ANDROLOGIA
23, 209-2 12 ( 199 1 )
ACCEPTED: APRIL12, 1991
The hemizona assay (HZA) as an experimental model to evaluate the inhibition of sperm binding to the murine zona pellucida by isolated zona pellucida protein M.-L. Windt, D. R. Franken, P. M. de Beer, P. J. D. Bouic and T . F. Kruger Key words. Zona pellucida
-
hemizona assay
~
sperm receptor
Summary. Compelling evidence has demonstrated that zona binding represents gamete recognition by sperm binding with high affinity and specificity to complex glycoproteins of the zona pellucida. In the present study we evaluated the hemizona assay (HZA) in the investigation of the interaction of mouse spermatozoa with unfertilized murine oocytes and hemizonae after exposure to solubilized murine zonae pellucidae proteins. The zonae pellucidae were isolated from ovarian tissue following described mincing techniques. The sperm binding characteristics of murine spermatozoa were studied by using SDS-PAGE isolated ZP2 ( +_ 120 Kd) and ZP3 ( 83 Kd) components of the zona pellucida. Sperm receptor activity was examined in a competitive gamete binding fashion using the HZA as an indicator of sperm/zona interaction. The results illustrated that isolated, solubilized ZP2 and ZP3 glycoprotein moieties of the zona pellucida inhibited sperm binding to hemizonae and oocytes when compared to controls, and that the HZA can be utilized as an internally controlled homologous bioassay to evaluate the effects of zona pellucida proteins on tight binding of spermatozoa to the zona pellucida.
~
oocyte binding
-
mousc.
Introduction The zona pellhcida is an acellular glycoprotein coat surrounding the plasma membrane of the fully grown oocyte and is synthesized and secreted by growing oocytes (Wassarman, 1988). In mice the glycoprotein consists of dimeric filaments of ZP2 and ZP3, crosslinked to one another by ZPI (Wassarman, 1988). It has been shown that murine ZP3 (Mr = 83 Kd) serves as the species-specificsperm receptor and also induces the acrosome reaction (Bleil & Wassarman, 1983). ZP2 (Mr = 120 Kd) facilitates secondary binding of the sperm to the zona where it plays a role in the maintenance of binding of acrosome reacted sperm (Bleil et al., 1988). Isolated and purified zona pellucida proteins can be very useful in the study of the regulation of fertilization, i.e. sperm-oocyte binding, acrosome reaction and polyspermy. Previous studies showed that ZP3 from unfertilized oocytes inhibited the binding of spermatozoa to oocytes, thereby proving that ZP3 is the receptor protein for mouse sperm (Bleil & Wassarman, 1980). In this ongoing study our purpose was to investigate the use of the HZA (Burkman et al., 1988) as an experimental model to study the effect of electrophoretically separated zona proteins on mouse sperm-oocyte binding (Bleil & Wassarman, 1980).
Materials and methods Isolation o f zonae pellucidae Reproductive Biology Unit, Ilept. of Obstetrics and Gynaecology, Dept. Internal Medicine, University of Stellenbosch, Immunology Section, Dept. Medical Microbiology, Tygerberg Hospital, Tygerberg, Republic of South Africa. Correspondence: M.-L. Windt, Department of Obstetrics and Gynaecology, Tygerberg Hospital, 3rd Floor, Tygerberg 7505, Republic of South Africa.
Ovaries were obtained from Balb/c sacrificed female mice. Eighty ovaries were processed at a time. The ovaries were cleaned of all excess fat tissue and masserated. A Dounce homogenizer was used to homogenize the ovarian tissue thereby releasing the zonae pellucidae. Homogenization was performed
210
M.-L. WINDTET
AL.
on ice in 1% Triton in PBS (Moller & Wassarman, 1989). The homogenized sample was then washed thoroughly (4 x ) by centrifugation (22 000 g, 5 min) with 0.1 yo polyvinylpyrrolidone (PVP) in PBS at 4°C. The washed pellet was resuspended in 0.1 yo PVP + PBS and zona pellucida fragments were removed microscopically with a mouth-operated micropipette. Zonae pellucidae were then centrifuged for 20 minutes in a microfuge (22 000 9). Supernatants were removed and pelleted zonae solubilized in 5 mM Na,HPO, (pH 2.5) (Florman & Wassarman, 1985; Moller & Wassarman, 1989). Solubilized zonae protein from 228 and 170 ovaries were finally electrophoresed for the first and second HZA respectively. In the experiment to test the purity of the ZP2 fraction, zona pellucida protein from 320 ovaries were electrophoresed.
Electrophoresis of zona pellucida protein SDS-polyacrylamide gel electrophoresis of solubilized zona pellucida proteins was carried out under non-reducing conditions according to the method of Laemmli (1970) using a 10% separating gel. A section of the gel containing molecular weight markers and solubilized zona pellucida protein was stained with silver stain (Oakley et al., 1980) to identify the zona proteins ZP2 (Mr = 120 Kd) and ZP3 (Mr = 83 Kd). Each corresponding protein band was excised from the remaining, unstained gel and electroeluted separately in SDS electrophoresis buffer at 200 V for 15 hours. Electroeluted materials were dialyzed extensively (12 h) against 7 M urea, followed by extensive dialysis (24 h) against several changes of distilled water and finally lyophilized (Florman & Wassarman, 1985). Lyophilized zona protein fractions were stored at - 20°C until further use. A gel slice without any protein was subjected to the same procedures and was used as a control in competition studies (control 1 ) . In the experiment to test for ZP2 purity, the electroeluted ZP2 fraction was dialyzed, divided into two equal volumes and lyophilized separately. After reconstitution, the two fractions were electrophoresed under reducing (5%, 2-mercaptoethanol) and non-reducing conditions as described before.
Collection and culture of mouse gametes Superovulated female Balb/c mice were sacrificed and cumulus-enclosed oocytes removed from the oviducts. Cumulus was removed by treatment with hyaluronidase (0.1yo)and oocytes washed in Ham's F10 medium and kept at 37"C, 5% CO, in air. Mouse sperm were collected in Ham's F10 + 32 mg ml-I BSA (bovine serum albumin) from minced caudae epididymae of male Balb/c mice, allowing spermatozoa to swim free into the medium for 30
minutes at 37"C, 5% CO, in air. Supernatants containing motile spermatozoa were removed, counted and diluted to 0.5 x lo6 spermatozoa/ml in 32 mg BSA/ml Ham's F10.
Hemizona-competition assay Lyophilized samples (ZP2, ZP3, control 1 ) were reconstituted in 100 p1 Ham's F10 + 32 mg BSA/ml. 80 p1 of the reconstituted protein were incubated with 80 pl of mice spermatozoa (0.5 x 106/ml)for 60 minutes at room temperature. A second control (control 2) where spermatozoa were subjected only to reconstituting medium was also included. Washed, superovulated oocytes were microbisected into equal hemizonae for use in the hemizona assay as described previously (Franken et al., 1989). Hemizonae were placed into 20 pl drops of the incubated sperm combinations to determine any inhibition of sperm-oocyte binding caused by zona proteins ZP3 and ZP2. The assay was repeated twice with different zona protein preparations. Combinations examined were (in duplicate) : Control 1 vs control 2; control 2 vs ZP2; control 2 vs ZP3. Whole eggs were also included in all drops. Drops were covered with liquid parafin and incubated for 4 hours at 37". Zonae and eggs were removed, rinsed in Ham's F 10 and tightly bound spermatozoa were counted under an inverted microscope (Burkman et al., 1988; Franken et al., 1989).
Results The results of the inhibition studies are presented in Table 1 and Figures 1 and 2.
Hemizonae binding As can be seen from Table 1 and Figure 1, both control 1 and 2 had no influence on the binding of murine spermatozoa to hemizonae. An average of 33.3 ( & 1.8) tightly bound spermatozoa per hemizona was counted. There was, however, a marked
Table 1. The effect of solubilized, isolated murine zona pellucida proteins on the binding of spermatozoa (mean & SD) to murine hemizonae and oocytes Hemizonal binding HZ1 Control 1 , 33 ( + 13) Control 2 29 ( + 16) Control 2 29 ( + 16)
HZ2 Control 2 29 ( + 16) ZP3 2 ( + 2) ZP2 2 ( + 1)
Oocyte binding 1 Control 1 27 ( k 15) Control 2 28 ( + 11) Control 2 28 ( + 11)
2 Control 2 28 ( + 11) ZP3 2 ( + 2) ZP2 2 ( + 2)
ANDROLOGIA 23, 209-212 (1991)
HEMIZONA ASSAY
MEAN NUMBER OF SPERM BOUND/HZ
40
L J 29
30
30
20
10
0
E 3 CONTROL
1
CONTROL 2
0ZP 2
mZP 3
Figure 1. Histograms of mean sperm binding to murine hemizonae after sperm pre-incubation with Ham’s FIO + BSA (control 2), a SDS gel strip (control I ) , ZP2 and ZP3. Control 1, ZP2 and ZP3 were electrophoresed, electroeluted, dialized and lyophilized.
MEAN NUMBER OF SPERM BOUND/OOCYTE
40
of ZP2. However, no bands corresponding to 83 Kd molecular weight could be demonstrated. When the same preparation of ZP2 was electrophoresed under reducing conditions, no new band at 83 Kd could be detected thereby indicating the presence of either ZP3 contamination or ZP2-ZP3 complexes in the original ZP2 preparation.
Discussion In previous studies it was reported that pre-treatment of murine spermatozoa with solubilized murine zona pellucida prevented the binding of these spermatozoa to unfertilized eggs (Bleil & Wassarman, 1980; Florman et al., 1984). Furthermore, when the individual, purified zona pellucida glycoproteins, ZP3, ZP2 and ZP1 were tested in analogous experiments, it was shown that only ZP3 prevented sperm binding; ZP2 and ZP1 had no effect (Bleil & Wassarman, 1980, 1986). It was concluded from other studies that ZP3 functions as the murine receptor for spermatozoa and also that it induces the acrosome reaction following binding to spermatozoa with intact acrosomes (Bleil & Wassarman, 1980, 1983, 1986; Florman & Wassarman, 1985; Wassarman, 1987, 1988). O n the other hand, ZP2 was shown to bind to the inner acrosomal membrane of acrosome reacted spermatozoa (Bleil & Wassarman, 1986) acting as a secondary sperm receptor following completion of the acrosome reaction (Bleil et al., 1988). Furthermore it appears that the receptor activity of ZP3 is destroyed following fertilization (Bleil & Wassarman, 1980) and that ZP2 also undergoes modification thus probably playing a role in the block to polyspermy (Bleil et al., 1981). Since the development of the hemizona assay (HZA) by Burkman et al., (1988) it has been used in several controlled studies, mainly to test fertilization potential of human spermatozoa (Franken et al., 1989). We utilized the HZA to investigate the inhibition potential of electrophoresed, electroeluted, dialysed, lyophilized murine zona pellucida glycoproteins. Although microbisecting of mouse oocytes proved to be more difficult than that of human oocytes (mouse oocytes are smaller and zone pellucidae thinner and more fragile) it was still possible. We included intact oocytes to guarantee results in case of lost zonae. The inhibition of sperm binding to hemizonae of spermatozoa pre-incubated with the isolated ZP3 fraction, was in agreement with findings of previous investigators (Bleil & Wasserman, 1980; Florman et al., 1984). Contrary to other studies though (Bleil & Wassarman, 1980, 1986), binding of the ZP2 treated spermatozoa was also inhibited.
flI
30
20
10
0
=
E 3 Control I E Z Control 2 0ZP 2 ZP 3 Figure 2. Histograms of mean sperm binding to murine oocytes after sperm pre-incubation with Ham’s F10 + BSA (control Z), a SDS gel strip (control l ) , ZP2 and ZP3, Control 1, ZP2 and ZP3 were electrophoresed, electroeluted, dialized and lyophilized.
decrease in binding when spermatozoa were preincubated in either ZP2 or ZP3, when compared to control 2 (93% decrease for both ZP2 and ZP3). Oocyte binding Results for oocyte binding (Fig. 2, Table 1) were similar to that of hemizona binding. Spermatozoa incubated with both control 1 and 2 showed an average binding of 27.7 (k 0.58) tightly bound spermatozoa per oocyte. A decrease in binding was shown for ZP2 and ZP3 incubated spermatozoa (93% and 89%, respectively) when compared to control 2. Purity of ZP2 fraction Electrophoresis of the electroeluted ZP2 fraction under non-reducing conditions revealed a diffuse band at approximately 120 Kd, confirming the presence ANDROLOGIA 23, 209-212 (1991)
211
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AL.
Purified ZP2 was shown to contain as much as 3% ZP3 as contaminant (Bleil et al., 1988) and since nanomolar concentrations of ZP3 can prevent spermatozoa from binding (Wassarman, 1987), we assumed that contamination of our ZP2 fraction by ZP3 could be responsible for this inhibition. Also, it appears that ZP2 and ZP3 can form dimers of 180 Kd (Wassarman, 1988). Based on this observation, we speculated that our preparation of ZP2 could contain a small amount of these dimers and that the inhibition observed with the ZP2 preparation could be explained in terms of the presence of ZP2-ZP3 dimers. Furthermore, it was reported in an autoradiographic study by Bleil & Wassarman (1986) that ZP2 can bind to some extent to acrosome intact spermatozoa. These authors suggested that ZP2 has a specific, but low affinity for the plasma membrane of acrosome intact spermatozoa. In another study by the same authors (Bleil & Wassarman, 1983), it was shown that ZP2 could also induce the acrosome reaction to some extent. The proposed contamination of the ZP2 fraction by ZP3 in our study could have contributed to the induction of the acrosome reaction, resulting in the inhibition of sperm binding. However, the integrity of the acrosome was not investigated in this study. In order to rule out the problem of ZP2 contamination by either ZP3 or ZP2-ZP3 dimers, the isolated ZP2 fraction was again electrophoresed under reducing and non-reducing conditions. The diffuse band at 120 Kd confirmed the presence of ZP2 but no ZP3 could be shown at 83 Kd. Although zona material from 160 ovaries (for reduced and nonreduced, respectively) was electrophoresed, the concentration of ZP3 in this sample was probably too low to detect with the silver stain method. Possible contamination by SDS, urea or polyacrylamide thereby causing inhibition was ruled out by the inclusion of control 2 (a SDS gel strip subjected to procedures similar to that of the glycoprotein containing fractions). We therefore conclude that the HZA can be used in murine studies, and that receptor activity of isolated murine ZP3 was indicated in our study. The expertise and information gained in the current murine study will be used in future studies on porcine and human zona pellucida glycoproteins, hopefully helping us to gain more information on human infertility and contraception.
Acknowledgements The authors wish to thank the Medical Superintendent of Tygerberg Hospital for permission to publish as well as the Medical Research Council of South Africa for financial support.
References Bleil JD, Wassarman PM (1980) Mammalian sperm-egg interaction: identification of a glycoprotein in mouse egg zonae pellucidae possessing receptor activity for sperm. Cell 202373882 Bleil JD, Wassarman PM (1983) Sperm-egg interactions in the mouse: sequence of events and induction of the acrosome reaction by a zona pellucida glycoprotein. Dev Biol 95:317-324 Bleil JD, Wassarman PM ( 1986) Autoradiographic visualization of the mouse egg’s sperm receptor bound to sperm. J Cell Biol 102:1363-137 1 Bleil JD, Beall CF, Wassarman PM (1981) Mammalian spermegg interaction: fertilization of mouse eggs triggers modification of the major zona pellucida glycoprotein, ZP2. Dev Biol 86: 189- 197 Bleil JD, Greve JM, Wassarman PM (1988) Identification of a secondary sperm receptor in the mouse egg zona pellucida: role in maintenance of binding of acrosome reacted sperm 10 eggs. Dev Biol 128:376-385 Burkman LJ, Coddington CC, Franken DR, Kruger TF, Rosenwaks Z, Hodgen G D (1988) The hemizona assay (HZA): development of a diagnostic test for the binding of human spermatozoa to the human hemizona pellucida to predict fertilization potential. Fertil Steril 49:688-697 Florman H M , Wassarman PM ( 1985) 0-linked oligosaccharides of mouse egg ZP3 account for its sperm receptor activity. Cell 41:313-324 Florman HM, Bechtol KB, Wassarman PM (1984) Enzymatic dissection of the functions of the mouse egg’s receptor for sperm. Dev Biol 106:243-255 Franken DR, Burkman LJ, Oehninger SC, Coddington CC, Veeck I,L, Kruger TF, Rosenwaks Z, Hodgen G D (1989) Hemizona assay using salt-stored human oocytes: evaluation of zona pellucida capacity for binding human spermatozoa. Gamete Res 22:15-22 Laemmli U K (1970) Cleavage of structural proteins during the assembly of the head of the bacteriophage T4. Nature 227:68&685 Moller CC, Wassarman PM (1989) Characterization of a proteinase that clcaves zona pellucida glycoprotein ZP2 following activation of mouse eggs. Dev Biol 132:103- 112 Oakley BR, Kirsch DR, Morris NR (1980) A simplified ultrasensitive silver stain for detecting proteins in polyacrylamide gels. Analyt Biochem 105:361-363 Wassarman PM (1987) The biology and chemistry of fcrtilization. Science 235:553-560 Wassarman PM (1988) Zona pellucida glycoproteins. Ann Rev Biochem 57:415-442
ANDROLOGIA 23, 209-2 12 ( 1 991 )
23, 209-2 12 ( 199 1 )
ACCEPTED: APRIL12, 1991
The hemizona assay (HZA) as an experimental model to evaluate the inhibition of sperm binding to the murine zona pellucida by isolated zona pellucida protein M.-L. Windt, D. R. Franken, P. M. de Beer, P. J. D. Bouic and T . F. Kruger Key words. Zona pellucida
-
hemizona assay
~
sperm receptor
Summary. Compelling evidence has demonstrated that zona binding represents gamete recognition by sperm binding with high affinity and specificity to complex glycoproteins of the zona pellucida. In the present study we evaluated the hemizona assay (HZA) in the investigation of the interaction of mouse spermatozoa with unfertilized murine oocytes and hemizonae after exposure to solubilized murine zonae pellucidae proteins. The zonae pellucidae were isolated from ovarian tissue following described mincing techniques. The sperm binding characteristics of murine spermatozoa were studied by using SDS-PAGE isolated ZP2 ( +_ 120 Kd) and ZP3 ( 83 Kd) components of the zona pellucida. Sperm receptor activity was examined in a competitive gamete binding fashion using the HZA as an indicator of sperm/zona interaction. The results illustrated that isolated, solubilized ZP2 and ZP3 glycoprotein moieties of the zona pellucida inhibited sperm binding to hemizonae and oocytes when compared to controls, and that the HZA can be utilized as an internally controlled homologous bioassay to evaluate the effects of zona pellucida proteins on tight binding of spermatozoa to the zona pellucida.
~
oocyte binding
-
mousc.
Introduction The zona pellhcida is an acellular glycoprotein coat surrounding the plasma membrane of the fully grown oocyte and is synthesized and secreted by growing oocytes (Wassarman, 1988). In mice the glycoprotein consists of dimeric filaments of ZP2 and ZP3, crosslinked to one another by ZPI (Wassarman, 1988). It has been shown that murine ZP3 (Mr = 83 Kd) serves as the species-specificsperm receptor and also induces the acrosome reaction (Bleil & Wassarman, 1983). ZP2 (Mr = 120 Kd) facilitates secondary binding of the sperm to the zona where it plays a role in the maintenance of binding of acrosome reacted sperm (Bleil et al., 1988). Isolated and purified zona pellucida proteins can be very useful in the study of the regulation of fertilization, i.e. sperm-oocyte binding, acrosome reaction and polyspermy. Previous studies showed that ZP3 from unfertilized oocytes inhibited the binding of spermatozoa to oocytes, thereby proving that ZP3 is the receptor protein for mouse sperm (Bleil & Wassarman, 1980). In this ongoing study our purpose was to investigate the use of the HZA (Burkman et al., 1988) as an experimental model to study the effect of electrophoretically separated zona proteins on mouse sperm-oocyte binding (Bleil & Wassarman, 1980).
Materials and methods Isolation o f zonae pellucidae Reproductive Biology Unit, Ilept. of Obstetrics and Gynaecology, Dept. Internal Medicine, University of Stellenbosch, Immunology Section, Dept. Medical Microbiology, Tygerberg Hospital, Tygerberg, Republic of South Africa. Correspondence: M.-L. Windt, Department of Obstetrics and Gynaecology, Tygerberg Hospital, 3rd Floor, Tygerberg 7505, Republic of South Africa.
Ovaries were obtained from Balb/c sacrificed female mice. Eighty ovaries were processed at a time. The ovaries were cleaned of all excess fat tissue and masserated. A Dounce homogenizer was used to homogenize the ovarian tissue thereby releasing the zonae pellucidae. Homogenization was performed
210
M.-L. WINDTET
AL.
on ice in 1% Triton in PBS (Moller & Wassarman, 1989). The homogenized sample was then washed thoroughly (4 x ) by centrifugation (22 000 g, 5 min) with 0.1 yo polyvinylpyrrolidone (PVP) in PBS at 4°C. The washed pellet was resuspended in 0.1 yo PVP + PBS and zona pellucida fragments were removed microscopically with a mouth-operated micropipette. Zonae pellucidae were then centrifuged for 20 minutes in a microfuge (22 000 9). Supernatants were removed and pelleted zonae solubilized in 5 mM Na,HPO, (pH 2.5) (Florman & Wassarman, 1985; Moller & Wassarman, 1989). Solubilized zonae protein from 228 and 170 ovaries were finally electrophoresed for the first and second HZA respectively. In the experiment to test the purity of the ZP2 fraction, zona pellucida protein from 320 ovaries were electrophoresed.
Electrophoresis of zona pellucida protein SDS-polyacrylamide gel electrophoresis of solubilized zona pellucida proteins was carried out under non-reducing conditions according to the method of Laemmli (1970) using a 10% separating gel. A section of the gel containing molecular weight markers and solubilized zona pellucida protein was stained with silver stain (Oakley et al., 1980) to identify the zona proteins ZP2 (Mr = 120 Kd) and ZP3 (Mr = 83 Kd). Each corresponding protein band was excised from the remaining, unstained gel and electroeluted separately in SDS electrophoresis buffer at 200 V for 15 hours. Electroeluted materials were dialyzed extensively (12 h) against 7 M urea, followed by extensive dialysis (24 h) against several changes of distilled water and finally lyophilized (Florman & Wassarman, 1985). Lyophilized zona protein fractions were stored at - 20°C until further use. A gel slice without any protein was subjected to the same procedures and was used as a control in competition studies (control 1 ) . In the experiment to test for ZP2 purity, the electroeluted ZP2 fraction was dialyzed, divided into two equal volumes and lyophilized separately. After reconstitution, the two fractions were electrophoresed under reducing (5%, 2-mercaptoethanol) and non-reducing conditions as described before.
Collection and culture of mouse gametes Superovulated female Balb/c mice were sacrificed and cumulus-enclosed oocytes removed from the oviducts. Cumulus was removed by treatment with hyaluronidase (0.1yo)and oocytes washed in Ham's F10 medium and kept at 37"C, 5% CO, in air. Mouse sperm were collected in Ham's F10 + 32 mg ml-I BSA (bovine serum albumin) from minced caudae epididymae of male Balb/c mice, allowing spermatozoa to swim free into the medium for 30
minutes at 37"C, 5% CO, in air. Supernatants containing motile spermatozoa were removed, counted and diluted to 0.5 x lo6 spermatozoa/ml in 32 mg BSA/ml Ham's F10.
Hemizona-competition assay Lyophilized samples (ZP2, ZP3, control 1 ) were reconstituted in 100 p1 Ham's F10 + 32 mg BSA/ml. 80 p1 of the reconstituted protein were incubated with 80 pl of mice spermatozoa (0.5 x 106/ml)for 60 minutes at room temperature. A second control (control 2) where spermatozoa were subjected only to reconstituting medium was also included. Washed, superovulated oocytes were microbisected into equal hemizonae for use in the hemizona assay as described previously (Franken et al., 1989). Hemizonae were placed into 20 pl drops of the incubated sperm combinations to determine any inhibition of sperm-oocyte binding caused by zona proteins ZP3 and ZP2. The assay was repeated twice with different zona protein preparations. Combinations examined were (in duplicate) : Control 1 vs control 2; control 2 vs ZP2; control 2 vs ZP3. Whole eggs were also included in all drops. Drops were covered with liquid parafin and incubated for 4 hours at 37". Zonae and eggs were removed, rinsed in Ham's F 10 and tightly bound spermatozoa were counted under an inverted microscope (Burkman et al., 1988; Franken et al., 1989).
Results The results of the inhibition studies are presented in Table 1 and Figures 1 and 2.
Hemizonae binding As can be seen from Table 1 and Figure 1, both control 1 and 2 had no influence on the binding of murine spermatozoa to hemizonae. An average of 33.3 ( & 1.8) tightly bound spermatozoa per hemizona was counted. There was, however, a marked
Table 1. The effect of solubilized, isolated murine zona pellucida proteins on the binding of spermatozoa (mean & SD) to murine hemizonae and oocytes Hemizonal binding HZ1 Control 1 , 33 ( + 13) Control 2 29 ( + 16) Control 2 29 ( + 16)
HZ2 Control 2 29 ( + 16) ZP3 2 ( + 2) ZP2 2 ( + 1)
Oocyte binding 1 Control 1 27 ( k 15) Control 2 28 ( + 11) Control 2 28 ( + 11)
2 Control 2 28 ( + 11) ZP3 2 ( + 2) ZP2 2 ( + 2)
ANDROLOGIA 23, 209-212 (1991)
HEMIZONA ASSAY
MEAN NUMBER OF SPERM BOUND/HZ
40
L J 29
30
30
20
10
0
E 3 CONTROL
1
CONTROL 2
0ZP 2
mZP 3
Figure 1. Histograms of mean sperm binding to murine hemizonae after sperm pre-incubation with Ham’s FIO + BSA (control 2), a SDS gel strip (control I ) , ZP2 and ZP3. Control 1, ZP2 and ZP3 were electrophoresed, electroeluted, dialized and lyophilized.
MEAN NUMBER OF SPERM BOUND/OOCYTE
40
of ZP2. However, no bands corresponding to 83 Kd molecular weight could be demonstrated. When the same preparation of ZP2 was electrophoresed under reducing conditions, no new band at 83 Kd could be detected thereby indicating the presence of either ZP3 contamination or ZP2-ZP3 complexes in the original ZP2 preparation.
Discussion In previous studies it was reported that pre-treatment of murine spermatozoa with solubilized murine zona pellucida prevented the binding of these spermatozoa to unfertilized eggs (Bleil & Wassarman, 1980; Florman et al., 1984). Furthermore, when the individual, purified zona pellucida glycoproteins, ZP3, ZP2 and ZP1 were tested in analogous experiments, it was shown that only ZP3 prevented sperm binding; ZP2 and ZP1 had no effect (Bleil & Wassarman, 1980, 1986). It was concluded from other studies that ZP3 functions as the murine receptor for spermatozoa and also that it induces the acrosome reaction following binding to spermatozoa with intact acrosomes (Bleil & Wassarman, 1980, 1983, 1986; Florman & Wassarman, 1985; Wassarman, 1987, 1988). O n the other hand, ZP2 was shown to bind to the inner acrosomal membrane of acrosome reacted spermatozoa (Bleil & Wassarman, 1986) acting as a secondary sperm receptor following completion of the acrosome reaction (Bleil et al., 1988). Furthermore it appears that the receptor activity of ZP3 is destroyed following fertilization (Bleil & Wassarman, 1980) and that ZP2 also undergoes modification thus probably playing a role in the block to polyspermy (Bleil et al., 1981). Since the development of the hemizona assay (HZA) by Burkman et al., (1988) it has been used in several controlled studies, mainly to test fertilization potential of human spermatozoa (Franken et al., 1989). We utilized the HZA to investigate the inhibition potential of electrophoresed, electroeluted, dialysed, lyophilized murine zona pellucida glycoproteins. Although microbisecting of mouse oocytes proved to be more difficult than that of human oocytes (mouse oocytes are smaller and zone pellucidae thinner and more fragile) it was still possible. We included intact oocytes to guarantee results in case of lost zonae. The inhibition of sperm binding to hemizonae of spermatozoa pre-incubated with the isolated ZP3 fraction, was in agreement with findings of previous investigators (Bleil & Wasserman, 1980; Florman et al., 1984). Contrary to other studies though (Bleil & Wassarman, 1980, 1986), binding of the ZP2 treated spermatozoa was also inhibited.
flI
30
20
10
0
=
E 3 Control I E Z Control 2 0ZP 2 ZP 3 Figure 2. Histograms of mean sperm binding to murine oocytes after sperm pre-incubation with Ham’s F10 + BSA (control Z), a SDS gel strip (control l ) , ZP2 and ZP3, Control 1, ZP2 and ZP3 were electrophoresed, electroeluted, dialized and lyophilized.
decrease in binding when spermatozoa were preincubated in either ZP2 or ZP3, when compared to control 2 (93% decrease for both ZP2 and ZP3). Oocyte binding Results for oocyte binding (Fig. 2, Table 1) were similar to that of hemizona binding. Spermatozoa incubated with both control 1 and 2 showed an average binding of 27.7 (k 0.58) tightly bound spermatozoa per oocyte. A decrease in binding was shown for ZP2 and ZP3 incubated spermatozoa (93% and 89%, respectively) when compared to control 2. Purity of ZP2 fraction Electrophoresis of the electroeluted ZP2 fraction under non-reducing conditions revealed a diffuse band at approximately 120 Kd, confirming the presence ANDROLOGIA 23, 209-212 (1991)
211
212
M.-L. WINDTET
AL.
Purified ZP2 was shown to contain as much as 3% ZP3 as contaminant (Bleil et al., 1988) and since nanomolar concentrations of ZP3 can prevent spermatozoa from binding (Wassarman, 1987), we assumed that contamination of our ZP2 fraction by ZP3 could be responsible for this inhibition. Also, it appears that ZP2 and ZP3 can form dimers of 180 Kd (Wassarman, 1988). Based on this observation, we speculated that our preparation of ZP2 could contain a small amount of these dimers and that the inhibition observed with the ZP2 preparation could be explained in terms of the presence of ZP2-ZP3 dimers. Furthermore, it was reported in an autoradiographic study by Bleil & Wassarman (1986) that ZP2 can bind to some extent to acrosome intact spermatozoa. These authors suggested that ZP2 has a specific, but low affinity for the plasma membrane of acrosome intact spermatozoa. In another study by the same authors (Bleil & Wassarman, 1983), it was shown that ZP2 could also induce the acrosome reaction to some extent. The proposed contamination of the ZP2 fraction by ZP3 in our study could have contributed to the induction of the acrosome reaction, resulting in the inhibition of sperm binding. However, the integrity of the acrosome was not investigated in this study. In order to rule out the problem of ZP2 contamination by either ZP3 or ZP2-ZP3 dimers, the isolated ZP2 fraction was again electrophoresed under reducing and non-reducing conditions. The diffuse band at 120 Kd confirmed the presence of ZP2 but no ZP3 could be shown at 83 Kd. Although zona material from 160 ovaries (for reduced and nonreduced, respectively) was electrophoresed, the concentration of ZP3 in this sample was probably too low to detect with the silver stain method. Possible contamination by SDS, urea or polyacrylamide thereby causing inhibition was ruled out by the inclusion of control 2 (a SDS gel strip subjected to procedures similar to that of the glycoprotein containing fractions). We therefore conclude that the HZA can be used in murine studies, and that receptor activity of isolated murine ZP3 was indicated in our study. The expertise and information gained in the current murine study will be used in future studies on porcine and human zona pellucida glycoproteins, hopefully helping us to gain more information on human infertility and contraception.
Acknowledgements The authors wish to thank the Medical Superintendent of Tygerberg Hospital for permission to publish as well as the Medical Research Council of South Africa for financial support.
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