Memory and PTPIP51--a new protein in hippocampus and cerebellum.

Previously the expression of Protein Tyrosine Phosphatase Interacting Protein 51 (PTPIP51) in mouse brain was reported. Here, we investigated PTPIP51 mRNA and protein in two of the brain regions namely the hippocampus and the cerebellum of mouse brains. On a cellular level both the protein and the mRNA were related to the pyramidal cells of the hippocampal formation, the granular cells of the dentate gyrus and the cells of the adjacent strata. In the cerebellum PTPIP51 was traced in Purkinje cells, the cells of the molecular layer and the granular layer. On a subcellular level only partial co-localization was seen for the endoplasmic reticulum, but not with mitochondria. In addition the interactome of PTPIP51 was analysed. In hippocampal cells a strong interaction with PTP1B and vesicle-associated membrane protein-associated protein B (VAPB) was detected. A somewhat differing interaction profile was found in the cerebellum, where high interaction levels were found for 14-3-3, diacylglycerol kinase α (DGKα), NFκB and PTP1B. These interaction partners represent specific signalling pathways linked to building memory. PTPIP51 can be associated with nerve growth factor signalling, dendritic and axonal growth, synaptogenesis, and all processes needed for memory formation. Moreover, in HT-22 mouse hippocampal cells PTPIP51 expression was induced by administrating the fibroblast growth factor 1 (FGF-1), which is known to take part in learning/memory processes. Knocking down p38-MAPK also led to an up-regulation of PTPIP51 probably resembling a compensative mechanism. Thus, a possible connection to the processing of memories can be anticipated. Differences in the interaction profile in both regions may be attributed to the actual/local differences in memory formation.

Mixed testicular atrophy related to atherosclerosis: first lessons from the ApoE(-/-)/ LDL receptor(-/-) double knockout mouse model.

Age-related testicular changes are associated with declining spermatogenesis and testosterone levels. A relationship to atherosclerosis has never been investigated systematically. The ApoE(-/-)/LDL receptor(-/-) double knockout mouse model, providing a remarkable homology to human atherosclerosis, is an ideal tool to investigate spermatogenetic alterations in this context. Testes (n = 10) from ApoE(-/-)/LDL receptor(-/-) double knockout mice at the age of 80 weeks were perfused in vivo with contrast agent, harvested and scanned with micro-CT at (4.9 µm³) voxel size. Testes (n = 8) of C57/BL mice at the same age served as controls. Testis volume (mm³) and total vascular volume fraction (mm³) were quantified using micro-CT. Serum testosterone levels were determined. Testicular histology and epididymal sections were analysed for tubular structure, spermatogenetic scores and sperm count. The expression of protamine 2 as a marker for elongated spermatids, inflammation markers (CD4, F4/80) and hypoxia inducible factor 1 alpha (HIF1 alpha) were investigated using immunohistochemistry. ApoE(-/-)/LDL receptor(-/-) double knockout mice exhibit diminished testis and vascular volume fraction with respect to that of controls (p < 0.001). These findings were associated with a reduction of testosterone levels (p < 0.001). Mixed atrophy was present in 41% of the seminiferous tubuli in ApoE(-/-)/LDL receptor(-/-) double knockout mice at the age of 80 weeks. Sperm counts from the epididymis demonstrated a significant decrease in ApoE(-/-)/LDL receptor(-/-) double knockout mice (p < 0.001). In addition, sperm specific protamine 2 expression was decreased in testicular tissue and epididymis of ApoE(-/-)/LDL receptor(-/-) double knockout mice compared with that of control mice. Peritubular inflammatory infiltration and the expression of the hypoxia related marker was observed. Mixed testicular atrophy in ApoE(-/-)/LDL receptor(-/-) double knockout mice is linked to reduced testis volume, vascular volume fraction and low testosterone serum levels, suggesting a direct relation between atherosclerosis and disturbed spermatogenesis.

PTPIP51 interaction with PTP1B and 14-3-3ß in adipose tissue of insulin-resistant mice.

OBJECTIVE: We investigated the expression of protein tyrosine phosphatase-interacting protein 51 (PTPIP51) and its interaction with protein tyrosine phosphatase 1B (PTP1B) and 14-3-3ß in mice exhibiting insulin resistance and obesity. DESIGN: A total of 20 mice were included in the study. Eight control animals were fed a normal standard diet, six animals were fed a high-fat diet and six animals were submitted to a treadmill training parallel to the feeding of a high-fat diet. After 10 weeks, a glucose tolerance test was performed and abdominal adipose tissue samples of the animals were collected. RESULTS: PTPIP51 protein was identified in the adipocytes of all samples. PTPIP51 interacted with PTP1B and with 14-3-3ß protein. Compared with untrained mice fed a standard diet, the interaction of PTPIP51 with PTP1B was reduced in high-fat diet-fed animals. The highest interaction of PTPIP51 with 14-3-3ß was seen in trained animals on high-fat diet, whereas untrained animals on high-fat diet displayed lowest values. CONCLUSION: PTPIP51 is expressed in adipose tissue of humans, rats and mice. Obesity with enhanced insulin resistance resulted in a reduction of PTPIP51 levels in adipocytes and influenced the interactions with PTP1B and 14-3-3ß. The interaction of PTPIP51 with PTP1B suggests a regulatory function of PTPIP51 in insulin receptor signal transduction. The interaction of PTPIP51 with 14-3-3ß, especially in trained individuals, hints to an involvement of PTPIP51 in the downstream regulation of insulin action.

Apoptotic gene expression in potentially fertile and subfertile men.

Human sperm contain similar amounts of protamine-1 (P1) and protamine-2 (P2). Although an aberrant protamine ratio have been observed in subfertile men, functional evidence is provided by protamine knockout mice exhibiting male infertility. As sperm DNA integrity is known to be linked with DNA fragmentation and apoptosis, we investigated whether the DNA fragmentation factor 40 (DFF40) ratio or caspase (Casp4, Casp6) and tumor necrosis factor superfamily member 10 (TNFSF10) ratio together with the P1/P2 ratio represents a reliable biomarker to discriminate between fertile and subfertile men. Real-time quantitative RT-PCR was used for amplification of P1, P2 and DFF40 in 49 testicular biopsies. Casp4, Casp6 and TNFSF10 have been selected from a PCR apoptosis array and were further investigated in another group of testicular biopsies (22 subfertile men versus 11 potentially fertile men). Using Spearman's rank correlation coefficient analysis, we did not find a correlation between DFF40 and P1, P2, P1/P2, score, fertilization rate and age. In addition, logistic regression analysis demonstrated no statistically significant effect of the analyzed variables on pregnancy. A two-way analysis of variance with repeated measures of relative expression of Casp4, Casp6 and TNFSF10 versus P1 or P2 in potentially fertile men and subfertile patients demonstrated statistically significant differences between both groups, all tested gene combinations and the interaction between two genes and both groups in all cases analyzed. Furthermore, significant differences in the expression of Casp4 and TNFSF10 between the groups of potentially fertile and subfertile men could be demonstrated. In addition, the means of differences of selected gene combinations revealed that the protamine to apoptotic gene ratio is statistically different between both groups. Our data suggest that Casp4, Casp 6 and TNFSF10 are differentially expressed in potentially fertile and subfertile men and represent useful biomarkers for predicting male fertility in combination with P1 and P2.

Prognostic markers for competent human spermatozoa: fertilizing capacity and contribution to the embryo.

Management of male infertility largely depends on our understanding of cellular and molecular aspects of spermatogenesis as well as sperm function. Apart from standardized and comprehensive semen analysis, prognostic markers estimating the fertilizing capacity of either ejaculated spermatozoa or testicular spermatids are required. While there is general agreement that correct replacement of DNA-binding histones by protamines represents a prerequisite for achieving competent spermatozoa, especially in intracytoplasmic sperm injection (ICSI) where natural selection mechanisms are bypassed, the function of a variety of transcripts within the spermatozoa's cytoplasm and of remaining highly acetylated histones is still a matter of debate. Hence, this review brings the up-to-date research on mammalian spermatozoal chromatin composition into focus, which is discussed in conjunction with the paternal role on epigenetic reprogramming of the zygote following fertilization. As paternal transcripts have been demonstrated to be transmitted to the oocyte, it is now accepted that they represent more than solely remnants of previous transcriptional activity. Acetylation of histones, normaly a characteristic of transcriptional activity, was for a long time a miracle, as spermatozoa are transcriptionally inactive cells, but is now suggested to represent epigenetic marks that are transmitted to the oocyte and play an important role in the regulation of early gene expression in the developing embryo.

Presence of histone H3 acetylated at lysine 9 in male germ cells and its distribution pattern in the genome of human spermatozoa.

During spermatogenesis, approximately 85% of histones are replaced by protamines. The remaining histones have been proposed to carry essential marks for the establishment of epigenetic information in the offspring. The aim of the present study was to analyse the expression pattern of histone H3 acetylated at lysine 9 (H3K9ac) during normal and impaired spermatogenesis and the binding pattern of H3K9ac to selected genes within ejaculates. Testicular biopsies, as well as semen samples, were used for immunohistochemistry. Chromatin immunoprecipitation was performed with ejaculated sperm chromatin. HeLa cells and prostate tissue served as controls. Binding of selected genes was evaluated by semiquantitative and real-time polymerase chain reaction. Immunohistochemistry of H3K9ac demonstrated positive signals in spermatogonia, spermatocytes, elongating spermatids and ejaculated spermatozoa of fertile and infertile men. H3K9ac was associated with gene promoters (CRAT, G6PD, MCF2L), exons (SOX2, GAPDH, STK11IP, FLNA, PLXNA3, SH3GLB2, CTSD) and intergenic regions (TH) in fertile men and revealed shifts of the distribution pattern in ejaculated spermatozoa of infertile men. In conclusion, H3K9ac is present in male germ cells and may play a role during the development of human spermatozoa. In addition, H3K9ac is associated with specific regions of the sperm genome defining an epigenetic code that may influence gene expression directly after fertilisation.

Recognising the Sertoli-cell-only (SCO) syndrome: a case study.

Total Sertoli-cell-only (SCO) syndrome is often confused with a focal SCO picture, in which testicular illness caused damage to seminiferous tubules and compromised the Sertoli cell range of maturation and functions, but from which still some spermatozoa can be retrieved for assisted reproductive techniques. Here, a possibly new SCO syndrome phenotype is reported exhibiting complete lack of germ cells despite normal architecture of the seminiferous tubules with presence of mature Sertoli cells and normal Leydig cells in the intertubular tissue. Sertoli cells are immunonegative for the prepubertal differentiation markers cytokeratin-18, anti-Muellerian hormone and M2A antigen, but reveal a positive signal for the gap junctional protein connexin 43 known to be expressed in Sertoli cells with an adult type of differentiation. The complete lack of germ cells in combination with fully differentiated adult-type Sertoli cells in this case is in contradiction with known SCO subtypes and with the current hypothesis of reciprocal regulation of Sertoli and germ cell differentiation.

Protamine-1 represents a sperm specific gene transcript: a study in Callithrix jacchus and Bos taurus.

Spermatozoa are transcriptionally inactive cells, but contain acetylated histones, normally a characteristic of transcriptionally active cells. Acetylgroups are thought to represent epigenetic marks that are transmitted to the oocyte and are involved in starting gene expression in the zygote and in regulating gene expression during early embryogenesis. We performed reverse transcription polymerase chain reaction (RT-PCR) in the common marmoset monkey (Callithrix jacchus) and in bovine spermatozoa, oocytes, zygotes, two- and four-cell embryos to evaluate the presence of specific transcripts known to play a role during fertilisation and early embryo development, namely protamine-1 (PRM1), protamine-2 (PRM2), histone H1 (H1), histone H3 (H3), histone H4 (H4), cAMP-responsive element modulator (CREM), DNA methyltransferase-1 (DNMT1), TATA box-binding protein (TBP). All transcripts tested were present in spermatozoa of the common marmoset, while bull spermatozoa lack PRM2. Marmoset oocytes exhibited transcripts for H1, H3, H4 and TBP, whereas bovine oocytes revealed H1, H3, H4, CREM, DNMT and TBP mRNAs. In zygotes, we amplified H1, H4, TBP (marmoset) and PRM1, H1, H3, H4, CREM, DNMT1 and TBP (bovine). Two-cell embryos showed PCR products for H1, H3 and TBP in the marmoset. In the bovine, all transcripts could be observed except PRM2. In four-cell embryos, PCR signals were obtained for PRM1, H1, H3, H4 and TBP in the marmoset. In the bovine, all transcripts were detected except PRM2. Our data suggest that, in both C. jacchus and Bos taurus, PRM1 transcripts are delivered by the spermatozoon to the oocyte.

Validation of reference genes in human testis and ejaculate.

Beta-actin (ACTB), glyceraldehyde-3-phosphate-dehydrogenase (GAPD), Heat Shock Protein 1, beta (HSPCB) and Adenosine Triphosphate subunit 5 beta (ATP5B) with distinct functional characteristics and expression patterns were investigated as suitable references for gene expression studies. We determined the expression stability of the four reference genes in ejaculates, cryopreserved as well as fixed and paraffin-embedded testicular tissue (from fertile and subfertile men) applying real-time qRT-PCR and statistical analysis. The mean gene expressions (mean Ct value) were compared for each gene between the fertile and subfertile donors by using the Wilcoxon-Mann-Whitney test. We did not observe significant statistical differences between variability of genes. To detect random effects, we used the two-way analysis of variance with a hierarchical model. The results show no significant statistical differences between proband and repetition within the probands. Taken together, we concluded that ACTB, GAPD, HSPCB and ATP5B have a variable expression within these samples, but this variability is not statistically significant. This finding demonstrated that all these genes could be appropriated for further studies on gene expression in ejaculate and testis tissue. Therefore, the selection of the suitable reference genes is highly specific for a particular experimental model and validation for each situation, on an individual basis, is a crucial requirement.

Aberrant mRNA expression of chromatin remodelling factors in round spermatid maturation arrest compared with normal human spermatogenesis.

During human spermiogenesis, chromatin condensation is associated with replacement of histones by protamines. This exchange is supported by acetylated histones and chromatin remodelling factors. Ten chromatin remodelling factor protein families are known. This study aims to analyse whether a different chromatin remodelling factor expression pattern exists between normal spermatogenesis and round spermatid maturation arrest as potential reason for impaired spermatogenesis and idiopathic male infertility. Laser capture microdissection was used to excise seminiferous tubules from testicular biopsies with normal spermatogenesis and round spermatid maturation arrest. RNA was isolated, first strand cDNA synthesis and pre-amplification were performed using Epigenetic Chromatin Remodelling Factors PCR arrays with 84 genes. Applying hierarchical cluster analysis, three gene expression clusters with six subgroups were identified. The expression pattern ranges from a few high expressed genes in round spermatid maturation arrest to a multitude of genes (74) which are more highly expressed in normal spermatogenesis than in maturation arrest. A total of 22 genes showed a significant difference between normal spermatogenesis and round spermatid maturation arrest (1 gene was up-regulated and 21 genes were down-regulated in the developmental arrest). The significantly different expression of chromatin remodelling factors between normal spermatogenesis and round spermatid maturation arrest may lead to impaired epigenetic information and aberrant transcription during sperm development representing one possible reason for developmental arrest of round spermatids.

Advanced glycation end products accumulate in the reproductive tract of men with diabetes.

Light microscopic studies comparing sperm parameters show little association between diabetes and male fertility. However, with the introduction of new analytical techniques, evidence is now emerging of previously undetectable effects of diabetes on sperm function. Specifically, a recent study has found a significantly higher sperm nuclear DNA fragmentation in diabetic men. As advanced glycation end products (AGEs) are important instigators of oxidative stress and cell dysfunction in numerous diabetic complications, we hypothesized that these compounds could also be present in the male reproductive tract. The presence and localization of the most prominent AGE, carboxymethyl-lysine (CML), in the human testis, epididymis and sperm was determined by immunohistochemistry. Parallel ELISA and Western blot analyses were performed to ascertain the amount of CML in seminal plasma and sperm from 13 diabetic and nine non-diabetic subjects. CML immunoreactivity was found throughout the seminiferous epithelium, the nuclei of spermatogonia and spermatocytes, in the basal and principle cells cytoplasm and nuclei of the caput epididymis and on most sperm tails, mid pieces and all cytoplasmic droplets. The acrosomal cap, especially the equatorial band, was prominently stained in diabetic samples only. The amount of CML was significantly higher (p = 0.004) in sperm from non-diabetic men. Considering the known detrimental actions of AGEs in other organs, the presence, location and quantity of CML, particularly the increased expression found in diabetic men, suggest that these compounds may play a hitherto unrecognized role in male infertility.

[TESE and mTESE. Therapeutic options in male infertility due to testicular azoospermia]. / TESE und M-TESE. Therapieoption bei infertilen Männern mit testikulärer Azoospermie.

Modern techniques of testicular sperm extraction (TESE) make it possible for an infertile man to father a child. The operations are standardized to a large extent and the underlying morphological alterations of spermatogenesis also appear to be sufficiently known. Current research is focused on prognostic factors for the testicular material that determine the sperm retrieval rate and success rates after in vitro fertilization/intracytoplasmic sperm injection (IVF-ICSI).TESE and microTESE are accepted standard operations for testicular sperm retrieval for IVF/ICSI. Predictions for effective sperm recovery are addressed.

eat-11 encodes GPB-2, a Gbeta(5) ortholog that interacts with G(o)alpha and G(q)alpha to regulate C. elegans behavior.

In C. elegans, a G(o)/G(q) signaling network regulates locomotion and egg laying [1-8]. Genetic analysis shows that activated Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) is suppressed by perturbations of this network, which include loss of the GOA-1 G(o)alpha, DGK-1 diacylglycerol kinase, EAT-16 G protein gamma subunit-like (GGL)-containing RGS protein, or an unidentified protein encoded by the gene eat-11 [9]. We cloned eat-11 and report that it encodes the Gbeta(5) ortholog GPB-2. Gbeta(5) binds specifically to GGL-containing RGS proteins, and the Gbeta(5)/RGS complex can promote the GTP-hydrolyzing activity of Galpha subunits [10, 11]. However, little is known about how this interaction affects G protein signaling in vivo. In addition to EAT-16, the GGL-containing RGS protein EGL-10 participates in G(o)/G(q) signaling; EGL-10 appears to act as an RGS for the GOA-1 G(o)alpha, while EAT-16 appears to act as an RGS for the EGL-30 G(q)alpha [4, 5]. We have combined behavioral, electrophysiological, and pharmacological approaches to show that GPB-2 is a central member of the G(o)/G(q) network and that GPB-2 may interact with both the EGL-10 and EAT-16 RGS proteins to mediate the opposing activities of G(o)alpha and G(q)alpha. These interactions provide a mechanism for the modulation of behavior by antagonistic G protein networks.

Kruppel-like factor 4 expression in normal and pathological human testes.

Krüppel-like factor 4 (KLF4) is a transcription factor involved in many cellular and developmental processes such as terminal differentiation of cells and carcinogenesis. Mice lacking KLF4 die post-natally due to skin barrier deficiencies and exhibit several additional cellular defects. The adult rodent testis expresses high levels of Klf4 mRNA. Using in situ hybridization, we previously localized most of the Klf4 mRNA to round spermatids in mice. Moreover, in rodent Sertoli cells, Klf4 is strongly inducible by FSH. Here, we show by northern blot analysis that the human testis also strongly expresses KLF4. Applying immunohistochemistry, we localized KLF4 protein to the nuclei of round spermatids during normal spermatogenesis stages II-IV. Analysing round spermatid maturation arrests, strong cytoplasmic staining could be seen in two samples. We failed to detect KLF4 in human Sertoli cells. Most human Leydig cells expressed KLF4 at high levels in the nucleus. However, some individual Leydig cells lacked KLF4, suggesting different functional states of the Leydig cells. The strong expression of KLF4 in the human testis and the importance of KLF4 in several mouse tissues suggest a significant role for KLF4 in the human testis. A first hint at a role for KLF4 during spermiogenesis could be the altered subcellular localization of the protein during arrested spermiogenesis.

Resistance mechanisms in human sarcoma mutants derived by single-step exposure to paclitaxel (Taxol).

A fluctuation analysis experiment was performed by exposing 15 expanded populations of MES-SA sarcoma cells to paclitaxel (Taxol) at a concentration of 10 nM for 7 days. The mutation rate was approximately 8 multiplied by 10(-7)/cell generation. ANOVA supports a stochastic cell survival mechanism of spontaneous mutation rather than induction of an adaptive response under these selection conditions. Surviving colonies were found in 12 populations, 9 of which had clones that remained resistant to paclitaxel after a 2-month period of propagation. Analysis of mdr1 gene expression by reverse transcription PCR demonstrated positive clones in 4 of the 9 populations with stable resistance. Accumulation of [(3)H]paclitaxel was decreased in these clones but not in the mdr1-negative clones compared with parental cells. A high degree of resistance to paclitaxel (36- to 93-fold) was selected by this single drug exposure in all 9 stably resistant mutants. Those with mdr1 activation demonstrated a broad cross-resistance to vinblastine, doxorubicin, and etoposide, whereas the other 6 mutants were cross-resistant only to the Vinca alkaloids. Because tubulins are the target molecules for paclitaxel cytotoxicity, we evaluated total tubulin content by immunoblotting and performed semiquantitative reverse transcription PCR analysis for expression of the alpha-tubulin isotypes B alpha 1, K alpha 1 and H alpha 44, the beta-tubulin isotypes M40, beta9, 5beta, beta2 and beta4, and gamma-tubulin. Total tubulin content was decreased significantly in one of the single-step mutants. All surviving clones, both resistant and sensitive to paclitaxel, displayed reduced expression of the 5beta and beta 4 beta-tubulin isotype transcripts in comparison with the parental cell line. These data suggest that stringent exposure to paclitaxel selected clones with reduced transcript levels of 5beta and beta4 beta-tubulin isotypes, but that these reduced levels were not directly involved in the resistance of the clones to paclitaxel. The results suggest an important role for non-multidrug-resistant mechanisms of resistance to paclitaxel. These mechanisms do not involve reduced drug accumulation and provide cross-resistance among both paclitaxel and tubulin depolymerizing agents.

The impact of sperm protamine deficiency and sperm DNA damage on human male fertility: a systematic review and meta-analysis.

Existing literature suggests evidence that protamine deficiency is related to DNA damage and male fertility. In this meta-analysis, we analyzed the relationship between the ratio of protamine-1 and protamine-2 with male fertility and the association of protamine deficiency with sperm DNA damage. Quality of available cohort studies was evaluated using the Newcastle-Ottawa Scale checklist. Summary effect estimates with 95% confidence intervals (CI) were derived using a random effects model. The effect of the protamine ratio on male fertility was analyzed in nine studies demonstrating a significantly higher value of the protamine ratio in subfertile men (n = 633) when compared with controls (n = 453, SMD = 0.46, 95% CI 0.25-0.66, Z = 4.42, p < 0.00001). Both protamine mRNA (SMD = 0.45, 95% CI 0.11-0.79, Z = 2.63, p = 0.009) and protein ratio (SMD = 0.46, 95% CI 0.25-0.68, Z = 4.22, p < 0.0001) showed significantly increased values in subfertile patients. The association between protamine deficiency and DNA damage was analyzed in 12 studies (n = 845) exhibiting a combined overall correlation coefficient (COR) of 0.53 (95% CI 0.28-0.71, Z = 3.87, p < 0.001). Protamine deficiency measured by CMA3 staining was significantly associated with sperm DNA damage (COR = 0.71, 95% CI 0.48-0.85, Z = 4.87, p < 0.001), whereas the P1/P2 ratio was not (COR = 0.17, 95% CI -0.16 to 0.46, Z = 0.99, p = 0.33). It is concluded that the protamine ratio represents a suitable biomarker for the assessment of sperm quality and protamine deficiency is closely related with sperm DNA damage.

A comprehensive investigation of sperm DNA damage and oxidative stress injury in infertile patients with subclinical, normozoospermic, and astheno/oligozoospermic clinical varicocoele.

One of the main pathogeneses of varicocoele and infertility is oxidative stress (OS), nevertheless, the oxidative damaged DNA in infertile patients with varicocoele remains poorly clarified. The objective of this study was to comprehensively investigate whether sperm DNA damage and OS injury were related with different issues of varicocoele. According to the varicocoele practice guidelines, surgical treatment was not indicated in the infertile patients with subclinical (SubVc, n = 15) and normozoospermic clinical varicocoele (NCVc, n = 22), the infertile astheno/oligozoospermic patients with clinical varicocoele (AOCVc, n = 51) would receive microsurgerical varicocoelectomy. Normozoospermic healthy donors with proven fertility (n = 25) were recruited as controls. Thiobarbituric acid and sperm chromatin structure assay (SCSA) methods were preformed to analyze seminal lipid peroxidation product malondialdehyde (MDA) and sperm DNA fragmentation index (DFI). We found that AOCVc and NCVc, except SubVc, could significantly elevate sperm DFI and seminal MDA levels. Varicocoelectomy could substantially improve semen parameters, and reduce sperm DFI and seminal MDA levels in the AOCVc patients. However, the non-operative NCVc patients would possibly suffer a severe deterioration of semen parameters accompanied by aberrantly higher levels of sperm DFI and seminal MDA, whereas no differences occurred in the non-operative SubVc patients. Sperm DFI level in the pregnant group was much lower compared to the non-pregnant group (AOCVc, p < 0.01; NCVc, p < 0.05) with the best cutoff value of 19.73%, while no differences in seminal MDA (p > 0.05) could be observed. Finally, a strong positive correlation was found between sperm DFI and seminal MDA (Rs = 0.504, p < 0.01), and they were also closely correlated with crucial semen parameters except normal morphology. Therefore, sperm DNA damage in clinical varicocoele, but not in SubVc, might be associated with the role of seminal reactive oxygen species (ROS) in mediating such damage. Varicocoelectomy could be beneficial for reducing OS injury and sperm DFI, and males with low sperm fragmented-DNA level had more opportunities to become pregnant.

Invasive floating macrophytes reduce greenhouse gas emissions from a small tropical lake.

Floating macrophytes, including water hyacinth (Eichhornia crassipes), are dominant invasive organisms in tropical aquatic systems, and they may play an important role in modifying the gas exchange between water and the atmosphere. However, these systems are underrepresented in global datasets of greenhouse gas (GHG) emissions. This study investigated the carbon (C) turnover and GHG emissions from a small (0.6 km(2)) water-harvesting lake in South India and analysed the effect of floating macrophytes on these emissions. We measured carbon dioxide (CO2) and methane (CH4) emissions with gas chambers in the field as well as water C mineralization rates and physicochemical variables in both the open water and in water within stands of water hyacinths. The CO2 and CH4 emissions from areas covered by water hyacinths were reduced by 57% compared with that of open water. However, the C mineralization rates were not significantly different in the water between the two areas. We conclude that the increased invasion of water hyacinths and other floating macrophytes has the potential to change GHG emissions, a process that might be relevant in regional C budgets.

The induction of ret by Wnt-1 in PC12 cells is atypically dependent on continual Wnt-1 expression.

Wild type PC12 pheochromocytoma cells that had been infected with a Wnt-1-carrying virus and thus express Wnt-1 (PC12/Wnt-1) are known to acquire the same flat cell phenotype as that of spontaneously occurring PC12 flat cell variants except that the latter do not presently express Wnt-1. Flat cell variants of PC12 cells exhibit markedly altered morphology and gene expression. In order to assess the possibility that the spontaneously occurring flat cell variants could have been induced in wild type PC12 cells by previous transient expression of the cell's endogenous Wnt-1, we have isolated PC12/Wnt-1 cells expressing little or no Wnt-1. In spite of absent Wnt-1 expression, they retained their flat cell morphology, glutamate/aspartate transporter activity, increased neu mRNA levels and lack of both norepinephrine transporter activity and nerve growth factor-induced differentiation. Thus, Wnt-1 expression is not required to maintain the flat cell phenotype. However, we identified one gene, ret, whose mRNA level in PC12 was not only increased by Wnt-1 expression, but whose increased mRNA level was also dependent on continual Wnt-1 expression. This finding suggests that the induction of ret by Wnt-1 can be used to elucidate the Wnt-induced signalling pathway in mammalian cells.

Neuronal expression of the NADPH oxidase NOX4, and its regulation in mouse experimental brain ischemia.

Ischemia-induced neuronal damage has been linked to elevated production of reactive oxygen species (ROS) both in animal models and in humans. NADPH oxidase enzymes (NOX-es) are a major enzymatic source of ROS, but their role in brain ischemia has not yet been investigated. The present study was carried out to examine the expression of NOX4, one of the new NADPH oxidase isoforms in a mouse model of focal permanent brain ischemia. We demonstrate that NOX4 is expressed in neurons using in situ hybridization and immunohistochemistry. Ischemia, induced by middle cerebral artery occlusion resulted in a dramatic increase in cortical NOX4 expression. Elevated NOX4 mRNA levels were detectable as early as 24 h after the onset of ischemia and persisted throughout the 30 days of follow-up period, reaching a maximum between days 7 and 15. The early onset suggests neuronal reaction, while the peak period corresponds to the time of neoangiogenesis occurring mainly in the peri-infarct region. The occurrence of NOX4 in the new capillaries was confirmed by immunohistochemical staining. In summary, our paper reports the presence of the ROS producing NADPH oxidase NOX4 in neurons and demonstrates an upregulation of its expression under ischemic conditions. Moreover, a role for NOX4 in ischemia/hypoxia-induced angiogenesis is suggested by its prominent expression in newly formed capillaries.