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RESEARCH |
Institute of Zoology, Johannes Gutenberg University, 55099 Mainz, Germany
Correspondence should be addressed to G Kamp; Email: kamp{at}uni-mainz.de
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionAcknowledgementsReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionAcknowledgementsReferences |
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Sperm hyperactivity has been observed in vitro in various mammalian species especially if capacitation of spermatozoa was induced with capacitation media containing Ca2+ (for review see Yanagimachi 1994). Rapid hyperactivation was reported when Ca2+ influx was triggered by a Ca2+ ionophore (Suarez et al. 1987). The analysis of hyperactive boar spermatozoa is complicated because spermatozoa tend to agglutinate (head-to-head) in capacitation media containing Ca2+ (Harayama et al. 1998) and the Ca2+ ionophore A23187 [GenBank] tends to inactivate sperm (Suarez et al. 1987). Suarez and colleagues (1987 (1992) tried to overcome this problem by pulse stimulation in which they exposed sperm to the ionophore for only a short period and then absorbed the excessive ionophore by adding bovine serum albumin (BSA). In this paper we present a novel and simple method for inducing hyperactivity in boar spermatozoa by using low concentrations of Ca2+ and a constant concentration of the Ca2+ ionophore A23187 [GenBank] . Both head-to-head agglutination as well as early inactivation are substantially prevented, and hyper-activity of boar spermatozoa can be studied for 40 min or more.
Computer-assisted sperm analysis (CASA) is a powerful tool for the objective assessment of sperm motility and is hence frequently used for evaluating the quality of semen from man and animals (Mortimer 1997, 2000, Verstegen et al. 2002). However, the quantitative evaluation of semen by CASA is based on kinematic parameters of individual spermatozoa that are reconstructed merely from the movements of their heads, because CASA instrumentation does not usually allow the direct analysis of flagellar movements that generate sperm hyperactivity (Mortimer 1997). Hence, to detect hyperactive spermatozoa by CASA requires kinematic parameters of sperm head movements that are correlated with characteristic changes in flagellar movements. For the accurate assessment of hyperactive spermatozoa by CASA, the set of parameters must be defined for a particular species, taking into account the image sampling frequency (Mortimer & Maxwell 1999).
Threshold kinematic parameters to differentiate between hyperactive and non-hyperactive spermatozoa have been reported for mouse (Neill & Olds-Clarke 1987), man (for review and citations see Mortimer 1997), ram (Mortimer & Maxwell 1999) and rat (Cancel et al. 2000). Hyperactivation in boar spermatoza has been mentioned several times (Saxena et al. 1986, Blottner et al. 1989, Hamano et al. 1989), but an analysis of movement patterns in ejaculated boar spermatozoa and in spermatozoa flushed from the oviducts of pigs has only been reported by Suarez et al.(1992). A detailed investigation of hyperactivated boar spermatozoa using CASA has not yet been reported. Therefore, we compared the two characteristic parameters of sperm flagellar movement, the flagellar beat angle (FBA) and the flagellar curvature ratio (FCR) with kinematic parameters of CASA in order to define objective kinematic thresholds that can be used to identify hyperactive boar spermatozoa. This identification and quantification of hyperactive spermatozoa is necessary for analyzing the energy metabolism of hyperactivity now in progress. We propose that hyperactivity depends on glycolytic ATP production in the principal piece of the flagellum (Westhoff & Kamp 1997, Bone et al. 2000).
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionAcknowledgementsReferences |
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Collection and storage of semen
Semen, collected from fertile boars kept for commercial artificial insemination, was provided by GFS (Genossenschaft zur Föderung der Schweinehaltung, Ascheberg, Germany). The semen was diluted with the same volume of BTS and stored at 17°C until use (non-cryopreserved). All experiments were performed with semen stored liquid for only 1 day. The final concentration of boar spermatozoa was about 20 x 106 per ml in all experiments.
Media and reagents
BTS containing 250 mg gentamicin l–1 was used for diluting boar semen. TALP-HEPES medium (pH 7.4) (Bavister & Yanagimachi 1977) comprised 6 mmol l–1 glucose, 113 mmol l–1 NaCl, 2.7 mmol l–1 KCl, 0.5 mmol l–1 MgCl2, 0.4 mmol l–1 NaH2PO4, 20 mmol l NaHCO3, 1 mmol l pyruvate, 20 mmol l–1 lactate, 20 mmol l–1 HEPES and 0.6% BSA. The Ca2+ ionophore A23187
[GenBank]
was added from a stock solution (1 mmol l–1 dissolved in DMSO). The final concentration of the Ca2+ ionophore was 5 µmol l–1 in all experiments. Equivalent amounts of DMSO were added to controls.
Seminal plasma is the supernatant of boar semen after centrifugation at 10 000 g for 10 min at 4 °C.
Hyperactivation of spermatozoa
Hyperactivation was induced either in the absence or in the presence of seminal plasma. For hyperactivation in the absence of seminal plasma, prediluted semen stored at 17 °C was centrifuged at 500 g for 2 min, the supernatant discarded and the sedimented spermatozoa were resuspended in TALP-HEPES. After addition of 50 µmol l–1 CaCl2 and 5 µmol l –1 of the Ca2+ ionophore A23187
[GenBank]
, the suspension was incubated at 37 ° C for 15 min and analyzed by CASA. For hyperactivation in the presence of seminal plasma, the sperm sediment was resuspended in a medium comprising 50% TALP-HEPES, 25% BTS and 25% pooled undiluted seminal plasma that had been collected from 12 boars. CaCl2 (700 µ mol l–1) was added and samples were analyzed 15 min after the addition of 5 µ mol l–1 Ca2+ ionophore (incubated at 37 ° C).
Analysis of sperm motility
Aliquots of 2.2 µl sperm suspension were pipetted into disposable counting chambers (standard count 4-chamber 20 micron slides, Leja, Nieuw Vennep, the Netherlands) that had been prewarmed to 37 ° C on a heated microscope stage. Motility was observed using a Leica inverted microscope (Leitz DM IRB, Leica, 64625 Bensheim) with a 20-times magnification objective for negative phase contrast. Spermatozoa were monitored with an analogous camera (Kappa CF 8/4, 37130 Gleichen) using either a 1:1 or 1:0.5 C-mount (Leica, 64625 Bensheim) and 50 pictures per s (50 Hz) were recorded via a video cassette recorder (Panasonic NV-HS800). A TV-screen (Sony KV-M1430D) was used for monitoring sperm motility.
Analysis of flagellar movement
The method of Mortimer and Maxwell (1999) for analyzing ram spermatozoa was slightly modified. For flagellar movement analysis, videotapes were replayed and projected (Panasonic PT-L795E LCD projector) on white paper attached to a magnetic rotatable table. Flagellar bends were directly copied on to the paper with respect to the position of the sperm head. The angle between the tangents of the outer flagellar waves was determined manually resulting in the flagellar beat angle (FBA, cf. Fig. 1
). Additionally, flagellar curvature ratio (FCR) was measured manually from the flagellar movements drawn for FBA analysis. FCR is the straight line distance from the neck (junction between the head and the mid piece) to the first inflection point of the tail divided by the distance on the curve between the same points (cf. Fig. 1; Suarez 1988). The inflection point was estimated as the intersection of the flagellar wave with a parallel straight line amidst two parallel tangents drawn on the outer flagellar waves.
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For each trajectory, 50–60 images collected at 50 Hz over 1 to 1.2 s were analyzed. The following kinematic parameters (for definitions see Mortimer 1997) were measured for each trajectory: curvilinear velocity (VCL, µm s–1), straight line velocity (VSL, µm s–1), average path velocity (VAP, µm s–1), mean lateral head displacement (LHDmean, µm) and beat cross frequency (BCF, Hz). From the kinematic parameters, linearity (LIN = VSL x 100/VCL), straightness (STR = VSL x 100/VAP) and wobble (WOB = VAP x 100/VCL) were calculated.
Statistics
To tell non-hyperactive from hyperactive spermatozoa, threshold values with the sensitivity and specificity at highest accuracy were calculated for different parameters using ROC (receiver operating characteristic) curve analysis and the computer program MedCalc for Windows (Medcalc, Mariakerke, Belgium). One hundred per cent sensitivity corresponds to the probability of detecting all actually hyperactive spermatozoa. Similarly, 100% specificity corresponds to the probability of detecting all non-hyperactive spermatozoa. Values are given as means±S.D., and significant differences between means were calculated with Microsoft Excel. According to the result of a variance ratio analysis (F-test, P < 0.01), an unpaired t-test for either homoscedastic or heteroscedastic variances was performed (P < 0.0001).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionAcknowledgementsReferences |
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Analysis of flagellar movement in hyperactive boar spermatozoa
Incubation of boar spermatozoa at 37 °C with TALP-HEPES in the presence of 25% seminal plasma, 700 µmol l–1 Ca2+ and 5 µmol l–1 Ca2+ ionophore resulted, after 15 min, in an almost synchronous change to vigorous motility with whiplash-like flagellar movements as is typical of hyperactivity (Fig. 1
). The FBA increased from 49° ± 12° in the control group to 200° ± 36° in the hyperactive spermatozoa while the FCR decreased from 0.89 ± 0.04 in controls to 0.47 ± 0.11 in the hyperactive group. Both effects were highly significant (P < 0.0001, n = 32). Hence, according to the definition of hyperactive spermatozoa from several species (Suarez & Osman 1987, Suarez 1988, Suarez et al. 1992, Mortimer et al. 1997, Mortimer & Maxwell 1999), the treatment stimulated spermatozoa to hyper-activity. The threshold values obtained from the ROC curve analysis to differentiate between non-hyperactive and hyperactive spermatozoa were 87° for FBA and 0.7 for FCR at 100% specificity and 100% sensitivity.
Kinematic parameters of CASA for hyperactivity
For computer-assisted quantification of hyperactive boar spermatozoa the kinematic parameters of hyperactive and non-hyperactive boar spermatozoa identified by FBA and FCR (Fig. 1) were determined by CASA (Table 1). VCL and LHDmean were significantly increased (P < 0.0001) while LIN, STR, WOB and BCF were significantly decreased (P < 0.0001) upon hyperactivation. The most pronounced change was found in LHDmean which increased about six-fold with hyperactivation from 1.3 to 8.0 µm.
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). The sensitivity and specificity are 100% at the threshold value (LHDmean > 3.5 µm for hyperactive spermatozoa). Additionally, WOB, VCL and LIN all showed approximately 100% sensitivity with high specificity (>50%). For a multiparametric definition of hyperactive boar spermatozoa, the following threshold values were used in combination: VCL >97 µm s–1, LHDmean >3.5 µm, LIN < 32% and WOB <71%.
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). This was due to by more false positive identifications by the monoparametric compared with the multiparametric analysis.
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). Below 30 µmol l–1 and above 60 µmol l–1 Ca2+ loss of motility and hyperactivity was observed in the presence of the calcium ionophore.
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). Calcium concentrations lower than 500 µmol l–1 did not induce hyperactivation and concentrations higher than 900 µmol l–1 resulted in immotility.
Time course of induced hyperactivity
Spermatozoa were incubated at 37 °C in TALP-HEPES media containing 50 or 700 µmol l–1 Ca2+ in the presence or absence of 25% seminal plasma respectively. About 15 min after the addition of the Ca2+ ionophore A23187
[GenBank]
, approximately 60% of spermatozoa had switched from non-hyperactive to hyperactive motility and maintained hyperactivity for at least 40 min (Fig. 3). Thereafter, the percentage of motile and hyperactive motile spermatozoa slowly decreased and sometimes head-to-head agglutination was observed in the absence of seminal plasma, while tail-to-tail agglutination was found in the presence of 25% seminal plasma.
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). The percentage of hyperactive spermatozoa was significantly (P < 0.0001) increased after induction of hyperactivity. In the absence of seminal plasma 63% of motile spermatozoa were hyperactive and in the presence of seminal plasma 58% were hyperactive.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionAcknowledgementsReferences |
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Hyperactivation and Ca2+
Hyperactivity is apparently induced during sperm capacitation in the female genital tract, but neither the extra-cellular trigger nor the pathway of signal transduction are known. As an intracellular signal, Ca2+ is involved in the control of sperm hyperactivity. Demembranated bull spermatozoa became motile and increasingly hyper-active at very low Ca2+ concentrations (>0.05 µmol l–1). About 70%–80% of sperm were hyperactive between 0.4 and 100 µmol l–1 Ca2+, but at 1 mmol l–1Ca2+ both the non-hyperactive and hyperactive motility decreased (Ho et al. 2002).
Hyperactivity has also been induced in intact spermatozoa of several mammalian species by capacitation media containing Ca2+ in the mM range (for review see Yanagimachi 1994). Capacitation is accompanied by changes in membrane structure thus increasing Ca2+ influx which triggers hyperactivity and, finally, the acrosome reaction. As a consequence of changes in membrane structure spermatozoa tend to aggregate head-to-head, especially in boars (Harayama et al. 1998). The motility of aggregated spermatozoa, however, cannot be analyzed by CASA and therefore capacitation with mM Ca2+ hampers the analysis of hyperactivity, at least in boar spermatozoa.
In intact mouse and boar spermatozoa, transient hyper-activity was also observed if Ca2+ influx was brought about by the Ca2+ ionophore A23187 [GenBank] (Suarez et al. 1987, 1992). The degree of hyperactivation seems to be dependent on various factors, particularly on the concentrations of Ca2+ and of the ionophore. Mouse spermatozoa were made hyperactive for several minutes by incubating them with 1 mmol l–1 Ca2+ and 1 µmol l–1 ionophore and attenuating the effect of the ionophore by albumin (Suarez et al. 1987). A similar procedure as in mice was used to hyperactivate boar spermatozoa (Suarez et al. 1992).
Unlike the approach above, we have varied the Ca2+ concentration and kept the concentration of the ionophore constant. Under these conditions a specific range of Ca2+ concentrations induces hyperactivity in boar spermatozoa. Using this method, boar spermatozoa remain hyperactive for at least 40 min which is long enough for studying the energy metabolism of hyperactive boar spermatozoa (see below). Sperm freed from seminal plasma by mild centrifugation (500 g, 2 min) and incubated in TALP-HEPES medium became hyperactive in the narrow range of 40 to 70 µmol Ca2+ l–1. High Ca2+ concentrations (>100 µmol l–1) resulted in a nearly complete loss of sperm motility and low Ca2+concentrations (<20 µmol l–1) prevented hyperactivity and reduced sperm motility. These responses of intact boar spermatozoa to Ca2+ in the presence of the ionophore are strikingly similar to those of demembranated bull spermatozoa (Ho et al. 2002). Hence, the use of the ionophore and a precisely adjusted Ca2+ concentration allow hyperactivation with less interference with sperm integrity than in demembranated sperm. However, the mechanism by which hyperactivation is triggered in vivo still remains obscure.
Hyperactivation and seminal plasma
Like sperm freed from seminal plasma, boar spermatozoa can be induced to hyperactivity in a relatively narrow range of calcium concentrations if seminal plasma (25%) is present. The range of Ca2+ concentration required for hyperactivity, however, is about 10 times higher (600 to 900 µmol l–1). This is likely due to the high concentrations of citrate in the seminal plasma (about 10 mmol l–1; Mann & Lutwak-Mann 1981, Kamp & Lauterwein 1995) which chelates Ca2+. As in the absence of seminal plasma, high Ca2+ concentrations (>1 mmol l–1) caused dramatic loss of both non-hyperactive and hyperactive motility. While the percentage of hyperactive boar spermatozoa tends to be slightly more constant in the presence than in the absence of seminal plasma, the disadvantage of using seminal plasma rests in its unknown composition (e.g. decapacitation factors) and its individual variation. Therefore the calcium concentration for inducing hyperactivity had to be adjusted with respect to the seminal plasma used. To reduce individual variation, seminal plasma from several boars was pooled, divided into aliquots and stored frozen until use.
Human seminal plasma contains factors inhibiting sperm capacitation (Cross 1996). There is no definitive definition of capacitation in the literature, and several authors equate capacitation to the events leading to the acrosome reaction. However, if capacitation is more widely defined, comprising all processes of sperm maturation that make sperm ready for fertilization, and if hyper-activation is considered to be essential for fertilization, both hyperactivation and the acrosome reaction are two parts of capacitation (Yanagimachi 2003). There is some evidence that hyperactivation and acrosome reaction are induced by different extracellular factors. For example, Neill and Olds-Clarke (1987) have shown in mouse sperm that hyperactivation proceeds in the absence of BSA while acrosome-related processes are not observed (chlortetracycline assay). On the other hand, Mortimer et al.(1998) reported that hyperactivation of human spermatozoa is inhibited by 5% seminal plasma during capacitation in human tubal fluid whereas acrosome-related processes are observed (chlortetracycline assay). Intracellularly, however, both hyperactivation and the acrosome reaction seem to be triggered by the same signal (Ca2+; Yanagimachi 1994) possibly supported by other second messengers (e.g. cAMP).
Flagellar beat analysis
Flagellar beat angle (FBA) and the flagellar curvature ratio (FCR) seem to be apt parameters for defining hyperactive spermatozoa. The average FBA values for hyperactive boar spermatozoa (200°) are similar to those of hyperactive human (175°) and lower than those for ram spermatozoa (~330°). Hyperactive and non-hyperactive boar spermatozoa are well discriminated by the threshold values of FBA (87°) and FCR (0.7). The FBA threshold value is the same for boar and human spermatozoa (this work and Mortimer et al. 1997), but it is considerably higher for ram spermatozoa (128°; Mortimer & Maxwell 1999). The higher value for ram spermatozoa may be accounted for by the fact that the maximum amplitude develops closer to the head (Mortimer & Maxwell 1999). A significant decrease in FCR from 0.93 to 0.49 upon hyperactivation has been reported for boar spermatozoa (Suarez et al. 1992), which is in good agreement with the threshold value we obtained.
Multiparametric definition of hyperactive boar sperm for CASA
CASA requires threshold values to differentiate between non-hyperactive and hyperactive spermatozoa. The values reported for human (Mortimer & Swan 1995; for review see Mortimer 1997), ram (Mortimer & Maxwell 1999) and rat (Cancel et al. 2000) spermatozoa are different and reflect the species-specific patterns of sperm movements. Nevertheless, the commonly used parameters to identify hyperactive spermatozoa are a decrease in curvilinear velocity (VCL) and linearity (LIN) as well as an increase in lateral head displacement (LHD) (cf. Mortimer 1997). For boar spermatozoa a decrease in VSL and an increase in VCL, resulting in a reduced linearity, were reported upon hyperactivation (Suarez et al. 1992). Although there are some differences in absolute values, especially in VCL after hyperactivation, the tendency of the changes upon hyperactivation has been confirmed in the present study.
ROC analysis of selected spermatozoa suggests that the mean lateral head displacement (LHDmean) alone is sufficient to discriminate between hyperactive and non-hyperactive spermatozoa. However, this monoparametric criterion neither considers the increased curvilinear velocity generated by deeper flagellar bending nor the decreased linearity due to the asymmetric beat pattern (Burkman 1990). Therefore, like Burkman (1990) we prefer a multiparametric definition including, at least, VCL, LIN and LHD. The wobble (WOB) serves as an additional parameter due to its high sensitivity and specificity in ROC curve analysis.
Energy metabolism of boar spermatozoa
Hyperactivity increases the mechanical power of spermatozoa (Mortimer 1997) which is required for penetrating the zona pellucida of oocytes and consequently is vital for fertile mammalian spermatozoa (Yanagimachi 1994, Stauss et al. 1995, Mortimer 1997, Bedford 1998). The ATP supply of hyperactivity seems to require glycolysis. In the absence of glucose, hyperactivity is prevented in mouse spermatozoa even if lactate is present and oxidized in the mitochondria for ATP production (Cooper 1984, Bone et al. 2000). Lactate enables motility but cannot fuel hyperactivity. We assume that this effect is a result of the compartmentation of mitochondria in the short midpiece of the flagellum and the insufficient transport capacity for mitochondrial ATP along the flagellum (Kamp et al. 1996). The ATP supply in the long mitochondria-free part of the flagellum (principal piece) is likely met by glycolysis. Sufficient glycolytic capacity in the principal piece seems to be secured by sperm-specific glycolytic enzymes, some of which are tightly bound to the fibrous sheath surrounding the dense fibres and the axoneme along the whole principal piece (Westhoff & Kamp 1997, Eddy et al. 2003). Because hyperactivity is essential for sperm fertility, the regulation of hyperactivity and glycolysis are very likely coordinated and should both be studied in context. Inducing and quantifying hyperactivity in boar spermatozoa as presented in this paper will allow us to follow this line of research.
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Acknowledgements |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionAcknowledgementsReferences |
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Footnotes |
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionAcknowledgementsReferences |
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) spermatozoa was determined by multiparametric CASA. One hundred percent refers to the total number of spermatozoa analyzed (immotile, local motile and motile spermatozoa). The percentage of hyperactive spermatozoa was highest at 0.05 mmol–1 Ca2+. (b) Prediluted boar semen was centrifuged (500 g, 2 min) and the spermatozoa were resuspended in TALP-HEPES (50% v/v), BTS (25% v/v) and seminal plasma (25% v/v) yielding a final concentration of about 20 x 106 spermatozoa per ml. Hyperactivation was induced and analyzed as in (a). After 15 min incubation (37 °C), the percentage of motile (•) and hyperactive (•) spermatozoa was determined as in (a). The highest percentage of hyperactive spermatozoa was seen at 0.8 mmol l–1 Ca2+.