Fluorometric Assessments of Acrosomal Integrity and Viability in Cryopreserved Bovine Spermatozoa

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1 BIOLOGY OF REPRODUCTION 56, (1997) Fluorometric Assessments of Acrosomal Integrity and Viability in Cryopreserved Bovine Spermatozoa Cheryl A. Thomas, 3 Duane L. Garner, 2 ' 3. Mel DeJarnette,4 and Clifton E. Marshall 4 School of Veterinary Medicine,/ University of Nevada, Reno, Select Sires Inc.,4 Plain City, Ohio ABSTRACT The combination of specific fluorometric staining and flow cytometry provides a rapid and precise means of assessing the functional status of cells. We sought to utilize this approach to quantify two important seminal characteristics, acrosomal integrity and sperm viability, and to compare these with classical microscopic measurements of acrosomal integrity and sperm motility. Samples of thawed, cryopreserved sperm packaged in 0.5-mi French straws were obtained from 12 Holstein bulls. Classical acrosomal assessments and sperm motility estimates were made using differential interference contrast microscopy. Fluorescent acrosomal probes included LysoTracker Green DND-26 (LYSO-G), fluorescein-labeled peanut agglutinin, the biotinylated isocoumarin serine protease inhibitor Bi-Aca-Aca- OMe-IC that was secondarily labeled with fluorescein-avidin, and rabbit antibodies to bovine acrosin that were secondarily labeled with fluoresceinated anti-rabbit immunoglobulin. The fluorescent probes for sperm viability were a combination of SYBR-14 and propidium iodide (PI) or of SYTO-17 and P. Significant differences were found among methods and among bulls, but not among straws (n = 3). All four fluorescent measures of acrosomal integrity showed highly significant correlations with both classical measurements. These data indicated that the quality of cryopreserved bovine sperm samples could be readily quantified using a variety of organelle-specific fluorescent staining techniques. INTRODUCTION There is a strong relationship between fertility, post-thaw motility [ 1, 2], and the percentage of normal acrosomes [2, 3]. A spermatozoon must retain a normal acrosome so that the acrosome reaction can occur at the proper time to facilitate fertilization. The vesiculation of the acrosomal and plasma membranes occurs during cell death. This is termed a false acrosome reaction. A true acrosome reaction, which precedes fertilization, occurs only in live, intact spermatozoa. Thus, it is important not only to analyze semen for sperm viability but also to determine the normalcy of the acrosomes simultaneously [2]. Such a dual assessment, however, can be especially difficult when spermatozoa are highly motile [4]. The percentage of spermatozoa with normal acrosomes can be effectively determined in fixed specimens using differential interference contrast microscopy (DIC) to identify the ridge along the rostral edge of the acrosome. This evaluation is sometimes coupled with an independent microscopic assessment of the percentage of progressively motile spermatozoa as a measure of sperm Accepted November 18, Received August 29, 'Supported in part by USDA NRIGP grant and the Nevada Agricultural Experiment Station Hatch project Correspondence: Duane L. Garner, School of Veterinary Medicine/ 202, University of Nevada, Reno, Reno, NV FAX: (702) ; dgarner@scs.unr.edu 991 Nevada viability. Although useful, these classical techniques are time consuming and are inherently variable. Flow cytometry has certain advantages over classical laboratory tests for evaluation of spermatozoa. This automated system has the ability to quantify spermatozoa in less than 2 min while simultaneously examining individual spermatozoa for viability and acrosomal status [5, 6]. Also, it can provide information on the quality and metabolic states of the cells on a nearly online time scale [7]. Such an automated approach also improves the repeatability of the assay because a much larger number of cells can be analyzed relative to the few hundred spermatozoa analyzed using visual microscopic methods. The objective of this study was to determine whether the various fluorometric measures of acrosomal status differed among themselves and also whether they differed from the classical assessment of acrosomal integrity, the percentage of normal acrosomes (NAR) as determined by DIC. A secondary objective was to compare fluorometric measures of sperm viability and to ascertain whether they differed from the classical assessment of viability, the percentage of motile spermatozoa (MOT). We examined five fluorescent probe combinations specific for intra-organelle ph, acrosin both enzymatically and immunologically, lectin binding, and sperm viability. The acidic nature of the intact acrosome [8] suggests that acidophilic probes such as LysoTracker Green DND-26 (LY- SO-G) may be useful in assessing the integrity of the acrosome. The recent development of irreversible serine protease inhibitors [9] has provided another useful fluorescent measure of acrosomal integrity that can be quantified [6]. Exposed acrosin can also be readily quantified using secondarily labeled anti-acrosin antibodies [10]. The glycoprotein character of the acrosomal components provides the fourth means of measuring acrosomal integrity by fluorescently labeling the acrosomal matrix of acrosome-reacted spermatozoa with tagged lectins [5]. The percentages of dead spermatozoa were quantified simultaneously with the acrosomal assessments by staining with propidium iodide (PI). The sperm samples were also examined to determine the proportion of viable spermatozoa separately from acrosomal assessments. This study utilized acrosomal probes [5, 6, 9, 10] and a viability probe [11] as well as the two novel probes LYSO-G and SYTO-17, which identified acrosomal integrity and viability, respectively. MATERIALS AND METHODS Reagents Reagents were obtained from the following sources. Molecular Probes, Inc. (Eugene, OR) was the supplier of PI (P-1304); LYSO-G (L-7526); SYTO-17 (S-7579); avidin- NeutraLite (A-2662); fluorescein goat anti-rabbit IgG (H + L) conjugate (F-2765); and SYBR-14, available together with PI as LIVE/DEAD Kit (L-7011). Sig-

2 992 THOMAS ET AL. ma Chemical Co. (St. Louis, MO) was the source of Tyrode's Salt Solution (T-2397), fluorescein-labeled lectin from Arachis hypogaea (PNA-FITC; L-7381), Triton X-100 (T-9284), Hepes (H-3375), BSA (fraction V, A-4503), and para-aminobenzamidine (A-7148). Anhydrous dimethyl sulfoxide (DMSO) was purchased from Aldrich Chemical Co. (Milwaukee, WI; 27,685-5). The biotinylated isocoumarin serine protease inhibitor Bi-Aca-Aca-OMe-IC (acrosin/bic) was developed in and obtained from the Powers laboratory (Georgia Institute of Technology, Atlanta, GA) [9]. Anti-acrosin, which was produced in this laboratory in 1974 [10], was reconstituted from lyophilized immunoglobulins. Semen Samples Ejaculated semen was collected from 12 bulls housed at Select Sires, Inc. (Plain City, OH). The ejaculates were diluted, processed, and cryopreserved in a 20% egg, 2.9% sodium citrate medium and packaged in 0.5-ml French straws at a concentration of 35 x 106 sperm/ml. The samples were collected and cryopreserved over a period of months before being selected to provide a range of semen quality. Aliquots of these selected samples (three straws) were analyzed for the classical seminal quality parameters while the remainder were shipped to Reno, Nevada, in liquid nitrogen (- 196 C) for flow cytometric analyses. Microscopic Examinations of Acrosomal Integrity and At Select Sires, the percentage of spermatozoa with normal acrosomal ridges (NAR) was determined using the methods of Saacke and Marshall [12] and Marshall [13]. These were classified in triplicate on 300 spermatozoa from the thawed, cryopreserved samples using DIC. These assessments were made after the three straws were thawed and again after they were incubated for 3 h at 37 C. The percentages of progressively motile spermatozoa (MOT), also in triplicate, were estimated visually using a thermostatically warmed stage (37 C) and light microscopy at x 10 magnification. Motility estimates were made immediately after the straws were thawed in 37 C water and again after 3 h of incubation at 37 C. Fluorescent Examinations of Acrosomal Integrity At the Flow Cytometry Center at the University of Nevada, Reno, spermatozoa were analyzed using fluorescent staining and flow cytometry. Three straws from each bull were thawed at 37 C in a CITO Warm Water Thaw (CITO Products, Inc., Watertown, WI) and put into warmed microcentrifuge tubes. The 0.5-ml assay volumes consisted of 167.l 1 thawed semen dispensed into 333 p. 1 of warmed Hepes containing 0.1% BSA (Hepes/BSA) [14] and the appropriate fluorescent stains. The experiments described hereafter refer to the fluorometric assessments that were all conducted at 37 C. Acrosomal ph levels. In this experiment, acrosomal integrity was examined through the use of two stains to quantify the green population of spermatozoa with intact acrosomes and those dead spermatozoa that stained red with PI. LYSO-G (FW ), which is an acidotropic probe that stains acidic organelles green, identified living spermatozoa with intact acrosomes. PI (2 mg/ml Tyrode's Salt Solution) stained the nuclei of spermatozoa having degenerate membranes (dead spermatozoa). A stock solution containing stain of 9.0 L 1 I PI and 5.0 pi 1 mm LYSO-G per milliliter Hepes/BSA was prepared and warmed to 37 C. After addition of semen, samples were incubated at 37 C for 30 min before analyses. Acrosin activity. Acrosomal integrity was investigated using the serine protease inhibitor, BIC, and the green fluorescein probe, avidin-neutralite. The serine protease, acrosin, irreversibly binds BIC. Avidin-NeutraLite was then used to localize the biotinylated inhibitor. Thus, BIC serves as a specific connector, binding acrosin to the fluoresceinlabeled avidin. Because acrosin is accessible only when the integrity of the acrosomal membranes has been breached, a second aliquot from each straw was treated with Triton X-100 to permeabilize the membranes of all spermatozoa. Sperm nuclei were identified by counterstaining with PI. In a previous study using this combination [6], semen was extended in a milk-based medium. We found that eggbased medium interfered with the binding of avidin- NeutraLite, therefore necessitating a wash step to isolate the spermatozoa from the medium (data not shown). Percoll gradient centrifugation [14] allowed separation with minimal damage to the spermatozoa. Percoll gradients (0-62.5%) were prepared in microcentrifuge tubes and briefly warmed to 37 C. One-half milliliter of thawed semen diluted 1:3 in Hepes/BSA was applied to the top of the gradients and centrifuged for 20 min at 620 x g. The supernatant was aspirated, and the pelleted spermatozoa were resuspended in 0.6 ml warmed Hepes/BSA. From this 0.6 ml, two 0.25-ml aliquots were transferred into two new microcentrifuge tubes for comparison of thawed, stained fractions with their detergent-treated counterparts. After the additions of 0.5.1I para-aminobenzamidine and 1.5 pi. PI, samples were incubated for 15 min at 37 C. Then 0.17 p.1 of BIC was added to all samples, with the treated samples receiving of Triton X-100. After 30 min of incubation, avidin-neutralite was added, and incubation was performed for another 30 min. Lectin binding. In order to determine which sperm populations appearing on cytograms were those labeled by the new tri-staining combination SYTO- 17, PI, and PNA-FITC, a live:freeze-killed standard curve was constructed. The SYTO-17 is a red nucleic acid stain specific for living cells (0.5 M diluted 1:10 in DMSO). While PI is specific for dead cells and is also a red stain, its fluorescence intensity was much greater than that of SYTO-17; thus two separate. definable red-labeled populations were produced (Fig. 1, left). When green stain was added (1 mg PNA-FITC/ml Tyrode's Salt Solution), three distinct populations appeared: red live, red dead, and red dead with green acrosomes (Fig. 1, right). For the standard curve, semen was filtered in glass wool/ sephadex columns to remove the dead spermatozoa and debris [11]. Half of the filtrate was freeze-killed three times in liquid nitrogen vapor. As a control, cytometric analyses were done on 100:0, 50:50, and 0:100 live:killed percentages of spermatozoa (Fig. 1). In the 12-bull assay using the tri-staining protocol, each straw was split into two fractions to determine the differences in the distributions of stained sperm populations in thawed samples and freeze-killed samples (Fig. 2). These differences showed the proportion of living spermatozoa possessing intact acrosomes. Microcentrifuge tubes of 1 ml Hepes/BSA were warmed to 37 C, to which 0.5 ml semen from each thawed straw was added. From these, 0.5-ml paired samples were designated as thawed or freeze-killed. All samples received 1.0

3 ACROSOMAL INTEGRITY AND VIABILITY OF BOVINE SPERMATOZOA 993,a 1 para-aminobenzamidine to prevent proteolysis from the enzyme, acrosin, which leaked out of degraded acrosomes in dying or dead spermatozoa. Each sample also received 3.0 V1 PI, 2.0 I.l SYTO-17, and 0.5 pl 1 PNA-FITC. While the stained, thawed samples were incubating for 15 min, their freeze-killed counterparts were exposed to liquid nitrogen vapors for 5 min, thawed, and freeze-killed two more times before being stained and incubated for 15 min at 37 0 C. Immunological assessment of acrosomal integrity. In this trial, rabbit anti-acrosin antibody [10] was used to bind exposed acrosin molecules. The antibodies were then secondarily labeled with fluorescein isothiocyanate (FITC)-labeled anti-rabbit IgG. PI and SYTO-17 were added as counterstains to identify dead and living sperm populations, respectively. A stock solution of stained Hepes/BSA containing 3.0 L 1 para-aminobenzamidine, 6.0 R1 SYTO-17, and 9.0 pl1 PI per milliliter Hepes/BSA was warmed to 37 C. The stock solution was used to dilute semen 1:3 for 0.25-ml final sample volumes for paired thawed and freeze-killed portions. After the treated samples were freeze-killed as described above for PNA-FITC, samples were treated with anti-acrosin and incubated for 15 min. Four microliters of goat anti-rabbit acrosin/igg was added, and samples were incubated 30 min. FIG. 1. Dot plots showing the effects of the stain combinations SYTO-1 7 plus PI, and SYTO-1 7/PI plus PNA-FITC. These combinations produced cytograms exhibiting two and three populations, respectively. Semen from the control bull was used to construct a standard curve by combining sephadex-filtered (live) and filtered freeze-killed semen in 100:0, 50:50, and 0:100 proportions for both the dual- and tri-stained samples. Fluorescent Staining to Assess In this experiment, the percentages of viable spermatozoa were quantified using the green fluorescent stain, SYBR-14, to identify living spermatozoa. Living spermatozoa are defined as those spermatozoa that stain with SYBR-14 while resisting the uptake of PI that stains the nuclei of killed or dead spermatozoa red. Because this loss in permeability is gradual as spermatozoa die, a small portion of spermatozoa are doubly stained with both SYBR-14 and PI. A stock solution was prepared containing 0.8 p.1 SYBR-14 and 9.0 p1i PI per milliliter Hepes/BSA. This was warmed to 37 C and used to dilute semen 1:3. Stained spermatozoa were incubated at 37 C for 15 min. Flow Cytometry Information on events was collected in list mode on a FACS Analyzer flow cytometer (Becton-Dickinson, Sunnyvale, CA). The generated data were then analyzed using a Hewlett Packard Consort 30 (Beckton-Dickinson), a 200-series computer, and SuperCyt Analyst 3 software (Sierra Cytometry, Reno, NV). The flow cytometer was equipped with 1) a standard FITC and phycoerythrin dichroic filter set; 2) LP 400 long-pass and DF 485/22 bandpass excitation filters at 485 nm; 3) a DM-560 dichroic mirror to separate fluorescent signals; 4) photomultiplier tube 1 (FL1) collecting 530 nm light through a DF 530/30 band-pass filter; and 5) FL2 collecting light through an LP 570 filter. Compensation was used to minimize spillover of FIG. 2. Dot plots (left) and histograms (right) showing populations of spermatozoa from thawed and freeze-killed samples from bulls 1 and 12. Shown are spermatozoa labeled with SYTO-17, PI, and IgG-FITC. Each histogram displays both the thawed and freeze-killed frequency distributions of the dot-plot populations. The black area represents the amount of change in the frequency distribution that occurs in a thawed sample after the freeze-kill treatment.

4 994 THOMAS ET AL. TABLE 1. Mean percentages ( SD) obtained by microscopic and fluorometric methods of assessing semen quality in 12 bulls. Microscopic examinations Acrosomal Fluorescent examinations status Viability Acrosomal status Viability Bull # NARI" MOT' MOT3 d LYSO-G, Acrosin/BIC PNA-FITCg Acrosin/lgGh SYBR ^ ± ± 4.8CD " 72 ± 2.9 Be 76 ± 1.2AB ± 9.0A ± " A A " ± 0.6 A : 77 ± _ ' _ " " ± A 71 ± 2.8 A B 73 ± 1.6A _ 6.7" ± ' A 61 ± 2.7 B 70 ± 0.6AB 62 ± " 77 ± A l K' 76 ± 1.1" 54 ± ' 70 _ " 62 ± 2.2' 68 ± " ± ± " 65 ± ) 9 57 ± ± ± ± 6.8AB AB 71 ± 2.4A 63 ± ± 5.0K : ± 2.8B 39 _ 1.6' 36 ± 1.4" 41 ± 1.6" ± 7.5" 37 ± ' 50 ( 6.6" 28 ± 0.9B" 35 ± ± _ 4.7' 27 _ A 23 ± 0.9" " Mean ±SEM ± d Among assessments for normal acrosomes, means within bull (row) with the same capitalized superscript do not differ (p > 0.05). 'The proportion of spermatozoa with normal acrosomal ridges. The proportion of spermatozoa with progressive forward motility immediately after thawing. 'A The proportion of spermatozoa with progressive forward motility 3 h after thawing. ' The proportion of spermatozoa that were stained with LysoTracker Green. 'The proportion of spermatozoa that bound the serine-protease inhibitor BIC. g The proportion of spermatozoa that stained with PNA-FITC. hthe proportion of spermatozoa that were tagged with anti-rabbit antibody to bovine acrosin and secondarily labeled with fluoresceinated anti-rabbit IgG. The proportion of viable spermatozoa as indicated by staining with SYBR-14. red fluorescence into the green channels. All analyses utilized this filter combination. Cytogram Analyses Two methods of cytogram analysis were used. For the acrosin/bic, acrosin/igg, and PNA-FITC experiments, the difference in the distributions of green fluorescence in histograms of thawed and freeze-killed (or Triton X-100-treated) samples was computed using the "Overlay Histograms" option of the SuperCyt Analyst 3 program (Sierra Cytometry) (Fig. 2). This function is based on the Kolmogorov-Smirnov two-sample test for the analysis of histograms from flow systems [15]. The LYSO-G and SYBR-14 experiments used the gating function to quantify the percentage of spermatozoa in the live green and dead red populations. Statistical Analyses Semen from each bull, for all five experiments, represented a homogenous pool from which 0.5-ml aliquots were packaged and cryopreserved in straws. The experimental design was a split plot in randomized complete block design (RCBD). The main plot included bulls 1 through 12, with treatments (trt: NAR, LYSO-G, acrosin/bic, PNA- FITC, acrosin/igg) as the subplots within bulls. Straw*bull was used to calculate the error term to partition out the effects of bulls. Bulls were chosen for the main plot where the degree of precision required was less than that of the subplots, because differences among bulls have been well established. Since the experimental objective was to compare methods that examine acrosomal integrity, treatments were assigned to the split plots to provide greater precision. The experimental units (replications) were straws, and the sampling units were individual spermatozoa. Within-bull ANOVA were conducted using an RCBD experimental design blocking straws to test the effects of treatments. Duncan's Multiple Range test grouped treatments that were not different (p > 0.05). The General Linear Models procedure of Statistical Analysis Systems (SAS) adjusted the sums of squares for outliers that were deleted from the data set. The least-squares means procedure of SAS calculated adjusted means and standard deviations for data with missing observations. All data were arcsine-transformed to remedy the problem of unequal variances. The relationships among variables were evaluated using correlation coefficients generated by SAS [16]. RESULTS Microscopic Examinations of Acrosomal Integrity and For all analyses, variables were randomized. However, for clarity of presentation, bulls have been rank ordered by percentages of spermatozoa with normal acrosomes as determined by DIC. All other methods of assessing acrosomal status and viability were compared against this standard. Mean percentage NAR, initial motility (MOT), and motility after 3 h (MOT3) for each bull, along with overall means and standard errors, are listed in Table 1. Bulls 10, 11, and 12 consistently ranked lowest in sperm quality across all methods. Bulls I and 4 most frequently ranked highest. The remaining bulls ranked in varying order among methods. Duncan's Multiple Range test of mean MOT indicated that bulls 1-9 did not differ and that bulls did not differ (p > 0.05). Within other methods, more complex groupings were observed. All microscopic methods showed a high degree of correlation with fluorometric methods (Table 2). Among values obtained with microscopic methods, NAR was most highly correlated with motility after 3 h (r = 0.99; data not shown).

5 ACROSOMAL INTEGRITY AND VIABILITY OF BOVINE SPERMATOZOA 995 TABLE 2. Correlations among microscopic and fluorometric methods of assessing semen quality in 12 bulls. Microscopic examinations Fluorescent examinations Fluorescent NARa MOTb MOT3c LYSO-Gd Acrosin/BICe PNA-FITC Acrosin/lgGg examinations (r) (r) (r) (r) (r) (r) (r) LYSO-Gd Acrosin/BICe PNA-FITC f Acrosin/lgGg SYBR-1 4h The proportion of spermatozoa with normal acrosomal ridges. b The proportion of spermatozoa with progressive forward motility immediately after thawing. c The proportion of spermatozoa with progressive forward motility 3 h after thawing. d The proportion of spermatozoa that were stained with LysoTracker Green. The proportion of spermatozoa that bound the serine-protease inhibitor BIC. The proportion of spermatozoa that stained with PNA-FITC. e The proportion of spermatozoa that were tagged with anti-rabbit antibody to bovine acrosin and secondarily labeled with fluoresceinated anti-rabbit IgG. fthe proportion of viable spermatozoa as indicated by staining with SYBR-14. Fluorescent Examinations of Acrosomal Integrity and Microscopically, spermatozoa identified with LYSO-G stained a dull green over the entire sperm head and the mitochondria. Within approximately 30 sec after the beginning of sperm death, the green fluorescence was replaced by red fluorescence from the PI, with the acrosome being the last part of the spermatozoa to lose green fluorescence (Fig. 3a). Those fluorescent probes that attached to the spermatozoa by means of enzymatic or ligand binding, acrosin/ BIC, PNA-FITC, and acrosin/igg, all appeared as a green fluorescent cap over a red sperm head (Fig. 3, b-d). The probe used for detecting viable spermatozoa, SYBR-14, stained a bright green over the sperm head and a less intense green over the mitochondria and tail portion (Fig. 3e). When sperm death began, the process of SYBR-14 replacement by PI took somewhat longer (> than 30 sec) than observed in LYSO-G-stained samples. The elevation of acrosomal ph, which would diminish the green fluorescence, apparently occurred more rapidly than did the replacement of SYBR-14 by PI. Statistical analyses showed that after the data were arcsine-transformed, assumptions for equal variance were met, and scaling was adequate. However, three outliers were detected. Data were reanalyzed with these three values deleted. There were differences among tests and bulls (p < 0.001), but straws did not differ (p = 0.20). There were significant interactions between bulls and treatments (p < 0.001); the comparison of treatment means (methods of assessing normal acrosomes) depended on individual bulls, and the comparison of bulls depended on treatment. Therefore, overall means could not be compared. The mean square errors (MSE) of arcsine-transformed data for bulls and treatments were 7.7 X 10-4 and 8.3 X 10-4, respectively. Within-bull ANOVA showed differences among the tests for acrosomal status (p < 0.001) but not among straws (p > 0.05). Duncan's Multiple Range test showed dissimilar groupings within bulls, as noted in Table 1. Table 1 displays mean percentages ± SD (untransformed data) of microscopic and fluorometric assessments of acrosomal status and sperm viability. It should be noted that the mean values of NAR reported for bulls 1, 7, and 12 include outliers. Adjusted least-squares means for these three bulls were , , and , respectively. Among tests for viability, interactions were not significant (p = 0.29). The mean percentage of MOT, quantified using the microscopic method, and the mean percentages of fluorometric estimates of viability, SYBR-14, LYSO-G, and SYTO-17, were 66%, 51%, 48%, and 42%, respectively. Both MOT and SYTO-17 differed from values obtained with all other methods. The mean percentages of LYSO-G-stained spermatozoa and SYBR-14-stained spermatozoa did not differ. The cytograms of Figure 1 show that the percentages of live, SYTO-17-stained spermatozoa in ratio groups of 100: 0, 50:50, and 0:100 live:killed were 84%, 37%, and 4%, respectively. The corresponding groups of PI-stained and PNA-FITC-labeled populations were 7%, 6%, 3% and 9%, 57%, and 93%, respectively. Presumably, the 4% of spermatozoa that were gated for SYTO-17 in the 0:100% live: killed fraction were unstained; microscopic examination did not reveal any living spermatozoa in this fraction. The PI/SYTO-17 combination was also used in the acrosin/igg experiment and showed the same dot-plot patterns as the PNA-FITC labeling. Bull 1 (Fig. 2, left) represented a bull of high semen quality with 69% living spermatozoa and 16% of spermatozoa exhibiting degraded acrosomes. In contrast, bull 12 represented a bull of low semen quality. His semen showed a small percentage of live spermatozoa (5%) and a large percentage that had degraded acrosomes (65%). After freeze-killing, the proportions of living spermatozoa were similarly low in the two bulls. DISCUSSION Microscopic Examinations of Acrosomal Integrity and In an effort to improve the precision and reliability of semen analysis, new fluorophores are continually being examined with respect to their suitability for assessing seminal quality. In this investigation, we compared several staining techniques for determining acrosomal status, along with sperm viability, with the microscopic methods that are routinely used in commercial bovine semen-processing organizations. The standard NAR evaluation of acrosomal integrity by DIC was compared with the fluorescent methods as shown in Figure 4. Generally, the percentage of spermatozoa with normal acrosomes, relative to the values obtained using fluorometric methods, appeared to be understated in bulls

6 996 THOMAS ET AL. ^^ so80 a 60 e U) so. 40 ' Bull FIG. 4. Line graphs comparing the estimates of mean percentages of spermatozoa with normal acrosomes (n = 3), obtained using microscopic and fluorometric methods. The microscopic method of estimating the proportion of spermatozoa with normal acrosomal ridges (NAR) was performed using DIC microscopy and visually evaluating 300 spermatozoa after incubation at 37 C. The remaining four line graphs were produced using four different fluorophores that label intact acrosomes (LYSO-G) or degraded acrosomes (PNA-FITC, acrosin/bic, and acrosin/lgg). Percentages represent the proportion of spermatozoa that were quantified using flow cytometry. Among values obtained with microscopic methods, NAR was most highly correlated with motility after 3 h (r = 0.99; data not shown). This is probably the case because some of the acrosomal degeneration noted in moribund spermatozoa was not readily apparent when sperm motility was examined initially (MOT). FIG. 3. Fluorescence microscopy displaying spermatozoa stained with fluorescent probes to identify acrosomal status and sperm viability. a) Spermatozoa with intact acrosomes analyzed using LYSO-G. The spermatozoa are in the process of cell death and thus replacement by red fluorescence from PI. The acrosome is the last portion of the spermatozoa to lose green fluorescence. b) A spermatozoon with a degenerated acrosome identified by the green fluorescein probe, avidin-neutralite, attached to the spermatozoa by means of the serine protease inhibitor BIC. The dead spermatozoa lacks intact membranes, and therefore the nucleus is stained red with PI. c) Dead spermatozoa with degenerated acrosomes, identified by PNA-FITC appearing as a green cap on the red-stained nucleus. d) Rabbit anti-acrosin antibody bound to exposed acrosin molecules. The antibodies are secondarily labeled green with anti-rabbit IgG conjugated to FITC. PI has stained the nucleus of the dead spermatozoa red. e) Sperm viability assessed using the nuclear-specific probe, SYBR- 14. Live spermatozoa with intact membranes are identified readily by the bright green fluorescence. Dead spermatozoa (not shown) are identified by PI-stained red nuclei. x1000. producing semen of low quality and overstated in bulls producing semen of high quality. Thus, the subjective nature of the microscopic methods tended to bias the visual examinations. Likewise, initial motility values obtained through DIC appeared inflated relative to flow cytometric values from LYSO-G, SYTO-17, or SYBR-14 staining (Fig. 5). Because variability tends to increase as sample size decreases, these apparent differences likely resulted from the much smaller number of spermatozoa (300 vs ) that were evaluated using the microscopic method as compared to flow cytometry c 60 E 50 o 40 I 30 n 30 OG ,,-- -,,-. ''-.. nitia '. ax. e Initial Motilit. --- Motility after 3 hr \ - - LYSO-G -- SYTO-17, - -- SYBR-14 I I I I I I I I FIG. 5. Line graphs comparing the estimates of mean percentages of viable spermatozoa (n = 3), obtained using microscopic and fluorometric methods. The microscopic method of estimating the proportion of spermatozoa with forward motility, after thawing and after 3-h incubation at 37 C, was done by using DIC microscopy and visually evaluating 300 spermatozoa. The remaining three line graphs were produced using three different fluorophores that label intact, viable spermatozoa. Percentages represent the proportion of spermatozoa that were quantified using flow cytometry. Bull

7 ACROSOMAL INTEGRITY AND VIABILITY OF BOVINE SPERMATOZOA 997 Fluorescent Examinations of Acrosomal Integrity and Of the fluorescent methods tested for assessing acrosomal status, the LYSO-G protocol was the quickest and easiest to use. It also displayed the highest degree of correlation with the microscopic method NAR (r = 0.966). LY- SO-G was also correlated highly with post-thaw motility MOT (r = 0.969) and SYBR-14 staining (r = 0.981), suggesting that it may be useful as a viability test as well as for acrosomal status. The combined use of SYBR-14 and PI produced dot plots with a small percentage of moribund spermatozoa that doubly stained both green and red; LY- SO-G, however, did not exhibit this third population. It is likely that this was due to the compartmentalization of the acrosome and the nuclear chromatin. Microscopically, spermatozoa appeared to lose LYSO-G and take up PI within about 30 sec after motility ceased. Although the uptake of PI by LYSO-G-stained spermatozoa was readily identified visually (Fig. 3a), flow cytometry was not able to distinguish this population. Possibly the lower fluorescence intensity of the LYSO-G compared to that of SYBR-14 made this population indiscernible under the conditions used for the flow analysis. The PNA/SYTO-17/PI system utilized the fluoresceinlabeled peanut lectin, Arachis hypogaea, together with live/ dead staining to simultaneously evaluate acrosomal status and viability. This and another lectin, Pisum sativum agglutinin (PSA), [5] bind preferentially to the acrosomal region in bovine spermatozoa and appeared specific for intracellular, acrosome-associated glycoconjugates [17]. Cross and Watson [17] found that living, intact spermatozoa bound little or no PNA, but there was much labeling of permeabilized spermatozoa. This was consistent with the results of PSA studies reported by Graham et al. [5]. In the present study, PSA binding was not very evident (data not shown), whereas PNA bound over the entire acrosome (Fig. 3b). The egg yolk-based medium in which the samples in the current experiments were stored perhaps interfered with the binding of PSA to the acrosomal matrix. This is likely because Graham et al. [5] and Cross and Watson [17] observed a high degree of PSA binding when they stained fresh samples diluted in medium based on Tyrode's albumin lactate pyruvate. Egg yolk also markedly inhibited the secondary binding of the avidin to the protease inhibitor, BIC, necessitating an extra wash step to isolate the spermatozoa. In human spermatozoa, PNA appeared to bind to the outer acrosomal membrane [18] while PSA reportedly bound to the acrosomal contents [19]. The immunological assessment of acrosomal status showed that bull 1 produced high quality semen, just as the other methods showed. There was a large difference (78%) between a sample that had simply been cryopreserved and thawed once and a sample that had been repeatedly frozen and thawed (Fig. 2, right). Repeated freeze-thawing degraded the membranes enclosing the acrosome, exposing it for direct binding with anti-acrosin and secondary labeling with FITC-labeled anti-igg. In contrast, the semen of bull 12 was of poor quality. Consequently, the frozen-thawed semen from this bull contained a relatively large proportion of spermatozoa with degraded acrosomes. After the repeated freeze-killing, the difference between the control (not freeze-killed) and the treated aliquot from bull 12 was relatively small (29%). Rabbit anti-bovine IgG did show binding of the acrosomal contents in the presence of egg yolk medium, although it appeared to be limited to the anterior portion of the cap (Fig. 3d). Thus, the acrosomes appeared to be somewhat detached. This was likely due to the rigor of the repeated freeze-thawing treatment, possibly resulting in the loss of some of the acrosin. This was in contrast to the binding observed over the entire acrosome in methanolfixed bovine spermatozoa [10]. The assessment of sperm viability using SYBR-14/PI or SYTO-17/PI showed similar results. The prestained buffer was easily prepared, expediting the processing of the semen samples for analyses. Samples were ready within 5-15 min of staining. The green/red, SYBR-14/PI-stained spermatozoa were very bright and easily distinguished when examined using the fluorescent microscope (Fig. 3e). The red/ red, SYTO-17/PI-stained spermatozoa, however, could not be readily distinguished microscopically. This novel system does, however, have the potential for flow cytometric applications requiring a separate green label that could be specific for some cellular attribute other than viability. It is important to note that all four measures of acrosomal integrity were highly correlated (Table 2), suggesting that any one of the techniques could yield similar information. One must conclude that the simplest procedure would be the assay of choice. Furthermore, the experiments utilizing probes specific for the acrosomal contents (BIC, PNA, and IgG) 1) required relatively complex procedures that are not accommodated by use of a prestained buffer system, or the so-called "batch" approach, 2) were correlated with the microscopic method NAR slightly less than LYSO-G, and 3) labeled spermatozoa with degenerating acrosomes. In addition, neither the BIC nor anti-acrosin antibodies are commercially available. In contrast, LYSO-G, which is specific for acidotropic organelles such as the acrosome, 1) can easily be used in a batch staining method, 2) showed the highest correlation among methods with NAR, and 3) labeled spermatozoa with intact acrosomes. Since it was highly correlated with post-thaw motility, as well (r = 0.97), it could be used with or without PI to provide a reasonable estimate of the percentage of viable spermatozoa with intact acrosomes. Therefore, LYSO-G appears to have the greater utility for assessing seminal quality because of its simplicity. REFERENCES I. Linford E, Glover FA, Bishop C, Stewart DL. The relationship between semen evaluation methods and fertility in the bull. J Reprod Fertil 1976; 47: Saacke RG, Vinson WE, O'Connor ML, Candler JE, Mullins J, Amann RP, Marshall CE, Wallace RA, Vincel WN, Kellgren HC. The relationship of semen quality and fertility: a heterospermic study. In: Proc 8th NAAB Tech Conf Artif Insem Reprod; 1980; Milwaukee, WI. Pps Berndtson WE, Olar TT, Pickett BW. Correlations between post-thaw motility and acrosomal integrity of bovine sperm. J Dairy Sci 1981; 64: Aalseth EP, Saacke RG. Vital staining and acrosomal evaluation of bovine sperm. 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