Effect of Casein Phospho Peptides and Ca 2+ on Penetration of Boar Spermatozoa into Pig Oocytes Matured In Vitro'

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1 BIOLOGY OF REPRODUCTION 55, (1996) Effect of Casein Phospho Peptides and Ca 2+ on Penetration of Boar Spermatozoa into Pig Oocytes Matured In Vitro' T. Mori, 2, 3 M. Hirayama, 4 K. Suzuki, 3 H. Shimizu, 3 and T. Nagai s Department of Animal Science, 3 Faculty of Agriculture, Hokkaido University, Sapporo 060, Japan Bio Science Laboratories,4 Meiji Seika Ltd., Saitama , Japan Department of Animal Production, s Tohoku National Agricultural Experiment Station, Iwate , Japan ABSTRACT To investigate the promoting effect of casein phospho peptides (CPP) on the penetration of ejaculated boar spermatozoa into in vitro-matured oocytes, preincubated spermatozoa were coincubated with in vitro-matured oocytes in fertilization medium with or without CPP for min. Sperm penetration into zona-intact oocytes was observed within 90 min of coincubation. The penetration rate reached a maximum earlier in the presence of CPP than in the absence of CPP. Furthermore, spermatozoa preincubated in fertilization medium with CPP before coincubation with matured oocytes retained the ability to penetrate oocytes for a longer time than spermatozoa preincubated in fertilization medium without CPP. In fertilization medium containing CPP, Ca 2' uptake by spermatozoa increased within 90 min of incubation, with a peak of 113 nm/i x 107 cells. On the other hand, in fertilization medium with no CPP, the intracellular Ca 2* concentration remained constant or gradually decreased until 180 min of incubation. When preincubated spermatozoa were coincubated with oocytes for 5 h in a fertilization medium containing CPP and/or EGTA, or with no addition, EGTA decreased the rate of oocytes penetrated by spermatozoa and the number of spermatozoa penetrating each oocyte. On the other hand, when CPP was added to the medium containing EGTA, the inhibitory effect of EGTA was neutralized. These results indicate that CPP promotes Ca 2' uptake of boar spermatozoa, resulting in a more rapid and longer-lasting shift of a subpopulation of spermatozoa that can penetrate oocytes; the findings also suggest that CPP neutralizes the inhibitory effect of EGTA on sperm penetration. INTRODUCTION Calcium ions play a consequential role in fertilization in both invertebrates and vertebrates [1]. As the plasma membrane of spermatozoa begins to modify, extracellular calcium ions are incorporated into their cytoplasm; and when a sufficient concentration of intracellular Ca 2+ has accumulated, capacitation of spermatozoa is accomplished [2-5]. Nagai et al. [6] reported that CPP enhance the penetration of frozen-thawed pig epididymal spermatozoa through a synergistic effect with caffeine. CPP are calcium-binding proteins [7, 8] that stabilize Ca 2+ in solution [9-11] and enhance Ca 2+ incorporation into various types of cells [7, 10]. CPP also enhanced Ca 2 + uptake of boar spermatozoa, resulting in a high penetration rate [6], although details of the mechanism are still unclear. The aim of this study was to investigate the mechanism Accepted March 26, Received December 11, 'This work was supported in part by a Grant-in-Aid (Bio-Media Program) from the Ministry of Agriculture, Forestry and Fisheries, Japan (BMP- 96-V-2-1). 2Correspondence. FAX: ; tamori@a2.hines.hokudai.ac.jp 364 of the promoting effect of CPP on the penetration of ejaculated spermatozoa into oocytes (experiments 1-3), as well as the effect of Ca 2+ on the penetration of boar spermatozoa, by changing the Ca 2+ concentration in fertilization medium containing CPP (experiment 4). MATERIALS AND METHODS In Vitro Maturation of Oocytes Ovaries were collected just after slaughter and were transported in thermos flasks (at about 37 C) from a local slaughterhouse to our laboratory within 1 h. After several washings with sterile warm saline, follicular oocytes were collected by aspiration with an 18-gauge needle attached to a 5-ml syringe and then pooled in 10-ml test tubes placed in a water bath (37 C) for settling. About 3 ml of the lower portion of collected fluid containing cumulus-oocyte complexes (COC) was transferred to a petri dish. Oocytes with compact cumulus cells and a dense cytoplasm were collected under dissection microscopy and washed two times with handling medium. After washing with maturation medium, selected COCs were incubated in droplets (200 p.l for each 20 COCs) of the maturation medium under mineral oil (Squibb & Sons Inc., Princeton, NJ) in a plastic culture dish (Falcon 1008; Becton, Dickinson and Co., Rutherford, NJ) at 39 C under an atmosphere of 5% CO 2 in air for h. Sperm Preparation The concentrated portion of freshly ejaculated semen was collected from boars (boar A in experiments 1-3 and boar B in experiment 4) by the gloved-hand method and stored in the dark for 18 h at 15C. About 1 ml of semen was washed with 9 ml of handling medium by centrifugation (500 x g, 3 min); and after another washing with preincubation medium, concentrated spermatozoa (1-2 x 108 cells/ml) were incubated in droplets of the preincubation medium covered with mineral oil under an atmosphere of 5% CO 2 in air at 37 C for h [12]. In Vitro Fertilization (IVF) At the end of incubation for maturation, oocytes with expanded cumulus cells were transferred to droplets (100,ul for 10 oocytes) of fertilization medium after being washed twice with the medium. A suspension of preincubated sperm was diluted with fertilization medium and introduced into the droplets containing oocytes; coincubation was then carried out under an atmosphere of 5% CO 2 in air at 37C for various times depending on the experiments. Observation of Spermatozoa-Penetrated Oocytes After coincubation with spermatozoa, the eggs were washed and incubated in droplets of culture medium. At

2 EFFECT OF CPP AND Ca 2+ ON PENETRATION OF BOAR SPERMATOZOA 365 the end of culture, the eggs were fixed with acetic alcohol (1:3) and 3-4 days later were stained with 1% aceto-orcein for observation of sperm penetration under a phase-contrast microscope. Oocytes with swollen sperm heads or male pronuclei with tails were identified as penetrated. Medium The handling medium was 25 mm Hepes-buffered (ph 7.4 in air) tissue culture medium 199 (TCM199) with Earle's salts (Nissui Seiyaku, Tokyo, Japan) supplemented with 3 mg/ml BSA (fraction V; Sigma Chemical Company, St. Louis, MO) and antibiotics. The maturation medium was almost the same as that described by Yoshida et al. [13]: Waymouth medium MB 752/1 (ICN Biomedicals, Costa Mesa, CA) supplemented with 10% (v:v) fetal bovine serum (FBS; Filtron, Brooklyn, NSW, Australia), 10% (v: v) pig follicular fluid (pff) collected from immature follicles (2-5 mm in diameter) of prepubertal pig ovaries, mg/ml porcine (p)fsh (UCB-Bioproducts, Brain-I'Alleud, Belgium), 1 mg/ml plh (UCB-Bioproducts), 50 mg/ml glutamine, and antibiotics. The sperm preincubation medium was modified TCM199 as described by Nagai et al. [14]: 0.1 mg/ml sodium pyruvate, 0.9 mg/ml calcium lactate, and 0.55 mg/ml D-glucose added to TCM199 with Earle's Salts, with the final ph adjusted to 7.6, in an atmosphere of 5% CO 2 in air. The fertilization medium was a Brackett and Oliphant's (BO) solution [15] supplemented with 5 mm caffeine and with the final ph of the medium adjusted to 7.6, in an atmosphere of 5% CO 2 in air [16]. CPP (Meiji Seika Ltd., Tokyo, Japan; 1 mg/ml; experiments 1-3) and/or EGTA (final concentration of 3.3 mm; experiment 4) was added to the fertilization medium. CPP was produced from milk casein according to the method of Hirayama et al. [17]. Chatot-Ziomek-Bavister (CZB) medium [18] was used as an embryo culture medium after coincubation of oocytes with spermatozoa. Measurement of Intracellular Ca 2 + Concentration The intracellular free Ca 2 + in spermatozoa during incubation in the fertilization medium was determined with a fluorescent Fura2/AM according to the method described by Zhou et al. [19]. Briefly, spermatozoa preincubated in a 5-ml round-bottom cryogenic vial (Falcon 4805; Becton, Dickinson and Co.) were centrifuged; the pellet was resuspended in a physical medium containing 1 mm Fura2/AM (Dojin, Kumamoto, Japan) to a final concentration of 2 X 107 cells/ml and then incubated at 39 C for 1 h with the cap tightly closed. After the loading of Fura2/AM, 3 ml of the sperm sample was transferred to a quartz cuvette, and fluorescence was measured with a luminescence spectrometer (Hitachi ; Hitachi, Tokyo, Japan). The intracellular Ca 2+ concentration was calculated by the following formula, described by Grynkiewcz et al. [20]: Experiment [CA 2 +]i = kd(f-fmin)/(fma-f), kd = 224 nm. To investigate the effect of CPP on the duration of coincubation needed for spermatozoa to penetrate oocytes, matured oocytes were coincubated with preincubated spermatozoa in the fertilization medium with or without CPP (1 mg/ml). The ability of the spermatozoa to attach directly to and penetrate the oolemma in the fertilization medium with or without CPP was also investigated through use of zona-free oocytes treated with a medium containing 0.01% Pronase (w:v) (Sigma) for 2-3 min. The sperm concentration for coincubation was adjusted to 1 x 105 cells/mi. After coincubation for min, the oocytes were transferred to CZB, and spermatozoa that had attached to the zona pellucida were removed by pipetting with a fine-bore pipette. Zona-free oocytes with spermatozoa attached were washed by gentle pipetting. After culture in CZB for up to 9-10 h after coincubation, all the oocytes were fixed and examined for penetration of spermatozoa. The intracellular Ca 2+ concentration in spermatozoa incubated in the fertilization medium with or without CPP was also measured in three replicates each. To avoid inconsistency with the results of IVF caused by characteristic deviation among ejaculates, measurements were performed at the same time as IVF, using the same ejaculates. Experiment 2 To investigate whether CPP has the effect of prolonging the ability of spermatozoa to penetrate oocytes in a fertilization medium, preincubated spermatozoa were incubated (prefertilization incubation) in droplets of the fertilization medium, with or without CPP, before coincubation with oocytes. The sperm concentration was the same as that in experiment 1. After a prefertilization incubation of spermatozoa for min, oocytes were introduced into droplets of spermatozoa and 60-min coincubation was carried out. At the end of each coincubation, all spermatozoa were removed from the surface of coincubated oocytes by pipetting. After washing, these oocytes were transferred to the fertilization medium without CPP. All the oocytes were subsequently incubated in a medium for which the total duration of culture was 180 min, except that oocytes cultured with spermatozoa were incubated for 240 min. After culture in CZB for 4-5 h after coincubation, the eggs were fixed for observation of sperm penetration. Experiment 3 To explore the mechanism of the CPP effect on penetration of spermatozoa into oocytes, IVF was performed with use of a fertilization medium containing CPP (0 or 1 mg/ml) and/or EGTA (0 or 3.3 mm). The penetration rate and the number of spermatozoa per penetrated oocyte were investigated in a 2 x 2 factorial experiment. Experiment 4 To investigate the effect of Ca 2 + in fertilization medium with and without CPP on sperm penetration, IVF was performed with use of fertilization medium containing 0, 1.125, 2.25, and 4.5 mm CaC1 2 in the presence or absence of CPP. The concentration of spermatozoa in coincubation was adjusted to cells/mi. Statistical Analysis of Data Differences in the rate of oocytes penetrated by spermatozoa, rate of monospermic oocytes, and rate of polyspermic oocytes among treatments were tested by chisquare analysis. Differences in the mean number of spermatozoa that penetrated an oocyte were determined by ANOVA (Fisher's predicted least significant difference). RESULTS Experiment As shown in Table 1 and Figure 1, sperm penetration into zona-intact oocytes was observed within 90 min of

3 366 MORI ET AL. TABLE 1. Effect of CPP on penetration of boar spermatozoa into oocytes with zona pellucida. Duration of coin- With (+) No. of No. of cuba- or with- oocytes oocytes No. of No. of sperm tion out (-) insem- pene- monospermic penetrated/ (min) CPP inated* trated (%) oocytes (%) oocytet (3) 2 (2.5) ' 2 (100) (3) 2 (2.6)0 2 (100) (3) 21 ( )b 12 (57.1) 1.7 ± (3) 12 (13.9) b 8 (66.7) 1.4 ± 0.7a (3) 40 (50.6)' 20 (50.0) 1.8 ± 1.1 b - 84 (3) 33 (39.2) c 23 (69.7) 1.4 ± (3) 78 ( 95.1)d 12(15.4) 4.4 ± (3) 57 (72.1) 27 (47.4) 2.2 ± 1.50 f (3) 75 (100) 4 (5.3) 8.5 ± 4. 6 d - 82 (3) 74 (90.2) d 11 (14.9) 4.8 ± (3) 82 (100)' 1 (1.2) 14.5 ± 6.7" - 75 (3) 73 (97.3) d 0 (0.0) 8.8 ± 4.7 d t Mean SD. There are significant differences between the values with TABLE 2. Effect of CPP on penetration of boar spermatozoa into zonafree oocytes. Duration of No. of No. of coin- With (+) No. of No. of mono- sperm cuba- or with- oocytes oocytes spermic penetion out (-) insemi- pene- oocytes trated/ (min) CPP nated* trated (%) (%) oocyte' (2) 1 (2.6) 1 (100) (2) 1 (2.6) a 1 (100) (2) 3 (8.6) a 3 (100) 1.0 ± (2) 3 (7.9) 2 (66.7) 1.5 ± (3) 7 (11.3) b 4 (57.1) 1.4 ± (3) 1 (2.7) 1 (100) (2) 21 (44.7)' 12 (57.1) (3) 7 ( 1 7.1)b 5 (71.4) (3) 38 ( 6 9.1)d 17 (30.9) (3) 21 (38.9) c 9 (42.9) 2.1 ± (2) 30 ( )d 3 (10.0) (2) 19 (45.2)1 6 (31.6) I Mean SD. There are significant differences between the values with coincubation, and the penetration rate later increased in fertilization medium both with and without CPP. However, the penetration rate reached a plateau earlier in the presence of CPP (150 min) than in the absence of CPP (180 min). The mean number of spermatozoa that penetrated each oocyte increased significantly (p < 0.05) after 120 or 150 min postcoincubation in fertilization medium with and without CPP, respectively. Although the number of spermatozoa penetrating an oocyte continued to increase until the end of coincubation (240 min), a significantly higher number (p < 0.05) of spermatozoa penetrated oocytes in the fertilization medium with CPP (Table 1). When zona-free oocytes were coincubated with spermatozoa, the rate of oocytes penetrated by spermatozoa significantly (p < 0.05) increased after 120 min, reaching a plateau after 180 min and remaining constant thereafter. Although zona-free oocytes were penetrated by fewer spermatozoa than zona-intact oocytes, the penetration rates obtained in the medium with CPP were significantly (p < 0.05) higher than those in the medium without CPP ( % and %, respectively) after 120 min of coincubation with oocytes (Table 2 and Fig. 1). As shown in Figure 2, in the fertilization medium containing CPP the Ca 2+ uptake by spermatozoa reached a peak of 123 nm/1 x 107 cells at 90 min of incubation and then decreased to the same level as in medium without CPP by 150 min. On the other hand, in the fertilization medium with no CPP, the intracellular Ca 2 + concentration remained constant or gradually decreased up until 180 min. The intracellular Ca 2+ concentration of spermatozoa increased thereafter in both media. Experiment ~ r 0 It In As shown in Table 3, spermatozoa had the ability to penetrate oocytes up until 150 min of prefertilization in- LoU U M +1b.E _ I I Duration of Incubation in the Fertilization Medium (min) Duration of coincubation (min) FIG. 1. Changes in the penetration rate of ejaculated spermatozoa of boars during coincubation with oocytes matured in vitro. Preincubated spermatozoa were coincubated for various times ( min) with oocytes, with zona pellucida (ZP+) or without zona pellucida (ZP-), in a fertilization medium with CPP (CPP+) or without CPP (CPP-). FIG. 2. Changes in the intracellular Ca 2+ concentration in boar spermatozoa during incubation in fertilization medium with CPP (CPP+) or without CPP (CPP-). After incubation for various times (0-240 min) in both fertilization media, spermatozoa were loaded with a fluorescent Fura2/AM (1 mm) for 60 min, and the intracellular Ca 2+ concentration was measured according to the procedures described in Materials and Methods. Each point is the mean SEM.

4 EFFECT OF CPP AND Ca 2 + ON PENETRATION OF BOAR SPERMATOZOA 367 TABLE 3. Effect of CPP on penetration of boar spermatozoa during coincubation with oocytes for 60 min after prefertilization incubation. Duration of prefertilization No. of No. of incu- With (+) No. of No. of mono- sperm ba- or with- oocytes oocytes spermic penetion out (-) insemi- pene- oocytes trated/ (min) CPP nated* trated (%) (%) oocyte t (2) 27 (100)a 2 (7.4) a - 27 (2) 25 (92.5)a 4 (16.0) b (5) 59 ( 8 4.2)b 8 (13.6) (5) 47 (55.2) c 17 (36.2) d (4) 42 (82.3)b 13 (31.0) e - 73 (5) 40 (54.7)c 17 (42.5) ' * Mean SD. There are significant differences between the values with cubation. However, the penetration rate decreased for spermatozoa that were incubated for a longer period before coincubation with oocytes. A greater decline in the penetration rates was observed in the medium without CPP than in the medium with CPP. Experiment 3 As shown in Table 4, EGTA in the fertilization medium lowered the penetration rate of spermatozoa. On the other hand, when CPP was added to the fertilization medium containing EGTA, the penetration rate and the mean number of spermatozoa that penetrated an oocyte were the same as for the control (using fertilization medium with no additions). Experiment 4 As shown in Table 5, even if the medium contained no CaC1 2 (0.0 mm), the penetration rate was 50% with CPP but only 13% without CPP. When the CaC1 2 concentration was increased to mm, both the penetration rate and the mean number of spermatozoa that penetrated an oocyte increased. Although further increases of CaC1 2 to 4.5 mm resulted in a decrease in the rate and mean number of penetrating spermatozoa in the fertilization medium without CPP, such decline was not observed in the medium with CPP Spermatozoa with a higher mobility were observed during coincubation in the medium containing a higher concentration of CaC1 2, while there seemed to be no differences in mobility between the spermatozoa in media with and without CPP. DISCUSSION CPP is a mixture of trypsin-hydrolysis subunits of milk casein (a-cpp from oasl-casein and -CPP from B-casein); it has the ability to stabilize Ca 2 + in solution [9, 10] and enhances the incorporation of Ca 2+ into various types of tissues cultured both in vitro [7, 10] and in vivo [8, 21]. This property of CPP depends on its Ca 2 +-chelating ability, similar to that of EGTA. However, their association constants are different (104 kb[m] for CPP vs. 108 kb[m] for EGTA), and CPP associates with Ca 2 + more weakly than EGTA. Experiment 3 shows that while the ability of EGTA TABLE 4. Effect of CPP on penetration of boar spermatozoa during coincubation with oocytes in fertilization medium containing 3.3 mm EGTA and no EGTA. With (+) or No. of with- With No. of No. of monoout (+) or oocytes oocytes spermic No. of sperm (-) without insemi- penetrated oocytes penetrated/ EGTA (-) CPP nated* (%) (%) oocyte (2) 100 (95.2)a 12 (12.0) (2) 25 ( )b 19 (76.0) 1.4 _ 0.8 b (1) 40 (100), 0 (0.0) c - 94 (2) 89 (94.7)a 5 (5.6) t Mean + SD. There are significant differences between the values with to associate with Ca 2 + was too strong to transport Ca 2 + to spermatozoa, a weaker CPP association was able to transport Ca 2+ to spermatozoa. Although the intracellular Ca 2 + concentration of spermatozoa incubated in fertilization medium with EGTA was less than that of preincubated (O min) spermatozoa, the intracellular Ca 2+ concentration of spermatozoa incubated in the medium with CPP increased significantly (data not shown). In fertilization medium containing both CPP and EGTA, the penetration rate was the same as that obtained in the medium with only CPP, and the number of spermatozoa that penetrated into a single oocyte was higher than in the medium containing only EGTA. It is suggested that a small portion of Ca 2 + released from EGTA molecules may be transported temporarily to the membrane of spermatozoa with the aid of CPP. This transportation would be effective if CPP was present at a position very peripheral to the membrane. It is possible that CPP molecules may bind to the membrane of spermatozoa. Experiments using a monoclonal antibody to CPP are presently being designed to determine whether or not CPP molecules exist on the membrane. Intracellular Ca 2 + is reported to play a critical role in the capacitation and acrosome reaction of sperm [1]. As the intracellular Ca 2 + concentration of individual spermatozoa TABLE 5. Effect of CaCI 2 concentration in a fertilization medium with or without CPP on penetration of boar spermatozoa during coincubation with oocytes matured in vitro. Concentration of CaCI 2 No. of in the With (+) No. of No. of monomedi- or with oocytes oocytes spermic No. of sperm um out (-) insemi- penetrated oocytes penetrated/ (mm) CPP nated* (%) (%) oocyte' (4) 58 (50.4)a 30 (51.7) a a (4) 15 ( )b 13 ( )b 1.1 ± 0.4a (4) 103 (92.0)c 18 (1 7.5)cde b (4) 103 (88.8)c 32 (31.1)f ' (4) 105 (92.9)c 8 (7.6) ge b' (4) 94 (93.1) C 9 (9.6)g d b (4) 107 (98.2)c 14 (13.1)d c (4) 71 (61.7)a 21 ( 2 9.6)f b t Mean SD. There are significant differences between the values with

5 368 MORI ET AL. was not measured in this experiment, changes in the intracellular Ca 2 + concentration could be considered only as changes in the total values of the sperm population tested. When the subpopulation of spermatozoa that incorporate Ca 2 + is dominant over the subpopulation that release Ca 2 + from their cytoplasm, the total intracellular Ca 2 + concentration increases, and vice versa. The results of experiment 1 (Fig. 2) suggest that increases in the total intracellular Ca 2 + concentration observed between 60 and 90 min of incubation could be attributed to the increase in the relative size of the population spermatozoa that incorporated Ca 2+. The results showed that more spermatozoa incorporated Ca 2+ in a shorter period when incubated in the fertilization medium with CPP than in the medium without CPP. The decline in the total intracellular Ca 2+ concentration following this increase may have been due to a shift in the main population of spermatozoa from the spermatozoa that had incorporated Ca 2+ to those releasing Ca 2 +. The spermatozoa at this stage might exocytose their acrosomal contents as well as Ca 2 +. The decline in the total intracellular Ca 2+ concentration may coincide with the increase in spontaneously acrosomereacted spermatozoa. The results for IVF of zona-free oocytes in fertilization medium with CPP also showed that the penetration rate increased after 120 min of coincubation compared with the penetration rate in fertilization medium without CPP. It was thought that compared to spermatozoa incubated in medium without CPP, spermatozoa incubated in medium with CPP incorporated sufficient Ca 2+ during a shorter incubation period to penetrate oocytes or to react spontaneously. There was a second increase in the total intracellular Ca 2+ concentration in spermatozoa incubated in both media after 180 min of incubation. Although the precise mechanism of this increase is unclear, one possibility is that the permeability of Ca 2+ to the cytoplasm of spermatozoa might increase after the acrosome reaction and lead to an increase in intracellular Ca 2 + concentration. At the usual Ca 2+ concentration (2.25 mm) in BO medium, penetration rates seem to vary among ejaculates even from the same boar. However, when the Ca 2 + concentration in medium with CPP was increased, the penetration rates become constant at a high level. Fraser [2] also reported that increments of extracellular Ca 2+, from 1.8 mm to 3.6 mm, promoted sperm penetration in mice, and it was suggested that this was attributable to an elevated intracellular Ca 2 + concentration. Therefore, in order to obtain constantly high penetration rates in an IVF system, it may be crucial to generate a high spermatozoa intracellular Ca 2 + concentration before or during the encounter with oocytes. It has been shown that when the fertilization medium contains no CaCl 2, penetration rates decline severely even if the medium contains CPP (Table 5). CPP, however, contains calcium in its molecules ( % of weight); this would give rise to an increase of approximately mm of Ca 2+ in a medium containing CPP. This slight Ca 2 + contamination from CPP might enhance the ability of spermatozoa to penetrate oocytes even in a medium deprived of CaC1 2. Nagai et al. [6] reported that the concentration of free Ca 2 + in a fertilization medium with CPP was less than that in a medium containing extra CaCl 2 in place of Ca 2 + derived from CPP. It was thought that the free Ca 2 + concentration was in an equilibrated state in the presence of CPP, and that CPP molecules tended to incorporate with Ca 2 + in a high CaCl 2 solution while Ca 2 + was freed from CPP molecules in medium deprived of CaCl 2. In the preparation of fertilization medium containing 4.5 mm CaCI 2 without CPP, precipitation was observed. This precipitation was thought to be calcium phosphate, which may decrease the concentration of free Ca 2+ in the medium. This may result in a lower penetration rate than in medium containing mm CaCl 2. However, in the presence of CPP, Ca 2 + at a high concentration in the medium was stabilized and supplied to spermatozoa. Acrosome reaction is necessary in order for spermatozoa to penetrate the zona pellucida and fuse with the oolemma [22]. Since the zona pellucida induces the acrosome reaction of boar spermatozoa [23], removal of the zona pellucida resulted in the loss of a reaction site. Therefore, only spontaneously reacted spermatozoa could fuse with the plasma membrane of oocytes without the zona pellucida. Table 1 shows findings for spermatozoa that completed penetration into zona-intact oocytes at 150 min of incubation in fertilization medium with CPP. Assuming that the majority of spermatozoa started the acrosome reaction at 90 min of incubation in the medium with CPP, and attached to zona pellucida, it is reasonable to speculate that those spermatozoa took less than 60 min to traverse the zona pellucida. However, the rate of sperm penetration to zonafree oocytes reached its maximum at 180 min of incubation even in medium with CPP. It is thought that spermatozoa obtain the ability to fuse with the oolemma when they traverse through the zona [22]. Therefore, a time lag of more than 30 min might be needed for spermatozoa to change their membrane or to complete the acrosome reaction to fuse with the oolemma without traversing the zona pellucida. The results also showed lower rates of sperm penetration into zona-free oocytes. Therefore, with incubation in a fertilization medium without exposure to the zona pellucida, the rate of spermatozoa that completed the acrosome reaction in order to fuse with the oolemma was lower than that for spermatozoa that traversed the zona pellucida. Even so, CPP enhanced sperm penetration to oocytes without the zona pellucida relative to penetration by spermatozoa incubated without CPP. From these results, it was concluded that CPP has the effect of enhancing the ability of spermatozoa to fuse with the oolemma by completing their acrosome reaction. It has been reported that preincubated spermatozoa undergo spontaneous acrosome reactions serially when introduced into the fertilization medium [24, 25], and a spermatozoon whose acrosome has reacted before there is contact with a zona-intact oocyte may be unable to penetrate the zona pellucida [26]. Therefore, it is thought that in our experiments the penetration rates may have decreased in spermatozoa incubated for a longer time before coincubation with zona-intact oocytes. In fact, the penetration rates into zona-intact oocytes decreased when spermatozoa were incubated for more than 180 min before insemination in fertilization medium. However, CPP supplementation to the fertilization medium had the effect of slowing the decline in penetration rates. Two possible ways in which CPP may affect sperm penetration can be considered: one is by maintaining the ability of the spermatozoon to penetrate oocytes and the other is by promoting a subpopulation of spermatozoa ready to penetrate oocytes successively. It can be deduced from the changes in the intracellular Ca 2 + concentration that CPP in the medium enhanced sperm membrane instabilization or the acrosome reaction by stimulating Ca 2 + uptake until 90 min of incubation, and that thereafter intracellular Ca 2 + was released with increased permeability of the sperm membrane. Thus, after 150 min of prefertilization incubation with CPP, the majority of spermatozoa must

6 EFFECT OF CPP AND Ca 2+ ON PENETRATION OF BOAR SPERMATOZOA 369 have undergone an acrosome reaction and as such may not have been able to penetrate zona-intact oocytes [26]. Therefore, it is unlikely that CPP prolongs the penetrability of spermatozoa. Alternatively, considering the heterogeneity in penetrability among individual spermatozoa, as well as among ejaculates and boars, it is possible that CPP promotes a subpopulation of spermatozoa having an intact acrosome that are ready to undergo the acrosome reaction in order to penetrate oocytes successively. In conclusion, CPP has an effect on the Ca 2+ uptake in boar spermatozoa that results in the faster shift of a subpopulation of spermatozoa that can penetrate oocytes and thereafter aids other potential subpopulations to shift more effectively. ACKNOWLEDGMENTS We are grateful to the staff of the Ebetsu Meat Inspection Office for their help in the collection of porcine ovaries and to Mrs. Y. Kato-Mori for manuscript preparation. 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