Fluorescein Isothiocyanate-conjugated Pisum Sativum Agglutinin Research Articles

Practical Techniques for Bovine Sperm Simultaneous Fluorimetric Assessment of Plasma, Acrosomal and Mitochondrial Membranes

This experiment was performed to develop and validate practical techniques for simultaneous evaluation of the integrity of plasma and acrosomal membranes, as well as mitochondrial function in bovine spermatozoa using associations of fluorescent probes. Four protocols of fluorescent probes association were defined: protocol 1: propidium iodide (PI), fluorescein isothiocyanate-conjugated Pisum sativum agglutinin (FITC-PSA) and rhodamine 123; protocol 2: PI, FITC-PSA and MitoTracker Green FM (MITO); protocol 3: PI, Hoechst 33342 (H342), FITC-PSA and CMXRos; and protocol 4: PI, H342, FITC-PSA and JC-1. Three ejaculates from each of the four bulls (n = 12) were utilized, showing sperm motility >/=80% and abnormal morphology </=10%. The semen was diluted in Modified Tyrode's medium (TALP) (25 x 10(6) spermatozoa/ml) and split into two aliquots, one sample was flash-frozen in liquid nitrogen and thawed. Samples for three treatments were prepared with the following ratio of fresh semen : flash-frozen semen: 100 : 0, 50 : 50 and 0 : 100. Samples were stained in all four protocols and evaluated by epifluorescence microscopy. Protocol 1 did not result in a satisfactory stain, so it could not be validated. Protocols 2, 3 and 4 were validated and showed high determination coefficient to plasma membrane integrity (R(2) = 0.95, 0.93 and 0.92, respectively), acrosome integrity (R(2) = 0.95, 0.92 and 0.91, respectively) and mitochondrial function (R(2) = 0.84, 0.93 and R(2) = 0.93, respectively). These techniques are efficient for the simultaneous integrity evaluation of plasma and acrosomal membranes and mitochondrial function in bovine spermatozoa. However, JC-1 has an advantage over MITO and CMXRos, as it separates two cell populations with high and low mitochondrial membrane potential.

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.

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.

Cryopreservation-induced acrosomal vesiculation in live spermatozoa from cynomolgus monkeys (Macaca fascicularis).

Cryopreserved spermatozoa are known to undergo accelerated capacitation and require a shorter incubation time for fertilization. However, details of their acrosomal membranes following cryopreservation remain unclear. Percoll density gradient centrifugation was used to remove dead spermatozoa; thus >90% live spermatozoa were recovered after cryopreservation, and acrosomal status was compared among non-incubated and incubated fresh and cryopreserved spermatozoa. Transmission election microscopy (TEM) using microwave methods and fluorescein isothiocyanate-conjugated Pisum sativum agglutinin (FITC-PSA) staining revealed that 21.1 and 61.6% respectively of non-incubated, cryopreserved spermatozoa were intact, whereas 97.6% (TEM) or 91.9% (FITC-PSA) of non-incubated fresh spermatozoa were intact. TEM revealed that 28.8% of the cryopreserved spermatozoa were swollen, and probably included among those counted as intact by FITC-PSA staining. The non-incubated cryopreserved spermatozoa had fused plasma and outer acrosomal membranes, and 36.4% of them had vesiculation when observed by TEM. FITC-PSA staining indicated that 22% of the live spermatozoa were acrosome reacted. Acceleration of the acrosome reaction was evident by both TEM and FITC-PSA. Incubation of cryopreserved spermatozoa for 2 h accelerated vesiculation to a state similar to that of fresh spermatozoa that had been incubated for 8 h. These results reveal that in cryopreserved spermatozoa, the process of acrosome reaction begins before incubation.

Soybean trypsin inhibitor as a probe for the acrosome reaction in motile cynomolgus macaque sperm.

Soybean trypsin inhibitor (SBTI) inhibits the catalytic activity of serine proteases, and has been shown to bind to acrosin, an acrosomal hydrolase which is not exposed on the surface of macaque sperm until after the acrosome reaction. Following activation with caffeine and dibutyryl cAMP, cynomolgus macaque sperm were induced to acrosome react with calcium ionophore A23187 in the presence of SBTI and were fixed for ultrastructural observation. Transmission electron microscopy (TEM) revealed secondary labelling of anti-SBTI-IgG with colloidal gold in association with the acrosomal matrix and fused membranes of sperm undergoing the acrosome reaction, but gold labelling was not observed on acrosome-intact sperm. When SBTI was conjugated with the fluorochrome Alexa 488, labelled (acrosome-reacted) sperm showed bright fluorescence that ranged from a patchy or punctate appearance to solid labelling over the region of the acrosomal cap. Following treatment with ionophore, the percentages of total acrosome-reacted sperm (motile and non-motile) as assessed with Alexa-SBTI, fluorescein isothiocyanate-conjugated Pisum sativum agglutinin (FITC-PSA), and TEM were 54.6%, 51.6% and 61.5%, respectively. Measures of acrosomal status with FITC-PSA and Alexa-SBTI were highly correlated (r = 0.94; n = 3). Macaque zonae pellucidae were co-incubated with activated sperm for 1 min and then rinsed in medium containing Alexa-SBTI and immediately observed with epifluorescence microscopy. The mean percentage of Alexa-SBTI-labelled (acrosome-reacted) motile sperm bound to the zona was 45.7 +/- 14 (range: 22-80.4%; n = 4). Fewer than 1% of the motile sperm in suspension surrounding the zonae were acrosome-reacted. Alexa-SBTI had no effect on sperm motility, survival, or zona binding capability.

Two-color fluorescence staining of lectin and anti-CD46 antibody to assess acrosomal status

Objective: To examine potential methods for distinguishing between the acrosome reaction and acrosomal loss. Design: Prospective randomized study. Setting: Department of Obstetrics and Gynecology, Osaka University Hospital, Suita, Japan. Patient(s): Five healthy volunteers and 34 patients with normozoospermia who were participating in an IVF program. Intervention(s): Semen samples were collected from the volunteers before the hamster egg penetration assay and from the patients at the time of IVF. Main Outcome Measure(s): The numbers of oocytes penetrated and spermatozoa bound were determined with the hamster egg penetration assay. Acrosomal status was assessed with two-color fluorescence staining using fluorescein isothiocyanate–conjugated Pisum sativum agglutinin (FITC-PSA) and MH61 (anti-CD46 monoclonal antibody) with Texas red–conjugated antimouse immunoglobulin G antiserum. Result(s): The MH61 monoclonal antibody inhibited the penetration of human spermatozoa into hamster oocytes but did not reduce the number of spermatozoa bound to the zona-free hamster oocytes. Two-color fluorescence staining revealed four staining patterns of the acrosomal region. The percentage of PSA-negative/CD46-positive spermatozoa increased to a greater extent than that of PSA-negative/CD46-negative spermatozoa with an increase in the incubation time. Conclusion(s): Two-color fluorescence staining with FITC-PSA and the anti-CD46 monoclonal antibody may be useful for distinguishing between the acrosome reaction and acrosomal loss.

Induction of acrosome reaction in human spermatozoa accelerates the time of pronucleus formation of hamster oocytes after intracytoplasmic sperm injection

To assess the relationship between the incidence of acrosome reaction (AR) and the timing of pronucleus (PN) formation after intracytoplasmic sperm injection (ICSI). Prospective study. Infertility Research Center, Jeil Women's Hospital. Human semen obtained from fertile donors was prepared by one of the following methods: washing only (washed control); Percoll gradient; pentoxifylline; human follicular fluid (FF); pentoxifylline + FF; or platelet-activating factor (PAF) treatment. The AR of each group was assessed by fluorescein isothiocyanate-conjugated Pisum sativum agglutinin or Arachis hypogea agglutinin. Spermatozoa of washed control, pentoxifylline + FF, and PAF treated groups, with significantly higher AR rate than others, were injected into mature hamster oocytes. Spermatozoon-injected oocytes were cultured for 6, 9, 12, or 15 hours. Then they were stained with Toluidine blue for PN formation examination under a light microscope. Acrosome reaction rates of washed control, Percoll gradient, pentoxifylline, FF, pentoxifylline + FF, and PAF treated groups were 10.5% +/- 2.6%, 10.3% +/- 1.7%, 16.4% +/- 1.8%, 24.8% +/- 5.6%, 28.4% +/- 3.8%, and 33.3% +/- 5.2%, respectively. Pronuclear formation rate in washed control, pentoxifylline + FF, and PAF treated groups were 5.6% (3/54), 19.0% (11/58), and 18.9% (10/53) at 6 hours; 32.7% (18/55), 51.8% (29/56), and 57.4% (31/54) at 9 hours; 36.1% (22/61), 53.6% (30/56), and 50.0% (27/54) at 12 hours; and 47.2% (25/53), 64.8% (35/54), 53.6% (30/56) at 15 hours after ICSI. Pronuclear formation rate was significantly higher in pentoxifylline + FF, and PAF treated groups than that in the washed control group at 6 and 9 hours after ICSI. Pronuclear formation of oocytes takes place faster on those that were injected with acrosome-reacted spermatozoon than those injected with acrosome-intact spermatozoon. It could be concluded that induction of the AR of spermatozoa accelerates the time of PN formation and early development of the embryo in ICSI.

Factors affecting fertilization: endometrial placental protein 14 reduces the capacity of human spermatozoa to bind to the human zona pellucida

To examine whether placental protein 14 (PP14) may affect directly those sperm functions crucial to fertilization and early embryo development. In these prospective studies, we evaluated semen samples of fertile men incubated under capacitating conditions with and without PP14. Academic tertiary institution. Biologically active PP14 was purified from human midtrimester amniotic fluid by anion exchange and immunoaffinity chromatography. After separation of the motile fraction, spermatozoa were incubated for 30 minutes with or without PP14 (concentration range of 0.01 to 100 micrograms/mL), washed, and then aliquots were prepared for use in the different assays. Human sperm-zona pellucida (ZP) binding was assessed using the hemizona assay (HZA) in a 4-hour gametes coincubation period. Sperm motility parameters were evaluated using a computerized semen analyzer. The acrosome reaction (AR) was determined by fluorescein isothiocyanate-conjugated Pisum sativum agglutinin and indirect immunofluorescence. Sperm-ZP binding, sperm motility patterns, and AR. Preincubation of sperm (and not the hemizonae) with PP14 produced a significant and dose-dependent inhibition of binding in the HZA. Monoclonal antibodies generated against PP14 showed no direct effect in the HZA and partially neutralized the inhibitory activity of PP14 in the HZA. Insulin-like growth factor binding protein-1 (IGFBP-1), an endometrial stromal cell product, showed no effect in the HZA. Neither PP14 nor IGFBP-1 interfered with sperm motility parameters or the AR. Placental protein 14 produced a potent, fast, and dose-dependent inhibition of binding of human spermatozoa to the human ZP without affecting other prefertilization events (i.e., hyperactivated motility or AR). The detrimental effect on sperm-zona interaction seems to be specific for this endometrial epithelial protein (not observed with an endometrial stromal product) and may have fundamental bearance to the fertilization process thus providing a mechanism for endometriosis-related infertility.

Platelet activating factor enhances the acrosome reaction, fertilization in vitro by subzonal sperm injection and resulting embryonic development in the rabbit.

This study was conducted to investigate the effect of platelet activating factor (PAF) on the acrosome reaction and fertilizing capacity of spermatozoa, and development of the resulting embryos in the rabbit. Rabbit spermatozoa were exposed to PAF, lyso-PAF, or high ionic strength medium (HIS) prior to subzonal sperm injection (SUZI) into 326 mature oocytes, or morphological assessment of the acrosome reaction. The rates of fertilization and blastocyst formation were compared among the three treatment groups. Acrosome reaction was assessed by fluorescein isothiocyanate-conjugated Pisum sativum agglutinin (FITC-PSA) staining and electron microscopy. PAF-treated spermatozoa fertilized the oocytes at a significantly higher rate (56.1%) than did lyso-PAF-(36.8%, P < 0.01) or HIS- (38.2%, P < 0.05) treated spermatozoa. The embryos produced by PAF-treated spermatozoa showed significantly higher blastocyst formation rates (34.0%) than lyso-PAF- (8.6%, P < 0.05) or HIS-(8.8%, P < 0.05) treated spermatozoa. FITC-PSA staining demonstrated a significantly higher incidence of acrosome reaction in PAF-treated spermatozoa (45.8%) than in lyso-PAF- (28.0%, P < 0.01) or HIS- (34.9%, P < 0.01) treated spermatozoa. Acrosome reaction of PAF-treated spermatozoa was also confirmed by electron microscopy. PAF treatment of spermatozoa enhances fertilizing capacity for SUZI possibly by augmenting the acrosome reaction. Enhanced embryonic development was also found in the oocytes fertilized by SUZI of PAF-treated spermatozoa.

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The aim of this study was to assess extenders for cooling equine semen at 5°C and to be used in assisted reproductive technologies (ARTs). Four ejaculates were obtained from each of four stallions. Gel-free semen was diluted in three different extenders: (1) SMK, an opaque skim milk–based extender; (2) SMT, a skim milk (65%) and Tyrode medium (35%); and (3) BSAG, a clear extender containing 1% bovine serum albumin. Samples were packaged (10 mL; 50 × 106 sperm/mL) and stored in a cooling device at 5°C for 12 hours. Analyses were done at 0, 4, 8, and 12 hours after cooling. Semen was analyzed for sperm motility characteristics using a computer-assisted sperm analysis, for plasma membrane and acrosome integrity and mitochondrial membrane potential, using fluorescent probes (propidium iodide, Hoechst 33342, fluorescein isothiocyanate–conjugated Pisum sativum agglutinin, and 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolyl carbocyanine iodide [JC-1]). Morphology was evaluated with differential interference contrast microscopy and sperm chromatin integrity by the toluidine blue technique. Data were analyzed by analysis of variance and by Tukey test, with time as a repeated measure (SAS, 1998), when P < .05 was significant. In general, milk-based extenders (SMT and SMK) showed improved maintenance of semen quality compared with BSAG. Finally, the addition of skim milk to equine semen extender for cooling at 5°C for 12 hours seems to play a crucial role in sperm preservation. Although, optically clearer extenders are desired for use in ARTs, such as sperm sexing, the milk-free extender (BSAG) is less efficient for cooled-stored equine semen.