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.

Postnatal cadmium administration affects the presence and distribution of carbohydrates in the sperm membrane during maturation in the epididymis in adult Wistar rats.

Cadmium (Cd) is a heavy metal related to a decrease in sperm parameters. The transit of spermatozoa through the epididymis is necessary to generate changes in the sperm membrane, such as the assembly of various carbohydrates that are added to the spermatazoan's surface to prepare it for successful fertilisation of the oocyte. No studies have yet analysed whether Cd alters the presence and distribution of these carbohydrates. We aimed to evaluate the changes induced by Cd in the distribution pattern of N-acetylglucosamine, sialic acid, mannose and fucose on the sperm membrane in the epididymis (e.g. caput, corpus, cauda) and if it alters the epididymal epithelium. Male Wistar pups were treated with Cd doses (0.125, 0.25 and 0.5mg/kg) on postnatal days 1-49. At postnatal day 90, they were humanely killed, sperm samples were obtained from the epididymis and tissue samples were taken for histological analysis. Cd concentrations in the blood and epididymis increased in proportion to the dose administered and decreased the serum testosterone levels and sperm quality. Histological analysis revealed alterations in the epithelium in all Cd-treated groups. Cd altered the distribution patterns of carbohydrates and fluorescence indices. All these alterations affected the structure and functioning of sperm.

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.

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.