Amsp3 may act upstream of Amdnmt3 in female caste differentiation in the honeybee (Apis mellifera).

In honey bees, the process of producing two female castes, including queens and workers, is nutritionally controlled by differential feeding royal jelly to newly emerged larvae. Although they have almost identical genetic blueprints, these castes show striking differences in their morphologies, longevities and reproductive capabilities. DNA methyltransferase 3 (Amdnmt3) gene is involved in the regulatory network for honeybee caste differentiation. Due to the role of two zinc fingers containing transcription factors, SP1 and SP3 in controlling mammalian Dnmts, this study aimed to determine a similar interaction of SPs with Amdnmt3 in the honeybee. We confirmed that the promoter region of Amdnmt3 contained multiple predicted SP1/SP3 binding sites and then investigated the role of AmSP3 in queen-worker differentiation network. We observed that the expression level of Amsp3 was significantly higher in worker larvae than that in queen larvae at 48 h, 84 h and 120 h. Knockdown of Amsp3 expression by RNAi in worker larvae significantly reduced the expression level of Amdnmt3 and caused morphological changes in adult bees towards a queen-like phenotype. However, the expression levels of Amsp3 and Amdnmt3 were repressed by juvenile hormone (JH). Our results suggest that AmSP3 is an important part of the queen-worker differentiation network and supports the role of Amdnmt3 in determining the phenotypic outcome of developing larvae.

Sensory feedback - Dependent neural de-orchestration: The effect of altered sensory feedback on Musician's Dystonia.

PURPOSE: Musician's dystonia (MD) is a task-specific movement disorder related to extensive expert music performance training. Similar to other forms of focal dystonia, MD involves sensory deficits and abnormal patterns of sensorimotor integration. The present study investigated the impaired cortical sensorimotor network of pianists who suffer from MD by employing altered auditory and tactile feedback during scale playing with multichannel EEG. METHODS: 9 healthy professional pianists and 9 professional pianists suffering from right hand MD participated in an experiment that required repeated scale playing on a MIDI piano under altered sensory feedback while EEG was measured. RESULTS: The comparison of EEG data in healthy pianists and pianists suffering from MD revealed a higher degree of inter-regional phase synchronisation between the frontal and parietal regions and between the temporal and central regions in the patient group and in conditions that are relevant to the long-trained auditory-motor coupling (normal auditory feedback and complete deprivation of auditory feedback), but such abnormalities decreased in conditions with delayed auditory feedback and altered tactile feedback. CONCLUSIONS: These findings support the hypothesis that the impaired sensorimotor integration of MD patients is specific to the type of overtrained task that the patients were trained for and can be modified with altered sensory feedback.

Detection of indoxyl sulfate levels in dogs and cats suffering from naturally occurring kidney diseases.

Indoxyl sulfate (IS), a protein-bound uraemic toxin, has been found to accumulate in the serum of people with renal diseases and is associated with free radical induction, nephrotoxicity cardiovascular toxicity, and osteoblast cytotoxicity. Although IS has been studied in humans and in experimental models, the role of IS in dogs and cats with kidney disease has not been investigated. A high performance liquid chromatography system was applied to detect plasma IS concentrations in non-azotaemic animals (63 dogs, 16 cats) and in animals with renal azotaemia (66 dogs, 69 cats). The IS levels of azotaemic animals were significantly higher (P <0.01) than those of non-azotaemic animals (median [IQR] 20.4 (9.5) mg/L vs. 7.2 (8.8) mg/L for dogs; median [IQR] 21 (18.9) mg/L vs. 14.8 (12.3) mg/L for cats). The IS level was significantly correlated with blood urea nitrogen, serum creatinine and phosphate concentrations. Dogs with acute kidney injury had significantly higher IS levels (P <0.01) than those with chronic kidney diseases (CKD) (median [IQR] 57.7 (40.8) mg/L vs. 17.7 (25.1) mg/L). When CKD was graded using the International Renal Interest Society (IRIS) staging system, IS levels were correlated with CKD severity in both dogs and cats. The IS concentration is directly related to loss of renal function. Further studies are necessary to determine whether measurement of IS provides any additional diagnostic or prognostic information in dogs and cats with kidney disease.

Expression of matrix metalloproteinases (MMP-2, MMP-9) and their inhibitors (TIMP-1, TIMP-2) in canine testis, epididymis and semen.

This study aims to investigate the role of matrix metalloproteinases (MMPs) in determining semen quality and to evaluate the expression and cellular localization of MMP-2, MMP-9, tissue inhibitor of metalloproteinase-1 (TIMP-1) and TIMP-2 in the testes, epididymis and ejaculated spermatozoa. Gelatinase activities between normal (n = 21) and abnormal (n = 25) semen samples showed a significant, sixfold increase in proMMP-2 and MMP-2 activity in high than low sperm concentration samples (p < 0.001). ProMMP-9 and MMP-9 levels were significantly elevated in samples with low sperm counts compared to those with high sperm density (p < 0.001). High levels of proMMP-2 and MMP-2 were associated with high sperm motility (≥70%, p < 0.001). Sperm-rich fraction showed significantly (eight-fold) higher proMMP-9 enzymatic activity compared with prostatic fraction. The mRNA expressions of MMP-2, MMP-9, TIMP-1 and TIMP-2 were confirmed in testicular and epididymal tissues. Immunohistochemical staining illustrated the MMP-2-specific strong immunoreactivity in the head of mature spermatids during spermatogenesis, whereas MMP-9, TIMP-1 and TIMP-2 were absent in these cells. Matrix metalloproteinase-9 immunoreactivity was observed in the spermatocyte and round spermatid, whereas TIMP-1 was only exhibited in the residual bodies. Immunolabeling of epididymal and ejaculated sperm demonstrated MMP-2 localization along acrosomal region of sperm, while MMP-9, TIMP-1 and TIMP-2 localization was merely limited to the flagella. In conclusion, spermatozoa initially acquire MMP-2 during their formation at testicular level, and the presence of this protein persists through the epididymal transit and up to ejaculate. The enzymatic activity of MMP-2 and MMP-9 may serve as an alternative biomarker in determining semen quality.

The combinatorial effect of different Equex STM paste concentrations, cryoprotectants and the straw-freezing methods on the post-thaw boar semen quality.

This study was to evaluate the combinatorial effect (14 treatments, A-N) of different Equex STM paste concentrations, cryoprotectants and the straw-freezing method on the post-thaw boar semen quality. Two ejaculates were collected from each of nine boars (three boars from each of three breeds). Semen was diluted in extenders with different concentrations of Equex STM paste and different cryoprotectants [glycerol or dimethylacetamide (DMA)] before cryopreserving via liquid nitrogen or dry ice. Motility, viability, percentage of spermatozoa with intense acrosomal staining and with normal morphology of post-thaw sperm were evaluated. The qualities of thawed semen were best preserved in treatment H (extender with 0.5% Equex STM paste and 5% glycerol and freezing by dry ice) and were worst in treatment B (extender with 0% Equex STM paste and 5% DMA and freezing by dry ice). Significant difference (p < 0.05) was present in post-thawed sperm motility (63% vs 27%), sperm viability (70% vs 33%) and sperm acrosomal integrity rate (68% vs 29%) between treatments H and B. However, sperm proportion with normal morphology showed no significant difference among treatments (66% vs 66%; p > 0.05). Moreover, statistical analysis suggests that no significant difference was present in semen quality among breed or individual donors (p > 0.05). These findings suggest that Equex STM paste improved the cryosurvival efficiency of boar sperm, and the favourable straw-freezing method changes between glycerol and DMA.

Antikinectin autoantibody in Behçet's disease and several other autoimmune connective tissue diseases.

OBJECTIVE: Antikinectin autoantibody has recently been identified as a potential biomarker in Behçet's disease (BD). Here, we established an enzyme-linked immunosorbent assay (ELISA) and an indirect immunofluorescent assay (IFA) for detecting this antibody. The clinical significance of antikinectin was investigated. METHODS: Partial or full-length cloning for human kinectin in prokaryotic or eukaryotic system was carried out. Three fragments covered kinectin coding sequence were used to establish ELISA. Full-length kinectin overexpressed HepG2 cells were used as a substrate for IFA. Serum samples from BD (n = 46), systemic lupus erythematosus SLE, n = 16), rheumatoid arthritis (RA, n = 160, ankylosing spondylitis (AS, n = 14), primary Sjörgen syndrome (pSS, n = 12), mixed connective tissue disease (MCTD, n = 8), and healthy donors (n = 51) were examined. RESULTS: Good measurement consistency between IFA and ELISA (p < 0.001) and previous immunoprecipitation assay (p = 0.011) was found. Antikinectin was found not only in 32.6% (IFA) to 41.3% (ELISA) BD patients but was also identified in pSS, SLE, MCTD, and RA with prevalence ranging from 12.5% to 25%. Nevertheless, the titer of antikinectin (ELISA) is statistically higher in BD compared to other autoimmune connective tissue diseases (p = 0.0286). Antikinectin was found exclusively among complete form of BD (p < 0.001), but there was no correlation with specific clinical manifestations. CONCLUSIONS: We confirmed the previous observation that antikinectin is related to BD, especially in the complete form of disease. The specificity and predictive values are moderate.

Study on the species-specificity of Isospora michaelbakeri by experimental infection.

Isospora michaelbakeri is one of the Isospora species most commonly found in the wild field, which can cause severe infection and mortality in young sparrows. In this study, we selected I. michaelbakeri (Chung Hsing strain) as a pathogen to orally inoculate russet sparrows (Passer rutilans), spotted munia (Lonchura punctulata), canary (Serinus canaria), Java sparrows (Padda oryzivora), chicken (Gallus domesticus), ducks (Anas platyrhynchos) and BALB/c mice. The results indicated that I. michaelbakeri infected only russet sparrows. Infected sparrows displayed lethargy, muscular weakness and fluffy feathers, followed by rapid death. Liver and spleen enlargement was seen in the infected birds. Schizonts were identified in thin smears from the venous blood, enlarged livers and spleens. Histopathological examination revealed schizonts and merozoites from the liver and spleen of infected russet sparrows, but not from other species experimentally inoculated with I. michaelbakeri in the present study.

Annual variation in semen characteristics of pigeons (Columba livia).

The purpose of this study was to evaluate annual semen characteristics of pigeons (Columba livia). Ten selected male pigeons, aged 2 to 5 yr were housed under natural environmental conditions, and semen collection was conducted via a digital massage twice weekly throughout the year. The success rate of semen collection in a total of 920 attempts was 40% (371/920) over the whole year. The highest success rate was 69% (55/80) in March followed by 66% (53/80) in November, whereas the lowest rates were in August (13%,10/80) and September (13%, 8/60) (P < 0.01). Volume of the ejaculate averaged 11.0 +/- 0.9 microL (mean +/- SEM). The greatest volume was obtained in November (13.5 +/- 1.0 microL), whereas the least was obtained in August (7.0 +/- 1.0 microL). The average sperm motility was 72 +/- 2% of all ejaculates, of which the highest motility (82 +/- 2%) was observed in March, whereas the least motility (48 +/- 3%) was in August. Sperm viability and sperm motility were positively correlated (r = 0.91; P < 0.01). Maximum sperm concentration was 4.9 +/- 0.4 x 10(9) sperm/mL noted in March, whereas the minimum was 3.8 +/- 0.2 x 10(9) sperm/mL observed in October. Donors generally exhibited susceptible (54%) or dull submission (43%), whereas resistance to handling was rarely observed (3%). During collections, a red (47%) or pink (44%) cloacal membrane was often observed, whereas during only 9% of the collections, the cloacal membrane was pale. When the ambient temperature decreased below 15 C, semen could not be obtained (0/80). A high amount of semen (>10 microL) was obtained when the temperature ranged between 19 and 24 C. Optimal sperm motility (approximately 80%) and viability (>85%) was observed when the temperature was between 18 and 24 C. At temperatures greater than 28 C, sperm motility and viability decreased. Sperm concentration was not significantly influenced by temperature fluctuations. In summary, annual variation in semen characteristics exhibited two peaks per year with mean motility and viability reaching peak annual values in March and November. Both of these months had mean ambient temperatures between 19 and 24 C, a range associated with maximal ejaculatory volumes.

Effect of sperm diluents on the acrosome reaction in canine sperm.

In this study we investigated the influence of sperm diluting media and temperature on the incidence of the acrosome reaction in dog sperm. Ejaculates were collected from 5 dogs, diluted with six different media and then incubated at 37 degrees C and 20 degrees C. Fluorescein isothiocynate conjugated peanut agglutinin (FITC-PNA) and ethidium homodimer as a vital stain were used in combination to determine the acrosomal status of viable spermatozoa, the technique was validated using electron microscopy. The outer acrosomal membrane of dog spermatozoa was shown to be the specific binding site for FITC-PNA. After 6 h of incubation, ejaculates diluted in media with a high Ca2+ concentration showed a significantly higher percentage (means +/- SD) of acrosome reacted spermatozoa [64 +/- 7 and 58 +/- 9 in sperm capacitation medium with (SP-TALP-1) and without BSA (SP-TALP-2), respectively] than those diluted in media with a low Ca2+ concentration [36 +/- 5, 39 +/- 4, 18 +/- 2 and 20 +/- 4 in Canine Capacitation Medium (CCM), Egg Yolk Tris dog semen extender (EXT-1), Modified Egg Yolk Tris extender (EXT-2) and Modified CCM (MCCM), respectively]. The increase in the percentage of acrosome reaction (AR) was slower at 20 degrees C than at 37 degrees C. In addition, the percentage of viable acrosome reacted spermatozoa increased significantly from 19 +/- 5 and 22 +/- 3 in non-bound sperm to 27 +/- 4 and 30 +/- 6 in zona pellucida bound sperm (diluted in EXT-2 and MCCM, respectively). We conclude that the composition of the spermatozoa diluent has a marked effect on the incidence of the acrosome reaction. Therefore, both the media used to dilute dog sperm and the temperature at which the spermatozoa are handled are important factors to consider when processing spermatozoa for artificial insemination, IVF procedures or preservation.

Detection of calcium ionophore induced membrane changes in dog sperm as a simple method to predict the cryopreservability of dog semen.

The sensitivity of dog sperm cells for extracellular Ca(2+)/Ca(2+)-ionophore challenge was compared to the detrimental effects of an optimized freeze/thawing protocol. Three sperm-rich fractions of ejaculates from 9 dogs were obtained, and one aliquot of each ejaculate was washed in a modified Tyrode's medium (HBT containing 0.1 mM Ca(2+)), without (control sample) and with 2.5 microM Ca(2+)-ionophore (induced sample) and incubated for 60 min at 38 degrees C in humidified atmosphere. Another aliquot from the same semen fractions was diluted, washed in a Tris buffer, and packed into 0.5-ml straws with a Tris buffer containing 7.5 vol % glycerol. The samples were stored for 1 week in liquid nitrogen after a computer-driven three-step freeze protocol and subsequently thawed for 50 sec in a 37 degrees C water bath and reconstituted into HBT. The acrosome integrity was determined using fluorescein-conjugated peanut agglutinin (PNA-FITC) as an acrosomal marker, while the vitality of the sperm cells was simultaneously assessed with the membrane impermeable DNA supravital stain ethidium homodimer 1 (EthD-1) using fluorescence microscopy and flow cytometry. The motility of frozen/thawed sperm samples was evaluated by microscopic as well as computerized motility analyses. Remarkably, the percentage sperm cells that underwent acrosome reactions induced by Ca(2+)-ionophore correlated very positively (r = 0.93) with the amount of acrosome damage observed in cryopreserved sperm samples. Furthermore, the degree of cellular damage induced by Ca(2+)-ionophore treatment correlated very negatively (r = -0.99) with the relative amount of sperm cells that remained motile after cryopreservation. Such clear correlations between Ca(2+)-ionophore induced acrosome reaction and motility parameters for frozen/thawed dog sperm cells were not found, suggesting that the generation of acrosome leakage and sperm immotility are two independent detrimental processes occurring during cryopreservation. From these results it can be concluded that Ca(2+)-ionophore treatment followed by simultaneous determination PNA-FITC and EthD-1 staining can be used to predict the cryopreservability of ejaculates from individual dogs used as donors.

Exposure of progesterone receptors on the plasma membranes of stallion spermatozoa as a parameter for prediction of fertility.

Subfertility in stallions is attributed to the inability of spermatozoa to undergo the acrosome reaction in response to progesterone. In the present study, it was assessed whether there is a correlation between stallion fertility, defined on the basis of first cycle foaling rate and first cycle 'non-return rate', and the proportion of spermatozoa with exposed progesterone receptors on their plasma membranes. Semen from Dutch Warmblood (n=10) and Friesian (n=4) stallions was analysed. Progesterone 3-(o-carboxymethyl) oxime-BSA coupled with fluorescein isothiocyanate was used as a progesterone receptor probe and ethidium homodimer was used as a supravital stain. A high correlation was observed between the proportion of spermatozoa with exposed progesterone receptors and stallion fertility (r > 0.70; P < 0.01). This result indicates that exposure of progesterone receptors is a potential parameter for predicting stallion fertility.

Progesterone-induced acrosome reaction in stallion spermatozoa is mediated by a plasma membrane progesterone receptor.

The aim of the present study was to investigate whether the induction of stallion sperm acrosome reaction (AR) by progesterone is mediated by binding of progesterone to a receptor on the sperm plasma membrane or to an intracellular progesterone receptor. Progesterone-BSA conjugate labeled with fluorescein isothiocyanate (P-BSA-FITC) in combination with a vital stain, ethidium homodimer, was applied to visualize the presence of the progesterone receptor on living spermatozoa. Alternatively, an indirect immunofluorescence technique employing a monoclonal antibody (C-262) against human intracellular progesterone receptor was conducted to validate the presence of the progesterone receptor. Immunogold labeling techniques enabled ultrastructural localization of P-BSA-FITC or C-262 with transmission electron microscopy. The dynamic changes in labeling patterns were monitored for sperm cells, using fluorescence microscopy and flow cytometry during a 5-h capacitation period. An increasing number of viable cells showed affinity for P-BSA-FITC or C-262 at the acrosomal plasma membrane region of the sperm head, while a decreasing number of viable cells were not labeled. In contrast, almost all deteriorated cells were labeled in the cytosol of the postequatorial region of the sperm head. Incubation with P-BSA-FITC resulted in the induction of AR but to a lesser extent than that for sperm incubated with free progesterone. Therefore, coupling of progesterone to its receptor on the sperm plasma membrane appears to be an important step in the induction of the AR.

Progesterone in mare follicular fluid induces the acrosome reaction in stallion spermatozoa and enhances in vitro binding to the zona pellucida.

The aim of this study was to investigate whether mare follicular fluid (FF) induces the acrosome reaction (AR) in stallion spermatozoa and, if so, to identify the component in FF responsible for it. Furthermore, the effect of this component on sperm-zona binding and the subsequent AR was studied. Pooled FF, aspirated from the preovulatory follicles of mares in oestrous, was used and aliquots of the fluid were treated with charcoal to remove steroids (CFF). Charcoal treatment reduced the progesterone concentration in FF from 153 to < 2 ng/mL. Spermatozoa from fertile stallions collected by a swim-up procedure were preincubated in modified Tyrode's medium for 5 h and then incubated for 30 min at 37 degrees C with either (1) 50% FF + 50% CFF, (2) 50% FF + 50% CFF + 150 ng/mL progesterone, (3) 50% CFF + 150 ng/mL progesterone, (4)150 ng/mL progesterone or (5) modified Tyrode's medium alone. The sperm-hemizona assay was applied: (a) to compare the number of spermatozoa bound to a hemizona in the presence and absence of 1.5, 15 or 150 ng/mL progesterone after 1 h co-incubation of spermatozoa and hemizonae, (b) to compare the incidence of the AR in sperm-hemizona complexes incubated for 1 h in the presence and absence of 1 microgram/mL progesterone. Both spermatozoa in suspension and bound to a hemizona were treated with the supravital dye Ethidium homodimer and fixed. Their plasma membranes were permeabilized, and the outer acrosomal membranes were labelled with FITC-PNA. Viable spermatozoa without the outer acrosomal membrane were considered as physiologically acrosome-reacted. Results showed that (1) FF induced a higher percentage of AR than did CFF or modified Tyrode's medium, (2) addition of 150 ng/mL progesterone to CFF restored 77% of the AR-inducing activity and (3) CFF and modified Tyrode's medium both induced the AR to a similar extent when supplemented with 150 ng/mL progesterone. Neither FF nor progesterone treatment affected sperm viability severely. The number of spermatozoa bound to a hemizona in the presence of 15 and 150 ng/mL progesterone was significantly higher (p < 0.05) than the number of spermatozoa bound in the absence of progesterone. A higher incidence of the AR was found in sperm-hemizona complexes incubated in the presence of progesterone (55.6 +/- 3.4% vs. 27.1 +/- 4.3%, in the presence and absence of progesterone, respectively) (n = 15, p < 0.05). It is concluded that mare FF can induce the AR in stallion spermatozoa. Progesterone is the physiological component responsible for this AR-inducing capacity. Progesterone enhances sperm-zona binding activity and exerts an additive effect on the zona-induced AR.

Acrosome-intact boar spermatozoa initiate binding to the homologous zona pellucida in vitro.

The acrosome reaction is a prerequisite for zona pellucida penetration by mammalian spermatozoa. In some species, the sperm undergo the acrosome reaction before binding to the zona pellucida, and in other species only acrosome-intact sperm can initiate binding to the zona. In the present investigation, we addressed the question whether acrosome-intact or acrosome-reacted boar sperm initiate binding to the pig zona pellucida by studying the acrosomal status of sperm bound to zonae pellucidae. Our approach was to vary the percentage of acrosome-intact sperm in suspension by long preincubation before incubation with hemizonae for 1 min. We hypothesized that if only acrosome-intact sperm are able to initiate binding to the zona pellucida, the majority of the sperm on the zona surface would be acrosome-intact regardless of the percentage of acrosome-reacted sperm in suspension. Fluorescein isothiocyanate-conjugated peanut agglutinin (Arachis hypogaea; FITC-PNA) in combination with optical sectioning by confocal laser-scanning microscopy was used to study the acrosomal status of sperm bound to the hemizona. Electron microscope studies showed that the FITC-PNA binding site is mainly limited to the outer acrosomal membrane of boar sperm, thus validating the use of FITC-PNA as an accurate probe for studying boar sperm acrosome reaction. Over 90% of the sperm bound to a hemizona were acrosome-intact irrespective of whether the majority of sperm in the suspension were acrosome-intact, acrosome-reacting, or acrosome-reacted. There was a significant difference (Kruskal-Wallis test, p < 0.05) in the mean +/- SEM number of sperm bound to the outer side, inner side, and edge of a hemizona (48 +/- 8, 14 +/- 3, and 7 +/- 2; n = 58; respectively). The acrosomal status of sperm bound to the various surfaces of hemizonae was similar. Taking the respective zona pellucida surface area into consideration, it was calculated that an average of 1.9 +/- 0.3, 1.0 +/- 0.2, and 1.5 +/- 0.3 spermatozoa were bound per 1000 microm2 of outer side, inner side, and edge of a hemizona, respectively (mean +/- SEM, n = 38). These observations indicate that acrosome-intact boar spermatozoa initiate binding to the pig zona pellucida. A gradient of sperm binding sites also exists, decreasing from the outside to the inside of the zona pellucida.

Induction of acrosome reaction in dog sperm by calcium ionophore.

The sensitivity of the plasma membrane to calcium ionophore (A23187) challenge was studied in dog sperm using fluorescein lectin staining for the assessment of acrosomal status and viability. Second fraction ejaculates from 5 dogs were washed, resuspended in Ca(2+)-free (EDTA-treated), 50, 100, 500, 1000 and 2000 microM/l Ca(2+)-containing Sp-TALP medium and induced with 50, 250, 500, 1000, 2500 and 5000 nM/l calcium ionophore. Samples were collected from each aliquot after 30 and 60 min of induction to assess the percentage of acrosome reacted sperm cells (AR rate), viability and motility by fluorescein isothiocyanate conjugated peanut agglutinin (FITC-PNA) and ethidium-homodimer combined staining. On each slide, 200 sperm cells were assessed under epifluorescence microscope (x 1250) in a blind manner. The response to ionophore challenge (AR rate, viability, motility) varied with Ca2+ and ionophore concentration in the suspension. A significantly higher AR rate was detected in samples containing 100, 500, 1000 and 2000 microM/L Ca2+ (> 40%) than in that containing 50 microM/L. Acrosome reaction could not be successfully induced in the EDTA-treated sample and in any of the aliquots in which 50, 250 and 500 nM/L ionophore concentrations were used for induction. Motility decreased drastically in all of the treated samples and stopped in that sample where as significant AR rate could be detected. Viability remained high (> 75%) during the incubation and did not differ significantly in the treated and the control groups.

Use of peanut agglutinin to assess the acrosomal status and the zona pellucida-induced acrosome reaction in stallion spermatozoa.

Peanut agglutinin (PNA) was used to assess the sperm acrosomal status and the acrosome reaction during gamete interaction in the equine species. PNA exclusively binds to the outer acrosomal membrane of stallion spermatozoa, as was established by transmission electron microscopy. Fluorescein isothiocyanate-PNA (FITC-PNA) labeling was used to monitor sperm acrosomal changes during a prolonged incubation period of 24 hours and during a 2-hours incubation in the presence of 5 microM calcium ionophore A23187. In addition, after a 4-hours preincubation in SP-TALP medium, sperm samples were incubated with matching hemizonae for 1 minute (onset binding) followed by a 60-minute incubation (1-hour binding) of the sperm-hemizona complexes in sperm-free medium to assess the acrosomal status of the bound spermatozoa. For acrosome assessment, spermatozoa and washed sperm-hemizona complexes were air dried onto microscope slides, fixed, permeabilized in ethanol, stained with FITC-PNA, and counterstained with the DNA dye ethidium homodimer. Both zona-bound and non-bound spermatozoa showed similar staining patterns. Acrosome-intact spermatozoa displayed intensively green fluorescence over the acrosomal cap, whereas reacting spermatozoa showed a patchy disrupted image of fluorescence. Sperm cells that completed the acrosome reaction were principally stained on the equatorial segment or not stained at all. During prolonged incubation and during the calcium ionophore treatment, the proportion of spermatozoa with an acrosomal modification (reacting) and a complete breakdown of the acrosome (reacted) increased noticeably. Significant induction of the acrosome reaction was observed within 60 minutes of sperm-zona contact (P < 0.001). In conclusion, a rapid and reliable assessment of the acrosomal status and the incidence of the acrosome reaction of stallion spermatozoa at the zona surface were demonstrated in this study.