Patterns of cells and extracellular material of the sea urchin Lytechinus variegatus (Echinodermata; Echinoidea) embryo,from hatched blastula to late gastrula

Scanning electron microscopy of six stages of Lytechinus variegatus embryos from hatching through gastrulation reveals changes in the shapes of the ectodermal cells and morphological changes in the extracellular material (ECM) in relation to the locations and migratory activities of mesenchyme cells. The classical optical patterns in the blastular wall (Okazaki patterns) are due to differential orientations of the cells, which bend and extend sheet-like lamellipodia over adjoining cells toward the eventual location of the primary mesenchymal ring. The blastocoelic surfaces of the blastomeres become covered with a thin basal lamina (BL) composed of fibers and nonfibrous material. During primary mesenchyme cell (PMC) ingression, a web-like ECM is located in the blastocoel overlying the amassed PMCs. This ECM becomes sparse in migratory mesenchyme blastulae, and is confined to the animal hemisphere. Localized regions of intertwining basal cell processes in the blastular wall are also present during PMC migration. While a distinct BL is present during early and midgastrulation, blastocoelic ECM is absent. Late gastrulae, on the other hand, have an abundance of blastocoelic ECM concentrated near secondary mesenchyme cell protrusive activity. ECM appearing at both the early mesenchyme and late gastrula stages are probably remnants of degraded BL and intercellular matrix preserved by fixation for SEM. Thus, early mesenchyme ECM is formed of BL material whose degradation is necessary for entry of PMCs into the blastocoel. Late gastrula ECM is apparently a degradation product of BL and intercellular material whose destruction is required for fusion of the gut with oral ectoderm in formation of the mouth.     

Can Camera Traps Monitor Komodo Dragons a Large Ectothermic Predator?

Camera trapping has greatly enhanced population monitoring of often cryptic and low abundance apex carnivores. Effectiveness of passive infrared camera trapping, and ultimately population monitoring, relies on temperature mediated differences between the animal and its ambient environment to ensure good camera detection. In ectothermic predators such as large varanid lizards, this criterion is presumed less certain. Here we evaluated the effectiveness of camera trapping to potentially monitor the population status of the Komodo dragon (Varanus komodoensis), an apex predator, using site occupancy approaches. We compared site-specific estimates of site occupancy and detection derived using camera traps and cage traps at 181 trapping locations established across six sites on four islands within Komodo National Park, Eastern Indonesia. Detection and site occupancy at each site were estimated using eight competing models that considered site-specific variation in occupancy (ψ)and varied detection probabilities (p) according to detection method, site and survey number using a single season site occupancy modelling approach. The most parsimonious model [ψ (site), p (site*survey); ω = 0.74] suggested that site occupancy estimates differed among sites. Detection probability varied as an interaction between site and survey number. Our results indicate that overall camera traps produced similar estimates of detection and site occupancy to cage traps, irrespective of being paired, or unpaired, with cage traps. Whilst one site showed some evidence detection was affected by trapping method detection was too low to produce an accurate occupancy estimate. Overall, as camera trapping is logistically more feasible it may provide, with further validation, an alternative method for evaluating long-term site occupancy patterns in Komodo dragons, and potentially other large reptiles, aiding conservation of this species.     

Epididymal SPAM1 is a marker for sperm maturation in the mouse

Sperm adhesion molecule 1 (SPAM1), is a glycosyl phoshatidylinositol-linked sperm membrane protein that is dually expressed in testis and epididymis. Epididymal SPAM1 is secreted in all three regions of the epididymis in all mammalian species studied, including humans. It shares the same molecular mass and neutral hyaluronidase activity as the testicular and sperm isoforms that are responsible for the penetration of the cumulus during fertilization. Using wild-type (W/T) sperm and those from mice homozygous for either a null (Spam1-/-) or mutant Spam1 allele, which results in decreased mRNA and protein, we demonstrate that sperm binding of epididymal SPAM1 occurs in vitro after exposure to W/T sperm-free epididymal luminal fluid (ELF). Binding or adsorption that occurred after incubation at room temperature or 32 degrees C was detected immunocytochemically and confirmed quantitatively using flow cytometry. The localization of SPAM1 on the plasma membrane of Spam1-null sperm mimicked that seen in the W/T. The remarkable increase in binding on W/T caudal sperm indicates that they are not fully saturated with SPAM1 during storage, and suggests that uptake of epididymal SPAM1 in vivo augments testicular SPAM1. Spam1-null sperm exposed to W/T ELF for 45-60 min during in vitro capacitation to allow epididymal SPAM1 binding showed a highly significant (P < 0.001) increase in cumulus penetration after 6-7 h compared to those incubated in ELF from null males. Similarly, the number of cumulus-free oocytes was also highly significantly greater (P < 0.001) than that for sperm capacitated in W/T SPAM1-antibody-inhibited ELF. Because epididymal SPAM1 uptake significantly increases cumulus penetration, we conclude that it is a marker of sperm maturation.     

CASK interacts with PMCA4b and JAM-A on the mouse sperm flagellum to regulate Ca2+ homeostasis and motility

Deletion of the highly conserved gene for the major Ca²⁺ efflux pump, Plasma membrane calcium/calmodulin‐dependent ATPase 4b (Pmca4b), in the mouse leads to loss of progressive and hyperactivated sperm motility and infertility. Here we first demonstrate that compared to wild‐type (WT), Junctional adhesion molecule‐A (Jam‐A) null sperm, previously shown to have motility defects and an abnormal mitochondrial phenotype reminiscent of that seen in Pmca4b nulls, exhibit reduced (P < 0.001) ATP levels, significantly (P < 0.001) greater cytosolic Ca²⁺ concentration ([Ca²⁺]_c) and ～10‐fold higher mitochondrial sequestration, indicating Ca²⁺ overload. Investigating the mechanism involved, we used co‐immunoprecipitation studies to show that CASK (Ca²⁺/calmodulin‐dependent serine kinase), identified for the first time on the sperm flagellum where it co‐localizes with both PMCA4b and JAM‐A on the proximal principal piece, acts as a common interacting partner of both. Importantly, CASK binds alternatively and non‐synergistically with each of these molecules via its single PDZ (PDS‐95/Dlg/ZO‐1) domain to either inhibit or promote efflux. In the absence of CASK–JAM‐A interaction in Jam‐A null sperm, CASK–PMCA4b interaction is increased, resulting in inhibition of PMCA4b's enzymatic activity, consequent Ca²⁺ accumulation, and a ～6‐fold over‐expression of constitutively ATP‐utilizing CASK, compared to WT. Thus, CASK negatively regulates PMCA4b by directly binding to it and JAM‐A positively regulates it indirectly through CASK. The decreased motility is likely due to the collateral net deficit in ATP observed in nulls. Our data indicate that Ca²⁺ homeostasis in sperm is maintained by the relative ratios of CASK–PMCA4b and CASK–JAM‐A interactions. J. Cell. Physiol. 227: 3138–3150, 2012. © 2011 Wiley Periodicals, Inc.     

Identification of a catalytic exosite for complement component c4 on the serine protease domain of c1s

The classical pathway of complement is crucial to the immune system, but it also contributes to inflammatory diseases when dysregulated. Binding of the C1 complex to ligands activates the pathway by inducing autoactivation of associated C1r, after which C1r activates C1s. C1s cleaves complement component C4 and then C2 to cause full activation of the system. The interaction between C1s and C4 involves active site and exosite-mediated events, but the molecular details are unknown. In this study, we identified four positively charged amino acids on the serine protease domain that appear to form a catalytic exosite that is required for efficient cleavage of C4. These residues are coincidentally involved in coordinating a sulfate ion in the crystal structure of the protease. Together with other evidence, this pointed to the involvement of sulfate ions in the interaction with the C4 substrate, and we showed that the protease interacts with a peptide from C4 containing three sulfotyrosine residues. We present a molecular model for the interaction between C1s and C4 that provides support for the above data and poses questions for future research into this aspect of complement activation.     

Investigating the role of murine epididymosomes and uterosomes in GPI-linked protein transfer to sperm using SPAM1 as a model

Sperm uptake of glycosyl phosphatidylinositol (GPI)-linked proteins from luminal fluids has been shown to occur in male and estrous female reproductive tracts. In males, this is attributed to membranous vesicles secreted into the epididymis and prostate. While epididymosomes have been characterized, there have been no reports of the presence of vesicles in female luminal fluids. Here we report the presence of vesicles, characterized as "uterosomes," in the murine estrous female reproductive fluid; and use Sperm Adhesion Molecule 1 (SPAM1/PH-20), a well-known hyaluronidase found in male and female fluids, as a model to investigate vesicle-mediated GPI-linked protein transfer to sperm. Epididymosomes and uterosomes isolated after ultracentrifugation of epididymal (ELF) and uterine luminal fluid (ULF) were analyzed by electron microscopy and shown to be approximately 10-70 and approximately 15-50 nm in diameter. The structural integrity of uterosomes was confirmed by their resistance to hypo-osmotic and freeze/thaw stresses; and immunogold labeling localized SPAM1 to their outer membrane surface, as was the case for epididymosomes. SPAM1 was acquired by caudal sperm during incubation in epididymosomes and uterosomes; uptake was abolished when the GPI anchor was enzymatically cleaved. Sperm analyzed by confocal and transmission electron microscopy (TEM) after incubation in fluorescently labeled vesicles revealed the label on the membrane over the acrosome and midpiece of the flagella, where SPAM1 normally resides. High magnification TEM images demonstrated vesicles juxtaposed to the sperm plasma membrane potentially transferring SPAM1. Taken together, these results implicate vesicular docking as the mechanism of vesicle-mediated GPI-linked protein transfer to sperm from murine reproductive fluids.     

Effects of human activities on Komodo dragons in Komodo National Park

Understanding how threatened wildlife can coexist with humans over the long term is a central issue in conservation and wildlife management. Komodo National Park in Eastern Indonesia, harbors the largest extant populations of the endemic Komodo dragon (Varanus komodoensis). Consistent with global trends, this species is expected to be increasingly exposed to human activities and in particular growing ecotourism activities. Here we comprehensively evaluated how human activities affected individual and population level attributes of Komodo dragons. We compared Komodo dragons phenotypic (behaviour, body size, and body condition) and demographic (age structure, sex ratio, survival, and density) responses to variation in human activities across national park. Komodo dragons were found to exhibit pronounced responses to high human activity level relative to sites with low and negligible human activities. Komodo dragons exposed to ecotourism exhibited significantly less wariness, larger body mass, better body condition, and higher survival. These results are entirely consistent with ecotourism activities that provided Komodo dragons with long-term and substantial nutritional subsidies as a consequence of feeding and human food refuse. However, we also noted the potential negative consequences of altered behaviour and adult-biased populations in ecotourism areas which may influence demographic processes through intraspecific competition or predation. To address this issue, we recommend that three management strategies to be implemented in future include: (1) removal of human-mediated nutritional subsidies, (2) alternative ecotourism, and (3) spatial regulation of ecotourism. Furthermore, we advocate the development of approaches to achieve a socio–ecological sustainability that benefits both people and wildlife conservation.     

Plasma membrane calcium ATPase 4 (PMCA4) co‐ordinates calcium and nitric oxide signaling in regulating murine sperm functional activity

Reduced sperm motility (asthenospermia) and resulting infertility arise from deletion of the Plasma Membrane Ca²⁺‐ATPase 4 (Pmca4) gene which encodes the highly conserved Ca²⁺ efflux pump, PMCA4. This is the major Ca²⁺ clearance protein in murine sperm. Since the mechanism underlying asthenospermia in PMCA4's absence or reduced activity is unknown, we investigated if sperm PMCA4 negatively regulates nitric oxide synthases (NOSs) and when absent NO, peroxynitrite, and oxidative stress levels are increased. Using co‐immunoprecipitation (Co‐IP) and Fluorescence Resonance Energy Transfer (FRET), we show an association of PMCA4 with the NOSs in elevated cytosolic [Ca²⁺] in capacitated and Ca²⁺ ionophore‐treated sperm and with neuronal (nNOS) at basal [Ca²⁺] (ucapacitated sperm). FRET efficiencies for PMCA4‐eNOS were 35% and 23% in capacitated and uncapacitated sperm, significantly (p < 0.01) different, with the molecules being <10 nm apart. For PMCA4‐nNOS, this interaction was seen only for capacitated sperm where FRET efficiency was 24%, significantly (p < 0.05) higher than in uncapacitated sperm (6%). PMCA4 and the NOSs were identified as interacting partners in a quaternary complex that includes Caveolin1, which co‐immunoprecipitated with eNOS in a Ca²⁺‐dependent manner. In Pmca4^?/? sperm NOS activity was elevated twofold in capacitated/uncapacitated sperm (vs. wild‐type), accompanied by a twofold increase in peroxynitrite levels and significantly (p < 0.001) increased numbers of apoptotic germ cells. The data support a quaternary complex model in which PMCA4 co‐ordinates Ca²⁺ and NO signaling to maintain motility, with increased NO levels resulting in asthenospermia in Pmca4^?/? males. They suggest the involvement of PMCA4 mutations in human asthenospermia, with diagnostic relevance.