In Vivo and In Vitro Fertilization of Hamster, Rat and Mouse Eggs after Treatment with Anti-hamster Ovary Antiserum Y. TSUNODA AND M. C. CHANG Worcester Foundation for Experimental Biology, Shrewsbury, Massachusetts 01545
ABSTRACT Rabbit antiserum against hamster ovary was examined on agargel diffusion plates against several hamster tissues, and also against rat and mouse ovarian extracts. Unabsorbed anti-hamster ovary antiserum showed eight to nine precipitin bands for hamster ovary and four to eight bands for other tissue extracts, but no bands against sperm antigens. Anti-hamster ovary antiserum also showed three to four bands for rat and one to two bands for mouse ovarian extracts. The present experiments confirmed previous reports for the hamster and mouse that treatment of eggs with anti-ovary antiserum blocked in vitro fertilization and that the extent of the inhibition was related to the formation of a precipitate on the zona pellucida. A single injection of anti-hamster ovary antiserum inhibited fertilization in mice but not in rats. In vitro fertilization of mouse eggs was also inhibited in the presence of such antiserum.
Recent studies on the antigenicity of mammalian ovaries have shown that heteroimmunization with ovarian tissue will yield antibodies (Ownby and Shivers, '72; Sacco and Shivers, '73a,b,c,d; Jilek and Pavlok, '75), and that these antibodies can block sperm penetration of the zona pellucida in vitro in hamsters (Shivers et al., '72), mice (Jilek and Pavlok, '75; Tsunoda and Chang, '76d, submitted) and rats (Tsunoda and Chang, '76c). Inhibition of pregnancy by isoimmunization with ovarian extract in mice was also reported (Porter, '65; Tsunoda and Chang, '76b). Antisera against mouse (Shahani et al., '72; Jilek and Pavlok, '75), rat and hamster ovary (Tsunoda and Chang, Ms in preparation) will also inhibit fertilization in vivo after injection with antiserum. Shivers and his colleagues (Shivers, '74) concluded that the zona antigen in mammalian eggs was species-specific. But recently, Tsunoda and Chang ('76c) found that rabbit anti-rat ovary antiserum drastically inhibited the fertilization of rat and mouse eggs in vitro and in vivo, but only slightly inhibited the fertilization of hamster eggs. The present study was undertaken to examine the effect of rabbit anti-hamster J. ExP. ZOOL.,1 9 5 : 409416.
ovary antiserum on the fertilization of hamster, rat and mouse eggs both in vitro and in vivo. MATERIALS A N D METHODS
Preparation and assay of antisera Antiserum against hamster ovary was prepared according to the procedure reported elsewhere (Tsunoda and Chang, '76c); 2 ml saline extract of ovarian tissues to give a protein concentration of 40 mg/ml was injected with Freunds complete adjuvant into rabbits via the subscapular route; the second and third booster injection was given with Freund's incomplete adjuvant at an interval of 6 to 14 days. Serum was obtained 10 to 15 days after the last injection. Normal serum was taken from the same rabbits before injection. Serum was obtained by centrifugation and heated at 56°C for 30 minutes, and then stored at -20°C. Titration of antiserum was done by the passive haemagglutination test (Herbert '67). The agar-gel double diffusion technique (Ouchterlony, '49) was used to detect precipitatin antibodies (Tsunoda and Chang, '76c). The specificity of antisera was examined 409
410
Y . TSUNODA AND M. C. CHANG
by using sera absorbed with hamster tissue extract. Absorption of antiserum was done according to Isojima and Stepus ('59): one portion of tissue in four portions of phosphate buffer saline (PBS) was homogenized. The homogenate was centrifuged at 10,000 rpm and washed until the supernatant fluid was clear (usually centrifuged more than 6 times). These washed tissue homogenates and antisera in a ratio of 1:1 were allowed to react at 37°C for 60 minutes and then centrifuged at 10,000 rpm for 15 minutes. This procedure was repeated twice to allow for the total absorption of antisera, and was used as absorbed antiserum when no bands were detected in agar-gel plates between absorbed antiserum and absorbing tissue.
In uitro and i n uiuo fertilization The media for fertilization, preparation and treatment of sperm suspensions, method of insemination and examination of eggs for fertilization followed procedures reported elsewhere (Tsunoda and Chang, '75a,b, '76a). To examine the effect of rabbit anti-hamster ovary antiserum on fertilization in rats and mice, 0.3 ml of antiserum was injected intraperitoneally to females subjected to a procedure of superovulation, but mice were allowed to mate, whereas rats were inseminated into the uterus (Tsunoda and Chang, '76a,c). RESULTS
Antigencity of antiserum The passive haemagglutination titre of the antiserum was more than 212.The numbers of precipitin bands formed between the antiserum and tissue extracts in agarTABLE 1
Numbers of precipitin b a n d s f o r m e d b y tissue extracts reacted against rabbit anti-hamster ovary a n t i s e r u m in agar-gel diffusion plates Tissue extracts (antigens)
Hamster
Sperm Serum epididymis Testis ovary Liver Small intestine Lung Uterus Kidney Heart ovary Ovary
+
Rat Mouse
Antiserum to ovary
0 5-6 5-7 8-9 6-8 5-7 6-8 4 3 5-7 5-7 3 4 1-2
gel diffusion plates are presented in table 1. Unabsorbed anti-hamster ovary antiserum showed many precipitin bands against various hamster tissue extracts, but did not react with sperm antigens. Anti-hamster ovary antiserum, however, showed three to four precipitin bands against rat ovarian tissue and one to two bands against mouse ovary (fig. 1). Effect of antiserum o n fertilization of hamster eggs i n uitro Table 2 presents the results concerning fertilization of hamster eggs in vitro. Fertilization failed after adding either 0.1 ml anti-ovary antiserum absorbed with liver and kidney tissue in 0.4 ml preincubated sperm suspensions 30 minutes before the introduction of eggs. However, 34% of the eggs were fertilized after adding anti-ovary antiserum absorbed with ovary as compared with 72% fertilized eggs after adding normal serum. The difference between normal and anti-ovary antiserum absorbed with ovary is significant (P < 0.001). Precipitation on the zona pellucida was heavy in the presence of anti-ovary antiserum or that absorbed with the kidney and liver. By contrast, the precipitate was absent after treatment with anti-ovary antiserum absorbed with the ovary. Eggs with or without follicular cells were incubated in a mixture of Tyrode's solution and anti-ovary antiserum (1:l) for 30 minutes and then introduced into preincubated sperm suspensions. Although 59% to 89% of the eggs were fertilized by treatment of eggs with normal serum, no eggs were fertilized by treatment with anti-ovary antiserum (fig. 2 ) . The results obtained by intrauterine insemination of spermatozoa exposed to antisera are presented in table 3 . After treatment of sperm suspensions with normal rabbit serum, all inseminated females had a high proportion of fertilized eggs. By treatment with anti-hamster ovary antiserum, seven of nine hamsters (78%) had fertilized eggs but the fertilization rate was lower (P < 0.05) than after treatment with control serum (54% vs. 72%). Effect of antiserum o n fertilization of mouse and rat eggs in viuo and in uitro The results obtained by a single intraperitoneal injection of unabsorbed anti-hamster ovary antiserum to female mice and
41 1
ANTI-OVARY ANTISERUM ON FERTILIZATION TABLE 2
In vitro fertilization of hamster eggs in the presence of anti-hamster ovary antiserum No. of eggs penetrated by more than one sperm
Number of eggs Treatment of Penetrated % !
93
%
%
Normal Anti-ovary Anti-ovary absorbed withovary Anti-ovary absorbed with liver and kidney Normal Anti-ovary
46 128
36(78) 0
33(72)
24 (67)
13(36)
+-
41
15(37)
14 (34)
2 (13)
0
-
68 37 42
0 37 (100) 0
0 33(89) 0
37 (100)
30 (81)
+ +
Normal Anti-ovary
109 51
69(63) 0
64(59) 0
36(52)
30(44)
-
Serum
Sperm 2 suspensions
Eggs with follicular cells 3 Denuded eggs 4
Fertilized
Zona precip itation
Examined 1
Polyspermic
Total 5
0
+
Eggs were examined 5-11 hours after exposure to sperm suspensions. 2 0.1 serum was added to 0.4 ml preincubated sperm suspensions 30 minutes before introduction of eggs with follicular cells. 3 Eggs with follicular cells were treated with normal serum or antiserum in Tyrode’s solution (1 : I ) for 30 minutes, then introduced into the sperm suspensions that had beenpreincubated in 20% normal serum in Tyrode’s solution for four hours. 4 Denuded eggs were treated with normal or anti-ovary serum in Tyrode’s solution ( 1 : l ) for 30 minutes before being introduced into sperm suspensions that had been preincubated in 20% normal serum in Tyrode’s solution for 75 minutes. 5 Including eggs with more than one perivitelline spermatozoon and polyspermic eggs. 1
TABLE 3
Fertilization of hamster eggs following intrauterine insemination of epididymal spermatozoa treated with normal serum OT anti-ovary antiserum 1 No. of eggs penetrated by more than one sperm
Number of eggs Serum No. of females inseminated 2
Recovered
Penehated
Fertilized
%
%
%
%
Normal
4 (4)
67
48(72)
48(72)
1 (2)
0
Antiserum
9 (7)
113
63(56)
61 (54)
Total
Polyspermic
3
2 (3) ~~
0 ~
~~
Spermatozoawereexposed tonormal serumor antiserumin themedium(l:4) justbeforeintrauterineinsemination. 2 Figures in parentheses denote the number of females that had fertilized eggs. 3 The same convention as in table 2. 1
rats are presented in table 4. The proportion of fertilized eggs recovered from mice injected with anti-hamster ovary antiserum was significantly lower (P < 0.001) than those treated with normal serum (57% vs. 100%). But zona precipitation was not observed on the eggs. By contrast, the injection with anti-hamster ovary antiserum did not inhibit fertilization of rat eggs following intrauterine insemination (85% vs. 73%). The effect of antiserum to hamster ovary on in vitro ferilization of mouse eggs was tested either by introduction of eggs with follicular cells to preincubated sperm sus-
pension, 30 minutes after adding 0.1 ml antiserum to 0.4 ml sperm suspension, or by treatment of eggs in a mixture of antiserum and medium (1: 1) for 30 minutes before being introduced to preincubated sperm suspension. It was found that 91% of 58 eggs were penetrated by treatment with normal serum and none of 108 eggs was penetrated by treatment with antiserum. DISCUSSION
The results of this study have confirmed the report of Ownby and Shivers (’72) that by the agar-gel double diffusion technique,
412
Y . TSUNODA AND M. C. CHANG TABLE 4
Effect of a single intraperitoneal injection of anti-hamster ovary antiserum on the fertilization of mouse and rat eggs No. of eggs penetrated by more than one sperm
Number of eggs Animal Serum
No. of females used 1
Recovered
Penetrated O/o
Mouse
Rat 1 2
Normal Antiserum
4
Normal Antiserum
Fertilized
I
Total 2
Polyspermic
%
%
Zona precipitation
-
5
81 87
81 (100) 51 (59)
81 (100) 50(57)
8 (10) 5(10)
0
-
3 3
60 62
46 (77)
44 (73) 5 3 (85)
1 1 (24) 9 (17)
1(2) 0
-
53(85)
0
All females had some fertilized eggs The same convention as in table 2.
unabsorbed anti-hamster ovary antiserum contains at least seven salinesoluble antigens. Porter et al. ('70a,b) showed that the guinea pig ovary and testis had common antigens which were located on the sperm acrosome, the zona pellucida, and within atretic ovarian follicles. In the present study, anti-hamster ovary antiserum did not react against hamster spermatozoa but did react with testis and epididymis extract, presumably containing common tissue antigens. In another experiment, antihamster sperm antiserum reacted only against spermatozoa (Tsunoda and Chang, Ms in preparation). Anti-hamster ovary antiserum also showed three to four precipitin bands against rat ovary and one to two bands against mouse ovary (table 1, fig. 1).This finding indicates the presence of a common antigen between hamster, rat and mouse ovarian tissues. We have also confirmed the reports by Shivers et al. ('72) for the hamster and Jilek and Pavlok ('75) for the mouse and have shown that the antibody acted to prevent sperm penetration. Moreover, the degree of fertilization suppression was related to the formation of a precipitate on the surface of the zona pellucida. Using the technique of intrauterine insemination with spermatozoa exposed to antiserum the fertilization rate was slightly depressed (P < 0.05) in the group treated with antiserum compared with those treated with normal serum (table 3). The reason for such slight inhibition of fertilization is not clear, but it may be due to an adverse effect on the spermatozoa. Regarding species-specificity of the antibody, Sacco and Shivers ('73d) reported that
antiserum to rabbit ovary did not crossreact with mouse, rat, guinea pig, or hamster ovary but did cross-react with the ovary of two other breeds of rabbits. Garavagno et al. ('74) reported that anti-hamster ovary antiserum did not cross-react with mouse zona antigen, precipitation on the zonae of mouse and rat eggs was not observed, and that the time required for removal of the zonae was the same after treatment with antiserum. In the present experiment, antiserum to hamster ovary cross-reacted with mouse and rat ovary (table 1, fig. 1), and anti-hamster ovary antiserum inhibited the fertilization of mouse eggs in vivo and in vitro but did not inhibit the fertilization of rat eggs. However, it must be emphasized that although a precipitate was not formed on the zona pellucida, in vitro fertilization of mouse eggs was prevented in the presence of anti-hamster ovary antiserum. A zona precipitate was absent similarly on rat eggs where fertilization wasinhibitedfollowing treatment with anti-rat ovary antiserum (Tsunoda and Chang, '76c) that anti-rat ovary antiserum inhibited the fertilization of rat eggs but strong precipitation was not observed on the zona pellucida. On the other hand, in some species there is a close relationship between the extent of inhibition of fertilization and the formation of precipitation on the zona pellucida of eggs after treatment with anti-ovary serum, thus further investigation concerning this point is required. Jnjection(s) of anti-ovary antiserum can prevent the fertilization after mating or artificial insemination in the mouse (Shahani et al., '72; Jilek and Pavlok, '75), rat and hamster (Tsunoda and Chang, M s in preparation). In this situa-
ANTI-OVARY ANTISERUM ON FERTILIZATION
tion, it seems that inhibition of fertilization was due to the effect of heteroimmunized anti-ovary antiserum on the zona pellucida (Jilek and Pavlok, '75). The finding concerning species-specificityof ovarian tissues confirmed earlier results of Tsunoda and Chang ('76c) that antiserum to rat ovary drastically inhibited the fertilization of rat and mouse eggs but only slightly inhibited the fertilization of hamster eggs in vitro and in vivo. ACKNOWLEDGMENTS
This work was supported by a grant from NICHD (HD 03003) and a grant from the Ford Foundation. One of the authors (M. C. C.) is a recipient of a Research Career Award (HD 18,334). The authors are most grateful to Dr. W. A. Stylos for his advice concerning immunological procedures. Thanks are also due to Mrs. Rose Bartke and Miss Dorothy M. Hunt for assistance, and to Dr. R. H. F. Hunter for reading the manuscript. LITERATURE CITED Garavagno, A., J. Posada, C. Barros and C. A. Shivers 1974 Some characteristics of the zona pellucida antigen in the hamster. J. Exp. Zool., 189: 37-50. Herbert, W. J . 1967 Passive Haemagglutination. In: Handbook of Experimental Immunology. D. M. Weir, ed. Blackwell Scientific Publications, Oxford and Edinburgh, pp. 720-744. Isojima, S., and S. Stepus 1959 Antigenicity of guinea pig testis and ovary. Int. Arch. Allergy Appl. Immunol., 15: 350-359. Jilek, A,, and A. Pavlok 1975 Antibodies against mouse ovaries and their effect on fertilization in vitro and in vivo in the mouse. J. Reprod. Fertil., 42: 377-380. Ouchterlony, 0. 1949 Antigen-antibody reactions in gels. Acta Pathol. Microbiol. Scand., 26: 507515. Ownby, C . L., and C. A. Shivers 1972 Antigens of the hamster ovary and effects of anti-ovary serum on eggs. Biol. Reprod., 6: 3 1 0 4 1 8 . Porter, C. W. 1965 Ovarian antibodies in female guinea pigs. Int. J. Fertil., 10: 257-260.
413
Porter, C. W., D. Highfill and R. Winovich 1970a Guinea pig ovary and testis: Demonstration of common gonad specific antigens in the ovary and testis. Int. J. Fertil., 1 5 : 171-176. - 1970b Guinea pig ovary and testis: Localization of common gonad specific .antigens. Int. J. Fertil., 15: 177-181. Sacco, A. G., and C. A. Shivers 1973a Effect of reproductive tissue-specific antisera on rabbit eggs. Biol. Reprod., 8: 4 8 1 4 9 0 . 1973b Antigens of the rabbit ovary, oviduct and uterus. J. Reprod. Fertil., 32: 403414. 1973c Localization of tissue-specific antigens in the rabbit ovary, oviduct and uterus by the fluorescent antibody technique. J. Reprod. Fertil., 32: 415420. - 1973d Comparison of antigens in the ovary, oviduct and uterus of the rabbit and other mammalian species. J. Reprod. Fertil., 32: 421427. Shahani, S. K., J. R. Padbidri and S. S. Rao 1972 Immunological studies with the reproductive organs, adrenals and spleen of the female mouse. Int. J. Fertil., 17: 161-165. Shivers, C. A. 1974 Immunological interference with fertilization. In: Immunological Approaches to Fertility Control. E. Diczfalusy, ed. Karolinska Inst., Stockholm, pp. 223-244. Shivers, C. A,, A. B. Dudkiewicz, L.E. Franklin and E. N. Fussell 1972 Inhibition of sperm-egg interaction by specific antibody. Science, 178: 1211-1213. Tsunoda, Y., and M. C. Chang 1975a Penetration of mouse eggs in vitro: Optimal sperm concentration and minimal number of spermatozoa. J. Reprod. Fertil., 44: 139-142. - 1975b I n vitro fertilization of rat and mouse eggs by ejaculated sperm and the effect of energy sources on in vitro fertilization of rat eggs. J. Exp. Zool., 193: 79-86. 1976a Spermatozoa of rats and mice treated with a-chlorohydrin and tested for in vitro and in vivo fertilization. J. Reprod. Fertil., in press. 1976b Reproduction i n rats and mice isoimmunized with homogenates of ovary testis with epididymis or sperm suspension. J. Reprod. Fertil., in press. 1976c Effect of anti-rat ovary antiserum on the fertilization of rat, mouse and hamster eggs in vivo and in vitro. Biol. Reprod., in press. (1976d, submitted) Effects of anti-mouse ovary antiserum on fertilization of mouse, rat and hamster eggs i n vivo and in vitro. J. Exp. zool.
PLATE 1 EXPLANATION O F FIGURES
414
1
Precipitin bands formed in Ouchterlony plate when anti-hamster antiserum (center well) reacted to mouse ( I ) , rat ( 2 ) and hamster (3) ovary extract.
2
Denuded hamster eggs treated with anti-hamster ovary serum before being introduced to sperm suspension. The egg after staining was photographed under a phase-contrast microscope. It was not fertilized and there was heavy precipitate on the zona pellucida. X 250.
ANTI-OVARY ANTISERUM O N FERTILIZATION Y. Tsunoda and M. C . Chang
PLATE I
415
ABSTRACT Rabbit antiserum against hamster ovary was examined on agargel diffusion plates against several hamster tissues, and also against rat and mouse ovarian extracts. Unabsorbed anti-hamster ovary antiserum showed eight to nine precipitin bands for hamster ovary and four to eight bands for other tissue extracts, but no bands against sperm antigens. Anti-hamster ovary antiserum also showed three to four bands for rat and one to two bands for mouse ovarian extracts. The present experiments confirmed previous reports for the hamster and mouse that treatment of eggs with anti-ovary antiserum blocked in vitro fertilization and that the extent of the inhibition was related to the formation of a precipitate on the zona pellucida. A single injection of anti-hamster ovary antiserum inhibited fertilization in mice but not in rats. In vitro fertilization of mouse eggs was also inhibited in the presence of such antiserum.
Recent studies on the antigenicity of mammalian ovaries have shown that heteroimmunization with ovarian tissue will yield antibodies (Ownby and Shivers, '72; Sacco and Shivers, '73a,b,c,d; Jilek and Pavlok, '75), and that these antibodies can block sperm penetration of the zona pellucida in vitro in hamsters (Shivers et al., '72), mice (Jilek and Pavlok, '75; Tsunoda and Chang, '76d, submitted) and rats (Tsunoda and Chang, '76c). Inhibition of pregnancy by isoimmunization with ovarian extract in mice was also reported (Porter, '65; Tsunoda and Chang, '76b). Antisera against mouse (Shahani et al., '72; Jilek and Pavlok, '75), rat and hamster ovary (Tsunoda and Chang, Ms in preparation) will also inhibit fertilization in vivo after injection with antiserum. Shivers and his colleagues (Shivers, '74) concluded that the zona antigen in mammalian eggs was species-specific. But recently, Tsunoda and Chang ('76c) found that rabbit anti-rat ovary antiserum drastically inhibited the fertilization of rat and mouse eggs in vitro and in vivo, but only slightly inhibited the fertilization of hamster eggs. The present study was undertaken to examine the effect of rabbit anti-hamster J. ExP. ZOOL.,1 9 5 : 409416.
ovary antiserum on the fertilization of hamster, rat and mouse eggs both in vitro and in vivo. MATERIALS A N D METHODS
Preparation and assay of antisera Antiserum against hamster ovary was prepared according to the procedure reported elsewhere (Tsunoda and Chang, '76c); 2 ml saline extract of ovarian tissues to give a protein concentration of 40 mg/ml was injected with Freunds complete adjuvant into rabbits via the subscapular route; the second and third booster injection was given with Freund's incomplete adjuvant at an interval of 6 to 14 days. Serum was obtained 10 to 15 days after the last injection. Normal serum was taken from the same rabbits before injection. Serum was obtained by centrifugation and heated at 56°C for 30 minutes, and then stored at -20°C. Titration of antiserum was done by the passive haemagglutination test (Herbert '67). The agar-gel double diffusion technique (Ouchterlony, '49) was used to detect precipitatin antibodies (Tsunoda and Chang, '76c). The specificity of antisera was examined 409
410
Y . TSUNODA AND M. C. CHANG
by using sera absorbed with hamster tissue extract. Absorption of antiserum was done according to Isojima and Stepus ('59): one portion of tissue in four portions of phosphate buffer saline (PBS) was homogenized. The homogenate was centrifuged at 10,000 rpm and washed until the supernatant fluid was clear (usually centrifuged more than 6 times). These washed tissue homogenates and antisera in a ratio of 1:1 were allowed to react at 37°C for 60 minutes and then centrifuged at 10,000 rpm for 15 minutes. This procedure was repeated twice to allow for the total absorption of antisera, and was used as absorbed antiserum when no bands were detected in agar-gel plates between absorbed antiserum and absorbing tissue.
In uitro and i n uiuo fertilization The media for fertilization, preparation and treatment of sperm suspensions, method of insemination and examination of eggs for fertilization followed procedures reported elsewhere (Tsunoda and Chang, '75a,b, '76a). To examine the effect of rabbit anti-hamster ovary antiserum on fertilization in rats and mice, 0.3 ml of antiserum was injected intraperitoneally to females subjected to a procedure of superovulation, but mice were allowed to mate, whereas rats were inseminated into the uterus (Tsunoda and Chang, '76a,c). RESULTS
Antigencity of antiserum The passive haemagglutination titre of the antiserum was more than 212.The numbers of precipitin bands formed between the antiserum and tissue extracts in agarTABLE 1
Numbers of precipitin b a n d s f o r m e d b y tissue extracts reacted against rabbit anti-hamster ovary a n t i s e r u m in agar-gel diffusion plates Tissue extracts (antigens)
Hamster
Sperm Serum epididymis Testis ovary Liver Small intestine Lung Uterus Kidney Heart ovary Ovary
+
Rat Mouse
Antiserum to ovary
0 5-6 5-7 8-9 6-8 5-7 6-8 4 3 5-7 5-7 3 4 1-2
gel diffusion plates are presented in table 1. Unabsorbed anti-hamster ovary antiserum showed many precipitin bands against various hamster tissue extracts, but did not react with sperm antigens. Anti-hamster ovary antiserum, however, showed three to four precipitin bands against rat ovarian tissue and one to two bands against mouse ovary (fig. 1). Effect of antiserum o n fertilization of hamster eggs i n uitro Table 2 presents the results concerning fertilization of hamster eggs in vitro. Fertilization failed after adding either 0.1 ml anti-ovary antiserum absorbed with liver and kidney tissue in 0.4 ml preincubated sperm suspensions 30 minutes before the introduction of eggs. However, 34% of the eggs were fertilized after adding anti-ovary antiserum absorbed with ovary as compared with 72% fertilized eggs after adding normal serum. The difference between normal and anti-ovary antiserum absorbed with ovary is significant (P < 0.001). Precipitation on the zona pellucida was heavy in the presence of anti-ovary antiserum or that absorbed with the kidney and liver. By contrast, the precipitate was absent after treatment with anti-ovary antiserum absorbed with the ovary. Eggs with or without follicular cells were incubated in a mixture of Tyrode's solution and anti-ovary antiserum (1:l) for 30 minutes and then introduced into preincubated sperm suspensions. Although 59% to 89% of the eggs were fertilized by treatment of eggs with normal serum, no eggs were fertilized by treatment with anti-ovary antiserum (fig. 2 ) . The results obtained by intrauterine insemination of spermatozoa exposed to antisera are presented in table 3 . After treatment of sperm suspensions with normal rabbit serum, all inseminated females had a high proportion of fertilized eggs. By treatment with anti-hamster ovary antiserum, seven of nine hamsters (78%) had fertilized eggs but the fertilization rate was lower (P < 0.05) than after treatment with control serum (54% vs. 72%). Effect of antiserum o n fertilization of mouse and rat eggs in viuo and in uitro The results obtained by a single intraperitoneal injection of unabsorbed anti-hamster ovary antiserum to female mice and
41 1
ANTI-OVARY ANTISERUM ON FERTILIZATION TABLE 2
In vitro fertilization of hamster eggs in the presence of anti-hamster ovary antiserum No. of eggs penetrated by more than one sperm
Number of eggs Treatment of Penetrated % !
93
%
%
Normal Anti-ovary Anti-ovary absorbed withovary Anti-ovary absorbed with liver and kidney Normal Anti-ovary
46 128
36(78) 0
33(72)
24 (67)
13(36)
+-
41
15(37)
14 (34)
2 (13)
0
-
68 37 42
0 37 (100) 0
0 33(89) 0
37 (100)
30 (81)
+ +
Normal Anti-ovary
109 51
69(63) 0
64(59) 0
36(52)
30(44)
-
Serum
Sperm 2 suspensions
Eggs with follicular cells 3 Denuded eggs 4
Fertilized
Zona precip itation
Examined 1
Polyspermic
Total 5
0
+
Eggs were examined 5-11 hours after exposure to sperm suspensions. 2 0.1 serum was added to 0.4 ml preincubated sperm suspensions 30 minutes before introduction of eggs with follicular cells. 3 Eggs with follicular cells were treated with normal serum or antiserum in Tyrode’s solution (1 : I ) for 30 minutes, then introduced into the sperm suspensions that had beenpreincubated in 20% normal serum in Tyrode’s solution for four hours. 4 Denuded eggs were treated with normal or anti-ovary serum in Tyrode’s solution ( 1 : l ) for 30 minutes before being introduced into sperm suspensions that had been preincubated in 20% normal serum in Tyrode’s solution for 75 minutes. 5 Including eggs with more than one perivitelline spermatozoon and polyspermic eggs. 1
TABLE 3
Fertilization of hamster eggs following intrauterine insemination of epididymal spermatozoa treated with normal serum OT anti-ovary antiserum 1 No. of eggs penetrated by more than one sperm
Number of eggs Serum No. of females inseminated 2
Recovered
Penehated
Fertilized
%
%
%
%
Normal
4 (4)
67
48(72)
48(72)
1 (2)
0
Antiserum
9 (7)
113
63(56)
61 (54)
Total
Polyspermic
3
2 (3) ~~
0 ~
~~
Spermatozoawereexposed tonormal serumor antiserumin themedium(l:4) justbeforeintrauterineinsemination. 2 Figures in parentheses denote the number of females that had fertilized eggs. 3 The same convention as in table 2. 1
rats are presented in table 4. The proportion of fertilized eggs recovered from mice injected with anti-hamster ovary antiserum was significantly lower (P < 0.001) than those treated with normal serum (57% vs. 100%). But zona precipitation was not observed on the eggs. By contrast, the injection with anti-hamster ovary antiserum did not inhibit fertilization of rat eggs following intrauterine insemination (85% vs. 73%). The effect of antiserum to hamster ovary on in vitro ferilization of mouse eggs was tested either by introduction of eggs with follicular cells to preincubated sperm sus-
pension, 30 minutes after adding 0.1 ml antiserum to 0.4 ml sperm suspension, or by treatment of eggs in a mixture of antiserum and medium (1: 1) for 30 minutes before being introduced to preincubated sperm suspension. It was found that 91% of 58 eggs were penetrated by treatment with normal serum and none of 108 eggs was penetrated by treatment with antiserum. DISCUSSION
The results of this study have confirmed the report of Ownby and Shivers (’72) that by the agar-gel double diffusion technique,
412
Y . TSUNODA AND M. C. CHANG TABLE 4
Effect of a single intraperitoneal injection of anti-hamster ovary antiserum on the fertilization of mouse and rat eggs No. of eggs penetrated by more than one sperm
Number of eggs Animal Serum
No. of females used 1
Recovered
Penetrated O/o
Mouse
Rat 1 2
Normal Antiserum
4
Normal Antiserum
Fertilized
I
Total 2
Polyspermic
%
%
Zona precipitation
-
5
81 87
81 (100) 51 (59)
81 (100) 50(57)
8 (10) 5(10)
0
-
3 3
60 62
46 (77)
44 (73) 5 3 (85)
1 1 (24) 9 (17)
1(2) 0
-
53(85)
0
All females had some fertilized eggs The same convention as in table 2.
unabsorbed anti-hamster ovary antiserum contains at least seven salinesoluble antigens. Porter et al. ('70a,b) showed that the guinea pig ovary and testis had common antigens which were located on the sperm acrosome, the zona pellucida, and within atretic ovarian follicles. In the present study, anti-hamster ovary antiserum did not react against hamster spermatozoa but did react with testis and epididymis extract, presumably containing common tissue antigens. In another experiment, antihamster sperm antiserum reacted only against spermatozoa (Tsunoda and Chang, Ms in preparation). Anti-hamster ovary antiserum also showed three to four precipitin bands against rat ovary and one to two bands against mouse ovary (table 1, fig. 1).This finding indicates the presence of a common antigen between hamster, rat and mouse ovarian tissues. We have also confirmed the reports by Shivers et al. ('72) for the hamster and Jilek and Pavlok ('75) for the mouse and have shown that the antibody acted to prevent sperm penetration. Moreover, the degree of fertilization suppression was related to the formation of a precipitate on the surface of the zona pellucida. Using the technique of intrauterine insemination with spermatozoa exposed to antiserum the fertilization rate was slightly depressed (P < 0.05) in the group treated with antiserum compared with those treated with normal serum (table 3). The reason for such slight inhibition of fertilization is not clear, but it may be due to an adverse effect on the spermatozoa. Regarding species-specificity of the antibody, Sacco and Shivers ('73d) reported that
antiserum to rabbit ovary did not crossreact with mouse, rat, guinea pig, or hamster ovary but did cross-react with the ovary of two other breeds of rabbits. Garavagno et al. ('74) reported that anti-hamster ovary antiserum did not cross-react with mouse zona antigen, precipitation on the zonae of mouse and rat eggs was not observed, and that the time required for removal of the zonae was the same after treatment with antiserum. In the present experiment, antiserum to hamster ovary cross-reacted with mouse and rat ovary (table 1, fig. 1), and anti-hamster ovary antiserum inhibited the fertilization of mouse eggs in vivo and in vitro but did not inhibit the fertilization of rat eggs. However, it must be emphasized that although a precipitate was not formed on the zona pellucida, in vitro fertilization of mouse eggs was prevented in the presence of anti-hamster ovary antiserum. A zona precipitate was absent similarly on rat eggs where fertilization wasinhibitedfollowing treatment with anti-rat ovary antiserum (Tsunoda and Chang, '76c) that anti-rat ovary antiserum inhibited the fertilization of rat eggs but strong precipitation was not observed on the zona pellucida. On the other hand, in some species there is a close relationship between the extent of inhibition of fertilization and the formation of precipitation on the zona pellucida of eggs after treatment with anti-ovary serum, thus further investigation concerning this point is required. Jnjection(s) of anti-ovary antiserum can prevent the fertilization after mating or artificial insemination in the mouse (Shahani et al., '72; Jilek and Pavlok, '75), rat and hamster (Tsunoda and Chang, M s in preparation). In this situa-
ANTI-OVARY ANTISERUM ON FERTILIZATION
tion, it seems that inhibition of fertilization was due to the effect of heteroimmunized anti-ovary antiserum on the zona pellucida (Jilek and Pavlok, '75). The finding concerning species-specificityof ovarian tissues confirmed earlier results of Tsunoda and Chang ('76c) that antiserum to rat ovary drastically inhibited the fertilization of rat and mouse eggs but only slightly inhibited the fertilization of hamster eggs in vitro and in vivo. ACKNOWLEDGMENTS
This work was supported by a grant from NICHD (HD 03003) and a grant from the Ford Foundation. One of the authors (M. C. C.) is a recipient of a Research Career Award (HD 18,334). The authors are most grateful to Dr. W. A. Stylos for his advice concerning immunological procedures. Thanks are also due to Mrs. Rose Bartke and Miss Dorothy M. Hunt for assistance, and to Dr. R. H. F. Hunter for reading the manuscript. LITERATURE CITED Garavagno, A., J. Posada, C. Barros and C. A. Shivers 1974 Some characteristics of the zona pellucida antigen in the hamster. J. Exp. Zool., 189: 37-50. Herbert, W. J . 1967 Passive Haemagglutination. In: Handbook of Experimental Immunology. D. M. Weir, ed. Blackwell Scientific Publications, Oxford and Edinburgh, pp. 720-744. Isojima, S., and S. Stepus 1959 Antigenicity of guinea pig testis and ovary. Int. Arch. Allergy Appl. Immunol., 15: 350-359. Jilek, A,, and A. Pavlok 1975 Antibodies against mouse ovaries and their effect on fertilization in vitro and in vivo in the mouse. J. Reprod. Fertil., 42: 377-380. Ouchterlony, 0. 1949 Antigen-antibody reactions in gels. Acta Pathol. Microbiol. Scand., 26: 507515. Ownby, C . L., and C. A. Shivers 1972 Antigens of the hamster ovary and effects of anti-ovary serum on eggs. Biol. Reprod., 6: 3 1 0 4 1 8 . Porter, C. W. 1965 Ovarian antibodies in female guinea pigs. Int. J. Fertil., 10: 257-260.
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Porter, C. W., D. Highfill and R. Winovich 1970a Guinea pig ovary and testis: Demonstration of common gonad specific antigens in the ovary and testis. Int. J. Fertil., 1 5 : 171-176. - 1970b Guinea pig ovary and testis: Localization of common gonad specific .antigens. Int. J. Fertil., 15: 177-181. Sacco, A. G., and C. A. Shivers 1973a Effect of reproductive tissue-specific antisera on rabbit eggs. Biol. Reprod., 8: 4 8 1 4 9 0 . 1973b Antigens of the rabbit ovary, oviduct and uterus. J. Reprod. Fertil., 32: 403414. 1973c Localization of tissue-specific antigens in the rabbit ovary, oviduct and uterus by the fluorescent antibody technique. J. Reprod. Fertil., 32: 415420. - 1973d Comparison of antigens in the ovary, oviduct and uterus of the rabbit and other mammalian species. J. Reprod. Fertil., 32: 421427. Shahani, S. K., J. R. Padbidri and S. S. Rao 1972 Immunological studies with the reproductive organs, adrenals and spleen of the female mouse. Int. J. Fertil., 17: 161-165. Shivers, C. A. 1974 Immunological interference with fertilization. In: Immunological Approaches to Fertility Control. E. Diczfalusy, ed. Karolinska Inst., Stockholm, pp. 223-244. Shivers, C. A,, A. B. Dudkiewicz, L.E. Franklin and E. N. Fussell 1972 Inhibition of sperm-egg interaction by specific antibody. Science, 178: 1211-1213. Tsunoda, Y., and M. C. Chang 1975a Penetration of mouse eggs in vitro: Optimal sperm concentration and minimal number of spermatozoa. J. Reprod. Fertil., 44: 139-142. - 1975b I n vitro fertilization of rat and mouse eggs by ejaculated sperm and the effect of energy sources on in vitro fertilization of rat eggs. J. Exp. Zool., 193: 79-86. 1976a Spermatozoa of rats and mice treated with a-chlorohydrin and tested for in vitro and in vivo fertilization. J. Reprod. Fertil., in press. 1976b Reproduction i n rats and mice isoimmunized with homogenates of ovary testis with epididymis or sperm suspension. J. Reprod. Fertil., in press. 1976c Effect of anti-rat ovary antiserum on the fertilization of rat, mouse and hamster eggs in vivo and in vitro. Biol. Reprod., in press. (1976d, submitted) Effects of anti-mouse ovary antiserum on fertilization of mouse, rat and hamster eggs i n vivo and in vitro. J. Exp. zool.
PLATE 1 EXPLANATION O F FIGURES
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1
Precipitin bands formed in Ouchterlony plate when anti-hamster antiserum (center well) reacted to mouse ( I ) , rat ( 2 ) and hamster (3) ovary extract.
2
Denuded hamster eggs treated with anti-hamster ovary serum before being introduced to sperm suspension. The egg after staining was photographed under a phase-contrast microscope. It was not fertilized and there was heavy precipitate on the zona pellucida. X 250.
ANTI-OVARY ANTISERUM O N FERTILIZATION Y. Tsunoda and M. C . Chang
PLATE I
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