Altered ureido protein modification profiles in seminal plasma extracellular vesicles of non-normozoospermic men.

Introduction: Extracellular vesicles (EVs) have been recognized as key players in numerous physiological functions. These vesicles alter their compositions attuned to the health and disease states of the organism. In men, significant changes in the proteomic composition(s) of seminal plasma EVs (sEVs) have already been found to be related to infertility. Methods: Methods: In this study, we analyze the posttranslational configuration of sEV proteomes from normozoospermic (NZ) men and non-normozoospermic (non-NZ) men diagnosed with teratozoospermia and/or asthenozoospermia by unbiased, discovery-driven proteomics and advanced bioinformatics, specifically focusing on citrulline (Cit) and homocitrulline (hCit) posttranscriptional residues, both considered product of ureido protein modifications. Results and discussion: Significant increase in the proteome-wide cumulative presence of hCit together with downregulation of Cit in specific proteins related to decisive molecular functions have been encountered in sEVs of non-NZ subjects. These findings identify novel culprits with a higher chance of affecting fundamental aspects of sperm functional quality and define potential specific diagnostic and prognostic non-invasive markers for male infertility.

Cumulus Cell DNA Damage as an Index of Human Oocyte Competence.

The determination of oocyte quality is crucial for achieving effective syngamy post-sperm injection and embryonic development. Cumulus cells (CCs) have been proposed as biomarkers of oocyte quality because of their close bio-dynamic relationship with the oocyte. To determine the quality of the oocyte, CCs were sampled during oocyte preparation for ICSI to determine a CC DNA fragmentation index (CCDFI) of each individual oocyte using a variant of the chromatin dispersion test. One hundred and thirty oocytes were selected and studied from two Spanish fertility clinics, 90 of which were fertilized and developed to embryos. Significant differences were found between the CCDFI of unfertilized and fertilized oocytes (p < .001) and between the CCDFI of embryos that were discarded and those that developed suitable for transfer or cryopreservation (p < .001). Oocyte quality was negatively correlated with CCDFI (Spearman's rho = - 0.45; p < .001). Receiver operator characteristics curves (ROC) suggested that a cut-off value of 24% CCDFI was able to discriminate the capacity of the gametes to result in syngamy with a sensitivity and specificity of 75.6% and 65%, respectively. This cut-off supports the application of CCDFI as potential index for the evaluation of the reproductive potential of oocytes prior to fertilization.

Evaluation of oxidative stress: Nanoparticle-based electrochemical sensors for hydrogen peroxide determination in human semen samples.

We have developed electrochemical sensors for the determination of H2O2 in a complex matrix such as human semen as a method to evaluate oxidative stress related to male infertility. Our sensors are based on the modification of conventional electrode surfaces with nanoparticles. We used diamond nanoparticles (DNp) either on glassy carbon or gold surfaces (GC/DNp and Au/DNp sensors, respectively), and copper nanoparticles electrochemically generated directly on glassy carbon surfaces (GC/CuNp). The morphology of the modified electrode surfaces was characterized by Atomic Force Microscopy (AFM), and the H2O2 determination performance evaluated by chronoamperometric measurements at different applied potentials. The best results are obtained for GC/DNp at +1.0 V, Au/DNp at -0.6 V and GC/CuNp at +0.2 V with detection limits (LD) of 1.1 µM, 2.4 µM and 2.6 µM, respectively. The analysis of H2O2 in doped synthetic semen using the GC/CuNp sensor shows the best recoveries, reaching a mean value of 103%. The GC/CuNp sensor was successfully applied to H2O2 analysis in real human semen. In this case, a H2O2 concentration of 1.42 ± 0.05 mM is found and recoveries of 102% on average are obtained.

Association of polymorphisms in genes coding for antioxidant enzymes and human male infertility.

PURPOSE: Although oxidative stress is thought to be an important cause of male infertility, primarily due to DNA and cell membrane damage, little is known about the genetic causes underlying suboptimal function of the seminal enzymatic antioxidant system. The aim of this study was to investigate the relationship of four potentially functional polymorphisms associated with oxidative stress pathway genes (superoxide dismutase-SOD2 lle58Thr and SOD2 rs4880, catalase-CAT C-262T, glutathione peroxidase 1-GPX1 Pro200Leu) and two null variants of the glutathione S transferase (GSTT and GSTM) genes and infertility risk. METHODS: A case control study was conducted on 313 infertile patients and 80 fertile donors. Each ejaculate was subjected to a seminal analysis that included the classical parameters seminal volume, sperm concentration, sperm motility, and sperm morphology, as well as sperm DNA fragmentation (patients only). Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and PCR multiplex methods were carried out for genotyping. RESULTS: Statistically significant differences were found between fertile donors and infertile patients for SNP CAT C-262T; the CC genotype was related with a twofold increased risk of infertility (odds ratio [OR] = 2.262; 95% confidence interval [CI] = 1.369-3.733; P = 0.001), whereas the CT genotype was associated with a protective effect (OR = 0.401; 95% CI = 0.241-0.667; P = 0.001). Surprisingly, the SOD2 Ile58ssThr SNP was not represented in the sample population, so its frequency in the current population frequenting fertility clinics in Madrid may be very low. CONCLUSIONS: Our results suggest that the CAT SNP C-262T is potentially associated with an increased risk of male infertility.

DNA Damage and Repair in Human Reproductive Cells.

The fundamental underlying paradigm of sexual reproduction is the production of male and female gametes of sufficient genetic difference and quality that, following syngamy, they result in embryos with genomic potential to allow for future adaptive change and the ability to respond to selective pressure. The fusion of dissimilar gametes resulting in the formation of a normal and viable embryo is known as anisogamy, and is concomitant with precise structural, physiological, and molecular control of gamete function for species survival. However, along the reproductive life cycle of all organisms, both male and female gametes can be exposed to an array of "stressors" that may adversely affect the composition and biological integrity of their proteins, lipids and nucleic acids, that may consequently compromise their capacity to produce normal embryos. The aim of this review is to highlight gamete genome organization, differences in the chronology of gamete production between the male and female, the inherent DNA protective mechanisms in these reproductive cells, the aetiology of DNA damage in germ cells, and the remarkable DNA repair mechanisms, pre- and post-syngamy, that function to maintain genome integrity.

Priming Equine Bone Marrow-Derived Mesenchymal Stem Cells with Proinflammatory Cytokines: Implications in Immunomodulation-Immunogenicity Balance, Cell Viability, and Differentiation Potential.

Mesenchymal stem cells (MSCs) have a great potential for treating equine musculoskeletal injuries. Although their mechanisms of action are not completely known, their immunomodulatory properties appear to be key in their functions. The expression of immunoregulatory molecules by MSCs is regulated by proinflammatory cytokines; so inflammatory priming of MSCs might improve their therapeutic potential. However, inflammatory environment could also increase MSC immunogenicity and decrease MSC viability and differentiation capacity. The aim of this study was to assess the effect of cytokine priming on equine bone marrow-derived MSC (eBM-MSC) immunoregulation, immunogenicity, viability, and differentiation potential, to enhance MSC immunoregulatory properties, without impairing their immune-evasive status, viability, and plasticity. Equine BM-MSCs (n = 4) were exposed to 5 ng/mL of TNFα and IFNγ for 12 h (CK5-priming). Subsequently, expression of genes coding for immunomodulatory, immunogenic, and apoptosis-related molecules was analyzed by real-time quantitative polymerase chain reaction. Chromatin integrity and proliferation assays were assessed to evaluate cell viability. Trilineage differentiation was evaluated by specific staining and gene expression. Cells were reseeded in a basal medium for additional 7 days post-CK5 to elucidate if priming-induced changes were maintained along the time. CK5-priming led to an upregulation of immunoregulatory genes IDO, iNOS, IL-6, COX-2, and VCAM-1. MHC-II and CD40 were also upregulated, but no change in other costimulatory molecules was observed. These changes were not maintained 7 days after CK5-priming. Viability and differentiation potential were maintained after CK5-priming. These findings suggest that CK5-priming of eBM-MSCs could improve their in vivo effectiveness without affecting other eBM-MSC properties.

Types, causes, detection and repair of DNA fragmentation in animal and human sperm cells.

Concentration, motility and morphology are parameters commonly used to determine the fertilization potential of an ejaculate. These parameters give a general view on the quality of sperm but do not provide information about one of the most important components of the reproductive outcome: DNA. Either single or double DNA strand breaks can set the difference between fertile and infertile males. Sperm DNA fragmentation can be caused by intrinsic factors like abortive apoptosis, deficiencies in recombination, protamine imbalances or oxidative stress. Damage can also occur due to extrinsic factors such as storage temperatures, extenders, handling conditions, time after ejaculation, infections and reaction to medicines or post-testicular oxidative stress, among others. Two singular characteristics differentiate sperm from somatic cells: Protamination and absence of DNA repair. DNA repair in sperm is terminated as transcription and translation stops post-spermiogenesis, so these cells have no mechanism to repair the damage occurred during their transit through the epididymis and post-ejaculation. Oocytes and early embryos have been shown to repair sperm DNA damage, so the effect of sperm DNA fragmentation depends on the combined effects of sperm chromatin damage and the capacity of the oocyte to repair it. In this contribution we review some of these issues.

Design of vectors for transgene expression: The use of genomic comparative approaches.

The design of transgenes has always been limited by the extent of available information on the endogenous locus whose expression pattern had to be replicated. Those genes whose expression domain had not been entirely documented resulted, usually, in transgenes with an unpredictable expression patterns and suboptimal performance in transgenic animals. The use of genomic comparative approaches, highlighting evolutionary conserved homologous DNA sequences, helps to identify crucial regulatory elements that are associated to a given expression domain. The inclusion of these conserved regulatory sequences in transgenic constructs would normally result in optimal expression levels of transgenes in recipient animals. The use of artificial chromosome-type transgenes usually ensures the inclusion of these preserved regulatory elements that are required for the faithful expression of the gene. These constructs could also contain insulators, a subset of regulatory sequences whose application is being addressed in transgenesis. Therefore, the generation of transgenic animals with genomic-type constructs is the recommended approach to achieve optimal transgene expression, according to the expected pattern of the corresponding endogenous locus.

The g.763G>C SNP of the bovine FASN gene affects its promoter activity via Sp-mediated regulation: implications for the bovine lactating mammary gland.

Fatty acid synthase (FASN) is an enzyme that catalyzes de novo synthesis of fatty acids in cells. The bovine FASN gene maps to BTA 19, where several quantitative trait loci for fat-related traits have been described. Our group recently reported the identification of a single nucleotide polymorphism (SNP), g.763G>C, in the bovine FASN 5' flanking region that was significantly associated with milk fat content in dairy cattle. The g.763G>C SNP was part of a GC-rich region that may constitute a cis element for members of the Sp transcription factor family. Thus the SNP could alter the transcription factor binding ability of the FASN promoter and consequently affect the promoter activity of the gene. However, the functional consequences of the SNP on FASN gene expression are unknown. The present study was therefore directed at elucidating the underlying molecular mechanism that could explain the association of the SNP with milk fat content. Three cellular lines (3T3L1, HepG2, and MCF-7) were used to test the promoter and the transcription factor binding activities by luciferase reporter assays and electrophoretic mobility shift assays, respectively. Band shift assays were also carried out with nuclear extracts from lactating mammary gland (LMG) to further investigate the role of the SNP in this tissue. Our results demonstrate that the SNP alters the bovine FASN promoter activity in vitro and the Sp1/Sp3 binding ability of the sequence. In bovine LMG, the specific binding of Sp3 may account for the association with milk fat content.

Papel de la caveolina-1 en la regeneración hepática tras hepatectomía parcial. Mecanismos de señalización y función en la regulación de la proliferación hepática / Role of caveolin 1 on liver regeneration after partial hepatectomy. Mechanisms of signalling and effect on the regulation of hepatocyte proliferation

Las caveolas participan en múltiples procesos celulares tales como el transportevesicular, homeostasis del colesterol, regulación de la señalización intracelular,por integrinas y proliferación celular. Sin embargo, su función en el hígado no estábien establecida. La expresión de caveolina 1 (Cav), la proteína más abundante enlas caveolas, está bien descrita en el hígado y en varias líneas de hepatocitos y enhígado cirrótico humano y en carcinoma hepatocelular. Sin embargo, el papel deCav-1 en la fisiopatología hepática es controvertido, ya que se ha propuesto un papel crítico en el proceso de regeneración tras hepatectomía parcial (HP). Contrariamentea esta observación, nuestros datos sugieren que Cav-1 aumenta en elhígado regenerante, con una re-distribución de la proteína desde las caveolas haciadominios no caveolares. Además, la Cav-1 localizada en estas fracciones está fosforiladaen la tirosina 14. A pesar de ello, el gen de la Cav-1 es dispensable parala regeneración hepática tras HP, tal como se deduce de animales que carecen deeste gen. En conjunto, estos datos muestran un papel dinámico de la Cav-1 en laproliferación hepática tras HP y en líneas hepáticas en cultivo, pero con mínimasimplicaciones en el proceso regenerativo

Although caveolae participate in many cellular processes such as vesicular transport,cholesterol homeostasis, regulation of signal transduction, integrin signalingand cell growth, their role in liver remains elusive. Expression of caveolin 1 (Cav),the most abundant protein of caveolae, has been reported in liver and in differenthepatocyte cell lines, in human cirrhotic liver and in hepatocellular carcinomas.However, the role of Cav-1 in liver pathophysiology remains controversial and acritical role in regeneration after partial hepatectomy (PH) has been reported.Opposite to this observation, our data support the view that Cav-1 increases inliver after PH with a redistribution of the protein from the caveolae enricheddomain to the noncaveolar fraction. Moreover, the Cav-1 located in the noncaveolarfraction is phosphorylated in tyrosine 14 (Tyr14). Even though, the Cav-1 geneis dispensable for liver regeneration after PH as deduced from data obtained withcommercially available animals lacking this gene. Taken together these resultssupport a dynamic role for Cav-1 in liver proliferation both in vivo after PH, andin vitro in cultured hepatic cell lines, but with minimal implications in the liverregeneration process

Dispensability and dynamics of caveolin-1 during liver regeneration and in isolated hepatic cells.

UNLABELLED: Caveolae participate in several cellular processes such as vesicular transport, cholesterol homeostasis, regulation of signal transduction, integrin signaling, and cell growth. The expression and functional role of caveolin (Cav), the most abundant protein of caveolae, has been reported in liver and in different hepatocyte cell lines, in human cirrhotic liver, and in hepatocellular carcinomas. The role of Cav-1 in liver regeneration after partial hepatectomy (PH) has been investigated as a model of liver proliferation in vivo. Our results show that Cav-1 increases in liver after PH with a redistribution of the protein from the caveola-enriched domain to the noncaveolar fraction. Moreover, the Cav-1 located in the noncaveolar fraction is phosphorylated in tyrosine 14, even though the Cav-1 gene is dispensable for liver regeneration after PH, as deduced from data obtained with commercially available animals lacking this gene. In addition to this, the proinflammatory stimulation of hepatocytes induces Cav-1 translocation to a noncaveolar fraction and tyrosine 14 phosphorylation mainly through the activation of tyrosine kinases such as Src. CONCLUSION: These results support a dynamic role for Cav-1 in liver proliferation both in vivo after PH and in vitro in cultured hepatic cell lines, but with minimal implications for the liver regeneration process.

Developmentally regulated activation of a SINE B2 repeat as a domain boundary in organogenesis.

The temporal and spatial regulation of gene expression in mammalian development is linked to the establishment of functional chromatin domains. Here, we report that tissue-specific transcription of a retrotransposon repeat in the murine growth hormone locus is required for gene activation. This repeat serves as a boundary to block the influence of repressive chromatin modifications. The repeat element is able to generate short, overlapping Pol II-and Pol III-driven transcripts, both of which are necessary and sufficient to enable a restructuring of the regulated locus into nuclear compartments. These data suggest that transcription of interspersed repetitive sequences may represent a developmental strategy for the establishment of functionally distinct domains within the mammalian genome to control gene activation.

Protection against Fas-induced liver apoptosis in transgenic mice expressing cyclooxygenase 2 in hepatocytes.

UNLABELLED: Cyclooxygenase-2 (COX-2) is upregulated in many cancers, and the prostanoids synthesized increase proliferation, improve angiogenesis, and inhibit apoptosis in several tissues. To explore the function of COX-2 in liver, transgenic (Tg) mice were generated containing a fusion gene (LIVhCOX-2) consisting of human COX-2 cDNA under the control of the human ApoE promoter. Six lines were developed; all of them expressed the LIVhCOX-2 transgene selectively in hepatocytes. The Tg mice exhibited a normal phenotype, and the increased levels of PGE2 found were due to the constitutively expressed COX-2. Histological analysis of different tissues and macroscopic examination of the liver showed no differences between wild-type (Wt) and Tg animals. However, Tg animals were resistant to Fas-mediated liver injury, as demonstrated by low levels of plasmatic aminotransferases, a lesser caspase-3 activation, and Bax levels and an increase in Bcl-2, Mcl-1, and xIAP proteins, when compared with the Wt animals. Moreover, the resistance to Fas-mediated apoptosis is suppressed in the presence of COX-2-selective inhibitors, which prevented prostaglandin accumulation in the liver of Tg mice. CONCLUSION: These results demonstrate that expression of COX-2-dependent prostaglandins exerted a protection against liver apoptosis.