Behavior of bull spermatozoa in bovine uterine tube epithelial cell co-culture: an in vitro model for studying the cell interactions of reproduction

Freshly ejaculated bull semen was centrifuged and spermatozoa were resuspended in modified sperm TALP. Bovine uterine tube epithelial cell monolayers (BUTC) were obtained from cows in the periovulatory phase of estrus. In Experiment 1, sperm aliquots were assigned to culture wells containing either BUTC, BUTC-conditioned TALP, or control TALP. Sperm heads attached to the monolayers within 1 h of co-culture. Attached spermatozoa showed vigorous tail motion. At 5, 8 and 11 h of incubation at 39 degrees C, the percentage of unattached sperm cells with intact acrosome membranes and percentage of motility of these cells was measured. Sperm-BUTC co-cultures were also fixed in situ for electron microscopy. Unattached spermatozoa in co-culture had more (P<0.05) acrosomal membrane loss, showed hyperactive motion and had an overall decrease in motility as compared to sperm cells in control or conditioned medium. Evaluation by electron microscopy showed BUTC attached spermatozoa to behave in the co-culture system similar to reports for spermatozoa found in uterine tubes in vivo. Microvilli of the BUTC appeared to actively entrap the spermatozoa. Mucus-type granules could be seen on acrosomal regions and vesiculation of acrosomal membranes was seen in some cells. In Experiment 2, 43% of the 12 x 10(6) sperm cells added to 2-cm(2) BUTC bound within 4 h of co-culture. By 7 h of co-culture 19% of the previously bound sperm cells had been released from the BUTC. Released cells had limited motility and were mostly dead (73%). Sperm cells remaining on the monolayer at 7 h showed vigorous tail motion and were gradually released from the BUTC over 48 h. Spermatozoa in co-culture interacted with the BUTC in a manner much like that seen in vivo, and sperm capacitation changes were stimulated by this interaction.     

L-arginine promotes capacitation and acrosome reaction in cryopreserved bovine spermatozoa

Sperm capacitation and acrosome reaction are essential for fertilization and they are considered as part of an oxidative process involving superoxide and hydrogen peroxide. In human spermatozoa, the amino acid L-arginine is a substrate for the nitric oxide synthase (NOS) producing nitric oxide (NO*), a reactive molecule that participates in capacitation as well as in acrosome reaction. L-arginine plays an important role in the physiology of spermatozoa and has been shown to enhance their metabolism and maintain their motility. Moreover, L-arginine has a protective effect on spermatozoa against the sperm plasma membrane lipid peroxidation. In this paper, we have presented, for the first time, the effect of L-arginine on cryopreserved bovine sperm capacitation and acrosome reaction and the possible participation of NOS in both processes. Frozen-thawed bovine spermatozoa have been incubated in TALP medium with different concentrations of L-arginine and the percentages of capacitated and acrosome reacted spermatozoa have been determined. L-arginine induced both capacitation and acrosome reaction. NO* produced by L-arginine has been inhibited or inactivated using NOS inhibitors or NO* scavengers in the incubation medium, respectively. Thus, the effect of NOS inhibitors and NO* scavengers in capacitated and non-capacitated spermatozoa treated with L-arginine has also been monitored. The data presented suggest the participation of NO*, produced by a sperm NOS, in cryopreseved bovine sperm capacitation and acrosome reaction.     

Capacitation and acrosomal exocytosis are enhanced by incubation of stallion spermatozoa in a commercial semen extender

Preserved stallion semen often has decreased spermatozoal motility and fertility that can vary significantly between individual stallions. It is not known whether the medium used for extending equine sperm contributes to these decreases by inducing premature capacitation during storage. If spermatozoa undergo capacitation or acrosome reaction prior to insemination, this could result in a diminished capacity to penetrate the cumulus mass and fertilize the egg. We hypothesized that skim milk-based semen extenders, similar to those used in cooled storage, stabilize sperm membranes and prolong sperm motility and longevity. However, this could decrease the efficiency of sperm to undergo subsequent capacitation in vivo. This study was designed to evaluate the effects from two media on sperm function. Spermatozoal motility was analyzed, intracellular calcium was measured, and the ability of sperm to undergo acrosome reaction was compared after incubation in a skim milk extender (SME) and Tyrode's medium containing albumin, lactate, and pyruvate (TALP) at 37 degrees C. Results suggest that the SME facilitated capacitation as detected by an increase in both intracellular calcium and acrosome reactions, and a decrease in motility, as compared to TALP. Our data support a shortened functional lifespan for equine sperm in skim milk extender, which indicates that further refinements in cooled semen preservation are required to improve fertility of transported equine semen.     

The effect of antioxidants on motility, viability, acrosome integrity and DNA integrity of frozen-thawed epididymal cat spermatozoa

Antioxidants partially ameliorated the negative effects of reactive oxygen species (ROS) produced during cryopreservation. The objective of the present study was to investigate the effect of cysteine and a water-soluble vitamin E analogue on the quality of frozen-thawed epididymal cat spermatozoa. Epididymal spermatozoa were collected from eight male cats and divided into three aliquots; these were resuspended with a tris egg yolk extender I (EE-I), or the same extender supplemented with 5mM dl-cysteine (EE-C) or with 5mM of a water-soluble vitamin E analogue (EE-Ve). Prior to the freezing step, sperm suspensions were added to the extender with Equex STM paste (EE-II). Sperm motility, progressive motility, membrane integrity, and acrosome status were evaluated at collection, after cooling, and at 0, 2, 4, and 6h post-thaw. Sperm DNA integrity was evaluated at 0 and 6h post-thaw. Relative to the control group, supplementation with vitamin E improved (P<0.05) post-thaw motility (69.4+/-5.6%), progressive motility (3.9+/-0.3), and membrane integrity (65.1+/-8.1%) immediately after thawing, whereas cysteine supplementation improved (P<0.05) post-thaw motility after 2h of incubation (53.8+/-12.2%) and DNA integrity after 6h (84.1+/-4.4%). However, neither antioxidant significantly increased the acrosome integrity of frozen-thawed spermatozoa. In conclusion, cysteine or vitamin E supplementation of tris egg yolk extender improved motility, progressive motility and integrity of the sperm membrane and DNA of frozen-thawed epididymal cat spermatozoa.     

Tyrphostin-A47 inhibitable tyrosine phosphorylation of flagellar proteins is associated with distinct alteration of motility pattern in hamster spermatozoa

To acquire fertilizing potential, mammalian spermatozoa must undergo capacitation and acrosome reaction. Our earlier work showed that pentoxifylline (0.45 mM), a sperm motility stimulant, induced an early onset of hamster sperm capacitation associated with tyrosine phosphorylation of 45-80 kDa proteins, localized to the mid-piece of the sperm tail. To assess the role of protein tyrosine phosphorylation in sperm capacitation, we used tyrphostin-A47 (TP-47), a specific protein tyrosine kinase inhibitor. The dose-dependent (0.1-0.5 mM) inhibition of tyrosine phosphorylation by TP-47 was associated with inhibition of hyperactivated motility and 0.5 mM TP-47-treated spermatozoa exhibited a distinct circular motility pattern. This was accompanied by hypo-tyrosine phosphorylation of 45-60 kDa proteins, localized to the principal piece of the intact-sperm and the outer dense fiber-like structures in detergent treated-sperm. Sperm kinematic analysis (by CASA) of spermatozoa, exhibiting circular motility (at 1st hr), showed lower values of straight line velocity, curvilinear velocity and average path velocity, compared to untreated controls. Other TP-47 analogues, tyrphostin-AG1478 and -AG1296, had no effect either on kinematic parameters or sperm protein tyrosine phosphorylation. These studies indicate that TP-47-induced circular motility of spermatozoa is compound-specific and that the tyrosine phosphorylation status of 45-60 kDa flagellum-localized proteins could be key regulators of sperm flagellar bending pattern, associated with the hyperactivation of hamster spermatozoa.     

Evaluation of in vitro capacitation of stallion spermatozoa

The primary aim of this study was to establish a flow cytometric technique for determining the capacitation status of stallion spermatozoa. To this end, a flow cytometric technique that demonstrates changes in plasma membrane fluidity; namely, merocyanine 540 staining, was compared with the more conventional Ca(2+)-dependent fluorescence microscopic technique, chlortetracycline (CTC) staining, for assessing capacitation status. In addition, the effect of bicarbonate/CO(2) on the progress of capacitation and the acrosome reaction (AR) and on temporal changes in sperm motility, with particular regard to hyperactivation, was analyzed. For the study, fresh semen was washed and then incubated for 5 h in bicarbonate-containing or bicarbonate-free medium, with or without Ca(2+) ionophore to induce the AR, and at intervals during incubation aliquots were taken and analyzed for capacitation and acrosome status. The AR was assessed using both the CTC and fluorescein isothiocyanate-peanut agglutinin (FITC-PNA) staining techniques with similar results. In brief, it was found that merocyanine 540 detects capacitation-related changes much earlier than CTC does (0.5 h versus approximately 3 h), and that flow cytometry for evaluation of capacitation and AR was a quicker (10 sec per sample) and more accurate (10,000 cells counted) technique than fluorescence microscopy. Furthermore, it was observed that Ca(2+) ionophore could not induce the AR in the absence of bicarbonate, but that the ionophore synergized the bicarbonate-mediated induction of the AR as detected by CTC (although it was not significant when evaluated using FITC-PNA). The percentage of hyperactive sperm in each sample was not affected by time of incubation under the experimental conditions studied. In conclusion, merocyanine 540 staining is a better method than CTC staining for evaluating the early events of capacitation for stallion spermatozoa incubated in vitro. Furthermore, bicarbonate sperm activation clearly plays a vital role in the induction of the AR in stallion spermatozoa.     

Chlortetracycline staining patterns of frozen-thawed bull spermatozoa treated with beta-cyclodextrins, dibutyryl cAMP and progesterone

Efforts to achieve complete chemical definition of media used for in vitro capacitation of bovine spermatozoa including removal of heparin purified from porcine intestinal mucosa are presented. Fluorescent staining with chlortetracycline (CTC), known to reflect changes coincident with sperm capacitation in certain species, was studied following treatments of frozen-thawed bull spermatozoa with beta-cyclodextrins, dibutyryl cAMP (dbcAMP) and progesterone in comparison with heparin. The CTC staining patterns (F, B and AR) were confirmed to correlate with known conditions that effectively prepare cryopreserved bull spermatozoa for fertilisation in vitro. In the absence of glucose, the routinely employed heparin-containing capacitating medium caused an increase in spermatozoa displaying the AR pattern. Both progesterone (100 microM) and dbcAMP (0.01-0.1 mM) were able to increase the proportion of B pattern stained sperm cells more than after exposure to control (mDM) conditions without a significant reduction in motility. Exposure to either dbcAMP or beta-cyclodextrins was accompanied by an increase in proportions of spermatozoa displaying the AR pattern over those seen in controls. Exposure to beta-cyclodextrins did not increase the proportion of B pattern stained spermatozoa. Comparison of spermatozoa from two bulls revealed differential responses of spermatozoa from different males to treatments with heparin and progesterone. In vitro fertilisation results demonstrated that previously cryopreserved bull spermatozoa could be capacitated in chemically defined conditions devoid of heparin or other biological components.     

Calcium requirement and increased association with bovine sperm during capacitation by heparin

The requirement for external Ca+2 during capacitation of ejaculated bovine sperm with heparin and changes in sperm-associated 45Ca+2 during capacitation were investigated in vitro. Sperm capacitation was evaluated by ability to undergo an acrosome reaction (AR) upon exposure to lysophosphatidylcholine. The percentage of sperm which were capacitated during a 4 h incubation with heparin increased exponentially with increased exposure time to 2 mM Ca+2. When sperm were incubated with or without heparin in the presence of 45CaCl2, there was no difference in the amount of 45Ca+2 associated with sperm initially or at 1 h of incubation. Incubation with heparin resulted in a greater amount of sperm-associated 45Ca+2 at 2, 3, and 4 h as compared to sperm incubated without heparin. The amount of 45Ca+2 associated with sperm during capacitation was unaffected by washing with 2 mM EGTA-5 mM LaCl3. Glucose (5 mM) inhibited the effects of heparin on sperm-associated 45Ca+2 and on capacitation. The inhibitory effects of glucose could be overridden by 8-bromo-cAMP. The results suggest that the requirement for external Ca+2 during capacitation with heparin may be related to an increased association of external Ca+2 with sperm.     

肝素处理山羊精子体外获能的研究

系统研究了作用浓度、时间和温度以及输卵管上皮细胞和卵丘细胞对肝素处理山羊精子体外获能后的精子活力、质膜完整性、顶体完整率、获能比例及受精和卵裂的影响,为改善山羊精子体外获能效果和研究获能机理提供了必要的数据。主要实验结果如下：1、在获能液中添加5、10、25、50和100μg/mL肝素处理45min时,添加50和100μg/mL肝素精子获能比率最高（分别为55%和56%）,但添加100μg/mL肝素处理后顶体完整率明显（P<0.05）低于对照组。说明山羊精子获能的最佳肝素浓度为50μg/mL。2、肝素作用时间（0, 10, 20, 30, 45, 60 和120 min）的延长,获能精子比例逐渐提高。其中,肝素处理45～120 min各组的获能精子比例差异不显著（P>0.05）,处理120 min组的精子活力和质膜完整率显著低于其它各组。说明50μg/mL肝素处理精子获能的最佳时间是45～60 min。3、在42℃和38.5℃下处理时,获能精子比例显著高于15℃和37℃,但42℃处理后精子活力和顶体完整率显著低于其它温度。因此,385℃为山羊精子获能的最佳温度。4、与输卵管上皮细胞共培养获能精子比例显著高于对照组和卵丘细胞组,但精子活力、质膜完整率和顶体完整率差异不显著。输卵管上皮组的受精率（91.3％）和卵裂率（72.2％）显著高于对照组（81.2％,65.0％）。说明与输卵管上皮细胞共培养能显著提高肝素处理山羊精子体外获能的效果。     

Separate and combined effects of caffeine and dbcAMP on olive baboon (Papio anubis) sperm

Background Improvement of baboon sperm capacitation is necessary for achieving high in vitro fertilization (IVF) rates in baboons. In this study, we evaluated separate and combined effects of caffeine and dbcAMP on baboon sperm capacitation. Methods Sixteen male baboons (n = 16) were electroejaculated. Each sperm sample was divided into two aliquots: one for chemical activation and the other untreated control. Group 1: dbcAMP (n = 6); Group 2: caffeine (n = 6) and Group 3: combination of caffeine and dbcAMP (n = 4). In each aliquot, sperm motility after 30 minutes of incubation was evaluated as well as zona pellucida (ZP) binding ability after overnight incubation with 4–5 ZP from unfertilized human oocytes. Results Sperm motility and ZP binding ability in all chemically activated groups increased significantly as compared to their respective controls (P < 0.05). Conclusion Combined and separate effects of caffeine and dbcAMP increases baboon sperm motility and ZP binding ability and may improve baboon IVF.     

Rabbit sperm function after co-culture with uterine epithelial cells

The effects of spermatozoa:uterine epithelial cell interactions in vitro on various sperm functions were studied using monolayers of uterine epithelial cells, endometrial stromal cells and fetal fibroblasts. Epithelial and stromal cells were isolated from uteri of rabbits in estrus, while fibroblasts were derived from 12-d-old rabbit fetuses. Twenty-nine to 31 h after culture initiation, washed, ejaculated rabbit spermatozoa were incubated with epithelial cells, uterine stromal or fetal fibroblastic cells, medium or conditioned medium. Sperm viability and loss of acrosome were measured after 10 to 20 h of incubation. Progressive sperm motility and fertilizing ability, which was assessed by an in vivo fertilization assay, were determined after 12 h co-culture. Sperm viability decreased throughout the culture period and was not affected by treatment. Sperm co-cultured with epithelial cells or incubated in medium had fewer acrosomes after 20 h than after 10 h. Fewer sperm co-cultured with stromal or fibroblastic cells lost their acrosomes. Progressive motility was positively affected by sperm-epithelium interaction as 39% of the co-cultured sperm were motile compared with 18% of the sperm incubated in media. In vivo fertilization experiments suggested that sperm incubated with epithelial cells or in medium had similar fertilizing ability. The co-culture of sperm with uterine cells provides an in vitro model to evaluate the effect of gamete-genital tract interaction on sperm function.     

Sperm function and production of bovine embryos in vitro after swim-up with different calcium and caffeine concentration.

Frozen semen from bulls was used in artificial insemination programs was submitted to swim-up in Sperm Talp media containing different calcium (1.8, 2.6, 3.6 mM) or caffeine (2.5, 5.0, 7.5 microM) concentrations. The following sperm variables were evaluated: sperm recovery, motility, vigor, morphology, alterations in the pattern of capacitation by chlortetracycline (CTC) staining, and alterations in lysophosphatidyl choline (LPC)-induced acrosome reaction (AR). Sperm obtained from swim-up under different conditions were also tested for in vitro embryo production. No significant differences in the variables motility, vigor, morphology, and LPC-induced AR were observed among the treatments. However, the use of caffeine resulted in greater frequency of sperm with the capacitated pattern by CTC staining, compared to controls without caffeine. The greatest frequency of capacitated sperm (53%) was observed with 7.5 microM caffeine. Different calcium and caffeine concentrations in swim-up resulted in no significant differences in the cleavage rate and embryo development. In summary, micromolar concentrations of caffeine in Sperm Talp may stimulate sperm capacitation.     

Direct contact is required between serum albumin and hamster spermatozoa for capacitation in vitro

A dialysis unit was used to test whether direct physical contact between serum albumin and hamster spermatozoa is required for capacitation and/or the acrosome reaction. Sperm and bovine serum albumin (BSA) were incubated cither together (direct incubation) or separated by a dialysis membrane (indirect incubation). Sperm viability was supported with “sperm motility factors” (hypotaurine and epinephrine) and polyvinylalcohol (PVA). Spermatozoa became capacitated and underwent acrosome reactions when directly incubated in medium containing BSA (TALP-PVA), but did not undergo acrosome reactions when indirectly incubated with BSA (medium TLP-PVA). When sperm were first incubated for 4 hr indirectly with BSA, followed by 4 hr direct incubation with BSA, capacitation did not occur during indirect incubation. These findings indicate that an “intimate association” is necessary between serum albumin and spermatozoa to support capacitation under in vitro incubation conditions. The data are consistent with the concept of direct transfer of compounds from sperm to albumin and/or vice versa during sperm capacitation.     

The effect of Equex STM paste and sperm morphology on post-thaw survival of cat epididymal spermatozoa

Cryopreservation of epididymal spermatozoa is a potentially valuable tool for preserving genetic material from individuals of endangered species that die accidentally. Improvement of sperm-freezing protocols would increase the efficacy of gene banking from endangered felids, and the domestic cat can be used as a model for the wild felids. Addition of the detergent Equex STM paste to semen freezing extenders has been found to improve post-thaw survival and longevity of spermatozoa from various species but has never been tested for cat spermatozoa. Spermatozoa from cats with a high percentage of morphologically abnormal spermatozoa are more susceptible for cold injury and osmotic stress than spermatozoa from normozoospermic cats. Therefore, the aims of this study were to investigate: (a) if addition of Equex STM paste to a semen freezing extender would improve post-thaw sperm survival, and (b) if there is a relation between the percentage of morphologically normal spermatozoa and cryopreservation induced damage in cat epididymal spermatozoa. Spermatozoa harvested from epididymides of 10 male cats were frozen in a Tris egg yolk extender with or without the addition of Equex STM paste (0.5%, v/v). Sperm motility, membrane integrity and acrosomal status were evaluated immediately after harvesting, and at 0, 2, 4 and 6 h post-thaw. Sperm membrane integrity and acrosomal status were also evaluated after cooling to 4 degrees C, just before freezing. Cooling did not cause significant damage to the spermatozoa, whereas freezing damaged sperm membranes and acrosomes. Addition of Equex to the freezing extender had a significant positive effect on the percentage of intact acrosomes immediately after thawing (P > 0.05), but had a negative effect on the longevity of the spermatozoa; the percentages of membrane intact and motile spermatozoa being significantly lower in the presence of Equex than in the controls at 6h after thawing. The percentage of morphologically normal spermatozoa was not found to be correlated with either cryopreservation induced acrosome or plasma membrane damage, or with post-thaw motility (P > 0.05). The results clearly show that addition of Equex STM paste in the freezing extender protects the acrosomes of cat epididymal spermatozoa during the freezing--thawing process, but reduces the sperm longevity during in vitro incubation at 38 degrees C. Our results also indicate that the percentage of morphologically normal epididymal spermatozoa is not correlated with cryopreservation induced sperm damage using the described freezing protocol.     

猕猴精子优选及体外获能的研究

选择活率高的精子并进行体外获能是开展猕猴体外受精研究的必要程序, 是研究猕猴受精生物学的重要手段。本实验采用上浮法和Percoll 梯度离心法对猕猴精液进行了优选, 并对处理后的精子形态正常率、精子活率、密度及受精率作了比较, 发现二者差异不显著; 用dbcAMP 和咖啡因使精子获能, 发现只有两种获能剂同时存在才能使猕猴精子获能并使卵母细胞受精。结论为: 上浮法和Percoll 法都是有效的精子优选法, 对受精率的影响差异不显著; dbcAMP 和咖啡因在猕猴精子体外获能时缺一不可。     

Capacitation status of frozen–thawed spermatozoa from wild ruminant species

Semen from Barbary sheep (Ammotragus lervia), Bighorn sheep (Ovis canadensis), Mouflon sheep (Ovis musimon), Fallow deer (Dama dama), collected by electroejaculation, and semen from Wildebeest antelope (Connochaetes taurinus), collected post mortem, were frozen using a standardized technique and a commercial freezing medium (Triladyl). Sperm quality was assessed by measuring their capacitation status (chlortetracycline assay) in addition to classic assessment: motility, viability (plasma membrane integrity), and acrosome integrity. Sperm cryosurvival measured in terms of recovery (from the initial, pre-freeze values) of motility, viability, and acrosome integrity was relatively high in most species. However, proportion of premature-capacitated spermatozoa (B + AR patterns) increased many times in relation to that of fresh semen: from 8% to 78% in Barbary sheep; from 27% to 61% in Bighorn sheep; and from 12% to 68% in Mouflon sheep. The use of a standardized technique for freezing of spermatozoa from wild ruminant species produced, in most of the cases, acceptable results. This approach may be extensively used to carry out sperm cryopreservation in field conditions. Chlortetracycline assay allowed identifying the same fluorescent patterns observed in spermatozoa from domestic animals and produced additional information on sperm cryosurvival. That is, it revealed those cells that survive freeze–thawing and are potentially fertile: non-capacitated (F pattern) and capacitated acrosome-intact spermatozoa (B pattern).     

Zona pellucida binding ability and responsiveness to ionophore challenge of cryopreserved dog spermatozoa after different periods of capacitation in vitro

The present study was aimed to evaluate the functional status of cryopreserved dog spermatozoa after different periods (2, 8 and 24 h) of capacitation in vitro. Sperm motility, viability and binding capacity to the zona pellucida of canine oocytes derived from frozen-thawed ovaries were evaluated at each time point. Sperm viability was assessed by flow cytometry using the Ca(2+)-sensitive indicator Fluo 3 AM and PI, to simultaneously detect the proportion of live spermatozoa and the existence of live sperm subpopulations with different intracellular Ca(2+) content. In addition, the acrosome reaction frequency in ionophore-treated aliquots of spermatozoa incubated in capacitating (CCM) versus non-capacitating (NCM) medium, were evaluated by using eosin-nigrosin staining at the same time intervals. The number of spermatozoa bound to the zona pellucida decreased in about 50% (from 18.61 +/- 14.40 to 7.7 +/- 6.97) when sperm incubation was prolonged from 2 to 8h, however, sperm motility, viability and the subpopulation of live spermatozoa with higher intracellular Ca(2+) concentration decreased in lower extent (10-15%). In CCM-incubated samples, the rate of acrosomal exocytosis in response to ionophore challenge was high (>80%), independently of the evaluation period. NCM-incubated sperm were not affected by ionophore treatment, however, their intracellular Ca(2+) concentration was no different than that observed in CCM-incubated spermatozoa. It was concluded that, after being capacitated, motile and viable spermatozoa seem to lose their ability to bind to the zona pellucida, but this loss is not accompanied by a reduced response to ionophore challenge and it may not be related with changes in the intracellular Ca(2+) concentration of spermatozoa.     

Evaluation of cryopreserved stallion semen from Tori and Estonian breeds using CASA and flow cytometry

Methods to evaluate the quality of frozen-thawed stallion semen are still needed, particularly those considering the sperm function. The present study evaluated sperm motility, membrane and acrosome integrity and the capacitation status of frozen-thawed spermatozoa from seven Tori and six Estonian breed stallions by way of computer assisted sperm analysis (CASA), a triple fluorophore stain combination and Merocyanine 540, respectively, the latter ones using flow cytometry. Two ejaculates from each stallion were cryopreserved using the Hannover method in 0.5 ml plastic straws. Two straws per ejaculate per stallion were thawed at 37 degrees C for 30s. Motility was analysed with CASA immediately after thawing, while for flow cytometry spermatozoa were cleansed by 70:40% Percoll discontinuous density gradient separation before analysed for sperm viability, acrosome integrity (stained with SNARF, PI and FITC-PSA) and capacitation status (stained with Merocyanine 540/Yo-Pro-1). Results (as least square means) were as follows: the motility of frozen-thawed semen was 43.4% for Tori stallions and 42.3% for Estonian stallions (P>0.05). After Percoll separation 79.3% of the spermatozoa from Tori stallions had intact acrosomes and 1.7% of them showed early signs of capacitation. The same parameters for Estonian stallions were 84.5 and 2.3%, respectively. There were no statistically significant differences between breeds or ejaculates within breed for any evaluated parameter. We conclude that triple staining and flow cytometry are valuable techniques to evaluate frozen-thawed stallion spermatozoa, and that no differences in quality of frozen semen were registered between Tori and Estonian breed stallions, allowing implementation of this technology in the Estonian horse population.     

Binding of the anti-human sperm monoclonal antibody HS-11 to bull spermatozoa is correlated with fertility in vitro

The present study aimed to determine if there is bull to bull variation in the binding of the anti-human sperm monoclonal antibody (MAb) HS-11 to bull spermatozoa, and to investigate if there is any correlation between HS-11 binding to spermatozoa and in vitro fertility of the bulls tested. Semen samples of a single collection (split frozen in 0.5-ml straws) from 8 dairy bulls were used. Swim-up separated motile spermatozoa were incubated in 90-microl drops of capacitation medium (TALP+10 microg/ml heparin) at 39 degrees C, 5% CO2, 95% air. At 0, 2, 4 and 6 h of incubation HS-11 was added (1:1000 final concentration), and the MAb binding was assessed by indirect immunofluorescence assay (IIFA). The HS-11 binding was indicated by a bright green fluorescence of the sperm acrosome region. In vitro-matured, good quality bovine oocytes were randomly allocated to spermatozoa of each bull for in vitro fertilization. Sperm samples of 2 to 3 bulls were used in each trial until 4 replicates per bull were attained for IVF (n approximately 100 oocytes/bull) and IIFA experiments. Sperm capacitation status was assessed simultaneously using an egg yolk lysophosphatidylcholine- (LC) induced acrosome reaction assay. The binding of HS-11 to spermatozoa was maximum at 4 h of incubation in most (6/8) of the bull semen samples. Significant (P < 0.01) differences were observed between bulls in the binding of HS-11 to their spermatozoa (range 22 +/- 8 to 52 +/- 5%) at 4 h, but not within replicates. Similarly, variations (P < 0.05) in the cleavage rate were also seen (range 22 +/- 9 to 58 +/- 7%) between bulls. The HS-11 binding and cleavage were significantly correlated (r = 0.43; n = 32; P < 0.05). The highest percentage of spermatozoa underwent acrosome reaction in response to LC treatment at the 4-h incubation period. This and the linear relationship between HS-11 binding and the cleavage rate observed in the present study together strengthen our earlier suggestion that the binding of the monoclonal antibody HS-11 to bull spermatozoa on a time-dependent manner, may indicate capacitation changes. We conclude that 1) between-bull differences exist in HS-11 binding to spermatozoa, and in the cleavage rate, and 2) HS-11 binding to spermatozoa is correlated with fertility, as determined by the cleavage of bovine oocytes matured and fertilized in vitro.     

Roles of bicarbonate, cAMP, and protein tyrosine phosphorylation on capacitation and the spontaneous acrosome reaction of hamster sperm.

Capacitation is a prerequisite for successful fertilization by mammalian spermatozoa. This process is generally observed in vitro in defined NaHCO3-buffered media and has been shown to be associated with changes in cAMP metabolism and protein tyrosine phosphorylation. In this study, we observed that when NaHCO3 was replaced by 4-(2-hydroxyethyl)1-piperazine ethanesulfonic acid (HEPES), hamster sperm capacitation, measured as the ability of the sperm to undergo a spontaneous acrosome reaction, did not take place. Addition of 25 mM NaHCO3 to NaHCO3-free medium in which spermatozoa had been preincubated for 3.5 h, increased the percentage of spontaneous acrosome reactions from 0% to 80% in the following 4 h. Addition of anion transport blockers such as 4,4'-diiso thiocyano-2, 2'-stilbenedisulfonate (DIDS) or 4-acetomido-4'-isothiocyanatostilbene-2,2'-disulfonic acid (SITS) to the NaHCO3-containing medium inhibited the acrosome reaction, with maximal inhibition at 600 microM, and with an EC50 of 100 microM. Increasing either extracellular or intracellular pH did not induce the acrosome reaction in NaHCO3-free medium. In contrast, addition of 500 microM dibutyryl cAMP (dbcAMP), alone or together with 100 microM 1-methyl-3-isobutylxanthine (IBMX), induced the acrosome reaction in spermatozoa incubated in NaHCO3-free medium. These compounds also partially reversed the inhibition of the acrosome reaction caused by the DIDS or SITS in complete medium. In contrast to these results, IBMX or dbcAMP did not induce acrosome reactions in cells incubated in Ca2+-free medium. When hamster sperm were incubated in the absence of NaHCO3 or in the presence of NaHCO3 and DIDS, cAMP concentrations were significantly lower than the values obtained from sperm incubated in complete medium. Protein tyrosine phosphorylation has also been shown to be highly correlated with the onset of capacitation in many species. During the first hour of capacitation, an increase in protein tyrosine phosphorylation was observed in complete medium. In the absence of NaHCO3, the increase in protein tyrosine phosphorylation was delayed for 45 min, and this delay was overcome by the addition of dbcAMP and IBMX. The induction of the acrosome reaction by calcium ionophore A23187 in NaHCO3-free medium was delayed 2 h, as compared with control medium. This delay was not observed in the presence of dbcAMP and IBMX. Taken together, these results suggest that a cAMP pathway may mediate the role of NaHCO3 in the capacitation of hamster spermatozoa and that protein tyrosine phosphorylation is necessary but not sufficient for complete capacitation.