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Cross-Reactivity of Sperm-Binding Proteins from Chicken, Turkey, and Duck Oocytes1
Cross-Reactivity of Sperm-Binding Proteins from Chicken, Turkey, and Duck Oocytes1 R. K. BRAMWELL and B. HOWARTH Department of Poultry Science, University of Georgia, Athens, Georgia 30602
1992 Poultry Science 71:1927-1932
INTRODUCTION The presence of spermatozoa receptors located on the mammalian zona pellucida (ZP) has been well documented (Gwatkin and Williams, 1977; Bleil and Wassarman, 1980; Wassarman et al, 1985). In the mouse and hamster, contact between spermatozoa and receptors on the ZP causes the intact spermatozoon to bind to the ZP (Wassarman, 1987). The bound spermatozoa then undergo an acrosome reaction and digest a pathway through the ZP to fuse with the egg plasma membrane (Yanagimachi and Noda, 1970a,b; Yanag-
Received for publication February 28, 1992. Accepted for publication July 30, 1992. Supported by state of Georgia and Hatch funds allocated to the Georgia Agricultural Experiment Stations, University of Georgia.
imachi, 1977, 1981). Spermatozoa recognition of the ZP overlying the ovum is accomplished, in part, by spermatozoon receptors on the ZP itself (O'Rand, 1988). Species-specific sperm-egg interaction has been studied in mammals. Lambert (1984) and Roldan et al (1985) studied sperm-egg interaction in heterologous crosses of mouse species and reported that the ZP of the heterologous combinations bound fewer spermatozoa than homologous species combinations. Hanada and Chang (1972) also reported that the ZP appeared to be the major block against interspecies fertilization in three rodent species. More recently, Roldan and Yanagimachi (1989), studying two hamster species, suggested that the ZP block of interspecies fertilization is not present in all interspecies crosses. Hartmann et al. (1972) discussed two steps involved in mammalian in vitro sperm-ZP interaction,
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ABSTRACT Binding and penetration of spermatozoa through the perivitellirte layer (PL) overlying the hen's ovum have been studied more frequently in the chicken than in other domesticated avian species. Species-specific action of the binding process was tested using an in vitro competition assay in which spermatozoa from the cock, torn, and drake were pretreated with solubilized PL protein (PL-P) from the chicken, turkey, and duck ovum. Spermatozoa were pretreated with PL-P for 20 min at 39 C and co-incubated in vitro with a .5 cm2 section of intact PL from the homologous sperm donor species for an additional 10 min at 39 C. Effectiveness of PL-P pretreatment was assessed quantitatively by the number of spermatozoa bound to the PL, and was expressed as a percentage of the control [minimum essential medium (MEM) pretreated sperm = 100%] binding. Pretreatment of cock spermatozoa with chicken, turkey, or duck PL-P resulted in 21,40, and 48% binding, respectively. Similarly, pretreatment of torn spermatozoa with PL-P from chicken, turkey, or duck resulted in 45,51, and 39% binding, and that of drake spermatozoa resulted in 38, 32, and 21% binding, respectively. Incubation of spermatozoa with PL-P from chicken, turkey, and duck ova indicated cross-reactivity and suppression of binding between avian spermatozoa and PL that was not species-specific. (Key words: sperm-egg interaction, species-specific binding, chicken, turkey, duck)
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BRAMWELL AND HOWARTH
MATERIALS AND METHODS Virgin White Leghorn hens were euthanatized by cervical dislocation at 20 min postoviposition and the body cavity opened to remove recently ovulated ova before their entrance into the reproductive tract. Each ovum was placed in deionized water and a small incision made in its outer wall, through the perivitelline layer (PL). Five minutes after being placed in water, the PL was removed from the ovum with forceps and stored frozen (to be solubilized). Nicholas Large White turkey hens were anesthetized by the administration of 15 to 20 mL equithesine (Gandal, 1956) into the brachial vein at 20 min postoviposition. It was necessary to anesthetize the turkey hens for PL collection because of their large body size and the need to keep the birds still to protect the recently ovulated ova from breaking. After the turkey hens
2
Donated by Scott Frazur, Feather Creek Farm, Inc., Grants Pass, OR 97526. 3 Beckman Instruments, Inc., Fullerton, CA 92634-3100. 4 Model No. SVC-100H, Savant Instruments, Inc., Farmingdate, NY 11735. 5 Gibco Laboratories, Life Technologies, Inc., Grand Island, NY 14072.
were anesthetized, the body cavity was opened for retrieval of the recently ovulated ova prior to its entrance into the infundibulum. Each turkey hen was euthanatized with an intravenous overdose of equithesine after collection of the ovum. The turkey PL was removed from the ovum and preserved as previously described for the chicken PL. Khaki Campbell ducks2 were killed by cervical dislocation at 10 min postoviposition, after which the recently ovulated ova were collected and PL removed and treated as previously described for the chicken. The PL from the chicken, turkey, and duck were acid solubilized by incubating for 2 h at 39 C with 300, 500, or 400 uL 5 mM NaH2PC»4 solution (acidified to pH 2.5 with HC1), respectively. The difference in volumes of acid buffer was based on size differences in the PL from the different species. Following incubation, any remaining intact PL was removed and the solution centrifuged for 10 min at 12,000 x g. Following centrifugation, the supernatant was removed and protein concentration measured by ultraviolet absorption on a Beckman DUr-65 spectrophotometer3 according to the method of Warburg and Christian (1942). The solubilized PL protein (PL-P) was divided into tubes labeled by species, with each tube containing 175 jig protein. The PL-P samples were lyophilized using a Savant speedvac concentrator,4 and frozen until resuspended in minimum essential medium 5 (MEM; Howarth, 1981) at the time of use. Three separate experiments were conducted to test for species specificity of sperm-egg interaction in the chicken, turkey, and duck. Cock Sperm Pretreatment Pooled cock semen was collected from Peterson broiler males, washed by diluting 2:1 in MEM and centrifuged for 10 min at 600 x g. Sperm concentration was determined by assessing packed cell volume of the semen sample (Maeza and Buss, 1976), after which the sample was diluted to a concentration of 1 x 106 sperm per milliliter. To test spermatozoa receptor activity, an in vitro competition assay was used in which the spermatozoa and PL-P were added to
Downloaded from http://ps.oxfordjournals.org/ at Florida International University on June 23, 2015
attachment and binding, and they suggested that attachment, the first step, was not species-specific whereas the binding step was. There is less known about avian interspecies sperm-egg interaction than about their mammalian counterparts. Studying the male gamete, Chiva et al. (1988) reported that by cytochemical criteria the basic proteins of spermatozoa in birds remain remarkably constant. Morris et al. (1987) found a closer relationship in Wolffian duct proteins from the chicken, turkey, and quail than were found in the duck and pigeon. Morris et al. (1987) also suggested that species-specific interaction in birds exists to a lesser degree than that found in mammals. The present study was conducted to determine whether, and to what extent, sperm-egg interaction in three avian species (chicken, turkey, and duck) was species-specific.
SPERM-EGG INTERACTION
Tom Sperm Pretreatment Semen was collected from Nicholas breeder toms, diluted 2:1 in MEM, and pooled. The pooled semen was washed in MEM as previously described and sperm concentration determined using packed cell volume. A sperm concentration of 1 x 106 was used for this experiment. The in vitro competition assay was conducted as previously described for cock sperm pretreatment with fresh intact turkey PL sections (.5 cm2) incubated with pretreated torn spermatozoa. Drake Sperm Pretreatment Semen was collected from Khaki Campbell drakes, diluted 2:1 in MEM, and
^Sigrna Chemical Co., St. Louis, MO 63178-9916. 7 A11 SDS-polyacrylamide solvents were obtained from Bethesda Research Laboratories, Life Technologies, Inc., Gaithersburg, MD 20877. 8 BIO-RAD Laboratories, Richmond, CA 94804.
pooled. Pooled spermatozoa were washed as previously described and the concentration determined by hemocytometer counts. Sperm concentrations used for pretreating drake spermatozoa were 5 x 10^ sperm per milliliter. The in vitro competition assay was conducted as previously described with fresh intact duck PL sections (.5 cm2) incubated with pretreated drake spermatozoa. In each of the previous three experiments, nonspecific binding was tested using chicken serum albumin6 and chicken egg albumen (ovalbumin)6 pretreatment of spermatozoa in the same manner as PL-P pretreatment was conducted. Data from cock, torn, and drake sperm pretreatment underwent an arc sine transformation and were evaluated by analysis of variance. Differences between treatment means were analyzed by Duncan's multiple range test, using the General Linear Models procedure of SAS® software (SAS Institute, 1985). Electrophoretic Analysis of Perivitelline Layer Protein An SDS-PAGE7 of SDS PL-P from each species was carried out according to the methods of Laemmli (1970) using a 4 to 25% gradient gel. The gels were stained for protein with Coomassie brilliant blue 8 and for carbohydrate with periodic acid-Schiff reagent6 (Fairbanks et al, 1971). The SDS solubilized PL was carried out by incubating PL from each species in 1% SDS (5 mL per intact PL) at 39 C for 18 h, the solution was centrifuged, and protein concentration determined as previously described. RESULTS AND DISCUSSION The competition assay employed in the current study was based on the methods used by Gwatkin and Williams (1977) and Bleil and Wassarman (1980). Using Schiff's reagent to stain the intact PL, as performed by Bramwell and Howarth (1991), pathways caused by the penetrating spermatozoa (Okamura and Nishiyama, 1978) could be seen under a light microscope. These pathways through the PL have also been observed using scanning electron microscopy (Howarth, 1984). The PL over-
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each tube and the total volume adjusted to 100 uL by adding additional MEM. Spermatozoa and PL-P from each species were individually co-incubated for 20 min at 39 C, after which the samples were centrifuged at 600 x g for 10 min after which 60 uL of supernate was removed and discarded. Control tubes containing spermatozoa that had not been pretreated with PL-P were prepared with MEM only but subjected to the same procedures as the pretreated sperm samples. Fresh MEM (60 uL) was added back to the spermatozoa along with a fresh (collected on the same day) intact section of chicken PL (.5 cm2). The intact PL and spermatozoa were incubated together for 10 min at 39 C. Each intact PL section was removed, rinsed in 20% formalin, placed on a microscope slide, and stained with several drops of Schiff's reagent prior to addition of the coverslip (Bramwell and Howarth, 1991). Holes in the membrane caused by the binding and penetration of spermatozoa were counted under a light microscope in a .269 mm 2 area and expressed as a percentage of the control (MEM pretreated = 100%) binding. Six replicates of each species PL-P were performed.
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BRAMWELL AND HOWARTH
TABLE 1. Effect of sperm penetration of the perivitelline layer (PL) due to pretreatment of cock, torn, and drake spermatozoa with solubilized PL protein (PL-P) from the chicken, turkey, and duck
PL-P
n
Sperm donor species binding1 Tom
Cock
Drake
"",,}
Control Chicken Turkey Duck
6 6 6 6
100.0 20.93 40.01 48.13
± 0* ± 11.0s ± 5.4b ± 18.2"
100.0 44.61 50.51 38.66
± ± ± ±
03 18./* 11.7" 9.8b
100.0 38.12 31.86 21.36
± ± ± ±
0* 6.2b 14.5b 6.9s
"Column values (3c ± SEM) with no common superscripts differ significantly (P £ .05). Values are expressed as a percentage of the control binding and penetration of the PL.
matozoon receptor activity was not entirely species-specific and that some crossreactivity between species does exist. This would indicate that some of the structural characteristics of the sperm-binding receptors has been conserved in these species so as to allow partial, but not complete, recognition of the spermatozoon and PL receptors. Chicken serum albumin and chicken egg albumen pretreatment of spermatozoa did not suppress sperm-egg binding when used at four times the protein level used for pretreatment of spermatozoa with PLP. The lack of inhibition from these nonspecific proteins indicates that spermegg binding is a specific reaction, and that recognition of certain (to date, unknown) characteristics of the spermatozoon and the PL receptors is necessary. Recently, Kido and Doi (1988) reported that the PL of the chicken ovum consisted of three major glycoproteins. The current results from SDS-PAGE indicated extreme similarities in the molecular weights of the glycoproteins present in the chicken, turkey, and duck SDS-solubilized PL (Figure 1). The largest of the two major bands found in each species had a molecular weight (MW) of approximately 167,000 Da and the smaller of the two major bands had a MW of approximately 35,000 Da as calculated from the standard. The smallest protein band by weight does not appear to be a glycoprotein, as it failed to stain with periodic acid-Schiff reagent. Therefore, it seems likely that the small MW band protein does not play a major role in sperm-egg binding and recognition due to what is known about the involvement of
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lying the female ovum, which is analogous to the mammalian ZP, has spermreceptors that recognize spermatozoa and initiate binding of the spermatozoon to the PL sperm-binding receptors. Spermreceptor activity has been shown to remain intact in the PL of recently ovulated ova as well as in PL-P (Howarth, 1990; Bramwell and Howarth, 1991). Cock spermatozoa pretreated with chicken PL-P exhibited only 21% binding as compared with the control (Table 1). Suppression of cock spermatozoa binding to the PL when pretreated with chicken PL-P was expected based on the principles of this assay. As spermatozoa and PL receptors recognized each other and bound, the sites on the spermatozoon that were occupied competitively inhibited further binding with intact PL sections. However, when cock spermatozoa was pretreated with turkey and duck PL-P, .sperm-PL binding was also decreased to 40 and 48% of control binding, respectively. The turkey and duck PL-P was partially effective in suppressing cock sperm-egg receptor activity, although not to the same extent as chicken PL-P. Tom spermatozoa pretreated with turkey PL-P decreased the number of holes in the intact PL to 51% of the control (not pretreated sperm) (Table 1). Tom spermatozoa pretreated with chicken and duck PL-P resulted in 45 and 39% binding, respectively. Pretreatment of drake spermatozoa with duck PL-P resulted in 21% binding as compared with 38 and 32% binding for pretreatment with chicken and turkey PL-P, respectively. Results from the previous three experiments indicated sper-
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SPERM-EGG INTERACTION
C
200
D
C
T
D
— «•"»
116 —
—
97 —
•<••
6 6 -
—
43-
«•
31 -
*.
• • up
UP
•I
It
FIGURE 1. The SDS-PAGE of the SDS-soluble fraction of the perivitelline layer from the chicken (C), turkey (T), and duck (D). The molecular mass of a low and high molecular weight standard (S) are labeled in daltons. The four lanes on the left were stained for protein with Coomassie brilliant blue. The three lanes on the right were stained for carbohydrate with periodic-acid Schiff's reagent. The gel was dried prior to photographing, which resulted in several cracks.
carbohydrate in this reaction (Edge et al., 1981; Florman and Wassarman, 1985; Howarth, 1992). The overall similarities found between PL from each of these species using SDS-PAGE support the results from the in vitro competition assay employed in the present study. The PL glycoproteins from each species were similar enough to interact with spermatozoon from the other species in the study to suppress binding to the intact PL. To date, little is known regarding what these similarities may be in the avian system. Carbohydrate moieties of glycoproteins play a role in sperm-egg recognition (Florman and Wassarman, 1985; Wassarman, 1988; Howarth, 1992). Howarth (1992), using trifluoromethanesulfonic acid (TFMS), removed carbohydrates from the glycoproteins of chicken PL-P before pretreating cock spermatozoa. Chicken PL-P treated with TFMS had no inhibitory effect on cock spermatozoa attachment and digestion of intact chicken PL. This indicates that removal of both n- and olinked oligosaccharides from components
of the PL receptor results in elimination of its sperm receptor activity, thus suggesting the importance of the carbohydrate portion of the spermatozoon receptors on the PL. It is likely that some of the components of the oligosaccharides differ between species in the current study, but are similar enough for gamete interaction to occur. Based upon a competition assay, pretrearment of spermatozoa with PL-P from each species had a suppressive effect on the ability of the spermatozoa to bind to and penetrate intact PL from its homologous species as well as from other species in the study, thus indicating crossreactivity of sperm-binding glycoproteins from chicken, turkey, and duck oocytes.
REFERENCES Bleil, J. D., and P. M. Wassarman, 1980. Mammalian sperm-egg interaction: Identification of a glycoprotein in mouse egg zonal pellucidae possessing receptor activity for sperm. Cell 20: 873-882. Bramwell, R. K., and B. Howarth, 1991. Preferential attachment of cock spermatozoa to the perivitelline layer directly over the germinal disc of the h e n ' s o v u m . P o u l t r y Sci. 70(Suppl. 1): 149.(Abstr.) Chiva, M., H. E. Kasinsky, M. Mann, and J. A. Subirana, 1988. On the diversity of sperm basic proteins in the vertebrates: VI. Cytochemical and biochemical analysis in birds. J. Exp. Zool. 245:304-317. Edge, A.S.B., C. R. Faltynek, L. Hof, L. E. Reichert, and P. Weber, 1981. Deglycosylation of glycoproteins by trifluoromethanesulfonic acid. Anal. Biochem. 118:131-137. Fairbanks, G., T. L. Steck, and D.F.H. Wallach, 1971. Electrophoretic analysis of the major polypeptides of the human erythrocyte membrane. Biochemistry 10:2606-2617. Florman, H. M., and P. M. Wassarman, 1985. Olinked oligosaccharides of mouse egg ZP3 account for its sperm receptor activity. Cell 41: 313-324. Gandal, C. P., 1956. Satisfactory general anesthesia in birds. J. Am. Vet. Med. Assoc. 128:332-334. Gwatkin, R.B.L., and D. T. Williams, 1977. Receptor activity of the hamster and mouse solubilized zona pellucida before and after the zona reaction. J. Reprod. Fertil. 49:55-59. Hanada, A., and M. C. Chang, 1972. Penetration of Zona-free eggs by spermatozoa of different species. Biol. Reprod. 6:300-309. Hartmann, J. F., R.B.L. Gwatkin, and C. F. Hutchison, 1972. Early contact interactions between mammalian gametes in vitro: Evidence that the vitellus influences adherence between sperm and zona pellucida. Proc. Nat. Acad. Sci. USA 69:2767-2769.
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BRAMWELL AND HOWARTH Roldan, E.R.S., A. D. VituUo, M. S. Merani, and I. Von Lawzewitsch, 1985. Cross fertilization in vivo and in vitro between three species of vesper mice, Calomys (Rodentia, Cricetidae). J. Exp. Zool. 233:433-442. Roldan, E.R.S., and R Yanagimachi, 1989. Crossfertilization between Syrian and Chinese hamsters. J. Exp. Zool. 250:321-328. SAS Institute, 1985. SAS® User's Guide: Basics. Version 5 Edition. SAS Institute Inc., Cary, NC. Warburg, O., and W. Christian, 1942. Isolierung und Kristallisation des garungsferments enolase. Biochem. Z. 310:384-421. Wassarman, P. M., 1987. Early events in mammalian fertilization. Annu. Rev. Cell Biol. 3:109-142. Wassarman, P. M., 1988. Fertilization in mammals. Sci. Am. (Dec.):78-84. Wassarman, P. M., H. M. Florman, and J. M. Greve, 1985. Receptor mediated sperm-egg interaction in mammals. Pages 341-360 in: Biology of Fertilization, Vol. 2. C. B. Metz and A. Mouroy ed. Academic Press, New York, NY. Yanagimachi, R, 1977. Specificity of sperm-egg interaction. Pages 225-295 in: Immunobiology of Gametes. M. Edidin and M. Johnson, ed. Cambridge University Press, Cambridge, England. Yanagimachi, R, 1981. Mechanisms of fertilization in mammals. Pages 81-82 in: Fertilization and Embryonic Development In Vitro. L. Mastroianni and J. D. Biggers, ed. Plenum Publishers, New York, NY. Yanagimachi, R, and Y. D. Noda, 1970a. Ultrastructural changes in the hamster sperm head during fertilization. J. Ultrastruct. Res. 31:465-485. Yanagimachi, R, and Y. D. Noda, 1970b. Physiological changes in the post-nuclear cap region of mammalian spermatozoa: A necessary preliminary to the membrane fusion between sperm and egg cells. J. Ultrastruct. Res. 31:486-493.
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Howarth, B., Jr., 1981. Preservation of the fertilizing capacity of cock semen incubated in vitro at 41 C. Poultry Sci. 60:1075-1078. Howarth, B., Jr., 1984. Maturation of spermatozoa and mechanism of fertilization. Pages 61-174 in: Reproductive Biology of Poultry. F. J. Cunningham, P. E. Lake, and D. Hewitt, ed. British Poultry Science Ltd., Longman-Group, Harlow, England. Howarth, B., 1990. Research Note: Avian sperm-egg interaction: Perivitelline layer possesses receptor activity for spermatozoa. Poultry Sci. 69: 1012-1015. Howarth, B., 1992. Carbohydrate involvement in sperm-egg interaction in the chicken. J. Recept. Res. 12(2):255-265. Kido, S., and Y. Doi, 1988. Separation and properties of the inner and outer layers of the vitelline membrane of hen's eggs. Poultry Sci. 67: 476-486. Laemmli, U. K., 1970. Cleavage of structural proteins during assembly of the head of bacteriophage T4. Nature 227:680-685. Lambert, H., 1984. Role of sperm-surface glycoproteins in gamete recognition in two mouse species. J. Reprod. Fertil. 70:281-284. Maeza, A. S., and E. G. Buss, 1976. Sperm concentration and sperm numbers as related to fertility in chickens. Poultry Sci. 55:2059.(Abstr.) Morris, S. A., B. Howarth, Jr., J. W. Crim, S. Rodriguez de Cordoba, P. Esponda, and J. M. Bedford, 1987. Specificity of sperm-binding wolffian duct proteins in the rooster and their persistence on spermatozoa in the female host glands. J. Exp. Zool. 242:189-198. Okamura, F., and H. Nishiyama, 1978. The passage of spermatozoa through the vitelline membrane in the domestic fowl, Gallus gallus. Cell Tissue Res. 188:497-508. O'Rand, M. G., 1988. Sperm-egg recognition and barriers to interspecies fertilization. Gamete Res. 19:315-328.
1992 Poultry Science 71:1927-1932
INTRODUCTION The presence of spermatozoa receptors located on the mammalian zona pellucida (ZP) has been well documented (Gwatkin and Williams, 1977; Bleil and Wassarman, 1980; Wassarman et al, 1985). In the mouse and hamster, contact between spermatozoa and receptors on the ZP causes the intact spermatozoon to bind to the ZP (Wassarman, 1987). The bound spermatozoa then undergo an acrosome reaction and digest a pathway through the ZP to fuse with the egg plasma membrane (Yanagimachi and Noda, 1970a,b; Yanag-
Received for publication February 28, 1992. Accepted for publication July 30, 1992. Supported by state of Georgia and Hatch funds allocated to the Georgia Agricultural Experiment Stations, University of Georgia.
imachi, 1977, 1981). Spermatozoa recognition of the ZP overlying the ovum is accomplished, in part, by spermatozoon receptors on the ZP itself (O'Rand, 1988). Species-specific sperm-egg interaction has been studied in mammals. Lambert (1984) and Roldan et al (1985) studied sperm-egg interaction in heterologous crosses of mouse species and reported that the ZP of the heterologous combinations bound fewer spermatozoa than homologous species combinations. Hanada and Chang (1972) also reported that the ZP appeared to be the major block against interspecies fertilization in three rodent species. More recently, Roldan and Yanagimachi (1989), studying two hamster species, suggested that the ZP block of interspecies fertilization is not present in all interspecies crosses. Hartmann et al. (1972) discussed two steps involved in mammalian in vitro sperm-ZP interaction,
1927
Downloaded from http://ps.oxfordjournals.org/ at Florida International University on June 23, 2015
ABSTRACT Binding and penetration of spermatozoa through the perivitellirte layer (PL) overlying the hen's ovum have been studied more frequently in the chicken than in other domesticated avian species. Species-specific action of the binding process was tested using an in vitro competition assay in which spermatozoa from the cock, torn, and drake were pretreated with solubilized PL protein (PL-P) from the chicken, turkey, and duck ovum. Spermatozoa were pretreated with PL-P for 20 min at 39 C and co-incubated in vitro with a .5 cm2 section of intact PL from the homologous sperm donor species for an additional 10 min at 39 C. Effectiveness of PL-P pretreatment was assessed quantitatively by the number of spermatozoa bound to the PL, and was expressed as a percentage of the control [minimum essential medium (MEM) pretreated sperm = 100%] binding. Pretreatment of cock spermatozoa with chicken, turkey, or duck PL-P resulted in 21,40, and 48% binding, respectively. Similarly, pretreatment of torn spermatozoa with PL-P from chicken, turkey, or duck resulted in 45,51, and 39% binding, and that of drake spermatozoa resulted in 38, 32, and 21% binding, respectively. Incubation of spermatozoa with PL-P from chicken, turkey, and duck ova indicated cross-reactivity and suppression of binding between avian spermatozoa and PL that was not species-specific. (Key words: sperm-egg interaction, species-specific binding, chicken, turkey, duck)
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BRAMWELL AND HOWARTH
MATERIALS AND METHODS Virgin White Leghorn hens were euthanatized by cervical dislocation at 20 min postoviposition and the body cavity opened to remove recently ovulated ova before their entrance into the reproductive tract. Each ovum was placed in deionized water and a small incision made in its outer wall, through the perivitelline layer (PL). Five minutes after being placed in water, the PL was removed from the ovum with forceps and stored frozen (to be solubilized). Nicholas Large White turkey hens were anesthetized by the administration of 15 to 20 mL equithesine (Gandal, 1956) into the brachial vein at 20 min postoviposition. It was necessary to anesthetize the turkey hens for PL collection because of their large body size and the need to keep the birds still to protect the recently ovulated ova from breaking. After the turkey hens
2
Donated by Scott Frazur, Feather Creek Farm, Inc., Grants Pass, OR 97526. 3 Beckman Instruments, Inc., Fullerton, CA 92634-3100. 4 Model No. SVC-100H, Savant Instruments, Inc., Farmingdate, NY 11735. 5 Gibco Laboratories, Life Technologies, Inc., Grand Island, NY 14072.
were anesthetized, the body cavity was opened for retrieval of the recently ovulated ova prior to its entrance into the infundibulum. Each turkey hen was euthanatized with an intravenous overdose of equithesine after collection of the ovum. The turkey PL was removed from the ovum and preserved as previously described for the chicken PL. Khaki Campbell ducks2 were killed by cervical dislocation at 10 min postoviposition, after which the recently ovulated ova were collected and PL removed and treated as previously described for the chicken. The PL from the chicken, turkey, and duck were acid solubilized by incubating for 2 h at 39 C with 300, 500, or 400 uL 5 mM NaH2PC»4 solution (acidified to pH 2.5 with HC1), respectively. The difference in volumes of acid buffer was based on size differences in the PL from the different species. Following incubation, any remaining intact PL was removed and the solution centrifuged for 10 min at 12,000 x g. Following centrifugation, the supernatant was removed and protein concentration measured by ultraviolet absorption on a Beckman DUr-65 spectrophotometer3 according to the method of Warburg and Christian (1942). The solubilized PL protein (PL-P) was divided into tubes labeled by species, with each tube containing 175 jig protein. The PL-P samples were lyophilized using a Savant speedvac concentrator,4 and frozen until resuspended in minimum essential medium 5 (MEM; Howarth, 1981) at the time of use. Three separate experiments were conducted to test for species specificity of sperm-egg interaction in the chicken, turkey, and duck. Cock Sperm Pretreatment Pooled cock semen was collected from Peterson broiler males, washed by diluting 2:1 in MEM and centrifuged for 10 min at 600 x g. Sperm concentration was determined by assessing packed cell volume of the semen sample (Maeza and Buss, 1976), after which the sample was diluted to a concentration of 1 x 106 sperm per milliliter. To test spermatozoa receptor activity, an in vitro competition assay was used in which the spermatozoa and PL-P were added to
Downloaded from http://ps.oxfordjournals.org/ at Florida International University on June 23, 2015
attachment and binding, and they suggested that attachment, the first step, was not species-specific whereas the binding step was. There is less known about avian interspecies sperm-egg interaction than about their mammalian counterparts. Studying the male gamete, Chiva et al. (1988) reported that by cytochemical criteria the basic proteins of spermatozoa in birds remain remarkably constant. Morris et al. (1987) found a closer relationship in Wolffian duct proteins from the chicken, turkey, and quail than were found in the duck and pigeon. Morris et al. (1987) also suggested that species-specific interaction in birds exists to a lesser degree than that found in mammals. The present study was conducted to determine whether, and to what extent, sperm-egg interaction in three avian species (chicken, turkey, and duck) was species-specific.
SPERM-EGG INTERACTION
Tom Sperm Pretreatment Semen was collected from Nicholas breeder toms, diluted 2:1 in MEM, and pooled. The pooled semen was washed in MEM as previously described and sperm concentration determined using packed cell volume. A sperm concentration of 1 x 106 was used for this experiment. The in vitro competition assay was conducted as previously described for cock sperm pretreatment with fresh intact turkey PL sections (.5 cm2) incubated with pretreated torn spermatozoa. Drake Sperm Pretreatment Semen was collected from Khaki Campbell drakes, diluted 2:1 in MEM, and
^Sigrna Chemical Co., St. Louis, MO 63178-9916. 7 A11 SDS-polyacrylamide solvents were obtained from Bethesda Research Laboratories, Life Technologies, Inc., Gaithersburg, MD 20877. 8 BIO-RAD Laboratories, Richmond, CA 94804.
pooled. Pooled spermatozoa were washed as previously described and the concentration determined by hemocytometer counts. Sperm concentrations used for pretreating drake spermatozoa were 5 x 10^ sperm per milliliter. The in vitro competition assay was conducted as previously described with fresh intact duck PL sections (.5 cm2) incubated with pretreated drake spermatozoa. In each of the previous three experiments, nonspecific binding was tested using chicken serum albumin6 and chicken egg albumen (ovalbumin)6 pretreatment of spermatozoa in the same manner as PL-P pretreatment was conducted. Data from cock, torn, and drake sperm pretreatment underwent an arc sine transformation and were evaluated by analysis of variance. Differences between treatment means were analyzed by Duncan's multiple range test, using the General Linear Models procedure of SAS® software (SAS Institute, 1985). Electrophoretic Analysis of Perivitelline Layer Protein An SDS-PAGE7 of SDS PL-P from each species was carried out according to the methods of Laemmli (1970) using a 4 to 25% gradient gel. The gels were stained for protein with Coomassie brilliant blue 8 and for carbohydrate with periodic acid-Schiff reagent6 (Fairbanks et al, 1971). The SDS solubilized PL was carried out by incubating PL from each species in 1% SDS (5 mL per intact PL) at 39 C for 18 h, the solution was centrifuged, and protein concentration determined as previously described. RESULTS AND DISCUSSION The competition assay employed in the current study was based on the methods used by Gwatkin and Williams (1977) and Bleil and Wassarman (1980). Using Schiff's reagent to stain the intact PL, as performed by Bramwell and Howarth (1991), pathways caused by the penetrating spermatozoa (Okamura and Nishiyama, 1978) could be seen under a light microscope. These pathways through the PL have also been observed using scanning electron microscopy (Howarth, 1984). The PL over-
Downloaded from http://ps.oxfordjournals.org/ at Florida International University on June 23, 2015
each tube and the total volume adjusted to 100 uL by adding additional MEM. Spermatozoa and PL-P from each species were individually co-incubated for 20 min at 39 C, after which the samples were centrifuged at 600 x g for 10 min after which 60 uL of supernate was removed and discarded. Control tubes containing spermatozoa that had not been pretreated with PL-P were prepared with MEM only but subjected to the same procedures as the pretreated sperm samples. Fresh MEM (60 uL) was added back to the spermatozoa along with a fresh (collected on the same day) intact section of chicken PL (.5 cm2). The intact PL and spermatozoa were incubated together for 10 min at 39 C. Each intact PL section was removed, rinsed in 20% formalin, placed on a microscope slide, and stained with several drops of Schiff's reagent prior to addition of the coverslip (Bramwell and Howarth, 1991). Holes in the membrane caused by the binding and penetration of spermatozoa were counted under a light microscope in a .269 mm 2 area and expressed as a percentage of the control (MEM pretreated = 100%) binding. Six replicates of each species PL-P were performed.
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BRAMWELL AND HOWARTH
TABLE 1. Effect of sperm penetration of the perivitelline layer (PL) due to pretreatment of cock, torn, and drake spermatozoa with solubilized PL protein (PL-P) from the chicken, turkey, and duck
PL-P
n
Sperm donor species binding1 Tom
Cock
Drake
"",,}
Control Chicken Turkey Duck
6 6 6 6
100.0 20.93 40.01 48.13
± 0* ± 11.0s ± 5.4b ± 18.2"
100.0 44.61 50.51 38.66
± ± ± ±
03 18./* 11.7" 9.8b
100.0 38.12 31.86 21.36
± ± ± ±
0* 6.2b 14.5b 6.9s
"Column values (3c ± SEM) with no common superscripts differ significantly (P £ .05). Values are expressed as a percentage of the control binding and penetration of the PL.
matozoon receptor activity was not entirely species-specific and that some crossreactivity between species does exist. This would indicate that some of the structural characteristics of the sperm-binding receptors has been conserved in these species so as to allow partial, but not complete, recognition of the spermatozoon and PL receptors. Chicken serum albumin and chicken egg albumen pretreatment of spermatozoa did not suppress sperm-egg binding when used at four times the protein level used for pretreatment of spermatozoa with PLP. The lack of inhibition from these nonspecific proteins indicates that spermegg binding is a specific reaction, and that recognition of certain (to date, unknown) characteristics of the spermatozoon and the PL receptors is necessary. Recently, Kido and Doi (1988) reported that the PL of the chicken ovum consisted of three major glycoproteins. The current results from SDS-PAGE indicated extreme similarities in the molecular weights of the glycoproteins present in the chicken, turkey, and duck SDS-solubilized PL (Figure 1). The largest of the two major bands found in each species had a molecular weight (MW) of approximately 167,000 Da and the smaller of the two major bands had a MW of approximately 35,000 Da as calculated from the standard. The smallest protein band by weight does not appear to be a glycoprotein, as it failed to stain with periodic acid-Schiff reagent. Therefore, it seems likely that the small MW band protein does not play a major role in sperm-egg binding and recognition due to what is known about the involvement of
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lying the female ovum, which is analogous to the mammalian ZP, has spermreceptors that recognize spermatozoa and initiate binding of the spermatozoon to the PL sperm-binding receptors. Spermreceptor activity has been shown to remain intact in the PL of recently ovulated ova as well as in PL-P (Howarth, 1990; Bramwell and Howarth, 1991). Cock spermatozoa pretreated with chicken PL-P exhibited only 21% binding as compared with the control (Table 1). Suppression of cock spermatozoa binding to the PL when pretreated with chicken PL-P was expected based on the principles of this assay. As spermatozoa and PL receptors recognized each other and bound, the sites on the spermatozoon that were occupied competitively inhibited further binding with intact PL sections. However, when cock spermatozoa was pretreated with turkey and duck PL-P, .sperm-PL binding was also decreased to 40 and 48% of control binding, respectively. The turkey and duck PL-P was partially effective in suppressing cock sperm-egg receptor activity, although not to the same extent as chicken PL-P. Tom spermatozoa pretreated with turkey PL-P decreased the number of holes in the intact PL to 51% of the control (not pretreated sperm) (Table 1). Tom spermatozoa pretreated with chicken and duck PL-P resulted in 45 and 39% binding, respectively. Pretreatment of drake spermatozoa with duck PL-P resulted in 21% binding as compared with 38 and 32% binding for pretreatment with chicken and turkey PL-P, respectively. Results from the previous three experiments indicated sper-
1931
SPERM-EGG INTERACTION
C
200
D
C
T
D
— «•"»
116 —
—
97 —
•<••
6 6 -
—
43-
«•
31 -
*.
• • up
UP
•I
It
FIGURE 1. The SDS-PAGE of the SDS-soluble fraction of the perivitelline layer from the chicken (C), turkey (T), and duck (D). The molecular mass of a low and high molecular weight standard (S) are labeled in daltons. The four lanes on the left were stained for protein with Coomassie brilliant blue. The three lanes on the right were stained for carbohydrate with periodic-acid Schiff's reagent. The gel was dried prior to photographing, which resulted in several cracks.
carbohydrate in this reaction (Edge et al., 1981; Florman and Wassarman, 1985; Howarth, 1992). The overall similarities found between PL from each of these species using SDS-PAGE support the results from the in vitro competition assay employed in the present study. The PL glycoproteins from each species were similar enough to interact with spermatozoon from the other species in the study to suppress binding to the intact PL. To date, little is known regarding what these similarities may be in the avian system. Carbohydrate moieties of glycoproteins play a role in sperm-egg recognition (Florman and Wassarman, 1985; Wassarman, 1988; Howarth, 1992). Howarth (1992), using trifluoromethanesulfonic acid (TFMS), removed carbohydrates from the glycoproteins of chicken PL-P before pretreating cock spermatozoa. Chicken PL-P treated with TFMS had no inhibitory effect on cock spermatozoa attachment and digestion of intact chicken PL. This indicates that removal of both n- and olinked oligosaccharides from components
of the PL receptor results in elimination of its sperm receptor activity, thus suggesting the importance of the carbohydrate portion of the spermatozoon receptors on the PL. It is likely that some of the components of the oligosaccharides differ between species in the current study, but are similar enough for gamete interaction to occur. Based upon a competition assay, pretrearment of spermatozoa with PL-P from each species had a suppressive effect on the ability of the spermatozoa to bind to and penetrate intact PL from its homologous species as well as from other species in the study, thus indicating crossreactivity of sperm-binding glycoproteins from chicken, turkey, and duck oocytes.
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