Effect of normobaric hypoxia on the testis in a murine model.

High-altitude hypoxia generates spermiogram impairment due to germinal epithelium, Leydig cells, sperm and seminal plasma alterations, but precise mechanisms involved are unknown. The objective of this work was to analyse the effect of normobaric hypoxia on the morphology of testicular interstitium and some associated molecular and hormonal factors. Twenty-four mice were exposed to normobaric hypoxia (8.1% inspired oxygen fraction) during 20 days. The effects on body weight, testicular weight, vascularisation, testosterone, HIF1-α and VEGF were analysed at different periods of exposure and compared to controls. Hypoxic mice had lower body weight than mice kept in normoxia. Testicular weight raised significantly the 1st day, but remained normal during the rest of experiment. Number of blood vessels per field and mean diameter of vessels were higher in hypoxic mice. Plasmatic and testicular testosterone raised during first 24 h of hypoxia, but decreased on the 5th day. Vascular/interstitial ratio (proportion of interstice occupied by blood vessels) duplicated at the end of the experiment. Most substantial early effects of hypoxia were testicular oedema, increase in number and diameter of blood vessels and elevation of plasmatic and testicular testosterone. Normobaric hypoxia generates similar effects to those induced by hypobaric hypoxia.

Competency assessment of residents of Intensive Care Medicine through a simulation-based objective structured clinical evaluation (OSCE). A multicenter observational study.

OBJECTIVES: The current official model of training in Intensive Care Medicine (ICM) in Spain is based on exposure to experiences through clinical rotations. The main objective was to determine the level of competency (I novice to V independent practitioner) achieved by the residents at the end of the 3rd year of training (R3) in ICM through a simulation-based OSCE. Secondary objectives were: (1) To identify gaps in performance, and (2) To investigate the reliability and feasibility of conducting simulation-based assessment at multiple sites. DESIGN: Observational multicenter study. SETTING: Thirteen Spanish ICU Departments. PARTICIPANTS: Thirty six R3. INTERVENTION: The participants performed on five, 15-min, high-fidelity crisis scenarios in four simulation centers. The performances were video recorded for later scoring by trained raters. MAIN VARIABLES OF INTEREST: Via a Delphi technique, an independent panel of expert intensivists identified critical essential performance elements (CEPE) for each scenario to define the levels of competency. RESULTS: A total of 176 performances were analyzed. The internal consistency of the check-lists were adequate (KR-20 range 0.64-0.79). Inter-rater reliability was strong [median Intraclass Correlation Coefficient across scenarios: 0.89 (0.65-0.97)]. Competency levels achieved by R3 were: Level I (18.8%), II (35.2%), III (42.6%), IV/V (3.4%). Overall, a great heterogeneity in performance was observed. CONCLUSION: The expected level of competency after one year in the ICU was achieved only in half of the performances. A more evidence-based educational approach is needed. Multiple center simulation-based assessment showed feasibility and reliability as an evaluation method of competency. TRIAL REGISTRATION: COBALIDATION. NCT04278976. (https://register. CLINICALTRIALS: gov).

Transplantation of Human Amniotic Membrane over the Liver Surface Reduces Hepatic Fibrosis in a Cholestatic Model in Young Rats.

PURPOSE: Biliary atresia precedes liver cirrhosis and liver transplantation. Amniotic membrane (AM) promotes tissue regeneration, inhibits fibrosis, and reduces inflammation. Here, we test amniotic membrane potential as a therapeutic tool against cholestatic liver fibrosis. METHODS: Three groups of rats were used: sham surgery (SS), bile duct ligature (BDL), and bile duct ligature plus human amniotic membrane (BDL + AM). After surgery, animals were sacrificed at different weeks. Biochemical and histopathological analyses of liver tissue were performed. Collagen was expressed as a percentage of total liver tissue area. qPCR was performed to analyse gene expression levels of transforming growth factor-ß1 (Tgfb1) and apelin (Apln). Statistical analysis performed considered p < 0.05 was significant. RESULTS: Groups undergoing BDL developed cholestasis. Biochemical markers from BDL + AM group improved compared to BDL group. Ductular reaction, portal fibrosis, and bile plugs were markedly reduced in the BDL + AM group compared to BDL group. Collagen area in BDL + AM group was statistically decreased compared to BDL group. Finally, expression levels of both Apln and Tgfb1 mRNA were statistically downregulated in BDL + AM group versus BDL group. CONCLUSION: AM significantly reduces liver fibrosis in a surgical animal model of cholestasis. Our results suggest that AM may be useful as a therapeutic tool in liver cirrhosis.

DNA polymerase lambda (Pol lambda), a novel eukaryotic DNA polymerase with a potential role in meiosis.

A new gene (POLL) encoding a novel DNA polymerase (Pol lambda) has been identified at mouse chromosome 19. Murine Pol lambda, consisting of 573 amino acid residues, has a 32% identity to Pol beta, involved in nuclear DNA repair in eukaryotic cells. It is interesting that Pol lambda contains all the critical residues involved in DNA binding, nucleotide binding and selection, and catalysis of DNA polymerization, that are conserved in Pol beta and other DNA polymerases belonging to family X. Murine Pol lambda, overproduced in Escherichia coli, displayed intrinsic DNA polymerase activity when assessed by in situ gel analysis. Pol lambda also conserves the critical residues of Pol beta required for its intrinsic deoxyribose phosphate lyase (dRPase) activity. The first 230 amino acid residues of Pol lambda, that have no counterpart in Pol beta, contain a BRCT domain, present in a variety of cell-cycle check-point control proteins responsive to DNA damage and proteins involved in DNA repair. Northern blotting, in situ hybridization analysis and immunostaining showed high levels of Pol lambda specifically expressed in testis, being developmentally regulated and mainly associated to pachytene spermatocytes. These first evidences, although indirect, suggest a potential role of Pol lambda in DNA repair synthesis associated with meiosis.

XYbp, a novel RING-finger protein, is a component of the XY body of spermatocytes and centrosomes.

RING-finger proteins participate in developmental processes, including gametogenesis. A fetal oocyte cDNA library was used to select genes expressed during male germ-cell differentiation. A novel RING-finger protein, XYbp (XY body protein), participating in mouse spermatogenesis has been identified. This novel gene generates a ubiquitously expressed transcript of 4.2 kb and a testis-specific one of 2.8 kb, processed by an alternative polyadenylation mechanism from a non-canonical polyadenylation signal. Transcription of XYbp is regulated during spermatocyte differentiation. The antiserum raised against the XYbp peptide demonstrated that XYbp is localised mainly in the XY bivalent of spermatocytes (XY body) and in the centrosomes of somatic and germ cells in all phases of the cell cycle. These studies indicate that we have identified a new member of the RING-finger family of proteins associated with the XY meiotic bivalent during spermatogenesis development and with the centrosomes of all cells.

Ilf2 is regulated during meiosis and associated to transcriptionally active chromatin.

Analysis of gene expression during testis development demonstrated accumulation of Ilf2 mRNA in pachytene spermatocytes. In these cells, the protein was localized in the nucleus, but it was absent from chromatin of the XY pachytene bivalent, in which there is no transcriptional activity. Nucleolar signal is inmmunolocalized in spermatogonia, Sertoli cells and oocytes. By in situ hybridisation, Ilf2 expression is detected in proliferative cells of adult ovary and a defined pattern is also exhibited in different tissues of embryos. The presence of ILF2 in active chromatin is corroborated in NIH3T3 cultured cells after transfection with Ilf2-EGFP constructs.

Fhx (Foxj2) expression is activated during spermatogenesis and very early in embryonic development.

FHX (FOXJ2) is a recently characterized human fork head transcriptional activator that binds DNA with a dual sequence specificity. We have cloned the cDNA for the mouse orthologue Foxj2 and characterized its expression in the gonads and along the early pre-implantation development of the mouse. In the testis, Foxj2 is expressed from pachytene spermatocytes to round spermatids, but not in spermatogonia. In addition to the germ lineage, only Sertoli cells of the testis showed expression of Foxj2. In the ovary, only granulosa cells of the follicles express the factor. Neither mature spermatozoa nor oocytes showed expression of Foxj2. Foxj2 expression is early activated in zygotic development, being detected since as early as 8-cell stage embryos. Both cell layers of the blastocyst: the trophectoderm (TE) and the inner cell mass (ICM), express Foxj2.

Differential expression of Ran GTPase during HMBA-induced differentiation in murine erythroleukemia cells.

Murine erythroleukemia (MEL) cells undergo erythroid differentiation in vitro when treated with hexamethylene bisacetamide (HMBA). To identify genes involved in the commitment of MEL cells to differentiate, we screened a cDNA library constructed from HMBA-induced cells by differential hybridization and isolated GTPase Ran as a down-regulated gene. We observed that Ran was expressed in a biphasic mode. Following a decrease in mRNA level during the initial hours of induction, Ran re-expressed at 24-48 h, and gradually declined again. To investigate the role of Ran during MEL differentiation we constructed MEL transfectants capable to express or block Ran mRNA production constitutively. No effects were observed on cell growth and proliferation. Blockage of Ran, however, interfered with MEL cell differentiation resulting in a decrease of cell survival in the committed population.

Quetiapine treatment of children with Tourette's syndrome: report of two cases.

Two children with Tourette's syndrome and comorbid disorders were treated with quetiapine, an atypical antipsychotic successfully used in patients with psychoses and schizophrenia with low incidence of extrapyramidal side effects. Clinical observations and standardized rating scales suggested that this drug produced beneficial effects on tics and other symptoms. Adverse effects (at low doses) were minimal. Because it was suggested that tic efficacy of the newer antipsychotics was related to higher D2 occupancy (with the exception of quetiapine and clozapine, which have relatively low D2 activity), it is hypothesized that tic patients are D2 sensitive and need lower doses of medications. These children were treated naturalistically and were reported retrospectively because of their encouraging outcomes. However, these findings should be interpreted with caution, because no contrast groups, drug withdrawal, or placebo were utilized. Controlled studies are needed to determine the efficacy of quetiapine in the treatment of Tourette's syndrome.

Serum protein concentrations in horses with severe liver disease: a retrospective study and review of the literature.

The present retrospective study was undertaken to determine the frequency of hypoproteinemia and hypoalbuminemia in horses with natural occurring severe liver disease. The study represents a review of case records and laboratory data of 84 horses presented with acute or chronic liver disease to the University of California Veterinary Medical Teaching Hospital between 1973 and 1991. Forty horses (48%) had serum protein concentrations above the maximum reference value (7.7 g/dL). The increase in serum protein concentration was associated with hyperglobulinemia (P = .00005, R2 = .80). Only 13% (11/84) of the horses had serum albumin concentrations below the minimum reference range (2.5 g/dL), and hypoproteinemia was found in only 1 of these horses. Of these, 18% (9/51) of the horses with chronic liver disease and 6% (2/33) of the horses with acute liver disease had albumin concentrations below the minimum reference value. Globulin concentrations in 64% of the horses (54/84) were above the maximum reference value (4.0 g/dL). The present study indicates that hypoproteinemia and hypoalbuminemia are not common features in horses with severe liver disease.

A clinical trial of probiotic administration for prevention of Salmonella shedding in the postoperative period in horses with colic.

The purpose of this study was to evaluate the effects of probiotic administration on the prevalence of fecal shedding of Salmonella, the prevalence of postoperative diarrhea, the length of antimicrobial therapy, and the length of the hospitalization stay during the postoperative period in horses with colic. Two commercially available probiotics for horses were used in a double-blind prospective study of 200 horses undergoing surgery for colic. Probiotic or placebo was administered PO once a day for 7 days postoperatively, and fecal cultures for Salmonella were obtained daily for 10 days. After selection of 186 patients completing the treatment protocol, the results indicated that the commercial probiotic formulations had no effect on Salmonella shedding, prevalence of diarrhea, length of antimicrobial therapy, or length of hospitalization (P > .05). Twenty percent of the horses yielded 1 or more positive fecal cultures for Salmonella; of these horses, 74% were classified as asymptomatic shedders. Twenty-six percent of all horses had fluid diarrhea postoperatively, with only 12% of these horses having positive fecal cultures for Salmonella. The most common isolate was Salmonella krefeld (24 of 39 isolates). Among the different gastrointestinal disorders, horses with feed and sand impactions appeared to be more prone to shed Salmonella.

Total serum bile acids and the bile acid profile as tests of liver function.

Total serum bile acid assay for the evaluation of liver function has been available for many years but its application has been limited primarily by factors such as methodology, equipment and cost. New and improved methods for bile acid assay such as the radioimmunoassay or the hydroxysteroid dehydrogenase techniques have brought the assay for bile acids into the realm of the clinical laboratory. The efficacy of bile acids for clinical diagnostic use in the evaluation of liver function has not been firmly established. Newer methods using high pressure liquid chromatography to develop a profile of the different bile acids may clarify its usefulness and define its role among the many available tests of liver function in animals.

Stat3 and Socs3 expression patterns during murine placenta development.

Signal transducers and activators of transcription 3 (Stat3) has been identified as an important signal transducer in the invasive phenotype of the trophoblasts cells in in vitro studies. However, the in situ distribution and patterns of expression of this molecule in trophoblast cells during the development of the placenta are still under-elucidated. Mice uteri of gestational ages between 7 and 14 days of pregnancy (dop) were fixed in methacarn and processed with immunoperoxidase techniques for detection of Stat3 and its phosphorylation at serine (p-ser727) residues, as well as the suppressor of cytokine signaling 3 (Socs3) expression. Stat3 was observed at 7 through 9 dop in both the antimesometrial and mesometrial deciduas, while continued immunoreactivity between 10 and 13 dop was seen only in the mesometrial decidua. In the placenta, Stat3 was detected in the cytotrophoblast cells of labyrinth and giant trophoblast cells between 10 and 14 dop. Immunoreactivity for Stat3 was also seen in trophoblast cells surrounding the maternal blood vessels. On days 10 and 11 of pregnancy, p-ser727 was detectable in the mesometrial decidua and in giant trophoblasts, while during 12-14 dop in the spongiotrophoblast region. In addition, Socs3 was immunodetected in maternal and placental tissues, principally in the giant trophoblast cells during the whole period of the study. The present in situ study shows the distribution of Stat3, its serine activation and Socs3 in different maternal and fetal compartments during murine placental development, thus further supporting the idea that they play a role during physiological placentation in mice. 

Immunotoxicological effects of inorganic arsenic on gilthead seabream (Sparus aurata L.).

Arsenic (As) has been associated with multitude of animal and human health problems; however, its impact on host immune system has not been extensively investigated. In fish, there are very few works on the potential risks or problems associated to the presence of arsenic. In the present study we have evaluated the effects of exposure (30 days) to sub-lethal concentrations of arsenic (5 µM As2O3) in the teleost fish gilthead seabream (Sparus aurata), with special emphasis in the innate immune response. The arsenic concentration was determined using atomic fluorescence spectrometry (AFS) in liver and muscle of exposed fish showing As accumulation in the liver after 30 days of exposure. The hepatosomatic index was increased at significant extent after 10 days but returned to control values after 30 days of exposure. Histological alterations in the liver were observed including hypertrophy, vacuolization and cell-death processes. Focusing on the immunological response, the humoral immune parameters (seric IgM, complement and peroxidase activities) were no affected to a statistically significant extent. Regarding the cellular innate parameters, head-kidney leucocyte peroxidase, respiratory burst and phagocytic activities were significantly increased after 10 days of exposition compared to the control fish. Overall, As-exposure in the seabream affects the immune system. How this might interfere with fish biology, aquaculture management or human consumers warrants further investigations. This paper describes, for the first time, the immunotoxicological effects of arsenic exposure in the gilthead seabream, which is a species with the largest production in Mediterranean aquaculture.

Phosphoprotein enriched in astrocytes-15 is expressed in mouse testis and protects spermatocytes from apoptosis.

Phosphoprotein enriched in astrocytes (PEA-15) is a 15 kDa acidic serine-phosphorylated protein expressed in different cell types, especially in the CN. We initially detected the expression of PEA-15 in primary cultures of Sertoli cells. To assess the presence and localization of PEA-15 in the mouse testis, we studied the expression pattern of the PEA-15 protein by immunohistochemistry and mRNA by in situ hybridization. Both the protein and the mRNA of PEA-15 were localized in the cytoplasm of Sertoli cells, all types of spermatogonia, and spermatocytes up till zygotene phase of the meiotic prophase. Subsequently, with ongoing development of the spermatocytes, the expression decreased and was very low in the cytoplasm of diplotene spermatocytes. To analyze the possible role of PEA-15 in the developing testis, null mutants for PEA-15 were examined. As the PEA-15 C terminus contains residues for ERK binding, we studied possible differences between the localization of the ERK2 protein in wild type (WT) and PEA-15(-/-)mice. In the WT testis, ERK2 was localized in the cytoplasm of Sertoli cells, B spermatogonia, preleptotene, leptotene, and zygotene spermatocytes, whereas in the KO testis, ERK2 was primarily localized in the nuclei of these cells and only little staining remained in the cytoplasm. Moreover, in PEA-15-deficient mice, significantly increased numbers of apoptotic spermatocytes were found, indicating an anti-apoptotic role of PEA-15 during the meiotic prophase. The increased numbers of apoptotic spermatocytes were not found at a specific step in the meiotic prophase.

Expression of stress inducible protein 1 (Stip1) in the mouse testis.

Phthalate esters are considered endocrine disruptors that interfere with the endocrine balance and development of the mammalian testis. Mono-2-ethylhexyl phthalate (MEHP), the active metabolite of the ubiquitously used plasticizer di-2-ethylhexyl phthalate (DEHP), acts upon Sertoli cells as initial target. By subtractive cDNA libraries we identified genes deregulated as response to MEHP in primary cultures of mouse Sertoli cells. The expression of mouse stress inducible protein 1 (Stip1) was detected as upregulated as a result of MEHP exposure. Stip1 is a cochaperone protein that is homologous to the human heat shock cognate protein 70 (hsc70)/heat shock protein 90 (hsp90)-organizing protein (Hop). To assess the presence and localization of Stip1 in mouse testis and its potential role in stress defense, we studied the expression pattern of the Stip1 protein by immunohistochemistry and of the mRNA by in situ hybridization. Both the protein and the mRNA of Stip1 were mainly found in the cytoplasm of all types of spermatogonia and spermatocytes up till zygotene, the expression decreased during late pachytene and was very weak in diplotene spermatocytes and round spermatids. Interestingly, this expression pattern resembled the pattern of stress sensitivity of spermatogenic cells in that the most sensitive cell types show the weakest expression of Stip1. This suggests an important role for Stip1 in the ability of germ cells to survive in stress conditions including high temperatures.

Lysophosphatidylcholine treatment allows interspecies fertilization of hamster oocytes with zona pellucida by human spermatozoa.

The study of interspecific gamete interactions has been improved by the use of in vitro fertilization techniques. Zona pellucida is at the cellular level the main barrier to cross-fertilization. Moreover, there are several cases in which the heterologous spermatozoa fail to enter the cytoplasm of zona-free oocytes, emphasizing the notion that another important barrier exists to cross-fertilization at the egg plasma membrane level. We report here a simple method to fertilize hamster oocytes with zona pellucida by human spermatozoa. We used lysophosphatidylcholine (LPC), a fusogenic agent, to treat spermatozoa, spermatozoa and zona-intact oocytes, or zona-intact oocytes alone. This molecule allows the penetration of hamster oocytes zona by human spermatozoa. Our results suggest that LPC modifies the zona pellucida of oocytes and the plasma membrane of both gametes in vitro, favouring sperm penetration through the zona pellucida and gamete fusion.

Role of phospholipase A2 in mammalian sperm-egg fusion: development of hamster oolemma fusibility by lysophosphatidylcholine.

Phospholipase A2 (PLA2), its localization on human sperm and its involvement in sperm-egg interaction, was investigated. Sperm-egg interaction was examined using an in vitro assay of the interaction between human sperm and zona-free or zona-intact hamster egg. PLA2- specific antibodies and/or lysophosphatidylcholine (LPC) were added to the coincubation medium. PLA2 was localized on the anterior tip of the human sperm head by an immunogold silver staining method in light microscopy (IGSS) and TEM. PLA2-specific antibodies inhibited human sperm-zona-free oocyte fusion significantly. LPC treatment allows interspecies fertilization of zona-intact hamster oocytes. PLA2 plays an important role in membrane-fusion events. This statement is supported by the fact that PLA2 is localized in the region where an exocytotic event, such as the acrosome reaction, occurs in the spermatozoon. PLA2-specific antibodies inhibited sperm-egg fusion, but not sperm-oolemma adhesion. LPC may stimulate the fertilizing ability of spermatozoa and induce changes on the zona pellucida and on the oolemma promoting in sperm-egg fusion. Based on these findings, it is suggested that sperm PLA2 and one of its modulators, the LPC, may contribute to membrane-fusion events in mammalian fertilization.

Study of the acrosome reaction and the fertilizing ability of hamster epididymal cauda spermatozoa treated with antibodies against phospholipase A2 and/or lysophosphatidylcholine.

The present report describes experiments in vitro that were designed to evaluate the involvement of phospholipase A2 (PLA2) in the acrosome reaction of mammalian sperm and the interaction of gametes. Hamster spermatozoa were incubated in a defined medium (TALP) to induce capacitation and the acrosome reaction. This medium was supplemented with antibodies against porcine pancreatic PLA2 and/or lysophosphatidylcholine (LPC). For in vitro fertilization, spermatozoa and/or oocytes were incubated in TALP medium that contained PLA2-specific antibodies, LPC, or antibodies plus LPC. The antibodies inhibited the acrosome reaction in a dose-dependent manner, without any effect on sperm motility or hyperactivation. These antibodies also inhibited fertilization in vitro. LPC, a product of the reaction catalysed by PLA2, speeds up and synchronizes the acrosome reaction and facilitates penetration of the zona pellucida by spermatozoa, the fusion process and polyspermy. The results of addition of the antibodies plus LPC showed that LPC is able to reverse the inhibitory effects of the antibodies on the acrosome reaction and fertilization. It is possible that endogenous PLA2 plays a role in the final stages of the acrosome reaction and the interaction of gametes, perhaps through one of its reaction products, LPC. The role of LPC might be to stimulate the fertilizing ability of spermatozoa, as well as to induce changes in the zona pellucida and the oolemma that allow sperm-egg fusion. Thus, it seems possible that PLA2 and one of its reaction products might contribute to membrane-fusion events during mammalian fertilization.