bluuebunny/crossref_metadata_2025 huggingface dataset:
Osmolyte permeability in molluscan red cells is regulated by Ca2+ and membrane protein phosphorylation: The present perspective
Authors: Pierce Sidney K.
Published: June 2005

During the past several years, a number of studies on a variety of cell types have demonstrated the importance of Ca2+ in the control of the mechanism that produces cell volume recovery following a hypoosmotic stress. We have been studying the details of this control using the red blood cell from the bivalve mollusc, Noetia ponderosa, as a model. Volume recovery by these molluscan cells is a Ca2+‐sensitive process that involves initial Ca2+ entry, followed by phosphorylation of certain membrane‐associated proteins and taurine efflux. This paper reviews the present state of those studies and presents a working hypothesis of the nature of the mechanism. © 1994 Wiley‐Liss, Inc.
Quantitative immunoblot detection of rare proteins in whole cell extracts using biotin‐strepavidin reagents
Authors: Stevenson Bruce R. , Richards Cheryl L. , Howarth Andrew G. , Maraj Vikram A. , Hibbard Jennifer G.
Published: June 2005

A novel immunoblotting method designed for quantitative detection of low copy‐number proteins in crude cell extracts is presented. This technique can be used with either mono‐ or poly‐clonal antibodies and utilizes the sensitivity and amplification of the high affinity interaction between biotin and strepavidin. Radioactive iodination of the strepavidin moiety allows for rapid and accurate quantification of proteins bound to nitrocellulose. This biotin/125I‐strepavidin technique is highly reproducible and can detect as little as 1 ng of protein. In addition, use of biotinylated secondary antibodies directed against a specific type of primary antibody avoids the problem of low affinity recognition of immunoglobulins from certain species by protein A. Finally, the methodology is simple and convenient, and secondary and tertiary reagents are commercially available. The application of this technique is illustrated in the determination of relative quantities of the tight junction‐associated protein ZO‐1, present in very small amounts in epithelial cells. This same technique can also be used for the quantitative analysis of relatively more abundant cellular constituents or purified protein. © 1994 Wiley‐Liss, Inc.
Morphodynamic hair bundles arising from sensory cell/supporting cell complexes frequency‐tune nematocyst discharge in sea anemones
Authors: Mire‐Thibodeaux Patricia , Watson Glen M.
Published: June 2005

Discharge of nematocysts from cnidocytes occurs in response to appropriate chemical and mechanical stimulation. In sea anemone tentacles, activating chemoreceptors for N‐acetylated sugars shifts maximal discharge into vibrating targets to low frequencies corresponding to prey movements and induces hair bundles to elongate by approximately 1–2 μm. Until now, only indirect, correlative evidence linked these two events. Using cytochalasin D, we provide evidence that bundle elongation is necessary for the frequency shift. Moreover, we find that only bundles associated with sensory cell/supporting cell complexes elongate with chemosensitization, and not bundles associated with cnidocyte/supporting cell complexes as was previously thought. Cytochemical labeling of sensory cells, purported to be bipolar neurons connected to the nerve net, suggests that sensory cells may interconnect with each other and with cnidocytes. Taken together, these findings are incompatible with the classical view that cnidocytes are independent effectors of nematocyst discharge and, furthermore, implicate the involvement of morphodynamic neurons in fine‐tuning vibration‐dependent discharge of nematocysts into swimming prey. © 1994 Wiley‐Liss, Inc.
Endogenous ionic currents and voltages in amphibian embryos
Authors: Metcalf M. E. Mourey , Shi Riyi , Borgens Richard B.
Published: June 2005

Using a noninvasive vibrating electrode for the measurement of extracellular current, we show that a polarized ionic current traverses the embryo for many hours in the anuran, perhaps days in the urodele following gastrulation. The voltage driving these ionic currents is an internally positive transepithelial potential (TEP) normally expressed across embryonic integuments. Current is driven out of the lateral walls of the neural folds and the blastopore and enters most of the rest of the embryo's body surface. The magnitude of the TEP is transitorily dependent on external sodium and can be reduced by the embryo's immersion in Na+depleted media or by treatment with 50 μM amiloride. Both treatments fail to chronically reduce externally detected currents, however. The pattern of currents traversing the embryo suggests they would be associated with rostral‐caudal and medial‐lateral gradients of voltage within the embryo. By sampling the distribution of TEPs in axolotl embryos, we provide measurements of the former—an internal, caudally negative, potential gradient beneath the neural plate ectoderm. The magnitude of these endogenous fields is on the order of 10 to 20 mV/mm and is within a range of potential known to affect the shape and migration of a variety of embryonic cell types in vitro. We suggest that endogenous currents and voltages in the vertebrate embryo may provide gross cues for cell movement and emerging developmental pattern. © 1994 Wiley‐Liss, Inc.
Characterization of striped bass growth hormone receptors by disulfide‐bond reduction and cross‐linking studies
Authors: Gray Elisabeth S. , Tsai Robert W.
Published: June 2005

Growth hormone (GH) receptors were analyzed in striped bass (Morone saxatilis) by addition of disulfide‐bond reducing agents to radioreceptor assays and by cross‐linking both striped bass and coho salmon (Oncorhynchus kisutch) crude membrane preparations to radiolabeled hormone. Dithiothreitol (DTT) caused a dose‐dependent increase in specific binding of 125I‐tilapia (Oreochromis mossambicus) GH to striped bass membrane preparations. Maximal enhancement of 3.4‐fold was obtained with 1 mM DTT and 0.03 trypsin inhibitor units/ml of aprotinin. Addition of N‐ethylmaleimide (NEM), which binds covalently to free sulfhydryl groups, decreased specific binding. Scatchard analysis of striped bass membrane preparations indicated a single class of GH receptors. Addition of DTT with aprotinin increased GH‐binding site concentration from 278 to 507 fmol/mg, while the dissociation constant of 0.56 nM remained unchanged. Cross‐linking 125I‐tilapia GH to striped bass hepatic membrane preparations and 125I‐salmon GH to coho salmon membrane preparations yielded two to three specifically labeled proteins on sodium dodecyl sulfate (SDS)‐polyacrylamide gel electrophoresis. Endoglycosidase H treatment was without effect on specifically labeled proteins from either species. Following digestion with N‐glycosidase F, relative molecular weights of specifically labeled 125I‐GH complexes were reduced, suggesting that hepatic GH‐binding proteins in striped bass and salmon are N‐linked glycoproteins. © 1994 Wiley‐Liss, Inc.
Identification of trypsin‐like activity in sturgeon spermatozoa
Authors: Ciereszko Andrzej , Dabrowski Konrad , Lin Feng , Doroshov Serge I.
Published: June 2005

We documented amidase activity in lake sturgeon (Acipenser fulvescens) and white sturgeon (Acipenser transmontanus) spermatozoa. This activity was inhibited by benzamidine and soybean trypsin inhibitor (SBTI), and N‐α‐p‐tosyl‐L‐lysine chloromethyl ketone‐HCl (TLCK), but not by N‐tosyl‐L‐phenylalanine chloromethyl keton (TPCK), indicating the presence of trypsin‐like activity in sturgeon spermatozoa. Higher activity occurred at pH 8.5 and 9.0 than at 8.0. No effect of 10 mM KCl and 100 mM NaCl on trypsin‐like activity was found. Calcium ions inhibited the activity at 10 and 50 mM. Similar activities were documented in neat semen and washed sperm suspension. Presence of trypsin‐like activity corresponds to the presence of acrosomes and acrosome reaction in spermatozoa of these fishes. © 1994 Wiley‐Liss, Inc.
Effect of hexylene glycol‐altered microtubule distributions on cytokinesis and polar lobe formation in fertilized eggs of Ilyanassa obsoleta
Authors: Conrad Abigail H. , Stephens Andy P. , Conrad Gary W.
Published: June 2005

Some effects of gravity on early morphogenesis are correlated with microtubule locations within cells. During first cleavage in Ilyanassa obsoleta embryos, a transitory polar lobe constriction forms and then relaxes, allowing the polar lobe to merge with one daughter cell. If the polar lobe is equally divided or removed, morphogenesis is severely disrupted. To examine microtubule locations during early Ilyanassa development, eggs were fixed and stained for polymerized α‐tubulin during first cleavage. The mitotic apparatus assembles at the animal pole. The cleavage furrow forms between the asters, constricting to a stabilized intercellular bridge encircling midbodybound microtubules, whereas the polar lobe constriction forms below and parallel to the spindle, constricting to a transitory intercellular bridge encircling no detectable microtubules. At metaphase an α‐tubulin epitope is distributed throughout the spindle, whereas a β‐tubulin epitope is present predominantly in the asters. Incubation in hexylene glycol, a drug that increases microtubule polymerization, during mitosis causes the polar lobe constriction to tighten around polymerized α‐tubulin and remain stably constricted. If hexylene glycol is removed, α‐tubulin staining disappears from the polar lobe constriction, which relaxes, whereas microtubules remain in the cleavage furrow, which remains constricted. These observations suggest that asymmetric distribution of microtubules affects early Ilyanassa cleavage patterns, and that continued presence of microtubules extending through an intercellular bridge is important for stabilization of the bridge constriction prior to completion of cytokinesis. These data provide the basis for further analysis of the role of microtubules in possible microgravity disruptions of Ilyanassa development. © 1994 Wiley‐Liss, Inc.
Pre‐metatarsal skeletal development in tissue culture at unit‐ and microgravity
Authors: Klement Brenda J. , Spooner Brian S.
Published: June 2005

Explant organ culture was used to demonstrate that isolated embryonic mouse pre‐metatarsal mesenchyme is capable of undergoing a series of differentiative and morphogenetic developmental events. Mesenchyme differentiation into chondrocytes, and concurrent morphogenetic patterning of the cartilage tissue, and terminal chondrocyte differentiation with subsequent matrix mineralization show that cultured tissue closely parallels in vivo development. Whole mount alizarin red staining of the cultured tissue demonstrates that the extracellular matrix around the hypertrophied chondrocytes is competent to support mineralization. Intensely stained mineralized bands are similar to those formed in pre‐metatarsals developing in vivo. We have adapted the culture strategy for experimentation in a reduced gravity environment on the Space Shuttle. Spaceflight culture of pre‐metatarsals, which have already initiated chondrogenesis and morphogenetic patterning, results in an increase in cartilage rod size and maintenance of rod shape, compared to controls. Older pre‐metatarsal tissue, already terminally differentiated to hypertrophied cartilage, maintained rod structure and cartilage phenotype during spaceflight culture. © 1994 Wiley‐Liss, Inc.
Polarity and body pattern in ascidian chimeras formed between normal (situs normale viscerum) and reversed (situs inversus viscerum) bodies
Authors: Ishii Teruhisa , Taneda Yasuho , Saito Yasunori
Published: June 2005

In the compound ascidian Polyandrocarpa misakiensis, when two anterior or two posterior body fragments of zooids are fused together at their cut surfaces, a double‐zooid monster is formed. On the other hand, fusion of an anterior fragment and a posterior fragment results in a single‐zooid chimera. In the present study, we tried to make chimeric zooids between normal (situs normale viscerum) and mirror‐image, or reversed (situs inversus viscerum), adult bodies. In order to distinguish between the two partners in a chimera, zooids of two strains that differed in color pattern were used (Ishii et al., [1993] Zool. Sci. 10:977–982). Using normal zooids of the spotless strain and reversed zooids of the white‐spot strain, we formed chimeras using the anterior fragment of a zooid and the posterior fragment in four combinations between these two colormorphs. In all the combinations, chimeras were obtained. Chimeras obtained from the combinations of a normal anterior fragment and a reversed anterior fragment appeared as double‐zooid monsters. Analysis of the body pattern of these chimeras revealed that the polarity of a reversed anterior fragment returned to normal polarity. Chimera derived from a normal posterior fragment and a reversed posterior fragment also resulted in a double‐zooid monster, but the polarity of a reversed posterior fragment did not change. Chimeras obtained from the combination of a normal anterior fragment and a reversed posterior fragment or from the combination of a reversed anterior fragment and a normal posterior fragment exhibited either of two distinct patterns of body regeneration: they became a malformed double‐zooid monster, or a single‐zooid chimera with normal polarity. Thus, in these single‐zooid chimeras, the reversed polarity of the anterior portion became normal. Furthermore, buds that were formed from the anterior portion of these single‐zooid chimeras had normal polarity. © 1994 Wiley‐Liss, Inc.
Influence of ocular and extraocular photoreception on spermatogenesis in Gambusia affinis holbrooki (Teleostei: Poeciliidae)
Authors: de Miguel María P. , Fraile Benito , Sáez Francisco J. , Vicentini Carlos A. , Paniagua Ricardo
Published: June 2005

Non‐blinded and blinded male mosquito‐fish (Gambusia affinis holbrooki) were subjected to three different photoperiods (a short photoperiod of 8 hr light daily, a simulated natural photoperiod that was increased from 12.25 to 14.50 hr of light, and a long photoperiod of 16 hr of light) at a constant mild temperature (20°C) during the spermatogenesis period (March–May). Afterwards, the testes were removed and studied by quantitative histological methods, along with the testes from initial and final field controls. Fish exposed to simulated natural or long photoperiod (16L:8D) developed complete spermatogenesis like the final controls. Fish exposed to short photoperiod developed spermatogenesis up to the primary spermatocyte stage. Photoperiodic effects on spermatogenesis were similar in both blinded and intact males subjected to the same photoperiodic conditions. Thus, extraocular photoreception must be involved in photoperiodic effects on reproduction, and the presence of the eyes is not required for spermatogenesis in this species. © 1994 Wiley‐Liss, Inc.
In vivo and in vitro induction of germinal vesicle breakdown in a freshwater bivalve, the zebra mussel Dreissena polymorpha (Pallas)
Authors: Fong Peter P. , Kyozuka Keiichiro , Abdelghani Haitham , Hardege Jörg D. , Ram Jeffrey L.
Published: June 2005

Oocyte maturation and germinal vesicle breakdown (GVBD) was induced in zebra mussel (Dreissena polymorpha) oocytes by in vivo and in vitro application of serotonin (5‐hydroxytryptamine, 5‐HT), and in vitro application of 8‐hydroxydipropylaminotetralin hydrobromide (8‐OH‐DPAT, a 5‐HT1A receptor agonist). Oocytes initiated GVBD approximately 30 minutes after exposure to 5‐HT (10−3 M) at 23°C, and by 40–50 minutes after exposure, most oocytes lacked a germinal vesicle. An exposure time to 5‐HT as brief as five to ten minutes was required to trigger the maturation process, which terminates in spawning of fertilizable oocytes in nearly all mussels. But, with an exposure time of less than five minutes, spawning was reduced by application of 10−4 M methiothepin (a potent blocker of 5‐HT‐induced spawning in zebra mussels). Thus, the sequence of oocyte maturation events in zebra mussels was determined. Oocytes are arrested at the germinal vesicle stage (prophase I) within the ovary. 5‐HT reinitiates the maturation process, including GVBD and spawning of metaphase I oocytes, which are further arrested until fertilization. To our knowledge this is the first demonstration of oocyte maturation induction by serotonergic ligands in a freshwater bivalve. © 1994 Wiley‐Liss, Inc.
Evidence in support of a role for Ca2+‐activated K+ channels in the hamster sperm acrosome reaction
Authors: Llanos Miguel N.
Published: June 2005

The sperm acrosome reaction (AR) is a crucial step for mammalian fertilization. This work describes experiments to test the effect of the cesium ion (Cs+) and charybdotoxin (ChTX) on the Ca2+ or Na+/K+ ionophores stimulated hamster sperm AR in vitro. Cs+ and ChTX, a polypeptide toxin from the venom of the scorpion Leirus quinquestriatus, are considered blockers of Ca2+‐activated K+ channels in several somatic cells. Both agents inhibited the AR by 55–66%. The inhibition was completely reversed by the Na+/K+ ionophore nigericin, but not by the Ca2+ ionophore A‐23187. Results give evidence in support of a role for Ca2+‐activated K+ channels in K+ influx required for the occurrence of the hamster sperm acrosome reaction. © 1994 Wiley‐Liss, Inc.
Development of rat × mouse hybrid embryos produced by microsurgery
Authors: Waksmundzka Malgorzata
Published: June 2005

Experimental production of hybrid embryos between Mus musculus L. and Rattus norvegicus L. was achieved by nuclear transplantation using both ovulated oocytes in metaphase II and pronuclear zygotes. Recipient egg‐cells were of mouse origin in all cases. The developmental potential of hybrids was examined in vivo.

Nucleo‐cytoplasmic hybrids resulting from the introduction of rat metaphase II chromosomes into enucleated mouse oocytes, which were subsequently activated, were regularly blocked at the 1‐ or 2‐cell stage. Nuclear (genetic) hybrids produced by transfer of a rat nucleus (in the form of metaphase II chromosomes or a pronucleus) into a nucleated mouse recipient (oocyte or zygote) were capable of development to the 5‐ to 8‐cell stage. Transplantation of rat cytoplasm alone to intact metaphase II oocytes, followed by oocyte activation, generated cytoplasmic hybrids which developed to the morula stage. In control experiments (nuclear transfer between mouse oocytes or zygotes), a high proportion of embryos formed morulae and blastocysts. These results demonstrate that the rat nucleus is incapable of functioning in mouse cytoplasm, that introduction of the rat genome into intact mouse egg‐cells impairs normal development, and that transfer of foreign (rat) cytoplasm into mouse egg‐cells affects preimplantation development of manipulated embryos. © 1994 Wiley‐Liss, Inc.
Secondary circulation of the vascular heat exchangers in skipjack tuna, Katsuwonus pelamis
Authors: Dewar Heidi , Brill Richard W. , Olson Kenneth R.
Published: June 2005

Vascular corrosion replicas of the counter current heat exchanger microcirculation in skipjack tuna, Katsuwonus pelamis, were examined by scanning electron microscopy. Smallbore (7–10 μm diameter) vessels, with characteristics typical of those associated with the secondary circulation, were abundant in the central and lateral heat exchangers. These vessels arise from the large‐bore (70 μm diameter) retial arterioles and, after a tortuous path, anastomose repeatedly to form 50–200 μm diameter secondary collecting vessels which ultimately exit from either pole of the rete. © 1994 Wiley‐Liss, Inc.
Temperature‐dependent sex determination: A mechanistic approach
Authors: Wibbels Thane , Bull James J. , Crews David
Published: June 2005

A variety of sex determination systems exists in reptiles, including temperature‐dependent sex determination (TSD). While the precise physiological basis of TSD is unknown, numerous past studies have provided a general characterization of this phenomenon. The period of thermosensitivity encompasses the middle one third to one half of embryonic development. It has been shown that in several species of reptiles this period starts prior to (histologically defined) gonadal differentiation and extends to a time when sex specific changes are becoming evident in the gonads. During the thermosensitive period, sex determination is sensitive to both the magnitude and duration of a given incubation temperature. Further, the treatment of developing eggs with specific steroid hormones (in particular, estrogens) has been shown to override the effects of male‐producing temperatures and induce female sex determination. This has led to a variety of studies investigating whether the production of estrogens may be naturally involved in TSD. The results from several studies are consistent with the hypothesis that temperature could be affecting endogenous estrogen production by controlling the production of aromatase. However, the results from other studies are not consistent with this hypothesis. Current studies by a variety of research laboratories are now addressing the estrogen hypothesis as well as TSD in general at various physiological and molecular levels. As such, these studies should provide a wealth of data on the physiological basis of TSD. © 1994 Wiley‐Liss, Inc.
Studies on sex determination in the American alligator alligator mississippiensis
Authors: Lance Valentine A. , Bogart Mark H.
Published: June 2005

Estradiol‐17β was measured in the plasma and urogenital tissues of male and female alligator embryos. Hormone levels were generally very low and there was no significant differences between the sexes. There were no significant changes in estrogen levels during the period of gonadal differentiation in either sex. Alligator embryos incubated at male producing temperatures were feminized by small doses of estrogen applied to the egg shell. The antiestrogen, tamoxifen, masculinizes turtle and bird embryos, but, paradoxically, feminized alligator embryos at male producing temperatures. The contraceptive steroid, norethindrone, a progestin that is claimed to block estrogen synthesis, is the most potent estrogenic steroid tested on alligator embryos. A single dose of norethindrone applied to the egg shell caused massive hypertrophy of the Müllerian duct and feminized embryos at male producing temperatures. The androgen, dihydrotestosterone, had no detectable effect on male or female embryos at the doses tested. Undifferentiated urogenital tracts of embryos were cultured at 30 and 33°C in the presence of steroids, tamoxifen, or antibodies to steroids. None of the treatments had any effect on tissue differentiation. Tissues survived for up to 6 weeks, but there was no evidence of gonadal differential in vitro. © 1994 Wiley‐Liss, Inc.
Chromosomal and temperature‐dependent sex determination: The search for a conserved mechanism
Authors: Coriat Anne‐Marie , Valleley Elizabeth , Ferguson Mark W. J. , Sharpe Paul T.
Published: June 2005

To test the hypothesis that the same (or similar) genes may be involved in chromosomal sex determination and temperature dependent sex determination we investigated the expression of the mammalian testis determining gene SRY in the American alligator (Alligator mississippiensis), a species with temperature dependent sex. We have cloned Alligator SRY‐related genes (A‐Srys) which constitute three major gene classes with high degrees of sequence homology (45–87%) to mammalian SRY and related genes. Expression analysis and cellular localisation studies using staged embryonic material from male and female incubation temperatures are underway to assess the role of A‐Sry genes in temperature dependent sex determination. © 1994 Wiley‐Liss, Inc.
Growth and differentiation of the secondary palate in a teleostean fish, Oncorhynchus kisutch
Authors: Shah Ravindra M. , Young Alan V. , Feeley Elizabeth J. E. , Donaldson Edward M.
Published: June 2005

A study was undertaken to examine the growth and differentiation of secondary palate in a teleostean fish, Oncorhynchus kisutch. The rate, pattern, and time of synthesis of various macromolecules, which play a crucial role during palate development in higher vertebrates were examined in the developing palate of fish. A spurt in DNA synthesis during midmorphogenesis of fish palate appeared to be related to temporal regulation of palate development. RNA synthesis was high during the time of primordial appearance and increased again from day 4 post hatching (PH) to correspond with differentiation of palate. Protein synthesis remained low initially but its trend paralleled that of RNA synthesis after day 4 PH. Glycosaminoglycan synthesis increased initially with the cartilaginous growth and then with the appearance of mucous cells. An increase in collagen synthesis corresponded with the thickening of collagen layer in the basement membrance. Cyclic AMP activity increased initially prior to the increase in DNA synthesis and subsequently remained high indicating its involvement in both growth and differentiation of fish palate. These profiles of synthesis of various macromolecules in teleostean fish differ considerably from that seen in higher vertebrates. On the basis of comparative analysis, it was suggested that the timing of mesenchymal differentiation may be one of the features in determining the characteristics of palate development in different classes of vertebrates. © 1995 Wiley‐Liss, Inc.
Paracellular, transepithelial permeation of macromolecules in the body wall epithelium of starfish embryos
Authors: Dan‐Sohkawa Marina , Kaneko Hiroyuki , Noda Koichi
Published: June 2005

The body wall epithelium of starfish gastrulae change the barrier property of the septate junction against large molecules in response to hypertonic environment caused by small molecules such as glycine, arabinose, urea, and NaCl. Ultrastructural obesrveations reveal that the septal portion of the junction either becomes diffuse or disappears altogether while the opposing junctional membranes remain unaltered. Under such conditions, Molecules as large as IgG and IgM can penetrate the body wall without causing morphological abnormalities to the embryo. We have devised a method to detect the paracellular permeation by applying fluorescein‐labeled IgG into the stimulation medium and monitoring the fluorescence which penetrated into the blastocoel. Micropreciptiates of LaCl3 were found, in thin sections of embryos treated with glycine, to lie along the intercelluar spaces, showing, although indirectly, that macromolecules flow through this pathway instead of a transcellular one.

The possible role of the septal plates in the barrier function of the septate junction is discussed. © 1995 Wiley‐Liss, Inc.
Notochord formation in amphibians: Two directions and two ways
Authors: Novoselov Vladimir V.
Published: June 2005

The formation, structure, and rearrangements of the notochord were studied in Pleurodeles waltlii and Xenopus laevis embryos. We measured length and cross‐sectional area and calculated volume of the notochord at different stages of development. In both species, the notochord emerges as a condensation and a columnarization of dorsal mesoderm along the midsagittal plane in the midgastrula. In P. waltlii embryos, the notochord segregates from archenteron roof by apical cell surface shrinking. The wave of shrinkage begins in the middle of the rudiment and simultaneously extends cranially and caudally. In X. laevis embryos, the notochord elongates in two directions also, but the mode of notochord formation is not identical in anterior and posterior parts of embryo. While anterior development looks similar to that found in P. waltlii, posterior development features closer binding of the notochord rudiment to ectoderm within existing delaminating boundaries from somite mesoderm. Rates of elongation in the different directions are approximately the same in the two species studied: It is 45–47% for the anterior and 53–55% for the posterior direction compared to total length of the notochord. © 1995 Wiley‐Liss, Inc.
Histidine‐rich protein B of embryonic feathers is present in the transient embryonic layers of scutate scales
Authors: Barnes George L. , Sawyer Roger H.
Published: June 2005

Based on its amino acid composition and N‐terminal sequence, a polypeptide (HRP‐B) has been identified as a member of the avian histidine‐rich protein (HRP) family. An antiserum against HRP‐B has been used to localize this polypeptide in developing feathers and scales of chick embryos. HRP‐B was first detectable in the barb ridge cells of feathers at 13 days of incubation and progressively appeared in the distal/proximal and peripheral/central gradients observed previously for the feather‐type β keratins in developing feathers. The HRP‐B polypeptide was detected only in the embryonic layers of scutate scales. It first appeared at 16 days of incubation and not found in the differentiated beta strata of these scales. At no time during the development of reticulate scales or apteric skin regions did the epidermal cells or cells of the embryonic layers express HRP‐B. The transient expression of HRP‐B by the embryonic layers of the scutate scale epidermis is discussed in light of the feather‐forming potential of the presumptive epidermis of the scutate scale‐forming region. © 1995 Wiley‐Liss, Inc.
Enhancing zona penetration by spermatozoa from a teratospermic species, the cheetah (Acinonyx jubatus)
Authors: Roth Terri L. , Swanson William F. , Blumer Evan , Wildt David E.
Published: June 2005

Cheetahs (Acinonyx jubatus) produce poor quality ejaculates that can limit the efficiency of standard assisted reproduction including artificial insemination (AI) and in vitro fertilization (IVF). The purpose of this study was to: (1) further study sperm‐oocyte interaction in this teratospermic species by examining the ability of malformed sperm to interact with various oocyte barriers; and (2) assess the potential of zona piercing for assisting IVF in a teratospermic felid. Zonae of salt‐stored (SS), domestic cat oocytes were mechanically pierced (ZnPd) three times each. Semen was collected by electroejaculation from six male cheetahs and ejaculates were processed for IVF. Sperm aliquots from each ejaculate were assessed for a sperm motility index (SMI) over time. Zona‐intact (ZnIn‐SS) oocytes (n=78) and ZnPd‐SS oocytes (n=74) were coincubated with spermatozoa in vitro for 6 h. The proportion of morphologically abnormal spermatozoa per ejaculate was high for all males (range 81.5% to 95.9% ). SMI values at 0 and 6 h were variable, ranging from 50 to 75 and 0 to 40, respectively. Spermatozoa from all ejaculates bound to and penetrated the outer zona pellucida of ZnIn‐SS and ZnPd‐SS oocytes similarly (P > 0.05). The proportion of oocytes containing spermatozoa within the inner zona layer and the average number of spermatozoa per oocyte in this region were greater (P < 0.05) for the ZnPd‐SS than ZnIn‐SS oocytes (39.2% and 1.0 versus 12.8% and 0.2, respectively). Although zona piercing enhanced sperm penetration, there was no increase (P > 0.05) in pleiomorphic spermatozoa penetrating the inner zona pellucid or PVS. Penetration of both ZnIn‐SS and ZnPd‐SS oocytes was positively correlated (P < 0.05; r =0.91) with SMI at 6 h but not to the proportion of the pleiomorphic spermatozoa ejaculated (P > 0.05). In summary, altering the integrity of the zona pellucida by creating artificial channels increases the number of cheetah spermatozoa entering the inner zona region, but not the PVS. This phenomenon occurs without increasing the number of pleiomorphic sperm entering the zona/oocyte interface, reinforcing the role of the zona pellucida, especially the inner region, as a powerful filter for malformed sperm. © 1995 Wiley‐Liss, Inc.
Cryoprotective effects of thermal hysteresis protein on survivorship of frozen gut cells from the freeze‐tolerant centipede Lithobius forficatus
Authors: Tursman Donald , Duman John G.
Published: June 2005

Thermal hysteresis proteins (THPs), which depress the freezing point of water by a non‐colligative process, are common in freeze‐avoiding polar marine fish and terrestrial arthropods where they function as antifreezes. However, some freeze‐tolerant arthropods and plants also produce THPs in winter, raising the question of whether these proteins are involved in the abilities of these organisms to survive the freezing of their extracellular fluids. The centipede Lithobius forficatus is one of these freeze‐tolerant, THP‐producing organisms.

Cells isolated from the midgut of both winter‐ and summer‐collected L. forficatus were frozen at various temperatures, either with or without the addition of THPs purified from the beetle Dendroides canadensis, and the survival of the cells was evaluated. The presence of THPs (0.02 mg/ml) in the bathing medium produced a statistically significant increase in cell survival, indicating a cryoprotective function for the THPs. The LT50 (temperature of 50% survival) of cells for summer centipedes was shifted from −8.2°C to −15.0°C, and that of cells of winter centipedes was shifted from −12.1°C to −15.1°C by addition of THPs. Also, cells from summer‐collected centipedes which had been incubated in media containing THPs and then washed in buffer without THPs to remove the protein from the media prior to freezing demonstrated significantly increased survivorship after freezing (LT50 = −14.5). Immunofluorescence studies of these cells indicated that the THPs were present in the cells and/or on the cell membrane. The presence of THPs in the medium also provided some protection to cells frozen under conditions designed to promote the potentially damaging process of recrystallization. (THPs are well known for their ability to inhibit recrystallization.) Therefore, THPs do appear to function as a component in the suite of adaptations which permit freeze tolerance in L. forficatus. © 1995 Wiley‐Liss, Inc.
The effect of dexamethasone on the adrenal gland in fetal and neonatal rats
Authors: Hristić M. , Kalafatić D. , Plećaš B. , Jovanović V.
Published: June 2005

The development and regeneration of the adrenal glands were examined by stereological and morphological methods in 20‐day‐old fetal, as well as 3‐day‐ and 14‐day‐old neonatal male rats born to dams treated with dexamethasone (Dx) on day 16 of gestation. In the fetuses and 3‐day‐old rats, zona fasciculata (ZF) and zona reticularis (ZR) were analyzed as one inner zone (IZ), while in 14‐day‐old animals they were analyzed separately. Single Dx treatment (1.5 mg/kg b.w.) of the dams led to atrophic changes in the adrenal cortex of the fetuses. These changes were visible to a certain degree up to the 14th neonatal day.

Administration of Dx to pregnant rats induced a significant decrease of both adrenal weight and volume, as well as the volume of zona glomerulosa + capsule (ZG + C) and IZ, both in fetuses and 3‐day‐old rats. This was due to a decrease in the number but not the volume of cortical cells. Also, necrotic cortical cells, infiltrations and resorption zones accompanied by the presence of macrophages, giant cells and lymphocytes were observed. In 14‐day‐old animals, the degree of atrophic changes in the adrenal cortex was reduced. Changes were observed only in ZR which was decreased in volume resulting from both a significant decrease of the volume and number of cortical cells. Then number of macrophages was somewhat increased, while giant cells were not present. However, the total number of parenchyma cells in ZG was increased, pointing to the possibility of renewal of cortical cells within this zone.

The results of the present study demonstrate that even a single Dx dose given to pregnant rat during the period critical for the development of the hypothalamo‐pituitary‐adrenal system in the fetuses leads to marked changes in the structure and function of the fetal adrenal glands which are partially maintained up to the 14th day of postnatal life. © 1995 Wiley‐Liss, Inc.
Characterization of adrenal cytochrome P450c21 from the blacktip shark (Carcharhinus limbatus)
Authors: Nunez B. Scott , Trant John M.
Published: June 2005

The characteristics of cytochrome P450c21 (21 hydroxylase; E.C. 1.14.99.10) were determined in interrenal tissue from blacktip shark (Carcharhinus limbatus). Consistent with other vertebrates, 21‐hydroxylase activity was enriched in the microsomal fraction of the interrenal and was dependent upon a source of reducing equivalents. The reduced form of nicotinamide adenine dinucleotide phosphate supported the highest level of activity. Specific activity increased with increasing temperature (Q10 = 2.5) with a maximum at 35°C. At 30°C, maximal 21‐hydroxylase activity reached a plateau around pH 7.5. Elasmobranchs maintain high concentrations of intracellular nitrogenous compounds known to disturb protein function. While urea (400 mM) had no apparent effect upon 21‐hydroxylase activity, trimethylamine oxide (200 mM) inhibited conversion of progesterone to 11‐deoxycorticosterone by 23%. This inhibition was not reversed by urea. Potassium chloride (150 mM) inhibited 21‐hydroxylase activity by 33%. Kinetic parameters of shark 21‐hydroxylase were determined for [3H]progesterone (Km = 80 nM; Vmax = 2.94 nmol/min/mg protein) and [3H]17α‐hydroxyprogesterone (Km = 90 nM; Vmax = 0.89 nmol/min/mg protein). Substituted progestins were used to determine the steric specificity of this enzyme. Of thirteen steroid substrates studied, nine were metabolized, indicating a relatively broad specificity. The addition of trimethylamine oxide did not alter the substrate specificity. Although the blacktip shark homolog of cytochrome P450c21 is similar in some respects to other vertebrate forms, its temperature dependence, preferential use of progesterone and broad substrate specificity are unusual characteristics. © 1995 Wiley‐Liss, Inc.
Glycoconjugates of the surface of the spermatozoa of Drosophila melanogaster: A qualitative and quantitative study
Authors: Perotti Maria‐Elisa , Pasini Maria E.
Published: June 2005

The important roles played by glycoconjugates in the recognition of gametes and in fertilization are well documented. In the present study, the nature and distribution of carbohydrate residues of the plasma membrane of spermatozoa of Drosophila melanogaster were characterized by use of FITC‐conjugated lectins as probes. The plasma membrane bound agglutinins from Concanavalia ensiformis (Con A) and Pisum sativum (PSA), native and succinylated agglutinins from wheat germ (WGA and s‐WGA), the A4 isoform of agglutinin‐I from Griffonia simplicifolia (GSA‐I A4), and, to a lesser extent, the lectins from Dolichus biflorus (DBA), Lotus tetragonolobus (LTA), and Glycine maximus (SBA). Each lectin gave a specific pattern of binding. The extent of binding of Con A, WGA, s‐WGA, and GSA‐I A4 over the acrosomal region was greater than over nonacrosomal regions, indicating the concentration of α‐mannose/α‐glucose, β‐N‐acetylglucosamine, and α‐N‐acetylgalactosamine residues in this area of the plasma membrane. The surface of the sperm failed to react with lectins from Ricinus communis (RCA‐I), Limulus polyphemus (LPA), and Limax flavus (LFA) and with the B4 isoform of agglutinin‐I from Griffonia simplicifolia‐I (GSA‐I B4). The plasma membrane over the nucleus did not react with any of the lectins tested.

Quantitative analysis of binding of Con A, s‐WGA, and GSA‐I A4 to spermatozoa showed that only Con A bound consistently to the sperm surface, showing high affinity for the acrosomal area of the plasma membrane. The other lectins tested bound only to limited and variably sized fractions of the total population of sperm. Therefore, only residues of α‐mannose/α‐glucose are a constitutive component of the plasma membrane, and they are characteristic of the acrosomal area. Con A can be used as a marker of the acrosome portion of sperm from Drosophila for visualization and assessment of acrosome status; labelling with FITC‐conjugated Con A provides a simple and reliable method for visualization of the acrosome of Drosophila sperm that is otherwise detectable only by ultrastructural examination. © 1995 Wiley‐Liss, Inc.
Regulatory volume decrease in neurons of Aplysia brasiliana
Authors: Scemes Eliana , Cassola Antônio C.
Published: June 2005

Aplysia brasiliana is an osmoconforming mollusc; the osmolality of its extracellular fluid follows that of the external media. This investigation analyzes the volume changes of isolated neuron bodies exposed to hypotonic shocks. The abdominal ganglion was isolated, and the volumes of neuron bodies as well as intracellular K+ concentration were measured. Hypotonic shocks induce a rapid swelling followed by a slower restoration of cell volume. The regulatory volume decrease (RVD) restores cell volume to its control value. Ba2+ slows RVD without blocking it. Furosemide (in the μM range) completely and reversibly blocks the regulatory volume decrease. On return to isotonic solution after RVD, cell shrinkage was only detected when furosemide was present. It is concluded that neuron bodies of A. brasiliana display efficient processes for volume regulation, based on a conductive pathway and a furosemide‐sensitive transport mechanism, which is probably the Na‐2Cl‐K cotransporter. A model simulation provides additional support to this hypothesis. © 1995 Wiley‐Liss, Inc.
Light pulses and injection of IP3 induce mating ability in Paramecium bursaria
Authors: Miwa Isoji , Wada Tsuneya
Published: June 2005

Populations of cells of Paramecium bursaria exposed to a light‐dark cycle (LD; 12:12 hr) show mating reactivity during the light period but not during the dark period. After transfer to constant light (LL) or constant darkness (DD), they continue to show a circadian rhythm of mating reactivity. When cells in the non‐reactive phase of DD were exposed to light pulse (from 5 sec to 6 hr in duration), they expressed mating ability 1.5 hr after the light pulse. Three wavelengths (416 nm, 547 nm, 626 nm) of light (5‐sec pulse) were most effective for inducing the mating ability. Light pulses of more than 2 hr were sufficient to shift the phase of the circadian mating rhythm in DD, but phase shifts did not occur after light pulses of less than 1 hr. We obtained a phase response curve (PRC) for light pulses of 6‐hr duration.

To investigate the intracellular light‐transduction pathway, we examined whether inositol triphosphate (IP3) could induce the mating activity in DD. Cells in the non‐reactive phase of DD were injected with 10 pl of a solution of IP3 (120 μM) under dim green light (480 nm, 8 μW/cm2) which did not induce mating ability. The cells begin to show mating activity 30 min after the injection and the extent of the activity was greater than that obtained by exposing cells to a 3 hr light pulse. However, injection of IP3 did not cause phase shifting in DD. Thus, circadian clock appeared not to be influenced by the injection of IP3. © 1995 Wiley‐Liss, Inc.
Histogenesis of antlerogenic tissues cultivated in diffusion chambers in vivo in red deer (Cervus elaphus)
Authors: Li Chunyi , Waldrup Kenneth A. , Corson Ian D. , Littlejohn Roger P. , Suttie James M.
Published: June 2005

In a previous study we showed that formation of deer pedicle and first antler proceeded through four ossification pattern change stages: intramembranous, transition, pedicle endochondral, and antler endochondral. In the present study antlerogenic tissues (antlerogenic periosteum, apical periosteum/perichondrium, and apical perichondria of pedicle and antler) taken from four developmental stages were cultivated in diffusion chambers in vivo as autografts for 42–68 days. The results showed that all the cultivated tissues without exception formed trabecular bone de novo, irrespective of whether they were forming osseous, osseocartilaginous, or cartilaginous tissue at the time of initial implant surgery; in two cases in the apical perichondria from antler group, avascularized cartilage also formed. Therefore, the antlerogenic cells, like the progenitor cells of somatic secondary type cartilage, have a tendency to differentiate into osteoblasts and then form trabecular bone. Consequently, the differentiation pathway whereby antlerogenic cells change from forming osteoblasts to forming chondroblasts during pedicle formation is caused by extrinsic factors. Both oxygen tension and mechanical pressure are postulated to be the factors that cause this alteration of the differentiation pathway. © 1995 Wiley‐Liss, Inc.
Spontaneous maturation in Bufo arenarum oocytes: Follicle wall involvement, respiratory activity, and seasonal influences
Authors: Zelarayán L. I. , Oterino J. , Bühler M. I.
Published: June 2005

The various somatic cell layers (surface epithelium, theca, and follicle cells) that envelope the amphibian oocyte were removed one by one. The various follicular preparations thus obtained were tested for their ability to undergo germinal vesicle breakdown (GVBD) in the presence or absence of progesterone during different seasonal periods. Intact follicles, follicles without epithelium, or defolliculated oocytes (without epithelium and theca layers) did not undergo GVBD when cultured in Ringer solution, unless progesterone was added to the culture medium. In contrast, denuded oocytes (lacking all follicular layers) cultured in a medium with no hormones underwent GVBD in percentages similar to those of denuded oocytes incubated with progesterone. This ability of denuded oocytes to undergo spontaneous maturation was found to be related to the respiratory activity of the oocyte, which is in turn a seasonal variable in Bufo arenarum. The different respiratory activity of the full‐grown oocyte according to the seasonal period was expressed by the respiratory control quotient (around 1.0 during winter and 4.0 during summer). In fact, only those oocytes with a respiratory control quotient over 2.0 were able to undergo spontaneous nuclear maturation. The maturation obtained without exogenous hormonal stimulus was genuine as indicated by the ability of these oocytes to induce pronuclear formation after sperm injection. © 1995 Wiley‐Liss, Inc.
Proliferation of newt spermatogonia by mammalian FSH via Sertoli cells in vitro
Authors: Maekawa Kohtaro , Ji Zai‐Si , Abé Shin‐Ichi
Published: June 2005

Our previous studies showed that mammalian follicle‐stimulating hormone (FSH) stimulated the proliferation and differentiation of secondary spermatogonia into primary spermatocytes in organ culture of testes fragments from Cynops pyrrhogaster. To elucidate how FSH stimulates spermatogonial proliferation, we studied the interaction of spermatogonia with somatic cells in the presence or absence of FSH in cultures of aggregated cells derived from fractionated cell populations.

When spermatogonia or those with somatic cells were cultured, they formed aggregates with themselves or with somatic cells, respectively. Most of the somatic cells in the aggregates were Sertoli cells, judged by the lipid droplets in their cytoplasm.

[3H]thymidine incorporation into aggregates and autoradiography demonstrated spermatogonial proliferation that was enhanced in the presence of FSH and somatic cells (most probably Sertoli cells), but not in the absence of FSH or in the presence of luteinizing hormone (LH).

To examine whether direct contact between spermatogonia and Sertoli cells is indispensable for the stimulation of spermatogonial proliferation by Sertoli cells, the two cell types were separated by a 0.4 μm pore filter in two compartments of a bicameral chamber. In this case, Sertoli cells did not stimulate spermatogonial proliferation in the presence of FSH, indicating that direct contact between spermatogonia and Sertoli cells is indispensable for the stimulation of spermatognial proliferation by Sertoli cells.

Finally, Sertoli cells isolated from the testes from the primary spermatocyte stage did not stimulate spermatogonial proliferation in the presence of FSH. This result indicated that the function of Sertoli cells with respect to their stimulation of proliferation was stage‐specific. © 1995 Wiley‐Liss, Inc.