Journal of Assisted Reproduction and Genetics, Vol. 9, No. 5, 1992
ANIMAL INVESTIGATIONS
Effects of Platelet Activating Factor on Mouse Sperm Function KAZUO SENGOKU, l'z MUTSUO ISHIKAWA, 1 KENICHI TAMATE, 1 and TETSUYA SHIMIZU 1
Submitted: March i1, 1992 Accepted: August 21, 1992
implantation process since PAF is produced by preimplantation embryos and PAF antagonists inhibit embryo implantation (3,4). Recently PAF has been found in rabbit, mouse, and human spermatozoa (5-7). Furthermore, it has been suggested that PAF has a beneficial effect on the motility and the acrosome reaction in human spermatozoa (8,9). Minhas et al. (10) and Kuzan et al. (6) demonstrated that the addition of PAF enhanced in vitro fertilization rates and PAF antagonists inhibited fertilization in mice. However, the effect of PAF on capacitation and the acrosome reaction in mouse spermatozoa has not been determined. The present study was designed to investigate the effect of PAF on capacitation and the induction of the acrosome reaction using in vitro fertilization of zona-intact and zona-free oocytes with preincubated spermatozoa.
Platelet activating factor (PAF) has been implicated in a variety o f reproductive processes. This study was designed to investigate the effect o f P A F and the specific P A F receptor antagonist, CV-3988, on capacitation and the acrosome reaction in mouse spermatozoa using an in vitro fertilization (IVF) system. When spermatozoa were preineubated f o r 30 min in medium containing P A F (10-7 to 10 -11 M), a significant increase in the fertilization rate with both cumulus-free and zona-free oocytes was observed. In contrast, treatment o f the spermatozoa with 10 -5 M CV-3988 caused a significant decrease in both sperm motility and fertilization rates with zona-intact and zona-free oocytes. This suppression was reversed by the addition o f PAF. Furthermore, the acrosome reaction was enhanced by P A F treatment o f spermatozoa in a dose-dependent manner. This stimulation o f the acrosome reaction by P A F required the presence o f calcium ions in the medium. While 10 -5 M CV-3988 inhibited the acrosome reaction, the inhibition was also reversed by the addition o f PAF. These results suggest that P A F can stimulate not only the capacitation process but also the acrosome reaction, both o f which are dependent on extracellular calcium.
MATERIALS AND METHODS Media Dulbecco's phosphate-buffered saline (PBS) was used for the collection of oocytes. The culture medium used for sperm and oocyte incubation was modified Krebs Ringer bicarbonate (m-KRB) containing 4 mg/ml bovine serum albumin (Fraction V, Sigma Chemical Co, St Louis, MO) equilibrated with 5% CO2 in air at 37°C. Calcium-deficient medium was prepared by omitting CaC12. Calcium concentrations were adjusted by the addition of stock CaCI2 solutions.
KEY WORDS: platelet activating factor; spermatozoa; mouse; in vitro fertilization.
INTRODUCTION Platelet activating factor (PAF) is one of the most biologically active phospholipids and has been implicated in a variety of reproductive processes. Administration of PAF antagonists, BN-52021 and CV3988, inhibited follicular rupture and this inhibition could be reversed by administration of PAF in rats and mice (1,2). PAF may also play a role in the
Chemicals PAF ( 1 - o - a l k y l - 2 - a c e t y l - s n - g l y c e r o - 3 - p h o s p h o r y l choline) was purchased from Avanti Polar Lipid Co., dissolved in chloroform at a stock concentration of 10 raM, and stored at -20°C. Working solutions were prepared by evaporating the chloroform under a stream of nitrogen and then redissolving the residue in m-KRB medium at the desired
1 Department of Obstetrics and Gynecology, Asahikawa Medical College, Nishikagura, 4 Sen 5 Gou, Asahikawa, Japan 078. z To whom correspondence should be addressed.
447
1058-0468/92/1000-0447506.50/0 © 1992PlenumPublishingCorporation
448
concentration. The PAF receptor antagonist, CV3988, was a gift form Takeda Co. (Tokyo). Working dilutions were prepared directly in warmed culture media.
Sperm Preparations Epididymal spermatozoa were collected from mature, 10- to 12-week-old ICR mice, by puncturing the caudal epididymides with an 18-gauge needle and allowing spermatozoa to migrate into 1 ml of m-KRB medium for 5 min. Sperm suspensions were diluted to a concentration of 50 x 106/ml and incubated for 30 min in the presence or absence of PAF or CV-3988. In the experiments for evaluating the role of calcium in the PAF-induced acrosome reaction, spermatozoa were initially collected in calcium-free medium and then preincubated in medium containing appropriate calcium concentrations.
In Vitro Fertilization ICR female mice (6 to 8 weeks old) were superovulated with an i.p. injection of 5 IU of pregnant mare serum gonadotropin (PMSG; Serotropin, Teikokuzouki, Japan), followed 48 hr later by an i.p. injection of 5 IU of human chorionic gonadotropin (hCG; Sigma). Sixteen hours after hCG injection, the oocytes enclosed in cumulus cells were released by dissecting the ampullae. Cumulus cells were dispersed by treatment with 0.1% hyaluronidase (Sigma, Type IV-S, 1150 NF U/mg) in PBS. Cumulus-free oocytes were washed three times in PBS and then transferred to the fertilizing medium (m-KRB). Oocytes were rendered zona-free by sequential treatment with 0.1% hyaluronidase and 0.2% pronase (Sigma, protease Type XIV, 4.5 U/mg) in PBS. The zona-free oocytes were rinsed several times before addition of the spermatozoa. The final sperm concentration was adjusted to 105/ ml by adding an aliquot of the incubated sperm suspension to 1 ml of medium containing oocytes. Oocytes and spermatozoa were incubated together for 20 min. The oocytes were rinsed three times after sperm exposure and cultured for an additional 6 hr in fresh m-KRB. After incubation, oocytes were mounted on a glass slide, fixed with 2.5% glutaraldehyde, stained with acetolacmoid, and assessed for fertilization with an inverted microscope (11). Oocytes were considered fertilized if two pronuclei and a sperm tail were identified.
SENGOKU, ISHIKAWA, TAMATE, AND SHIMIZU
Assessment of Motility and Acrosome Reaction Sperm motility was evaluated every 1.5 hr for 6 hr. The percentage motility (in triplicate samples of the control and each of experimental culture media containing PAF or CV-3988) was estimated and then averaged. Acrosome loss was evaluated by the dual stain procedure of Didion et al. (12). Briefly, live and dead sperm were first differentiated using trypan blue (0.1% for 10 min at 37°C). Sperm suspensions were then washed two times in albuminfree mKRB medium. A 20-~1 aliquot of the sperm suspension was mounted on a slide, air-dried, and stained with 10% Giemsa solution in distilled water (40 min, at 37°C). Slides were examined at 400x magnification. Dead spermatozoa stained blue in the postacrosomal region, while live spermatozoa were unstained. Acrosomal regions of live sperm with intact acrosome were stained purple. In contrast, spermatozoa without acrosomes were stained white. This staining pattern enables distinction between true and false acrosome reactions. More than 200 spermatozoa were examined in each sample after 30 and 120 min of preincubation. The percentage acrosome reactions was calculated as the live acrosome-reacted spermatozoa divided by the total live spermatozoa.
Statistical Analysis Statistical significance of the data was determined by the chi-square test and Student's t test. Differences were considered significant at a level of P < 0.05.
RESULTS Effect of PAF and CV-3988 on Sperm Motility The effects of PAF and CV-3988 on the motility of spermatozoa are shown in Table I. The introduction of PAF (10 - 7 and 10 - 9 M) had no significant effect on the motility of spermatozoa at each time point up to 6 hr. Spermatozoa treated with CV-3988 at doses of 10 - 7 and 10 - 9 M showed no change in motility. However, high concentrations of CV-3988 (10 -5 M) exhibited a significant reduction of motility and 10 -3 M CV-3988 caused an immediate loss of all motility. The inhibition of motility of CV-3988 (10 -5 M) was reversed by the addition of PAF. Journal of Assisted Reproduction and Genetics, Vol. 9, No. 5, 1992
PLATELET ACTIVATING FACTOR AND MOUSE SPERM
449
Table I. Effect of P A F and CV3988 on the Motility of M o u s e S p e r m a t o z o a ~ Percentage motility
Treatment N o n e (control) PAF 10 - 7 M 10 - 9 M CV3988 10 -3 M 10 -5 M 10-7M 10 - 9 M
P A F , 10 -7 M, + CV3988, 10 -5 M
1.5-hr incubation
3-hr incubation
4.5-hr incubation
6-hr incubation
69.3 --- 2.8
61.7 --- 1.7
51.7 +- 8.3
41.6 --- 7.6
68.3 -+ 1.6 68.5 ± 2.1
60.9 --- 1.5 61.0 -+ 2.1
58.2 -+ 4.3 56.6 - 3.8
52.2 --- 5.3 51.1 --- 4.6
0 50.0 -+ 3.1" 67.8 -+ 2.7 68.3 -+ 2.5
0 31.3 +- 2.5* 58.3 ± 4.3 60.1 +- 5.1
0 9.9 ± 3.1" 51.3 ± 2.9 52.0 -+ 3.2
0 2.1 +-- 2.9* 31.3 --- 5.1 32.3 ± 7.1
67.1 ± 1.5
52.0 + 3.1
30.1 -+ 4.2
25.0 -+ 4.6
Data are m e a n s -+ SE. * P < 0.05; different from the control at each interval.
Effect of PAF on in Vitro Fertilization The fertilization rates, with cumulus-free and zona-free oocytes, of PAF- and CV-3988-treated spermatozoa are shown in Tables II and III. When spermatozoa were preincubated for only 30 min instead of 90 to 120 min (normal capacitation time) and exposed to cumulus-free oocytes for 20 min, a decrease in fertilization rate was observed (43.9 and 78.2%, respectively). Spermatozoa treated with PAF showed a significant increase in the fertilization rate with cumulus-free oocytes. However, a dose-dependent effect was not observed at doses of 10 - 7 , 10 - 9 , and 10 - 1 1 M (72.2, 64.8, and 62.2%, respectively). With zona-free oocytes, PAF also enhanced the fertilizing ability of spermatozoa as well as that with cumulus-free oocytes. On the other hand, inhibition of fertilization with the cumulusfree oocytes was observed with 10 -5 M CV-3988treated spermatozoa. Levels of 10 - 7 and 10 - 9 M CV-3988 showed no effect on the fertilizing ability of spermatozoa (Table III). Similarly, spermatozoa treated with 10 -5 M CV-3988 showed a significantly
lower fertilization rate with the zona-free oocytes, and 10 - 7 and 10 -9 M CV-3988 had no effect on fertilization in zona-free oocytes. The inhibition of fertilization of CV-3988 (10 -5 M) was reversed by the addition of 10 - 7 M PAF concomitantly with CV-3988. To evaluate whether the small amount of PAF and CV-3988 affected the fertilizing ability of the oocytes, cumulus-free oocytes were preincubated with PAF and CV-3988 for 15 rain and then mixed with untreated spermatozoa. Exposure of oocytes to PAF and CV-3988 did not show a significant effect on the fertilization rate. Effect of PAF on the Acrosome Reaction The effect of PAF on the acrosome reaction is shown as a function of PAF concentration in the incubation medium for the 30- and 120-min time points in Fig. 1. A low percentage (19.4%) of spermatozoa underwent the acrosome reaction during the first 30 min of incubation in control medium, but this percentage increased to 45.2 at 120 min. In contrast, a significant increase in the acrosome-reacted
Table II. In Vitro Fertilization of C u m u l u s - F r e e and Z o n a - F r e e O o c y t e s by S p e r m a t o z o a Preincubated for 30 min in Media with P A F Cumulus-free oocytes Treatment Control PAF 10 - 7 M 10 - 9 M
10-11 M
Zona-free o o c y t e s
No. of eggs
No. of fertilized eggs (%)
No. of eggs
114
50 (43.9)
73
36 (49.3)
151 108 74
109 (72.2)** 70 (64.8)* 46 (62.2)*
62 70 66
48 (77.4)** 52 (74.2)** 46 (69.7)*
* P < 0.05 c o m p a r e d with control. ** P < 0.01 c o m p a r e d with control.
Journal of Assisted Reproduction and Genetics, Vol. 9, No. 5, 1992
No. of fertilized eggs (%)
450
SENGOKU, ISHIKAWA, TAMATE, AND SHIMIZU
Table III. In Vitro Fertilization of Cumulus-Free and Zona-Free Oocytes by Spermatozoa Preincubated for 30 min in Media with CV3988 Zona-free oocytes
Cumulus-free oocytes No. of eggs
Treatment Control CV3988 10 -5 M 10-7M 10 -9 M CV3988, 10 -5 M + PAF, 10 -7 M
No. of fertilized eggs (%)
No. of eggs
No. of fertilized eggs (%)
92
44 (47.8)
73
36 (49.3)
92 77 66
30 (32.6)* 37 (48.1) 38 (53.5)
90 74 84
28 (31.1)* 32 (43.2) 47 (56.0)
78
45 (57.7)
73
44 (60.3)
* P < 0.05 compared with control.
spermatozoa was observed in PAF-treated spermatozoa compared to nontreated samples. These values increased even more by 120 min. The percentage of acrosome-reacted spermatozoa increased as the PAF concentration increased, suggesting a dose-dependent response (30.2 and 54.7% after 30 and 120 min of incubation, respectively, at 10- 11 M , 38.7 and 58.4% at 10 - 9 M, respectively; and 45.4 and 62.6% at 10 - 7 M, respectively). While 10 - 7 M CV-3988 had no effect on the acrosome reaction, 10-5 M CV-3988 inhibited acrosome loss compared with the control at 30 and 120 min of incubation (Fig. 2). This inhibition was reversed by the addition of PAF. To determine the interaction between
and PAF, the stimulatory effect of PAF on the acrosome reaction with decreasing calcium concentrations in the medium was examined. For this purpose, the 90-min time point with 10 - 7 M PAF was chosen. Decreasing Ca 2+ in the medium without PAF reduced the percentage of acrosome reaction in a dose-dependent manner from 37.2 to 0.9%. The addition of PAF to the medium enhanced acrosomal loss, even when C a 2+ concentrations were as low as 0.25 mM. However, absence of C a 2 + blocked the PAF-induced acrosome reaction at the level of 3.2% (Table IV). Ca 2+
DISCUSSION When using an in vitro fertilization system to evaluate the effects of experimental treatment on
(%) 70-
(%)
*
60.
60. tO O t~ O L
E o O
50' 50.
40.
~ 40" L
30.
O
ANIMAL INVESTIGATIONS
Effects of Platelet Activating Factor on Mouse Sperm Function KAZUO SENGOKU, l'z MUTSUO ISHIKAWA, 1 KENICHI TAMATE, 1 and TETSUYA SHIMIZU 1
Submitted: March i1, 1992 Accepted: August 21, 1992
implantation process since PAF is produced by preimplantation embryos and PAF antagonists inhibit embryo implantation (3,4). Recently PAF has been found in rabbit, mouse, and human spermatozoa (5-7). Furthermore, it has been suggested that PAF has a beneficial effect on the motility and the acrosome reaction in human spermatozoa (8,9). Minhas et al. (10) and Kuzan et al. (6) demonstrated that the addition of PAF enhanced in vitro fertilization rates and PAF antagonists inhibited fertilization in mice. However, the effect of PAF on capacitation and the acrosome reaction in mouse spermatozoa has not been determined. The present study was designed to investigate the effect of PAF on capacitation and the induction of the acrosome reaction using in vitro fertilization of zona-intact and zona-free oocytes with preincubated spermatozoa.
Platelet activating factor (PAF) has been implicated in a variety o f reproductive processes. This study was designed to investigate the effect o f P A F and the specific P A F receptor antagonist, CV-3988, on capacitation and the acrosome reaction in mouse spermatozoa using an in vitro fertilization (IVF) system. When spermatozoa were preineubated f o r 30 min in medium containing P A F (10-7 to 10 -11 M), a significant increase in the fertilization rate with both cumulus-free and zona-free oocytes was observed. In contrast, treatment o f the spermatozoa with 10 -5 M CV-3988 caused a significant decrease in both sperm motility and fertilization rates with zona-intact and zona-free oocytes. This suppression was reversed by the addition o f PAF. Furthermore, the acrosome reaction was enhanced by P A F treatment o f spermatozoa in a dose-dependent manner. This stimulation o f the acrosome reaction by P A F required the presence o f calcium ions in the medium. While 10 -5 M CV-3988 inhibited the acrosome reaction, the inhibition was also reversed by the addition o f PAF. These results suggest that P A F can stimulate not only the capacitation process but also the acrosome reaction, both o f which are dependent on extracellular calcium.
MATERIALS AND METHODS Media Dulbecco's phosphate-buffered saline (PBS) was used for the collection of oocytes. The culture medium used for sperm and oocyte incubation was modified Krebs Ringer bicarbonate (m-KRB) containing 4 mg/ml bovine serum albumin (Fraction V, Sigma Chemical Co, St Louis, MO) equilibrated with 5% CO2 in air at 37°C. Calcium-deficient medium was prepared by omitting CaC12. Calcium concentrations were adjusted by the addition of stock CaCI2 solutions.
KEY WORDS: platelet activating factor; spermatozoa; mouse; in vitro fertilization.
INTRODUCTION Platelet activating factor (PAF) is one of the most biologically active phospholipids and has been implicated in a variety of reproductive processes. Administration of PAF antagonists, BN-52021 and CV3988, inhibited follicular rupture and this inhibition could be reversed by administration of PAF in rats and mice (1,2). PAF may also play a role in the
Chemicals PAF ( 1 - o - a l k y l - 2 - a c e t y l - s n - g l y c e r o - 3 - p h o s p h o r y l choline) was purchased from Avanti Polar Lipid Co., dissolved in chloroform at a stock concentration of 10 raM, and stored at -20°C. Working solutions were prepared by evaporating the chloroform under a stream of nitrogen and then redissolving the residue in m-KRB medium at the desired
1 Department of Obstetrics and Gynecology, Asahikawa Medical College, Nishikagura, 4 Sen 5 Gou, Asahikawa, Japan 078. z To whom correspondence should be addressed.
447
1058-0468/92/1000-0447506.50/0 © 1992PlenumPublishingCorporation
448
concentration. The PAF receptor antagonist, CV3988, was a gift form Takeda Co. (Tokyo). Working dilutions were prepared directly in warmed culture media.
Sperm Preparations Epididymal spermatozoa were collected from mature, 10- to 12-week-old ICR mice, by puncturing the caudal epididymides with an 18-gauge needle and allowing spermatozoa to migrate into 1 ml of m-KRB medium for 5 min. Sperm suspensions were diluted to a concentration of 50 x 106/ml and incubated for 30 min in the presence or absence of PAF or CV-3988. In the experiments for evaluating the role of calcium in the PAF-induced acrosome reaction, spermatozoa were initially collected in calcium-free medium and then preincubated in medium containing appropriate calcium concentrations.
In Vitro Fertilization ICR female mice (6 to 8 weeks old) were superovulated with an i.p. injection of 5 IU of pregnant mare serum gonadotropin (PMSG; Serotropin, Teikokuzouki, Japan), followed 48 hr later by an i.p. injection of 5 IU of human chorionic gonadotropin (hCG; Sigma). Sixteen hours after hCG injection, the oocytes enclosed in cumulus cells were released by dissecting the ampullae. Cumulus cells were dispersed by treatment with 0.1% hyaluronidase (Sigma, Type IV-S, 1150 NF U/mg) in PBS. Cumulus-free oocytes were washed three times in PBS and then transferred to the fertilizing medium (m-KRB). Oocytes were rendered zona-free by sequential treatment with 0.1% hyaluronidase and 0.2% pronase (Sigma, protease Type XIV, 4.5 U/mg) in PBS. The zona-free oocytes were rinsed several times before addition of the spermatozoa. The final sperm concentration was adjusted to 105/ ml by adding an aliquot of the incubated sperm suspension to 1 ml of medium containing oocytes. Oocytes and spermatozoa were incubated together for 20 min. The oocytes were rinsed three times after sperm exposure and cultured for an additional 6 hr in fresh m-KRB. After incubation, oocytes were mounted on a glass slide, fixed with 2.5% glutaraldehyde, stained with acetolacmoid, and assessed for fertilization with an inverted microscope (11). Oocytes were considered fertilized if two pronuclei and a sperm tail were identified.
SENGOKU, ISHIKAWA, TAMATE, AND SHIMIZU
Assessment of Motility and Acrosome Reaction Sperm motility was evaluated every 1.5 hr for 6 hr. The percentage motility (in triplicate samples of the control and each of experimental culture media containing PAF or CV-3988) was estimated and then averaged. Acrosome loss was evaluated by the dual stain procedure of Didion et al. (12). Briefly, live and dead sperm were first differentiated using trypan blue (0.1% for 10 min at 37°C). Sperm suspensions were then washed two times in albuminfree mKRB medium. A 20-~1 aliquot of the sperm suspension was mounted on a slide, air-dried, and stained with 10% Giemsa solution in distilled water (40 min, at 37°C). Slides were examined at 400x magnification. Dead spermatozoa stained blue in the postacrosomal region, while live spermatozoa were unstained. Acrosomal regions of live sperm with intact acrosome were stained purple. In contrast, spermatozoa without acrosomes were stained white. This staining pattern enables distinction between true and false acrosome reactions. More than 200 spermatozoa were examined in each sample after 30 and 120 min of preincubation. The percentage acrosome reactions was calculated as the live acrosome-reacted spermatozoa divided by the total live spermatozoa.
Statistical Analysis Statistical significance of the data was determined by the chi-square test and Student's t test. Differences were considered significant at a level of P < 0.05.
RESULTS Effect of PAF and CV-3988 on Sperm Motility The effects of PAF and CV-3988 on the motility of spermatozoa are shown in Table I. The introduction of PAF (10 - 7 and 10 - 9 M) had no significant effect on the motility of spermatozoa at each time point up to 6 hr. Spermatozoa treated with CV-3988 at doses of 10 - 7 and 10 - 9 M showed no change in motility. However, high concentrations of CV-3988 (10 -5 M) exhibited a significant reduction of motility and 10 -3 M CV-3988 caused an immediate loss of all motility. The inhibition of motility of CV-3988 (10 -5 M) was reversed by the addition of PAF. Journal of Assisted Reproduction and Genetics, Vol. 9, No. 5, 1992
PLATELET ACTIVATING FACTOR AND MOUSE SPERM
449
Table I. Effect of P A F and CV3988 on the Motility of M o u s e S p e r m a t o z o a ~ Percentage motility
Treatment N o n e (control) PAF 10 - 7 M 10 - 9 M CV3988 10 -3 M 10 -5 M 10-7M 10 - 9 M
P A F , 10 -7 M, + CV3988, 10 -5 M
1.5-hr incubation
3-hr incubation
4.5-hr incubation
6-hr incubation
69.3 --- 2.8
61.7 --- 1.7
51.7 +- 8.3
41.6 --- 7.6
68.3 -+ 1.6 68.5 ± 2.1
60.9 --- 1.5 61.0 -+ 2.1
58.2 -+ 4.3 56.6 - 3.8
52.2 --- 5.3 51.1 --- 4.6
0 50.0 -+ 3.1" 67.8 -+ 2.7 68.3 -+ 2.5
0 31.3 +- 2.5* 58.3 ± 4.3 60.1 +- 5.1
0 9.9 ± 3.1" 51.3 ± 2.9 52.0 -+ 3.2
0 2.1 +-- 2.9* 31.3 --- 5.1 32.3 ± 7.1
67.1 ± 1.5
52.0 + 3.1
30.1 -+ 4.2
25.0 -+ 4.6
Data are m e a n s -+ SE. * P < 0.05; different from the control at each interval.
Effect of PAF on in Vitro Fertilization The fertilization rates, with cumulus-free and zona-free oocytes, of PAF- and CV-3988-treated spermatozoa are shown in Tables II and III. When spermatozoa were preincubated for only 30 min instead of 90 to 120 min (normal capacitation time) and exposed to cumulus-free oocytes for 20 min, a decrease in fertilization rate was observed (43.9 and 78.2%, respectively). Spermatozoa treated with PAF showed a significant increase in the fertilization rate with cumulus-free oocytes. However, a dose-dependent effect was not observed at doses of 10 - 7 , 10 - 9 , and 10 - 1 1 M (72.2, 64.8, and 62.2%, respectively). With zona-free oocytes, PAF also enhanced the fertilizing ability of spermatozoa as well as that with cumulus-free oocytes. On the other hand, inhibition of fertilization with the cumulusfree oocytes was observed with 10 -5 M CV-3988treated spermatozoa. Levels of 10 - 7 and 10 - 9 M CV-3988 showed no effect on the fertilizing ability of spermatozoa (Table III). Similarly, spermatozoa treated with 10 -5 M CV-3988 showed a significantly
lower fertilization rate with the zona-free oocytes, and 10 - 7 and 10 -9 M CV-3988 had no effect on fertilization in zona-free oocytes. The inhibition of fertilization of CV-3988 (10 -5 M) was reversed by the addition of 10 - 7 M PAF concomitantly with CV-3988. To evaluate whether the small amount of PAF and CV-3988 affected the fertilizing ability of the oocytes, cumulus-free oocytes were preincubated with PAF and CV-3988 for 15 rain and then mixed with untreated spermatozoa. Exposure of oocytes to PAF and CV-3988 did not show a significant effect on the fertilization rate. Effect of PAF on the Acrosome Reaction The effect of PAF on the acrosome reaction is shown as a function of PAF concentration in the incubation medium for the 30- and 120-min time points in Fig. 1. A low percentage (19.4%) of spermatozoa underwent the acrosome reaction during the first 30 min of incubation in control medium, but this percentage increased to 45.2 at 120 min. In contrast, a significant increase in the acrosome-reacted
Table II. In Vitro Fertilization of C u m u l u s - F r e e and Z o n a - F r e e O o c y t e s by S p e r m a t o z o a Preincubated for 30 min in Media with P A F Cumulus-free oocytes Treatment Control PAF 10 - 7 M 10 - 9 M
10-11 M
Zona-free o o c y t e s
No. of eggs
No. of fertilized eggs (%)
No. of eggs
114
50 (43.9)
73
36 (49.3)
151 108 74
109 (72.2)** 70 (64.8)* 46 (62.2)*
62 70 66
48 (77.4)** 52 (74.2)** 46 (69.7)*
* P < 0.05 c o m p a r e d with control. ** P < 0.01 c o m p a r e d with control.
Journal of Assisted Reproduction and Genetics, Vol. 9, No. 5, 1992
No. of fertilized eggs (%)
450
SENGOKU, ISHIKAWA, TAMATE, AND SHIMIZU
Table III. In Vitro Fertilization of Cumulus-Free and Zona-Free Oocytes by Spermatozoa Preincubated for 30 min in Media with CV3988 Zona-free oocytes
Cumulus-free oocytes No. of eggs
Treatment Control CV3988 10 -5 M 10-7M 10 -9 M CV3988, 10 -5 M + PAF, 10 -7 M
No. of fertilized eggs (%)
No. of eggs
No. of fertilized eggs (%)
92
44 (47.8)
73
36 (49.3)
92 77 66
30 (32.6)* 37 (48.1) 38 (53.5)
90 74 84
28 (31.1)* 32 (43.2) 47 (56.0)
78
45 (57.7)
73
44 (60.3)
* P < 0.05 compared with control.
spermatozoa was observed in PAF-treated spermatozoa compared to nontreated samples. These values increased even more by 120 min. The percentage of acrosome-reacted spermatozoa increased as the PAF concentration increased, suggesting a dose-dependent response (30.2 and 54.7% after 30 and 120 min of incubation, respectively, at 10- 11 M , 38.7 and 58.4% at 10 - 9 M, respectively; and 45.4 and 62.6% at 10 - 7 M, respectively). While 10 - 7 M CV-3988 had no effect on the acrosome reaction, 10-5 M CV-3988 inhibited acrosome loss compared with the control at 30 and 120 min of incubation (Fig. 2). This inhibition was reversed by the addition of PAF. To determine the interaction between
and PAF, the stimulatory effect of PAF on the acrosome reaction with decreasing calcium concentrations in the medium was examined. For this purpose, the 90-min time point with 10 - 7 M PAF was chosen. Decreasing Ca 2+ in the medium without PAF reduced the percentage of acrosome reaction in a dose-dependent manner from 37.2 to 0.9%. The addition of PAF to the medium enhanced acrosomal loss, even when C a 2+ concentrations were as low as 0.25 mM. However, absence of C a 2 + blocked the PAF-induced acrosome reaction at the level of 3.2% (Table IV). Ca 2+
DISCUSSION When using an in vitro fertilization system to evaluate the effects of experimental treatment on
(%) 70-
(%)
*
60.
60. tO O t~ O L
E o O
50' 50.
40.
~ 40" L
30.
O
