bluuebunny/crossref_metadata_2025 huggingface dataset:
Energy transport and cell polarity: Relationship of phosphagen kinase activity to sperm function
Authors: Tombes Robert M. , Shapiro Bennett M.
Published: June 2005

The energy required for motility of sea urchin sperm is transported from the mitochondrion to the flagellum by a phosphocreatine shuttle involving diffusion of phosphocreatine (PCr) between isozymes of creatine kinase (CrK) localized at the two sites (Tombes and Shapiro, Cell, 41:325, '85; Tombes et al., Biophys. J., 52:75, '87). The present studies demonstrate that high sperm CrK (various echinoderms; sea squirt, bristle worm, salmon) or arginine kinase (mollusc, barnacle, moth) activity is seen in several species with sperm of a primitive morphology (mitochondrion at the base of the head, relatively long flagellum). In contrast, CrK activity is 10–100‐fold less abundant in sperm of other species (frog, mouse, rooster, rabbit, bull, and human) that either possess a modified morphology (mitochondria that extend along the flagellum) and/or utilize glycolytic metabolism. We interpret these findings as support for the use of phosphagen kinase‐dependent energy transport in cells in which the production of adenosine triphosphate (ATP) by the mitochondrion is distant from its utilization, leading to a form of metabolic polarization. Two other cell types, frog photoreceptors and rabbit oviduct cells, whose morphology and function also suggest that they exhibit metabolic polarization, contain relatively high CrK activity. The presence of high phosphagen kinase activity in metabolically polarized gametes and somatic cells further substantiates the role of such enzymes in facilitating energy transport.
Free amino acids and cell volume regulation in the euryhaline ciliate Paramecium calkinsi
Authors: Cronkite D. L. , Pierce S. K.
Published: June 2005

Paramecium calkinsi was isolated from a tidal marsh in which the salinity fluctuated widely on a daily basis. In the laboratory, this ciliate survived for days in sea water ranging in osmotic strength from 10 to 2,000 mOsm and divided in nutritive media of 1,000 mOsm or less. When transferred from 750 to 250 mOsm, cells swelled but regained 78% of the original volume within 60 min and the original volume within 1 day. Cells acclimated to 250 mOsm and transferred to 750 mOsm shrank, regained 40% of the original volume in 60 min, and regained little more volume during the next 24 hr. Free amino acids (FAA), principally proline and alanine, are osmo‐lytes in P. calkinsi. In cells that have been acclimated for more than 1 month Pro is undetectable at 10 mOsm but at 250 mOsm is present in substantial amounts and is still higher at 750 mOsm. Ala is found in cells at all three salinities and increases dramatically with increasing salinity. A complex pattern of amino acid changes occurs during the 4 hr following a transfer from 250 to 750 mOsm, resulting in a marked increase in Ala but no change in Pro. Thus the metabolic changes that lead to the increased FAA levels of acclimated cells are apparently long‐term and complex. After transfer of cells from 750 to 250 mOsm there is a rapid and selective loss of Pro and Ala from the cells to the medium.
Cryoprotective capacity of end products of anaerobic metabolism
Authors: Loomis S. H. , Carpenter J. F. , Anchordoguy T. J. , Crowe John H. , Branchini B. R.
Published: June 2005

The cryoprotective capacity of end products of anaerobic metabolism was measured using resonance energy transfer studies and stabilization of freeze‐labile enzymes during freeze‐thaw. The most effective end products of anaerobic metabolism at preventing fusion of small unilamellar vesicles during freezing were alanopine, alanine, proprionate, acetate, and lactate. Octopine and strombine were less effective, and ethanol resulted in fusion. Lactate dehydrogenase or phosphofructokinase were protected during freeze‐thaw by all of the end products tested. The possible mechanisms of protection of lipid bilayers and enzymes as well as the implications of these results for the survival of intertidal bivalve molluscs during winter are discussed.
Cellular contribution to symmetrical forelimbs from triploid‐marked “polarizing region” in the embryo of the axolotl, Ambystoma mexicanum
Authors: Tank Patrick W.
Published: June 2005

Grafts of posterior tissue placed anterior to the limb bud in the salamander embryo exert a polarizing influence. To explain this result, the idea that the anteroposterior axis of the developing forelimb is polarized by a diffusible morphogen has been proposed [J. Slack, (1977) J. Embryol. Exp. Morphol., 39:169–182]. An alternative hypothesis, and the working hypothesis of the present study, is that the polarization of the developing salamander forelimb is accomplished by short‐range cellular interactions resulting in intercalation rather than by the more global influence of a diffusible morphogen. One prediction of this intercalation hypothesis is that cells will be contributed to the limb from the “polarizing tissue”. To test this idea, grafts of triploid marked polarizing tissue were implanted anterior to the limb bud in 82 diploid axolotl embryos at stages 32–34 of development. A total of 27 (33%) of the limbs that resulted were symmetrical and ranged in complexity from one to seven digits. Histological analysis of a subgroup of the original symmetrical limbs revealed that mesodermally derived tissues in the anterior side of these limbs (the side which formed as a duplication in response to the influence of the graft) contained high percentages of trinucleolate cells (muscle, 12.1%; connective tissue, 12.5%; and cartilage, 13.4%) when compared to similar tissues in the posterior side of the same symmetrical limbs (muscle, 1.8%; connective tissue, 0.7%; and cartilage, 0.6%). When symmetrical limbs were amputated, 73% regenerated symmetrical limbs. When these regenerated limbs were again amputated, 63% formed symmetrical secondary regenerates. Histological analysis of the first generation of regenerated limbs revealed that the pattern of distribution of trinucleolate cells in each regenerate was similar to the pattern seen in the original symmetrical limb. These results indicate that there is considerable cellular contribution to the anterior side of the symmetrical forelimb from the mesoderm of grafted “polarizing tissue”. This result supports the idea that short‐range cellular interactions are sufficient for formation of symmetrical forelimbs in salamander embryos.
Spontaneous maturation of follicular oocytes in Rana dybowskii in vitro: Seasonal influences, progesterone production, and involvement of cAMP
Authors: Kwon Hyuk B. , Lim Young K. , Choi Mee J. , Ahn Ryun S.
Published: June 2005

Seasonal and hormonal influences regulating oocyte maturation (germinal vesicle breakdown, GVBD) in ovarian follicles of Rana dybowskii were investigated. During the early winter (Dec.–Jan.) GVBD occurred at a low incidence following in vitro culture of intact follicles. Addition of progesterone or frog pituitary homogenate (FPH) to such follicles induced oocyte maturation, whereas IBMX or forskolin inhibited hormone‐induced oocyte maturation. The time course of spontaneous in vitro maturation varied markedly with the seasons and between animals. Follicles isolated from the ovaries in early February required 21–24 hours of culture to mature spontaneously, and addition of FPH or progesterone to the culture medium markedly accelerated the time course of GVBD. In contrast, follicles isolated in late February matured very rapidly (< 6 hours), and FPH or progesterone were ineffective in accelerating the time course of GVBD. IBMX and forskolin separately or in combination stimulated follicular progesterone production, which resembled that seen following FPH stimulation. FPH addition to such follicles shifted the steroid peak to the left (accelerated) and increased the absolute amount of hormone detected in latematuring follicles (50% GVBD, about 18 hours) but not in rapidly maturing follicles (50% GVBD, 3 hours). In contrast to other amphibians, a high incidence of spontaneous oocyte maturation occurred during in vitro culture. Essentially all animals exhibited spontaneous maturation during the normal breeding season, even those animals collected in the early winter and kept in artificial hibernation at 4°C for extended periods.
Reception of photoperiodic information by fetal siberian hamsters: Role of the mother's pineal gland
Authors: Elliott Jeffrey A. , Goldman Bruce D.
Published: June 2005

The rate of reproductive development in juvenile male Siberian hamstersis strongly influenced by daylength (photoperiod). Recent studies indicate that reception of photoperiodic cues begins during fetal life. The present experiments yielded a further demonstration that developing male Siberian hamsters receive information about the photoperiod to which their mother is exposed during pregnancy. The possibility that photoperiodic information is transmitted from mother to young after birth was investigated by cross‐fostering young gestated on 12L and 16L to mothers from the other photoperiod. Litters were cross‐fostered on the day of birth and then were transferred, along with their foster mothers, to 14L. We found no influence of the mother after birth, indicating that transmission of photoperiodic information from mother to young must occur during gestation. To determine if the pineal gland of the mother is required for this response, adult females were pinealectomized or sham‐operated and paired with intact males in 12L, 14L, or 16L. After parturition parents and offspring were exposed to 14L. The influence of prenatal photoperiod on postnatal testicular development in 14L was blocked by pinealectomy of the mother. Postnatal testicular development was retarded in offspring that experienced a photoperiod transfer from either 15L to 14L or 8L to 12L at birth. In contrast, the inhibitory effect of a transfer from 16L to 14L at birth was abolished when juvenile males were exposed to a single long photoperiod (16.3 h light) at age 17–21 days and then were returned to 14L.
Effects of short‐term 17β‐estradiol treatment on the properties of t3‐binding proteins in the plasma of immature rainbow trout, salmo gairdneri
Authors: Cyr Daniel G. , Eales J. G.
Published: June 2005

To determine the effects of 17β‐estradiol (E2) on theproperties of the plasma proteins that bind 3,5,3′‐triiodo‐L‐thyronine (T3), immature rainbow trout, Salmo gairdneri, were intraperitoneally injected on days 0 and 3 with 0.5 mg E2‐3‐benzoate/100 g body weight, and plasma was sampled on days 4, 7, or 12. Control trout received peanut oil alone. E2 increased significantly the proportion of [125I]T3 bound to plasma‐binding sites. Separation of plasma proteins of control trout by agarose filtration on Bio‐Gel A‐1.5 m revealed that T3 bound to two main sites corresponding to molecular weights of 130 kDa (presumed prealbumin) and 55 kDa (presumed β‐globulin). Addition of a calculated amount of unlabeled T3 displaced [125I]T3 from the 55 kDa site, indicating its role as the T3 high‐affinity and low‐capacity site. E2 treatment initiated production of vitellogenin, which did not bind T3. Saturation analysis of [125I]T3 binding conducted on miniature G‐25 Sephadex columns indicated that E2 increased the capacity of the low‐affinity, high‐capacity T3‐binding site without effect on the high‐affinity, low‐capacity T3‐binding site. In conclusion, T3 binds to two main plasma sites and E2 causes an increase in the capacity of the low‐affinity site, resulting in a decrease in free T3 and in possible changes in T3 availability to tissues.
Ecdysteroid biosynthesis in the crustacean Y‐organ and control by an eyestalk neuropeptide
Authors: Spaziani Eugene , Watson R. Douglas , Mattson Mark P. , Chen Z.‐F.
Published: June 2005

The endocrine axis is described that controls growth, molting, and regeneration in crustaceans: The ecdysial glands (Y‐organs), sources of ecdysteroid hormones, and eyestalk neurosecretory centers, sources of a putative peptide (molt‐inhibiting hormone, MIH) that exerts negative control of Y‐organ function. Current knowledge of the physiology of these components is briefly reviewed, and in this context Y‐organs are discussed as useful models for study of steroidogenesis generally in animals at the cellular and molecular levels. Data are emphasized that demonstrate the utilization of cholesterol by Y‐organs for biosynthesis of at least two ecdysteroid secretion products, and suppression of the process by MIH. Evidence from radioactive tracer experiments is presented to show that (a) cholesterol in circulating hemolymph and in vitro in the presence of hemolymph serum is quantitatively bound to high‐density lipoprotein (HDL), (b) Y‐organs take up cholesterol by an active process, and (c) uptake is greatly accelerated in the absence of MIH (eyestalks absent) or inhibited by eyestalk extract. Results of in vitro experiments with 125I‐labeled HDL suggest that the HDL‐cholesterol complex is taken into Y‐organ cells mediated by receptors for the HDL apoprotein. Measured as a function of HDL concentration in the medium, cellular acid‐precipitable radioactivity (HDL uptake), acid‐soluble radioactivity (HDL degradation), and ecdysone secretion each exhibited saturation kinetics. Also, at saturation levels of external HDL, uptake, degradation, and secretion were significantly higher in Y‐organs from de‐eyestalked crabs than in glands from intact crabs.
Absence of temperature‐dependent sex determination in congeneric sexual and parthenogenetic Cnemidophorus lizards
Authors: Crews David
Published: June 2005

Eggs from congeneric gonochoristic and parthenogenetic whiptail lizards were incubated at various temperatures. There was no significant deviation from a sex ratio of one‐half in the sexual species at any temperature, whereas no males were produced in the parthenogenetic species. Temperature‐dependent sex determination appears to be completely absent in these lizards.
Effects of electric fields on fibroblast contractility and cytoskeleton
Authors: Harris Albert K. , Pryer Nancy K. , Paydarfar David
Published: June 2005

We used silicone rubber substrata and fluorescent staining of cytoskeletal components to study the mechanisms by which electrical voltage gradients cause reorientation of embryonic chick fibroblasts in tissue culture. No evidence was found for a direct stimulation of cell contractility, either parallel or perpendicular to the voltage gradient. Instead, there was a gradual weakening in cell contractility in the axis parallel to this gradient, accompanied by progressive retraction of lamellae oriented along this axis, apparently due to selective weakening of cell‐substratum adhesions. The cells then elongated perpendicular to the electric field, and strengthened their contractility in that axis.

Fluorescence microscopy showed that cytoplasmic actin stress fibers and microtubules oriented perpendicular to the imposed voltage gradient. Many more cases were observed in which cell morphology had reoriented, but the actin fibers had not, as compared to the converse (cytoskeleton oriented, but not morphology). This disparity further supports the interpretation that the redirection of cell contractility is a consequence of morphological reorientation, rather than its cause.

We also studied the effects of reversing the polarity of the electric fields at constant intervals (of as long as 1 minute). Fibroblasts failed to orient in response to such alternating fields, even after long exposure, but these same cells did reorient in response to pulsed currents in a consistent direction separated by “rest periods” (with no current). This combination of results is more consistent with an electrophoretic mechanism than with one depending on voltage‐induced changes in membrane permeabilities.
Steroidogenesis in Fundulus heteroclitus: III. Evidence for involvement of cAMP and protein synthesis in the gonadotropic modulation of ovarian steroid production and aromatase activity
Authors: Petrino Teresa R. , Hoch Kathryn L. , Lin Yu‐Wai P. , Wallace Robin A.
Published: June 2005

The role of cAMP and the involvement of protein synthesis in ovarian steroidogenesis and aromatase activity of the killifish Fundulus heteroclitus were examined by using isolated prematurational follicles (1.2–1.4 mm diameter) cultured in vitro. Addition of dibutyryl cAMP (dbcAMP) or cAMP in combination with an inhibitor of its catabolism [3‐isobutyl‐1‐methylxanthine (IBMX)] promoted a dose‐dependent increase of media 17α‐hydroxy,2β‐dihydroprogesterone (17α‐OH,20β‐DHP), testosterone (T), and 17β‐estradiol (E2) levels, comparable to the response invoked by a F. heteroclitus pituitary extract (FPE). In addition to steroid production, there was a two‐fold increment in follicular 3H‐leucine incorporation following FPE treatment, probably due to stimulation of protein synthesis. Cycloheximide blocked both 3H‐leucine incorporation and steroid production in a dose‐related manner. FPE also promoted a dose‐related enhancement (more than an additive effect) of E2 accumulation when T was included in the culture media. Results obtained with dbcAMP or cAMP + IBMX in combination with T were essentially the same as with FPE and T, indicating that FPE or increasing levels of cAMP are able to modify aromatase activity. When the endogenous production of E2 caused by FPE or cAMP (dbcAMP or cAMP + IBMX) treatment was suppressed by the use of trilostane (an inhibitor of Δ5‐3β‐hydroxysteroid dehydrogenase), the conversion of exogenous T to E2 was still significantly enhanced. However, no such stimulatory effect on aromatase activity was observed under the same conditions in the presence of cycloheximide. Taken together these results suggest that FPE‐stimulated follicular steroid production by large, prematurational follicles is likely mediated by an intracellular increase in cAMP levels and involves protein synthesis, including that of aromatase. Also, the production of maturation‐initiating steroid by F. heteroclitus follicles does not appear to be affected by physiological levels of E2, unlike the situation reported for other fish and amphibians.
Influence of pH on the regulatory properties of aerobic and anoxic forms of pyruvate kinase in a marine whelk
Authors: Michaelidis Basile , Storey Kenneth B.
Published: June 2005

The effects of pH on the kinetic properties of pyruvate kinase were determined for both the aerobic and anoxic (16 hours exposure to N2‐bubbled seawater) enzyme forms from foot muscle and gill of the marine whelk, Busycotypus canaliculatum. The two enzyme forms from each tissue had distinctly different properties; compared to the aerobic enzyme, the anoxic enzyme form had significantly increased values for S0.5 phosphoenolpyruvate and Ka fructose‐1,6‐bisphosphate and greatly reduced I50 values for L‐alanine. Effects of pH on kinetic constants were assessed at pH 6.6, 7.0, and 7.5. Both the aerobic and anoxic forms of pyruvate kinase in whelk foot showed minimal values for S0.5 PEP and Ka FBP at pH 7.0; these rose by up to twofold at the higher or lower pH values. I50 values for alanine and Mg.ATP were strongly affected by low pH; I50 Mg.ATP fell by tenfold at pH 6.6 compared to pH 7.0, whereas I50 ALA dropped eightfold for the aerobic and 2.5‐fold for the anoxic enzyme form over the same pH change. The qualitative effects of pH change were similar for the anoxic enzyme form from gill. PK‐aerobic from gill behaved differently, however; as pH decreased, S0.5 PEP increased, whereas Ka FBP and I50 ALA both decreased. Furthermore, inhibition by Mg‐ATP occurred only at low pH. Although anoxia‐induced phosphorylation of PK is clearly the primary mechanism for changing the activity state of PK during anaerobiosis, the results suggest that the progressive fall in intracellular pH that occurs during anoxia would enhance the inactivation of PK activity in whelk organs. In particular, the effect of low pH in increasing the sensitivity of the anoxic enzyme forms to inhibitors could facilitate the anoxia‐induced changes in carbon flow at the phosphoenolpyruvate branchpoint.
Excess retinoid acts through the stroma to produce mucous glands from newborn hamster cheek pouch in vitro
Authors: Covant H. A. , Hardy Margaret H.
Published: June 2005

Many morphogenetic processes are modified or initiated by retinoids. The cheek pouch of the newborn hamster can be induced to develop mucous glands in vitro by adding excess retinoid. The objective of this study was to determine whether the retinoid acted through the epithelium or the mesenchymal stroma. Explants of cheek pouch were grown for 7 days in either standard medium, or medium supplemented with 6 μg/ml retinyl acetate (RAc; 1.8 × 10−5M). After separation of most explants into epithelium and mesenchyme by trypsinization, the separated tissues were recombined in all possible ways and cultured for a further 1–2 weeks in standard medium. All explants were analysed histologically and/or histochemically from complete serial paraffin sections. No glands were formed in 30 recombinants containing stroma that had not been exposed to RAc, but four of 25 recombinants containing previously exposed stroma had glands, as well as four of 18 unseparated explants exposed to RAc. Exposure of epithelium to RAc did not result in the incidence of glands. It was concluded that RAc acting through the stroma was responsible for the instructive interaction with the epithelium for gland formation. A molecular mechanism is suggested.
Effect of intermittent GnRH analogue treatments on hormonal levels and spermatogenesis in photostimulated red‐winged blackbirds (Agelaius phoeniceus)
Authors: Lacombe Diane , Matton Pierre , Cyr André
Published: June 2005

In photostimulated red‐winged blackbirds (Agelaius phoeniceus) receiving intermittent injections of 10.0 μg of a potent GnRH analogue (GnRH‐A), the pituitary gland was not desensitized, the androgen response was not clearly impaired, and the diameter of the seminiferous tubules was not modified in GnRH‐A‐treated birds compared to saline‐injected redwings. However, the thickness of the germinal epithelium was significantly reduced in redwings receiving 10.0 μg of GnRH‐A every 4th day and every 7th day compared to the saline‐injected redwings. Surprisingly, the group receiving the maximum number of injections (treated every 2nd day) had a thickness of the germinal epithelium and a proportion of individuals with normal spermatogenesis similar to the saline‐injected group and other GnRH‐A‐treated groups. The proportion of redwings with disturbed spermatogenesis was significantly higher in groups receiving GnRH‐A every 4th day and every 7th day compared to saline‐injected redwings. Impairment of spermatogenesis in redwings treated with GnRH‐A at different frequencies occurred without pituitary desensitization and the clear failure of the androgen response, suggesting that factors affecting Sertoli cell and Leydig cell function are involved.
Sex‐specific DNA in reptiles with temperature sex determination
Authors: Demas Suzanne , Duronslet Marcel , Wachtel Stephen , Caillouet Charles , Nakamura Dean
Published: June 2005

Banded krait minor (“Bkm”) satellite DNA, originating in the W‐chromosome of the snake Bungarus fasciatus, has been found in the genome of diverse eukaryotic species including fruit fly, quail, and horse. Concentrations of Bkm have been found in the presumptive W‐chromosome of snakes with isomorphic sex chromosomes and in the male‐determining region of the Y‐chromosome in mouse and man. We therefore asked whether Bkm‐related DNA might be present in quantitative excess in DNA from males or females in two related species of sea turtle, Chelonia mydas, in which sex is determined by the temperature of the incubating egg, and Lepidochelys kempi, in which the critical sex‐determining temperature has recently been described. Filter hybridization with the Bkm 2(8) probe revealed male‐specific fragments in both species; female‐specific fragments were also revealed in C. mydas. Sex‐specific DNA sequences in temperature‐sex‐determined species such as Kemp's ridley and the green turtle were unexpected, but could be explained if there were an underlying genetic mode of sex determination in these animals, or alternatively, if temperature‐influenced sex determination involved structural modifications in DNA adjacent to, or directly concerned with, the sex‐determining genes. If these results are confirmed across a broader sample of sea turtles, the techniques described in this paper might be used routinely to identify gender in the young of these endangered animals, in which male and female are grossly indistinguishable.
Differentiation of the vitelline coat and the polarized site of sperm entrance in the egg of Unio elongatulus (Mollusca, Bivalvia)
Authors: Focarelli Riccardo , Rosa Domenico , Rosati Floriana
Published: June 2005

We studied the differentiation of the polarized site of sperm entrance in the egg of a freshwater bivalve, Unio elongatulus. As previously shown, in this egg model the property of sperm recognition and binding is restricted to a region characterized by a wrinkled surface which surrounds a truncated cone or crater region, at the vegetal pole of the egg. The crater is formed during oogenesis at a site opposite the occyte attachment to the ovarian wall. It first appears as a small bleb, and later detaches, leaving in the oocyte an open narrow process filled with an orderly array of microtubules. Here the vitelline coat differentiates from the rest of the oocyte. The role of the microtubules in the formation of the region and the differential distribution of the vitelline coat components is discussed. The synthesis during development of fucosyl containing glycoproteins and their presence in an electrophoretic pattern of isolated vitelline coats were also studied using Lotus tetragonolobus (LTA) conjugated with fluorescein and peroxidase.
Study of the differentiation of secretory cells in the golden hamster oviductal epithelium by use of a monoclonal antibody
Authors: Abe Hiroyuki , Oikawa Taneaki
Published: June 2005

The ontogeny of an oviductal zona pellucida glycoprotein, designated ZP‐0, in the golden hamster oviductal epithelium was investigated by means of light and electron microscopic immunolabeling studies using a monoclonal antibody. Light microscopic immunohistochemistry showed that ZP‐0 appeared in the oviductal epithelium from 9.5 to 10.5 days after birth. At first, ZP‐0 appeared in the Golgi region of the epithelial cells, and then increased in amount as development progressed to fill the supranuclear cytoplasm. Electron microscopic observations showed that rough endoplasmic reticulum and Golgi apparatus were well developed in non‐ciliated cells of the ampullar and isthmic segments at 10.5 days after birth. Secretory granules appeared near the Golgi apparatus and markedly increased in number until 15.5 days after birth. Ultrastructural immunocytochemistry showed that ZP‐0 appeared in the well‐developed Golgi apparatus and then was stored within the secretory granules of developing secretory cells. ZP‐0 was never present in the ciliated cells. These results indicate a coincidence between the beginning of ZP‐0 biosynthesis and the morphological cytodifferentiation of secretory cells in the golden hamster oviduct during postnatal development. This leads us to the conclusion that ZP‐0 is a possible differentiation marker of the golden hamster oviductal secretory cell.
Patterning of the head in hydra as visualized by a monoclonal antibody: III. The dynamics of head regeneration
Authors: Javois Lorette C.
Published: June 2005

The dynamics of the early patterning processes leading to the regeneration of a head in tissue excised from the body column of Hydra oligactis were examined by using a monoclonal antibody, CP8. This antibody displays position‐specific binding, labeling the head ectodermal epithelial cells. During regeneration of a head, antibody labeling is present well before morphological signs of the head, at a time correlated with the determination of the tissue (Javois et al., Dev. Biol., 117:607–618, '86). By quantifying antibody labeling during regeneration of three different pieces of tissue excised from the body column, it was found that the dynamics of the early patterning processes as visualized by CP8 labeling varied. The pattern of labeling observed as well as the spread of labeled tissue suggested that the amount and geometry of apical tissue in the regenerate played a critical role in the patterning processes. Contrary to the labeling pattern observed in heads which formed during bud development or which regenerated following decapitation (Javois et al., '86), not all the CP8+ tissue was confined to the head structures in these regenerates. Several alternative explanations for this surprising result are presented. The usefulness of these data in refining pattern formation models by more explicitly constraining their parameters is discussed.
A search for immunoreactive substance P and other neural peptides in the limb regenerate of the newt Notophthalmus viridescens
Authors: Globus Morton , Alles Paul
Published: June 2005

Immunochemical studies demonstrate that the undecapeptide substance P (SP) may be detected by radioimmunoassay in newt limb regenerates and that SP is localized in the blastemal epidermis by immunofluorescence and peroxidase‐antiperoxidase staining. Immunoreactive SP is predominantly distributed at the periphery of epidermal cells, suggesting the presence of SP binding sites on the cell surface; the basal germinative layer of the epidermis and blastemal mesenchyme cells remain unreactive. The pattern of SP immunoreactivity in the blastema was compared with that of four other tachykinin‐family peptides (eledoisin, kassinin, substance K, and neuromedin K) and with three non‐tachykinin neural peptides (bombesin, neurotensin, and metenkephalin). With the exception of neurotensin, which showed weak staining in the basal layer but an absence in the peripheral layers of the epidermis, none of the peptides examined exhibited immunoreactivity in the blastema epidermis comparable to that of SP.
A model for studying isolation mechanisms in parasite populations: The genus Lepeophtheirus (Copepoda, Caligidae)
Authors: De Meeus T. , Renaud F. , Gabrion C.
Published: June 2005

In the Mediterranean, the parasitic copepod Lepeophtheirus thompsoni Baird, 1850 specifically infests turbot (Psetta maxima L., 1758), whereas L. europaensis Zeddam, Berrebi, Renaud, Raibaut, and Gabrion, 1988 infests brill (Scophthalmus rhombus L., 1758) and flounder (Platichthys flesus L., 1758). Experimental infestation of turbot by copepods from each of the three fish species showed an absence of any physiological incompatibility preventing natural development of the two parasite species, at least on one host species, i.e., the turbot. Moreover, interspecific hybrids were obtained experimentally, which implies that 1) there is no strict genetic barrier between the two species and 2) the natural prezygotic isolation results from a choice of the most favorable habitat. We discuss the origin and possible consequences of the presence, in the Mediteranean, of L. europaensis on brill and flounder, two hosts separated by their taxonomic status and ecobiology.
Natural freeze tolerance in the terrestrial turtle, Terrapene carolina
Authors: Costanzo Jon P. , Claussen Dennis L.
Published: June 2005

To investigate freeze tolerance in box turtles (Terrapene carolina), we froze 19 adults to body temperatures as low as −3.6°C under controlled laboratory conditions. Our data reveal that box turtles tolerate the freezing of at least 58% of their body water and can remain frozen, without injury, for at least 73 hr. Supercooling occurred in 63% of the turtles but only lasted from 0.1 to 2.0 hr. Supercooling points (X ± SEM = −1.1 ± 0.3°C) were high, and thus supercooling is not an effective strategy for freeze avoidance in T. carolina. Body ice contents (7–58% of total body water) of turtles frozen for 0.7–50 hr, determined calorimetrically, were inversely related to core body temperature. Significant thermal gradients occurred within the turtles' bodies during prefreeze cooling, freezing, and thawing. Since T. carolina hibernates in shallow terrestrial burrows, where exposure to subzero temperatures is likely, freeze tolerance represents a remarkable adaptation that probably enhances winter survival. These turtles are the largest (up to 0.5 kg) animals, by nearly an order of magnitude, for which natural freeze tolerance has been demonstrated.
Patterns of survival at subzero temperatures by hatchling painted turtles and snapping turtles
Authors: Packard Gary C. , Packard Mary J.
Published: June 2005

Neonatal painted turtles (Chrysemys picta) do not emerge from their nest immediately upon hatching but instead remain inside the nest cavity until the following spring. Because the nest chamber is only 7–12 cm beneath the surface of the ground, hatchlings in northerly populations may be exposed to subzero temperatures during their first winter of life. We studied the pattern of survival by hatchling painted turtles exposed to subzero temperatures in a controlled laboratory experiment and discovered that 50% of the animals in our samples could withstand exposure to approximately −8.6°C for 18 hr. In contrast, hatchling snapping turtles (Chelydra serpentina) died at −2°C. This extraordinary ability for hatchling painted turtles to withstand exposure to subzero temperatures is a key feature in the unusual life history of the species and distinguishes painted turtles from other species occurring at high latitudes.
ENU‐induced allele of brachyury (Tkt1) exhibits a developmental lethal phenotype similar to the original brachyury (T) mutation
Authors: Justice Monica J. , Bode Vernon C.
Published: June 2005

New alleles of brachyury (Tkt1, Tkt4) were induced in the mouse complete tw5 haplotype by ethylnitrosourea (ENU). Like the original brachury (T) mutation, the new alleles cause a short‐tailed phenotype in heterozygotes, and interact with the t complex tail interaction factor (tct) in trans to cause phenotypically tailless mice. Because ENU is mainly a point mutagen, it is important to determine that the new alleles are homozygous embryonic lethal mutations like the original T allele, and to characterize their embryonic lethal phenotype. Moreover, the Tkt1 mutation maps to an inverted position relative to quaking (qk) in t haplotypes as compared with its position on normal chromosome 17. The Tkt1 allele was separated from the resident tw5 lethal gene, tclw5, by recombination, allowing embryology studies to be performed. Embryological analyses show that the Tkt1 allele is nearly identical to the classic T allele. At 9 and 10 days of development, homozygous Tkt1/Tkt1 embryos are grossly abnormal with properties including 1) irregular, disorganized somite pairs, 2) a shortened posterior end of the embryo, 3) an irregular neural tube, and 4) an abnormal notochord. In addition, 10 day‐old abnormal embryos have anterior limb buds that point dorsally rather than ventrally, and are smaller than normal littermates. We conclude that the Tkt1 mutation is a valuable allele for both mapping and molecular characterization of the brachyury locus.
Induction of acrosomal filament formation in the sperm of Sicyonia ingentis
Authors: Griffin Fred J. , Clark Wallis H.
Published: June 2005

The acrosome reaction (AR) in the sperm of Sicyonia ingentis, which consists of the exocytosis of an acrosomal vesicle followed by the formation of an acrosomal filament, can be induced in vitro when sperm are incubated in egg water (Griffin et al., '87). Egg water (EW) was determined to possess a protein/carbohydrate ratio of approximately 4:1 and contain trypsin‐like proteolytic activity (based on the hydrolysis of synthetic peptidyl‐MCA substrates and the results of protease activity SDS‐PAGE). Results of this study demonstrate that the two phases of the AR are elicited by separate inducers or inducing activities within EW and suggest a role for an EW trypsin‐like proteolytic activity in filament formation. The protein concentration of EW (within the range of 20–80 μg/ml) correlated with acrosomal filament formation in sperm but not with acrosomal exocytosis. EW‐induced filament formation was inhibited in the presence of soybean trypsin inhibitor and p‐aminobenzamidine, although acrosomal exocytosis was not. Bovine trypsin, chymotrypsin, and Pronase induced filament formation in the small number of sperm that had spontaneously undergone acrosomal exocytosis in sea water (not incubated in EW), but did not induce additional sperm to undergo exocytosis.
In vitro fertilization of Siberian hamster oocytes
Authors: Parkening T. A.
Published: June 2005

Siberian hamsters were superovulated and various media were tested in an effort to fertilize the recovered oocytes in vitro. The highest percentage of fertilized ova was achieved by using a modified Tyrode's medium, designated MT (Bavister, J. Reprod. Fertil., 18:544–545, '69), previously formulated to fertilize Syrian hamster ova in vitro. Spermatozoa incubated in this medium in a concentrated state overnight (14 hr) and then diluted (1 hr) fertilized 39% of the ova. Similar results (40%) were obtained with this medium by adding 20% human follicular fluid to fresh concentrated sperm for 30 min and then diluting the sperm for 2–3 hr prior to the addition of ova. Ova fertilized in vitro cleaved to the two‐cell stage but failed to develop any further in culture. Two‐cell embryos recovered from mated hamsters and cultured did not undergo additional cleavage. Four‐cell embryos collected from mated females and cultured cleaved to the six‐ to eight‐cell stage and stopped. Techniques and media used for fertilizing large numbers of Syrian and Chinese hamster ova in vitro will have to be modified to achieve the same degree of success in the Siberian hamster.
Changes in density and viscosity of chicken egg albumen and yolk during incubation
Authors: Meuer Hans‐Jürgen , Egbers Christoph
Published: June 2005

In early stages of avian development, respiratory gas exchange takes place mainly through the blood vessels of the yolk sac membrane. Therefore, a short distance between the yolk sac membrane and the eggshell is required to ensure a sufficient oxygen supply of the embryo. In freshly laid eggs the yolk sphere is located approximately in the center of the egg, but soon after the beginning of incubation it moves to the upper pole right beneath the eggshell. The cause for this displacement is not known. To quantify the forces acting on the yolk sphere of chicken eggs during incubation, we measured the densities of albumen, yolk and subembryonic fluid and the viscosity of the liquid albumen. We found that between day 0 and day 4 of incubation the calculated buoyancy due to density differences between the egg components increases only threefold. This is probably too small to overcome the yield stress of the gel‐like thick albumen that forms a capsule around the yolk sphere. A different process responsible for the yolk displacement is proposed. Under reduced gravity, the movements of the yolk are influenced substantially by the fluid properties of the thin albumen, especially yield stress. Furthermore, acceleration peaks present in a space laboratory will probably displace the yolk sphere away from the eggshell. This is strong evidence that an early chick embryo will not be able to survive in a space laboratory in ovo.
Lampbrush and mitotic chromosomes of the hemiclonally reproducing hybrid Rana esculenta and its parental species
Authors: Bucci Stefania , Ragghianti Matilde , Mancino Giorgio , Berger Leszek , Hotz Hansjürg , Uzzell Thomas
Published: June 2005

Mitotic chromosomes of the European water frogs Rana ridibunda and Rana lessonae, the parental species of Rana esculenta, differ significantly in their centromeric regions: when C‐banded or when made fluorescent, the centromeres of R. ridibunda (and of the ridibunda chromosomes in R. esculenta) are visible as a conspicuous dark granule or as a conspicuous fluorescent spot; the centromeres of R. lessonae (and of the lessonae chromosomes in R. esculenta) are inconspicuous or not fluorescent. Lampbrush chromosomes of these three taxa are described in detail for the first time; those of R. ridibunda and R. lessonae differ significantly in morphostructural characters such as conspicuousness of centromeres and number, form, and location of giant loops as well as in chiasma frequency. Chromosomes of the two parental species can thus be distinguished when present in lampbrush complements of hybrids. Reproduction in both sexes of natural R. esculenta lineages is hemiclonal: only the unrecombined genome of one parental species, usually R. ridibunda, is transmitted to haploid gametes (hybridogenesis). In 18 hybrids from natural populations of Poland, somatic tissues had allodiploid complements with chromosomes from each parental species. In contrast, spermatocytes I of five males and oocytes I of seven of eight females (221 of 222 oocytes) were autodiploid and contained only R. ridibunda chromosomes that formed n bivalents. These 12 hybrids thus were hybridogenetic. A single female hybrid had oocytes I (33 of 34) with genomes of both parental species; they showed various disturbances including tetraploidy, reduced number of chiasmata, and incomplete synapsis resulting in univalents. This individual thus was not hybridogenetic. The irregular lampbrush patterns indicate that such hybrids will have severely reduced fertility and most of their successful gametes will result in allotriploid progeny.
Improved developmental potential of rabbit oocytes fertilized by sperm microinjection into the perivitelline space enlarged by hypertonic media
Authors: Yang Xiangzhong , Chen Jingbo , Chen Yu Qi , Foote Robert H.
Published: June 2005

The objectives of the present study were: 1) to develop a simple and more efficient technique for sperm microinjection than is currently available, using the rabbit as a model, and 2) to evaluate the development of rabbit oocytes fertilized by single or multiple sperm microinjection. Hyperosmotic sucrose in phosphate‐buffered saline (SPBS) was employed to dehydrate oocytes to increase the perivitelline space for sperm microinjection and prevent possible injury to the vitellus. In the first experiment, 58% (n = 29) oocytes treated with 0.5 M SPBS developed to morulae following multiple sperm microinjection compared, respectively, to 47% (n = 34) and 60% (n = 15) for control IVF with or without sucrose exposure (P > 0.05). Blastocyst development from microinjected oocytes, however, was much lower (P < 0.05) than that of controls (14% vs. 42% and 40%, respectively). Sham operation by puncturing the zona pellucida of the sucrose‐treated oocytes with the microinjection pipette did not increase parthenogenesis (P > 0.05). In Experiment 2 a smallersize injection pipette and shorter sucrose exposure time after sperm microinjection resulted in 41% (n = 42) of the oocytes developing into blastocysts for the microinjection group, whereas only 21% (n = 24) developed to blastocysts in the control IVF group (P < 0.05). When relatively older oocytes (17 hr post ovulation injection) were used to test if microinjection could reduce the time to fertilization and cleavage (Expt. 3), an average of 27% (n = 63) blastocysts resulted from microinjection vs. 0% (n = 28) for the control IVF group.
Low temperature tolerance of the gemmules of Eunapius fragilis
Authors: Boutselis Nicholas J. , Fell Paul E. , Loomis Stephen H.
Published: June 2005

This present study has shown that hydrated gemmules of Eunapius fragilis (Leidy) are able to survive brief exposure to temperatures as low as −100°C. However, the rates at which the gemmules are cooled and warmed have a marked effect on their survival. Hydrated gemmules exhibited good survival when cooled to −30°C at 0.1°C/min or 0.5°C/min. On the other hand, gemmules cooled at 1.0°C/min or 10.0°C/min did not survive. The effect of warming rate on the survival of gemmules exposed to −30°C was less pronounced than that of the cooling rate. However, gemmules which were warmed very slowly (0.1°C/min) showed a low level of germination. Hydrated gemmules also survived exposure to −72°C for at least 30 days. It appears that the time of exposure to low temperature is far less important in determining survival than the rates at which the gemmules are cooled to such low temperatures and then subsequently warmed.
Two types of striated muscle suggest two‐geared swimming in the pteropod mollusc Clione limacina
Authors: Satterlie Richard A. , Goslow G. E. , Reyes Arjun
Published: June 2005

Forward locomotion in the pteropod mollusc Clione limacina occurs in two distinct speeds, slow and fast. Fast swimming can include an initial ballistic escape phase following proper stimulation. Escape swimming is always followed by a period of fast swimming (both are forms of fast swimming), but changes from slow to fast swimming can occur without activation of the escape phase. Light and electron microscopical examination of the swim musculature reveals two types of striated muscles, suggesting that the neuromuscular system is designed to operate with two distinct “gears”. Histochemical staining for myosin ATPase (alkaline preincubation) and NADH diaphorase suggests that swim musculature includes slow‐twitch, fatigue‐resistant fibers and fast‐twitch fatigable fibers. Combined electrophysiological and force transducer recordings support the twitch speed dichotomy, as fast‐twitch motor units were found to have twitch times of 20 ms (time to peak tension) while slow‐twitch motor unit twitch times were found to be more than three times as long. Fatigue tests were used to simulate both slow and fast swimming. Fatigue rates during simulated fast swimming suggest that the change from escape swimming to fast swimming may be partly explained by fatigue in the fast‐twitch motor units, but other factors, possibly including peripheral modulation of muscle activity, must also contribute to the change.
Apolipoprotein A‐I synthesis during the acclimatization of the carp (Cyprinus carpio)
Authors: Inostroza Juan , Vera Maria Ines , Goicoechea Oscar , Amthauer Rodolfo , Krauskopf Manuel
Published: June 2005

Total protein and apo A‐I synthesis were studied in suspensions of isolated hepatocytes from winter‐ and summer‐acclimatized carp. The overall protein synthesis in liver is significantly higher in summer‐adapted fish. Although apo A‐I synthesis accounts for the same fraction of the total protein synthesis when assayed at the seasonal environmental temperature in both adaptive states, winter‐acclimatized fish synthesize apo A‐I at 10°C at a higher rate than the summer fish. Thus, reprogramming of apo A‐I synthesis accompanies the adaptative compensatory response.

Differences were found in the apo A‐I content in liver of carp acclimatized to the cold and warm seasons. The low apo A‐I content found in the liver of winter‐acclimatized carp reverses upon treatment of the fish with insulin. This apo A‐I enhancement leads to hepatocytes resembling those of the summer‐acclimatized state.