bluuebunny/crossref_metadata_2025 huggingface dataset:
Selective growth and serial passage of mouse melanocytes from Neonatal epidermis in a medium supplemented with bovine pituitary extract
Authors: Hirobe Tomohisa
Published: June 2005

Suspensions of disaggregated epidermal cells from skins of newborn C57BL/10JHir mice were plated in a growth medium that consisted of Ham's F‐10 plus bovine pituitary extract (BPE), insulin, and transferrin. Fetal bovine serum (FBS) was added to the culture medium at a concentration of 4% at the time of plating. On the second day of culture, a small number of melanocytes was randomly distributed among large sheets of keratinocytes. From the third day onward, FBS was excluded from the culture medium to prevent the proliferation of keratinocytes and fibroblasts. The melanocytes began to grow preferentially, and after 12 days pure and enriched populations of melanocytes could be harvested. In the absence of the proliferation of keratinocytes and fibroblasts, melanocytes could be serially passaged in the growth medium supplemented with a conditioned medium (CM) prepared from keratinocyte‐enriched cultures, namely, those at the early stages of the primary culture. FBS was added at a concentration of 1% for the first day. These results suggest that both BPE and keratinocyte CM contain growth factors required for proliferation of melanocytes.
Production of identical twin and triplet mice by nuclear transplantation
Authors: Kono T. , Tsunoda Y. , Nakahara T.
Published: June 2005

Transplantation of a single nucleus from two‐ or four‐cell embryos into one of the enucleated blastomeres of a two‐cell embryo resulted in successful production of identical triplet and twin mice. The proportion of reconstituted embryos that developed in blastocysts was 71% (84/118) when four‐cell embryos were used as donors of nuclei; 10 sets of quadruplet and nine sets each of triplet and twin blastocysts were obtained by this technique. After transfer to recipients, 30% (18/61) developed to term, and one set of identical triplet and four sets of identical twin mice were obtained. When two‐cell embryos were used as donors of nuclei, 79 (95%) sets of twin embryos developed to blastocysts. Of 38 twin blastocysts transferred to recipients, 21 sets (55%) developed to term as identical twin mice. These results demonstrate that the enucleated two‐cell embryo develops in vitro after transfer of a nucleus from a two‐ or four‐cell embryo and the resultant blastocyst has high potential for development to term after transfer to a recipient.
Effects of lamellolysin from a parasitoid wasp on Drosophila blood cells in vitro
Authors: Rizki R. M. , Rizki T. M.
Published: June 2005

Female parasitoid Leptopilina heterotoma inject a factor, lamellolysin, along with their eggs into the host hemocoel to destroy selectively host hemocytes that encapsulate foreign objects. In parasitized Drosophila melanogaster larvae, these hemocytes (lamellocytes) change from discoidal cells to bipolar cells that no longer adhere to each other to form capsules. To study the effects of lamellolysin on Drosophila lamellocytes in vitro, a giant strain of D. melanogaster was constructed to yield hemolymph with an abundance of lamellocytes. The effect of lamellolysin on the adhesivity of lamellocytes in vitro was demonstrated when the cells were gently rotated in the culture medium. Under these conditions, the bipolar shape of the affected lamellocytes resembled that of lamellocytes in parasitized hosts. When lamellocytes were exposed to lamellolysin in stationary culture medium, the elongation of the bipolar cells continued until they became threadlike. Lamellocytes fragmented in both stationary and rotating culture medium in the presence of lamellolysin, although loss of cellular material was more pronounced in the latter. This study demonstrates that lamellolysin acts directly and destructively on lamellocytes.
Protamines in prosobranchian gastropods (mollusca) vary with different modes of reproduction
Authors: Daban Montserrat , Chiva Manel , Rosenberg Ellen , Kasinsky Harold E. , Subirana Juan A.
Published: June 2005

We have studied the protamines from different families of prosobranch gastropods. In those with external fertilization we find a simple pattern of sperm nuclear basic proteins, consisting of one major protamine. This protamine has between 75 and 105 amino acid residues in the ten species of the order Archaeogastropoda that we have examined. In six species of the order Patellogastropoda (Kozloff, Marine Invertebrates of the Pacific Northwest, Univ. of Wash. Press, Seattle, pp. 193–232, 1987), the protamine‐like component contains 220 ± 10 amino acid residues. However, the protamines from the archaeogastropod Monodonta turbinata and the patellogastropod Patella cerulea show a very similar amino acid composition. Cytochemically, four species of archaeogastropods and three species of patellogastropods show intermediate type 3 sperm basic proteins in the classification scheme of Bloch (Genetics 61:93–111, 1969; Handbook of Genetics, Plenum, New York, pp. 136–167, 1976). The sperm basic protein profile in six internally fertilizing species of the orders Mesogastropoda and Neogastropoda is more complex since several protamine bands are found. In all cases the major protamines have high electrophoretic mobilities and the number of amino acid residues is estimated to be from 40 to 60. The combined set of major protamines in the mesogastropod Littorina neritoides and the neogastropod Murex brandaris display a similar amino acid composition that differs significantly from that in the externally fertilizing gastropods. In one mesogastropod and three neogastropod species cytochemical staining indicates the possible presence of type 2 stable protamines. Protamines from a neogastropod have a capacity for compacting DNA that is similar to that of an externally fertilizing archaeogastropod. We propose that changes in protamine features amongst different prosobranch gastropods are correlated with changes in nuclear condensation patterns and the fertilization biology of these mollusks.
Isolation of highly acidic and vanadium‐containing blood cells from among several types of blood cell from ascidiidae species by density‐gradient centrifugation
Authors: Michibata Hitoshi , Iwata Yukinari , Hirata Junko
Published: June 2005

The reported pH values of ascidian blood cells vary considerably. It is likely that one or two of several different types of blood cell must have a highly acidic solution within their vacuoles, in which vanadium ions would be present in a reduced state. Thus, one of the reasons for the variation in reported pH values may be that measurements of pH were made without fractionation of the total population of cells. In the present experiments, the combination of fractionation of whole cells by density‐gradient centrifugation, use of a microelectrode that allows measurements of pH under anaerobic conditions, and atomic absorption spectrometry for determinations of vanadium revealed that the contents of signet ring cells, recently identified as vanadocytes, of three ascidian species, Ascidia gemmata, A. ahodori, and A. sydneiensis samea, had low pH values of 2.4, 2.7, and 4.2, respectively. Results obtained by ESR (electron spin resonance) spectrometry confirmed the values of pH obtained with the microelectrode.
Expression of the 9G1 antigen in the apical cap of axolotl regenerates requires nerves and mesenchyme
Authors: Onda Hiroaki , Tassava Roy A.
Published: June 2005

Monoclonal antibody 9G1 (mAb 9G1) is reactive to the wound epithelium of axolotl larvae and therefore provided the opportunity to examine the interaction between the wound epithelium, nerves, and blastemal mesenchyme during axolotl limb regeneration. In unamputated limbs, mAb 9G1 is reactive to most or all cells of the dermis, skeletal elements, blood vessels, and nerves, to a few unidentified cells in muscle, and to none in epidermis. During regeneration of axolotl limbs, mAb 9G1 reacts strongly to an intracellular antigen of the blastemal mesenchyme and of the distal‐most portion of the wound epithelium, the so‐called apical epithelial cap (AEC). Because this thickened wound epithelium of regenerating amphibian limbs has been suggested as functioning in a manner similar to the apical ectodermal ridge (AER) of embryonic limb buds, it was of interest to further examine the reactivity of mAb 9G1 during various stages of regeneration. Whether mAb 9G1 reactivity in the AEC depended on mesenchyme and/or nerves was also tested. Monoclonal antibody 9G1 reactivity appears in the AEC of regenerating limbs prior to outgrowth of the blastema and persists throughout blastemal stages. Apical epithelial cap reactivity to mAb 9G1 is nerve dependent during early stages of blastema development and becomes nerve‐independent at later stages. When epithelium‐free blastemal mesenchyme is grafted onto injured flank musculature, ectopic limb regeneration occurs and the AEC derived from flank epidermis exhibits mAb 9G1 reactivity. These results show that a mAb 9G1 reactive AEC is characteristic of regenerating limbs and that expression of the 9G1 antigen by the AEC is dependent upon underlying blastemal mesenchyme and nerves.
Effects of thyroid hormone deficiency in eggs on early development of the medaka, Oryzias latipes
Authors: Tagawa Masatomo , Hirano Tetsuya
Published: June 2005

To examine the biological significance of thyroid hormones contained in eggs, the hormones were depleted from eggs by treating the mother fish with thiourea. Constantly spawning medaka (Oryzias latipes) were reared in fresh water containing 0.03% thiourea. The plasma thyroxine (T4) and triiodothyronine (T3) concentrations in the mother fish were about 8 and 5 ng/ml, respectively, before the treatment, and decreased to 1/5 of the initial levels within 1 day. Initial concentrations of T4 and T3 in eggs just after fertilization were about 4 and 1 ng/g, respectively, and decreased to less than 1/4 when the mother was treated with thiourea for 7 days. Minimum effective concentration of thiourea to decrease the hormone concentrations in mother's plasma and the eggs was 0.003%. During the course of normal development, egg thyroid hormones are maintained at the initial levels during the first 4 days, and continuously decrease toward hatching (10 days after fertilization). There was no difference between the normal eggs (derived from the mother fish without thiourea treatment) and hormone‐deficient eggs (hormone concentrations were less than 1/10 of the control) in hatchability, the time of hatching, and survival under starvation. No difference was seen between the two groups in body weight, body length, condition factor, and survival rate 16 days after hatching. These findings indicate that more than 90% of the hormones contained in eggs are not essential for early development in medaka.
Evidence for a role of Na+,K+‐ATPase in the hydration of Atlantic croaker and spotted seatrout oocytes during final maturation
Authors: LaFleur G. J. , Thomas P.
Published: June 2005

During final maturation the oocytes of many marine teleosts swell four to five times their original size due to uptake of water. The involvement of active inorganic ion transport and Na+,K+‐ATPase in oocyte hydration in Atlantic croaker (Micropogonias undulatus) and spotted seatrout (Cynoscion nebulosus), marine teleosts which spawn pelagic eggs, was investigated by examining changes in the inorganic ion content of ovarian follicles containing mainly oocytes, by performing in vitro incubations of the follicles with ion channel blockers, and by assaying membrane preparations of ovaries containing hydrating and non‐hydrating oocytes for Na+,K+‐ATPase activity and content. There were marked increases in the contents of K+, Mg++, and Ca++, but not Na+, in oocytes of M. undulatus and C. nebulosus during hydration. Incubation of follicle‐enclosed oocytes in K+‐free medium or with ouabain or amiloride, inhibitors of Na+, K+‐ATPase and Na+ channels, respectively, blocked gonadotropin‐induced oocyte hydration in M. undulatus. In addition, Na+,K+‐ATPase activity increased threefold and the concentration of the enzyme increased 50% in ovarian tissue during oocyte hydration. These results strongly suggest a major role for active ion regulation by a ouabain‐sensitive Na+,K+‐ATPase system in oocyte hydration in two species of sciaenid fishes.
Sources of calcium, magnesium, and phosphorus for embryonic softshell turtles (Trionyx spiniferus)
Authors: Packard Mary J. , Packard Gary C.
Published: June 2005

Eggs of softshell turtles (Trionyx spiniferus) were incubated on wet (water potential = −150 kPa) and dry (water potential = −1,500 kPa) substrates at 29°C. The rigid, heavily calcified eggshells restricted exchanges of water between eggs and their environment, but eggs exposed to wet substrates nonetheless contained more water at the end of incubation than eggs in the drier environment. However, differences in the amount of water contained by eggs had no demonstrable effect on patterns of metabolism and growth by embryos. The embryos in both treatments obtained about 75% of their calcium from the eggshell, but acquired all of their magnesium and phosphorus from the yolk. Residual yolk removed from hatchlings contained minimal quantities of all three elements. Thus, all the calcium mobilized during development was deposited in the carcass, and yolk was depleted of almost its entire store of this element. This pattern of mobilization and deposition of calcium is similar to that observed in embryos of other chelonians and in those of oviparous lepidosaurinas. In contrast, embryonic crocodilians and birds store some of the calcium mobilized from the eggshell in the yolk, and residual yolk consequently contains a substantial reserve of calcium. Our results indicate that the pattern of mobilization and deposition of calcium documented for birds and crocodilians probably is an attribute of the archosaurian lineage and not a simple result of embryos developing in rigid, heavily calcified eggshells.
Early devlopmental profile of ornithine decarboxylase in the frog, Microhyla ornata and its regulation by polyamines
Authors: Joseph Kusumam , Baby T. G.
Published: June 2005

Ornithine decarboxylase (ODC) activity and polyamine levels were measured during early development of the frog, Microhyla ornata. ODC activity was found to be high and it showed three major peaks during the first 60 hr of development. Putrescine and spermidine levels increased gradually during the above period with little change in spermine. Treatment of developing embryos with exogenous putrescine and spermidine prevented the normal increase in ODC activity. Spermine did not have any significant effect. Addition of ornithine also prevented the increase in ODC activity. Experiment using exogenous ornithine and α‐methylornithine revealed that formation of putrescine and/or spermidine from ornithine is necessary for the suppression of ODC to occur. Suppression of ODC takes place even if conversion of putrescine to spermidine is blocked, indicating that putrescine, independent of its conversion to spermidine, also plays a role in ODC regulation.
Purinergic receptors as a regulatory process of luminescence in Porichthys' luminous organs
Authors: Rees J. F. , Kaouass M. , Baguet F.
Published: June 2005

The effects of exogenous adenosine and adenine phosphorylated derivatives were tested on the adrenergic luminescence of isolated luminous organs of the fish Porichthys. Adenosine triphosphate, at concentrations ranging from 10−5M to 10−2M, strongly inhibited the two peaks of the biphasic‐shaped light emission induced by adrenalin. Adenosine diphosphate mimicked the effects of ATP, whereas adenosine monophosphate (AMP) and cyclic‐AMP were ineffective. Adenosine had no inhibitory effect but potentiated the first peak of light whose intensity was five times increased. It is concluded that P2‐purinergic receptors might be present on photocytes and exert a negative control on the luminescent system. Adenosine‐induced potentiation might be related to an intracellular effect of this compound.
Hepatic 5′‐deiodinase activity of Japanese quail using reverse‐T3 as substrate: Assay validation, characterization, and developmental studies
Authors: Freeman Thomas B. , McNabb F. M. Anne
Published: June 2005

Using rT3 as substrate, an in vitro 5′D assay was validated for use with liver tissue from adult Japanese quail, by defining conditions under which activity is proportional to enzyme (protein) concentration and is linear with incubation time. Activity was measured as the release of 125I from labeled rT3. Using validated assay conditions we found the following 5′D characteristics: maximal activity from 10 to 50 mM dithiothreitol (cofactor), an apparent Km of 0.52 μM rT3, pH optimum of 7.6‐8.5, complete inhibition by 1 mM propylthiouracil and by 1.0 mM iopanic acid, and substrate “preference” of rT3 > T4 > T3. Based on these characterizations the quail hepatic 5′D activity is like the Type I 5′D activity found in mammalian liver and kidney and embryonic chicken liver. To determine how previous unvalidated assays, that used high tissue and relatively low substrate (T4) concentrations, influenced 5′D studies we reevaluated 5′D development using an assay validated for each developmental stage with rT3 as substrate. We found extreme quantitative differences in the activities measured and in the proportional relationships between stages, and only limited qualitative similarity in the pattern of 5′D development when unvalidated T4 assay results were compared with validated rT3 assay results. Our data in this paper show good correspondence between whole liver 5′D activity per unit body weight and plasma T3/T4 ratios for the developmental stages sampled.
Reversal of postmortem degeneration of mouse oocytes during meiotic maturation in vitro
Authors: Schroeder Allen C. , Johnston Daniel , Eppig John J.
Published: June 2005

The developmental capacity of oocytes matured in vitro following isolation at the germinal vesicle stage from freshly killed mice (control) was compared with that of oocytes isolated from the carcasses of mice killed 3, 6, 9, and 12 hr earlier. The yield of intact, cumulus cell‐enclosed oocytes decreased as the interval between death of the animal and removal of the ovary increased. After 15–16 hr of culture of medium containing follicle‐stimulating hormone, the frequency of germinal vesicle breakdown, extrusion of a polar body, and cumulus expansion was equivalent in oocytes of all groups. The frequency of development of inseminated ova to 2‐cell stage embryos in the control, 3, and 6 hr postmortem groups was the same but declined markedly in the 9 and 12 hr groups. There was also no difference in the frequency of blastocyst development from 2‐cell stage embryos between the control, 3, 6, and 9 hr postmortem groups, but the 2‐cell embryos in the 12 hr postmortem group did not develop to blastocysts. Thirty‐six percent of the 2‐cell stage embryos from the 6 hr postmortem group did not develop to blastocysts. Thirty‐six percent of the 2‐cell stage embryos from the 6 hr postmortem group developed to live young after transfer to foster mothers. Follicles of 6 hr postmortem ovaries showed degeneration manifested as prominent crystalline inclusions within the oocytes and many pyknotic granulosa cells. The crystals disappeared within 1 hr of culture and the secondary oocytes appeared normal. The cultured oocyte–cumulus cell complexes, therefore, reversed degenerative changes induced by the death of the animal. This study demonstrates the feasibility of recovering developmentally competent oocytes from valuable recently deceased zoological, agricultural, and endangered mammals.
Trehalose accumulation in the tardigrade Adorybiotus coronifer during anhydrobiosis
Authors: Westh Peter , Ramløv Hans
Published: June 2005

Changes in the trehalose level in the tardigrade Adorybiotus coronifer were observed during induction of, and arousal from, anhydrobiosis. A trehalose accumulation surpassing 1.6% dry weight (d.w.) in anhydrobiotic animals collected dry on their biotope was rapidly reduced on rehydration, reaching the level of active animals (0.1–0.2% d.w.) after 6 hours. Tardigrades dried while embedded in sand in the laboratory accumulated trehalose from 0.1% to 2.3% d.w. within 5–7 hours.

Induction of aerobic acidosis in arousing tardigrades by CO2 perfusion reversibly arrested reactivation for at least 36 hours and induced a reduction in anabolic and catabolic activities, measured as a significant reduction in trehalose degradation, and a sevenfold reduction in the rate of protein synthesis.

These data support the hypotheis that trehalose generally serves a protective role in desiccationtolerant Metazoa, but indicate that tardigrades require only a moderate level for efficient protection.
Culture of intact sertoli/germ cell units and isolated seroli cells from Squalus testis: I. Evidence of stage‐related functions in vitro
Authors: Dubois Wilfrid , Callard Gloria V.
Published: June 2005

As part of an ongoing program of research using the testis of the dogfish shark (Squalus acanthias) to characterize morphologic and functional changes during spermatogenesis, we have developed procedures for culturing intact spermatocysts (germ cell/Sertoli cell clones) and isolated Sertoli cells from premeiotic, meiotic, and postmeiotic stages of development. Phase contrast and light microscopy confirmed the stage and cellular composition of spermatocysts and showed that they retained their closed, spherical configuration for at least 15 d in culture. Stagerelated variations in [3H] thymidine incorporation (premeiotic ≫ meiotic = postmeiotic) were observed, a pattern that was the same quantitatively and qualitatively after one or seven days of culture. [3H] Leucine‐labeled protein synthesis was twofold greater in cultures with premeiotic spermatocysts than in cultures with more mature stages, whether medium or cysts were analyzed. Sertoli cells isolated from spermatocysts of different stages differred in size, shape, cytological appearance, ability to from flattened monolayers, and rate of DNA synthesis. One day after seeding, [3H] thymidine labeling of Sertoli cells corresponded to the pattern obtained with intact spermatocysts (premeiotic ≫ meiotic = postmeiotic); however, 7 days in culture effected a 40‐ to 200‐fold increase in this parameter and altered the stage‐dependent pattern (premeiotic = meiotic < postmeiotic). Also, when [3H]leucine‐labeled macromolecules secreted by Sertoli cells from premeiotic versus meiotic stages were analyzed by polyacrylamide gel electrophoresis (PAGE), banding patterns differed. Initial results demonstrate the feasibility and potential of this in vitro system for studying qualitative and quantitative changes during spermatogenesis.
The meiotic response of cumulus cell‐enclosed mouse oocytes to follicle‐stimulating hormone in the presence of different macromolecules
Authors: Downs Stephen M. , Dow Mark P. D. , Fagbohun C. Funsho
Published: June 2005

Experiments were carried out to determine the effect of different macromolecules on the follicle‐stimulating hormone (FSH)‐induced maturation of mouse oocytes in culture. Cumulus cell‐enclosed oocytes (CEO) were isolated from gonadotropin‐primed mice and maintained in meiotic arrest for 17–18 h with the cAMP analogue, dibutyryl cAMP (dbcAMP). Germinal vesicle breakdown (GVB) was stimulated by the addition of FSH. Medium was supplemented with either no macromolecule or with varying concentrations of polyvinylpyrrolidone (PVP), polyvinylalcohol (PVA), crystallized bovine serum albumin (BSA), or fetal bovine serum (FBS). Oocyte maturation in all FSH‐free cultures occurred at a frequency of about 30% or below. High frequencies of maturation were achieved when FSH was added to macromolecule‐free medium or to cultures containing PVP, PVA, or BSA. Crystallized BSA was the most effective of these in supporting stimulation of maturation (94% GVB at 3 mg/ml, compared with 72–74% with synthetic polymer‐supplemented or macromolecule‐free media). The BSA effect was not due to contaminating fatty acids, and a less pure fraction V BSA was not as effective in supporting FSH‐induced maturation. FBS suppressed FSH stimulation of maturation in a dose‐dependent fashion. Sera from pigs, goats, horses, and rats were also inhibitory, but bovine calf serum (BCS) permitted a high maturation frequency (80% GVB). When added to medium containing either FBS or BCS, crystallized BSA had no effect on FSH‐stimulated maturation, but fraction V BSA suppressed maturation in both serum‐supplemented media. Under no conditions did FSH stimulate maturation in cumulus cell‐free oocytes. These results demonstrate that hormone‐induced oocyte maturation is supported in vitro by nonprotein polymers as well as BSA and that the behavior of the oocyte–cumulus cell complex depends on the purity of the BSA sample. In addition, serum contains inhibitory factors that suppress the positive response to FSH. Thus, the choice of macromolecular supplement is of critical importance when testing the hormone responsiveness of isolated cumulus cell‐enclosed oocytes in culture.
Trypsin inhibitors prevent the progesterone‐initiated increase in intracellular calcium required for the human sperm acrosome reaction
Authors: Pillai Muralidharan C. , Meizel Stanley
Published: June 2005

Inhibitors of trypsin‐like enzymes, benzamidine hydrochloride and 4′‐acetamidophenyl 4‐guanidinobenzoate (also an inhibitor of other serine proteases), were tested for their effects on the acrosome reaction (AR) of human sperm initiated by progesterone or the calcium ionophore ionomycin. The AR was assayed by indirect immunofluorescence and transmission electron microscopy. The trypsin inhibitors, when added 10 min prior to stimulation by progesterone, significantly inhibited the AR in comparison with progesterone treatement alone. Transmission electron microscopic examination of the sperm after progesterone treatment indicated that the inhibitors blocked the membrane fusion events of the AR. By contrast, when ionomycin (at final concentrations of 3μM) was added to sperm preincubated in inhibitors, sperm underwent morphologically normal AR, acrosomal matrix loss was not inhibited, and the percentage of acrosomereacted sperm was the same as that obtained in the absence of inhibitors. Using the cell calcium indicator fura‐2, we further demonstrated that both trypsin inhibitors prevented the progesterone stimulated rise in intracellular Ca2+ ([Ca2+]int) required for the AR, but did not affect [Ca2+]int in unstimulated sperm. These results suggest that sperm trypsin‐like activity may be directly or indirectly involved in increasing sperm [Ca2+]int during stimulation by progesterone.
Egg exudate‐induced reduction of sperm lysin sensitivity in the vitelline coat after fertilization of Bufo japonicus and its participation in polyspermy block
Authors: Yamasaki Hisashi , Katagiri Chiaki
Published: June 2005

The jellyless eggs of Bufo japonicus or those from which the vitelline coats (VCs) had been removed (denuded eggs) were electrically activated. The exudate that accompanied egg activation (AEX) was collected to study its role in preventing polyspermy. When dejellied (but VC intact) eggs were treated with AEX, the eggs lost not only fertilizability but also the sensitivity of their VCs to the sperm lysin. By contrast, denuded eggs treated with AEX were fertilizable; even activated eggs were highly fertilizable, provided they were deprived of their VCs and inseminated 30 min after activation. The loss of sensitivity to sperm lysin occurred in VCs 3–5 min after activation either in De Boer's or 1/20 De Boer's solution. The activity of AEX to reduce the sensitivity of VCs to sperm lysin was heat‐sensitive and dependent on Ca2+, but it was not affected at all by the variety of protease inhibitors used. The activity was lost by the preincubation of AEX with fragmented VCs in the presence of Ca2+, suggesting Ca2+‐dependent binding of AEX molecules to the VC at fertilization. Immunocytochemical studies employing anti‐AEX rabbit serum showed that the pertinent antigens were localized in the cortical granules of unfertilized eggs and in both the inner surface of VCs and the perivitelline space of fertilized eggs. We conclude that the AEX‐induced loss of lysin sensitivity in VCs and the deposition of cortical granule materials on the inner wall of VCs constitute a slow and permanent block to polyspermy.
The distribution of tenascin and its transcript in the developing avian central nervous system
Authors: Tucker Richard P.
Published: June 2005

I have studied the temporal and spatial regulation of the expression of the extracellular matrix protein tenascin in the developing chick brain using immunological and in situ hybridization methods. Tenascin is barely detectable by immuno‐slot blot analysis in the E4 telencephalon, but increases rapidly in abundance until E14, when the level of expression tapers off. The late appearance of tenascin was confirmed by immunohistochemistry. Tenascin can not be detected at E4, but at E7 is distributed homogeneously in most parts of the central nervous system. Tenascin is much less abundant in the retina than in other brain areas. As the avian retina is devoid of both vasculature and fibronectin, this supports the notion that tenascin may mask fibronectin from migrating growth cones and cell bodies. I have also used in situ hybridization with a cDNA probe to determine the origins of tenascin in the developing nervous system. Tenascin transcript is found in regions of glial proliferation and the cell bodies of radial glia. In contrast, tenascin appears to be made by neurons in the retina. As a low‐molecular weight splice variant of tenascin is detected on western blots of retina homogenates, different cell types may be responsible for generating the multiple forms of tenascin found in whole brain homogenates.
The striated body: A new structure in the sperm head of the rabbit spermatozoon
Authors: Courtens J. L. , Biggiogera M. , Gabbiani G. , Fakan S.
Published: June 2005

The striated body, a new structure lying in the perinuclear substance of rabbit spermatozoa, is described. It is composed of protein cords displaying a perpendicular periodic arrangement of dark and light bands. It appears in late spermatids, immediately prior to spermiation and is still present in epididymal spermatozoa, mostly, but not exclusively close to the perforatorium. The nature of this component has been analysed using a series of cytochemical techniques, as well as various antibodies and immunocytochemistry. The absence of RNA and of DNA favours a nonviral origin for this structure. The striated body is devoid of actin, differing in that respect from the rest of the perinuclear substance.
Nitrogen excretion is enhanced during urine recycling in two species of terrestrial crab
Authors: Wolcott Donna L.
Published: June 2005

Terrestrial crabs confront the problems of salt and water balance and nitrogen excretion in a terrestrial environment with organ systems evolved to function while immersed in an aquatic environment. Their antennal glands produce urine that is essentially isosmotic to the blood, and contains very little nitrogenous waste, yet terrestrial crabs are excellent iono‐ and osmoregulators in nature. Because voided urine is passed to the branchial chamber for reclamation of ions, the gills, the major organ for ion regulation in aquatic crabs, are still able to perform their ion‐regulating role even in air‐exposed individuals. The present study demonstrates that, during urine recycling, the gills retain their function as the major avenue for nitrogen excretion as well. In two species of terrestrial crab, Gecarcinus lateralis and Cardisoma guanhumi, the concentration of ammonia increases more than 10‐fold in urine during recycling at the gill, greatly enhancing the amount of nitrogen that can be excreted for a given volume of water lost in the urine. Additional results show that without the ability to reclaim ions from the urine by recycling it at the gills, G. lateralis would be unable to remain in osmotic equilibrium.
Labeling of developing vascular endothelium after injections of rhodamine‐dextran into blastomeres of Xenopus laevis
Authors: Rovainen Carl M.
Published: June 2005

The goal of this work has been to label endothelial cells with flurescent marker and to record their behavior during angiogenesis in vivo. Single blastomeres in 16–128‐cell‐stage embryos of pigment‐deficient Xenopus laevis were injected intracelluarly with 5% tetramethylrhodamine dextran. Subsequently, the embryos and tadpoles were examined with an epifluorescence microscope, a silicon‐intensified target (SIT) camera, and video recordings. Clones that would include endothelium could be selected as early as stages 33–36 on the basis of heavy labeling in the ventral mesodermal core of the tail. Strands of fluorescent cells and early vessels appeared in the tail at stages 39–41. Subsequently, groups of endothelial cells were followed in case histories in the tail and in the aortic arches and gills of tadpoles.

Two main results were that the patterns of fluorescent endothelial cells were stable in established arteries, veins, and capillaries for at least 2–12 days, and that labeled endothelial cells migrated distally in elongating sprouts.

In addition, it was inferred that endothelium was derived from multiple blastomeres, probably in the ventral vegetal regions. Only small fractions of total endothelium were labeled from any single blastomere. None of the early blastomeres produced exclusive clones of vascular endothelium; other labeled cell types in various clones included muscle fibers, lymphatics, mesodermal stellate cells, blood cells, gut, proctodeum, and some epidermis, in addition to endothelial cells.

The method of intracellular marking of blastomeres is recognized as a direct approach for charting lineage and fate tables in embryos of Xenopus and other species. The present study extends the period of observation in vivo for up to 2 weeks in the growing tadpole and focuses on endothelial cells during angiogenesis. Even though fluorescent dextran was apparently packaged in vesicles and metabolized, individual cells and small groups could be identified and followed with time. This method provides excellent opportunities for addressing problems in vascular development in the living animal.
Transverse ionic passages through the cuticular ductules from the haemocoel to the glandular cavities and the lumen of the spermathecal duct in female crickets (Gryllus bimaculatus)
Authors: Yasuyama Kouji , Kimura Tetsuya , Yamaguchi Tsuneo
Published: June 2005

In the spermathecal duct of the female cricket Gryllus bimaculatus, the glandular cells, each of which has a central cavity, and the cuticular ductules, extending from the cuticular intima of the spermathecal duct, are widely distributed along the duct's length, except in the proximal and distal regions. The cuticular ductules approach the glandular cavities along a tortuous course and enter them, or run down towards them to terminate in the region of the basement membrane. When an isolated spermathecal duct was incubated in an ionic lanthanum solution, deposits of lanthanum were found in the lumina of the cuticular ductules, the secretory cavities of the glandular cells, and in the openings of the cuticular ductules to the lumen of the spermathecal duct, but also in the intercellular space, and the surface of muscle fibers surrounding the spermathecal duct. This finding suggests that each cuticular ductule is a tripartite passage between the haemocoel, the glandular cavity, and the lumen of the spermathecal duct.
Effect of temperature on the splenic mechanism of respiratory compensation in the newt
Authors: Frangioni Giuliano , Borgioli Gianfranco
Published: June 2005

The spleen of the newt Triturus cristatus carnifex (Laurenti), an urodele amphibian, hoards erythrocytes when the animal is well‐oxygenated and releases them into the bloodstream when the animal is in a hypoxic condition. Between the extremes of congestion and decongestion the weight of the spleen varies up to a ratio of 6:1. However, the degree of the phenomenon depends on the temperature, which, in ectotherms, regulates metabolic rate and thus oxygen demand. In order to assay the degree of hypertrophy reached by congested spleens in standard respiratory conditions, groups of eight animals—previously anesthetized by immersion for 20 minutes in a 0.2% solution of chlorbutol in tap water at 18°C—were exposed to 100% humid air in a thermostatic chamber set at a temperature of 6, 12, 18, 24, 27, 30, or 33°C. Each animal remained at the set temperature until surpassing the minimum time necessary for maximum hypertrophy to be reached, equal to the time necessary for 4,000 heartbeats. At 33°C the spleens are almost completely bloodless, while at the other temperatures their hypertrophy from congestion increases linearly as the temperature diminishes, reaching maximum values at 6°C. Though the progressive congestion of the spleen as the temperature descends is due mainly to blood cells and only very slightly to plasma, erythrocyte concentration in the blood remains constant at all the lower temperatures up to that of anesthesia (18°C); this phenomenon implies variability of blood volume in the newt. Instead, above 18°C, the values of the hematic parameters increase as the temperature rises, reaching a maximum increment of about 30% by 27°C.
Subdivisions of the terminal nerve in Xenopus laevis
Authors: Hofmann Michael H. , Meyer Dietrich L.
Published: June 2005

The majority of recent studies on the terminal nerve (nt) in various vertebrates either involved tracer injections into the nasal cavity or made use of the LHRH‐/FMRFamide‐like immunoreactivity (ir) of a portion of its fibers. The present investigation was designed to determine the extent of overlap between data rendered by the two methods in Xenopus. The findings reveal no overlap of nt projections visualized by the two experimental techniques. This result sheds doubt on the validity of current definitions of the nt system.
Sex differences in hepatic monooxygenases in winter flounder (Pseudopleuronectes americanus) and scup (Stenotomus chrysops) and regulation of P450 forms by estradiol
Authors: Gray Elisabeth Snowberger , Woodin Bruce R. , Stegeman John J.
Published: June 2005

Details concerning the endogenous regulation of hepatic cytochrome P450 mono‐oxygenases in teleosts, and the features of this regulation common among fish species, are poorly known. Gonadally mature female winter flounder (Pseudopleuronectes americanus) have been reported to have severalfold lower levels of microsomal cytochromes P450 and b5 and NADPH cytochrome c reductase than do males (Stegeman and Woodin ('84) Mar. Environ. Res., 14:422–425). These strong sex differences prompted more detailed study of P450 regulation in winter flounder liver, and a comparison with sex differences in another marine teleost, scup (Stenotomus chrysops). Ethoxyresorufin O‐deethylase (EROD) activity/nmol P450 was less in gonadally mature females than in males of both species. Immunoblot analysis with MAb 1‐12‐3 to P450E (the EROD catalyst) showed that the content of P450E counterpart was also much less in females of both species. Aminopyrine N‐demethylase (APND) and testosterone 6β‐hydroxylase (6β‐OHase) activities per nmol P450 were higher in gonadally mature female than in mature male flounder, differences not seen in scup. Polyclonal antibodies to scup P450A were shown to detect proteins in a number of teleosts. The levels of anti‐P450A cross‐reacting protein were greater in mature female than in male flounder, but as with 6β‐OHase activity, the content of this protein was not sexually differentiated in scup. Estradiol treatment of winter flounder depressed the rates of EROD, APND, 6β‐OHase, and estradiol 2‐OHase activities per mg protein, but APND and 6β‐OHase activities per nmol P450 were unchanged. Thus, E2 promotes general decreases in some hepatic P450‐catalyzed activities, but in achieving sex differences there is also specific regulation of the P450E counterpart, and possibly of the 6β‐OHase (P450A?). Other factors, temporal or hormonal, can modify the effect of E2 treatment, and may contribute to the specific regulation of P450 forms in naturally maturing fish, and to species differences in this regulation.
Resumption of meiosis in pig oocytes cultured with cumulus and parietal granulosa cells: The effect of protein synthesis inhibition
Authors: Motlik Jan , Nagai Taku , Kikuchi Kazuhiro
Published: June 2005

In denuded and cumulus‐enclosed pig oocytes, puromycin at concentrations 5, 10, and 25 μg/ml did not lower the rate of germinal vesicle breakdown (GVBD) after 24 h of culture. GVBD was prevented in 50, 75, and 100 μg/ml of puromycin. After 40 h of culture, 5 and 10 μg puromycin/ml impaired significantly incidence of metaphase II (42 and 30%), respectively. Concetrations of 25 and 50 μg puromycin/ml absolutely prevented the first polar body (I PB) expulsion. The results indicated that GVBD in pig oocytes is far less sensitive to puromycin than I PB expulsion.

Culture of cumulus‐enclosed pig oocytes isolated with a piece of membrana granulosa (C+P oocytes) did not allow GVBD after 24 and 32 h in control medium. After 24 h of culture, GVBD occurred in 43 and 56% of C + P oocytes in the medium supplemented with 17 and 25 μg puromycin/ml. GV was broken down in 80 and 68% of C + P oocytes cultured in 17 and 25 μg puromycin/ml for 32 h. It is concluded that inhibition of protein synthesis by puromycin released pig oocytes from the block exerted by granulosa cells.
Subzero temperature adaptations in arthropods from the summit of Mauna Kea, Hawaii
Authors: Duman John G. , Montgomery Steven L.
Published: June 2005

A preliminary study of subzero temperature adaptations was made of arthropods from the summit (4,200 m) of Mauna Kea, Hawaii. This barren, alpine stone desert aeolian habitat commonly experiences snow and nightly subzero temperatures in spite of its subtropical location. In mid‐December, collections were made of a wolf spider (Lycosa sp.). the lygaeid bug (Nysius wekiuicola), and a noctuid caterpillar (Agrotis sp.). The Agrotis caterpillars were freeze tolerant. They had quite high supercooling points (SCP = −4.8°C) and survived a 24 hr frozen exposure at −6.2°C. The site of nucleation is apparently not the hemolymph, as the hemolymph SCP was −15.1°C. The spiders and wekiu bugs were not freeze tolerant. The SCPs of the spiders (X = −5.8°C) were not particularly low, but these highly mobile arthropods may be able to seek thermally buffered microhabitats when the need arises. The wekiu bugs showed greater supercooling abilities, with some individuals supercooling to −18°C, although the SCPs were quite variable. The subzero temperature adaptations of the arthropods from such an isolated subtropical site are remarkable adaptive capabilities.
Hepatic ulstrastructure changes associated with the parr‐smolt transformation of Atlantic salmon (Salmo salar)
Authors: Robertson John C. , Bradley Terence M.
Published: June 2005

Liver samples of two groups of juvenile Atlantic salmon (Salmo salar) were collected fall through spring and examined by light and transmission electron microscopy. One group of fish was maintained under simulated natural photoperiod (SNP), the other under an 8 month advanced, phase‐shifted photoperiod (8MA). As indicated by changes in branchial Na+/K+ATPase activity, salinity tolerance, and morphological characteristics, fish in the SNP group underwent parr‐smolt transformation in the spring. Smoltification‐associated changes in ultrastructure were observed in liver parenchymal cells only. Hepatocytes of SNP smolts were smaller with markedly decreased glycogen content as compared to cells of SNP parr. An increase in mitochondrial size and a decrease in numbers of mitochondria per hepatocyte were also seen during smoltification. In smolt hepatocytes, the nucleus and mitochondria occupied a greater percent of the cell volume than in parr liver parenchymal cells. Rough and smooth endoplasmic reticulum, dense bodies, and Golgi profiles also appeared to occupy more of the cell volume of smaller hepatocytes seen during smoltification. No smoltification‐associated changes were seen in hepatocyte nuclear size, chromatin state, or nucleolar frequency. During the fall, Atlantic salmon in the 8MA group underwent some morphological and osmoregulatory changes associated with smoltification. Hepatic fine structure of these fish in the fall resembled that seen in SNP smolts. In spring, 8MA fish exhibited no indications of smoltification, and hepatocyte ultrastructure of these fish was similar to that of SNP parr. Biological implications of observations are discussed, particularly in light of other investigations concerning the liver and smoltification.
Axon composition of the proprioceptive PD nerve during growth and regeneration of lobster claws
Authors: Cooper R. L. , Govind C. K.
Published: June 2005

A prominent chordotonal organ spanning the propus and dactyl (PD) joint in the chelipeds of the lobster Homarus americanus has the somata of its sensory cells embedded in an elastic strand while their axons collectively travel in a nerve to the ganglion. Electron microscopic examination of the PD nerve revealed several large and many small axon profiles denoting few movement‐sensitive and many position‐sensitive cells. This axon composition typified the nerve in the normally occurring, paired asymmetric crusher and cutter claws, as well as in the experimentally induced, paired symmetric cutter claws. In the asymmetric condition, the crusher nerve had many more axons than the cutter while in the symmetric condition, the numbers were equivalent between the paired cutter claws. In a size‐graded series of lobsters with paired asymmetric claws there was an increase in the number and size of axons in keeping with an increase in claw size. In a newly regenerated claw, the PD nerve was a miniature version of its pristine counterpart with smaller and fewer axons. Moreover, in newly regenerated paired asymmetric claws, the crusher nerve did not have a larger number of axons than the cutter, suggesting that PD nerve has not fully differentiated. The distribution of axons according to size was, however, similar to the pristine nerve in that there were few large axons and many small ones, suggesting that the ratio of large, movement‐sensitive cells to small, position‐sensitive ones in the PD organ is rigidly specified during regeneration and growth.
Lens formation from the cornea following implantation into hindlimbs of larval Xenopus laevis: The influence of limb innervation and extent of differentiation
Authors: Filoni Sergio , Albanesi Cristina , Bernardini Sergio , Cannata Stefano M.
Published: June 2005

Corneal fragments of larval Xenopus laevis at stage 48 (according to Nieuwkoop and Faber, ′56), were implanted into sham denervated unamputated hindlimbs, denervated unamputated hindlimbs, amputated and sham denervated hindlimbs, and amputated and denervated hindlimbs of larvae at stages 52 and 57.

The results show that unamputated limbs at stage 52, either innervated or denervated, manifest a weak capacity to promote the first lens‐forming transformations of the outer cornea. This capacity is absent in both limb types at stage 57. After amputation, limbs of both early and late stages form a regenerative blastema and support lens formation from the outer cornea. Denervation of early stage limbs has no appreciable effect on blastema formation and lens‐forming transformation of corneal implants. However, denervation of late stage limbs inhibits both processes.

These results indicate that the limb tissues of the early stage limbs contain non‐neural inductive factors at a low level and that after limb amputation and blastema formation the level of these factors becomes high enough to promote lens formation from implanted cornea, even after denervation. In contrast, the limb tissues of late stage limbs do not contain a suitable level of non‐neural inductive factors.