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Inhibition of pig oocyte in vitro fertilization by the action of components of the zona pellucida
Zheriogenology 42:227-234,1994
INHIBITION
OF PIG OOCYTE IN VITRO FERTILIZATION BY THE ACTION OF COMPONENTS OF THE ZONA PELLUCIDA
R. Fierro, E. Bonilla, E. Casas, I. Jimenez, Y. Ducolomb and M. Betancourt Departamento de Ciencias de la Salud Universidad Autonoma Metropolitana-Iztapalapa CP.09340 Mexico, D.F., Mexico Received for publication: August 27, 1993 Accepted: June 10, 1994 ABSTRACT The aim of the present study was to determine whether the previous addition of porcine zona pellucida (ZP) components to spermatozoa of the same species has an inhibitory effect on in vitro fertilization (IVF). Boar spermatozoa were exposed to whole porcine solubilized zona pellucida (SZP), ZP glycoproteins (55 kDa and 90 kDa) and peptides (37 kDa, 40 kDa and 68kDa). Doses tested were 40, 70 and 100 ug/ml. In vitro fertilization was clearly inhibited by each component when the oocytes were compared with those fertilized with untreated spermatozoa. All the components had an effect in a dose dependent manner. Key words: Zona pellucida, IVF-inhibition, oocyte, glycoproteins, boar spermatozoa INTRODUCTION The ZP is the extracellular glycoprotein structure that surrounds the mammalian oocyte. Since one of the main ZP timctions is to serve as a receptor site for spermatozoa during fertilization, several studies have been conducted to identify the responsible molecules for the sperm-oocyte interaction (8,9). The ZP of pig oocytes is basically composed of 3 glycoprotein families identifled by an apparent molecular weight as 90kDa, 55kDa and 55kDa (11). Chemical deglycosylation of these glycoproteins using trifluoromethanesulfonic acid and anisole yields 3 core peptides with Mr values of 68, 37 and 40kDa, respectively (12).
Acknowledgments: The authors wish to thank Demetrio Ambriz, Alejandro Cordova and Carmen Prado for their technical assistance; ABC Slaughterhouse, Texcoco, Mexico, for providing the porcine ovaries; and the Veterinary School of the National University of Mexico for providing the semen samples. This work was partially supported by CONACYT (Mexico) grant 1505M9207.
Copyright 8 1994 Buttetworth-Heinemann
Theriogenology
228
In mice, in vitro studies have shown that sperm pretreatment with SZP or one of its glycoproteins (ZP3) with an Mr of 83 kDa prevents binding of these spermatooza to unfertilized ova (20). These results indicate that ZP3 alone accounts for all sperm receptor activity present in the ZP of oocytes. In pigs, when the ZP or its glycoproteins are added to spermatozoa, sperm-oocyte binding is inhibited to different degrees by several components, indicating that in more than one glycoprotein family could be involved in the sperm binding (3). Noguchi and Nakano (18) and Yurewicz et al. (21) have demonstrated that sugar chains from the pig ZP3 glycoprotein 55kDa are involved in the receptor sperm-ZP mechanisms during the first step of the fertilization process. Since the above studies only analyzed binding to or sperm penetration of the ZP, our aim in the present study was to determine whether the previous addition of porcine ZP components to spermatozoa of the same species also has an inhibitory effect on in vitro fertilization (IVF). MATERIALS AND METHODS Oocyte Isolation and Purification of the ZP Components The collection of oocytes from porcine ovaries and subsequent isolation and purification of the ZP glycoproteins by preparative 2D-PAGE and electrophoretic elution has been described previously by Hedrick and Wardrip (12). Zona pellucida deglycosylation was performed with trifluoromethanesulfonic acid according to the method described by Karp et al. (16). Glycoprotein purification by preparative SDS-PAGE and electrophoretic elution from the gels was performed according to the method described by Gerton et al. (10). The purity of the components was verified upon electrophoresis, when the presence of only 1 glycoprotein or peptide was observed after silver staining. Purified components were sterilized through filtration using a 0.22~uM millipore membrane. Protein concentrations from ZP preparations were determined by the Lowry method (17). In Vitro Fertilization For the present study we used the IVF technique proposed by Bavister et al. (2) with some modifications. Oocyte Maturation Porcine oocytes were collected from 6-mo-old Yorkshire female pigs at a slaughterhouse and were transported in less than 2 h to the laboratory in 0.157 M NaCl solution at 37’C. Oocytecumulus complexes were recovered by aspiration from nonatretic follicles (3 to 6 mm diameter) with a 21-gauge needle (Becton Dickinson, Mexico City, Mexico.), rinsed twice with maturation medium (TCM- 199; Microlab, Mexico City, Mexico), supplemented with 10% heat-inactivated fetal calf serum (Microlab); 2 IU/ml FSH and LH (Serono, Mexico City, Mexico); 1 ug/ml l3estradiol (Sigma, St. Louis, MO, USA); 1 mg/ml glucose (Baker, Mexico City, Mexico); 0.25 mM sodium pyruvate (Sigma); and 50 &ml gentamycin (Scheramex, Mexico City, Mexico).
Theriogenology
Osmolarity was 280 to 290 mOsm/kg. Ten to 15 oocyte-cumulus complexes were placed in a 4wells plate (Nunc, Roskilde, Denmark) with a 60-ul drop of the maturation medium. The drops were overlaid with paraflin oil (Sigma), and oocyte cultures were incubated at 37°C for 48 h in 5% CO2 air with 100% relative humidity. Sperm Capacitation Fresh semen was obtained by ejaculation from I-yr-old boars (Hampshire or Duroc) by the gloved hand method. It was filtered through a double gauze to remove the gel particles and then kept at 2S’C until the assay was begun (approximately 1 h). Spermatozoa were incubated in TALP-HEPES medium supplemented with 1 mM sodium pyruvate, 6 mg/ml BSA fraction \ (Sigma), and 50 ug/ml gentamycin (medium osmolarity was 290 to 300 mOsm/kg), at a concentration of 8 x lo6 sperm/ml. The spermatozoa were incubated at 37’C for 3 h in 5% COz in air. Aliquots containing spermatozoa with 80% motility and rapid, linear forward movement were used for the fertilization trials. After sperm capacitation, SZP, SSkDa, 9OkDa glycoproteins, 37kDa, 40kDa and 68kDa peptides were added for 20 min at a final concentration of 40, 70 and 100 &ml In order to verify that fertilization was not inhibited by any of the reagents used during the purification of ZP components, as well as to discard unespecific inhibition, BSA was processed on SDS-PAGE electrophoresis and, after its elution, fertilization inhibition was tested using this protein in the same doses and conditions used for experiments where ZP components were tested. In Vitro Fertilization Matured oocytes with an expanded cumulus mass were placed in a 4-wells plate with a 60ul drop of fertilization medium: TALP medium supplemented with 0.25 mM sodium pyruvate, 6 mg/ml fatty acid-free BSA (Sigma) and 50 @ml gentamycin (280 to 300 mOsrn/kg,) and were then overlaid with paraRin oil and incubated at 37°C for 2 h of equilibration. The ZP componenttreated spermatozoa or untreated spermatozoa was added to the droplets containing the matured oocytes at a concentration of 5 x lo4 sperm/drop. Twenty-four hours after insemination, the oocytes were fixed in a methanol/acetic acid solution (3: 1, v/v) for 24 h at room temperature and stained with 1% acetic-orcein. Oocytes with 2 pronuclei were considered fertilized. Each ZP component was tested in triplicate.
Statistical Analysis Data were statistically analyzed by the Chi-square test. A probability of PcO.05 was considered to be significant. RESULTS Pig oocyte maturation and fertilization techniques previously established in our laboratory (6) have yielded a fertilization efficiency of 60 to 80%. A similar percentage was found in the
230
Theriogenology
control group in this study (Table 1). All sperm samples had motility rates higher than 80%. The quality of the semen samples varied considerably among donor pigs, as has been previously discussed by Berger et al. (3). The experiments were carried out in triplicate with 10 to 12 oocytes in each experiment. Table 1 shows different degrees of IVP inhibition obtained with the 55kDa, 90kDa glycoproteins, 68kDa, 4OkDa, 37kDa peptides and SZP, according to the dosage used. Statistical analysis showed a significant difference between the percentage of fertilization in samples treated with ZP components and the control group @O.OOl). There was a correlation between the concentration of the protein and the percentage of fertilization inhibition for all the ZP components (Figure 1). The SZP, 90 kDa, S&Da, 68 kDa, and 37 kDa had an r=O.9, while the 40 kDa component had an 1=0.8; The highest inhibition was shown at the lowest concentration.
Table 1, The percentage of in vitro fertilized pig oocytesa inseminated with boar spermatozoa treated with different zona pellucida components. The values are average of 3 experiments f SDb Concentration (&ml) Component
0
40
70
100
SZPC
68*8
44k8
47*12
21*7
55kDa
.53*5
3W4
1WlO
21*6
9OkDa
71k4
35k7
3til6
16+7
68kDad
68+3
37*3
3 I+5
18&S
40kDae
62+3
228
2W3
224
37kDae
66+4
38+9
26*6
12*3
al 0 to 12 oocytes were analyzed in each experiment. btil values from treated samples differ significantly from the control (P
231
Theriogenology 60
T
70
$50 2 K50 Ei w ‘1 330 L 20
10 340 0 ~ WZP
1
55kDa
9OkDa
40kDa
69kDa
I
37kDa
ZP components (glycoproteins and peptides)
n oughl
n 4O”ghl
7ougM
0
1ooughl
Figure 1 Histogram representing the mean number of fertilized oocytes with different concentrations of porcine ZP components. All components showed great inhibition compared with the paired controls (p
Table 2. Inhibition index (15O%)a for different porcine zona pellucida components Component
150% (WW.
szpb
78.5
55kDa
45.5
90kDa
60.0
68kDac
57.7
40kDad
43.7
37kDad
54.4
a1500/~represents the component quantity, necessary to inhibit IVF to 50%. bSZF? Whole solubilized zona pellucida. C68kDa peptide is produced by deglycosylation of the 90kDa glycoprotein. QO and 37kDa peptides are produced by deglycosylation of the 55kDal3 and 55kDaa glycoproteins, respectively.
232
Theriogenology
When pig spermatozoa were incubated with electrophoresis-resolved and eluted BSA, using the same doses and conditions tested for the ZP component experiments, IVF was not inhibited (control = 75%, 4Oug = 83.3%, 7Oug = 73.6%, 1OOug= 93.3%). To compare the efficiency of different ZP components, the inhibition index (I 50%) was obtained, which represents the component quantity necessary to inhibit IVF to 50% (13). Table 2 shows that in order to obtain this percentage of inhibition, 78.5ug of SZP, 45.5ug of 55kDa and 60 pg of 90kDa were required; in regard to the peptides, 54.4ug of 37kDa, 43.7ug of 40 kDa and 57.7 ug of 68kDa were needed. It is evident that each of the components was more effective for inhibiting fertilization than the SZP (P
233
Theriogenology
These data differ from those of Berger et al. (3) who studied the inhibition of sperm binding to pig ZP by solubilized components of the ZP. They found that only the whole zona and glycoproteins had an inhibitory effect at a 10 to 15ug, dose, whereas peptides did not have an inhibitory effect at a dose of 5 to 15 pg. We observed that the SZP inhibits they are taken separately (Table 2). The inhibitory effect than the 90K glycoprotein inhibition, even at the lowest concentration
fertilization less efficiently than its components when 55 kDa glycoprotein and its peptides showed higher and its peptide. The 40 kDa peptide showed maximum (40 &ml).
According to the results obtained in the present study, the in vitro interaction of ZP components and spermatozoa greatly inhibits IVF. This inhibition may be due to the ZP component binding to the sperm membrane, thus blocking the sperm receptor site for the ZP However, since all the ZP components inhibited IVF (Table l), sperm-binding blockage is likely due to a steric hindrance rather than to a specific interaction between the ZP and sperm receptors. Finally, IVF inhibition could also be due to the induction of acrosomal reaction. When spermatozoa are coincubated with certain ZP components, such a reaction is partially induced, 14% with the 55kDa component and 9% with 90 kDa (4). Since IVF inhibition by ZP components can be explained by specific interactions with the sperm-receptor, by steric hindrance or by acrosomal reaction, further research is needed. REFERENCES 1 Bamezai AK, Mahi-Brown CA, Talwar, GP. Inhibition of penetration of canine zonae pelhmidae by homologous spermatozoa in vitro using monoclonal antibodies raised against porcine zonae. J Reprod Biol 1988;14:177-189. 2 Bavister BD, Leibfried ML, Lieberman G. Development of preimplantation embryos of the golden hamster in a defined culture medium. Biol Reprod 1983;28:235-247. 3. Berger T, Davis A, Wardrip NJ, Hedrick JL. Sperm binding to the pig zona pellucida and inhibition of binding by solubilized components of the zona pellucida. J Reprod Fertil 1989;86:559-565. 4 Berger T, Turner KO, Meizel S, Hedrick JL Zona Pellucida induced acrosome reaction in boar sperm Biol Reprod 1989;40:525-530 5. Betancourt M, Bonilla E, Casas E, Fierro R, Serrano, H. Especificidad de anticuerpos policlonales en contra de peptidos de la zona pelhcida de ovocitos de ccrdo Mem Acad Invest Biol Rep (Mexico) 1991;16.84-101 6 Betancourt, M, Fierro, R, Ambriz, D. In vitro fertilization of pig oocytes matured in vitro Theriogenol 1993;40:1155-1160. 7. Cahova M, Draber P. Inhibition of fertilization by a monoclonal antibody recognizing the oligosacchar-ide sequence GalNAcB1+4GalB1-+4 on the mouse zona pellucida J Reprod Immunol 1992,21:241-256 8. Dunbar B, Prasad S, Timmons T. Comparative structure and function of the mammalian zonae pellucidae In. Dunbar B, O’Rand M (eds), A Comparative Overview of hlammalian Fertilization. Xew York, Plcr:ur,~ Press, 1991, 97-116 9 Florman, H M and Wassarman, P.M. O-linked oligosaccharides of mouse egg ZP3 account for its sperr-1 receptor activity Cell 1935;41:313-324
234
Theriogenology
10. Gerton GL, Wardrip NJ, Hedrick JL A gel eluter for recovery of proteins separated by polyacrylamide gel electrophoresis. Anal Biochem 1982; 126: 116-12 1. 11.Hedrick JL, Wardrip NJ. Isolation of the zona pellucida and purification of its glycoprotein families from pig oocytes. Anal Biochem 1986;157:63-70. 12. Hedrick JL, Wardrip NJ. On the macromolecular composition of the zona pellucida from porcine oocytes. DevBiol 1987,121:478-488. 13. Hedrick JL The pig zona pellucida: Sperm binding ligands, antigens, and sequence homologies. In: Dondero F, Jhonson PM, (eds), Reproductive Immunology. New York, Serono Symposio Publishers, Raven Press, 1993; 59-65. 14. Henderson CJ, Hulme MJ, Aitken RJ. Contraceptive potential of antibodies to zona pellucida. J Reprod Fertil 1988;83:325-343. antibodies to porcine zona 15. Isojima S, Koyama K, Hasegawa A, Tsunoda Y, Hanada A. Monoclonal pellucida antigens and their inhibitory effects on fertilization. J Reprod Immunol 1984;6:77-87. 16. Karp DR, Atkinson JP, Schreffler DC. Genetic variation in glycosilation of the fourth component murine complement. J Biol Chem 1982;257:7330-7335. 17. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ Protein measurement with the folin phenol reagent. J Biol Chem 1951;193:265. 18. Noguchi S, Nakano, M Structure of the acidic N-linked carbohydrate chains of the 55-kDa glycoprotein family (PZP3) from porcine zona pellucida. Eur J Biochem 1992;209:883-894. 19. Paterson M, Koothan PT, Morris KD, O’byme KT, Braude P, Williams A, Aitken RJ. Analysis of the contraceptive potential of antibodies against native and deglycosylated porcine ZP3 in vivo and in vitro. Biol Reprod 1992;46:523-534. 20. Wassarman PM, Bleil JD, Florman, HM, Greve JM, Roller RJ, Salzmann GS, Samuels FG. The mouse egg’s receptor for sperm: What is it and how does it work? Cold Spring Harbor Symposium Quant Biol 1985;50:11-19. 21. Yurewicz EC, Pack BA, Sacco AG. Isolation, composition, and biological activity of sugar chains of porcine oocyte zona pellucida 55K glycoproteins. Mol Rep Devel 1991;30:126-134.
INHIBITION
OF PIG OOCYTE IN VITRO FERTILIZATION BY THE ACTION OF COMPONENTS OF THE ZONA PELLUCIDA
R. Fierro, E. Bonilla, E. Casas, I. Jimenez, Y. Ducolomb and M. Betancourt Departamento de Ciencias de la Salud Universidad Autonoma Metropolitana-Iztapalapa CP.09340 Mexico, D.F., Mexico Received for publication: August 27, 1993 Accepted: June 10, 1994 ABSTRACT The aim of the present study was to determine whether the previous addition of porcine zona pellucida (ZP) components to spermatozoa of the same species has an inhibitory effect on in vitro fertilization (IVF). Boar spermatozoa were exposed to whole porcine solubilized zona pellucida (SZP), ZP glycoproteins (55 kDa and 90 kDa) and peptides (37 kDa, 40 kDa and 68kDa). Doses tested were 40, 70 and 100 ug/ml. In vitro fertilization was clearly inhibited by each component when the oocytes were compared with those fertilized with untreated spermatozoa. All the components had an effect in a dose dependent manner. Key words: Zona pellucida, IVF-inhibition, oocyte, glycoproteins, boar spermatozoa INTRODUCTION The ZP is the extracellular glycoprotein structure that surrounds the mammalian oocyte. Since one of the main ZP timctions is to serve as a receptor site for spermatozoa during fertilization, several studies have been conducted to identify the responsible molecules for the sperm-oocyte interaction (8,9). The ZP of pig oocytes is basically composed of 3 glycoprotein families identifled by an apparent molecular weight as 90kDa, 55kDa and 55kDa (11). Chemical deglycosylation of these glycoproteins using trifluoromethanesulfonic acid and anisole yields 3 core peptides with Mr values of 68, 37 and 40kDa, respectively (12).
Acknowledgments: The authors wish to thank Demetrio Ambriz, Alejandro Cordova and Carmen Prado for their technical assistance; ABC Slaughterhouse, Texcoco, Mexico, for providing the porcine ovaries; and the Veterinary School of the National University of Mexico for providing the semen samples. This work was partially supported by CONACYT (Mexico) grant 1505M9207.
Copyright 8 1994 Buttetworth-Heinemann
Theriogenology
228
In mice, in vitro studies have shown that sperm pretreatment with SZP or one of its glycoproteins (ZP3) with an Mr of 83 kDa prevents binding of these spermatooza to unfertilized ova (20). These results indicate that ZP3 alone accounts for all sperm receptor activity present in the ZP of oocytes. In pigs, when the ZP or its glycoproteins are added to spermatozoa, sperm-oocyte binding is inhibited to different degrees by several components, indicating that in more than one glycoprotein family could be involved in the sperm binding (3). Noguchi and Nakano (18) and Yurewicz et al. (21) have demonstrated that sugar chains from the pig ZP3 glycoprotein 55kDa are involved in the receptor sperm-ZP mechanisms during the first step of the fertilization process. Since the above studies only analyzed binding to or sperm penetration of the ZP, our aim in the present study was to determine whether the previous addition of porcine ZP components to spermatozoa of the same species also has an inhibitory effect on in vitro fertilization (IVF). MATERIALS AND METHODS Oocyte Isolation and Purification of the ZP Components The collection of oocytes from porcine ovaries and subsequent isolation and purification of the ZP glycoproteins by preparative 2D-PAGE and electrophoretic elution has been described previously by Hedrick and Wardrip (12). Zona pellucida deglycosylation was performed with trifluoromethanesulfonic acid according to the method described by Karp et al. (16). Glycoprotein purification by preparative SDS-PAGE and electrophoretic elution from the gels was performed according to the method described by Gerton et al. (10). The purity of the components was verified upon electrophoresis, when the presence of only 1 glycoprotein or peptide was observed after silver staining. Purified components were sterilized through filtration using a 0.22~uM millipore membrane. Protein concentrations from ZP preparations were determined by the Lowry method (17). In Vitro Fertilization For the present study we used the IVF technique proposed by Bavister et al. (2) with some modifications. Oocyte Maturation Porcine oocytes were collected from 6-mo-old Yorkshire female pigs at a slaughterhouse and were transported in less than 2 h to the laboratory in 0.157 M NaCl solution at 37’C. Oocytecumulus complexes were recovered by aspiration from nonatretic follicles (3 to 6 mm diameter) with a 21-gauge needle (Becton Dickinson, Mexico City, Mexico.), rinsed twice with maturation medium (TCM- 199; Microlab, Mexico City, Mexico), supplemented with 10% heat-inactivated fetal calf serum (Microlab); 2 IU/ml FSH and LH (Serono, Mexico City, Mexico); 1 ug/ml l3estradiol (Sigma, St. Louis, MO, USA); 1 mg/ml glucose (Baker, Mexico City, Mexico); 0.25 mM sodium pyruvate (Sigma); and 50 &ml gentamycin (Scheramex, Mexico City, Mexico).
Theriogenology
Osmolarity was 280 to 290 mOsm/kg. Ten to 15 oocyte-cumulus complexes were placed in a 4wells plate (Nunc, Roskilde, Denmark) with a 60-ul drop of the maturation medium. The drops were overlaid with paraflin oil (Sigma), and oocyte cultures were incubated at 37°C for 48 h in 5% CO2 air with 100% relative humidity. Sperm Capacitation Fresh semen was obtained by ejaculation from I-yr-old boars (Hampshire or Duroc) by the gloved hand method. It was filtered through a double gauze to remove the gel particles and then kept at 2S’C until the assay was begun (approximately 1 h). Spermatozoa were incubated in TALP-HEPES medium supplemented with 1 mM sodium pyruvate, 6 mg/ml BSA fraction \ (Sigma), and 50 ug/ml gentamycin (medium osmolarity was 290 to 300 mOsm/kg), at a concentration of 8 x lo6 sperm/ml. The spermatozoa were incubated at 37’C for 3 h in 5% COz in air. Aliquots containing spermatozoa with 80% motility and rapid, linear forward movement were used for the fertilization trials. After sperm capacitation, SZP, SSkDa, 9OkDa glycoproteins, 37kDa, 40kDa and 68kDa peptides were added for 20 min at a final concentration of 40, 70 and 100 &ml In order to verify that fertilization was not inhibited by any of the reagents used during the purification of ZP components, as well as to discard unespecific inhibition, BSA was processed on SDS-PAGE electrophoresis and, after its elution, fertilization inhibition was tested using this protein in the same doses and conditions used for experiments where ZP components were tested. In Vitro Fertilization Matured oocytes with an expanded cumulus mass were placed in a 4-wells plate with a 60ul drop of fertilization medium: TALP medium supplemented with 0.25 mM sodium pyruvate, 6 mg/ml fatty acid-free BSA (Sigma) and 50 @ml gentamycin (280 to 300 mOsrn/kg,) and were then overlaid with paraRin oil and incubated at 37°C for 2 h of equilibration. The ZP componenttreated spermatozoa or untreated spermatozoa was added to the droplets containing the matured oocytes at a concentration of 5 x lo4 sperm/drop. Twenty-four hours after insemination, the oocytes were fixed in a methanol/acetic acid solution (3: 1, v/v) for 24 h at room temperature and stained with 1% acetic-orcein. Oocytes with 2 pronuclei were considered fertilized. Each ZP component was tested in triplicate.
Statistical Analysis Data were statistically analyzed by the Chi-square test. A probability of PcO.05 was considered to be significant. RESULTS Pig oocyte maturation and fertilization techniques previously established in our laboratory (6) have yielded a fertilization efficiency of 60 to 80%. A similar percentage was found in the
230
Theriogenology
control group in this study (Table 1). All sperm samples had motility rates higher than 80%. The quality of the semen samples varied considerably among donor pigs, as has been previously discussed by Berger et al. (3). The experiments were carried out in triplicate with 10 to 12 oocytes in each experiment. Table 1 shows different degrees of IVP inhibition obtained with the 55kDa, 90kDa glycoproteins, 68kDa, 4OkDa, 37kDa peptides and SZP, according to the dosage used. Statistical analysis showed a significant difference between the percentage of fertilization in samples treated with ZP components and the control group @O.OOl). There was a correlation between the concentration of the protein and the percentage of fertilization inhibition for all the ZP components (Figure 1). The SZP, 90 kDa, S&Da, 68 kDa, and 37 kDa had an r=O.9, while the 40 kDa component had an 1=0.8; The highest inhibition was shown at the lowest concentration.
Table 1, The percentage of in vitro fertilized pig oocytesa inseminated with boar spermatozoa treated with different zona pellucida components. The values are average of 3 experiments f SDb Concentration (&ml) Component
0
40
70
100
SZPC
68*8
44k8
47*12
21*7
55kDa
.53*5
3W4
1WlO
21*6
9OkDa
71k4
35k7
3til6
16+7
68kDad
68+3
37*3
3 I+5
18&S
40kDae
62+3
228
2W3
224
37kDae
66+4
38+9
26*6
12*3
al 0 to 12 oocytes were analyzed in each experiment. btil values from treated samples differ significantly from the control (P
231
Theriogenology 60
T
70
$50 2 K50 Ei w ‘1 330 L 20
10 340 0 ~ WZP
1
55kDa
9OkDa
40kDa
69kDa
I
37kDa
ZP components (glycoproteins and peptides)
n oughl
n 4O”ghl
7ougM
0
1ooughl
Figure 1 Histogram representing the mean number of fertilized oocytes with different concentrations of porcine ZP components. All components showed great inhibition compared with the paired controls (p
Table 2. Inhibition index (15O%)a for different porcine zona pellucida components Component
150% (WW.
szpb
78.5
55kDa
45.5
90kDa
60.0
68kDac
57.7
40kDad
43.7
37kDad
54.4
a1500/~represents the component quantity, necessary to inhibit IVF to 50%. bSZF? Whole solubilized zona pellucida. C68kDa peptide is produced by deglycosylation of the 90kDa glycoprotein. QO and 37kDa peptides are produced by deglycosylation of the 55kDal3 and 55kDaa glycoproteins, respectively.
232
Theriogenology
When pig spermatozoa were incubated with electrophoresis-resolved and eluted BSA, using the same doses and conditions tested for the ZP component experiments, IVF was not inhibited (control = 75%, 4Oug = 83.3%, 7Oug = 73.6%, 1OOug= 93.3%). To compare the efficiency of different ZP components, the inhibition index (I 50%) was obtained, which represents the component quantity necessary to inhibit IVF to 50% (13). Table 2 shows that in order to obtain this percentage of inhibition, 78.5ug of SZP, 45.5ug of 55kDa and 60 pg of 90kDa were required; in regard to the peptides, 54.4ug of 37kDa, 43.7ug of 40 kDa and 57.7 ug of 68kDa were needed. It is evident that each of the components was more effective for inhibiting fertilization than the SZP (P
233
Theriogenology
These data differ from those of Berger et al. (3) who studied the inhibition of sperm binding to pig ZP by solubilized components of the ZP. They found that only the whole zona and glycoproteins had an inhibitory effect at a 10 to 15ug, dose, whereas peptides did not have an inhibitory effect at a dose of 5 to 15 pg. We observed that the SZP inhibits they are taken separately (Table 2). The inhibitory effect than the 90K glycoprotein inhibition, even at the lowest concentration
fertilization less efficiently than its components when 55 kDa glycoprotein and its peptides showed higher and its peptide. The 40 kDa peptide showed maximum (40 &ml).
According to the results obtained in the present study, the in vitro interaction of ZP components and spermatozoa greatly inhibits IVF. This inhibition may be due to the ZP component binding to the sperm membrane, thus blocking the sperm receptor site for the ZP However, since all the ZP components inhibited IVF (Table l), sperm-binding blockage is likely due to a steric hindrance rather than to a specific interaction between the ZP and sperm receptors. Finally, IVF inhibition could also be due to the induction of acrosomal reaction. When spermatozoa are coincubated with certain ZP components, such a reaction is partially induced, 14% with the 55kDa component and 9% with 90 kDa (4). Since IVF inhibition by ZP components can be explained by specific interactions with the sperm-receptor, by steric hindrance or by acrosomal reaction, further research is needed. REFERENCES 1 Bamezai AK, Mahi-Brown CA, Talwar, GP. Inhibition of penetration of canine zonae pelhmidae by homologous spermatozoa in vitro using monoclonal antibodies raised against porcine zonae. J Reprod Biol 1988;14:177-189. 2 Bavister BD, Leibfried ML, Lieberman G. Development of preimplantation embryos of the golden hamster in a defined culture medium. Biol Reprod 1983;28:235-247. 3. Berger T, Davis A, Wardrip NJ, Hedrick JL. Sperm binding to the pig zona pellucida and inhibition of binding by solubilized components of the zona pellucida. J Reprod Fertil 1989;86:559-565. 4 Berger T, Turner KO, Meizel S, Hedrick JL Zona Pellucida induced acrosome reaction in boar sperm Biol Reprod 1989;40:525-530 5. Betancourt M, Bonilla E, Casas E, Fierro R, Serrano, H. Especificidad de anticuerpos policlonales en contra de peptidos de la zona pelhcida de ovocitos de ccrdo Mem Acad Invest Biol Rep (Mexico) 1991;16.84-101 6 Betancourt, M, Fierro, R, Ambriz, D. In vitro fertilization of pig oocytes matured in vitro Theriogenol 1993;40:1155-1160. 7. Cahova M, Draber P. Inhibition of fertilization by a monoclonal antibody recognizing the oligosacchar-ide sequence GalNAcB1+4GalB1-+4 on the mouse zona pellucida J Reprod Immunol 1992,21:241-256 8. Dunbar B, Prasad S, Timmons T. Comparative structure and function of the mammalian zonae pellucidae In. Dunbar B, O’Rand M (eds), A Comparative Overview of hlammalian Fertilization. Xew York, Plcr:ur,~ Press, 1991, 97-116 9 Florman, H M and Wassarman, P.M. O-linked oligosaccharides of mouse egg ZP3 account for its sperr-1 receptor activity Cell 1935;41:313-324
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Theriogenology
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