Embryonic Membranes Research Articles

Regulation of maternal phospholipid composition and IP3-dependent embryonic membrane dynamics by a specific fatty acid metabolic event in C. elegans

Natural fatty acids (FAs) exhibit vast structural diversity, but the functional importance of FA variations and the mechanism by which they contribute to a healthy lipid composition in animals remain largely unexplored. A large family of acyl-CoA synthetases (ACSs) regulates FA metabolism by esterifying FA to coenyzme A. However, little is known about how particular FA-ACS combinations affect lipid composition and specific cellular functions. We analyzed how the activity of ACS-1 on branched chain FA C17ISO impacts maternal lipid content, signal transduction, and development in Caenorhabditis elegans embryos. We show that expression of ACS-1 in the somatic gonad guides the incorporation of C17ISO into certain phospholipids and thus regulates the phospholipid composition in the zygote. Disrupting this ACS-1 function causes striking defects in complex membrane dynamics, including exocytosis and cytokinesis, leading to early embryonic lethality. These defects are suppressed by hyperactive IP(3) signaling, suggesting that C17ISO and ACS-1 functions are necessary for optimal IP(3) signaling essential for early embryogenesis. This study shows a novel role of branched chain FAs whose functions in humans and animals are unknown and uncovers a novel intercellular regulatory pathway linking a specific FA-ACS interaction to specific developmental events.

Development of the spinal cord and peripheral nervous system in platypus (Ornithorhynchus anatinus) and short-beaked echidna (Tachyglossus aculeatus)

The modern monotremes (platypus and echidnas) are characterized by development of their young in a leathery egg that is laid into a nest or abdominal pouch. At hatching, the young are externally immature, with forelimbs capable of digitopalmar prehension, but hindlimbs little advanced beyond limb buds. The embryological collections at the Museum für Naturkunde in Berlin were used to examine the development of the spinal cord and early peripheral nervous system in developing monotremes and to correlate this with known behavioural development. Ventral root outgrowth to the bases of both the fore- and hindlimbs occurs at 6.0 mm crown–rump length (CRL), but invasion of both limbs does not happen until about 8.0–8.5 mm CRL. Differentiation of the ventral horn precedes the dorsal horn during incubation and separate medial and lateral motor columns can be distinguished before hatching. Rexed's laminae begin to appear in the dorsal horn in the first week after hatching, and gracile and cuneate fasciculi emerge during the first two post-hatching months. Qualitative and quantitative comparisons of the structure of the cervicothoracic junction spinal cord in the two monotremes with that in a diprotodont marsupial (the brush-tailed possum, Trichosurus vulpecula) of similar size at birth, did not reveal any significant structural differences between the monotremes and the marsupial. The precocious development of motor systems in the monotreme spinal cord is consistent with the behavioural requirements of the peri-hatching period, that is, rupture of embryonic membranes and egg, and digitopalmar prehension to grasp maternal hair or nest material.

Effects of DL-3-n-Butylphthalide on Vascular Dementia and Angiogenesis

3-n-Butylphthalide (NBP) is a compound extracted from Chinese celery and is used as an anti-hypertensive herbal medicine for treating stroke patients. The aim of this study is to demonstrate the effects and mechanisms of this compound through in vitro and in vivo experiments. Culture experiments were performed by adding hydrogen peroxide (H(2)O(2)) to SH-SY5Y cells. From the MTT assay result, enhanced cell survival was observed with DL-NBP treatment, regardless of whether they are added before, simultaneously with or after the addition of H(2)O(2). For the in vivo experiment, Spontaneously Hypertensive rats and Wistar Kyoto control rats with chronic cerebral ischemia, which were induced by bilateral transection of the common carotid arteries, were given DL-NBP. Their performances in the place navigation test and spatial probe test in the Morris Water Maze have significantly improved compared with the DL-NBP untreated animals, indicating an improvement in spatial learning and memory in the ischemic-animals. In addition, in the chick embryonic chorioallantoic membrane assay, angiogenesis was more vigorous under the effects of DL-NBP, together with increased expression of growth factors, VEGF, VEGF-receptor and bFGF. All these suggested that one of the mechanisms of DL-NBP might be ameliorating vascular dementia and promoting angiogenesis.

Characterization of yolk sac proteins of Bos indicus cattle embryos

The yolk sac is an embryonic membrane that is essential for the embryo's initial survival in many mammals. It also plays an important role in the production of proteins necessary for development. We studied proteins of the yolk sac in bovine embryos at up to 40 days of gestation. We examined the yolk sac of 17 bovine embryos at different gestational periods, measuring α-fetoprotein, α-1-antitrypsin, and transferrin. This experiment was carried out by Western blot technique, associated with electrophoresis on a 6% sodium dodecyl sulfate polyacrylamide gel. Mouse monoclonal antibody anti-human-α-fetoprotein, mouse antibody anti-human-transferrin and rabbit polyclonal anti-human-α-1-antitrypsin were used as primary antibodies, and conjugated peroxidase as a secondary antibody. We detected the three proteins in some of the yolk sac samples; however, the bands in some specimens (samples) were weak, maybe a result of poor antigen-antibody reaction, since the antibodies used in this study were not specific to bovine proteins. The fact that weak bands appeared might be due to a weak cross-reaction.

Functional analysis of a fatty acid binding protein produced by Aphidius ervi teratocytes

Aphidius ervi (Hymenoptera, Braconidae) is an endophagous parasitoid of various aphid species, including Acyrthosiphon pisum (Homoptera, Aphididae), the model host used in the present study. Parasitized hosts show a marked increase of their nutritional suitability for the developing parasitoid larvae. This alteration of the biochemical and metabolic profile is due to a castration process mediated by the combined action of the venom, injected at the oviposition, and of the teratocytes, cells deriving from the dissociation of the embryonic membrane. Teratocytes produce and release in the host haemocoel two parasitism-specific proteins, which are of crucial importance for the development of their sister larvae. One of the proteins is a fatty acid binding protein (Ae-FABP), which shows a high affinity for C14–C18 saturated fatty acids (FAs) and for oleic and arachidonic acids. To better define the possible nutritional role of this protein, we have studied its immunolocalization profile in vivo and the impact on FA uptake by the epidermal and midgut epithelia of A. ervi larvae.During the exponential growth of A. ervi larvae, Ae-FABP is distributed around discrete lipid particles, which are abundantly present in the haemocoel of parasitized host aphids and in the midgut lumen of parasitoid larvae. Moreover, a strong immunodetection signal is evident on the surface of the two larval epithelia involved in nutrient absorption: the parasitoid midgut epithelium and the external epidermal layer. These two epithelia can effectively absorb radiolabelled myristic acid, but the FA transport rates are not affected by the presence in the medium of Ae-FABP. The protein appears to act essentially as a vector in the host haemolymph, transferring FAs from the digestion sites of host lipids to the growing parasitoid larvae. These data indicate that the proteins produced by A. ervi teratocytes may play complementary roles in the nutritional exploitation of the host.

Gene cloning, expression, and localization of antigen 5 in the life cycle of Echinococcus granulosus

Antigen 5 (Ag5) has been identified as a dominant component of cyst fluid of Echinococcus granulosus and is considered as a member of serine proteases family, which in other helminth, plays an important role in the egg hatch and larva invasion. However, whether Ag5 is expressed and secreted in all life stages is unknown. In this study, according to the sequence in GenBank, we cloned and sequenced the open reading frame (ORF) of Ag5 gene from the protoscolices of E. granulosus isolated from the sheep in Qinhai Province of China, and found several substitutions and a base insert and deletion in a short region near the stop code, leading to a frameshift mutation which is conserved with the homologue of other cestode. The ORF is 1,455 bp in length, encoding 484 amino acids with a secretory signal peptide. Bioinformatics analysis predicted several phosphorylation and myristoylation sites and a N-glycosylation site and a species-specific linear B epitope in the protein. The ORF was cloned into the plasmid pET28a(+) vector and expressed in Escherichia coli . The recombinant protein was purified by affinity chromatography. Anti-rEgAg5 antiserum was prepared in rats and used to analyze the localization of Ag5 in protoscolex and adult worm by immunofluorescence technique. Results demonstrated that the Ag5 is strongly expressed in the tegument of protoscolex and the embryonic membrane of egg and surface of oncosphere; meanwhile, it is also weakly expressed in tegument of the adult. This study showed that Ag5 is expressed in all stages of life cycle, secreted from the surface of the worm and may be anchored in membrane by its myristoylation sites; these characteristics make it a candidate antigen for diagnosis and vaccine for both intermediate and definitive hosts.

Extensive Determination of Glycan Heterogeneity Reveals an Unusual Abundance of High Mannose Glycans in Enriched Plasma Membranes of Human Embryonic Stem Cells

Most cell membrane proteins are known or predicted to be glycosylated in eukaryotic organisms, where surface glycans are essential in many biological processes including cell development and differentiation. Nonetheless, the glycosylation on cell membranes remains not well characterized because of the lack of sensitive analytical methods. This study introduces a technique for the rapid profiling and quantitation of N- and O-glycans on cell membranes using membrane enrichment and nanoflow liquid chromatography/mass spectrometry of native structures. Using this new method, the glycome analysis of cell membranes isolated from human embryonic stem cells and somatic cell lines was performed. Human embryonic stem cells were found to have high levels of high mannose glycans, which contrasts with IMR-90 fibroblasts and a human normal breast cell line, where complex glycans are by far the most abundant and high mannose glycans are minor components. O-Glycosylation affects relatively minor components of cell surfaces. To verify the quantitation and localization of glycans on the human embryonic stem cell membranes, flow cytometry and immunocytochemistry were performed. Proteomics analyses were also performed and confirmed enrichment of plasma membrane proteins with some contamination from endoplasmic reticulum and other membranes. These findings suggest that high mannose glycans are the major component of cell surface glycosylation with even terminal glucoses. High mannose glycans are not commonly presented on the surfaces of mammalian cells or in serum yet may play important roles in stem cell biology. The results also mean that distinguishing stem cells from other mammalian cells may be facilitated by the major difference in the glycosylation of the cell membrane. The deep structural analysis enabled by this new method will enable future mechanistic studies on the biological significance of high mannose glycans on stem cell membranes and provide a general tool to examine cell surface glycosylation.

Brooding and embryonic development in the crustacean Paragnathia formica (Hesse, 1864) (Peracarida: Isopoda: Gnathiidae)

The crustacean family Gnathiidae Leach, 1814 (Peracarida: Isopoda) comprises 12 genera known mostly from marine environments. Juvenile gnathiid isopods are fish ectoparasites, feeding on blood and tissue fluids in order to complete their life cycles. Gnathiid juvenile development generally includes three moults, the last involving metamorphosis to non-feeding, adult stages. The blood meal ingested by juveniles provides resources for adult survival, reproduction and embryological development. Reproductive biology in the brackish water gnathiid, Paragnathia formica (Hesse, 1864), is unusual amongst crustaceans, since brooding females have paired internal uterine sacs, rather than an external brood pouch. Known embryological development for P. formica includes three post gastrulation stages. In the current study, brooding and embryological development in this gnathiid were reexamined using histological and fluorescence methods, and by scanning electron microscopy. Novel observations were made of the blastodisc and germ cell migration within developing eggs, release of Stage 2 embryos by rupture of embryonic membranes, the in utero moult of Stage 2 to Stage 3 embryos, and the asynchronous development of the brood within the paired uterine sacs. These findings highlight the remarkable nature of brooding in P. formica and expand the paucity of knowledge of embryological development in gnathiids in general.

Analysis of 4D DIC Microscopic Data to Determine Cell Contacts in Caenorhabditis elegans Embryos

The Caenorhabditis elegans embryo is particularly amenable to microscopy and embryological studies because of its short developmental time, transparent shell, and nonpigmented cells. Identification of cell contacts is important for understanding how cells within the embryo can be polarized by their neighbors. This protocol describes a technique for identifying cell membranes and potential contacts between different blastomeres in the embryo and for following those contacts through development. This protocol involves manual segmentation of the membrane of each blastomere from four-dimensional (4D) differential interference contrast (DIC) data sets. Although this technique is described for use with C. elegans embryos, it will work with any 4D DIC data set. The recent development of a pleckstrin homology (PH)-domain tagged::GFP expressed in C. elegans embryos simplifies this analysis; however, many organisms lack appropriate green fluorescent protein (GFP) transgenes. The use of the GFP transgene improves on laser-mediated techniques for labeling embryonic cell membranes with fluorescent dye. Such methods require that a laser be used to carefully permeabilize the eggshell for entry of the dye into the embryo. The technique described in this protocol requires only easily obtainable 4D DIC data sets.

Folic Acid Remodels Chromatin on Hes1 and Neurog2 Promoters during Caudal Neural Tube Development

The mechanism(s) behind folate rescue of neural tube closure are not well understood. In this study we show that maternal intake of folate prior to conception reverses the proliferation potential of neural crest stem cells in homozygous Splotch embryos (Sp(-/-)) via epigenetic mechanisms. It is also shown that the pattern of differentiation seen in these cells is similar to wild-type (WT). Cells from open caudal neural tubes of Sp(-/-) embryos exhibit increased H3K27 methylation and decreased expression of KDM6B possibly due to up-regulation of KDM6B targeting micro-RNAs such as miR-138, miR-148a, miR-185, and miR-339-5p. In our model, folate reversed these epigenetic marks in folate-rescued Sp(-/-) embryos. Using tissue from caudal neural tubes of murine embryos we also examined H3K27me2 and KDM6B association with Hes1 and Neurog2 promoters at embryonic day E10.5, the proliferative stage, and E12.5, when neural differentiation begins. In Sp(-/-) embryos compared with WT, levels of H3K27me2 associated with the Hes1 promoter were increased at E10.5, and levels associated with the Neurog2 promoter were increased at E12.5. KDM6B association with Hes1 and Neurog2 promoters was inversely related to H3K27me2 levels. These epigenetic changes were reversed in folate-rescued Sp(-/-) embryos. Thus, one of the mechanisms by which folate may rescue the Sp(-/-) phenotype is by increasing the expression of KDM6B, which in turn decreases H3K27 methylation marks on Hes1 and Neurog2 promoters thereby affecting gene transcription.

P15. Statistical analysis of heterogeneous motility of embryonic stem cells under a microfluidic flow

Cell membrane motility describes cellular dynamics such as adhesion, extension and migration, pertaining to intracellular molecular dynamics. Therefore, cell shape and motility have been identified as key features on cellular physicochemistry. We suggested the free peripheral membrane length ratio ( L fr ) as a new parameter and ANOVA-incorporated analysis strategy to characterize cell’s shape and motility. L fr means the ratio of free peripheral membrane length unattached by other cells ( L f ) to the cell’s peripheral length. With the help of statistical analysis of embryonic stem cell membrane motilities under a microfluidic flow, we demonstrated L fr has a positive correlation with cellular area ( A r ) and L f but negative correlation with cellular roundness ( R n ). Acknowledgement This work was supported by INHA UNIVERSITY Research Grant ( INHA-40875 ).

Pipe-Dependent Ventral Processing of Easter by Snake Is the Defining Step in Drosophila Embryo DV Axis Formation

The establishment of Drosophila embryonic dorsal-ventral (DV) polarity relies on serine proteolytic activity in the perivitelline space between the embryonic membrane and the eggshell. Gastrulation Defective cleaves and activates Snake, which processes and activates Easter, which cleaves Spätzle to form the activating ligand for the Toll receptor. Ventral restriction of ligand formation depends on the Pipe sulfotransferase, which is expressed in ventral cells of the follicular epithelium surrounding the developing oocyte. Pipe modifies components of the developing eggshell to produce a ventral cue embedded in the vitelline membrane. This ventral cue is believed to promote one or more of the proteolysis steps in the perivitelline space. By examining the processing of transgenic, tagged versions of the perivitelline proteins during DV patterning, we find that the proteolysis of Easter by Snake is the first Pipe-dependent step and therefore the key ventrally restricted event in the protease cascade. We also find that Snake and Easter associate together in a complex in both wild-type and pipe mutant-derived embryos. This observation suggests a mechanism in which the sulfated target of Pipe promotes a productive interaction between Snake and Easter, perhaps by facilitating conformational changes in a complex containing the two proteins.

Embryonated Eggs as an Alternative Infection Model To Investigate Aspergillus fumigatus Virulence

Infection models are essential tools for studying microbial pathogenesis. Murine models are considered the "gold standard" for studying in vivo infections caused by Aspergillus species, such as A. fumigatus. Recently developed molecular protocols allow rapid construction of high numbers of fungal deletion mutants, and alternative infection models based on cell culture or invertebrates are widely used for screening such mutants to reduce the number of rodents in animal experiments. To bridge the gap between invertebrate models and mice, we have developed an alternative, low-cost, and easy-to-use infection model for Aspergillus species based on embryonated eggs. The outcome of infections in the egg model is dose and age dependent and highly reproducible. We show that the age of the embryos affects the susceptibility to A. fumigatus and that increased resistance coincides with altered chemokine production after infection. The progress of disease in the model can be monitored by using egg survival and histology. Based on pathological analyses, we hypothesize that invasion of embryonic membranes and blood vessels leads to embryonic death. Defined deletion mutant strains previously shown to be fully virulent or partially or strongly attenuated in a mouse model of bronchopulmonary aspergillosis showed comparable degrees of attenuation in the egg model. Addition of nutrients restored the reduced virulence of a mutant lacking a biosynthetic gene, and variations of the infectious route can be used to further analyze the role of distinct genes in our model. Our results suggest that embryonated eggs can be a very useful alternative infection model to study A. fumigatus virulence and pathogenicity.

Terpestacin Inhibits Tumor Angiogenesis by Targeting UQCRB of Mitochondrial Complex III and Suppressing Hypoxia-induced Reactive Oxygen Species Production and Cellular Oxygen Sensing

Cellular oxygen sensing is required for hypoxia-inducible factor-1alpha stabilization, which is important for tumor cell survival, proliferation, and angiogenesis. Here we find that terpestacin, a small molecule previously identified in a screen of microbial extracts, binds to the 13.4-kDa subunit (UQCRB) of mitochondrial Complex III, resulting in inhibition of hypoxia-induced reactive oxygen species generation. Consequently, such inhibition blocks hypoxia-inducible factor activation and tumor angiogenesis in vivo, without inhibiting mitochondrial respiration. Overexpression of UQCRB or its suppression using RNA interference demonstrates that it plays a crucial role in the oxygen sensing mechanism that regulates responses to hypoxia. These findings provide a novel molecular basis of terpestacin targeting UQCRB of Complex III in selective suppression of tumor progression.

γ-Secretase Dependent Production of Intracellular Domains Is Reduced in Adult Compared to Embryonic Rat Brain Membranes

Backgroundγ-Secretase is an intramembrane aspartyl protease whose cleavage of the amyloid precursor protein (APP) generates the amyloid β-peptide (Aβ) and the APP intracellular domain. Aβ is widely believed to have a causative role in Alzheimer's disease pathogenesis, and therefore modulation of γ-secretase activity has become a therapeutic goal. Besides APP, more than 50 substrates of γ-secretase with different cellular functions during embryogenesis as well as adulthood have been revealed. Prior to γ-secretase cleavage, substrates are ectodomain shedded, producing membrane bound C-terminal fragments (CTFs).Principal FindingsHere, we investigated γ-secretase cleavage of five substrates; APP, Notch1, N-cadherin, ephrinB and p75 neurotrophin receptor (p75-NTR) in membranes isolated from embryonic, young or old adult rat brain by analyzing the release of the corresponding intracellular domains (ICDs) or Aβ40 by western blot analysis and ELISA respectively. The highest levels of all ICDs and Aβ were produced by embryonic membranes. In adult rat brain only cleavage of APP and Notch1 could be detected and the Aβ40 and ICD production from these substrates was similar in young and old adult rat brain. The CTF levels of Notch1, N-cadherin, ephrinB and p75-NTR were also clearly decreased in the adult brain compared to embryonic brain, whereas the APP CTF levels were only slightly decreased.ConclusionsIn summary our data suggests that γ-secretase dependent ICD production is down-regulated in the adult brain compared to embryonic brain. In addition, the present approach may be useful for evaluating the specificity of γ-secretase inhibitors.

Comparative in vivo antiangiogenic effects of calreticulin from Trypanosoma cruzi and Homo sapiens sapiens

Angiogenesis is a complex multi-step process of neovascularization arising from preexisting blood vessels whose generation is regulated by pro- and anti-angiogenic factors. Both Trypanosoma cruzi calreticulin (TcCRT) and its human counterpart (HuCRT) are antiangiogenic. This is the first report where the TcCRT and HuCRT anti-angiogenic properties are compared in vivo. In the chick embryonic chorioallantoid membrane assay (CAM) and at equimolar concentrations, TcCRT displayed significantly higher antiangiogenic activities than its human counterpart. LPS had marginal effects at the concentrations present in the recombinant protein preparations and the TcCRT antiangiogenic effects were largely inhibited by specific polyclonal antibodies, thus, reinforcing the fact that the observed TcCRT effects can be attributed to the parasite-derived molecule and not to the endotoxin. The antiangiogenic TcCRT effects correlate with its anti-tumor in vivo effects, as recently shown in our laboratory.

055. EVEN REPTILES DO IT, THE STRUCTURE AND FUNCTION OF PLACENTAE IN VIVIPAROUS LIZARDS

Live birth (viviparity) has evolved independently more than 100 times in squamate reptiles (lizards and snakes). Most viviparous lineages are characterised by a simple placenta with squamous epithelia on both maternal and embryonic sides, and the remnants of an eggshell persist for some, if not all, of pregnancy. The embryos are predominantly lecithotrophic, with maternal-embryonic exchanges being limited mostly to inorganic ions, water and respiratory gases. Nevertheless, there is differentiation of a chorioallantoic placenta and a yolk sac (omphalo) placenta in all species. Complex placentae have evolved in the Squamata in only four or five lineages, all in the lizard family Scincidae (skinks). In species with complex placentation, the uterus differentiates to allow different functions in association with each embryonic membrane type. Species with complex placentae are characterised by a hypertrophy of maternal and embryonic cells, elaboration of the maternal surface, a reduction in yolk with a concomitant increase in placentotrophy and, in some, regional differentiation of the choriallantois into a placentome and a paraplacentome. Both the placentome and omphaloplacenta are organs of embryonic nutrition, but they transport nutrients by different mechanisms; the paraplacentome is a specialised gas exchange organ. The most placentotrophic species are in the South American genus Mabuya, where the females produce micro-lecithal eggs and the placenta is more complex than in any mammal, with four specialised structures for nutrient exchange and one for gas exchange. The number of independent origins of viviparity and the range of placental complexities exhibited by skinks enables us to infer the evolutionary trajectories that have resulted in microlecithal eggs and an almost complete reliance of placentotrophy from oviparous ancestors.

Improved Membrane Proteomics Coverage of Human Embryonic Stem Cells by Peptide IPG-IEF

Protein biomarkers are fundamental tools for the characterization of stem cells and for tracking their differentiation and maturation down developmental lineages. Technology development allowing increased coverage of difficult cellular proteomes should allow for the discovery of new and novel membrane protein biomarkers for use by the stem cell research community. The amphipathic and highly hydrophobic nature and relative low abundance of many membrane proteins present significant analytical challenges. These difficulties are amplified when the source material (tissue or cells) is only available in limited quantities (e.g., embryonic stem cells). Recent advances in enrichment for purer membrane fractions, the enzymatic and chemical digestion of membrane proteins in the presence of solvents or chaotropes, and the use of "shotgun" proteomics methodologies have gradually resulted in increased membrane proteome coverage with numbers of predicted integral membrane proteins now in excess of 1000 being routinely reported. We have recently demonstrated the advantages of using peptide isoelectric focusing in the first dimension on immobilized pH gradients (peptide IPG-IEF) followed by reversed phase chromatography and tandem MS to increase membrane proteome coverage. This study looked at achieving a similar level of membrane proteome coverage using modifications to reported methodologies while restricting the number of characterized human embryonic stem cells to 10(7) cells. Two-thousand two-hundred and ninety-two (2292) nonredundant proteins were identified with two or more high accuracy peptide matches from 260 mug of a human embryonic stem cell membrane enriched fraction with a false discovery rate of 0.32%. Gene Ontology (GO) mapping predicted 1279 (44.9%) of this list to be membrane proteins of which 395 proteins were predicted to be derived from the plasma membrane compartment. The TMHMM algorithm predicted 904 integral membrane proteins with up to 16 transmembrane helices. Collectively, we assert that the substantial membrane proteome coverage achieved using these procedures will enable rapid advances in the identification and quantitation of novel membrane proteins as markers of differentiation status and/or genetic mutation from relatively low numbers of cultured embryonic stem cells.

Observations on the Nesting Ecology of the Mississippi Mud Turtle,Kinosternon Subrubrum HippocrepisGray

Abstract Surface nesting is common among tropical turtles, but is an uncommon and poorly documented event in temperate species. Here we report two separate instances of surface nesting in Kinosternon subrubrum (Eastern Mud Turtle), in a Louisiana swamp in November, 2001 and March, 2002. The clutches, each consisting of a single egg, were found on man-made earthen structures within a Dwarf Palmetto-hardwood floodplain. The adaptive significance of ovipositing eggs on the surface of compact clay soils is possibly related to the need for oxygen exchange between the eggshell and embryonic membranes as well as avoidance of high water-table conditions. The two nests were laid in winter months, unlike the typical May or June nesting of northern females.

Stage-specific changes in membrane microviscosity in Misgurnus fossilis embryos

Natural and probe fluorescence as well as membrane microviscosity was studied in eggs and embryos of Misgurnus fossilis by fluorescence microscopy. The lateral mobility of the probe (pyrene) increased in loach embryos from early to late blastula, which indicates a decrease in plasma membrane microviscosity. At the later stage of mid-gastrula, the microviscosity remained largely invariant. Considering that the embryo exposure to different temperatures changes the quantum yield of fluorescence and the degree of pyrene excimerization, one can gain information about both the temperature-induced structural changes and changes in membrane microviscosity in the embryos. Natural and probe fluorescence of embryonic membranes is proposed as at tool to study morphogenetic mechanisms.