Capacitation of Bovine Sperm by Heparin1

Transcription

1 BIOLOGY OF REPRODUCTION 38, (1988) Capacitation of Bovine Sperm by Heparin1 J. J. PARRISH, J. SUSKO-PARRISH, M. A. WINER, and N. L. FIRST2 Department of Meat and Animal Science University of Wisconsin Madison, Wisconsin 5376 ABSTRACT Capacitation of bovine sperm was evaluated by determining the ability of sperm to fertilize bovine oocytes in vitro and to undergo an acrosome reaction upon exposure to lysophosphatidylcholine (LC). Incubation of sperm with heparin (1,g/ml) increased the percentage of oocytes fertilized, but this required exposing sperm to heparin for at least 4 h before adding them to oocytes. There was no effect on the percentage of motile or acrosome-reacted sperm after exposure of noncapacitated sperm to 1 pg/ml LC for 15 mm. When sperm were incubated for 4 h with beparin, exposure to 1,4g/ml LC for 15 mm had no effect on the percentage of sperm that were motile, but the percentage of acrosome-reacted sperm increased from less than 1% to over 7%. The acrosome reactions (ARs) induced by LC were synchronous, reached maximal levels within 15 mm, and differed (p-<.oo1) between sperm incubated under capacitating (with heparin) and noncapacitating conditions (without heparin). The time course required for heparin to capacitate sperm as judged by in vitro fertilization and to render sperm sensitive to LC induction of the AR were found to be similar. The percentage of ARs induced by LC and percentage of oocytes fertilized by sperm were found to be heparin-dose-dependent, with the maximum responses occurring at 5-1 Mg/mI heparin. The correlation between the mean fertilization and LCinduced AR percentages was.997 (p<.1). These studies demonstrate capacitation of bovine sperm by heparin requires at least a 4-h exposure of sperm to heparin and suggest that plasma membrane changes prior to an AR can be detected by exposure of bovine sperm to LC. INTRODUCTION Mammalian sperm are not immediately capable of fertilizing oocytes, rather they must undergo a period of preparation that normally occurs in the female reproductive tract (Austin, 1951; Chang, 1951). The changes that occur in sperm involve at least two components: an initial sperm membrane alteration (Yanagimachi, 1981; O Rand, 1982; Ahuja, 1984; Langlais and Roberts, 1985; Wolf et a!., 1986) that allows the sperm to undergo the second phase, the fusion of the plasma membrane and outer acrosomal membrane (Yanagimachi and Usui, 1974). The first phase is considered to be the period of capacitation, and the second phase is the acrosome reaction (AR). Two approaches have been used in a number of species to differentiate between factors that capacitate sperm and factors that directly induce the AR. Accepted January 27, Received March 17, This research was supported by the College of Agricultural and Life Sciences, University of Wisconsin, Madison and NICHD Grant No. HD Reprint requests. 2 The first involves incubation of sperm under capacitating conditions, followed by exposure of sperm to oocytes for short periods of time (mouse: Fraser, 1983; hamster: Bavister, 1979; Bavister et a!., 1976; Cornett et a!., 1979; guinea pig: Fleming and Yanagimachi, 1982a). If sperm are capacitated after the initial incubation, they will then fertilize oocytes within the time constraints imposed (Bavister, 1986). The second approach has been to expose sperm (15-2 mm) to substances that will induce an AR only in capacitated sperm. Ideally, this substance would be the in vivo AR-inducing agent, which may be the zona pellucida-as described for mice (Bleil and Wassarman, 1983; Florman et a!., 1984)-or other components of the cumu!us/oocyte complex. Because the in vivo AR-inducing substance is unknown in most species, correct doses of fusogenic lipids (Ohzu and Yanagimachi, 1982; L!anos and Meizel, 1983), Ca2 ionophores (Byrd and Wolf, 1986) or external manipulation of Ca2 concentrations (Fleming and Yanagimachi, 1982b;Yanagimachi and Usui, 1974) have been used as suitable alternatives. Previously we have shown that bovine sperm incubated in vitro with heparin will acrosome-react (Handrow et a!., 1982; Parrish et a!., 1985) and 1171

2 1172 PARRISH ET AL. fertilize a higher percentage of bovine oocytes than sperm incubated without heparin (Parrish et a!., 1985, 1986a). The long sperm incubations with oocytes (18-24 h) used in those studies made it difficult to determine whether the effects of heparin were on capacitation or the AR. The purpose of the present studies was to identify which phase was effected by heparin. To accomplish this, capacitation tests utilizing both the ability of sperm to fertilize oocytes in vitro and the sensitivity of sperm to the fusogenic lipid,!ysophosphatidy!choline (LC), were used. Heparin was found to effect capacitation of bovine sperm, butdirect effects on the AR could not be Culture evaluated. Media MATERIALS AND METHODS The basis for many of the media used was a modified Tyrodes medium (TALP) described by Bavister and Yanagimachi (1977). Modifications of TALP used for sperm cultures (Sp-TALP), working with oocytes in air (low bicarbonate-talp), and for in vitro fertilization (IVF, Fert-TALP) are shown in Table 1. It is important to note that glucose is not present in Sp-TALP, because glucose inhibits the effects of heparin on bovine sperm (Parrish et al., 1985, 1986b; Susko-Parrish et al., 1985). Glucose was used in Fert-TALP to minimize further effects of heparin on sperm during coincubation with oocytes. Energy substrates were present in the form of lactate and pyruvate in a!! media. The medium used for in vitro maturation of bovine oocytes was that described by Critser et a!. (1986) and consisted of TC-199 with Hanks salts supplemented with.2 mm pyruvate, 1% heat-treated fetal calf serum,. 12 units/ml NIH-oLH (lot 24),.1 units/ml NIH-oFSH (lot 15) and 1 pg/ml estradiol-17a. A!! media were sterilized by filtering through.22-pm filters before use. For maturation medium, estradio!-17/3 in ethanol was added after filtering. Sperm Culture Ejaculated bovine sperm provided by American Breeders Service (ABS, DeForest, WI) were diluted 1:1 in an egg-yolk/sodium-citrate extender (Pace et al., 1981) to protect them during transit to the laboratory (2 h at 2-25#{176}C). The extender was the same as that used by ABS for freezing semen except glycerol was omitted. Sperm were washed by diluting 2 ml of semen with 8 ml of Sp-TALP, centrifuging at 275 g for 1 mm (2-25#{176}C), resuspending the sperm pellet in 1 ml SP-TALP, and repeating the centrifugation. The resulting sperm pellet was resuspended to 5 X 16 sperm/mi in Sp-TALP and.5-rn! aliquots were placed in 1 X 55 mm capped plastic tubes. Sperm samples were gassed with 5% CO2 in air and incubated for.25-4 h in a water bath at 39#{176}C.This temperature was chosen because it is the body temperature of cattle, and JYF with bovine gametes has been shown to be temperaturedependent with an optimum at 39#{176}C(Lenz et al., TABLE 1. Concentration of TALP constituents. Ingredient Units Sp-TALP Low bicarbonate-talp Fert-TALP NaCI mm KCI mm NaHCO, mm NaH,PO4 mm.3,3.3 Lactate (sodium salt) mm CaCI2 mm MgCl, mm HEPES mm Pyruvate mm Glucose mm Bovine serum albumin mg/mi 6.oa 3.o 6b Penicillamine MM Hypotaurine M Epinephrine MM Gentamycin Mg/mI acohns fraction V. b Fatty-acid-free

3 CAPACITATION OF BOVINE SPERM a). At the end of incubation, LC was added at -4 pg/rn!, and the sperm were incubated for an additional.25 h. The percentage of sperm that were motile was then estimated by an experienced observer at 2 x magnification with bright-field microscopy. The percentage of sperm that were acrosome-reacted was determined on air-dried sperm smears with a napthol yellow-erthyrosin B-staining procedure, which has been described previously (Lenz et a!., 1982), and validated with electron microscopy to detect bovine sperm that have vesiculated (Lenz et a!., 1983b). The ultrastructure of the LC-induced AR was determined with electron microscopy. Sperm were fixed in 4% glutaraldehyde in.1 M cacodylate buffer, post-fixed in 1% osmium tetraoxide, dehydrated with a graded series of ethanols, treated with propylene oxide, and embedded in Eponate 812. Thin sections were stained with uranyl acetate and lead citrate and viewed with a Hitachi H-6 transmission-electron microscope. In Vitro Fertilization Cumulus/oocyte complexes were aspirated from 1- to 5-mm antra! follicles of bovine ovaries obtained from an abattoir (Ball et a!., 1983; Leibfried-Rut!edge et a!., 1987). Isolated cumulus/oocyte complexes were washed three times in low bicarbonate-talp, placed in maturation medium at 1 complexes per 5-pl drop, and incubated under oil for h at 39#{176}C (Critser et a!., 1986). Cumulus cells were removed by placing up to 1 cumulus/oocyte complexes in a 1.5-mi microcentrifuge tube containing 2 p1 of low bicarbonate-talp and vortexing for 5 mm. Cumulusfree oocytes were recovered, washed three times in low bicarbonate-talp, and placed in Fert-TALP at five oocytes per 5-p 1 drop under oil. Sperm were added at 1 X 16 /ml and coincubated 4 h at 39#{176}C with oocytes. At the end of incubation, loosely bound sperm were removed from oocytes by passing the oocytes in and out of a pipette just slightly larger than the oocytes until no more sperm were removed. Oocytes were fixed and stained as previously described (Parrish et al., 1985). Oocytes were considered penetrated if a sperm head was detected in the oocyte cytoplasm. Often the oocyte had been activated and chromatin was at anaphase II or telophase II of meiosis when metaphase II oocytes were penetrated. Metaphase II was determined by chromatin at metaphase and the presence of a polar body. Oocytes that had not yet reached metaphase II by the time of sperm addition were penetrated by sperm during IVF, as evidenced by decondensed sperm heads in metaphase I oocytes. Oocytes that were not fertilized were either at metaphase I or II of meiosis. Only degenerate obcytes were not included in determination of fertilization, since we have found that bovine sperm will penetrate and decondense in bovine oocytes at all stages of maturation from germinal vesicle to metaphase II (Parrish, unpublished results). Lysophosphatidylcholine Preparation Egg yolk LC (Sigma Chemical Co., St. Louis, MO) was either dissolved in water and lyophilized in 1- to 1-mg aliquots or dissolved in methanol at 5 mg/mi. Both preparations were then stored at -2#{176}C. The day of use, for the methanol-dissolved sample, an aliquot was removed, evaporated under nitrogen gas, and resuspended with Sp-TALP. Lyophilized samples were also resuspended with SP-TALP. The concentration. of LC in that working stock solution was 1 times the concentration to which sperm were to be exposed. There was no difference between methods of preparing LC in effects on bovine sperm. Heparin Preparation Hog intestinal mucosal heparin as a sodium salt was obtained from Sigma Chemical Co. (Lot #79B-169, 17 units/mg) and was dissolved in saline as a 1 X stock. Vials (2 p1) of heparin stock were stored frozen at -2#{176}C until used. Statistical Analysis Data were analyzed with analysis of variance and planned comparisons of means were tested with linear contrasts or through use of Duncan s multiple range test. Time Course of IVF RESU LTS The percentage of oocytes penetrated by sperm preincubated.25 to 4 h with heparin is shown in Figure 1. The highest percentage of oocytes penetrated by sperm occurred when sperm were preincubated 4 h with heparin. While less than 1% of oocytes were penetrated by sperm preincubated without heparin for either.25 or 4 h, addition of heparin during IVF resulted in a small increase in the percentage of oocytes penetrated by sperm

4 1174 PARRISH ET AL. 1 The concurrent decline in percentage of sperm that were motile and increase in percentage of sperm that 8 UI I- I- 6 UI. ( 4 U TIME FIG. 1. Effect of length of heparin exposure on penetration of bovine oocytes by bovine sperm. Washed ejaculated sperm were resuspended in Sp-TALP and incubated with 1 Mg/mI heparin for.25 to 4 h at 39#{176} C. Sperm were then diluted 5-fold into drops of Fert-TALP that contained oocytes. Further procedures of in vitro fertilization (IVF) and methods for evaluation of oocytes are described in Materials and Methods. The experiment was repeated three times, each with an ejaculate from a different bull. The number of oocytes tested per time point in each replicate ranged from 15 to 5. The values presented are the means ± SE for the percentage of oocytes penetrated by sperm initially incubated with heparin (.), and as controls, no exposure of sperm to heparin before or during IVF (o) or heparin exposure only during IVF (.). Values with an asterisk differ from the control for sperm exposure to heparin only during IVF, p<.1. (h) were acrosome-reacted suggests that exposure of noncapacitated sperm to greater than 1 pg/mi LC results in overall membrane damage. At least a 4-h exposure of sperm to heparin appears to be required to cause capacitation of bovine sperm (Fig. 1). Sperm from the same ejaculates used to obtain the data displayed in Figure 2 were incubated 4 h with or without heparin. Subsequently, sperm were exposed for 15 mm to the same treatments previously used (Fig. 2). Results for percentage of sperm that were motile and percentage of sperm that a were acrosome-reacted are shown in Figure 3. The overall trends for percentage of motile sperm in Figure 3 are similar to those illustrated in Figure 2, except there were more motile sperm when sperm were incubated with heparin and exposed to 2 pg/rn! LC than when incubated without heparin (p<.1). LC had no effect on the percentage of sperm that were motile at doses up to 1 pg/mi whether sperm were incubated 4 h with or without heparin (p>.5). When sperm were incubated without heparin, the dose response of LC on percen- 1 (p<.5). There was no difference in the percentage of oocytes penetrated by sperm preincubated.25 to 3 h with heparin or when heparin was added only during IVF. Only when sperm were preincubated 4 h with heparin was the percentage of oocytes penetrated by sperm increased above levels for sperm with heparin added only during IVF U, -U, MO11LE SPERM Induction of the AR by LC The effect of LC on noncapacitated sperm is shown in Figure 2. Addition of or 1 pg/mi heparin had no effect on the percentage of sperm that were motile or acrosome-reacted, and data were therefore pooled for presentation. The percentage of sperm that were motile or acrosome-reacted were not different for noncapacitated sperm exposed for 15 mm to, 5, or 1 pg/m! LC (p>.5). However, exposure of sperm to higher levels of LC, 2 or 4 pg/ml, resulted in a decline in percentage of sperm that were motile (p<.1) and an increase in percentage of sperm that were acrosome-reacted (p<o.o1). 2 ACROSOME REACTED SPERM -#{149} LYSOPHOSPHATIDYLCHOLINE (uglml) FhG. 2. Effect of lysophosphatidylcholine (LC) on noncapacitated bovine sperm. Ejaculated sperm (noncapacitated) were washed and resuspended in Sp-TALP (n = 3). Heparin ( or 1 sg/ml) was added and sperm were exposed to LC ( to 4 Mg/mI) for 15 mm at 39#{176}C.The percentage of sperm that were motile (.) and percentage of sperm that were acrosome-reacted (.) were then determined. Concentration of heparin had no effect on percentage of motile (p>.5) or percentage of acrosome-reacted (p>.5) sperm, and data were therefore pooled. Values represent the mean ± SE, with * * or * signifying a difference from no LC, 9<.1 or p<.ool, respectively.

5 CAPACITATION OF BOVINE SPERM N UI. ( SQ LYSOPHOSPHATIDYLCHOLINE (ug/mi) FIG. 3. Effect of lysophosphatidylcholine (LC) on bovine sperm incubated 4 h with or without heparin. Ejaculated sperm were washed and resuspended in Sp-TALP (n = 3). Heparin ( or 1 Mg/ml) was added to.5 ml-aliquots of sperm and incubated 4 h at 39#{176}Cunder 5% CO, in air. At the end of incubation, LC was added to each aliquot of sperm to yield to 4 Mg/mI final LC concentrations. After a further 15-mm incubation, the percentage of sperm that were motile (a, #{149}) and percentage of sperm that were acrosome-reacted (o,.) were determined. The closed values (a,.) represent sperm incubated with heparin, and the open values (a, o) represent sperm incubated without heparin. Values with * * or * * * differ from no heparin within the same concentration of LC, 9<.1 or 9<.1, respectively. sperm incubated with heparin and exposed to or 1 pg/mi LC. Had the LC been damaging, we would have expected adenosine triphosphate to leak out of the sperm tail, resulting in the cessation of motility. A qualitative change in the motility pattern of sperm did occur when 1 pg/ml LC was added to sperm incubated with heparin. There was an apparent increase in the amplitude of the sperm tail beats, which was similar to activated motility observed in hamsters or guinea pigs. Some sperm exhibited figure 8 movements, but the majority were acrosomereacted and thus were stuck, head first, to the glass slide when observed under the microscope. Electron micrographs of sperm exposed to or 1 pg/m! LC are shown in Figures 4-9. After incubation at 39#{176}Cfor 4 h, the acrosoma! plasma membranes were intact for sperm incubated with or without heparin (Figs. 4 and 6). There was intensive head-to-head agglutination seen in sperm treated with heparin. In the absence of heparmn, there was no change in sperm membranes after LC addition (Fig. tage of sperm that acrosome-reacted was similar to that shown for noncapacitated sperm (Fig. 2). No change in the percentage of sperm that were acrosome-reacted occurred when sperm were incubated without heparin and exposed to, 5, or 1 pg/rn! LC (p>.5), but the percentage of sperm with an AR increased when sperm were exposed to higher doses of LC, 2 or 4 pg/rn! (p<.1). Results for sperm incubated under capacitating conditions with heparin (Fig. 3) were different from the previous results for noncapacitated sperm (Fig. 2). When sperm were incubated 4 h with heparin and then exposed to 1 pg/rn! LC for 15 mm, the percentage of sperm that were acrosome-reacted increased (p<.1), as compared with sperm incubated with heparin but not exposed to LC. This did not occur for sperm incubated 4 h with heparmn and exposed to 5 pg/mi LC or incubated 4 h without heparin and exposed to 5 or 1 pg/m! LC. The ability of sperm incubated 4 h with heparin to acrosome-react upon exposure to 1 pg/ml LC does not appear to be an overall membrane-damaging effect, since no difference in percentage of sperm that were motile occurred for.#{176} S H.!#{149} #{149} : :- _ - S.. FIG. 4. Electron micrograph of sperm incubated 4.25 h in sperm- TALP. X 8.

6 1176 PARRISHETAL. r 5 because we had demonstrated (Fig. 3) that bovine ejaculated sperm incubated 4 h without heparin do not acrosome-react upon exposure to 1 pg/m! LC. At various intervals after the start of incubation, heparin was added and the LC challenge done at the end of the 4 h incubation. There was no effect of LC on the percentage of sperm that were acrosomereacted when sperm were incubated with heparin for up to 3 h (p>.5). However, when sperm were incubated 4 h with heparin, the percentage of sperm - that acrosome-reacted upon exposure of sperm to LC I increased. Since percentage of sperm that were acrosome-reacted was not different for sperm incubated 4 h without heparin and exposed to or 1 pg/mi LC (Figs. 2 and 3), the incubation of.sperm without heparin for various lengths of time before addition of heparln would probably not have affected results The time course for induction of the AR by 1 pg/ml LC in sperm incubated 4 h with heparin is shown in Figure 11. While the maximal percentage of FIG. 5. Electron micrograph of sperm incubated 4 h in sperm- TALP and then for.25 h with 1 Mg/mI lysophosphatidylcholine. X 12,. 5). Extensive vesiculation of the outer acrosoma! membrane and plasma membrane overlying it did occur when LC was added to sperm incubated with heparin (Figs. 7-9). The vesiculation was confined to the acrosomal region as seen by the lack of vesiculation in accompanying sperm tail cross-sections (Fig. 7) and the presence of the fusion point between plasma membrane and outer acrosoma! membrane along the anterior limit of the equitorial segment (Fig. 8). Fusion did not occur between adjacent speral agglutinated with heparin as seen in Figure 9. The acrosome reaction as induced by LC in sperm capacitated with heparin therefore appears to be morphologically normal. Requirements for LC Effects Figure 1 shows the effect of duration of sperm incubation with heparin on the ability of sperm to acrosome-react when challenged with 1 pg/mi LC. FIG. 6. Electron micrograph of sperm incubated 4.25 h in sperm- Al! sperm incubations were for a total length of 4 h TALP with 1 Mg/mI heparin. X 1,.

7 CAPACITATION OF BOVINE SPERM 1177 FIG. 7. Electron micrograph of sperm incubated 4 h in sperm-talp with 1 Mg/mi heparin, and then for.25 h with 1 Mg/rnl lysophosphatidylcholine. X 1,. FIG. 8. Electron micrograph of sperm incubated with heparin and exposed to lysophosphatidylchoiine as described in Figure 7. The arrow points to the fusion point of the outer acrosomal membrane with the plasma membrane along the anterior limit of the equitorial segment. The plasma membrane posterior to this point is not fused. X 35,. acrosome-reacted sperm obtained varied among bulls, a!! peaked by 15 mm of exposure to LC. The dose-response of heparmn concentration during a 4-h sperm incubation on the percentage of oocytes subsequently fertilized by these sperm and the percentage of sperm that were acrosome-reacted or remained motile after a 15-mm challenge with 1 pg/mi is shown in Figure 12. The percentage of sperm that were motile declined approximately 1% as heparin concentration increased from to 1 pg/mi. The apparent decline in the percentage of motile sperm was probably due to increased difficulty in estimation of motility. Heparin induced head-to-head agglutination of sperm, and this agglutination increased with increasing concentrations of heparin. The LC had no effect on the percentage of motile sperm (p>.5). The percentage of sperm with ARs that were induced by LC peaked at 5-1 pg/mi heparin, as did the percentage of oocytes that were penetrated by sperm. There was a high correlation between the percentage of sperm which acrosome-reacted in response to the LC challenge and the percentage of oocytes that were fertilized by sperm which had not been exposed to LC, both for means overall replications (r = 1.; p<.1, d.f. = 4) and for the individual values (r =.72, p<.1, d.f. = 22). In a subsequent and similar experiment (n = 3), higher levels of heparmn were used during the sperm incubation. Interestingly, the percentage of sperm that acrosomereacted in response to the LC exposure declined from a peak of 67 ± 12 at 7.5 pg/rn! heparmn, to 65 ± 11, 46 ± 6, and 34 ± 8, at 1, 5, and 1 pg/m! heparin, respectively. The percentage of sperm that were acrosome-reacted in the absence of LC was 6 ± 3, 8 ± 3, 6 ± 2, and 3 ± 1 at 7.5, 1, 5, and 1 pg/m! heparin. A similar biphasic response of sperm to heparin has been noted previously in the rabbit (Lenz et a!., 1983c). DISCUSSION In these studies, we provide evidence that the glycosaminog!ycan, heparin, affects capacitation of

8 1178 PARRISH ET AL. (I, z 2 I- U 4UI U I U) U 4 SQ #{149}1 - I EfiIN 3 4 INCUBATION WITH HEPARIN (h) t. - FIG. 9. Electron micrograph of sperm incubated with heparin and exposed to lysophosphatidylcholine as described in Figure 7. The sperm on the left is acrosome-reacted, and the vesiculation of the plasma membrane has no effect on and does not involve the plasma membrane of the acrosome-intact sperm on the right X 25,. p FIG. 1. Effect of length of heparin exposure on induction of the acrosome reaction by lysophosphatidylcholine (LC). Ejaculated sperm were washed and resuspended in Sp-TALP (n = 3). Sperm were incubated a total of 4 h with heparin added at, 1, 2, 3, or 3.75 h after the start of incubation, thereby exposing sperm to heparin for 4, 3, 2, 1, or.25 h. At the end of incubation, or 1 Mg/mI LC was added, and after a further 1 5-mm incubation, the percentage of sperm that were acrosome-reacted was determined. Values with white circle (o) represent no LC addition, and values with a black circle (.) represent addition of LC. Differences in sperm response between LC doses were evaluated within a time of heparin addition. Values with asterisks differ from no LC, 9<.1. ejaculated bovine sperm in vitro. An initial experiment demonstrated that incubation of sperm with heparin for at least 4 h prior to mixing sperm with oocytes was required to increase the percentage of oocytes penetrated by sperm (Fig. 1). By minimizing the time sperm and oocytes were together (4 h) and including controls for effects of heparin during fertilization, the major effects of heparin were shown to occur during the initial sperm incubation and appeared to involve capacitation. A test was developed using LC to detect sperm undergoing capacitation. The test was similar to that described for hamsters (Ohzu and Yanagimachi, 1982; Lianos and Meize!, 1983) and guinea pigs (Fleming and Yanagimachi, 1982b). The major difference was that the fusogenic properties of LC were exploited to probe for changes in sperm membranes that occurred during capacitation and altered the sensitivity of those membranes to fusion by LC. It was not the intent to induce fertilizability in sperm TIME (mln) FIG. 11. The time course of lysophosphatidylcholine (LC)-induced acrosome reaction. Ejaculated sperm were washed and incubated 4 h at 39#{176}C with 1 Mg/mI heparin. LC (1 pg/ml) was added, and the percentage of sperm that were acrosome-reacted determined from to 2 mm later. Each line represents results from a different bull.

9 CAPACITATION OF BOVINE SPERM 1 i79 D. MOTILE SPERM HEPARIN (ug/mi) FIG. 12. Effect of heparin concentration on sperm incubated 4 h. Washed ejaculated sperm (n = 4) were incubated with to 1 Mg/mI heparin for 4 h at 39#{176}C.At the end of incubation, a -M1 aliquot was removed and diluted 5-fold into a drop of Fert-TALP that contained oocytes. Further procedures of in vitro fertilization (IVF) and methods for evaluation of oocytes are described in Materials and Methods. The mean ± SE for the percentage of oocytes penetrated white star is presented with the number of oocytes tested per treatment in each replicate ranging from 2 to 41. To other aliquots of the sperm sample, to 1 Mg/mI lysophosphatidylcholmne (LC) was added, and after a further 1 5-mm incubation, the percentage of sperm that were motile (a, a) and the percentage of sperm that were acrosome-reacted (o,.) were determined. The open symbols (a, o) represent sperm not exposed to LC, and the closed symbols (a, #{149}) represent sperm treated with LC. All values represent the mean ± SE. with LC. Short exposure of noncapacitated bovine sperm to a maximum of 1 pg/rn! LC was not damaging to sperm membranes (Figs. 2 and 3). When sperm were incubated 4 h under capacitating conditions with heparin, 1 pg/rn! LC increased the percentage of sperm that were acrosome-reacted from less than 1% to over 7%, but had no effect on the percentage of sperm that were motile (Fig. 3). The ARs induced by LC also appeared to be ultrastructurally normal (Figs. 7-9). Both the dose (Fig. 12) and time (Fig. 1) effects of heparin on the ability of sperm to acrosome-react in response to 1 pg/rn! LC were found to correlate highly with the ability of sperm to penetrate oocytes (Figs. 1 and 12). These results suggest that the LC assay using 1 pg/ml LC detects the presence of membrane changes in sperm produced by heparin and, furthermore, that these changes occur concurrently with capacitation. We have previously demonstrated that capacitation of bovine sperm with heparin is blocked by addition of glucose and that, simultaneously, the ability of LC to induce ARs is also blocked (Susko- Parrish et a!., 1985; Parrish et al., 1986b). This lends support to the idea that membrane changes detected by LC are associated with capacitation. In contrast to the ability of LC to detect membrane changes associated with capacitation in bovine (Fig. 2-6), hamster (Ohzu and Yanagimachi, 1982; L!anos and Meize!, 1983), or guinea pig sperm (Fleming and Yanagimachi, 1982b), LC does not differentiate between capacitated and noncapacitated human sperm (Byrd and Wolf, 1986). However, another fusogenic agent, the Ca2 ionophore A (1 mm) does induce ARs only in capacitated human sperm. Tests designed to detect the presence of capacitated sperm in different species by using fusogenic agents may need to evaluate different fusogenic agents. Diffusion of the agents into different membrane domains or extrace!!u!ar media components may regulate the usefulness of a particular agent within a species. Although heparin was shown to capacitate bovine sperm, its possible direct effect on the AR cannot be evaluated since no other reliable method for capacitation of bovine sperm exists (First and Parrish, 1987). In hamster sperm, by contrast, we and others have shown that heparin appears to induce an AR in capacitated sperm but does not appear to effect capacitation (Handrow et a!., 1986; Meize! and Turner, 1986; Parrish and Susko-Parrish, 1986). Heparin has also been reported to affect sperm from the human (Roy et a!., 1985) and rabbit (Lenz et a!., 1983c). Because of this wide variation in heparin effects on sperm from a variety of species, formulating a unifying hypothesis of heparin s molecular effects will be difficult. It has been speculated that heparin may act by influencing multiple events during capacitation of bovine sperm (First and Parrish, 1987). Some of these events may be more closely associated with the AR in other species, explaining, in part, the apparent differences in heparmn effects between species. The present research has described the ability of heparin to capacitate ejaculated bovine sperm in vitro. Heparin was first shown to capacitate sperm as evidenced by ability of sperm incubated with heparin to penetrate oocytes within 4 h. This effect of heparin was also shown to require at least a 4-h incubation of sperm with heparin. An assay using LC was developed to detect membrane changes occurring during capacitation. This assay was shown to differentiate between bovine sperm populations incubated

10 118 PARRISH ET AL. under capacitating or noncapacitating conditions. LC would induce ARs within 15 mm in sperm incubated under capacitation conditions with heparin but had no effect on sperm incubated under noncapacitating conditions without heparin. The time of sperm incubation with heparin required for induction of ARs by LC correlated with the time course for capacitation of sperm by heparin. Furthermore, as heparin concentrations were increased, there was an increase in the percentage of sperm that acrosornereacted in response to LC and, simultaneously, a highly correlated increase in the percentage of oocytes fertilized by sperm not exposed to LC. The sensitivity of bovine sperm to LC should therefore prove useful in elucidating the mechanism of sperm capacitation by heparin. ACKNOWLEDGMENTS We are grateful to Brad Haley for recovery and transport of ovaries from the abattoir and Julie Busby for typing this manuscript. REFERENCES Ahuja KK, Lectin-coated agarose beads in the investigation of sperm capucitation in the hamster. Dcv Biol 14: Austin CR, Observations on the penetration of the sperm into the mammalian egg. Aust j Sci Res B 4: Ball GD, Leibfried ML, Lenz RW, Ax RL, Bavister BD, First NL, Factors affecting successful in vitro fertilization of bovine follicular oocytes. Biol Reprod 28: Bavister BD, Fertilization of hamster eggs in vitro at sperm:egg ratios close to unity. J Exp Zool 21: Bavister BD, Animal in vitro fertilization and embryo development. In: Gwatkin (ed.), Manipulation of Mammalian Development. New York: Plenum Publishing, pp Bavister BD, Yanagimachi R, The effects of sperm extract and energy sources on the motility and acrosome reaction of hamster sperm in vitro. Biol Reprod 16: Bavister BD, Yanagimachi R, Teichman RJ, Capacitation of hamster spermatozoa with adrenal gland extracts. Biol Reprod 14: Bleil JD, Wassarman PM, Sperm-egg interactions in the mouse; sequence of events and induction of the acrosome reaction by a zona pellucida glycoprotemn. Dcv Biol 95: Byrd W, Wolf DP, Acrosomal status in fresh and capacitated human ejaculated sperm. Biol Reprod 34: Chang MC, Fertilizing capacity of spermatozoa deposited into the fallopian tubes. Nature (Lond) 168: Cornett LE, Bavister BD, Meizel S, Adrenergic stimulation of fertilizing ability in hamster spermatozoa. BioI Reprod 2: Critser ES, Leibfried-Rutledge ML, Eyestone WE, Northey DL, First NL, Acquisition of developmental competence during maturation in vitro. Theriogenology 25:15 First NL, Parrish JJ, In vitro fertilization of ruminants. J Reprod Fertil 34: (Suppl.) Fleming AD, Yanagimachi R, 1982a. Fertile life of acrosome-reacted guinea pig spermatozoa. J Exp Zool 22: Fleming AD, Yanagimachi R, 1982b. Effects of various lipids on the acrosome reaction and fertilizing capacity of guinea pig spermatozoa with special reference to the possible involvement of lysophospholipids inthe acrosome reaction. Gamete Res 4: Florman HM, Bechtol KB, Wassarman PM, Enzymatic dissection of the functions of the mouse egg s receptor for sperm. Dcv Biol 16: Fraser LR, Mouse sperm capacitation assessed by kinetics and morphology of fertilization in vitro. J Reprod Fertil 69 : Handrow RE., Lenz RW, Ax RL, Structural comparisons among glycosaminoglycans to promote an acrosome reaction in bovine spermatozoa. Biochem Biophys Rca Common 17: Handrow RE., Parrish JJ, Susko-Parrish JL, Effect of glycosaininoglycans on capacitation and the acrosome reaction of bovine and hamster sperm. Biol Reprod 34:93 (Suppl.) Langlais J, Roberts KD, A molecular membrane model of sperm capacitation and the acrosome reaction of mammalian spermatozoa. Gamete Res 12: Leibfried-Rutledge ML, Critser ES, Eyestone WH, Northey DL, First NL, Developmental potential of bovine oocytes matured in vitro or in vivo. Biol Reprod 36: Lenz RW, Ax RL, Grimek HJ, First NL, Proteoglycan from bovine follicular fluid enhances an acrosome reaction in bovine spermatozoa. Biochem Biophys Res Commun 16: Lenz RW, Ball GD, Leibfried ML, Ax RL, First NL, 1983a. In vitro maturation and fertilization of bovine oocytes are temperaturedependent processes. Biol Reprod 29: Lenz RW, Ball GD, Lohse JK, First NL, Ax RL, 1983b. Chondroitin sulfate facilitates an acrosome reaction in bovine spermatozoa as evidenced by light microscopy, electron microscopy and in vitro fertilization. Biol Reprod 28:683-9 Lenz RW, Bellin ME, Ax RL, 1983c. Rabbit spermatozoa undergo an acrosome reaction in the presence of glycosaminoglycans. Gamete Res 8:11-19 Llanos MN, Meizel S Phospholipid increases during capacitation of golden hamster sperm in vitro. Biol Reprod 28: Meizel S, Turner KO, Glycosaminoglycans stimulate the acrosome reaction of previously capacitated hamster sperm. J Exp Zool 237: Ohzu E, Yanagimachi R, Acceleration of acrosome reaction in hamster spermatozoa by lysolecithin. J Exp Zool 224: O Rand MG, Modification of the sperm membrane during capacitation. Ann NY Acad Sci 383: Pace MM, Sullivan JJ, Elliott Fl, Graham EF, Cooker GH, Effects of thawing temperature, number of spermatozoa and spermatozoal quality on fertility of bovine spermatozoa packaged in.5-ml French straws. J Anim Sci 53: Parrish JJ, Susko-Parrish JL, Effects of sea urchin fucose sulfate on bovine and hamster sperm. Biol Reprod 34:64 (Suppl.) Parrish JJ, Susko-Parrish JL, First NL, Effect of heparin and chondroitin sulfate on the acrosome reaction and fertility of bovine sperm in vitro. Theriogenology 24: Parrish JJ, Susko-Parrish JL, First JL, 1986b. Capacitation of bovine sperm by oviduct fluid or heparmn is inhibited by glucose. J Androl 7:22 Parrish JJ, Susko-Parrish JL, Leibfried-Rutledge ML, Critser ES, Eyestone WH, First NL, 1986a. Bovine in vitro fertilization with frozen-thawed semen. Theriogenology 25:591-6 Roy JB, Hurst GG, Hooper G, Heparin catalysis of sperm capacitation in vitro. 41st Ann Meeting Am Fertil Soc, p. 75 Susko-Parrish JL, Parrish JJ, Handrow RR, Glucose inhibits the action of heparin by an indirect mechanism. Biol Reprod 32:8 (Suppl.) Wolf DE, Hagopian SS, Ishijima 5, Changes in sperm plasma membrane lipid diffusibility after hyperactivation during in vitro capacitation in the mouse. J Cell Biol 12: Yanagimachi R, Mechanisms of fertilization in mammals. In: Mastroianni L, Biggers J (eds.), Fertilization and Embryonic Development In Vitro. New York: Plenum Publishers, pp Yanagimachi R, Usui N, Calcium dependence of the acrosome reaction and activation of guinea pig spermatozoa. Exp Cell Res 89: