Journal of Experimental Zoology (J Exp Zool)

Publications in this journal

• Article: Early estrogen exposure induces abnormal development of Fundulus heteroclitus.

  Hiroshi Urushitani, Akio Shimizu, Yoshinao Katsu, Taisen Iguchi
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  ABSTRACT: Many chemicals released into the environment exhibit estrogenic activity, having the potential to disrupt development and the functioning of the endocrine system. In order to establish a model system to study the effects of such environmental chemicals on aquatic animals, we examined the effects of a natural estrogen, 17 beta-estradiol (E(2)), on early development of Fundulus heteroclitus. Embryos of F. heteroclitus were reared in seawater containing 10(-10), 10(-8), and 10(-6) M E(2) throughout the experiment. Hatching and survival rates decreased in a dose-dependent manner, and fry treated with 10(-6) M E(2) and 10(-8) M E(2) were dead by two weeks and 12 weeks after hatching, respectively. More than 85% of fry treated with 10(-8) M E(2) showed malformations: i.e., eye extrusion, crooked vertebral column, faded lateral-stripe pattern eight weeks after hatching. Body weight and head and body lengths were significantly reduced in E(2)-treated fry when compared to controls. Ossification was not completed in vertebrae, cranial bones, and other bones in fry treated with 10(-8) M E(2) even 12 weeks after hatching. Sex ratio of control fry was 57% male and 43% female, whereas fry treated with 10(-8) M E(2) were 100% female eight weeks after hatching. The present results demonstrate that exogenous estrogen induced death of embryos and fry, malformations, sex reversal, and incomplete ossification of vertebrae and cranial bones, which would result in shorter body and head lengths and in malformed vertebrae leading to a hunchback condition.
  Journal of Experimental Zoology 01/2003; 293(7):693-702.
• Article: Purification and identification of a second form of vitellogenin from ascites of medaka (Oryzias latipes) treated with estrogen.

  Munetaka Shimizu, Yumi Fujiwara, Haruhisa Fukada, Akihiko Hara
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  ABSTRACT: Estrogen treatment of medaka leads to accumulation of ascites, in which vitellogenin (Vg) and choriogenins (precursors to vitelline envelope) are abundant. Besides those female-specific proteins, we detected a new component in ascites that cross-reacts with antiserum against egg yolk proteins. We tentatively named it egg yolk-related protein (YRP). YRP was purified from ascites by hydroxylapatite chromatography followed by gel filtration. Purified YRP had a molecular mass of 460 kDa in intact state while 570 kDa for Vg. The molecular weight of purified YRP on SDS-PAGE under both reducing and nonreducing conditions was 130 kDa. YRP was confirmed to be a lipoglycophosphoprotein by staining with Sudan black, periodic acid-Schiff (PAS) and methyl green. Amino acid composition of YRP resembled that of Vg except for a relatively low content of serine. A specific antiserum against YRP was raised in a rabbit. Antiserum against YRP specifically immunostained its antigen but not Vg or choriogenins. YRP was detected as a female-specific protein in serum of breeding medaka. The antiserum also cross-reacted with a band at 29 kDa in egg extracts, which is not immunoreactive to antiserum against Vg. These data show that YRP is a precursor to some egg yolk proteins with differing antigenicity from Vg (Hamazaki et al. '87). We thus conclude that YRP is a second form of medaka Vg and rename YRP as Vg 2 while formerly reported Vg as Vg 1.
  Journal of Experimental Zoology 01/2003; 293(7):726-35.
• Article: Tropomyosin isoforms present in the sea anemone, Anthopleura japonica (Anthozoa, Cnidaria).

  Masakatsu Fujinoki, Toshiko Tomiyama, Tadashi Ishimoda-Takagi
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  ABSTRACT: Five isoforms of tropomyosin, designated as TMa, TMb, TMc, TMd, and TMe, were detected in the sea anemone, Anthopleura japonica. The apparent molecular weights of these isoforms were estimated to be approximately 30 kD to 37.5 kD, and their pI values were approximately 4.55 (TMa and TMb) and 4.65 (TMc, TMd, and TMe). Although sea anemone tropomyosin isoforms have the ability to bind to rabbit skeletal muscle actin, they preferably bind to actin at higher concentrations of Mg(2+) (10-20 mM) and slightly lower pH (6.2-7.2) than those used in conventional conditions. Antigenic properties of sea anemone tropomyosin seemed to be considerably specific to each isoform. Distribution of tropomyosin isoforms in the sea anemone body was somewhat portion-specific. TMa, TMb, and TMe were detected similarly in the extracts from tentacle, oral disc, column, mouth, and pedal disc. Although TMc and TMd were detected abundantly in the tentacle extract and moderately in the column and mouth extracts, these components were not contained in the pedal disc extract and detected only faintly in the oral disc extract.
  Journal of Experimental Zoology 01/2003; 293(7):649-63.
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  Available from: miami.edu

  Article: Studies on lipid metabolism in trout (Oncorhynchus mykiss) branchial cultures.

  Heinz J M Hansen, Scott P Kelly, Martin Grosell, Chris M Wood
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  ABSTRACT: Cultured branchial cell epithelia from freshwater rainbow trout were incubated with ((32)P)phosphate and ((14)C)acetate as lipid precursors under both symmetrical (L15 media apical/L15 media basolateral) and asymmetrical (freshwater apical/L15 media basolateral) culture conditions. Epithelia composed of pavement cells alone, or containing both pavement cells and chloride cells, were examined. Lipids (labeled with (32)P and (14)C) were isolated and assayed by thinlayer chromatography, and fatty acids (labeled with (14)C) were isolated and assayed by paper chromatography. The main goal was to see whether the loss of a major incorporation into ((32)P)phosphatidylethanolamine [((32)P)PE], previously seen in eel gills in vivo when the fish were transferred from an osmotic steady state to more dilute media, was the result of a hormonal regulation, i.e., did it only apply to gill tissue in vivo or could it also be seen in the absence of hormonal modulation after incorporation of ((32)P)phosphate in vitro? We likewise wished to see whether a major incorporation into ((32)P)PE was dependent upon the presence of chloride cells. Results show that it is possible to obtain a ((32)P)PE dominated incorporation pattern, even in the pavement cells alone, provided that ((32)P)phosphate is added specifically to freshwater on the apical side of epithelia bathed asymmetrically (freshwater/L15). This is identical to the pattern seen in vivo in trout adapted to freshwater. However, this pattern is not seen under symmetrical conditions (L15/L15) or when ((32)P)phosphate is added to the basolateral media. The shift from symmetrical (L15/L15) to asymmetrical (freshwater/L15) culture conditions thus leads to the establishment of a major incorporation into ((32)P)PE and not to the equivalent loss as seen in vivo in more dilute apical media. We conclude that hormonal control is not needed to change the pattern of short-term lipid formation but, nevertheless, the responses are not altogether the same in vitro and in vivo. Furthermore, cultured trout gill epithelia, in contrast to gills in vivo, do not exhibit a marked incorporation of ((14)C)acetate into palmitoleic acid.
  Journal of Experimental Zoology 01/2003; 293(7):683-92.
• Article: Factors affecting the efficiency of somatic cell nuclear transplantation in the fish embryo.

  Tong Ming Liu, Xiao Mu Yu, Yu Zhen Ye, Jian Feng Zhou, Zhong Wei Wang, Jing Gou Tong, Ching Jiang Wu
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  ABSTRACT: Procedures to improve somatic cell nuclear transplantation in fish were evaluated. We reported effects of nonirradiated recipient eggs, inactivated recipient eggs, different combinations between recipient eggs and donor cells, duration of serum starvation, generation number, and passage number of donor cells on developmental rates of nuclear transplant (NT) embryos. Exposure to 25,000 R of gamma-rays inactivated recipient eggs. Single nucleus of cultured, synchronized somatic cell from gynogenetic bighead carp (Aristichthys nobilis) was transplanted into nonirradiated or genetically inactivated unfertilized egg of gibel carp (Carassius auratus gibelio). There was no significant difference in developmental rate between nonirradiated and inactivated recipient eggs (27.27% vs. 25.71%, respectively). Chromosome count showed that 70.59% of NT embryos contained 48 chromosomes. It showed that most NT embryos came from donor nuclei of bighead carp, which was supported by microsatellite analysis of NT embryos. But 23.53% of NT embryos contained more than 48 chromosomes. It was presumed that those superfluous chromosomes came from nonirradiated recipient eggs. Besides, 5.88% of NT embryos were chimeras. Eggs of blunt-snout bream (Megalobrama amblycephala) and gibel carp were better recipient eggs than those of loach (Misgurnus anguillicaudatus) (25% and 18.03% vs. 8.43%). Among different duration of serum starvation, developmental rate of NT embryos from somatic nuclei of three-day serum starvation was the highest, reaching 25.71% compared to 14.14% (control), 20% (five-day), and 21.95% (seven-day). Cultured donor cells of less passage facilitated reprogramming of NT embryos than those of more passage. Recloning might improve the developmental rate of NT embryos from the differentiated donor nuclei. Developmental rate of fourth generation was the highest (54.83%) and the lowest for first generation (14.14%) compared to second generation (38.96%) and third generation (53.01%).
  Journal of Experimental Zoology 01/2003; 293(7):719-25.
• Article: Expression of the gene of interest fused to the EGFP-expressing gene in transgenic mice derived from selected transgenic embryos.

  Jun Kubo, Keitaro Yamanouchi, Kunihiko Naito, Hideaki Tojo
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  ABSTRACT: The present paper describes the expression of a target fusion gene, WAP/hGH fused to the EGFP-expressing gene in transgenic mice derived from the transfer of transgenic embryos selected because of their expression of enhanced green fluorescent protein (EGFP). The 6.7-kb fusion gene was microinjected as a single cassette gene construct into the pronuclei of mouse zygotes. The surviving embryos were cultured and were classified according to the EGFP expression patterns at the morula or blastocyst stage. After the transfer of embryos with uniform-expression or mosaic-expression of EGFP, transgenesis occurred in 85.7% to 86% or 44.1% to 44% of the pups, respectively. No transgenic pups were derived from EGFP negative embryos. In the transgenic females, EGFP was ubiquitously expressed under the control of the CAG promoter, and hGH was expressed under the control of the WAP promoter in an appropriate fashion: hGH was secreted into the milk of lactating transgenic females. The presence or absence of the expression of EGFP coincided with that of the hGH gene in the transgenic mice. The present cassette gene construct is a useful example for circumventing the routine analyses of DNA and RNA required for the generation and maintenance of transgenic lines.
  Journal of Experimental Zoology 01/2003; 293(7):712-8.
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  Available from: seaturtle.org

  Article: Identification of purebred Crocodylus siamensis for reintroduction in Vietnam.

  Nancy Nelson Fitzsimmons, Jason Craig Buchan, Phan Viet Lam, Gert Polet, Ton That Hung, Nguyen Quoc Thang, Jacob Gratten
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  ABSTRACT: Crocodylus siamensis, the Siamese crocodile, is a critically endangered species of freshwater crocodile previously distributed throughout much of SE Asia. Recovery plans call for reintroductions to the wild using founder individuals currently in captivity, mostly in commercial crocodile farms. On many farms C. siamensis has been intentionally hybridised with either Cuban crocodiles, C. rhombifer, or the estuarine crocodile, C. porosus, and hybrids may be difficult to distinguish morphologically. We report on the combined use of microsatellite and mtDNA genetic markers to determine the species status of potential founder individuals for reintroduction of C. siamensis. Genetic markers were used to characterise 103 captive and wild-caught individuals of C. siamensis, C. rhombifer and C. porosus in Vietnam and to distinguish purebred versus hybrid individuals. Although the microsatellite loci used had some overlap of allele sizes among species, assignment tests allowed differentiation. Four hybrids were identified, two of which had not been recognised morphologically as hybrids, and one of these was thought to be a C. siamensis suitable for reintroduction. Ten of the identified purebred C. siamensis were subsequently released into Cat Tien National Park in southern Vietnam.
  Journal of Experimental Zoology 01/2003; 294(4):373-81.
• Article: Generation of a sequence characterized amplified region probe for authentication of crocodilian species.

  Forrest Chung-Fai Yau, Ka-Lok Wong, Jun Wang, Paul Pui-Hay But, Pang-Chui Shaw
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  ABSTRACT: A 209-base pair (bp) crocodilian-specific sequence characterized amplified region (SCAR) was identified from a 425-bp randomly amplified polymorphic DNA (RAPD) fragment. The 209-bp SCAR was produced from amplifications of DNA extracted from fresh and/or dry meat samples from at least three species of Crocodylus, Caiman crocodylus, and Alligator mississippiensis. No amplification was observed from DNA of other common animal species. The use of SCAR opens the way for quick authentication of crocodilian samples for conservation biology and trade regulation.
  Journal of Experimental Zoology 01/2003; 294(4):382-6.
• Article: Variation in plasma lipids during the reproductive cycle of male and female desert tortoises, Gopherus agassizii.

  Valentine A Lance, Allen R Place, Janice S Grumbles, David C Rostal
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  ABSTRACT: Plasma triacylglycerol, phospholipid, cholesterol, cholesterol esters, fatty acids, and total lipids were measured in 30 female and 20 male desert tortoises (Gopherus agassizii) during the annual reproductive cycle in the eastern Mojave desert, Nevada. Blood samples were collected at monthly intervals from April to October. All lipid fractions, with the exception of free fatty acids, were significantly higher in female plasma than in male plasma in all months of the year. In contrast, free fatty acids were higher in male plasma than in female plasma in all months. The seasonal pattern in estradiol secretion mirrored that of triacylglycerol, phospholipid, cholesterol, and total lipid, all of which showed a significant correlation with the hormone. Estradiol and the vitellogenesis-associated lipids were all significantly higher in August, September, October, and April than in June. The seasonal variation in cholesterol ester levels in females did not correlate with any of the reproductive events and did not appear to be involved in yolk precursor formation. Total lipid in males showed a negative correlation with testosterone and spermatogenesis. Individual fatty acids in the June and August samples (at the highest and lowest estradiol levels) were compared in male and female plasma. The percent of C18:3n3, C18:2n6, C18:1n9, C20:5n3, and C22:5 were significantly higher in the June female plasma sample than in the August sample. Docosahexanoic (C22:6n3) acid was barely detectable in female plasma in either month.
  Journal of Experimental Zoology 01/2003; 293(7):703-11.
• Article: Oxidative, heat and anthelminthic stress responses in four species of Trichinella: comparative study.

  Javier Martinez, Jorge Perez-Serrano, W E Bernadina, Filomena Rodriguez-Caabeiro
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  ABSTRACT: The aim of this study was to compare levels of stress proteins in four Trichinella species when exposed to different stressors. Heat shock protein (HSP) 60, 70 and 90 responses were evaluated in infective larvae (L(1)) of four classic Trichinella species following exposure to oxidative, anthelminthic and thermal stress. Larvae of T. nativa, T nelsoni, T. pseudospiralis and T. spiralis were exposed to peroxide shock (0.2%, 1%, or 2% H(2)O(2)for 2h), high temperatures (40 degrees C or 45 degrees C for 2h), or 0.1 microg/ml of the benzimidazole anthelminthics: mebendazole (MBZ), albendazole (ALB) or thiabendazole (TBZ) for 4h. Following exposures, the L(1) were tested for induced morphological changes. Those observed were: (i) no change (in all species exposed to 40 degrees C) (ii) aberrant forms (in all species exposed to anthelminthics, in T. nativa, T. nelsoni and T. spiralis exposed to 45 degrees C, and in T. spiralis and T. nelsoni exposed to 0.2% H(2)O(2)) and (iii) severe degradation or death (in T. nativa and T. pseudospiralis exposed to 0.2% H(2)O(2), and in all species at 1% and 2% H(2)O(2)). In Western blot analyses, L(1) proteins were probed with monoclonal antibodies (mAbs) specific for the three HSPs. Greater changes in HSP levels occurred following H(2)O(2) exposure than with other stresses in all Trichinella species, while accumulation of a 50 kDa HSP was only observed in T. spiralis and T. pseudospiralis. Anthelminthic stress only caused decreased HSP levels in T. nativa. Thermal stress caused no significant changes in the HSP response of any species. It is suggested that other stress proteins (e.g., glucose-regulated proteins) may be involved in adaptation to thermal stress.
  Journal of Experimental Zoology 01/2003; 293(7):664-74.
• Article: Dynamics of vitellogenin mRNA expression and changes in hemolymph vitellogenin levels during ovarian maturation in the giant freshwater prawn Macrobrachium rosenbergii.

  Vidya Jayasankar, Naoaki Tsutsui, Safiah Jasmani, Hisako Saido-Sakanaka, Wei-Jun Yang, Atsuro Okuno, Thi Thanh Hien Tran, Katsumi Aida, Marcy N Wilder
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  ABSTRACT: The dynamics of vitellogenin mRNA expression during ovarian maturation in Macrobrachium rosenbergii were examined by measuring hemolymph vitellogenin (Vg) levels and Vg mRNA expression in the hepatopancreas and ovary at differing reproductive stages in both intact and eyestalk ablated animals. Vg mRNA was quantified using real-time RT-PCR and hemolymph Vg was measured by enzyme immunoassay. In intact animals, Vg mRNA levels in the hepatopancreas and hemolymph Vg levels showed a gradual increase during the molt cycle concomitant with increasing gonadosomatic index (GSI), with Vg levels decreasing prior to ecdysis although GSI continued to increase. Eyestalk ablation was seen to accelerate Vg synthesis as well as ovarian maturation, although it did not alter the overall pattern of Vg expression. Vg mRNA expression was negligible in the ovary of both intact and eyestalk ablated animals, confirming that the hepatopancreas is the principal site of Vg synthesis in M. rosenbergii with the ovary being only a minor contributor. This study has shown that Vg synthesis is correlated to ovarian maturation and the molt cycle in M. rosenbergii.
  Journal of Experimental Zoology 01/2003; 293(7):675-82.
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  Available from: seaturtle.org

  Article: Studies on the molecular evolution of the Crocodylia: footprints in the sands of time.

  Herbert C Dessauer, Travis C Glenn, Llewellyn D Densmore
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  ABSTRACT: A reasonably large number of studies focusing on the molecular evolution of crocodilians have been completed during the past 100 years. Proteins were initially studied before DNA was known to carry the genetic information of cells and organisms, and were subsequently studied to infer changes at the DNA level. More recently, studies on the DNA itself have been completed. We have had the pleasure of taking part in or facilitating many studies conducted over the past 50 years, especially several of the earliest studies done using newly developed molecular techniques. We provide a review of the molecular genetic studies on crocodilians, summarizing the findings of these studies as well as the context in which they were undertaken. This review is a personal look at the history of molecular studies on the evolutionary biology of crocodilians. Our excuse for this focus is that our professors, our students and we have had the opportunity to be among the first to apply many new techniques to studies of crocodilians since 1950, when one of us (HCD) was a graduate student of Roland Coulson and Tom Hernandez. Although we will review much of the material in this subject area, we do not claim that it is complete. Instead, we focus our presentation on work in which we have participated or with which we are particularly familiar. We especially focus on materials relevant to the research presented at the 2(nd) International Crocodilian DNA Workshop, 7-9 November, 2001, at the San Diego Zoo. Thus, the following review also stands as a tribute to our mentors, students, and colleagues.
  Journal of Experimental Zoology 01/2003; 294(4):302-11.
• Article: Distribution and gene expression of cytoskeletal proteins in two-cell rat embryos and developmental arrest.

  Hiromichi Matsumoto, Jin-Yi Jiang, Disuke Mitani, Eimei Sato
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  ABSTRACT: Previous observations in rat two-cell embryos suggested that distribution of microfilaments and microtubules are involved in developmental arrest. Therefore, we examined the distribution of cytoskeletal proteins, actin binding proteins, and microtubule-associated proteins in rat two-cell embryos. We also examined gene expression of beta-actin, alpha-tubulin, and cytoskeletal proteins that showed changes in their distributions. Distribution of cytoskeletal proteins was examined by immunocytochemistry. Although distributions of alpha-actinin, MAP1A, MAP1B/MAP5, and MAP2 were disturbed in arrested embryos, these abnormal distributions occurred following the initiations of developmental arrest and marked damage of microfilaments and microtubules. Gene expression of cytoskeletal proteins was examined by RT-PCR. Beta-actin and alpha-actinin mRNA was detected in normal late two-cell stage but not in arrested embryos. The difference occurred after zygotic gene activation. Expression of alpha-tubulin was detected in neither normal late two-cell stage nor arrested embryos. No MAP1A, MAP1B/MAP5, or MAP2 expression was detected in embryos during the two-cell stage. In conclusion, both distributions of microfilaments and microtubules are closely involved in rat developmental arrest, but other distributions of cytoskeletal proteins, actin binding proteins, and microtubule-associated proteins do not appear to have major roles in two-cell arrest. Furthermore, mRNA expression patterns are different between microfilaments and microtubules. Both distribution and mRNA transcription of microfilaments are involved in rat developmental arrest, whereas only distribution of maternal microtubules is disturbed in arrested embryos.
  Journal of Experimental Zoology 01/2003; 293(7):641-8.
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  Available from: ncsu.edu

  Article: Juvenoid hormone methyl farnesoate is a sex determinant in the crustacean Daphnia magna.

  Allen W Olmstead, Gerald A Leblanc
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  ABSTRACT: Daphnids (Daphnia magna) utilize cyclic parthenogenesis as a reproductive strategy. During periods of abundant resources, these organisms reproduce asexually. In response to environmental cues that signal the onset of environmental adversity, daphnids produce males and reproduce sexually. The environmental cues that stimulate the sexual reproductive phase are well known; however, the endocrine signals that transduce these environmental cues remain unknown. The present study was undertaken to test the hypothesis that the crustacean juvenoid hormone, methyl farnesoate, is a male sex determinant in this species. Continuous exposure to aqueous concentrations of methyl farnesoate greater than approximately 30 nM stimulated a concentration-dependent production of male-containing broods of organisms. Short-term exposures to methyl farnesoate during periods of egg and embryo maturation revealed that male sex determination occurred during a specific 12-hour period of ovarian egg development. Exposure of eggs to 400 nM methyl farnesoate during this sensitive developmental period resulted in the production of all-male broods of offspring, while exposure to concentrations as low as 52 nM produced mixed broods of males and females. This active concentration range of methyl farnesoate is consistent with levels measured in the hemolymph of some decapod crustaceans. These results demonstrate that methyl farnesoate is capable of programming daphnid embryos to develop into males and is likely the endocrine factor responsible for initiating the sexual reproductive phase in these organisms.
  Journal of Experimental Zoology 01/2003; 293(7):736-9.
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  Available from: lsu.edu

  Article: The crocodilian mitochondrial control region: general structure, conserved sequences, and evolutionary implications.

  David Alfred Ray, Llewellyn Densmore
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  ABSTRACT: We present the first comprehensive analysis of the crocodilian control region. We have analyzed sequences from all three families of Crocodylia (Crocodylidae, Gavialidae, Alligatoridae), incorporating all genera except Paleosuchus and Melanosuchus. Within the control region of other vertebrates, several sequence motifs and their order appear to be conserved. Herein, we compare aligned crocodilian D-loop sequences to homologous sequences from other vertebrates ranging from fish to birds. Among other findings, we have discovered that while domain I tends to be shorter than the same region in mammals and birds, it contains sequences similar in structure to both the goose-hairpin and termination associated sequences (TAS). Domain II is highly conservative with regard to size among the taxa examined and contains several of the conserved sequence boxes characterized in other vertebrates. Domain III contains several interesting sequence motifs including tandemly repeated sequences, a long poly-A region in the Crocodylidae, and possible bidirection promoter sequences.
  Journal of Experimental Zoology 01/2003; 294(4):334-45.