Percoll-washed Spermatozoa Research Articles

2099 IMPAIRED CLEARANCE OF APOPTOTIC GERM CELLS CAUSES IMPAIRED SPERMATOGENESIS ASSOCIATED WITH LEYDIG CELL HYPERPLASIA AND INCREASED TESTOSTERONE LEVELS

changes in human sperm head using cytochemical stains and to confirm that CAP/AR do not include DNA damage. METHODS: Percoll-washed spermatozoa were incubated in BWW / fetal cord serum ultrafiltrate (10%;3.5h;CAP) and, subsequently, with lysophosphatidylcholine (2.5 M; 30 min, AR). Then, ethanol-fixed spermatozoa were used for: PSA-FITC (for AR), aniline blue (AB, for histones), chromomycin A3 (CMA3, for protamines), toluidine blue (TB, for chromatin compaction), iodoacetamide-fluorescein (IAF, for sulfhydryl groups), induced decondensation, sperm chromatin structure assay (DNA fragmentation index, DFI) and immunoblotting (for histone, H2B). RESULTS: CAP/AR was associated with similar increases in staining for AB ( 75%) and TB ( 50%) but had no inflence on CMA3. The increase ( 55%) in IAF staining observed during CAP was abscent after AR. CAP/AR did not damage DNA (DFI remained low) nor affect histone content. CAP, and even more AR, primed sperm heads to decondense ( 100% and 160% increases, respectively) when challenged with SDS (1%) DTT (0.1 mM). Interestingly, induced decondensation correlated with all other tests CAP, AB, TB, IAF; also CAP was related to AB and TB stains. CONCLUSIONS: The data strongly support human sperm chromatin remodeling during CAP/AR, these modifications are interlinked and probably help prepare chromatin for after-fertilization events.

2098 REMODELING OF HUMAN SPERM CHROMATIN DURING IN VITRO CAPACITATION AND ACROSOME REACTION: EVIDENCE FROM CYTOCHEMICAL TESTS AND CORRELATIONS BETWEEN ASSAYS

You have accessJournal of UrologyInfertility: Physiology, Pathophysiology, Basic Research1 Apr 20122098 REMODELING OF HUMAN SPERM CHROMATIN DURING IN VITRO CAPACITATION AND ACROSOME REACTION: EVIDENCE FROM CYTOCHEMICAL TESTS AND CORRELATIONS BETWEEN ASSAYS Eve de Lamirande, Maria San Gabriel, Naif Alhathal, and Armand Zini Eve de LamirandeEve de Lamirande Montreal, Canada More articles by this author , Maria San GabrielMaria San Gabriel Montreal, Canada More articles by this author , Naif AlhathalNaif Alhathal Montreal, Canada More articles by this author , and Armand ZiniArmand Zini Montreal, Canada More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2012.02.2265AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Capacitation (CAP) and acrosome reaction (AR), are sequential processes of sperm activation. Beside the known ionic, membrane and transduction events and final release of proteolytic enzymes to help sperm movement towards the egg, changes to chromatin are also expected. These should involve remodeling rather than DNA damage. Our aims were evaluate the changes in human sperm head using cytochemical stains and to confirm that CAP/AR do not include DNA damage. METHODS Percoll-washed spermatozoa were incubated in BWW +/− fetal cord serum ultrafiltrate (10%;3.5h;CAP) and, subsequently, with lysophosphatidylcholine (2.5 μM; 30 min, AR). Then, ethanol-fixed spermatozoa were used for: PSA-FITC (for AR), aniline blue (AB, for histones), chromomycin A3 (CMA3, for protamines), toluidine blue (TB, for chromatin compaction), iodoacetamide-fluorescein (IAF, for sulfhydryl groups), induced decondensation, sperm chromatin structure assay (DNA fragmentation index, DFI) and immunoblotting (for histone, H2B). RESULTS CAP/AR was associated with similar increases in staining for AB (∼75%) and TB (∼50%) but had no inflence on CMA3. The increase (∼55%) in IAF staining observed during CAP was abscent after AR. CAP/AR did not damage DNA (DFI remained low) nor affect histone content. CAP, and even more AR, primed sperm heads to decondense (∼100% and ∼160% increases, respectively) when challenged with SDS (1%) + DTT (0.1 mM). Interestingly, induced decondensation correlated with all other tests CAP, AB, TB, IAF; also CAP was related to AB and TB stains. CONCLUSIONS The data strongly support human sperm chromatin remodeling during CAP/AR, these modifications are interlinked and probably help prepare chromatin for after-fertilization events. © 2012 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 187Issue 4SApril 2012Page: e845-e846 Advertisement Copyright & Permissions© 2012 by American Urological Association Education and Research, Inc.MetricsAuthor Information Eve de Lamirande Montreal, Canada More articles by this author Maria San Gabriel Montreal, Canada More articles by this author Naif Alhathal Montreal, Canada More articles by this author Armand Zini Montreal, Canada More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Direct contact between boar spermatozoa and porcine oviductal epithelial cell (OEC) cultures is needed for optimal sperm survival in vitro

Oviductal epithelial cell (OEC) co-culture prolongs sperm viability and motility in vitro in a number of species including humans and horses. This study has sought to determine the effects of homologous OEC co-culture on boar sperm function. To determine whether the effects on spermatozoa were specifically caused by co-culture with or by OEC secretions, or by both factors together, a number of co-culture and cell-conditioned medium (CM) experiments were conducted. Firstly, Percoll-washed spermatozoa were co-cultured with OECs and pig kidney epithelial (LLC-PK1) cells, and in medium without cells. Secondly, Percoll-washed spermatozoa were incubated with CM derived from both OECs and LLC-PK1 cells and in unconditioned medium. A number of sperm function parameters were assessed after 5, 30, 60, 90, 120, and 180 min, and 24 h of co-culturing or incubation with CM. Of all the sperm function parameters investigated, the percentage (%) viability data yielded the most interesting results. OECs (mean ± S.E.M.; 31.2 ± 1.10) were better than LLC-PK1 cells (24.3 ± 0.93) at prolonging the viability of unbound spermatozoa after 24 h of co-culturing ( P < 0.05). Also after 24 h, the viability of spermatozoa bound to the OECs (77.6 ± 1.83) was significantly higher than in the case of the LLC-PK1 cells (53.5 ± 1.43; P < 0.001). Other sperm function parameters, e.g., capacitation and motility, were also influenced by OEC co-culturing and incubation with CM, although to a lesser degree. In conclusion, porcine homologous OEC co-culture and CM incubation specifically affect sperm function. However, we propose that it is OEC co-culturing, rather than OEC-CM, that has the greater influence.

Direct effect of macrophage migration inhibitory factor on sperm function: possible involvement in endometriosis-associated infertility

To evaluate the effect of macrophage migration inhibitory factor (MIF) on sperm capacitation, a maturational process that occurs in the female reproductive tract and enables spermatozoa to become fully competent at fertilizing the oocyte. Incubation of Percoll-washed spermatozoa with varying concentrations of human recombinant MIF or fetal cord serum (positive control). Human reproduction research laboratories. Fresh semen samples obtained from healthy volunteers after a minimum of 2 days of sexual abstinence. Protein tyrosine phosphorylation by Western blotting, the acrosomal status upon binding to the Pisum sativum agglutinin conjugated to fluorescein isothiocyanate, and sperm motility by computer-assisted sperm analysis. MIF displayed a dose-dependent effect on the phosphotyrosine content of p105 and p81, the two major tyrosine-phosphorylated proteins associated with human sperm capacitation. A significant induction of tyrosine phosphorylation was seen at 2 ng/mL of MIF for both p105 and p81, but a trend for a down-regulation of the basal tyrosine phosphorylation level was noted at elevated concentrations (12-24 ng/mL). MIF pretreatment of spermatozoa resulted in a dose-dependent change in the acrosome reaction induced by the Ca(2+) ionophore A23187. After being increased at 1-4 ng/mL MIF with a statistically significant effect observed at 4 ng/mL, the acrosome reaction gradually decreased and fell below the control levels at higher concentrations. Furthermore, a significant decrease in the motility of spermatozoa was observed after exposure to an elevated concentration of MIF (12 ng/mL). The present data indicate that MIF may play a physiological role in sperm capacitation but may have deleterious effects on sperm function at abnormal pathophysiological levels, which suggests a role in endometriosis-associated infertility.

Protein tyrosine phosphorylation, hyperactivation and progesterone-induced acrosome reaction are enhanced in IVF media: an effect that is not associated with an increase in protein kinase A activation

Sperm capacitation is a prerequisite for successful in vitro fertilization (IVF) and therefore a focus of sperm preparation in IVF laboratories. The technology of IVF is, therefore, potentially valuable in advancing our understanding of the molecular processes that occur during sperm capacitation. We have investigated sperm capacitation induced by a commercial IVF medium compared to that occurring in standard capacitating medium (CM) typically used in a nonclinical setting. Percoll-washed spermatozoa were resuspended in Cook Sydney IVF medium, Cook Sydney IVF sperm buffer, Earle's balanced salt medium (capacitating medium) or a modified Earle's balanced salt medium [non-capacitating medium (NCM)] for up to 120 min at 37 degrees C and, if applicable, in the presence of 5% CO2 in air. Sperm protein kinase A (PKA) activity, PKA-dependent serine/threonine phosphorylation, tyrosine phosphorylation, hyperactivation and progesterone-induced acrosome reaction were evaluated. IVF medium was shown to accelerate sperm capacitation (compared with capacitating medium) as determined by tyrosine phosphorylation, sperm hyperactivation and progesterone-induced acrosome reaction. This effect was not associated with enhanced activation of PKA or increased levels of serine/threonine phosphorylation. In contrast, IVF sperm buffer (used for sperm preparation) did not stimulate sperm capacitation when incubated for up to 90 min. We have shown that different capacitating media vary strikingly in their efficacy and that this difference reflects activation of a pathway other than the well-characterized activation of soluble adenylyl cyclase/cAMP/PKA.

The cyclic GMP-specific phosphodiesterase inhibitor, sildenafil, stimulates human sperm motility and capacitation but not acrosome reaction.

Capacitation is the series of transformations that spermatozoa undergo in the female genital tract in order to bind to the zona pellucida, initiate the acrosome reaction, and fertilize an egg. Cyclic adenosine monophosphate (cAMP) plays an important role in this process and its levels are regulated by 2 key enzymes, adenylyl cyclase and cyclic nucleotide phosphodiesterase (PDE), the latter being involved in cAMP degradation. Evidence was provided for the involvement of PDE in sperm motility and capacitation. Of the 10 gene families of PDE that exist in mammalian tissues, the calcium-calmodulin-dependent (type 1) and the cAMP-specific (type 4) have been found in human spermatozoa. Using sildenafil, we investigated a highly potent cyclic guanosine monophosphate (cGMP)-specific PDE (type 5) inhibitor and whether this PDE is present in human spermatozoa and is involved in sperm functions. Sildenafil inhibited PDE activity of Percoll-washed spermatozoa with an IC50 of 97+/-3 and 33+/-3 microM when cAMP and cGMP, respectively, were used as substrates. Because the IC50 of sildenafil obtained for PDE type 5 is much lower (2 to 6 nM) than that obtained with sperm PDE, the data suggest that PDE type 5 represents only a small fraction of the whole PDE activity of spermatozoa. Sildenafil causes dose-dependent increases in sperm cAMP levels and capacitation, which are associated with an increase in the levels of tyrosine phosphorylation of 2 fibrous sheath proteins (p105/81). Sperm velocity, amplitude of lateral head displacement, and hyperactivation were increased at 30-180 minutes. Sildenafil did not trigger the acrosome reaction in capacitated spermatozoa. These results suggest that under our experimental conditions, sildenafil triggers human sperm motility and capacitation, probably via its inhibitory action on PDE activity other than type 5 with a resultant rise in cAMP levels.

Involvement of reactive oxygen species in human sperm arcosome reaction induced by A23187, lysophosphatidylcholine, and biological fluid ultrafiltrates.

Although recent evidence indicated that the production of reactive oxygen species (ROS) by human spermatozoa may be involved in the regulation of capacitation, very little is known about the role of ROS in the acrosome reaction. To address this issue, Percoll-washed spermatozoa were incubated in Ham's F-10 medium in the absence (no capacitation) or presence (capacitation) of fetal cord serum ultrafiltrate (FCSu) or progesterone. The effects of the ROS scavengers, superoxide dismutase (SOD), and catalase were then tested on the acrosome reaction induced by lysophosphatidylcholine (LPC), A23187, and ultrafiltrates from follicular fluid (FFu) and FCSu, as well as on the protein tyrosine phosphorylation associated with this process. 2-Methyl-6-(p-methoxyphenyl)-3,7-dihydroimidazo [1,2-a] pyrazin-3-one (MCLA)-amplified chemiluminescence was used to determine the extracellular superoxide (O2.-) production from spermatozoa. The observations that both SOD and catalase reduced (in the case of LPC) or totally prevented (in the other cases) the acrosome reaction of capacitated spermatozoa and that hydrogen peroxide (H2O2) or ROS generated by the combination of xanthine and xanthine oxidase (O2.-, which dismutates to H2O2) triggered the acrosome reaction indicated the involvement of ROS in this process. In fact, capacitated spermatozoa in which the acrosome reaction was induced by LPC, A23187, and FFu produced more O2.- than noncapacitated spermatozoa treated with the same agents. A23187 and LPC had minor effects on protein tyrosine phosphorylation of noncapacitated spermatozoa. However, these inducers caused a decrease in tyrosine phosphorylation of Triton-soluble proteins (mainly those of 37, 42, and 47 kDa) from capacitated spermatozoa, a decrease more pronounced in the presence of SOD. On the other hand, there was a marked increase in tyrosine phosphorylation of few proteins (70 to 105 kDa) from the Triton-insoluble fraction, which was partly reversed by SOD (in the case of LPC and A23187) or catalase (in the case of A23187), or abolished in the presence of the two antioxidants (in the case of A23187). These data indicate that the acrosome reaction is associated with an extracellular O2.- generation by spermatozoa and that both O2.- and H2O2 may be involved in the regulation of this process. The mechanism by which these ROS act is unknown but may involve tyrosine phosphorylation of sperm proteins.

Human epididymal secreted protein CD52 on ejaculated spermatozoa: correlations with semen characteristics and the effect of its antibody.

CD52 is a glycosylphosphatidylinositol-anchored glycoprotein secreted by the epididymis where it is incorporated into sperm membranes. The antigen is common to spermatozoa and lymphocytes, and its role is not known. Quantitative analysis using immunostaining with the monoclonal antibody CAMPATH-1G and flow cytometry indicated positive signals from approximately 80% of viable, washed ejaculated spermatozoa. Staining intensity increased after capacitation overnight, and decreased after short incubation with high density lipoprotein. After incubation of Percoll-washed spermatozoa with CAMPATH-1G, motility was reduced from 83 to 74% after 5 min and from 73 to 52% after 3.5 h. Swimming velocities were reduced by approximately 30% and linearity by 15% in 5 min, but no further decreases were observed over 3.5 h. After 20 min incubation with the antibody, cell viability was decreased by 10 and 20% in freshly washed and capacitated spermatozoa respectively. Comparison of fertile and infertile patients revealed no difference in the percentages of immunostained spermatozoa or in staining intensity and there were no differences in sperm labelling between normozoospermia and teratozoospermia. Compared with normozoospermia, percentages of stained spermatozoa were lower by 20 and 27% in asthenozoospermia and oligozoospermia respectively, whereas staining intensity in asthenozoospermia was less than half that in oligozoospermia. The correlation of percentages of stained spermatozoa with percentages of motile cells suggests the involvement of epididymal CD52 in the maturation of sperm function.

Low levels of nitric oxide promote human sperm capacitation in vitro.

The influence of nitric oxide on human sperm hyperactivation and capacitation, as well as its mechanism of action and its possible origin from spermatozoa were studied. Percoll-washed spermatozoa from healthy volunteers were incubated in Ham's F-10 medium supplemented or not with the nitric oxide-releasing agents, diethylamine-NONOate or spermine-NONOate, in combination or not with superoxide dismutase or catalase (scavengers for the superoxide anion and for hydrogen peroxide, respectively), or with sodium nitrate, sodium nitrite, or preincubated NONOates. Sperm hyperactivation, capacitation, and nitric oxide synthase activity were determined. High concentrations (0.3 to 1 mM) of NONOates reduced sperm motility. However, a lower concentration (0.1 mM) of the two NONOates had no effect on the percentage of sperm motility or of hyperactivation but resulted in a significant increase in sperm capacitation (24% +/- 4%) when compared to that of control spermatozoa (Ham's F-10 alone, 12% +/- 2%). Nitric oxide released by the NONOates appeared responsible for this effect because sodium nitrate or nitrite or preincubated NONOates (to exhaust the formation of nitric oxide) had no influence on sperm capacitation. Catalase, but not superoxide dismutase, abolished the capacitating action of the NONOates. No nitric oxide synthase activity was detected in spermatozoa, whether they were in their basal state or already capacitated. Furthermore, the nitric oxide synthetase inhibitor L-NG nitroarginine methyl ester did not block sperm capacitation induced by fetal cord serum ultrafiltrate. It is therefore concluded that, although spermatozoa do not possess detectable nitric oxide synthase activity, low levels of nitric oxide induce human sperm capacitation, and this action likely involves hydrogen peroxide.

Increased reactive oxygen species formation in semen of patients with spinal cord injury

To evaluate reactive oxygen species production of semen samples and Percoll-washed spermatozoa from men with spinal cord injuries and to determine if there is a relationship between this reactive oxygen species production and sperm motility.Semen samples from healthy volunteers and infertile patients were collected by masturbation.Semen samples from men with a spinal cord injury were obtained by electroejaculation or by masturbation after treatment with physostigmine.Motility was measured using the CellSoft computer-assisted analysis system (Cryo Resources Ltd., Montgomery, NY). Luminol-amplified chemiluminescence was used to measure reactive oxygen species production.Semen samples and Percoll-washed spermatozoa from men with a spinal cord injury produced reactive oxygen species at much higher frequency and levels than equivalent preparations from infertile men or healthy volunteers. There was an inverse relationship between the percentage of motility and reactive oxygen species production in Percoll-washed spermatozoa from men with a spinal cord injury.Semen samples and Percoll-washed spermatozoa from men with spinal cord injury produce high levels of reactive oxygen species that may be related to the low sperm motility and infertility observed in these men.

Human sperm hyperactivation and capacitation as parts of an oxidative process

Capacitation of spermatozoa is essential for fertilization ans is visually characterized by hyperactivated motility. Previous reports have shown that foetal cord serum (FCS) and superoxide anion, O 2 ⋅ , can trigger human sperm hyperactivation (HA) and capacitation and that superoxide dismutase (SOD) could prevent these processes. We investigated further the role of O 2 ⋅ and FCS components in human spern HA and capacitation. Percoll-washed spermatozoa were incubated, at 37°C, in Ham's F-10 medium with 75% of FCS, dialyzed FCS (> 12kdD), ultrafiltrate from FCS (FCSu;<3kD), or xanthine + xanthine oxidase + catalase (X + XO + cat). Spermatozoa incubated with FCSu were also supplemented with catalse to prevent the loss of motility often after 2–3 h of incubation. FCS and dialyzed FCS induced significant levels of HA (10 ±1% and 7.7 ±0.7%, respectively) that were, however, lower than those observed with FCSu (19 ± 1%) or X + XO + cat (16 ± 2%). Similar results were obtained when the lysophorphatidylcholine-induced acrosome raction (LPC-AR, a measure of sperm capacitation) was evaluated. The presence of SOD in the incubation medium blocked the induction of HA and capaciatation by FCS. FCSu, X + XO + cat, as well as the spontaenous HA and capacitation. The enzymatic activity of SOD was needed for the prevention of these processes. Desferioxamine, up to 100 μM, had no effect on HA and LPC-AR iniduced by FCSu and X + XO + cat. Addition of SOD to already hyperactivated spermatozoa reversed the HA. These data suggest that spermatozoa need a sustaned O 2 ⋅ generation to maintain HA and proceed to capacitation. We hypothesize that FDSu or the O 2 ⋅ generated by X + XO + cat activate enzymeds, possible a reduced nicotinamide adeninie dinucleotide dinucleotide phosphate [NAD(P)H] oxidase at the level of sperm membrane.

Reactive oxygen species generated by human neutrophils inhibit sperm motility: protective effect of seminal plasma and scavengers

To investigate whether activated poly-morphonuclear neutrophils (PMN) can affect sperm motility and to assess the capacity of seminal plasma and of scavengers of reactive oxygen species to prevent any loss of motility. Blood PMN were isolated, and their capacity to generate reactive oxygen species was investigated. The effect of activated PMN was tested on the motility of Percoll-washed spermatozoa in the presence and absence of reactive oxygen species scavengers or seminal plasma (whole or fractionated). Fertile volunteers and patients attending the Infertility Clinic at the Royal Victoria Hospital. The production of reactive oxygen species by activated PMN occurred in the 1st hour after stimulation and was linearly correlated with the concentration of PMN. Percoll-washed spermatozoa had reduced motility in the presence of PMN, and the drop in motility was more severe as the concentration of PMN was increased. Catalase and dimethylsulfoxide reduced the toxic effect of PMN on sperm motility, whereas superoxide dismutase was without effect. Seminal plasma (50%, vol/vol) allowed a highly variable protective effect against the loss of sperm motility that appeared to originate predominantly from the low molecular weight (< 10 kd) fraction but also from the high molecular weight (> 12 kd) fraction of seminal plasma. These data suggest that [1] the presence of PMN, even at 0.6 x 10(6) cells/mL, in semen can be hazardous for spermatozoa, [2] H2O2 and .OH are responsible for the loss of motility, and [3H] seminal plasma confers a variable protection due to high and low molecular weight factors.

Formation of reactive oxygen species in spermatozoa of infertile patients

To determine the incidence of reactive oxygen species formation in semen of a population of patients consulting for infertility. The incidence of reactive oxygen species formation in whole semen and in washed spermatozoa was studied. The values obtained were correlated with semen parameters. The effect of the type of sperm washing on reactive oxygen species formation was also investigated. Semen samples from patients consulting for infertility and control subjects were obtained by masturbation after 3 days of sexual abstinence. Reactive oxygen species formation was measured in whole semen, sperm suspension washed by Percoll gradients, or repeated centrifugations. Sperm motility parameters were measured by computer-aided sperm analysis. Fertile control men and an unselected population of patients consulting for infertility. None. Reactive oxygen species formation by fresh semen specimen or washed spermatozoa was measured in a computer-driven LKB 1251 Luminometer (LKB-Wallac, Turku, Finland). Reactive oxygen species formation was detected in 40% of the semen with spermatozoa from infertile patients, whereas none was found in 6 azoospermic men and 10 control men. The level of reactive oxygen species formation was inversely correlated to the semen volume, the percentage of motile spermatozoa, and sperm linearity both in semen and in Percoll-washed spermatozoa. Washing by repeated centrifugation-resuspension increased 20- to 50-fold sperm reactive oxygen species formation. This enhancement was caused by the centrifugation itself and by the removal of seminal plasma. Both morphologically normal and abnormal spermatozoa produced reactive oxygen species. The data suggest that reactive oxygen species formation by spermatozoa may be a significant cause for male infertility.