Polar Cytoplasm Research Articles

Staufen1, Kinesin1 and microtubule function in cyclin B1 mRNA transport to the animal polar cytoplasm of zebrafish oocytes

In zebrafish oocytes, cyclin B1 mRNAs are transported to the animal polar cytoplasm. To elucidate the molecular basis of cyclin B1 mRNA transport, we analyzed zebrafish Staufen1, a protein known to play a central role in mRNA transport to the vegetal pole of Xenopus oocytes. Zebrafish Staufen1 interacts with cyclin B1 mRNA throughout oocyte growth. Both cyclin B1 mRNA and Staufen1 are evenly distributed in the cytoplasm of young oocytes but are co-localized to the animal polar cytoplasm in later stages. Real-time imaging showed that the plus ends of oocyte microtubules are free in the cytoplasm in early stages but anchored to the animal polar cytoplasm in later stages. Transport of cyclin B1 reporter mRNA to the animal polar cytoplasm was inhibited by disruption of microtubules and injection of antibodies against Staufen1 or Kinesin1, a plus-end-directed microtubule motor that interacts with Staufen1, indicating that the transport depends on movement along microtubules toward the plus ends. Reporter mRNAs with an element required for the vegetal localization of vg1 mRNA in Xenopus oocytes were localized to the animal polar cytoplasm in zebrafish oocytes, indicating that the element is functional for animal polar localization in zebrafish oocytes. Our findings suggest that cyclin B1 mRNA-Staufen1 protein complexes are transported toward the animal pole of zebrafish oocytes by the plus-end-directed motor protein Kinesin1 along microtubules and that a common mRNA transport machinery functions in zebrafish and Xenopus oocytes, although its transport direction is opposite due to different organizations of microtubules.

Variability of morphological parameters as a criterion to forecast efficiency of cryopreserved human oocytes.

Background. Cryopreservation of oocytes becomes increasingly important in the programs of assisted reproductive technologies, as it avoids the ethical and legal problems associated with the cryopreservation of embryos. Objective. The purpose of the work was to study the morphological parameters of the oocytes and their impact on the effectiveness of cryopreservation. Methods. This is a retrospective data analysis of IVF cycles using vitrified oocytes. The 724 mature oocytes with dysmorphism and normal morphology were vitrified using CryoTec by vitrification method. A total of 724 mature oocytes were vitrified. P-level critical value was 0,05. Results It was shown that evaluation and selection of the most promising cell material prior to freezing can be carried out using the qualitative morphological criteria, as there has been revealed the relationship between these criteria and the success of cryopreservation. Qualitative parameters to select the cryoresistant oocytes are as follows: state of the first polar body, Zona pellucida and cytoplasm. The results showed that classification based on oocytes morphology revealed a significant correlation with survival after cryopreservation, fertilization rate and in vitro development of embryos to blastocyst stage. Conclusion. The proposed criteria for assessing the morphofunctional state of oocytes prior to cryopreservation contribute to the effectiveness of the treatment of infertility by means of in vitro fertilization.

Formation of mos RNA granules in the zebrafish oocyte that differ from cyclin B1 RNA granules in distribution, density and regulation

Many translationally repressed mRNAs are deposited in the oocyte cytoplasm for progression of the meiotic cell cycle and early development. mos and cyclin B1 mRNAs encode proteins promoting oocyte meiosis, and translational control of these mRNAs is important for normal progression of meiotic cell division. We previously demonstrated that cyclin B1 mRNA forms RNA granules in the zebrafish and mouse oocyte cytoplasm and that the formation of RNA granules is crucial for regulating the timing of translational activation of the mRNA. However, whether the granule formation is specific to cyclin B1 mRNA remains unknown. In this study, we found that zebrafish mos mRNA forms granules distinct from those of cyclin B1 mRNA. Fluorescent in situ hybridization analysis showed that cyclin B1 RNA granules were assembled in dense clusters, while mos RNA granules were distributed diffusely in the animal polar cytoplasm. Sucrose density gradient ultracentrifugation analysis showed that the density of mos RNA granules was partly lower than that of cyclin B1 mRNA. Similar to cyclin B1 RNA granules, mos RNA granules were disassembled after initiation of oocyte maturation at the timing at which the poly(A) tail was elongated. However, while almost all of the granules of cyclin B1 were disassembled simultaneously, a fraction of mos RNA granules firstly disappeared and then a large part of them was disassembled. In addition, while cyclin B1 RNA granules were disassembled in a manner dependent on actin filament depolymerization, certain fractions of mos RNA granules were disassembled independently of actin filaments. These results suggest that cytoplasmic regulation of translationally repressed mRNAs by formation of different RNA granules is a key mechanism for translational control of distinct mRNAs in the oocyte.

Intracellular ROS protection efficiency and free radical-scavenging activity of quercetin and quercetin-encapsulated liposomes

Quercetin (3,5,7,3’,4’-pentahydroxyflavone) is a natural bio-flavonoid originating from fruits, vegetables, seeds, berries, and tea. The antioxidant activity of quercetin and its protective effects against cardiovascular disorders, anti-cancer, anti-inflammatory, and anti-viral activities have been extensively documented; however, the clinical request of quercetin in cancer treatment is significantly limited due to its very poor delivery features. In order to increase the hydrophilicity and drug delivery capability, we encapsulated quercetin into liposomes. Our data indicated that liposomal quercetin can significantly improve the solubility and bioavailability of quercetin and can be used as an effective antioxidant for ROS protection within the polar cytoplasm, and the nano-sized quercetin encapsulated by liposomes enhanced the cellular uptake (cancer cell human MCF_7). Quercetin has many pharmaceutical applications, many of which arise from its potent antioxidant properties. The present research examined the antioxidant activities of quercetin in polar solvents by a comparative study using reduction of ferric iron in aqueous medium, intracellular ROS/toxicity assays, and reducing DPPH assays. Cell viability and ROS assays demonstrated that quercetin was able to penetrate into the polar medium inside the cells and to protect them against the highly toxic and deadly belongings of cumene hydroperoxide. The purpose of this study was to determine whether a liposomal formulation of quercetin can suggestively improve its solubility and bioavailability and can be a possible request in the treatment of tumor. The authors encapsulated quercetin in a liposomal delivery system. They studied the in vitro effects of this compound on proliferation using human MCF-7 carcinoma cells. The activity of liposomal quercetin was equal to or better than that of free quercetin at equimolar concentrations. Our data indicated that liposomal quercetin can significantly improve the solubility and bioavailability of quercetin and can be a potential application in the treatment of tumor.

Possible involvement of insulin-like growth factor 2 mRNA-binding protein 3 in zebrafish oocyte maturation as a novel cyclin B1 mRNA-binding protein that represses the translation in immature oocytes

In immature zebrafish oocytes, dormant cyclin B1 mRNAs localize to the animal polar cytoplasm as aggregates. After hormonal stimulation, cyclin B1 mRNAs are dispersed and translationally activated, which are necessary and sufficient for the induction of zebrafish oocyte maturation. Besides cytoplasmic polyadenylation element-binding protein (CPEB) and cis-acting elements in the 3′ untranslated region (UTR), Pumilio1 and a cis-acting element in the coding region of cyclin B1 mRNA are important for the subcellular localization and timing of translational activation of the mRNA. However, mechanisms underlying the spatio-temporal control of cyclin B1 mRNA translation during oocyte maturation are not fully understood. We report that insulin-like growth factor 2 mRNA-binding protein 3 (IMP3), which was initially described as a protein bound to Vg1 mRNA localized to the vegetal pole of Xenopus oocytes, binds to the 3′ UTR of cyclin B1 mRNA that localizes to the animal pole of zebrafish oocytes. IMP3 and cyclin B1 mRNA co-localize to the animal polar cytoplasm of immature oocytes, but in mature oocytes, IMP3 dissociates from the mRNA despite the fact that its protein content and phosphorylation state are unchanged during oocyte maturation. IMP3 interacts with Pumilio1 and CPEB in an mRNA-dependent manner in immature oocytes but not in mature oocytes. Overexpression of IMP3 and injection of anti-IMP3 antibody delayed the progression of oocyte maturation. On the basis of these results, we propose that IMP3 represses the translation of cyclin B1 mRNA in immature zebrafish oocytes and that its release from the mRNA triggers the translational activation.

A cis-acting element in the coding region of cyclin B1 mRNA couples subcellular localization to translational timing

Subcellular localization of messenger RNAs (mRNAs) to correct sites and translational activation at appropriate timings are crucial for normal progression of various biological events. However, a molecular link between the spatial regulation and temporal regulation remains unresolved. In immature zebrafish oocytes, translationally repressed cyclin B1 mRNA is localized to the animal polar cytoplasm and its temporally regulated translational activation in response to a maturation-inducing hormone is essential to promote oocyte maturation. We previously reported that the coding region of cyclin B1 mRNA is required for the spatio-temporal regulation. Here, we report that a sequence, CAGGAGACC, that is conserved in the coding region of vertebrate cyclin B1 mRNA is involved in the regulation. Like endogenous cyclin B1 mRNA, reporter mRNAs harboring the sequence CAGGAGACC were localized to the animal polar cytoplasm of oocytes, while those carrying mutations in the sequence (with no change in the coding amino acids) were dispersed in the animal hemisphere of oocytes. Furthermore, translational activation of the mutant mRNAs was initiated at a timing earlier than that of endogenous and wild-type reporter mRNAs during oocyte maturation. Interaction of CAGGAGACC with proteins in vitro suggests that this sequence functions in collaboration with a trans-acting protein factor(s) in oocytes. These findings reveal that the sequence in the coding region of cyclin B1 mRNA plays an important role as a cis-acting element in both subcellular localization and translational timing of mRNA, providing a direct molecular link between the spatial and temporal regulation of mRNA translation.

Intracellular ROS Protection Efficiency and Free Radical-Scavenging Activity of Curcumin

Curcumin has many pharmaceutical applications, many of which arise from its potent antioxidant properties. The present research examined the antioxidant activities of curcumin in polar solvents by a comparative study using ESR, reduction of ferric iron in aqueous medium and intracellular ROS/toxicity assays. ESR data indicated that the steric hindrance among adjacent big size groups within a galvinoxyl molecule limited the curcumin to scavenge galvinoxyl radicals effectively, while curcumin showed a powerful capacity for scavenging intracellular smaller oxidative molecules such as H2O2, HO•, ROO•. Cell viability and ROS assays demonstrated that curcumin was able to penetrate into the polar medium inside the cells and to protect them against the highly toxic and lethal effects of cumene hydroperoxide. Curcumin also showed good electron-transfer capability, with greater activity than trolox in aqueous solution. Curcumin can readily transfer electron or easily donate H-atom from two phenolic sites to scavenge free radicals. The excellent electron transfer capability of curcumin is because of its unique structure and different functional groups, including a β-diketone and several π electrons that have the capacity to conjugate between two phenyl rings. Therfore, since curcumin is inherently a lipophilic compound, because of its superb intracellular ROS scavenging activity, it can be used as an effective antioxidant for ROS protection within the polar cytoplasm.

Transgenic zebrafish reveals novel mechanisms of translational control of cyclin B1 mRNA in oocytes

Temporal translation control of localized mRNA is crucial for regulating various cellular and developmental processes. However, little is known about the mechanisms of temporal translation control of localized mRNA due to the limitation in technology. cyclin B1 mRNA at the animal polar cytoplasm of immature zebrafish oocytes is translationally repressed, and its activation is temporally regulated during maturation. Mechanisms of cyclin B1 translation in oocytes were analyzed using transgenic zebrafish in which reporter mRNAs are produced from transgenes introduced into the genome through transcription in the nucleus followed by transport to the cytoplasm, as in endogenous mRNAs. Real-time imaging of the site and timing of translation showed that mRNAs containing the full-length cyclin B1 sequence precisely mimic the localization and translation of endogenous cyclin B1 mRNA. However, mRNAs containing cyclin B1 3′ untranslated region but lacking open reading frame (ORF) underwent abnormal localization and precocious translational activation, indicating the significance of the ORF in translational control of cyclin B1 mRNA. Our genetic approach in combination with real-time imaging of the translation site and timing provides a novel insight into the mechanisms of temporal control of translation.

Electron paramagnetic resonance study of amphiphiles partitioning behavior in desiccation-tolerant moss during dehydration

Desiccation tolerance is a crucial characteristic for desert moss surviving in arid regions. Desiccation procedure always induces amphiphiles transferring from the polar cytoplasm into lipid bodies. The behavior of amphiphiles transferring can contribute to the enhancement of desiccation tolerance and the reduction of plasma membrane integrity simultaneously. The effects of amphiphiles partitioning into the lipid phase during water loss has been studied for pollen and seeds using electron paramagnetic resonance (EPR) spectroscopy. However, desiccation-tolerant high plants occur among mosses, several angiosperms and higher plants seeds or pollens. They have different strategies for survival in dehydration and rehydration. A desiccation-tolerant moss Tortula desertorum was used to investigate the behaviors of amphiphilic molecules during drying by spin label technology. There are small amount of amphiphilic probes partitioning into membrane during moss leaves dehydration, comparing with that in higher plants. Cytoplasm viscosity changed from 1.14 into glass state only dehydration less than 60 min. Moss leaves lost plasma membrane integrity slightly, from 0.115 to 0.237, occurred simultaneously with amphiphiles partition. The results showed the more advantages of mosses than higher plants in adapting fast dehydration. We propose that EPR spin label is feasible for studying the amphiphiles partitioning mechanisms in membrane protection and damage for desiccation-tolerant mosses.

59 IN VITRO DEVELOPMENT OF FELINE CLONED EMBRYOS RECONSTRUCTED WITH PREADIPOCYTES

The technique of somatic cell nuclear transfer (NT) in domestic cats is expected to contribute to the conservation of wildcats, for which extinction is a concern. In this study, we examined in vitro developmental ability of cloned embryos produced using the preadipocytes of domestic cats as nuclear donors. Primary cultures of preadipocytes were established as reported previously (Yagi et al. 2004 Biochem. Biophys. Res. Commun. 321, 967–974). Briefly, fat tissue (2–3 g) was excised from an adult female cat and digested using 0.1% collagenase for 1 h at 37�C followed by centrifugation. Only mature adipocytes that were floating near the surface of the supernatant were collected and placed in a 12.5-cm2 culture flask filled with DMEM containing 20% FBS. The flask was filled with medium, tightly capped, and cultured upside down for 7–10 days, so that the floating adipocytes attached to the inner ceiling surface of the flask. When firm attachment of the cells to the ceiling surface of the flask was confirmed, the flask was then inverted and culture was continued using the routine cell culture technique for adherent cells. In vivo-matured oocytes were collected from the ovaries of domestic cats superovulated by eCG and hCG. IVM oocytes were obtained by culturing cumulus–oocyte complexes from the ovaries collected at local veterinary clinics in TCM199-based medium for 24 to 30 h. In vivo-matured and IVM oocytes were enucleated by aspirating the first polar body and adjacent cytoplasm using a bevelled pipette in the presence of 7.5 µg mL–1 cytochalasin B. The nuclei of the donor cells were transferred to enucleated in vivo-matured and IVM oocytes by means of electric fusion (300 V mm–1, 30 µs, twice). The reconstructed embryos were activated electrically (125 V mm–1, 60 µs, twice), followed by treatment with 10 µg mL–1 cycloheximide and 5 µg mL–1 cytochalasin B. The cloned embryos were cultured in vitro for 7 days in MK-1 so that their developmental ability might be examined. The fusion rate of donor cells was similar between in vivo-matured and IVM oocytes (56.8%, 21/37 v. 54.5%, 42/77). The developmental ability of NT embryos reconstructed with in vivo-matured oocytes was similar to that of NT embryos reconstructed with IVM oocytes (cleavage: 52.4%, 11/21 v. 42.9%, 18/42; development to blastocysts: 9.5%, 2/21 v. 11.9%, 5/42). The results indicate that cloned feline embryos reconstructed using preadipocytes can develop in vitro into blastocysts.

Parental exposure to low-dose X-rays in Drosophila melanogaster induces early emergence in offspring, which can be modulated by transplantation of polar cytoplasm

In recent years there has been growing concern over the biological effects of low-dose X-rays, but few studies have addressed this issue. Our laboratory had observed flies ( Drosophila melanogaster) irradiated with low-dose X-rays tend to emerge earlier than normal flies. This observation led us to quantitatively examine the effects of low-dose X-irradiation on development in the fly. Following exposure of prepupal (day 5) flies to 0.5 Gy X-rays, the time to emergence was slightly shorter than in the sham controls. This tendency was increased when the X-ray exposure came during the pupal stage (day 7). In these flies, the time to eclosion decreased significantly, by an average of 30 h sooner than sham controls. A further experiment examined whether such radiation effects could be observed in the unexposed F1 generation of exposed individuals. Greater radiation effects on early F1 emergence were seen when the time between exposure and mating was 3 days, indicating an effect on early spermatid development. Early F1 emergence was also observed after exposure of female flies to X-rays during late previtellogeny. Furthermore, rapid emergence could be induced in the F1 embryos of unexposed parents by transferring the polar cytoplasm (precursor cells of the germ cell line) from F1 embryos of exposed flies. These results show that radiation-induced effects can be transmitted to the next generation through the germ cell line.

Factors Affecting Enucleation Rates of Bovine and Porcine Oocytes After Removal of Cumulus Cells by Vortexing.

In vitro matured bovine and porcine oocytes were enucleated by aspiration of the first polar body and adjacent cytoplasm after removing the cumulus cells by vortexing and the factors affecting enucleation rate were investigated. The results indicate that vortexing cumulus-oocyte complexes for long periods in hyperosmotic media moves the first polar body and reduces the enucleation rate. Removal of 20% cytoplasmic volume increased enucleation rate compared with that of 10% and showed no deleterious effect on subsequent developmental capacity of reconstructed bovine embryos. Aging of oocytes could also reduce enucleation efficiency by inducing the migration of the chromatin material to the center of the oocyte. Therefore, we recommend to enucleate newly matured bovine and porcine oocytes by removing the first polar body and adjacent 20% of ooplasm after the removal of cumulus cells by vortexing for a short period using a hypo-osmotic media to achieve a high enucleation rate .

Evidence for polar cytoplasm/nuage in rat oocytes

In many organisms oocytes contain dark-staining material, termed nuage, that is concentrated at one pole of the oocyte cytoplasm and that influences the further development of the oocyte after fertilization. In mammalian oocytes, ultrastructural studies have detected small patches of nuage-like material, but thus far no nuage-rich zone of polar cytoplasm has been reported. Here, we report that when large sections of rat ovary embedded in methacrylate resin are stained with toluidine blue and surveyed, many oocytes contain a narrow, sharply defined, basophilic zone of polar cytoplasm that appears analogous to the polar cytoplasm of Xenopus and other non-mammalian species. This basophilic polar cytoplasm was common in multilaminar follicles and was not visible in smaller, primordial follicles. In one out of five oocytes stimulated with hCG to complete the first meiotic division, a relatively faint region of cortical basophilia was detectable. Further studies will be needed to ascertain if this nuage-like material has an influence upon the development of oocytes similar to that seen in non-mammalian species.

Basal cells in human bronchial epithelium.

The morphology and distribution of basal cells were investigated in adult human bronchial epithelium. In both smokers and non-smokers typical basal cells were more numerous than atypical basal cells, which were distinguished by their spindle-shaped nucleus, polar cytoplasm, and processes that extended along the basal lamina. The nucleus of the atypical basal cell was consistently closer to the muco-ciliary surface than was the nucleus of the typical basal cell. In cross-sections of bronchi the numbers of typical basal cells/mm epithelium were greatest in large airways; in smaller bronchi (i.e., generations 7-16) the frequency declined. The numbers of atypical basal cells/mm epithelium were similar throughout the bronchial tree. Throughout the bronchial tree both types of basal cells contributed to the maintenance of epithelial cohesion by providing desmosomal attachment for columnar cells. The importance of typical basal cells in this role was indicated by their greater numbers, which collectively presented a large surface for epithelial cell attachment by desmosomes. The surface presented by the cell body and processes of each atypical basal cell for attachment of columnar cells by desmosomes was extensive. If the basal cell is the most important progenitor of bronchial epithelium and the cell at risk in the development of lung cancer, the presence of more basal cells in upper airways, where many lung cancers originate, may be significant. The basal cell populations in larger bronchi are perhaps the greatest concentration of cells with proliferative capability and potential for neoplastic transformation in the human bronchial tree.

Early embryonic development of the grey short‐tailed opossum, Monodelphis domestica, in vivo and in vitro

Eggs and embryos (up to the unilaminar blastocyst stage) were collected from grey short‐tailed opossums, Monodelphis domestica, at known time intervals after mating. Some were fixed immediately for examination by light and electron microscopy while others were maintained in culture at 37 °C in 5% CO2 in Ham's F10 medium containing foetal calf serum. Cleavage to the early blastocyst stage was obtained in vitro although most embryos displayed arrested development at the eight‐cell stage. From gross morphological and ultrastructural observations a pattern of embryogenesis to the unilaminar blastocyst was constructed. Comparisons were made with the events of early development in other marsupials.SummaryOocytes were fertilized soon after ovulation and their zonae pellucidae surrounded by a mucoid layer originating from mucopolysaccharide secretion of oviductal epithelial cells. Within 24 hours zygotes passed into the uterus and shell membrane formation commenced. The early embryonic development in vivo and in culture of the grey short‐tailed opossum, Monodelphis domestica, was investigated by light and electron microscopy. The following timetable of events was constructed: The first cleavage at about 28 hours after ovulation (54 hours after mating) was at right angles to the equator of the zygote and was accompanied by the vesiculation of yolk mass. The resulting two blastomeres were composed of peripheral polar cytoplasm. The second cleavage (44 hours) and third cleavage (approx. 72 hours) produced small blastomeres with no cell contact. Yolky vesicles surrounded the blastomeres. The fourth cleavage division (approx. 96 hours) occurred in the same equatorial plane as the second resulting in two tiers of cells which subsequently flattened against the inner surface of the zona pellucida. Tight junctions formed between adjacent blastomeres. Unilaminar blastocysts were formed about 5 days after fertilization. Development in vitro followed a similar time course to that in vivo, however, most embryos were arrested at the 8‐cell stage.

INCOMPLETE CYTOKINESIS IN FUNARIA STOMATA

The development of the one‐celled condition in Funaria (Musci) stomata was investigated using light and electron microscopy. The guard cell parent cell is unusual in that it undergoes karyokinesis but incomplete cytokinesis. The septal wall, and the cell plate from which it forms, have incurved edges in contact with the polar cytoplasm. No evidence was found to support Haberlandt's claim that the stomate is initially two celled but undergoes wall resorption. Preprophase microtubule bands appear to be present in nonstomatal epidermal cells with normal cytokinesis, but the possibility is raised that they are absent in guard cell parent cells.

PROTEIN SYNTHESIS IN NORMAL AND LOBELESS GASTRULAE OF ILYANASSA OBSOLETA.

Several hundred proteins synthesized by normal and lobeless Ilyanassa gastrulae were identified by the two-dimensional electrophoresis of polypeptides labeled in vivo with 35S-methionine. Acidic proteins were separated in the first dimension by isoelectric focusing and basic proteins by non-equilibrium pH gradient electrophoresis (NEPHGE). No qualitative differences were detected among either the acidic or basic polypeptides produced by normal or lobeless gastrulae. These findings show, for those peptides detected in this analysis, that (1) the stage-specific changes in protein synthesis that occur in Ilyanassa embryos by gastrulation (Collier and McCarthy, 1981) are not polar lobe dependent, and (2) the polar lobe cytoplasm does not qualitatively affect the expression of either maternal or embryonic mRNAs during gastrulation.

Regulation of Polypeptide Synthesis During Early Embryogenesis of Ilyanassa obsoleta

We have shown by two-dimensional electrophoresis of in vivo labeled polypeptides that stage-specific changes in polypeptide synthesis occurred during the early embryogenesis of the Ilyanassa embryo, and that more than 98% of the 350 polypeptides synthesized during this time were translated from maternal mRNAs. The removal of the polar lobe had no effect on polypeptide synthesis during early cleavage, but by the mesentoblast-stage there had occurred in the lobeless embryo a change in the rate of accumulation of 11 polypeptides, all of which were translated from maternal mRNAs. We have found no unequivocal evidence for the differential localization of mRNAs in the polar lobe. Our observations on polypeptide synthesis in the isolated polar lobe have indicated that the cytoplasm of the polar lobe contains both repressors of translation and activators of mRNPs. In embryos treated with actinomycin D we have observed an increased rate of accumulation of 14 polypeptides at the mesentoblast-stage of development. We have interpreted this as an example of ‘superinduction’ that resulted from transcriptionally dependent events repressed by actinomycin D. We have postulated that this actinomycin D-sensitivity indicates the transcription of regulatory genes. These genes produce translational repressors that decrease the rate of translation of these 14 polypeptides in the normal embryo. Because the synthesis of six of these polypeptides is also increased by removing the polar lobe, we suggest that the transcription of the regulatory genes controlling the translation of these six peptides is induced by the polar lobe cytoplasm.

Cytokinesis: Cleavage furrow establishment in cylindrical sand dollar eggs

AbstractBy confining fertilized, denuded sand dollar (Echinarachnius parma) eggs in short lengths of capillary tubing so that they were reshaped into elongate cylinders it was possible to control the orientation of both the mitotic apparatus and the subsequent cleavage furrow as well as the distance from the mitotic apparatus to the cylindrical cell surface. The technique left the polar regions accessible to experimentation and was used before first and second cleavages.The time when the equatorial surface becomes capable of forming a complete, permanent furrow which can function independently of the mitotic apparatus was determined by aspirating the mitotic apparatus from one of a pair of cylindrical cells at second cleavage and timing the interval between the operation and the appearance of the furrow in the control cell. When the mitotic apparatus is removed 4 minutes or less before control furrowing, the operated cell cleaves, but when the same operation is accomplished 5 minutes or more before control division, furrows do not appear in the operated cell. The results imply that the necessary interaction between the mitotic apparatus and the surface is not completed until 4 minutes before the furrow initiates function, and that thereafter the mitotic apparatus is dispensable. The distance between astral centers and the extent of their radiate structure continue growing after the position of the furrow is established.Experiments performed before the mitotic apparatus becomes dispensable, during the period of its interaction with the surface, revealed that agitation of the poles by vibration and repeated manipulation did not interfere with division when imposed continuously until the furrow appeared. Changing the shape of the poles did not affect cleavage; neither did aspiration of polar cytoplasm and polar portions of asters before both first and second cleavage. Cleavage also occurred despite sequestration of the polar portions of both asters in large‐bore pipettes. Repeated skewering of the mitotic apparatus along the length of its midline often altered its appearance but did not prevent division.

Differential responses to ultraviolet irradiation of the polar cytoplasm ofDrosophila eggs: II. Response to dose.

The posterior pole cytoplasm of early embryos ofDrosophila melanogaster was irradiated with increasing doses of U.V. light at a wave length specific for nucleic acids 35 and 75 min after ovoposition, when nuclei were still inside the yolk mass. Probit analysis showed that:Sterility was higher in irradiations performed at 35 min.Mortality was higher in the experiment performed at 75 min.After treatment at 35 min, sterility and mortality were independent phenomena.The numbers of targets involved in germ cell determination were of the order of tens and hundreds at 35 and 75 min respectively.The numbers of targets involved in viability were of several orders of magnitude in both developmental stages.The irradiation, even at very low doses, caused a background effect which was probably due to "old" embryos. The sex ratio was distorted in favour of females at high doses and of males at low doses.