Head Membranes Research Articles

A novel class of insecticidal alkylsulfones are potent inhibitors of vesicular acetylcholine transport

Pyridine alkylsulfone derivatives typified by oxazosulfyl (Sumitomo Chemical Company Ltd.) and compound A2 (Syngenta) represent a new class of insecticides, with potent activity against several insect orders. Whilst the MOA of this class has been attributed to interaction with the voltage-gated sodium channel (VGSC), here we present strong evidence that their toxicity to insects is mediated primarily through inhibition of the vesicular acetylcholine transporter (VAChT). Alkylsulfone intoxication in insects is characterised by (i) a reduction in cholinergic synaptic transmission efficiency demonstrated by a depression of cercal afferent activity in giant-interneurone preparations of American cockroach (Periplaneta americana), (ii) selective block of cholinergic-transmission dependent post-synaptic potentials in the Drosophila giant-fibre pathway and (iii) abolition of miniature excitatory post-synaptic currents (mEPSCs) in an identified synapse in Drosophila larvae.Ligand-binding studies using a tritiated example compound ([3H]-A1) revealed a single saturable binding-site, with low nanomolar Kd value, in membrane fractions of green bottle fly (Lucilia sericata). Binding is inhibited by vesamicol and by several examples of a previously identified class of insecticidal compounds known to target VAChT, the spiroindolines. Displacement of this binding by analogues of the radioligand reveals a strong correlation with insecticidal potency. No specific binding was detected in untransformed PC12 cells but a PC12 line stably expressing Drosophila VAChT showed similar affinity for [3H]-A1 as that seen in fly head membrane preparations. Previously identified VAChT point mutations confer resistance to the spiroindoline class of insecticides in Drosophila by Gal-4/UAS directed expression in cholinergic neurones and by CRISPR gene-editing of VAChT, but none of these flies show detectable cross-resistance to this new chemical class.Oxazosulfyl was previously shown to stabilise voltage-gated sodium channels in their slow-inactivated conformation with an IC50 value of 12.3μM but inhibits binding of [3H]-A1 with approximately 5000 times greater potency. We believe this chemistry class represents a novel mode-of-action with high potential for invertebrate selectivity.

Analyzing the impact and mechanism of bisphenol A on testicular lipid metabolism in Gobiocypris rarus through integrated lipidomics and transcriptomics

Bisphenol A (BPA) is one of the most common environmental endocrine chemicals, known for its estrogenic effects that can interfere with male spermatogenesis. Lipids play crucial roles in sperm production, capacitation, and motility as important components of the sperm plasma membrane. However, limited research has explored whether BPA affects lipid metabolism in the testes of male fish and subsequently impacts spermatogenesis. In this study, we employed Gobiocypris rarus rare minnow as a research model and exposed them to environmentally relevant concentrations of BPA (15 μg/L) for 5 weeks. We assessed sperm morphology and function and analyzed changes in testicular lipid composition and transcriptomics. The results demonstrated a significant increase in the sperm head membrane damage rate, along with reduced sperm motility and fertilization ability due to BPA exposure. Lipidomics analysis revealed that BPA increased the content of 11 lipids while decreasing the content of 6 lipids in the testes, particularly within glycerophospholipids, glycerolipids, and sphingolipid subclasses. Transcriptomics results indicated significant up-regulation in pathways such as cholesterol metabolism, peroxisome proliferator-activated receptor signaling, and fat digestion and absorption, with significant alterations in key genes related to lipid metabolism, including apolipoprotein A-I, apolipoprotein C-I, and translocator protein. These findings suggest that BPA exposure can induce testicular lipid metabolism disruption in rare minnows, potentially resulting in abnormalities in rare minnow spermatogenesis.

Study on the application of 3D printing head film fixation technology in cranial radiotherapy.

Objective: To investigate the use of 3D printing technology to customize individualized precise radiotherapy head masks for cranial radiotherapy patients. Through the comparison with thermoplastic head film, evaluate the effect of this material on deep dose attenuation and body surface dose, and evaluate its positioning accuracy and repeatability for clinical application. Methods: Thirty patients with head and neck radiotherapy were divided into the control group and the experimental group. The control group used the traditional thermoplastic head film fixation technique for body position fixation, and the experimental group used the 3D printing head film fixation technique. The patient setup was verified by kV-CBCT scanning to obtain the translational setup error and rotational setup error in the X, Y, and Z directions. Results: At a depth of 5 cm, both materials have a radiation attenuation rate of <1%. At the surface location, the body surface dose of control group increased by approximately 27%. With a 3D printing head film, the body surface dose increased by approximately 18%. The positioning of two groups of patients was verified by the kV-CBCT, and a total of 232 data sets were obtained. The average translation positioning errors in the X, Y, and Z direction of control group and experimental group were 1.29 mm, 1.42 mm, 1.38 mm and 1.16 mm, 1.24 mm, 1.16 mm, respectively. The average rotation positioning error in the X, Y, and Z direction of control group and experimental group were 1.29°, 1.02°, 1.01° and 1.08°, 0.96°, 1.00°, respectively. The translational setup errors in the Y and Z directions and rotational setup errors in the X direction significantly differed between the control and experimental groups (all p<0.05), but no statistical significance was found in the other directions (all p>0. 05). Conclusion: Compared to the traditional thermoplastic head membranes, 3D printing head membranes has shown a reliable and reproducible interactional positioning accuracy. Of course, further investigations are needed before the new technology can be used on a regular basis.

Bisphenol A regulates apolipoprotein A1 expression through estrogen receptors and DNA methlylation and leads to cholesterol disorder in rare minnow testis.

Bisphenol A (BPA) is a well-known plasticizer that widely distributed in the aquatic environment. BPA has many adverse effects on reproduction. However, few studies have investigated the mechanism of BPA affecting reproduction from the perspective of lipid metabolism. Apolipoprotein A1 (ApoA1) is the major component of high-density lipoprotein (HDL), and plays critical roles in reverse cholesterol transport (RCT). In this study, in order to investigate the effect and molecular mechanism of BPA on testicular ApoA1 and the role of ApoA1 in BPA induced abnormal spermatogenesis, adult male rare minnow Gobiocypris rarus were exposed to 15μg/L of BPA for 1, 3 and 5 weeks. Results showed that BPA could significantly affect testicular ApoA1 mRNA and protein levels, testicular cholesterol levels, plasmatic sex hormone levels and the integrity of sperm head membrane. The main mechanism of BPA regulating ApoA1 expression is to alter Esr recruitment and CpG sites DNA methylation in ApoA1 promoter. The induced ApoA1 up-regulated high density lipoprotein cholesterol levels and enhanced RCT, and finally decreased the testicular free cholesterol levels. This is likely a key mechanism by which BPA induces sex hormone disorder and sperm head membrane damage. The present study reveals the mechanism by which BPA interferes with spermatogenesis from the perspective of cholesterol transport.

In vitro effect of ferrous sulphate on bovine spermatozoa motility parameters, viability and Annexin V-labeled membrane changes.

The aim of this study was to assess the dose- and time-dependent in vitro effects of ferrous sulphate (FeSO4.7H2O) on the motility parameters, viability, structural and functional activity of bovine spermatozoa. Spermatozoa motility parameters were determined after exposure to concentrations (3.90, 7.80, 15.60, 31.20, 62.50, 125, 250, 500 and 1000 μM) of FeSO4.7H2O using the SpermVisionTM CASA (Computer Assisted Semen Analyzer) system in different time periods. Cell viability was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide (MTT) assay, and the Annexin V-Fluos was applied to detect the membrane integrity of spermatozoa. The initial spermatozoa motility showed increased average values at all experimental concentrations compared to the control group (culture medium without FeSO4.7H2O). After 2 h, FeSO4.7H2O stimulated the overall percentage of spermatozoa motility at the concentrations of ≤ 125 μM. However, experimental administration of 250 μM of FeSO4.7H2O significantly (P < 0.001) decreased the spermatozoa motility but had no negative effect on the cell viability (P < 0.05) (Time 2 h). The lowest viability was noted after the addition of ≥ 500 μM of FeSO4.7H2O (P < 0.001). The concentrations of ≤ 62.50 μM of FeSO4.7H2O markedly stimulated (P < 0.001) spermatozoa activity after 24 h of exposure, while at high concentrations of ≥ 500 μM of FeSO4.7H2O the overall percentage of spermatozoa motility was significantly inhibited (P < 0.001) and it elicited cytotoxic action. Fluorescence analysis confirmed that spermatozoa incubated with higher concentrations (≥ 500 μM) of FeSO4.7H2O displayed apoptotic changes, as detected in head membrane (acrosomal part) and mitochondrial portion of spermatozoa. Moreover, the highest concentration and the longest time of exposure (1000 μM of FeSO4.7H2O; Time 6 h) induced even necrotic alterations to spermatozoa. These results suggest that high concentrations of FeSO4.7H2O are able to induce toxic effects on the structure and function of spermatozoa, while low concentrations may have the positive effect on the fertilization potential of spermatozoa.

Investigation of epididymal proteins and general sperm membrane characteristics of Formosan pangolin (Manis pentadactyla pentadactyla)

BackgroundFormosan Pangolin (Manis pentadactyla pentadactyla) is one of the three subspecies of Chinese pangolins, it is also an isolated sub-species naturally habitat in Taiwan. Despite earlier report on successful breeding of Sunda (Manis javanica) pangolin, breeding of Formosan pangolins in zoo captive populations is still challenging due to unknown reproductive characterizations of this species in both male and female populations.ResultsWe characterized for the first time, reproductive tract of male Formosan pangolin. We showed pangolin epididymis was a collagen-enriched organ with apparent segmented sub-regions similar to other mammals. However, unlike most mammals exhibited two V-ATPase subunits, Formosan pangolin exhibited only V-ATPase subunit 2. This specific V-ATPase subunit extended its cellular localization throughout the cytoplasm of epididymal clear cells, suggesting pH regulation of luminal microenvironment might be different from other mammals. Electron micrographs showed rod-shaped pangolin sperm cells with multi-lamellar membrane structure at the sperm head. Similar to well-defined capacitation and acrosome reaction membrane changes in other mammals, we reported three distinct patterns (homogenous, punctuated and faded) of pangolin sperm head membrane changes. The concurrent increase in phosphotyrosine protein expression detected at the sperm mid-piece/tail and the emergence of punctuated membrane aggregates likely representing three sperm activation stages, namely inactivated, capacitated and acrosome reacted status of pangolin sperm.ConclusionBy revealing unique epididymal V-ATPase distribution and sperm membrane dynamics in Formosan pangolin, we would understand better the fundamental aspects of reproduction parameters of Formosan pangolin.

Investigation of hydrostatic pressure-induced stress preconditioning of boar semen using modified cryopreservation.

The aim of this study was to investigate the effect of varying hydrostatic pressure treatments (HP) on the boar semen quality during the modified cryopreservation. In Experiment I, combinations of pressure level (20/40/80MPa) and duration of application (40/80/120min) were used. Before freezing, only the magnitude but not the duration influenced the total (TM%) and progressive motilities (PM%). The 20/40MPa levels yielded a significant (p<0.05) improvement compared to control samples (atmospheric), but the 80MPa was detrimental. The post-freezing-thawing (FT) motilities were influenced significantly by both the HP level and its duration. For TM%, the 40MPa:120min gave the highest post-FT result (54.8%±3.3%); however, the 40MPa:80min (41.0%±3.1%) application showed the largest and significant improvement (18.4%±3.1%) compared to its control (22.6%±3.1%) and compared to the improvement (12.9%±3.6%) achieved by 40MPa:120min. For PM%, the improvement with the 40MPa:120min application was slightly larger than with the 40MPa:80min one (15.2%±4.2% vs. 13.8%±3.3%); furthermore, the difference was not significant. In Experiment II, the 40MPa:80min combination was tested at four different stages of the semen handling. By pressurization after dilution with the freezing extender without glycerol, significantly higher post-FT values (TM%, intact acrosome% and head membrane%) were obtained. The two experiments demonstrated possible improvement in post-FT semen quality achievable through the appropriate application of HP to boar semen during cryopreservation.

Study of radiotherapy positioning repeatability of kyphosis patients with head and neck cancer

Objective To compare the repeatability of radiotherapy placement between negative pressure vacuum pad (A group) and common soft pillow (B group) combined with a thermoplastic membrane immobilization technology in severe kyphosis patients with head and neck cancer. Methods From September 2013 to December 2014, 22 cases of severe kyphosis patients with primary or secondary head and neck cancer who received radiotherapy were randomly divided into group A and group B. Padding waist hips with negative pressure vacuum pad and ordinary soft pillow were used so that the patients could lay in CT-Sim bed, and the head cushion was set on the headrest. After the fixing of thermoplastic head and neck shoulder membrane, CT-Sim was located. Every five times of radiotherapy were accompanied by CT-Sim school position once. In the direction of the last layer of right styloid process center point as the reference point, CT-sim software was used to measure the location change with left and right sides, head, and under direction (x, y, z, respectively) in patients, and the data were analyzed. Results Group A significantly reduced the osseous placement error between tags compared with group B (P < 0.05). The average placement errors were as follows, x: (0.68±0.14) mm vs (1.15±0.77) mm, y: (0.48±0.23) mm vs (1.49±1.24) mm, z: (0.76±0.54) mm vs (2.11±1.02) mm, while the average transfer errors were as follows, x: (0.70±0.21) mm vs (1.15±0.93) mm, y: (0.50±0.31) mm vs (1.50±1.28) mm, z: (0.85±0.26) mm vs (1.77±0.88) mm. Conclusion Vacuum suction pad combined with thermoplastic membrane has good repeatability on radiotherapy positioning for severe kyphosis patients with head and neck cancer. Key words: Head and neck neoplasms; Vacuum pad, head and neck shoulder membrane; Radiotherapy positioning repeatability; Errors; Hunchback

Role of Membrane Lipid Fatty Acids in Sperm Cryopreservation

Lipid is an important constituent of cell membrane. Membrane lipid composition of spermatozoa has been correlated to different function. Many researchers have related membrane lipid with survival success after cryopreservation or cold shock. Sperm maturation and acrosome reactions are natural phenomenon, but cryopreservation or cold shock is not. Therefore, sperm cells are not programmed for such change and undergo stress. So the change in membrane lipid composition due to cold shock or cryopreservation may be looked upon as response of spermatozoa to a certain stressed condition. A significant body of research worked on the relationship between membrane lipid and fatty acid composition and ability of cell to tolerate adverse change in temperature. However, as the approach of different research groups was different, it is very difficult to compare the changes. Studies have been done with different species, ejaculated/seminal or epididymal sperm. Lipid analyses have been done with whole cell membrane isolated by different methods. Fatty acids estimated were from whole cell, plasma membrane, head membrane, or phospholipids. The cryopreservation condition, media composition, and diluents/cryoprotectants were also different. At this onset a comprehensive review is needed to cover changes of sperm membrane lipid composition of different species under different cryopreservation conditions.

N-Butanol extract of Rhynchosia volubilis Lour: a potent spermicidal agent In Vitro.

Rhynchosia volubilis Lour has been a major drug in a folk prescription for contraception in China, whereas its mechanism remains unknown. Its antifertility effects on male mice and antimicrobial activities on sexually transmitted infection (STI) pathogens were previously reported. This study was undertaken to develop the n-Butanol extract of Rhynchosia volubilis Lour (BERVL) as a spermicidal agent with STI prevention. The spermicidal activities of BERVL with different doses were assessed using selected high-motile sperms of normal human semen samples, and their inhibitory effects on Lactobacillus acidophilus were determined. The mechanism of the spermicidal activity was explored by aqueous Eosin Y and Hoechst 33342/PI staining. The results showed spermicidal activities and inhibitory effects of BERVL on Lactobacillus acidophilus were dose-dependent. Dose of 90 mg/mL BERVL terminated all progressive sperm motility within 2 min, and had slight inhibitory effect on Lactobacillus acidophilus, suggesting it was an effective and safe dose for contraception use. About 80% sperms exposed to BERVL displayed changes consistent with high permeability of head membrane. It is concluded that BERVL as spermicide has advantages over N-9 with strong ability to instantaneously kill human sperm and possesses light inhibitory effect on Lactobacillus acidophilus.

A boundary element model for acoustic responses in the ear canal and its statistical validation and updating

A boundary element method (BEM) model for acoustic responses in individual ear canals was developed, and its validity was assessed and updated using a statistical approach. The BEM model was developed using high resolution computed tomography (CT) scanning from a healthy male adult. The BEM model, which was discretized adaptively for a frequency band of up to 22 kHz, included the head, pinna, ear canals, and tympanic membranes. The variability of the BEM model due to the uncertain acoustic boundary conditions and measurement locations was predicted using the eigenvector dimension reduction (EDR) method. Then, the likelihood function estimation approach was introduced in order to measure the agreement between the acoustic responses of the simulation model and experimental results. In order to enhance the simulation model performance, the acoustic boundary conditions of the BEM model were updated using a statistical calibration approach that maximizes the likelihood function value between the calculated probability density functions (PDFs) of the simulation model and the measurement data. The results of the validation and calibration procedures applied to the BEM model demonstrated that the proposed method provides a very effective method of verifying the model validity and enhancing the performance of the simulation model.

Human sperm anatomy and endocrinology in varicocele: role of androgen receptor

The study of androgens involved in male reproduction has been object of intense efforts, while their reported action on human male gametes is limited. We previously described the presence of androgen receptor (AR) in sperm with a role related to the modulation of the PI3K pathway. In the present study, we investigated the expression of AR and its ultrastructural location in normal sperm as well as in spermatozoa obtained from varicocele patients. We observed a reduced AR content in varicocele sperm with respect to healthy sperm by western blot analysis and transmission electron microscopy (TEM). The ultrastructural location of AR was detected mainly on the head membrane as well as in the nucleus, neck, and mitochondria. Influence of dihydrotestosterone (DHT) treatment on cholesterol efflux was increased in normal sperm, while it was reduced or absent in varicocele sperm. To better understand DHT/AR significance in human male gametes, we evaluated triglyceride content and lipase, acyl-CoA dehydrogenase, and glucose-6-phosphate dehydrogenase activities upon DHT treatment. The metabolic outcome glimpsed in normal sperm was an increased metabolic rate, while 'varicocele' sperm economized energy. Taken together, our results reveal DHT and AR as new players in sperm endocrinology, indicating that varicocele sperm may have difficulty in switching to the capacitated status. A decreased AR expression and a consequent reduced responsiveness to DHT in sperm may represent molecular mechanisms involved in the pathophysiology of varicocele leading to male infertility. This study revealed new detrimental effects of varicocele on sperm at the molecular level.

Insecticidal 3-benzamido-N-phenylbenzamides specifically bind with high affinity to a novel allosteric site in housefly GABA receptors

γ-Aminobutyric acid (GABA) receptors (GABARs) are an important target for existing insecticides such as fiproles. These insecticides act as noncompetitive antagonists (channel blockers) for insect GABARs by binding to a site within the intrinsic channel of the GABAR. Recently, a novel class of insecticides, 3-benzamido-N-phenylbenzamides (BPBs), was shown to inhibit GABARs by binding to a site distinct from the site for fiproles. We examined the binding site of BPBs in the adult housefly by means of radioligand-binding and electrophysiological experiments. 3-Benzamido-N-(2,6-dimethyl-4-perfluoroisopropylphenyl)-2-fluorobenzamide (BPB 1) (the N-demethyl BPB) was a partial, but potent, inhibitor of [3H]4′-ethynyl-4-n-propylbicycloorthobenzoate (GABA channel blocker) binding to housefly head membranes, whereas the 3-(N-methyl)benzamido congener (the N-methyl BPB) had low or little activity. A total of 15 BPB analogs were tested for their abilities to inhibit [3H]BPB 1 binding to the head membranes. The N-demethyl analogs, known to be highly effective insecticides, potently inhibited the [3H]BPB 1 binding, but the N-methyl analogs did not even though they, too, are considered highly effective. [3H]BPB 1 equally bound to the head membranes from wild-type and dieldrin-resistant (rdl mutant) houseflies. GABA allosterically inhibited [3H]BPB 1 binding. By contrast, channel blocker-type antagonists enhanced [3H]BPB 1 binding to housefly head membranes by increasing the affinity of BPB 1. Antiparasitic macrolides, such as ivermectin B1a, were potent inhibitors of [3H]BPB 1 binding. BPB 1 inhibited GABA-induced currents in housefly GABARs expressed in Xenopus oocytes, whereas it failed to inhibit l-glutamate-induced currents in inhibitory l-glutamate receptors. Overall, these findings indicate that BPBs act at a novel allosteric site that is different from the site for channel blocker-type antagonists and that is probably overlapped with the site for macrolides in insect GABARs.

Cycloxaprid Insecticide: Nicotinic Acetylcholine Receptor Binding Site and Metabolism

Cycloxaprid (CYC) is a novel neonicotinoid prepared from the (nitromethylene)imidazole (NMI) analogue of imidacloprid. In this study we consider whether CYC is active per se or only as a proinsecticide for NMI. The IC50 values (nM) for displacing [(3)H]NMI binding are 43-49 for CYC and 2.3-3.2 for NMI in house fly and honeybee head membranes and 302 and 7.2, respectively, in mouse brain membranes, potency relationships interpreted as partial conversion of some CYC to NMI under the assay conditions. The 6-8-fold difference in toxicity of injected CYC and NMI to house flies is consistent with their relative potencies as in vivo nicotinic acetylcholine receptor (nAChR) inhibitors in brain measured with [(3)H]NMI binding assays. CYC metabolism in mice largely involves cytochrome P450 pathways without NMI as a major intermediate. Metabolites of CYC tentatively assigned are five monohydroxy derivatives and one each of dihydroxy, nitroso, and amino modifications. CYC appears be a proinsecticide, serving as a slow-release reservoir for NMI with selective activity for insect versus mammalian nAChRs.

Centrifugation on Percoll density gradient enhances motility, membrane integrity andin vitrofertilizing ability of frozen–thawed boar sperm

The effects of Percoll density gradient centrifugation on sperm quality, in vitro fertilizability and developmental capacity of frozen-thawed boar sperm were evaluated. Two-step density gradient centrifugation by Percoll enhanced significantly the motility parameters of sperm compared with a simple centrifugation procedure. Percentages of motile sperm and sperm with intact plasma and acrosome membranes after Percoll separation were significantly greater than those after simple centrifugation. The rates of penetration, cleavage and blastocyst formation after in vitro fertilization were significantly improved by Percoll separation compared with simple centrifugation and were influenced positively by the intactness of sperm head membranes, but not any sperm motility parameters. However, insemination with increased concentrations of sperm prepared by Percoll gradient centrifugation did not improve the success of fertilization and embryo development in vitro. Our results indicate that the integrity of sperm head membranes after Percoll separation is important for successful embryo development in vitro, more so than sperm motility.

New GABA/Glutamate Receptor Target for [3H]Isoxazoline Insecticide

The highly effective and selective isoxazoline insecticide A1443 is known to potently displace [(3)H]ethynylbicycloorthobenzoate ([(3)H]EBOB) binding to house fly head membranes with an IC50 of 0.2 nM in a manner characteristic of GABA-gated chloride channel antagonists. To further define its mode of action, we prepared phenyl-labeled [(3)H]A1443 as described with a specific activity of 14 Ci/mmol. This new radioligand with an apparent IC50 of about 0.4 nM is poorly displaced by most insecticides acting at the [(3)H]EBOB site. Interestingly, the isoxazoline binding site is directly coupled to the avermectin GABA/glutamate chloride channel activator site. These findings revive interest in the insect GABA/glutamate receptor as an insecticide target.

ANALYTICAL ASPECTS REGARDING THE FLAVOR COMPOUNDS IN BEER

Contemporary population shows a great tendency to consume food capable of generating an intense feeling. Each food contains substances that generate sensations of taste and aroma. Beer is a food with specific flavor, which is mostly appreciated if, is fresh. It is considered that the beer flavor is dependent on the nature and the concentration of carbonyl compounds that are formed during the fermentation of the malt; the most important is the diacetyl (butandione). Carbonyl compounds in beer can be analyzed after a selective preconcentration. The methods of partial isolation as head space method and membrane process are designed to provide only qualitative information. For quantitative determination of carbonyl compounds present in beer, full extraction methods are used. The gas chromatographic separation method is used in order to study the mixtures obtained after the isolation of carbonyl compounds in beer. When the electron capture detector is used, only vicinal diketones can be identified and dosed in beer. The mixtures of 2,4-dinitrophenylhidrazone precipitates can be analyzed by spectrophotometric study or by liquid chromatographic separation. The analytical focus is the identification and dosing of the butandione and pentandione that have a key role in generating the sensation of flavor of beer. Brewers need rapidly and accurate information on the concentration of diacetyl in beer. In order to help the process, a neural sensor was proposed. It is used to determine the intensity of flavor sensation and it should provide the possibility of differentiating the products that come from different beer suppliers.

Prostasomal DNA characterization and transfer into human sperm

Human prostasomes, exosome-like microvesicles secreted by acinar cells of the prostate gland, contain chromosomal DNA. Agarose gel electrophoresis of DNA from seminal prostasomes displayed fragments of over 12 kb and smaller, with a distinct band around 1 kb that was excised, cloned, and sequenced. The sequences showed 8 out of 25 clones (32%) originating from genes. We elaborated the concept further by carrying out a genome-wide DNA copy number analysis of prostasomal DNA, hypothesizing that human prostasomes contain fragments of DNA randomly selected from the entire genome. Acridine orange-stained prostasomes were incubated with freshly prepared sperm for different times, and a transfer of acridine orange-stained prostasomal DNA to sperm (preferentially the head region) was observed. Fluorescence microscopy of slices in the center of 14 optical slides of the sperm head displayed an even fluorescence rather than a halo-like one, indicating DNA-uptake rather than just binding along the sperm head membrane.

In vitroeffect of nickel on bovine spermatozoa motility and annexin V‐labeled membrane changes

In this study the effect of in vitro culture of bovine spermatozoa with nickel (NiCl(2)) on spermatozoa motility and membrane changes was analyzed. The spermatozoa motility significantly decreased after 120 min of culture at the concentration of 1000 μM Ni ml(-1) (P < 0.05) and after 240 min of culture at the concentration of 500 and 1000 μM Ni ml(-1) (P < 0.001) as compared with control. The progressive motility was the highest in the control group and in the groups with the lowest nickel concentrations (7.8 and 125 μM Ni ml(-1)). The progressive spermatozoa motility was significantly altered even after 30 min of culture in the group with the highest nickel concentration (1000 μM Ni ml(-1)). A significant decrease in progressive motility from the concentration of 250 μM Ni ml(-1) was detected after 240 min of culture. Concentrations from 125 μM Ni ml(-1) in various time periods of culture stimulated spermatozoa motility after 30 min (P < 0.001), but later an inhibitory effect was noted. After 240 min of in vitro spermatozoa culture with 125 μM Ni ml(-1) a typical Annexin V fluorescence reaction was detected. Fluorescence was detected in mitochondrial segment of bovine spermatozoa. In spermatozoa exposed to higher nickel concentrations the Annexin V-positive reaction was detected also on the spermatozoa head membrane. In the group with the highest concentration and the longest time of exposure (1000 μM Ni ml(-1); 240 min) the apoptotic Annexin-positive regions were detected not only in the mitochondrial part, but also in the spermatozoa head (acrosomal and postacrosomal part), showing significant alteration of spermatozoa membrane integrity.

Effect of seminal plasma on functional integrity of rabbit sperm membranes during storage at 4ºC or freezing.

The effect of semen plasma removal either by simple centrifugation or by separation through a Percoll gradient on the integrity of plasma membranes of rabbit spermatozoa during storage at 4ºC and freezing was evaluated in two successive experiments. A modifi ed hypo-osmotic swelling test procedure combined with supravital staining was employed to evaluate simultaneously membrane integrity of head and tail membranes of sperm cells. In the fi rst experiment, the impact of semen plasma on membrane integrity of sperm cells was examined in Tris-citric acid-glucose extender at 4ºC for 96 h. The percentage of sperm cells with disintegrated tail and head membranes increased in all groups in correlation with the length of storage. After storage for 96 h, removal of semen plasma, irrespective of the method of removal, resulted in signifi cant increase (P&lt;0.01) in the percentage of sperm cells with disintegrated plasma membranes. The adverse effect of storage and removal of semen plasma was more prominent on the tail membranes, and especially during the fi rst 24 h of the storage period. In the second experiment, the impact of semen plasma on membrane integrity of sperm cells undergoing freezing was examined. A total of three groups were arranged as described in the fi rst experiment, and semen samples were frozen in straws using an extender including acetamide and methyl cellulose. Freezing of semen drastically reduced the percentage of sperm cells with intact plasma membranes in postthaw samples. However, removal of semen plasma, irrespective of the method of removal, did not affect the proportion of sperm cells with intact plasma membranes. In conclusion, the effect of semen plasma on plasma membranes varied signifi cantly relative to the preservation temperature of sperm cells. Although it exerted a protective infl uence during storage at 4ºC, no protective impact was monitored during freezing.