Biochemical characterization and membrane fluidity of membranous vesicles isolated from boar seminal plasma

Mammalian seminal plasma contains membranous vesicles (MV), which differ in composition and origin. Among these particles, human prostasomes and equine prostasome-like MV have been the most studied. The aim of the present work is to characterize the biochemical composition and membrane fluidity of MV isolated from boar seminal plasma. The MV from boar seminal plasma were isolated by ultracentrifugation and further purification by gel filtration on Sephadex G-200. The MV were examined by electron microscopy (EM), amount of cholesterol, total phospholipid, protein content, and phospholipid composition were analyzed. Membrane fluidity of MV and spermatozoa were estimated from the electron spin resonance (ESR) spectra of the 5-doxilstearic acid incorporated into the vesicle membranes by the order parameter (S). The S parameter gives a measure of degree of structural order in the membrane and is defined as the ratio of the spectral anisotropy in the membranes to the maximum anisotropy obtained in a rigidly oriented system. The S parameter takes into consideration that S = 1 for a rapid spin-label motion of about only one axis and S = 0 for a rapid isotropic motion. Intermediate S values between S = 0 and S = 1 represents the consequence of decreased membrane fluidity. The EM revealed the presence of bilaminar and multilaminar electron-dense vesicles. Cholesterol to phospholipid molar ratio from the isolated MV was 1.8. Phospholipid composition showed a predominance of sphingomyelin. The S parameter for porcine MV and for boar spermatozoa was 0.73 +/- 0.02 and 0.644 +/- 0.008, respectively, with the S for MV being greater (p < 0.001) than the S for spermatozoa. The high order for S found for boar MV was in agreement with the greater cholesterol/phospholipids ratio and the lesser ratio for phosphatidylcholine/sphingomyelin. Results obtained in the present work indicate that MV isolated from boar semen share many biochemical and morphological characteristics with equine prostasome-like MV and human prostasomes. The characteristics of the porcine MV of the seminal plasma, however, differed from those of boar sperm plasma membranes.     

Structural and ultrastructural features of boar seminal vesicles

The morphological features of boar seminal vesicles were examined by light and transmission microscopy. Boar seminal vesicles consist of glandular tissue arranged in multiple lobules containing a system of ramified secretory tubules. The secretory tubules are composed of a mucosa formed by an epithelium and an underlying lamina propria and, are surrounded by a muscular layer. The epithelium is made up of columnar cells and occasional basal cells. Mast cells are frequently found among epithelial cells. Three types of columnar cells, considered different stages of the secretory cell cycle, are present: principal cells, clear cells and dense cells. Principal cells are functionally differentiated cells characterised by abundant mitochondria, great development of the rough endoplasmic reticulum and presence of secretory granules in their cytoplasm. The apical surface of many principal cells shows apical blebs filled with PAS-positive material. No acid mucosubstances are detected. Microvilli cover the apical surface except in the apical blebs. Dense cells, arranged between principal cells, are also functional differentiated cells but with signs of cellular degeneration. Clear cells are an initial differentiated stage of columnar cells and are characterised by the presence of a poorly developed rough endoplasmic reticulum and by the absence of secretory granules. Proliferating cells are present among columnar cells. Basal cells contain scarce cytoplasm, few organelles and no secretory granules. The lack of mitotic activity in these cells suggests that they do not act as precursors of columnar cells.     

Expression and analysis of two novel rat organic cation transporter homologs, SLC22A17 and SLC22A23

The organic cation transporter (OCT, SLC22) family is a family of polyspecific transmembrane proteins that are responsible for the uptake or excretion of many cationic drugs, toxins, and endogenous metabolites in a variety of tissues. Many of the OCTs have been previously characterized, but there are a number of orphan genes whose functions remain unknown. In this study, two novel rat SLC22 genes, SLC22A17 (BOCT1) and SLC22A23 (BOCT2), were cloned and characterized. Northern blot analysis showed that BOCT1 and BOCT2 mRNA was expressed in a wide variety of tissues. BOCT1 was strongly expressed in brain, primary neurons and brain endothelial cells, with highest expression in choroid plexus. BOCT2 was also abundantly expressed in brain, as well as in liver. To characterize the products of these genes, BOCT1 cDNA was isolated from a rat blood-brain barrier cDNA library, and BOCT2 cDNA was isolated from rat brain capillary and from cultured neurons using PCR techniques. Plasmids expressing BOCT1 and BOCT2 were transfected into HEK-293 cells, as were control cDNAs for OCT1 and OCTN2. Recombinant cell surface protein was verified by western blot and fluorescence microscopy. Transport activity of BOCT1 and BOCT2 was evaluated using radioisotope uptake assays. The OCT1- and OCTN2-expressing cells transported the canonical substrates, 1-methyl-4-phenyl-pyridinium (MPP(+)) and carnitine, respectively. However, BOCT1 and BOCT2-expressing cells did not show transport activity for these substrates or a number of other SLC22 substrates. These novel family members have a nonconserved amino terminus, relative to other OCTs, that may preclude typical SLC22 transport function.     

Polymorphisms in the organic cation transporter genes SLC22A4 and SLC22A5 and Crohn's disease in a New Zealand Caucasian cohort

Polymorphisms in the organic cation transporter (OCTN) genes SLC22A4 (OCTN1; polymorphism 1672C/T) and SLC22A5 (OCTN2; polymorphism -207G/C) at the inflammatory bowel disease (IBD) 5 locus comprise a two-allele haplotype (SLC22A-TC) associated with increased risk for Crohn's disease (CD). In this study, we examined the contribution of the disease susceptibility haplotype SLC22A-TC to CD in a New Zealand Caucasian population. The frequencies of the gene polymorphisms 1672C/T and -207G/C were examined in 182 patients with CD and 188 ethnically matched controls by PCR-RFLP analysis. There was a significant difference in the allele frequency (0.444 vs 0.519; P = 0.041) of the 1672T polymorphism in the SLC22A4 gene between controls and patients with CD. In contrast, there was no significant difference (0.497 vs 0.552; P = 0.135) for the -207C polymorphism in the SLC22A5 gene. The homozygote SLC22A-TC diplotype was significantly associated with an increased risk for CD (odds ratio 2.19), and the SLC22A-TC haplotype was associated with increased risk (P = 0.0007) of ileocolonic involvement. The population-attributable risk for the SLC22A-TC haplotype is 15.1%. Thus, SLC22A-TC is associated with an increased risk of CD and disease phenotype in our New Zealand CD cohort.     

Association analysis of the SLC22A11 (organic anion transporter 4) and SLC22A12 (urate transporter 1) urate transporter locus with gout in New Zealand case-control sample sets reveals multiple ancestral-specific effects

Introduction

There is inconsistent association between urate transporters SLC22A11 (organic anion transporter 4 (OAT4)) and SLC22A12 (urate transporter 1 (URAT1)) and risk of gout. New Zealand (NZ) Māori and Pacific Island people have higher serum urate and more severe gout than European people. The aim of this study was to test genetic variation across the SLC22A11/SLC22A12 locus for association with risk of gout in NZ sample sets.

Methods

A total of 12 single nucleotide polymorphism (SNP) variants in four haplotype blocks were genotyped using TaqMan® and Sequenom MassArray in 1003 gout cases and 1156 controls. All cases had gout according to the 1977 American Rheumatism Association criteria. Association analysis of single markers and haplotypes was performed using PLINK and Stata.

Results

A haplotype block 1 SNP (rs17299124) (upstream of SLC22A11) was associated with gout in less admixed Polynesian sample sets, but not European Caucasian (odds ratio; OR = 3.38, P = 6.1 × 10^-4; OR = 0.91, P = 0.40, respectively) sample sets. A protective block 1 haplotype caused the rs17299124 association (OR = 0.28, P = 6.0 × 10^-4). Within haplotype block 2 (SLC22A11) we could not replicate previous reports of association of rs2078267 with gout in European Caucasian (OR = 0.98, P = 0.82) sample sets, however this SNP was associated with gout in Polynesian (OR = 1.51, P = 0.022) sample sets. Within haplotype block 3 (including SLC22A12) analysis of haplotypes revealed a haplotype with trans-ancestral protective effects (OR = 0.80, P = 0.004), and a second haplotype conferring protection in less admixed Polynesian sample sets (OR = 0.63, P = 0.028) but risk in European Caucasian samples (OR = 1.33, P = 0.039).

Conclusions

Our analysis provides evidence for multiple ancestral-specific effects across the SLC22A11/SLC22A12 locus that presumably influence the activity of OAT4 and URAT1 and risk of gout. Further fine mapping of the association signal is needed using trans-ancestral re-sequence data.     

Measurement of immunoglobulin G, A and M concentrations in boar seminal plasma

How the immune system relates to the boar reproductive tract is not well defined. This is an important area of study because disease-causing agents may be transmitted through boar semen. We have previously identified porcine reproductive and respiratory syndrome virus (PRRSV) in boar semen and wanted to identify PRRSV-specific antibodies within seminal plasma. However, literature documenting total immunoglobulin concentration or the predominant immunoglobulin isotype in boar semen was not available. Therefore, we developed a sandwich enzyme-linked immunoassay (ELISA) to quantitate total IgG, IgA and IgM in seminal plasma from 16 healthy, nonvaccinated, adult boars (n = 102 semen samples). In seminal plasma, IgG was the predominant isotype followed by IgA and IgM. Mean levels +/- the standard deviation followed by the 95% confidence interval of IgG, IgA and IgM were 23.2 +/- 14 microg/mL (15.5 to 31.0), 4.8 +/- 2.5 microg/mL (3.5 to 6.2) and 3.7 +/- 1.7 microg/mL (2.7 to 4.7), respectively. These concentrations of immunoglobulins in seminal plasma were considerably lower than in other swine secretions, which might allow for the survival of infectious agents in boar semen.     

Association of SNPs in zinc transporter genes on seminal plasma zinc levels in humans

BioMetals - This study is to examine the effects of single nucleotide polymorphisms (SNPs) of SLC30A and SLC39A on seminal plasma zinc concentration. Blood and seminal plasma samples were collected...     

Proteinase inhibitors in aggregated forms of boar seminal plasma proteins

Boar seminal plasma proteins were separated by gel chromatography on Sephadex G-75 into five fractions (I–V). Serine proteinase inhibitors were found mainly in the protein fraction with relative molecular weight 5–25 kDa. Small amounts of these inhibitors were also found in the high molecular weight protein fraction (M_r>100 kDa). The protein fraction containing most of the proteinase inhibitory activity was further separated by RP HPLC. Isolated proteins were characterized by SDS electrophoresis and immunoblotting, N-terminal amino acid sequencing and by determination of the proteinase inhibitory activity. In the fraction containing proteinase inhibitors, also β-microseminoprotein (β-MSP), AQN 1 and lactoferrin were identified. The possible existence of complexes of protein components in the fraction with relative molecular weight 5–25 kDa was studied in detail using gel chromatographic separation on Sephadex G-50. A part of proteinase inhibitors with M_r 8 kDa was eluted together with AQN 1 spermadhesin. An interaction of isolated spermadhesin AQN 1 and proteinase inhibitor was shown.     

Separation, characterization and identification of boar seminal plasma proteins

Methods used for the isolation, separation and characterization of boar seminal plasma proteins are discussed, as well as techniques applied to study their binding properties. Attention is paid to interactions of these proteins with different types of saccharides and glycoconjugates, with membrane phospholipids, and to interactions between proteins. Boar seminal plasma contains different types of proteins: spermadhesins of the AQN and AWN families; DQH and PSP proteins belong to the most abundant. Some of these proteins are bound to the sperm surface during ejaculation and thus protein-coating layers of sperm are formed. Sperms coated with proteins participate in different types of interactions occurring in the course of the reproduction process, e.g. formation of the oviductal sperm reservoir, sperm capacitation, oocyte recognition and sperm binding to the oocyte.     

Localization and characterization of glutathione peroxidase (GPx) in boar accessory sex glands, seminal plasma, and spermatozoa and activity of GPx in boar semen

Boar ejaculate owes its characteristic large volume mainly to accessory sex gland (ASG) secretions. These are main contributors to the protective functions of seminal plasma, especially against oxidative damage. Numerous antioxidants have been detected in ASG secretions, and, respectively, in seminal plasma. However, as regards one key antioxidant protector -- the Se-dependent enzyme glutathione peroxidase (GPx) -- there is no agreement yet among researchers as to its presence in boar seminal plasma. Nevertheless, the beneficial effect of dietary Se supplementation on male fertility has been widely recognized. The aim of the present study was to investigate the localization and characterization of GPx in boar ASGs, seminal plasma, and spermatozoa, as well as to evaluate GPx activity in boar semen. Immunohistochemical assays demonstrated GPx presence in the epithelial cells, vacuole membranes, and vascular endothelium of boar seminal vesicle, prostate and bulbourethral glands. Western blot analysis demonstrated the presence of a monomer form of GPx with MW 20 kDa in lysates from seminal vesicle, prostate, bulbourethral glands, and spermatozoa, but not in seminal plasma. Surprisingly, peroxidase activity detected in seminal plasma from normal ejaculates was nearly three times as high as in spermatozoa. Our findings confirmed the presence of immunoreactive GPx in the boar reproductive tract, while further investigation is still warranted to uncover the exact protein forms involved and their function.     

Purification and properties of alkaline phosphatase from boar seminal plasma

An alkaline phosphatase was purified from boar seminal plasma using adsorption to calcium phosphate gel, gel filtration, and ion-exchange chromatography. The preparation gave a single band on SDS polyacrylamide electrophoresis. The enzyme was a non-specific alkaline phosphatase that hydrolysed pyrophosphate slowly and had no phosphodiesterase activity. The pH optimum was 10 and the Km was approximately 0.2 mM with p-nitrophenyl phosphate as substrate. The enzyme was a zinc metalloenzyme as indicated by the loss of activity when treated with o-phenanthroline and the restoration of activity by zinc and magnesium ions. It also lost activity when treated with thiols. Molecular weight estimates from SDS polyacrylamide gel electrophoresis and gel filtration suggest that the enzyme is a tetramer of identical subunits, each of which has a molecular weight of 68,000.     

Suppression of glutathione S-transferases in rat seminal vesicles or pituitary glands

We have studied the tissue-specific expression of GSH S-transferases in rat seminal vesicles and pituitary glands by in vitro translation and immunoprecipitation. The major GSH S-transferase subunit expressed in rat seminal vesicles belongs to the Yb mobility class whose expression diminishes when the rats are treated with pentobarbital. The pattern of GSH S-transferase expression in the pituitary gland is very similar to that of the rat brain with Yb size subunit(s) predominant. The Y beta size subunit is also expressed together with the Yc and Y delta subunits. The expression of GSH S-transferases was drastically reduced in pituitary gland poly(A) RNAs from diethylstilbestrol-treated, ovariectomized female rats. Xenobiotics such as phenobarbital, 3-methylcholanthrene, and trans-stilbene oxide induce rat liver GSH S-transferase activities, especially the Ya- and Yb-subunit containing isozymes. Induction of GSH S-transferases by a combination of the three xenobiotics is neither additive nor synergistic, however. Our results clearly demonstrate that GSH S-transferase expression in seminal vesicles and pituitary glands can be suppressed by phenobarbital and diethylstilbestrol, respectively. Our findings suggest that different GSH S-transferase isozymes respond differently to various xenobiotics. Both induction and suppression occur in rats treated with xenobiotics. This notion helps to explain the lack of additive or synergistic induction in rats treated with more than one xenobiotic.     

Myelinosome-like vesicles in human seminal plasma: A cryo-electron microscopy study

Seminal plasma is particularly rich in extracellular vesicles. Myelinosomes are membranous organelles described throughout the seminiferous epithelium of the testis but never reported in semen. Our aim was to determine the presence of myelinosomes in human seminal plasma.Transmission electron microscopy and cryo electron microscopy analysis of standard myelinosome preparation from TM4 Sertoli cells and human seminal plasma samples.We have specified by cryo-EM the morphological aspect of “standard” myelinosomes isolated from the culture media of TM4 Sertoli cells. Vesicles with the same morphological appearance were revealed in human seminal plasma samples.Human seminal plasma contains a population of large EV (average diameter 200 nm) whose morphological appearance resemble those of myelinosomes. Defining the specific biomarkers and functionalities of myelinosome in human seminal plasma are the concerns to be addressed in our further research.     

Evidence of glutathione transporter in rat brain synaptosomal membrane vesicles

Glutathione (GSH) transport was studied in synaptosomal membrane vesicles (SMV) of rat cerebral cortex. The present study shows that GSH uptake into SMV occurs very quickly in a time-dependent manner into an osmotically active intravesicular space. The initial rate of transport followed Michealis-Menten saturation kinetics with a K_m 4.5±0.8 μM that shows a high affinity of the transporter for GSH. Therefore GSH uptake in SMV occurs by a mediated transport system which can be activated by either an inward gradient of cations, like Na⁺ or K⁺, or membrane depolarization. These results, together with those obtained by valinomycin-induced K⁺ diffusion potential, indicate that GSH synaptosomal transport is electrogenic by a negative charge transfer. The increase of GSH uptake measured by trans-stimulation experiments confirms a GSH bidirectional mediated transport which seems susceptible of modulation by changes in ionic fluxes and in the membrane potential. These results may indicate a possible involvement of this transporter in the role suggested for GSH in synaptic neurotransmission; also considering that GSH precursor of neuroactive aminoacids (glyeine, glutamate), may contribute to regulate their level in synapses. Finally, a GSH transporter in synaptosomes may contribute to maintaining the GSH homeostasis in cerebral cortex, where decreases of GSH levels have been related to susceptibility to neuropathologies.     

Occurrence of prostasome-like membrane vesicles in equine seminal plasma

Equine seminal plasma was shown to contain membrane vesicles that are similar to the well characterized prostasomes in human seminal plasma. Determination of nucleoside and nucleotide concentrations of these particles have shown that ATP, ADP and adenosine are the main components of the nucleotidic pool. 5' nucleotidase, endopeptidase and dipeptidyl peptidase i.v. activities have been found on the surface of the particles. The interaction between these prostasome-like vesicles and spermatozoa was demonstrated by electron micrograph scans which revealed the steps of a fusion-like process leading to mixing of the membranes. In addition, endopeptidase activity, a marker enzyme of these seminal vesicles that is normally absent from equine spermatozoa, was shown to be acquired by these cells after interaction with the vesicles. The addition of these vesicles to equine spermatozoa resulted in the modification of adenylate catabolism. Therefore, a role in stabilizing the energy charge of the spermatozoa thus allowing longer viability is proposed for these organelles.     

Selenium and glutathione peroxidase in seminal plasma of men and bulls

High levels of selenium and glutathione peroxidase (GSH-Px) were found in bull seminal plasma but low concentrations in human seminal plasma. In man the seminal plasma selenium was associated with two macromolecules separable by gel filtration, but no GSH-Px was found in the same fractions. Selenium in bull seminal plasma was associated with two proteins, which could be separated by gel filtration and anion exchange chromatography. Both macromolecules coeluted with GSH-Px activity and had identical optima at pH 7.0. Their responses to thermal treatment, however, differed. Seminal vesicle secretory fluid in the bull contained both these proteins, while the larger molecule was also found in fractionations of ampulla, prostate and Cowper's glands. The larger enzyme form is evidently a tetramer of the smaller one. Both enzyme forms were extremely sensitive to heavy metals and some divalent metal ions. GSH caused an activation while other reducing agents were suppressive. Triton X-100 had no effect, while sodium deoxycholate was inhibitory. These properties are typical for a phospholipid hydroperoxide GSH-Px. It is concluded that this selenium-dependent enzyme may be important in the protection of bovine spermatozoa against damage caused by oxygen radicals, while in man such a mechanism is not functional.     

Characterization of the organic cation transporter SLC22A16: a doxorubicin importer

Specific efflux transporters, such as P-glycoprotein, have been shown to confer drug resistance by decreasing the intracellular accumulation of anticancer drugs. Understanding influx transporters, as well as efflux transporters, is essential to overcome this resistance. We report the expression profile and pharmacological characterization of an organic cation transporter, SLC22A16. The results of our experiments indicate that SLC22A16 is a mediator of doxorubicin uptake in cancer cells. Quantitative real-time RT-PCR analyses show that SLC22A16 is expressed in primary samples taken from patients with acute leukemia. Xenopus oocytes injected with SLC22A16 cRNA import doxorubicin, a widely used anticancer drug for hematological malignancies, in a saturable and dose-dependent manner. The apparent Km value for doxorubicin import was 5.2+/-0.4 microM. In cytotoxic assays, stable transfectants of leukemic Jurkat cells overexpressing SLC22A16 cells became significantly more sensitive to doxorubicin (2 microM) treatment. Characterization of SLC22A16 will help in designing novel therapies targeting hematological malignancies.