Maintenance Of Sperm Motility Research Articles

Effects of substrate availability and mitochondrial disruption on oxidative metabolism and sperm motility in fertile dogs.

In several mammalian species, the measurement of mitochondrial oxygen consumption (MITOX) under different metabolic conditions has demonstrated a positive correlation with sperm motility and may be a sensitive indicator of mitochondrial health. In general, the maintenance of sperm motility and many key sperm functions and fertilizing events are heavily energy-dependent processes, and some species-specific substrate preferences exist. Although canine sperm have been known to undergo capacitation and maintain motility with supplementation of a wide range of energy substrates, the relationship between mitochondrial function, and the maintenance of oxidative metabolism and sperm motility remain unclear. The objective of this study was to explore the metabolic flexibility of canine sperm, and to investigate the relationship between mitochondrial function, and maintenance of motility under differing nutrient conditions. We explored substrate preferences and the bioenergetics underlying maintenance of canine sperm motility by monitoring mitochondrial oxidative function and sperm kinematics in the presence of mitochondrial effector drug treatments: FCCP, antimycin (ANTI), and oligomycin (OLIGO). We hypothesized that canine sperm possess the ability to use compensatory pathways and utilize diverse nutrient sources in the maintenance of motility. Oxygen consumption (change in pO2, oxygen partial pressure) and sperm kinematics (CASA) were measured concurrently (t0-t30) to assess the relationship between oxidative metabolism and maintenance of sperm motility in dogs. Four media were tested: containing glucose, lactate, and pyruvate (GLP), containing glucose (G), fructose (F), or lactate and pyruvate (LP). In the absence of pharmacological inhibition of the electron transport chain, energetic substrate had no effect on sperm kinematics in fertile dogs. Following mitochondrial disruption by ANTI and OLIGO, mitochondrial oxygen consumption was negatively correlated with several sperm motility parameters in GLP, G, F, and LP media. In every media, FCCP treatment quickly induced significantly higher oxygen consumption than in untreated sperm, and spare respiratory capacity, the maximal inducible oxidative metabolism, was high. With respiratory control ratios RCR >1 there was no indication of bioenergetic dysfunction in any media type, indicating that sperm mitochondria of fertile dogs have a high capacity for substrate oxidation and ATP turnover regardless of substrate. Our results suggest MITOX assessment is a valuable tool for assessing mitochondrial functionality, and that canine sperm employ flexible energy management systems which may be exploited to improve sperm handling and storage.

Pharmacological perturbation of peroxisome-proliferator-activated receptor gamma alters motility and mitochondrial function of bovine sperm.

Sperm transit through the female reproductive relies upon maintenance of sperm motility. Peroxisome-proliferator-activated receptor gamma (PPARγ) is a ligand-activated nuclear transcription factor with roles in glucose metabolism and reproductive processes including placental function. PPARγ roles in the mammalian postejaculatory sperm function are incompletely defined. Determine expression, localization, and functions of PPARγ in postejaculatory bovine sperm. Frozen-thawed bovine sperm from three to four different bulls were pooled and subjected to immunofluorescence and western blot for detection and localization of PPARγ. Functions in sperm energetics were explored through the addition of pharmacological inhibition (GW; GW9662) and activation (Ros; Rosiglitazone) in the culture medium at 0 and 24h under non-capacitating conditions. Samples were analyzed for sperm kinematics (CASA) and mitochondrial membrane potential (MMP; JC-1 fluorophore). PPARγ was detected in bovine sperm and co-localized to the acrosome with re-localization to the equatorial region in acrosome-compromised sperm. The addition of Ros 50µM for 24 h maintained superior total and progressive motility of sperm compared to vehicle control (VC-DMSO 0.01%). The PPARγ antagonist GW 1µM was detrimental to both total and progressive motility. A challenge experiment (Ros + GW) partially rescued total and progressive motility phenotypes observed with GW incubation. GW-treated samples had a lower number of sperm with high MMP at 24 h compared to Ros or VC. The negative GW MMP phenotype was reversed with the addition of Ros + GW. Likewise, GW-treated samples had more sperm with low MMP compared to VC and Ros, and this phenotype was partially restored with Ros + GW. PPARγ is expressed in post-ejaculatory bovine sperm with regulatory roles in sperm motility and MMP. These findings implicate PPARγ as a novel regulator of postejaculatory mammalian sperm energetics through non-canonical signaling mechanisms.

Role of Ion Channels in the Maintenance of Sperm Motility and Swimming Behavior in a Marine Teleost.

In oviparous marine fishes, the hyperosmotic induction of sperm motility in seawater (SW) is well established, however, the potential function of ion channels in the maintenance of post activated spermatozoon swimming performance remains largely unknown. Here, we investigated the influence of ion channels on the spermatozoon swimming parameters using the gilthead seabream (Sparus aurata) as a model for modern marine teleosts. Our data show that the SW-induced activation of seabream sperm motility requires three concomitant processes, the hyperosmotic shock, an ion-flux independent increase of the intracellular concentration of Ca2+ ([Ca2+]i), but not of [K+]i or [Na+]i, and the alkalization of the cytosol. The combination of all three processes is obligatory to trigger flagellar beating. However, the time-course monitoring of sperm motion kinetics and changes in the [Ca2+]i, [K+]i and [Na+]i in SW or in non-ionic activation media, showed that the post activated maintenance of spermatozoa motility is dependent on extracellular Ca2+ and K+. A meta-analysis of a seabream sperm transcriptome uncovered the expression of multiple ion channels, some of which were immunolocalized in the head and/or tail of the spermatozoon. Selective pharmacological inhibition of these ion channel families impaired the long-term motility, progressivity, and velocity of SW-activated spermatozoa. The data further revealed that some antagonists of K+-selective or Ca2+-selective channels, as well as of stretch-activated and mechanosensitive channels, altered the trajectory of spermatozoa, suggesting that these ion channels are likely involved in the control of the swimming pattern of the post activated spermatozoon. These combined findings provide new insight into the signaling pathways regulating spermatozoon activation and swimming performance in marine fishes.

In vitro storage of boar spermatozoa increases the demand of adenosine triphosphate for reactivation of motility.

Prolonging the shelf-life of liquid-preserved semen without compromising its fertilizing capacity may increase the efficiency of artificial insemination in pigs. Many fertilization-relevant processes are adenosine triphosphate dependent. The impact of semen storage and rewarming to body temperature on the energy status of spermatozoa is as yet unknown. To investigate the energy status of boar spermatozoa during storage and subsequent rewarming and to reveal the potential role of mitochondrial function for reactivation and maintenance of sperm motility. Extended semen samples (n=7 boars) were used. Spermatozoa were challenged by storage at 17°C for 7 days and incubation at 38°C for 180min. The adenosine triphosphate concentration and energy charge in semen samples and lactate concentration in the extracellular medium were assessed. Viability and mitochondrial activity were determined by flow cytometry, and clustered single-cell analysis of motility parameters was performed. The energy status was not affected by semen storage (p>0.05). Rewarming resulted in a net reduction in adenosine triphosphate concentration, which increased with storage time (maximum Day 5: -24.2±10.3%) but was not accompanied by a loss in viability, motility, or mitochondrial activity. Blocking glycolysis with 2-deoxy-d-glucose prevented the re-establishment of motility and mitochondrial activity after rewarming. Mitochondrial activity gradually subsided in virtually all spermatozoa during incubation at 38°C, while adenosine triphosphate and energy charge remained high. Concomitantly, extracellular lactate levels rose, and sperm populations with lower velocity, increased linearity, and low lateral head displacement grew larger. Size changes for major sperm subpopulations correlated with the percentage of viable spermatozoa with high mitochondrial activity (r=0.44-0.70 for individual subpopulations, p<0.01). Storage of boar spermatozoa increases the demand of adenosine triphosphate for reactivation of spermatozoa towards fast, non-linear, and hyperactivation-like motility patterns upon rewarming. Maintenance of glycolysis seems to be decisive for sperm function after long-term storage in vitro.

Use of a mitochondrial-specific live sperm stain to confirm penetration of equine oocytes after in vitro fertilization

Intracytoplasmic sperm injection (ICSI) is an important research and clinical tool. However, work with standard in vitro fertilization (IVF) is needed to explore sperm capacitation and the early events of fertilization. Repeatable methods for equine IVF have not yet been reported. Definitive evidence of fertilization, such as presence of the sperm tail within the cytoplasm, is critical when assessing potential methods for IVF. We have previously stained live sperm with the mitochondrial-specific stain, MitoTracker, and successfully visualized the sperm tail in the oocyte cytoplasm after ICSI. In the present study, we evaluated whether staining sperm with MitoTracker would A) be compatible with maintenance of sperm motility in culture and B) allow visualization of sperm tails within the COC or oocyte after co-culture with sperm. Semen was collected from one fertile stallion, and 200 µl of extended semen was layered under 1 mL of MOPS-buffered medium containing 10 or 100 nM Mitotracker Red CMXRos, in a 5-mL tube. The tube was incubated at a 45 o angle at 38 °C for 20 min. The uppermost 750 µL of the medium was collected, washed, centrifuged, and the pellet resuspended in modified TALP. Sperm were added to 50-µL droplets of modified TALP to a concentration of 1 million sperm/mL and these were incubated in 5% CO 2 in air. Sperm motility was assessed visually after 1, 6 and 22 h in culture. At 22 h, sperm were removed, counterstained with DAPI, and evaluated under fluorescence microscopy. In some replicates, in vitro-maturedequine cumulus-oocyte complexes (COCs) were added to the sperm droplets and co-incubated for 1 to 9 h. To assess sperm progress toward oocyte penetration, oocytes were denuded, fixed, and counterstained with DAPI. Some COCs were stained intact for confocal assessment of chromatin, sperm mitochondria, and acrosome status. These were fixed, permeabilized with 0.1% Triton-X 100, labeled with fluorescein-conjugated peanut agglutinin, then stained with Hoechst 33342. A minimum of three replicates were performed with each MitoTracker concentration. MitoTracker-stained sperm showed progressive motility after 1, 6 and 22 h incubation (∼80%, 50%, and 15% respectively) but at 22 h appeared to have a higher proportion of immotile sperm, and less vigorous motility, than did unstained sperm (∼25% progressive at 22 h). Staining with 10 nM MitoTracker supported more vigorous motility than did 100 nM. Both concentrations enabled clear visualization of the sperm tail on fluorescence microscopy. In penetrated oocytes, the sperm tail was clearly visualized in the oocyte cytoplasm, associated with sperm chromatin. On confocal microscopy, acrosomal ghosts were visualized at the periphery of the zona pellucida, indicating that sperm underwent the acrosome reaction in response to the zona. We conclude that staining of live sperm with 10 nM MitoTracker during swim-up provides a simple method to allow confirmation of sperm penetration of oocytes after sperm-oocyte coincubation.

139 Effect of protein and calcium ionophore A23187 concentration on hyperactivated motility and acrosome status of stallion sperm

In vitro fertilization does not occur readily in the horse. Fertilization can be achieved using sperm treated with the calcium ionophore A23187 (CaI), but rates are low and variable. In order to fertilize, it is thought that the sperm must show hyperactivated motility and undergo the acrosome reaction. The presence of protein in the media is thought to suppress the effect of CaI, but protein is needed for maintenance of sperm motility. Therefore, the objective of this study was to assess the effect of CaI in the presence or absence of protein on the acrosome and on hyperactivated motility of equine sperm. For this purpose, sperm from 4 stallions were exposed for 10min at 37°C to vehicle or to 1 (C1), 5 (C5) or 10 (C10) μM CaI, with (BSA) or without (N) 7mg mL−1 BSA. The sperm were then washed and incubated at 37°C for 2h. Total and hyperactivated motility were measured by computer-assisted semen analysis. Sperm were considered hyperactivated if curvilinear velocity was &amp;gt;180μm s−1, amplitude of lateral head displacement was &amp;gt;12μm, linearity was &amp;lt;30% and fractal dimension value was &amp;gt;1.3. The percentage of live acrosome-reacted sperm was measured by flow cytometry after staining with propidium iodide and Pisum sativum agglutinin. Data were analysed by repeated-measures 2-way ANOVA. Results were expressed as mean±standard error. Total motility in C5 and C10 treatments was significantly decreased in relation to control (BSA-vehicle) starting at 30min of incubation (35.42±13.57 to 28.20±13.10% v. 71.72±9.21%, respectively; P&amp;lt;0.05). Hyperactivated motility was significantly lower in C10, C5 and N-C1 than in control after 2h of incubation (1.46±0.64v. 3.10±0.58%, respectively). Live acrosome-reacted sperm were significantly higher (P&amp;lt;0.05) for BSA-C5 (14.04±1.99%) and BSA-C10 (14.85±2.52%) than for control (7.50±1.62%) after 2h of incubation. The exposure to sperm of concentrations ≥5μM CaI was associated with loss of motility from 30min of incubation on. However, 2h of incubation after ≥5 μM CaI in the presence of BSA were needed to increase the percentage of live acrosome-reacted sperm. This mismatch between motility and acrosome response helps to clarify the reasons for the variable effect of sperm CaI treatment on equine IVF. Further studies measuring calcium influx and assessing the effect of sperm pre-incubation on CaI response are needed to explore mechanisms for equine in vitro sperm capacitation.

Effect of L-carnitine supplementation to bulls on sperm motility maintenance under high environmental temperatures

Effect of L-carnitine supplementation to bulls on sperm motility maintenance under high environmental temperatures

Role and regulation of Glycogen synthase kinase-3 beta in bovine spermatozoa.

The serine/threonine kinase Glycogen synthase kinase 3 (GSK-3) is a master switch that regulates a multitude of cellular pathways, including the acrosome reaction in sperm. In epididymal sperm cells, for example, GSK-3 activity correlates with inhibition of motility-yet no direct pathways connecting GSK-3 activation with loss of motility have been described. Indeed, the details of how GSK-3 is regulated during sperm capacitation and the acrosome reaction remains obscure. To this end, we addressed the involvement of the GSK-3 beta isoform in several known pathways that contribute to motility and the acrosome reaction. We established that Protein kinase A (PKA) is the main regulator of GSK-3β in sperm, as pre-treatment of cells with a GSK-3 inhibitor prior to addition of H89, an inhibitor of PKA, attenuated the motility loss induced by blocking PKA activity. Both induced and spontaneous acrosome reactions also occurred less frequently in sperm treated with GSK-3 inhibitors. Finally, we observed a slow decline in phosphorylation of GSK-3β on Ser 9, which represents an inhibited state, during sperm capacitation; this phenotype is reversed during the induced acrosome reaction, in parallel to activation of Protein phosphatase 1. These results suggest that maintenance of sperm motility and acrosome reaction timing are mediated by PKA through the regulation of GSK-3 beta activity. Mol. Reprod. Dev. 84: 8-18, 2017. © 2016 Wiley Periodicals, Inc.

Plasticity of gene expression according to salinity in the testis of broodstock and F1 black-chinned tilapia, Sarotherodon melanotheron heudelotii.

The black-chinned tilapia Sarotherodon melanotheron heudelotii Rüppell 1852 (Teleostei, Cichlidae) displays remarkable acclimation capacities. When exposed to drastic changes of salinity, which can be the case in its natural habitat, it develops quick physiological responses and keeps reproducing. The present study focused on the physiological impact of salinity on male reproductive capacities, using gene expression as a proxy of acclimation process. Two series of experimental fish were investigated: the first one was composed of fish maintained in freshwater for several generations and newly acclimated to salinities of 35 and 70, whereas the second one consisted of the descendants of the latter born and were raised under their native salinity. Expression patterns of 43 candidate genes previously identified from the testes of wild males was investigated in the three salinities and two generations. Twenty of them showed significant expression differences between salinities, and their predicted function revealed that most of them are involved in the osmotic tolerance of sperm cells and/or in the maintenance of sperm motility. A high level of expression variation was evidenced, especially for fish maintained in freshwater. In spite of this, gene expression patterns allowed the differentiation between fish raised in freshwater and those maintained in hypersaline water in both generations. Altogether, the results presented here suggest that this high variability of expression is likely to ensure the reproductive success of this species under varying salinities.

Sperm nuclear vacuoles in relation to acrosome reactions and sperm motility.

We investigated sperm nuclear vacuolation in relation to acrosome reactions and the maintenance of sperm motility. Thirty male patients who visited our Male Infertility Clinic were enrolled. These patients underwent conventional semen analyses, Acrobeads tests, and high-magnification observation of the sperm head to evaluate the degree of nuclear vacuolation on the Acrobeads test scoring after 24 hours of incubation. The presence of acrosome reactions was evaluated using the Acrobeads test. The spermatozoa were classified into three groups: (I) those bound to MH61-beads, (II) motile spermatozoa that did not bind to MH61-beads, and (III) immotile spermatozoa that did not bind to MH61-beads. The percentage of spermatozoa with large nuclear vacuoles (%LNV) was compared between the three groups. The degree of sperm nuclear vacuolation was evaluated in 17,992 ejaculated spermatozoa. The mean %LNVs were 2.4% in group I, 5.8% in group II, and 9.8% in group III. These values were significantly different from each other (P < 0.001, paired t-test). There were no correlations between the %LNV values and the Acrobeads scores. In conclusion, the degree of sperm nuclear vacuolation was significantly lower in the acrosome-reacted spermatozoa and spermatozoa with maintained motility, and higher in the immotile spermatozoa that did not bind to MH61-beads.

Subcellular Localization of Selectively Permeable Aquaporins in the Male Germ Line of a Marine Teleost Reveals Spatial Redistribution in Activated Spermatozoa1

In oviparous vertebrates such as the marine teleost gilthead seabream, water and fluid homeostasis associated with testicular physiology and the external activation of spermatozoa is potentially mediated by multiple aquaporins. To test this hypothesis, we isolated five novel members of the aquaporin superfamily from gilthead seabream and developed paralog-specific antibodies to localize the cellular sites of protein expression in the male reproductive tract. Together with phylogenetic classification, functional characterization of four of the newly isolated paralogs, Aqp0a, -7, -8b, and -9b, demonstrated that they were water permeable, while Aqp8b was also permeable to urea, and Aqp7 and -9b were permeable to glycerol and urea. Immunolocalization experiments indicated that up to seven paralogous aquaporins are differentially expressed in the seabream testis: Aqp0a and -9b in Sertoli and Leydig cells, respectively; Aqp1ab, -7, and -10b from spermatogonia to spermatozoa; and Aqp1aa and -8b in spermatids and sperm. In the efferent duct, only Aqp10b was found in the luminal epithelium. Ejaculated spermatozoa showed a segregated spatial distribution of five aquaporins: Aqp1aa and -7 in the entire flagellum or the head, respectively, and Aqp1ab, -8b, and -10b both in the head and the anterior tail. The combination of immunofluorescence microscopy and biochemical fractionation of spermatozoa indicated that Aqp10b and phosphorylated Aqp1ab are rapidly translocated to the head plasma membrane upon activation, whereas Aqp8b accumulates in the mitochondrion of the spermatozoa. In contrast, Aqp1aa and -7 remained unchanged. These data reveal that aquaporin expression in the teleost testis shares conserved features of the mammalian system, and they suggest that the piscine channels may play different roles in water and solute transport during spermatogenesis, sperm maturation and nutrition, and the initiation and maintenance of sperm motility.

A preliminary search for alternatives to albumin as a medium supplement for the culture of human sperm

Non-animal macromolecules (Select Phytone™ UF, wheat peptone, dextran 40, hydroxyethyl starch and methyl cellulose) as an alternative medium supplement for human spermatozoa were compared to bovine serum albumin. Select Phytone™ UF and wheat peptone discolored the medium and smelled like broth, making them unlikely to be acceptable for clinical use, whilst the others were colorless and odorless. All supplements were effective in the yield of spermatozoa isolated by swim-up technique, and maintenance of sperm motility. In summary, there are non-animal macromolecules that will support short-term sperm culture.

Evidence for the Involvement of Aquaporins in Sperm Motility Activation of the Teleost Gilthead Sea Bream (Sparus aurata)1

The expression of aquaporins in the spermatozoa of the marine teleost gilthead sea bream (Sparus aurata) and their involvement in the motility activation process were investigated. Sperm motility was activated by a hyperosmotic shock, but it was completely inhibited by 10 microM HgCl(2), such inhibition being partially recovered by beta-mercaptoethanol (ME). Conventional RT-PCR using primers specific for S. aurata aquaglyceroporin (glp) and aquaporin 1a (aqp1a) demonstrated the presence of both mRNAs in spermatozoa. Heterologous expression in Xenopus laevis oocytes showed that 10 and 100 microM HgCl(2) equally inhibited water and solute transport through S. aurata aquaporin 1a and S. aurata aquaglyceroporin, but treatment with ME only recovered aquaporin 1a-mediated water permeability. Western blot analysis using isoform-specific antisera on protein extracts from spermatozoa revealed bands that corresponded to the predicted molecular mass of S. aurata aquaglyceroporin (31 kDa) and S. aurata aquaporin 1a (28 kDa). The antisera also demonstrated that both aquaporins were localized in the head and flagellum of the spermatozoa. However, the immunoreaction at the plasma membrane of the spermatozoa head was more intense after the hyperosmotic activation, suggesting the translocation of both aquaporin 1a and aquaglyceroporin into the plasma membrane after the osmotic shock. This study therefore provides the first direct demonstration for the presence of aquaporins in fish sperm. The different sensitivities of S. aurata aquaporin 1a and S. aurata aquaglyceroporin to ME may explain the failure of this reducing agent to fully recover the mercurial inhibition of sperm motility, suggesting that these aquaporins may play different physiological roles during the activation and maintenance of sperm motility in sea bream.

Effect of L-carnitine supplementation to bulls on sperm motility maintenance under high environmental temperature

Effect of L-carnitine supplementation to bulls on sperm motility maintenance under high environmental temperature

Relationship between sperm-hyaluronic acid (HA) binding assay scores and HA-mediated increase in maintenance of sperm motility and velocity in IVF conditions

OBJECTIVE: Hyaluronic acid (HA) substantially increases maintenance of human sperm motility and velocity. Also, HA presence improved IVF pregnancy rates. We studied sperm motility and velocity in Vitrolife G-IVF media with and without added HA.DESIGN: Washed sperm fractions of the same semen samples were incubated with or without added 0.1 mg/mL HA. Sperm motility and velocity were assayed by CASA (Hamilton–Thorne, Beverly, MA) a time-related fashion.MATERIALS AND METHODS: Semen was purified with 45% Isolate gradients (600xg, 20 min). The sperm were washed, and re-suspended in Vitrolife G-IVF media or in G-IVF containing 0.1 mg/mL HA in duplicates. Sperm attributes were assayed by CASA (two different drops, four fields each), after 0, 30, 90, 150 minutes, and overnight incubation, including sperm motility, curvilinear velocity(VCL), pathway velocity(VAP), progressive velocity(VSL), cross-beat frequency(BCF), amplitude head displacement(AHL), and proportions of rapid and medium velocity sperm. The sperm HA-binding scores (proportion of motile sperm bound to HA) were determined in chambers of the sperm-HA binding assay (HBA, MidAtlantic Diagnostics, NJ), in the initial semen, and after 90 minutes and overnight incubation. Statistical analyses were performed by paired-t test and Pearson's correlation using the Sigma-Stat 3.0 software.RESULTS: We studied 30 semen samples (concentration: 116±10.9x106 sperm; motility: 59.9±2.5%, mean±SEM). The HA-mediated improvements in G-IVF-HA vs. G-IVF were as follows: At 90 minutes, increases in VCL (p<0.001), VAP (p<0.001), and in proportions of rapid (p<0.001) and medium (p<0.001) velocity sperm. After the overnight incubation there were respective increases in sperm motility (p<0.05), VCL (p<0.05), VAP (p<0.05) and in rapid velocity sperm (p<0.05). In line with our hypothesis of a potential relationship between sperm HA-receptors (degree of maturity, as it measured by the HA-binding score) and HA-mediated increase in sperm velocity: HA-score vs. VSL (r=0,54, p<0.01), vs. BCF (r=0.47, p<0.05), and vs. rapid/medium velocity sperm (r=0.49 p<0.01).CONCLUSIONS: 1) HA improves the retention of sperm motility and velocity even after 24 hours in IVF conditions. 2) As hypothesized, the HA-related improvement was related to sperm maturity and consequential higher density of HA receptors. 3)s. 4) The HBA-slide test could facilitate selection of husbands who may benefit from addition of HA during IVF. OBJECTIVE: Hyaluronic acid (HA) substantially increases maintenance of human sperm motility and velocity. Also, HA presence improved IVF pregnancy rates. We studied sperm motility and velocity in Vitrolife G-IVF media with and without added HA. DESIGN: Washed sperm fractions of the same semen samples were incubated with or without added 0.1 mg/mL HA. Sperm motility and velocity were assayed by CASA (Hamilton–Thorne, Beverly, MA) a time-related fashion. MATERIALS AND METHODS: Semen was purified with 45% Isolate gradients (600xg, 20 min). The sperm were washed, and re-suspended in Vitrolife G-IVF media or in G-IVF containing 0.1 mg/mL HA in duplicates. Sperm attributes were assayed by CASA (two different drops, four fields each), after 0, 30, 90, 150 minutes, and overnight incubation, including sperm motility, curvilinear velocity(VCL), pathway velocity(VAP), progressive velocity(VSL), cross-beat frequency(BCF), amplitude head displacement(AHL), and proportions of rapid and medium velocity sperm. The sperm HA-binding scores (proportion of motile sperm bound to HA) were determined in chambers of the sperm-HA binding assay (HBA, MidAtlantic Diagnostics, NJ), in the initial semen, and after 90 minutes and overnight incubation. Statistical analyses were performed by paired-t test and Pearson's correlation using the Sigma-Stat 3.0 software. RESULTS: We studied 30 semen samples (concentration: 116±10.9x106 sperm; motility: 59.9±2.5%, mean±SEM). The HA-mediated improvements in G-IVF-HA vs. G-IVF were as follows: At 90 minutes, increases in VCL (p<0.001), VAP (p<0.001), and in proportions of rapid (p<0.001) and medium (p<0.001) velocity sperm. After the overnight incubation there were respective increases in sperm motility (p<0.05), VCL (p<0.05), VAP (p<0.05) and in rapid velocity sperm (p<0.05). In line with our hypothesis of a potential relationship between sperm HA-receptors (degree of maturity, as it measured by the HA-binding score) and HA-mediated increase in sperm velocity: HA-score vs. VSL (r=0,54, p<0.01), vs. BCF (r=0.47, p<0.05), and vs. rapid/medium velocity sperm (r=0.49 p<0.01). CONCLUSIONS: 1) HA improves the retention of sperm motility and velocity even after 24 hours in IVF conditions. 2) As hypothesized, the HA-related improvement was related to sperm maturity and consequential higher density of HA receptors. 3)s. 4) The HBA-slide test could facilitate selection of husbands who may benefit from addition of HA during IVF.

Sperm proteome reflects pregnancy outcome

OBJECTIVE: Mapping of the sperm proteome has already revealed the complexity of spermatogenesis and sperm function. Proteins have been identified to be involved in proteosome turnover, transcription, protein synthesis and protein transport. It has been suggested that sperm proteins are essential for normal syngamy and early embryonic development. This study therefore investigated sperm proteomic fingerprints in correlation with infertility and the relationship to reproductive outcome.DESIGN: Proteomic analysis of human sperm.MATERIALS AND METHODS: Infertile couples (n=22) undergoing IVF treatment donated sperm, with consent, following ICSI. The mean maternal and paternal age was 37±3.9 and 39.6±5.4 years respectively. Sperm samples ranged from 12%-74%, mean 54.5±17.4% for motility and 2-181million/ml, mean 65.5±50.6 for concentration. Each washed sperm sample was further diluted to 1 million/ml prior to centrifugation and 2 washes in PBS. The final pellet was resuspended in protein lysis buffer and stored at -80°C until analysis. Samples were processed using surface enhanced laser desorption ionization time-of-flight mass spectrometry to determine a sperm proteomic fingerprint. Implantation outcome was monitored.RESULTS: On average 15.1±6.9 oocytes were retrieved with a fertilization rate of 57.8±19.4%. 14 out of the 22 infertile patients resulted in successful 100% implantation with the remaining 8 infertile patients with 0% implantation. Proteomic fingerprints were generated for each of the 22 sperm samples. Statistical analysis was performed according to implantation outcome and identified a novel set of six sperm proteins across a mass range of 2.9-4.6 kDa that exhibited higher levels of expression with 100% implantation (P < 0.01). Further investigation of these proteins revealed a potential 3.9kDa ID candidate to be hepatocyte growth factor (HGF) activator. HGF is a pleiotropic cytokine that influences mitogenesis, motility and differentiation and has a positive effect on the maintenance of sperm motility.CONCLUSIONS: As proteins are responsible for cellular function, identification and quantification of the sperm proteome will gain new insights into the causes and potential treatment of male infertility. In this study the contribution and expression of a novel set of sperm proteins to the resulting embryo could contribute to reproductive outcome. The ability to diagnostically assay sperm prior to infertility treatment should assist in patient management. OBJECTIVE: Mapping of the sperm proteome has already revealed the complexity of spermatogenesis and sperm function. Proteins have been identified to be involved in proteosome turnover, transcription, protein synthesis and protein transport. It has been suggested that sperm proteins are essential for normal syngamy and early embryonic development. This study therefore investigated sperm proteomic fingerprints in correlation with infertility and the relationship to reproductive outcome. DESIGN: Proteomic analysis of human sperm. MATERIALS AND METHODS: Infertile couples (n=22) undergoing IVF treatment donated sperm, with consent, following ICSI. The mean maternal and paternal age was 37±3.9 and 39.6±5.4 years respectively. Sperm samples ranged from 12%-74%, mean 54.5±17.4% for motility and 2-181million/ml, mean 65.5±50.6 for concentration. Each washed sperm sample was further diluted to 1 million/ml prior to centrifugation and 2 washes in PBS. The final pellet was resuspended in protein lysis buffer and stored at -80°C until analysis. Samples were processed using surface enhanced laser desorption ionization time-of-flight mass spectrometry to determine a sperm proteomic fingerprint. Implantation outcome was monitored. RESULTS: On average 15.1±6.9 oocytes were retrieved with a fertilization rate of 57.8±19.4%. 14 out of the 22 infertile patients resulted in successful 100% implantation with the remaining 8 infertile patients with 0% implantation. Proteomic fingerprints were generated for each of the 22 sperm samples. Statistical analysis was performed according to implantation outcome and identified a novel set of six sperm proteins across a mass range of 2.9-4.6 kDa that exhibited higher levels of expression with 100% implantation (P < 0.01). Further investigation of these proteins revealed a potential 3.9kDa ID candidate to be hepatocyte growth factor (HGF) activator. HGF is a pleiotropic cytokine that influences mitogenesis, motility and differentiation and has a positive effect on the maintenance of sperm motility. CONCLUSIONS: As proteins are responsible for cellular function, identification and quantification of the sperm proteome will gain new insights into the causes and potential treatment of male infertility. In this study the contribution and expression of a novel set of sperm proteins to the resulting embryo could contribute to reproductive outcome. The ability to diagnostically assay sperm prior to infertility treatment should assist in patient management.

Effects of phosphodiesterase 5 inhibitors on sperm parameters and fertilizing capacity

The aim of this review study is to elucidate the effects that phosphodiesterase 5 (PDE5) inhibitors exert on spermatozoa motility, capacitation process and on their ability to fertilize the oocyte. Second messenger systems such as the cAMP/adenylate cyclase (AC) system and the cGMP/guanylate cyclase (GC) system appear to regulate sperm functions. Increased levels of intracytosolic cAMP result in an enhancement of sperm motility and viability. The stimulation of GC by low doses of nitric oxide (NO) leads to an improvement or maintenance of sperm motility, whereas higher concentrations have an adverse effect on sperm parameters. Several in vivo and in vitro studies have been carried out in order to examine whether PDE5 inhibitors affect positively or negatively sperm parameters and sperm fertilizing capacity. The results of these studies are controversial. Some of these studies demonstrate no significant effects of PDE5 inhibitors on the motility, viability, and morphology of spermatozoa collected from men that have been treated with PDE5 inhibitors. On the other hand, several studies demonstrate a positive effect of PDE5 inhibitors on sperm motility both in vivo and in vitro. In vitro studies of sildenafil citrate demonstrate a stimulatory effect on sperm motility with an increase in intracellular cAMP suggesting an inhibitory action of sildenafil citrate on a PDE isoform other than the PDE5. On the other hand, tadalafil's actions appear to be associated with the inhibitory effect of this compound on PDE11. In vivo studies in men treated with vardenafil in a daily basis demonstrated a significantly larger total number of spermatozoa per ejaculate, quantitative sperm motility, and qualitative sperm motility; it has been suggested that vardenafil administration enhances the secretory function of the prostate and subsequently increases the qualitative and quantitative motility of spermatozoa. The effect that PDE5 inhibitors exert on sperm parameters may lead to the improvement of the outcome of assisted reproductive technology (ART) programs. In the future PDE5 inhibitors might serve as adjunct therapeutical agents for the alleviation of male infertility.

Hepatocyte growth factor modulates in vitro survival and proliferation of germ cells during postnatal testis development

The hepatocyte growth factor (HGF) is a pleiotropic cytokine that influences mitogenesis, motility and differentiation of many different cell types by its tyrosine kinase receptor c-Met. We previously demonstrated that the c-Met/HGF system is present and functionally active during postnatal testis development. We found also that spermatozoa express c-Met and that HGF has a positive effect on the maintenance of sperm motility. In the present paper, we extend our study on the germ cells at different stages of differentiation during the postnatal development of the testis. We demonstrate that c-met is present in rat spermatogonia, pachytene spermatocytes and round spermatids and that HGF significantly increases spermatogonial proliferation in 8- to 10-day-old pre-pubertal rats. At this age HGF does not affect Sertoli cells and peritubular myoid cells proliferation. In addition, we studied the effect of the factor on germ cell apoptosis and we show that HGF prevents the germ cell apoptotic process. We also studied the effect of HGF on 18- to 20-day-old and 28- to 30-day-old rat testes. At these ages also the factor significantly increases germ cell duplication and decreases the number of apoptotic cells. However, the effect on programmed cell death is higher in the 8- to 10-day-old rats and declines in the older animals. In conclusion, we report that rat germ cells (spermatogonia, pachytene spermatocytes and round spermatids) express c-met and that HGF modulates germ cell proliferating activity and apoptosis in vitro. These data indicate that the c-Met/HGF system is involved in male germ cell homeostasis and, consequently, has a role in male fertility.

Pathophysiology of sperm motility

Mammalian spermatozoa acquire the ability to swim during their transit from the testis to the oviduct under the control of several external and intracellular factors. These factors play also a pivotal role in regulating acquisition of hyperactivated motility and during the process of chemotaxis. This review summarizes the involvement of such factors in acquisition and maintenance of sperm motility, hyperactivation and chemotaxis, focusing in particular on the molecular bases of asthenozoospermia, a pathology of seminal plasma characterized by reduced sperm motility, which is one of the main causes of male infertility. Current in vitro treatments of asthenozoospermia are shown, together with the most recent findings on pharmacological and physiological molecules capable of stimulating sperm motility. The structure, function and mechanism of sperm flagellum responsible for the development of active motility are also analyzed in details.

Concerted action between Ca 2+ and hyperosmolality initiates sperm motility in amphioxus Branchiostoma belcheri tsingtauense

This study investigated the effects of different environmental conditions on the initiation and maintenance of sperm motility in amphioxus Branchiostoma belcheri tsingtauense. The findings were that: (1) hyperosmolality in the absence of Ca 2+ ions did not initiate amphioxus sperm motility; (2) addition of Ca 2+ into EGTA-containing Ca 2+-free artificial sea water (ASW), in which no sperm were motile, restored sperm motility; (3) Ca 2+ failed to induce sperm motility under conditions of hypoosmolality; (4) K + channel blockers quinine and 4-aminopiridine did not suppress the initiation of sperm motility; and (5) changes in pH did not cause sperm motility in a solution isotonic to seawater without Ca 2+. In conclusion, we inferred that a concerted action between Ca 2+ and hyperosmolality was essential to initiate motility of amphioxus sperm, whereas K + and pH were indispensable to maintain motility.