Sperm–zona pellucida interaction and immunological infertility

Abstract

Immune reactions against gametes appear to be physiologically important for the maintenance of homeostasis in reproduction. In contrast, aberration of the immune homeostasis might give rise to ‘immunological infertility’. Antisperm antibodies cause infertility by blocking fertilization. The mechanism can be explained as inhibiting the acrosome reaction of sperm by their blocking effect on capacitation through inhibiting an increase of fluidity of the sperm membrane. Autoantibodies against zona pellucida also cause infertility by blocking sperm–zona pellucida interaction, though the definitive mechanism has not been elucidated.

Pretreatment of spermatozoa with D‐mannnose completely inhibited sperm penetration through, but not binding to, the zona pellucida. Furthermore, very rapid kinetics between sperm extracts and D‐mannnose by a BIAcore apparatus suggest that a D‐mannose ligand of the sperm surface is easy to bind to and dissociate from a D‐mannose residue in the sperm receptor site on the zona pellucida. Thus, D‐mannnose on the human zona pellucida might be an essential molecule acting as a second sperm receptor, through which sperm penetrate into the zona pellucida.

Because these antibodies appear to not cause any deleterious clinical symptoms, sperm and zona pellucida antigens are promising candidates in the development of an immunocontraceptive. (Reprod Med Biol 2006; 5: 95–104)

IMMUNOLOGICAL INFERTILITY

NONSPECIFIC AND SPECIFIC immune reactions against gametes or embryos appear to be physiologically important for the maintenance of homeostasis in reproduction. ¹ For example, in the male and female reproductive tracts, activation of the complement system by immature and aged sperm can enhance their destruction and clearance during the selection of active motile sperm. This mechanism of sperm selection is physiologically important, whereby increasing the opportunity of sperm to fertilize the ova. ² Furthermore, the present study has shown that the occurrence of cellular and/or humoral immune reaction against sperm antigens expressed on the surface of embryos appears to augment their receptivity by the uterus. ³

In contrast, aberration of the immune homeostasis might give rise to ‘immunological infertility’. The majority of subjects with asthenozoospermia and/or oligozoospermia have a higher incidence of low complement‐inhibiting activity and a reduced level of complement regulatory proteins, such as membrane cofactor protein (MCP, CD46) and decay accelerating factor (DAF, CD55) in their seminal plasma. This can lead to non‐specific activation of the alternative complement pathway, thus, inflicting injury to sperm. ⁴

An immune reaction acting adversely against gametes or hormones is a mechanism that contributes to the development of ‘immunological infertility’. A previous study reported the first case of an endocrine disorder that was the result of the production of an autoantibody to testosterone. ⁵ Autoantibodies against zona pellucida (ZP) might cause infertility by blocking sperm–zona pellucida interaction. ⁶ , ⁷ These antibodies are found in approximately 6% of infertile women with indeterminate etiology; ⁷ whereas the incidence of infertility with associated antisperm antibodies is higher (approximately 18%).

The presence of these antibodies in blood, follicular and uterine fluids, and cervical mucus can impair reproductive processes that are crucial for a successful pregnancy. Because these antibodies appear to not cause any deleterious clinical symptoms yet be associated with infertility, sperm and ZP antigens are promising candidates in the development of an immunocontraceptive. Thus, immunologically defined factors, especially associated with gametes, are important to shed light on the process of human fertilization and to develop a contraceptive vaccine.

ANTISPERM ANTIBODY

SPERM CAN ELICIT an immune response in women as an alloantigen and in men as an autoantigen. Antisperm antibodies might cause infertility by (i) impairing sperm motility; (ii) blocking penetration of cervical mucus by sperm; (iii) interfering with sperm–egg interaction; (iv) interfering with the implantation of blastocysts; and (v) inhibiting the growth and development of embryos.

To date, reports on the cytotoxic effects of antisperm antibodies on embryonic growth have been restricted to studies with experimental animals. In contrast, naturally occurring antisperm antibodies found in the sera of immunologically infertile women do not appear to inhibit embryo growth or implantation of blastocysts. The clinical data clearly show that high implantation rate and subsequently high pregnancy rate are achieved by in vitro fertilization (IVF) and embryo transfer treatment of infertile women possessing sperm immobilizing antibodies in their circulation. ³ , ⁸

The authors of the present study were the first to show that naturally occurring sperm immobilizing antibodies can block penetration of the sperm through the human ZP. ⁹ This observation has been confirmed subsequently by many investigators ¹⁰ and further substantiated by our clinical data. ³ , ¹¹ Although the definitive mechanism whereby antisperm antibodies block sperm–zona interaction has not been elucidated, the capacity of antisperm antibodies to inhibit the acrosome reaction of sperm, an indispensable process for successful fertilization, is well established. ¹⁰ , ¹² It is noteworthy that the inhibition is reversible. Sperm preincubated with antisperm antibody recover their ability to penetrate the ZP with subsequent washing in antibody‐free medium (Fig. 1). ¹ Recovery from the blocking effect of antisperm antibodies on the acrosome reaction is also observed after incubation of the antibody‐bound sperm in antibody‐free medium. ¹² The inhibitory effects of the antibodies on the acrosome reaction might be explained by their blocking effects on capacitation ¹³ through inhibiting an increase of fluidity of the sperm membrane. ¹⁴

Figure 1.

Time course results of zona penetration test using sperm exposed to serum containing sperm immobilizing antibody (cited from ¹ ). (▪) Serum containing sperm immobilizing antibody; (□) control serum.

AUTOANTIBODY TO ZONA PELLUCIDA

FROM CIRCUMSTANTIAL EVIDENCE, the production of autoantibodies against ZP has been implicated in the development of immunological infertility in some women. ⁶ , ⁷ , ¹⁵ , ¹⁶ , ¹⁷ , ¹⁸ , ¹⁹ , ²⁰ , ²¹ , ²² , ²³ , ²⁴ , ²⁵ , ²⁶ , ²⁷ Because the ZP plays an important role in such reproductive processes as expression of the sperm receptor site(s) and protection against polyspermy and zona shedding on implantation, the presence of antibodies impairing such functions could result in infertility.

However, there has been serious controversy over the actual existence of such antibodies because antizona activities have been detected in the sera of both infertile and fertile women and men, and these activities disappeared after thorough adsorption of the sera with porcine red blood cells. ²⁸ , ²⁹ In many previous reports showing the presence of antizona activities, indirect immunofluorescence (IF) method using porcine ZP as the target instead of human ZP was used as a screening test, because the ZP from these two sources show antigenic cross‐reactivity and the availability of human ZP is limited. However, IF test using intact porcine ZP was proven to have some methodological problems on account of binding of non‐specific immunoglobulin and hetero‐hemagglutinin against porcine red blood cells present in human serum. ¹⁶ , ¹⁷ False positive reactions and disappearance after absorption of the sera with porcine erythrocytes have made the actual existence of such antibodies confusing. Thus, the final conclusion on the existence of specific antibodies to human zona antigen should be drawn only after confirmation by another more specific detection methods and of reproducibility on human ZP. The clinical significance of autoantibodies to ZP as a potential cause of human female infertility must also be substantiated in view of their biological activities in human fertilization.

Passive hemagglutination reaction for detection of antizona activities

We have developed a passive hemagglutination reaction (PHAR) in place of IF using porcine ZP for screening antizona activities. ⁶ PHAR is known to be a sensitive serologic test and widely used for detection of autoantibodies, such as rheumatoid factor, anti‐DNA and anti‐ENA antibodies. The success in isolating an adequate amount of zona substance from porcine ovaries ³⁰ enabled the development of the PHAR. A comparable sensitivity of the PHAR with that of IF was checked at the maximum dilution times at which the positive reaction appeared against the rabbit antiporcine ZP antiserum. The results showed that the sensitivity of PHAR was 1 : 32 000 and this was almost the same as IF.

A comparative study on antizona antibody activities in the sera from clinically defined categories of patients registered at the World Health Organization Reference Bank for Reproductive Immunology was carried out. ¹⁹ , ²⁰ A total of 318 samples from 14 different categories were measured in blind tests by PHAR. In our study using PHAR, a positive reaction was obtained with 3/96 (3.1%) serum samples from infertile women but with only 2/222 (0.9%) samples from the control subjects. It was noted that no positive samples were found in sera from males of different categories and that there was no discrepancy in the results between each of the two identical serum samples that had been included in duplicate.

Antizona activities of sera from infertile women and control subjects detected by the PHAR

As shown in Table 1, positive PHAR with titers of up to 1 : 32 were detected in 45/1827 (2.4%) serum samples from women with infertility of different causes, but in only three samples (0.5%) from 592 control subjects, two from non‐pregnant women and one from a pregnant woman. Moreover, 18/45 (40%) infertile women with a positive PHAR were diagnosed as having unexplained infertility. This incidence was much greater than the overall incidence in our clinic (approximately 10%). Indeed, the incidence of positivity in women with unexplained infertility (5.6%) was significantly higher than that in women with anovulation or infertility from other causes (1.7%, P < 0.01) or in the control group of non‐pregnant and pregnant women (1.5%, P < 0.05). None of the serum samples obtained from men and children gave positive PHAR. Importantly, none of the serum samples with a positive PHAR contained antisperm antibodies screened by the sperm immobilization test.

Table 1.

Incidence of antizona activities in sera from infertile women and control subjects measured by the passive hemagglutination reaction

Clinical diagnosis	No. samples	No. positive reaction	% positive reaction
Infertile women
Unexplained	320	18	5.6*,**
Known causes	1552	27	1.7*
Total	1872	45	2.4
Age‐matched control women	193	3	1.5**
Other control subjects
Postmenopausal women		0	0
Fertile men	39	0	0
Infertile men	253	0	0
Children	100	0	0
Total	292	0	0

^* P < 0.01;

^** P < 0.05.

Follow‐up study on pregnancies of patients with positive antizona activities

We followed up 30 patients who showed positive PHAR in routine examinations on the first attendance at our Infertility Clinic for a minimum of 2 years. As shown in Table 2, no pregnancy occurred in 11 patients who had a consistently positive PHAR during a minimum of a 2‐year period of treatment, whereas three pregnancies were achieved in 19 patients with fluctuating positive PHAR during the same follow‐up period.

Table 2.

Follow‐up study of patients with positive antizona activities

Antizona activity	Clinical diagnosis	No. patients	No. pregnancy
	Unexplained	5	0
Consistent	Anovulation	3	0
positive	Tubal obstruction	2	0
PHAR†	Sperm abnormality	1	0
	Total	11	0
	Unexplained	8	1‡
Fluctuating	Anovulation	4	0
positive	Tubal obstruction	2	1§
PHAR	Sperm abnormality	5	1¶
	Total	19	3

^† Passive hemagglutination reaction;

^‡ ‡controlled ovarian hyperstimulation;

^§ in vitro fertilization‐embryo transfer; artificial insemination with washed and concentrated husband sperm.

Inhibitory effects of sera with positive PHAR on human sperm–zona interaction

Four of seven serum samples from patients whose sera consistently gave positive PHAR produced strong or faint IF on human ZP even after absorption with human AB red blood cells. Because of the limited availability of human oocytes, the test was carried out in four separate experiments with controls each time. As shown in Table 3, in experiment 1, considerable numbers of sperm bound to and penetrated through the human ZP on exposure of the test ovum to control serum. In contrast, fewer sperm bound to and penetrated through the human ZP after exposure of the test ova to the two serum samples (Nos. 1 and 2) that gave strong positive IF on human ZP. The numbers of sperm bound to and penetrating through the human ZP were slightly reduced compared with the control after treatment with the sera from three other patients that gave faint or negative IF on human ZP. Complete block of sperm–zona interaction by sera from the same two patients (Nos. 1 and 2) were observed in experiments 2 and 3. In experiment 4, two ova were used for each test and control serum. The test serum (No. 3) strongly blocked sperm–zona interaction in both human ZP.

Table 3.

Effects of passive hemagglutination reaction‐positive serum on sperm binding to (penetrating through) human zona pellucida in vitro

IF† on human zona	Clinical diagnosis (age)	Experiment
		1	2	3	4
Strongly	Unexplained (31)	8 (2)	0 (0)	0 (0)	–
positive	Unexplained (23)	5 (0)	0 (0)	0 (0)	–
	Unexplained (34)	–	–	–	3 (1)  3 (0)
Faint or	Anovulation (20)	–	6 (1)	–	–
negative	Anovulation (20)	2 (23)	18 (3)	–	–
	Unexplained (32)	2 (10)	–	–	–
	Unexplained (33)	12 (13)	0 (0)	–	–
Control		17 (47)	4 (1)	10 (5)  8 (4)	34 (6) 42 (5)

^† Immunofluorescence.

It is noteworthy that in the case No. 3, none of the 13 matured ova (five collected in the first attempt and eight in the second) were fertilized by the husband's sperm in IVF trials even when fetal cord serum instead of autoserum was used to avoid possible deteriorating effects of autoantibodies to ZP. Repeated semen analysis and morphological examination failed to show any abnormalities of the husband's sperm.

Autoantibodies as a causal factor for the development of immunological infertility

The above described results of a significantly higher incidence of positive PHAR in women with unexplained infertility, resistancy to the treatment for a relatively longer period in infertile women, women with consistent positive antizona activities and the inhibitory action of autoantibodies against human ZP on human sperm–egg interaction clearly establish the pathogenetic concept that antizona autoantibodies are one causal factor responsible for unexplained infertility in women. The evidence that self zona antigen can elicit autoantibodies against ZP and produce long‐term contraception in female mice has clearly been shown by Miller et al. ³¹ Moreover, Lou and Tung ³² showed the lack of B cell tolerance to self zona antigen(s) by an experiment that found that immunization of mice with the epitope of ZP3 peptide recognized by T cell can elicit autoantibodies against the zona protein antigen outside the original T cell‐recognized epitope and this amplification of production of autoantibody was not detected in ovariectomized mice. Furthermore, they also showed the existence of a strong correlation between autoantibody titers and fertility reduction. ³³

Human ZP contains four glycoproteins, which are highly conserved among species. ³⁴ If antibodies detected by PHAR are directed to the common antigen(s), then the question arises of whether these antibodies can cause infertility at such a low titer (up to 1 : 32), and, if not, whether the serum giving a positive PHAR possesses autoantibodies against antigen(s) appropriate to human ZP. The answer to the former question seems to be arguable, because murine monoclonal antibodies against a common antigen of porcine and human ZP are required at high titers to inhibit sperm penetration across the human ZP in vitro. ³⁵ As for the answer to the latter question, it is likely that autoantibodies against the antigen(s) appropriate to human ZP probably coexist with those reacting with the common antigen(s). Derangement of the immune system against autologous zona antigens might result in the production of autoantibodies against both the antigen(s) appropriate to human ZP and those against the antigen(s) common to porcine and human ZP. In fact, it has been reported that monkeys immunized with human ZP3 peptide developed T‐cell responses to the immunizing peptide and produced antibodies reacting with native ZP. ³⁶

With regard to infertility in women with antizona activities, it is important to note that human sperm‐egg interaction in an in vitro system was dramatically reduced in terms of sperm binding to and penetration through human ZP in the presence of sera giving a positive PHAR and IF on human ZP. Of particular interest was our finding that in one case (No. 3), complete failure of fertilization was observed in her IVF trials twice with five mature ova in the first trial and eight in the second trial, respectively. Although, no abnormalities were found on analysis of the sperm used in terms of concentration, motility and sperm morphology. In our IVF clinic, the fertilization rate of matured eggs is at least 80% on insemination with sperm, if the motility of the latter is more than 20% in the original semen and the total number of motile sperm after swim‐up treatment is more than 0.2 × 10⁶ (unpubl. data). Liu et al. ³⁷ also reported that complete failure of IVF (no fertilization of more than three eggs inseminated) was not found when more than 40% of the sperm had normal morphology. The absolute inability of these eggs to be fertilized is presumably the result of exposure to autoantibodies to ZP in vivo, because the serum of patient No. 3 stained human ZP strongly and blocked sperm‐zona interaction in vitro. In recent reports, significant positive correlations between the presence of anti‐ZP antibodies and the degree of fertilization failure were shown in IVF trials. ²³ , ²⁵ , ²⁶ , ²⁷ Judging from these laboratory and clinical observations, antibodies against the antigen(s) appropriate to human ZP is likely to be closely related to infertility.

The cases that conceived during follow‐up studies in the group with fluctuating antizona activities might also be interpreted by recruitment of fertilizable ova developed at times not influenced by autoantibodies to ZP. This explanation seems to be supported by a prospective study, ²² in which antizona antibody titers were attenuated in most patients with subsequent successful pregnancies.

What kinds of molecules are the target of anti‐ZP autoantibodies?

Interestingly, a radioimmunoassay (RIA) using iodine‐labeled porcine zona antigen was not reactive with autoantibodies to human zona antigens, but with murine monoclonal antibody against antigen shared by porcine and human ZP. However, the autoantibodies measured by PHAR found to be detectable by an RIA system using a solid phase sensitized with solubilized porcine zona antigen and iodine‐labeled protein A (data not shown). Because radioactive iodine is known to be incorporated mainly into tyrosine residues of peptide antigen and not into carbohydrates or lipids, carbohydrates might be the epitopes of antigenic molecules of the autoantibodies to ZP, as demonstrated in sperm antigens.

ROLE OF CARBOHYDRATE ON SPERM–ZONA INTERACTION

IN THE INITIAL phase of fertilization, capacitated sperm reversibly bind to the surface of the ZP, and this reversible binding is followed by an irreversible binding and penetration of the ZP. Since Oikawa and coworkers ³⁸ first demonstrated that wheat germ agglutinin blocked fertilization, there has been an accumulation of evidence concerning the role of carbohydrates in various sperm–zona pellucida interactions in many mammalian species. ³⁹ , ⁴⁰ , ⁴¹ , ⁴² , ⁴³

In 1989, we first showed, by using human zona penetration test, that d‐mannose on the ZP is an essential molecule acting as a ligand that binds to lectine‐like molecules on the sperm surface. ⁴⁴ As shown in Table 4, when human ZP were pretreated with concanavalin A (Con A), wheat germ agglutinin, or Ricinous communis agglutinin‐I, no sperm were found to bind and penetrate through the ZP. Penetration of sperm was restored when the ZP pretreated with wheat germ agglutinin and Ricinous communis agglutinin‐I were rinsed with N‐acetyl‐D‐glucosamine (wheat germ agglutinin inhibitor) and d‐galactose (Ricinous communis agglutinin‐I inhibitor), respectively. Conversely, the inhibitory effect of Con A on sperm penetration was not reversed by treatment with d‐mannose (Con A inhibitor). Furthermore, pretreatment of sperm with d‐mannose completely inhibited sperm penetration through but not binding to the ZP. However, sperm penetration was clearly shown when the ZP was pretreated with d‐mannose (Table 5). We also demonstrated that d‐mannose did not show a suppressive effect on the acrosome reaction of human sperm. ⁴⁵ These data suggest that the binding of a d‐mannose ligand of the sperm surface to a d‐mannose residue in the sperm receptor site on the ZP is mandatory in human fertilization, as the second step after sperm–egg recognition.

Table 4.

Effects of exposure of human oocytes to various lectins on sperm binding to and penetration through the zona pellucida

	Total no. sperm bound (No. eggs examined)	Total no. sperm penetrating (No. eggs examined)
Control	41 (3)	18 (3)
Con A	0 (3)	0 (3)
WGA	0 (3)	0 (3)
RCA‐I	0 (3)	0 (3)
DBA	15 (1)	20 (1)
LPA	9 (1)	3 (1)
PNA	15 (1)	15 (1)
SBA	10 (1)	11 (1)
UEA‐I	13 (1)	8 (1)

Table 5.

Effects of treatment of sperm or the zona pellucida with sugars on zona penetration test

Treatment of	Total no. sperm bound (No. eggs examined)	Total no. sperm penetrating (No. eggs examined)
Sperm
Control	93 (8)	48 (8)
D‐mannose	35 (6)	0 (6)
N‐acetyl‐D‐glucosamine	10 (1)	6 (1)
D‐galactose	9 (1)	4 (1)
L‐fucose	45 (3)	21 (3)
Zona pellucida
Control	52 (4)	19 (4)
D‐mannose	67 (4)	22 (4)

Biomolecular interaction between mannose residues and mannose ligand of sperm extracts

Surface plasmon resonance by a BIAcore apparatus

Surface plasmon resonance (SPR) is a very useful and sensitive method for analyzing biomolecular interaction, the intensity of binding in real‐time and kinetics. Namely, a change in the resonance angle occurs when the mass on the surface of the sensor chip increases as a result of the molecular interaction. The resonance angle is expressed in resonance units (RU). A response of 1000 RU represents a change in the surface protein concentration of about 1 ng/mm². The response time represents a speed of binding or dissociation.

Interaction between mannose residues and Con A

As mannosyl residues, α‐D‐mannosylated‐biotin‐albumin (mannose‐BSA) and mannopentaose‐DI‐(N‐Acetyl‐D‐glucosamine)‐Asparagine ([man]₅‐[ClcNAc]₂‐Asp, mannopentaose) were immobilized on a SA and CM5 sensor chip, respectively. Figure 2 shows the relative response curve for Con A binding to each sensor chip. The amount of binding was estimated by subtracting RU of the control from RU of the sample 40 s after injection. Although the amount of ConA that bound to mannose‐BSA and mannopentaose were 195 RU and 113 RU, respectively, the SPR responses after the injection were quite different. The SPR signal in the mannose‐BSA‐fixed chip slowly decreased, whereas that in the mannopentaose‐fixed chip decreased rapidly. However, the SPR signal during injection onto the mannopentaose‐fixed chip was much larger than that onto the mannose‐BSA‐fixed chip.

Figure 2.

Sensorgram showing the interaction of concanavalin A (Con A) with (a) immobilized α‐D‐mannosylated‐biotin‐albumin and (b) mannopentaose‐DI‐(N‐Acetyl‐D‐glucosamine)‐Asparagine. Con A (20 µL) at a concentration of 200 µg/mL was injected onto the sensor chip.

Analysis of sperm samples

Two sperm extracts were used in the present study. ⁴⁶ Crude acid extract was prepared according to the method used by NagDas et al. ⁴⁷ The extract was further purified by applying to a CM Sepharose column. The bound fraction contained bands showing protease activity at molecular weights of approximately 35 kDa and 50 kDa by gelatine‐SDS‐PAGE (protease fraction).

The SPR signal during the injection of acid extracts and protease fractions onto the SA sensor chip immobilizing mannose‐BSA was not obviously changed compared with that of the control. However, when sperm samples were injected onto the mannopentaose‐fixed sensor chip, the SPR response during the injection was very intense (more than 1500 RU) compared with that in the control (Fig. 3). Furthermore, rapid increase and decrease of the SPR signal suggest that a d‐mannose ligand of the sperm surface is easy to bind to and dissociate from a d‐mannose residue in the sperm receptor site on the ZP. These kinetics between sperm and ZP seem to be a rationale for the second step, in which sperm penetrate through the ZP.

Figure 3.

Sensorgram showing the interaction of acid extracts (a) and protease fractions (b) with immobilized mannopentaose‐DI‐(N‐Acetyl‐D‐glucosamine)‐Asparagine. Acid extracts and protease fractions (20 µL) at a concentration of 500 µg/mL were injected onto the sensor chip.

IMMUNOCONTRACEPTION

Sperm antigens

WE HAVE IDENTIFIED three sperm antigens; rSMP‐B, YWK‐II ⁴⁸ and 80 kDa human sperm antigen (80 kDa HSA). ⁴⁹ The use of peptides as contraceptive vaccine offers distinct advantages because they can be synthesized readily and in sufficient amounts for biological testing. Also a region of the polypeptide specific to germ cells can be selected; thereby avoiding potential unexpected immunological cross‐reactivity of induced antibodies with somatic cells of the testis and other tissues. YAL‐198, corresponding to residues 21–36 of the YWK‐II polypeptide significantly (9/12) reduced fertility. Active immunization with rSMP‐230, residues 1–28 of rSMP‐B protein induced infertility in 83% of the treated female rats. Without adjuvant, the peptides conjugated to tetanus toxoid as carrier protein reduced the fertilization rate by 38% with YAL‐198 and 45% with rSMP‐230. It is noteworthy that antibody titers against each peptide were higher in the non‐pregnant groups than those in the pregnant groups.

Another approach is mucosal immunization using recombinant Salmonella dublin expressing YAL‐198. The microorganism was administered orally or vaginally to female rats. Both routes of immunization induced significant levels of antibody titers in sera and vaginal secretions associated with infertility in 60% (3/5) and 80% (4/5) of treated animals. ⁵⁰

The synthetic peptides of 80 kDa HSA immunologically mimicked the native protein and impaired infertility after passive administration of antipeptide antibodies in male and female rats.

Zona pellucida antigens

Autoantibodies to ZP could cause the development of immunological infertility in women by inhibiting sperm penetration across ZP. Furthermore, they appear not to be systematically detrimental. These two tenets are the basis for the development of an immunocontraceptive using ZP antigens. ⁵¹ In fact, female animals including non‐human primates immunized with ZP antigens resulted in infertility. However, the infertility was associated with disturbances in follicular development but not with block in fertility. Use of purified or recombinant ZP glycoproteins minimized contamination of other ovarian‐associated proteins, but failed to avoid the development of ovarian histopathology even in homologous animal models. Efforts should be made to isolate B cell epitope of ZP antigen that can elicit antibodies in high titer without oophoritogenic T cell responses. At present, ZP‐based contraceptive vaccines have been used for controlling wildlife populations.