A Sea Anemone Lebrunia neglecta Venom Fraction Decreases Boar Sperm Cells Capacitation: Possible Involvement of HVA Calcium Channels.

Sea anemones produce venoms characterized by a complex mixture of low molecular weight compounds, proteins and peptides acting on voltage-gated ion channels. Mammal sperm cells, like neurons, are characterized by their ion channels. Calcium channels seem to be implicated in pivotal roles such as motility and capacitation. In this study, we evaluated the effect of a low molecular weight fraction from the venom of the sea anemone Lebrunia neglecta on boar sperm cells and in HVA calcium channels from rat chromaffin cells. Spermatozoa viability seemed unaffected by the fraction whereas motility and sperm capacitation were notoriously impaired. The sea anemone fraction inhibited the HVA calcium current with partial recovery and no changes in chromaffin cells' current kinetics and current-voltage relationship. These findings might be relevant to the pharmacological characterization of cnidarian venoms and toxins on voltage-gated calcium channels.

Perfluorooctane sulfonate and perfluorooctanoic acid induce plasma membrane dysfunction in boar spermatozoa during in vitro capacitation.

Spermatozoa require the capacitation, a series of biochemical events, to perform fertilization. Many toxic compounds can interfere in this process, including perfluorooctane sulfonate (PFOS) and perfluorooctanoic acid (PFOA), which belong to the perfluoroalkyl substances (PFAS). Since both substances are found in many everyday materials and are highly persistent, they accumulate in organisms where they have been associated with fertility problems. This study analyzes the effects of PFOS and PFOA on the functionality of boar spermatozoa, and changes in the plasma membrane (PM) during capacitation. The median lethal concentrations (LC50) of PFOS and PFOA were 460 and 1894 µM, respectively, while the mean inhibitory concentrations of capacitation (ICC50) were 274 µM and 1458 µM, respectively. The ICC50 of PFOA was insufficient to reduce the capacitation, but 950 µM (½ LC50) of PFOA and the ICC50 of PFOS significantly reduced the number of capacitated spermatozoa. PFOS and PFOA also impeded the progesterone (P4)-induced acrosomal reaction (iAR). These effects occur despite the accumulation of [Ca2+]i under capacitating conditions. The accumulation of [Ca2+]i produces saturation, which prevents its entry through ionophore A23187 and P4 in the presence of PFOS. Membrane potential (Emv) was deregulated. Both PFAS affected lipid membrane conductance mediated by valinomycin. The spermatozoa presented 49% and 47% of membrane dysfunction with PFOS and PFOA, respectively. By causing membrane damage, both substances prevented the release of cholesterol and altered the organization of membrane microdomains (MMDs). Data indicate that both PFAS caused alterations in PM functionality.

Beneficial and deleterious effects of sitagliptin on a methionine/choline-deficient diet-induced steatohepatitis in rats.

Non-alcoholic fat liver disease (NAFLD) is the most common chronic liver disease in the world. NAFLD is a spectrum of diseases ranging from simple steatosis to hepatic carcinoma. The complexity of pathomechanisms makes treatment difficult. The oral antidiabetic agents, dipeptidyl peptidase four inhibitors (DPP-4i) have been proposed as possible therapeutic agents. This study was performed using a well-established NAFLD model in rats to elucidate whether sitagliptin could prevent steatohepatitis. Rats were fed a methionine/choline-deficient (MCD) diet with or without sitagliptin treatment for six weeks. Liver tissue was examined to estimate sitagliptin's effect on the development of NASH. The MCD diet decreased the SAM/SAH ratio, and increased plasma levels of homocysteine, free fatty acids, and long-chain acylcarnitines in the MCD rats. MMP2 and Col1A2 expression also increased under the MCD diet. Sitagliptin treatment did not reverse these effects and increased steatosis and long-chain acylcarnitines. In conclusion, sitagliptin was ineffective to prevent from NAFLD in the MCD rat model. This result challenges previous data reporting beneficial effects and is consistent with the clinical trials' negative results.

Boar sperm incubation with reduced glutathione (GSH) differentially modulates protein tyrosine phosphorylation patterns and reorganization of calcium in sperm, in vitro fertilization, and embryo development depending on concentrations.

The sperm in the female's reproductive tract undergo changes to fertilize the oocyte (sperm capacitation). These changes are regulated by redox system. However, some assisted reproductive technologies require sperm capacitation under in vitro conditions, though this increases the generation of ROS. Therefore, the aim of this study was to evaluate the effect of GSH as an antioxidant agent during the capacitation of boar sperm [evaluated by calcium compartmentalization, tyrosine phosphorylation (Tyr-P), motility, viability, and acrosomal integrity], in vitro fertilization (evaluated by penetration, monospermy, and efficiency %), and later embryo development (evaluated by cleavage and blastocyst rates, total number of cells per blastocyst and blastocyst diameter). Four experimental groups with different GSH concentrations (0-control, 0.5, 1, and 5 mM) were formed. When 1-GSH was added to the medium, the percentage of capacitated sperm increased after 4 h of incubation; the localization of Tyr-P was modified at 1 h and 4 h of incubation depending on the GSH concentration. Percentages of total and progressive sperm motility also increased at 4 h of incubation, but only in the 5-GSH group compared to control. Viability, acrosomal integrity, and general Tyr-P (Western blot) not differ among the experimental groups. The addition of GSH during gamete interaction increased penetration, monospermy, and efficiency rates in the 1-GSH group compared to the others. However, the effect of GSH was not observed in cleavage and blastocyst rates compared to the control. In conclusion, adding GSH modulates sperm capacitation (by means of calcium compartmentalization and tyrosine phosphorilation pattern) depending on its concentration, and improves IVF output at 1-GSH during gamete interaction.

Perfluorooctane Sulfonate (PFOS) and Perfluorohexane Sulfonate (PFHxS) Alters Protein Phosphorylation, Increase ROS Levels and DNA Fragmentation during In Vitro Capacitation of Boar Spermatozoa.

Perfluorooctane sulfonate (PFOS) and perfluorohexane sulfonate (PFHxS) are toxic and bioaccumulative, included in the Stockholm Convention's list as persistent organic pollutants. Due to their toxicity, worldwide distribution, and lack of information in spermatozoa physiology during pre-fertilization processes, the present study seeks to analyze the toxic effects and possible alterations caused by the presence of these compounds in boar sperm during the in vitro capacitation. The spermatozoa capacitation was performed in supplemented TALP-Hepes media and mean lethal concentration values of 460.55 µM for PFOS, and 1930.60 µM for PFHxS were obtained. Results by chlortetracycline staining showed that intracellular Ca2+ patterns bound to membrane proteins were scarcely affected by PFOS. The spontaneous acrosome reaction determined by FITC-PNA was significantly reduced by PFOS and slightly increased by PFHxS. Both toxic compounds significantly alter the normal capacitation process from 30 min of exposure. An increase in ROS production was observed by flow cytometry and considerable DNA fragmentation by the comet assay. The immunocytochemistry showed a decrease of tyrosine phosphorylation in proteins of the equatorial and acrosomal zone of the spermatozoa head. In conclusion, PFOS and PFHxS have toxic effects on the sperm, causing mortality and altering vital parameters for proper sperm capacitation.

Identification of lipid raft glycoproteins obtained from boar spermatozoa.

The surface of the spermatozoa is coated with glycoproteins the redistribution of which during in vitro capacitation plays a key role in the subsequent fertilization process. Lipid rafts are membrane microdomains involved in signal transduction through receptors and include or recruit specific types of proteins and glycoproteins. Few studies have focused on identifying glycoproteins resident in the lipid rafts of spermatozoa. Proteins associated with lipid rafts modify their localization during capacitation. The objective of the study was to identify the glycoproteins associated with lipid rafts of capacitated boar spermatozoa through a lectin-binding assay coupled to mass spectrometry approach. From the proteomic profiles generated by the raft proteins extractions, we observed that after capacitation the intensity of some bands increased while that of others decreased. To determine whether the proteins obtained from lipid rafts are glycosylated, lectin blot assays were performed. Protein bands with a good resolution and showing significant glycosylation modifications after capacitation were analyzed by mass spectrometry. The bands of interest had an apparent molecular weight of 64, 45, 36, 34, 24, 18 and 15 kDa. We sequenced the 7 bands and 20 known or potential glycoproteins were identified. According to us, for ten of them this is the first time that their association with sperm lipid rafts is described (ADAM5, SPMI, SPACA1, Seminal plasma protein pB1, PSP-I, MFGE8, tACE, PGK2, SUCLA2, MDH1). Moreover, LYDP4, SPAM-1, HSP60, ZPBP1, AK1 were previously reported in lipid rafts of mouse and human spermatozoa but not in boar spermatozoa. We also found and confirmed the presence of ACR, ACRBP, AWN, AQN3 and PRDX5 in lipid rafts of boar spermatozoa. This paper provides an overview of the glycosylation pattern in lipid rafts of boar spermatozoa before and after capacitation. Further glycomic analysis is needed to determine the type and the variation of glycan chains of the lipid rafts glycoproteins on the surface of spermatozoa during capacitation and acrosome reaction.

Rac1 is necessary for capacitation and acrosome reaction in guinea pig spermatozoa.

Actin cytoskeleton remodeling is a critical process for the acquisition of fertilizing capacity by spermatozoa during capacitation. However, the molecular mechanism that regulates this process has not been fully elucidated. In somatic cells, Ras-related C3 botulinum toxin substrate 1 protein (Rac1) promotes the polymerization of actin by participating in the modeling of two structures: lamellipodia and adhesion complexes linked with the plasma membrane. Rac1 is expressed in mammalian spermatozoa; however, the role of Rac1 in sperm physiology is unknown. This study aimed to elucidate the participation of Rac1 in capacitation and acrosome reaction (AR). Rac1 was found to be dispersed throughout the acrosome and without changes in the middle piece. After 60 minutes of capacitation, Rac1 was found in the apical region of the acrosome only, which concurred with an increase in Rac1-GTP. Rac1 inhibition prevented such changes. In the middle piece, Rac1 localization remained unchanged. Besides, Rac1 inhibition blocked capacitation and AR. The present study demonstrates that Rac1 participates only in the actin cytoskeleton remodeling that occurs in the acrosomal apical region during capacitation, a region where a large amount of actin is polymerized and shaped in a diadem-like structure. Our data also show that this actin cytoskeleton organized by Rac1 interacts with filamin-1, and such interaction was blocked by the inhibition of Rac1, which led to a different organization of the actin cytoskeleton. All these outcomes imply that the formation of an F-actin cytoskeleton in the acrosomal apical region is a necessary event for capacitation and AR, and which is Rac1 driven.

Potential of the HSP70 protein family as biomarker of Crassostrea virginica under natural conditions (Ostreoida: Ostreidae)

The HSP70 proteins are used as exposure biomarkers, and the oyster Crassostrea virginica is considered as a bioindicator organism in environmental assessment. According to the season, the level of expression of the HSP70 family in wild oysters has not been characterized before. The aim of this work was to analyze the expression of the HSP70 family as an exposure biomarker using C. virginica gills from individuals and groups of oysters from the Tampamachoco Lagoon under natural conditions. Ninety oyster samples were collected at locations from the Tampamachoco Lagoon during the dry and "north winds" seasons. One group of oysters was maintained under laboratory conditions and exposed to thermal stress. The pH, temperature, salinity, and dissolved oxygen (DO2) were measured inside the brackish Tuxpam-Tampamachoco system. Salinity and DO2 were outside the limits recommended for brackish systems in both seasons. The electrophoresis and immunodetection assay of HSP70 were performed using proteins from oyster gills. The HSP73, HSP72 and HSP69 isoforms were expressed in dry season tissue samples, while only the HSP73 and HSP72 isoforms were detected in north winds season tissue samples and positive control tissue samples. The HSP73 isoform has not been previously reported in C. virginica. To evaluate the expression of HSP70 protein family at individual and group levels from wild animals, it is also important to determine a seasonal baseline expression.

Las proteínas HSP70 se utilizan como biomarcadores de exposición y el ostión Crasostrea virginica es considerado como un organismo bioindicador en evaluaciones ambientales. De acuerdo con la estación, el nivel de expresión de la familia de las HSP70 en ostiones silvestres no ha sido caracterizado. El objetivo de este trabajo fue analizar la expresión de la familia de las proteínas HSP70 como un biomarcador de exposición utilizando branquias de C. virginica de manera individual y grupal bajo condiciones naturales. Noventa muestras de ostión se recolectaron en un sitio dentro de la Laguna Tampamachoco durante las estaciones de secas y "nortes". Un grupo se mantuvo bajo condiciones de laboratorio y expuesto a estrés térmico. El pH, la temperatura, la salinidad y el oxígeno disuelto (DO2) se midieron dentro del sistema salobre Tuxpan-Tampamachoco. La salinidad y el DO2 estuvieron fuera de los límites recomendados para sistemas salobres en ambas estaciones. La electroforesis y el ensayo de inmunodetección se llevaron a cabo con branquias de ostiones. Las isoformas HSP73, HSP72 y HSP69 se expresaron en tejido de las muestras de la época de secas, mientras que solamente las isoformas HSP73 y HSP72 se detectaron en tejido de las muestras de época de nortes y control positivo. La isoforma HSP73 no ha sido reportada previamente en C. virginica. Para evaluar la expresión de la familia de las proteínas HSP70 a nivel individual y grupal de organismos silvestres es importante determinar una línea de base de expresión estacional.

Prevalence and genotypes of the adenovirus infection as well detection of co-infection with bocavirus in Mexican immunosuppressed and non-immunosuppressed children with pneumonia.

BACKGROUND: Adenovirus (AdV) causes respiratory infection; recent observations suggest that some subtypes have more ability to develop fatal disease. AdV infection has been associated with co-infection with human bocavirus (HBoV). We analysed the frequency of AdV infection, its subtypes and the presence of co-infection with HBoV, as well the clinical characteristics of such co-infection in Mexican paediatric immunosuppressed (IP) and non-immunosuppressed patients (non-IP) diagnosed with pneumonia. METHODS: A total of 5185 nasopharyngeal swabs from two groups of children with pneumonia, one IP and the other non-IP, were analysed for the detection of AdV by immunofluorescence and confirmed by PCR and culture. HBoV was identified by PCR. Positive samples for AdV and AdV/HBoV were typed using PCR sequencing, the clinical characteristics of the AdV/HBoV co-infection were analysed. RESULTS: Thirty-seven of the 5185 (0.71%) samples were positive for AdV, of those 27/37 (73%) were detected in non-IP and 10/37 (27%) in the IP group. Twelve were typed as follows: 9/12 (75%) as Species B1 subtype 3, of those 8/9 (88.9%) in non-IP and 1/9 in the IP group. One of twelve AdV2 subtype B11a was identified in one non-IP and the remaining two out of 12 successfully typed, were identified as Species C subtypes 2 and 6 in the group of non-IP. The presence of both AdV and HBoV1 in co-infection was observed in 2/37 (5.4%) non-IP with a syndrome like influenza. CONCLUSIONS: In this 5 year analysis of samples from non-IP and IP hospitalized paediatric patients with a diagnosis of pneumonia, a low incidence of AdV was found. B1 was the most frequent subtype and frequently found in non-IP, and two cases of co-infection AdV/HBoV1 were detected in two non-IP with a influenza-like syndromes. This is the first report of HBoV and AdV co-infection in Mexico. The frequency of AdV and HBoV co-infection was lower than that reported in other populations.

Effect of porcine follicular fluid proteins and peptides on oocyte maturation and their subsequent effect on in vitro fertilization.

The follicular fluid (FF) is a microenvironment that contains molecules involved in oocyte maturation, ovulation, and fertilization. Characterizing the proteins and peptides present in the FF could be useful for determining which proteins and peptides to use as a supplement for culture media. Biologically active peptides produced during the maturation or degradation of functional proteins are called cryptides. The aim of this study was to identify the proteins and cryptides in porcine FF that could stimulate porcine oocyte in vitro maturation (IVM) and in vitro fertilization (IVF) when added to culture maturation medium. Five FF protein fractions (F1-F5) were obtained by ionic exchange chromatography, resolved by SDS-PAGE, and identified by tandem mass spectrometry. These fractions had effects on IVM and/or IVF. The F1 fraction, which was composed of immunoglobulin fragments, cytokeratin, transferrin, and plasminogen precursor increased IVM and IVF. The F2, F3, and F4 fractions reduced the percentage of oocytes in first metaphase. Additionally, the F3 fraction, which was composed of immunoglobulins and transthyretin, interfered with germinal vesicle breakdown. The F5 fraction, which was mainly composed of serum albumin and keratin, favored germinal vesicle breakdown and promoted IVM. Most of the 31 proteins which were associated with the immune response and inflammatory processes could be related to oocyte maturation and fertilization. Some of the identified proteins were present in more than one fraction; this could be explained by a change in their isoelectric points, because of the loss of part of the amino acid sequence or a change in the glycosylation status of the protein. Improved oocyte IVM and IVF will increase embryo production, which in turn will contribute to the efficiency of assisted reproduction in various mammalian species.

Molecular cloning and functional analysis of the FSH receptor gene promoter from the volcano mouse (Neotomodon alstoni alstoni).

To gain further insights on the genetic divergence and the species-specific characteristics of the follicle-stimulating hormone receptor (FSHR), we cloned 946 bp of the 5'-flanking region of the FSHR gene from the volcano mouse (Neotomodon alstoni alstoni), and compared its features with those from other mammalian species. The sequence of neotomodon FSHR (nFSHR) gene from the translation initiation site to -946 is 74, 71, 64, and 59% homologous to rat, mouse (129/J), human, and sheep, respectively. The nFSHR 5'-flanking region exhibits new interesting putative cis-regulatory elements including those for the SRY transcription factor, which had not been previously related to the FSHR gene. The transcriptional regulation properties of nFSHR gene were studied in mouse Sertoli (MSC-1) and non-Sertoli (H441) cell lines, and compared with those obtained with similar 129/J constructs. All constructs tested were more active in H441 than in MSC-1 cells. The low transcription levels detected in MSC-1 cells probably reflect the recruitment of Sertoli cells-specific nuclear factors that repress transcription of the FSHR gene. In H441 cells, 129/J constructs were more active than their neotomodon counterparts, indicating important species-specific differences in their transcription pattern. Functional analysis of a series of progressive 5'-deletion mutants identified regions involved in positive and negative transcriptional regulation as well as the strongest minimal promoter spanning 260 bp upstream the translation initiation site. The identification of inhibitory nuclear transcription factors, which are apparently expressed in MSC-1 cells, may contribute to a better understanding of the transcriptional regulation of the FSHR gene.

Effects of atrazine and fenoxaprop-ethyl on capacitation and the acrosomal reaction in boar sperm.

Atrazine is a herbicide of the chloro-s-triazine family. It inhibits photosynthesis in plants and is an endocrine disruptor, but its effects on human health are controversial. Fenoxaprop-ethyl, an aryloxy phenoxyalkanoic acid herbicide, inhibits the biosynthesis of fatty acids and provokes depolarization of membranes. The aim of this study is to evaluate the in vitro effects of both herbicides on capacitation, spontaneous acrosome reaction (SAR) and progesterone-induced acrosome reaction (PIAR) in boar sperm. Sperm capacitation is done in TALP-HEPES media for 4 hours. Capacitation and SAR are evaluated immediately; PIAR, 30 minutes later. LC50 for fenoxaprop-ethyl is 60 micromolar [corrected] and 40 micromolar [corrected] for atrazine. Fenoxaprop-ethyl induces capacitation at 60 micromolar [corrected] and SAR at all concentrations, also increases significantly PIAR. Atrazine decreased capacitation whereas increase significantly SAR and PIAR at all concentrations. It seems that fenoxaprop-ethyl and atrazine accelerate the capacitation and the acrosomal reaction, possibly via plasma membrane destabilization.

Carbohydrate affinity chromatography indicates that arylsulfatase-A from capacitated boar sperm has mannose and N-acetylglucosamine/sialic acid residues.

Carbohydrate residues on membrane proteins from sperm are important in gamete interaction. In recent years, Arylsulfatase A (AS-A) has been acquiring an important role from the various putative gamete interaction responsibles in sperm. The aim of this study was to determine if the capacitated boar sperm Arylsulfatase-A (AS-A), contains D-mannose, N-acetylglucosamine and/or sialic acid residues by its purification using affinity chromatography with Concanavalia ensiformis Agglutinin(Con-A) or Wheat Germ Agglutinin (WGA) as ligands. Sperm samples were capacitated in TALP-HEPES medium. Protein extract was added to the affinity columns. Sequencing of retained proteins was done after SDS-PAGE. Total capacitated sperm proteins electrophoresis showed molecular masses between 14 kDa and 102 kDa. A major band of 68 kDa, and 2 minor bands of 52 kDa and 47 kDa were observed. They were AS-A, hyaluronidase and lactadherin, respectively. The Con-A-retained proteins (RP) pattern showed bands from 14 to 98 kDa. After sequencing and BLAST analysis, the 62 kDa band corresponded to Arylsulfatase-A. The WGA RP fraction showed bands from 14 to 100 kDa. The 65 kDa band corresponded to AS-A. This study showed that AS-A has mannose, N-acetylglucosamine and/or sialic acid residues as part of its glycosilation. In this study AS-A was isolated from boar capacitated sperm by affinity chromatography using separately Con-A and WGA, indicating that there are mannose, N-acetylglucosamine and/or sialic acid residues in its glycosilation. AS-A is a membrane protein of capacitated sperm. Further investigation is needed to fully characterize the glycosidic residues bore by AS-A and to determine its function.

Changes in the distribution of lectin receptors during capacitation and acrosome reaction in boar spermatozoa.

Sperm glycocalyx modifications are known to occur during capacitation and the acrosome reaction (AR). These changes are very important for gamete recognition and fertilization in mammals but are not fully understood. The purpose of this study was to determine the distribution of surface carbohydrates in boar spermatozoa during capacitation and the AR. These processes may be associated with specific changes in the content and distribution of surface carbohydrates. Thirty-nine ejaculates from fertile boars of various breeds were analyzed. N-Acetylglucosamine and sialic acid, mannose and fucose residues were detected by fluorescence microscopy and flow cytometry using FITC-conjugated lectins. Triticum vulgaris agglutinin (WGA) bound on the head and tail of fresh sperm, and fluorescence intensity (FI) decreased in capacitated sperm (6751 to 5621 fluorescence units (FU), P<0.05), and decreased further in acrosome-reacted sperm (5240 FU, P<0.05). Concanavalia ensiformis agglutinin (Con-A) bound homogeneously on the head and the midpiece of fresh sperm with a FI of 5335 FU, and increased in capacitated sperm (5957 FU, P<0.05) mainly on the acrosomal region. In acrosome-reacted sperm, fluorescence was concentrated on the border of the acrosomal region (5608 FU, P<0.05). It was not possible to detect Ulex europaeus agglutinin (UEA) by fluorescence microscopy. However, flow cytometry revealed UEA receptors (187 FU), with a nonsignificant decreased number in capacitated (142 FU) and AR sperm (142 FU). Labeling patterns were similar in all breeds. Sperm glycocalyx modifications observed in this study provide insights to the molecular modifications accompanying capacitation and the AR. This kind of study could improve the diagnosis of reproductive problems of subfertile boars and males of other species.