Pisum Sativum Agglutinin Research Articles

Evaluation on Sperm Acrosome Integrity of Infertile Men with Varicocele

Objective To determine the sperm acrosome integrity of samples from infertile men with varicocele. Methods Forty-nine infertile men with varicocele were divided into three groups according to the grade of varicocele. Group A (grade I), B (grade II), and C (grade III) consisted of 15, 18, and 16 cases, respectively. Besides, 15 semen samples from normospermic donors were used as the control. The acrosome integrity of sperm was examined with fluorescein-labeled Pisum sativum agglutinin. Acrosomal ultrastructure was observed with transmission electron microscopy. Results In three varicocele groups, most samples had high sperm abnormal morphol-ogy rates. There were significant differences in acrosome integrity rates between each varicocele group and the control (P Conclusions Infertile men with varicocele had low level of acrosome integrity. Severe varicocele for infertile men might be associated with severe acrosomal defects. Evalu-ating sperm acrosome should aid the understanding of the sperm structural state and benefit the treatment for infertile men.

Effects that bovine sperm cryopreservation using two different extenders has on sperm membranes and chromatin

The process of cryopreservation impairs sperm cell function, potentially leading to a reduction in fertility. The objectives of the present study were to evaluate the effects that cryopreservation using two different extenders has on sperm motility and mitochondrial function, as well as on the integrity of plasma membranes, acrosomal membranes and chromatin, using practical and objective techniques. The focus of the present study was to identify correlations between alterations in sperm membranes and sperm motility in cryopreserved bovine spermatozoa. Seven ejaculates were collected from eight Simmental bulls ( n = 56). After collection, semen volume and concentration were assessed for purposes of dilution. Sperm motility was evaluated subjectively and by computer-assisted semen analysis, morphological characteristics were evaluated by differential interference microscopy, the integrity of plasma and acrosomal membranes, as well as mitochondrial function, were determined using a combination of fluorescent probes containing fluorescein isothiocyanate– Pisum sativum agglutinin, propidium iodide or 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolcarbocyanine iodide. Chromatin integrity was evaluated using the acridine orange technique. The semen was subsequently divided into two aliquots and diluted with one of two extenders (Bioxcell ® or Botu-Bov ®), after which both were packaged in 0.5 mL straws and frozen using an automated system. Two straws of semen from each treatment were thawed, and the semen parameters were evaluated as described above. Cryopreservation of sperm reduced motility, damaging plasma and acrosomal membranes, as well as decreasing mitochondrial function. The Botu-Bov ® extender was more effective in preserving sperm motility and membrane integrity than was the Bioxcell ® extender.

Cryopreservation of European Mouflon (Ovis Gmelini Musimon) Semen During the non‐Breeding Season is Enhanced by the Use of Trehalose

The influence of trehalose on European mouflon spermatozoa cryopreservation during the non-breeding season was tested. Semen was frozen in two different extenders: (a) recommended Tris-based ram extender (CTR); (b) CTR extender supplemented with trehalose 0.147 mm (TRH). Sperm viability and acrosome integrity were assessed using propidium iodide and fluorescein isothiocynate labelled Pisum Sativum agglutinin. Trehalose significantly enhanced sperm viability after thawing compared with CTR extender (62.7% vs 51.8%; p < 0.05), whereas no differences were observed on acrosome integrity (42.9% vs 42.1%). Trehalose influence was also evidenced in the in vitro fertility test performed with sheep oocytes matured in vitro. Both fertilization rates (60.9% TRH vs 43.6% CTR; p < 0.05) and cleavage rates (58% TRH vs 39.8% CTR; p < 0.001) were higher for trehalose frozen semen compared with control extender frozen semen. A higher percentage of zygotes resulting from fertilization with trehalose cryopreserved semen presented the first cleavage earlier if compared with the group fertilized with control semen (48.7% vs 31.5%, respectively; p < 0.01). This result was confirmed by embryo kinetic development. Fertilization with trehalose cryopreserved semen leaded to an higher percentage of blastocysts (40.2% vs 27.8% CTR; p < 0.05), and enhanced in particular the number of blastocysts that developed on the day 6th of culture (28.6% vs 17% CTR; p < 0.05). Our data demonstrated that, during mouflon non-breeding season, trehalose extender enhances spermatozoa viability and its in vitro fertilizing capacity, allowing the production of an higher number of blastocysts.

Fluorescent Stain Method for the Simultaneous Determination of Mitochondrial Potential and Integrity of Plasma and Acrosomal Membranes in Boar Sperm

The purpose of this study was to validate a technique for simultaneous evaluation of the plasma, acrosomal and mitochondrial membranes in boar spermatozoa, using an association of fluorescent probes: Propidium iodide (PI), fluorescein isothiocyanate-conjugated Pisum sativum agglutinin (FITC-PSA) and JC-1. Three ejaculates from each of four different boars, all showing motility >or=80% and abnormal morphology <or=10%, were diluted in TALP medium and split into two aliquots. One of the aliquots was flash frozen in liquid nitrogen and thawed in three continuous cycles, to induce damage in cellular membranes and to disturb mitochondrial function. Three treatments were prepared with the following fixed ratios of fresh semen : flash frozen semen; 100 : 0 (T100), 50 : 50 (T50), and 0 : 100 (T0). The samples were then submitted to a stain technique. To a 150-microl aliquot of diluted semen it was added 3 microl of PI (0.5 mg/ml), 2 microl of JC-1 (153 microm) and 50 microl of FITC-PSA (100 microg/ml). Samples were incubated at 38.5 degrees C for 8 min, in the dark. An 8-microl sample was put on a slide, coverslipped and immediately evaluated by epifluorescent microscopy. The association of fluorescent probes was divided into eight cell classes, according to plasma membrane integrity, intact acrosome and mitochondrial function. For plasma membrane integrity, detected by PI probe, the equation: (p < 0.0001) and R(2) = 0.97 was obtained. The intact acrosome, verified by the FITC-PSA probe, produced the equation: (p < 0.0001) and R(2) = 0.98. The mitochondrial potential, marked by JC-1, was estimated by the equation: (p < 0.001) and R(2) = 0.99. The group of spermatozoa with combined intact plasma membrane, intact acrosome and high mitochondrial potential (IPIAH), was estimated by the equation: (p < 0.0001) and R(2) = 0.97. The resulting linear equations demonstrate that this technique is efficient and practical for the simultaneous evaluations of the plasma, acrosomal and mitochondrial membranes in boar spermatozoa.

Ginsenoside Re promotes human sperm capacitation through nitric oxide‐dependent pathway

The regulation of sperm capacitation is important for successful fertilization. Ginsenosides, the biologically effective components of ginseng, have been found to enhance intracellular nitric oxide (NO) production and the latter has recently been indicated to play a significant role in modulation of sperm functions. We investigated the effect of Ginsenoside Re on human sperm capacitation in vitro and the mechanism by which the Ginsenosides play their roles. Spermatozoa were separated by Percoll and incubated with 0, 1, 10, or 100 microM of Ginsenoside Re. The percentages of spontaneous and lysophosphatidylcholine (LPC)-induced acrosome reaction (AR), as a measure of sperm capacitation, were assayed with fluorescein isothiocyanate-conjugated Pisum sativum agglutinin (FITC-PSA). The intracellular cGMP level was measured by [(3)H] cGMP radioimmunoassay system. The results showed that the percentages of both spontaneous and LPC-induced AR and intracellular cGMP level were significantly enhanced by Ginsenoside Re with a concentration-dependent manner. Sodium nitroprusside (SNP, 100 nM), a NO donor, mimicked the effects of Ginsenoside Re. And pretreatment with a NOS inhibitor N(omega)-nitro-l-arginine methyl ester (L-NAME, 100 microM) or a NO scavenger N-acetyl-l-cysteine (LNAC, 1 mM) completely blocked the effects of Ginsenoside Re. Furthermore, the AR-inducing effect of Ginsenoside Re was significantly reduced in the presence of the soluble guanylate cyclase inhibitor LY83583 or cGMP-dependent protein kinase (PCK) inhibitor KT5823, whereas addition of the cGMP analogue 8-Br-cGMP significantly increased the AR of human spermatozoa. Data suggested that Ginsenoside Re is beneficial to sperm capacitation and AR, and that the effect is accomplished through NO/cGMP/PKG pathway.

Acrosome‐reacted human sperm in insemination medium do not bind to the zona pellucida of human oocytes

In the literature there is still confusion whether acrosome-reacted sperm in medium can initiate primary binding to human zona pellucida (ZP). The ability of acrosome-reacted sperm to bind to ZP in vitro can be deduced by measuring the acrosome reaction (AR) of ZP-bound sperm compared with sperm in medium after incubation under different conditions inhibiting the ZP-induced AR. Motile sperm from fertile men, normospermic men and infertile men diagnosed with disordered ZP-induced AR (DZPIAR) were selected by swim-up (2 x 10(6) in 1 mL medium) and incubated for 1-2 h with four oocytes from failed in vitro fertilization (IVF). The acrosome status of sperm was assessed using pisum sativum agglutinin labelled with fluorescein. The ZP-induced AR was inhibited in experiments using sperm from DZPIAR patients, hyper-osmotic medium (400 mOsm/kg) and medium containing soybean trypsin inhibitor (SBTI; 4 mg/mL). Pre-treatment with calcium ionophore was used to create a sperm population with elevated AR. In all experiments with factors inhibiting the ZP-induced AR, the AR was significantly lower for ZP-bound sperm compared with sperm in medium: DZPIAR patients 4% vs. 15%, hyper-osmotic medium 3% vs. 12%, SBTI 2% vs. 12% and SBTI 3% vs. 23% after treatment with calcium ionophore. In conclusion, acrosome-reacted sperm in vitro have significantly reduced, in fact probably zero ability to bind to the ZP.

Evaluation of Coomassie blue staining of the acrosome of equine and canine spermatozoa

To evaluate Coomassie blue staining of the acrosome of equine and canine spermatozoa. Spermatozoa of 5 mixed-breed male dogs and 3 Thoroughbred stallions. Various proportions of intact and acrosome-damaged spermatozoa were fixed in 2% phosphate-buffered formaldehyde or 4% paraformaldehyde, smeared onto glass slides, and stained with Coomassie blue stain. Acrosomal status (damaged vs intact) was also assessed by use of flow cytometry after staining with fluorescein isothiocyanate-conjugated Pisum sativum agglutinin (FITC-PSA) and propidium iodide. Comparisons were made between percentages of expected and observed acrosome-intact spermatozoa in different proportions of live and flash-frozen samples; the percentages of acrosome-intact spermatozoa as determined by use of Coomassie blue staining and flow cytometry were also compared. Strong correlations were found between the expected and observed distributions of acrosome-intact spermatozoa when fixed in 4% paraformaldehyde (r2 = 0.93 and 0.89 for canine and equine spermatozoa, respectively) as well as between Coomassie blue-stained cells and those stained with FITC-PSA and assessed by use of flow cytometry (r2 = 0.96 and 0.97 for canine and equine spermatozoa, respectively). However, in canine samples that were fixed in 2% phosphate-buffered formaldehyde, these correlations were weak. Staining with Coomassie blue stain was a simple and accurate method to evaluate the acrosome in equine and canine spermatozoa after fixation in 4% paraformaldehyde. This assay should be useful in routine evaluation of semen samples from these species.

138 EFFECT OF SEMINAL PLASMA ON CHILLING AND FREEZING OF CANINE SPERMATOZOA

Seminal plasma (SP) is usually removed from semen that is to be cryopreserved. However, some reports indicate that SP has beneficial effects on spermatozoa during chilling and freezing (Barrios et al. 2000 Biol. Reprod. 63, 1531–1537; Moore et al. 2005 Theriogenology 63, 2372–2381; Vadnais et al. 2005 Anim. Reprod. Sci. 87, 121–132). The purpose of this study was to determine the effect on sperm survival of adding SP to the extender before cooling and freezing canine spermatozoa. In replicate experiments, ejaculates obtained from 4 healthy dogs (3–4 years old) of various breeds were pooled and centrifuged at 300g for 10 min at 25�C; the supernatant of seminal plasma was decanted. Spermatozoa were suspended in egg yolk-Tris (EYT) buffer. The study comprised 2 experiments: Exp. 1: Sperm were suspended in EYT extender containing 0%, 20%, 50%, 80%, or 100% SP, and were slowly cooled to 4�C for 2 h or held at 25�C as controls. Exp. 2: Sperm samples, each of which contained 1 � 108 sperm mL-1, were assigned to 5 groups to be frozen. In group 1, sperm in EYT + 20% SP were cooled to 4�C, diluted to contain final concentrations of 5% glycerol + 10% SP in EYT, and then frozen. In the 4 other groups, sperm in EYT alone were first cooled slowly to 4�C, then diluted to contain 5% glycerol plus 0%, 20%, 40%, or 50% SP in EYT, and then frozen. Spermatozoa were frozen at 25�C min in plastic straws that were suspended above liquid nitrogen and thawed in water at 38�C for 30 s. Sperm survival was assayed by determining progressive motility and integrity of plasma and acrosome membranes. Progressive motility was determined by microscopic examination at 400� magnification. Membrane integrity was assessed by use of a double fluorescent dye, and acrosome integrity by staining sperm with Pisum sativum agglutinin. The results of the first experiment showed that 20%, 50%, 80%, or 100% SP did not improve motility, membrane integrity, or acrosome integrity of spermatozoa chilled to 4�C compared to those chilled without SP (P &amp;gt; 0.05). Survival of spermatozoa suspended in EYT + 20% SP and maintained at 25�C was significantly higher than for those that were chilled (P &amp;lt; 0.05). The results of the second experiment showed that spermatozoa suspended in EYT + 20% SP and then diluted at 4�C to contain 5% glycerol + 10% SP exhibited the highest progressive motility and membrane integrity after being frozen and thawed (P &amp;lt; 0.05). In summary, although seminal plasma did not affect spermatozoa that were only chilled, addition of seminal plasma did significantly improve survival of canine spermatozoa that were frozen and thawed.

Identification of peptides binding to Pisum sativum agglutinin from a phage-displayed random peptide library

To obtain peptides binding specifically to Pisum sativum agglutinin (PSA) from a phage-displayed random peptide library. (1) A phage-displayed random hexapeptide library was screened with PSA as target. (2) Dot blot was used to analyze the influence of the alpha-Met-D-mannoside on binding between PSA and phage-displayed peptides. (3) Three peptides (RMWSF, RYDYSY, LRLRQL) were selectively synthesized, and different concentrations were used to inhibit PSA and ConA binding to the HRP. The enrichment occurred obviously after three rounds of screening. The insert sequences of amino acids, displayed on 22 phage DNAs from the third round of screening, were divided into three groups. The binding of phage-displayed peptides to PSA was specific as shown by dot blot and could be inhibited by alpha-Met-D-mannoside. LRLRQL was not dissolved in water. ARMWSF and RYDYSY inhibited binding of PSA to HRP, but failed to inhibit binding ConA to HRP. The binding site of peptides ARMWSF and RYDYSY is different to that of alpha-Met-D-mannoside.

The in vitro effect of emergency contraception doses of levonorgestrel on the acrosome reaction of human spermatozoa

Introduction The aim of this study was to evaluate the effect of three concentrations of levonorgestrel (LNG) comparable to the levels found in serum following ingestion of LNG as emergency contraception (EC) on the acrosome reaction (AR) of capacitated and noncapacitated spermatozoa of fertile men. Materials and Methods A total of 24 semen samples from three fertile men were evaluated. The spermatozoa were selected by Percoll gradient. Twelve samples were subsequently incubated with human tubal fluid medium supplemented with bovine serum albumin (HTF/BSA) for 20 h under capacitating conditions. The capacitated spermatozoa and the spermatozoa from the remaining 12 samples were exposed to LNG at 1, 10 and 100 ng/mL, to follicular fluid (FF) (20 %v/v) and to HTF medium. The ratio of live to dead spermatozoa was assessed after 1, 2 and 3 h of incubation at 37°C and 5% CO 2. After 30 min of exposure to the different LNG concentrations, aliquots were divided into two parts. In the first part, spermatozoa were immediately stained with Hoescht 33258 and fluorescein isothiocyanate–pisum sativum agglutinin (FITC-PSA) in order to assess AR rate and to repeat evaluation of the live-to-dead ratio. After 3 h of incubation, the remaining part of the aliquots were submitted to the same procedures. Each concentration of LNG was then compared with FF and HTF medium as positive and negative controls, respectively. Results The results showed that in vitro exposure to the three different LNG concentrations did not induce AR. Conclusion This study failed to show any in vitro effect on AR of LNG concentrations similar to those found in serum following intake of LNG as EC. If this effect exists or if there is any other that influences sperm fertilizing capacity, in vitro experiments are probably not an appropriate way of testing it.

Induction of human sperm capacitation and protein tyrosine phosphorylation by endometrial cells and interleukin-6*

In order to become fully competent at fertilizing the oocyte, spermatozoa must undergo the maturational process of capacitation during their journey in the female reproductive tract. Endometrial cells secrete an array of growth factors that can affect spermatozoa. Among these factors, it has been previously demonstrated that interleukin-6 (IL-6) affects the fertilizing capacity of human spermatozoa. As the expression of this cytokine varies throughout the menstrual cycle and increases during the periovulatory period, the involvement of IL-6 in human sperm capacitation was investigated, with emphasis on the signal transduction cascade triggered by this agent in sperm cells. Spermatozoa were treated with recombinant human IL-6. Protein phosphotyrosine content and localization of the phosphotyrosine containing proteins were evaluated by western blot and indirect immunofluorescence, respectively, using a monoclonal anti-phosphotyrosine antibody. The acrosomal status was evaluated on IL-6 treated spermatozoa before or after challenge with the ionophore A23187 according to the fluorescent pattern observed upon binding to the Pisum sativum agglutinin conjugated to fluorescein isothiocyanate. In the present study, it is shown that, as for endometrial cell-conditioned media, IL-6 induces human sperm capacitation. The IL-6 effects most likely occur through binding to its receptor, IL-6Ralpha, whose presence in the sperm is also reported in this study. As for the IL-6 receptor, this is the first report on the presence of the tyrosine kinase JAK1 in the spermatozoa. Moreover, this kinase becomes phosphorylated on tyrosine residues upon sperm treatment with recombinant IL-6, which suggests its activation by the cytokine. Taken together, our results demonstrate that the IL-6 intracellular signalling machinery is present in human spermatozoa and might be involved in the acquisition of sperm fertilizing ability, also known as the capacitation process.

Correlation between tyrosine phosphorylation intensity of a 107 kDa protein band and A23187-induced acrosome reaction in human spermatozoa

This study, performed using semen samples from 10 men, investigated the relationship between sperm protein tyrosine phosphorylation and acrosomal status in conditions supporting in vitro capacitation. Percoll-selected spermatozoa (cells from the 95% fraction) were incubated for 3 h at 37 degrees C under an atmosphere of 5% CO2 in air, in a polyvinyl alcohol (1 mg ml(-1)) containing Biggers-Whitten-Whittingham's medium, nonsupplemented or supplemented with either bovine serum albumin (BSA; fatty acid free, 3 mg ml(-1)) or 2-hydroxy-propyl-beta-cyclodextrin (2-OH-p-beta-CD; 0.5, 1, 2 mmol l(-1)). Sperm suspension in each medium was split into two aliquots. The first was used to evaluate the acrosomal status by staining with the fluorescein isothiocyanate Pisum sativum agglutinin after induction of the acrosome reaction (AR) for 45 min with 10 micromol l(-1) of A23187 calcium ionophore. The second aliquot was used for sodium dodecyl sulphate polyacrylamide gel electrophoresis and immunoblotting, followed by a densitometric analysis. Compared with the nonsupplemented medium, BSA- or 2-OH-p-beta-CD-supplementation induced an increase in both the percentage of live acrosome-reacted sperm and the tyrosine phosphorylation intensity of the main phosphorylated 107 kDa protein. A correlation between the percentage of live acrosome-reacted sperm and the 107-kDa protein phosphotyrosine intensity was observed. Therefore, the 107 kDa protein-phosphotyrosine level measurement would bring additional information to conventional semen parameters in the assessment of the human sperm functionality.

Integrated Lectin Affinity Microfluidic Chip for Glycoform Separation

Lectin affinity chromatography was miniaturized into a microfluidic format, which results in improvement of performance, as compared to the conventional method. A lectin affinity monolith column was prepared in the microchannel of a microfluidic chip. The porous monolith was fabricated by UV-initiated polymerization of ethylene dimethacrylate (EDMA) and glycidyl methacrylate (GMA) in the presence of porogeneities, followed by immobilization of pisum sativum agglutinin (PSA) on the monolith matrix. Using electroosmosis as the driven force, lectin affinity chromatographies of three kinds of glycoprotein, turkey ovalbumin (TO), chicken ovalbumin (CO), and ovomucoid (OM), were carried out on the microfluidic system. All the glycoproteins were successfully separated into several fractions with different affinities toward the immobilized PSA. The integrated system reduces the time required for the lectin affinity chromatography reaction to approximately 3%, thus, the overall analysis time from 4 h to 400 s. Only 300 pg of glycoprotein is required for the whole separation process. Moreover, troublesome operations for lectin affinity chromatography are simplified.

Prediction of outcomes of assisted reproductive technology treatment by calcium ionophore-induced acrosome reaction

Conventional semen analysis is not a good predictor of successful outcome of in vivo insemination, intrauterine insemination (IUI) or hysteroscopic intra-tubal insemination (HIT), or a predictor of complete fertilization failure (CFF) in conventional IVF (IVF). We investigated whether calcium ionophore induced acrosome reaction provides an additional indicator of pregnancy among various assisted reproduction modalities as well as an indicator of complete fertilization failure in IVF, independent of conventional semen parameters, sperm concentration and motility. prospective study Subjects included infertile couples with no female infertility factors (n=478) and women with tubal obstruction (n=32). Procedures included conventional IVF (IVF): n=148, ICSI: n=72, HIT: n=245, IUI: n=61. They all underwent a standard protocol of history-taking, physical examination and investigations including conventional semen analysis as well as a test of the sperm acrosome reaction following ionophore challenge. Every patient gave an informed consent prior to participating in this study, which was approved by the local Institutional Review Board, Faculty of Medicine. Calcium ionophore induced acrosomal reactivity was assessed in spermatozoa selected by swim-up. Spermatozoa on the slide were subjected to staining with fluorescent isothiocyanate-conjugated pisum sativum agglutinin (FITC-PSA) and with Hoescht 33258. Spermatozoa were considered viable when their heads were unstained or stained only lightly with Hoechst 33258. When the equatorial segment strongly stained with FITC-PSA, the spermatozoon was considered to have reacted. The percentage of acrosome-reacted spermatozoa to viable spermatozoa (%AR) was calculated. At least 300 spermatozoa per sample were examined to determine the %AR. We excluded abnormal or deformed spermatozoa, as well as unclassifiable fluorescence patterns. Multiple logistic regression analysis and receiver-operator characteristic (ROC) curve analysis were performed to determine the best predictive variables and cut-off values in the prediction of fertilization rate and pregnancy in each modality. χ-square analysis was used to compare the different fertilization and pregnancy rates. P<0.05 was considered significant. Cut-off value of %AR for IVF was 21% and the value was independent from the parameters of conventional semen analysis in predicting fertilization rate and pregnancy. Fertilization rate on IVF was higher for those with %AR over 21% compared to that of those with a lower percentage (63.6% vs. 35.4%; p<0.01). The pregnancy rate was also higher (43.1% vs. 18.9%; p<0.01). CFF was lower for those with %AR over 21% (24.5% vs. 4.2%,p<0.01). Fertilization rate, pregnancy rate, and CFF on ICSI did not differ for those with %AR over 21%. Pregnancy rates were higher for those with %AR over 21% than for those with a lower percentage on IUI (28.0% vs. 5.6%, p<0.05) and on HIT (23.9% vs. 6.2%, p<0.01). These data shows that %AR may be a novel parameter independent of conventional semen parameters in choosing assisted reproduction modalities. Thus, patients with acrosomal reactivity below 21% could not achieve pregnancy by IUI, HIT, or IVF with high rates. With reactivity below 21%, a low fertilization rate for initial IVF, ICSI or split fertilization is indicated.

202FERTILIZING ABILITY OF EPIDIDYMAL CAT SPERMATOZOA AFTER CRYOPRESERVATION OR STORAGE AT 4°C.

The possibility of harvesting cat epididymal spermatozoa from excised testis represents a potentially important tool for preserving valuable genetic material from males that die unexpectedly. The purpose of this study was to evaluate plasma membrane integrity, acrosomal status and in vitro fertilizing capability of epididymal cat sperm after short- and long-term storage at 4°C and after cryopreservation. Spermatozoa were collected by flushing the cauda epididymis and the vasa deferentia removed from adult cats undergoing orchiectomy. Spermatozoa were split into three aliquots: A-fresh, B-frozen, and C-cooled. The A portion was immediately tested on the day of collection (control), the B portion was frozen in pellets in Test Yolk Buffer (TYB) with 8% glycerol, while the C portion was extended in TYB and stored at 4°C for 1 day, 2 days or 7 days. Cat oocytes recovered from minced ovaries were matured for 24h in TCM 199+1UI/mL hCG+0.5UI/mL FSH+0.3% BSA at 38.5°C in 5% CO2. In vitro fertilization (IVF) of in vitro-matured (IVM) oocytes was performed using spermatozoa from portions A, B or C in modified Tyrode’s solution supplemented with 0.6% BSA at 38.5°C in 5% in air. Before IVF, aliquots of sperm samples from the three portions were stained simultaneously with fluorescein isothiocyanate-labelled Pisum Sativum agglutinin (FITC-PSA) and propidium iodide to evaluate the percentage of plasma membrane-intact spermatozoa with acrosomes present. At least 200 spermatozoa were counted in duplicate for each sample. After 24h of incubation, to evaluate fertilization rate, the oocytes were stained with aceto-lacmoid. Oocytes with two pronuclei, presumably male and female, were classified as fertilized. The percentages of spermatozoa maintaining plasma membrane integrity and intact acrosomes after 1 day or 2 days of storage at 4°C were 75% and 65%, respectively, and were similar to that of fresh epididymal spermatozoa (78%). However, the percentages of spermatozoa with intact plasma membranes that had intact acrosomes after 7 days of storage at 4°C (52%) or after cryopreservation (48%) were significantly lower (P&amp;lt;0.01, ANOVA) than those of samples stored for 1 or 2 days at 4°C. As shown in the Table, fertilization rates of oocytes inseminated with fresh spermatozoa and spermatozoa stored for 1 or 2 days were similar. In contrast, the fertilization rates of oocytes inseminated with spermatozoa that had been cryopreserved or stored for 7 days were significantly lower than those obtained with spermatozoa used immediately after collection or storage for 1 day. From our results, we suggest that storage of epididymal spermatozoa at 4°C may be a potentially useful method for temporary storage of spermatozoa from endangered cats, and may allow more efficient use and long-distance transport of genetically important germplasm. This work was supported by MIUR (ex 40%). Table 1 In vitro fertilization of in vitro-matured cat oocytes with fresh, stored at 4°C and cryopreserved epididymal cat spermatozoa

Role of capsule in serotypic differences and complement fixation by Lactococcus garvieae

Seventeen geographically distinct isolates of Lactococcus garvieae, isolated from diseased fish, were compared serologically using antiserum raised against the various isolates in rainbow trout. Sera raised against a capsule deficient isolate did not agglutinate capsulated isolates, regardless of origin. In contrast, all antisera raised against capsulated isolates cross reacted strongly with non-capsulated isolates. Antisera raised against capsulated Japanese isolates cross reacted with other capsulated Japanese isolates including isolates from geographically distinct prefectures within Japan (Ehime and Oita). However, antisera against these virulent capsulated isolates did not cross react with European capsulated isolates. Antisera raised against European capsulated isolates cross reacted with other European isolates, regardless of origin within Europe (UK, Italy, Spain), but did not cross-react with Japanese capsulated isolates. Agglutination assays performed with a range of fifteen lectins revealed differences in surface carbohydrate structure: capsule deficient isolates agglutinated with concanavalin A, Ricinis communis agglutinin, Pisum sativum agglutinin, Lens culinaris agglutinin, wheat germ agglutinin and succinylated wheat germ agglutinin. European capsulated isolates agglutinated with concanavalin A only. The Japanese capsulated isolates were not agglutinated by any of the lectins used in this study. Representative isolates from each group (Japanese capsulated and non-capsulated, European capsulated and non-capsulated) were investigated for their ability to fix complement. Non-capsulated isolates fixed complement regardless of origin, and antibody did not markedly enhance complement fixation. In contrast, the capsulated isolates were less efficient at fixing complement, but complement fixation was markedly increased by homologous antibody.

Effects of diadenosine polyphosphates and seminal fluid vesicles on rabbit sperm cells.

Membrane vesicles were isolated from rabbit seminal plasma. Electron microscopy analyses showed the presence of numerous small, round vesicles with a diameter of about 70 nm. Determination of enzyme activities was carried out by high performance liquid chromatography and showed that the vesicles can degrade the diadenosine polyphosphates (ApnA), Ap3A and Ap4A and ATP and ADP, but not AMP. Studies of the degradation of diadenosine compounds by the vesicles present in seminal fluid showed an increasing production of AMP as the by-product and a time-dependent generation of dephosphorylated products consistent with the presence of ecto-ATP diphosphophosphatase (ecto-apyrase). In the presence of rabbit spermatozoa, AMP did not accumulate because 5'nucleotidase and adenosine deaminase, present at the surface of sperm cells, transformed AMP into adenosine and inosine. The effects of seminal fluid vesicles and diadenosine compounds on the acquisition of fertilizing capacity by rabbit spermatozoa were evaluated by Pisum sativum agglutinin fluorescein isothiocyanate conjugated staining. The results obtained with uncapacitated spermatozoa showed that the capacitating effector BSA could be substituted efficiently by the addition of diadenosine compounds and vesicles previously incubated for 2 h to the capacitative medium. Under these experimental conditions, the spontaneous acrosome reaction rate was not increased. Capacitated rabbit spermatozoa did not undergo acrosome reaction when l-alpha-lysophosphatidylcholine was substituted by diadenosine compounds previously incubated with vesicles. In conclusion, this study has shown that rabbit seminal fluid vesicles can degrade diadenosine compounds to AMP and that the addition of the vesicles and diadenosine compounds to uncapacitated rabbit spermatozoa favours the acquisition of the fertilizing capacity.

Functional changes and motility characteristics of Japanese Black bull spermatozoa separated by Percoll

Spermatozoa from two Japanese Black bulls (Bull-ATF and Bull-KTG) were separated by centrifugation at 700× g for 15 min in modified TALP with or without 45–90% Percoll. Control washed spermatozoa and those collected from the bottom of 45 and 90% Percoll fractions were examined for viability and membrane integrity (using Hoechst bis-benzimide 33258 or propidium iodide and 6-carboxyfluorescein diacetate (PI-CFDA)), acrosomal status (using fluorescence isothiocyanate (FITC) conjugated Pisum Sativum agglutinin (PSA) and Peanut agglutinin (PNA), Naphthol Yellow S and Erythrosin B (NE) or triple staining (TS)), capacitation status (using chlortetracycline (CTC)), motility characteristics (using a computer-assisted sperm motion analysis system (CASA)) and for in vitro fertility. Percoll-separated spermatozoa showed greater viability and membrane integrity than controls, as determined by supravital staining. Differences were observed in the results regarding viability and acrosomal status of spermatozoa among sperm staining methods. Bull-ATF, which showed significantly greater in vitro fertility than Bull-KTG ( P<0.05), showed a significantly higher rate of CTC-B-pattern (capacitated) spermatozoa ( P<0.01) than Bull-KTG. The motility characteristics of control washed spermatozoa and those separated by 45–90% Percoll were analyzed by CASA. More motile and progressively motile spermatozoa were observed in the fraction at the bottom of the 90% Percoll solution than in the 45% Percoll fraction or in controls ( P<0.01). Moreover, the spermatozoa of Bull-KTG, which showed lower in vitro fertility than Bull-ATF, did not show significant differences in motility from those of Bull-ATF. These results provided basic information about Japanese Black bull spermatozoa, and suggested that spermatozoa with greater motility and viability can be obtained by Percoll separation than without separation. However, Percoll separation did not enhance their in vitro fertility.

Superoxide dismutase affects the viability of thawed European mouflon (Ovis g. musimon) semen and the heterologous fertilization using both IVF and intracytoplasmatic sperm injection.

This study evaluated the effects of superoxide dismutase (SOD) on viability and acrosome integrity of European mouflon spermatozoa after cryopreservation and on the fertilization rates of sheep oocytes after i.v.f. or intracytoplasmatic sperm injection (i.c.s.i.). Frozen semen was thawed and washed with synthetic oviduct fluid supplemented with 0.6% bovine serum albumin. After centrifugation, the spermatozoa pellet was split into two culture systems: (i) without SOD; and (ii) in the presence of 1500 IU mL(-1) SOD. Sperm viability and acrosome integrity were evaluated simultaneously, immediately after thawing and after 3, 6 and 9 h of culture (5% CO2, 39 degrees C, 90% humidity), by incubating sperm with propidium iodide and fluorescein isothiocyanate-labelled Pisum sativum agglutinin. At the same time, sperm were assessed for motility using a standard scoring system (independent operators' observation of sperm) that graded degree of motility (i.e. 1 = immotile to 10 = maximum motility, as observed at the moment of thawing). For i.v.f., frozen-thawed semen derived from the two culture systems was placed in culture together with in vitro-matured sheep oocytes. For i.c.s.i., semen derived from the same culture systems as that for i.v.f. was used, and incubated for 1 h under standard conditions. The results showed a marked difference (P < 0.01) between the percentages of live spermatozoa in medium with SOD and those obtained in medium alone, after 3, 6 and 9 h of culture. The percentages of intact acrosome spermatozoa were higher in medium with SOD after 6 h (P = 0.05) of culture. Spermatozoa motility decreased significantly in SOD containing medium at 3 and 6 h of culture compared with motility in control medium. Fertilization rates were significantly lower in medium with SOD than in medium alone, whereas in the i.c.s.i. system fertilization rates were significantly higher in the presence of SOD. The results indicate that the addition of SOD to the culture media enhances the viability rates and the acrosome integrity of cryopreserved mouflon spermatozoa.

Computer-assisted analysis of sperm morphology with the aid of lectin staining

Lectins are useful for staining the acrosome, which is a pre-requisite for the assessment of acrosome reaction in vitro. We tested wheat germ agglutinin, peanut agglutinin and pisum sativum agglutinin. The determination of the categories of normal and abnormal spermatozoa as defined by the World Health Organization, including an additional category 'acrosome-less cell', was performed with the aid of a system of computer-assisted semen analysis (CASA) in comparison with visual estimation. The acrosome reaction in vitro was induced by calcium ionophore A23187. Incubation with A23187 decreased the percentage of normal sperm heads and increased the number of acrosome-less sperm heads in comparison with the control samples. This shift was demonstrable with all three lectin staining procedures. No significant differences were observed in the comparison of sperm classes obtained by visual assessment or determination by CASA with two of the lectin staining procedures. After staining with pisum sativum agglutinin the classes of normal and of acrosome-less spermatozoa were significantly different between visual and CASA estimation. Our results indicate that estimation of the acrosomal status in vitro is possible by CASA when lectin staining is used.