Pattern recognition receptors involved in the immune system of hagfish (Eptatretus burgeri).

The initial defense against invading pathogenic microbes is the activation of innate immunity by binding of pattern recognition receptors (PRRs) to pathogen associated molecular patterns (PAMPs). To explain the action of PRRs from hagfish, one of the extant jawless vertebrates, we purified the GlcNAc recognition complex (GRC) from serum using GlcNAc-agarose. The GRC comprises four proteins of varying molecular masses: 19 kDa, 26 kDa, 27 kDa, and 31 kDa. Exposure of Escherichia coli to the GRC led to the phagocytic activation of macrophages, revealing the opsonic function of the GRC. The GRC in serum formed a large complex with a molecular mass of approximately 1200 kDa. The GRC bound to Escherichia coli but not to rabbit red blood cells, despite both having GlcNAc on their surface. These structural and binding properties are similar to those of mannose-binding lectin (MBL). The amino acid sequence of a portion of the 31 kDa protein in the GRC matched the amino acid sequence of variable lymphocyte receptor (VLR)-B in some place. According to the Western blot analysis, the 31 kDa protein was recognized by the anti-hagfish VLR-B antiserum. Based on the results, it appears that the GRC functions as a PRR like MBL and that its 31 kDa protein has a structure similar to that of VLR-B.

Distribution of XTdrd6/Xtr protein during oogenesis and early development in Xenopus laevis: Zygotic translation begins only in germ cells that have entered the genital ridge.

We previously identified Xenopus tudor domain containing 6/Xenopus tudor repeat (Xtdrd6/Xtr), which was exclusively expressed in the germ cells of adult Xenopus laevis. Western blot analysis showed that the XTdrd6/Xtr protein was translated in St. I/II oocytes and persisted as a maternal factor until the tailbud stage. XTdrd6/Xtr has been reported to be essential for the translation of maternal mRNA involved in oocyte meiosis. In the present study, we examined the distribution of the XTdrd6/Xtr protein during oogenesis and early development, to predict the time point of its action during development. First, we showed that XTdrd6/Xtr is localized to germinal granules in the germplasm by electron microscopy. XTdrd6/Xtr was found to be localized to the origin of the germplasm, the mitochondrial cloud of St. I oocytes, during oogenesis. Notably, XTdrd6/Xtr was also found to be localized around the nuclear membrane of St. I oocytes. This suggests that XTdrd6/Xtr may immediately interact with some mRNAs that emerge from the nucleus and translocate to the mitochondrial cloud. XTdrd6/Xtr was also detected in primordial germ cells and germ cells throughout development. Using transgenic Xenopus expressing XTdrd6/Xtr with a C-terminal FLAG tag produced by homology-directed repair, we found that the zygotic translation of the XTdrd6/Xtr protein began at St. 47/48. As germ cells are surrounded by gonadal somatic cells and are considered to enter a new differentiation stage at this phase, the newly synthesized XTdrd6/Xtr protein may regulate the translation of mRNAs involved in the new steps of germ cell differentiation.

Viable offspring obtained from Prm1-deficient sperm in mice.

Protamines are expressed in the spermatid nucleus and allow denser packaging of DNA compared with histones. Disruption of the coding sequence of one allele of either protamine 1 (Prm1) or Prm2 results in failure to produce offspring, although sperm with disrupted Prm1 or Prm2 alleles are produced. Here, we produced Prm1-deficient female chimeric mice carrying Prm1-deficient oocytes. These mice successfully produced Prm1(+/-) male mice. Healthy Prm1(+/-) offspring were then produced by transferring blastocysts obtained via in vitro fertilization using zona-free oocytes and sperm from Prm1(+/-) mice. This result suggests that sperm lacking Prm1 can generate offspring despite being abnormally shaped and having destabilised DNA, decondensed chromatin and a reduction in mitochondrial membrane potential. Nevertheless, these mice showed little derangement of expression profiles.

Asymmetrical allocation of JAK1 mRNA during spermatogonial stem cell division in Xenopus laevis.

During Xenopus spermatogenesis, each primary spermatogonium (PG), the largest single cell in the testis, undergoes mitotic divisions with a concomitant decrease in size to produce smaller differentiating spermatogonia. The spermatogonial stem cells (SSCs) occur in this PG population. Taking advantage of identifiable and isolatable properties of Xenopus SSCs, we examined JAK1 gene expression during the spermatogenesis because there have been reports on the important role of JAK/STAT pathway in regulating the status of SSCs in Drosophila and mouse. Surprisingly, in situ hybridization revealed the presence of JAK1 mRNA in the differentiating spermatogonia and primary spermatocytes as well as some PGs. Inhibition of JAK1 activity in the testis caused a decrease in percentage of BrdU-incorporating spermatogonia, suggesting that JAK1 was at least involved in regulation of spermatogonial proliferation. Interestingly, single cell reverse transcription-polymerase chain reaction (RT-PCR) clearly showed two different types of SSCs: SSCs with JAK1 mRNA (JAK1+ ) or without JAK1 mRNA (JAK1- ). Since JAK1- SSC level was increased by induction of testis regeneration, self-renewing SSCs were thought to be JAK1- . In addition, we found barrel-shaped PGs, in which JAK1 mRNA was localized asymmetrically to one half of the cell. The stainability with propidium iodide and morphology of two nuclei in the barrel-shaped PG were similar to those of PG nucleus. Based on the above observations, we propose the hypothesis that JAK1+ SSC is preparing for production of PGs destined to differentiate (destined PGs) and the accumulated JAK1 mRNA in the SSC is distributed exclusively into the destined PGs through mitotic division.

Hagfish C1q: its unique binding property.

Hagfish C1q (HaC1q) was identified and characterized as a pattern-recognition molecule (PRM) in the hagfish complement system. The serum from hagfish, Eptatretus burgeri, was applied to a GlcNAc-agarose column and eluted sequentially with GlcNAc and EDTA. Four (31, 27, 26, and 19 kDa) and one (26 kDa) proteins were detected as bound molecules in the GlcNAc- and the EDTA-eluates, respectively. Among these, the 26 kDa protein from the EDTA eluate was found to be a homologue of mammalian C1q through cDNA analysis. HaC1q had an ability to bind to various microbes in a Ca(2+)-dependent manner and its target ligands on the microbes were lipopolysaccharide, lipoteichoic acid, and peptidoglycan. The binding of HaC1q to GlcNAc-agarose was not inhibited by an excess amount of monosaccharide such as GlcNAc. While HaC1q bound to Sepharose 6B with a matrix of GlcNAc-agarose (polymer of agarobiose), it did not bind to Sepharose 4B that contained lower concentration of agarobiose than Sepharose 6B. Therefore, the target of HaC1q on GlcNAc-agarose was concluded to be agarobiose and high density of the target moiety seemed to be required for the stable binding. This finding was in accordance with the known behavior of other lectins involved in the complement system. We have concluded that HaC1q recognizes agarobiose-like structures present on the surface of microbes and acts as a pattern-recognition molecule in the process for elimination of invading microbes.

Xtr, a plural tudor domain-containing protein, is involved in the translational regulation of maternal mRNA during oocyte maturation in Xenopus laevis.

Xtr in the fertilized eggs of Xenopus has been demonstrated to be a member of a messenger ribonucleoprotein (mRNP) complex that plays a crucial role in karyokinesis during cleavage. Since the Xtr is also present both in oocytes and spermatocytes and its amount increases immediately after spematogenic cells enter into the meiotic phase, this protein was also predicted to act during meiotic progression. Taking advantage of Xenopus oocytes' large size to microinject anti-Xtr antibody into them for inhibition of Xtr function, we examined the role of Xtr in meiotic progression of oocytes. Microinjection of anti-Xtr antibody into immature oocytes followed by reinitiation of oocyte maturation did not affect germinal vesicle break down and the oscillation of Cdc2/cyclin B activity during meiotic progression but caused abnormal spindle formation and chromosomal alignment at meiotic metaphase I and II. Immunoprecipitation of Xtr showed the association of Xtr with FRGY2 and mRNAs such as RCC1 and XL-INCENP mRNAs, which are involved in the progression of karyokinesis. When anti-Xtr antibody was injected into oocytes, translation of XL-INCENP mRNA, which is known to be repressed in immature oocytes and induced after reinitiation of oocyte maturation, was inhibited even if the oocytes were treated with progesterone. A similar translational regulation was observed in oocytes injected with a reporter mRNA, which was composed of an enhanced green fluorescent protein open reading frame followed by the 3' untranslational region (3'UTR) of XL-INCENP mRNA. These results indicate that Xtr regulates the translation of XL-INCENP mRNA through its 3'UTR during meiotic progression of oocyte.

Xtr, a plural tudor domain-containing protein, coexists with FRGY2 both in cytoplasmic mRNP particle and germ plasm in Xenopus embryo: its possible role in translational regulation of maternal mRNAs.

Xtr is present exclusively in early embryonic and germline cells. We have previously shown that loss-of-function of the Xtr in embryos causes arrest of karyokinesis progression. Since Xtr contains plural tudor domains, which are known to associate with target proteins directly, we examined Xtr-interacting proteins by immunoprecipitation with an anti-Xtr monoclonal antibody and detected a few RNA-binding proteins such as FRGY2, a component of messenger ribonucleoprotein (mRNP) particle. The coexistence of Xtr with FRGY2 by constituting an mRNP particle was further confirmed by gel filtration assay. Search of mRNAs in the immunoprecipitate with Xtr suggested that the Xtr-associated molecules included several mRNAs, of which translational products were known to play crucial roles in karyokinesis progression (RCC1, XRHAMM, and so on) and in germ cell development (XDead end). Immunohistochemical observation clearly showed the co-localization of Xtr with FRGY2 also in germ plasm, in which XDead end mRNA has been shown to be localized specifically. Taken together, we proposed the possible role of Xtr in translational activation of the maternal mRNAs repressed in mRNP particle.

Functional demonstration of the ability of a primary spermatogonium as a stem cell by tracing a single cell destiny in Xenopus laevis.

In Xenopus, although primary spermatogonium (PG), the largest cell in the testis, is believed to be spermatogonial stem cell by histological observations, functional evidence has never been obtained. In the present study, we first indicated that culture of juvenile testis in a medium supplemented with follicle stimulating hormone resulted in no proliferation of PG. In this culture system, early secondary spermatogonia could undergo mitotic divisions with a concomitant decrease in their size, so that they became distinguishable in size from PG. Because the subcutaneous environment of juveniles permitted aggregates of the dissociated testicular cells to reconstruct the normal testis structure, we next inserted a genetically marked PG isolated from cultured testes into the aggregate and transplanted it subcutaneously. In this system, 73.9% of the aggregates contained a marked PG. When we observed the aggregates 12 weeks after transplantation, most aggregates (70.0%) contained marked PG that had self-renewed. Among these, fully growing aggregates contained many spermatogenic cells at the later developmental stage. These results suggested that isolated PG from the cultured testes had the ability as stem cells, and that purification of the spermatogenic stem cells became reliable in Xenopus.

Tdrd1/Mtr-1, a tudor-related gene, is essential for male germ-cell differentiation and nuage/germinal granule formation in mice.

Embryonic patterning and germ-cell specification in mice are regulative and depend on zygotic gene activities. However, there are mouse homologues of Drosophila maternal effect genes, including vasa and tudor, that function in posterior and germ-cell determination. We report here that a targeted mutation in Tudor domain containing 1/mouse tudor repeat 1 (Tdrd1/Mtr-1), a tudor-related gene in mice, leads to male sterility because of postnatal spermatogenic defects. TDRD1/MTR-1 predominantly localizes to nuage/germinal granules, an evolutionarily conserved structure in the germ line, and its intracellular localization is downstream of mouse vasa homologue/DEAD box polypeptide 4 (Mvh/Ddx4), similar to Drosophila vasa-tudor. Tdrd1/Mtr-1 mutants lack, and Mvh/Ddx4 mutants show, strong reduction of intermitochondrial cement, a form of nuage in both male and female germ cells, whereas chromatoid bodies, another specialized form of nuage in spermatogenic cells, are observed in Tdrd1/Mtr-1 mutants. Hence, intermitochondrial cement is not a direct prerequisite for oocyte development and fertility in mice, indicating differing requirements for nuage and/or its components between male and female germ cells. The result also proposes that chromatoid bodies likely have an origin independent of or additional to intermitochondrial cement. The analogy between Mvh-Tdrd1 in mouse spermatogenic cells and vasa-tudor in Drosophila oocytes suggests that this molecular pathway retains an essential role(s) that functions in divergent species and in different stages/sexes of the germ line.

cDNA cloning of a mannose-binding lectin-associated serine protease (MASP) gene from hagfish (Eptatretus burgeri).

Hagfish, agnathan cyclostome, is the most primitive extant vertebrate and its complement (C) system seems to be a primordial system in comparison with a well-developed C system in gnathostome vertebrates. From a phylogenic perspective of defense mechanisms, we have isolated complement C3 from the serum of hagfish (Eptatretus burgeri). In this study, we first attempted to identify a hagfish Bf or C2 as a C3 convertase by RT-PCR using degenerative primers designed on the basis of the conserved amino acid stretches among the several kinds of serine proteases. Contrary to our expectation, homology search of cloned RT-PCR product suggested that there was a partial cDNA encoding the homologue of neither Bf nor C2 but a mannose-binding lectin-associated serine protease (MASP). Analyses of a full-length cDNA clone isolated from a hagfish liver cDNA library by using the partial cDNA as a probe indicated that this cDNA encoded hagfish MASP 1. This evidence strongly suggests that the hagfish defends itself against pathogens at least by the complement system composed of lectin pathway.

Transgenic frogs expressing the highly fluorescent protein venus under the control of a strong mammalian promoter suitable for monitoring living cells.

To easily monitor living cells and organisms, we have created a transgenic Xenopus line expressing Venus, a brighter variant of yellow fluorescent protein, under the control of the CMV enhancer/chicken beta-actin (CAG) promoter. The established line exhibited high fluorescent intensity not only in most tissues of tadpoles to adult frogs but also in germ cells of both sexes, which enabled three-dimensional imaging of fluorescing organs from images of the serial slices of the transgenic animals. Furthermore, by using this transgenic line, we generated chimeric animals by brain implantation and importantly, we found that the brain grafts survived and expressed Venus in recipients after development, highlighting the boundary between fluorescent and nonfluorescent areas in live animals. Thus, Venus-expressing transgenic frogs, tadpoles, and embryos would facilitate their use in many applications, including the tracing of the fluorescent cells after tissue/organ transplantation.

Involvement of Xtr (Xenopus tudor repeat) in microtubule assembly around nucleus and karyokinesis during cleavage in Xenopus laevis.

We have previously shown that the transcriptional product of the novel gene, Xenopus tudor repeat (Xtr), occurred exclusively in germline cells and early embryonic cells and that the putative Xtr contained plural tudor domains which are thought to play a role in the protein-protein interactions. To understand the role of Xtr, we produced an antibody against a polypeptide containing Xtr tudor domains as an antigen and investigated the distribution and the function of the Xtr. Immunoprecipitation/Western blot and immunohistochemical analyses indicated a similar occurrence of the Xtr to the mRNA except for a slightly different profile of its amount during spermatogenesis. In spite of a large amount of Xtr mRNA at late-secondary spermatogonial stage, the amount of Xtr was kept at a low level until this stage and increased after entering into the meiotic phase. Depletion of the Xtr function in the activated eggs by injection of the anti-Xtr antibody caused the inhibition both of microtubule assembly around nucleus and of karyokinesis progression after prophase, but not of the oscillation of H1 kinase activity. These results suggest that the karyokinesis of at least early embryonic cells are regulated by unique mechanisms in which the Xtr is involved.

Mouse Tudor Repeat-1 (MTR-1) is a novel component of chromatoid bodies/nuages in male germ cells and forms a complex with snRNPs.

Characteristic ribonucleoprotein-rich granules, called nuages, are present in the cytoplasm of germ-line cells in many species. In mice, nuages are prominent in postnatal meiotic spermatocytes and postmeiotic round spermatids, and are often called chromatoid bodies at the stages. We have isolated Mouse tudor repeat-1 (Mtr-1) which encodes a MYND domain and four copies of the tudor domain. Multiple tudor domains are a characteristic of the TUDOR protein, a component of Drosophila nuages. Mtr-1 is expressed in germ-line cells and is most abundant in fetal prospermatogonia and postnatal primary spermatocytes. The MTR-1 protein is present in the cytoplasm of prospermatogonia, spermatocytes, and round spermatids, and predominantly localizes to chromatoid bodies. We show that (1) an assembled form of small nuclear ribonucleoproteins (snRNPs), which usually function as spliceosomal complexes in the nucleus, accumulate in chromatoid bodies, and form a complex with MTR-1, (2) when expressed in cultured cells, MTR-1 forms discernible granules that co-localize with snRNPs in the cell plasm during cell division, and (3) the deletion of multiple tudor domains in MTR-1 abolishes the formation of such granules. These results suggest that MTR-1, which would provide novel insights into evolutionary comparison of nuages, functions in assembling snRNPs into cytoplasmic granules in germ cells.

Two novel genes expressed in Xenopus germ line: characteristic features of putative protein structures, their gene expression profiles and their possible roles in gametogenesis and embryogenesis.

We compared the secondary spermatogonia and the primary spermatocytes of Xenopus for the proteins in their microsomal fractions and identified a newly synthesized protein (94 kDa) and three other proteins (99, 85, and 72 kDa) which increased their amount after entering the meiotic phase. These four proteins were used as antigens to produce polyclonal antibody which was found to react with the four proteins as well as two other proteins (208 and 60 kDa). Immunoscreening of Xenopus testis cDNA library with this polyclonal antibody yielded two cDNA clones (Xmegs and Xtr) encoding novel proteins. Xmegs mRNA was specifically expressed in the spermatogenic cells from the mid-pachytene stage to completion of two meiotic divisions. The putative Xmegs protein contained 19 tandem repeats of 26 amino acid residues rich in proline as well as potential phosphorylation sites (i.e., serine and threonine residues). Around this repetitive area, we found five PEST sequences known as a proteolytic signal to target protein for degradation. The presence of PEST sequences was believed to allow protein levels to closely parallel mRNA abundance. These results suggested the possible role of this novel protein in the regulation of two meiotic divisions specific to the spermatogenesis in a phosphorylation- and/or dephosphorylation-dependent manner. On the other hand, Xtr mRNA was expressed in both spermatogenic and oogenic cells except for round spermatids and the later stage cells. This mRNA was also expressed in the early stage embryos and its amount was kept constant from the St. I oocyte to the gastrula stage and decreased thereafter. The putative Xtr protein contained four complete and one partial tudor-like domains that were discovered in Drosophila tudor protein which plays an important role in PGC differentiation and abdominal segmentation. The characteristic expression profile of Xtr and the protein structure similar to the Drosophila tudor protein suggested its possible role in the progression of meiosis and PGC differentiation.

Oviductin, the oviductal protease that mediates gamete interaction by affecting the vitelline coat in Bufo japonicus: its molecular cloning and analyses of expression and posttranslational activation.

Previous studies indicated that the acquisition of egg fertilizability during transit through the pars recta portion of the oviduct in Bufo japonicus is accompanied by hydrolytic conversion of the vitelline coat 40- to 52-kDa components to 39-kDa components induced by a 66-kDa serine protease, "oviductin." In this study, we cloned a 3028-bp cDNA that contained an open reading frame encoding 974 amino acids with a calculated molecular mass of 107.6 kDa, including two protease domains and three repeats of CUB domains. Sequence analysis indicated that the catalytically active 66-kDa protein comprised an N-terminally located oviductin protease and two CUB domains. The oviductin gene was transcribed as a part of 6-kb mRNA that was expressed specifically in the cells lining the bottom of epithelial folds in the oviductal pars recta, and this expression was highly accelerated when the pars recta fragments were cultured in the presence of hCG. Western blot analyses using antibodies against a protease domain revealed that the catalytically inactive 102-kDa proteins in the pars recta granules yield 66-kDa catalytically active and 82- and 59-kDa inactive molecules. We propose that the oviductin translated as 107.6-kDa precursors are processed both N- and C-terminally to give rise to a 66-kDa active form comprising a serine protease and two CUB domains.

Relative amounts of basic nuclear proteins SP4 and SP5 in Xenopus laevis sperm correlate with gene copy number.

Mature sperm of an anuran amphibian Xenopus laevis contain six sperm-specific basic nuclear proteins (SP 1-6). In an attempt to understand how the amounts of SP produced are regulated during transcription and translation, the relative amounts of proteins and messenger RNA (mRNA), and the gene number in nuclei were compared between SP4 and SP5. Measurements of the peak areas of proteins separated by reversed-phase high performance liquid chromatography (HPLC) indicated that SP4 is present in about a five times greater amount than SP5. Nuclease S1 protection assays showed the existence of SP4 mRNA in about a five times greater amount than SP5 mRNA. Southern hybridization analyses of restriction enzyme-digested genomic DNA indicated a single copy of SP5 gene in the haploid genome, as contrasted with the five SP4 genes found in our previous study. Thus the deposition of different amounts of SP4 and SP5 in sperm nuclei reflects the relative number of these genes in the genome.

Genes for Sperm-specific Basic Nuclear Proteins in Bufo and Xenopus Are Expressed at Different Stages in Spermatogenesis: (sperm-specific basic nuclear protein/gene expression/spermatogenesis/in situ hybridization/amphibians).

The expression of the genes for sperm-specific basic nuclear proteins was examined, using the cDNA clones encoding protamine (P2) of Bufo japonicus and SP4 of Xenopus laevis as probes. Northern analyses showed that the mRNAs for these proteins were present only in the testes. Analyses with total RNA extracted from testicular cells at various spermatogenic stages revealed that in Bufo the transcripts of protamine genes are present in the spermatids, while in Xenopus the mRNAs for SP4 are present in both primary spermatocytes and spermatids. In situ hybridization studies with radiolabeled antisense RNA probes generated from cDNAs indicated that the Bufo protamine mRNAs accumulated first in round spermatids, while the Xenopus SP4 mRNAs did so in the pachytene stage of primary spermatocytes and thereafter.

Nuclear Basic Proteins of Xenopus laevis Sperm: Their Characterization and Synthesis during Spermatogenesis: (Nuclear basic proteins/Cell separation/Spermatogenesis/Xenopus laevis).

Nuclear basic proteins from morphologically and functionally mature sperm of Xenopus laevis were analyzed by acid/urea/Triton X-100 polyacrylamide gel electrophoresis (AUT-PAGE). Six sperm-specific proteins (SP1-6) were identified in addition to somatic histones H3, H4 and smaller amount of H2A and H2B, but not H1. Of these, SP3-6 were unique in containing 33-41% arginine and having very low lysine/arginine ratios, while SP2 was more similar to H3 and H4 in having a lower arginine and higher lysine content. Fractionations of testicular cells at different spermatogenic stages by unit gravity sedimentation showed that primary spermatocytes and acrosomal vesicle spermatids possess typical somatic type histones but no SPs. Injection of [14 C]-arginine into the testis and its tracing by fluorography on AUT-PAGE gels indicated that all somatic histones are synthesized during the stages between spermatogonia and primary spermatocytes, whereas SPs are synthesized at differentially regulated rates during the stages after acrosomal vesicle formation. In indirect immunofluorescence studies with anti-SP3-5 rabbit antiserum, a positive reaction was observed in the last step of spermiogenesis after the commencement of nuclear coiling.

The Properties of the Oviducal Pars Recta Protease which Mediates Gamete Interaction by Affecting the Vitelline Coat of a Toad Egg: (oviduct/trypsin-like enzyme/vitelline coat/gamete interaction/toad).

SDS-PAGE analyses of the vitelline coats (VCs) of coelomic eggs (CEVC) and uterine eggs (UEVC) of Bufo japonicus revealed that the UEVC lacks the 40K-52K molecular weight components present in the CEVC; this is concomitant with the increased stainability of a 39K component and the appearance of a 36K component. These macromolecular alterations, accompanied by the acquisition of egg fertilizability, were induced when coelomic eggs were treated with the contents of secretory granules obtained from the oviducal pars recta (PRG). Gel-filtration of PRG in combination with hydrolytic assays employing either fluorescamine-labeled CEVC or a variety of synthetic substrates showed that the CEVC to UEVC alterations are ascribable to the action of a protease hydrolyzing specifically peptidyl-Arg-MCAs in a highly Ca2+ -dependent way. This enzyme, which has an optimal pH of 8.0-8.2, is inhibited by soybean trypsin inhibitor and leupeptin, as well as by such serine protease inhibitors as DFP and p-APMSF. On the basis of a SDS-PAGE analysis, its molecular weight is estimated to be 66K. Treatment of coelomic eggs with the partially purified PR protease did not render the eggs fertilizable, although CEVC to UEVC macromolecular alterations were effected. We conclude that the action of this oviducal protease in partially hydrolyzing the VC is a prerequisite but insufficient in itself to render the coelomic eggs fully accessible to a fertilizing sperm.

Genesis of Epidermal Action Potentials in Cytokinesis Arrested Xenopus Embryos: (amphibian embryo/epidermal action potential/cytokinesis/cytochalasin B/cell differentiation).

When Xenopus embryos were treated continuously with cytochalasin B (3-10 µg/ml) from the 8 cell stage, cleavage arrested embryos in various degrees were observed. In 3-5 µg/ml cytochalasin B, cytokinesis was inhibited at the midblastula stage and pigment granules remained at the cell cortex of the animal pole. These cells showed epidermal like action potentials when the control embryos (St. 26/28) generated epidermal action potentials. In 5-7 µg/ml cytochalasin B, furrows, following their formation at early cleavage stages, regressed and no further cleavage from the 16 cell stage to morula stage took place. The pigment granules were dispersed throughout the interior of the cytoplasm. These cells showed no epidermal action potentials. Thus, it is considered that cytokinesis per sé, following the midblastula stage, is not a prerequisite for the genesis of epidermal action potentials, and that chronological times corresponding to the tailbud larva stage and a stable structure of the cellular cortex are required to bring about these potentials.