated with various enzymes and was injected into the unfertilized eggs, then the aster formation in first division was observed after fertilization.
Treatment with 1 μg/ml or higher concentration of trypsin inhibited the centriolar activity for aster induction, whereas the treatment with 50 μg/ml of DNase 1, 80 μg/ml of RNase A, 40 μg/ml of RNase T1, or 0.1 μg/ml of trypsin had no inhibitory effect to induce asters. Injection of 0.5 μg/ml of RNase A or 1 mUg/ml of RNase T1 into the egg caused the detention of mitosis at the streak stage. To examine the temperature effect for aster induction, the centriolar fraction was pre-treated with boiling temperature, and it was found that the fraction became incapable to induce any aster.
Results obtained suggest that the effective components of the sperm centriolar fraction to induce asters in the fertilized sea urchin eggs are the proteins but not the nucleic acids. The aster inducing activity is destroyed by heat treatment.     

The Cell Cycle Governs the Onset of Spherulation of Xenopus Eggs Fused by an Electric Field

The mechanism of electric field-induced fusion has been studied in detail (Zimmermann, Biochim. Biophys. Acta. 694 (1982) 227), but little is known about the process by which the two fused cells become a single entity, a process we term spherulation. We observe a clear difference between activated and unactivated Xenopus eggs in the time after electic field (EF) application when spherulation starts, and in the time required for spherulation to be completed. In unactivated eggs, spherulation started 7 min after EF application and was completed within 5 1/2 min. In activated eggs, the lag between EF application and the start of spherulation increased with the cell cycle. At the end of the first cell cycle spherulation started 78 min after EF application and was competed 30 min later. The lag period is not due to delayed fusion, for electric coupling between activated eggs can be recorded before the start of spherulation. The morphology of the contact zone between paired eggs, as observed by light and electron microscopy, is also described. We suggest that the difference in the timing of spherulation reflects a difference in the lability of the cytoskeleton through the cell cycle.     

Experiments Pertaining to the Suppression of Accessory Sperm in Fertilized Newt Eggs*

In the physiologically polyspermic eggs of the newt, Cynops pyrrhogaster, a number of accessory sperm undergo pronuclear formation along with a concomitant DNA synthesis, but degenerate after zygote nucleus formation. When denuded eggs were divided into two halves at various post-fertilization stages, the andromerogons produced before zygote nucleus formation but not after that stage cleaved at a high frequency. The accessory sperm were unable to participate in the cleavage when they were located in the half of the egg which was connected with the diploid merogon by a cytoplasmic bridge higher than 100 μm in height. The removal of the egg nucleus or the retardation of early post-fertilization nuclear events by treatment with cycloheximide resulted in the induction of multipolar cleavage. Continuous exposure of the fertilized eggs to aphidicolin showed that in the appreciable absence of the DNA synthesis many eggs underwent a first cleavage cytokinesis of a mostly abortive type, but failed to initiate the following cytokinesis at all. Cytological examinations in association with these experiments suggest that the observed suppression of accessory sperm includes the inhibition of centriolar replication under the influence of the zygote nucleus, resulting in the failure of cytasters corporating with nuclear-independent activity of cortical cytoplasm.     

Surrogate Production of Eggs and Sperm by Intrapapillary Transplantation of Germ Cells in Cytoablated Adult Fish

Germ cell transplantation (GCT) is a promising assisted reproductive technology for the conservation and propagation of endangered and valuable genetic resources. In teleost fish, GCT in adult gonads has been achieved only in male recipients, limiting greatly the usefulness of this technique in situations where both sexes need equal and timely attention for conservation and/or propagation. Here we describe a simplified GCT approach that ultimately leads to production of donor-derived eggs and sperm in considerably short time. Donor germ cells isolated from young pejerrey Odontesthes bonariensis (Atherinopsidae) were transplanted non-surgically through the genital papilla into the sexually mature gonads of Patagonian pejerrey O. hatcheri recipients whose gonads have been depleted of endogenous GCs by heat (26°C) and chemical treatment (four doses of Busulfan at 30 mg/kg and 40 mg/kg for females and males, respectively). Transplanted spermatogonial and oogonial cells were able to recolonize the recipients'' gonads and produce functional donor origin eggs and sperm within 7 months from the GCT. We confirmed the presence of donor-derived gametes by PCR in 17% and 5% of the surrogate O. hatcheri fathers and mothers, respectively. The crosses between surrogate fathers and O. bonariensis mothers yielded 12.6–39.7% pure O. bonariensis and that between a surrogate mother and an O. bonariensis father yielded 52.2% pure O. bonariensis offspring. Our findings confirm that transplantation of germ cells into sexually competent adult fish by non-surgical methods allows the production of functional donor-derived eggs and sperm in a considerably short time. The methods described here could play a vital role in conservation and rapid propagation of endangered fish genetic resources.     

Identification of Surface Antigens of Sarcocystis muris (Coccidia)

Zoites of Sarcocystis muris were recovered from the skeletal muscles of infected mice by trypsin digestion. Extracts of zoites prepared by freeze-thaw, Triton X-100 (0.1%), or a combination of the two treatments contained antigenic components. Testing of these antigens by agar gel diffusion and immunoelectrophoresis against sera from infected mice showed one major precipitin band. SDS-polyacrylamide-gel electrophoresis (SDS-PAGE) of the extracts revealed at least eight detectable polypeptides ranging in molecular weight from 10,000 to 220,000. The antigenic components of the extract were identified by labeling the parasite surface with [¹²⁵I] and precipitation of the [¹²⁵I]-labeled antigens with immune sera. Analysis of the immunoprecipitates by SDS-PAGE and autoradiography revealed three antigens with molecular weights of 27,500, 43,000 and 90,000. The smallest of these was the predominant antigen as suggested by labeling intensity.     

Response to Sperm Penetration of the Cortex of Eggs of the Fish, Plecoglossus altivelis

The response of the egg to sperm penetration was examined in eggs of the fish, Plecoglossus altivelis , by scanning electron microscopy. Eggs responded to sperm penetration by forming a fertilization cone at a "sperm entry site", which is a specialized structure in the egg surface under the micropyle. Within one minute, the fertilization cone showed dramatic morphological changes from its earliest appearance, through full two-storied growth to its marked recession. The sperm entry site in the egg surface is discussed as a morphologically specialized organ responsible for the entrance of a fertilizing spermatozoon. The morphological characteristics of the egg and sperm are also described.     

Sperm Penetration of the Vitelline Envelope of Sicyonia ingentis Eggs is Mediated by a Trypsin-like Lysin of Acrosomal Vesicle Origin

The sperm of the decapod crustacean Sicyonia ingentis are nonmotile, unistellate cells. At spawning, mated females release both stored sperm and eggs. The sperm bind, via the tip of their anterior appendage, to the egg's vitelline envelope (VE), rapidly undergo acrosomal exocytosis, and penetrate the VE. In the present study we used protease inhibitors to show that sperm penetration of the VE is due to the activity of a sperm trypsin-like protease(s). Sperm extracts contained several proteases when examined using gelatin-substrate SDS-PAGE, with two major bands of relative molecular weight 46 kD and 30 kD. Using fluorescent peptidyl-MCA substrates, sperm extract showed trypsin-like and aminopeptidase-like activities, but no chymotrypsin-like activity. Sperm extracts were found to degrade isolated VEs. Using soybean trypsin inhibitor and anti-inhibitor antibodies, protease was localized at the light and electron microscope levels to the acrosome remnants of reacted sperm.     

Selective degradation of the IκB kinase （IKK） by autophagy

Proteasome-mediated degradation and autophagy are the two major pathways mediating the turnover of cellular proteins. The proteasomal pathway is known to be a highly specific and regulated process mediating the degradation of short-lived proteins such as many important factors involved in cellular signaling. In contrast, it is generally thought that autophagy is rather nonselective as it is responsible for the bulk degradation of long-lived proteins and organelles. Challenging this general view, in this issue of Cell Research, Qing et al. report that selective degradation of the IκB kinase （IKK） triggered by the loss of Hsp90 function is mediated by autophagy [1].     

用原子力显微镜观察人类精子的表面结构

目的:探讨用原子力显微镜观察精子表面结构的方法。方法:经常规洗涤正常人精液后进一步除去精子表面和生理溶液中的蛋白质,直接用原子力显微镜观察人类精子的表面形态。结果:获得了人类精子表面的细微结构和精子头的三维数据。精子全长约47μm,精子头约4.6μm,顶体位于精子头前端1/2~2/3,顶体前端扁平。精子赤道区有两圈环形凸起。结论:不需特殊处理,用原子力显微镜能直接观察精子表面的超微结构,并获得量化的三维数据。     

Echinoderm faunas from the Lower Cretaceous (Aptian–Albian) of Alexander Island,Antarctica

Abstract: Strata assigned to the Fossil Bluff Group on Alexander Island, Antarctica, contain Aptian to Albian high‐latitude echinoderm faunas that lived at palaeolatitudes greater than 60 degrees south. The Pluto Glacier Formation, of essentially Aptian age, yields a deep‐water assemblage that includes two ophiuroids, an ophiacanthid and a representative of the ophiolepidid genus Mesophiomusium, both represented by partially articulated specimens. The echinoid fauna includes a new genus of diadematoid, Australidiadema, and a new genus of disasteroid, Notidisaster, which extend the record of both groups into the southern hemisphere. The overlying Neptune Glacier Formation, of late Albian age, yields only spatangoids which are common but rarely well enough preserved to be identified even to genus level, although at least some belong to the genus Hemiaster.     

Selective cell death by water-soluble Fe-porphyrins with superoxide dismutase (SOD) activity

We investigated the effect on cell death of reactive oxygen species induced by [[5,10 (or 5,15)-bis(N-methyl-4-pyridyl)-15,20 (or 10,20) diphenyl]porphinato]iron (cis-FeMPy(2)P(2)P or trans-FeMPy(2)P(2)P) with SOD activity. The SOD activities of the cis-FeMPy(2)P(2)P and trans-FeMPy(2)P(2)P were measured using stopped-flow kinetic analysis. The cell viability of four cell lines treated with cis-Fe-porphyrin, trans-Fe-porphyrin, mitomycin c (MMC), or cisplatin was estimated by the alamar blue exclusion assay of the modified MTT method. The amount of cis-FeMPy(2)P(2)P and trans-FeMPy(2)P(2)P in the Walker 256 cultured for 24 h was 4.0 and 2.6 fmolcell(-1), respectively, indicating that the plasma membrane permeability of the Fe-porphyrins depended on their structure. Cis-FeMPy(2)P(2)P selectively killed Walker 256 and H-4-II-E as cancer cells but not FR and BRL-3A as normal cells and showed a significant cytotoxicity for the cancer cells compared with trans-FeMPy(2)P(2)P, MMC and cisplatin. We believe that cis-FeMPy(2)P(2)P as an SOD mimic converts intracellular O(2)(*-) to H(2)O(2) and that H(2)O(2) or *OH causes DNA damage and induces cell death. This result suggests that for the SOD mimic, O(2)(*-) may be useful as a target molecule to induce selective cell death between cancer and normal cells and that a metalloporphyrin having SOD activity is a new class of anticancer agents.     

Pre-loading of mouse oocytes with DNA-specific fluorochrome (Hoechst 33342) permits rapid detection of sperm-oocyte fusion

Mouse oocytes exposed to 1 microgram Hoechst 33342 (H-33342)/ml and then fertilized in vitro developed normally into blastocysts and blastocyst outgrowths. After penetration of the zona, the fertilizing spermatozoon showed intense fluorescence upon fusion with the vitelline membrane. Due to fluorochrome leakage from the perivitelline space a faint fluorescence was detected in zona-bound spermatozoa. This fluorescence of zona-bound spermatozoa intensified with increased fluorochrome concentration (10 micrograms/ml), obscuring the fluorescence of the fertilizing spermatozoa. Spermatozoa added to zona-free mouse oocytes (pre-loaded with 1 or 10 micrograms H-33342/ml) fluoresced within 10 min of insemination, provided the zonae were removed mechanically. Removal by protease digestion induced leakage of fluorochrome, so that all spermatozoa in the vicinity of an oocyte pre-loaded with 10 micrograms H-33342/ml became labelled. This leakage was not visibly apparent when protease-treated oocytes were exposed to only 1 microgram H-33342/ml. The technique could not be applied to zona-free hamster oocytes under our conditions, since the fluorochrome leaked freely from the oocytes whether the zona was removed mechanically or enzymically.     

Identification of Sporozoite Surface Proteins and Antigens of Eimeria nieschulzi (Apicomplexa)

Sodium dodecyl sulfate polyacrylamide gel electrophoresis, immunoblotting, lectin binding, and ¹²⁵I surface labeling of sporozoites were used to probe sporozoites of the rat coccidian, Eimeria nieschulzi. Analysis of silver stained gels revealed >50 bands. Surface iodination revealed about 14 well labeled, and about 10 weakly labeled but potential, surface proteins. The most heavily labeled surface proteins had molecular masses of 60, 53–54, 45, 28, 23–24, 17, 15, 14, 13, and 12 kD. Following electrophoresis and Western blotting, 2 of the 12 ¹²⁵I labeled lectin probes bound to two bands on the blots, which collectively indicated that two bands were glycosylated. Concanavalin A (ConA) specifically recognized a band at 53 kD, which may represent a surface glycoprotein, and a lectin derived from Osage orange (MPA) bound to a single band at 82–88 kD, that may also be a surface molecule. Immunoblotting using sera collected from rats inoculated orally with oocysts, as well as sera from mice hyperimmunized with sporozoites, revealed that many surface molecules appear to be immunogenic.     

Cloning of Fatso (Fto), a novel gene deleted by the Fused toes (Ft) mouse mutation

The Fused toes (Ft) mouse mutation was created by insertional mutagenesis, resulting in the deletion of several hundred kb of genomic sequences of mouse Chromosome (Chr) 8. Mice heterozygous for the Ft mutation are characterized by partial syndactyly of forelimbs and massive thymic hyperplasia indicating that programmed cell death is affected. Homozygous Ft/Ft embryos die at midgestation and show severe malformations of craniofacial structures. Furthermore, establishment of left-right asymmetry is random. Here we report on the positional cloning of a novel gene by exon trap analysis of a genomic clone encoding wild-type sequences corresponding to parts of the deletion in Ft mutants. RT-PCR experiments demonstrated that the newly identified gene, Fatso (Fto), is expressed throughout embryonic development. Wide expression was also found in tissues of adult mice. We show that expression of Fto is completely absent in mouse embryonic fibroblasts homozygous for the Ft mutation. In addition, we isolated the full-length cDNA which encodes a putative 58-kDa protein showing no similarities to known proteins or protein motifs. The expression data of Fto define it as a candidate gene involved in processes such as programmed cell death, craniofacial development, and establishment of left-right asymmetry. Received: 5 May 1999 / Accepted: 9 June 1999     

Sperm ultrastructure of the honey bee (Apis mellifera) (L) (Hymenoptera, Apidae) with emphasis on the nucleus-flagellum transition region

The flagellum of Apis mellifera (Hymenoptera, Apidae) consists of two mitochondrial derivatives, an axoneme and two accessory bodies. The mitochondrial derivatives are of unequal size and lie parallel to the axoneme. In the larger derivative four regions can be distinguished while in the smaller, only three. The region occurring only in the larger derivative consists of paracystalline material. The smaller mitochondrial derivative terminates anterior to the larger one. An extremely long centriolar adjunct is observed between the nucleus and the smaller mitochondrial derivative. This adjunct is compact, very electron dense and gradually tapers from base toward apex, finishing at the anterior extremity of the axonemal microtubules. In this flagellar region, there is only one accessory body present between the larger mitochondrial derivative and the axoneme. Anteriorly, the tips of the axonemal microtubules are inserted in a well developed mass of granular appearance. This material surrounds the nuclear base, separating it from the anterior end of the larger mitochondrial derivative. We believe that the structure identified here as a centriolar adjunct is homologous to that observed in Formicidae, Ichneumonoidea and Symphyta. Therefore, very probably, it is common to most Hymenoptera.