Showing papers on "Sperm plasma membrane published in 2014"

Oxidative stress and male reproductive health

Abstract: One of the major causes of defective sperm function is oxidative stress, which not only disrupts the integrity of sperm DNA but also limits the fertilizing potential of these cells as a result of collateral damage to proteins and lipids in the sperm plasma membrane. The origins of such oxidative stress appear to involve the sperm mitochondria, which have a tendency to generate high levels of superoxide anion as a prelude to entering the intrinsic apoptotic cascade. Unfortunately, these cells have very little capacity to respond to such an attack because they only possess the first enzyme in the base excision repair (BER) pathway, 8-oxoguanine glycosylase 1 (OGG1). The latter successfully creates an abasic site, but the spermatozoa cannot process the oxidative lesion further because they lack the downstream proteins (APE1, XRCC1) needed to complete the repair process. It is the responsibility of the oocyte to continue the BER pathway prior to initiation of S-phase of the first mitotic division. If a mistake is made by the oocyte at this stage of development, a mutation will be created that will be represented in every cell in the body. Such mechanisms may explain the increase in childhood cancers and other diseases observed in the offspring of males who have suffered oxidative stress in their germ line as a consequence of age, environmental or lifestyle factors. The high prevalence of oxidative DNA damage in the spermatozoa of male infertility patients may have implications for the health of children conceived in vitro and serves as a driver for current research into the origins of free radical generation in the germ line.

Reprotoxicity of gold, silver, and gold–silver alloy nanoparticles on mammalian gametes

Abstract: Metal and alloy nanoparticles are increasingly developed for biomedical applications, while a firm understanding of their biocompatibility is still missing. Various properties have been reported to influence the toxic potential of nanoparticles. This study aimed to assess the impact of nanoparticle size, surface ligands and chemical composition of gold, silver or gold–silver alloy nanoparticles on mammalian gametes. An in vitro assay for porcine gametes was developed, since these are delicate primary cells, for which well-established culture systems exist and functional parameters are defined. During coincubation with oocytes for 46 h neither any of the tested gold nanoparticles nor the gold–silver alloy particles with a silver molar fraction of up to 50% showed any impact on oocyte maturation. Alloy nanoparticles with 80% silver molar fraction and pure silver nanoparticles inhibited cumulus–oocyte maturation. Confocal microscopy revealed a selective uptake of gold nanoparticles by oocytes, while silver and alloy particles mainly accumulated in the cumulus cell layer surrounding the oocyte. Interestingly sperm vitality parameters (motility, membrane integrity and morphology) were not affected by any of the tested nanoparticles. Only sporadic association of nanoparticles with the sperm plasma membrane was found by transmission electron microscopy. In conclusion, mammalian oocytes were sensitive to silver containing nanoparticles. Likely, the delicate process of completing meiosis in maternal gametes features high vulnerability towards nanomaterial derived toxicity. The results imply that released Ag+-ions are responsible for the observed toxicity, but the compounding into an alloy seemed to alleviate the toxic effects to a certain extent.

Oviduct Binding and Elevated Environmental pH Induce Protein Tyrosine Phosphorylation in Stallion Spermatozoa

Abstract: Sperm-oviduct binding is an essential step in the capacitation process preparing the sperm for fertilization in several mammalian species. In many species, capacitation can be induced in vitro by exposing spermatozoa to bicarbonate, Ca 2+ ,a nd albumin; however, these conditions are insufficient in the horse. We hypothesized that binding to the oviduct epithelium is an essential requirement for the induction of capacitation in stallion spermatozoa. Sperm-oviduct binding was established by coincubating equine oviduct explants for 2 h with stallion spermatozoa (2 3 10 6 spermatozoa/ml), during which it transpired that the highest density (per mm 2 ) of oviduct-bound spermatozoa was achieved under noncapacitating conditions. In subsequent experiments, sperm-oviduct incubations were performed for 6 h under noncapacitating versus capacitating conditions. The oviduct-bound spermatozoa showed a timedependent protein tyrosine phosphorylation response, which was not observed in unbound spermatozoa or spermatozoa incubated in oviduct explant conditioned medium. Both oviductbound and unbound sperm remained motile with intact plasma membrane and acrosome. Since protein tyrosine phosphorylation can be induced in equine spermatozoa by media with high pH, the intracellular pH (pH i ) of oviduct explant cells and bound spermatozoa was monitored fluorometrically after staining with BCECF-AM dye. The epithelial secretory cells contained large, alkaline vesicles. Moreover, oviduct-bound spermatozoa showed a gradual increase in pH i , presumably due to an alkaline local microenvironment created by the secretory epithelial cells, given that unbound spermatozoa did not show pH i changes. Thus, sperm-oviduct interaction appears to facilitate equine sperm capacitation by creating an alkaline local environment that triggers intracellular protein tyrosine phosphorylation in bound sperm.

Antimicrobial host defence peptide, LL-37, as a potential vaginal contraceptive

Abstract: STUDY QUESTION Does antimicrobial peptide, LL-37, inhibit sperm fertilizing ability? SUMMARY ANSWER Our results indicate that LL-37 inhibits mouse and human sperm fertilizing ability. WHAT IS KNOWN ALREADY LL-37, a cationic antimicrobial peptide, exerts its microbicidal effects through the disruption of microbial cytoplasmic membranes following its interaction with microbial surface anionic phospholipids. ALL-38 (an LL-37 close analogue: LL-37 + Ala at the N-terminus) is produced in the vagina 2-6 h post-intercourse from its precursor hCAP-18, a seminal plasma component. At this time, motile sperm have already swum into the uterine cavity, thus unexposed to ALL-38. Since sperm contain a substantial amount of acidic sulfogalactosylglycerolipid (SGG) on their surface, treatment of sperm with LL-37 may cause their membrane disruption in an analogous manner to that occurring on microbial membranes. STUDY DESIGN, SIZE AND DURATION Mouse/human sperm treated (2-30 min) with LL-37 in a physiological concentration range (up to 10.8 µM) were assessed for SGG-dependent LL-37 binding, and parameters relevant to fertilizing ability, namely motility and intactness of the sperm acrosome and plasma membrane. Ability of mouse sperm to fertilize eggs in vitro was also evaluated. Each study was performed with greater than or equal to three different sperm samples. The efficacy of LL-37 to inhibit sperm fertilizing ability in vivo was determined in female mice (n = 26 each for LL-37 treatment and no treatment), using sperm retrieved from 26 males. PARTICIPANTS/MATERIALS, SETTING, METHODS Human sperm samples were donated by fertile men. LL-37 was chemically synthesized and was biotinylated for sperm binding studies. Sperm motility was assessed by videomicroscopy and the acrosomal status by Coomassie blue staining of acrosome-intact mouse sperm or the exposure of CD46, an inner acrosomal membrane protein, of acrosome reacted human sperm. Sperm membrane permeabilization/disruption was assessed by the loss of hypo-osmotic swelling response, an incorporation of Sytox Green (a membrane impermeable fluorescent DNA dye), and electron microscopy. Mouse IVF was scored by the presence of two pronuclei in eggs 6 h post-insemination. Ability of mouse sperm to fertilize eggs in vivo was determined by the pregnancy outcome of female mice injected transcervically with sperm with or without LL-37. MAIN RESULTS AND THE ROLE OF CHANCE Biotinylated LL-37 bound to both mouse and human sperm and the binding was partially dependent on sperm surface SGG. Mouse and human sperm became immotile and underwent a premature acrosome reaction upon treatment with LL-37 at 3.6 and 10.8 µM, respectively. The initial action of LL-37 on both mouse and human sperm appeared to be through permeabilization/disruption of sperm surface membranes evidenced by the loss of hypo-osmotic swelling response, Sytox Green staining and electron microscopy revealing ultrastructural damage. Mouse sperm treated with 3.6 µM LL-37 lost the ability to fertilize eggs both in vitro and in vivo. All 26 female mice inseminated with sperm and LL-37 did not become pregnant. No apparent damage to the reproductive tract was observed as revealed by histological characterization in LL-37-inseminated mice and these females resumed fecundity following mating with fertile males. LIMITATIONS, REASONS FOR CAUTION Direct demonstration that LL-37 treated human sperm fail to fertilize eggs was limited by legal restrictions on obtaining human eggs for such use. WIDER IMPLICATIONS OF THE FINDINGS Our results reveal selective inhibitory effects of LL-37 on sperm fertilizing ability in mice without apparent impairment to the female reproductive tract. LL-37 is therefore a promising candidate to be developed into a vaginal contraceptive with microbicidal activity. STUDY FUNDING/COMPETING INTEREST(S) This work was supported by Grand Challenges Explorations grant from the Bill & Melinda Gates Foundation (OPP1024509), Canadian Institutes of Health Research (MOP119438 & CCI82413) and International Collaboration and Exchanges NSFC of China (No.30611120525). There are no competing interests to declare.

Sperm maturation in dogs: sperm profile and enzymatic antioxidant status in ejaculated and epididymal spermatozoa.

Abstract: Spermatozoa become more susceptible to the attack of reactive oxygen species during maturation. To avoid oxidative damage, the epididymis must provide the necessary antioxidant protection. The aim of this study was to compare the canine sperm profile and the enzymatic antioxidant status of the ejaculated fractions and samples collected from the different segments of the epididymis (caput, corpus and cauda). Five adult dogs were used, and after 1-3 weeks, subsequently to bilateral orchiectomy and epididymal storage, sperm samples were collected from the different segments of the epididymis. Samples were evaluated for conventional microscopy and computer-assisted motility analysis: sperm plasma membrane permeability and the activity of the antioxidant enzymes catalase, glutathione peroxidase (GPx) and superoxide dismutase (SOD). Samples collected from the caput and corpus showed lower values for most of the motility variables evaluated, indicating different levels of immaturity. Catalase activity was observed only in ejaculated samples. Conversely, GPx activity was higher in the cauda epididymidis. Correlations were found between SOD and GPx and SOD and sperm motility in the epididymal cauda and corpus, highlighting the importance of the enzymes for the protection of spermatozoa during the transit along the epididymis.

The Effect of Curcumin on Intracellular pH (pHi), Membrane Hyperpolarization and Sperm Motility.

Abstract: Background: Curcumin has shown to affect sperm motility and function in vitro and fertility in vivo. The molecular mechanism(s) by which curcumin affects sperm motility has not been delineated. Since modulation of intracellular pH (pHi) and plasma membrane polarization is involved in sperm motility, the present study was conducted to investigate the effect of curcumin on these sperm (human and murine) parameters. Methods: The effect of curcumin on sperm forward motility was examined by counting percentages of forward moving sperm. The effect of curcumin on intracellular pH (pHi) was measured by the fluorescent pH indicator 2,7-bicarboxyethyl-5,6carboxyfluorescein-acetoxymethyl ester (BCECF-AM). The effect of curcumin on plasma membrane polarization was examined using the fluorescence sensitive dye bis (1,3-dibarbituric acid)-trimethine oxanol [DiBAC4(3)]. Results: Curcumin caused a concentration-dependent (p<0.05) decrease in forward motility of both human and mouse sperm. It also caused a concentration-dependent decrease in intracellular pH (pHi) in both human and mouse sperm. Curcumin induced significant (p<0.05) hyperpolarization of the plasma membrane in both human and mouse sperm. Conclusion: These findings indicate that curcumin inhibits sperm forward motility by intracellular acidification and hyperpolarization of sperm plasma membrane. This is the first study to our knowledge which examined the effect of curcumin on sperm pHi and membrane polarization that affect sperm forward motility. These exciting findings will have application in deciphering the signal transduction pathway involved in sperm motility and function and in development of a novel non-steroidal contraceptive for infertility.

Arachidic Acid in Extender Improves Post-thaw Parameters of Cryopreserved Nili-Ravi Buffalo Bull Semen

Abstract: Cryopreservation process reduces lipids and phospholipids from buffalo bull spermatozoa. It was therefore hypothesized that supplementation of fatty acid to extender may improve the post-thaw quality of buffalo semen. The objective was to evaluate the effect of arachidic acid supplementation in extender on post-thaw quality of buffalo bull (Bubalus bubalis) spermatozoa. Semen was collected from three adult Nili-Ravi buffalo bulls of similar age group with artificial vagina (42°C) for 3 weeks (replicate). Qualified semen ejaculates (n = 18) were split into four aliquots and diluted in tris-citric acid extender containing 0.0 (control), 5.0, 10.0 and 20.0 ng/ml at 37°C having approximately 50 × 10(6) spermatozoa/ml. Diluted semen was cooled to 4°C in 2 h and equilibrated for 4 h at 4°C. Cooled semen was filled in 0.5-ml straws at 4°C, kept on liquid nitrogen vapours for 10 min and plunged in liquid nitrogen for storage. Thawing of frozen semen was performed after 24 h at 37°C for 30 s. Sperm progressive motility (%) was improved in a dose-dependent manner by supplementing arachidic acid at 5.0, 10.0 and 20.0 ng/ml compared with control. Structural and functional integrity of sperm plasma membrane (%), number of acrosome-intact live sperm (%) and sperm chromatin integrity (%) were better (p 0.05) from those at 5.0 ng/ml. Further improvement in structural and functional integrity of sperm plasma membrane, number of acrosome-intact live sperm and chromatin integrity was observed at 20.0 ng/ml of arachidic acid in extender. In conclusion, arachidic acid supplementation in extender improved the post-thaw quality parameters of cryopreserved Nili-Ravi buffalo bull spermatozoa. Among the arachidic acid concentrations studied, maximum improvement in post-thaw semen quality parameters was observed at 20.0 ng/ml.

A microarray-based analysis of gametogenesis in two Portuguese populations of the European clam Ruditapes decussatus.

Abstract: The European clam, Ruditapes decussatus is a species with a high commercial importance in Portugal and other Southern European countries. Its production is almost exclusively based on natural recruitment, which is subject to high annual fluctuations. Increased knowledge of the natural reproductive cycle of R. decussatus and its molecular mechanisms would be particularly important in providing new highly valuable genomic information for better understanding the regulation of reproduction in this economically important aquaculture species. In this study, the transcriptomic bases of R. decussatus reproduction have been analysed using a custom oligonucleotide microarray representing 51,678 assembled contigs. Microarray analyses were performed in four gonadal maturation stages from two different Portuguese wild populations, characterized by different responses to spawning induction when used as progenitors in hatchery. A comparison between the two populations elucidated a specific pathway involved in the recognition signals and binding between the oocyte and components of the sperm plasma membrane. We suggest that this pathway can explain part of the differences in terms of spawning induction success between the two populations. In addition, sexes and reproductive stages were compared and a correlation between mRNA levels and gonadal area was investigated. The lists of differentially expressed genes revealed that sex explains most of the variance in gonadal gene expression. Additionally, genes like Foxl2, vitellogenin, condensing 2, mitotic apparatus protein p62, Cep57, sperm associated antigens 6, 16 and 17, motile sperm domain containing protein 2, sperm surface protein Sp17, sperm flagellar proteins 1 and 2 and dpy-30, were identified as being correlated with the gonad area and therefore supposedly with the number and/or the size of the gametes produced.

Increasing Extender Viscosity Improves the Quality of Cooled Boar Semen

Abstract: The use of several types of gelling extenders for the storage of semen from several domestic species in the solid state has been shown to have beneficial effects on some semen quality parameters. The objective of this study was to evaluate the effect of a new high-viscosity semen extender, Zoosperm ND-5 3D ® (Import-Vet, Centelles, Spain), on the the quality of boar spermatozoa at preserved at 17oC for 7 days. Sodium alginate was used for the first time to increase the viscosity of the extender for the liquid storage of boar semen. The same extender, but without increased viscosity, was used as a control extender (Zoosperm ND-5 ® , Import-Vet, Centelles, Spain). Sixteen ejaculates from four Pietrain boars were evaluated for motility (by the CASA system), and for viability, acrosome status, plasma membrane fluidity, externalization of phosphatidylserine at the plasma membrane of the spermatozoa and mitochondrial membrane potential (by flow cytometry). In samples diluted with the Zoosperm ND-5 3D ® viscous extender, the STR (straightness) parameter and the number of progressively motile spermatozoa were higher compared to those of the non-viscous extender (p < 0.05). In addition, the number of spermatozoa with damaged acrosomes, an unstable sperm plasma membrane and externalization of phosphatidylserine at the plasma membrane was lower in samples treated with the viscous extender (p < 0.05). In conclusion, an increase in extender viscosity improves quality of boar spermatozoa following long-term storage.

Bactericidal/permeability-increasing protein originates in both the testis and the epididymis and localizes in mouse spermatozoa.

Abstract: Bactericidal/permeability-increasing protein (BPI) is an endogenous antibiotic protein with activity against gram-negative bacteria. In the present study, we examined the expression of BPI in postnatal mouse testes and epididymides as well as the subcellular localization within epididymal spermatozoa. Our results showed that, BPI mRNA was expressed in testis and epididymis independently. Throughout the epididymis, the BPI protein level gradually decreased in the epididymal epithelium in a spatial manner, specialized within the cytoplasm of clear cells in the cauda part. We detected BPI proteins in intact acrosome, implying its testicular origin; on the other hand, after the acrosome reaction, BPI proteins were observed dispersed across the entire sperm head, especially enriched at the equatorial segment. Our findings suggested a dual origin of the BPI that generated both in the testis and epididymis, and associated with mouse spermatozoa. BPI protein might be involved in the dynamics modification of the sperm plasma membrane and also the fertilization process.

Impact of Reactive Oxygen Species on Spermatozoa: ABalancing Act between Beneficial and Detrimental Effects

Abstract: Reactive oxygen species (ROS) plays an important role in sperm motility. The physiological generation at low concentration induces beneficial effects on sperm functions and plays a significant role in sperm metabolism. Meanwhile, the excessive generation of reactive oxygen species can overwhelm protective mechanism and triggers changes in lipid and protein layers of sperm plasma membrane, which induces lipid damage, protein damage, DNA damage, motility impairment and alteration in capacitation and acrosome reaction. Reactive oxygen species (ROS) can be measured by variable biomarkers as melondialdehyde (MDA) concentration, individual free radicals, etc. The quantification of free radicals in livestock semen is also briefly reported.

Significance of egg's zona pellucida glycoproteins in sperm-egg interaction and fertilization.

Abstract: Mammalian fertilization is a highly programmed process by which sperm and egg unite to form a zygote, a cell with somatic chromosome numbers. To fertilize an egg, the capacitated (acrosome-intact) spermatozoa recognize and bind to the egg's extracellular glycocalyx coat, the zona pellucida (ZP). The tight and irreversible binding of the opposite gametes in the mouse and many other species studied, including man, results in the opening of Ca2+ channels on sperm plasma membrane (PM) and influx of Ca2+. The transient rise in Ca2+ and other second messengers, such as cAMP and IP3, initiates a cascade of signaling events that elevate sperm pH and triggers the fusion of the sperm PM and underlying outer acrosomal membrane at multiple sites (induction of the acrosomal reaction). The fusion of the two membranes results in the exocytosis of acrosomal contents at the site of sperm-egg adhesion. The hydrolytic action of the acrosomal enzymes (glycohydrolases, proteinases, esterases, sulfatases etc), released at the site of sperm-egg adhesion, along with the enhanced thrust generated by the hyperactivated spermatozoon, are important factors that regulate the penetration of the ZP and the fusion of the acrosome-reacted spermatozoon with the egg. Evidence accumulated over the past two decades strongly suggests that glycan units of the ZP have a significant role in the recognition and adhesion of the opposite gametes and induction of the AR. In this review article, we intend to highlight well programmed molecular events that results in the sperm-egg adhesion and fertilization. Our intention is also to discuss the increasing controversy on the role of ZP glycan chains in sperm-egg interactions.

Modified hypoosmotic swelling test for the assessment of boar and bull sperm sensitivity to cryopreservation

Abstract: Routine methods for the evaluation of sperm quality are not sufficiently useful to determine the sensitivity of sperm cells to cold shock. The aim of our preliminary study was to determine whether the sperm plasma membrane integrity evaluated by modified hypoosmotic swelling test based on simple hypoosmotic swelling test (HOS test) and eosin-nigrosin staining could be helpful in predicting the degree of boar sperm survivability during semen cryopreservation. Ejaculates collected from 24 boars and 20 bulls were used in the experiment. Fresh ejaculates were evaluated by routine sperm analysis and a modified HOS test, and subsequently frozen. Sperm cryosurvivability was defined as the percentage of motile spermatozoa that survived the freezing process. A higher percentage of sperm was recovered after the thawing of semen with a higher percentage of HOS-positive and eosin-negative sperm (P < 0.01). Both indicators were found to be correlated (r = 0.707 and r = 0.705, respectively; P < 0.01). Moreover, the percentage of HOS-positive and eosin-negative sperm was similar to the percentage of viable sperm after thawing as determined by traditional eosin-nigrosin staining in boars (50.90 ± 9.88% and 49.31 ± 11.63%, respectively) and bulls (55.91 ± 10.34% and 55.63 ± 6.64%, respectively) and both indicators showed a positive correlation (r = 0.558 and r = 0.504, respectively; P < 0.05). In conclusion, based on the obtained results, we can assume that the modified HOS test can detect differences in sperm membrane resistance which allows assessment of semen quality from the perspective of its further use, e.g. cryopreservation. Ejaculate, membrane, viability, survival test, freezing, semen analysis As the use of artificial insemination with preserved semen forms a large part of reproductive technologies, production of high-quality insemination doses is of major interest. Various approaches to improving the quality of frozen-thawed sperm have been used in relation to changes in the holding times before freezing; new packaging systems; addition of various additives; use of intrauterine artificial insemination etc. However, there is great variability among individual animals in sustaining sperm cryopreservation. Cryopreservation of semen is associated with different injuries to the spermatozoa, such as cold shock, osmotic stress, cryoprotectant intoxication and intracellular ice crystal formation during freezing and thawing. Therefore, the cryopreservation process results in reduced fertility compared to fresh semen. Boar spermatozoa, especially, are characterized by marked inter-individual differences in their resistance to cold shock and freezing (Holt et al. 2005). In comparison with other farm animals, boar spermatozoa have a higher unsaturated/saturated fatty acid ratio and lower cholesterol content, and thus their response to decreasing temperatures is stronger (Parks and Lynch 1992). We need to identify those studs and/or ejaculates that will better survive the preservation process. The question that motivated our study was as to whether we were able to predict the ability of sperm to survive the preservation process merely on the basis of the assessment of fresh semen. However, routine methods for the evaluation of semen quality are considered insufficient to determine the sensitivity of sperm cells to cold shock (Amann 1989; Roca et al. 2006; Casas et al. 2010). ACTA VET. BRNO 2014, 83: 313–319; doi:10.2754/avb201483040313 Address for correspondence: MVDr. Petra Přinosilova, Ph.D. Laboratory for Spermatology and Andrology Department of Genetics and Reproduction Veterinary Research Institute, Hudcova 70, 621 00 Brno Phone: +420 777 786 828 E-mail: prinosilova@vri.cz http://actavet.vfu.cz/ Sperm plasma membrane integrity is commonly evaluated by intravital staining tests documenting a change in the permeability of the plasma membrane or by a test of membrane tolerance and resistance to hypo-osmotic conditions (HOS test). Simple HOS test, introduced by Jeyendran et al. (1984) is still widely used for the evaluation of both human and animal semen. The rationale of the test is based on the assumption that after the passage of fluid into an undamaged membrane of a live sperm under hypoosmotic conditions, coiling of sperm tails occurs. However, poor quality spermatozoa with borderline membrane integrity will exhibit swelling in simple HOS test, thereby yielding false positive results. By combining supravital staining with HOS test, based on eosin-nigrosin staining of semen samples that underwent HOS test and eosin-nigrosin staining, the number of false positive results can be reduced (Chan et al. 1991). Moreover, modified HOS test (mHOST) brings more information about the integrity of the whole sperm membrane because HOS test gives information regarding the sperm tail membrane integrity, while the sperm viability test is more specific in evaluating the integrity of the sperm head membranes. It has been shown that the membranes in the head and tail compartments have different lability (Hammerstedt 1979). Accordingly, based on the above mentioned facts, the aim of our preliminary study was to investigate whether plasma membrane integrity, evaluated by mHOST, can be helpful in predicting the degree of boar and bull sperm survivability during semen cryopreservation. Materials and Methods Fresh bull (Experiment 1) and boar (Experiment 2) ejaculates were evaluated by routine sperm analysis and by modified HOS test and were then frozen in straws. The standard fresh semen analysis included total sperm count, total sperm motility and progressive sperm motility, and plasma membrane integrity (viability) – evaluated by eosin nigrosin staining (World Health Organization 2010). Semen volume was measured using a graduated vial and concentration was measured in a Burker chamber. Sperm motility (and progressive motility) was analysed under an optical microscope at × 200 magnification. Sperm morphology was evaluated according to Tygerberg’s strict criteria (Kruger et al. 1986). Samples were stained for sperm morphology analysis according to Farelly (smears were fixed in 3.5% formalin and stained with 5% aniline blue for 10 s and 0.5% crystal violet for 6 s) and evaluated with the use of the SASMO computer program (Strict Analysis of Sperm Morphology; Veznik et al. 2001). The analysis of sperm motility and viability was carried out also after the semen samples were thawed. The principle of modified HOS test is based upon the addition of 0.1 ml of fresh semen to 1 ml of a hypoosmotic solution equilibrated to 37 °C (World Health Organization 2010). The sample was then incubated at 37 °C for 30 min. After incubation and mixing, the sample was smeared on a slide. Additional staining with 0.5% eosin and 10% nigrosin was carried out before microscopic evaluation at × 1000 magnification, using oil immersion. At least 200 spermatozoa were evaluated. Having performed modified HOS test, we obtained the following main three sperm categories: 1) HOS-positive and eosin-negative, i.e. live spermatozoa with good membrane resistance, 2) HOS-negative and eosin-positive, i.e. dead spermatozoa and 3) HOS-positive and eosin-positive, i.e. viable spermatozoa but with reduced plasma membrane resistance (Plate V, Fig. 1). Experiment 1: Determination of boar sperm sensitivity to cryopreservation The ejaculates of 24 boars (Pietren and Large White breed of proven fertility) were collected by the glovedhand technique. An aliquot of each semen sample was gently mixed with BTS medium (Beltsville thawing solution, Minitube, Tiefenbach, Germany) at a 1:1 ratio (v/v) and transported in an insulated container at 17 °C within 1 h to the laboratory and immediately assessed. Only ejaculates with sperm motility higher than 60% were used in the experiment. Ejaculates were frozen using the standard procedure as described by Westendorf et al. (1975) and modified by Thurston et al. (1999). Briefly, after 20 h storage at 17 °C the extended semen was centrifuged at 800 × g for 10 min. The supernatant was discarded and the pellet was resuspended in a cooling extender (0.24 M lactose, 20% egg yolk (v/v) and 100 μg/ml kanamycin sulfate) at a ratio of 3/5 of the final volume and cooled to 5 °C within 2 h. Subsequently, the freezing extender (0.24 M lactose, 20% egg yolk (v/v), 7.5% glycerol (v/v), 1.3% Equex STM (v/v), Minitube, Tiefenbach, Germany, and 100 μg/ml kanamycin sulfate) was added at a ratio of 2/5 of the final volume, thus giving final glycerol concentration of 3%. The final sperm concentration was 0.5 × 109 spermatozoa/ ml. The semen was loaded into 0.5 ml straws (Minitube, Tiefenbach, Germany), sealed and placed in liquid nitrogen vapour at 4 cm above the nitrogen level for 20 min, then plunged into liquid nitrogen and stored until use. After at least 2–4 weeks of storage, samples were thawed in a water bath at 38 °C for 30 s and emptied into BTS medium to reach the final sperm concentration of 100 × 106 spermatozoa/ml. 314

Effect of Bovine Serum Albumin on Motility, Plasmalemma, Viability and Chromatin Integrity of Buffalo Bull Spermatozoa

Abstract: The present work was designed to study the effect of different concentrations of bovine serum albumin (BSA) in extender on post-thaw quality of buffalo spermatozoa. Semen was collected from three buffalo bulls with artificial vagina (42°C) for three weeks (replicates). Qualifying semen ejaculate from each bull was split into five aliquots and diluted in tris-citric egg yolk extender containing either 0.0 (control) or 1.0 or 2.0 or 3.0 or 4.0 mg/ml BSA. Diluted semen was cooled to 4C in 2 h and equilibrated for 4 h at the same temperature. Cooled semen was filled in 0.5 ml plastic straws at 4C, kept over liquid nitrogen vapours (5cm) for 10 min and then plunged in the liquid nitrogen for storage. Frozen straws were thawed at 37°C for 30 seconds in duplicate for the assessment of sperm motility, plasma membrane integrity, viability and chromatin damage at 0, 2 and 4 h post-thaw. Sperm motility and chromatin damage remained similar in all experimental extenders at 0, 2 and 4 h post-thaw. However, higher (P>0.05) values of sperm chromatin damage were observed in extender without BSA supplementation. Sperm plasma membrane integrity and viability were observed higher (P<0.05) in extenders containing BSA compared to control at 0 h post-thaw. However, aforementioned parameters were similar in all extenders at 2 and 4 h post-thaw. The results of this study suggest protective role of BSA in cryopreservation of buffalo semen and needs further investigations.

Identification of peroxiredoxin-5 in bovine cauda epididymal sperm

Abstract: Developing spermatozoa require a series of posttesticular modifications within the luminal environment of the epididymis to achieve maturation; this involves several surface modifications including changes in plasma membrane lipids, proteins, carbohydrates, and alterations in the outer acrosomal membrane. Epididymal maturation can therefore allow sperm to gain forward motility and fertilization capabilities. The objective of this study was to identify maturation-dependent protein(s) and to investigate their role with the production of functionally competent spermatozoa. Lectin blot analyses of caput and cauda sperm plasma membrane fractions identified a 17.5 kDa wheat germ agglutinin (WGA)-binding polypeptide present in the cauda sperm plasma membrane not in the caput sperm plasma membrane. Among the several WGA-stained bands, the presence of a 17.5 kDa WGA-binding polypeptide band was detected only in cauda epididymal fluid not in caput epididymal fluid suggesting that the 17.5 kDa WGA-binding polypeptide is secreted from the cauda epididymis and binds to the cauda sperm plasma membrane during epididymal transit. Proteomic identification of the 17.5 kDa polypeptide yielded 13 peptides that matched the sequence of peroxiredoxin-5 (PRDX5) protein (Bos Taurus). We propose that bovine cauda sperm PRDX5 acts as an antioxidant enzyme in the epididymal environment, which is crucial in protecting the viable sperm population against the damage caused by endogeneous or exogeneous peroxide.

Cholesterol efflux from sperm: approaches and applications

Abstract: Following ejaculation, sperm appear morphologically mature, but functionally they are inactive. To attain functional capability or capacitation in vivo, sperm must reside in the female reproductive tract for a certain period of time before fertilization. In the female reproductive tract, cholesterol efflux from the sperm plasma membrane is considered vital for the initiation of capacitation. A number of in vitro studies have used biological and nonphysiological means to understand the phenomenon of cholesterol efflux and capacitation. In this review, we present a short overview of the role of cholesterol efflux in regulating sperm capacitation using various agents. This review provides insight to researchers designing new procedures for sperm preparation to be used in assisted reproductive technology.

Confronting the fissions and fusions of human fertilization

Abstract: Two year ago, an article appeared in JARG documenting and confirming for the first time a fast block to polyspermy in the human (Mio et al., Possible mechanism of polyspermy block in human oocytes observed by time-lapse cinematography, 2012 J Assist Reprod Genet, 29:951–956, DOI 10.1007/s10815-012-9815). That the fusion of sperm and egg plasma membranes launches the program of development from which organisms arise remains a central tenet in plant and animal biology. And, as has been appreciated for many years, the ability of the oocyte to fend off supernumerary sperm and assure a diploid composition of chromosomes in the resulting zygote relies on the establishment of a block to polyspermy through mechanisms that we now understand to be of both fast and slow varieties. While the existence of fast and slow blocks to polyspermy has been well documented in divergent metazoan organisms, controversy remained as to the presence of a fast block in the human oocyte—that is, until the publication of Mio and colleagues—mainly because of the lack of imaging strategies permitting the tracking multiple sperm during the process of in vitro fertilization (IVF). Even though the problems associated with polyspermy persist in the practice of human ARTs, and the introduction of ICSI in the 1990s has greatly reduced the incidence of this type of genetic imbalance during the generation of human embryos, it remains a serious problem for clinics that continue to use IVF in their daily practices. Most scientists and clinicians would argue for the continuation of investigations into the basic mechanisms underscoring human fertilization even in the face of opposition from governments and societies uncomfortable with such lines of research. At the heart of all such studies has been the lingering fascination with the various fissions and fusions featured at the level of the plasma membrane of gametes. Whether your fancy be the acrosome reaction, intermingling of sperm and egg membranes, or the burst of exocytosis of cortical granules that stages the so-called slow block to polyspermy, the precision and celerity of membrane dynamics that defines successful fertilization stands alone as one of the most spectacular biological phenomena with which we remain obsessed—and for good reason! So important was the discovery of the molecular machinery that guides membranes to and through their appointments with fission and fusion that it was recognized as worthy of the 2013 Nobel Prize in Physiology and Medicine. It turns out, and not so surprisingly, that proteins have evolved for the expressed purpose of consummating the intimate contacts made between a variety of apposed membranes (be they of cells or their internal organelles) effecting true fusion. Viruses have been known for years to deploy such molecules, also known as fusogens, to invade cells and more in line with the world of reproductive medicine are fusogens believed to participate in the formation of syncytial trophoblast. A central candidate by the name of SYNCYTIN 1 is the gene product now widely suspected to mediate this aspect of placental development in the human and other species. This month, JARG features a series of papers pertaining to male infertility and sperm physiology. At the top of our list is the paper by Bjerregaard and colleagues from Copenhagen entitled “Syncytin-1 and its receptor is present in human gametes” (10.1007/s10815-014-0224-1). This work alerts our readership generally, and those fertilization aficionados here and beyond, to the demonstration of plausible functional cooperativity for the fusion of human sperm and eggs. Drawing upon the sensitivity qRT-PCR, both sperm specific expression of SYNCYTIN 1 and oocyte-specific expression of the syncytin receptor ASCT-2 mRNAs are documented, positioning these candidate fusion partners in a gender-specific context with clear implications for human ARTs. Moreover, the paper goes on to show a SYNCYTIN 1 pattern of protein expression in the equatorial segment of acrosome reacted sperm, the specialized domain of the sperm plasma membrane long recognized to be the site of sperm–egg fusion (see this month’s cover). What findings like this will mean to the daily practice of human ARTs and our understanding of failures in fertilization will require further experimentation and analysis. And, lest we forget, the identification of molecules mediating sperm–egg fusion may spark a new wave of contraceptive development research upon which even larger problems in reproductive health could be confronted for the overall benefit of humankind. Besides focusing on issues of male reproductive health this month, we are offering a template for discourse on the importance of reproductive science and medicine in the larger arena of scientific publishing. A study conducted by Duncan, Woodruff, and colleagues at Northwestern University prompts this topic with the following question: why is it that important papers in the field of reproduction rarely appear in the pages of the top journals? (Duncan et al., A small field for fertile science: the low visibility of reproductive science in high impact journals, 10.1007/s10815-014-0205-4 ). It comes as no great surprise that part of the answer derives from our small stature relative to other disciplines. And who could ignore the vestigial Victorian undercurrents that some societies have retained effectively thwarting advances in the fields of embryonic stem cells and regenerative medicine, as we have recognized in past issues of JARG. This paper, and the accompanying editorial (Woodruff et al., Keeping reproductive science visible, viable, and valuable—a call to rethink how we publish, 10.1007/s10815-014-0206-3), are timely contributions that we hope will prompt debate in the coming months, and we welcome your comments and insights into this matter. Along these lines, it is important to keep in mind what leaders in the scientific enterprise globally are perceiving for the future of scientific publishing. It is therefore of interest to note that last December, the day before accepting the 2013 Nobel prize in Physiology and Medicine, Randy Schekman from the University of California San Francisco aired his sentiments regarding the influence of high impact journals on the perceptions and practice of science. His commentary, entitled “How journals like Nature, Cell and Science are damaging science,” draws parallels between the dark side of incentivizing on the banking and finance industries and the practices top journals commonly deploy to advance themselves and the authors whose work bears their trademarks (www.theguardian.com/commentisfree/2013/dec/09/how-journals-nature-science-cell-damage-science). Schekman’s message draws upon what he believes has transformed the foundations of scientific integrity accorded to “discovery” into a business model rife with spurious and misleading commercial interests. From their lofty perch, he notes that these journals use the impact factor as more of a “gimmick” than a metric worthy of adoption in achieving academic rank or obtaining grants, a reliance that far too many promotion committees and funding agencies have become reliant upon for making big decisions for forging ahead within the purview of the biomedical research enterprise. He argues further that more and more of the very best publications are finding their way into open access journals and that this medium may return the field of scientific publication to a level of legitimacy it so deserves. Maybe our shared fascinations with the building blocks of human reproduction need a little more interdisciplinary fission, fusion, and recycling before moving discovery into the arena of translational medicine. Journals continue to influence funding agendas, academic advancement, and therapeutic direction for better or worse. The time has arrived for reproductive science to assume a larger identity that separates impact blurred by commercial interests or egos from the laudable goal of improving reproductive health for everyone.