Clin. exp. Immunol. (1977) 28, 163-172.

Human and guinea-pig cross-reacting spermatozoa autoantigens and autoantibodies MARTA D 'AL ME IDA* & G. A. VO I S IN* (Centre d'Immuno-Pathologie de l'Association Claude Bernard et de l'INSERM, Hdpital Saint-Antoine, Paris, France

(Received 13 September 1976) SUMMARY

Cross-reactivity between human and guinea-pig spermatozoa autoantigens has been studied in two systems: (1) guinea-pig anti-autoantigens S, P and T immune sera reacting on human spermatozoa and (2) activity of human anti-sperm sera on guinea-pig spermatozoa. These assays, as well as the results of cross-absorptions in the two systems, revealed a close antigenic relationship between the two species of spermatozoa concerning the guinea-pig autoantigen S. However, there were some differences in its localization and serological manifestations in both species. The guinea-pig autoantigen T bore a more distant, though definite, relationship with human spermatozoa. No clear evidence of cross-reactivity between the two species was found for autoantigen P. At variance with autoimmune (or isoimmune) human antibodies, 'natural' human antibodies active on guinea-pig spermatozoa were absorbed by guinea-pig spleen cells. INTRODUCTION Spermatozoa autoantigens are very interesting in many respects. In humans, they are linked to infertility syndromes (Rumke & Hellinga, 1959). In guinea-pigs, they can induce autoimmune aspermatogenic orchitis (AIAO) (Voisin, Delaunay & Barber, 1951; Freund, Lipton & Thompson, 1953). Four autoantigens have been described in guinea-pig spermatozoa, named S, P) T and Z (Voisin & Toullet, 1968). The first three have been extensively studied (Toullet, Voisin & Nemirovsky, 1973; Toullet & Voisin, 1974; Voisin, Toullet & d'Almeida, 1974) and shown to have different immunological, physicochemical and pathological properties. Female guinea-pigs react against S, P and T autoantigens, making antibodies that cannot be distinguished from male autoantibodies directed against the same autoantigens. Other authors have also defined several testicular autoantigens in the guinea-pig (Hagopian et al., 1976). The situation is much less clear for human spermatozoa, although several autoantigens are likely to exist (hyaluronidase, Metz, 1973; swollen sperm heads, Kolk, Samuel & Rumke, 1974). On the other hand, cross reactions have been described between spermatozoa autoantigens of guineapig, rabbit, rat and mouse (Voisin, Toullet & Maurer, 1958; Toullet, Voisin & Nemirovsky, 1973) and also, less specifically, between testicular and spermatozoa antigens of rodents (Otani & Behrman, 1963; Barth & Russel, 1964). Cross reactions between rodents and primates are controversial. While Otani & Behrman (1963) failed to find any cross-reaction between guinea-pig and human or monkey testis, Katsh (1962) concluded that a common antigenicity exists between human and guinea-pig testis and the common antigen(s) is only revealed after extraction. More recently, Hansen (1972) found an antigenic relationship between human, bull and rabbit spermatozoa, using immunofluorescence methods. *

Team associated to the CNRS (ERA 149).

Correspondence: Dr Marta d'Almeida, Centre d'Immuno-Pathologie de I'Association Claude Bernard et de 1'INSERM, Hbpital Saint-Antoine, 75012 Paris, France.

163

164

Marta d'Almeida & G. A. Voisin

In view of the preceding facts, it was considered of interest to look for antigenic relationships between human

and

guinea-pig spermatozoa autoantigens, especially

S,

and

T

and whether human

equivalents

could be identified, also if human and guinea-pig anti-sperm sera would react on the same autoantigens in one or the other species. The experimental design was first to make use of guinea-pig anti-S, -P or -T immune sera and to react them with human spermatozoa, utilizing three techniques (immunofluorescence, spermagglutination and spermatotoxicity). Absorption of the same specific immune sera with human spermatozoa was also attempted. Conversely, sera from infertile men containing autoantibodies against human spermatozoa as well as sera from infertile women containing antibodies against human spermatozoa were tested against guinea-pig spermatozoa. Absorption of the same sera was attempted with guinea-pig spermatozoa and

guinea-pig spermatozoal autoantigens. Several patterns of cross-reactivity were actually observed in the two directions and using several techniques. MATERIALS AND METHODS Specific sera. Guinea-pig immune sera were raised by immunizing castrated male guinea-pigs with one of the three autoantigens (S, P or T) extracted from guinea-pig epididymal spermatozoa. The preparation of these autoantigens and immune sera have been described earlier (Toullet, Voisin & Nemirovsky, 1973). Human sera (male and female) came from infertile couples attending the clinic for immunological causes of sterility. Only those samples providing clear spermatotoxic and/or spermagglutinating activities were included in this study. Collection of spermatozoa. Guinea-pig spermatozoa were collected from excised epididymis by retroperfusion of the vas deferens and epididymis cauda with PBS at pH 7-4 as in Toullet et al., 1973. Epididymal human spermatozoa were obtained by a similar technique from a young man who died from trauma. Ejaculated human spermatozoa were collected from healthy donors of 0 blood group. Fresh samples, simply diluted, or diluted after two washings in PBS at pH 7*4, were used for immunological reactions. Frozen washed spermatozoa were

employed for absorption experiments. Test reactions. Immunofluorescent staining (indirect method)

was

performed on air-dried, formalin-fumes-fixed

or

methan-

ol-fixed smears of human or guinea-pig spermatozoa, as described by Hjort & Hansen (1971). Fluorescent rabbit immune serum against human or guinea-pig globulins prepared by the Pasteur Institute were used at the dilutions of 1/50 and 1/40

respectively. The spermatotoxic and

spermagglutinating techniques employed with guinea-pig spermatozoa have been already described

(Toullet & Voisin, 1974). For human spermatozoa we used a modification of the original Franklin & Dukes (1964) tubeslide agglutination test and the spermatotoxic method of Hamerlynck&Rumke (1968) adapted toourexperimental conditions.

These two methods have been recently detailed (Rose et al., 1976). In the procedure of passive haemagglutination and haemagglutination-inhibition reactions, chromium chloride-treated SRBC were coated with the soluble guinea-pig spermatozoa autoantigens S and P. Absorption experiments. Absorptions were performed with: frozen epididymal guinea-pig spermatozoa (2 x x for 107-2109 ml of undiluted serum); frozen washed human ejaculated spermatozoa(108-109 for0-1 ml of undiluted serum) and washed spleen cells dissociated from human or guinea-pig frozen spleens (9 x 10'-109 for0-1 ml of undiluted serum). In absorption studies sera were diluted with the antigenic suspension in order to obtain their highest positive dilution and 1 the mixture left to incubate for 4°C. hr at After centrifugation, the supernatants were tested the same day.

0-1

RESULTS Reactions of human spermatozoa with guinea-pig immune

sera

directed against guinea-pig spermatozoa

autoantigens Properties of the immune sera when tested against guinea-pig spermatozoa S. Anti-S staining the acrosome and the head

cap

or autoantigens (Table 1). Antiof guinea-pig spermatozoa (Fig. Ib) giving anaphylactic

FIG. 1. Immunofluorescent reactions of guinea-pig anti 5, P and T immune sera on human and guinea-pig spermatozoa. (a) Anti-S staining of the acrosome plus the neck and adjacent portions of the head and the middle piece of human spermatozoa (non fixed). (Magnification x 2040). (b) Anti-S staining of the acrosome plus the head-cap of guinea-pig spermatozoa (non-fixed). (Magnification x 680.) (c) Anti-T staining of the acrosome of human spermatozoa (non-fixed). (Magnification x 2040.) (d) Anti-T staining of the acrosome plus the postnuclear cap of guinea-pig spermatozoa (formalin-fixed). (Magnification x 680.) (e) Anti-P negative reaction on human spermatozoa (non-fixed). (Magnification x 2040.) (f) Anti-P staining of the acrosome of guinea-pig

spermatozoa (formalin-fixed). (Magnification

x

680.)

FIG. I

Marta d'Almeida & G. A. Voisin

166

TABLE 1. Reactions with guinea-pig spermatozoa and/or autoantigens

Guinea-pig immune sera

Immunofluorescence

Anti-S

Acrosome + head cap + Acrosome Acrosome plasma membrane

Anti-P Anti-T

Anaphylaxis

Passive haemagglutination*

Complement fixation

+ (S)

-

+-

Spermatotoxicity

Precipitation

-

(P)

+

+

Anti-ovalbumine *

Chromium chloride-treated SRBC coated with the autoantigens S or P.

reactions (PDC) and passive haemagglutination with S-coated SRBC, and almost no complement fixation, no spermatotoxicity and no precipitation. Anti-P. Anti-P staining the acrosome (only the anterior part) of guinea-pig spermatozoa (Fig. if) giving anaphylaxis, passive haemagglutination with P-coated SRBC, a strong complement fixation but no spermatotoxicity and precipitation. Anti-T. Anti-T staining the acrosome but also all the cytoplasmic membrane of guinea-pig spermatozoa (Fig. Id) and immature germinal cells (at least spermatids), fixing complement and the only one to be spermatotoxic. Properties of the guinea-pig immune sera when tested against human spermatozoa. The same anti-S, anti-T, anti-P and anti-OVA guinea-pig sera were tested against human spermatozoa by four techniques: immunofluorescence, spermagglutination, spermatotoxicity and complement-fixation (Table 2). Anti-S sera gave a constant and bright fluorescence of the acrosome of human spermatozoa, often accompanied by a staining of the neck and adjacent portions of the head and middle pieces (Fig. la). These sera were found to agglutinate the human spermatozoa but not the homologous ones. The agglutinating titres varied from 8-128 in good correlation with the haemagglutinating titres (antigen-S-coated SRBC). The human spermatozoa agglutinated mostly by the middle piece and the posterior part of the head (Fig. 2). These reactions were considered specific as they could be absorbed exclusively by the spermatozoa of either of the two species (Table 3). The immunofluorescent staining produced by the anti-T sera on human spermatozoa was localized mainly on the acrosome (Fig. Ic) rarely on the tail. With some sera, a limited fluorescence of the middle piece was seen. These staining patterns vanished when the immune sera were incubated with guinea-pig spermatozoa. Incubation with human spermatozoa resulted in a diminished fluorescence (Table 3). No spermagglutination was detected with any of the anti-T immune sera tested on ejaculated human spermatozoa. *TABLE 2. Action of guinea-pig immune sera* on human spermatozoa Nature of guinea-pig immune sera* and results of the tests Test reactions with human spermatozoa

Immunofluorescence

Spermagglutination Spermatotoxicity Complement fixation

Anti-S

(16/16)t l (12/13) ± (4/10)

Anti-P

+ (3/10)

Anti-T

Anti-oxalbumine

(12/12) +(

3/9) -

* Guinea-pigs weere immunized against autoantigens S, P or T and oxalbumine. t Number positive/number tested sera.

Guinea-pig and human spermatozoa

FIG. 2.

Guinea-pig anti-autoantigen

(Magnification

x

S

agglutination

guinea-pig autoantigen P immune tinating activity on human spermatozoa. activity

sera on

mostly by

the

mniddle-piece.

sera

did

show immunofluorescent

not

(30-40%/) of all groups of guinea-pig autoimmune

sera

(Fig. le)

tested showed

a

or

weak

spermagglu-

spermatotoxic

human spermatozoa.

Absorption ofguinea-pig spermatozoa

Guinea-pig anti-S, anti-P human

of human spermatozoa,

480)

The

Some

167

autoantigens

spleen

autoantibodies

by

and anti-T autoimmune

cells and made

human spermatozoa

sera were

(Table 4)

incubated with human spermatozoa and

guinea-pig spermatozoa. with human spermatozoa led to disappearance of the bright guinea-pig fluorescence observed on guinea-pig spermatozoa acrosomes and head-cap. Incubation of the same sera with human spleen cells resulted in no decrease of the observed fluorescent staining. Furthermore, the passive haemagglutination of S-coated SRBC given by guinea-pig anti-S sera could be inhibited by a Incubation of

saline

The tion

extract same

to react

anti-S

with

sera

of human spermatozoa.

saline

extract

of human spermatozoa

of BGG-coated red cells

nation and the

by

passive haemolysis

The immunofluorescent

(otherwise unable to serum) also

the guinea-pig anti-BGG

reaction

of P-coated SRBC caused

of these

sera on

guinea-pig

by

inhibit the passive

haemagglutinapassive haemagglutianti-autoantigen P sera.

inhibited the

the guinea-pig

acrosome was

solely

but

specifically

dimin-

ished after incubation with human spermatozoa.

TABLE 3.

Activity

of guinea-pig spermatozoa autoantibodies

on

human spermatozoa.

Absorption

on

guinea-pig

and

human spermatozoa

Reaction of guinea-pig immune sera with human spermatozoa after incubation with:

Guinea-pig

Tests

immune-sera Saline

Anti-S Anti-T

Immunofluorescence Spermagglutination Immunofluorescence

+ + A-

Human Guinea-pig Human Guinea-pig spermatozoa spermatozoa spleen cells spleen cells -

-

+ +

+

-

IV-

+ + a

Marta d'Almeida & G. A. Voisin

168

TABLE 4. Absorption of guinea-pig spermatozoa autoantibodies by human spermatozoa

Reaction of guinea-pig immune sera with guinea-pig spermatozoa and autoantigens after incubation with:

Guinea-pig immune sera

Tests and final serum dilutions

Anti-S

Saline

Immunofluorescence (1/20) Passive haemagglutination (1/100) Immunofluorescence (1/10) Passive haemagglutination (1/2000) Immunofluorescence (1/80) Spermagglutination (1/20) Spermatotoxicity (1/200)

Anti-P Anti-T

Human Human spermatozoa spleen cells

+

-

+

+ +

+

+

+ + + +

+ + +

+ + +

In the absorption experiments performed with guinea-pig anti-autoantigen T sera, as many as 7 x 109 human spermatozoa (per 0 1 ml of nondiluted serum) had no effect on the action of any of these sera on guinea-pig spermatozoa.

Reactions of sera from infertile

men

and women (containing anti-human

sperm

antibodies) with guinea-pig

spermatozoa

Sera from two groups of persons were tested against guinea-pig spermatozoa: (1) fertile couples: sera collected from fifteen fertile men and fourteen fertile women from 20-40 years; (2) infertile couples: fifteen men and fourteen women of the same age group as above were chosen on account of the reactivity of their sera with human spermatozoa in, at least, two of the three tests: immunofluorescence, spermaggluwere

tination and

spermatotoxicity.

These fifty-eight human sera were tested by the afore-mentioned tests against guinea-pig spermatozoa and in passive haemagglutination with S-coated SRBC. Passive haemagglutination with P-coated SRBC was invariably negative. TABLE 5. Reactions of human sera with guinea-pig spermatozoa

Percentage of positive reactions with guinea-pig spermatozoa or S antigen in the following tests:

Origin of sera

Sterile men* Fertile ment Sterile women* Fertile woment

Number of sera studied 15 15 14 14

Immunofluorescence 73 P