Genetic Variation in Circadian Rhythm Genes CLOCK and ARNTL as Risk Factor for Male Infertility

Background

The circadian system has a major role in maintaining homeostasis and proper body functions including reproductive capacity. The aim of this study was to examine whether there is an association between genetic variability in the primary clock genes CLOCK and ARNTL and male infertility in humans.

Methodology/Principal Findings

We performed a case-control study, where we searched for an association between polymorphisms of CLOCK and ARNTL genes and male infertility in 961 Slovenian and Serbian Caucasian men. The study group consisted of 517 patients with idiopathic infertility and a control group of 444 fertile men. A statistically significant difference was found in genotype distribution between the two groups in the CLOCK gene: rs11932595 (p = 6·10⁻⁵, q = 4·10⁻⁴, OR equaled 1.9 with 95% CI 1.4–2.7), rs6811520 (p = 2·10⁻³, q = 8·10⁻³, OR = 1.7 with 95% CI 1.2–2.2) and rs6850524 (p = 0.01, q = 0.02, OR = 1.4 with 95% CI 1.1–1.9). Further analyses of haplotypes were consistent with genotyping results.

Conclusions/Significance

We provide evidence that genetic variability in the CLOCK gene might be associated with male infertility warranting further confirmation and mechanistic investigations.     

USE AND ABUSE OF NASOGASTRIC INTUBATION

The value of nasogastric intubation in the treatment of paralytic ileus and in some cases of mechanical obstruction, as well as in the preparation of obstructed patients for operation, cannot be denied. However, it is felt that intubation is oftentimes employed unnecessarily, and that the complications of this procedure are not fully appreciated. Fluid and electrolyte loss, sinusitis, parotitis, laryngeal obstruction, esophagitis, knotting and difficulty in withdrawing tubes and perforations of the gastrointestinal tract are complications that can occur when nasogastric intubation is employed.Two hundred consecutive operations on the gallbladder and bile ducts were reviewed, and the need for intubation in these cases was evaluated. It was needed in only 7.5 per cent of the cases in the series. In light of the hazards and the rather rare necessity for nasogastric intubation, “routine” use should be eschewed.     

Lead optimization of a pyridine-carboxamide series as DGAT-1 inhibitors

The structure–activity relationship studies of a novel series of carboxylic acid derivatives of pyridine-carboxamides as DGAT-1 inhibitors is described. The optimization of the initial lead compound 6 based on in vitro and in vivo activity led to the discovery of key compounds 10j and 17h.     

Purification and characterization of regenerating mouse L929 karyoplasts

Within 72–96 hr after preparation, about 10% of the karyoplasts made from mouse L929 cells regenerated to reform whole viable cells. As soon as 30 hr after preparation, however, nearly all of the remaining 90% of karyoplasts were dead. By separating living and dead karyoplasts at 30 hr, therefore, that fraction destined to complete regeneration was effectively purified. Complete separation was accomplished by sedimentation through Ficoll-paque (Pharmacia), a patented preparation originally developed for the separation of monocytes from whole blood. With the addition of this technique to the previously reported purification scheme for karyoplasts, various biochemical and morphological studies were attempted. Of particular importance are results indicating that karyoplasts that regenerate do not initially contain any more cytoplasm than the average karyoplast in a preparation—that is, about 10% of the cytoplasm within a whole cell. Electron microscopy of karyoplasts immediately after preparation indicated an unequal partitioning of cytoplasmic organelles at the time of enucleation. For example, karyoplasts initially contained about 11.4% of the mitochondrial volume of whole cells, but only 2.9% of the Golgi apparatus. The size of the karyoplasts and the volume occupied by a variety of organelles was followed throughout the process of regeneration. Although there was an approximately linear increase in the diameter of regenerating karyoplasts, there appeared not to be a simple concordant increase in the volume occupied by all cellular organelles. An extensive investigation was performed to determine whether or not karyoplasts contained centrioles. Immediately after enucleation, 15,000 random thin sections through karyoplasts, which represented about 100 complete bodies, were examined for the presence or absence of centrioles. No centrioles were observed. Examination of the cytoplasts revealed that they contained a sufficient number of centrioles to account for all of the centrioles that were present in the whole cells before enucleation. Centrioles were first detected in karyoplasts at 24 hr after preparation, about the same time that karyoplasts regained the ability to adhere to the surface of tissue culture dishes. At this time, however, the average karyoplast had less than one centriole. By 72 hr, the regenerated karyoplasts had approximately the same number of centrioles as whole cells.     

High-Resolution Melting Curve Analysis for Identification of Pasteurellaceae Species in Experimental Animal Facilities

Pasteurellaceae are among the most prevalent bacterial pathogens isolated from mice housed in experimental animal facilities. Reliable detection and differentiation of Pasteurellaceae are essential for high-quality health monitoring. In this study, we combined a real-time PCR assay amplifying a variable region in the 16S rRNA sequence with high-resolution melting curve analysis (HRM) to identify and differentiate among the commonly isolated species Pasteurella pneumotropica biotypes “Jawetz” and “Heyl”, Actinobacillus muris, and Haemophilus influenzaemurium. We used a set of six reference strains for assay development, with the melting profiles of these strains clearly distinguishable due to DNA sequence variations in the amplicon. For evaluation, we used real-time PCR/HRM to test 25 unknown Pasteurellaceae isolates obtained from an external diagnostic laboratory and found the results to be consistent with those of partial 16S rRNA sequencing. The real-time PCR/HRM method provides a sensitive, rapid, and closed-tube approach for Pasteurellaceae species identification for health monitoring of laboratory mice.     

Cenomanian ostracods of the Tarfaya upwelling region (Morocco) as palaeoenvironmental indicators

Changes in ostracod occurrence and assemblage composition from the Cenomanian of the Tarfaya Basin (Morocco) are investigated in order to evaluate their potential for palaeoenvironmental analyses of Cretaceous upwelling regions. Most frequent genera found at Tarfaya are Brachycythere, Veenia, Reticulocosta, Cytherelloidea, and Nigeroloxoconcha. Assemblages found at Tarfaya are characterised by low diversities. Successive appearance of assemblages dominated by these genera represents a depth gradient, corresponding to the results of the performed correspondence analysis. Ostracods are most abundant if associated either with moderate food supply and sufficient oxygenation or high food supply and high oxygenation. They disappear almost completely during permanent oxygen deficiency and are, contrary to benthic foraminifera, less frequent during periods of enhanced food supply. Species of the genus Brachycythere appear to be more sensitive to oxygen deficiency than other genera. Reticulocosta and Cytherelloidea avoid strong food pulses and are probably better adapted to oxygen depletion than other genera. Ostracods appear to be powerful environmental indicators for sea-level changes, oxygenation or food supply in Late Cretaceous upwelling settings, such as in the Cenomanian Tarfaya Basin.     

Regulation by repression of urease biosynthesis in Proteus rettgeri

Measuring the specific enzyme activity in cells of Proteus rettgeri it was shown that urease formation is controlled by repression through ammonia. Derepressed synthesis of the enzyme, as initiated by the absence of ammonia, required an external nitrogen source, which may not only be urea, but also nitrate, glutamate or nutrient broth. In contradiction to earlier reports the observations indicated that urea is not required for the synthesis of this enzyme, and that, therefore, urease is not an inducible enzyme in this microorganism.     

Fibrils Colocalize Caspase-3 with Procaspase-3 to Foster Maturation

Most proteases are expressed as inactive precursors, or zymogens, that become activated by limited proteolysis. We previously identified a small molecule, termed 1541, that dramatically promotes the maturation of the zymogen, procaspase-3, to its mature form, caspase-3. Surprisingly, compound 1541 self-assembles into nanofibrils, and localization of procaspase-3 to the fibrils promotes activation. Here, we interrogate the biochemical mechanism of procaspase-3 activation on 1541 fibrils in addition to proteogenic amyloid-β(1–40) fibrils. In contrast to previous reports, we find no evidence that procaspase-3 alone is capable of self-activation, consistent with its fate-determining role in executing apoptosis. In fact, mature caspase-3 is >10⁷-fold more active than procaspase-3, making this proenzyme a remarkably inactive zymogen. However, we also show that fibril-induced colocalization of trace amounts of caspase-3 or other initiator proteases with procaspase-3 dramatically stimulates maturation of the proenzyme in vitro. Thus, similar to known cellular signaling complexes, these synthetic or natural fibrils can serve as platforms to concentrate procaspase-3 for trans-activation by upstream proteases.     

Le sperme « inflammatoire »: ses relations avec la fertilité

Objectives

Sperm inflammation is caused by bacterial or viral infection of the male genitourinary tract; it is often clinically asymptomatic. There is a dilemma about the causal relationship between leukocytes as markers of inflammation and poor semen quality. We were interested in sperm changes at molecular level caused by inflammation.

Material and methods

This study was based on a literature review and personal data. In 200 male partners of infertile couples with normal semen analysis, the percentage of sperm with DNA denaturation and the level of reactive oxygen species (ROS) were determined by flow cytometric analysis, after acridine orange and dihydroethidium stainings, and correlated with seminal plasma elastase levels.

Results

In the literature, a positive relationship between inflammation and increased sperm apoptosis was found with increased necrosis and decreased mitochondrial membrane potential. We found a positive correlation between the percentage of sperm with denatured DNA and elastase levels. The percentage increased from 8.6% at elastase level 0–100 μg/l to 15.7% at elastase level 100–250 μg/l; this increase was not dependent on ROS production. The percentage of sperm with denatured DNA normalized at elastase levels above 600 μg/l.

Discussion and conclusion

Changes in sperm DNA or membranes do not necessarily affect classical semen characteristics or reduce fertility in males. They can, however, have a negative effect on capacitation and acrosomal reaction, resulting in failed fertilization or poor embryo development. Before treatment, we must take into account the location and the duration of the inflammation as well as the damage done to sperm.     

Mouse Wnt receptor gene Fzd5 is essential for yolk sac and placental angiogenesis

Wnts are secreted signaling molecules implicated in various developmental processes and frizzled proteins are the receptors for these Wnt ligands. To investigate the physiological roles of frizzled proteins, we isolated and characterized a novel mouse frizzled gene Fzd5. Fzd5 mRNA was expressed in the yolk sac, eye and lung bud at 9.5 days post coitum. Fzd5 specifically synergized with Wnt2, Wnt5a and Wnt10b in ectopic axis induction assays in Xenopus embryos. Using homologous recombination in embryonic stem cells, we have generated Fzd5 knockout mice. While the heterozygotes were viable, fertile and appeared normal, the homozygous embryos died in utero around 10.75 days post coitum, owing to defects in yolk sac angiogenesis. At 10.25 days post coitum, prior to any morphological changes, endothelial cell proliferation was markedly reduced in homozygous mutant yolk sacs, as measured by BrdU labeling. By 10.75 days post coitum, large vitelline vessels were poorly developed, and the capillary plexus was disorganized. At this stage, vasculogenesis in the placenta was also defective, although that in the embryo proper was normal. Because Wnt5a and Wnt10b co-localized with Fzd5 in the developing yolk sac, these two Wnts are likely physiological ligands for the Fzd5-dependent signaling for endothelial growth in the yolk sac.