THE JOURNAL OF EXPERIMENTAL ZOOLOGY 215:35-46 11981)

In Vitro Capacitation and the Chemically Induced Acrosome Reaction in Bovine Spermatozoa

WILLIAM BYRD Department of Zoology and Physiology, Louisiunn State University, Baton Rouge, Louisiana 70803

ABSTRACT The effect of bovine serum albumin (BSA), calcium, and iono- phore A23187 on in vitro capacitation and the induction ofthe acrosome reaction in bovine spermatozoa was examined using light, fluorescent, and electron micros- copy and spectrofluorometry. Transmission electron microscopy of fixed sper- matozoa labeled with the plant lectin concanavalin A indicated that a significant redistribution of lectin binding sites requires a t least three hours of incubation in a capacitating medium that contains BSA and calcium. Spermatozoa underwent capacitation and the acrosome reaction following a minimum incubation time of 2-3 hours incapacitating medium in the continuous presence of calcium ionophore A23187 (1-10 pm). The induction of the acrosome reaction was determined by light microscopy of fixed-stained cells as well as transmission electron microscopy. These data suggest that a minimum of 2-3 hours is required for in vitro capacita- tion of bovine spermatozoa. Capacitation was also examined using the tetracy- cline-HC1 (T-HC1) binding assay of Ericsson ('67). Measurement of fluorescence by spectrofluorometry and by fluorescence microscopy demonstrated that the level of fluorescent T-HC1 bound to spermatozoa was dependent upon the concentration of BSA, the presence of calcium, and time of incubation. However, the loss of bound T-HCl does not coincide with the development of the capacitated state determined by lectin binding and induction of the acrosome reaction. It is suggested that this loss of fluorescence may represent either a preliminary step in capacitation or result from nonspecific binding of fluorescent label to BSA.

Mammalian spermatozoa obtained directly from the caudal epididymis or from fresh ejacu- lates are incapable of fertilizing eggs of the same species. Spermatozoa normally undergo a maturation step (capacitation) in the female reproductive tract which is an essential pre- requisite for penetration of the zona pellucida and fusion with the egg plasma membrane (Gwatkin, '76). Capacitation consists of a series of changes both structural and metabolic (Chang and Hunter, '75).

To mimic in vitro the physiological conditions that induce capacitation in vivo, it is necessary to include calcium and often bovine serum al- bumin (BSA) in the capacitating medium (Rog- ers, '78). Calcium, which has been implicated in several membrane related fusion events, is required for capacitation (Reyes et al., '781, sperm motility (Davis, '781, and sperm binding to eggs (Saling et al., '78)-as well as the induc- tion of the acrosome reaction in vitro (Yanagimachi and Usui, '74; Talbot et al., '76;

Green, '78). Blood serum proteins, in particular BSA, may play an important role in in vitro capacitation and the induction of the acrosome reaction (Barros and Garavagno, '70; Yanagi- machi, '70; Lui and Meizel, '77; Oliplant et al., '77).

Characterization of the physical changes in the spermatozoan surface during capacitation is important since capacitation leads to egg rec- ognition by the spermatozoan and fusion. Pre- vious studies of successful capacitation have demonstrated that capacitation does not alter the ultrastructural features of the spermatozoa (Bedford, '74). However, spermatozoa do un- dergo modification at the plasma membrane during capacitation and exhibit altered surface properties such as decrease in the net negative charge of the surface (Vaidya et al., '691, disap- pearance of surface bound fluorescent tetracy- cline-HC1 (T-HCL) (Ericsson, '67), and removal or modification of surface bound components of the plasma membrane over the acrosome (Oli-

0022-104X/81/2151-0035$02.30O 1981 ALAN R. LISS. INC.

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phant and Brackett, '73a; Gordon et al., '75; Kinsey and Koehler, '78). These changes sug- gest that capacitation, through the removal or alteration of surface bound components, pre- cedes membrane destabilization and induction of the acrosome reaction.

The basic physiological and morphological changes leading to capacitation and the acro- some reaction in vivo and in vitro have been defined for relatively few species. In this paper we describe the conditions for capacitation and induction of the acrosome reaction using cal- cium ionophore A23187 with ejaculated bovine spermatozoa in vitro and the requirement for BSA and calcium in this reaction. We initially assayed for capacitation in vitro using the fluo- rescent T-HC1 technique of Ericsson ('67). The value of the antibiotic tetracycline as a probe is that it binds to cations on biological mem- branes (Caswell and Hutchinson, '71a) and po- tentially serves as a means of examining ionic processes occurring in the membrane during capacitation. Using this technique, Goodeaux et al. ('78) reported a decrease in fluorescence bound to treated bovine spermatozoa in vitro suggesting that capacitation had occurred. Since there seems to be some controversy as to the reliability of this procedure as a test for capacitation in vivo (Vaidya et al., '691, we also examined capacitation in vitro by analysis of binding sites of the plant lectin concanavalin A on the sperm surface by transmission electron microscopy. Chemical induction of the acro- some reaction in capacitated spermatozoa was accomplished using the ionophore A23187. Pre- liminary reports of this work have already been presented (Byrd et al., '79a,b).

METHODS AND MATERIALS

Animals Ejaculated bull sperm were obtained by sex-

ual preparation from fertile dairy cattle from the Louisiana Animal Breeders Cooperative. The ejaculates were layered over 10 volumes of Tyrodes solution (pH 7.4) a t 37" and concen- trated by centrifugation at 750g for 5 minutes a t 23". The sperm were resuspended in 10 vol- umes of Tyrodes and centrifuged as above. After resuspension in a small volume of Ty- rodes at 37", the sperm were counted with a hemocytometer, and the concentration was adjusted to 200 x lo6 sperm/ml.

Fluorescence assay Tetracycline HCl (T-HC1) in Tyrodes solution

(10 mg/ml stock, pH 5.5) was added to sperm at a final concentration of 10 Fg/ml per 100 x lo6

sperm/ml (Goodeaux et al., '78). After incuba- tion for 10 minutes a t 37", sperm were washed three times in Tyrodes. Sperm treated with T-HC1 do not display altered motility, morphol- ogy, or fertility (Ericsson, '67). Concentrated sperm were resuspended in either Tyrodes or Tyrodes with 6% BSA and incubated at 37". At indicated times, 2 ml aliquots were taken and fixed with paraformaldehyde (0.1% final). After fixation sperm were washed three times with 10 volumes of Tyrodes and centrifuged at 15,OOOg for 10 minutes to remove any unbound tetracycline. Quantitative measurement of sperm fluorescence was determined using an Aminco-Bowman spectrophotofluorometer. Fluorescence was measured at an excitation wavelength of 430 nm and an emission wave- length of 525 nm. Due to the initial turbidity of the samples, readings were not taken until one minute after addition to the spectropho- tometer. There was no change in fluorescence of samples from one to three minutes after addi- tion. Optical densities of the sperm suspensions taken a t 340 nm showed that there was no significant loss of spermatozoa during the washing process. Sperm were also examined by light microscopy to insure that the loss of fluo- rescence was not due to the loss of the acro- soma1 caps. Qualitative assessment of fluores- cence change during the incubation period was measured by examining the sperm suspensions in Figure 1 using a Leitz fluorescent micro- scope. Aliquots of the sperm suspensions were mounted and examined under oil a t 100 x magnification. The excitation wavelength used was 300-500 nm (Zeiss BG-12 filter), while the emission wavelength was 510 nm.

In Vitro Effect of BSA and Calcium on Fluorescence Loss

After incubation in T-HC1, as above, cells were washed and resuspended in BSA ranging in concentration from 0.75% to 1% (w/v), incu- bated at 37", and measured as above for fluores- cence.

To determine if calcium ions were required for the decrease in fluorescence, sperm were washed in calcium-free Tyrodes after incuba- tion in tetracycline. Sperm were then resus- pended in Tyrodes (t- calcium) or Tyrodes with 6% BSA (2 calcium) and processed as above.

Chemical Induction of the Acrosome Reaction by Zonophore A23187

BSA and calcium were assayed for their ability to induce capacitation as measured by the induction of the acrosome reaction using

CAPACITATION IN BOVINE SPERMATOZOA 37

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Fig. 1. Measurement of fluorescence loss in the presence or absence of BSA. To measure sperm surface alterations during incubation, sperm were incubated with tetracycline-HCI, washed and resuspended in Tyrodes (0) or Tyrodes with 6% BSA (0). Samples were fixed in paraformaldehyde, washed, and the fluorescence of the samples was measured on a spectrofluo- rometer (excitation wavelength of 430 nm and emission wavelength of 525 nm). Arrows indicate time points of samples processed for fluorescent microscopy seen in Figure 2.

ionophore A23187 (supplied by Eli Lilly Co.). Aliquots of sperm were washed and treated as before, final concentration of sperm was ad- justed to 100 x lo6 sperm/ml. On the basis of preliminary experiments a final concentration of 10 WM ionophore was used. Samples were incubated in the continuous presence of iono- phore, followed by fixation in 0.1% paraformal- dehyde. At each time point sperm were assayed for percent motility and percent acrosome reac- tion. These experiments were carried out for 4-5 hours.

The induction of the acrosome reaction was assayed by light microscopy using the proce- dure of Bryan and Akruk ('77). Fixed sper- matozoa were smeared on protamine covered slides, dried at room temperature, and stained with naphthol yellow S and erythrosin B. Acro- soma1 caps stained cherry-red while the post- nuclear caps stained a pale pink. After mount- ing, the loss of the acrosomal caps was deter- mined by phase-contrast microscopy at 100 x magnification by counting at least 400 sperm on each slide in a blind control. A certain

number of sperm not treated with BSA or iono- phore were observed to have lost their acro- somes, perhaps due to mechanical damage. To determine if the low percentage of intact caps in treated spermatozoa was due to a true acrosome reaction, spermatozoa were fixed for transmis- sion electron microscopy.

Transmission Electron Microscopy Sperm were fixed after incubation in vitro

with 1% paraformaldehyde (pHCHO), pH 7.8, to prevent gelling of BSA, if present. After washing, sperm were then fixed in 2.5% glutar- aldehyde (0.1 M phosphate buffer, pH 7.4) for one hour. Sperm were washed, post-fixed with 1% osmium tetroxide in 0.05% phosphate (pH 7.4) for 45 minutes on ice. Samples were washed, stained with 0.5% uranyl acetate, washed, dehydrated, and embedded in Spurr's resin. Sections were post stained with uranyl acetate and lead citrate.

Labeling of Prefixed Cells To determine the presence of concanavalin A

(Con A) receptors, sperm were prepared using a

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modified method of Gordon e t al. ('75). After incubation, spermatozoa were prefixed in 1% pHCHO (pH 7.81, washed in Tyrodes, and then fixed in 2.5% glutaraldehyde in 0.1 M phos- phate buffer (pH 7.3) for one hour and washed in buffer. Cells were then incubated in two changes of 0.2 M glycine-phosphate buffer for one hour each and then resuspended in Tyrodes (dextrose free) with 0.6 mg/ml of concanavalin A. Sperm were then incubated for 20 minutes at room temperature, washed in Tyrodes (dex- trose free), and reacted with 20 pglml of horse- radish peroxidase for 15 minutes, and rinsed again. After washing, sperm were incubated in diaminobenzidine (0.5 mg/ml in 0.5 M Tris buf- fer, pH 7.2, with 0.01% hydrogen peroxide) for 15 minutes. After incubation, sperm were washed and then fixed and processed as above for transmission electron microscopy.

RESULTS

Measurement of Capacitation by Fluorescent- Tetracycline HCl Loss

Using a spectrofluorometer to quantitate the loss of fluorescence, we established the time required for bovine spermatozoa to undergo ca- pacitation (i.e., fluorescence loss) in Tyrodes (+6% BSA). Bovine spermatozoa were incu- bated in T-HC1 as described in Methods. Incu- bation in T-HC1 did affect initial motility of spermatozoa, but full motility returned within minutes after washing in Tyrodes.

Figure 1 shows the typical results of incubat- ing identical samples of spermatozoa. Each point in Figure 1 represents the average of two different samples, and each sample was the av- erage of at least three readings. T-HC1-labeled spermatozoa incubated in Tyrodes alone did not exhibit loss of fluorescence after 60 minutes (A) and 240 minutes (B) of incubation. Sper- matozoa incubated in Tyrodes with 6% BSA showed a sharp decrease in fluorescence which reached a minimum by 30 minutes of incuba- tion (D). We did not observe a complete loss of fluorescence in any of the samples. This may be due to a residual amount of label bound to the tail, unbound label in the medium, or label bound to dead or immotile spermatozoa. Micro- scopic examination of fixed-stained sper- matozoa using the technique of Bryan and Akruk ('77) shows that there is less than a 5% increase in spermatozoa lacking their acro- soma1 caps during the first 30 minutes in all samples. This demonstrates that the abrupt de- crease in fluorescence in the presence of BSA is not due to the loss of the acrosomal caps result- ing from mechanical damage during washing

or fixation or to a possible acrosome reaction. When samples A-D from Figure 1 were ob-

served by fluorescence microscopy (Fig. 21, there was a correlation with the loss of fluores- cence as measured by spectrofluorometry. La- beled spermatozoa were fluorescent over the entire surface of the cell with the midpiece being the brightest area. T-HC1-labeled sam- ples, incubated in Tyrodes alone, exhibited a strong fluorescence a t 60 minutes of incubation (Fig. 2a) and were still fluorescent by 240 min- utes of incubation (Fig. 2b). Samples incubated in Tyrodes (+6% BSA) showed a decrease in fluorescence by 15 minutes (Fig. 2c) and almost a complete absence by 30 minutes of incubation (Fig. 2d). There was a very faint fluorescence in Figure 2d which did not reproduce (see insert).

The decrease in fluorescence in Tyrodes supplemented with BSA was found to be de- pendent upon the concentration of BSA and the time of incubation (Fig. 3). At concentrations of 1.5% to 12%, BSA fluorescence decreases within 35 minutes of incubation. Below this concentration there is a substantial increase in the time required to lose fluorescence. In fur- ther studies, a WO BSA was used as the stan- dard concentration.

Fluorescence loss is also calcium dependent. The minimal calcium concentration required to cause loss of fluorescence in the presence of 6% BSA was 0.51 mM. Below this concentration there was no decrease by 330 minutes of incu- bation in the presence of BSA. Increasing con- centrations of calcium had no effect on the tim- ing of fluorescence loss.

Evaluation of Capacitation by Redistribution of Lectin Binding Sites

To determine the distribution of lectin bind- ing sites we labeled fixed spermatozoa with Con A using the procedure of Gordon et al. ('75). Alternately, we conjugated live spermatozoa prior to incubation with Con A. This latter technique was not successful due to the low motility observed in spermatozoa after incuba- tion with the lectin. No capacitation was ob- served in this latter group.

Figure 4 a-d shows the distribution of Con A binding sites on spermatozoa after incubation in Tyrodes (+6% BSA). Spermatozoa that have been incubated in BSA for 30 seconds and then fixed exhibit a dense, uniform labeling over the entire acrosome (Fig. 4a). After incubation in Tyrodes (t BSA) both samples show a uniform labeling over the entire head at 60 minutes (Figs. 2b, c). By four hours of incubation in the presence of 6% BSA, Con A labeling is almost

CAPACITATION IN BOVINE SPERMATOZOA 39

Fig. 2. Fluorescence microscopy of samples in Figure 1. Aliquots of the same sperm suspension used in Figure 1. (Points a-d) were examined by fluorescence microscopy. The excitation wavelength used was 300-500 nm while the emission wavelength was 510 nm. a) Samples were incubated in Tyrodes solution and fixed after 60 minutes of incubation. b) Samples were incubated in Tyrodes solution and fixed after 240 minutes of incubation. c) Samples were incubated in Tryodes solution with 6% BSA and fmed after 15 minutes of incubation. d) Samples were incubated in Trycdes solution with 6% BSA and fixed after 30 minutes of incubation. Insert: sperm sample without emission filter demonstrating presence of a spermatozoan.

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Fig. 3. Effects of different BSA concentrations on fluores- cence loss. After incubation in Tyrodes with tetracycline- HCl, cells were washed and resuspended in Tyrodes with BSA ranging in concentration from 0 to 12% (w/v) and incu- bated at 37". Samples were fixed and measured as in Figure 1. The ordinate in Figure 3 shows the mean times (? the standard error) shown for five ejaculates to reach the greatest reduction in fluorescence as exhibited in Figure 1, point D.

absent from the surface of the spermatozoa (Fig. 4d). Loss of Con A binding was observed after three hours of incubation. In the absence of BSA there is still a uniform layer of Con A covering the surface, suggesting that the redis- tribution of binding sites has not occurred by three to four hours of incubation.

Chemical Induction of the Acrosorne Reaction by Ionophore A23187

The acrosome reaction was induced in vitro using the calcium ionophore A23187. Initial attempts a t inducing the acrosome reaction with A23187 were unsuccessful. Dilution of bovine spermatozoa to concentrations of less than 10 x lo6 s p e d m l resulted in decreased motility (less than 5% by 90 minutes of incuba- tion) in the presence of ionophore. We found that the optimum concentration for long periods of incubation with high motility was 100 x lo6 sperm/ml. At this concentration washed spermatozoa would remain motile for several hours. We also found that short pulses of ionophore Us30 minutes) a t concentrations of 1-10 pM were insufficient to induce an acro- some reaction after spermatozoa had been in-

cubated in Tyrodes (+BSA) for five hours. Con- tinuous exposure of spermatozoa in the pres- ence of BSA at a final concentration of 10 pM ionophore resulted in the induction of an acro- some reaction in greater than 90% of the sper- matozoa observed following an incubation period of two hours or more. The results of a representative experiment are seen in Figure 5. Figure 5 shows that spermatozoa incubated in Tyrodes with BSA in the continuous pres- ence of A23187 lose 93% of their acrosomal caps after 120 minutes of incubation. The remaining 7% of unreacted spermatozoa at five hours of incubation may represent either immotile or dead sperm. Two hours represents the mini- mum time to achieve the acrosome reaction using ionophore. In other experiments, at least three hours, and in one case four hours, were required before a significant increase (i.e., > 90%) in the percentage of reacted acrosomes had occurred.

There is always a small percentage of sper- matozoa that do not have intact acrosomes after washing and incubation. In this experiment 18% of the spermatozoa were lacking their acrosomal caps immediately after washing and before incubation. Transmission electron mi- croscopy shows that there is no vesiculation or fusion of the overlying plasma membrane with the outer acrosomal membrane. These sper- matozoa have either discharged their caps in- tact or else rounded off membranous whorls. Spermatozoa incubated in Tyrodes with di- methyl-sulfoxide (solvent for A23187) or in Ty- rodes with ionophore, but lacking either cal- cium or BSA, also show an increased number of reacted acrosomes with time. However, this may be due to membrane destabilization by DMSO which is present in each of these sam- ples. It is also apparent that for the acrosome reaction to take place, it is necessary for exter- nal calcium to be present in addition to the calcium ionophore.

Transmission electron microscopy of samples missing their acrosomal caps (only those greater than 9oo/o reacted) demonstrated that they have undergone an acrosome reaction (Fig. 6) . After two hours of treatment with ionophore (samples taken from the experiment represented in Fig. 5 ) , spermatozoa in Tyrodes with BSA and A23187 exhibit the typical vesiculation associated with a true acrosome reaction. These vesicles are formed by the fu- sion of the outer acrosomal membrane and the plasma membrane at multiple points (Russell et al., '79). There was no evidence of the acro- some reaction in the other samples.

Fig. 4. Labeling of sperm surfaces with concanvalin A. Fixed sperm were conjugated with concanvalin A to demonstrate the presence of lectin receptors using a modified method of Gordon, e t al.. 1975. X 24,500 a) Longitudinal section of a spermatozoan fixed in the presence of 6% BSA after 30 seconds of incubation a dense, uniform layer of reaction product seen over the head covering the plasma membrane (pm-Con A). bJ Sperm fixed after 60 minutes of incubation in the presence of 6% BSA. c) Sperm fixed after 60 minutes of incubation in the absence of BSA. In both b and c there is still a uniform layer of reaction product visible over the entire head. dJ Sperm fixed after three hours of incubation in the presence of 6% BSA. The localization of reaction product is not as pronounced and appears patchy over the entire head.

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Fig. 5. Chemical induction of the acrosome reaction using ionophore A23187. Washed ejaculates (100 x 10' sperm/ml) were incubated in the presence or absence of BSA in Tyrodes (+ calcium) at 37". Ionophore (10 p m final; 0.0% dimethysulf- oxide, DMSO) or DMSO alone was present in some samples during the entire incubation period. At indicated times samples were fixed in 0.1% paraformaldehyde (pH 7.8) and then assayed for the acrosome reaction by light micorscopy using the procedure of Bryan and Akruk, '77. Each sample represents at least 400 sperm counted by phase-contrast microscopy at 100 x.

DISCUSSION

We have found that bovine spermatozoa can undergo capacitation within two to three hours in vitro in a defined medium containing BSA. Capacitation was determined by two criteria: 1) The redistribution of lectin binding sites; 2) the acrosome reaction in the presence of cal- cium ionophore A23187. This in vitro timing of two to three hours is in close agreement with the only published time for capacitation of bovine spermatozoa in vivo of three hours (Iri- tani et al., '75). The time required to achieve capacitation was not given in an earlier report of bovine capacitation in vitro induced by hypertonic salt treatment (Brackett et al., '78). Gwatkin and Williams ('78) found that bovine spermatozoa displayed altered surface charac- teristics as determined by their agglutination with soybean agglutinin after treatment for one hour with the hypertonic salt medium of

Brackett and Oliphant ('73b, '75). Agglutina- tion by this method has been associated with the in vitrocapacitation of guinea pig sperma- tozoa (Talbot and Franklin, '78).

Ericsson ('67) reported a fluorometric tech- nique for assaying capacitation of spermatozoa in vivo. Capacitation was operationally defined as the loss of fluorescent T-HCl bound to the surface of rabbit spermatozoa. The tetracycline antibiotics bind preferentially to cations on the membrane surface and are useful as fluores- cent probes of biological membranes (Caswell and Hutchinson, '71a, b; Saling and Storey, '79). Using this technique, Goodeaux et al. ('78) found that the loss of T-HC1 occurs in bovine spermatozoa after incubation in vitro for 30 minutes in a medium containing BSA. We have observed this same fluorescence loss as re- ported by Goodeaux et al. ('78) under identical conditions using fluorescence microscopy and spectrofluorometry .

CAPACITATION IN BOVINE SPERMATOZOA 43

Fig. 6. Transmission electron micrograph of bovine spermatozoa after ionophore induced acrosome reaction. Samples were fixed after incubation in ionophore (from Fig. 5) after two hours of incubation in the presence of 6% BSA. A chain of vesicle (v) composed of the outer acrosomal membrane and the plasma membrane surrounds the inner part of the acrosome (a) and nucleus (n). The nucleus (n) is covered by the acrosome ac (Ad, while the marginal ridge of the acrosome (MR) is visible to the left.

In this study (Figure 21, and that of Saling and Storey ('79), there is a noticeable localiza- tion of the antibiotic in the midpiece region. These observations are in agreement with the calcium distribution in bull spermatozoa as demonstrated by Babcock et al. ('78). By use of electron microanalysis they found that the midpiece region had several times the back- ground levels of calcium which would account for the higher levels of the antibiotics bound to this region.

However, visualization of surface changes on the spermatozoa by loss of fluorescent T-HC1 did not correlate with the time to undergo ca- pacitation as established by other criteria. Ear- lier work by Vaidya et al. ('69) indicates that the removal of fluorescence from spermatozoa is not coincidental with the development of the capacitated state in vivo in the rabbit. Under their conditions, capacitation required 10

hours in the fallopian tube, but only two hours were required for removal of T-HC1. Vaidya et al. ('69) suggest that spermatozoa may become partially capacitated without losing fluores- cence, for they find no fluorescence loss in spermatozoa remaining in a pseudopregnant uterus for as long as 24 hours, although ca- pacitation is definitely initiated within 1420 hours (Bedford, '67). We suggest two possible explanations for the loss of fluorescence during incubation in the presence of BSA. First, that fluorescence loss could represent or precede a n initial stage of capacitation. Second, tetracy- cline may be binding to the BSA resulting in a loss of fluorescence from the sperm.

The loss or redistribution of lectin binding sites seen here in bovine spermatozoa during capacitation has been reported in other species (Gordon e t al., '75; ORand, '77; Friend et al., '77; Kinsey and Koehler, '78; Courtens and

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Gournier-Delpech, '79). These findings suggest that the cell surface changes occurring prior to the acrosome reaction include modification of carbohydrate containing components of the plasma membrane covering the acrosome. A probable explanation of the production of lec- tin-free areas on the acrosomal cap is that the receptors are lost or shed from the surface rather than masked or displaced (Oliphant and Brackett, '73a). The loss of lectin receptors may signal a change in membrane ultrastructure in such a way as to favor membrane fusion.

Early removal of T-HCl from the spermato- zoan surface as well as the late redistribution of lectin binding sites and induction of the acro- some reaction are dependent upon medium supplemented with BSA. The necessity for BSA-supplemented media for inducing ca- pacitation in several systems i n vitro is well documented (Rogers, '78). However, is there a physiological role or need for the presence of albumin in vivo? Capacitation i n vitro can be induced using Fallopian tube fluid (Barros and Austin, '67; Barros, '68), follicular fluid (Ya- nagimachi, '691, and blood sera (Barros and Garavagno, '70; Yanagimachi, '70). The factor in blood sera appears to be a high molecular weight substance associated with the albumin fraction (Yanagimachi, '70; Toyoda et al., '71; Miyamoto and Chang, '73). In a subsequent study, Lui et al. ('77) isolated the fraction of bovine follicular fluid responsible for the i n vitro acrosome reaction of hamster sper- matozoa. Serum albumin was identified in this study as the causative agent in inducing the reaction. This suggests that BSA in vitro may be mimicking conditions i n uivo.

Recent work by Davis et al. ('79) suggests a mechanism by which BSA induces capacita- tion. They have evidence that there is transfer of phosphatidylcholine and cholesterol between BSA and cauda epididymal rat spermatozoa in uitro, They propose that the lipid exchange in- duces membrane destabilization and increased calcium permeability which can then promote fusion and the acrosome reaction.

Iwamatsu and Chang ('69) reported that cal- cium ions also affect capacitation and in vitro fertilization of mouse ova in the presence of bovine follicular fluid. In support of this work, Yanagimachi and Usui ('74) found that guinea pig spermatozoa failed to fertilize ova in a cal- cium-free medium. Spermatozoa incubated in a calcium-free medium underwent capacitation but were arrested until the addition of calcium ions, which then induced activation and the acrosome reaction in three to six minutes.

Chemical induction of the acrosome reaction using A23187 results in formation of a sheath of vesicles covering the forward portion of the spermatozoan head extending back to the equatorial segment. Morphologically this acro- some reaction closely resembles the chemically induced reaction seen in human spermatozoa (Russell et al., '79). There are no microfila- ments or tubule-like elements as seen in other spermatozoa (Peterson et al., '78; Stambaugh and Smith, '78). The absence of these elements may be due to the time of fixation after induc- tion of the acrosome or improper fixation of these structures.

Treatment with A23187 causes an initial de- crease in sperm motility and a subsequent fur- ther slow decrease over several hours. There is a species variability in the response of sper- matozoa to A23187. Concentrations of A23187 up to 10 pM have little effect on human sper- matozoa (Russell et al., '791, while guinea pig spermatozoa (Green, '78) are rapidly rendered immotile in calcium ionophore. The variation in timing to induce the acrosome reaction A23187 must be a result of the physiological condition of the spermatozoa in each of the ejaculates used. The initial motility of un- washed ejaculates is important to the success of these experiments. Unwashed spermatozoa with an initial motility of less than 25% will show a dramatic increase in motility (greater than 90%) after washing and resuspension. However, these spermatozoa lose motility rap- idly and do not respond well to ionophore treatment.

The capacity of an ionophore to induce the acrosome reaction in media containing calcium makes it seem likely that the reaction is a con- sequence of an increase in free calcium in the spermatozoa. Direct evidence that the uptake of calcium in bovine spermatozoa is due to A23187 has been presented by Babcock et al. ('78). The requirement for calcium in the induc- tion of the acrosome reaction and membrane fusion is well documented in mammals (Yanagimachi, '78) as well as in invertebrate sperm (Dan, '67; Collins and Epel, '77).

The successful capacitation of bovine sper- matozoa in vitro must be tested further to de- termine its relevance to the situation in utero. The procedure of Brackett and Oliphant ('75) for capacitation of rabbit spermatozoa with hypertonic salt medium does not appear to pro- duce a capacitated state comparable to that in- duced in viuo (Bedford, '79). This raises the pos- sibility that spermatozoa may become ca- pacitated through more than one mechanism.

CAPACITATION IN BOVINE SPERMATOZOA 45

Much additional information remains to be ac- quired concerning sperm capacitation and in- duction of the acrosome reaction, including en- hancement of the capacitation medium with energy sources such as lactate and pyruvate.

ACKNOWLEDGMENTS

The author would like to thank Drs. J. Cap- rio, T. Dietz, and E. Weidner for reading this manuscript. The technical assistance of W.L. Steffens and L.A. Fabre is gratefully acknowl- edged. I would also thank Jane Anderson, Allen Wisegold, and Dr. C. Baham of the Louisiana Animals Breeder Cooperative who generously provided bovine specimens. Partial support for this work was provided by the Graduate Re- search Council of L.S.U.

Babcock, D.R., D.M. S G e r h o r n , and T. Hutchinson (1978) Calcium redistribution in individual cells correlated with ionophore action on motility. J. Exp. Zool. 204:391-400.

Barros, C. (1968) In vitro capacitation of golden hamster spermatozoa with fallopian tube fluid of the mouse and rat. J. Reprod. Fertil. 17t20S206.

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