bluuebunny/crossref_metadata_2025 huggingface dataset:
Origin of anuran calling: Description of toad releasing
Authors: Schmidt Robert S. , Kemnitz Christopher P. , Hudson Wayne R.
Published: June 2005

Electromyographic electrodes were implanted in forelimb muscles of male American toads (Bufo americanus). Toads then were allowed to clasp an artificial clasp object incorporating a pair of solenoids that, when activated, produced a stimulus simulating the side vibrations normally associated with release signalling by conspecific males. Electromyographic activity, in response to the release stimulus, was recorded. The adductor muscle contracted immediately after stimulus onset (causing intensified clasping), responding to the stimulus as a displacement threat. Then the abductor muscle contracted (causing rapid unclasping, or releasing), responding to the stimulus as a release signal. These techniques have provided a better understanding of releasing than has been possible with natural objects (i.e., living males).
Growth rate and plasma corticosterone levels in juvenile alligators maintained at different stocking densities
Authors: Elsey Ruth M. , Joanen Ted , McNease Larry , Lance Valentine
Published: June 2005

Sixty‐six captive‐reared, juvenile alligators maintained in environmentally controlled chambers were randomly assigned to four treatments consisting of stocking densities of 0.35, 0.18, 0.12, and 0.09 m2/alligator. Alligators were maintained at these densities for 3.5 months. At approximately 4 week intervals the alligators were weighed, measured, and bled via cardiac puncture. Duplicate aliquots of plasma were assayed for corticosterone. All of the alligators continued to grow during the experiment, but alligators maintained at the lowest stocking density were significantly heavier and grew significantly faster (P < 0.01) and had significantly lower plasma corticosterone (P < 0.01) than alligators maintained at the highest stocking density. These results indicate that crowding (i.e., high‐density rearing) of juvenile alligators inhibits maximum growth and that the subsequent stress associated with this crowding results in chronically elevated plasma corticosterone. Plasma corticosterone showed a strong negative correlation with change in body weight; the faster the rate of growth, the lower the hormone levels. These results also show that a single blood sample taken at monthly intervals is sufficient to monitor chronically elevated plasma corticosterone in this species.
Indomethacin inhibits ovarian PGE secretion and gonadotropin‐induced ovulation in a reptile (Anolis carolinensis)
Authors: Jones Richard E. , Orlicky D. J. , Austin Harriet B. , Rand Matthew S. , Lopez Kristin H.
Published: June 2005

Although prostaglandins (PGs) have been shown to play an important role in gonadotropin‐induced ovulation in mammals, birds, and fishes, nothing is known about the role of PGs in reptilian ovulation. Females of the lizard Anolis carolinensis, each with a large vitellogenic ovarian follicle, were pretreated with an inert subcutaneous pellet or one containing indomethacin (an antiprostaglandin) at a delivery rate of 2.38 μg/lizard/day. Females in each pretreatment group were treated with subcutaneous injections, 7 h apart, of either vehicle alone or 25 μg porcine FSH. They then were sacrificed at 9, 12, and 24 h after the initial injection. After noting whether ovulation had occurred, ovaries from animals with an unovulated large (>5 mm diameter) follicle were incubated in 5 ml Anolis Ringer's for 1 h. The follicles then were examined histologically, and levels of PGE in the incubation medium were determined by radioimmunoassay. FSH significantly induced ovulation in a majority of females by 12 h, and indomethacin significantly reduced the number of FSH‐induced ovulations at 12 h but not at 24 h. Thus, indomethacin delayed FSH‐induced ovulation. Indomethacin also inhibited the preovulatory changes in histology of the follicular wall seen after FSH treatment alone and lowered PGE secretion at all time periods. Secretion of PGE was not influenced by FSH. Thus, prostaglandins may mediate or permit, at the ovarian level, the ovulatory influence of gonadotropin in this reptile.
Adaptive changes of liver composition and structure in golden ide during winter acclimatization
Authors: Segner Helmut , Braunbeck Thomas
Published: June 2005

The present investigation was designed to study hepatic alterations in fish during the winter period and to compare them with acclimation phenomena in fish subjected to fluctuating environmental variables in the laboratory. Immature young‐of‐the‐year golden ide,Leuciscus idus melanotus, were kept in an outdoor pond from September to April. Liver response was analyzed using histological, morphometrical, and biochemical methods.

In a first phase from September to March, hepatocellular activity was reduced without evidence of disturbance of liver functions. Cell size, energy reserves, and protein contents as well as mitochondrial number and volume decreased significantly; RNA contents were moderately reduced. Cellular compartmentation was preserved, and gross pathological alterations were lacking. The reduced metabolic capacity of the hepatocyte was apparently compensated for by an increase of total liver mass, resulting in maximum values of all parameters investigated in March.

The second phase from March to April was characterized by a decrease of all liver parameters investigated and severe alterations in hepatocellular structure. These observations are interpreted as evidence of drastic structural and functional reorganization in spring.

Comparison with data from laboratory fish reveals that part of the wintering phenomena can be attributed to processes of temperature and nutritional adaptation. However, the overall adaptive response observed in golden ide in the outdoor pond profoundly differed from that of fish subjected to selected environmental variables in the laboratory.

Since golden ide is in use as a test fish for the assessment of chemical toxicity, the observation of naturally occurring changes in the condition of golden ide may be of relevance with regard to the reproducibility of test results throughout the year.
Vitellin synthesis in relation to oogenesis in in vitro‐incubated ovaries of Penaeus semisulcatus (crustacea, decapoda, penaeidae)
Authors: Browdy C. L. , Fainzilber M. , Tom M. , Loya Y. , Lubzens E.
Published: June 2005

Ovaries of the penaeid shrimp Penaeus semisulcatus in various stages of oocyte development were incubated in vitro with 14C or 3H leucine. Protein synthesis was measured by TCA precipitation and vitellin specific synthesis by radioimmunoprecipitation. The proteins synthesized were subjected to native or SDS denaturative gel electrophoresis and visualized by fluorography. The results led to the following conclusions: 1) Protein synthesis was found to depend on the concentration of leucine in the culture media. 2) A relatively constant level of vitellin synthesis was observed in ovaries with oocytes ranging in size between 100 and 380 μm. 3) The vitellogenic stage was found to occur in ovaries with oocytes ranging between 100 and 300 μm in size. During this period, 40–60% of the in vitro–synthesized proteins were found to be immunologically identical to vitellin. 4) A decrease in the relative amount of vitellin synthesized was found with the appearance of cortical crypts in oocytes exceeding 300 μm in diameter. 5) The protein immunologically identical to vitellin, which was synthesized in vitro by the ovaries, was found to consist of the subunits found in purified native vitellin. 6) A bioassay system for testing the effect of various factors on vitellin synthesis is suggested by in vitro incubations of several pieces removed from the ovaries of one female.
Aerobic metabolism is maintained in animal tissue during rapid sulfide oxidation in the symbiont‐containing clam Solemya reidi
Authors: Anderson A. E. , Felbeck H. , Childress J. J.
Published: June 2005

Solemya reidi is a gutless clam that contains intracellular, symbiotic sulfur‐oxidizing chemoautotrophic bacteria in its gills. It lives in burrows in highly reducing muds rich in sulfide, a compound toxic at low levels to aerobic respiration. In these habitats there is wide variation in levels of O2 and sulfide, both of which are metabolic requirements for this symbiosis. In order to assess whether the animal tissues utilize aerobic or anaerobic ATP‐generating pathways in the presence of sulfide, and to determine the sulfide concentrations that might induce a shift to anaerobiosis, clams were incubated in varying levels of sulfide and oxygen. Symbiont‐free foot tissues from clams incubated in various levels of sulfide (50–500 μM) were analyzed for a common substrate (aspartate) and product (succinate) of invertebrate anaerobic metabolism. The concentrations of these metabolites were compared with those found in tissues of aerobic and anaerobic controls. Aerobic metabolism was maintained in animal tissues in the presence of up to 100 μM sulfide, levels at which maximum autotrophy has been previously reported; however, at higher concentrations, where inhibition of autotrophy has been reported, the onset of anaerobic pathways was evident. This use of anaerobic pathways was not due to the absence of O2, the classical definition of anaerobiosis, but rather to the inhibitory effect of sulfide on aerobic metabolism. The metabolic flexibility of this species in regard to energy metabolism is clearly advantageous living as it does at the interface between a highly oxic environment and an anoxic, highly reducing one.
Comparative kinematics of terrestrial prey capture in Salamanders and newts (Amphibia:Urodela:Salamandridae)
Authors: Miller Brian T. , Larsen John H.
Published: June 2005

High‐speed cinematography was used to compare the kinematics of terrestrial prey capture among postmetamorphic individuals of the family Salamandridae that have a) terrestrial (Salamandra, Salamandrina, and Tylototriton), b) amphibious (Pleurodeles, Cynops, and Paramesotriton), and c) aquatic (Pachytriton) lifestyles. Tongue projection was common during prey capture attempts in Salamandra, Salamandrina, and Tylototriton, occurred frequently in Cynops and Paramesotriton, but was absent during terrestrial feeding by Pachytriton. The gape cycle consisted of four distinct phases that were associated with discrete movements of the hyolingual apparatus. In all salamandrids that projected the tongue, the gape 1) increased rapidly while the tongue was elevated from the floor of the mouth in preparation for protraction, 2) was stabilized while the tongue was protracted, 3) increased to maximum during retraction and 4) decreased rapidly while the tongue transported the prey into the oral cavity. The absence of tongue projection during some (Cynops, Paramesotriton), or all prey capture attempts (Pachytriton) resulted in gape profiles distinctly different from those described above. Most notably, these profiles lacked the gape stabilization component characteristic of tongue projection. The maximum distance that the tongue was projected beyond the mandible approached 6% snout‐vent length (SVL) in most salamandrids, but reached 20% SVL in Salamandrina. Several elements of the feeding system in Salamandrina are functionally related to the evolution of its specialized tongue projection apparatus. These include a gape cycle of relatively long duration (200 ms) and the absence of a head‐body surge and of eye retraction during the strike.
Differences in the responses of two mudskippers, Boleophthalmus boddaerti and Periophthalmus chrysospilos to changes in salinity
Authors: Chew S. F. , Ip Y. K.
Published: June 2005

When mudskippers were fully submerged in 100% seawater (SW), the mortality of Boleophthalmus boddaerti was higher than that of Periophthalmus chrysospilos. However, this trend was reversed when mudskippers were submerged in dechlorinated tap water or in 10% SW. The plasma osmolality of P. chrysospilos exposed to 80% SW was significantly higher than that of control fish free to be in or out of 50% SW and that of fish submerged in 10% or 50% SW. The muscle and liver of the fish exposed to 80% SW also contained significantly higher levels of ninhydrin‐positive substances (NPS). In addition, the aminating activity of glutamate dehydrogenase (GDH) in the extracts from the liver and muscle of P. chrysospilos submerged in 80% SW for 7 days was significantly higher than those of fish submerged in 10% SW for the same period. However, the levels of NPS in the muscle and liver of B. boddaerti were unchanged by changes in salinity. Furthermore, the aminating and deaminating activities of GDH in the extracts from the liver and muscle of B. boddaerti, after fish had been submerged in 80% SW for 7 days, were not significantly different from those of fish submerged in 10% SW for a similar period of time. Such differences between the two species of mudskippers in response to changes in salinity may be correlated to the differences in the environmental conditions in their natural habitats.
Concurrent protein synthesis is required for in vivo chitin synthesis in postmolt blue crabs
Authors: Horst Michael N.
Published: June 2005

Chitin synthesis in crustaceans involves the deposition of a protein‐polysaccha‐ride complex at the apical surface of epithelial cells which secrete the cuticle or exoskeleton. The present study involves an examination of in vivo incorporation of radiolabeled amino acids and amino sugars into the cuticle of postmolt blue crabs, Callinectes sapidus. Rates of incorporation of both 3H leucine and 3H threonine were linear with respect to time of incubation. Incorporation of 3H threonine into the endocuticle was inhibited greater than 90% in the presence of the protein synthesis inhibitor, puromycin. Linear incorporation of 14C glucosamine into the cuticle was also demonstrated; a significant improvement of radiolabeling was achieved by using 14C‐N‐acetylglu‐cosamine as the labeled precursor. Incorporation of 3H‐N‐acetylglucosamine into the cuticle of postmolt blue crabs was inhibited 89% by puromycin, indicating that concurrent protein synthesis is required for the deposition of chitin in the blue crab. Autoradiographic analysis of control vs. puromycin‐treated crabs indicates that puromycin totally blocks labeling of the new endocuticle with 3H glucosamine. These results are consistent with the notion that crustacean chitin is synthesized as a protein‐polysaccharide complex. Analysis of the postmolt and intermolt blue crab cuticle indicates that the exoskeleton contains about 60% protein and 40% chitin. The predominant amino acids are arginine, glutamic acid, alanine, aspartic acid, and threonine.
Either chick embryo dermis or retinoid‐treated mouse dermis can initiate glandular morphogenesis from mammalian epidermal tissue
Authors: Hardy Margaret H. , Dhouailly Danielle , Törmä Hans , Vahlquist Anders
Published: June 2005

Excess retinoids can cause developing mouse vibrissa follicles to be transformed into mucous glands in organ culture. The objective was to test the hypothesis that retinoids act in this system by altering morphogenetic properties of the dermis. After inititation by retinoic acid (RA) in organ culture, glands were shown to develop further in embryonic skin grafted to the chick chorioallantoic membrane (CAM). Recombinants of 12.5 day mouse epidermis with untreated or RA‐treated mouse or chick dermis were then grafted to CAM for 7 days. For homospecific recombinants, 13.5 day mouse dermis originated from 11.5 day skin cultured for 2 days, with or without 5.2 μg/ml RA. For heterospecific recombinants, 12 day dermis came from chick embryos, previously injected with 250 μg RA. Glands were absent from the homospecific recombinants including untreated mouse dermis, but appeared in 26% of those with RA‐treated dermis. Among heterospecific recombinants, 75% of those with RA‐treated chick dermis and 29% of those with untreated dermis had glands. Untreated 10–12 day chick skin contained two forms of endogenous vitamin A, retinol (4.5 μg/g protein) and dehydroretinol (3.7 μg/g protein), while 13–14 day mouse skin contained only retinol (1.8 μg/g protein), as shown by high performance liquid chromatography. RA injection increased retinol and dehydroretinol in chick skin, while RA was undetectable. Thus RA can act through mouse dermis to form epithelial glands and through chick dermis to increase the incidence of glands. The glands in recombinants with untreated chick dermis may result from the higher levels of endogenous retinoids in chick skin, compared with mouse skin.
Radioreceptor Assay for growth hormone in coho salmon (Oncorhynchus kisutch) and its application to the study of stunting
Authors: Gray Elisabeth S. , Young Graham , Bern Howard A.
Published: June 2005

Binding sites for native chum salmon growth hormone (sGH) in coho salmon (Oncorhynchus kisutch) hepatic membranes were analyzed by radioreceptor assay. Displaceable (specific) binding represented up to 25% of total radiolabeled sGH added. Binding was dependent on buffer pH and membrane protein concentration, and was complete by 24 hours at 15°C. Specific binding was greatest in liver membranes, and was also detected in rr.uscle, ovary, gill, kidney, and brain. Scatchard analyses indicated a single class of hepatic binding sites that were specific for sGH. In stunts, abnormal seawater salmon with elevated plasma GEI levels and inhibited growth, specific binding of sGH to liver membranes was three times lower thiin in normal seawater smolts. The concentration of salmon GH binding sites was decreased in stunt livers by 60%, while their affinity for sGH was unchanged. Down‐regulation of hepatic GH receptors by high plasma GH levels may explain in part the low sGH binding in stunts.
Evidence for phosphorylation/dephosphorylation control of phosphofructokinase from organs of the Anoxia‐Tolerant sea mussel Mytilus edulis
Authors: Michaelidis Basile , Storey Kenneth B.
Published: June 2005

The transition from aerobic to anoxic conditions induced stable modifications of the kinetic and regulatory properties of 6‐phosphofructo‐1‐kinase (PFK) from the posterior adductor muscle and hepatopancreas of the marine bivalve mollusc Mytilus edulis L. Anoxia resulted in a reduction in the maximal activity of PFK, a decrease in affinity for fructose‐6‐P (S0.5 increased by 60% in muscle and twofold in hepatopancreas), reduced effects of activators (AMP, fructose‐2,6‐P2), and reduced inhibition by ATP (I50 increased 2–2.4 fold). Organ‐specific differences in PFK properties were greatest for enzyme activators, with the hepatopancreas enzyme showing much lower Ka values for AMP and fructose‐2,6‐P2 as well as activation by inorganic phosphate (Ka 0.07 mM aerobic, 0.11 mM anoxic). By contrast, the muscle enzyme was not affected by Pi and neither enzyme was inhibited by citrate. To determine if the anoxia‐induced modifications of PFK were due to covalent modification of the enzyme protein, PFK preparations were incubated with ATP + Mg2+ + protein kinase second messengers (cAMP, cGMP, or Ca2+ plus phorbol 12‐myristate 13‐acetate) or with Mg2+ + alkaline phosphatase. Protein kinase action on posterior adductor muscle PFK increased enzyme Vmax, and affinity for fructose‐6‐P. cAMP‐ or cGMP‐stimulated protein kinase had the same action on hepatopancreas PFK; but incubation with stimulators of protein kinase C had the opposite effect on the enzyme. Alkaline phosphatase treatment reversed the effects of in vitro phosphorylation. The data suggest that the glycolytic rate depression (reverse Pasteur effect) that occurs with the transition to anoxia in these facultative anaerobes is facilitated by reversible phosphorylation control over the activity of PFK.
Heterogeneity of purified actin in the electric organ of the electric eel Electrophorus electricus
Authors: AyresSá Lilian , Neto Vivaldo Moura , de Oliveira Mécia M. , Chagas Carlos
Published: June 2005

Actin extracted from the electric organ and epaxial muscle of the electric eel Electrophorus electricus was characterized by several biochemical techniques. Isoelectric focusing gels revealed two isoforms of actin in the electric organ, in contrast to the unique form of actin present in the epaxial muscle of this eel and the rabbit leg muscle, which were used as controls. Proteolytic digestion and immunoblotting analysis confirmed that these isoforms were composed of actin. When actin from the electric organ was mixed with actin from the epaxial muscle or from rabbit muscle and subjected to isoelectric focusing gel, it was possible to identify an alpha‐actin isoform in the electric organ as well as a more basic actin that migrated in the same manner as cytoplasmic actin or gamma actin from smooth muscle. The data are interpreted in terms of the differentiation of the electric organ of E. electricus.
Vitellogenin purification and development of assay for vitellogenin receptor in oocyte membranes of the tilapia (Oreochromis niloticus, linnaeus 1766)
Authors: Chan S. L. , Tan C. H. , Pang M. K. , Lam T. J.
Published: June 2005

An assay has been successfully developed for vitellogenin receptors in isolated oocyte membranes of the tilapia (Oreochromis niloticus). Vitellogenin produced by estrogen stimulation of the male tilapia was purified by anion‐exchange chromatography on DEAE‐Sephacel and identified by its incorporation of 32P‐orthophosphate and 3H‐leucine in vivo. Only one molecular form of vitellogenin was found to be induced by estrogen treatment. After iodination with Iodogen, the 125I‐vitellogenin was shown to be immunologically similar to the unlabeled protein and was used to develop an assay for vitellogenin receptors. For such assays, the optimal conditions for binding were pH 7.4, 6‐h incubation at 23°C with 7.5 mM Mg2+ and 10 mM Ca2+. The magnitude of the specific binding was dependent on the amount of oocyte membranes and was saturable. The binding shows both tissue and protein specificity: the 125I‐vitellogenin binds specifically to only oocyte membranes and not to those of the brain, muscles, liver, intestine, and testis; its binding was displaceable only by unlabeled vitellogenin and a yolk protein extract. Saturation studies and Scatchard analyses revealed only a single class of binding sites. These studies provide unequivocal evidence that the binding sites for vitellogenin in the tilapia oocytes are specific receptors. The number and affinity (KD) of these receptors increase from the previtellogenic (9.98 × 109/oocyte) to the vitellogenic stage (3.65 × 1012/oocyte), and remained unchanged at the preovulatory stage (3.53 × 1012/oocyte), at which time the affinity of the receptors was also highest (0.3 μM).
Fate of haploid parthenogenetic cells in mouse chimeras during development
Authors: Ito Michihiko , Kaneko‐Ishino Tomoko , Ishino Fumitoshi , Matsuhashi Michio , Yokoyama Minesuke , Katsuki Motoya
Published: June 2005

The developmental capability of haploid parthenogenetic cells was investigated by studies on haploid parthenogenetic ⟷ fertilized mouse chimeras. Two chimeras were born. One female chimera was smaller at birth and grew slower than its littermates. The distribution of haploid‐derived cells in the chimeras was analyzed 11 months after their birth. Cells derived from haploid embryos were found only in the brain, eyes, pigment cells in hair follicles, and spleen, in which they constituted 30%, 20%, 10%, and <5%, respectively, of the cells. The correlation between the parthenogenetic contribution to the brain and growth retardation is discussed. All of the cells examined in these chimeric organs (brain and eyes) contained a diploid amount of DNA, suggesting that diploidization of the haploid parthenogenetic cells occurred during development. Possibly, the haploid state is not sufficient for cell growth, even in chimeras with fertilized embryos.
Expression of the cell adhesion molecules, L‐CAM and N‐CAM during avian scale development
Authors: Shames Rose B. , Jennings Anita G. , Sawyer Roger H.
Published: June 2005

To examine the involvement of cell adhesion molecules in the inductive epithelial‐mesenchymal interactions during avian scale development, a study of the spatiotemporal distribution of L‐CAM and N‐CAM was undertaken. During scutate scale development, L‐CAM and N‐CAM are expressed together in cells of the transient embryonic layers destined to be lost at hatching. The ongoing linkage of the cells of these layers by both CAMs sets them apart, early in development, as unique cell populations. L‐CAM and N‐CAM were also expressed simultaneously at the basal surface of the early germinative cells where signal transduction is presumed to occur. In spite of the differences in cell shape, adhesion, density and proliferative state between populations of epidermal placode and interplacode cells, the expression of L‐CAM and N‐CAM appeared to be uniform and nondiscriminating for these discrete cell lineages. The same pattern of L‐CAM and N‐CAM expression was observed during morphogenesis of reticulate scales that develop without placode formation. While L‐CAM and N‐CAM are present during the early stages of scale development and most likely function in cell adhesion, the data do not support a role for these adhesion molecules in the formation of the morphogenetically critical placode and interplacode cell populations.

In both scale types, L‐CAM became predominantly epithelial, and N‐CAM became predominantly dermal as histogenesis occurred. Initially, N‐CAM was concentrated near the basal lamina where it may be involved in the reciprocal epidermal‐dermal interactions required for morphogenesis. However, as development of the scales progressed, N‐CAM disappeared from the tissues. L‐CAM expression continued in the epidermis and was intense on all suprabasal cells undergoing differentiation into either an αstratum or β‐stratum. However, L‐CAM was more prevalent on the basal cells of α‐keratinizing regions than on the basal cells of β‐keratinizing regions.
Fibroblast growth factor and culture in monolayer rescue mesoderm cells destined to die in the developing avian wing
Authors: MacCabe J. A. , Blaylock R. L. , Latimer J. L. , Pharris L. J.
Published: June 2005

In an effort to elucidate control mechanisms for developmentally programmed cell death, conditions were sought that rescue the cells destined to die. Three areas of mesodermal cell death in the chick wing were examined: the posterior necrotic zone (PNZ), the opaque patch (OP), and apical mesoderm. The PNZ and OP are areas of normally programmed cell death, whereas the apical mesoderm undergoes cell death only after the overlying apical ectodermal ridge is excised. Cell death in vitro was quantitated using the chromium‐release assay. While these tissues undergo apparently normal cell death in organ culture, in monolayer culture almost all are rescued. In addition, the cells are rescued by the addition of fibroblast growth factor to organ cultures. Since fibroblast growth factor is present in decreasing amounts in the limb at this stage of development, normal cell death may occur upon withdrawal of growth factor.
Effects of olfactory tract lesions on gonadotropin and milt responses to the female sex pheromone, 17α, 20β‐dihydroxy‐4‐pregnen‐3‐one, in male goldfish
Authors: Dulka Joseph G. , Stacey Norman E.
Published: June 2005

For approximately 10 h before ovulation, female goldfish release the gonadal steroid 17α,20β‐dihydroxy‐4‐pregnen‐one (17,20β‐P) to the water where it functions as a potent sex pheromone that induces in males a rapid (15‐min) increase in blood gonadotropin (GtH) and a subsequent (4–6 h) increase in milt (sperm and seminal fluid) volume. To determine which olfactory pathways mediate responses to pheromonal 17,20β‐P, this study examined the effects of various olfactory tract (OT) lesions on the 17,20β‐P‐induced GtH and milt responses in male goldfish. Males remained intact (INTACT) or received either a sham operation (SHAM), complete bilateral OT section (OTX), or bilateral lesions to the lateral (LOTX) or medial (MOTX) subdivisions of the paired OTs. At both 2 days and 1 week after surgery, one‐half of each treatment group was exposed to either ethanol (control) or water‐borne 17,20β‐P for 1 h and then blood sampled for GtH determination. Milt was stripped and collected from all fish 7–8 h after the exposures had commenced. Exposure to water‐borne 17,20β‐P induced increases in GtH and milt volume in INTACT, SHAM, and LOTX animals but failed to evoke these responses in OTX and MOTX fish. The results indicate that central projections of the medial olfactory tracts constitute a functional neuroanatomical pathway by which pheromonal 17,20β‐P stimulates GtH and milt production in goldfish. In addition, functional and anatomical similarities between this pathway and the mammalian vomeronasal system are discussed.
Potassium dependence for sperm–egg fusion in mice
Authors: Boldt Jeffrey , Casas Adela , Whaley Elizabeth , Creazzo Tony , Lewis Jill B.
Published: June 2005

In this study, we examined the potassium requirements for sperm–egg fusion in mouse. Zona‐free mouse eggs prepared by the method described by Boldt and Wolf were inseminated with capacitated sperm in culture media containing 0‐6 mM extracellular K+, and scored for penetration. Penetration of zona‐free eggs was dependent on extracellular K+, with no penetration observed under K+ ‐free conditions. Media transfer experiments indicated that the lack of penetration observed was due to effects on fusion, and not on postpenetration events such as sperm head decondensation. To analyze whether the K+ effect was attributable to an effect on the sperm (i.e., occurrence of acrosome reactions), sperm were treated with the Ca2+ ionophore A23187 before insemination. Less than 5% of zona‐free eggs were penetrated with ionophore‐treated sperm under K+ ‐free conditions, suggesting that K+ is required for fusion per se. Addition of ionophore to insemination cultures similarly did not overcome the block to fusion observed under K+ ‐free conditions. The potassium channel blockers 4‐aminopyridine (0.1‐5 mM) and tetraethyl ammonium chloride (5–50 mM) had no inhibitory effect on fusion. These data indicate that extracellular K+ is required for sperm‐egg fusion and that this requirement may not involve membrane K+ channels.
Culture medium and protein supplementation influence in vitro fertilization and embryo development in the domestic cat
Authors: Johnston L. A. , Donoghue A. M. , O'brien S. J. , Wildt D. E.
Published: June 2005

The influence of culture medium and protein supplements on in vitro fertilization (IVF) and morula‐to‐blastocyst development in culture was examined in the domestic cat. In Study I, follicular oocytes were fertilized and cultured in 1) modified Krebs Ringer bicarbonate (mKRB); 2) modified Tyrode's solution (TALP) without phosphate or glucose; or 3) Ham's F10. All media contained bovine serum albumin (BSA). Fertilization rates were similar (P>.05) among mKRB (75.0%), TALP (70.6%), and Ham's F10 (80.0%) treatments. Compared to TALP (77.8%), more (P<.05) embryos in Ham's F10 (95.0%) developed to the morula stage; development of mKRB embryos (88.9%) was intermediate and not different (P>.05). Study II evaluated the effects of protein availability and type on IVF and embryo growth. Ham's F10 was supplemented with polyvinylalcohol (PVA, 2 mg/ml), BSA (4 mg/ml), fetal calf serum (FCS, 5%), or estrous cat serum (ECS, 5%) and used to fertilize and culture embryos. Fertilization was enhanced (P<.05) using either FCS (84.0%) or ECS (85.2%) compared to PVA (67.3%). Oocytes exposed to BSA fertilized at a rate (76.1%) similar (P>.05) to other treatments. Embryos exposed to all four treatment groups (PVA, 82.8%; BSA, 82.8%; FCS, 92.9%; or ECS, 97.8%) were equally capable (P>.05) of becoming morulae. However, more FCS‐ or ECS‐supplemented morulae continued to the early blastocyst stage (30.8%, 22.2%, respectively, P<.05) than PVA‐ (10.3%) or BSA‐ (13.8%) exposed morulae. In the domestic cat IVF system, the type of medium and protein supplement used appears to have a greater impact on embryo development in vitro than on fertilization. There also is a morula‐to‐blastocyst developmental block for cat embryos resulting from IVF which is partially circumvented by adding serum to the culture medium. Ham's F10 containing FCS or ECS produces the highest incidence of fertilization (∼85%), and ∼2‐fold more of these embryos develop in culture to blastocysts compared to embryos supplemented with PVA or BSA.
Dependence of body growth on thyroid activity in turtles
Authors: Denver Robert J. , Licht Paul
Published: June 2005

We surgically thyroidectomized (Tx) or sham‐operated hatchling slider turtles—Trachemys (Pseudemys) scripta—and measured their growth (mass and carapace length) for 5 to 7 months. Autopsy at the end of the experiments revealed that up to 30% of the thyroidectomized animals had regenerated thyroid follicles (thyroids had few but enlarged follicles). Therefore, we segregated thyroidectomized turtles a posteriori into two groups: those with observable thyroid tissue at autopsy, greater than 3 ng/ml plasma thyroxine (T4), and normal plasma T4 binding capacity were designated partial Tx (PTx), and others with no observable thyroid tissue, less than 3 ng/ml plasma T4, and low plasma T4 binding capacity were designated Tx. Growth rate was significantly reduced 6–8 weeks after thyroidectomy in the Tx animals; length‐specific growth rate was affected sooner than mass‐specific growth rate. Growth of Tx animals continued to diverge from sham turtles through the end of the experiment; PTx animals did not differ from shams at any time. In a second experiment growth rate was reduced in Tx turtles maintained under both varibale (14L:10D, thermal gradient of 19 to 40°C) and constant (constant light and temperature—30°C) environmental conditions (i.e., reduced growth rate was probably not related to a thermoregulatory dysfunction). However, all animals maintained under the constant conditions stopped growing after 3 months, whereas turtles kept under varibale conditions continued to grow to the end of the experiment (4.5 months). Thyroxine (T4) replacement therapy of long‐term Tx turtles restored growth after 6 weeks.

Plasma thyrotropin (TSH) was greatly elevated in Tx turtles (301 ± 48.7 ng/ml) and at low to nondetectable levels in controls (⩾ 1 ng/ml). T4 replacement therapy was effective in reducing TSH levels in Tx turtles. Plasma T4 and TSH concentrations showed the expected inverse relationship. However, although PTx animals had elevated plasma T4 levels that were in the lower range of the sham controls (PTx: 36.6 ± 8.2 vs. 112.7 ± 17.1 ng/ml in sham), their plasma TSH was also significantly elevated (36.5 ± 9.3 ng/ml vs. 0.8 ± 0.2 ng/ml in sham). These results demonstrate the requirement of an intact thyroid gland for normal growth of turtles, and provide the first direct evidence for a role for the thyroid in body growth of a reptile. They also reveal that the set‐point for the feedback between the thyroid and pituitary secretion can be altered by perturbation of thyroid activity.
Identification of epididymal proteins associated with hamster sperm
Authors: Robitaille Gilles , Sullivan Robert , Bleau Gilles
Published: June 2005

The electrophoretic analysis of the proteins that were extracted from immature caput and mature cauda sperm showed evidence of accumulation of several proteins during the epididymal transit of the sperm. An antiserum, raised against detergent‐extracted proteins from mature spermatozoa, immunostained six epididymal proteins with apparent molecular masses of 16, 22.5, 26, 37, 60, and 80 kDa on Western blots of epididymal fluid. Of these proteins, only the 26 kDa protein was significantly immunodetected in proximal caput epididymal fluid. Its biosynthesis by caput epididymis was confirmed by immunoprecipitation of an in vitro translated product of caput poly (A) RNA. The homology of the 26 kDa epididymal protein with the 26 kDa sperm protein was verified by epitope mapping. The other epididymal proteins were found in the fluid of the more distal portions of the organ. Their presence in the epididymal fluid coincided with their detection on the sperm. These epididymal proteins were considered to be sperm‐coating proteins.
Comparative aspects of estrus synchronization, ovulation induction, and embryo cryopreservation in the scimitar‐horned oryx, bongo, eland, and greater kudu
Authors: Schiewe Mitchel C. , Bush Mitchell , Phillips Lyndsay G. , Citino Scott , Wildt David E.
Published: June 2005

Comparative studies were conducted to evaluate the efficacy of estrus synchronization, ovulation induction, nonsurgical embryo collection, and cryopreservatin procedures in the scimitar‐horned oryx (Oryx dammah), bongo (Tragelaphus euryceros), eland (Taurotragus oryx), and greater kudu (Tragelaphus strepsiceros). In the scimitar‐horned oryx, a 6α‐methyl‐17α‐acetoxyprogesterone (MAP) pessary vs. prostaglandin F2α (PGF2α) estrus synchronization treatment was compared; only PGF2α was tested in the other species. Ovarian responsiveness to follicestimulating hormone (FSH‐P, b.i.d. for 5 days) or pregnant mare's serum gonadotropin (PMSG, single injection) was evaluated in all species. MAP and PGF2α were equally effective (P < 0.05) for synchronizing estrus in scimitar‐horned oryx. Signs of estrus, including mounting, were observed in 15 of 33 (45.4%) oryx donors, but copulation was observed on only 8 occasions. Mean number of CL/ovulating bongo and eland female was enhanced (P < 0.05) by FSH‐P compared to PMSG (4.5 ± 1.8 vs. 1.3 ± 0.3 CL/bongo and 10.6 ± 4.6 vs. 1.0 ± 0.6 CL/eland, respectively) treatment. The difference in mean (± SEM) CL number between FSH‐P‐ and PMSG‐treated oryx donors approached significance (4.6 ± 1.6 vs. 2.7 ± 0.8, respectively; P = 0.08). Embryos and ova were recovered nonsurgically from 10 of 19 (52.6%) of the ovulating oryx, 9 of 10 (90%) bongo, 6 of 10 (60%) eland, and both (100%) greater kudu. A high proportion (60%) of cryopreserved oryx embryos maintained an excellent‐or good‐quality grade (QG) after thawing. A higher proportion (P < 0.05) of embryos frozen in 1.5 M dimethyl sulfoxide (6 of 10) or 1.5 M glycerol (6 of 10) solutions maintained their prefreeze QG compared to the propylene glycol counterparts (3 of 10). The transfer of thawed embryos to synchronized oryx recipients (n = 9) resulted in no pregnancies. Conventional techniques for synchronizing ovarian activity, inducing ovulation, and flushing the uterine content of farm livestock largely are adaptive to these African antelope species. However, the overall efficiency of these strategies for consistently promoting superovulation and large numbers of embryos is relatively low. For these species, the primary limitations to effective embryo recovery and transfer appear to be: 1) a lack of estrous behavior which contributes to a low incidence of mating and, thus, poor fertilization and recipient synchrony, and 2) a resilience of the ovaries to conventional gonadotropic preparations.
In vitro fertilization of lobster oocytes
Authors: Talbot P. , Poolsanguan W. , Poolsanguan B. , Al‐Hajj H.
Published: June 2005

The feasibility of fertilizing preovulatory lobster oocytes was examined in vitro under various experimental conditions. Large (1.6 mm diameter) and small (0.6 mm diameter) oocytes were compared in fertilization trials. Small oocytes were surrounded by thin envelope thought to correspond to envelope 1A of mature oocytes. Large oocytes were surrounded by a fully formed vitelline envelope comprised of two distinct sublayers, 1A and 1B. Although sperm bound very effectively to the coat surrounding small oocytes, none penetrated the coat or fertilized the oocytes. Large diameter oocytes that were removed from follicles by dissection were fertilized by sperm from the proximal vas deferens of males and by sperm from the seminal receptacle of females. Fertilized oocytes showed a high degree of polyspermy. Higher numbers of sperm bound to, penetrated, and fertilized large diameter oocytes when inseminations were done in a saline solution (2.5LSH) than when done in artificial sea water (ASW). Sperm failed to bind to large diameter oocytes that were induced to ovulate in vitro by collagenase treatment. Contaminating enzymes may have destroyed the sperm receptor in envelope 1A of collagenase treated oocytes. Our in vitro fertilization method will allow the process of fertilization to be studied experimentally in lobsters, and it may be applicable to other decapods.
Modulation of the human sperm acrosome reaction by effectors of the adenylate cyclase/cyclic AMP second‐messenger pathway
Authors: De Jonge C. J. , Han H.‐L. , Lawrie H. , Mack S. R. , Zaneveld L. J. D.
Published: June 2005

The acrosome reaction of spermatozoa appears to be analogous to various somatic cell exocytotic events which involve cascade reactions, i.e., transmission of an external signal across the cell membrane resulting in activation of an “amplifier” enzyme and the generation of a second messenger. Using a synchronous acrosome reaction system (De Jonge et al., J. Androl., 10:232–239, ′89a), it was found that analogues of the second‐messenger cAMP, dibutyryl cAMP (dbcAMP) and 8‐bromo cAMP, stimulated the acrosome reaction of capacitated spermatozoa. Additionally, treatment of spermatozoa with either xanthine or non‐xanthine phosphodiesterase inhibitors induced a significant (P < 0.05) increase in the percent acrosome reaction after a period of capacitation in comparison to untreated controls. These results indicate that analogues of cAMP or inhibitors which prevent cAMP hydrolysis can induce the human sperm acrosome reaction. Subsequent experiments were conducted to test whether the amplifier enzyme in the cascade reaction, adenylate cyclase, has a role in the acrosome reaction. Forskolin, an adenylate cyclase stimulator, caused a significant (P < 0.01) increase in the percent acrosome reaction in comparison to controls. Modulators of adenylate cyclase—adenosine, 2′‐0‐methyladenosine, and 2′,3′‐dideoxyadenosine—significantly (P < 0.01) inhibited the forskolin‐induced acrosome reaction. dbcAMP was able to overcome the inhibition by adenosine. Two inhibitors of protein kinase A, the Walsh inhibitor and H‐8, caused'a significant (P < 0.01) inhibition of the dbcAMP‐induced acrosome reaction. Finally, in the absence of extracellular calcium, dbcAMP induced a significant (P < 0.01) increase in the acrosome reaction in contrast to A23187. These results suggest that: 1) a molecular mechanism for the human sperm acrosome reaction involves the cAMP second‐messenger system; i.e., activation of adenylate cyclase, the amplifier enzyme that produces cAMP, production of cAMP as a second messenger, and activation of cAMP‐dependent kinase A; and that 2) activation of adenylate cyclase occurs after calcium influx.
Anatomic and enzymatic responses of the three‐spined stickleback, Gasterosteus aculeatus to thermal acclimation and acclimatization
Authors: Vézina Daphné , Guderley Helga
Published: June 2005

The three‐spined stickleback (Gasterosteus aculeatus) from the St. Lawrence Estuary prefers temperatures between 9 and 12°C, but encounters temperatures ranging from 0 to 30°C. To determine whether despite this preference for cool temperatures this species retains a capacity for metabolic compensation during thermal acclimation, adult sticklebacks were acclimated to 4°C and 20°C for 6 weeks at various ration levels. When food rations were maximal, PFK, CPT, and CS activities were twofold higher in cold‐acclimated sticklebacks, particularly in the pectoral muscle. This pattern of thermal compensation is similar to that shown by eurythermal fish with considerably higher thermal optima. However, at sub‐maximal rations, warm‐acclimated sticklebacks can undergo a decline in condition which confounds the compensatory response. In the field, even though food is abundant, the condition of male sticklebacks also declined as temperatures rose. When the condition factor declined by 30%, the pectoral and axial muscle indices decreased by 40% and 55%, respectively, and the activities of glycolytic and oxidative enzymes in the pectoral and axial muscles dropped by up to 60%. Clearly, a temperature‐induced increase in metabolic rate can confound a compensatory response to temperature.
Goblet cell alkaline phosphatase in silkworm midgut epithelium: Its entity and role as an ATPase
Authors: Azuma Masaaki , Takeda Susumu , Yamamoto Hiroshi , Endo Yasuhisa , Eguchi Masaharu
Published: June 2005

The characteristics of two alkaline phosphatases (ALP) in the midgut epithelium of the silkworm Bombyx mori (Insecta: Bombycidae) have been investigated further. Distributions of the soluble ALP and membrane‐bound ALP were determined quantitatively by rocket immuno‐electrophoresis with monospecific antibody against the purified soluble ALP and membrane‐bound ALP, respectively. More than 70% of soluble ALP was distributed in the posterior midgut, and the rest of it in the anterior and middle midguts. The membrane‐bound ALP existed at similar levels in all regions of midgut except the most anterior portion. Enzyme‐histochemistry of ALPs could be detected at the apical cell surface of goblet and columnar cells as shown in the previous paper on the immunohistochemistry of ALPs (Azuma, M., and M. Eguchi (1989) J. Exp. Zool. 251:108–112). Furthermore, it was shown that the soluble ALP (goblet cell ALP) required Mg2+ to express its maximum activity, but the membrane‐bound ALP (columnar cell ALP) was insensitive to Mg2+. By using ATP as the substrate, the purified soluble ALP possessed ATPase action with Mg2+ ‐ and/or HCO‐dependent manner. From these results, it is concluded that the soluble ALP functions as one of the ATPase at the goblet cell surface, differing from the K+‐ATPase. This ATPase may be a possible component in generating the alkalinization of midgut lumen characteristic of lepidopteran insects.
Behavioral thermoregulation of the toad, Bufo marinus: Effects of air humidity
Authors: Malvin Gary M. , Wood Stephen C.
Published: June 2005

Two experiments were performed. The first tested the hypothesis that the toad, Bufo marinus, will select a lower ambient temperature under dry environmental conditions. This behavioral response would reduce evaporative water loss and facilitate survival on land. The second experiment measured the effects of temperature on evaporative water loss. In the first experiment, toads were placed in a thermal gradient (11–40°C) for 3 days. On days 1 and 3, water‐filled dishes were placed along the temperature gradient and humid air was circulated through the chamber. On day 2, water dishes were removed, and dry air was circulated through the chamber. Body temperature (Tb) was recorded with a cloacal thermistor. Selected Tb was approximately 8.6°C lower during the dry conditions than during the humid conditions. The behavioral hypothermia took about 6 h to develop. In the second experiment, a reduction in Tb from 17.7 to 12.°C reduced evaporative water loss by 42%. Consequently, behavioral hypothermia of the toad is an important adaptation to dry environmental conditions.
Evidence of luminous bacterial symbionts in the light organs of myctophid and stomiiform fishes
Authors: Foran David
Published: June 2005

The myctophids and stomiiforms represent two common groups of luminous fishes, but the source of luminescence in these animals has remained undetermined. In this study, labeled luciferase gene fragments from luminous marine bacteria were used to probe DNA isolated from specific fish tissues. A positive signal was obtained from skin DNA in all luminous fishes examined, whereas muscle DNA gave a weaker signal and brain DNA was negative. This observation is consistent with luminous bacteria acting as the light source in myctophids and stomiiforms and argues against the genes necessary for luminescence residing on the fish chromosomes. To confirm the location of this signal, a bacterial probe was hybridized in situ to sections of a stomiiform. A strong signal was generated directly over specific regions of the fish light organs, whereas no signal was found over other internal or epidermal tissues of the fish. Taken together, these data provide the first indication that luminous bacterial symbionts exist in myctophids and stomiiforms and that these symbionts account for luminescence in these fishes.