bluuebunny/crossref_metadata_2025 huggingface dataset:
The effects of amino acids, cumulus cells, and bovine serum albumin on in vitro fertilization and first cleavage of hamster eggs
Authors: Juetten Jacquline , Bavister Barry D.
Published: June 2005

Three factors were studied for their effects on the first cleavage division of in vitro fertilized hamster eggs. Eggs from superovulated females were inseminated with precapacitated epididymal hamster sperm in medium containing either fatty acid‐free (FAF) or fraction V (V) bovine serum albumin (BSA), in the presence or absence of cumulus cells. After incubation for 3 to 4 h with sperm, eggs were transferred to culture medium containing FAF‐ or V‐BSA, with or without amino acids (glutamine, isoleucine, methionine, and phenylalanine), and the percentages of eggs cleaving to two cells were recorded after a further 20 h of incubation. Fatty acid‐free BSA was found to support a significantly higher percentage of eggs cleaving than V‐BSA and was used in all further experiments. The presence of cumulus cells during fertilization was found to have a beneficial effect on cleavage when no amino acids were added to the culture medium, but when amino acids were included, eggs fertilized with or without cumulus present cleaved equally well. These results represent another step toward defining the optimal environmental conditions for obtaining the first cleavage division of hamster eggs in vitro.
Changes in body water and plasma constituents during bullfrog development: Effects of temperature and hormones
Authors: Brown Stephen C. , Horgan Elizabeth A. , Savage Lynne M. , Brown Patricia Stocking
Published: June 2005

The osmoregulatory responses to warmer temperatures and hormone treatment in cold‐adapted (5° C) Rana catesbeiana tadpoles and newly metamorphosed frogs were examined. Tadpoles transferred to 11° C and 18° C and left for 5 days lost 7% and 10% of their body weight. Plasma [Na+] was elevated 28% and 21%, respectively. Control (5° C) animals maintained their body weight and plasma [Na+] constant. Daily treatment with either ovine prolactin (oPRL) or ovine growth hormone (oGH) prevented the weight loss and the increase in extracellular [Na+] that occurred when tadpoles were transferred to 18° C. Neither propylthiouracil (PTU) nor arginine vasotocin (AVT) were effective in countering temperature‐induced weight loss in tadpoles. Newly metamorphosed frogs transferred to 18° C also lost weight; this was not prevented by daily treatment with saline, oPRL, oGH or PTU. However, in frogs treated daily with AVT, initial BW was regained by day 6. When warm‐adapted (18° C) tadpoles were treated daily for 18 days with saline, bPRL, bGH, thyroxine (T4), ergocornine, cortisol, or cortisol + T4, bPRL was most effective in retarding weight loss and maintaining body water content, whereas T4 + cortisol caused the greatest loss of weight and body water. By day 20, the correlations between weight loss and both body water content and hematocrit were highly significant. These data suggest that reported increases in plasma solute concentrations in larval amphibians may actually reflect decreases in extracellular fluid volume, rather than increased amounts of solutes, per se.
Events in the movement of newt epidermal cells across implanted substrates
Authors: Mahan James T. , Donaldson Donald J.
Published: June 2005

Pieces of coverslip glass, polycarbonate filters, or coverslip plastic, coated with fibrinogen or type I collagen, were implanted under one edge of a fresh skin wound on adult newt hind limbs so that the implant served as wound bed for migrating epidermal cells as they attempted to form a wound epithelium. Migratory events were then analyzed by phase contrast and electron microscopy. Phase‐contrast microscopy revealed two types of lamellipodia on leading edge cells: one which was attached broadly to the cell body and one attached by a long, thin stalk. Stalkless forms were by far the most common type and we believe they provide the motive force for cell movement. Stalked forms often moved at distinct angles to the direction of sheet movement, suggesting that they may be sensory appendages. Phase photographs of the leading edge of migrating sheet 4 hours and 8 hours after implantation showed that all cells that were on the leading edge at 4 hours continued to advance for the next 4 hours, demonstrating clearly that under these circumstances the distalmost cells do not become immobile upon contact with the substrate as others have suggested. TEM revealed that migrating sheets were modified monolayers and that regardless of proximodistal location in the sheet, and even in the intact skin adjoining a wound, each epidermal cell adjacent to the substrate puts forth a lamellipodium which underlaps the cell in front. This and the behavior of sheets as they were teased or pulled from the implant suggest strongly that all basal cells contribute to movement of the sheet by interacting with the substrate. Based on TEM, this interaction may be by either focal or close contacts.
Oxidation of amino acids, Krebs cycle intermediates, fatty acids and ketone bodies by Raja erinacea liver mitochondria
Authors: Moyes Christopher D. , Moon Thomas W. , Ballantyne James S.
Published: June 2005

The oxidation of amino acids, Krebs cycle intermediates, fatty acids and ketone bodies was examined in a well‐coupled mitochondrial preparation from the liver of the little skate, Raja erinacea. Glutamate was the preferred amino acid on the basis of both the respiratory control ratio (RCR; 7.97 ± 0.60 SE) and the state 3 rate (12.50 (± 1.16 SE) nmoles 0 min−1 (mg protein−1). Other amino acids, including sarcosine and β‐alanine, were oxidized at less than one third the rate of glutamate with RCRs less than 4. Oxidation of ornithine and arginine was not detected. Of the Krebs cycle intermediates tested, isocitrate had the best RCR and state 3 rate. Malate enhanced the oxidation of tricarboxylic acids (isocitrate and citrate) and α‐ketoglutarate, probably by stimulation of transport into the mitochondria. Oxaloacetate and succinyl CoA were not oxidized at detectable rates. Pyruvate oxidation was low, even at high concentrations (10 mM). Fatty acids (as carnitine esters) were oxidized at high rates, especially hexanoyl (C‐6), octanoyl (C‐8), decanoyl (C‐10), lauroyl (C‐12), and myristoyl (C‐14) carnitine. Acetoacetate oxidation was not detected, but high rates of oxygen consumption were observed in response to 1 mM β‐hydroxybutyrate. Oxidation of this ketone body was malonate‐sensitive, although 14CO2 production from 3 (14C)‐β‐hydroxybutyrate was minimal, indicating that ketone body carbon was not entering the Krebs cycle. The malonate sensitivity of the oxidation process was due to direct inhibition of β‐hydroxybutyrate dehydrogenase (I50 for malonate = 0.558 (± 0.031 SE) mM). The liver of this skate appears to be well suited to utilize fatty acids rather than ketone bodies or carbohydrate.
Cardiovascular responses to catecholamines at 12°C in the American bullfrog (Rana catesbeiana)
Authors: Herman Ceil A. , Robleto Daniel O. , Mata Paula L. , Heller R. Scott
Published: June 2005

The effects of epinephrine, norepinephrine, phenylephrine, and isoproterenol on blood pressure and heart rate were studied in cannulated American bullfrogs, Rana catesbeiana. The bullfrogs were chronically cannulated with a T cannula in the right sciatic artery. In warm‐acclimated (22°C) bullfrogs, preinjection mean systemic arterial pressure (SAP) prior to experimental treatment was 13.1 ± 0.7 mm Hg. Preinjection heart rate was 34.8 ± 1.8 beats per minute. These parameters were lower in cold‐acclimated (12°C) bullfrogs. Cold‐acclimated animals had mean SAP values of 8.2 ± 0.3 mm Hg, and heart rate was 11.1 ± 1.1 beats per minute. Epinephrine, norepinephrine, and phenylephrine increased blood pressure to an equivalent degree in warm‐and cold‐acclimated animals. Dose‐related decreases in heart rate in response to these catecholamines were observed in warm‐ but not in cold‐acclimated bullfrogs. Warm‐acclimated animals were more responsive to isoproterenol from 0.03 μg/kg body weight (bw) to 10 μg/kg bw than were cold‐acclimated animals. The response to isoproterenol was effectively blocked by propranolol (5 mg/kg bw) in both warm‐ and cold‐acclimated animals. Propranolol alone decreased mean SAP in both warm‐ and cold‐acclimated animals, suggesting blockade of endogenous sympathetic activity. Beta receptor response thus appears diminished, but not absent at 12°C. However, the alpha receptors responsible for elevation of blood pressure equally responsive at 12° and 22°C.
Analysis and function of organic matrix from sea urchin tests
Authors: Swift Debbie M. , Sikes C. Steven , Wheeler A. P.
Published: June 2005

The organic matrix from sea urchin tests was extracted using 2% acetic acid. This material contained 81–85% protein, 14–19% carbohydrate, and < 1% phosphate. The whole matrix was separated into aqueous soluble and insoluble fractions to determine their individual characteristics and functional activities. Electrophoresis of the soluble matrix (SM) resulted in a single band of approximately 170kD. The SM was capable of inhibiting in vitro CaCO3 precipitation (at nanomolar concentrations) in solutions supersaturated with Ca2+ and CO32‐. It was also capable of inhibiting CaCO3 spicule formation in sea urchin larvae at 10 μg SM per ml of larvae as measured by 14C incorporation. The insoluble matrix (IM) fraction had a higher protein content than the SM component and a lower PO4 content.

That the SM from urchin tests is capable of inhibiting both in vitro crystallization and organismal calcification lends credence to its probable role as a mineralization regulator. What role it plays in vivo can only be surmised at this point, and whether or not this role is the same when it acts in conjunction with other components of matrix is unknown.
A new, early, morphological indication of sex differentiation in anura, Rana japonica and R. nigromaculata
Authors: Shirane Terukiyo
Published: June 2005

The genital ridges and gonads of normally developing and testosterone‐treated young larvae of Rana japonica and R. nigromaculata were examined histologically. Two classes of primitive gonads were recognized in both species, one with large germ cells and the other with small germ cells. Testosterone‐treated animals all had gonads with small germ cells. From this and other observations, it is concluded that gonads with large germ cells differentiate into ovaries and those with small germ cells differentiate into testes.
Isolation and partial characterization of the plasma membrane from human spermatozoa
Authors: Mack S. R. , Everingham J. , Zaneveld L. J. D.
Published: June 2005

Ejaculated human spermatozoa were subjected to nitrogen cavitation (600 psi for ten min) to remove the plasma membrane (PM). Electron microscopic examination of the cavitated cells revealed that 33% of the PM was removed from the sperm which includes both the head and tail regions.

The released membrane was separated from the cavitated cells by centrifugation followed by a discontinuous sucrose density gradient centrifugation. A single membrane population was resolved at the 1.0 M sucrose interface. Examination of the isolated membranes by electron microscopy revealed vesicles of various sizes displaying unit membrane structures. Biochemical analysis of the isolated membranes showed a threefold enrichment in the surface membrane marker 5′ nucleotidase and also suggested little contamination by enzymes from the cytosol (lactate dehydrogenase) or mitochondria (cytochrome oxidase). Analytical lipid analysis of the isolated membranes revealed a 26‐fold enrichment in the distribution of cholesterol, an 11‐fold enrichment of phospholipids, and a cholesterol:phospholipid molar ratio of 0.83. Also found was a twofold increase in glycosphingolipids which are ubiquitous components of PM in eukaryotic cells. These data indicate that the membrane vesicles isolated after nitrogen cavitation are primarily PM.
The action of acetylcholine upon heart rate changes markedly with development in bullfrogs
Authors: Burggren Warren , Doyle Michael
Published: June 2005

Dose‐response curves for the action of acetylcholine (ACh) and its antagonist, atropine, upon heart rate in situ were made for three larval stages and adults of the bullfrog, Rana catesbeiana. The absolute magnitude of heart rate inhibition at any ACh concentration progressively increased throughout larval development. Unexpectedly, metamorphosis to the adult resulted in a sharp decrease in cholinergic cardiac sensitivity down to levels similar to the earliest larvae. Thus, cholinergic sensitivity of heart rate, reflecting properties of the cardiac pacemaker, is greatest immediately before metamorphosis and lowest in early larvae and adults.
Sensory projection patterns of supernumerary legs and aristae in D. melanogaster
Authors: Lienhard Markus C. , Stocker Reinhard F.
Published: June 2005

The central projection patterns of afferents from normal and duplicated legs and antennal aristae in D. melanogaster were established by horseradish peroxidase or cobalt labelling. Duplications were obtained genetically, by using l(1)ts726 and engrailed mutants, which are known to produce supernumerary legs and aristae, respectively. In normal legs, afferents from singly innervated mechanoreceptive (M‐) bristles terminate in the lateral and medial periphery of the corresponding leg neuromere. The medial periphery is also occupied by terminals of multiply innervated chemoreceptive (C‐) bristles; another target of C‐bristles is the anterior margin of the neuromere. The sensory projection patterns of homologous M‐bristles on different leg segments are overlapping, suggesting that terminals are not ordered according to the proximo‐distal location of their cell bodies on the leg. The sensory projections of duplicated legs or aristae overlap the corresponding normal projections. However, when stimulating supernumerary legs in the proboscis extension paradigm, the frequency and intensity of reflex activity are significantly reduced compared to normal legs. These data suggest that normal and supernumerary afferents compete with one another in a subtle way which is not accompanied by gross morphological changes of the projection patterns.
Anatomy of axolotl flank integument during limb bud development with special reference to a transcutaneous current predicting limb formation
Authors: Borgens Richard B. , Callahan Linda , Rouleau M. F.
Published: June 2005

We have compared the anatomy of immature axolotl integument from limb‐forming regions with adjacent non‐limb‐forming regions of the flank, concentrating on the earliest stages of limb bud development. We have extended these observations to include prominent buds just prior to their differentiation. At the ultrastructural level, we note striking differences between these two regions of skin, including a complete loss of hemidesmosomes and tonofilaments in the basal cells of the epidermis; a marked deterioration of the basal lamella; and focal areas of desquamating cells in the apical regions of the bud—all characteristics of limb‐forming regions. These observations were made in the same larvae which provided measurements of a steady endogenous electric (ionic) current that either was coincident with or predicted the area of limb bud outgrowth (Borgens et al.: J. Exp. Zool. 228:491–503, 1983). We discuss these physiological measurements, the changes in the anatomy of the bud‐forming region, and the relevance of these observations to our theory of early limb formation.
Stage‐specific expression of β‐1, 3‐glucanase in sea urchin embryos and hybrids
Authors: Peeler Margaret Truschel , Chambers Scott A. , Wang Chun‐Yen , McClay David R.
Published: June 2005

In some species of sea urchin, such as Lytechinus variegatus β‐1, 3‐glucanase activity is present at two distinct developmental stages (in the unfertilized egg and again following gastrulation). There is a different form of the enzyme β‐1, 3‐glucanase specific to each stage, and these forms can be distinguished immunologically and biochemically. The distinguishing characteristics of the egg and embryonic enzymes were used to analyze the forms of β‐1, 3‐glucanase present in other species that express activity at only one of the two stages. The enzyme present in Tripneustes esculentes, which has activity only early in development, is an 80,000 dalton enzyme antigenically similar to the egg enzyme in Lytechinus. The enzyme present following gastrulation in Echinometra lucunter, however, is distinct from the egg enzyme and is similar to the embryonic enzyme in Lytechinus, despite the absence of activity in the eggs of Echinometra. These results indicate the two forms of β‐1, 3‐glucanase (egg and embryonic) are expressed independently and in a stage‐specific manner, suggesting they are not functionally equivalent. β‐1, 3‐glucanase ase activity was also analyzed in interspecific hybrids. Tripneustes esculentes (which normally lacks the embryonic form) suppressed the expression of the embryonic enzyme in crosses with Lytechinus variegatus (which normally does express it). This suppression occurred whether the Tripneustes genetic material was maternal or paternal in origin. However, the embryonic enzyme normally expressed by Echinometra was expressed in crosses of this species with Tripneustes, i.e, Tripneustes did not suppress enzyme expression in these hybrids. This differential effect on the expression of the embryonic stage enzyme in these hybrids suggests that the mechanisms leading to the expression of this enzyme differ in Lytechinus and Echinometra, despite the biochemical similarities of the enzyme.
Development of spontaneous motility in the guppy embryo (Lebistes reticulatus) and the effect of spinal transection
Authors: Richards Cheryl , Pollack Emanuel D.
Published: June 2005

Guppy (Lebistes reticulatus) embryos pass through a distinct sequence of motor behaviors that leads to swimming capability during the course of their development. We have characterized these activities in order of appearance, with several corresponding morphological features, as belonging to the coil stage, tail‐twitch stage, S‐movement stage, and swimming stage. A primary feature of development was an increase in the amount of activity per unit of time over these four stages. The developmental pattern of motility was not interrupted by spinal transection until the onset of swimming, implying that supraspinal information is not required for the occurrence of the primitive behaviors that precede swimming. Elimination of swimming by spinal transection did not elicit a reversion to less complex activities, suggesting that once the cerebral control for swimming is developed, it represents a hardwired system not behaviorally reducible to antecedent components.
Comparative thyroid function in adult Japanese quail and Ring doves: Influence of dietary iodine availability
Authors: McNichols M. J. , McNabb F. M. Anne
Published: June 2005

Thyroid function was studied in Japanese quail, Coturnix japonica, and Ring doves, Streptopelia risoria, when both were fed the same dietary iodine (I; 930 μg I/kg). We also compared thyroid function in groups of doves receiving low I (< 100 μg I/kg) or moderate I (930 μg I/kg). We measured thyroid gland (TG) weight, TG stable I content, TG 125I uptake, and 125I labeling of thyroid hormones. Triiodothyronine (T3) and thyroxine (T4) concentrations in TGs and serum were also determined. Our results indicate that doves and quail receiving the same dietary I show similar serum T3 (the presumed metabolically active hormone) and TG functional state but that there are some differences between the species in the way in which this functional state is achieved.

We also assessed the effects of differences in I availability on thyroid function in doves. With low dietary I doves show decreases in some measures of thyroid function (reduced serum T4 and TG‐hormone stores) compared to doves with moderate I but maintain a comparable level of serumT3. This regulation of T3 appears to be independent of serum T4 or TG‐hormone stores.
Photoperiodic variation of Leydig cell numbers in the testis of the golden hamster: A possible mechanism for their renewal during recrudescence
Authors: Hardy Matthew P. , Mendis‐Handagama S. M. L. C. , Zirkin Barry R. , Ewing Larry L.
Published: June 2005

Golden hamster testes regress after short day exposure. The present study asks: 1) are Leydig cell numbers depleted during short days, and 2) if so, how are they replenished during recrudescence. Control hamsters were shown 14 h of light and 10 h of dark (LD 14:10) for 10 weeks (n = 12). Testicular regression was induced by LD 6:18 for 10 weeks (n = 4), and recrudescence by switching regressed hamsters to LD 14:10 for 3 and 5 weeks (n = 8 for each group). All hamsters were injected with [3H]thymidine [3 μCi/gm body wt., intraperitoneally (i.p.)] 1 h or 2 weeks before sacrifice. Leydig cell number per testis was determined by stereological analysis of sections of perfusion‐fixed testes, and labeling indices were determined by autoradiography. Leydig cell numbers were reduced significantly from 18.2 × 106 in control to 9.0 × 106 in regressed testes (p < 0.05); then increased to 14.0 × 106 and 17.9 × 106 in 3‐ and 5‐week recrudesced hamsters. The labeling index was nondetectable (n.d.) for regressed hamsters. In control and recrudescing hamsters the labeling index was measured at two times (t1 = 1 h vs. t2 = 2 weeks post‐injection): in controls, t1 = 0.22 ± 0.15% (mean ± SEM) vs. t2 = 0.28 ± 0.22%; in 1 week recrudesced, n.d. vs. 1.92 ± 0.77% (p < 0.05); at 3 wk, n.d. vs. 4.58 ± 1.74% (p < 0.05); at 5 weeks, 1.92 ± 0.61% vs. 2.25 ± 0.59%. These results are indicative of the existence of interstitial precursor cells that divide, then differentiate, and thus replenish the Leydig cell population during testicular recrudescence.
Adenylate cyclase and cyclic AMP in the ciliate Euplotes crassus: Involvement in both cell cycle and sexual reproduction
Authors: Verni F. , Rosati G.
Published: June 2005

In this paper the presence and the possible activity of adenylate cyclase‐cyclic AMP (AC‐cAMP) system has been investigated through cell cycle and sexual reproduction of the ciliate Euplotes crassus. For this purpose cytochemical studies for AC demonstration at the ultrastructural level were carried on. Moreover, “in‐vivo” experiments were performed by adding different substances to the cell cultures: alloxan (inhibitor of AC), theophylline (inhibitor of phosphodiesterase), exogenous cAMP, and cholera toxin (which maintains AC in an active state). The results obtained are indicative that cAMP is a “regulatory factor” acting during cell cycle as E. crassus cells are not able to undergo macronuclear DNA synthesis in the presence of theophylline. As concerns sexual reproduction both cytochemical observation and “in‐vivo” experiments are suggestive of a rapid activation of AC upon mixing strains of different mating types. This activation, which lasts only for the initial part of the waiting period, appears to be necessary for pair formation.
Antigenic comparison of five species of mammalian zonae pellucidae
Authors: Maresh Grace A. , Dunbar Bonnie S.
Published: June 2005

A comparison of glycoproteins of the zona pellucida (ZP) of five different mammalian species (cat, dog, rabbit, pig, and rat) has been made using polyclonal and monoclonal antibodies. In an enzyme‐linked immunosorbent assay (ELISA), polyclonal antisera against total rabbit and pig ZP recognize their homologous ZP to the greatest extent but also detect crossreactive antigenic determinants in the ZP of all other species tested. Polyclonal antibodies against each of two purified rabbit ZP glycoproteins or one purified pig ZP glycoprotein also show some recognition of heterologous (pig, cat, and dog) ZP, but not rat ZP. Monoclonal antibodies (McR5‐rabbit ZP protein determinant; McPSI‐determinant associated with post‐translational modifications of pig ZP proteins such as carbohydrates) further demonstrate that specific determinants are shared between some but not all of these mammalian species. For example, both of these antibodies recognize distinct determinants which are most abundant in pig and cat ZP. However, McR5 recognizes a determinant on all species of ZP except the rat, while McPSI does not recognize either the rabbit or rat ZP. Collectively, these studies suggest that the molecules of the pig, dog, and cat ZP are more closely related to each other than to those of the rabbit ZP, while there is little similarity with rat ZP molecules. Immunoblot analysis of ZP glycoproteins separated by two‐dimensional polyacrylamide gel electrophoresis was used to identify antigenic relationships among four different species. The polyclonal antisera show that all of the major proteins of pig, rabbit, cat, and dog ZP are antigenically related. These studies provide information that is important for the comparison of structural and functional similarities and dissimilarities in the molecular components of the mammalian zona pellucida that are involved in the fertilization process.
Methionine and iron as growth factors for rat embryos cultured in canine serum
Authors: Flynn Thomas J. , Friedman Leonard , Black Thomas N. , Klein Norman W.
Published: June 2005

Development of headfold‐staged rat embryos cultured in canine serum containing various supplements was compared with development in rat serum to seek suitable alternatives to rat serum in rodent embryo culture and to identify nutritional factors for cultured rodent embryos that may have relevance for normal mammalian embryonic growth and development. Supplementation of canine serum with glucose, methionine, and a lipophilic iron chelate allowed growth and development of cultured rat embryos, approximating those obtained with rat serum. These findings suggest that properly supplemented canine serum can serve as a suitable rodent embryo culture medium and that glucose, iron, and methionine may be important nutrients in mammalian embryonic development.
Hyperactivated motility induced in mouse sperm by calcium ionophore A23187 is reversible
Authors: Suarez Susan S. , Vincenti Leila , Ceglia Matthew W.
Published: June 2005

The reversibility of hyperactivated motility was tested in caudal epididymal mouse sperm by treating them with 1 μM calcium ionophore A23187 in dimethyl sulfoxide (DMSO), followed 2 min later by the addition of medium containing high levels of bovine serum albumin (BSA) (final concetrations: 0.5 μM A23187, 22 mg/ml BSA). Controls received DMSO alone, followed by BSA. Immediately following treatment with A23187, motility was weak and vibratory. Two minutes after the addition of high levels of BSA, motility was hyperactivated, as determined by videotape analysis of linearity of trajectory and acuteness of flagellar bending. Ten minutes after the addition, the movement pattern returned to that of fresh, uncapacitated epididymal sperm. Control sperm retained the linear swimming pattern of fresh caudal epididymal sperm during the 10 min of observation. Ninety minutes later, however, both control and treated sperm became hyperactivated. The percentage of motile sperm was not affected by treatment or time. Thus, ionophoreinduced hyperactivation is reversible and does not interfere with the normal development of hyperactivation during incubation under capacitating conditions in vitro.
Intracellular voltage recordings in the opercular epithelium of Fundus heteroclitus
Authors: Zadunaisky J. A. , Curci S. , Schettino T. , Scheide J. I.
Published: June 2005

Opercular epithelial cells of Fundus heteroclitus were investigated using conventional microelectrodes. The area of interest was the cells lining the inside of the opercular epithelium closest to the gill arches, an area with a high density of chloride cells. Only one cell type could be discerned from the values of 60 opercular cells measured with the opercular epithelium in open circuit conditions. A mean apical voltage of −18.0 ± 0.6 mV was observed with intracellular values ranging from −10 to −30 mV. The predicted intracellular chloride content was 59 mM/liter. Apical fractional resistance (f) was 0.78 ± 0.02. The intracellular potential measurements were typically difficult to maintain for extended periods (longer than 3 min). The opercular cells depolarize with serosal isoproterenol treatment (10−6 M) corresponding to the increase in opercular transepithelial potential. The opercular cell apical fR decreased with isoproterenol treatment. These data indicate the observed opercular cells were involved in opercular chloride transport.
Light‐induced release of Artemia dried embryos from diapause: Analysis of metabolic status
Authors: Van Der Linden A. , Blust R. , Van Laere A. J. , Decleir W.
Published: June 2005

Commercially obtained Artemia cysts were hydrated and incubated for prolonged periods in aerobic conditions, but deprived from light. A part of these cysts refused to hatch and remained in diapause until they were activated by illumination. During a dark incubation period of 20 days, the light‐induced hatchability remained constant, while the respiration rate and the ATP concentrations slowly decreased to the very low values of 0.01 μl O2/hour mg WW and 0.3 nmol ATP/mg protein, respectively. The endogenous trehalose reserves did not measurably decrease and glycerol was not synthesized during this treatment.

Furthermore, we have demonstrated that light, acting as a diapause breaker, induced, in an irreversible manner, an enhancement of respiration and a concomitant breakdown of trehalose. The fact that a very short period of illumination was sufficient to induce hatching and that the induction was irreversible demonstrated that we were definitely not dealing with an exogeneous dormancy, whereby the environment causes the arrest of development. Light acted as a transient signal suspending diapause and triggering development.
Thyroid hormones act at the level of the pituitary to regulate thyrotropin and growth hormone secretion in hatchling slider turtles (Pseudemys scripta elegans)
Authors: Denver Robert J. , Licht Paul
Published: June 2005

Pituitaries from hatchling turtles (Pseudemys scripta elegans) were incubated with different doses of L‐thyroxine (T4: 1–500 ng/ml) or 3,5,3′‐triiodothyronine (T3: 1–50 ng/ml) for various times (1–48 hr) to assess their effects on basal thyrotropin (TSH) and growth hormone (GH) secretion and responsiveness to thyrotropin‐releasing hormone (TRH: 10 ng/ml). Pretreatment with a pharmacological dose of T4 (500 ng/ml) for 1 hr inhibited the TSH response to TRH, and physiological doses were effective after slightly longer times. The TRH effect was reduced by exposure to 50 ng/ml T4 for 1 hr, and abolished after 4 hr. Pretreatment with 50 ng/ml T4 or T3 for 6 hr did not affect basal TSH secretion, but the attenuation of the TRH response persisted for at least 20 hr after removal of thyroid hormones. Longer exposure (16 hr) to 50 ng/ml T4 or T3 reduced both TSH basal secretion and the response to TRH. A low dose (1 ng/ml) of T4 or T3 for the same amount of time had no effect on basal TSH secretion and only slightly reduced the TRH response; T3 was more potent than T4. Basal secretion and TRH responses of glands pretreated with 5 ng/ml T4 were not different from controls after 4 and 6 hr, but were significantly reduced after 24 hr of exposure; 15 ng/ml T4 reduced the TRH response after 6 hr, but basal secretion was not affected until 24 hr. Treatment with 2 ng/ml T4 for 48 hr reduced the response to TRH but did not affect the baseline. Basal and TRH‐stimulated GH secretion was also inhibited by thyroid hormones, but the onset of these effects were delayed (16 to 24 hr after exposure) and required a relatively high dose (50 ng/ml).

These data are consistent with an earlier report from this laboratory (Denver and Licht: J. Exp. Zool. [in press], '88) that in vivo treatment with thyroid hormone or a goitrogen alters in vitro pituitary hormone secretion in turtles and provide evidence for a direct action of thyroid hormones at the level of the pituitary.
Sperm nuclear enlargement in fertilized hamster eggs is related to meiotic maturation of the maternal chromatin
Authors: Wright Shirley J. , Longo Frank J.
Published: June 2005

Experiments were conducted to test the hypothesis that sperm nuclear expansion in fertilized hamster eggs is correlated with meiotic processing of the maternal chromatin. In vitro fertilized hamster eggs were fixed at regular intervals following insemination, stained with the DNA specific fluorochrome, Hoechst 33342, and the extent of sperm nuclear expansion measured. Sperm nuclei enlarged in multiple, distinct phases (A–E) that were temporally correlated with meiotic processing of the maternal chromosomes: phase A, metaphase II; phase B, early anaphase II; phase C, late anaphase II, and phases D and E, female pronuclear development and expansion. During phase A, sperm nuclei were unchanged but enlarged at different rates during phases B (272 μm2/min), D (106 μm2/min) and E (29 μm2/min), and condensed during phase C (rate = −102 μm2/min). Area increases of both sperm nuclei and female pronuclei during phase D were significantly less in polyspermic and polygynic zygotes. If sperm nuclear enlargement and the status/activity of the maternal chromatin were correlated, it would be anticipated that alterations in the normal progression of meiotic maturation would be manifested in sperm nuclear expansion. The following agents affected both meiotic maturation and sperm nuclear enlargement: colchicine, antimycin A, and puromycin. These observations suggest that processes attending sperm nuclear transformations, the completion of maternal meiotic maturation, and development of the female pronucleus are coupled and may be linked by common regulatory agents.
Effect of sodium dodecyl sulfate on polar lobe formation and function in Ilyanassa obsoleta embryos
Authors: Render Joann
Published: June 2005

Polar lobes, anucleate vegetal pole protrusions formed by Ilyanassa obsoleta embryos, serve as a mechanism for shunting morphogenetic determinants to one cell during the first two cleavages. Polar lobe material becomes segregated in the CD cell during first cleavage and in the D cell during second cleavage, resulting in a very unequal four‐cell stage. Larval structures including external shell, foot, operculum, statocysts, and eyes develop only when polar lobe material is present. Treatment with the anionic detergent sodium dodecyl sulfate (SDS) before and during the first cleavage inhibited polar lobe formation and equalized cleavage, as the lobe material was distributed to two cells. No polar lobes formed during second cleavage in SDS‐equalized embryos, and the four‐cell stage consisted of four equal cells with reduced cell contacts. SDS irreversibly inhibited polar lobe formation without affecting cytokinesis. Although 27% of the larvae from SDS‐equalized embryos had one or more lobe‐dependent structures duplicated, morphogenesis was impaired: more than 40% of such larvae failed to form shell and/or statocysts. When cells were separated after equalized first cleavage and raised as pairs, the pairs of resulting larvae duplicated lobe‐dependent structures with the same frequency as whole equalized embryos. Possible explanations for impaired morphogenesis in SDS‐treated embryos are discussed.
Effects of capture and temperature stresses on plasma steroid concentrations in male tuatara (Sphenodon punctatus)
Authors: Cree Alison , Guillette Louis J. , Cockrem John F. , Joss Jean M. P.
Published: June 2005

The effects of capture and temperature stresses on plasma concentrations of testosterone (T) and progesterone (P) were examined in wild male tuatara (Sphenodon punctatus), the only living sphenodontid reptile. Free‐roaming male tuatara on Stephens Island, New Zealand, had low levels (0.3–0.5 ng/ml) of plasma T in July (winter), and high levels (15–17 ng/ml) in January (summer). Levels in November appeared intermediate (5 ng/ml) but were measured with reference to standards prepared in human rather than tuatara plasma. These seasonal variations are consistent with available information on the annual cycle of spermatogenesis and reproductive behavior in male tuatara. In November, plasma T was unchanged in males bled at 0, 3, and 12 h after capture but had fallen significantly to 57% of the initial value by 24 h after capture (experiment 1). In January, males held in collecting bags for 3 h prior to blood sampling had levels of plasma T similar to those of free‐roaming males sampled at the same time (experiment 2). In July, an acute heat stress (12.8°C elevation in body temperature over 30 min) had no effect on plasma T (experiment 3). Significant changes in plasma P were seen prior to changes in plasma T in experiment 1: plasma P was elevated at 3, 12, and 24 h after capture. No difference in plasma P was observed between free‐roaming and captive males in experiment 2. In experiment 3, no change in plasma P occurred in response to heat stress. Free‐roaming males showed no evidence of a diurnal cycle in plasma T or plasma P in November. These data indicate that plasma T in male tuatara is unaffected by short‐term (3 h) capture; however, longer‐term (24 h) capture combined with repeated blood sampling causes a significant reduction in plasma T, as previously reported for other male reptiles. Changes in plasma P suggest that activity of the adrenal gland may be sensitive to captivity stress in this species. These data provide the first values for circulating steroid levels in free‐roaming wild male tuatara and help define conditions of captivity that influence plasma steroid concentrations, a prerequisite for further studies of steroid cycles in this species.
Germ cell deficiency causes testis cord differentiation in reconstituted mouse fetal ovaries
Authors: Hashimoto Naohiro , Kubokawa Reiko , Yamazaki Kimie , Noguchi Motoko , Kato Yoshihiro
Published: June 2005

Sex‐reversal in fetal ovaries was studied by using a dissociation‐reconstitution technique. Gonads of 12.5 gestation‐day male and female mouse fetuses were dissociated into single cells. To eliminate germ cells, the dissociated cells were cultured for 14 h, and then somatic cells attached to culture dishes were harvested and aggregated by gyratory culture for 24 h. The aggregates were then transplanted into ovarian bursa in ovary‐ectomized nude mice. The recovered explants were examined histologically. Male somatic cells developed into testes containing Sertoli cells, Leidig cells, and tunica albuginea. Female somatic cells formed testis cords and differentiated into Sertoli cells, but they did not differentiate into other testis components or ovarian tissues. However, aggregates consisting of both female and male somatic cells differentiated into well‐developed testes containing Leidig cells and tunica albuginea as well as Sertoli cells. Enzyme marker analysis showed significant contributions of female cells in these organized testes. In contrast, aggregates containing both female germ cells and somatic cells developed into ovaries and did not differentiate into any testicular tissues.

The results indicate that female somatic cells in fetal gonads at 12.5 gestation day have the potency to form testis cords and differentiate into Sertoli cells. The subsequent steps in testis development require the contributions of male cells. The present study also suggests that testicular differentiation is independent of germ cells but ovarian development involves the interaction between germ cells and somatic cells.
Carbonic anhydrase in branchial tissues of osmoregulating shore crabs, Carcinus maenas
Authors: Böttcher K. , Siebers D. , Becker W.
Published: June 2005

Activities of carbonic anhydrase (CA) were determined in various organs of shore crabs, Carcinus maenas, acclimated to salinities of 10, 20, 30, 40, and 50‰. In addition, some properties of the branchial enzyme (effects of detergents, storage conditions, and specific inhibitors) were tested. Subcellular distribution of gill CA was analyzed by means of centrifugation procedures. In hemolymph no CA activity could be detected. In all other organs except gills activities were low and insensitive to salinity change. High activities of CA were found in the gills. Activities in posterior gills were—in comparison with anterior gills—more than twofold increased. Acclimation of crabs to reduced ambient salinities resulted in significant increases in CA only in posterior gills. In the gill enzyme specific activities and stability during storage could be increased by means of the detergent Triton X‐100 used at 1% (v/v) in the homogenizing medium. Sulfonamide inhibitors reduced CA activities effectively. Inhibition constants Ki of the gill enzyme were 7.0 × 10−9 M for acetazolamide and 3.1 × 10−8 M for methazolamide. Measurements of CA activities in supernatants and pellets obtained by differential centrifugation and in fractions obtained by centrifugation of 7,500g supernatants in a sucrose density gradient indicated that the major portion of CA activity in the gill (ca. 75%) was bound to cellular particulates including plasma membranes and ca. 25% occurred in a soluble form in the cytoplasm. Both the bound and the soluble forms of CA were increased in crabs acclimated to low salinity, suggesting that CA in crab gills, besides its known role in CO2 excretion and acid‐base balance, functions in osmoregulatory processes.
Seawater inflow through the madreporite and internal body regions of a starfish (Leptasterias hexactis) as demonstrated with fluorescent microbeads
Authors: Ferguson John C.
Published: June 2005

Since the tube feet of starfish can continue to function by osmotic inflation after being separated from the madreporite, doubt has been raised in recent years that this organ is significant for keeping fluid balance. To clarify this question, small specimens of Leptasterias hexactis were exposed to a suspension of fine fluorescent beads for 48 hr and then studied as cryostatic sections under UV light. Abundant beads were found in tube feet and ampullae, clearly showing that madreporite uptake does help sustain their activity under normal circumstances. Even larger accumulations of beads were located in Tiedemann's bodies, indicating that they filter high‐pressure water from the stone canal to generate perivisceral coelomic fluid. Since beads were found only in the upper axial sinus, near madreporitic pores and an opening to the stone canal, perihemal fluid is probably drawn up the axial sinus into the low‐pressure end of the stone canal where it is mixed with incoming seawater and recirculated. In addition to contributing to tube feet inflation, these flows probably form part of a previously unrecognized pattern of fluid replenishment, purification, and circulation in starfish involving the stone canal, Tiedemann's bodies, perivisceral coelom, and distal perihemal spaces and return via the axial sinus.
Nuclear transplantation in the pig embryo: Nuclear swelling
Authors: Prather R. S. , Sims M. M. , First N. L.
Published: June 2005

The transfer of nuclei from cleavage stage embryos to enucleated activated meiotic metaphase II oocytes results in a reprogramming of the transferred nucleus such that it behaves as a zygotic nucleus. One estimator of nuclear reprogramming is nuclear swelling after nuclear transfer. The diameter of nuclei after nuclear transfer was not found to be dependent upon the amount of cytoplasm transferred with the donor cell or the amount of cytoplasm in the recipient cell. Nuclei from 4‐, 8‐, and 16‐cell stage embryos swelled to a similar diameter after nuclear transfer (26.9, 27.3, and 27.2 μm, respectively) and this was significantly different from the diameter of contemporary donor embryos (18.3, 14.3, and 13.0 μm, respectively). This is a swelling of 47, 91, and 109%, respectively. Since the degree of nuclear swelling does not appear to be related to cytoplasmic volume it is concluded that the components mediating nuclear swelling are not in a limiting supply.
Steroid production by amphibian (Rana nigromaculata) ovarian follicles at different developmental stages
Authors: Kwon Hyuk B. , Choi Han H. , Ahn Ryun S. , Yoon Yong D.
Published: June 2005

Rana ovarian follicles at different developmental stages were obtained from frogs collected at different seasons. Isolated follicles were sorted into five different classes (Class I–V) by size and cytoplasmic pigmentation. They were incubated for 6 hours in amphibian Ringer in the presence or absence of frog pituitary homogenate (FPH, 0.05 pituitary/ml) and changes in steroid production by follicles were assessed by measuring estradiol (E2), testosterone (T), and progesterone (P4) in media and follicles by radioimmunoassay. Small follicles (size range 0.22–0.7 mm) secreted very low or non‐detectable levels of the three steroids in the presence or absence of FPH. Medium‐sized vitellogenic follicles (0.70–1.11 mm) actively secreted all the three types of steroids tested (E2, P4, and T). Larger follicles (1.15–1.58 mm), isolated during September–February, secreted much more T than E2 and P4. In contrast, fully grown follicles (> 1.50 mm) collected late in the hibernation period secreted more P4 and less T than the large follicles and very low levels of E2; however, follicles (Class V) collected in breeding season (May) secreted solely P4 and negligible amounts of T and E2. All types of follicles produced very low levels of the steroids without FPH stimulation. Intrafollicular levels of steroids essentially reflected those which accumulated in the medium. Oocyte responsiveness to exogenous hormone, as evidenced by germinal vesicle breakdown (GVBD), increased as the breeding season approached. The data indicate that only the medium‐sized follicles produced considerable amounts of E2 while larger follicles produced mainly T and full‐grown follicles collected during breeding season produced only P4.
Sex pheromones in a marine polychaete: Biologically active compounds from female Platynereis dumerilii
Authors: Zeeck E. , Hardege J. D. , Bartels‐Hardege H. , Willig A. , Wesselmann G.
Published: June 2005

A sex pheromone from Platynereis dumerilii females was extracted from the coelomic fluid of ripe heteronereids by a closed loop stripping technique. It was identified by coupled gas chromatography and mass spectroscopy and synthesis as 3,5‐octadien‐2‐one. In an electrophysiological bioassay, the two isomers of this substance were found to be active on both sexes. In contrast, in behavioural bioassays no effect was found on males while female heteronereids were induced to release eggs.
A method for obtaining a pure population of t6/t6 mouse embryos prior to developmental arrest
Authors: Lu Rong , Palter Karen , Hillman Nina
Published: June 2005

Developmental delay, as the result of ovariectomy, causes mouse blastocyst embryos obtained from +/t6 inter se matings to separate into two distinct populations when placed into outgrowth medium. One population remains as free floating embryos for a significantly longer period of time than the other population. Based upon their phenotypic expression following attachment and outgrowth, the former population was considered to be composed entirely of t6/t6 embryos and the latter, to be composed of +/+ and +/t6 embryos (Nadijcka et al., '81). In the present study, two‐dimensional gel electrophoresis identified the embryos which were delayed in attachment as t6/t6 embryos since they synthesize only p63/6.9a, a product of the Tcp‐1a locus which is unique to t‐haplotypes. The early attaching embryos, assumed to be +/+ and +/t6, synthesize both p63/6.9a and b. The p63/6.9b protein is coded for by Tcp‐1b on the wild‐type homologous chromosome. Control +/+ blastocyst embryos synthesize only p63/6.9b. The data show that t6/t6 embryos can be identified prior to their lethal period and, thus, subjected to comparative studies to determine the cause of their lethality. Developmental delay is the first method established to enable one to unambiguously identify t6/t6 embryos prior to developmental arrest.