Modification of Experimental Protocols for a Space Shuttle Flight and Applications for the Analysis of Cytoskeletal Structures During Fertilization, Cell Division , and Development in Sea Urchin Embryos

To explore the role of microgravity on cytoskeletal organization and skeletal calcium deposition during fertilization, cell division, and early development, the sea urchin was chosen as a model developmental system. Methods were developed to employ light, immunofluorescence, and electron microscopy on cultures being prepared for flight on the Space Shuttle. For analysis of microfilaments, microtubules, centrosomes, and calcium-requiring events, our standard laboratory protocols had to be modified substantially for experimentation on the Space Shuttle. All manipulations were carried out in a closed culture chamber containing 35 ml artificial sea water as a culture fluid. Unfertilized eggs stored for 24 hours in these chambers were fertilized with sperm diluted in sea water and fixed with concentrated fixatives for final fixation in formaldehyde, taxol, EGTA, and MgCl2(exp -6)H2O for 1 cell to 16 cell stages to preserve cytoskeletal structures for simultaneous analysis with light, immunofluorescence, and electron microscopy, and 1.5 percent glutaraldehyde and 0.4 percent formaldehyde for blastula and plueus stages. The fixed samples wre maintained in chambers without degradation for up to two weeks after which the specimens were processed and analyzed with routine methods. Since complex manipulations are not possible in the closed chambers, the fertilization coat was removed from fixation using 0.5 percent freshly prepared sodium thioglycolate solution at pH 10.0 which provided reliable immunofluorescence staining for microtubules. Sperm/egg fusion, mitosis, cytokinesis, and calcium deposition during spicule formatin in early embryogenesis were found to be without artificial alterations when compared to cells fixed fresh and processed with conventional methods

Annual variation in the levels of transcripts of sex-specific genes in the mantle of the common mussel, Mytilus edulis

Mytilus species are used as sentinels for the assessment of environmental health but sex or stage in the reproduction cycle is rarely considered even though both parameters are likely to influence responses to pollution. We have validated the use of a qPCR assay for sex identification and related the levels of transcripts to the reproductive cycle. A temporal study of mantle of Mytilus edulis found transcripts of male-specific vitelline coat lysin (VCL) and female-specific vitelline envelope receptor for lysin (VERL) could identify sex over a complete year. The levels of VCL/VERL were proportional to the numbers of sperm/ova and are indicative of the stage of the reproductive cycle. Maximal levels of VCL and VERL were found in February 2009 declining to minima between July - August before increasing and re-attaining a peak in February 2010. Water temperature may influence these transitions since they coincide with minimal water temperature in February and maximal temperature in August. An identical pattern of variation was found for a cryptic female-specific transcript (H5) but a very different pattern was observed for oestrogen receptor 2 (ER2). ER2 varied in a sex-specific way with male > female for most of the cycle, with a female maxima in July and a male maxima in December. Using artificially spawned animals, the transcripts for VCL, VERL and H5 were shown to be present in gametes and thus their disappearance from mantle is indicative of spawning. VCL and VERL are present at equivalent levels in February and July-August but during gametogenesis (August to January) and spawning (March to June) VCL is present at lower relative amounts than VERL. This may indicate sex-specific control mechanisms for these processes and highlight a potential pressure point leading to reduced reproductive output if environmental factors cause asynchrony to gamete maturation or release

Postcopulatory sexual selection

The female reproductive tract is where competition between the sperm of different males takes place, aided and abetted by the female herself. Intense postcopulatory sexual selection fosters inter-sexual conflict and drives rapid evolutionary change to generate a startling diversity of morphological, behavioural and physiological adaptations. We identify three main issues that should be resolved to advance our understanding of postcopulatory sexual selection. We need to determine the genetic basis of different male fertility traits and female traits that mediate sperm selection; identify the genes or genomic regions that control these traits; and establish the coevolutionary trajectory of sexes

Radon and risk of extrapulmonary cancers: results of the German uranium miners' cohort study, 1960–2003

Data from the German miners' cohort study were analysed to investigate whether radon in ambient air causes cancers other than lung cancer. The cohort includes 58 987 men who were employed for at least 6 months from 1946 to 1989 at the former Wismut uranium mining company in Eastern Germany. A total of 20 684 deaths were observed in the follow-up period from 1960 to 2003. The death rates for 24 individual cancer sites were compared with the age and calendar year-specific national death rates. Internal Poisson regression was used to estimate the excess relative risk (ERR) per unit of cumulative exposure to radon in working level months (WLM). The number of deaths observed (O) for extrapulmonary cancers combined was close to that expected (E) from national rates (n=3340, O/E=1.02; 95% confidence interval (CI): 0.98–1.05). Statistically significant increases in mortality were recorded for cancers of the stomach (O/E=1.15; 95% CI: 1.06–1.25) and liver (O/E=1.26; 95% CI: 1.07–1.48), whereas significant decreases were found for cancers of the tongue, mouth, salivary gland and pharynx combined (O/E=0.80; 95% CI: 0.65–0.97) and those of the bladder (O/E=0.82; 95% CI: 0.70–0.95). A statistically significant relationship with cumulative radon exposure was observed for all extrapulmonary cancers (ERR/WLM=0.014%; 95% CI: 0.006–0.023%). Most sites showed positive exposure–response relationships, but these were insignificant or became insignificant after adjustment for potential confounders such as arsenic or dust exposure. The present data provide some evidence of increased risk of extrapulmonary cancers associated with radon, but chance and confounding cannot be ruled out

Residential Radon and Brain Tumour Incidence in a Danish Cohort

BACKGROUND: Increased brain tumour incidence over recent decades may reflect improved diagnostic methods and clinical practice, but remain unexplained. Although estimated doses are low a relationship between radon and brain tumours may exist. OBJECTIVE: To investigate the long-term effect of exposure to residential radon on the risk of primary brain tumour in a prospective Danish cohort. METHODS: During 1993-1997 we recruited 57,053 persons. We followed each cohort member for cancer occurrence from enrolment until 31 December 2009, identifying 121 primary brain tumour cases. We traced residential addresses from 1 January 1971 until 31 December 2009 and calculated radon concentrations at each address using information from central databases regarding geology and house construction. Cox proportional hazards models were used to estimate incidence rate-ratios (IRR) and 95% confidence intervals (CI) for the risk of primary brain tumours associated with residential radon exposure with adjustment for age, sex, occupation, fruit and vegetable consumption and traffic-related air pollution. Effect modification by air pollution was assessed. RESULTS: Median estimated radon was 40.5 Bq/m(3). The adjusted IRR for primary brain tumour associated with each 100 Bq/m(3) increment in average residential radon levels was 1.96 (95% CI: 1.07; 3.58) and this was exposure-dependently higher over the four radon exposure quartiles. This association was not modified by air pollution. CONCLUSIONS: We found significant associations and exposure-response patterns between long-term residential radon exposure radon in a general population and risk of primary brain tumours, adding new knowledge to this field. This finding could be chance and needs to be challenged in future studies

Coevolution of Interacting Fertilization Proteins

Reproductive proteins are among the fastest evolving in the proteome, often due to the consequences of positive selection, and their rapid evolution is frequently attributed to a coevolutionary process between interacting female and male proteins. Such a process could leave characteristic signatures at coevolving genes. One signature of coevolution, predicted by sexual selection theory, is an association of alleles between the two genes. Another predicted signature is a correlation of evolutionary rates during divergence due to compensatory evolution. We studied female–male coevolution in the abalone by resequencing sperm lysin and its interacting egg coat protein, VERL, in populations of two species. As predicted, we found intergenic linkage disequilibrium between lysin and VERL, despite our demonstration that they are not physically linked. This finding supports a central prediction of sexual selection using actual genotypes, that of an association between a male trait and its female preference locus. We also created a novel likelihood method to show that lysin and VERL have experienced correlated rates of evolution. These two signatures of coevolution can provide statistical rigor to hypotheses of coevolution and could be exploited for identifying coevolving proteins a priori. We also present polymorphism-based evidence for positive selection and implicate recent selective events at the specific structural regions of lysin and VERL responsible for their species-specific interaction. Finally, we observed deep subdivision between VERL alleles in one species, which matches a theoretical prediction of sexual conflict. Thus, abalone fertilization proteins illustrate how coevolution can lead to reproductive barriers and potentially drive speciation

Molecular Characterization of a Novel Intracellular ADP-Ribosyl Cyclase

Background. ADP-ribosyl cyclases are remarkable enzymes capable of catalyzing multiple reactions including the synthesis of the novel and potent intracellular calcium mobilizing messengers, cyclic ADP-ribose and NAADP. Not all ADP-ribosyl cyclases however have been characterized at the molecular level. Moreover, those that have are located predominately at the outer cell surface and thus away from their cytosolic substrates. Methodology/Principal Findings. Here we report the molecular cloning of a novel expanded family of ADP-ribosyl cyclases from the sea urchin, an extensively used model organism for the study of inositol trisphosphate-independent calcium mobilization. We provide evidence that one of the isoforms (SpARC1) is a soluble protein that is targeted exclusively to the endoplasmic reticulum lumen when heterologously expressed. Catalytic activity of the recombinant protein was readily demonstrable in crude cell homogenates, even under conditions where luminal continuity was maintained. Conclusions/Significance. Our data reveal a new intracellular location for ADP-ribosyl cyclases and suggest that production of calcium mobilizing messengers may be compartmentalized

The genome of the sea urchin Strongylocentrotus purpuratus

We report the sequence and analysis of the 814-megabase genome of the sea urchin Strongylocentrotus purpuratus, a model for developmental and systems biology. The sequencing strategy combined whole-genome shotgun and bacterial artificial chromosome (BAC) sequences. This use of BAC clones, aided by a pooling strategy, overcame difficulties associated with high heterozygosity of the genome. The genome encodes about 23,300 genes, including many previously thought to be vertebrate innovations or known only outside the deuterostomes. This echinoderm genome provides an evolutionary outgroup for the chordates and yields insights into the evolution of deuterostomes