bluuebunny/crossref_metadata_2025 huggingface dataset:
Growth of fetal rat gastro‐intestinal epithelial cells is region‐specifically controlled by growth factors and substrata in primary culture
Authors: Fukamachi Hiroshi , Ichinose Masao , Tsukada Shinko , Kurokawa Kiyoshi , Shiokawa Koichiro , Miki Kazumasa , Takeuchi Shigeo
Published: October 2003

The mammalian gastro‐intestinal tract can be divided into three parts: esophagus and forestomach, glandular stomach, and intestine. We have previously reported primary culture systems for duodenal and glandular stomach epithelial cells in which the cells express tissue‐specific marker proteins. However, the effects of growth factors and substrata on cell growth have not been fully investigated. In this study a primary culture system was established for forestomach epithelial cells and the mechanism by which the growth of gastro‐intestinal epithelial cells is controlled in primary culture was examined. Forestomach, glandular stomach and duodenal epithelial cells proliferated rapidly in culture, increasing their numbers about 30‐, 20‐and 10‐fold, respectively, in the first 5 days. Scanning electron microscopy showed that these three types of epithelial cells exhibited region‐specific morphologies in culture. Results on the effects of growth factors and substrata on the proliferation of the epithelial cells revealed that the culture conditions required to induce maximal epithelial growth differed. Forestomach and glandular stomach epithelial cells required similar combinations of growth factors to proliferate, and these were quite different from those required for duodenal epithelial cells. Glandular stomach and duodenal epithelial cells could proliferate in a serum‐free condition while forestomach epithelial cells could not. Thus, glandular stomach epithelial cells exhibited intermediate characteristics between forestomach and duodenal epithelial cells regarding their growth factor requirement. Glandular stomach and duodenal epithelial cells could not proliferate on plastic without collagen substrata while forestomach epithelial cells could. Duodenal epithelial cells proliferated faster on collagen gels than on collagen films, and forestomach epithelial cells faster on collagen films than on collagen gels. Glandular stomach epithelial cells proliferated similarly on both substrata. Thus again, glandular stomach epithelial cells exhibited intermediate characteristics between forestomach and duodenal epithelial cells regarding their substratum dependency. We conclude that the growth of gastro‐intestinal epithelial cells is affected by both growth factors and substrata, and that glandular stomach epithelial cells exhibit intermediate characteristics between forestomach and duodenal epithelial cells in responding to these factors. These results suggest that a head‐to‐tail gradient exists in the gastro‐intestinal tract which controls the epithelial response to growth factors and substrata.
Disorganization of actin bundles of ectoplasmic specialization
Authors: Sakai Yasuhiro , Yamashina Shohei
Published: October 2003

The degradation of ectoplasmic specialization consisting of bundles of actin sandwiched between the plasma membrane and endoplasmic reticulum of the Sertoli cell, occurs just before spermiation. For elucidation of the processes involved in this degradation, changes in fibrous actin of the rat testis were analyzed using BODIPY‐phalloidin by fluorescence and electron microscopy.

Before step 17, the fluorescence of BODIPY‐phalloidin was evenly distributed around the spermatid head. When the spermatids became positioned at the luminal surface, the fluorescence had condensed on the concave side of the spermatid head. At step 19, lines of fluorescence distributed at regular intervals projected at right angles from the head. Ultrastructural observation showed that the tubulobulbar complex was formed at step 19 and electron‐dense material accumulated around thin tubules of the tubulobulbar complex. Immunohistochemical examination of BODIPY‐phalloidin showed that the electron dense materials around the thin tubules of the tubulobulbar complex had the capacity to bind to phallotoxin. Therefore the pattern of fluorescence in the spermatid at step 19 corresponds to that of the tubulobulbar complex.

Actin bundles of the ectoplasmic specialization would thus appear to de‐polymerize into actin monomers via electron dense materials around the thin tubules of the tubulobulbar complex. The tubulobulbar complex may contribute to the disorganization of actin bundles.
VEB4: Early zygotic mRNA expressed asymmetrically along the animal‐vegetal axis of the sea urchin embryo
Authors: Nasir Adnan , Reynolds Susan D. , Angerer Lynne M. , Angerer Robert C.
Published: October 2003

We have analyzed a gene, designated VEB4, that is expressed transiently in very early blastulae of the sea urchin, Strongylocentrotus purpuratus. Sequence analysis of the complete open reading frame shows that VEB4 encodes an unusual, highly charged protein with a pl of 9.55. We show here that VEB4 mRNA accumulate in a spatial pattern that is indistinguishable from that of two other recently described genes encoding metallo‐endoproteases, SpAN, related to astacin and SpHE, the hatching enzyme (Reynolds et al. 1992). VEB4 and other members of this gene set encode the earliest strictly zygotic gene products that have been identified. The asymmetric accumulation of VEB4 mRNA in non‐vegetal blastomeres of the 16 cell embryo and their descendants reflects the animal‐vegetal maternal developmental axis.
Spiculogenesis in the sea urchin embryo: Studies on the SM30 spicule matrix protein
Authors: Brown Martin F. , Partin Jacqueline S. , Killian Christopher E. , Lennarz William J.
Published: October 2003

When proteins isolated from spicules of Strongylocentrotus purpuratus embryos were examined by western blot analysis, a major protein of approximately 43 kDa was observed to react with the monoclonal antibody, mAb 1223. Previous studies have established that this antibody recognizes an asparagine‐linked, anionic carbohydrate epitope on the cell surface glycoprotein, msp130. This protein has been shown to be specifically associated with the primary mesenchyme cells involved in assembly of the spicule. Moreover, several lines of evidence have implicated the carbohydrate epitope in Ca2+ deposition into the growing spicule. The 43 kDa, spicule matrix protein detected with mAb 1223 also reacted with a polyclonal antibody to a known spicule matrix protein, SM30. Further characterization experiments, including deglycosylation using PNGaseF, two‐dimensional electrophoresis, and immunoprecipitation, verified that the 43 kDa spicule matrix protein had a pl of approximately 4.0, contained the carbohydrate epitope recognized by monoclonal antibody mAb 1223 and reacted with anti‐SM30. Electron microscopy confirmed the presence of proteins within the demineralized spicule that reacted with mAb 1223 and anti‐SM30. We conclude that the spicule matrix protein, SM30, is a glycoprotein containing carbohydrate chains similar or identical to those on the primary mesenchyme cell membrane glycoprotein, msp130.
Pluripotency of mouse embryonic cells on germline at 3.5–8.5 and 11.5 days post‐coitum after aggregation with precompacted embryos
Authors: Kato Yoko , Tsunoda Yukio
Published: October 2003

Albino mouse embryonic cells (Gpi‐la/a) at 3.5–8.5 and 11.5 days were aggregated with zona cut 8–16 cell stage embryos from F1 females (Gpi‐1 b/b), respectively. The aggregated embryos were transferred to pseudopregnant female mice. The recipients were allowed to go to term or were dissected at mid‐gestation to assess the donor contribution in the conceptuses using glucose phosphate isomerase (GPI) analysis. The donor cells, which were previously labeled with fluorescent latex microparticles, were aggregated with embryos, and the allocation of the donor cells at the compacted morula and blastocyst stages were observed under a fluorescence microscope. When 3.5 and 45 day old inner‐cell‐mass (ICM) cells were used, fertile chimeric mice were obtained (50 and 19%, respectively), and when 5.5 days old primitive ectoderm cells were aggregated, they did not form chimeras but contributed to the fetuses, placenta and membrane after 13.5 days of pregnancy. However, cells from further stages never contributed to the conceptuses even though they were analyzed after 10.5 days of pregnancy. The labeled donor cells at these stages were not positively incorporated in the interior part of the compacted morula and the ICM of the blastocyst stage unlike the ICM at 3.5 days post‐coitum after overnight culture.
Responses to localized UV‐laser irradiation are different between Bombyx and Drosophila eggs
Authors: Myohara Maroko
Published: October 2003

It has been found previously that in the silkworm, Bombyx mori, localized ultra‐violet (UV) irradiation of eggs at as early as the fertilization stage could induce cuticle defects in resultant larvae. To compare the responses to the UV irradiation between Bombyx and Drosophila, embryos at various stages were irradiated with a scanning UV‐laser microbeam (355 nm) over an area of 1.4–1.6% of the total egg surface. The results revealed that: (i) in Bombyx, embryonic mortality after irradiation was always low and independent of the stage of embryos at irradiation, while in Drosophila, the mortality was high at the cleavage stage and decreased as the development proceeded; (ii) in Bombyx, frequency of defective larvae after the irradiation was always high and independent of the stage of embryos at irradiation, whereas in Drosophila, high defect frequencies could be obtained only when irradiations were performed at or after the syncytial blastoderm stage; (iii) in Bombyx, the hypoderm surrounding the site of defect was always fused together to produce a continuous cuticle, while in Drosophila, the surrounding hypoderm was rarely fused together thus producing a hole in the cuticle; (iv) in Bombyx, the extent of the defects was constant from the fertilization through to the cellularization stage, whereas in Drosophila, the extent of the defects was largest at the cleavage stage and decreased as the development proceeded. These results suggest that there may be differences between the two insects in the mechanisms and/or the timing of embryonic pattern specification.
Phosphatidylinositol transfer protein in murine embryonal carcinoma cells during retinoic acid‐induced differentiation
Authors: de Wit I. Suzanna C. , de Laat Siegfried W. , Snoek Gerry T. , Wirtz Karel W. A.
Published: October 2003

Phosphatidylinositol transfer protein (PI‐TP) was studied in P19 embryonal carcinoma (EC) cells at different stages of retinoic acid (RA) induced differentiation. Western blot analysis indicated an increased expression of PI‐TP (35 kDa) during differentiation. Western blots of isoelectric focusing gels showed that the 35 kDa band consisted of the PI‐carrying form of PI‐TP (pl 5.5) and of a novel, more acidic form of PI‐TP (pl 5.4), levels of both of which increased during differentiation. These increased levels were not reflected in the in vitro PI‐transfer activity of the cytosolic fraction nor in the mRNA levels as analyzed by northern blotting. By using indirect immunofluorescence it was shown that PI‐TP is localized in the cytoplasm and associated with perinuclear Golgi structures and that this distribution is slightly affected during RA‐induced differentiation. Immunoprecipitation of PI‐TP from [32P]Pi labeled cells demonstrated that the level of phosphorylation of PI‐TP is high in undifferentiated P19 EC cells and low after 5 days of RA‐induced differentiation. These results strongly suggest that changes in the levels of PI‐TP are intimately connected with changes in the growth characteristics of P19 EC cells during RA‐induced differentiation. It remains to be established to what extent this connection is governed by the recent finding that PI‐TP is an essential cytosolic factor in stimulating phospholipase C activity.
Characterization and localization of tropomyosin proteins in Xenopus embryos with specific antibodies
Authors: Tanaka Tetsuya S. , Tatsuta Takashi , Ikenishi Kohji
Published: October 2003

In the process of monoclonal antibody (mAb) production against the 38kDa protein which is lacking in the gastrula arrested mutant embryos in Xenopus we incidentally obtained two kinds of mAb (designated as B11 and 2D10 antibodies, respectively) recognizing tropomyosin (TM) proteins in Xenopus embryos. The characterization of the corresponding antigens to those mAb was performed by immunoblotting and silver staining for two‐dimensional (2‐D) gels in the present study. The localization of the antigens in Xenopus embryos was also investigated by fluorescent microscopy.

By 2‐D immunoblots with those mAb, three distinct protein spots or TM isoforms were recognized in Xenopus embryos; a 38 kDa spot with a pl of approximately 4.8 reacted with both antibodies in embryos at stages later than the mid‐tailbud (stage 28) and two 30 kDa spots, which are probably isomers, with a pl of approximately 4.8 were detected with 2D10 antibody in embryos at stages extending from the fertilized to the mid‐neurula (stage 20). By immunofluorescent microscopy, B11 antibody was shown to react mainly with muscle cells and their precursor cells. In contrast, 2D10 antibody stained the cytoplasm of almost all cells in embryos at stages from the fertilized to the tadpole.

Judging from the results obtained with immunoblotting and fluorescent microscopy, it is likely that the 38 kDa spot is a skeletal muscle TM isoform and the two 30 kDa spots are non‐muscle TM isoforms.
Sea urchin fertilization stimulates CaM kinase‐II (multifunctional [type II] Ca2+/CaM kinase) activity and association with p34cdc2
Authors: Tombes Robert M. , Peppers Lee S.
Published: October 2003

Upon fertilization, the sea urchin egg synthesizes proteins which impart a Ca2+ dependence to M‐phase onset. A potential target of this Ca2+ dependence may be CaM kinase‐II (the multifunctional [type II] Ca2+/calmodulin [CaM]‐dependent protein kinase) which is necessary for nuclear envelope breakdown in fertilized sea urchin eggs. This study was intended to determine whether sea urchin CaMK‐II is activated after fertilization and whether it interacts with other known M‐phase regulators, such as p34cdc2. We report that total CaMK‐II activity, measured by solution assays, increases after fertilization, peaking just prior to cleavage. Interestingly, total CaMK‐II activity continues to fluctuate, peaking again prior to second and third cleavage. Gel assays also reveal enhanced levels of the 56 and 62 kDa potential CaMK‐II phosphoproteins after fertilization. Finally, CaMK‐II activity and only the 62 kDa phosphoprotein physically associate with p34cdc2, but again only after fertilization. These changes in CaMK‐II activity and p34cdc2‐association after fertilization may ensure that Ca2+ signals are targeted to the M‐phase machinery at the appropriate developmental times.
Sequence motifs shared by the 5′ flanking regions of two epidermis‐specific genes in the ascidian embryo
Authors: Ueki Tatsuya , Satoh Noriyuki
Published: October 2003

HrEpiA, B, C and D are epidermis‐specific genes of the ascidian embryo, which show different temporal expression patterns during embryogenesis. In this study we investigated the putative cis‐regulatory elements shared by HrEpiB and HrEpiD. Fusion genes were constructed by ligating the 5′ upstream regions of the genes with bacterial lacZ. Microinjection of the constructs into fertilized eggs revealed that the 5′ flanking region from −345 to + 200 of HrEpiB, regarding the transcription initiation site as +1, was sufficient for the epidermis‐specific expression of lacZ, and that from −166 to +108 of HrEpiD was also sufficient for the epidermis‐specific expression of the reporter gene. A comparison of the sequences of these two regions demonstrated several conserved sequences for DNA‐binding regulatory proteins; some were shared by both genes and some were exclusive to each. In addition, the 5′ flanking regions of the genes shared several sequence motifs. These common and uncommon sequences may be responsible for their proper spatial and temporal expression during the differentiation of epidermal cells of the ascidian embryo.
Transgenesis of newt with exogenous gene expression facilitated by satellite 2 repeats
Authors: Makita Ryosuke , Kondoh Hisato , Okamoto Mitsumasa
Published: October 2003

Genetiç transduction of newt was undertaken in search of conditions allowing high exogenous gene expression in stages from late embryo to larva when regeneration experiments, which are advantageous for this group of animals, are possible. DNA of a β‐galactosidase‐encoding gene miwZ was injected in the cytoplasm of fertilized eggs in different molecular forms, and its fate and gene expression were studied in individual embryos. When DNA was injected in a linear form, DNA concatemerized quickly and the majority of the exogenous sequences were localized in the nuclear fraction from early gastrula onwards. By contrast, when circular DNA was injected, the exogenous DNA sequence largely remained in the cytoplasmic fraction, and the entry of the sequence into the nuclear fraction was delayed to mid‐gastrula stage. Reflecting these differences, β‐galactosidase expression in an embryo occurred earlier and more efficiently by linear DNA injection. Whole mount staining for β‐galactosidase activity in tail bud embryos showed only spotty staining with circular DNA injection while spreading staining was also observed with linear DNA, suggesting difference in clone sizes of expressing cells depending on the DNA form. Expression of β‐galactosidase, however, became low in the hatching stage. To augment and stabilize β‐galactosidase expression to this stage, repeats of satellite 2 sequence, which is a transcriptionally active middle repetitive sequence highly conserved among urodeles, were added to the DNA in a way to bracket the miwZ gene. Injection of this DNA in linear form resulted in one order of magnitude higher β‐galactosidase expression compared to without satellite 2 in the neurula embryos, and in high and persistent expression in the hatching larvae, without significant increase in the copy number of the exogenous DNA. Satellite 2 sequence probably provided a genetic environment favorable for transcription of a gene.
Whole embryo culture and the study of postimplantation mammalian development
Authors: Eto Kazuhiro , Osumi‐Yamashita Noriko
Published: October 2003

In the past decade, striking advances have been made in the field of gene introducing/disrupting technology including generation of transgenic and knockout mice, which have enabled us to elucidate roles of specific genes in development. In this technology, embryos introduced with exogenous genes or chimeric embryos aggregated/injected with embryonic stem (ES) cells carrying targeted genes are allowed to develop in the uterus of foster mothers. The uterus, however, is like a black box for researchers investigating postimplantation development of mammalian embryos. Embryo culture is one of the powerful techniques that can open this black box. In this review, we focus on the applicable aspects of the whole embryo culture in the study of mammalian development and discuss the future possibilities of this technique.
Multiple GTP‐binding proteins in sea urchin sperm: Evidence for Gs and small G‐proteins
Authors: Cuéllar‐Mata Patricia , Martínez‐Cadena Guadalupe , Castellano Laura E. , Aldana‐Veloz Gustavo , Vargas Irma , Darszon Alberto , García‐Soto Jesús
Published: October 2003

Sea urchin sperm plasma membranes isolated from heads and flagella were used to examine the presence of Gs (stimulatory guanine nucleotide‐binding regulatory protein) and small G‐proteins. Flagellar plasma membranes incubated with [32P]NAD and cholera toxin (CTX) displayed radiolabeling in a protein of 48 kDa, which was reactive by immunoblotting with a specific antibody against mammalian Gs. CTX‐catalyzed [32P]ADP‐ribosylation in conjunction with immunoprecipitation with anti‐Gs, followed by electrophoresis and autoradiography, revealed one band of 48 kDa. Head plasma membranes, in contrast, did not show substrates for ADP‐ribosylation by CTX. In flagellar and head plasma membranes pertussis toxin (PTX) ADP‐ribosylated the same protein described previously in membranes from whole sperm; the extent of ADP‐ribosylation by PTX was higher in flagellar than in head membranes. Small G‐proteins were investigated by [32P]GTP‐blotting. Both head and flagellar plasma membranes showed three radiolabeled bands of 28, 25 and 24 kDa. Unlabeled GTP and GDP, but not other nucleotides, interfered with the [α‐32P]GTP‐binding in a concentration‐dependent manner. A monoclonal antibody against human Ras p21 recognized a single protein of 21 kDa only in flagellar membranes. Thus, sea urchin sperm contain a membrane protein that shares characteristics with mammalian Gs and four small G‐proteins, including Ras. Gs, Gi and Ras are enriched in flagellar membranes while the other small G‐proteins do not display a preferential distribution along the sea urchin sperm plasma membrane. The role of these G‐proteins in sea urchin sperm is presently under investigation.
Specific inhibition of sperm β‐N‐acetylglucosaminidase by the synthetic inhibitor N‐acetylglucosaminono‐1,5‐lactone O‐(phenylcarbamoyl)oxime inhibits fertilization in the ascidian, Phallusia mammillata
Authors: Godknecht Alexander J. , Honegger Thomas G.
Published: October 2003

Increasing evidence has evolved from studies in ascidians and mammals that sperm β‐N‐acetylglucosaminidase (GlcNAc'ase) plays a crucial role in fertilization. In the ascidian Phallusia mammillata, GlcNAc'ase is the predominant sperm‐bound glycosidase and N‐acetylglucosamine (GlcNAc) is the prevailing glycoside residue on the vitelline coat. We report here that the GlcNAc'ase inhibitor O‐(2‐acetamido‐2‐deoxy‐D‐glucopyrano‐sylidene)‐amino‐N‐phenylcarbamate (PUGNAC) is a potent competitive inhibitor of sperm‐bound GlcNAc'ase in P. mammillata. The inhibitor constant Ki for the isolated enzyme is 47 nmol/L. Fertilization of eggs is inhibited by PUGNAC in a dose dependent competitive manner with 50% inhibition at an inhibitor concentration of 85 μmol/L. Further experiments, in which intact eggs possessing an egg coat were mixed with eggs from which the coat had been removed, showed that only fertilization of intact eggs was inhibited by PUGNAC. This finding suggests that PUGNAC prevents the binding of the sperm‐associated GlcNAc'ase to terminal GlcNAc residues on the vitelline coat, thus inhibiting sperm binding and subsequently fertilization. Furthermore and most importantly, it shows that treatment with PUGNAC does not affect the viability of sperm and that the process of sperm‐egg fusion is not affected.
Ca2+ release from intracellular stores by thapsigargin in sea urchin eggs: Relationship to larval development and relevance in egg activation
Authors: Payan Patrick , Bourgeade Vincent , De Renzis Guy , Girard Jean‐Pierre
Published: October 2003

Thapsigargin (Tg), an inhibitor of microsomal Ca2+ ATPase, is used as a tool to study the changes in Ca2+ sequestration in sea urchin eggs and their relationship to embryonic development. Micromolar amounts of Tg inhibit ATP‐dependent Ca2+ sequestration in a dose‐dependent and non‐reversible manner, depending on the bulk of biological material used. IC5O values are 1 nmol/L and 1–10μmol/L, respectively, in the cortical Ca2+ stores (isolated cortices preparation) and in digitonin‐permeabilized eggs, a preparation giving access to the deeper reticulum compartment. Micromolar Tg does not induce Ca2+ release from 45Ca pre‐loaded cortices but leads to a loss of 25% of the total Ca2+ content from the cortical area. Using microspectrofluorimetry of fura‐2‐loaded eggs, we found that 10 μmol/L Tg induced a moderate rise in cytosolic Ca2+ activity as compared with the fertilization‐induced Ca2+ transient whether eggs were fertilized or not. Early events related to fertilization as, for example, elevation of the fertilization envelope, proton excretion and sustained increase of amino acid uptake, are triggered by 10μmol/L Tg but with a delayed onset relative to sperm‐induced effects. The present findings indicate that although it triggers most fertilization‐related events, Tg cannot be considered as a true mitotic agent in sea urchin eggs. When added after fertilization, Tg affects cleavage and the further embryonic development giving rise to abnormalities comparable to the animalized larvae obtained with other compounds responsible for the inhibition of reticular Ca2+ sequestration.
Regional difference in the responsiveness to the inductive influence of the gizzard mesenchyme among the endoderms of various small intestinal segments of the chick embryo
Authors: Matsushita Susumu
Published: October 2003

The differentiation of the endoderms of duodenal, jejunal and ileal segments of the small intestine of 6 day old chick embryos cultured in recombination with the gizzard mesenchyme of 6 day chick embryos was examined. Only the duodenal endoderm differentiated in a mesenchyme‐dependent fashion into gizzard‐like mucous epithelium forming tubular glands that expressed no sucrase‐antigen, while jejunal and ileal endoderms tended to become the sucrase‐antigen‐positive epithelium most likely according to their developmental fates. The analysis on the differentiation of the duodenal and gizzard endoderms in the presence of various digestive‐tract mesenchymes confirmed that the duodenal endoderm had the tendency to differentiate into intestine‐type and was different from the gizzard endoderm, which showed the differentiation tendency into gizzard‐type. Thus, among the segments of small intestine, only the endoderm of duodenum that was situated next to the gizzard was found to have an ability to respond to the inductive influence of the gizzard mesenchyme and to change its developmental fate.
Dependence of removal of sperm‐specific proteins from Xenopus sperm nuclei on the phosphorylation state of nucleoplasmin
Authors: Ohsumi Keita , Shimada Arata , Okumura Eiichi , Kishimoto Takeo , Katagiri Chiaki
Published: October 2003

In Xenopus laevis, nucleoplasmin from fully grown oocytes is not highly phosphorylated, but is more extensively phosphorylated during oocyte maturation to retain this state until mid‐blastula transition. Incubation of demembranated sperm with nucleoplasmin from oocytes or mature eggs revealed that egg nucleoplasmin is twice as potent as oocyte nucleoplasmin in removing sperm‐specific basic proteins from chromatin (protamine‐removing activity: PRA). Dephosphorylation of egg nucleoplasmin by alkaline phosphatase induced a remarkable decline of PRA in nucleoplasmin. Treatment of oocyte nucleoplasmin with cdc2 protein kinase induced an increase of the extent of phosphorylation, but to a level lower than that exhibited by egg nucleoplasmin, suggesting the involvement of other unspecified kinase(s) in phosphorylating nucleoplasmin during oocyte maturation. Incubation of sperm with cdc2 kinase induced selective phosphorylation of sperm‐specific basic proteins, accompanied by their enhanced removal from sperm chromatin upon exposure to high‐salt solutions. These results suggest that removal of sperm‐specific basic proteins from sperm chromatin in fertilized eggs is facilitated by phosphorylation of both nucleoplasmin and sperm‐specific basic proteins.
Elimination of heparan sulfate by heparitinases induces abnormal mesodermal and neural formation in Xenopus embryos
Authors: Furuya Shigeki , Sera Miho , Tohno‐oka Rie , Sugahara Kazuyuki , Shiokawa Koichiro , Hirabayashi Yoshio
Published: October 2003

The expression of heparan sulfate glycosaminoglycan (HS‐GAG) was examined in Xenopus embryos during the developmental stages. Chemical analysis showed the existence of HS‐GAG in the 35S‐labeled embryos. By western blot analysis using a specific anti‐HS monoclonal antibody, HS‐GAG related epitope was found after the neurulation on two protein bands, whose molecular weights were approximately 90 kDa and 100 kDa, respectively. Immunohistochemistry revealed that HS‐GAG occurred exclusively in the animal hemisphere in early gastrulae, and then appeared predominantly on the sheath of the neural tube, the notochord and epithelium. To address whether HS‐GAG chains contribute to Xenopus embryonic development, we eliminated the embryonic HS‐GAG by injecting purified Flavobacterium heparitinases (HSase) into their blastocoels. Most of the injected embryos were aberrant in mesodermal and neural formation, and became acephalic. Histological examination showed that these embryos were completely devoid of the central nervous system and the mesodermal tissues. Neither heat‐inactivated heparitinase nor chondroitinase showed such abnormality. The HS‐GAG‐eliminated embryos showed decreased expression of both muscular and neural‐specific markers. These results suggest that HS‐GAG plays an indispensable role in establishing the fundamental body plan during early Xenopus development.
Presence of a Tetrahymena growth promoting activity in fetal bovine serum
Authors: Hosoya Hiroshi , Matsuoka Tatsuhiko , Hosoya Natsumi , Takahashi Tadao , Kosaka Toshikazu
Published: October 2003

Treatment of cultures of the ciliate Tetrahymena with fetal bovine serum (FBS) enhanced the rate of cell proliferation. The growth promoting activity was partially purified from FBS as a high Mr complex including four components with apparent Mr values of 180 kDa, 68 kDa, 60 kDa and 30 kDa by a 4‐step procedure. The 180 kDa component was identified by amino acid sequencing as α2‐macroglobulin. The addition of purified α2‐macroglobulin from bovine plasma to cultures of Tetrahymena was also found to enhance the rate of cell proliferation. This report is the first dealing with the direct identification of a mammalian factor which promotes the growth of free‐living protozoa.
Differential distribution of ganglioside GM1 and sulfatide during the development of Xenopus embryos
Authors: Kubo Hideo , Kotani Masaharu , Ozawa Hideki , Kawashima Lkuo , Tai Tadashi , Suzuki Akemi
Published: October 2003

A frozen section technique for frog oocytes was developed without using any organic solvent. It was applied to examine the distribution of acidic glycosphingolipids (ganglioside GM1 and sulfatide) in Xenopus oocytes, eggs and embryos by indirect immunofluorescence microscopy with specific monoclonal antibodies against the acidic glycolipids. Although glycolipids are generally present on the cell surface, GM1 and sulfatide were distributed in the cytoplasm of animal and vegetal hemispheres, respectively, of the fully grown oocytes and oviposited and fertilized eggs. In blastula, GM1 was present on the cell boundaries and in the Golgi of the blastomeres of animal hemisphere and marginal zone, whereas the staining of the outermost layer of animal blastomeres became faint or negligible at stage 9. Sulfatide in blastula was still observed in vegetal blastomeres. In gastrula, GM1 was distributed in the inner layer of ectoderm and the involuting mesoderm. In neurula, GM1 was concentrated in the dorsal midline including the closing neural tube, notochord and somites, while sulfatide was present in endoderm. The unique distribution of GM1 and sulfatide in oocytes, eggs and early embryos may help to elucidate one aspect of the biochemical bases laid on the animal–vegetal polarity.
Sperm‐activating proteins from unfertilized eggs of the Pacific herring, Clupea pallasii
Authors: Oda Shoji , Igarashi Yoshihiko , Ohtake Hideki , Sakai Kosuke , Shimizu Nobuyoshi , Morisawa Masaaki
Published: October 2003

Ripe unfertilized eggs of the Pacific herring, Clupea pallasii, release sperm‐activating proteins into seawater at the time of fertilization. Five species of herring sperm‐activating proteins (HSAP) with different pl values (4.8, 4.9, 5.0, 5.1 and 5.4) were purified from the egg‐conditioned medium by gel filtration and isoelectric focusing. Molecular mass of the HSAP (pl = 5.1), the major species of the five HSAP, was determined to be 8.1 kDa by mass spectrometry. Molecular weights of all of the HSAP were estimated to be 7700 by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS‐PAGE). The first 20 amino acid sequences from N‐terminal ends of three HSAP (pl = 4.9, 5.0 and 5.1) were almost identical, suggesting that the HSAP have similar structures.
Lineage‐specific alteration in cell cycle structure in early Tubifex embryos
Authors: Shimizu Takashi
Published: October 2003

Embryos of the freshwater oligochaete Tubifex exhibit asynchrony in division timing as early as the second cleavage; this cleavage asynchrony becomes pronounced as development proceeds. The present study was undertaken to elucidate the composition and duration of the cell cycles of early Tubifex embryos, with special reference to their cell lineages. No significant variations in lengths of cleavage cycles were found among early embryos. In all blastomeres up to the eighth cleavage cycle, the M phase was followed directly by a 30 min S phase, which suggested that early embryos lack G1 phase. The durations of the M phase did not change during this period of development, but did differ between cell lines. The M phase in the A and B cell lines lasted for about 130 min, while the M phase in the C and D cell lines lasted for about 95 min. An examination of chromosome cycles showed that this difference in M phase durations resulted from a longer stay by the A/B cell lines in prometaphase. Only G2 phase lengthened during early development. After several rounds of G2 phase extension, three classes of G2 phase duration were established: the most extended G2 phase (∼6 h) in the first quartette of micromeres (cells 1 a–1 d), the shortest G2 phase (∼1.58 h) in teloblasts, and an intermediate G2 phase (∼2.4 h) in the progeny of macromeres (i.e. endodermal cells). Experiments with syncytial blastomeres showed that the timing of entry into the M phase, hence the duration of the G2 phase, was affected by cytoplasmic compositions. The shortest G2 phase correlated closely with the presence of yolk‐free cytoplasm called pole plasm.
Specific induction of self‐discrimination by follicle cells in Ciona intestinalis oocytes
Authors: Pinto Maria Rosaria , De Santis Rosaria , Marino Rita , Usui Noriko
Published: October 2003

Self‐incompatibility, a mechanism that prevents self‐fertilization in ascidians, is based on the ability of the oocyte vitelline coat to distinguish and accept only heterologous spermatozoa. In Ciona intestinalis self‐discrimination is established during late oogenesis and is contributed or controlled by products of the overlying follicle cells. In this study we have further investigated the role of the follicle cells in the onset of self‐discrimination by using in vitro maturation of ovarian oocytes deprived of the follicle cells and incubated with either autologous or heterologous follicle cells. Fertilization assays demonstrate that the action of the follicle cells is exerted even when they are detached from the vitelline coat and that only autologous follicle cells can promote the induction of self‐sterility on the egg coat. Electron microscopy of the oocytes during maturation reveals that the switch from self‐fertility to self‐sterility is accompanied by the appearance of a thin electron‐dense layer on the outer surface of the vitelline coat. We suggest that the formation of this layer is the result of the interaction between products of the follicle cells and the autologous vitelline coat.
The ter mutation first causes primordial germ cell deficiency in ter/ter mouse embryos at 8 days of gestation
Authors: Sakurai Takayuki , Iguchi Taisen , Moriwaki Kazuo , Noguchi Motoko
Published: October 2003

The ter (teratoma) mutation causes primordial germ cell (PGC) deficiency in ter/ter embryos at 9.5–12.5 days of post‐coitum (dpc) in mouse strains 129/Sv‐ter and LTXBJ‐ter. To study the effects of the ter mutation on the PGC development more precisely, we examined the PGC number and distribution in 7.5–12.5 dpc embryo of ter congenic C57BL/6J‐ter strain using their complete serial sections. The ter genotypes of embryos were identified by the polymerase chain reaction (PCR) polymorphisms of the microsatellite DNA of the Grl‐1 locus mapped near the ter locus. Results showed that: (i) the PGC number in ter/ter embryos was similar to those of + /ter and + / + embryos at 7.5 dpc, and did not increase at 8.0–12.5 dpc, although those of normal littermates did usually; (ii) the PGC migration to genital ridges was never affected in all embryos; and (iii) the ter genotype difference in the PGC numbers was not recognized between + /ter and + / + embryos. We concluded that the ter mutation does not affect the PGC appearance around 7.5 dpc, but first causes PGC deficiency around 8.0 dpc at the beginning of their migration and proliferation, suggesting that the normal function of the ter gene may be essential for the proliferation or survival mechanisms of PGC.
Onset of competence to respond to activin A in isolated eight‐cell stage Xenopus animal blastomeres
Authors: Kinoshita Kei , Bessho Tomoko , Asashima Makoto
Published: October 2003

Single animal hemisphere blastomeres isolated from the eight‐cell stage Xenopus embryos differentiate into mesoderm when treated with activin A, whereas when cultured without activin they form atypical epidermis. The mesoderm tissue induced by activin is different between dorsal and ventral blastomeres. In the present study, the duration and timing of activin treatment was varied, in order to identify the critical stage when animal blastomeres acquire competence to respond to activin A. It was shown that the critical time was 45 min after blastomere isolation, which corresponds approximately to NF stage 6 (32‐cell stage) of normal development. The competence gradually increased during the morula stages.
Self‐ and non‐self‐recognition in bisexual mating of Dictyostelium discoideum
Authors: Higuchi Issei , Kanemura Yasuko , Shimizu Hajime , Urushihara Hideko
Published: October 2003

Cell recognition plays a central part in the sexual process. Although cell‐surface molecules involved in gamete recognition have been identified in several organisms, our knowledge of the molecular basis of sexual cell recognition is still limited. We have been studying molecular mechanisms of sexual cell fusion using the lower eukaryote Dictyostelium discoideum. There are homothallic, heterothallic, bisexual and asexual strains in D. discoideum, and how they distinguish between each other to find out proper partners is an interesting and important question. However, analytical studies of sexuality in D. discoideum have been carried out mostly on heterothallic strains, and the polymorphism of the mating system has not yet been thoroughly investigated. In the present study, we extended our analysis to the bisexual mating phenomenon paying special attention to the mechanism of self‐incompatibility. We showed that a bisexual strain WS2162 was self‐incompatible at the step of sexual cell fusion. Results of antibody inhibition of cell fusion and detection of gp138, a cell‐fusion‐related protein found in heterothallic strains, suggest that a molecular basis for bisexual and heterothallic mating are common. We propose two models to clarify the mechanisms of self‐ and non‐self discrimination in bisexual mating patterns of D. discoideum.
Timing of initiation of muscle‐specific gene expression in the ascidian embryo precedes that of developmental fate restriction in lineage cells
Authors: Satou Yutaka , Kusakabe Takehiro , Araki Lsato , Satoh Noriyuki
Published: October 2003

The lineage of muscle cells in the ascidian embryo is well documented. Of the B‐line blastomeres, B7.4 cells become restricted to give rise to muscle at the 44‐cell stage, whereas B7.8 and B7.5 cells become restricted to muscle at the 64‐cell stage. In this study we addressed three issues concerning the timing of initiation of muscle‐specific structural gene transcription: (i) whether the initiation of the transcription of muscle‐specific genes is correlated to that of developmental fate restriction; (ii) if there is a relationship in the timing, whether it is applicable to B7.4‐, B7.5‐ and B7.8‐sublineages; and (iii) whether two muscle‐specific genes, one for actin and the other for the myosin heavy chain, show the same pattern of temporal expression.

Whole‐mount in situ hybridization revealed that hybridization signal of actin mRNA was first evident in B6.2 (the progenitor of B7.4) at the 32‐cell stage, in B7.8 at the 64‐cell stage, and in B7.5 around the 76‐cell stage, respectively, suggesting that the transcription of the gene is initiated prior to the developmental fate restriction in the B7.4‐sublineage. Presence of actin transcripts in the 32‐cell embryos was confirmed by means of reverse transcription‐polymerase chain reaction (RT‐PCR). The myosin heavy chain gene was expressed in the same manner as the actin gene. Therefore, we concluded that the timing of initiation of transcription of the muscle‐specific genes is not always associated with that of the fate restriction and that the temporal profile of the gene expression differs among the sublineages, even though two different genes have the same expression profile.
Immunohistochemical localization and possible physiological roles of tumor necrosis factor‐α in mouse cumulus‐oocyte complexes
Authors: Sato Eimei , Nakayama Taisuke , Kamio Kae , Takahashi Yuji , Toyoda Yutaka
Published: October 2003

Tumor necrosis factor‐α (TNF‐α), a cytokine which is produced by activated macrophages, has been shown to participate in the regulation of ovarian functions. In the course of our investigation on the mechanism of maturation, fertilization and degeneration of mouse oocytes, immunoreactivity to TNF‐α was found in the cytoplasm of the cells surrounding the maturing oocytes and of granulosa cells facing the antral cavity. Immunoblot analysis with the specific antibody to TNF‐α identified the 17 kDa Mr band in the extract of cumulusoocyte complexes. Various concentrations of TNF‐α (mouse, recombinant) and anti TNF‐α antiserum (polyclonal rabbit anti‐mouse recombinant TNF‐α) were then used to determine their effect on the germinal vesicle breakdown (GVBD), polar body extrusion, fertilization and fragmentation of mouse oocytes/eggs. TNF‐α at concentrations of 10 ng/mL or less and anti‐TNF‐α antiserum at concentrations of 10% or less, had no effect on the spontaneous GVBD and polar body extrusion of mouse oocytes in culture. Mouse follicular oocytes cultured for more than 72 h in modified Krebs‐Ringer solution in vitro undergo spontaneous fragmentation, which is a degenerative change to form ‘blastomeres’ with or without nuclear fragments or chromatin. Ghost‐like blastomeres were also identified in the space among fragmented ‘blastomeres’. The spontaneous fragmentation of mouse follicular oocytes was suppressed in the presence of TNF‐α at concentrations of 1 ng/mL or greater. Anti‐TNF‐α antiserum (1%) accelerated the induction of fragmentation of oocytes cultured in vitro. The addition of anti TNF‐α antiserum (10%) to the culture medium did not influence the fertilization rates of the eggs surrounded by the expanded cumulus. These results appear to indicate that the process of degeneration of mouse oocytes/eggs is modulated by TNF‐α accumulated in the expanded cumulus during oocyte maturation.
Connexins 26, 32 and 43 are expressed in virgin, pregnant and lactating mammary glands
Authors: Pérez‐Armendariz E. M. , Luna J. , Aceves C. , Tápia D.
Published: October 2003

Gap junctions (GJ) are formed by a number of homologous proteins termed connexins. Here expression of connexins Cx26, Cx32 and Cx43, was evaluated by immunofluorescence (IF) in mammary glands from virgin, pregnant and lactating rats. Cx26, Cx32 and Cx43 labeling was detected in epithelial parenchymal cells at all functional stages. Cx26 and Cx32 labeling was very low in glands from virgin animals, somewhat greater in glands from pregnant animals and significantly higher (in number and size) in lactating animals. In the last ones, Cx26 and Cx32 punctate labeling was localized to the basal and lateral membranes of alveolar epithelial cells and collecting ductules. Cx43 punctate labeling was restricted to the periphery of alveoli towards the basal pole of epithelial cells at all functional stages, and it enlarged slightly during lactation. At this localization, Cx43 may form GJ between myoepithelial cells and/or between epithelial and myoepithelial cells. Cx43 was also found to be steadily expressed in the connective tissue which surrounds and invades each parenchymal lobe, at all functional stages. At this localization, Cx43 may couple fibroblasts and/or adipose cells. IF studies in sections from lactating mice showed the same distribution of connexins. Immunoblots confirmed specificity of labeling and the presence of Cx32 and Cx43 in the mammary gland. The increase in connexin expression detected during pregnancy and lactation may be important for epithelial cell differentiation and secretion in the mammary gland.
Bone morphogenetic protein acts as a ventral mesoderm modifier in early Xenopus embryos
Authors: Suzuki Atsushi , Shioda Noriko , Ueno Naoto
Published: October 2003

Mesoderm of early vertebrate embryos gradually acquires dorsal–ventral polarity during embryogenesis. This specification of mesoderm is thought to be regulated by several polypeptide growth factors. Bone morphogenetic protein (BMP), a member of the TGF‐β family, is one of the regulators suggested to be involved in the formation of ventral mesoderm. In this paper, the nature of the endogenous BMP signal in dorsal–ventral specification was assessed in early Xenopus embryos using a dominant negative mutant of the Xenopus BMP receptor. In ectodermal explant assays, disruption of endogenous BMP signaling by the mutant receptor changed the competence of the explant cells to mesoderm‐inducing factors, activin and basic fibroblast growth factor (bFGF), and led to formation of neural tissue without mesoderm induction. This result suggests that endogenous BMP acts as a ventral mesoderm modifier rather than a ventral mesoderm inducer, and that interactions between endogenous BMP and mesoderm‐inducing factors may be important in dorsal–ventral patterning of embryonic mesoderm. In addition, the induction of neural tissue by inhibition of the BMP signaling pathway also suggests involvement of BMP in neural induction.
Characterization of gene of anuran cathepsin D as a metamorphosis‐associated enzyme
Authors: Mukai Masanori , Obara Masanobu , Yoshizato Katsutoshi
Published: October 2003

Cathepsin D is a member of the lysosomal aspartic proteinases and has been claimed to play a crucial role in the breakdown of larval tissues during anuran metamorphosis. The present study aimed to characterize the gene of anuran cathepsin D as an approach for elucidating the molecular mechanism of involution of larval tissues. Three overlapping cDNA of Xenopus cathepsin D were amplified by the polymerase chain reaction and were cloned, which covered 590 bp, 355 bp and 790 bp sequences of the region between two enzyme active sites, a 5'‐end and 3'‐end regions of the gene, respectively. Altogether they revealed 1.6 kbp encoding 422 amino acids of the enzyme. Xenopus cathepsin D showed about 60% homology to mammalian enzymes at the amino acid sequence level. Northern blot analyses revealed the gradual increase of cathepsin D transcripts in the tail during prometamorphosis, reaching a peak at stage 61 and 62. Thyroid hormone notably enhanced the signals of northern blotting of the tail. The in situ hybridization histochemistry showed that both epidermal cells in the basal layer and subepidermal mesenchymal cells embedded in the thick layer of collagen fibers produce high levels of mRNA of cathepsin D in response to thyroid hormone, suggesting active contribution of the cells closely located at the basement membrane to the breakdown of tail tissues.