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Hoechst 33258 staining
SCIENCE Animal Reproduction
Science 45 (1997) 299-309
Assessment of the acrosomal status of membrane-intact ram spermatozoa after freezing and thawing, by simultaneous lectin/Hoechst 33258 staining A. Valcircel
*,
M.A. de las Heras, L. Pkrez, D.F. Moses, H. Baldassarre
Centrn de Invesrigaciones Reproductiuus Pkrez Compunc, Au de Mayo 701. 1084 Buenos Aires, Argentina Accepted
23 May 1996
Abstract We have evaluated the effect of freezing and thawing on the acrosomal status of ram spermatozoa, especially those that withstood cryopreservation as assessed by membrane integrity. To this end, we performed simultaneous lectin/Hoechst 33258 staining, and compared the ability of three fluoresceinated lectins. Pisum sariuum lectin Ram spermatozoa were treated with fluorescein isothiocyanate-labelled (PSA), fluorescein isothiocyanate-labelled Aruchis hypogea lectin (PNA) and fluorescein isothiocyanate-labelled Triricum uufgaris lectin (WGA) and simultaneously with Hoechst 33258 for determination of membrane integrity and acrosomal status. In all cases, three forms were readily distinguished by their distribution pattern. For both PSA and PNA, the most abundant form found in fresh semen consisted of fluorescence on the acrosomal area. This form corresponds to acrosome-intact spermatozoa, as assessed by Differential Interference Contrast (DIG) microscopy. Two minor forms showed weak fluorescence on the equatorial segment or no fluorescence on the head. DIC microscopy revealed that both forms were associated with acrosome-lost spermatozoa. WGA labelling showed two forms, one of which consisted of fluorescence on the entire head, albeit more intensely on its anterior segment. Spermatozoa in this form were acrosome-intact by DIC. The other form lacked fluorescence on the acrosomal region, but still showed faint fluorescence in the posterior region. This form was acrosome-lost by DIC.
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037%4320/97/$17.00 Copyright 0 1997 Elsevier Science B.V. All rights reserved. PII SO378-4320(96)01586-2
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Incubation of fresh spermatozoa with calcium ionophore A23187 for up to 1 h significantly increased the percentage of those forms identified as acrosome-reacted as described above. This was confirmed by the time-dependent accumulation of these forms, as well ‘as by DIC microscopy. At all times, differences among values obtained using these three lectins were not significant. Freezing and thawing led to a decrease of both membrane integrity and acrosomal integrity, irrespective of the lectin used. However, almost all spermatozoa that withstood cryopreservation, as evaluated by Hcechst exclusion, showed intact acrosomes. In this case, no differences between fresh and frozen/thawed samples were observed. These results suggest that the structural integrity of ram spermatozoa is mostly unaffected after cryopreservation, suggesting that it is damage to the plasma membrane that is primarily responsi-
ble for the low fertility of cryopreserved samples. Keywords: Agglutinins; Semen; Cryopreservation;Sheep-male reproduction; Acrosomal status; Lectins
1. Introduction Cryopreservation of spermatozoa from many species leads to a series of alterations resulting in a marked decrease in their fertility (reviewed in Parks and Graham, 1992). Among these changes, damage to plasma/acrosomal membranes has been indicated as a major cause of function loss, due to leakage of cellular components and inactivation of crucial proteins. Damage to the plasma membrane can be measured by means of fluorescent vital or supravital dyes such as carboxyfluorescein diacetate (CFDA) and propidium iodide (PI). In ram spermatozoa, we have demonstrated that at least 30% of those sperrmatozoa that remained motile after freezing and thawing had damage to the plasma membrane &al&me1 et al., 1994); those spermatozoa rapidly lose their motility at 37°C and would therefore be nonfunctional in vivo. Hence, from a physiological view, determination of parameters of cellular structure or function is meaningful only when performed on membrane-intact spermatozoa, in particular after cryopreservation. An important aspect in the evaluation of semen quality is the assessment of the ability of spermatozoa to undergo the acrosome reaction. This process is associated to the activation and exocytotic release of proteolytic enzymes from the acrosome and is thought to play a role in the penetration of the egg investments (reviewed by Yanagimachi, 1994). In ram spermatozoa, it is often difficult to monitor the acrosomal status by conventional light microscopy (Shams-Borhan and Harrison, 1981; Watson et al., 1991); on the other hand, electron microscopy (Shams-Borhan and Harrison, 1981; Ben-Av et al., 1988; Williams et al., 1991) is not practical for routine purposes. Several methods have been recently described to evaluate the Percentage of acrosome-reacted ram spermatozoa, including triple stain (Gutierrez et al., 1993), naphtol yellow/erythrosin B
staining (Graham et al., 1987; Williams et al., 1991) and enzymic methods (Williams et al., 1991). However, for different reasons, these techniques has not been widely used. Pluorescein-conjugated lectins are being increasingly used for the determination of acrosomal status. In particular, agglutinins from Pisum sativum (PSA), Aruchis hypogea (PNA) and Triticum vulgaris (WGA) have been the most widely used. Dual staining aimed at the simultaneous determination of acrosomal status and membrane integrity offers further information on those spermatozoa that are potentially
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fertile. Supravital fluorescent stains such as Hoechst 33258 and propidium iodide (alone or combined with fluorescein diacetate), which enter the cell nucleus and bind DNA only when the plasma membrane is damaged and/or permeabilized, have proved particularly useful for the assessment of sperm integrity (Cross et al., 1986; Garner et al., 1986; Harrison and Vickers, 1990; Mendoza et al., 1992). In this paper we show, by means of a combined Hoechst 33258/fluoresceinated lectin staining, that acrosomal integrity is maintained in those spermatozoa that have withstood cryopreservation, as assessed by the most relevant alteration described so far, i.e. damage to the plasma membrane.
2. Materials and methods 2.1. Chemicals Fluorescein isothiocyanate-labelled lectins from pea (PSA), peanut (PNA) wheat germ (WGA), as well as other reagents of analytical grade, were supplied by Sigma Chemical Co (St Louis, MO, USA). 2.2. Semen collection and freezing Semen was collected from 12 adult Merino rams of proven fertility, by means of an artificial vagina. Immediately after collection, concentration and percent motility were assessed using a computerized semen analyzer (CellTrakTM, Motion Analysis, Santa Rosa, CA, USA) configured in our laboratory for its use with ram semen (Moses et al., 1995). Samples showing less than 70% motility were discarded. Semen was extended at a final concentration of 5 X 10’ spermatozoa ml -’ in a Tes-Tris-based diluent containing 20% (v/v) egg yolk and 4% (v/v> glycerol, pH 7.3, 340 mOsm kg-‘. After motility evaluation, diluted samples were slowly cooled to 4°C within 2 h, frozen in 0.2-ml pellets on dry ice and transferred to liquid nitrogen. Thawing was performed at 37°C in Kahn tubes. 2.3. Induction of acrosomal exocytosis by calcium ionophore A23187 Ten-p1 semen aliquots, containing 4-5 X 10’ spermatozoa were diluted 1:lO in phosphate-buffered saline (PBS), mixed with an equal volume of A23187 in PBS (prepared from a stock 5 mM solution in DMSO), such that final concentrations were 5 ,uM A23 187 and 0.1% (v/v) DMSO, and incubated at 37°C in a humidified incubator. Control samples contained 0.1% DMSO alone. After 30 and 60 min, aliquots were taken for lectin binding/membrane integrity assay and DIC microscopy. 2.4. Simultaneous assessment of lectin binding pattern and membrane integrity Ten-p1 semen aliquots, containing 4-5 X 10’ spermatozoa, were diluted in 90 ~1 3% (w/v> sodium citrate, mixed with 100 ~1 of a Hoechst 33258 solution (1 pg ml-’ in
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sodium citrate) and incubated at 37°C for 3 min. Excess dye was removed by centrifugation through 1 ml of a Ficoll solution (70 g 1-l Ficoll.400, 20 mM Hepes, 10.3 mM Tris, 285.2 mM fructose, 4 mM MgCl,, 100 mg 1-l kanamycin sulfate, pH 7.3, 340 mOsm kg-’ > at 15 600 g for 30 s. The supematant was carefully aspirated, avoiding contact with the pellets, which were further washed in 400 ~1 of PBS and fixed in 95% ethanol at 4°C (Cross et al., 1986) for 10 min. The suspensions were centrifuged, washed in PBS and finally resuspended in 90 ~1 PBS. For lectin labelling, 10 ~1 of a solution containing 100 pg ml -’ of one of each lectin in PBS were added. The suspensions were incubated 37°C for 8 min in the dark, washed in 1 ml of PBS and mounted on low-fluorescence glass slides using 90% (v/v) glycerol in PBS. At least 200 spermatozoa per slide were assessed for Hoechst and lectin fluorescence, in a Nikon Optiphot epifluorescence microscope, using UV and B-2A filters, respectively. Lectin specificity was determined by competition with different monosaccharides. For this purpose, stock lectin solutions were mixed with an equal volumen of 1 M o-methyl mannoside, D-galactose or N-acetyl-D-glucosamine. After 30 min, labelling was carried out as described above. 2.5. Assessment of acrosomal status by light microscopy Acrosomal morphology was assessed by differential interference contrast (DIG) microscopy, as described by Watson et al. (19911, with the exception that intermediate and late stages of acrosomal loss were considered as a whole. At least 100 spermatozoa per sample, in duplicates, were observed. 2.6. Statistical analysis
Differences were analyzed by Student’s t-test or by ANOVA and Tukey test, after angular transformation of data. Correlation was determined by linear regression analysis (Steel and Torrie, 1981).
3. Results We have compared the ability of three lectins to characterize acrosomal integrity of ram spermatozoa. Fig. 1, Fig. 2 and Fig. 3 show the labelling pattern of PSA, PNA and WGA. For both PSA and PNA, the most abundant form found in fresh semen consisted of fluorescence on the acrosomal area (Fig. la and Fig. 2a). This form corresponds to acrosome-intact spermatozoa, as assessed by DIC. Two minor forms showed weak fluorescence on the equatorial segment or no fluorescence on the head (Fig. lb and Fig. 2a). DIC microscopy revealed that both forms were associated with acrosome-lost spermatozoa. WGA labeling showed two forms, one of which consisted of fluorescence on the entire head, albeit more intensely on its anterior segment (Fig. 3a). Spermatozoa in this form were acrosome-intact by DIC. The other form lacked fluorescence on the acrosomal region, but still showed faint fluorescence in the posterior region (Fig. 3b).
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Fig. 1 PSA binding pattern in ram spermatozoa. (a) intact (lower) and acrosome-damaged (upper) spermatozoa, showing fluorescence on the acrosomal area. (b) acrosome-reacted spermatozoon, showing weak fluorescence on the equatorial region (arrow). Bar: 4 pm.
Fig. 2. PNA binding pattern in ram spermatozoa. (a) three intact spermatozoa, showing fluorescence on the acrosomal area, and one acrosome-reacted spermatozoon (arrow), with no fluorescence on the head. (b) acrosome-damaged spermatozoon. Bar: 4 pm.
This form was acrosomeless by DIC. Some spermatozoa showed partial damage to the acrosome readily evident by lectin staining (Fig. la, Fig. 2b and Fig. 3a). Membrane-damaged (non viable) spermatozoa were characterized by anomalous
Fig. 3. WGA binding pattern in ram spermatozoa. (a) acrosome-intact (upper) and damaged (lower) spermatozoa. showing fluorescence on the entire head, mainly on the acrosomal, and the principal piece of the flagellum. (b) acrosome-reacted spermatozoon, with no fluorescence on the acrosome. Bar: 4 pm.
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Table 1 Percentage of acrosome-reacted spermatozoaaf’terincubation with A23187, as evaluated by PSA, PNA and WGA labelling Lectin
Time (min)
PSA PNA
0 2*la l*l’
30 42&3 b 38i-3 b
60 66*4c 60*4c
WGA
2il’
361t4~
66*5c
Values represent mean f SD (n = 12). Values with different superscripts significantly differ (P < 0.001).
lectin distribution in all cases, including: (a> patch labelling on the acrosome, complete staining and postacrosomal/equatorial staining for PSA; (b) patch labelling on the acrosome for PNA; and (c) complete or patch labelling exclusively on the acrosome, equatorial labelling or no head fluorescence for WGA (data not shown). In all cases, 10-E% of non-viable spermatozoa showed intact acrosomes, and 45-50% showed acrosomal loss or damage, as assessed by DIC. In all subsequent experiments, only intact spermatozoa were included in the analyses. PSA labelling was abolished by preincubation of the lectin with 1 M o-methyl marmoside, whereas D-galactose and N-acetyl-D-glucosamine had no effect. Similarly, labelling of PNA and WGA was inhibited only in the presence of D-galactose and N-acetyl-D-glucosamine, respectively. Incubation of spermatozoa with 5 PM A23187 for up to 1 h significantly increased the percentage of those forms identified as acrosome-reacted as described above (Table 1). This was confirmed by the time-dependent accumulation of those forms, as well by DIC microscopy. At all times, differences among the values obtained using the three lectins were not significant (P > 0.05). Linear regression analysis indicated a strong correlation ability to detect acrosome reaction (Table 2).
between
lectins as for their
Table 3 shows the effect of cryopreservation on the percentage of acrosome-intact spermatozoa within the total population (damaged and intact spermatozoa) and on the percentage of acrosome-intact spermatozoa within the membrane-intact population. It is clear that freezing and thawing lead to a decrease of both membrane integrity and acrosomal integrity, irrespective of the lectin used. However, almost all spermatozoa that withstood cryopreservation, as evaluated by Hoechst exclusion, showed intact acro-
Table 2 Linear regression analysis among values obtained from PSA, PNA and WGA lectins Comparison PSA vs. PNA PSA vs. WGA PNA vs. WGA (n = 42) for each comparison.
r= 0.91 0.92 0.91
P
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Table 3 Effect of freezing and thawing on membrane integrity and acrosomal status of ram spermatozoa PSA Fresh
PNA F/T
Fresh
Acrosome-intact within the total population (%b) 72 f 4 a 43 k 10 b 70f6a Acrosome-intact within the membrane-intact 96f3 98i1 9652 population (W)
WGA F/T
Fresh
F/T
42f10b 94k2
69*6’ 95+3
42f10b 95f2
Values represent meanf SD (n = 12). Numbers with different superscripts significantly differ (P < 0.001). Percentage of membrane integrity and motility were 73&4 and 73&2, respectively, for fresh semen, and 44+ 10 and 38 f 5, respectively, for frozen/thawed (F/T) semen.
somes. In this case, no differences between fresh and frozen/thawed observed (Table 3).
samples were
4. Discussion The present results indicate that lectins can be effectively used to monitor acrosomal status after cryopreservation and induced acrosomal exocytosis in ram spermatozoa. In addition, a method was developed for the simultaneous assessment of membrane integrity and acrosomal status. Hence, the importance of this method lies in that it can distinguish the ‘true’ from the ‘false’ acrosome reaction, the latter being the consequence of degenerative changes of the acrosome, not necessarily related to the process of egg penetration (Bedford, 1970). The dual staining procedure described herein involves a first step at which damaged cells are labelled with a fluorescent supravital stain, followed by lectin labelling. The supravital dye Hoechst 33258 is widely used for the assessment of membrane integrity, since,under normal conditions it does not penetrate the plasma membrane, but it reaches the nucleus and binds DNA upon membrane damage or permeabilization (Visser, 1981). As subsequent lectin staining involves fixation with ethanol, which permeabilizes all cells, it is essential that the excess fluorochrome be thoroughly removed by washing, and that it remain within the cell after fixation. In our conditions, attempts to use propidium iodide, commonly used in combination with carboxyfluorescein acetate (ValcSrcel et al., 1994) were unsuccessful, as the dye appeared to diffuse from stained cells, thus labelling cells that were originally intact as well. This effect was also found in human spermatozoa (Centola et al., 1990). A possible explanation for the discrepancy between CFDA/PI and Hoechst 33258 methods as to their incidence of false negative results could be that the excess PI present in cells would be enough to label all cells after ethanol permeabilization, whereas the excess of Hoechst 33258 would not. Control experiments showed that after Hoechst staining, washing and permeabilization, the percentage of membrane integrity was not significantly different from that obtained after staining alone, thus validating this procedure. DIC microscopy was used to monitor the acrosomal status of spermatozoa showing
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the different patterns of lectin binding. For PNA and PSA, the most abundant form found in fresh semen exhibited acrosomal staining, whereas unstained spermatozoa and those showing equatorial labelling were associated with acrosome-reacted forms. Patch labelling on the acrosome was found in nonviable cells. PNA binding pattern coincided with that found in other species (Virtanen et al., 1984, Lee and Damjanov, 1985; Aitken et al., 1993; Vazquez et al., 1993; Cross and Watson, 1994). This was also the case for PSA (Cross et al., 1986; Fukuda et al., 1989; Centola et al., 1990; Mendoza et al., 1992; Casey et al., 1993; Kawakami et al., 1993; Tesarik et al., 1993). By homology with human spermatozoa (Talbot and Chacon, 1982) it is possible that both forms associated to acrosome loss represent different functional stages, as only forms showing equatorial labelling are able to bind the plasma membrane of the oocyte. These spermatozoa are thought to have just acrosome-reacted, whereas unlabelled spermatozoa have lost their acrosomes for a longer time, and are unable to interact with the oocyte (Tesarik et al., 1993). As for WGA, most spermatozoa showed intense labelling in the acrosomal region and weak postacrosomal labelling, as was found in human (Virtanen et al., 1984; Lee and Damjanov, 1985; Cross et al., 1986; Cross and Overstreet, 1987); bovine (Cross and Watson, 1994) and boar (Aguas and Pinto da Silva, 1983) spermatozoa. The calcium ionophore A23187 has been successfully used in ram spermatozoa to induce acrosomal exocytosis (Shams-Borhan and Harrison, 1981; Ben-Av et al,, 1988; Watson et al., 1991). We found 66%, 60% and 66% of reacted forms after 1 h of exposure to calcium ionophore, when PSA, PNA and WGA were used, respectively. The degree of acrosomal exocytosis is highly dependent on the incubating conditions and the capacitation stage, therefore it may be not fully comparable with the results obtained by other authors; nevertheless, incubation under identical conditions led to the same values of acrosomal exocytosis, irrespective of the lectin used. This does not seem to be the case in human spermatozoa, where PNA revealed a higher percentage of acrosome reaction than PSA (Aitken and Brindle, 1993). Cryopreservation of spermatozoa causes a decrease in motility (Keel et al., 1987), functionality (Check et al., 1991), membrane integrity (Halt et al., 1992; Valcarcel et al., 1994) and velocity (Moses et al., 1995). In addition, extensive damage to the,acrosome may contribute to the loss of acrosin (Pedersen and Lebech, 1971; Mack and Zaneveld, 1987; Centola et al., 1990; Cross and Hanks, 1991) and a consequent decrease in fertility. Much of the damage may occur during thawing (Woolley and Richardson, 1978) and physical damage to the acrosome may be secondary to cell death (Cross and Hanks, 1991). Nevertheless, our results indicate that almost all spermatozoa that have survived thawing, or at least remained membrane-intact, also maintained their acrosomes intact, in contrast with the results from Centola et al. (1990); McLaughlin et al. (1993) and Tao et al. (1993) who found that viable frozen/thawed human spermatozoa had a higher degree of acrosome damage than fresh spermatozoa. It must be taken into account that cryoresistance is related to the composition of the plasma membrane (Parks and Graham, 1992), and that acrosomal and plasma membranes may differ in their composition (Halt and North, 1985). Using dual staining with propidium iodide/carboxyfluorescein diacetate, we have found that about 18% of frozen/thawed spermatozoa with their plasma membrane damaged are acrosome-intact
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(Valcarcel et al., 1996). On the other hand, 14% of frozen/thawed spermatozoa that were positive to Hoechst staining were acrosome-intact as assessed by lectin binding (data not shown). These results, along with the fact that virtually all frozen/thawed intact spermatozoa display normal lectin binding, indicate that, at least in ram spermatozoa, resistance of the plasma membrane to cryodamage is lower than that of acrosome membranes, and is more critical to the success of cryopreservation. In conclusion, assessment of acrosomal status of ram spermatozoa by lectin binding is a fast, reliable and highly reproducible technique that can be used in a variety of conditions for the follow-up of in vitro reproductive procedures. In particular, this technique allowed us to show that acrosomal integrity is not the most relevant damage to ram spermatozoa by cryopreservation, so that other functions related to the integrity of the plasma membrane seem to be more critical for the maintenance of fertility.
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Assessment of the acrosomal status of membrane-intact ram spermatozoa after freezing and thawing, by simultaneous lectin/Hoechst 33258 staining A. Valcircel
*,
M.A. de las Heras, L. Pkrez, D.F. Moses, H. Baldassarre
Centrn de Invesrigaciones Reproductiuus Pkrez Compunc, Au de Mayo 701. 1084 Buenos Aires, Argentina Accepted
23 May 1996
Abstract We have evaluated the effect of freezing and thawing on the acrosomal status of ram spermatozoa, especially those that withstood cryopreservation as assessed by membrane integrity. To this end, we performed simultaneous lectin/Hoechst 33258 staining, and compared the ability of three fluoresceinated lectins. Pisum sariuum lectin Ram spermatozoa were treated with fluorescein isothiocyanate-labelled (PSA), fluorescein isothiocyanate-labelled Aruchis hypogea lectin (PNA) and fluorescein isothiocyanate-labelled Triricum uufgaris lectin (WGA) and simultaneously with Hoechst 33258 for determination of membrane integrity and acrosomal status. In all cases, three forms were readily distinguished by their distribution pattern. For both PSA and PNA, the most abundant form found in fresh semen consisted of fluorescence on the acrosomal area. This form corresponds to acrosome-intact spermatozoa, as assessed by Differential Interference Contrast (DIG) microscopy. Two minor forms showed weak fluorescence on the equatorial segment or no fluorescence on the head. DIC microscopy revealed that both forms were associated with acrosome-lost spermatozoa. WGA labelling showed two forms, one of which consisted of fluorescence on the entire head, albeit more intensely on its anterior segment. Spermatozoa in this form were acrosome-intact by DIC. The other form lacked fluorescence on the acrosomal region, but still showed faint fluorescence in the posterior region. This form was acrosome-lost by DIC.
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037%4320/97/$17.00 Copyright 0 1997 Elsevier Science B.V. All rights reserved. PII SO378-4320(96)01586-2
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Incubation of fresh spermatozoa with calcium ionophore A23187 for up to 1 h significantly increased the percentage of those forms identified as acrosome-reacted as described above. This was confirmed by the time-dependent accumulation of these forms, as well ‘as by DIC microscopy. At all times, differences among values obtained using these three lectins were not significant. Freezing and thawing led to a decrease of both membrane integrity and acrosomal integrity, irrespective of the lectin used. However, almost all spermatozoa that withstood cryopreservation, as evaluated by Hcechst exclusion, showed intact acrosomes. In this case, no differences between fresh and frozen/thawed samples were observed. These results suggest that the structural integrity of ram spermatozoa is mostly unaffected after cryopreservation, suggesting that it is damage to the plasma membrane that is primarily responsi-
ble for the low fertility of cryopreserved samples. Keywords: Agglutinins; Semen; Cryopreservation;Sheep-male reproduction; Acrosomal status; Lectins
1. Introduction Cryopreservation of spermatozoa from many species leads to a series of alterations resulting in a marked decrease in their fertility (reviewed in Parks and Graham, 1992). Among these changes, damage to plasma/acrosomal membranes has been indicated as a major cause of function loss, due to leakage of cellular components and inactivation of crucial proteins. Damage to the plasma membrane can be measured by means of fluorescent vital or supravital dyes such as carboxyfluorescein diacetate (CFDA) and propidium iodide (PI). In ram spermatozoa, we have demonstrated that at least 30% of those sperrmatozoa that remained motile after freezing and thawing had damage to the plasma membrane &al&me1 et al., 1994); those spermatozoa rapidly lose their motility at 37°C and would therefore be nonfunctional in vivo. Hence, from a physiological view, determination of parameters of cellular structure or function is meaningful only when performed on membrane-intact spermatozoa, in particular after cryopreservation. An important aspect in the evaluation of semen quality is the assessment of the ability of spermatozoa to undergo the acrosome reaction. This process is associated to the activation and exocytotic release of proteolytic enzymes from the acrosome and is thought to play a role in the penetration of the egg investments (reviewed by Yanagimachi, 1994). In ram spermatozoa, it is often difficult to monitor the acrosomal status by conventional light microscopy (Shams-Borhan and Harrison, 1981; Watson et al., 1991); on the other hand, electron microscopy (Shams-Borhan and Harrison, 1981; Ben-Av et al., 1988; Williams et al., 1991) is not practical for routine purposes. Several methods have been recently described to evaluate the Percentage of acrosome-reacted ram spermatozoa, including triple stain (Gutierrez et al., 1993), naphtol yellow/erythrosin B
staining (Graham et al., 1987; Williams et al., 1991) and enzymic methods (Williams et al., 1991). However, for different reasons, these techniques has not been widely used. Pluorescein-conjugated lectins are being increasingly used for the determination of acrosomal status. In particular, agglutinins from Pisum sativum (PSA), Aruchis hypogea (PNA) and Triticum vulgaris (WGA) have been the most widely used. Dual staining aimed at the simultaneous determination of acrosomal status and membrane integrity offers further information on those spermatozoa that are potentially
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fertile. Supravital fluorescent stains such as Hoechst 33258 and propidium iodide (alone or combined with fluorescein diacetate), which enter the cell nucleus and bind DNA only when the plasma membrane is damaged and/or permeabilized, have proved particularly useful for the assessment of sperm integrity (Cross et al., 1986; Garner et al., 1986; Harrison and Vickers, 1990; Mendoza et al., 1992). In this paper we show, by means of a combined Hoechst 33258/fluoresceinated lectin staining, that acrosomal integrity is maintained in those spermatozoa that have withstood cryopreservation, as assessed by the most relevant alteration described so far, i.e. damage to the plasma membrane.
2. Materials and methods 2.1. Chemicals Fluorescein isothiocyanate-labelled lectins from pea (PSA), peanut (PNA) wheat germ (WGA), as well as other reagents of analytical grade, were supplied by Sigma Chemical Co (St Louis, MO, USA). 2.2. Semen collection and freezing Semen was collected from 12 adult Merino rams of proven fertility, by means of an artificial vagina. Immediately after collection, concentration and percent motility were assessed using a computerized semen analyzer (CellTrakTM, Motion Analysis, Santa Rosa, CA, USA) configured in our laboratory for its use with ram semen (Moses et al., 1995). Samples showing less than 70% motility were discarded. Semen was extended at a final concentration of 5 X 10’ spermatozoa ml -’ in a Tes-Tris-based diluent containing 20% (v/v) egg yolk and 4% (v/v> glycerol, pH 7.3, 340 mOsm kg-‘. After motility evaluation, diluted samples were slowly cooled to 4°C within 2 h, frozen in 0.2-ml pellets on dry ice and transferred to liquid nitrogen. Thawing was performed at 37°C in Kahn tubes. 2.3. Induction of acrosomal exocytosis by calcium ionophore A23187 Ten-p1 semen aliquots, containing 4-5 X 10’ spermatozoa were diluted 1:lO in phosphate-buffered saline (PBS), mixed with an equal volume of A23187 in PBS (prepared from a stock 5 mM solution in DMSO), such that final concentrations were 5 ,uM A23 187 and 0.1% (v/v) DMSO, and incubated at 37°C in a humidified incubator. Control samples contained 0.1% DMSO alone. After 30 and 60 min, aliquots were taken for lectin binding/membrane integrity assay and DIC microscopy. 2.4. Simultaneous assessment of lectin binding pattern and membrane integrity Ten-p1 semen aliquots, containing 4-5 X 10’ spermatozoa, were diluted in 90 ~1 3% (w/v> sodium citrate, mixed with 100 ~1 of a Hoechst 33258 solution (1 pg ml-’ in
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sodium citrate) and incubated at 37°C for 3 min. Excess dye was removed by centrifugation through 1 ml of a Ficoll solution (70 g 1-l Ficoll.400, 20 mM Hepes, 10.3 mM Tris, 285.2 mM fructose, 4 mM MgCl,, 100 mg 1-l kanamycin sulfate, pH 7.3, 340 mOsm kg-’ > at 15 600 g for 30 s. The supematant was carefully aspirated, avoiding contact with the pellets, which were further washed in 400 ~1 of PBS and fixed in 95% ethanol at 4°C (Cross et al., 1986) for 10 min. The suspensions were centrifuged, washed in PBS and finally resuspended in 90 ~1 PBS. For lectin labelling, 10 ~1 of a solution containing 100 pg ml -’ of one of each lectin in PBS were added. The suspensions were incubated 37°C for 8 min in the dark, washed in 1 ml of PBS and mounted on low-fluorescence glass slides using 90% (v/v) glycerol in PBS. At least 200 spermatozoa per slide were assessed for Hoechst and lectin fluorescence, in a Nikon Optiphot epifluorescence microscope, using UV and B-2A filters, respectively. Lectin specificity was determined by competition with different monosaccharides. For this purpose, stock lectin solutions were mixed with an equal volumen of 1 M o-methyl mannoside, D-galactose or N-acetyl-D-glucosamine. After 30 min, labelling was carried out as described above. 2.5. Assessment of acrosomal status by light microscopy Acrosomal morphology was assessed by differential interference contrast (DIG) microscopy, as described by Watson et al. (19911, with the exception that intermediate and late stages of acrosomal loss were considered as a whole. At least 100 spermatozoa per sample, in duplicates, were observed. 2.6. Statistical analysis
Differences were analyzed by Student’s t-test or by ANOVA and Tukey test, after angular transformation of data. Correlation was determined by linear regression analysis (Steel and Torrie, 1981).
3. Results We have compared the ability of three lectins to characterize acrosomal integrity of ram spermatozoa. Fig. 1, Fig. 2 and Fig. 3 show the labelling pattern of PSA, PNA and WGA. For both PSA and PNA, the most abundant form found in fresh semen consisted of fluorescence on the acrosomal area (Fig. la and Fig. 2a). This form corresponds to acrosome-intact spermatozoa, as assessed by DIC. Two minor forms showed weak fluorescence on the equatorial segment or no fluorescence on the head (Fig. lb and Fig. 2a). DIC microscopy revealed that both forms were associated with acrosome-lost spermatozoa. WGA labeling showed two forms, one of which consisted of fluorescence on the entire head, albeit more intensely on its anterior segment (Fig. 3a). Spermatozoa in this form were acrosome-intact by DIC. The other form lacked fluorescence on the acrosomal region, but still showed faint fluorescence in the posterior region (Fig. 3b).
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Fig. 1 PSA binding pattern in ram spermatozoa. (a) intact (lower) and acrosome-damaged (upper) spermatozoa, showing fluorescence on the acrosomal area. (b) acrosome-reacted spermatozoon, showing weak fluorescence on the equatorial region (arrow). Bar: 4 pm.
Fig. 2. PNA binding pattern in ram spermatozoa. (a) three intact spermatozoa, showing fluorescence on the acrosomal area, and one acrosome-reacted spermatozoon (arrow), with no fluorescence on the head. (b) acrosome-damaged spermatozoon. Bar: 4 pm.
This form was acrosomeless by DIC. Some spermatozoa showed partial damage to the acrosome readily evident by lectin staining (Fig. la, Fig. 2b and Fig. 3a). Membrane-damaged (non viable) spermatozoa were characterized by anomalous
Fig. 3. WGA binding pattern in ram spermatozoa. (a) acrosome-intact (upper) and damaged (lower) spermatozoa. showing fluorescence on the entire head, mainly on the acrosomal, and the principal piece of the flagellum. (b) acrosome-reacted spermatozoon, with no fluorescence on the acrosome. Bar: 4 pm.
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Table 1 Percentage of acrosome-reacted spermatozoaaf’terincubation with A23187, as evaluated by PSA, PNA and WGA labelling Lectin
Time (min)
PSA PNA
0 2*la l*l’
30 42&3 b 38i-3 b
60 66*4c 60*4c
WGA
2il’
361t4~
66*5c
Values represent mean f SD (n = 12). Values with different superscripts significantly differ (P < 0.001).
lectin distribution in all cases, including: (a> patch labelling on the acrosome, complete staining and postacrosomal/equatorial staining for PSA; (b) patch labelling on the acrosome for PNA; and (c) complete or patch labelling exclusively on the acrosome, equatorial labelling or no head fluorescence for WGA (data not shown). In all cases, 10-E% of non-viable spermatozoa showed intact acrosomes, and 45-50% showed acrosomal loss or damage, as assessed by DIC. In all subsequent experiments, only intact spermatozoa were included in the analyses. PSA labelling was abolished by preincubation of the lectin with 1 M o-methyl marmoside, whereas D-galactose and N-acetyl-D-glucosamine had no effect. Similarly, labelling of PNA and WGA was inhibited only in the presence of D-galactose and N-acetyl-D-glucosamine, respectively. Incubation of spermatozoa with 5 PM A23187 for up to 1 h significantly increased the percentage of those forms identified as acrosome-reacted as described above (Table 1). This was confirmed by the time-dependent accumulation of those forms, as well by DIC microscopy. At all times, differences among the values obtained using the three lectins were not significant (P > 0.05). Linear regression analysis indicated a strong correlation ability to detect acrosome reaction (Table 2).
between
lectins as for their
Table 3 shows the effect of cryopreservation on the percentage of acrosome-intact spermatozoa within the total population (damaged and intact spermatozoa) and on the percentage of acrosome-intact spermatozoa within the membrane-intact population. It is clear that freezing and thawing lead to a decrease of both membrane integrity and acrosomal integrity, irrespective of the lectin used. However, almost all spermatozoa that withstood cryopreservation, as evaluated by Hoechst exclusion, showed intact acro-
Table 2 Linear regression analysis among values obtained from PSA, PNA and WGA lectins Comparison PSA vs. PNA PSA vs. WGA PNA vs. WGA (n = 42) for each comparison.
r= 0.91 0.92 0.91
P
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Table 3 Effect of freezing and thawing on membrane integrity and acrosomal status of ram spermatozoa PSA Fresh
PNA F/T
Fresh
Acrosome-intact within the total population (%b) 72 f 4 a 43 k 10 b 70f6a Acrosome-intact within the membrane-intact 96f3 98i1 9652 population (W)
WGA F/T
Fresh
F/T
42f10b 94k2
69*6’ 95+3
42f10b 95f2
Values represent meanf SD (n = 12). Numbers with different superscripts significantly differ (P < 0.001). Percentage of membrane integrity and motility were 73&4 and 73&2, respectively, for fresh semen, and 44+ 10 and 38 f 5, respectively, for frozen/thawed (F/T) semen.
somes. In this case, no differences between fresh and frozen/thawed observed (Table 3).
samples were
4. Discussion The present results indicate that lectins can be effectively used to monitor acrosomal status after cryopreservation and induced acrosomal exocytosis in ram spermatozoa. In addition, a method was developed for the simultaneous assessment of membrane integrity and acrosomal status. Hence, the importance of this method lies in that it can distinguish the ‘true’ from the ‘false’ acrosome reaction, the latter being the consequence of degenerative changes of the acrosome, not necessarily related to the process of egg penetration (Bedford, 1970). The dual staining procedure described herein involves a first step at which damaged cells are labelled with a fluorescent supravital stain, followed by lectin labelling. The supravital dye Hoechst 33258 is widely used for the assessment of membrane integrity, since,under normal conditions it does not penetrate the plasma membrane, but it reaches the nucleus and binds DNA upon membrane damage or permeabilization (Visser, 1981). As subsequent lectin staining involves fixation with ethanol, which permeabilizes all cells, it is essential that the excess fluorochrome be thoroughly removed by washing, and that it remain within the cell after fixation. In our conditions, attempts to use propidium iodide, commonly used in combination with carboxyfluorescein acetate (ValcSrcel et al., 1994) were unsuccessful, as the dye appeared to diffuse from stained cells, thus labelling cells that were originally intact as well. This effect was also found in human spermatozoa (Centola et al., 1990). A possible explanation for the discrepancy between CFDA/PI and Hoechst 33258 methods as to their incidence of false negative results could be that the excess PI present in cells would be enough to label all cells after ethanol permeabilization, whereas the excess of Hoechst 33258 would not. Control experiments showed that after Hoechst staining, washing and permeabilization, the percentage of membrane integrity was not significantly different from that obtained after staining alone, thus validating this procedure. DIC microscopy was used to monitor the acrosomal status of spermatozoa showing
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the different patterns of lectin binding. For PNA and PSA, the most abundant form found in fresh semen exhibited acrosomal staining, whereas unstained spermatozoa and those showing equatorial labelling were associated with acrosome-reacted forms. Patch labelling on the acrosome was found in nonviable cells. PNA binding pattern coincided with that found in other species (Virtanen et al., 1984, Lee and Damjanov, 1985; Aitken et al., 1993; Vazquez et al., 1993; Cross and Watson, 1994). This was also the case for PSA (Cross et al., 1986; Fukuda et al., 1989; Centola et al., 1990; Mendoza et al., 1992; Casey et al., 1993; Kawakami et al., 1993; Tesarik et al., 1993). By homology with human spermatozoa (Talbot and Chacon, 1982) it is possible that both forms associated to acrosome loss represent different functional stages, as only forms showing equatorial labelling are able to bind the plasma membrane of the oocyte. These spermatozoa are thought to have just acrosome-reacted, whereas unlabelled spermatozoa have lost their acrosomes for a longer time, and are unable to interact with the oocyte (Tesarik et al., 1993). As for WGA, most spermatozoa showed intense labelling in the acrosomal region and weak postacrosomal labelling, as was found in human (Virtanen et al., 1984; Lee and Damjanov, 1985; Cross et al., 1986; Cross and Overstreet, 1987); bovine (Cross and Watson, 1994) and boar (Aguas and Pinto da Silva, 1983) spermatozoa. The calcium ionophore A23187 has been successfully used in ram spermatozoa to induce acrosomal exocytosis (Shams-Borhan and Harrison, 1981; Ben-Av et al,, 1988; Watson et al., 1991). We found 66%, 60% and 66% of reacted forms after 1 h of exposure to calcium ionophore, when PSA, PNA and WGA were used, respectively. The degree of acrosomal exocytosis is highly dependent on the incubating conditions and the capacitation stage, therefore it may be not fully comparable with the results obtained by other authors; nevertheless, incubation under identical conditions led to the same values of acrosomal exocytosis, irrespective of the lectin used. This does not seem to be the case in human spermatozoa, where PNA revealed a higher percentage of acrosome reaction than PSA (Aitken and Brindle, 1993). Cryopreservation of spermatozoa causes a decrease in motility (Keel et al., 1987), functionality (Check et al., 1991), membrane integrity (Halt et al., 1992; Valcarcel et al., 1994) and velocity (Moses et al., 1995). In addition, extensive damage to the,acrosome may contribute to the loss of acrosin (Pedersen and Lebech, 1971; Mack and Zaneveld, 1987; Centola et al., 1990; Cross and Hanks, 1991) and a consequent decrease in fertility. Much of the damage may occur during thawing (Woolley and Richardson, 1978) and physical damage to the acrosome may be secondary to cell death (Cross and Hanks, 1991). Nevertheless, our results indicate that almost all spermatozoa that have survived thawing, or at least remained membrane-intact, also maintained their acrosomes intact, in contrast with the results from Centola et al. (1990); McLaughlin et al. (1993) and Tao et al. (1993) who found that viable frozen/thawed human spermatozoa had a higher degree of acrosome damage than fresh spermatozoa. It must be taken into account that cryoresistance is related to the composition of the plasma membrane (Parks and Graham, 1992), and that acrosomal and plasma membranes may differ in their composition (Halt and North, 1985). Using dual staining with propidium iodide/carboxyfluorescein diacetate, we have found that about 18% of frozen/thawed spermatozoa with their plasma membrane damaged are acrosome-intact
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(Valcarcel et al., 1996). On the other hand, 14% of frozen/thawed spermatozoa that were positive to Hoechst staining were acrosome-intact as assessed by lectin binding (data not shown). These results, along with the fact that virtually all frozen/thawed intact spermatozoa display normal lectin binding, indicate that, at least in ram spermatozoa, resistance of the plasma membrane to cryodamage is lower than that of acrosome membranes, and is more critical to the success of cryopreservation. In conclusion, assessment of acrosomal status of ram spermatozoa by lectin binding is a fast, reliable and highly reproducible technique that can be used in a variety of conditions for the follow-up of in vitro reproductive procedures. In particular, this technique allowed us to show that acrosomal integrity is not the most relevant damage to ram spermatozoa by cryopreservation, so that other functions related to the integrity of the plasma membrane seem to be more critical for the maintenance of fertility.
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