Objective evaluation of hyperactivated motility in rat spermatozoa using computer-assisted sperm analysis*

Aida M. Cancel1, Danelle Lobdell2, Pauline Mendola3 and Sally D. Perreault4,5

1 Toxicology Program, University of North Carolina, Chapel Hill, NC 27599, 2 Department of Social and Preventive Medicine, The State University of New York at Buffalo, Buffalo, NY 14214, 3 US EPA, Human Studies Division, Chapel Hill, NC 27599 and 4 US EPA, Reproductive Toxicology Division, Chapel Hill NC USA 27711, USA


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The aim of this study was to use computer-assisted sperm analysis (CASA) to examine changes in motion parameters of rat spermatozoa incubated under culture conditions that support IVF. Rat cauda epididymal spermatozoa were evaluated in six replicate experiments, at 0 and 4h of incubation. CASA was conducted at 60 Hz on digital 1s tracks (~100 spermatozoa/rat). Mean values of CASA parameters that describe the vigour of spermatozoa [curvilinear velocity (VCL), amplitude of lateral head displacement (ALH) and beat cross frequency (BCF)] increased, while those indicating progressiveness [straight line velocity (VSL), linearity (LIN) and straightness (STR)] decreased between 0 and 4 h. Visual inspection of sperm tracks after 4 h of incubation revealed classical hyperactivation patterns. Bivariate models were evaluated to objectively define the subpopulation of hyperactivated (HA) spermatozoa. Of all models considered, ALH and LIN, VCL and LIN, BCF and LIN, VCL and BCF, and VCL and ALH showed significant changes in the percentage of HA spermatozoa after the 4 h incubation period. The efficacy of detecting HA spermatozoa was evaluated using sperm tracks that were visually classified as HA or progressive. VCL and LIN provided the most accurate prediction of HA spermatozoa. It was concluded that analysis of CASA data using bivariate models could be used to detect and monitor hyperactivation in rat spermatozoa.

Key words: computer-assisted sperm analysis/hyperactivation/rat/spermatozoa


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Hyperactivated (HA) motion is a type of vigorous non-linear motion that mammalian spermatozoa exhibit as they progress through the female oviduct (Yanagimachi, 1994Go; ESHRE, 1996; Suarez, 1996Go). During hyperactivation, the pattern and vigour of the sperm track undergo dramatic changes, which are best characterized by movements in a random path and result in a non-progressive circular movement. These changes in motion are described as `whiplash' or `figure-8'. Several studies have suggested that HA allows spermatozoa to detach from the oviductal epithelium and provides increased thrust for penetration of the cumulus (Suarez et al., 1991Go; Suarez and Dai, 1992Go; Pacey et al., 1995Go). Recently, it has been shown that HA also facilitates penetration of spermatozoa through the oocyte zona pellucida (Stauss et al., 1995Go). Consequently, objective measures of HA can serve as biological end-points to evaluate the functional capabilities of spermatozoa.

Computer-assisted sperm analysis (CASA) provides the means for an objective classification of a given population of spermatozoa. Using digital images of each sperm track, CASA machines are able to analyse, by processing algorithms, the motion properties of spermatozoa. The commonly reported CASA parameters include curvilinear velocity (VCL), amplitude of lateral head displacement (ALH), beat cross frequency (BCF), average path velocity (VAP), straight line velocity (VSL), straightness (STR) and linearity (LIN). These CASA parameters have been modelled and refined mathematically to describe best the motion parameters of each spermatozoon as it travels through a microscopic field (Boyers et al., 1989Go).

Until recently, CASA of HA spermatozoa was difficult because of poor tracking efficiency. The relatively new use of 60 Hz combined with increased resolution of the optical system and better computers to allow longer tracking has made it possible to evaluate spermatozoa with high velocities and erratic tracks. Therefore vigour and pattern changes associated with HA spermatozoa can be detected using CASA. The tracks of HA spermatozoa would be expected to have increased amplitude of the ALH, increased VCL and decreased LIN. In addition, distribution based statistical methods are being applied successfully to characterize HA motility of hamster spermatozoa (S.D.Perreault, unpublished results). This distribution-based analysis has proven to be more powerful in determining changes in sperm motion that may not be detected using conventional mean-based statistical outcomes.

CASA has been used to characterize the motion of rat epididymal spermatozoa and changes in CASA parameters have been interpreted as indicators of testicular and epididymal toxicity (Toth et al., 1989Go; Slott et al., 1993Go; Perreault, 1998Go). As such, these CASA measures are made shortly (10–30 min) after collection and dilution of epididymal or ejaculated spermatozoa in medium that is not designed to support sperm capacitation and/or IVF. Freshly diluted rat epididymal spermatozoa swim in a progressive manner (fast and straight) and these are the end-points of interest in routine toxicology protocols (Perreault, 1998Go). However, examination of freshly diluted spermatozoa does not necessarily predict whether those spermatozoa can undergo HA and other changes required for successful fertilization. Therefore, it is desirable to develop assays for rat spermatozoa that can be reliable indicators of sperm function.

Hyperactivation of rat spermatozoa in the oviduct has been described (Shalgi and Phillips, 1988Go). However, limited information is available regarding the motion of rat spermatozoa during IVF and to date there are no published reports demonstrating HA motility of rat spermatozoa during in-vitro incubation. In this report, it was postulated that rat spermatozoa would exhibit HA motility after incubation under conditions that support IVF and that HA rat spermatozoa could be classified in an objective manner by using CASA parameters to identify those spermatozoa showing both increased vigour and decreased progression.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Animals
Sexually mature (>90 days old) Sprague–Dawley rats (Charles River Breeders, Raleigh, NC, USA) were allowed to acclimatize in an AAALAC-approved animal care facility (12 h light:12 h dark, 72°F) for at least 2 weeks prior to use. Animals were housed two per cage and provided access to Purina Laboratory Chow and tap water ad libitum. All investigations were conducted in accordance with the Guiding Principles for the Care and Use of Research Animals.

Preparation of rat spermatozoa and in-vitro culture conditions
Animals were killed by CO2 asphyxiation followed by cervical dislocation. The epididymis was removed, trimmed of fat and clamped at the corpus–cauda junction. Rat spermatozoa were obtained by puncturing the distal cauda epididymidis and allowing the spermatozoa to disperse in 3 ml of Armstrong's Rat Fertilization Medium (ARFM) supplemented with 4 mg/ml bovine serum albumin (BSA) (Fraction V: A-4503, Sigma Chemical Co., St Louis, MO, USA) (Evans and Armstrong, 1984Go). This Krebs–Ringer, a bicarbonate-buffered medium, has been shown to support rat IVF (Miyamoto and Chang, 1973Go; Toyoda and Chang, 1974Go; Perreault and Jeffay, 1993Go), and has been used successfully in hamster zona-free oocyte IVF using rat spermatozoa (A.M.Cancel, unpublished results). After culturing (37°C, 5% CO2 in air) for 30 min, to allow dispersion of the spermatozoa, the concentration of spermatozoa was determined by haemocytometer and aliquots of the sperm suspension (<100 µl) were transferred to 3 ml of fresh medium to give a final concentration of 3.3x104/ml. The incubation (37°C, 5% CO2 in air) was continued for 4 h, which is the minimum time for attachment of rat spermatozoa to and penetration of the zona pellucida during IVF (Shalgi et al. 1988). Aliquots of rat spermatozoa were withdrawn at the start (0 h) and end (4 h) of incubation, and analysed using CASA.

A complication that arises when studying HA spermatozoa during CASA analysis is that spermatozoa may stick to the glass cannulae producing tracking errors and preventing the accuracy of the motility analysis. Recommendations for using higher concentrations of BSA in the medium, to minimize sticking of spermatozoa to the glass cannula, have been published (ESHRE, 1998). To test whether a higher concentration of BSA in the medium may affect the detection and accurate measurement of HA motility, the rat fertilization medium was supplemented with 15 mg/ml BSA, which has been used for the evaluation of hyperactivation of hamster spermatozoa (Perreault and Jeffay, 1993Go).

CASA analysis
Six replicate experiments were conducted on 6 separate days. Suspensions of spermatozoa were loaded into flat 100 µm deep cannulae (HTR1099, VitroCom Inc., Mountain Lakes, NJ, USA) for CASA analysis using an HTM-IVOS motility analyser (Hamilton Thorne Research, Beverly, MA, USA), with software version 10.6. One-second tracks were captured at 60 Hz under x4 dark-field illumination, and saved as digital images to an optical disc. Instrument settings were: temperature = 37°C, video frequency = 60, magnification 0.82. Tracks were recalled later for analysis at 60 Hz, with minimum size = 7 pixels, minimum contrast = 65, low VSL cut-off = 20 µm/s, and low VAP cut-off = 30 µm/s. During analysis, the playback feature was used to identify and delete obviously mistracked spermatozoa as may occur due to collisions. Tracks of 30–60 points were accepted, while track fragments of <30 points were eliminated by setting the minimum track length at 30 points. This setting was justified because previous work has shown a high correlation between CASA measures based on 30 or 60 points for rat spermatozoa tracked under identical conditions (Dunson et al., 1999Go). Observations based on tracks with fewer than 30 points showed systematic biases. For each of six rats and each time-point, ~100 sperm tracks (mean, 96; range, 53–170) were imported into a Statistics Package for Social Sciences (SPSS) database for statistical analysis. The percent motile spermatozoa and percent HA spermatozoa were also determined subjectively using the playback option. To be classified HA by visual analysis, a spermatozoon had to clearly exhibit whiplash motion.

CASA parameters evaluated in this study were those provided by the HTM-IVOS motility analyser and defined as follows (HTM-IVOS Operation Manual): VAP: a smoothing of the path of the centre of brightness of the spermatozoon, which reduces the effect of the lateral head displacement; VSL: the distance between the first and last tracked point of the spermatozoon trajectory divided by the time elapsed; VCL: the sum of the distances between each centre of brightness, during each frame, divided by the time elapsed; ALH: the maximum value of the distance of any point on the track from the corresponding average path, multiplied by two; BCF: frequency with which the cell track crosses the cell path in either direction; STR: measures the departure of the cell path from a straight line. STR is derived from the ratio of VSL/VAP multiplied by 100; LIN: measures the straightness of the path. LIN is derived from the ratio of VSL/VCL multiplied by 100.

Analysis plan
Means for each CASA end-point were calculated for each rat and treatment at each time point. This preserved the rat as the unit of measure in the statistical analysis. These means were compared across time (0 versus 4 h) to detect motility changes in CASA end-points during in-vitro culture conditions in medium that has been shown to support rat IVF. Appropriate pairs of CASA end-points were selected for bivariate analysis, by including one measure of vigour (VCL, BCF or ALH) and one of progression (VSL, VAP, STR and LIN). Cut-off gates were selected based on the distribution of CASA data in six control samples (ARFM-4 mg/ml BSA) at 0 h. Since HA spermatozoa have high vigour and low progression, the cut-offs for vigour parameters were set at the 90th percentile and for progression at the 10th percentile. Spermatozoa were considered HA if they exceeded the cut-off for vigour and fell below the cut-off for progression. The percent HA spermatozoa for each bivariate model was defined as the number of spermatozoa that passed both criteria for the bivariate analysis divided by the number of tracked spermatozoa x100. Finally, the accuracy of track classification was examined using subjectively selected HA and progressive tracks (n = 35 each). Accuracy was defined as the percentage of HA (or progressive) tracks classified correctly by the bivariate model.

Statistical analysis
CASA measurements for each spermatozoon were imported into a statistical software package (SPSS 7.5 for Windows). Means for each treatment by time were calculated, as was the average change in each CASA parameter over time. A generalized linear model with repeated measures was used to analyse the main effects of time and treatment for each of the CASA parameters and for the bivariate indicators of hyperactivation. Paired sample t-tests were conducted to test the average change in each CASA parameter over time by treatment (4 mg/ml and 15 mg/ml BSA). Pearson correlation coefficients were calculated from the individual sperm tracks. Significance was set at P < 0.01 in consideration of the multiple comparisons in these analyses, unless otherwise specified.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Changes in individual CASA motility parameters
Motility parameters were measured in samples of spermatozoa at the beginning of the experiment (0 h) and after incubation (4 h) in ARFM with 4 mg/ml or 15 mg/ml BSA. The percentage of motile spermatozoa was maintained during the 4 h incubation. Mean values of CASA parameters changed as expected for spermatozoa with high vigour and low progression, characteristics of HA spermatozoa. The vigour parameters VCL, ALH and BCF increased, while progression parameters (VSL, VAP, STR and LIN) decreased (Tables I and IIGoGo). However, only mean BCF and LIN showed statistically significant changes over time. There was no significant difference in any parameter at either time when BSA was increased from 4 to 15 mg/ml.


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Table I. Mean CASA values of rat spermatozoa incubated in ARFM with 4 mg/ml or 15 mg/ml BSA
 

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Table II. Changes ({Delta}) in CASA parameters after 4 h incubation in ARFM
 
Subjective analysis of HA motion
Subjective evaluation of sperm tracks at 4 h yielded a small number of spermatozoa (~3%) exhibiting whiplash motion typical of hyperactivation (Figure 1CGo, Table IIIGo), but most of the spermatozoa either had progressive tracks typical of those visualized at 0 h (Figure 1AGo), or showed a transitional pattern (Figure 1BGo). In addition, it was common to see a spermatozoon switch between transitional and HA motion. This made it difficult to classify intermediate forms and suggests that classification based solely on whiplash motion may be overly strict. These subjectively determined HA sperm tracks were tallied and the percent of HA spermatozoa was determined for each animal. There was no significant difference in the percent HA spermatozoa between treatments with 4 mg/ml or 15 mg/ml BSA (Table IIIGo).



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Figure 1. Representative tracks and individual computer-assisted sperm analysis (CASA) parameters of rat spermatozoa during in-vitro capacitation. Sperm trajectories were reconstructed from the x,y co-ordinates obtained during CASA analysis.

VAP = average path velocity; VCL = curvilinear velocity; VSL = straight line velocity; STR = straightness; LIN = linearity; ALH = amplitude of lateral head movement; BCF = beat cross frequency.

 

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Table III. Mean ± SEM percentages of spermatozoa that meet the hyperactivated criteria using various bivariate models
 
Bivariate analysis of hyperactivated tracks
Bivariate models were applied in an attempt to use CASA-based end-points and provide an objective definition of hyperactivation for rat spermatozoa. Since CASA parameters LIN, VSL and STR had a high correlation coefficient among each other (r >= 0.80, P = 0.0001), and changes in the mean CASA values for LIN were statistically significant over time, only LIN was included in the bivariate analysis. Cut-off points for bivariate analyses using the remaining end-points were determined using distribution analysis of the spermatozoon tracks at 0 h (ARFM-4 mg/ml BSA). Gates were set at the 10th percentile for progression terms (LIN <=22% and VAP <=107.0 µm/s) and the 90th percentile for vigour terms (VCL >=388.7 µm/s, ALH >=29.6 µm and BCF >=33.3 Hz). Spermatozoa that exceeded the cut-off for vigour and fell below the cut-off for progressiveness were classified as HA, as illustrated in Figure 2Go.



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Figure 2. Bivariate model curvilinear velocity (VCL) and linearity (LIN). Representative sample of a bivariate analysis used to characterize hyperactivated rat spermatozoa. Open circles (m) represent CASA sperm values at the beginning of the experiment (t = 0 h) and closed circles (l) represent CASA sperm values after 4 h incubation. Lines represent 90th or the 10th percentile cut-off points. Hyperactivated spermatozoa located in the lower right quadrant exceeded the cut-off for VCL and fell below the cut-off for LIN.

 
Three bivariate models combining vigour and progression terms (LIN paired with VCL, ALH or BCF) showed statistically significant differences in the percentage of HA spermatozoa between 0 h and 4 h (Table IIIGo). Interestingly, bivariate models combining two vigour terms (`VCL and ALH' and `ALH and BCF') also showed significant increases in hyperactivation over time (Table IIIGo). The concentration of BSA did not significantly affect the percentage of HA spermatozoa at either 0 or 4 h.

The accuracy of these five bivariate models was evaluated against sperm tracks that were previously classified as HA or progressive (n = 35 each). Table IVGo shows the percent HA spermatozoa detected by these models. Models `LIN and BCF' and `ALH and BCF', identified only 42.9% HA spermatozoa; whereas `VCL and LIN', `ALH and LIN' and `ALH and VCL' correctly identified 94.3, 85.7 and 91.4% HA spermatozoa respectively.


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Table IV. Accuracy of bivariate models to correctly identify hyperactivated spermatozoa*
 

    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Hyperactivation has been described in spermatozoa from many species after incubation under in-vitro culture conditions, but not in the rat. Several investigators have studied the movement of rat spermatozoa during in-vitro culture conditions (Moore and Akhondi, 1996Go; Oberländer et al., 1996Go) but did not report on HA motility. This study provides the first evidence of hyperactivation of rat spermatozoa observed during in-vitro culture conditions typically used for rat IVF. A potentially important component of the analysis strategy was the use of a higher image sampling frequency (60 Hz) than was used in most previous studies (25 or 30 Hz). The increased image sampling frequency in the current studies provided improved track definition. Indeed, for uncapacitated rat spermatozoa, all CASA measures except VSL have been documented to be extremely sensitive to tracking rate, comparing 60 and 30 Hz (Dunson et al., 1999Go). Furthermore, it has been shown that the use of 60 Hz allows better discrimination than 30 Hz between forward progressive motility and HA motility in human spermatozoa (Mortimer and Swan, 1995).

In an attempt to define HA rat spermatozoa objectively, current CASA guidelines were followed which recommend the use of multiparametric kinematic definitions to classify individual spermatozoa into specific subpopulations (ESHRE, 1996, 1998). In doing so, it was postulated that one or more bivariate models that included both a vigour term and a progression term would provide a better definition of hyperactivation than reliance on a single parameter. Using a distribution approach, cut-off gates were defined for each CASA parameter in an objective manner; these were used in bivariate analysis and it was found that several bivariate models could be used to define and quantify a subpopulation of HA rat spermatozoa. The use of such a distribution-based analysis to characterize subtle changes in the motion characteristics of spermatozoa has been recommended previously (Toth et al., 1989Go; Gladen et al., 1991Go).

The model containing VCL and LIN had the highest accuracy for defining HA sperm tracks that agreed with visually classified whiplash sperm tracks. Models containing VAP, which represents the general trajectory of the spermatozoa corrected for time, did not identify changes in HA between 0 and 4 h incubation. Indeed, while CASA means for VAP decreased over time, VAP values from tracks of selected HA spermatozoa increased during the 4 h incubation. It is important to note that VAP may impact on other CASA measurements that are defined with reference to the average path of spermatozoa. The two CASA measurements most likely to be influenced by VAP are ALH and BCF. In addition, ALH is sometimes defined as the mean amplitude rather than the maximum amplitude of the track (Mortimer, 1997Go). Since VAP and ALH are defined by an instrument-specific algorithm, and VAP influences BCF and ALH, bivariate models containing these parameters may need to be verified when using other CASA instruments. Based on this study, use of the model containing VCL and LIN is recommended, since it is very accurate in defining HA tracks and is based on end-points that are less likely to vary across different CASA instruments.

The bivariate models described herein categorize as HA about 20% of the sperm tracks analysed after a 4 h incubation. These CASA-derived estimates of HA spermatozoa were higher than the subjective classification (~3%). The most likely explanation for this difference in percentage HA is that the subjective evaluation was based on the single, strict criterion that spermatozoa exhibit whiplash motion during the 1 s playback option. Therefore, rat spermatozoa exhibiting transitional motion were not scored as HA during the subjective classification. The transition from progressive to whiplash motion occurs as sperm tracks become circular and circumscribe a smaller and smaller circumference; at the same time, the amplitude of the track increases. Furthermore, spermatozoa may switch back and forth between transitional and whiplash motion (Suarez, 1996Go). Transitional forms of motion are well described in hamster and human spermatozoa (Suarez, 1988Go; Mortimer and Swan, 1995) and were clearly evident in rat spermatozoa during the conduct of this study. The bivariate analyses, with the cut-off points set at a statistically defined place, allow inclusion of transitional spermatozoa based on their kinematic characteristics. Indeed, a similar approach using two end-points has been used to evaluate hyperactivation of mouse (Neill and Olds-Clarke, 1987Go) and bovine (McNutt et al., 1994Go) spermatozoon tracks, while three or more end-points have been used to characterize HA primate (Mahony and Gwathmey, 1999Go), human (reviewed in Mortimer, 1997), boar (Abaigar et al., 1999Go) and ram (Vulcano et al., 1998Go) sperm tracks. The percentage of rat HA sperm tracks obtained was low relative to mouse and hamster, where >50% HA spermatozoa have been reported (Suarez et al., 1991Go; Suarez and Dai, 1992Go; Yanagimachi, 1994Go; Suarez, 1996Go). A low percentage of HA rat sperm was also observed at earlier and later time points up to 6 h (data not shown). However, the percentage of HA rat sperm was similar to that observed for human spermatozoa, where low percentages of HA spermatozoa can be found at any given time during in-vitro culture conditions.

Increasing the concentration of BSA to 15 mg/ml during in-vitro culture does not appear to alter the frequency of detection of HA spermatozoa during CASA analysis. Although BSA appears to be an important component for rat spermatozoa capacitation, it does not affect the percentage HA spermatozoa, indicating that a range of BSA concentrations can be used when studying hyperactivation.

In summary, it was shown that rat spermatozoa undergo changes in motion, during in-vitro culture conditions in media that has been shown to support rat IVF, that are characteristic of hyperactivation as described in other species. Using CASA analysis of rat spermatozoa, an increase in mean vigour and a decrease in mean progression over a 4 h period of time were found under conditions that also support rat IVF. Finally, it was demonstrated that bivariate models using two CASA parameters, one for progression (LIN) and one for vigour (VCL) can be used to define HA tracks in an objective and accurate manner. This subpopulation probably represents spermatozoa with fertilizing ability.

Significant relationships have been successfully determined between HA of human spermatozoa and IVF or pregnancy rates in donor insemination (Wang et al., 1993Go; Johnston et al., 1994Go). Indeed, evaluation of HA human spermatozoa has been suggested as a prognostic end-point to test sperm fertility (Mortimer, 1997Go). Standardization of methods, such as the one described here, allow accurate and objective identification of HA spermatozoa. These methods can now be further developed and applied to studies of sperm function and may be useful in toxicological studies after reproductive toxicant exposure.


    Acknowledgments
 
The authors thank Susan Jeffay for her technical assistance. We also thank Dr David F.Katz (Department of Biomedical Engineering, Duke University, Durham, NC) for his helpful discussions. This work was funded by the EPA/UNC Toxicology Research Program, Training Agreement CT902908, with the Curriculum in Toxicology, University of North Carolina at Chapel Hill.


    Notes
 
* The research described in this article has been reviewed by the Health Effects Research Laboratory, US Environmental Protection Agency, and approved for publication. Approval does not signify that the contents necessarily reflect the views and policies of the agency nor does mention of trade names or commercial products constitute endorsement or recommendation for use. Back

5 To whom correspondence should be addressed at: US EPA, NHEERL MD 72, Research Triangle Park, NC 27711, USA.E-mail: darney.sally{at}epa.gov Back


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 Results
 Discussion
 References
 
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Submitted on October 20, 1999; accepted on February 29, 2000.