Organelle‐specific probe JC‐1 identifies membrane potential differences in the mitochondrial function of bovine sperm

The mitochondrial probe 5,5′,6,6′‐tetrachloro‐1,1′,3,3′‐tetraethylbenzimidazolyl‐carbocyanine iodide (JC‐1) not only identifies mitochondria exhibiting low membrane potentials by the emission of green fluorescence (range, 510–520 nm) but also differentiates these from mitochondria exhibiting relatively high membrane potentials. This discrimination occurs because JC‐1 forms aggregates at high membrane potentials. These J‐aggregates emit a bright red‐orange fluorescence at 590 nm. In this study, JC‐1 was combined with the classical dead cell stain, propidium iodide (PI), to identify a spectrum of functional sperm along with degenerate sperm. Flow cytometric analysis of bull sperm showed that the aggregate:monomer ratio differed among bulls before cryopreservation (P < 0.001) but not afterwards (P > 0.05). The effects of stain equilibration time, sperm concentration, and live:dead ratios were examined. The addition of SYBR‐14 to the JC‐1 and PI combination enhanced the distinction between the red PI‐stained and red‐orange JC‐1–stained populations. This discrimination between J‐aggregates and the PI‐stained sperm was affected by sperm concentration. These studies show that JC‐1 can be useful in monitoring mitochondrial function in bovine sperm. Mol. Reprod. Dev. 53:222–229, 1999. © 1999 Wiley‐Liss, Inc.     

Synthesis of a New Series of 1H‐Imidazol‐1‐yl Substituted 8‐Phenylxanthines as Adenosine Receptor Ligands

A new series of 1H‐imidazol‐1‐yl substituted 8‐phenylxanthine analogs has been synthesized to study the effects of the imidazole group on the binding affinity of compounds for adenosine receptors. Competition binding studies of these compounds were carried out in vitro with human cloned receptors using [³H]DPCPX and [³H]ZM 241385 as radioligands at A₁ and A_2A adenosine receptors, respectively. The effect of the substitution pattern of the (imidazolyl)alkoxy group on various positions of the phenyl ring at C(8) was also studied. The xanthine derivatives displayed varying degrees of affinity and selectivity towards A₁ and A_2A receptor subtypes despite a common but variedly substituted Ar C(8).     

Morphology‐function relationships and repeatability in the sperm of Passer sparrows

Sperm performance is likely to be an important determinant of male reproductive success, especially when females copulate with multiple males. Understanding sperm performance is therefore crucial to fully understand the evolution of male reproductive strategies. In this study, we examined the repeatability of sperm morphology and motility measures over three breeding seasons, and we studied relationships between sperm morphology and function. We conducted this study in wild‐derived captive house sparrows (Passer domesticus) and Spanish sparrows (P. hispaniolensis). Results for the two species were similar. As predicted from results in other passerine species, total sperm length was highly repeatable across ejaculates, and repeatability for the length of other components was moderate. The repeatability of sperm swimming speed across ejaculates was lower, but statistically significant, suggesting that sperm velocity may be a relatively dynamic trait. Surprisingly, swimming speed did not correlate with the relative length of the midpiece, and it correlated negatively with the relative length of the flagellum and with total sperm length. This pattern is the opposite of what theory predicts and differs from what has been found in house sparrows before. Also contrary to previous work, we found no evidence that total sperm length correlates with sperm longevity. These results therefore highlight the need for a better understanding of relationships between sperm morphology and function in passerine birds. J. Morphol. 276:370–377, 2015. © 2014 Wiley Periodicals, Inc.     

1‐Aminobenzocyclobutene‐1‐phosphonic Acid and Related Compounds as Inhibitors of Phenylalanine Ammonia‐Lyase

Five new geminal aminocycloalkanephosphonic acids ( 4 – 8 ) containing both an aromatic ring and a cycloalkane ring were synthesized and evaluated as potential inhibitors of buckwheat phenylalanine ammonia‐lyase (PAL). Within the set of compounds which are related to 2‐aminoindane‐2‐phosphonic acid (AIP, 3 ), a known powerful inhibitor of PAL, racemic 1‐aminobenzocyclobutene‐1‐phosphonic acid ( 4 ), was six times weaker than AIP as an in vitro inhibitor of buckwheat PAL, but six times stronger than AIP as an in vivo inhibitor of phenylalanine‐derived anthocyanin synthesis in buckwheat.     

Synthesis of 8‐Substituted 2′‐Deoxyisoguanosines via Unprotected 8‐Brominated 2‐Amino‐2′‐deoxyadenosine

A variety of applications of 8‐alkynylated nucleosides has prompted the synthesis of new purine analogues. Bromination of unprotected 2‐amino‐2′‐deoxyadenosine with Br₂/AcOH/AcONa gives 2‐amino‐8‐bromo‐2′‐deoxyadenosine (87%). The brominated derivative is converted to 8‐alkynylated 2‐amino‐2′‐deoxyadenosines by palladium‐catalyzed Sonogashira cross‐coupling reaction via microwave assistance (81 – 95%). The resulting compounds are further transformed to 8‐alkynylated 2′‐deoxyisoguanosines (52 – 70%). The physical properties of new compounds are investigated.     

Spectrofluorimetric determination of aluminium using 2‐hydroxy‐1‐naphthylidene‐(8‐aminoquinoline)

A sensitive and selective spectrofluorimetric method has been developed for the rapid determination of aluminium. This method is based on the complex formation between aluminium and 2‐hydroxy‐1‐naphthylidene‐(8‐aminoquinoline) (HNAQ). The optimum conditions for the complex formation were a metal‐to‐ligand (M : L) stoichiometric ratio of 1:1, a pH of 5.5 and a 0.20 m acetate buffer. The fluorescence of the complex was monitored at an emission wavelength of 502 nm with excitation at 438 nm. Under these conditions, linear calibration curves were obtained in the ranges 0.05–1 and 1–5 ppm. The detection limit was 3.4 ppb for the former and 13.5 ppb for the latter. The maximum relative standard deviation of the method for an aluminium standard of 200 ppb was 1.5% (n = 5). This method was successfully applied for the determination of aluminium in drinking water, pharmaceutical antacid tablets and suspension samples. Copyright © 2010 John Wiley & Sons, Ltd.     

Bone marrow‐derived mesenchymal stem cells mitigate caspase‐3 and 8‐hydroxy proline induced via β‐adrenergic agonist in pulmonary injured rats

Estimating the ability of bone marrow‐derived mesenchymal stem cells (BM‐MSCs) to alleviate pulmonary injury induced via isoproterenol (ISP). ISP was injected in a dose of (100 mg/kg, subcutaneously twice at an interval of 24 h). One month post BM‐MSCs transplantation by intravenous injection, pulmonary oxidative stress was assessed, and Western blot analyses and histopathological investigations were conducted. Compared with the normal control group, BM‐MSCs transplantation significantly decreased the expression of pulmonary anti‐oxidative stress marker. Western blot analysis revealed that ISP significantly reduced the protein expression of the anti‐oxidative stress marker nuclear related factor‐2 (Nrf2). However, the apoptotic marker (caspase‐3) and collagen content marker (8‐hydroxyproline) were markedly elevated. These biochemical markers were confirmed by histopathological investigations. Finally, it was demonstrated that BM‐MSCs transplantation showed a superior effect in improving pulmonary function through alleviating oxidative stress, apoptosis, and collagen content.     

Automatic annular laser trapping: a system for high‐throughput sperm analysis and sorting

An automatic microscope system is designed to study the response of sperm motility to an annular laser trap. A continuous annular laser trap provides a parallel way to analyze and sort sperm based on their motility and to study the effects of laser radiation, optical force and external obstacles. In the described automatic microscope system, the phase contrast images of swimming sperm are digitized to the computer at video rates. The microscope stage is controlled in real‐time to relocate the sperm of interest to the annular trap with a normal or tangential entering angle. The sperm is continuously tracked and the swimming behavior is identified. Using this system, parallel sorting on human and gorilla sperm are achieved and threshold power levels separating the “fast” group and the “slow” group are compared for those two species. (© 2009 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Length rather than year‐round spawning,affects reproductive performance of RAS‐reared F‐generation pikeperch,Sander lucioperca (Linnaeus, 1758) – Insights from practice

The continuous production of large numbers of high quality gametes is essential for aquaculture, particularly in candidate species, such as pikeperch, Sander lucioperca (L.). The common practice of year‐round reproduction is under suspicion of inflicting adverse effects on the quality of the gametes through the disturbance of endogenous rhythms. We hypothesized that such perturbation does not affect RAS‐reared F‐generation broodstock. Reproductive performance (number of eggs) and gamete quality (fertilization and hatching rate) were assessed over the course of 3 years covering six independent, photothermal shifted spawning seasons in a commercial pikeperch hatchery (n = 31 egg batches of F‐generation fish in total). No substantial differences in fertilization or hatching rates could be detected between the individual spawning seasons. Fecundity varied, but there are indications for a size effect on female fecundity with intermediate sized females producing higher number of eggs (~65–70 cm). Low egg quality could be detected in batches of very large fish. In conclusion, size‐specific broodstock composition, but not year‐round reproduction of F‐generation pikeperch spawners affects the reproductive performance.     

Interleukin‐8 ‐251A/T gene polymorphism and lung cancer susceptibility: a meta‐analysis

Many studies have examined the association between the interleukin‐8 ‐251T/A ( rs4073 ) gene polymorphism and lung cancer risk in various populations, but the results have been inconsistent. In this meta‐analysis, PubMed was searched for case–control studies published through 01 December 2013. The data were extracted, and pooled odds ratios (OR) with 95% confidence intervals (CI) were calculated. We assessed six published studies on the association between the interleukin‐8 ‐251T/A polymorphism and lung cancer risk. The included studies yielded a total of 3265 lung cancer cases and 3607 controls. For the homozygous A/A and A allele carriers (T/A + A/A), the pooled ORs for all studies combining 3265 cases and 3607 controls were 1.03 (95% CI = 0.92–1.14; P = 0.235 for heterogeneity) and 1.07 (95% CI = 0.96–1.19; P = 0.245 for heterogeneity) when compared with the homozygous wild‐type genotype (T/T). When the analysis was stratified by ethnicity, significant risks were found among Asians for both the A allele carriers and the homozygous A/A individuals. However, no significant associations were found in non‐Asian populations using any of the genetic models. This meta‐analysis suggests that the interleukin‐8 ‐251A allele confer an increased risk for the development of lung cancer among Asians.     

Genitalia‐associated microbes in insects

In sexual reproduction different types of symbiotic relationships between insects and microbes have become established. For example, some bacteria have evolved almost exclusive vertical transmission and even define the compatibility of insect mating partners. Many strictly sexually transmitted diseases have also been described in insects. Apart from such rather specific relationships the role of opportunistic infections in the reproductive process has been widely neglected. Opportunistic microbes transmitted passively during mating might impose an energetic cost, as the immune system will need to be alert and will use resources to fight potential intruders. Through mating wounds and contaminated reproductive organs opportunistic microbes might be transferred to mating partners and even enter the body cavity. Females as the “receiving” sex are particularly likely to have evolved adaptations to avoid or reduce opportunistic infections. Males of several species show highly complex seminal fluids, which as well as containing components that influence a males’ fertilization success, also possess antimicrobial substances. The role of antimicrobials in the reproductive process is not well understood. Some evidence hints at the protection of sperm against microbes, indicating a role for natural selection in shaping the evolution of reproductive traits. By highlighting the potential importance of microbes in sexual selection and their role in reproduction in general I will make a case for studies in sexual selection, especially the ones investigating postcopulatory processes, that should incorporate environmental, as well as genotypic variation, in reproductive traits.     

MiR‐21‐5p/dual‐specificity phosphatase 8 signalling mediates the anti‐inflammatory effect of haem oxygenase‐1 in aged intracerebral haemorrhage rats

Intracerebral haemorrhage (ICH) is a severe neurological disorder caused by bleeding within the brain tissue. Inflammation has been implicated in ICH pathogenesis and is a potential therapeutic target for ICH. Haemin, an activator of haem oxygenase‐1 (HO‐1), rapidly increases HO‐1 protein expression and activity and has been shown to distinctly affect anti‐inflammatory functions after central nervous system (CNS) injury. However, less is known about the mechanisms that underlie the anti‐inflammatory effects of haemin in aged rats post‐ICH. Here, we performed microarray analysis to identify miRNAs that respond strongly to HO‐1 regulation in ICH rats and found that miR‐21‐5p induced the most significant change. Using Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment and Gene Ontology (GO) analysis, we focused on dual‐specificity phosphatase 8 (DUSP8) from the predicted miR‐21‐5p targets. Luciferase reporter assays confirmed that miR‐21‐5p bound directly to DUSP8. MiR‐21‐5p upregulation in vitro downregulated DUSP8 expression. Importantly, intracerebroventricularly injecting antagomir for miR‐21‐5p (A‐miR‐21‐5p), which was used to inhibit miR‐21‐5p in aged ICH rats, significantly reduced the neurological defects, repaired cognitive impairment, alleviated blood–brain barrier (BBB) permeability, inhibited neuronal apoptosis posthaemorrhage and accelerated haematoma absorption. In addition, serum miR‐21‐5p levels were notably elevated in patients relative to healthy individuals and were correlated with National Institutes of Health Stroke Scale (NIHSS) scores and clinical outcomes. In summary, A‐miR‐21‐5p increased HO‐1 expression in cerebral haematomas, thus eliciting the DUSP8‐modulated perifocal neuroprotective effect of haemin. MiR‐21‐5p with haemin therapy may be a potential therapy post‐ICH.     

Sexual rest and post‐meiotic sperm ageing in house mice

Fertilization by aged sperm can result in adverse fitness consequences for both males and females. Sperm storage during male sexual rest could provide an environment for post‐meiotic sperm senescence causing a deterioration in the quality of stored sperm, possibly impacting on both sperm performance (e.g. swimming ability) and DNA quality. Here, we compared the proportion of sperm with fragmented DNA, an indicator of structural damage of DNA within the sperm cell, among males that had been sexually rested for approximately 2 months, to that of males that had mated recently. We found no evidence of intra‐epididymal sperm DNA damage or any impairment in sperm performance, and consequently no evidence of post‐meiotic sperm senescence. Our results suggest that male house mice are likely to possess mechanisms that function to ensure that their sperm reserves remain stocked with ‘young’, viable sperm during periods of sexual inactivity. We also discuss the possibility that our experimental design leads to no difference in the age of sperm among males from the two mating treatments. Post‐meiotic sperm senescence is especially relevant under sperm competition. Thus, we sourced mice from populations that differed in their levels of post‐copulatory sexual selection, enabling us to gain insight into how selection for higher sperm production influences the rate of sperm ageing and levels of DNA fragmentation. We found that males from the population that produced the highest number of sperm also had the smallest proportion of DNA‐fragmented sperm and discuss this outcome in relation to selection acting upon males to ensure that they produce ejaculates with high‐quality sperm that are successful in achieving fertilizations under competitive conditions.     

Gender‐dependent regulation of G‐protein‐gated inwardly rectifying potassium current in dorsal raphe neurons in knock‐out mice devoid of the 5‐hydroxytryptamine transporter

Agonists at G‐protein‐coupled receptors in neurons of the dorsal raphe nucleus (DRN) of knock‐out mice devoid of the serotonin transporter (5‐HTT^?/?) exhibit lower efficacy to inhibit cellular discharge than in wild‐type counterparts. Using patch‐clamp whole‐cell recordings, we found that a G‐protein‐gated inwardly rectifying potassium (GIRK) current is involved in the inhibition of spike discharge induced by 5‐HT_1A agonists (5‐carboxamidotryptamine (5‐CT) and (±)‐2‐dipropylamino‐8‐hydroxy‐1,2,3,4‐tetrahydronaphthalene hydrobromide (8‐OH‐DPAT); 50 nM–30 μM) in both wild‐type and 5‐HTT^?/? female and male mice. These effects were mimicked by 5′‐guanylyl‐imido‐diphosphate (Gpp(NH)p; 400 μM) dialysis into cells with differences between genders. The 5‐HTT^?/? knock‐out mutation reduced the current density induced by Gpp(NH)p in females but not in males. These data suggest that the decreased response of 5‐HT_1A receptors to agonists in 5‐HTT^?/? mutants reflects notably alteration in the coupling between G‐proteins and GIRK channels in females but not in males. Accordingly, gender differences in central 5‐HT neurotransmission appear to depend—at least in part—on sex‐related variations in corresponding receptor‐G protein signaling mechanisms. © 2006 Wiley Periodicals, Inc. J Neurobiol, 2006