ACTH does not mediate divergent stress responsiveness in rainbow trout.

Two lines of rainbow trout selected for high (HR) and low (LR) responsiveness to a standardised confinement stressor displayed a sustained divergence in plasma cortisol levels during a 3-h period of confinement (max.: HR: 167+/-13 ng ml(-1); LR: 103+/-8 ng ml(-1); P<0.001). However, no significant difference in plasma ACTH levels was evident (max: HR: 153+/-9 pg ml(-1); LR: 142+/-7 pg ml(-1)). Dexamethasone (DEX) was administered to HR and LR fish to block endogenous adrenocorticotropin (ACTH) release. Administration of a weight-adjusted dose of ACTH to the DEX-blocked fish elevated plasma cortisol levels to a significantly greater extent in HR (233+/-24 ng ml(-1)) than LR (122+/-14 ng ml(-1)) fish (P<0.001). Plasma cortisol levels in DEX-blocked HR and LR fish after sham injection were low but also significantly different (HR: 6.7+/-1 ng ml(-1); LR: 2.2+/-0.2 ng ml(-1); P<0.001). These results indicate that modulation of cortisol responsiveness to stressors in HR and LR fish resides, at least in part, downstream of the hypothalamic-pituitary axis.     

Aspects of the structure and development of monotreme spermatozoa and their relevance to the evolution of mammalian sperm morphology

Summary The elongated spermatid nuclei of monotremes exhibit a circumferentially arranged spiral pattern of chromatin condensation, and ultimately form helical filiform sperm heads up to 50 m long and either circular or slightly oval in transverse section. The acrosome is formed by the collapse of the proacrosomal vacuole onto the rostral surface of the elongating nucleus. However, genesis of acrosomal material occurs in the absence of a prominent proacrosomal granule. The flagellum becomes inserted into the distal extremity of the nucleus, the most proximal mitochondria of the midpiece directly abutting the nuclear membrane, so that a prominent neck region is absent. The axoneme is simple and, in the midpiece, small dense peripheral fibres are closely applied to the outer surface of each of the nine microtubule doublets. The cortical fibrous sheath of the principal piece is an anastomosing spiral that lacks lateral columnar elements.The spermatozoal cytoplasmic droplet undergoes migration and is lost during epididymal passage.Monotreme spermatozoa exhibit a montage of features, some of these being also found in marsupials and some in sauropsidan vertebrates, as well as a number of their own distinctive features. It is concluded that monotreme spermatozoa also have a close affinity with the unspecialised spermatozoa of some eutherian mammals.We wish to thank Dr. Mervyn E. Griffiths for providing the Zaglossus material. Permits to collect these native mammals were provided by the National Parks and Wildlife Services, and State Fisheries Services of New South Wales and Queensland. Portion of this work was supported by grants from the Australian Research Grants Committee. The provision of skilled technical assistance and access to electron microscope facilities were kindly provided by Dr. C.D. Shorey, formerly Acting Director of the University of Sydney Electron Microscope Unit, and by Mr. John Hardy, Senior Lecturer-in-Charge of the University of Queensland Electron Microscope Centre. Members of the technical staff of the Departments of Anatomy and Veterinary Anatomy are also acknowledged for their assistance     

DO PHYSICAL FACTORS REGULATE PHYTOPLANKTON DISTRIBUTION PATTERNS IN LARGE, SHALLOW LAKES?

Factors that regulate phytoplankton dynamics in shallow, productive lakes are poorly understood, due to their predisposition for frequent algal blooms and sediment resuspension events. In Lake Apopka, greatest phytoplankton biomass reflects wind-induced resuspension of algae (meroplankton) that exists on the aphotic lake bottom in a layer approximately 5 cm thick; this assemblage is dominated by diatoms (>60% of total biomass) that can occur in resting stages. Once exposed to moderate light, meroplankton are capable of growth and photosynthetic rates comparable with surface populations. In Lake Okeechobee, remote sensing was used to assess the basin-wide distribution of suspended particles. Satellite reflectance values agreed well with in situ particle densities at 20 in-lake stations (average r²; LANDSAT = 0.81, AVHRR = 0.53), and maps of algal blooms (r² = 0.79, p ≤ 0.01). The greatest chlorophyll concentrations occurred in the vicinity of tributary nutrient inputs at the lake's perimeter, while turbidity increased towards the center of the lake reflecting predominant water circulation patterns. These results underscore the importance of physical-biological interactions in lakes.     

Binding and direct electrochemistry of OmcA, an outer-membrane cytochrome from an iron reducing bacterium, with oxide electrodes: A candidate biofuel cell system

Dissimilatory iron-reducing bacteria transfer electrons to solid ferric respiratory electron acceptors. Outer-membrane cytochromes expressed by these organisms are of interest in both microbial fuel cells and biofuel cells. We use optical waveguide lightmode spectroscopy (OWLS) to show that OmcA, an 85 kDa decaheme outer-membrane c-type cytochrome from Shewanella oneidensis MR-1, adsorbs to isostructural Al₂O₃ and Fe₂O₃ in similar amounts. Adsorption is ionic-strength and pH dependent (peak adsorption at pH 6.5-7.0). The thickness of the OmcA layer on Al₂O₃ at pH 7.0 [5.8 ± 1.1 (2σ) nm] from OWLS is similar, within error, to that observed using atomic force microscopy (4.8 ± 2 nm). The highest adsorption density observed was 334 ng cm⁻² (2.4 × 10¹² molecules cm⁻²), corresponding to a monolayer of 9.9 nm diameter spheres or submonolayer coverage by smaller molecules. Direct electrochemistry of OmcA on Fe₂O₃ electrodes was observed using cyclic voltammetry, with cathodic peak potentials of −380 to −320 mV versus Ag/AgCl. Variations in the cathodic peak positions are speculatively attributed to redox-linked conformation change or changes in molecular orientation. OmcA can exchange electrons with ITO electrodes at higher current densities than with Fe₂O₃. Overall, OmcA can bind to and exchange electrons with several oxides, and thus its utility in fuel cells is not restricted to Fe₂O₃.     

Observations of spermiogenesis and epididymal sperm maturation in the rufous hare wallaby, Lagorchestes hirsutus (Metatheria, Mammalia)

Acrosomal development in the early spermatid of the rufous hare wallaby shows evidence of formation of an acrosomal granule, similar to that found in eutherian mammals, the Phascolarctidae and Vombatidae. Unlike the other members of the Macropodidae so far examined, the acrosome of this species appears to be fully compacted at spermiation and extends evenly over 90% of the dorsal aspect of the nucleus. During spermiogenesis, the nucleus of the rufous hare wallaby spermatid showed evidence of uneven condensation of chromatin; this may also be related to the appearance of unusual nucleoplasm evaginations from the surface of the fully condensed spermatid. This study was unable to find evidence of the presence of Sertoli cell spurs or nuclear rotation during spermiogenesis in the rufous hare wallaby. The majority of spermatozoa immediately before spermiation had a nucleus that was essentially perpendicular to the long axis of the sperm tail. Nuclei of spermatozoa found in the process of being released or isolated in the lumen of the seminiferous tubule were rotated almost parallel to the long axis of the flagellum; complete parallel alignment occurred during epididymal maturation. At spermiation spermatozoa have characteristically small cytoplasmic remnants compared to those of other macropods. Unlike the majority of macropodid spermatozoa so far described, the spermatozoa of the rufous hare wallaby showed little evidence of morphological change during epididymal transit. There was no formation of a fibre network around the midpiece or of plasma membrane specializations in this region; the only notable change was a distinctive flattening of midpiece mitochondria and scalloping of the anterior mitochondrial sheath to accommodate the sperm head. Preliminary evidence from spermiogenesis and epididymal sperm maturation supports the classification of the rufous hare wallaby as a separate genus but also indicates that its higher taxonomic position may need to be re‐evaluated.     

Successful artificial insemination in the koala (Phascolarctos cinereus) using extended and extended-chilled semen collected by electroejaculation

Artificial insemination in the koala using chilled, electroejaculated semen provides for a marked improvement in the reproductive and genetic management of captive koala colonies in Australia and internationally, and makes available the option of using semen collected from wild populations to expand restricted gene pools. Dilution of koala semen for artificial insemination is complicated because koalas are induced ovulators, and it is thought that ovulating factors are present in the semen, so that semen extension for preservation purposes might be anticipated to result in a failure to induce ovulation. The first two experiments of this study were designed to determine whether artificial insemination using undiluted, extended, and extended-chilled semen collected by electroejaculation was capable of inducing a luteal phase and/or the production of pouch young. In Experiment 1, 1 ml undiluted electroejaculated semen, 2 ml diluted (1:1) semen, and 1 ml diluted (1:1) semen resulted in seven of nine, six of nine, and six of nine koalas showing a luteal phase, respectively; four pouch young were produced in each treatment. A second artificial insemination experiment was conducted in which 2 ml diluted (1:1) semen was administered in three groups of nine koalas. The first group received semen that had been collected and diluted immediately without chilling, the second group was deposited with semen stored chilled for 24 h, and the final group received semen that had been chilled for 72 h. In the first group, five females had a luteal phase, but none became pregnant. In group 2, two of the five females that had a luteal phase gave birth, whereas in group 3, four of the six females that had a luteal phase produced pouch young. In addition, experiment 3 was conducted to determine whether it was possible to produce pouch young by naturally mating koalas that were in the latter stages of their behavioral estrus; this information is important to the logistics of transporting koala semen for artificial insemination by establishing the maximum time frame in which females might be expected to shed a fertile oocyte. Of the 12 females mated on Day 8 of estrus, 6 gave birth, whereas only 3 of the 10 females naturally mated on Day 10 of estrus produced pouch young. The majority of females (21 of 22) in experiment 3 showed evidence of a luteal phase. Together, these experiments have shown that it is possible to use undiluted, extended, or extended-chilled semen to produce koala offspring up to Day 8 of estrus at conception rates similar to those achieved following natural mating. These findings represent a significant advancement in the use of reproductive technology in marsupials and provide the basis for the shipment of koala semen over long distances. The pouch young produced in this study represent the first marsupials born following artificial insemination of extended-chilled semen and bring the total number of koalas produced by artificial insemination to 31.     

A conformational change in heparan sulfate 3-O-sulfotransferase-1 is induced by binding to heparan sulfate

The 3-O-sulfation of glucosamine by heparan sulfate 3-O-sulfotransferase-1 (3-OST-1) is a key modification step during the biosynthesis of anticoagulant heparan sulfate (HS). In this paper, we present evidence of a conformational change that occurs in 3-OST-1 upon binding to heparan sulfate. The intrinsic fluorescence of 3-OST-1 was increased in the presence of HS, suggesting a conformational change. This apparent conformational change was further investigated using differential chemical modification of 3-OST-1 to measure the solvent accessibility of the lysine residues. 3-OST-1 was treated with acetic anhydride in either the presence or absence of HS using both acetic anhydride and hexadeuterioacetic anhydride under nondenaturing and denaturing conditions, respectively. The relative reactivity of the lysine residues to acetylation and [2H] acetylation in the presence or absence of HS was analyzed by measuring the ratio of acetylated and deuterioacetylated peptides using matrix-assisted laser desorption ionization mass spectrometry. The solvent accessibilities of the lysine residues were altered differentially depending on their location. In particular, we observed a group of lysine residues in the C-terminus of 3-OST-1 that become more solvent accessible when 3-OST-1 binds to HS. This observation indicates that a conformational change could be occurring during substrate binding. A truncated mutant of 3-OST-1 that lacked this C-terminal region was expressed and found to exhibit a 200-fold reduction in sulfotransferase activity. The results from this study will contribute to our understanding of the interactions between 3-OSTs and HS.     

The neurohypophysial hormones of the egg-laying mammals: identification of arginine vasopressin in the platypus (Ornithorhynchus anatinus)

Two neurohypophysial peptides have been purified from acetone desiccated posterior pituitary glands of the platypus (Ornithorhynchus anatinus) by molecular sieving and high-pressure liquid chromatography. A single pressor peptide, having an amino acid composition and a chromatographic retention time identical to those of arginine vasopressin, has been identified. A single oxytocic peptide has been isolated that ressembles oxytocin by its chromatographic retention time, but lack of material has prevented to obtain a correct amino acid composition. The pressor peptide is roughly four times more abundant than the oxytocic peptide. Neurohypophysial hormones of platypus seem similar to those of echidna, the other living prototherian, and to those of most placental mammals.