- Email: [email protected]
The effect on fertility of immunizing female sheep with ram sperm acrosin and hyaluronidase
Journal of Reproductive Immunology, 1 (1979) 61-73 © Elsevier/North-Holland Biomedical Press
61
THE E F F E C T ON F E R T I L I T Y OF IMMUNIZING FEMALE SHEEP WITH RAM SPERM ACROSIN AND HYALURONIDASE
D.B. MORTON * and P.A. McANULTY ** Strangeways Research Laboratory, Worts"Causeway, Cambridge CB1 4RN, U.K. (Received 10 November 1978; accepted 20 November 1978)
Female sheep were injected with highly purified and partially purified preparations of ram sperm acrosin and hyaluronidase. The fertility and immune response of the sheep were monitored. Fertility was not significantly reduced in any single group, though a positive correlation was observed between high ant~ody titres against acrosin and reduced fertility. Studies on the direct action of sera from the ewes on ejaculated ram spermatozoa did not show any evidence of sperm agglutination or immobilization. Similar studies with denuded spermatozoa (detergent induced 'acrosome reaction') sometimes resulted in agglutination and enzyme inhibition was also seen; there was no correlation between any of these parameters and pregnancy. INTRODUCTION Females injected with spermatozoa from males o f the same species show a reduction in fertility (Katsh, 1959; Metz, 1973). The cause o f the infertility is thought to be correlated with the immune response generated against specific, although as y e t unknown, sperm antigens. Goldberg (1974) has obtained some reduction (40%) in fertility by immunization with the sperm-specific enzyme lactate dehydrogenase X, so presumably other sperm antigens must be involved. The proposed roles for hyaluronidase and acrosin in sperm penetration between the cumulus cells and through the zona pellucida o f the egg, respectively, make these enzymes potential candidates, as antibody-mediated inhibition of enzyme activity might prevent fertilization. The following experiments were therefore carried out to determine if ram sperm hyaluronidase and acrosin could, on their own or together, act as infertility provoking immunogens in sheep. The sheep was chosen as the experimental species since sufficient numbers of spermatozoa could be amassed to provide the relatively large amount of purified enzymes needed to immunize the ewes (the yield o f purified enzyme is approximately 1 mg from 101 o spermatozoa).
* Present address: Department of Anatomy, Medical Sciences Building, Leicester University, Univer. sity Road, Leicester LE1 7RH, U.K. ** Present address: Department of Reproductive Studies, Life Science Research, Stock, Essex CM4 9PE, U.K.
62 MATERIALS AND METHODS
Animals 64 ewes of mixed breed, age and parity were used in the experiments. For most of the experimental period they were kept in open sheds, fed on hay and concentrates, and when sufficient grass was available, grazed during the day.
Fertility asssessment The study was carried out during two breeding seasons. During the first season, 19741975, fertility was measured by ovum cleavage, i.e. eggs were flushed from the oviduct or uterus 3 - 7 days after mating and examined for cleavage. The fertilization rate was expressed as the proportion of ewes with fertilized eggs. During the 1975-1976 season, ewes carried their lambs to term and fertility was measured by the lambing rate, i.e. the proportion of ewes bearing lambs.
Antigens used and the immunization programme Ewes were injected intramuscularly with an emulsion of Freund's Complete Adjuvant (FCA) and a solution of antigen in phosphate-buffered saline (PBS) ( in the ratio of 1 : 1) three times at 2-weekly intervals. The ewes were then run with the ram from two weeks after the third injection. The methods for preparing the sperm antigens are those used in the purification of ram sperm acrosin and hyaluronidase and have been described previously (Morton, 1976, 1977a, b). The descriptions below are brief outlines of the methods used. Because only small amounts of the highly purified enzymes were available, there was a possibility that not enough protein would be injected to ensure an adequate immune response. Consequenfly, two other groups of sheep were injected with partially purified solutions of enzyme which, although less pure, contained greater amounts of enzyme protein. Ewes were divided into seven groups and given one of the following antigens at each injection: (1) Partially purified hyaluronidase (DE-S): A 0.1% Triton X-100 aqueous extract of frozen-thawed ram semen was adjusted to pH 4.0, centrifuged, and the supernatant dialysed against 0.01 M Tris-HC1, pH 7.6. It was then chromatographed on diethylaminoethyl cellulose (DE-52, Whatman) equilibrated in the same buffer and the unabsorbed protein used in the experiments after dialysis against PBS. This peak contained approximately 70% of the total hyaluronidase activity in the sperm extract and had a specific activity of approximately 0.4 U/rag. On average, each ewe was given 0.5 mg of partially purified hyaluronidase at each injection in 2 ml of emulsion. (2) Hyaluronidase: Highly purified enzyme was prepared with a specific activity of between 10-20 U/mg. However this was still impure, as one other band was demonstrable on electrophoresis and also on immunodiffusion with a polyvalent antiserum. The contaminant was finally separated on immunodiffusion plates by running the enzyme against a monospecific antiserum to hyaluronidase raised in rabbits. Troughs were cut in agar plates and filled alternately with purified hyaluronidase or rabbit antiserum. The
63 resulting single precipitin lines were cut out of the gel, thoroughly washed with PBS and then mixed with adjuvant and emulsified by homogenisation with an Ultra-Turrax. This type of preparation had been used previously to raise specific antisera in rabbits and pigs. (3) Partially purified acrosin (CM-A3): A 2 M MgC12 extract was prepared from ram spermatozoa which had previously been extracted with 0.1% Triton X-100. The magnesium chloride extract was dialysed against 0.005 M sodium formate, pH 3.0, and then chromatographed on carboxymethyl cellulose (CM-52, Whatman) equilibrated with 0.04 M sodium acetate, pH 5.0. Acrosin was eluted by a 0.35 M NaC1 gradient, pooled and dialysed against PBS. The specific activity of this solution was approximately 4 U/mg with benzoyl DL-arginine-p-nitroanilide-HC1 as substrate. At each injection ewes were given 2.5-3 mg of protein in 2 ml of emulsion. (4) Acrosin: Highly purified acrosin with a specific activity of between 25 and 30 U/mg was prepared and dialysed against PBS. At each injection 121 gg of protein was given in 1 ml of emulsion. (5) Acrosin and hyaluronidase: Solutions of highly purified acrosin and hyaluronidase were used (see Treatments 2 and 4 above) and at each injection ewes received 121/gg of acrosin and 244/lg of hyaluronidase in 2 ml of emulsion. In this instance the hyaluronidase was not injected as an immune complex. [6J Control I: Sheep were injected with 1 ml PBS in 2 ml of emulsion. (7) Control IL" Sheep were injected with 0.5 mg purified rabbit IgG (prepared by ammonium sulphate precipitation and ion-exchange chromatography on DE-52) in PBS and given in 2 ml of emulsion. [8) Control III: During 1975-1976, the lambing rate of the main flock on the farm was calculated.
Measurement of immune response The immune response to the two defined antigens, hyaluronidase and acrosin, was measured. This was done either on immunodiffusion plates (I.D.) or by a two-step radioimmunoassay (R.I.A.) with labelled antigen. For the radioimmunoassay the two enzymes were radio-labelled with 1251 by the method described by Bolton and Hunter (1973). In the assay the test serum was serially diluted with an 80/2g/ml solution of sheep 7-globulin (prepared by ammonium sulphate fractionation of normal sheep serum) in 0.1 M phosphate buffer (PH 6.0) containing 0.5 M NaC1, 0.005 M EDTA, 0.01 M glycine and 0.2% Brij 35 (a non-ionic detergent). 50 pl of test serum was incubated with 10/21 of 12SI-labelted acrosin (specific radioactivity = 165 X 10 -9 Ci//2g, 67.5 ng in assay) or hyaluronidase (specific radioactivity = 41.8 X 10 -9 Ci//gg, 41.7 ng in assay) for 24 h at 37°C. All the sheep 7-globulin was then precipitated out by use of an antiserum raised in rabbits against sheep 7-globulin. This second step was carefully quantitated so that 100% of the sheep 7-globulin (at a concentration of 80/2g/ ml) was precipitated by the rabbit antibody. We did not differentiate between the 7globulin classes. 50/al of the rabbit antiserum (diluted 3 : 2 with the diluting buffer described above) were added, and after 24 h at 37°C the mixture was centrifuged at 8000 g for 15 min. The amount of unbound labelled enzyme remaining in solution was measured in a scintillation counter and so the amount precipitated could then be calculated,
64
?
25
7B (DE-S, ++ve I D)
(DE-S~+ve ID)
/ /
_c
~
/
15B (Control L-re ] D)
-~ 15 r'---~O~o~
o
i
E <
I
I
I
I
I
10-6
10-5
10-4
10-3
10-2
I
10-1
Serum dilution Fig. 1. Curves obtained with typical sera in hyaluronidase radioimmunoassay. Precipitation of 12si_ labelled hyaluronidase by serial dilutions of sera showing strong (e), weak (o) or no (A) precipitin lines on I.D. plates. The blank (. . . . . . ) was an 80/~g/ml solution of sheep y-globulin in buffer.
after allowing for non-specific precipitation by the sheep "r-globulin solution. There was an appreciable amount of non-specific binding by the "y-globulin solution and the non-immune controls gave comparable values (see Fig. 1). The results were expressed as the amount of labelled antigen precipitated at a serum dilution of 1/1000. The sera for acrosin radioimmunoassay were pretreated to acidic conditions (pH 4.0 with formate buffer for 30 rain) to remove any innate inhibitor activity which might compete with antibody binding (e.g. al-proteinase inhibitor and an c~2-macroglobulin). Furthermore, as acrosin is a proteolytic enzyme and can degrade y-globulin (see Fig. 2), immunoassays were carried out in the presence o f the acrosin inhibitor, benzamidine (0.02 M).
Assay o f sperm agglutinating and sperm immobilizing activities in serum from immunized ewes The antibody fraction was prepared from sera from ewes with a demonstrable immune response to the enzymes, judged by radioimmunoassay or immunodiffusion tests. The 7globulins were prepared by precipitating the sera three times with 0.67 serum volumes of 4 M ammonium sulphate (adjusted to pH 7). The final precipitate was made up to 0.5 serum volumes and the solution was exhaustively dialysed against Medium S (Harrison et al., 1978). The major protein component of this preparation would be IgG. 20 /A of freshly ejaculated ram semen was mixed with 1 ml of Medium S and 100/11 of this diluted semen was mixed with 5 #1 of fractionated sheep serum and incubated at 37°C. Aliquots
65 ~ii~ i i!~
4;; ~i~i
iiiii i! ~iii
i ii~!/~/~L ~
Uii!iii!!~!!i~
;; iii!!ii~i~iiii;i
IgG-~
~!'ili!~
!~!!!iii'ii!iii!i;
!!i!!~tlt
ii !i! ~
~-H
--L
1
2
3
4
5
6
Fig. 2. The proteolytic effect of acrosin on sheep IgG. Gels 1 and 4: sheep lgG alone; gels 2 and 5: IgG with acrosin; gels 3 and 6: IgG, acrosin and benzamidine. Gels 1 - 3 are non-reduced, gels 4 - 6 are reduced. UnlabeUed arrows point to the products of digestion. H and L are heavy and light chains, respectively.
were taken at 2, 10 and 30 min later, examined under a microscope and scored for sperm agglutination and motility. As there is evidence that acrosin and hyaluronidase are not normally exposed on the sperm surface but are located within the acrosome (Morton, 1976) a second series of experiments was conducted to investigate the effect of these sera on sperm agglutination using denuded spermatozoa. These denuded spermatozoa have their plasma membrane over the sperm head and the outer acrosomal membrane removed by freeze-thawing and detergent treatment (see Morton, 1977b) and are a model for acrosome-reacted spermatozoa. It is at this time, when spermatozoa have undergone the acrosome reaction, that one might expect antibodies to the enzymes to have their maximum effect.
Assay o f antibody-mediated enzyme inhibiting capacity of ewe sera using denuded spermatozoa Approximately 50 million detergent-treated ram spermatozoa, in 20 pl PBS were preincubated with 100 pl of fractionated ewe serum for 30 rnin. Aliquots of 50/A were then examined viscometrically for acrosin and hyaluronidase activities at 40°C. Hyaluronidase activity was measured using a 2 mg/ml solution of hyaluronic acid in PBS whereas acrosin activity was measured by using a 2 mg/ml solution of purified cartilage proteoglycan in the presence of 50 /ag/ml polyphloretin phosphate (both in PBS buffer). The poly-
66 phloretin phosphate will inhibit any hyaluronidase action on cartilage proteoglycan so that only the effect of acrosin on the polypeptide backbone of the macromolecule is measured. After 90 min incubation corrected flow times for substrate in the presence or absence of serum-treated spermatozoa were compared to evaluate the degree of inhibition: % inhibition = (Ts - Tb)/(Tc - Tb) × 100 where Tb = flow time for buffer (PBS) alone, T c = flow time for substrate mad buffer after 151 h, and T s = flow time for substrate and buffer after incubation with serumtreated spermatozoa for 1-~ h. Thus when no degradation of the substrate has occurred the flow times should be the same, giving 100% inhibition. But if the membrane-bound enzymes on the spermatozoa have degraded either of these macromolecules, then the viscosity should drop and the flow time should correspondingly decrease, giving a figure of less than 100%. The proteolytic action o f acrosin on IgG
Sheep IgG (1.34 mg/ml) was incubated at 37°C for 72 h either alone or with highly purified ram sperm acrosin (83 U/ml), or with acrosin and 0.01 M benzamidine (see Fig. 2). The products were then run on sodium dodecyl sulphate disc gels (Neville, 1971) under non-reduced and reduced conditions (with 2-mercaptoethanol). The inhibition o f ram sperm acrosin and hyaluronidase activities by immune and control globulins from rabbit sera
Highly purified preparations of ram sperm acrosin and hyaluronidase were prepared as described above. A constant amount of acrosin, 0.776 mU (specific activity 30 U/mg), was incubated with varying amounts of rabbit antibody (Fab monomer prepared from either a specific immune antiserum or a control serum) at 37°C for 30 min and then assayed for activity with benzoyl DL-arginine-p-nitroanilide. Similarily, 0.84 mU of hyaluronidase (specific activity 12 U/mg) was incubated with varying amounts of IgG (prepared from either a specific immune antiserum or a control serum) for 30 min at 40°C and then assayed for activity on hyaluronic acid. RESULTS A summary of the fertility results obtained in the experiment is given in Table I. It can be seen that no one group had significantly lowered fertility when analysed by a X2 test, although the partially purified acrosin group, CM-Aa, showed most tendency to deviate from the predicted value. The farm flock was not included in the statistical analysis as they had been handled far less during the experiments and had not been injected. In point of fact, its inclusion would have made no essential difference to the results. The immune response of the animals in any one group was variable. In general, animals showing a precipitin line on immunodiffusion would have a relatively high titre by radioimmunoassay (see Fig. 1 and Table 2). However, radioimmunoassay showed up more positives, partly as it was more sensitive, but also because this technique would have
67
~o~
O
o
~'~ ~1 ~il:~
~
~
o
~
i
~A
68 detected non-precipitating as well as precipitating antibodies. There were higher titres of antibody to acrosin and hyaluronidase in the ewes immunised with the partially purified enzyme preparations (Table 2) and this probably reflects the greater amount of protein in these preparations, giving a superior immune response. The immune response data from ewes in the two acrosin vaccinated groups (i.e. purified and partially purified) were compared to see if non-pregnant sheep had higher titres against acrosin than those that were pregnant. We found an average titre of 14.2 -+ 2.6 S.E.M. (n = 24; range 0 - 4 8 . 6 ) in the pregnant ewes compared with 28.8 +-8.1 (n = 6; range 0 . 7 - 5 0 . 1 ) in the non-pregnant ewes and the difference was statistically significant (Student's t t e s t , P < 0.01). The direct effect of circulating antibodies from the immunized ewes was examined by monitoring sperm motility, sperm agglutination and sperm enzyme inhibition (see Table 3). Motility and agglutination of intact ejaculated ram spermatozoa in the presence of antibody were unaffected whereas when denuded spermatozoa were used agglutination was observed and the degree of agglutination showed some correlation with antibody titre to acrosin. Furthermore, it was observed from the viscometry studies that the antibody fraction from some sera inhibited acrosin or hyaluronidase activities but it did not seem to be related to the titre. There was no difference between the treatment of denuded spermatozoa with saline and control serum, showing that endogenous inhibitors in the serum had no effect on the activity of the membrane-bound enzymes.
TABLE 2 The immune response of ewes treated with various sperm antigens measured against acrosin and hyaluronidase by radioimmunoassay (R.I.A.) and immunodiffusion(I.D.) Group
Hyaluronidase
Acrosin
R.I.A. a
R.I.A. b
I.D. c
R.I.A. a
R.I.A. b
I.D. c
Control I
0/3
0.0 + 0.0
0/8
3/6
0/8
Control II
1/3
0/3
0/3
DE-S
6/7
6/7
n.t.
n.t.
n.t.
Hyaluronidase
2/4
1/4
n.t.
n.t.
n.t.
CM-A3
n.t.
0.8 ± 0.8 (0.0-2.5) 5.1 +- 3.2 (0.0-23.5) 2.4 ± 1.6 (0.0-6.8) n.t.
0.2 ± 0.1 (O.O-0.4) 0.0 ± 0.0
n.t.
13/18
Acrosin
n.t.
n.t.
n.t.
14/19
Hyaluronidase + acrosin
2/2
5.1 +- 2.1 (3.0 and 7.2)
2/2
2/2
20.6 ± 4.3 6/18 (0.0-50.1) 8.8 ± 2.2 5/19 (0.0-34.1) 25.0 + 16.4 1/2 (8.6 and 41.4)
n.t., not tested. a Number of animals positive at serum dilution of 10-3. b Labelled antigen precipitated at a serum dilution of 10 -3 (ng -+S.E.M. with range in brackets). c Number of animals with precipitating antibodies.
0/3
69 e~
~
Z ~ Z Z
~
~
o
?,
{
e-
e,
~
I I
+~
~
++ +
+
+
+
,
+
÷
+
+÷I
0
Ill
II
I I I I
II
II
o 0 e~ 0 o
L~
0
E
0
2 0
bl
O
0
I
I
0
o
"
5
Z
I
<<
O
0
<+
.
o,~
70
0
0
20
40 60 80 100 120 ~g antibody/mu enzyme
I "~
I
140 160
Fig. 3. The inhibition of ram sperm acrosin and hyaluronidase activities by immune and control globulins from rabbit sera. a, acrosin with specific Fab ant~ody; A, acrosin with control Fab antibody; o, hyaluronidase with specific IgG antibody; •, hyalurortidase with control IgG antibody.
Overall there was no obvious correlation between pregnancy and sperm agglutinating, sperm immobilising or enzyme inhibiting activities of the sera. The experiments on the proteolytic action of acrosin on sheep IgG (Fig. 2) showed that over the period of the radioimmunoassay significant digestion of the IgG could take place and that the presence of 10 mM benzamidine prevented this effect. Whether the antigen binding capacity of the antibody was reduced was not determined but benzamidine was routinely used in the radioimmunoassay. It was possible to inhibit enzyme activity completely with antibodies isolated from specific antisera raised in rabbits against the purified enzymes (Fig. 3). In addition to the experiments reported here, we have shown that as with acrosin, specific monomeric Fab fragments will also inhibit hyaluronidase activity but the inhibition was not complete in the range tested (126 #g Fab/mU hyaluror.idase gave 80% inhibition). DISCUSSION The reduced fertility of females injected with spermatozoa is thought to be due to the immunological response to sperm antigens. The way in which the immune response has its effect on fertility is unknown. One obvious possibility is that the circulating antibodies will be secreted into the genital tract and, once there, have a variety of detrimental effects on spermatozoa. For instance, sperm agglutination could arise by antibody cross-linking surface antigens or sub-surface antigens which subsequently become exposed (such as acrosin after the acrosome reaction). The experiments on the effect of serum antibody fraction on ram spermatozoa showed that the spermatozoa denuded of the outer membranes were agglutinated, whereas intact spermatozoa were not (Table 3). This provides further evidence that acrosin and hyaluronidase are not superficial sperm antigens but are probably positioned, in part anyway,
71 on the inner acrosomal membrane; they may also be contained within the acrosomal contents. Thus, spermatozoa having undergone the acrosome reaction could be agglutinated by the antibody in the female tract, and the resultant aggregations might well interfere with their ability to reach the site of fertilization and to penetrate the egg. Spermatozoa might also be immobilized not only through agglutination but also through the action of antibody with complement, resulting in sperm death (Isojima, 1973; Russo and Metz, 1974). In the present experiments no evidence was obtained for any sperm immobilizing effect on intact cells, presumably as the antigens were not superficial. However, sperm immobilization might still occur with acrosome-reacted spermatozoa or in a complement-sufficient system. There is some evidence that antibody coating may render the sperm cell more susceptible to phagocytosis by neutrophils or macrophages (Symons, 1967) and to lymphocyte attack. This raises the possibility of cell-mediated immunity but there seems to be little evidence for lymphocyte migration into the lumen of the reproductive tract. Cellmediated immunity has been described associated with graft rejection and pregnancies (see W.H.O. Workshop on Immunological Response of the Female Reproductive Tract, 1976). A further way antibodies might act is to prevent spermatozoa from fertilizing the egg by inhibiting sperm enzymes needed for penetration through the various layers surrounding the egg. We have shown that some ewes produced antibodies which were able to inhibit acrosin and hyaluronidase activities. Interestingly, however, there is obviously some diversity between antibodies since, irrespective of the amount of antibody present, some sera give greater inhibition than others. Presumably this is due to a greater proportion of the antibodies binding close to the active site of the enzyme. This may be attributed to the genetic diversity in immune response between individuals of the same species treated under identical immunization schedules. In separate experiments it was also shown that rabbit antibodies, both IgG and Fab fragments, specific for sheep acrosin or hyaluronidase are able to inhibit the activity of these enzymes (Fig. 3). Bivalent IgG could cause apparent inhibition of enzyme activity simply through precipitating the enzyme out of solution. But the experiments with the monovalent Fab fragment, which would leave the enzyme in solution, show that this is not the case and the inhibition is more likely to be due to antibodies binding close to the active site, thus impeding substrate-enzyme interaction (see Cinader, 1966; Arnon, 1974). Inhibition of enzyme activity due to antibody has usually been assessed with soluble enzyme substrates. In the case of spermatozoa digesting a path through the zona pellucida, however, the 'native' substrate is insoluble as well as the enzyme. Consequently, it has to be considered that antibody binding farther away from the active site, or even antibody binding to other surface antigens in the acrosomal region, might prevent substrate-enzyme interaction and thereby inhibit sperm penetration of the egg. The foregoing discussion assumes that antibodies will be produced against 'the sperm antigen(s)', i.e. they are immunogenic. And secondly, that these antibodies will be secreted intact into the female reproductive tract (McAnulty and Morton, 1978). From the results shown in Table 2, it can be seen that circulating antibodies to hyaluronidase were found in both the I.D. and the R.I.A. test systems but no significant drop in fertility
72 was observed (Table 1). The lack of an effect on fertility after injection of this enzyme could be due to the nonessential role of hyaluronidase in fertilization or to an insufficient rate of antibody secretion into the tract in relation to the amount of sperm antigen present or passing through. Sperm transport in the sheep (and the human) can be considered to be a continual process with spermatozoa constantly passing from the cervix through the uterus and oviduct into the abdominal cavity. Thus, small amounts of antibody would be 'mopped up' by the early spermatozoa at the site of fertilization, leaving a relatively 'antibody-free' milieu for those arriving subsequently. However, it is relevant to note when considering hyaluronidase in the sheep that the cumulus cells are lost very shortly after ovulation (Moor, R.M., personal communication) and so in this species at least there may be no cumulus or corona cell barrier to sperm penetration. This raises the question of the role played by ram sperm hyaluronidase in fertilization. A similar analysis of the immune response for acrosin showed that, in general, antibody levels were higher than for hyaluronidase and there was a correlation between the level of circulating antibody and fertility; sheep with higher antibody titres to acrosin showed significantly lowered fertility. This suggests that sufficient anti-acrosin antibody was secreted into the tract but the effect of this is not clear. We have shown that purified Fab fragments of IgG from a specific rabbit antiserum to acrosin will inhibit enzyme activity (Fig. 3) and it is probable that sheep antibodies act in a similar way. However, the inhibitory capacity of the sera from immunized ewes was not correlated with their fertility (Table 3)and, therefore, other antibody effects, such as sperm agglutination, immobilization or phagocytosis may be more important than enzyme inhibition. Antibodies to hyaluronidase should also have been secreted into the tract and should equally have prevented fertilization in these ways but, as outlined in the preceeding paragraph, there may be a critical point at which the rate of antibody secretion in relation to the amount of antigen is too low, and only when there is 'antibody excess' does infertility result. Hence only ewes injected with acrosin which resulted in high titres had a reduced fertility. ACKNOWLEDGEMENTS The authors wish to express their thanks to Drs. H.M. Dott, R.M. Moor, S. Willadsen and A. Trounson for their help with the sheep experiments and to the World Health Organisation for their financial support. We would also like to thank Mrs. V. Curry for technical assistance, Mrs. C. Gibson for typing the manuscript and Dr. P. Roughley for kindly providing the cartilage proteoglycan. REFERENCES Arnon, R. (1974) Enzyme inh~ition by antibodies. In 7th Karolinska Symposium on Research Methods in Reproductive Endocrinology: ImmunologicalApproaches to Fertility Control, Geneva, 1974 (Diczfalusy,E., ed.), Suppl. No. 194, pp. 133-153, Karolinska Instituter, Stockholm. Bolton, A.E. and Hunter, W.M. (1973) The labelling of proteins to high specific radioactivifiesby conjugation to a 12Si.containing acylating agent. Application to the radioimmunoassay. Biochem. J. 133,529-438. Cinader, B. (1966) Antibodies to enzymes: a discussion of the mechanisms of inh~ition and activa-
73 tion. Antibodies to Biologically Active Molecules, Vol. 1, pp. 85-137. Pergamon Press. Oxford. Goldberg, E. (1974) Effects of immunization with LDH-X on fertility. In 7th Karolinska Symposium on Research Methods in Reproductive Endocrinology: Immunological Approaches to Fertility Control, Geneva, 1974 (Diczfalusy, E., ed.), pp. 2 0 2 - 2 2 2 . Karolinska Instituter, Stockholm. Harrison, R.A.P., Dott, H.M. and Foster, G.C. (1978) Effect of ionic strength, serum albumin and other macromolecules on the maintenance of motility and the surface of mammalian spermatozoa in a simple medium. J. Reprod. Fert. 52, 6 5 - 7 3 . Isojima, S. (1973) Transmission of antisperm antibody into the female genital tract. In Proceedings 2rid International Symposium on Immunology of Reproduction, Varna. pp. 284-288. Katsh, S. (1959) Immunology, fertility and infertility: a historical survey. Am. J. Obstet. Gynec. 77, 946-956. McAnulty, P.A. and Morton, D.B. (1978) The immune response of the female rabbit genital tract following intravaginal or systemic immunisation. J. Clin. Lab. Immunol., in press. Metz, C.B. (1973) Role of specific sperm antigens in fertilization. Fed. Prod. 32, 2057-2064. Morton, D.B. (1976) Lysosomal enzymes in mammalian spermatozoa~ In: Lysosomes in Biology and Pathology (Dingle, J.T. and Dean, R.T.), Vol. 5, pp. 2 0 3 - 2 5 5 . North-HoUand Publ. Co., Amsterdam. Morton, D.B. (1977a) The occurrence and function of proteolytic enzymes in the reproductive tract of mammals. In: Proteinases in Mammalian Cells and Tissues (Barrett, A.J., ed.), pp. 4 4 5 - 5 0 0 . North-Holland Publ. Co., Amsterdam. Morton, D.B. (1977b) Immunoenzymic studies on acrosin and hyaluronidase in ram spermatozoa. In Immunobiology of Gametes (Edidin, M. and Johnson, M.H., eds.), pp. 115-155. Cambridge University Press, Cambridge. Neville, D.M. (1971) Molecular weight determination of protein-dodecyl sulphate complexes by gel electrophoresis in a discontinuous buffer system. J. Biol. Chem. 246, 6328-6334. Russo, J. and Metz, C.B. (1974) The ultrastructural lesions induced by antibody and complement in rabbit spermatozoa. Biol. Reprod. 10, 293-308. Symons, D.B.A. (1967) Reaction of spermatozoa with uterine and serum globulin detected by immunofluorescence. J. Reprod. Fert. 14, 163-165. World Health Organization Workshop (1976) Immunological Response of the Female Reproductive Tract, Geneva 1975 (Cinader, B. and de Weck, A.). Scriptor, Copenhagen.
61
THE E F F E C T ON F E R T I L I T Y OF IMMUNIZING FEMALE SHEEP WITH RAM SPERM ACROSIN AND HYALURONIDASE
D.B. MORTON * and P.A. McANULTY ** Strangeways Research Laboratory, Worts"Causeway, Cambridge CB1 4RN, U.K. (Received 10 November 1978; accepted 20 November 1978)
Female sheep were injected with highly purified and partially purified preparations of ram sperm acrosin and hyaluronidase. The fertility and immune response of the sheep were monitored. Fertility was not significantly reduced in any single group, though a positive correlation was observed between high ant~ody titres against acrosin and reduced fertility. Studies on the direct action of sera from the ewes on ejaculated ram spermatozoa did not show any evidence of sperm agglutination or immobilization. Similar studies with denuded spermatozoa (detergent induced 'acrosome reaction') sometimes resulted in agglutination and enzyme inhibition was also seen; there was no correlation between any of these parameters and pregnancy. INTRODUCTION Females injected with spermatozoa from males o f the same species show a reduction in fertility (Katsh, 1959; Metz, 1973). The cause o f the infertility is thought to be correlated with the immune response generated against specific, although as y e t unknown, sperm antigens. Goldberg (1974) has obtained some reduction (40%) in fertility by immunization with the sperm-specific enzyme lactate dehydrogenase X, so presumably other sperm antigens must be involved. The proposed roles for hyaluronidase and acrosin in sperm penetration between the cumulus cells and through the zona pellucida o f the egg, respectively, make these enzymes potential candidates, as antibody-mediated inhibition of enzyme activity might prevent fertilization. The following experiments were therefore carried out to determine if ram sperm hyaluronidase and acrosin could, on their own or together, act as infertility provoking immunogens in sheep. The sheep was chosen as the experimental species since sufficient numbers of spermatozoa could be amassed to provide the relatively large amount of purified enzymes needed to immunize the ewes (the yield o f purified enzyme is approximately 1 mg from 101 o spermatozoa).
* Present address: Department of Anatomy, Medical Sciences Building, Leicester University, Univer. sity Road, Leicester LE1 7RH, U.K. ** Present address: Department of Reproductive Studies, Life Science Research, Stock, Essex CM4 9PE, U.K.
62 MATERIALS AND METHODS
Animals 64 ewes of mixed breed, age and parity were used in the experiments. For most of the experimental period they were kept in open sheds, fed on hay and concentrates, and when sufficient grass was available, grazed during the day.
Fertility asssessment The study was carried out during two breeding seasons. During the first season, 19741975, fertility was measured by ovum cleavage, i.e. eggs were flushed from the oviduct or uterus 3 - 7 days after mating and examined for cleavage. The fertilization rate was expressed as the proportion of ewes with fertilized eggs. During the 1975-1976 season, ewes carried their lambs to term and fertility was measured by the lambing rate, i.e. the proportion of ewes bearing lambs.
Antigens used and the immunization programme Ewes were injected intramuscularly with an emulsion of Freund's Complete Adjuvant (FCA) and a solution of antigen in phosphate-buffered saline (PBS) ( in the ratio of 1 : 1) three times at 2-weekly intervals. The ewes were then run with the ram from two weeks after the third injection. The methods for preparing the sperm antigens are those used in the purification of ram sperm acrosin and hyaluronidase and have been described previously (Morton, 1976, 1977a, b). The descriptions below are brief outlines of the methods used. Because only small amounts of the highly purified enzymes were available, there was a possibility that not enough protein would be injected to ensure an adequate immune response. Consequenfly, two other groups of sheep were injected with partially purified solutions of enzyme which, although less pure, contained greater amounts of enzyme protein. Ewes were divided into seven groups and given one of the following antigens at each injection: (1) Partially purified hyaluronidase (DE-S): A 0.1% Triton X-100 aqueous extract of frozen-thawed ram semen was adjusted to pH 4.0, centrifuged, and the supernatant dialysed against 0.01 M Tris-HC1, pH 7.6. It was then chromatographed on diethylaminoethyl cellulose (DE-52, Whatman) equilibrated in the same buffer and the unabsorbed protein used in the experiments after dialysis against PBS. This peak contained approximately 70% of the total hyaluronidase activity in the sperm extract and had a specific activity of approximately 0.4 U/rag. On average, each ewe was given 0.5 mg of partially purified hyaluronidase at each injection in 2 ml of emulsion. (2) Hyaluronidase: Highly purified enzyme was prepared with a specific activity of between 10-20 U/mg. However this was still impure, as one other band was demonstrable on electrophoresis and also on immunodiffusion with a polyvalent antiserum. The contaminant was finally separated on immunodiffusion plates by running the enzyme against a monospecific antiserum to hyaluronidase raised in rabbits. Troughs were cut in agar plates and filled alternately with purified hyaluronidase or rabbit antiserum. The
63 resulting single precipitin lines were cut out of the gel, thoroughly washed with PBS and then mixed with adjuvant and emulsified by homogenisation with an Ultra-Turrax. This type of preparation had been used previously to raise specific antisera in rabbits and pigs. (3) Partially purified acrosin (CM-A3): A 2 M MgC12 extract was prepared from ram spermatozoa which had previously been extracted with 0.1% Triton X-100. The magnesium chloride extract was dialysed against 0.005 M sodium formate, pH 3.0, and then chromatographed on carboxymethyl cellulose (CM-52, Whatman) equilibrated with 0.04 M sodium acetate, pH 5.0. Acrosin was eluted by a 0.35 M NaC1 gradient, pooled and dialysed against PBS. The specific activity of this solution was approximately 4 U/mg with benzoyl DL-arginine-p-nitroanilide-HC1 as substrate. At each injection ewes were given 2.5-3 mg of protein in 2 ml of emulsion. (4) Acrosin: Highly purified acrosin with a specific activity of between 25 and 30 U/mg was prepared and dialysed against PBS. At each injection 121 gg of protein was given in 1 ml of emulsion. (5) Acrosin and hyaluronidase: Solutions of highly purified acrosin and hyaluronidase were used (see Treatments 2 and 4 above) and at each injection ewes received 121/gg of acrosin and 244/lg of hyaluronidase in 2 ml of emulsion. In this instance the hyaluronidase was not injected as an immune complex. [6J Control I: Sheep were injected with 1 ml PBS in 2 ml of emulsion. (7) Control IL" Sheep were injected with 0.5 mg purified rabbit IgG (prepared by ammonium sulphate precipitation and ion-exchange chromatography on DE-52) in PBS and given in 2 ml of emulsion. [8) Control III: During 1975-1976, the lambing rate of the main flock on the farm was calculated.
Measurement of immune response The immune response to the two defined antigens, hyaluronidase and acrosin, was measured. This was done either on immunodiffusion plates (I.D.) or by a two-step radioimmunoassay (R.I.A.) with labelled antigen. For the radioimmunoassay the two enzymes were radio-labelled with 1251 by the method described by Bolton and Hunter (1973). In the assay the test serum was serially diluted with an 80/2g/ml solution of sheep 7-globulin (prepared by ammonium sulphate fractionation of normal sheep serum) in 0.1 M phosphate buffer (PH 6.0) containing 0.5 M NaC1, 0.005 M EDTA, 0.01 M glycine and 0.2% Brij 35 (a non-ionic detergent). 50 pl of test serum was incubated with 10/21 of 12SI-labelted acrosin (specific radioactivity = 165 X 10 -9 Ci//2g, 67.5 ng in assay) or hyaluronidase (specific radioactivity = 41.8 X 10 -9 Ci//gg, 41.7 ng in assay) for 24 h at 37°C. All the sheep 7-globulin was then precipitated out by use of an antiserum raised in rabbits against sheep 7-globulin. This second step was carefully quantitated so that 100% of the sheep 7-globulin (at a concentration of 80/2g/ ml) was precipitated by the rabbit antibody. We did not differentiate between the 7globulin classes. 50/al of the rabbit antiserum (diluted 3 : 2 with the diluting buffer described above) were added, and after 24 h at 37°C the mixture was centrifuged at 8000 g for 15 min. The amount of unbound labelled enzyme remaining in solution was measured in a scintillation counter and so the amount precipitated could then be calculated,
64
?
25
7B (DE-S, ++ve I D)
(DE-S~+ve ID)
/ /
_c
~
/
15B (Control L-re ] D)
-~ 15 r'---~O~o~
o
i
E <
I
I
I
I
I
10-6
10-5
10-4
10-3
10-2
I
10-1
Serum dilution Fig. 1. Curves obtained with typical sera in hyaluronidase radioimmunoassay. Precipitation of 12si_ labelled hyaluronidase by serial dilutions of sera showing strong (e), weak (o) or no (A) precipitin lines on I.D. plates. The blank (. . . . . . ) was an 80/~g/ml solution of sheep y-globulin in buffer.
after allowing for non-specific precipitation by the sheep "r-globulin solution. There was an appreciable amount of non-specific binding by the "y-globulin solution and the non-immune controls gave comparable values (see Fig. 1). The results were expressed as the amount of labelled antigen precipitated at a serum dilution of 1/1000. The sera for acrosin radioimmunoassay were pretreated to acidic conditions (pH 4.0 with formate buffer for 30 rain) to remove any innate inhibitor activity which might compete with antibody binding (e.g. al-proteinase inhibitor and an c~2-macroglobulin). Furthermore, as acrosin is a proteolytic enzyme and can degrade y-globulin (see Fig. 2), immunoassays were carried out in the presence o f the acrosin inhibitor, benzamidine (0.02 M).
Assay o f sperm agglutinating and sperm immobilizing activities in serum from immunized ewes The antibody fraction was prepared from sera from ewes with a demonstrable immune response to the enzymes, judged by radioimmunoassay or immunodiffusion tests. The 7globulins were prepared by precipitating the sera three times with 0.67 serum volumes of 4 M ammonium sulphate (adjusted to pH 7). The final precipitate was made up to 0.5 serum volumes and the solution was exhaustively dialysed against Medium S (Harrison et al., 1978). The major protein component of this preparation would be IgG. 20 /A of freshly ejaculated ram semen was mixed with 1 ml of Medium S and 100/11 of this diluted semen was mixed with 5 #1 of fractionated sheep serum and incubated at 37°C. Aliquots
65 ~ii~ i i!~
4;; ~i~i
iiiii i! ~iii
i ii~!/~/~L ~
Uii!iii!!~!!i~
;; iii!!ii~i~iiii;i
IgG-~
~!'ili!~
!~!!!iii'ii!iii!i;
!!i!!~tlt
ii !i! ~
~-H
--L
1
2
3
4
5
6
Fig. 2. The proteolytic effect of acrosin on sheep IgG. Gels 1 and 4: sheep lgG alone; gels 2 and 5: IgG with acrosin; gels 3 and 6: IgG, acrosin and benzamidine. Gels 1 - 3 are non-reduced, gels 4 - 6 are reduced. UnlabeUed arrows point to the products of digestion. H and L are heavy and light chains, respectively.
were taken at 2, 10 and 30 min later, examined under a microscope and scored for sperm agglutination and motility. As there is evidence that acrosin and hyaluronidase are not normally exposed on the sperm surface but are located within the acrosome (Morton, 1976) a second series of experiments was conducted to investigate the effect of these sera on sperm agglutination using denuded spermatozoa. These denuded spermatozoa have their plasma membrane over the sperm head and the outer acrosomal membrane removed by freeze-thawing and detergent treatment (see Morton, 1977b) and are a model for acrosome-reacted spermatozoa. It is at this time, when spermatozoa have undergone the acrosome reaction, that one might expect antibodies to the enzymes to have their maximum effect.
Assay o f antibody-mediated enzyme inhibiting capacity of ewe sera using denuded spermatozoa Approximately 50 million detergent-treated ram spermatozoa, in 20 pl PBS were preincubated with 100 pl of fractionated ewe serum for 30 rnin. Aliquots of 50/A were then examined viscometrically for acrosin and hyaluronidase activities at 40°C. Hyaluronidase activity was measured using a 2 mg/ml solution of hyaluronic acid in PBS whereas acrosin activity was measured by using a 2 mg/ml solution of purified cartilage proteoglycan in the presence of 50 /ag/ml polyphloretin phosphate (both in PBS buffer). The poly-
66 phloretin phosphate will inhibit any hyaluronidase action on cartilage proteoglycan so that only the effect of acrosin on the polypeptide backbone of the macromolecule is measured. After 90 min incubation corrected flow times for substrate in the presence or absence of serum-treated spermatozoa were compared to evaluate the degree of inhibition: % inhibition = (Ts - Tb)/(Tc - Tb) × 100 where Tb = flow time for buffer (PBS) alone, T c = flow time for substrate mad buffer after 151 h, and T s = flow time for substrate and buffer after incubation with serumtreated spermatozoa for 1-~ h. Thus when no degradation of the substrate has occurred the flow times should be the same, giving 100% inhibition. But if the membrane-bound enzymes on the spermatozoa have degraded either of these macromolecules, then the viscosity should drop and the flow time should correspondingly decrease, giving a figure of less than 100%. The proteolytic action o f acrosin on IgG
Sheep IgG (1.34 mg/ml) was incubated at 37°C for 72 h either alone or with highly purified ram sperm acrosin (83 U/ml), or with acrosin and 0.01 M benzamidine (see Fig. 2). The products were then run on sodium dodecyl sulphate disc gels (Neville, 1971) under non-reduced and reduced conditions (with 2-mercaptoethanol). The inhibition o f ram sperm acrosin and hyaluronidase activities by immune and control globulins from rabbit sera
Highly purified preparations of ram sperm acrosin and hyaluronidase were prepared as described above. A constant amount of acrosin, 0.776 mU (specific activity 30 U/mg), was incubated with varying amounts of rabbit antibody (Fab monomer prepared from either a specific immune antiserum or a control serum) at 37°C for 30 min and then assayed for activity with benzoyl DL-arginine-p-nitroanilide. Similarily, 0.84 mU of hyaluronidase (specific activity 12 U/mg) was incubated with varying amounts of IgG (prepared from either a specific immune antiserum or a control serum) for 30 min at 40°C and then assayed for activity on hyaluronic acid. RESULTS A summary of the fertility results obtained in the experiment is given in Table I. It can be seen that no one group had significantly lowered fertility when analysed by a X2 test, although the partially purified acrosin group, CM-Aa, showed most tendency to deviate from the predicted value. The farm flock was not included in the statistical analysis as they had been handled far less during the experiments and had not been injected. In point of fact, its inclusion would have made no essential difference to the results. The immune response of the animals in any one group was variable. In general, animals showing a precipitin line on immunodiffusion would have a relatively high titre by radioimmunoassay (see Fig. 1 and Table 2). However, radioimmunoassay showed up more positives, partly as it was more sensitive, but also because this technique would have
67
~o~
O
o
~'~ ~1 ~il:~
~
~
o
~
i
~A
68 detected non-precipitating as well as precipitating antibodies. There were higher titres of antibody to acrosin and hyaluronidase in the ewes immunised with the partially purified enzyme preparations (Table 2) and this probably reflects the greater amount of protein in these preparations, giving a superior immune response. The immune response data from ewes in the two acrosin vaccinated groups (i.e. purified and partially purified) were compared to see if non-pregnant sheep had higher titres against acrosin than those that were pregnant. We found an average titre of 14.2 -+ 2.6 S.E.M. (n = 24; range 0 - 4 8 . 6 ) in the pregnant ewes compared with 28.8 +-8.1 (n = 6; range 0 . 7 - 5 0 . 1 ) in the non-pregnant ewes and the difference was statistically significant (Student's t t e s t , P < 0.01). The direct effect of circulating antibodies from the immunized ewes was examined by monitoring sperm motility, sperm agglutination and sperm enzyme inhibition (see Table 3). Motility and agglutination of intact ejaculated ram spermatozoa in the presence of antibody were unaffected whereas when denuded spermatozoa were used agglutination was observed and the degree of agglutination showed some correlation with antibody titre to acrosin. Furthermore, it was observed from the viscometry studies that the antibody fraction from some sera inhibited acrosin or hyaluronidase activities but it did not seem to be related to the titre. There was no difference between the treatment of denuded spermatozoa with saline and control serum, showing that endogenous inhibitors in the serum had no effect on the activity of the membrane-bound enzymes.
TABLE 2 The immune response of ewes treated with various sperm antigens measured against acrosin and hyaluronidase by radioimmunoassay (R.I.A.) and immunodiffusion(I.D.) Group
Hyaluronidase
Acrosin
R.I.A. a
R.I.A. b
I.D. c
R.I.A. a
R.I.A. b
I.D. c
Control I
0/3
0.0 + 0.0
0/8
3/6
0/8
Control II
1/3
0/3
0/3
DE-S
6/7
6/7
n.t.
n.t.
n.t.
Hyaluronidase
2/4
1/4
n.t.
n.t.
n.t.
CM-A3
n.t.
0.8 ± 0.8 (0.0-2.5) 5.1 +- 3.2 (0.0-23.5) 2.4 ± 1.6 (0.0-6.8) n.t.
0.2 ± 0.1 (O.O-0.4) 0.0 ± 0.0
n.t.
13/18
Acrosin
n.t.
n.t.
n.t.
14/19
Hyaluronidase + acrosin
2/2
5.1 +- 2.1 (3.0 and 7.2)
2/2
2/2
20.6 ± 4.3 6/18 (0.0-50.1) 8.8 ± 2.2 5/19 (0.0-34.1) 25.0 + 16.4 1/2 (8.6 and 41.4)
n.t., not tested. a Number of animals positive at serum dilution of 10-3. b Labelled antigen precipitated at a serum dilution of 10 -3 (ng -+S.E.M. with range in brackets). c Number of animals with precipitating antibodies.
0/3
69 e~
~
Z ~ Z Z
~
~
o
?,
{
e-
e,
~
I I
+~
~
++ +
+
+
+
,
+
÷
+
+÷I
0
Ill
II
I I I I
II
II
o 0 e~ 0 o
L~
0
E
0
2 0
bl
O
0
I
I
0
o
"
5
Z
I
<<
O
0
<+
.
o,~
70
0
0
20
40 60 80 100 120 ~g antibody/mu enzyme
I "~
I
140 160
Fig. 3. The inhibition of ram sperm acrosin and hyaluronidase activities by immune and control globulins from rabbit sera. a, acrosin with specific Fab ant~ody; A, acrosin with control Fab antibody; o, hyaluronidase with specific IgG antibody; •, hyalurortidase with control IgG antibody.
Overall there was no obvious correlation between pregnancy and sperm agglutinating, sperm immobilising or enzyme inhibiting activities of the sera. The experiments on the proteolytic action of acrosin on sheep IgG (Fig. 2) showed that over the period of the radioimmunoassay significant digestion of the IgG could take place and that the presence of 10 mM benzamidine prevented this effect. Whether the antigen binding capacity of the antibody was reduced was not determined but benzamidine was routinely used in the radioimmunoassay. It was possible to inhibit enzyme activity completely with antibodies isolated from specific antisera raised in rabbits against the purified enzymes (Fig. 3). In addition to the experiments reported here, we have shown that as with acrosin, specific monomeric Fab fragments will also inhibit hyaluronidase activity but the inhibition was not complete in the range tested (126 #g Fab/mU hyaluror.idase gave 80% inhibition). DISCUSSION The reduced fertility of females injected with spermatozoa is thought to be due to the immunological response to sperm antigens. The way in which the immune response has its effect on fertility is unknown. One obvious possibility is that the circulating antibodies will be secreted into the genital tract and, once there, have a variety of detrimental effects on spermatozoa. For instance, sperm agglutination could arise by antibody cross-linking surface antigens or sub-surface antigens which subsequently become exposed (such as acrosin after the acrosome reaction). The experiments on the effect of serum antibody fraction on ram spermatozoa showed that the spermatozoa denuded of the outer membranes were agglutinated, whereas intact spermatozoa were not (Table 3). This provides further evidence that acrosin and hyaluronidase are not superficial sperm antigens but are probably positioned, in part anyway,
71 on the inner acrosomal membrane; they may also be contained within the acrosomal contents. Thus, spermatozoa having undergone the acrosome reaction could be agglutinated by the antibody in the female tract, and the resultant aggregations might well interfere with their ability to reach the site of fertilization and to penetrate the egg. Spermatozoa might also be immobilized not only through agglutination but also through the action of antibody with complement, resulting in sperm death (Isojima, 1973; Russo and Metz, 1974). In the present experiments no evidence was obtained for any sperm immobilizing effect on intact cells, presumably as the antigens were not superficial. However, sperm immobilization might still occur with acrosome-reacted spermatozoa or in a complement-sufficient system. There is some evidence that antibody coating may render the sperm cell more susceptible to phagocytosis by neutrophils or macrophages (Symons, 1967) and to lymphocyte attack. This raises the possibility of cell-mediated immunity but there seems to be little evidence for lymphocyte migration into the lumen of the reproductive tract. Cellmediated immunity has been described associated with graft rejection and pregnancies (see W.H.O. Workshop on Immunological Response of the Female Reproductive Tract, 1976). A further way antibodies might act is to prevent spermatozoa from fertilizing the egg by inhibiting sperm enzymes needed for penetration through the various layers surrounding the egg. We have shown that some ewes produced antibodies which were able to inhibit acrosin and hyaluronidase activities. Interestingly, however, there is obviously some diversity between antibodies since, irrespective of the amount of antibody present, some sera give greater inhibition than others. Presumably this is due to a greater proportion of the antibodies binding close to the active site of the enzyme. This may be attributed to the genetic diversity in immune response between individuals of the same species treated under identical immunization schedules. In separate experiments it was also shown that rabbit antibodies, both IgG and Fab fragments, specific for sheep acrosin or hyaluronidase are able to inhibit the activity of these enzymes (Fig. 3). Bivalent IgG could cause apparent inhibition of enzyme activity simply through precipitating the enzyme out of solution. But the experiments with the monovalent Fab fragment, which would leave the enzyme in solution, show that this is not the case and the inhibition is more likely to be due to antibodies binding close to the active site, thus impeding substrate-enzyme interaction (see Cinader, 1966; Arnon, 1974). Inhibition of enzyme activity due to antibody has usually been assessed with soluble enzyme substrates. In the case of spermatozoa digesting a path through the zona pellucida, however, the 'native' substrate is insoluble as well as the enzyme. Consequently, it has to be considered that antibody binding farther away from the active site, or even antibody binding to other surface antigens in the acrosomal region, might prevent substrate-enzyme interaction and thereby inhibit sperm penetration of the egg. The foregoing discussion assumes that antibodies will be produced against 'the sperm antigen(s)', i.e. they are immunogenic. And secondly, that these antibodies will be secreted intact into the female reproductive tract (McAnulty and Morton, 1978). From the results shown in Table 2, it can be seen that circulating antibodies to hyaluronidase were found in both the I.D. and the R.I.A. test systems but no significant drop in fertility
72 was observed (Table 1). The lack of an effect on fertility after injection of this enzyme could be due to the nonessential role of hyaluronidase in fertilization or to an insufficient rate of antibody secretion into the tract in relation to the amount of sperm antigen present or passing through. Sperm transport in the sheep (and the human) can be considered to be a continual process with spermatozoa constantly passing from the cervix through the uterus and oviduct into the abdominal cavity. Thus, small amounts of antibody would be 'mopped up' by the early spermatozoa at the site of fertilization, leaving a relatively 'antibody-free' milieu for those arriving subsequently. However, it is relevant to note when considering hyaluronidase in the sheep that the cumulus cells are lost very shortly after ovulation (Moor, R.M., personal communication) and so in this species at least there may be no cumulus or corona cell barrier to sperm penetration. This raises the question of the role played by ram sperm hyaluronidase in fertilization. A similar analysis of the immune response for acrosin showed that, in general, antibody levels were higher than for hyaluronidase and there was a correlation between the level of circulating antibody and fertility; sheep with higher antibody titres to acrosin showed significantly lowered fertility. This suggests that sufficient anti-acrosin antibody was secreted into the tract but the effect of this is not clear. We have shown that purified Fab fragments of IgG from a specific rabbit antiserum to acrosin will inhibit enzyme activity (Fig. 3) and it is probable that sheep antibodies act in a similar way. However, the inhibitory capacity of the sera from immunized ewes was not correlated with their fertility (Table 3)and, therefore, other antibody effects, such as sperm agglutination, immobilization or phagocytosis may be more important than enzyme inhibition. Antibodies to hyaluronidase should also have been secreted into the tract and should equally have prevented fertilization in these ways but, as outlined in the preceeding paragraph, there may be a critical point at which the rate of antibody secretion in relation to the amount of antigen is too low, and only when there is 'antibody excess' does infertility result. Hence only ewes injected with acrosin which resulted in high titres had a reduced fertility. ACKNOWLEDGEMENTS The authors wish to express their thanks to Drs. H.M. Dott, R.M. Moor, S. Willadsen and A. Trounson for their help with the sheep experiments and to the World Health Organisation for their financial support. We would also like to thank Mrs. V. Curry for technical assistance, Mrs. C. Gibson for typing the manuscript and Dr. P. Roughley for kindly providing the cartilage proteoglycan. REFERENCES Arnon, R. (1974) Enzyme inh~ition by antibodies. In 7th Karolinska Symposium on Research Methods in Reproductive Endocrinology: ImmunologicalApproaches to Fertility Control, Geneva, 1974 (Diczfalusy,E., ed.), Suppl. No. 194, pp. 133-153, Karolinska Instituter, Stockholm. Bolton, A.E. and Hunter, W.M. (1973) The labelling of proteins to high specific radioactivifiesby conjugation to a 12Si.containing acylating agent. Application to the radioimmunoassay. Biochem. J. 133,529-438. Cinader, B. (1966) Antibodies to enzymes: a discussion of the mechanisms of inh~ition and activa-
73 tion. Antibodies to Biologically Active Molecules, Vol. 1, pp. 85-137. Pergamon Press. Oxford. Goldberg, E. (1974) Effects of immunization with LDH-X on fertility. In 7th Karolinska Symposium on Research Methods in Reproductive Endocrinology: Immunological Approaches to Fertility Control, Geneva, 1974 (Diczfalusy, E., ed.), pp. 2 0 2 - 2 2 2 . Karolinska Instituter, Stockholm. Harrison, R.A.P., Dott, H.M. and Foster, G.C. (1978) Effect of ionic strength, serum albumin and other macromolecules on the maintenance of motility and the surface of mammalian spermatozoa in a simple medium. J. Reprod. Fert. 52, 6 5 - 7 3 . Isojima, S. (1973) Transmission of antisperm antibody into the female genital tract. In Proceedings 2rid International Symposium on Immunology of Reproduction, Varna. pp. 284-288. Katsh, S. (1959) Immunology, fertility and infertility: a historical survey. Am. J. Obstet. Gynec. 77, 946-956. McAnulty, P.A. and Morton, D.B. (1978) The immune response of the female rabbit genital tract following intravaginal or systemic immunisation. J. Clin. Lab. Immunol., in press. Metz, C.B. (1973) Role of specific sperm antigens in fertilization. Fed. Prod. 32, 2057-2064. Morton, D.B. (1976) Lysosomal enzymes in mammalian spermatozoa~ In: Lysosomes in Biology and Pathology (Dingle, J.T. and Dean, R.T.), Vol. 5, pp. 2 0 3 - 2 5 5 . North-HoUand Publ. Co., Amsterdam. Morton, D.B. (1977a) The occurrence and function of proteolytic enzymes in the reproductive tract of mammals. In: Proteinases in Mammalian Cells and Tissues (Barrett, A.J., ed.), pp. 4 4 5 - 5 0 0 . North-Holland Publ. Co., Amsterdam. Morton, D.B. (1977b) Immunoenzymic studies on acrosin and hyaluronidase in ram spermatozoa. In Immunobiology of Gametes (Edidin, M. and Johnson, M.H., eds.), pp. 115-155. Cambridge University Press, Cambridge. Neville, D.M. (1971) Molecular weight determination of protein-dodecyl sulphate complexes by gel electrophoresis in a discontinuous buffer system. J. Biol. Chem. 246, 6328-6334. Russo, J. and Metz, C.B. (1974) The ultrastructural lesions induced by antibody and complement in rabbit spermatozoa. Biol. Reprod. 10, 293-308. Symons, D.B.A. (1967) Reaction of spermatozoa with uterine and serum globulin detected by immunofluorescence. J. Reprod. Fert. 14, 163-165. World Health Organization Workshop (1976) Immunological Response of the Female Reproductive Tract, Geneva 1975 (Cinader, B. and de Weck, A.). Scriptor, Copenhagen.