bluuebunny/crossref_metadata_2025 huggingface dataset:
172. A MODEST INFLAMMATORY INSULT IN THE PRE-IMPLANTATION PERIOD ALTERS OVIDUCT CYTOKINE EXPRESSION AND PROGRAMS FETAL DEVELOPMENT
Authors: Chin P. Y. , Thompson J. G. , Robertson S. A.
Published: November 2010

The cytokine milieu surrounding the pre-implantation embryo contributes to programming optimal embryo development. Perturbations to the maternal environment such as infection and inflammation during the pre-implantation period can influence cytokine synthesis and may cause changes in embryo development that compromise pregnancy outcome. We aimed to investigate the effect of an inflammatory insult with LPS during the pre-implantation period on later fetal development and the role of oviduct cytokine expression in mediating this response. LPS (at various doses of 0.5–62.5 μg) was administered i.p. to C57Bl/6 mice on both day 3 and day 4 post coitum (pc). The effects of treatment on day 4 blastocysts and day 18 fetal development were analysed. Blastocysts from LPS-treated mothers showed reduced viability and smaller total cell number, but were only significant when doses of >12.5 μg LPS were administered. This was not a direct effect of LPS, as no effect of LPS on embryo development in vitro was seen, even at very high LPS concentrations (25 μg/mL). At day 18 pc, pregnancy rates and the number of viable fetuses, as well as fetal and placenta weights were all reduced after low dose LPS treatment (0.5 μg) compared to control PBS-treated females. qPCR analysis of day 4 oviduct tissue revealed that administration of 12.5ug of LPS resulted in increased mRNA expression of cytokines including IL6, TNFA, IL1B, IFNG, IL10 and LIF. Our findings show that a modest pro-inflammatory insult with LPS in the pre-implantation period programs the embryo for later adverse effects on fetal and placental development. The effects of LPS appear to be mediated indirectly via the maternal tract, and altered oviduct cytokine expression which impairs pre-implantation embryo development is implicated as the underlying mechanism. This model will now be utilised to investigate the potential role of specific inflammatory cytokines TNFA and IFNG in mediating this response.
173. TRP53-DEPENDENT EXPRESSION OF GENES REGULATING EMBRYO VIABILITY
Authors: Ganeshan L. , O'Neill C.
Published: November 2010

Embryos from inbred strains are sensitive to the stresses of culture in vitro compared to hybrid strains. This difference provides a powerful model for identifying genetic regulators of embryo viability in vitro. We showed that culture stress can cause the activation of the TRP53 stress response pathway, culminating in poor embryo viability.1 Directed genomic analysis of potential target genes was undertaken by comparing blastocysts cultured from zygotes of C57BL6.Trp53+/+, C57BL6.Trp53–/– and B6CBF2 genetic backgrounds. This comparison allowed genes with altered expression due to culture in susceptible strains to be identified and determined whether their changed expression was primarily due to the actions of TRP53. Culture had no effect on the expression of a number of house-keeping genes tested. Of known TRP53 target genes tested, only Bax showed a negative association with embryo viability. Interestingly, this was only partially reduced in C57BL6.Trp53–/–, indicating that other factors account for much of its up-regulation. Pluripotency genes showed a complex picture. Nanog was strongly negatively correlated with viability yet NANOG protein was strongly positively associated, indicating complex regulatory control; Oct4 showed no association yet OCT4 was strongly positively associated; Utf1 was the only pluripotency gene tested that showed Trp53 dependent down-regulation in cultured C57BL6.Trp53+/+. Markers of differentiation, Cdx2 (trophectodermal), Gata6 (endodermal) and Brachyury (mesodermal) did not differ between strains. The results identify Trp53-dependent alterations in gene expression in regulators of cell survival (Bax) and pluripotency (Utf1) that may account for the reduction in the capacity of susceptible blastocysts to form a proliferating inner cell mass. The study also identifies for the first time the possibility of complex post-transcriptional regulation of other pluripotency genes (Nanog and Oct4) that may shed future light on the regulation of pluripotency.
(1) Li A, et al. Biol. Reprod. 2007; 76: 362–367.
176. INHIBITION OF PORCINE OOCYTE NUCLEAR MATURATION IN VITRO USING A PHOSPHODIESTERASE INHIBITOR AND AN ADENYLATE CYCLASE ACTIVATOR
Authors: Bertoldo M. , Sellens T. , Grupen C. G.
Published: November 2010

Asynchronous nuclear and cytoplasmic maturation is thought to contribute to poor embryo production in vitro. Nuclear arrest is mediated by cAMP and can be maintained within the oocyte using non-specific phosphodiesterase inhibitors (3-isobutyl-1-methylxanthine ; IBMX) and the adenylate cyclase activator forskolin (FSK) (1). The aim of this study was to investigate the effect of IBMX and FSK supplementation on porcine oocyte nuclear maturation during COC recovery and IVM using a defined culture system. In all experiments, cAMP modulators were added to Hepes-buffered media held in collection tubes. COCs recovered from 3–5 mm diameter follicles of prepubertal ovaries were cultured in basic maturation media in the absence of FSH. Nuclear maturation was assessed using orcein dye. In Experiment 1, IVM media was supplemented with 0, 50 or 500 µM IBMX. In Experiment 2, IVM media was supplemented with 0, 5, 10, 50 and 100µM FSK. In Experiment 3, IVM medium was supplemented with combinations of IBMX and FSK to give the treatments; control, 50IBMX/50FSK, 50IBMX/100FSK, 500IBMX/50FSK and 500IBMX/100FSK. Nuclear maturation was assessed at 0, 2, 4 and 18 h after the onset of IVM. At 18 h of culture, there were no differences in the proportion of oocytes supplemented with 0, 50 or 500 µM IBMX reaching MII. Incubation with 10, 50 or 100 µM FSK resulted in 8-16% of oocytes at MII at 18 h compared to the other groups (25–29%; P < 0.001). The combinations of IBMX and FSK resulted in greater proportions (86–98%) of oocytes remaining at the GV stage at 18 h compared to the control (16%; P < 0.001). There were no differences in the proportion of oocytes remaining at the GV stage at the earlier time points (P > 0.05). The results demonstrate that these cAMP modulators, in combination, are highly effective in maintaining porcine oocyte meiotic arrest in vitro for an extended period.
(1) Albuz FK et al., Proceedings of the 25th Annual Meeting of ESHRE, Amsterdam, The Netherlands, 2009.
179. PURIFICATION AND CHARACTERISATION OF MOUSE TESTICULAR MACROPHAGES: GENE EXPRESSION RESPONSE TO LIPOPOLYSACCHARIDE ACTIVATION INDICATES AN IMMUNOSUPPRESSIVE PHENOTYPE
Authors: Winnall W. R. , Gould J. , Muir J. A. , Hertzog P. , Hedger M. P.
Published: November 2010

Studies on rat testicular macrophages (TMs) have indicated that these cells play an important role in testis function by supporting the immunosuppressive environment that protects developing germ cells and by responding to pathogens. By comparison, mouse TMs are essentially uncharacterised due to difficulties in isolating sufficient cells for study. We have established a technique for isolating 95% pure TMs from adult mice by differential adherence. Mouse TMs were cultured for 3h with saline, 10 or 100 ng/mL lipopolysaccharide (LPS) and compared with resident peritoneal macrophages (PMs) and bone marrow-derived macrophages (BMMs). Expression of inflammatory regulators was determined using real-time Q-PCR and AgilentTM microarray analysis. Microarray analysis indicated that each macrophage type displayed very distinct gene expression profiles. There were 526 genes uniquely expressed in TMs at basal levels compared with the other macrophages and 268 genes uniquely expressed by TMs after LPS treatment. Q-PCR determined that LPS induced expression of the anti-inflammatory cytokine interleukin (IL)-10 in each of the macrophage types, with BMMs the strongest responders. LPS stimulated IL-10 mRNA approximately 100-fold in TMs, but only 20-fold in PMs. The anti-inflammatory transforming growth factor-β1 was not significantly induced at this time-point in any macrophage type. In terms of pro-inflammatory mediators, the TM response to LPS was always lower compared to the BMMs. Compared to PMs, the responses of TMs were similar for the hallmark pro-inflammatory cytokine tumour necrosis factor- a, but 40% less for IL-1β. TMs were also deficient in production of IL-6 and cyclooxygenase-2 and IL-12. TMs were therefore relatively strong responders to LPS in terms of IL-10, but weak responders in terms of pro-inflammatory mediators, indicating an immunosuppressive phenotype. The isolation and gene measurement methods established in this study will allow us to use knockout and transgenic mouse models to determine the role for TMs in testicular inflammation/fibrosis models.
182. ROLE OF CRISP4 IN ION CHANNEL REGULATION AND MALE REPRODUCTION
Authors: Koppers A. J. , Gibbs G. M. , Reddy T. , McIntyre P. , O'Bryan M. K.
Published: November 2010

CRISPs are a group of 3 proteins found in mammals (4 in the mouse) which show a strong expression bias in the male reproductive organs. Whilst the function of most CRISPs are yet to be elucidated, mouse CRISP2 is a known regulator of the ion channel, ryanodine receptor. CRISP4 is most abundantly produced by the principal cells of the epididymis and are secreted into the lumen, where they adhere to sperm during epididymal transit. In this study we examined the role of CRISP4 ion channel regulation in mouse spermatozoa through cell assays and mouse models. The identification of the Transient Receptor Potential (TRP) ion channel, TRPM8 to interact with CRISP4 was confirmed using stably-transfected CHO cell lines. Production of CRISP4 KO mouse model, whilst males are fertile, they exhibit a subtle infertility phenotype characterized by a reduced ability to capacitate and undergo the acrosome reaction. This data is further emphasized by the ability of TRPM8 agonists, icillin and menthol, to inhibit the acrosome reaction in mouse spermatozoa that could be prevented by the addition of recombinant CRISP4 crisp domain. Corresponding to these data, CRISP4 is localized to the tail and head of mouse spermatozoa. In conclusion, we have demonstrated that CRISP4 is a regulator of TRPM8 in mouse spermatozoa, and due to its expression and localization pattern is an important protein in sperm epididymal maturation.
302. FIBROID ASSOCIATED HEAVY MENSTRUAL BLEEDING: CORRELATION OF CLINICAL SYMPTOMS, DOPPLER ULTRASOUND ASSESSMENT OF VASCULATURE AND TISSUE GENE EXPRESSION PROFILES
Authors: Tsiligiannis S. , Zaitseva M. , Coombs P. , Shekleton P. , Vollenhoven B. , Hickey M. , Rogers P.
Published: November 2010

Understanding of the mechanisms that cause fibroid associated heavy menstrual bleeding (HMB) is limited. Despite many fibroids having a highly vascular peri-fibroid myometrial (PFM) zone, angiogenic gene expression in this area has never been investigated. The aim of this study was to correlate clinical symptoms, ultrasound appearances and tissue gene expression profiles in women scheduled for hysterectomy due to symptomatic fibroids. We hypothesised that fibroid heterogeneity, colour flow and spectral Doppler resistive indices would correlate with differences in gene expression profiles. It was thought and that increased peri-fibroid gene expression of key angiogenic genes would correlate with increased peri-fibroid vascularity. N = 6 patients underwent B-mode, colour and spectral Doppler ultrasound assessment. Following hysterectomy tissue samples collected from three areas – fibroid, PFM and distant myometrium (DM) were analysed using quantitative RT-PCR and a customised angiogenesis PCR array. A higher mean peak systolic velocity (PSV) in the PFM region when compared to mean PSV within the fibroid (P < 0.001) was seen. Differences in angiogenic gene expression were consistent with the heterogeneity of the clinical data collected. One fibroid sample showed dissimilar gene expression to all other fibroids; at ultrasound and sample collection significant degenerative features were observed. Fibroid heterogeneity within a single uterus was also demonstrated, with two fibroids from the one uterus having significantly dissimilar gene profiles and ultrasound appearances. No differences in gene expression were found between PFM and DM. Despite this, gene interaction maps showed different interaction of genes between fibroid and PFM regions compared to genes between the fibroid and the DM. These are the first molecular data demonstrating that the PFM region may be functionally distinct from distant myometrium.
319. RELAXIN REGULATES AQUAPORIN EXPRESSION IN THE CERVIX OF LATE PREGNANT MICE
Authors: Soh Y. , Parry L. J.
Published: November 2010

Aquaporins (AQPs) have been implicated in the regulation of fluid balance in the cervix during pregnancy to promote hydration, a characteristic of cervical ripening in late gestation.There are four AQPs in the cervix; AQP3, 4, 5 and 8. Cervical fluid balance involves AQP5 and 8 in early pregnancy and AQP3 and 4 in late pregnancy [1]. However, the factors involved in the regulation of cervical AQPs are unknown. We propose that the ovarian peptide hormone relaxin regulates cervical AQPs because high circulating levels of relaxin correspond to changes in AQPs and it is involved in cervical ripening. To test this hypothesis, expression of aqp3, 5 and 8 was compared in the cervices of relaxin wildtype (Rln+/+) and relaxin knockout (Rln–/–) at various stages of pregnancy (day 14.5, 16.5 and 18.5 pregnancy) by quantitative PCR. In the Rln–/– mice, aqp3 expression was significantly lower on day 18.5 gestation compared to Rln+/+ littermates. Aqp5 and 8 expression did not change significantly between genotypes. To determine whether relaxin could restore the Rln+/+ phenotype, Rln–/– mice were implanted with Alzet osmotic minipumps on day 12.5 pregnancy to infuse either recombinant H2 human relaxin (200 μg/mL; Corthera Inc) or 0.9% saline as a control. Cervices were collected after 4 or 6 days of infusion for gene expression analysis. Relaxin infusion in pregnant Rln–/– mice increased cervical aqp3, and also decreased aqp5 expression compared with saline-controls in the 6-day infusion group. Additionally, relaxin treatment caused a 6-fold increase in cervix wet weight, dispersal of collagen fibres and a decrease in relaxin receptor (Rxfp1) expression. These data suggest that relaxin promotes cervical hydration through an action on AQPs.
(1) Anderson et al, 2006. Endocrinology 147(1): 130–140.
321. HEDGEHOG SIGNALLING COMPONENTS IN DEVELOPING RAT TESTIS
Authors: Sahin Z. , Szczepny A. , Ustunel I. , Loveland K.
Published: November 2010

Hedgehog (Hh) signalling regulates normal development of many tissues and is upregulated in some cancers. Mice which lack the testicular desert hedgehog (Dhh) ligand exhibit disrupted embryonic gonad formation and male infertility in adulthood. However, the roles and sites of Hedgehog (Hh) signalling activity in the developing rat testis are unknown. Transcripts encoding Hh pathway components in embryonic and juvenile rat testes were localised by in situ hybridization with DIG-labelled cRNA probes. On embryonic day (E) 17.5, Sertoli cells contained transcripts encoding Dhh and both gonocytes and Sertoli cells contained the Ptc2 and Smo receptor transcripts. The cytoplasmic regulators (fused [Fu], suppressor of fused [SuFu]) and the three Gli transcriptional mediators were also detected in gonocytes. On E21.5 and postnatal day 4, all transcripts were present in Sertoli cells, but not gonocytes. To test the function of Hh signalling at the onset of rodent spermatogenesis, the newborn (day 1) mouse testis was cultured in hanging drop cultures. Addition of the selective Hh signalling inhibitor, cyclopamine, yielded a significantly lower level of the Hh target, Gli1 mRNA, as measured by real time PCR compared to vehicle controls. This confirms Hh pathway activity and inhibition in this system. Meiotic markers, SYCP3 and Stra8, were consistently upregulated (n = 6) following cyclopamine addition, however the fold-change varied between experiments. Overall, the cellular expression data indicate that Hh signals are active in both embryonic and juvenile rodent testes. The finding that Hh genes analysed in this study are expressed in both Sertoli and germ cells shows that both cell types are potential Hh targets, depending on the developmental stage of testis. The upregulation of candidates for Dhh target genes in the juvenile mouse testis in vitro suggests that Hedgehog signaling downregulates the expression of meiotic genes in gonocytes via paracrine mechanisms.
323. MEASURING CHANGES IN TESTICULAR CELL POPULATIONS USING FLOW CYTOMETRY
Authors: Morin G. , Loveland K.
Published: November 2010

Spermatogenesis is first established during the first two weeks postpartum by the transition of undifferentiated (Kit–) into differentiated spermatogonia (Kit+). We recently showed that changes in the level of the growth factor activin alters the proportion of spermatogonial subtypes (1). However, detection of this transition by histology is unreliable. This project objective is to develop methods to efficiently measure changes in somatic and germ cell populations at the onset of spermatogenesis. Using surface (Kit receptor) and internal (mouse vasa homologue {MVH}) markers, we evaluated the proportion of differentiating germ cells in wild type Swiss mice by flow cytometry. Whole testes of mice at 7, 10, 14 days postpartum (dpp) were enzymatically dissociated and single cell suspensions were labelled with anti-Kit receptor antibody to detect Leydig cells and differentiating spermatogonia. These suspensions were then fixed and permeabilized in order to detect MVH, allowing spermatogonia to be distinguished from Leydig cells. Our present results show that combined Kit and MVH labelling is effective for evaluating the proportion of undifferentiating and differentiating germ cells. Our preliminary observations identified an elevation in the proportion of Kit+MVH+ cells between 7 and 10 days from 0.37 to 18%, indicating that spermatogonial differentiation advances dramatically between these ages. At day 14, the proportion of Kit+MVH+ cells decreased to 11%, as the emerging spermatocytes dilute spermatogonial numbers. These findings agree with published data (2). We have also used surface markers to discriminate between spermatogonia and Leydig cells without fixation or permeabilization, allowing us to isolate these cells for molecular and proteomics analysis. This will facilitate comparative profiling of germ cells with different levels of Kit, including those in mice with altered levels of growth factors (2) and hormones that govern the progression of testis development.
(1) Mithraprabhu, 2010 Biology of Reproduction.(2) Bellve, 1977 Journal of Cell Biology.
324. SLX4, A KEY REGULATOR OF MEIOSIS AND DNA REPAIR IN THE MALE GERMLINE
Authors: Bernstein I. R. , McLaughlin E. A. , O'Bryan M. K.
Published: November 2010

Genome integrity relies on the ability of DNA repair proteins to open, cleave and recombine DNA. Slx4 (synthetic lethal of unknown function) or BTBd12 (BTBD12 domain-containing protein 12) the human homologue is a DNA repair protein which has multiple independent cellular roles. Importantly, it is a component in Holliday junction homologous recombination (HR) where it interacts with other proteins to repair double strand breaks (DSB). SLX4 forms a heterodimer complex with SLX1, an endonuclease, in response to DNA damage. This complex works by recruiting several associated proteins including XPF- ERCC1 a 3′ flap endonuclease, to DSB allowing proper single strand annealing repair of the DNA. An essential part of meiosis in the male and female germ cell formation is DNA recombination which is initiated by DSB. Meiotic error at this stage can cause aneuploidy and infertility. As SLX4 acts as a key regulator of recombination we proposed that loss of this protein would cause dysfunction in germ cell development. Breeding pairs of SLX4+/– heterozygous mice were obtained from EUCOMM and germline transmission of the null allele confirmed. Adult mice were normal and healthy but analysis of the male reproductive tract confirmed abnormally small testes (wt+/– vs testes). Further investigation demonstrated hypospermatogenesis with major loss of meiotic spermatocytes and post meiotic and spermatids. Epididymal sperm retrieval confirmed that the SLX4–/– male does produce poorly motile sperm with significant acrosomal abnormalities and associated failure of sperm-zona pellucida binding (2.5 ± 1.6 vs 13.6 ± 3.6 sperm bound) when compared to heterozygote or wild type littermates. Interestingly the female SLX4 null mouse also has small ovaries compared with het and wt littermates (wt vs null). Histomorphological analysis reveals a significant reduction in the ovarian follicle pool compared to wild type littermates with concomitant increased levels of apoptosis. Early breeding trials confirm significant loss of fertility in null males and females probably due to deficient sperm development and oocyte dysfunction. Our study now aims to further examine the role of SLX4 in germ cell development in the testes and ovary and its impact on infertility.
325. INVESTIGATION INTO THE EXPRESSION OF HUMAN FRIENDLY BEHAVIOUR IN THE OSTRICH (STRUTHIO CAMELUS) - EFFECT OF IMPRINTING
Authors: Malecki I. A. , Rybnik-Trzaskowska P. K.
Published: November 2010

In birds, filial imprinting to humans occurs after artificial hatching, which may lead to human following and sexual imprinting. We investigated the effect of imprinting on expression of favourable to human behaviours in juvenile ostriches. For the first seven days from hatching, the birds were exposed to a dummy female. At 12 months of age, a year before ostriches reach sexual maturity, the males (n = 25) were given testosterone intramuscularly in five every second day injections to induce precocial sexual behaviour. Additional six males, not exposed to the dummy, received the vehicle. Two weeks after the first injection, behavioural data collection commenced and, a range of behaviours were scored in five times over the period of 2 weeks from the last injection. The behaviours were given a score of 1 (behaviour present) or 0 (behaviour not present). The males exposed to a dummy female (n = 11) scored less for friendly to human behaviours than those not exposed to a dummy. No courtship type behaviour was observed in all males. Plasma testosterone concentrations increased and reached maximum levels by day 6 post-injection in exposed and not exposed to a dummy males but the levels were not different from the control (vehicle). We conclude that exposure to a dummy led to less friendly to human behaviour, while absence of the dummy led to more friendly behaviour towards humans. Testosterone injections failed to induce precocial sexual behaviour possibly because the amount given was insufficient or the birds could not yet express sexual behaviour to humans. The levels of testosterone in blood plasma occurred coincidently as they were probably associated with pre-pubertal development.
326. SPIF - A NOVEL TESTIS-SPECIFIC GENE AND ITS INTERACTION WITH PKA
Authors: Tannock S. J. , McLaughlin E. A. , Aitken R. J. , Roman S. D.
Published: November 2010

The activation of protein kinase A (PKA) is strongly implicated in capacitation and sperm motility. However, the full pathway is yet to be elucidated. To identify potential PKA binding partners in sperm, a yeast two-hybrid assay was performed using the testis specific catalytic subunit (Cs) of PKA as the ‘bait’ to screen a mouse testis cDNA library. A novel cDNA clone termed Sperm PKA Interacting Factor (SPIF) was identified from the screen on three separate occasions. The interaction was confirmed by a protein pull-down using a C-terminal recombinant protein to SPIF and a PKACs antibody. During cloning and sequence analysis, SPIF was found to contain two isoforms; a full length (4770 bp) and a truncated form (2784 bp) with alternate start sites and an identical 3′ end, with only the full length isoform containing the PKA binding motif. SPIF was found to be testis specific using PCR and Northern Blotting with high expression levels in round spermatids and adult testis. The interaction between SPIF and PKA was further demonstrated with protein co-localisation in round spermatids and in the midpiece and flagellum of mouse sperm. In summary, we have identified a novel testis specific gene that in concert with PKA could prove to be an essential link in the incomplete capacitation pathway
339. THE RELATIONSHIP BETWEEN DNA FRAGMENTATION IN MOUSE OOCYTES AND GENITAL TRACT MICROORGANISMS
Authors: Harris J. E. , Pelzer E. S. , Allan J. A. , Whiteside E. , De Boer L. , Knox C. L.
Published: November 2010

Colonising bacteria detected within the follicular fluid of women undergoing assisted reproductive technology cycles has been associated with decreased embryo transfer rates and decreased pregnancy rates. The bacteria isolated from the follicular fluid of these women, at the time of trans-vaginal oocyte retrieval include Gram-positive anaerobic rods, Gram-negative anaerobic rods, streptococci, staphylococci, and lactobacilli. Some of these bacteria are opportunistic pathogens in the female genital tract. The expression of virulence factors including hyaluronidase and DNase, could affect the structural integrity of the oocyte and induce DNA fragmentation and apoptosis. Hyaluronin is a major carbohydrate component of the cumulus oocyte complex that could be a target for bacterial hyaluronidase. Other virulence factors associated with bacteria detected in cases of genital tract infections include the hydrolytic enzymes sialidase, β-galactosidase and β-N-acetylhexosaminidase (Howe 1999). Production of reactive oxygen species as a result of the presence of bacteria and bacterial heat shock proteins have been suggested as mechanisms responsible for increased DNA fragmentation and apoptotic progression of male gametes in colonised semen. Previous studies have not confirmed a direct relationship between sperm morphology and the level of sperm DNA fragmentation. DNA fragmentation in oocytes in relation to the presence of bacteria remains to be investigated. Oocyte quality is only determined by the assessment of cumulus cell morphology prior to in-vitro fertilisation. In-vitro testing of oocyte DNA integrity under various conditions may further the understanding of oocyte quality. In this study, mouse oocytes were used to investigate the structural and DNA integrity of oocytes after in vitro exposure to Lactobacillus spp., Streptococcus spp., and Staphylococcus spp. and determine whether the morphological appearance of the oocyte was predictive of the level of DNA fragmentation and whether DNA fragmentation can be attributed to certain bacterial species found colonising follicular fluid.
(1) Howe L (1999). Mucinase and sialidase activity of the vaginal microflora: implications for the pathogenesis of preterm labour. International Journal of STD and AIDS 10(7), 442–446.
340. ANALYSIS OF PROTEINS SECRETED BY THE PREIMPLANTATION MOUSE EMBRYO
Authors: Gentles R. , O'Neill C.
Published: November 2010

Many embryos generated by assisted reproductive technologies do not have the capacity for full development. A non-destructive test that allows the most viable embryos to be identified would allow development of a range of strategies to improve these technologies. Analysis of the proteinaceous secretome of the preimplantation mouse embryo by electrospray mass spectrometry of tryptic digests of embryo-conditioned culture media identified 20 peptides.1 Lactate dehydrogenase beta (LDH) and protein disulfide isomerase (PDI) were consistently among the most abundant peptides identified. PDI has been found to be secreted and surface expressed in a number of other cell types suggesting a biologically significant role. LDH-beta is a marker of plasma membrane integrity and is widely used in medicine for assessment of cardiac health. The aim of this study was to assess the expression of these proteins in the early embryo to gain some understanding of the potential significance of their presence within the embryonic secretome. Zygotes (B6CBF2) were collected 20–22 h post hCG. They were cultured in groups of 10 embryos for 96 h in 10 mL of modHTF 30 µg BSA/mL media. Embryonic protein was extracted for western blot analysis or embryos subjected were to immunolocalization studies for PDI and LDH. Western blot analysis revealed presence of proteins of expected molecular mass. Staining for both proteins occurred at high levels at each stage of development throughout pre-implantation development. Staining was predominantly cytoplasmic and excluded from nuclei. Cultured blastocysts had less LDH than fresh but the level for PDI was similar for both. Staining of non-permeabilized embryos revealed extracellular staining of PDI. The results show high levels of cellular expression of two proteins reported to be released by the embryo in vitro.
(1) Beardsley A, Li Y, O’Neill C (2010). Characterization of a diverse secretome generated by the mouse preimplantation embryo in vitro. Reproductive Biology and Endocrinology 8, 71.
The effects of parasitism on recapture rates of wood mice (Apodemus sylvaticus)
Authors: Coltherd Jennifer C. , Morgan Carys , Judge Johanna , Smith Lesley A. , Hutchings Michael R.
Published: November 2010

Context. In studies of population dynamics, disease ecology and prevalence, point-sampling of data is a widely used sampling technique and capture–mark–recapture (CMR) is the most popular method of point sampling. Population estimates based on CMR are sensitive to deviations from the assumptions of the models such as equal catchability of all animals. Although consistent deviations from these assumptions can be accounted for in a robust statistical framework (e.g. identification of trap-shy animals), transient effects cannot be adequately incorporated in the statistical models and thus are expected to affect the accuracy of model predictions. In natural animal populations, parasitism is often transient but inevitable and studies showing behavioural changes, e.g. foraging strategies, as a result of infection are numerous. Thus, parasitism may represent a transient source of heterogeneity of trapping probability in small mammal populations and may affect accuracy of wildlife sampling techniques.

Aims. The aim of the present study was to quantify the effect of parasitic status on capture rates of wood mice (Apodemus sylvaticus), and thus the potential for parasitism to act as a source of bias in wildlife sampling techniques such as CMR.

Methods. In total, 41 wood mice were successfully captured, marked and released from two study sites in two different years, with weight, sex and faecal egg counts (FEC; used as a measure of the level of parasitism) recorded at every capture.

Key results. In both studies, FEC was positively correlated with the capture rate of mice and the number of different traps in which an individual was caught.

Conclusions. We conclude that parasitism affects the rate of capture of wood mice and has the potential to represent a significant source of heterogeneity in trapping probability.

Implications. The consistent results of these two small studies suggest that wildlife point-sampling techniques may be biased by the parasitic status of the animals, which has potentially significant and far-reaching implications for wildlife population studies and disease-prevalence studies.
Dlg1 binds GKAP to control dynein association with microtubules, centrosome positioning, and cell polarity
Authors: Manneville Jean-Baptiste , Jehanno Muguette , Etienne-Manneville Sandrine
Published: November 2010

Centrosome positioning is crucial during cell division, cell differentiation, and for a wide range of cell-polarized functions including migration. In multicellular organisms, centrosome movement across the cytoplasm is thought to result from a balance of forces exerted by the microtubule-associated motor dynein. However, the mechanisms regulating dynein-mediated forces are still unknown. We show here that during wound-induced cell migration, the small G protein Cdc42 acts through the polarity protein Dlg1 to regulate the interaction of dynein with microtubules of the cell front. Dlg1 interacts with dynein via the scaffolding protein GKAP and together, Dlg1, GKAP, and dynein control microtubule dynamics and organization near the cell cortex and promote centrosome positioning. Our results suggest that, by modulating dynein interaction with leading edge microtubules, the evolutionary conserved proteins Dlg1 and GKAP control the forces operating on microtubules and play a fundamental role in centrosome positioning and cell polarity.
Length-selective retention of walleye pollock, Theragra chalcogramma, by midwater trawls
Authors: Williams Kresimir , Punt André E. , Wilson Christopher D. , Horne John K.
Published: November 2010

Williams, K., Punt, A. E., Wilson, C. D., and Horne, J. K. 2011. Length-selective retention of walleye pollock, Theragra chalcogramma, by midwater trawls. – ICES Journal of Marine Science, 68: 119–129. Midwater trawls are commonly used during acoustic surveys of fish abundance to determine species and length compositions of acoustically sampled fish aggregations. As trawls are selective samplers, catches can be unrepresentative of sampled populations and lead to biased abundance estimates. Length-dependent retention of walleye pollock was estimated using small recapture nets, so-called pocket nets, attached to the outside of the trawl. Experimental haul sets comprising eight hauls each were conducted in the Gulf of Alaska in 2007 and 2008 and in the eastern Bering Sea (EBS) in 2007. Pocket-net catches were then modelled by fitting parameters for selectivity and escapement location along the trawl. Within- and between-haul variability was jointly estimated using hierarchical Bayesian methods. There was significant undersampling of juvenile (<25 cm) pollock, with the length-at-50%-retention (L50) estimated between 13.5 and 26.1 cm among haul sets. In the EBS set, L50 values were correlated with light level, escapement being greater at night. Trawl selectivity may be a significant source of error in acoustic-survey estimates of the abundance of pollock.
Development of the EcoQO for the North Sea fish community
Authors: Greenstreet Simon P. R. , Rogers Stuart I. , Rice Jake C. , Piet Gerjan J. , Guirey Emma J. , Fraser Helen M. , Fryer Rob J.
Published: November 2010

Greenstreet, S. P. R., Rogers, S. I., Rice, J. C., Piet, G. J., Guirey, E. J., Fraser, H. M., and Fryer, R. J. 2011. Development of the EcoQO for the North Sea fish community. – ICES Journal of Marine Science, 68: 1–11. Development of the Ecological Quality Objective (EcoQO) for the North Sea demersal fish community is described. Size-based metrics were identified as the most effective indicators of the state of the community, but such metrics are also sensitive to environmental influence. Redefining the large fish indicator (LFI) produced a metric more sensitive to fishing-induced change and therefore more useful to managers. Fish stocks were thought to be exploited at a sustainable rate in the early 1980s, so in a process echoing the precautionary approach to fish stock management, this was considered the reference period for the LFI, suggesting a value of 0.3 as the appropriate EcoQO. The LFI declined from around 0.3 in 1983 to 0.05 in 2001, followed by a recovery to 0.22 in 2008. However, analyses of the longer-term groundfish survey data suggest that, even were fishing pressure to be reduced to early 20th century levels, the LFI would be unlikely to rise much above a value of 0.3. The response of the LFI to variation in fishing pressure suggested a more complex relationship than anticipated, underscoring the need for operational theoretical size-resolved multispecies fish community models to support management towards broader ecosystem objectives.
Limitation of Nocturnal ATP Import into Chloroplasts Seems to Affect Hormonal Crosstalk, Prime Defense, and Enhance Disease Resistance in Arabidopsis thaliana
Authors: Schmitz Gudrun , Reinhold Thomas , Göbel Cornelia , Feussner Ivo , Neuhaus H. Ekkehard , Conrath Uwe
Published: November 2010

When grown under short-day conditions at low light, leaves of an Arabidopsis thaliana (accession Col-0) mutant with defects in the two genes encoding plastid ATP/ADP antiporters (so-called ntt1-2 null mutants) display a variety of physiological changes. These include the formation of necrotic lesions and the accumulation of hydrogen peroxide in the leaves. Here, we show that, under short-day conditions, leaves of the ntt1-2 mutant display enhanced resistance to Hyaloperonospora arabidopsidis, Botrytis cinerea, and Pseudomonas syringae pv. tomato DC3000. Resistance to these pathogens was associated with constitutively elevated levels of the plant hormone salicylic acid and, eventually, jasmonic acid, and constitutive or primed activation after pathogen attack of various defense genes that are dependent on these hormones. In addition, the antagonistic crosstalk between the salicylic acid and jasmonic acid signaling pathways seems to be affected in ntt1-2. Because the enhanced resistance of ntt1-2 to H. arabidopsidis was not seen when the mutant was grown under long-day conditions, our findings argue that nocturnal ATP import into chloroplasts is crucial to keep A. thaliana from runaway activation of pathogen resistance.
Buprenorphine Enhances and Prolongs the Postoperative Analgesic Effect of Bupivacaine in Patients Receiving Infragluteal Sciatic Nerve Block
Authors: Candido Kenneth D. , Hennes Jason , Gonzalez Sergio , Mikat-Stevens Marianne , Pinzur Michael , Vasic Vladimir , Knezevic Nebojsa Nick
Published: November 2010

Background

Results from previous studies have shown favorable effects from the addition of buprenorphine to local anesthetics used for interscalene or axillary perivascular brachial plexus blocks. The main objective of the current study was to determine whether addition of buprenorphine could enhance bupivacaine analgesia after infragluteal sciatic nerve block.

Methods

One hundred and three consenting adult patients for elective foot and ankle outpatient surgeries were prospectively assigned randomly, in double-blind fashion, to one of three groups. Group 1 received 0.5% bupivacaine with epinephrine 1:200,000 for infragluteal sciatic block plus 1 ml normal saline intramuscularly. Group 2 received bupivacaine sciatic block along with intramuscular buprenorphine (0.3 mg). Group 3 received bupivacaine plus buprenorphine for infragluteal sciatic block and 1 ml normal saline intramuscularly.

Results

Although patients receiving buprenorphine either for sciatic block or intramuscularly had less pain in the postanesthesia care unit compared with patients receiving only bupivacaine, the individual pair-wise comparison of the analysis of variance model showed no statistical difference. However, only buprenorphine added to bupivacaine for sciatic block prolonged postoperative analgesia. Patients receiving a combination of buprenorphine and bupivacaine for sciatic block had lower numeric rating pain scores and received less opioid medication at home than patients in the other two groups.

Conclusions

The results show that buprenorphine may enhance and prolong the analgesic effect of bupivacaine when used for sciatic nerve blocks in patients undergoing foot and ankle surgery under general anesthesia but does not do so to the extent shown in previous studies using brachial plexus models with mepivacaine and tetracaine.
Defect characterization in graphene and carbon nanotubes using Raman spectroscopy
Authors: Dresselhaus M. S. , Jorio A. , Souza Filho A. G. , Saito R.
Published: November 2010

This review discusses advances that have been made in the study of defect-induced double-resonance processes in nanographite, graphene and carbon nanotubes, mostly coming from combining Raman spectroscopic experiments with microscopy studies and from the development of new theoretical models. The disorder-induced peak frequencies and intensities are discussed, with particular emphasis given to how the disorder-induced features evolve with increasing amounts of disorder. We address here two systems, ion-bombarded graphene and nanographite, where disorder is represented by point defects and boundaries, respectively. Raman spectroscopy is used to study the ‘atomic structure’ of the defect, making it possible, for example, to distinguish between zigzag and armchair edges, based on selection rules of phonon scattering. Finally, a different concept is discussed, involving the effect that defects have on the lineshape of Raman-allowed peaks, owing to local electron and phonon energy renormalization. Such effects can be observed by near-field optical measurements on the
G
′ feature for doped single-walled carbon nanotubes.
Single-layer and bilayer graphene superlattices: collimation, additional Dirac points and Dirac lines
Authors: Barbier Michaël , Vasilopoulos Panagiotis , Peeters François M.
Published: November 2010

We review the energy spectrum and transport properties of several types of one-dimensional superlattices (SLs) on single-layer and bilayer graphene. In single-layer graphene, for certain SL parameters an electron beam incident on an SL is highly collimated. On the other hand, there are extra Dirac points generated for other SL parameters. Using rectangular barriers allows us to find analytical expressions for the location of new Dirac points in the spectrum and for the renormalization of the electron velocities. The influence of these extra Dirac points on the conductivity is investigated. In the limit of
δ
-function barriers, the transmission
T
through and conductance
G
of a finite number of barriers as well as the energy spectra of SLs are
periodic
functions of the dimensionless strength
P
of the barriers,

, with
v
F
the Fermi velocity. For a Kronig–Penney SL with alternating sign of the height of the barriers, the Dirac point becomes a Dirac line for
P
=
π
/2+
nπ
with
n
an integer. In bilayer graphene, with an appropriate bias applied to the barriers and wells, we show that several new types of SLs are produced and two of them are similar to type I and type II semiconductor SLs. Similar to single-layer graphene SLs, extra ‘Dirac’ points are found in bilayer graphene SLs. Non-ballistic transport is also considered.
Fractional quantum Hall effect in suspended graphene probed with two-terminal measurements
Authors: Skachko I. , Du X. , Duerr F. , Luican A. , Abanin D. A. , Levitov L. S. , Andrei E. Y.
Published: November 2010

Recently, fractional quantization of two-terminal conductance was reported in suspended graphene. The quantization, which was clearly visible in fields as low as 2 T and persistent up to 20 K in 12 T, was attributed to the formation of an incompressible fractional quantum Hall state. Here, we argue that the failure of earlier experiments to detect the integer and fractional quantum Hall effect with a Hall-bar lead geometry is a consequence of the invasive character of voltage probes in mesoscopic samples, which are easily shorted out owing to the formation of hot spots near the edges of the sample. This conclusion is supported by a detailed comparison with a solvable transport model. We also consider, and rule out, an alternative interpretation of the quantization in terms of the formation of a p–n–p junction, which could result from contact doping or density inhomogeneity. Finally, we discuss the estimate of the quasi-particle gap of the quantum Hall state. The gap value, obtained from the transport data using a conformal mapping technique, is considerably larger than in GaAs-based two-dimensional electron systems, reflecting the stronger Coulomb interactions in graphene.
The cophylogeny of populations and cultures: reconstructing the evolution of Iranian tribal craft traditions using trees and jungles
Authors: Tehrani Jamshid J. , Collard Mark , Shennan Stephen J.
Published: November 2010

Phylogenetic approaches to culture have shed new light on the role played by population dispersals in the spread and diversification of cultural traditions. However, the fact that cultural inheritance is based on separate mechanisms from genetic inheritance means that socially transmitted traditions have the potential to diverge from population histories. Here, we suggest that associations between these two systems can be reconstructed using techniques developed to study cospeciation between hosts and parasites and related problems in biology. Relationships among the latter are patterned by four main processes: co-divergence, intra-host speciation (duplication), intra-host extinction (sorting) and horizontal transfers. We show that patterns of cultural inheritance are structured by analogous processes, and then demonstrate the applicability of the host–parasite model to culture using empirical data on Iranian tribal populations.
Magnesium-Agarose Electrophoretic Mobility Shift Assay (EMSA) of Transcription Factor IID Binding to DNA
Authors: Carey Michael F. , Peterson Craig L. , Smale Stephen T.
Published: November 2010

INTRODUCTION

The general transcription factor IID (TFIID) is a key target for regulation because its binding to a core promoter is the nucleating step in transcription complex assembly. Many eukaryotic activators stimulate recruitment of the TFIID when its concentration is made limiting at a promoter in vitro. Magnesium-agarose gels can separate large complexes containing TFIID, TFIIA (the DA complex), and TFIIB (the DAB complex) and permit a quantitative measurement of how activators stimulate assembly of such complexes. The advantage of the electrophoretic mobility shift assay (EMSA) is that the reactions can be performed under subsaturating conditions where a TFIID footprint might not be observed. Typically, the activator is incubated with a 32P-labeled DNA template, recombinant TFIIA purified from Escherichia coli, and immunopurified TFIID. After incubation, the samples are electrophoresed on magnesium-containing agarose gels, dried onto DEAE-cellulose paper, and autoradiographed. The DNA-protein complexes containing TFIID migrate with reduced mobility on magnesium-agarose gels both because of the large size of the complex and because the TATA-binding protein (TBP) subunit induces a sharp bend in the DNA, causing altered mobility. By comparing the binding of TFIID over a wide concentration range, with and without activator, one can assess whether the activator interacts with TBP or with one of the TBP-associated factors (TAFIIs). Additional factors such as TFIIA and TFIIB can be added subsequently to quantify their contributions to assembly of the transcription complex.