MOLECULAR REPRODUCTION AND DEVELOPMENT 32:23-27 (1992)
Assessment of Pisum sativum Agglutinin in Identifying Acrosomal Damage in Stallion Spermatozoa M.E. FARLIN, D.J. JASKO, J.K. GRAHAM, AND E.L. SQUIRES Animal Reproduction and Biotechnology Laboratory, Colorado State Uniuersity,Fort Collins, Colorado
ABSTRACT
The use of fluorescein-conjugated
Pisurn safivurn agglutinin (FITC-PSA) was evaluated for its
ability to distinguish acrosome-intact from acrosome-damaged stallion spermatozoa. Incubation of fresh (acrosomeintact) and frozen-thawed (acrosome-damaged) spermatozoa with FITC-PSA resulted in acrosome-intact spermatozoa that exhibited no fluorescence, while acrosome-damaged spermatozoa exhibited fluorescent staining over the rostra1 portion of the head and equatorial segment. Experiments using mixtures of various ratios of acrosome-intact and acrosome-damaged spermatozoa determined the precision (intrasample coefficient of variation), and linearity (increased percentage of spermatozoa with PSA binding, with increased percentage of frozenthawed spermatozoa in a sample)of FITC-PSAbinding. The binding of FITC-PSA increased in samples as the portion of frozen-thawed (acrosome-damaged)to fresh (acrosomeintact) spermatozoa increased. A positive correlation existed (r = 0.98, P < 0.05) between the percentage of FITC-PSA bound sperm and the proportion of damaged spermatozoa added to a sample. Location of PSA lectin binding on acrosome-damaged spermatozoa, determined by electron microscopy using gold-conjugated PSA, was to components of the outer acrosomal membrane and acrosomal matrix. These results demonstrate that FITC-PSA binding may be useful in determining acrosomal integrity of fresh and frozen-thawed stallion spermatozoa. Q 1992 Wiley-Liss, Inc.
Key Words: Assay, Lectin, binding, Fluorescence
INTRODUCTION Evaluation of the stallion spermatozoal acrosome via light microscopy has been reported after dual staining with trypan blue and Geisma (Didion e t al., 1989) and after triple staining with trypan blue, Bismarck brown, and rose Bengal (Varner et al., 1987). An assay utilizing chlortetracycline fluorescence has also been developed to visualize changes in the stallion spermatozoal acrosome (Varner et al., 1987). However, these methods required tedious staining procedures and resulted in subjective patterns. Specific binding of monoclonal antibodies to spermatozoal antigens in the acrosomal ground substance (Blach e t al., 1988) and to a n integral acrosomal mem-
0 1992 WILEY-LISS, INC.
brane component of spermatozoa (Zhang et al., 1990) has been used to evaluate integrity of the stallion spermatozoal acrosome. The specific binding of antibodies to antigens within the acrosome has been confirmed using immunocytochemical techniques and electron microscopy (Blach et al., 1988; Zhang et al., 1990). Although the use of monoclonal antibodies offered a specific assay for changes in the acrosome, monoclonal antibodies are difficult to obtain and require visualization by the binding of a second agent, usually a fluorescein-conjugated anti-antibody. Lectins that bind to glycoconjugates of the acrosomal matrix or outer acrosomal membrane have also been used to visualize the acrosome of human (Cross and Meizel, 1989), bull (Graham et al., 1990), and ram (Magargee et al., 1988) spermatozoa. Electron microscopy and immunocytochemical techniques identified the binding sites of some of these lectins within the acrosoma1 matrix and, possibly, the plasma and outer acrosoma1 membranes (Cross et al., 1986). Lectins found to be useful for the visualization of the acrosome of human spermatozoa include Ricinus comrnunis agglutinin (RCA),Pisum sativum agglutinin (PSA),Arachis hypogea agglutinin (PNA) (Cross and Meizel, 1989), and Concavalia ensiformis (ConA) (Holden et al., 1990).The PSA lectin has also been used for the visualization of the bull and ram acrosome (Graham et al., 1990). While spermatozoa with damaged plasma and acrosomal membranes have been reported to become fluorescently labeled over the anterior sperm head and equatorial regions, spermatozoa with intact membranes that were impermeable to these lectins did not fluoresce after exposure (Cross and Overstreet, 1987).This feature has allowed for the use of specific lectids) for the evaluation of acrosomal integrity in spermatozoa of several species. The use of lectins for the visualization of the stallion spermatozoal acrosome has not been previously reported. The objective of this study was to investigate the use of fluorescein isothiocynate-labeled Pisum sativ u m agglutinin (FITC-PSA) for the evaluation of acrosomal integrity of stallion spermatozoa. This paper
Received November 7,1991; accepted December 13,1991. Address reprint requests to David Jasko, DVM, PhD, Equine Sciences Program, Colorado State University, Fort Collins, CO 80523.
24
M.E. FARLIN ET AL.
reports the use of PSA for the visualization of the acrosome in intact (fresh) and damaged (frozen-thawed) stallion spermatozoa.
MATERIALS AND METHODS Spermatozoa Preparation Stallion semen was collected with a n artificial vagina, and spermatozoal concentration was determined photometrically (Densimeter, Model 543A Modl; Animal Reproduction Systems, Chino, CA). Semen was extended in a nonfat, milk-based extender (E-Z Mixin; Animal Reproduction Systems) a t a concentration of 50 x 10' cells/ml. Extended semen was centrifuged at 400g for 15 min to remove seminal plasma. The spermatozoal pellet was resuspended in BO medium (Brackett and Oliphant, 1975; containing 112 mM NaC1,4 mM KC1,2.25 mM CaCl,, 0.83 mM NaH,PO,, 0.52 mM MgCI,, 37 mM NaHCO,, 10 mM sodium lactate, 13.9 mM glucose, 1.25 mM pyruvic acid supplemented with 3 g/liter of bovine serum albumin) to a concentration of 50 X 10' cells/ml. PSA Lectin Staining for Microscopic Evaluation A 50 pl aliquot of spermatozoa (50 x 10' cells/ml) in BO medium was transferred to a 1.5 ml microcentrifuge tube and spermatozoa fixed by the addition of a n equal volume of 15% formalin in 0.9% saline. Spermatozoa were fixed for 1-2 hr and then pelleted by centrifugation for 30 sec in a microcentrifuge to remove aldehyde compounds. After removal of the supernatant, 200 ~1of 0.2 M glycine in phosphate-buffered saline (PBS) was added, and the suspension was incubated 10 min a t 25°C to block remaining free aldehydes from interfering with lectin binding. Spermatozoa were again pelleted by centrifugation for 30 sec. The supernatant was removed and 25 p1 of 100 pg/ml FITC-PSA (Sigma, St. Louis, MO) was added. The suspension was incubated for 30 min at 37°C. Following incubation, to reduce background fluorescence, unbound PSA was removed by adding 200 p1 of PBS and washing spermatozoa by centrifugation in a microcentrifuge for 30 sec. The supernatant was aspirated and the pellet resuspended and counterstained in 25 pl of 0.02 mg/ml Evan's blue in PBS. After incubation with PSA, samples were prepared for viewing by placing 6 pl of the spermatozoa suspension on a slide for evaluating sperm at ~ 1 , 0 0 0with epifluorescence (Leitz Laborlux 12) using a n H2 filter block for excitation at 3 9 0 4 9 0 nm, a RKP 510 beam splitting mirror, and a LP 515 suppression filter. Individual spermatozoa were classified according to the specific PSA fluorescence exhibited on the spermatozoal head. Spermatozoa lacking green fluorescence over the rostra1 portion of the head and equatorial segment (lack of FITC-PSA lectin binding), or minimal green fluorescence (faint, green outline not exceeding one-half of the spermatozoal head), were classified as acrosome intact. Spermatozoa classified as partially acrosome damaged were those that exhibited a green out-
line over one-half of the spermatozoal head, with additional faint o r patchy green fluorescence over the acrosome. Those spermatozoa that displayed a distinct homogeneous green fluorescence over the acrosomal region or a faint green fluorescence over the acrosomal region in combination with a distinct green fluorescence over the equatorial region were classified as acrosome damaged.
Evaluation of FITC-PSA Binding for Assessing Acrosomal Damage To validate the use of FITC-PSA lectin binding for the evaluation of acrosomal damage of stallion spermatozoa, proportions of acrosome-intact and acrosomedamaged spermatozoa were incubated with FITC-PSA. Acrosome-damaged spermatozoa were produced by plunging 1.5 ml spermatozoa extended in BO medium into liquid nitrogen and thawing a t 25°C. This procedure was repeated a second time, to produce theoretically 100%acrosome-damaged sperm. To determine the precision of PSA analysis of acrosomal damage (intrasample coefficient of variation), samples containing equal proportions of fresh and frozen-thawed spermatozoa were processed for microscopic evaluation of FITC-PSA binding. For each sample, 100 cells on each of five different slides were evaluated for the percentage of acrosome-intact, partially acrosome-damaged, and acrosome-damaged cells. Separate samples were prepared from single ejaculates from eight stallions, and the mean intrasample coefficient of variation was calculated for the percentage of 1)acrosome-intact, 2) partially acrosome-damaged, and 3) acrosome-damaged sperm using analysis of variance (SAS, 1988). The response of PSA binding to spermatozoa in Samples containing various percentages of acrosome-damaged spermatozoa was studied by making samples with aliquots of fresh and frozen-thawed spermatozoa at ratios of 4 9 , 3:1, 1:1, 1:3,and 0:4 (vo1:vol).Samples were fixed and processed for epifluorescent microscopic evaluation of the spermatozoal acrosome using FITC-PSA lectin binding a s previously described. A total of 200 cells for each sample were classified as acrosome intact, partially damaged, or acrosome damaged. Single ejaculates from eight stallions were separately processed and analyzed in this manner. Analysis of variance and regression analysis (SAS, 1988) were used to compare percentages of acrosome-intact, partially damaged, and acrosome-damaged spermatozoa, as determined by FITC-PSA binding, to the known proportion of frozenthawed spermatozoa in the samples. Location of PSA Binding to the Stallion Spermatozoa1 Acrosome To verify that the PSA lectin specifically bound to a n internal acrosomal component, samples were processed for electron microscopic evaluation. Spermatozoa were washed and resuspended in BO medium to a concentration of 50 x lo6 cells/ml. Acrosome-damaged spermatozoa were produced by snap freezing a sample in liquid nitrogen and subsequent thawing. Spermatozoa from
STALLION SPERM ACROSOME DAMAGE both the fresh and frozen-thawed samples were centrifuged in a microcentrifuge for 30 sec and resuspended in 0.5 ml colloidal gold-conjugated PSA (1:lO dilution in PBS, 15 nm; EY Laboratories, San Mateo, CA). After incubation for 2 h r a t 37°C (as described by Blach e t al., 19881, unbound PSA was diluted with PBS and removed with supernatant following centrifugation for 3 min in a clinical centrifuge. Spermatozoa were fixed and resuspended in 6% glutaraldehyde, washed in 0.1 M cacodylate buffer, and fixed in 1%osmium tetroxide in 0.1 M cacodylate buffer for 1.5 hr. Cells were washed with 0.1 M cacodylate buffer, dehydrated with ethanol, and embedded in 100% Spurr’s resin. Thin sections were cut, mounted on 200-mesh nickel grids, stained with alkaline lead citrate, and viewed with a J e o l l 2 0 0 electron microscope. One ejaculate was split among three treatments, which were processed simultaneously. Treatments were 1)frozen-thawed (acrosome-damaged) spermatozoa incubated with gold-conjugated PSA; 2) fresh (acrosome-intact) spermatozoa incubated with gold-conjugated PSA; and 3) frozen-thawed (acrosome-damaged) spermatozoa incubated with PSA lectin in the presence of 0.5 M a-mannoside, the lectin’s specific binding sugar. Treatments 2 and 3 were used as controls to ensure 1) that PSA did not bind to undamaged cells (treatment 2) and 2) that saturation of PSA with a competitive binding sugar in the medium would prevent PSA binding to damaged spermatozoa (treatment 3).
RESULTS The incubation of FITC-PSA lectin with fresh and frozen-thawed stallion spermatozoa resulted in staining patterns indicative of acrosomal membrane status. PSA was prevented from attaching to acrosomal contents in those spermatozoa with intact acrosomes but bound to acrosomal contents in spermatozoa with disrupted plasma and acrosomal membranes. All spermatozoa were easily identifiable by red fluorescence over the tail and midpiece due to the Evan’s blue counterstain. In acrosome-intact spermatozoa, no green fluorescence denoting PSA binding was evident over the spermatozoal head. Partially acrosome-damaged spermatozoa were those that exhibited a green outline over one-half of the spermatozoal head, with additional faint or patchy green fluorescence over the acrosome. Acrosome-damaged spermatozoa, however, exhibited homologous FITC-PSA fluorescence over the rostra1 part of the spermatozoal head and equatorial segment. In samples containing equal proportions of fresh and frozen-thawed spermatozoa incubated with FITC-PSA, specific fluorescent patterns for 1)acrosome-intact, 2) partially acrosome-damaged, and 3) acrosome-damaged spermatozoa were exhibited. A high degree of precision (sample repeatability) existed in PSA binding patterns based on low coefficients of variation obtained for pattern classifications (Table 1).Coefficient of variation was lowest for the classification of acrosome-damaged spermatozoa and was below 10% when as few a s
25
TABLE 1. Predicted Pooled Coefficient of Variation for Spermatozoa With Various PSA Fluorescent Patterns in Samples Containing Equal Proportions of Fresh and Frozen-Thawed Spermatozoa (n = 8)
Staining pattern Acrosome damaged Acrosome intact Partially damaged
Predicted coefficient of variation based on number of spermatozoa evaluated 100 200 300 400 500 9.8 16.0 24.0
7.0 11.3 17.0
5.7 9.3 14.0
4.9 8.6 12.0
4.4 7.2 10.7
100 spermatozoa were evaluated. As expected, coefficients of variation decreased with the evaluation of additional spermatozoa. The linearity of PSA binding was determined by the analysis of the percentage of acrosome-damaged spermatozoa (as identified by PSA binding) in samples containing higher proportions of frozen-thawed spermatozoa. An increase (P < 0.05) in the percentage of acrosome-damaged spermatozoa was evident in samples with greater frozen-thawed proportions (Table 2, Fig. 1). Concurrently, a decrease (P < 0.05) in the percentage of acrosome-intact spermatozoa occurred. A positive correlation (r = 0.98, P < 0.05) was determined between the proportion of acrosome-damaged (frozenthawed) spermatozoa added to a sample and the percentage of acrosome-damaged spermatozoa in the sample, as identified by FITC-PSA binding (Fig. 1). Electron microscopic evaluation of frozen-thawed (acrosome-damaged) spermatozoa indicated that goldconjugated PSA lectin bound to components of either the acrosomal matrix underlying the outer acrosomal membrane or to the acrosomal membrane itself (Fig. 2b). In addition, gold-conjugated PSA did not bind to components of the acrosomal membrane or matrix in the fresh (acrosome-intact) sample or in frozen-thawed (acrosome-damaged) samples with a competing sugar present in the medium (Fig. 2a, c).
DISCUSSION Conventional stains used to evaluate the acrosomal integrity of spermatozoa from various species have not been widely used for evaluating stallion spermatozoa. This has been due to the stallion spermatozoa’s small acrosome. Alternative methods for the evaluation of the spermatozoal acrosome using fluorescence microscopy have utilized either monoclonal antibodies (Blach et al., 1988) or fluorescently labeled lectins (Cross and Meizel, 1989; Graham et al., 1990). Microscopic evaluations utilizing fluorescent probes have had a distinct advantage over conventional stains as fluorescent probes provide greater contrast between acrosome-intact and acrosome-damaged spermatozoa (Cross and Meizel, 1988). This enhanced contrast has been useful for evaluating the small acrosome of the stallion spermatozoa (Blach et al., 1988; Zhang et al., 1990).
26
M.E. FARLIN ET AL. TABLE 2. Means (SD) of Spermatozoa Displaying Various PSA Fluorescent Patterns in Samples Containing Different Percentages of Frozen-Thawed Spermatozoa (n = 8)* FITC-PSA binding pattern (%) Acrosome Partially Percentage of frozenAcrosome thawed spermatozoa intact acrosome damaged damaged
0 25 50 75 100
52.2 (12.7)" 43.2 (9.7)b 33.3 (8.7)' 21.4 (6.5)d 11.1 (5.9)e
31.9 (13.0)a 24.3 (7.2)b 15.9 (6.3)' 11.8 (5.5)' 4.4 (2.3)d
15.9(5.0)a 32.4(4.7)b 50.6 (4.0)' 66.9 (4.3)' 84.5 (6.2)a
*Means within columns with different superscripts differ significantly (P< 0.05). of a cryoprotectant, has been previously shown to damage both the plasma and the outer acrosomal membranes of stallion spermatozoa (Blach et al., 1988). Staining patterns characteristic of FITC-PSA binding in fresh and frozen-thawed samples should be similar to patterns observed for acrosome-intact and acrosomedamaged spermatozoa, respectively. This study demonstrates the high degree of precision and linearity with which PSA accurately defined acrosome-intact and acrosome-damaged stallion spennatozoa (Table 1,Fig. 2 ) . However, a small percentage of spermatozoa identified as acrosome-damaged by PSA was present in fresh samples. This could be the result of spermatozoa in a fresh sample that may have had preexisting acrosomal damage. Such is the case with degenerative spermatozoa present in ejaculates. Conversely, frozen-thawed samples contained small per0 00 0.25 0.50 0.75 I .00 centages of PSA identified acrosome-intact spermatozoa. These spermatozoa may have represented cells in FRACTION DAMAGED SPERM Fig. 1. Scatterplot of fraction of damaged (frozen-thawed) sperma- which a complete loss of the acrosome and associated PSA binding sites occurred. tozoa and percentage of acrosome-damaged spermatozoa as determined via PSA binding. Solid line represents least squares regression As was reported for PSA labeling of human spermaline (Y = 68.6X + 15.8, r = 0.98, P < 0.05). Dashed lines enclose the tozoa (Cross and Overstreet, 1987; Cross et al., 19861, 95%confidence interval for predicted values (n = 8). PSA bound to both the anterior and equatorial portions of the acrosome. However, contradictory to the described binding of PSA to acrosomal contents in ethanol-permeabilized sperm, the majority (97%) of goldVisualization of the spermatozoa1 acrosome with lectins has several advantages over visualization utilizing conjugated PSA was bound to the outer acrosomal monoclonal antibodies. First, lectins are easily obtain- membrane in frozen-thawed stallion spermatozoa. able, and, second, they can be easily conjugated to sev- This difference may have been due to the difference in eral different fluorescent compounds; thus the need for methods used to permeabilize spermatozoa (ethanol or a n additional reagent (i.e. second fluorescent antibody) freeze-thaw), due to difference in the species of permefor visualization is eliminated. This study was con- abilized spermatozoa (human or equine), or due to difducted to determine if fluorescein isothiocynate labeled ferences in immunocytochemical techniques used to loPisum satiuum agglutinin (FITC-PSA) could be used to calize the binding of PSA. This paper has reported the characterization of evaluate the acrosomal integrity of stallion spermatoPisum sativum lectin binding for the evaluation of aczoa. FITC-PSA was characterized to distinguish three rosomal damage in stallion spermatozoa. The PSA lecseparate staining patterns; acrosome-intact, partially tin has several potential areas of use where the acrosodamaged, and acrosome-damaged spermatozoa. The ma1 status of spermatozoa is of interest. These include comparison of FITC-PSA binding patterns of acrosome- 1)evaluation of semen of stallions with suspected inferdamaged stallion spermatozoa, produced by snap freez- tility due to acrosomal abnormalities, 2) evaluation of ing in liquid nitrogen and subsequent thawing, to fresh the effect of cryopreservation on the acrosome, and 3) spermatozoa allowed acrosome-intact spermatozoa to evaluation of acrosomal status during in vitro capacitabe distinguished from acrosome-damaged spermatozoa. tion. The use of FITC-PSA for assessing acrosomal Such a freezing protocol, conducted without the benefit integrity in stallion spermatozoa is a n alternative 100
4
STALLION SPERM ACROSOME DAMAGE
27
Fig. 2. Electron micrographs of stallion spermatozoal head after incubation with gold-conjugated PSA. a: Fresh spermatozoa with intact acrosome and lack of gold-conjugated PSA binding. b: Frozenthawed spermatozoa with disrupted acrosome and gold-conjugated PSA binding to acrosomal matrix in association with outer acrosomal
membrane over rostra1 spermatozoal head. e: Frozen-thawed spermatozoa with disrupted acrosome and lack of gold-conjugated PSA binding after incubation with gold-conjugated lectin in the presence of a-mannoside.
to more complicated methods utilizing conventional stains or monoclonal antibodies.
cedures to detect viability and the true acrosome reaction in spermatozoa of various species. Gamete Res 225-57. Graham J K , Kunze E, Hammerstedt RH (1990): Analysis ofsperm cell viability, acrosomal integrity, and mitochondria1 function using flow cytometry. Biol Reprod 43:55-64. Holden CA, Ross VH, Sathananthan AH, Trounson A 0 (1990):Assessment of the human sperm acrosome reaction using concavalin A lectin. Mol Reprod Dev 25:247-257. Magargee SR, Kunze E, Hammerstedt RH (1988): Changes in lectinbinding features of ram sperm surfaces associated with epididymal maturation and ejaculation. Biol Reprod 38:667-685. Nolan JP, Graham JK, Hammerstedt RH: Artificial induction of exocytosis in bull sperm. Arch Biochem Biophys (in press). Parrish J J , Susko-Parrish MA, Winer, First NL (1988): Capacitation of bovine sperm by heparin. Biol Reprod 38:1171-1180. SAS Institute Inc. (1988):“SAS/STAT User’s Guide: Release 6.03 Edition.” Carey, NC: SAS Institute Inc. Shams-Borhan G, Harrison RAP (1981):Production, characterization, and use of ionophore-induced,calcium-dependent acrosome reaction in ram spermatozoa. Gamete Res 4:407-432. Varner DD, Ward CR, Bayard TS, Kenney RM (1987): Induction and characterization of acrosome-reaction in equine spermatozoa. Am J Vet Res 48:1383-1389. Zhang J , Boyle MS, Smith CA, Moore HDM (1990):Acrosome reaction of stallion spermatozoa evaluated with monoclonal antibody and zona-free hamster eggs. Mol Reprod Dev 27:152-158.
ACKNOWLEDGMENTS This study was supported by the Colorado State Experiment Station, Colorado State University.
REFERENCES Blach EL, Amann RP, Bowen RA, Sawyer HR, Hermenet MJ (1988): Use of a monoclonal antibody to evaluate integrity of the plasma membrane of stallion sperm. Gamete Res 21:233-241. Brackett BG, Oliphant G (1975): Capacitation of rabbit spermatozoa in vitro. Biol Reprod 12:260-274. Cross NL, Meizel S (1989): Methods for evaluating the acrosomal status of mammalian sperm. Biol Reprod 41:635-641. Cross NL, Morales P, Overstreet JW, Hanson FW (1986): Two simple methods for detecting acrosome-reacted human sperm. Gamete Res 15:213-226. Cross NL, Overstreet JW (1987): Glycoconjugates of the human sperm surface: Distribution and alterations that accompany capacitation in vitro. Gamete Res 16:23-35. Didion BA, Dobrinsky JR, Giles JR, Graves CN (1989): Staining pro-
Assessment of Pisum sativum Agglutinin in Identifying Acrosomal Damage in Stallion Spermatozoa M.E. FARLIN, D.J. JASKO, J.K. GRAHAM, AND E.L. SQUIRES Animal Reproduction and Biotechnology Laboratory, Colorado State Uniuersity,Fort Collins, Colorado
ABSTRACT
The use of fluorescein-conjugated
Pisurn safivurn agglutinin (FITC-PSA) was evaluated for its
ability to distinguish acrosome-intact from acrosome-damaged stallion spermatozoa. Incubation of fresh (acrosomeintact) and frozen-thawed (acrosome-damaged) spermatozoa with FITC-PSA resulted in acrosome-intact spermatozoa that exhibited no fluorescence, while acrosome-damaged spermatozoa exhibited fluorescent staining over the rostra1 portion of the head and equatorial segment. Experiments using mixtures of various ratios of acrosome-intact and acrosome-damaged spermatozoa determined the precision (intrasample coefficient of variation), and linearity (increased percentage of spermatozoa with PSA binding, with increased percentage of frozenthawed spermatozoa in a sample)of FITC-PSAbinding. The binding of FITC-PSA increased in samples as the portion of frozen-thawed (acrosome-damaged)to fresh (acrosomeintact) spermatozoa increased. A positive correlation existed (r = 0.98, P < 0.05) between the percentage of FITC-PSA bound sperm and the proportion of damaged spermatozoa added to a sample. Location of PSA lectin binding on acrosome-damaged spermatozoa, determined by electron microscopy using gold-conjugated PSA, was to components of the outer acrosomal membrane and acrosomal matrix. These results demonstrate that FITC-PSA binding may be useful in determining acrosomal integrity of fresh and frozen-thawed stallion spermatozoa. Q 1992 Wiley-Liss, Inc.
Key Words: Assay, Lectin, binding, Fluorescence
INTRODUCTION Evaluation of the stallion spermatozoal acrosome via light microscopy has been reported after dual staining with trypan blue and Geisma (Didion e t al., 1989) and after triple staining with trypan blue, Bismarck brown, and rose Bengal (Varner et al., 1987). An assay utilizing chlortetracycline fluorescence has also been developed to visualize changes in the stallion spermatozoal acrosome (Varner et al., 1987). However, these methods required tedious staining procedures and resulted in subjective patterns. Specific binding of monoclonal antibodies to spermatozoal antigens in the acrosomal ground substance (Blach e t al., 1988) and to a n integral acrosomal mem-
0 1992 WILEY-LISS, INC.
brane component of spermatozoa (Zhang et al., 1990) has been used to evaluate integrity of the stallion spermatozoal acrosome. The specific binding of antibodies to antigens within the acrosome has been confirmed using immunocytochemical techniques and electron microscopy (Blach et al., 1988; Zhang et al., 1990). Although the use of monoclonal antibodies offered a specific assay for changes in the acrosome, monoclonal antibodies are difficult to obtain and require visualization by the binding of a second agent, usually a fluorescein-conjugated anti-antibody. Lectins that bind to glycoconjugates of the acrosomal matrix or outer acrosomal membrane have also been used to visualize the acrosome of human (Cross and Meizel, 1989), bull (Graham et al., 1990), and ram (Magargee et al., 1988) spermatozoa. Electron microscopy and immunocytochemical techniques identified the binding sites of some of these lectins within the acrosoma1 matrix and, possibly, the plasma and outer acrosoma1 membranes (Cross et al., 1986). Lectins found to be useful for the visualization of the acrosome of human spermatozoa include Ricinus comrnunis agglutinin (RCA),Pisum sativum agglutinin (PSA),Arachis hypogea agglutinin (PNA) (Cross and Meizel, 1989), and Concavalia ensiformis (ConA) (Holden et al., 1990).The PSA lectin has also been used for the visualization of the bull and ram acrosome (Graham et al., 1990). While spermatozoa with damaged plasma and acrosomal membranes have been reported to become fluorescently labeled over the anterior sperm head and equatorial regions, spermatozoa with intact membranes that were impermeable to these lectins did not fluoresce after exposure (Cross and Overstreet, 1987).This feature has allowed for the use of specific lectids) for the evaluation of acrosomal integrity in spermatozoa of several species. The use of lectins for the visualization of the stallion spermatozoal acrosome has not been previously reported. The objective of this study was to investigate the use of fluorescein isothiocynate-labeled Pisum sativ u m agglutinin (FITC-PSA) for the evaluation of acrosomal integrity of stallion spermatozoa. This paper
Received November 7,1991; accepted December 13,1991. Address reprint requests to David Jasko, DVM, PhD, Equine Sciences Program, Colorado State University, Fort Collins, CO 80523.
24
M.E. FARLIN ET AL.
reports the use of PSA for the visualization of the acrosome in intact (fresh) and damaged (frozen-thawed) stallion spermatozoa.
MATERIALS AND METHODS Spermatozoa Preparation Stallion semen was collected with a n artificial vagina, and spermatozoal concentration was determined photometrically (Densimeter, Model 543A Modl; Animal Reproduction Systems, Chino, CA). Semen was extended in a nonfat, milk-based extender (E-Z Mixin; Animal Reproduction Systems) a t a concentration of 50 x 10' cells/ml. Extended semen was centrifuged at 400g for 15 min to remove seminal plasma. The spermatozoal pellet was resuspended in BO medium (Brackett and Oliphant, 1975; containing 112 mM NaC1,4 mM KC1,2.25 mM CaCl,, 0.83 mM NaH,PO,, 0.52 mM MgCI,, 37 mM NaHCO,, 10 mM sodium lactate, 13.9 mM glucose, 1.25 mM pyruvic acid supplemented with 3 g/liter of bovine serum albumin) to a concentration of 50 X 10' cells/ml. PSA Lectin Staining for Microscopic Evaluation A 50 pl aliquot of spermatozoa (50 x 10' cells/ml) in BO medium was transferred to a 1.5 ml microcentrifuge tube and spermatozoa fixed by the addition of a n equal volume of 15% formalin in 0.9% saline. Spermatozoa were fixed for 1-2 hr and then pelleted by centrifugation for 30 sec in a microcentrifuge to remove aldehyde compounds. After removal of the supernatant, 200 ~1of 0.2 M glycine in phosphate-buffered saline (PBS) was added, and the suspension was incubated 10 min a t 25°C to block remaining free aldehydes from interfering with lectin binding. Spermatozoa were again pelleted by centrifugation for 30 sec. The supernatant was removed and 25 p1 of 100 pg/ml FITC-PSA (Sigma, St. Louis, MO) was added. The suspension was incubated for 30 min at 37°C. Following incubation, to reduce background fluorescence, unbound PSA was removed by adding 200 p1 of PBS and washing spermatozoa by centrifugation in a microcentrifuge for 30 sec. The supernatant was aspirated and the pellet resuspended and counterstained in 25 pl of 0.02 mg/ml Evan's blue in PBS. After incubation with PSA, samples were prepared for viewing by placing 6 pl of the spermatozoa suspension on a slide for evaluating sperm at ~ 1 , 0 0 0with epifluorescence (Leitz Laborlux 12) using a n H2 filter block for excitation at 3 9 0 4 9 0 nm, a RKP 510 beam splitting mirror, and a LP 515 suppression filter. Individual spermatozoa were classified according to the specific PSA fluorescence exhibited on the spermatozoal head. Spermatozoa lacking green fluorescence over the rostra1 portion of the head and equatorial segment (lack of FITC-PSA lectin binding), or minimal green fluorescence (faint, green outline not exceeding one-half of the spermatozoal head), were classified as acrosome intact. Spermatozoa classified as partially acrosome damaged were those that exhibited a green out-
line over one-half of the spermatozoal head, with additional faint o r patchy green fluorescence over the acrosome. Those spermatozoa that displayed a distinct homogeneous green fluorescence over the acrosomal region or a faint green fluorescence over the acrosomal region in combination with a distinct green fluorescence over the equatorial region were classified as acrosome damaged.
Evaluation of FITC-PSA Binding for Assessing Acrosomal Damage To validate the use of FITC-PSA lectin binding for the evaluation of acrosomal damage of stallion spermatozoa, proportions of acrosome-intact and acrosomedamaged spermatozoa were incubated with FITC-PSA. Acrosome-damaged spermatozoa were produced by plunging 1.5 ml spermatozoa extended in BO medium into liquid nitrogen and thawing a t 25°C. This procedure was repeated a second time, to produce theoretically 100%acrosome-damaged sperm. To determine the precision of PSA analysis of acrosomal damage (intrasample coefficient of variation), samples containing equal proportions of fresh and frozen-thawed spermatozoa were processed for microscopic evaluation of FITC-PSA binding. For each sample, 100 cells on each of five different slides were evaluated for the percentage of acrosome-intact, partially acrosome-damaged, and acrosome-damaged cells. Separate samples were prepared from single ejaculates from eight stallions, and the mean intrasample coefficient of variation was calculated for the percentage of 1)acrosome-intact, 2) partially acrosome-damaged, and 3) acrosome-damaged sperm using analysis of variance (SAS, 1988). The response of PSA binding to spermatozoa in Samples containing various percentages of acrosome-damaged spermatozoa was studied by making samples with aliquots of fresh and frozen-thawed spermatozoa at ratios of 4 9 , 3:1, 1:1, 1:3,and 0:4 (vo1:vol).Samples were fixed and processed for epifluorescent microscopic evaluation of the spermatozoal acrosome using FITC-PSA lectin binding a s previously described. A total of 200 cells for each sample were classified as acrosome intact, partially damaged, or acrosome damaged. Single ejaculates from eight stallions were separately processed and analyzed in this manner. Analysis of variance and regression analysis (SAS, 1988) were used to compare percentages of acrosome-intact, partially damaged, and acrosome-damaged spermatozoa, as determined by FITC-PSA binding, to the known proportion of frozenthawed spermatozoa in the samples. Location of PSA Binding to the Stallion Spermatozoa1 Acrosome To verify that the PSA lectin specifically bound to a n internal acrosomal component, samples were processed for electron microscopic evaluation. Spermatozoa were washed and resuspended in BO medium to a concentration of 50 x lo6 cells/ml. Acrosome-damaged spermatozoa were produced by snap freezing a sample in liquid nitrogen and subsequent thawing. Spermatozoa from
STALLION SPERM ACROSOME DAMAGE both the fresh and frozen-thawed samples were centrifuged in a microcentrifuge for 30 sec and resuspended in 0.5 ml colloidal gold-conjugated PSA (1:lO dilution in PBS, 15 nm; EY Laboratories, San Mateo, CA). After incubation for 2 h r a t 37°C (as described by Blach e t al., 19881, unbound PSA was diluted with PBS and removed with supernatant following centrifugation for 3 min in a clinical centrifuge. Spermatozoa were fixed and resuspended in 6% glutaraldehyde, washed in 0.1 M cacodylate buffer, and fixed in 1%osmium tetroxide in 0.1 M cacodylate buffer for 1.5 hr. Cells were washed with 0.1 M cacodylate buffer, dehydrated with ethanol, and embedded in 100% Spurr’s resin. Thin sections were cut, mounted on 200-mesh nickel grids, stained with alkaline lead citrate, and viewed with a J e o l l 2 0 0 electron microscope. One ejaculate was split among three treatments, which were processed simultaneously. Treatments were 1)frozen-thawed (acrosome-damaged) spermatozoa incubated with gold-conjugated PSA; 2) fresh (acrosome-intact) spermatozoa incubated with gold-conjugated PSA; and 3) frozen-thawed (acrosome-damaged) spermatozoa incubated with PSA lectin in the presence of 0.5 M a-mannoside, the lectin’s specific binding sugar. Treatments 2 and 3 were used as controls to ensure 1) that PSA did not bind to undamaged cells (treatment 2) and 2) that saturation of PSA with a competitive binding sugar in the medium would prevent PSA binding to damaged spermatozoa (treatment 3).
RESULTS The incubation of FITC-PSA lectin with fresh and frozen-thawed stallion spermatozoa resulted in staining patterns indicative of acrosomal membrane status. PSA was prevented from attaching to acrosomal contents in those spermatozoa with intact acrosomes but bound to acrosomal contents in spermatozoa with disrupted plasma and acrosomal membranes. All spermatozoa were easily identifiable by red fluorescence over the tail and midpiece due to the Evan’s blue counterstain. In acrosome-intact spermatozoa, no green fluorescence denoting PSA binding was evident over the spermatozoal head. Partially acrosome-damaged spermatozoa were those that exhibited a green outline over one-half of the spermatozoal head, with additional faint or patchy green fluorescence over the acrosome. Acrosome-damaged spermatozoa, however, exhibited homologous FITC-PSA fluorescence over the rostra1 part of the spermatozoal head and equatorial segment. In samples containing equal proportions of fresh and frozen-thawed spermatozoa incubated with FITC-PSA, specific fluorescent patterns for 1)acrosome-intact, 2) partially acrosome-damaged, and 3) acrosome-damaged spermatozoa were exhibited. A high degree of precision (sample repeatability) existed in PSA binding patterns based on low coefficients of variation obtained for pattern classifications (Table 1).Coefficient of variation was lowest for the classification of acrosome-damaged spermatozoa and was below 10% when as few a s
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TABLE 1. Predicted Pooled Coefficient of Variation for Spermatozoa With Various PSA Fluorescent Patterns in Samples Containing Equal Proportions of Fresh and Frozen-Thawed Spermatozoa (n = 8)
Staining pattern Acrosome damaged Acrosome intact Partially damaged
Predicted coefficient of variation based on number of spermatozoa evaluated 100 200 300 400 500 9.8 16.0 24.0
7.0 11.3 17.0
5.7 9.3 14.0
4.9 8.6 12.0
4.4 7.2 10.7
100 spermatozoa were evaluated. As expected, coefficients of variation decreased with the evaluation of additional spermatozoa. The linearity of PSA binding was determined by the analysis of the percentage of acrosome-damaged spermatozoa (as identified by PSA binding) in samples containing higher proportions of frozen-thawed spermatozoa. An increase (P < 0.05) in the percentage of acrosome-damaged spermatozoa was evident in samples with greater frozen-thawed proportions (Table 2, Fig. 1). Concurrently, a decrease (P < 0.05) in the percentage of acrosome-intact spermatozoa occurred. A positive correlation (r = 0.98, P < 0.05) was determined between the proportion of acrosome-damaged (frozenthawed) spermatozoa added to a sample and the percentage of acrosome-damaged spermatozoa in the sample, as identified by FITC-PSA binding (Fig. 1). Electron microscopic evaluation of frozen-thawed (acrosome-damaged) spermatozoa indicated that goldconjugated PSA lectin bound to components of either the acrosomal matrix underlying the outer acrosomal membrane or to the acrosomal membrane itself (Fig. 2b). In addition, gold-conjugated PSA did not bind to components of the acrosomal membrane or matrix in the fresh (acrosome-intact) sample or in frozen-thawed (acrosome-damaged) samples with a competing sugar present in the medium (Fig. 2a, c).
DISCUSSION Conventional stains used to evaluate the acrosomal integrity of spermatozoa from various species have not been widely used for evaluating stallion spermatozoa. This has been due to the stallion spermatozoa’s small acrosome. Alternative methods for the evaluation of the spermatozoal acrosome using fluorescence microscopy have utilized either monoclonal antibodies (Blach et al., 1988) or fluorescently labeled lectins (Cross and Meizel, 1989; Graham et al., 1990). Microscopic evaluations utilizing fluorescent probes have had a distinct advantage over conventional stains as fluorescent probes provide greater contrast between acrosome-intact and acrosome-damaged spermatozoa (Cross and Meizel, 1988). This enhanced contrast has been useful for evaluating the small acrosome of the stallion spermatozoa (Blach et al., 1988; Zhang et al., 1990).
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M.E. FARLIN ET AL. TABLE 2. Means (SD) of Spermatozoa Displaying Various PSA Fluorescent Patterns in Samples Containing Different Percentages of Frozen-Thawed Spermatozoa (n = 8)* FITC-PSA binding pattern (%) Acrosome Partially Percentage of frozenAcrosome thawed spermatozoa intact acrosome damaged damaged
0 25 50 75 100
52.2 (12.7)" 43.2 (9.7)b 33.3 (8.7)' 21.4 (6.5)d 11.1 (5.9)e
31.9 (13.0)a 24.3 (7.2)b 15.9 (6.3)' 11.8 (5.5)' 4.4 (2.3)d
15.9(5.0)a 32.4(4.7)b 50.6 (4.0)' 66.9 (4.3)' 84.5 (6.2)a
*Means within columns with different superscripts differ significantly (P< 0.05). of a cryoprotectant, has been previously shown to damage both the plasma and the outer acrosomal membranes of stallion spermatozoa (Blach et al., 1988). Staining patterns characteristic of FITC-PSA binding in fresh and frozen-thawed samples should be similar to patterns observed for acrosome-intact and acrosomedamaged spermatozoa, respectively. This study demonstrates the high degree of precision and linearity with which PSA accurately defined acrosome-intact and acrosome-damaged stallion spennatozoa (Table 1,Fig. 2 ) . However, a small percentage of spermatozoa identified as acrosome-damaged by PSA was present in fresh samples. This could be the result of spermatozoa in a fresh sample that may have had preexisting acrosomal damage. Such is the case with degenerative spermatozoa present in ejaculates. Conversely, frozen-thawed samples contained small per0 00 0.25 0.50 0.75 I .00 centages of PSA identified acrosome-intact spermatozoa. These spermatozoa may have represented cells in FRACTION DAMAGED SPERM Fig. 1. Scatterplot of fraction of damaged (frozen-thawed) sperma- which a complete loss of the acrosome and associated PSA binding sites occurred. tozoa and percentage of acrosome-damaged spermatozoa as determined via PSA binding. Solid line represents least squares regression As was reported for PSA labeling of human spermaline (Y = 68.6X + 15.8, r = 0.98, P < 0.05). Dashed lines enclose the tozoa (Cross and Overstreet, 1987; Cross et al., 19861, 95%confidence interval for predicted values (n = 8). PSA bound to both the anterior and equatorial portions of the acrosome. However, contradictory to the described binding of PSA to acrosomal contents in ethanol-permeabilized sperm, the majority (97%) of goldVisualization of the spermatozoa1 acrosome with lectins has several advantages over visualization utilizing conjugated PSA was bound to the outer acrosomal monoclonal antibodies. First, lectins are easily obtain- membrane in frozen-thawed stallion spermatozoa. able, and, second, they can be easily conjugated to sev- This difference may have been due to the difference in eral different fluorescent compounds; thus the need for methods used to permeabilize spermatozoa (ethanol or a n additional reagent (i.e. second fluorescent antibody) freeze-thaw), due to difference in the species of permefor visualization is eliminated. This study was con- abilized spermatozoa (human or equine), or due to difducted to determine if fluorescein isothiocynate labeled ferences in immunocytochemical techniques used to loPisum satiuum agglutinin (FITC-PSA) could be used to calize the binding of PSA. This paper has reported the characterization of evaluate the acrosomal integrity of stallion spermatoPisum sativum lectin binding for the evaluation of aczoa. FITC-PSA was characterized to distinguish three rosomal damage in stallion spermatozoa. The PSA lecseparate staining patterns; acrosome-intact, partially tin has several potential areas of use where the acrosodamaged, and acrosome-damaged spermatozoa. The ma1 status of spermatozoa is of interest. These include comparison of FITC-PSA binding patterns of acrosome- 1)evaluation of semen of stallions with suspected inferdamaged stallion spermatozoa, produced by snap freez- tility due to acrosomal abnormalities, 2) evaluation of ing in liquid nitrogen and subsequent thawing, to fresh the effect of cryopreservation on the acrosome, and 3) spermatozoa allowed acrosome-intact spermatozoa to evaluation of acrosomal status during in vitro capacitabe distinguished from acrosome-damaged spermatozoa. tion. The use of FITC-PSA for assessing acrosomal Such a freezing protocol, conducted without the benefit integrity in stallion spermatozoa is a n alternative 100
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STALLION SPERM ACROSOME DAMAGE
27
Fig. 2. Electron micrographs of stallion spermatozoal head after incubation with gold-conjugated PSA. a: Fresh spermatozoa with intact acrosome and lack of gold-conjugated PSA binding. b: Frozenthawed spermatozoa with disrupted acrosome and gold-conjugated PSA binding to acrosomal matrix in association with outer acrosomal
membrane over rostra1 spermatozoal head. e: Frozen-thawed spermatozoa with disrupted acrosome and lack of gold-conjugated PSA binding after incubation with gold-conjugated lectin in the presence of a-mannoside.
to more complicated methods utilizing conventional stains or monoclonal antibodies.
cedures to detect viability and the true acrosome reaction in spermatozoa of various species. Gamete Res 225-57. Graham J K , Kunze E, Hammerstedt RH (1990): Analysis ofsperm cell viability, acrosomal integrity, and mitochondria1 function using flow cytometry. Biol Reprod 43:55-64. Holden CA, Ross VH, Sathananthan AH, Trounson A 0 (1990):Assessment of the human sperm acrosome reaction using concavalin A lectin. Mol Reprod Dev 25:247-257. Magargee SR, Kunze E, Hammerstedt RH (1988): Changes in lectinbinding features of ram sperm surfaces associated with epididymal maturation and ejaculation. Biol Reprod 38:667-685. Nolan JP, Graham JK, Hammerstedt RH: Artificial induction of exocytosis in bull sperm. Arch Biochem Biophys (in press). Parrish J J , Susko-Parrish MA, Winer, First NL (1988): Capacitation of bovine sperm by heparin. Biol Reprod 38:1171-1180. SAS Institute Inc. (1988):“SAS/STAT User’s Guide: Release 6.03 Edition.” Carey, NC: SAS Institute Inc. Shams-Borhan G, Harrison RAP (1981):Production, characterization, and use of ionophore-induced,calcium-dependent acrosome reaction in ram spermatozoa. Gamete Res 4:407-432. Varner DD, Ward CR, Bayard TS, Kenney RM (1987): Induction and characterization of acrosome-reaction in equine spermatozoa. Am J Vet Res 48:1383-1389. Zhang J , Boyle MS, Smith CA, Moore HDM (1990):Acrosome reaction of stallion spermatozoa evaluated with monoclonal antibody and zona-free hamster eggs. Mol Reprod Dev 27:152-158.
ACKNOWLEDGMENTS This study was supported by the Colorado State Experiment Station, Colorado State University.
REFERENCES Blach EL, Amann RP, Bowen RA, Sawyer HR, Hermenet MJ (1988): Use of a monoclonal antibody to evaluate integrity of the plasma membrane of stallion sperm. Gamete Res 21:233-241. Brackett BG, Oliphant G (1975): Capacitation of rabbit spermatozoa in vitro. Biol Reprod 12:260-274. Cross NL, Meizel S (1989): Methods for evaluating the acrosomal status of mammalian sperm. Biol Reprod 41:635-641. Cross NL, Morales P, Overstreet JW, Hanson FW (1986): Two simple methods for detecting acrosome-reacted human sperm. Gamete Res 15:213-226. Cross NL, Overstreet JW (1987): Glycoconjugates of the human sperm surface: Distribution and alterations that accompany capacitation in vitro. Gamete Res 16:23-35. Didion BA, Dobrinsky JR, Giles JR, Graves CN (1989): Staining pro-
