Pisum Sativum Agglutinin Staining Research Articles

Iberiotoxin and clofilium regulate hyperactivation, acrosome reaction, and ion homeostasis synergistically during human sperm capacitation.

Potassium channels play essential roles in the regulation of male fertility. However, potassium channels mediating K+ currents in human sperm (IKSper ) remain controversial. Besides SLO3, the SLO1 potassium channel is a potential candidate for human sperm KSper. This study intends to elucidate the function of SLO1 potassium channel during human sperm capacitation. Human sperm were treated with iberiotoxin (IbTX, a SLO1 specific inhibitor) and clofilium (SLO3 inhibitor) separately or simultaneously during in vitro capacitation. A computer-assisted sperm analyzer was used to assess sperm motility. The sperm acrosome reaction (AR) was analyzed using fluorescein isothiocyanate-conjugated Pisum sativum agglutinin staining. Sperm protein tyrosine phosphorylation was studied using western blotting. Intracellular Ca2+ , K+ , Cl- , and pH were analyzed using ion fluorescence probes. Independent inhibition with IbTX or clofilium decreased the sperm hyperactivation, AR, and protein tyrosine phosphorylation, and was accompanied by an increase in [K+ ]i , [Cl- ]i , and pHi , but a decrease in [Ca2+ ]i . Simultaneously inhibition with IbTX and clofilium lower sperm hyperactivation and AR more than independent inhibition. The increase in [K+ ]i , [Cl- ]i , and pHi , and the decrease in [Ca2+ ]i were more pronounced. This study suggested that the SLO1 potassium channel may have synergic roles with SLO3 during human sperm capacitation.

KCNQ1 Potassium Channel Expressed in Human Sperm Is Involved in Sperm Motility, Acrosome Reaction, Protein Tyrosine Phosphorylation, and Ion Homeostasis During Capacitation.

Potassium channels are involved in membrane hyperpolarization and ion homeostasis regulation during human sperm capacitation. However, the types of potassium channels in human sperm remain controversial. The voltage-gated ion channel KCNQ1 is ubiquitously expressed and regulates key physiological processes in the human body. In the present study, we investigated whether KCNQ1 is expressed in human sperm and what role it might have in sperm function. The expression and localization of KCNQ1 in human sperm were evaluated using Western blotting and indirect immunofluorescence. During capacitation incubation, human sperm were treated with KCNQ1- specific inhibitor chromanol 293B. Sperm motility was analyzed using a computer-assisted sperm analyzer. The acrosome reaction was studied using fluorescein isothiocyanate-conjugated Pisum sativum agglutinin staining. Protein tyrosine phosphorylation levels and localization after capacitation were determined using Western blotting and immunofluorescence. Intracellular K+, Ca2+, Cl−, pH, and membrane potential were analyzed using fluorescent probes. The results demonstrate that KCNQ1 is expressed and localized in the head and tail regions of human sperm. KCNQ1 inhibition reduced sperm motility, acrosome reaction rates, and protein tyrosine phosphorylation but had no effect on hyperactivation. KCNQ1 inhibition also increased intracellular K+, membrane potential, and intracellular Cl−, while decreasing intracellular Ca2+ and pH. In conclusion, the KCNQ1 channel plays a crucial role during human sperm capacitation.

266 BIOCOMPATIBILITY OF NANOCERIA IN RAM SPERM DURING 24 HOURS OF INCUBATION

In recent years, there has been increasing interest in nanoparticles, especially those widely present in our environment. Several studies have been performed to evaluate their potential toxic effect and their possible use for biomedical applications. Among others, cerium dioxide nanoparticles (nanoceria, CeO2 ENPs) have been recently investigated for their use in biomedicine, based on their potential antioxidant function, due to the presence of oxygen vacancies and redox transformations (Ce4+/Ce3+) occurring at the surface. However, little is known about the potential toxicity of nanoceria in the reproductive system and on gametes, and no information is available with regard to its biocompatibility and potential toxicity on male gametes. The aim of the present study was to investigate effects of increasing doses of CeO2 ENPs on ram spermatozoa during 24 h storage at 4°C, based on assessment of main kinematic parameters, membranes and DNA integrity, ROS production, mitochondrial activity, and CeO2 intracellular uptake. The ejaculates of 3 rams of proven fertility were pooled and incubated with increasing doses of nanoceria (0, 22, 44, and 220 µg mL–1) up to 24 h at 4°C. The experiment was conducted in 4 replicates. At 0, 2, and 24 h of incubation, the 4 groups were submitted to the following analyses: i) main kinematic parameters (total motility and progressive motility) through CASA (computer-assisted sperm analysis); ii) acrosome and membrane integrity (propidium iodide + Pisum sativum agglutinin staining, PI+PSA); iii) flow cytometry for sperm chromatin structure assay (SCSA, acridine orange staining), mitochondrial activity (Mitotracker Orange), and ROS production (H2DCFDA). Moreover, an aliquot of semen from each group in each time step was fixed and processed for transmission electron microscopy to assess intracellular uptake of CeO2 nanoparticles by spermatozoa. Increasing concentrations of nanoceria did not affect the main kinematic parameters of ram semen; there were no differences in total and progressive motility among groups at any time point during the 24 h of incubation (P > 0.05). Integrity of the acrosome and cytoplasmic membranes, assessed through PI+PSA staining, was not affected by nanoceria in any group (P > 0.05). Moreover, exposure to nanoparticles did not increase DNA fragmentation (P > 0.05), and there was no difference in the amount of ROS produced and mitochondrial activity within the 24 h of incubation with nanoceria (P > 0.05). Absence of internalization of the nanoparticles by spermatozoa and occasional interaction between the sperm surface and nanoceria were observed by transmission electron microscopy analysis. In the present study, exposure of ram spermatozoa to increasing doses of nanoceria was not cytotoxic; furthermore, high concentrations of these nanoparticles were well tolerated. These data open new perspectives on the biomedical use of nanoceria and provide more information about their impact on male gametes.

Pig Spermatozoa Defect in Acrosome Formation Caused Poor Motion Parameters and Fertilization Failure through Artificial Insemination and In vitro Fertilization.

The selection of morphologically normal spermatozoa is critical to obtain high breeding performances in boar breeding farms and artificial insemination (AI) centers. Parameters for the selection of semen mainly include total sperm motility, concentration, and morphology. However, these primary parameters are often not reliable for discriminating between normal and abnormal, non-fertilizable spermatozoa. The present study was designed to compare the motion characteristics, fertilization ability using in vitro fertilization (IVF), and acrosome formation of the semen from boars having low (boar number 2012) and normal (boar number 2004 and 2023) breeding performances. The ultimate goal was to identify additional simple and easy criteria for the selection of normal sperm. There was no significant difference between boar 2004 and boar 2023 sperm total motility in computer assisted sperm analysis. However, boar number 2012 semen presented a significantly reduced population of rapid moving spermatozoa and an increased population of slow moving spermatozoa compared to boar numbers 2004 and 2023. Analysis of detailed motion characteristics revealed that sperm from boar number 2012 had significantly reduced motility in progressiveness, average path velocity, straight-line velocity (VSL), curvilinear velocity (VCL), straightness, and linearity. The assessment of the fertilizing ability by IVF also showed that sperm from boar number 2012 showed a fertility rate of 3.4%, whereas sperm from boar number 2023 had a fertility rate of 75.45%. Interestingly, most of the sperm nuclei were found on the peripheral area of the oocytes, suggesting that the sperm from boar number 2012 lacked penetration ability into the oocyte zonapellucida. The acrosome formation analysis using Pisum sativum agglutinin staining demonstrated that the sperm from boar number 2012 had a defect in acrosome formation. Consequently, primary parameters for selecting semen before AI such as motility are not sufficient to select normal and fertilizable spermatozoa. In conclusion, the present study suggests that the acrosome staining and detailed motion characteristics such as progressiveness, VCL, and VSL should be included in determining semen quality together with primary parameters for successful AI and high breeding performance in the swine industry.

B7-H3 Promoted Sperm Motility in Humans

To determine whether seminal B7-H3 levels are correlated to semen parameters and affect human sperm functions. A total of 83 healthy donors of proven fertility (aged 22-37 years) and 176 infertile men (aged 21-38 years) were recruited. Computer-assisted semen analysis and enzyme-linked immunosorbent assay were used to assess the correlations between seminal B7-H3 levels and semen parameters. Flow cytometry and fluorescence microscopy were used to detect the putative receptor for B7-H3. Computer-assisted semen analysis and FITC-conjugated pisum sativum agglutinin staining were performed for assessing sperm motility, capacitation, and acrosome reaction (AR) after incubation with various concentrations of B7-H3 for 0-4 hours in vitro. Seminal B7-H3 level was significantly higher in the healthy donors than that in the infertile men (P <.05), and closely associated with sperm concentrations and progressive motility (all P <.05), but not the other parameters examined (all P >.05). A putative receptor for B7-H3 was detected on the surface of sperm, with no significant differences in expression between the healthy donors and infertile men (P >.05). Seminal B7-H3 promoted sperm progressive motility in a time- and dose-dependent manner in vitro, although having no significant influence on sperm capacitation and AR. B7-H3 showed a favorable effect on human sperm motility, without affecting sperm capacitation and AR.

Pregnancy outcome after intracytoplasmic sperm injection with strontium oocyte activation in a globozoospermic patient

Pregnancy outcome after intracytoplasmic sperm injection with strontium oocyte activation in a globozoospermic patient

Role of rabbit prostate granules on sperm viability and acrosome reaction evaluated with different methods

In the present study, the role of rabbit seminal granules was observed. Their influence on motility, capacitation and acrosome reaction, as well as the presence of apoptosis and the morphology of rabbit sperm, were compared in different conditions. Ejaculated sperm from five mature New Zealand White rabbit bucks during three series of collections were studied, comparing raw semen, Percoll-selected sperm and Percoll-selected sperm plus prostate granules. We observed sperm motility kinetic traits by computer-assisted sperm analyzer (CASA) analysis in each sample. Acrosome status was evaluated by FITC-labeled Pisum sativum Agglutinin staining and chlortetracycline fluorescence assay, phosphatidylserine translocation was determined by AnnexinV/Propidium iodide assay and sperm morphology was studied using transmission electron microscopy (TEM). All traits were observed after 30 min incubation at 37 °C in 5% CO2. Data showed that sperm motility and viability markedly improved in the presence of prostate granules, whereas capacitation, acrosome reaction and phosphatidylserine translocation were lowered. TEM confirmed these results. In conclusion, the role of granules was confirmed in synchronizing sperm capacitation and acrosome reaction with egg availability; indeed, rabbit ovulation occurs only 6 to 10 h after mating.

Effect of Dilution Temperature on Boar Semen Quality

As boar semen is very sensitive to cold shock and changes in temperature during semen processing can have a profound impact on semen quality, the effect of the extender temperature at the time of dilution was investigated in a two-step dilution protocol for boar semen being processed for liquid storage. Fifteen boars of different breeds and ages from a commercial artificial insemination centre were included. One ejaculate per boar was collected and processed with Beltsville Thawing Solution semen extender. Each ejaculate was diluted (1 : 1) at 30 °C, and subsequently, the samples were diluted (30 × 10(6) sperm/ml) with either preheated extender [29.3 °C ± 0.2 °C, group A (GA)] or extender at room temperature [22.7 °C ± 0.6 °C, group B (GB)]. Samples were transported to the Faculty of Veterinary Medicine (University of Ghent, Belgium) in two isotherm boxes (one per group), stored at 17 °C and investigated for three consecutive days (D0 to D2). At D0, D1 and D2, motility parameters [computer-assisted semen analysis (CASA)] and the per cent of sperm with intact membrane (% IM) by eosin nigrosin staining were evaluated. At D0 and D2, the % of sperm with intact acrosome (% IA) was studied by Pisum sativum agglutinin staining. The average temperature of the 1 : 1 dilution was 29.4 °C ± 1.1 °C immediately after extender addition. No significant differences were found between groups for per cent motility [79.3 ± 9.0 for GA and 81.1 ± 9.2 for GB (p = 0.372)], % progressive motility [56.5 ± 13.3 for GA and 58.4 ± 13.8 for GB (p = 0.737)] or any CASA parameter. No differences were found for % IM [85.1 ± 10.7 and 84.5 ± 3.8 for GA and GB, respectively (p = 0.761)] and % IA [72.2 ± 9.4 for GA and 68.3 ± 16.6 for GB (p = 0.792)]. In conclusion, when a two-step dilution is performed, preheating the extender for the second dilution to match the semen temperature did not result in better semen quality compared to a dilution at a moderate room temperature.

Sperm nuclear vacuoles, as assessed by motile sperm organellar morphological examination, are mostly of acrosomal origin

Microinjection of nuclear vacuole-free spermatozoa selected by motile sperm organellar morphological examination (MSOME) has been claimed to enhance assisted reproduction treatment outcome compared with intracytoplasmic sperm injection. However, the nature of these nuclear vacuoles is unclear, since their localization at the front of the sperm head suggests they might be of acrosomal origin. To study this hypothesis, acrosomal status was evaluated using Pisum sativum agglutinin staining on a smear, together with sperm organellar morphological examination using the same optics as for MSOME on 30 sperm samples from infertile patients, yielding >3200 spermatozoa. Vacuoles were present in 61% of spermatozoa when acrosomal material or intact acrosomes were observed, versus 29% when spermatozoa were acrosome reacted (P<0.0001). Induction of the acrosomal reaction by ionophore A23587 from 17.4% to 36.1% significantly increased the percentage of vacuole-free spermatozoa from 41.2% to 63.8% (P<0.001). These data suggest that most nuclear vacuoles are of acrosomal origin. Hence, the best spermatozoa selected by MSOME are mostly acrosome-reacted spermatozoa. As microinjection of spermatozoa with a persistent acrosome drastically hampers embryo development in animal models, this suggests that the improvement in pregnancy rates reported following intracytoplasmic injection of morphologically selected sperm might be due to the procedure allowing injection of acrosome-reacted spermatozoa.

Binding regulation of porcine spermatozoa to oviductal vesicles in vitro.

In vivo, en route to the site of fertilization, mammalian spermatozoa bind to oviduct epithelial cells (OECs). This binding may favor sperm survival and capacitation, but little is known about the regulation of detachment of sperm from the oviduct in vivo. Therefore, we studied sperm-oviduct interaction in vitro using vesicles formed from OECs in primary culture. Porcine oviducts were collected from gilts at the slaughterhouse. OECs were obtained by compressing the oviduct and culturing them in TCM-199 medium with 10% fetal calf serum for 48 hours. For the first experiment, to test the hypothesis that progesterone (P4) and estradiol (E2) affect sperm-OEC binding, OECs were pretreated for 48 hours with 100 ng/mL of P4 or E2. In the second experiment, porcine follicular fluid (pFF, 5%), caffeine (1 microM), calcium ionophore A23187 (1 microM), and DMSO (0.01%) were added to the incubation medium to provide insights on the mechanisms of sperm release from the oviduct. For both experiments, 50 x 10(6) sperm were coincubated with 50 microL of OEC vesicles in 1 mL of incubation medium for up to 24 hours at 37 degrees C and 5% CO2. After an initial 30 minutes of coincubation, the vesicles settled and a sample of the supernatant was removed to evaluate sperm release and acrosomal status; subsequent samples were removed after 2, 4, and 24 hours of coincubation. To evaluate the effect of the different treatments on sperm integrity, the acrosome status of the spermatozoa was determined using fluoresceinlabeled Pisum sativum agglutinin staining. Experiment 1 showed that P4 pretreatment of OECs interferes with sperm binding compared with pretreatment with E2 or controls (P < .05). In experiment 2, coincubation in the presence of A23187 increased sperm detachment compared with pretreatment with DMSO, pFF, caffeine, or controls (P < .05). For each experiment, the treatments did not affect the percentage of acrosome-reacted sperm compared with controls (P < .05).

Stimulation by n6-cyclopentyladenosine of A1 adenosine receptors, coupled to galphai2 protein subunit, has a capacitative effect on human spermatozoa.

The effects of selective A(1) receptor agonist on human spermatozoa were examined to verify physiological responses and to investigate the signal transduction pathway. N6-Cyclopentyladenosine on uncapacitated spermatozoa did not induce spontaneous acrosome reaction after 5 h capacitation, whereas the number of capacitated spermatozoa, assessed by lysophosphatidylcholine-induced acrosome reaction with Pisum sativum agglutinin staining, was significantly increased. N6-Cyclopentyladenosine was also added to capacitated human spermatozoa to find out whether the agonist could induce the acrosome reaction. Results, although statistically significant, could not be considered biologically significant. A1-Mediated capacitation was followed by the increase of tyrosine phosphorylation of a protein subset ranging between M(r) = 200 000 and 30 000. Stimulation of A1 receptor with the selective agonist elicited an agonist-induced inositol phospholipid hydrolysis leading to a transient rise of inositol triphosphate (IP3). This increase was not induced by A(1) receptor antagonist and was blocked by phospholipase C inhibitor. Coimmunoprecipitation experiments showed that the A(1) receptor is coupled to Galphai2 subunit suggesting that the activation of phospholipase C is mediated by betagamma subunits. In conclusion, the A(1) adenosine receptor in human spermatozoa is coupled to Galphai2, signals via IP3, and affects the capacitative status of ejaculated spermatozoa.

Simple histochemical stain for acrosomes on sperm from several species.

The acrosome reaction is an exocytotic process that enables a sperm to penetrate the zona pellucida and fertilize an egg. The process involves the fenestration and vesiculation of the sperm plasma membrane and outer acrosomal membrane releasing the acro somal contents. Many different methods have been devel oped to detect the acrosomal status of sperm. These techniques are sometimes complicated, costly, and can be used on only a few species. The aim of this study was to develop an efficient and inexpensive method to assess the acrosomal status of sperm from a variety of species. We prepared and fixed sperm from humans, cattle, swine, rabbits, guinea pigs, and mice and stained them with Coomassie G250. The acrosomes were stained intensely blue in color. Following capacitation, some sperm were incubated for 1 hr with 10 microM calcium ionophore A23187 to induce the acrosome reaction. They were also stained with Coomassie G-250. Ionophore-treated sperm lacked Coomassie staining over the acrosomal region. Differential interference contrast (DIC), bright field microscopy or Pisum sativum agglutinin staining confirmed that the acrosomes of sperm from these species were reacted in response to calcium ionophore treatment and the acrosome reaction frequencies matched results with Coomassie staining. These results demonstrate that the acrosomal status of mammalian sperm from several species can be determined easily and reliably using this simple Coomassie Blue G-250 staining method.

Ca(2+)-related changes in the capacitation state of human spermatozoa assessed by a chlortetracycline fluorescence assay.

Chlortetracycline (CTC) fluorescence patterns were used to assess Ca(2+)-related changes in the capacitation state of human spermatozoa incubated under conditions that would affect their intracellular Ca2+ levels. Initial experiments were designed to identify consistently occurring patterns and to correlate these with acrosomal status. Incubation for up to 1 h with the ionophore A23187 (10 mumol l-1), known to promote capacitation and acrosomal exocytosis, allowed the identification of three different CTC staining patterns which were very similar to those described for mouse spermatozoa. For this reason, they were given the same nomenclature: 'F'-characteristic of uncapacitated, acrosome-intact cells; 'B'-characteristic of capacitated, acrosome-intact cells; and 'AR'-characteristic of capacitated, acrosome-reacted cells. The distribution of the three patterns in the ionophore-treated suspensions was very different from that in control suspensions treated with dimethylsulfoxide only, with a significantly higher proportion of cells displaying the B and AR patterns and a significantly lower number of cells displaying the F pattern in the ionophore-treated group at all times. A strong concordance was found between the acrosomal status of cells determined using both CTC and fluorescein-conjugated Pisum sativum agglutinin (PSA) staining methods on the same cells. Verification of PSA staining patterns with acrosomal status was obtained by means of transmission electron microscopy. The proportion of cells with uniform fluorescence in the acrosomal region correlated with acrosome-intact cells; those with only equatorial segment staining correlated with fully-reacted cells, and those exhibiting equatorial fluorescence and patchy fluorescence over the rest of the acrosomal region correlated with cells in intermediate stages of exocytosis.(ABSTRACT TRUNCATED AT 250 WORDS)