bluuebunny/crossref_metadata_2025 huggingface dataset:
Early Morphogenesis of Chick Gonad in the Absence of Mesonephros
Authors: MERCHANT‐LARIOS HORACIO , POPOVA LILIA , REYSS‐BRION MARYSE
Published: January 2005

The development of the gonads in male and female chick embryos with induced unilateral mesonephric agenesis was studied using grafting, histoenzymology, and electron microscopy. As in embryos with a mesonephros, proliferation of the coelomic epithelium and its interaction with mesenchymal cells to form the medullary cords take place in the amesonephric gonads. In a similar manner, gonadal sexual differentiation and the differentiation of steroidogenic tissue, detectable by the presence of Δ5‐3β‐hydroxysteroid dehydrogenase, do not appear to be affected by the absence of an organized mesonephros. However, the initiation of gonadal development, further growth, and the onset of meiosis observable in developing ovaries are retarded. This delay appears to be reversible, as was demonstrated by experiments in which ovaries from chicks with complete mesonephric agenesis were transplanted into the coelomic cavity of male and female 3 1/2‐day‐old embryos. Meiosis finally occurred in the oocytes of all ovaries, regardless of the sex of the host. Therefore, the presence of a differentiated mesonephros in chick embryos is not required for the establishment of an undifferentiated gonad and sexual differentiation, or for initiation of meiosis.
Teratoid Tumors Derived from Mouse Embryos Grown in an Immunologically Privileged Site
Authors: CASTRO NOEMÍ , BARBIERI FRANCISCO D. , MICELI DORA C. , PASQUALINI CHRISTIANE DOSNE
Published: January 2005

Mouse early embryos and embryo fragments were transplanted into an immunologically privileged site, consisting of a glass cylinder previously implanted under the skin of adult mice in order to test their tumor producing potential, in allogeneic adult recipients. The highest yield of tumors was obtained upon transplantation of 6 1/2 day old embryos in toto. i.e., including the embryonic and extraembryonic areas. Histological examination showed teratomas composed of differentiated tissues derived from the three germ layers containing isolated foci of undifferentiated cells and nodules of trophoblast giant cells. Areas exhibiting the histological appearance of yolk sac carcinoma were also observed. Transplantation of the whole 6 1/2 day old egg cylinder, including the ectoplacental cone, and the isolated embryonic area produced a lower incidence of teratomas with a reduced variety of differentiated tissues. No yolk sac carcinoma was found in these grafts. The ectoplacental cone of 6 1/2 day embryos produced no tumors. Grafts of genital ridges from 12 1/2 day embryos gave rise to teratomas with well differentiated tissues of embryonic and extraembryonic origin. Areas ressembling yolk sac carcinoma were also observed. The life span of trophoblastic giant cells within the glass cylinder was significantly longer than in other experimental systems.
Ultrastructural Study of Asters Induced by Microinjection with Sperm Centriolar Fraction in Sea Urchin Eggs
Authors: HIRANO KEN‐ICHI , MASUDA MICHITAKA , SATO HIDEMI
Published: January 2005

Multiple asters can be artificially induced in sea urchin fertilized eggs by the microinjection of the centriolar fraction of sperm homogenate. Investigation was continued by the electron microscopy to determine whether the multi‐aster formation was due to the centrioles or the contaminants in the injected sperm fraction. Thirty three asters in 3 operated eggs were thoroughly examined, and we confirmed that the presence of centrioles in the central region of 26 asters. We considered that the rest of them might contained the centrioles in the sections lost during the preparation procedures. Fragmented axoneme, the plug of electron dense material, and the centriolar fossa, which were usually accompanied with the isolated centrioles, disappeared from the centrioles in these multiple asters. However, electron dense, amorphous materials were formed associating with the triplet blades and distributed around the centrioles. Many astral microtubules were terminated in these pericentriolar materials. Results obtained suggest that, although the pericentriolar material is acting as the microtubule organizing center, all multiple asters, except those derived from fertilization (2 asters per egg), are most likely induced by the injected centrioles and not by the contaminants.
Induction of the Synthesis of Melanin and Pteridine in Cells Isolated from the Axolotl Embryo
Authors: LØVTRUP SØREN , REHNHOLM ASTRID , PERRIS ROBERTO
Published: January 2005

It has previously been reported that when LiCl and tyrosine is added to ectodermal cells isolated from the blastula of Ambystoma mexicanum, then the synthesis of melanin is initiated in cells not normally engaged in this activity (mesenchyme cells, nerve cells and undifferentiated animal cells). In the present paper it has been shown that to obtain this effect tyrosine (0.02 mM) has to be present in the culture medium during at least one of the first seven days of culture, thus several days before melanin is produced. It is concluded that the added tyrosine is acting as an inductor of, and not as a substrate for the synthesis of melanin.

In the normal cultures it is possible to observe the spontaneous formation of yellow cells, indicating that they have produced pteridine. These cells are spherical, suggesting that they are undifferentiated embryonic cells. GTP is a precursor in the synthesis of pteridine, and in analogy with the observations made with tyrosine it was found that in the presence of LiCl a number of different cell types elaborate pteridine when GTP (0.1 mM) is added to the medium. Also in this case was it possible to show that GTP acts as an inductor, not as a substrate.
Effect of Aminopterin and Deoxyribonucleosides on the Cleavage and Embryogenesis of the Sea Urchin, Hemicentrotus pulcherrimus
Authors: NIIKURA KAZUAKI , FUJIWARA AKIKO , YASUMASU IKUO
Published: January 2005

In the embryos of the sea urchin, Hemicentrotus pulcherrimus, reared with 150 μM aminopterin from the time of fertilization, cessation of the development occurred at the blastula stage, at which the dTTP level became quite low. Another addition of thymidine to the embryo culture containing aminopterin resulted in an elevation of dTTP concentration in the embryos and allowed them to develop normally. Decrease in the dTTP level, resulting from the inhibition of thymidylate synthesis by aminopterin, probably causes a failure of egg cleavage and development. 5‐Bromo‐2′‐deoxyuridine (BUdR) also released the aminopterin‐inhibition of egg cleavage and allowed the treated embryos to develop to early gastrulae. Thereafter, the degeneration of archenteron occurred and these embryos became large permanent blastulae. Other deoxyribonucleosides failed to cancel the inhibition by aminopterin of egg cleavage. In the embryos kept with both BUdR and aminopterin, BUdR incorporation into DNA occurred at a similar rate as in thymidine incorporation in the embryos kept with thymidine and aminopterin, and was inhibited by another addition of thymidine. Without aminopterin treatment, BUdR incorporation hardly occurred and the embryos developed normally. BUdR incorporation into DNA in place of thymidine probably occurs in aminopterin‐treated embryos, resulting in abnormal development.
Differentiation Capacities of the Foreleg Imaginal Disc of Sarcophaga ruficornis Fabr (Diptera, Sarcophagidae): I. Fate Map
Authors: KUMAR K. , SINGH A. K.
Published: January 2005

When an imaginal disc of a mature larva is implanted into a host larva of the same age, it undergoes metamorphosis with the host. On the basis of the results obtained from the transplanted whole disc and disc fragments, a fate map of the foreleg disc of Sarcophaga ruficornis has been constructed. The fate map of S. ruficornis presented by us is basically similar to that of Drosophila but anterior and posterior rows of bristles of femur, apical, preapical and one long bristle of tibia have been precisely localized.
Role of Contents of the Germinal Vesicle in Male Pronuclear Development and Cleavage of Starfish Oocytes
Authors: YAMADA HISAFUMI , HIRAI SETSURO
Published: January 2005

During 1‐methyladenine induced germinal vesicle breakdown, contents of the germinal vesicle of starfish oocytes are mixed with the surrounding cytoplasm. Upon injection of contents of the germinal vesicle from immature (fully grown) oocytes into enucleated and inseminated oocytes, incorporated spermatozoa were not observed to change structurally. Alternatively, after treatment of the above oocytes with 1‐methyladenine, sperm asters and male pronuclei were developed and subsequent cleavage was also detected. From these results, it is concluded that both action of 1‐methyladenine and participation of contents of the germinal vesicle are indispensable for male pronuclear development and subsequent cleavage.
The Role of External Potassium Ion in Activation of Sea Urchin Spermatozoa
Authors: MITA MASATOSHI , YASUMASU IKUO
Published: January 2005

When sperm of the sea urchin, Hemicentrotus pulcherrimus, are diluted into K+‐free seawater, the pH of the suspension gradually decreases, whereas a rapid decline in pH is observed following dilution into regular seawater. Sperm motility and respiration are also activated after dilution into K+‐free seawater, but levels of activity are less than those observed following dilution into regular seawater. Upon addition of 10 mM K+ to K+‐free seawater, rapid acid release occurs and motility and respiratory rate in sperm are reactivated. The effect of K+ on respiration was competitive with respect to the external Na+ concentrations. Harmaline, a potent inhibitor of Na+/K+‐ATPase, causes a decrease in movement and respiration of the sperm. Harmaline does not inhibit the rapid decline in pH, although it depresses the release of acid from mitochondria. These results suggest that external K+ plays an important role in intracellular alkalinization of sea urchin sperm.
Fish Hereditary Melanoma Cell Lines of Different Degrees of Cell Differentiation
Authors: WAKAMATSU YUKO , OIKAWA ATSUSHI , OBIKA MASATAKA , HIROBE TOMOHISA , OZATO KENJIRO
Published: January 2005

Four cell lines including two sublines were established from hereditary melanomas in interspecific hybrids between platyfish (Xiphophorus maculatus) carrying the Sp gene and swordtails (X. helleri) and maintained in vitro for more than 34 months. Cells in each cell line grew randomly across each other with an apparent lack of contact inhibition of growth and at a population doubling time of 50 to 72 hr. They retained the characteristics of young pigment cells in regard to ultrastructure, tyrosinase activity, the DOPA and combined DOPA‐premelanin reactions. In the degree of differentiation, the cells of the three cell lines seemed comparable to early melanocytes close to melanoblasts, and those of the remaining one cell line seemed comparable to young melanocytes but were in a more differentiated state than the early melanocytes. Colony forming ability on plastic plates was at a level of 10% in the three cell lines but only 1% in the one cell line. All four cell lines failed to form colonies in soft agar. Chromosome analysis revealed that these four cell lines were heteroploid with many abnormal figures of chromosomes and double minute chromosomes. None of the cell lines showed transplantability to fish.
Effect of Phosphatidylcholine on Acrosome Breakdown and Fertilizing Capacity of Amphibian Spermatozoa
Authors: CABADA MARCELO , BLOJ BERNABÉ , de ORTIZ LUISA P. , VALZ‐GIANINET JORGE N. , FONTDEVILA MARINA DIAZ
Published: January 2005

The effect of vesicles of purified egg yolk phosphatidylcholine on the fertilizing capacity and acrosome breakdown of amphibian spermatozoa was studied. When Bufo arenarum spermatozoa were incubated with either small unilamellar vesicles (prepared by sonication) or with large unilamellar vesicles (prepared by reverse‐phase evaporation) a decrease in the fertilizing capacity of spermatozoa was found. At the same phosphatidylcholine concentration, large unilamellar vesicles were more inhibitory than small unilamellar vesicles. The inhibition was dependent upon the phospholipid concentration and the length of the incubation period. Small unilamellar vesicles did not modify the time course of acrosome breakdown in Leptodactylus chaquensis, while large unilamellar vesicles markedly accelerated the rate of acrosome breakdown. In both biossays, the charge of the vesicles (made either positive or negative by the addition of 5% stearylamine or 5% phosphatidic acid) did not influence their biological effect. Multilamellar vesicles did not alter the fertilizing capacity nor the acrosome breakdown. We conclude that the size and the structure of the vesicles are important parameters in determining the inhibitory capacity of phosphatidyl choline on amphibian fertilization.
Effects of pH on the Fertilization Response of the Medaka Egg
Authors: IWAMATSU TAKASHI
Published: January 2005

The effects of changing the intra‐ and extracellular pH on the cortical reaction and sperm penetration into eggs were examined in Oryzias latipes. When eggs were inseminated in a saline adjusted to various pH values with different buffers, fertilization took place normally over a wide range of external pH (pH0) from about 6 to 10 with a peak at 7 to 8.5. The primary causes of the reduced fertilization were impaired motility or immobility of the spermatozoa in the acidic saline and the swelling of the chorion in the alkaline saline. A rise in the intracellular pH (pHi) shortly after commencement of the cortical reaction was found by monitoring the color of a pH indicator micro‐injected into the cytoplasm. The experimental results obtained by microinjection of various pH buffers (pH 4–12) suggest that the intracellular membrane fusion of the plasma membrane with the cortical alveolar membrane (CABD) is affected by the acidic pHi while the propagation of the CABD and the intercellular membrane fusion between the plasma membranes of the egg and the spermatozoon are affected by the acidic pH0.
Subunits of Xanthine Dehydrogenase and Their Differential Appearance in Chick Tissues During Development
Authors: IRIE SHINKICHI , KATO YOSHIHIRO
Published: January 2005

Xanthine dehydrogenase (XDH) from adult chick liver comprises two polypeptide chains of different size in a molar ratio of 1: 1. The molecular weights of these subunits were estimated to be 155K (α) and 135K (β) daltons (1). However, XDH isolated from the liver of newly hatched chick was not found to represent the equimolar ratio of these two subunits; that is, the amount of subunit β was lower than that of subunit α. While examamining electrophoretically the change in the amounts of these subunits in the liver, the subunit α was found to appear earlier in the embryonic stage, but β only after hatching. In the kidney, however, both subunits were detected before hatching, being consistent with the fact that XDH exists before hatching in the kidney. The two subunits also appeared differentially in the kidney; i.e., subunit α appeared earlier than subunit β. In either tissue, the rate of increase in XDH activity corresponded to that of subunit β. Thus, the synthesis of two subunits of XDH are separately regulated at least until just after hatching.
The Possible Involvement of Cyclic AMP and Volatile Substance (s) in the Development of a Macrocyst‐Forming Strain of Dictyostelium mucoroides
Authors: AMAGAI AIKO , FILOSA MICHAEL F.
Published: January 2005

A mutant MF1 previously isolated from Dictyostelium mucoroides‐7 (Dm7) formed macrocysts with or without light when plated on agar at high cell dinsities. At lower cell densities, however, the MF1 cells formed only fruiting bodies. This failure to form macrocysts was shown to be due to the subthreshfold concentration of a volatile substance(s) required for macrocyst formation. Although ammonia is a volatile substance produced by both the Dm7 and MF1 cells, no evidence of its involvement in macrocyst formation was obtained. Mixing the Dm7 and MF1 in a one‐to‐one ratio resulted only in fruiting body formation suggesting that the Dm7 cells produced a factor which allowed MF1 cells to form fruiting bodies. This factor may be cyclic AMP (cAMP) since addition of cAMP to the medium directed development of MF1 cells to fruiting body formation. The effect of cAMP was exhibited most conspicuously when MF1 cells were exposed at the aggregation stage. Based on these results it is suggested that developmental pathway of the D. mucoroides macrocystforming strain Dm7 and its mutant MF1 may be determined by the relative concentrations of the volatile, macrocyst‐inducing substance(s) and cAMP at the aggregation stage.
Oogenesis in a Flesh Fly, Sarcophaga ruficornis
Authors: VERMA GANPATI PRASAD , ISHIKAWA MASARU
Published: January 2005

Maturation of the oocyte in the polytrophic ovariole of the flesh fly, Sarcophaga ruficornis was subdivided into 12 growing stages. The nurse cells increasing in volume during initial stages 1–6, shrink sharply in size from stage 7 onwards and finally disintegrate in stage 12. The oocyte continues to increase in volume till it reaches 1.2×1077μ3 in stage 12 before ovulation. The onset of vitellogenesis is marked by the appearance of protein‐containing granules in the peripheral ooplasm in stage 7. The follicular epithelial cells around the oocyte exhibit changes in their shape in different stages and perhaps facilitate as well as stop the transport of yolk precursors to the ooplasm. Finally, after the formation of egg membranes around the yolk‐filled oocyte, the follicular epithelium is sloughed off in stage 12.
A Cytochemical Analysis of Development of Yolk in the Growing Oocyte of the Guinea Pig
Authors: VERMA GANPATI PRASAD , ISHIKAWA MASARU
Published: January 2005

A cytochemical analysis reveals the development of fatty yolk (FY) in the early antral oocyte and proteid yolk (PY) in the late antral oocyte of the guinea pig. While the FY develops from the lipid elements of the oocyte (endogenous), the PY apparently develops from the protein‐positive precursor granules infiltrating into the oocyte (exogenous). Cytochemically the FY is composed of saturated triglycerides and the PY of tyrosine‐, tryptophan‐, histidine‐, arginine‐, and ‐SH and ‐NH2 groups‐containing protein. It has been found possible to conclude that the FY is used up by the growing oocyte, while the PY continues to be present in the mature pre‐ovulatory oocyte.
Further Evidence for the Presence of a Circadian Clock in the Prothoracic Glands of the Saturniid Moth Samia cynthia ricini: Decapitated Larvae Can Respond to Light‐Dark Changes
Authors: MIZOGUCHI AKIRA , ISHIZAKI HIRONORI
Published: January 2005

A small peak of haemolymph ecdysteroid titre precedes the gut purge that characterizes larval‐prepupal transition of the saturniid moth Samia cynthia ricini. This peak shifts its phase in parallel with the phase shifts of gut purge according to the changes in light‐dark conditions preceding gut purge. Decapitated larvae responded to these light‐dark changes as intact larvae did, as assessed by the phase shifts of the haemolymph ecdysteroid peak. This indicates that the brain‐centred PTTH clock is not prerequisite for realization of the circadian‐clock‐controlled timing in the initiation of prepupal development, and supports indirectly our previous notion that the prothoracic glands of Samia possess a circadian clock dictating gut purge timing.
Formation of the Neural Plate and the Mesoderm in Normally Developing Embryos of Xenopus laevis
Authors: IMOH HIROSHI
Published: January 2005

Normally developing embryos of Xenopus were fixed at various stages between the blastula and early tail bud stage, and their serial sections were examined. The marginal belt of the blastula was characterized by abundance of cells with RNA‐rich peripheral cytoplasm called mesoplasm. At the early gastrula stage, the marginal belt was folded into two layers giving rise to mesodermal material and marginal ectoderm. During gastrulation, the mesodermal material, which consisted of RNA‐rich cells, spread to enclose the blastocoel and the endoderm, and a large part of it was shifted to the dorsal side of the embryo. It gradually established the mesodermal layer. The notochord was formed on the dorsal lip of the blastopore by involution, separately from preformed mesodermal material. The RNA‐rich cells in the marginal ectoderm became columnar, forming a broad belt in the marginal zone. This belt was deformed and shifted to the dorsal side during gastrulation, eventually establishing the neural plate showing quantitative differentiation along the head‐tail axis. Possible mechanisms involved in the formation of the neural plate and mesoderm were discussed with reference to the organizer and the mesoplasm.
Changes in Osmotic Pressure and Swelling in Horseshoe Crab Embryos During Development
Authors: HAYAKAWA MASAHARU , TANIMOTO SATOMI , KONDOAND AKIO , NAKAZAWA TOHRU
Published: January 2005

Water influx accompanying the swelling of embryos during normal development of horseshoe crabs, Limulus polyphemus and Tachypleus tridentatus, following a rupture of the chorion, was analyzed. The increase in volume of perivitelline fluid during deveopment was about 90 percent of the increase in total embryo weight. Considerable water discharge was observed on drying the embryos in air and a reversible water influx occurred with a second immersion in sea water, even though the embryos died as a result of this treatment. Since the osmotic pressure of the perivitelline fluids decreased markedly during development until the end of swelling, a close correlation between swelling and osmotic pressure was recognized. These results indicate that certain osmoactive substances may be produced in the perivitelline fluid at the initial stage of swelling.
Scanning Electron Microscopy of the Extracellular Matrix of Amphibian Gastrulae by Freeze‐drying
Authors: KOMAZAKI SHINJI
Published: January 2005

In order to demonstrate the extracellular matrix (ECM) which tends to be missing from specimens prepared by ordinary procedures, a freeze‐drying method was applied to gastrulae of the newt (Cynops pyrrhogaster) without prefixation and the embryos were examined with a scanning electron microscope (SEM). It was revealed that a prominent amount of fibrillar ECM occupied the blastocoel and amorphous ECM covered the inner surface of the blastocoelic wall (BW) as well as the surface of migrating cells. These results were compared with those obtained from observations of specimens fixed chemically.
Inhibitory Effect of Calmodulin Antagonists and Calcium Antagonists on Fertilization‐Induced Cyanide‐Insensitive Respiration in Sea Urchin Eggs
Authors: TSUCHIDA EMI , FUJIWARA AKIKO , FUJINO YUKIO , YONAGA TAKARA , YASUMASU IKUO
Published: January 2005

During initial several minutes after fertilization, sea urchin eggs exhibited high rate of respiration which was only slightly inhibited by cyanide. This cyanide‐insensitive respiration was inhibited by calcium antagonists, diltiazem and verapamil, and calmodulin antagonists, N‐(6‐aminohexyl)‐5‐chloro‐1‐naphthalensulfonamide hydrochloride (W‐7), N‐(6‐aminohexyl)‐1‐naphthalenesulfonamide hydrochloride (W‐5) and chlorpromazine, which were added within 1 min after insemination. The inhibitory effect of W‐7 on cyanide‐insensitive respiration was higher than that of W‐5. Cyanide‐sensitive respiration of fertilized eggs observed after this initial period was not inhibited by these compounds. Ca2+ influx in eggs just after fertilization was inhibited by calcium antagonists but was rather enhanced by calmodulin antagonists. Fertilization‐induced stimulation of cyanide‐insensitive respiration probably results from calmodulin‐dependent reactions which are activated by Ca2+ influx.
Tissue‐ and Stage‐Specific Expression of Sericin Genes in the Middle Silk Gland of Bombyx mori
Authors: ISHIKAWA ETSUKO , SUZUKI YOSHIAKI
Published: January 2005

Sericin gene expression in the middle silk glands during Bombyx mori larval development was analyzed using probes from a genomic DNA clone for 10.5 kb sericin mRNA. The 10.5 kb mRNA, the most abundant in the fifth instar, is not detected in the third feeding, fourth feeding and fourth moulting stages. It becomes detectable at 2 days of the fifth instar, and accumulates rapidly. The second major mRNA in the late fifth instar, a 9.0 kb component having a similar sequence to the 10.5 kb mRNA, becomes detectable only at 6 and 7 days of the instar by use of the repetitious coding sequence probe of the sericin clone. Using the same probe about 20 kb RNAs with a fainter intensity than that of the major mRNAs are detected. They are present extremely faintly in the third and fourth feeding stages, disappear in the fourth moulting stage, and increase in the fifth instar. Two other faint poly(A)+ RNA components are detected by a DNA probe containing the 5′ end sequence of the sericin clone. One is 4.3 kb, and appears in the third, fourth and fifth feeding stages but not in the fourth moulting stage. The other is 3.0 kb, and it becomes detectable after 1 day of the fifth instar.
Establishment of the Neural Differentiation Pattern in the Prospective Prosencephalic Ectoderm of the Chick Embryo (Gallus domesticus)
Authors: KHARE M. K. , CHOUDHURY S.
Published: January 2005

Small graft pieces (size: 0.15 × 0.2 mm to 0.1 × 0.4 mm and 0.25 × 0.25 mm) were isolated from the prospective prosencephalic ectoderm of the definitive primitive streak (st. 4) and head process (st. 5) chick blastoderms, and cultured in vivo intracoelomically. In all 1437 graft pieces isolated from 448 donor blastoderms were transplanted into the coelom of 941 host embryos; 216 hosts died before reaching 12 days. The 725 surviving hosts, which carried 1241 implants, yielded 304 analysable grafts. The very small size of the isolates affected the recovery rate. The histological analysis of the recovered grafts reveals that the frequency of neuralization was higher in the grafts isolated from the central region of the prospective prosencephalic ectoderm than in those from its peripheral region. A centrifugal extension of differentiation tendencies of prosencephalic structures was observed from st. 4 to st. 5. At st. 5 frequency of telencephalic structures was high in the grafts of the anterior region, whereas that of diencephalic structures was high in those of the more posterior region. Eye structures (retina as well as tapetum) differentiated in an area measuring ca. 0.4 × 0.1 mm extending transversely about 0.1 mm anterior to the head process at st. 5. Structural elements of the lens were not observed in any graft.
Promotion of Myoblast Proliferation by Hypoxanthine and RNA in Chick Embryo Extract
Authors: ICHIO  , KIMURA ICHIR. , OZAWA EIJIRO
Published: January 2005

In the course of our attempt to clarify the growth‐promoting activities of chick embryo extract (EE), its heat‐stable activity was found to be due to hypoxanthine and its related substances including RNA. When added to a basal culture medium composed of Eagle's MEM, horse serum and Fe‐saturated ovotransferrin hypoxanthine or adenine (10 μM) markedly promoted quail myoblast proliferation. The concentration of hypoxanthine in EE was very high (274±34μM) and increased 2‐fold during incubation at 37°C, while that in horse serum was very low (<3 μM). Guanine, xanthine and pyrimidines were ineffective. The nucleosides and nucleotides of hypoxanthine and adenine were effective, but the deoxynucleosides strongly inhibited the proliferation of avian myoblasts. Further, RNA was also effective but DNA was not. Hypoxanthine and RNA also promoted rat myoblast proliferation and the deoxynucleosides did not inhibit rat myoblast proliferation. These findings suggest that a supply of raw materials for RNA synthesis is important for optimal proliferation of myoblasts.
Temperature‐Dependent Sex Determination in Gekko japonicus (Gekkonidae, Reptilia)
Authors: TOKUNAGA SHOJI
Published: January 2005

The effects of temperature on sexual differentiation in early development of the gekkonid lizard, Gekko japonicus were studied. The eggs were collected within 24 hr after the oviposition and were incubated at 20, 24, 28 and 32°C. The number of eggs hatched was 14 at 24°C, 20 at 28°C and 21 at 32°C. Hatching never occurred at 20°C. The hatched lizards without Müllerian ducts were judged as males. The sex of all lizards with Müllerian ducts were identified histologically. The sex ratios, male/(male+female), were 0.07 at 24°C, 0.75 at 28°C and 0.24 at 32°C. The disparities of the sex ratio from 1/2 were statistically significant and differences in the sex ratio with various incubation temperatures were also significant. These sex ratios can probably be best interpreted by a temperature‐dependent sex determination. The different sex ratios do not seem to be related to a predetermination of sex with a differential mortality.
Fertilization Cone of Carp Eggs as Revealed by Scanning Electron Microscopy
Authors: KUDO SHIGEHAR. , SATO ATSUHIKO
Published: January 2005

The process of formation of the fertilization cone in carp eggs was examined by scanning electron microscopy. The fertilized eggs responded to penetration of one sperm by primary and secondary steps of formation of a fertilization cone of unique morphology. In the primary step, the earliest fertilization cone was seen at the superior or anterosuperior part of a fused sperm head in inseminated eggs fixed 20 sec after immersion in fresh water. The cone reached a maximum of more than 10 μm in length and 3–4 μm in thickness by 40 sec, resulting in a transient plugging of the micropylar canal. In the secondary step, usually seen at 105–120 sec, a conformation reminiscent of a very small caldera volcano was formed, with the shortened earlier cone and part of the sperm tail at its top. By 2.5 min, the fertilization cone had become conical, and the sperm tail still extended from its top. At 3 min, the sperm tail was often not detectable, but a cytoplasmic eminence was still seen as a trace of the fertilization cone. The role of the earlier fertilization cone in blocking polyspermy is discussed.
Rôle du Système Nerveux Sur la Prolifération Cellulaire des Blastèmes de Régénération de Membre de Triton (Pleurodeles Waltlii Michah)
Authors: BOILLY BÉNONI , OUDKHIR MUSTAPHA , LASSALLE BERNARD
Published: January 2005

Cellular proliferation of both epidermal and mesenchymal cells of denerved cone stage limb blastemas (Pleurodeles waltlii Michah) was studied in vivo. The mitotic index and thymidine 3H incorporation (autoradiography following short term (1 hr) incubation and scintigraphy) show that denervation causes a significant decrease in the cellular proliferation (about 70% 4 days after denervation). Denervation affects first the mesenchymal cells, the proliferation of epidermal cells declines later. These results are discussed in term of cellular cycle.
Movements of Mitochondria Associated with Isolated Egg Cortex
Authors: SHIMIZU TAKASHI
Published: January 2005

Cortical tragments of Tubifex eggs undergoing ooplasmic segregation were isolated in plastic dishes with an oil droplet separating part of them from the dish. Except at the edges of fragments and at the base of the oil droplet, the nearly entire area of the cortex was firmly attached to the plastic surface. Many mitochondria were associated with the cortical actin lattice. On addition of ATP to the preparations, only nonadhering portions of the cortex exhibited contractions. At the base of oil droplets, some mitochondria moved in the same direction concurrently with cortical contractions. Contractile activities of isolated cortices were completely inhibited by their preincubation with N‐ethylmaleimide‐modified heavy meromyosin, suggesting the involvement of an actomyosin force‐generating mechanism in the cortical contractions during ooplasmic segregation.
The Differentiation of the Vitelline Envelope of Xenopus Oocytes*
Authors: PINTO MARIA ROSARIA , SANTELLA LUIGIA , CASAZZA GIANNA , ROSATI FLORIANA , MONROY ALBERTO
Published: January 2005

We have studied the differentiation of the vitelline envelope (V.E.) of the oocyte of the anuran Xenopus laevis. The V.E. precursor material is synthesized by the oocyte concomitantly with the onset of vitellogenesis, and its extrusion reaches a maximum at late vitellogenesis. Oocytes at different stages of growth were incubated in L‐[3H]fucose and the progress of incorporation was followed by kinetic and histoautoradiographic analysis. We found that the highest overall rate of incorporation was exhibited by the vitellogenic oocytes. These oocytes showed clusters of grains in the perinuclear and in the cortical areas. The highest accumulation of grains in the V.E. was associated with the late vitellogenic stage, when the differentiation of the V.E. was almost complete. L‐[3H]Fucose labelled glycoproteins have been identified by electrophoretic analysis of V.E. prepared from late vitellogenic stages.
Effect of Actinomycin D and Cycloheximide on Gonadotropin‐Induced 17α, 20β‐Dihydroxy‐4‐pregnen‐3‐one Production by Intact Ovarian Follicles and Granulosa Cells of the Amago Salmon, Oncorhynchus rhodurus*
Authors: NAGAHAMA YOSHITAKA , YOUNG GRAHAM , ADACHI SHINJI
Published: January 2005

The effect of actinomycin D and cycloheximide on gonadotropin (partially purified chum salmon gonadotropin, SGA)‐induced 17α, 20β‐dihydroxy‐4‐pregnen‐3‐one (17α, 20β‐diOHprog, a maturation‐inducing steroid in amago salmon) production was examined in intact ovarian follicles and granulosa cells of postvitellogenic amago salmon, Oncorhynchus rhodurus. Both actinomycin D and cycloheximide blocked gonadotropin‐induced 17α, 20β‐diOHprog production by intact follicles. In contrast, gonadotropin‐induced 17α‐hydroxyprogesterone production by intact follicles was not abolished by actinomycin D, but was abolished by cycloheximide, suggesting that postvitellogenic amago salmon ovarian follicles already contain the RNAs necessary for the synthesis of 17α‐hydroxyprogesterone.

In isolated granulosa cells, chum salmon gonadotropin was able to stimulate 17α, 20β‐diOHprog production only when a precursor, 17α‐hydroxyprogesterone was provided in the incubation medium, indicating that gonadotropin acts directly on granulosa cells to enhance the activity of 20β‐hydroxysteroid dehyrogenase (20β‐HSD). Total inhibition of 20β‐HSD enhancement in granulosa cells, judged by 17α, 20β‐diOHprog production, was achieved when actinomycin D was added between 1 hr before the start of incubation with 17α‐hydroxyprogesterone and gonadotropin to 6 hr after. With cycloheximide total inhibition was observed when added in the period of 1 hr before to 9 hr after the start of the incubation. These results suggest that chum salmon gonadotropin acts on granulosa cells to enhance the de novo synthesis of 20β‐HSD by a mechanism involving RNA synthesis.
Inhibition of Starfish Oocyte Maturation by Tumor‐Promoting Phorbol Esters*
Authors: KISHIMOTO TAKEO , YOSHIKUNI MICHIYASU , IKADAI HIROYUKI , KANATANI HARUO
Published: January 2005

Effect of tumor promoters including phorbol esters and teleocidin on 1‐methyladenine (1‐MeAde)‐induced oocyte maturation was studied in the starfish. When isolated immature oocytes were treated with 1‐MeAde and 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA), 1‐MeAde‐induced maturation was completely inhibited at more than 2.5 μg/ml. However, if TPA was added after the hormone‐dependent period (the minimum period wherein 1‐MeAde is required), such maturation‐inhibiting effect was no longer observed. Pretreatment with TPA for 5 min showed that its inhibitory action is irreversible. However, when TPA‐injected oocytes were treated with 1‐MeAde, all oocytes underwent germinal vesicle breakdown (GVBD). GVBD was induced in TPA‐treated oocytes upon injection of the cytoplasm of maturing oocytes containing maturation‐promoting factor (MPF). These facts show that TPA acts on the oocyte surface to inhibit the production of MPF. Retinoids including retinal, retinol and retinoic acid reversed the inhibitory effect of TPA on 1‐MeAde‐induced maturation. Experiments with various phorbol esters showed a good correlation between their maturation‐inhibiting activity and their known tumor‐promoting activity. Further, telecoidin, which is structurally unrelated to phorbol esters, inhibited 1‐MeAde action. Since both tumor‐promoting phorbol esters and teleocidin are known to activate Ca2+ ‐activated, phospholipid‐dependent protein kinase (protein kinase C) and their activation effect is inhibited by retinoids, it appears that the activation of protein kinase C by tumor promoters is involved in blocking of 1‐MeAde action.
Oocyte Competence to Maturation‐inducing Hormone. I. Breakdown of Germinal Vesicles of Small Oocytes in Starfish, Asterina pectinifera*
Authors: BULET PHILIPPE , KISHIMOTO TAKEO , SHIRAI HIROKO
Published: January 2005

1‐Methyladenine (1‐MeAde) is the endogenous maturation‐inducing substance (MIS) in starfish. However, small oocytes have no competence to 1‐MeAde even at the concentration of 10−5M. Furthermore, when they were injected with cytoplasm of fully‐grown (large) and maturing (1‐MeAde‐treated) oocytes, known to contain maturation‐promoting factor (MPF), they did not undergo germinal vesicle breakdown (GVBD). On the other hand, germinal vesicles (GV) of the small oocytes underwent nuclear breakdown when the small oocytes were fused with the large maturing oocytes. Therefore it is concluded that the GV of the small oocytes are capable of undergoing nuclear breakdown in the presence of the sufficient MPF, but that the small oocytes can not amplify the injected MPF. Fused cells displayed particular shape changes during the course of nuclear breakdown of both the large and the small oocytes.
One Millimeter large Oocytes as a Tool to Study Hormonal Control of Meiotic Maturation in Starfish: Role of the Nucleus in Hormone‐stimulated Phosphorylation of Cytoplasmic Proteins
Authors: PICARD ANDRÉ , PEAUCELLIER GÉRARD , LE BOUFFANT FRANCOISE , DOREE MARCEL
Published: January 2005

Single nuclei (germinal vesicles) manually isolated from large oocytes of the starfish Echinaster sepositus, as well as the complementary anucleated oocytes, were used to investigate the early changes of protein phosphorylation which occur from 1‐MeAde addition to germinal vesicle breakdown (GVBD). Stimulation of protein phosphorylation was already evident in the nucleus shortly after 1‐MeAde addition (18 min, thus about 0.40x the time required for GVBD), although it began first in the cytoplasm. No translocation of phosphoprotein across the nuclear envelope was detected before GVBD. Presence of the nucleus is not required for the hormone to stimulate protein phosphorylation in the remaining part of the oocytetin:fact the patterns of protein phosphorylation in enucleated oocytes were found to be identical, whether enucleation was performed after or before hormonal treatment. Cytoplasm taken at the time of GVBD from maturing Echinaster oocytes induces meiotic maturation when transferred in stage VI immature oocytes of the amphibian Xenopus laevis.
Calcium‐ and Cyclic AMP‐independent, Labile Protein Kinase Appearing during Starfish Oocyte Maturation: its Extraction and Partial Characterization*
Authors: SANO KIYOSHI
Published: January 2005

A burst of protein phosphorylation and an appearance of maturation‐promoting factor have been reported to occur shortly before germinal vesicle (nucleus) breakdown (GVBD) in 1‐methyladenine‐induced oocyte maturation of starfish. To detect if a protein kinase is activated before GVBD, protein kinase activity was compared in maturing oocytes which were just undergoing GVBD and immature oocytes of Asterina pectinifera. The oocytes were homogenized in a buffer modified from that used for extracting amphibian maturation‐promoting factor. When the supernatant protein of homogenized immature oocytes was used as a substrate, protein kinase activity in the supernatant of the maturing oocytes was 7‐fold higher than that of immature oocytes. The protein kinase in the supernatant of the maturing oocytes showed a high substrate specificity for histone H1 among the exogenous substrates examined, and the activity of the maturing oocytes for histone H1 was 6‐ to 7‐fold higher than that of immature oocytes. The protein kinase detected in the maturing oocytes was very labile and was inhibited neither by ethylene glycol bis(β‐aminoethyl ether)N, N, N′, N′‐tetraacetic acid nor by the heat‐stable inhibitor protein of cyclic AMP‐dependent protein kinase. These results indicate that a calcium‐ and cyclic AMP‐independent, labile “maturation‐specific protein kinase” appeared before GVBD in maturing oocytes, and suggest its participation in the phosphorylation burst in vivo. The possible correlation of this kinase with maturation‐promoting factor and chromosome condensation was discussed.
U.V. Irradiation Inhibits The Electrical Block to Polyspermy in Echinoderms*
Authors: DAVID C. , MOREAU M. , VILAIN J. P. , GUERRIER P.
Published: January 2005

Oocytes of the sea urchin Sphaerechinus granularis and the startish Marthasterias glacialis have been submitted to U.V. irradiation before fertilization. This treatment significantly increased the incidence and severity of polyspermy in the sea urchin and was also found effective on starfish oocytes. These were found more resistant to damage than sea urchin eggs and U.V. irradiation did not affect either their response to the hormone l‐methyladenine or the rate of elevation of the fertilization envelope, which assures the late and definitive block to polyspermy.

Electrophysiological measurements performed on M. glacialis oocytes definitively demonstrate that U.V. irradiation completely inactivates voltage‐dependent sodium channels, without altering the other main conductances, Cl−, K+ or Ca2+. After such a treatment, the relative permeability of the membrane to Na+ as compared to K+ shifted from 0.019±0.003 to 0.003±0.002 and only the calcium component of the action potentials could be observed. However, a fertilization potential, preceded by small sperm induced steps, is still present in these conditions, although its peak and plateau level are greatly reduced.

These new findings are discussed, which confirm the electrical nature of the fast block to polyspermy but question about the specificity of those sperm‐gated channels which are supposed to trigger the fertilization potential.
Ultrastructural and Morphometric Observations of Cortical Endoplasmic Reticulum in Arbacia, Spisula and Mouse Eggs*
Authors: LUTTMER SHIRLEY , LONGO FRANK J.
Published: January 2005

Results of morphometric investigations indicate that Arbacia eggs possess a network of cortical endoplasmic reticulum equal in volume and surface area to that within the subcortex. The cortical endoplasmic reticulum surrounds individual cortical granules and forms associations with the plasma membrane reminiscent of junctions shared by the sarcolemma and sarcoplasmic reticulum. Mouse eggs, which also exhibit a cortical granule reaction, possess endoplasmic reticulum that is associated with cortical granules and the plasmalemma. The same relative volume of cortical endoplasmic reticulum is present in mouse eggs as in Arbacia. Significantly less cortical endoplasmic reticulum is present in Spisula eggs which do not undergo cortical granule discharge upon activation. These observations are discussed in light of the hypothesis that the cortical endoplasmic reticulum transduces the interaction of the gametes into an intracellular calcium release which initiates the cortical granule reaction and the activation of development.
Cortical Granules of Sea Urchin Eggs do not Undergo Exocytosis at the Site of Sperm‐egg Fusion*
Authors: EPEL DAVID , PATTON CHRIS
Published: January 2005

Microscopic observations of sea urchin egg fertilization (phase contrast, Nomarski and transmission electron microscope) reveal that the cortical granules in the area of sperm egg‐fusion do not undergo exocytosis. These intact granules remain associated with the sperm, moving into the egg cytoplasm with the entering sperm. This sperm‐cortical granule association occurs before the sperm centriole affects microtubule organization and the sperm‐cortical granule association is not affected by cytochalasin D or griseofulvin. We discuss the possibility that a reorganization of the egg cytoplasm ensues from the sperm‐egg interaction at the site of sperm‐egg fusion. Other possibilities are that the retention of cortical granules is not related to egg reorganization, but is necessary for successful sperm incorporation or reflects an unrelated component of the activation process.
Periodicity of Cytoplasmic Cycle in Nonnucleate Fragments of Sea Urchin and Starfish Eggs*
Authors: YONEDA MITSUKI , YAMAMOTO KENYA
Published: January 2005

Fertilized eggs of the sea urchin, Hemicentrotus pulcherrimus and the starfish, Asterina pectinifera were separated by manual bisection to obtain pairs of nucleate and non‐nucleate fragments. Simultaneous observation on the pair revealed that the cyclic change in the cortical tension in the non‐nucleate fragment was definitely prolonged than the cleavage interval of nucleate partner by about 30%. Activated non‐nucleate cytoplasmic fragments derived from the unfertilized eggs and colchicine treated whole eggs of H. pulcherrimus still showed the same degree of prolongation.
Dihydrofolate Reductase in Starfish Oocytes and Embryos: Developmental Consequences of Its Inhibition by Methotrexate1
Authors: IKEGAMI SUSUMU , IMAYOSHI JUNJI , TAKAHASHI NOBUO , NAGANO HIROSHI
Published: January 2005

Dihydrofolate reductase in immature oocytes of the starfish, Asterina pectinifera, is estimated to be 12 pg per oocyte. After completion of meiosis, the quantity of the enzyme is approximately 20 pg per egg. The content of the enzyme in the egg is kept nearly constant at this value from fertilization to the beginning of blastulation. Methotrexate, an analogue of dihydrofolate, at 20 μM did not affect meiotic maturational process and fertilization, but inhibited embryonic development at the 512‐cell stage which corresponds to the beginning of blastulation. Incorporation of externally supplied deoxy[3H]uridine into DNA of the embryos cultured in the continuous presence of 20 μM of methotrexate stopped at the 256‐cell stage, suggesting that the cessassion of development of the embryo at the 512‐cell stage was caused by inhibition of DNA synthesis at the preceding stage. Uptake of [3H]methotrexate was low at early cleavage stages but increased just before blastulation. Externally supplied 1 mM of thymidine counteracted the inhibitory effect of methotrexate at 20 μM, suggesting that the starvation of the methotrexate‐treated embryo for thymidine nucleotides halted DNA synthesis at the beginning of blastulation.
Unique Pattern of Gene Expression in the Erythroid Precursor Cells
Authors: MISHINA YUJI , KATO TAKASHI , URABE AKIO , TAKAKU FUMIMARO , NATORI SHUNJI , OBINATA MASUO
Published: January 2005

Erythroid cells were fractionated by preformed Percoll density gradient from livers of 12.5 day old mouse fetuses. With combination of lysing of mature erythroid cells, the CFU‐E (colony forming unit of erythroid) was enriched as high as 30% pure. The mRNA levels of the rt‐genes previously cloned as genes expressed in the reticulocytes are estimated in the fractionated erythroid cells. These rt‐genes show a drastic change in expression during erythroid differentiation; Their expression was not detectable at the CFU‐E cell stage. But it reached to maximum at the polychromatic erythroblast (stage I) and then decreases with maturation. The result suggests that mRNA synthesis of these rt‐genes may be induced after the stimulation of erythropoietin.