Pentoxifylline induces capacitation and acrosome reaction and improves quality of motility in canine ejaculated spermatozoa

To evaluate effects of different concentrations of pentoxifylline, as phosphodiesterase inhibitor, on quality of motility, capacitation and acrosome reaction, Ejaculated spermatozoa were collected from crossbred dogs. The sperm were incubated at concentrations of 0.1, 1, 10 and 100 mM pentoxifylline for 2 h. Conventional assessment was also made on the percentage of motility and quality of motility of spermatozoa; values were expressed as sperm motility index (SMI). Capacitation and acrosome reaction were also evaluated by chlortetracycline fluorescence staining. SMI as quality index of sperm was significantly increased in concentrations of 10 and 100 mM pentoxifylline during 1 and 2 h compared to control. The number of capacitated or acrosome reacted spermatozoa significantly (P < 0.05) were higher than controls at high concentrations of pentoxifylline (10 and 100 mM) during 1 and 2 h. In conclusion, high concentration of pentoxifylline is able to induce capacitation and acrosome reaction and improves quality of motility in canine ejaculated spermatozoa.     

In vitro induction of the acrosome reaction in ovine spermatozoa by calcium ionophore A23187

The relationship between concentration of calcium ionophore A23187 and incubation time upon the proportion of spermatozoa undergoing acrosome reaction (AR) in vitro was investigated in rams from a commercial artificial insemination (AI) program. Two ejaculates were collected by artificial vagina from each of nine rams of three breeds (Finn Dorset, Charolais and Suffolk) aged 8-36 months. Each ejaculate was diluted in a skimmed milk extender. Spermatozoa were thereafter incubated for 45 or 60 min in modified Tyrode's medium (TALP) which contained either zero, 0.1 or 1.0 microM/l A23187. After fixing in 10% formaldehyde, the number of spermatozoa that had undergone AR was determined by phase contrast microscopy. In pre-incubation samples, 21.3 +/- 3.3% of spermatozoa had undergone AR. Percentages of acrosome reacted spermatozoa were significantly (P < 0.001) increased after incubation with A23187. After incubation with 0.1 microM/l A23187 for 45 and 60 min there were 22.4 +/- 3.0% and 31.7 +/- 4.3% acrosome reacted spermatozoa, respectively. After incubation with 1.0 microM/l A23187 for 45 and 60 min there were 46.2 +/- 6.5% and 53.8 +/- 5.9% acrosome reacted spermatozoa, whilst corresponding numbers in control samples were 17.0 +/- 2.7% and 22.3 +/- 4.2%. There was also a significant (P < 0.001) effect of individual animals upon the responses to different concentrations of A23187. These findings indicate that (i) A23187 can be used to assess the AR of ovine spermatozoa in vitro and (ii) there are effects of individual animals upon the proportion of spermatozoa undergoing AR.     

Induction and characterization of acrosome reaction in equine spermatozoa

Equine spermatozoa were incubated in a chemically defined medium for 8 hours. The medium preserved spermatozoal viability, as assessed by total spermatozoal motility, progressive spermatozoal motility, and spermatozoal exclusion of eosin stain. Effects of time and divalent cation ionophore, A23187, on the occurrence and character of the spermatozoal acrosome reaction were determined. Two light microscopic assays, a triple-stain technique and a chlortetracycline fluorescence assay, were calibrated with transmission electron microscopy for detection of the acrosome reaction. Incubation time and A23187 addition increased the percentage of acrosome reactions in sperm populations (P less than 0.05).     

Effect of relaxin on acrosome reaction and utilization of glucose in boar spermatozoa

Relaxin is a peptide hormone found in seminal plasma that has a physiological influence on sperm motility in some species. There are no reports on the effect of relaxin on acrosome reaction and utilization of glucose in boar spermatozoa. In this study, to investigate the effects of relaxin on sperm motility, acrosome reaction, and incorporation and oxidation of labeled glucose, boar spermatozoa were washed and preincubated for swim-up and then incubated (0-6 h) with 0, 20, or 40 ng/ml relaxin in mTALP medium. The results indicated that the addition of relaxin stimulated sperm motility significantly (P<0.05) during 1-4 h of incubation. The percentage of acrosome-reacted live spermatozoa was higher (P<0.05) when the spermatozoa were treated with 20 or 40 ng/ml relaxin. The rate of incorporation, and oxidation of glucose were also greater (P<0.05) in the spermatozoa incubated with relaxin compared to the control spermatozoa. The rate of incorporation and oxidation of (14)C-glucose were increased in correlation with acrosome reaction up to 4 h of incubation and then decreased in line with the increasing incubation period. In conclusion, the present study demonstrates that relaxin accelerates not only motility but also the acrosome reaction and utilization of glucose in boar spermatozoa.     

In vitro capacitation of stallion spermatozoa assessed by the lysophosphatidylcholine-induced acrosome reaction and the penetration rate into in vitro-matured, zona-free mare oocytes

The purpose of this study was to evaluate the ability of various chemicals to induce capacitation of stallion spermatozoa using 2 different assay systems. In Experiment 1, freshly ejaculated spermatozoa were treated for 0, 3 and 6 h with 10 μ g/ml heparin, 0.5 mM hypotaurine or 5 mM caffeine, or were incubated for 0, 3 and 6 h following 1 min exposure to 0.1 μ M ionophore A23187. The acrosome reaction (AR) in the capacitated spermatozoa was induced by 15 min challenge with 100 μ g/ml lysophosphatidylcholine (LPC). In the BO/BSA-control medium (Brackett and Oliphant medium with 0.3% BSA), mean percentage of AR spermatozoa at 0 h was 30%, and the AR rates increased to 40 and 48% after 3 and 6 h incubation, respectively. There was no significant further increase of the AR rates in the spermatozoa treated with heparin (50% at 6 h) and hypotaurine (58% at 6 h) when compared to the control. Caffeine had a beneficial effect on inducing sperm capacitation after 3 and 6 h incubation (AR rates; 61 and 66%, respectively, P<0.01). Immediately after ionophore A23187 treatment, the AR rate increased to 56%, and reached 68 and 67% after 3 and 6 h incubation, respectively (P<0.01). Spermatozoal motility at any time points did not differ between control and any chemical treatment groups, except one treatment (ionophore; 3 h group).In Experiment 2, frozen-thawed spermatozoa were treated with 4 different chemicals as described above. Aliquot of spermatozoa was added to a microdrop of BO/BSA medium in which 6 to 10 in vitro-matured, zona-free mare oocytes were placed, and the oocytes were fixed and stained 20 h after insemination. The penetration rate by BO/BSA-treated spermatozoa was 76%, which was comparable to the results with heparin (73%), hypotaurine (78%) and caffeine (58%). In contrast, treatment of spermatozoa with ionophore A23187 gave a significantly lower penetration rate (30%) than the control value. Surprisingly these two experiments had different conclusions in assessing capacitation of stallion spermatozoa.     

Agglutination and acrosome reaction of ram spermatozoa incubated in the bovine folicular fluid]

Fresh-ejaculated sperm of ram was incubated at a temperature of 38 degrees C in the bovine follicular fluid and homologous blood serum (blood plasma). The spermatozoa were studied in native state under a microscope with phase contrast and as ultra-thin sections under a transmission electron microscope. In the follicular fluid and in the blood serum, strong agglutination of spermatozoa occurred, with its maximum after about two hours. Only the heads of spermatozoa agglutinated, the flagella being loose were arranged parallelly. Although the progressive motility of spermatozoa was not observed, the motility of flagella was not affected. The investigation under the electron microscope showed that the agglutination occurred only in acrosomes of intact spermatozoa. It was also found out that the follicular fluid induced the acrosome reaction of spermatozoa. The course of the acrosome reaction is similar to that in the other mammals: first of all plasma membrane becomes undulated, then it fuses with the outer acrosome membrane, giving origin to vesicles within the entire acrosome, except the equatorial segment. The acrosome reaction was found in about 5% spermatozoa, and therefore it may be assumed that secretions of the oviduct and uterus play their role to induce the acrosome reaction.     

Severe tail defects in the spermatozoa ejaculated by an English bulldog

This paper describes the case of a 2-year-old English Bulldog with severe teratozoospermia that consisted mainly of "Dag-like" defects, which is strong coiling of the tail. Although libido, semen volume and sperm concentration were normal, sperm motility was 5.0%, and 93.3% of spermatozoa exhibited morphological abnormalities affecting the tail. Transmission electron microscopic examination of the spermatozoa revealed strong folding, coiling and fracture of sperm midpieces and tails, axonemal defects and the presence of swollen and unevenly distributed mitochondria. Taking into account the dog's history and examinations and the constantly high percentage of abnormal spermatozoa over time, the defect was considered to be genetic in origin.     

Glycosaminoglycans in ewe reproductive tracts and their influence on acrosome reactions in bovine spermatozoa in vitro

Glycosaminoglycans (GAG) promote acrosome reactions (AR) in bovine and rabbit spermatozoa in vitro. Female bovine reproductive tract secretions contained GAG and the concentrations and composition of those GAG varied with different regions and stages of the estrous cycle. This study was designed to evaluate the types of GAG found in reproductive tract secretions of ewes at different stages of the estrous cycle and the ability of the secretions to promote AR. Ewes (n = 48) were slaughtered at 0, 12, 24, 36, 72 and 144 h following observations of standing estrus. Reproductive tracts were flushed with a modified Tyrode's medium (TALP: 7 ml) not containing calcium. Concentrations and composition of GAG in the tract were determined by high performance liquid chromatography. Concentrations of GAG decreased anterior to the cervix: 2.28 to .74 mg/100 mg protein (P less than .05). Increases in heparin-like GAG during the estrual phase and chondroitin sulfate GAG during the luteal phase were noted. Bovine sperm were incubated in tract flushings standardized to contain 4 micrograms/ml of GAG and supplemented with calcium. Sperm incubated in estrual flushings for 9 h exhibited a higher incidence of AR than those incubated in luteal flushings or control, 50% vs 36% and 29%, respectively (P less than .0001). It is concluded that during the estrous cycle there were changes in concentrations and composition of GAG in ewe reproductive tracts and the potencies of those female secretions causing AR varied.     

Characterizing the in vitro capacitation of motile bull sperm by the hyamine-induced acrosome reaction

Described in this paper is a method to characterize capacitation of bull spermatozoa. Involved is the action of 0.005 per cent of hyamine 2389 on selected motile cells at 37 degrees C for 30 minutes to detect the number of spermatozoa capable of acrosomal reaction. The latter amount results from the percentual difference between acrosome-free spermatozoa prior to hyamine action and those after hyamine action. Acrosome-free spermatozoa can be easily identified by means of simple staining under optical light microscopy. The value thus obtained is related to the amount of spermatozoa in progressively motile in the unselected original suspension and is called "Percentage Acrosome Reaction Induction" (% ARI). The ARI capacitation-specific acts: Only small amounts of acrosome-free spermatozoa are recordable among incapacitated spermatozoa or from spermatozoa incubated in the presence of decapacitation factors (1.9 or 2.3 per cent, respectively). On the other hand, clearly increased values of 17.6 ot 26.1 per cent are obtainable from treatments with capacitation effect (high ionic strength or addition of heparin). The method is claimed to be suitable for adequate choice of conditions for improved capacitation and in vitro fertilization as well as for elucidation of specific individual differences in capacitation.     

Participation of phosphofructokinase,malate dehydrogenase and isocitrate dehydrogenase in capacitation and acrosome reaction of boar spermatozoa

The aim of this work was to determine the enzymatic activity of phosphofructokinase (PFK), malate dehydrogenase (MDH) and isocitrate dehydrogenase (IDH) in boar spermatozoa and study their participation in bicarbonate‐induced capacitation and follicular fluid‐induced acrosome reaction. Enzymatic activity of these enzymes was determined spectrophotometrically in extracts of boar spermatozoa. Sperm suspensions were incubated in the presence of bicarbonate (40 mM), a well‐known capacitation inducer, or follicular fluid (30%), as an acrosome reaction inducer, and different concentrations of oxoglutarate, oxalomalate and hydroxymalonate, inhibitors of PFK, IDH and MDH, respectively. Capacitation percentages were determined by the fluorescence technique of chlortetracycline (CTC), and true acrosome reaction was determined by trypan blue and differential–interferential contrast, optical microscopy. The activity of PFK in boar spermatozoa enzymatic extracts was 1.70 ± 0.19 U/10¹⁰ spermatozoa, the activity of NAD‐ and NADP‐dependent IDH was 0.111 ± 0.005 U/10¹⁰ and 2.22 ± 0.14 U/10¹⁰ spermatozoa, respectively, and the activity of MDH was 4.24 ± 0.38 U/10¹⁰ spermatozoa. The addition of the specific inhibitors of these enzymes prevented sperm capacitation and decreased sperm motility during capacitation and inhibited the acrosome reaction (AR), without affecting the sperm motility during this process. Our results demonstrate the participation of PFK, IDH and MDH in bicarbonate‐induced capacitation and follicular fluid‐induced acrosome reaction in boar spermatozoa, contributing to elucidate the mechanisms that produce energy necessary for these processes in porcine spermatozoa.     

Platelet-activating factor in Iberian pig spermatozoa: receptor expression and role as enhancer of the calcium-induced acrosome reaction

Platelet-activating factor (PAF) is a phospholipid involved in reproductive physiology. PAF receptor is expressed in some mammalian spermatozoa species where it plays a role in these germ-cell-specific processes. The aim of this study is to identify PAF receptor in Iberian pig spermatozoa and to evaluate PAF's effects on motility, viability and acrosome reaction. Semen samples from Iberian boars were used. PAF receptor identification was performed by Western blotting. Spermatozoa motility was analysed by computer-assisted sperm analysis system, whereas spermatozoa viability and acrosome reaction were evaluated by flow cytometry. Different PAF concentrations added to non-capacitating medium during 60 min have no effect on any spermatozoa motility parameter measured. Acrosome reaction was rapid and potently induced by 1 μm calcium ionophore A23187 showing an effect at 60 min and maximum at 240 min. PAF added to a capacitating medium is not able to induce spermatozoa acrosome reaction at any time studied. However, PAF, in the presence of A23187, significantly accelerates and enhances the calcium-induced acrosome reaction in a concentration-dependent manner in Iberian boar spermatozoa. Exogenous PAF does not affect at all spermatozoa viability, whereas slightly exacerbated the A23187-induced loss in viability. This work demonstrates that PAF receptor is expressed in Iberian pig spermatozoa and that its stimulation by PAF regulates the calcium-induced acrosome reaction. This work contributes to further elucidate the physiological regulation of the most relevant spermatozoa functions for successful fertilization: acrosome reaction.     

Effect of relaxin on the motility, acrosome reaction and utilization of glucose of fresh and frozen-thawed bovine spermatozoa

The study was conducted to investigate the effect of relaxin on motility, acrosome reaction (AR), viability and utilization of glucose in fresh and frozen‐thawed bovine spermatozoa. Both semen samples were washed twice through centrifugation (5 min at 600 g), and preincubated for 1 h at 39°C for swim up. The swim‐up separated spermatozoa were resuspended in a sperm Tyrode's albumin lactate pyruvate (Sp‐TALP) medium containing 0 (control) and 40 ng/mL porcine relaxin and incubated for 0–6 h. Sperm motility was determined on the basis of movement quality examined by a phase contrast microscope. Sperm viability and AR were evaluated by using the triple staining technique. The incorporation and oxidation of ¹⁴C‐glucose was assessed by a liquid scintillation counter. Motility was improved (P < 0.05) in both fresh and frozen‐thawed spermatozoa by the addition of relaxin to the Sp‐TALP medium, whereas relaxin showed no significant effect on viability in either fresh or frozen‐thawed spermatozoa. The percentage of AR increased (P < 0.05) when fresh or frozen‐thawed spermatozoa were incubated with relaxin. In contrast, the incorporation and oxidation of ¹⁴C‐glucose increased (P < 0.05) in both kinds of spermatozoa incubated with relaxin. Thus the results demonstrated that the addition of relaxin to the Sp‐TALP medium increased the motility, AR and utilization of glucose in fresh and frozen‐thawed bovine spermatozoa.