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A 28K-dalton cell surface autoantigen of spermatogenesis: Characterization using a monoclonal antibody
DEVELOPMENTAL
BIOLOGY
89,
(1982)
92-1~
A 28K-Dalton Cell Surface Autoantigen of Spermatogenesis: Characterization Using a Monoclonal Antibody STEPHEN J. GAUNT’ Department
of
Zoology, Received
University January
of
Oxford,
South
30, 1981; accepted
Parks
Road,
in revised
Oxford, form
OX1
August
3PS, United
Kingdom
5, 1981
In mammals, some differentiation antigens of spermatogenesis are autoantigens. One of these autoantigens has now been identified and characterized using a monoclonal antibody, lB3. The antibody-producing cells were prepared using the spleen cells of a mouse which had been immunized syngeneically with vas deferens spermatozoa. Evidence is presented that the lB3 antigen resides on a surface protein or glycoprotein of molecular weight 28K daltons. In absorption assays using a variety of different mouse tissues, the lB3 antigen was detected only on testicular cells and spermatozoa. The antigen appeared on the cell surface at a specific stage in spermatogenesis: it was not found on cells of the prepuberal, 8-day mouse testis, but was first seen on pachytene primary spermatocytes arising at 14 days. lB3 antigen was then retained on all postpachytene cells within the testis. By immunofluorescence, lB3 antigen was seen to be present over the entire cell surface of spermatozoa within the testis and caput epididymis, but cauda epididymal and vas deferens spermatozoa showed fluorescent lahelling only upon the anterior tip of the head. Although tissue specific, lB3 antigen is not species specific. It was found on testicular cells of rat, rabbit, vole, and guinea pig, but not of the rhesus monkey. Some possible applications of monoclonal antibodies to the study of spermatogenesis are discussed.
INTRODUCTION
Romrell, 1980). Thus, the differentiation antigens of spermatogenesis are autoantigens. This is a consequence of the development of spermatozoa within the “adlumenal compartment” of the testicular seminiferous tubules (Dym and Fawcett, 1970), a site which is isolated from the immune system by the blood-testis barrier (Kormano, 1967; Setchell, 1967), and which is therefore immunologically privileged (Johnson, 1973). Unfortunately, each autoantiserum may recognize several different antigenic determinants (O’Rand and Porter, 1979). Such antisera do not therefore allow the characterization of single antigens. Use of monoclonal antibodies (Kohler and Milstein, 1976) does, however, permit analysis of individual antigens, and the feasibility of preparing monoclonal antibodies to the differentiation antigens on spermatogenic cells has already been demonstrated by Bechtol et al., (1979). This paper describes a monoclonal antibody, lB3, which recognizes an autoantigen on spermatogenic cells. In the mouse, the lB3 antigen is specific to testicular cells, first appears on pachytene-stage primary spermatocytes, and resides on a 28K-dalton polypeptide.
During spermatogenesis, germ cells of the male, the spermatogonia, differentiate to form spermatozoa. Cell surface molecules specific to the process of spermatogenesis arise during this differentiation, and these are of interest for several reasons. First, we may gain an understanding at the molecular level of the cell surface phenomena which are peculiar to spermatogenic cells. These include interaction between germinal cells and Sertoli cells (Ziparo et al., 1980), capacitation of mature spermatozoa (Oliphant and Brackett, 19’73), fertilization of the egg, and a process whereby surface molecules on spermatozoa become extensively immobilized (Millette, 19’7’7; Millette and Bell&, 1977). Second, they provide markers to assess differentiation during the in vitro culture of germinal cells (Steinberger et al., 1970). Third, they provide some of the antigens which are responsible for the diseases of experimental allergic orchitis in animals, and autoimmune infertility in man (reviewed in Johnson, 1973; Johnson et al., 1975). Use of antisera to cell surface antigens has been a popular approach in the study of the cell surface during spermatogenesis. Antisera specific to the differentiation antigens of spermatogenesis may be prepared simply by immunizing an animal with its own testicular cells or spermatozoa (Tung et al., 1979; O’Rand and ’ Present address: A.R.C. tingdon Road, Cambridge,
Institute of Animal CB3 OJQ, U.K.
0012-1606/82/010092-09$02.00/O Copyright All rights
Q 1982 by Academic Press, Inc. of reproduction in any form reserved.
Physiology,
MATERIALS
AND
METHODS
Immunixation and cell fusion. A female C3H mouse was immunized with spermatozoa from C3H males. Immunizations were at 2-week intervals, and each immunizing dose consisted of the entire contents extruded
307 Hun-
9.2
STEPHEN
J. GAUNT
28K Autoantigen
from the vasa deferentia of a single male mouse. The first four injections were given intraperitoneally in Freunds complete adjuvant (GIBCO). The final injection was given intravenously in Alpha medium (Flow Laboratories Ltd.). Four days later, the mouse was killed and 5 X lo7 spleen cells, purified on Ficoll-Paque (Pharmacia), were mixed with 5 X lo6 NSl nonsecretor myeloma cells (Kohler and Milstein, 1976). The cells were washed in serum-free Alpha medium, centrifuged (100~ for 2 min) and then resuspended, for fusion, in 100 ~1 of 50% (w/v) polyethylene glycol 1500 (BDH Ltd.). After 1 min at room temperature, the mixture was diluted over a further 2 min with 10 ml Alpha medium. The fused cells were centrifuged, resuspended in Alpha medium with 10% (v/v) foetal calf serum and HAT supplement (Littlefield, 1964), and then dispensed as 96 separate cultures in four multiwell dishes (Flow Laboratories, Ltd.). At 2-day intervals, half of the medium in each well was replaced with fresh HAT medium. After 2-weeks, the supernatants of 50 wells which contained colonies were tested by indirect immunofluorescence for binding to vas deferens spermatozoa. Only one of the supernatants was positive. Cells from this well were plated sparsely (500 cells per 60-mm dish) into 0.25% (w/v) agarose (LGT agarose, Miles Ltd.) and allowed to grow as colonies. Of 10 colonies picked, 9 produced antibody. Repeating this procedure, one positive clone was recloned to obtain 12 subclones, all of which produced antibody. One subclone, designated lB3, was used in further investigations. Indirect immunojluorescence. For use in indirect immunofluorescence, lB3 culture supernatant was used either without prior concentration, or after fivefold concentration using an Amicon ultrafiltration device. A suspension of testicular cells was obtained by gentle disruption of mouse testes using a loose-fitting homogenizer. The cells (approximately 20 mg wet wt) were incubated in lB3 antibody (250 ~1) for 20 min at 4°C washed twice in PBS/BSA (phosphate-buffered saline plus 0.5% bovine serum albumen), incubated for 20 min at 4°C in fluorescein-conjugated rabbit anti-mouse IgG antiserum (Miles Ltd.), and then washed a further three times in PBEVBSA. All incubations were carried out in the presence of 0.1% azide. Cells were examined using a Zeiss RA microscope fitted with epifluorescent illumination. As judged by the proportion of testicular cells labelled, and their intensity of fluorescence, the lB3 antibody was always present in saturating concentrations. Dissociated ovarian cells and preimplantation mouse embryos were examined as above. Prior to immunofluorescent labelling, zonae were removed from embryos using pronase (Mintz, 1967) and the embryos were then
of Spermatogenesis
93
cultured for 12 hr in an attempt to permit recovery of the cell surface. Absorption assays. The amounts of lB3 antigen on different tissues were compared by absorption assays. For this, 280-~1 samples of a constant dilution of lB3 culture supernatant were incubated with serial dilutions of each of the tissues under test. After 1 hr at 4°C the absorption mixtures were centrifuged (5000~ for 20 min) and the supernatants collected. Duplicate, loo-p.1 aliquots of each supernatant were assayed for remaining lB3 activity by an indirect radioactive binding assay (Morris and Williams, 1975). For this assay, the aliquots were incubated for 30 min at 4°C with 3mg (wet weight) samples of testicular cells in roundbottom wells of a microtiter plate. Cells were washed twice in PBS/BSA, and then the cells of each well were incubated for a further 30 min in 30 ~1 of PBS/BSA containing lo5 cpm of ‘251-labelled F(ab’)z rabbit antimouse IgG (Jensenius and Williams, 1974). After three further washes in PBS/BSA solution, cells were dissolved in 2 M sodium hydroxide (100 ~1 per well) and then transferred to tubes for counting. The dilution of the lB3 antibody used in absorptions was derived by titration of the batch of supernatant against 3 mg of testicular cells and lo5 cpm ‘251-labelled second-layer antibody. The dilution chosen (l/32) gave 80% of the maximal binding activity. The conditions used in the assays therefore allowed quantitation of relative binding activity (Morris and Williams, 1975). Solubilixation of the lB3 antigen. Using the procedures described by Letarte-Muirhead et al. (1974), a variety of detergents were tested for their ability to solubilize the lB3 antigen. The antigen was solubilized either directly from testicular cells or, when using deoxycholate, from a crude testicular cell membrane fraction. To prepare the membrane fraction, testicular cells were first allowed to swell for 20 min at 4°C in a hypotonic buffer (10 mMTris-HCl, pH 8.0). The cells were then disrupted by means of a mechanically driven homogenizer, and the suspension was gently centrifuged (1OOg for 2 min) in order to pellet nuclei and unbroken cells. These procedures were repeated until such time as the supernatant appeared to be clear of nuclei and whole cells by phase-contrast microscopy. A pellet of crude membranes was then obtained by centrifugation of this supernatant for 30 min at 50,OOOg. Fifty milligrams (wet weight) of either testicular cells or their crude membranes were incubated at 0°C for 1 hr in 100 ~1 of 2% detergent in either PBS or, for deoxycholate, 0.9% NaCl, 10 mM Tris-HCl, pH 8.0 buffer. Alpha medium (100 ~1) was then added. For each detergent, duplicate samples of these mixtures were prepared. One sample was centrifuged (150,OOOg for 1
94
DEVELOPMENTALBIOLOGY
hr) and the supernatant, containing the detergent-solubilized material, was collected. lB3 culture supernatant and Alpha medium were then added to both samples of each duplicate set in order to obtain solutions which contained 0.25% detergent and a l/8 dilution of lB3 antibody. All samples were then centrifuged (50009 for 20 min) and the supernatants collected. Duplicate lOO-~1 aliquots of each of the supernatants were assayed for lB3 antibody activity by indirect radioactive binding assay as described above, but using testicular cells (3 mg/well) which had been fixed in 0.1% glutaraldehyde. The concentration of the lB3 antibody used in the absorption was found in a preliminary experiment to be a limiting concentration when working with glutaraldehyde-fixed target cells. Preparation
of mono&ma1 antibody affinity
column.
lB3 antibody was found to be an IgM when tested by immunodiffusion against immunoglobulin class- and subclass-specific antisera (Miles Ltd.). Ascites fluid, obtained from mice bearing lB3 cell tumours, was used as the source of lB3 antibody for the preparation of affinity columns. The mice were C3H X Balb/c F1 hybrids. Ascites fluid was clarified by centrifugation, and the IgM fraction, containing lB3 activity, was isolated by gel filtration as the first protein peak to elute from a Sephacryl S-300 (Pharmacia) column (80.0 X 4.0 cm), using 0.5 M NaCl, 0.1 M Tris-HCl, pH 8.0, buffer. The IgM preparation was dialysed against carbonate buffer (0.5 M NaCl, 0.1 M carbonate, pH 8.3). Sepharose 4B (Sigma) was activated by cyanogen bromide using the procedures developed by McMaster and Williams (1979). After activation, the Sepharose was washed with water, then carbonate buffer. For coupling to Sepharose beads, IgM was gently stirred with the activated Sepharose (2 mg protein/ml of beads) in carbonate buffer for 12 hr at 4°C. The beads were washed, then stirred for a further 2 hr in 1 M ethanolamineHCl, pH 9.0, in order to block any remaining active sites. After washing, conjugated beads were stored in 0.5 MNaCl, 0.1% azide, 10 mMTris-HCl, pH 8.0, buffer. A 2C5 monoclonal antibody column served as a control during affinity purification of the lB3 antigen. 2C5, a gift from B. J. Randle, is a monoclonal mouse IgM which binds to mouse teratocarcinoma cells, but not to testicular cells (unpublished). Preparation of 1B3 antigen by affinity chromatography. A solution of the lB3 antigen was prepared, as described above, by solubilizing the crude membrane pellet obtained from 20 mouse testes in 1 ml of deoxycholate solution (2% sodium deoxycholate, 0.9% NaCl, 10 mM Tris-HCl, pH 8.0). Material which remained insoluble was then pelleted (150,OOOg for 1 hr) and the clear supernatant was reduced to 1% deoxycholate so-
VOLUME 89,1982
lution by addition of an equal volume of NaCl/Tris buffer (0.9% NaCl, 10 mM Tris-HCl, pH 8.0). Phenylmethylsulphonyl fluoride (Sigma) was added to a concentration of 2 mM as a protease inhibitor. The affinity column was set up using 200 ~1 of antibody-conjugated beads. The beads were washed in 1% DOC/NaCl/Tris buffer (1% sodium deoxycholate, 0.9% NaCl, 10 mMTris-HCl, pH 8,O) prior to passage through the column of the detergent-solubilized material. The column was again washed by passing approximately 2 ml of 1% DOC/NaCl/Tris buffer prior to the elution of bound antigen by a high-pH buffer (1% deoxycholate, 0.9% NaCl, 0.05 M diethylamine-HCl, pH 11.5). After initial entry of elution buffer into the column, the first 50 ~1 of solution to be voided was discarded, and the following 300 ~1 was collected as the lB3 antigen preparation. This was immediately neutralised by addition of 50 ~1 of 1 M Tris-HCl, pH 8.0, and then dialysed against 0.5% DOC/NaCl/Tris buffer. Affinity-purified antigen was radiolabelled with ‘%I using the chloramine T method (Jensenius and Williams, 1974). The reaction was carried out in the presence of 0.5% deoxycholate. Labeled protein was separated from nonincorporated ‘%I by gel filtration in G25 Sephadex (Pharmacia) using 0.5% DOC/NaCl/Tris buffer. In some preparations, the labelled antigen was subjected to a second affinity-column purification step. Prior to electrophoresis, labelled antigen preparations were precipitated in 15% TCA at 4°C and precipitates were washed twice with cold acetone as described by Sunderland et al. (1979). Air-dried precipitates were then solubilized in SDS denaturing buffer, containing 1% P-mercaptoethanol, prior to electrophoresis in 15% polyacrylamide slab gels (Laemmli, 1970). After drying, gels were exposed to X-ray film to detect radioactivity. RESULTS Binding of lB3 Antibody Testicular Cells
to Mouse Spermatozoa and
Of the 50 independent hybrid cultures which were tested, only one produced antibody which bound to vas deferens spermatozoa. This culture, and its subclone lB3, made antibody which gave fluorescent labelling of most (approximately 70%), but not all, spermatozoa at the anterior tip of the head. These labelled spermatozoa (Figs. 2, 3) were seen to bear material, which was irregular in its form, along the acrosomal border. Spermatozoa not showing this material were never labelled (Fig. 1). The attached material showed speckled fluorescence (Fig. 3) but, in general, fluorescent labelling was mostly confined to the associated surface of the spermatozoon head.
STEPHEN
J. GAUNT
.%K Autoantigenof Spermatogenesis
AS judged by the pattern of fluorescent labelling, cauda epididymal spermatozoa were identical to vas deferens spermatozoa in their surface distribution of lB3 antigen. In contrast, all spermatozoa obtained from the caput epididymis bound lB3 antibody over their entire surface (Fig. 4). Forty percent of cells from the testis bound lB3 antibody when tested by indirect immunofluorescence. These cells included spermatozoa, spermatids (recognised by presence of the developing sperm head-Fig. 5), and round cells. All spermatozoa and spermatids were seen to be labelled. Spermatozoa from the testis, like those from the caput epididymis, were strongly fluorescent over their entire surface. Vas deferens spermatozoa were also tested by immunofluorescence after their in vitro incubation for 2 hr in Krebs-Ringer solution plus 10% BSA. These conditions, developed by Miyamoto and Chang (1973) to permit capacitation of mouse spermatozoa, did not result in any change in the distribution of the lB3 antigen on the cell surface. Tissue Distribution
95
Leydig cells) which are present at this time (Bellve et al., 19’77) do not bear surface lB3 antigen. The first cells to bind lB3 antibody were seen at 14 days (Fig. 6). These were among the largest cells present in the 14-day testis and they comprised about 15% of the total cell population. The time of appearance, and the size of these labelled cells suggest that they are pachytene-stage primary spermatocytes (Bell& et al., 1977), although these criteria cannot exclude the possibility that the antigen may first appear at a slightly earlier stage than true pachytene. Since all spermatids and spermatozoa of the adult testis were also found to be fluorescence positive, it seemed likely that the lB3 antigen, after first appearing on pachytene cells, was retained on all postpachytene stages of spermatogenesis within the testis. The foetal gonads of 15-day, female mouse embryos contain the pachytene stage of primary oocyte (Borum, 1961). None of the cells dissociated from these foetal gonads was seen, by immunofluorescence, to bear lB3 antigen. The antigen thus seems to be restricted to gametogenesis in the male.
of the lB3 Antigen
The distribution of the lB3 antigen throughout a variety of different mouse tissues was examined by absorption assays (Fig. 7). Of the tissues tested, only testicular cells and spermatozoa extruded from the vas deferens were found to absorb lB3 antibody activity. For 50% absorption, the weight of spermatozoa needed was approximately 25 times greater than that of testicular cells. Thus, the lB3 antigen in the spermatozoa preparations was only 1/25th as abundant as in the testis. Absorption of lB3 antibody by testicular cells was examined for several different strains of adult mice: 129, A2G, Balb/c, AKR, CFLP, and C57Bl. For each of these strains, the absorption was similar to that of C3H mice (data not shown). Ovarian cells from adult mice, mouse teratocarcinoma cells, and cells of the preimplantation mouse embryo were all negative for lB3 antigen as judged by indirect immunofluorescence. Time of Appearance of the lB3 Antigen During Spermatogenesis
In the mouse, spermatogenesis begins at 8 days of age and successive stages in meiosis appear at predictable times thereafter. Testes from mice of ages 8, 10, 12, and 14 days were examined for the presence of lB3 antigen-positive cells. Up to 12 days there were no cells positive for lB3 antigen as judged by immunofluorescence. It was therefore concluded that spermatogonia, and the somatic cells of the testis (including Sertoli and
Species Distribution
of the lB3 Antigen
The lB3 antigen was present, but at different concentrations, on testicular cells of the rabbit, rat, vole, and guinea pig. As judged by the weights of testicular cells required for 50% absorption of antibody (Fig. 8a), the concentration of lB3 antigen in the rat testis resembled that in mouse testis. The antigen concentrations in guinea pig and rabbit testis were, respectively, 36-fold less and 6-fold greater than in mouse testis. After indirect immunofluorescent labelling of cells dissociated from the guinea pig testis, lB3 antigen was not detectable on the majority of spermatids or spermatozoa. Patches of fluorescence were, however, seen on a few spermatozoa. In this species, round cells, which accounted for approximately 10% of the total cell population, were the only cell type seen to be clearly labelled. Like the mouse testis, the rabbit testis contained approximately 40% of cells which bore lB3 antigen. Rabbit spermatozoa, like mouse spermatozoa, retained lB3 antigen over the entire cell surface when in the testis and the caput epididymis (Fig. 9), but fluorescent labelling was confined to a small patch on the head of spermatozoa which had been taken from the cauda epididymis or vas deferens (Fig. 10). This fluorescent patch, present on approximately 80% of the cells, resembled that found on mouse spermatozoa: labelling was restricted to the anterior tip of the sperm head and, in general, was not detected unless fragments of material were seen to be attached to this region of the spermatozoon.
FIGS. contrast; FIGS. FIG. FIG. FIG.
l-6. Indirect immunofluorescent labelling of mouse spermatozoa and testicular cells using lB3 monoclonal right: uv illumination. 1-3. Spermatozoa from the vas deferens (arrows indicate material attached to the acrosomal region). 4. Spermatozoa from the &put epididymis. 5. A cluster of spermatids from the adult testis (arrow indicates the developing sperm heads). 6. Cells dissociated from the testes of 14-day-old mice. Bars, 10 pm. 96
antibody.
Left:
phase
STEPHEN
J. GAUNT
28K
Autoantigen
0
50
100 E fi
a -
i OS s
50
100
b. IL 625
125
25
5
1 IBoWl
Reciprocal
Dilution
of Tissue
FIG. ‘7. Tissue distribution of lB3 antigen in the mouse. A constant dilution of lB3 antibody was absorbed with serial dilutions of testis (a), kidney (A), spleen (A), heart (D), lung (e), liver (D), brain (0), or cells extruded from the vas deferens (0). In each case, the maximum weight of tissue used was 30 mg. Antibody remaining unabsorbed was then assayed, by an indirect binding assay, using testicular cells as targets. Percentage absorption values give the results of indirect binding assays using lB3 antibody without prior absorption (OS),
or using
no lB3
antibody
(100%).
In absorption assays, the lB3 antigen was not detected on testicular cells of the rhesus monkey (Fig. 8b). Characterization
of the lB3 Antigen
Incubation of testicular cells with proteases greatly reduced their ability to absorb activity of lB3 monoclonal antibody (Fig. 11). Therefore, it was considered most likely that the antigenic activity was carried by a protein or glycoprotein molecule. As a first step towards isolation of the molecule bearing the antigen, a variety of detergents were tested for their ability to solubilize the molecule with optimal retention of its antigenic activity (Table 1). As tested in absorption assays, the activity on cells or membrane fractions was retained in each of the detergents tested. Detergents differed, however, in their ability to solubilize the antigen. Solubilities varied from 4% in Tween 40 to 55% in deoxycholate. A 2% deoxycholate solution was chosen as the best solvent for the antigen prior to its purification. The lB3 antigen was solubilized, purified using a monoclonal antibody affinity column, and then radiolabelled using the procedures described under Materials and Methods. Of the total radioactivity incorporated, 90% was recovered after precipitation in 15% TCA and then washing of the precipitate in acetone to remove
of Sperma@mis
97
deoxycholate. After electrophoresis in SDS-polyacrylamide gels, most of the radioactivity present was found to run as a single band of molecular weight 28K daltons, but several less abundant molecules were also present (Fig. 12, Tracks B). As controls on this experiment, the procedure was repeated using columns of Sepharose 4B beads which either had no bound antibody, or had bound 2C5 monoclonal antibody. 2C5 is a monoclonal mouse IgM which has no binding to testicular cells. Neither of these two columns gave any purification of the 28K molecule (Fig. 12, Tracks A, C). These controls were carried out in order to ensure that the 28K product had not simply bound nonspecifically to either Sepharose beads or IgM. Incubation of ‘%I-labelled, affinity-purified antigen with proteases (0.5% papain or Pronase for 2 hr at 3’7°C) prior to acrylamide gel electrophoresis left only a trace of the labelled 28K molecule (data not shown). The most likely explanation for this finding is that the 28K molecule is a protein or glycoprotein. DISCUSSION
In recent years, several authors have used antisera, prepared by immunizing animals with testicular cells or spermatozoa, to examine the appearance of cell surface antigens during spermatogenesis (Toullet, et al., 1973; Millette and Bellve, 1977; O’Rand and Romrell, 1977, 1980; Tung and Fritz, 1978; Tung et al., 1979). Antisera prepared in this way have been found to recognize a multiplicity of antigens (Shen and Menge, 1971; O’Rand and Porter, 1979), and it remains unclear as to whether the same groups of cell surface molecules are being characterized by the different antisera used. Use of monoclonal antibodies will permit a very systematic analysis of the differentiation antigens of spermatogenesis, since for each antibody there is a single antigenie determinant, and since the molecule which bears this determinant can be identified. The present study describes the characterization of a differentiation antigen of spermatogenesis using a monoclonal antibody, lB3. The lB3 antigen is present on the germinal cells of male mice, rats, rabbits, guinea pigs, and voles. It appears to be specific to the germinal cells and tests upon adult mice showed that it was not detectable in somatic tissues. The antigen, solubilized in deoxycholate, was purified from mouse testes by use of an antibody affinity column. The only major component eluting from the column was a 28K-dalton polypeptide, and so it is probable that this molecule bears the lB3 antigenic site. Two pieces of evidence suggest that the lB3 differentiation antigen is an autoantigen. First, the antibody was
raised
following
a syngeneic
dure and, second, the antigen
immunization
proce-
did not appear on the
98
DEVELOPMENTAL
BIOLOGY
germinal cells of mice until they had reached the pachytene stage of primary spermatocyte, a cell type concealed from the immune response of the host by the blood-testis barrier (Dym and Fawcett, 1970). That the autoantigens of spermatogenesis first appear on pachytene cells is already known from previous studies using conventional antisera (O’Rand and Romrell, 1977,198O; Romrell and O’Rand, 1978; Tung and Fritz, 1978). Pachytene antigens, such as lB3 antigen, have been classified as “early autoantigens” (O’Rand and Romrell, 1980), distinguishing them from “late autoantigens” which first appear on spermatids (Toullet et al., 1973; Tung et al., 1979; O’Rand and Romrell, 1980). Like lB3 antibody, the monoclonal antibody XT-l, described by Bechtol et al. (1979), may recognize a pachytene-stage autoantigen of spermatogenesis. Thus, XT-l antigen was first detectable in mouse testes of 13 days of age, the approximate time of first appearance of pachytene cells (Bell+ et al, 1977). Since the authors did not identify the XT-l antigen, it is not known whether or not it is the 28K polypeptide now described. The lB3 antigen remained detectable on postpachytene stages of mouse germinal cells. With the maturation of spermatozoa within the epididymis, however, there was a marked change in the pattern of immunofluorescent cell surface labelling. Thus, while mouse spermatozoa within the testis and caput epididymis were labelled over their entire surface, cauda epididyma1 and vas deferens spermatozoa only showed labelling of the anterior head region of most, but not all, of the cells. Rabbit spermatozoa showed a similar change. It was obvious, from immunofluorescence studies, that there was an overall loss of lB3 antigen from spermatozoa within the epididymis. Two different expla-
VOLUME 89, 1982
0
b
7776
12%
216
36
6
1
K375mgl
Reciprocal
Dilution
of Tissue
8. Species distribution of lB3 antigen. lB3 antibody was absorbed with serial dilutions of testicular tissue taken from mouse (o), rat (0), rabbit (A), vole (W), guinea pig (a), or rhesus monkey (0). In each case, the maximum wet weight of tissue used was 375 mg. Unabsorbed antibody was then assayed, by indirect binding, using mouse testicular cells as targets. Values for 0 and 100% absorption were obtained as described in the legend to Fig. 7. FIG.
nations may account for this loss. First, it may be directly associated with the process of maturation within the epididymis. For example, adsorption of epididymal proteins onto the spermatozoon surface (Killian and Amann, 1973) may mask the antigen. A second explanation, however, is that loss of the antigen is merely due to shedding from the membrane, a phenomenon simply associated with aging of the cells. Loss of lB3
FIGS. 9, 10. Indirect immunofluorescent labelling of rabbit spermatozoa using lB3 monoclonal antibody. Left: phase contrast; right: uv illumination. The cells were obtained from the caput epididymis (Fig. 9), and the cauda epididymis (Fig. 10). Arrow indicates material attached to the acrosomal region of a cauda epididymal spermatozoon. Bars, 10 pm.
STEPHEN
J. GAUNT
28K Autoantigen
of Spermatogwis
99
FIG. 11. Sensitivity of lB3 antigen to proteases. Mouse testicular cells (300 mg in 10 ml of solution) were incubated for 1 hr at 37°C in PBS (a), 0.5% trypsin (A), or 0.5% papain (0). The cells were then washed extensively in foetal calf serum prior to their serial dilution for absorption assays. In each case, the maximum wet weight of tissue used was 100 mg. Unabsorbed antibody was assayed, by indirect binding, using mouse testicular cells as targets. Values for 0 and 100% absorption were obtained as described in the legend to Fig. 7.
antigen from germinal cells of the guinea pig was already evident by the time of spermatid and spermatozoon formation within the testis. It seems plausible that the rate of loss of lB3 antigen from the germinal cells might be approximately the same for all species, and that the essential difference lies in the amount of antigen which appears on the cell surface during pachytene. The fluorescent labelling at the anterior tip of mature spermatozoa, seen in both mouse and rabbit, occurred in association with attachment of debris in this region. The debris itself showed only speckled fluorescence, while the underlying spermatozoon surface was more intensely labelled. One possible explanation for this finding is that antigen-positive material present in the epididymal tract may have adsorbed on to the surface of the spermatozoon. It is unclear, however, as to why such adsorption should be limited to the head region of only some of the spermatozoa. As a second TABLE ACTIVITY
1
AND SOLUBILITY OF lB3 ANTIGEN OF DETERGENTS
IN A VARIETY
lB3 activity (W) Detergent
Fraction
Tween 40 Triton X-100 Berol Brij 96 Lubrol-PX Deoxycholate
C C C C C M
Detergent/ buffer 114 92 102 114 127 92
Soluble/ buffer 4 12 26 30 47 55
Note. Detergents were used in extractions at a concentration of 2%. Detergent/buffer: antigenic activity of cells (C) or membranes (M) in detergent expressed as a percentage of activity in buffer only. Soluble/buffer: activity in 150,OOOgdetergent supernatant expressed as a percentage of activity of cells or membranes in buffer only.
FIG. 12. Purification of lB3 antigen from a deoxycholate extract of mouse testes. SDS-polyacrylamide gel electrophoresis of ‘%I-labelled eluates from: Tracks A, control column of Sepharose beads without conjugated antibody; Tracks B, lB3 antibody column; Tracks C, 2C5 antibody column. In A, B, and C, there were threefold differences in the amount of radioactivity loaded onto adjacent lanes, and the maximum radioactivity loaded was the same in each case. The figure shows an autoradiograph of the dried gel.
explanation, the debris may result from detachment of the outer acrosomal membrane, such as that which occurs during acrosome vesiculation or vacuolation (Jones, 1975). Fluorescent labelling of the anterior sperm head would then indicate that the lB3 antigen is present within the acrosomal contents, or upon the inner acrosomal membrane. Support for this interpretation comes from the work of Toullet et al. (1973). Using an antiserum to guinea pig spermatozoa, these authors described “T antigen” which is present in both the plasma membrane and the acrosomal membrane. Within the acrosome, the lB3 antigen might be protected from whatever mechanism causes its loss from the surface plasma membrane during maturation of spermatozoa in the epididymis. The experiments described in this paper do not indicate the function of the 28K molecule which bears the lB3 antigenic site, However, the specificity of the antigen to the testis, and its presence on testicular cells of a variety of different mammalian species, suggest that it may play a role in a cell surface phenomenon which is peculiar to spermatogenic cells. Availability of a monoclonal antibody to a cell surface molecule provides an opportunity to examine the molecule’s function. This opportunity has been exploited in the field
DEVELOPMENTAL BIOLO~SY
100
of cellular immunology where monoclonal antibodies have been identified which block in vitro cell-to-cell interactions between lymphocytes (Webb et al., 1979; Reinherz, et al., 1980). Two in vitro systems of cellular interaction have so far been developed using spermatogenie cells: fertilization, and a specificity in adhesion to Sertoli cells shown by pachytene-stage primary spermatocytes (Ziparo et al., 1980). It will be of interest to test whether lB3 antibody can inhibit either of these two cell-to-cell interactions. I thank K. Chada, B. J. Randle, J. K. Heath, and C. F. Graham for their valuable comments. The work was supported by grants from the Medical Research Council and the Cancer Research Campaign. REFERENCES BECHTOL, K. B., BROWN, S. C., and KENNE’IT, R. H. (1979). Recognition of differentiation antigens of spermatogenesis in the mouse by using antibodies from spleen cell-myeloma hybrids after syngeneic immunization. Proc. Nut. Acad. Sci. USA 76, 363-367. BELLV~, A. R., CAVICCHIA, J. C., MILLETTE, C. F., O’BRIEN, D. A., BHATNAGAR, Y. M., and DYM, M. (197’7). Spermatogenic cells of the prepuberal mouse: Isolation and morphological characterization. J. Cell. BioL 74, 68-85. BORUM, K. (1961). Oogenesis in the mouse. Exp. Cell Res. 24.495-507. DYM, M., and FAWCET~, D. W. (1970). The blood-testis barrier in the rat and the physiological compartmentation of the seminiferous epithelium Biol. Repro& 3, 308-326. JENSENIUS, J. C., and WILLIAMS, A. F. (1974). The binding of antiimmunoglobulin antibodies to rat thymocytes and thoracic duct lymphocytes. Eur. J. Immunol. 4, 91-97. JOHNSON, M. H. (1973). Physiological mechanisms for the immunological isolation of spermatozoa. Advan Reprod Physiol6,279-324. JOHNSON, M. H., HEKMAN, A., and R~~MKE, P. H. (1975). The male and female genital tracts in allergic disease. In “Clinical Aspects of Immunology” (P. G. H. Gell, R. R. A. Coombs, and P. J. Lachmann, eds.), 3rd ed., pp. 1509-1544. Blackwell, London. JONES, R. C. (1975). Fertility and infertility in mammals in relation to sperm structure. In “The Biology of the Male Gamete” (J. G. Duckett and P. A. Racey, eds.), pp. 343-365. Academic Press, New York. KILLIAN, G. J., and AMANN, R. P. (1973). Immunoelectrophoretic characterization of fluid and sperm entering and leaving the bovine epididymis. Biol. Reprod 9,489-499. KOHLER, G., and MILSTEIN, C. (1976). Derivation of specific antibodyproducing tissue culture and tumour lines by cell fusion. Eur. J Immunol. 6.511-519. KORMANO, M. (1967). Dye permeability and alkaline phosphatase activity of testicular capillaries in the postnatal rat. Histochemie 9, 327-338. LAEMMLI, II. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London) 227, 680-685. LETARTE-MUIRHEAD, M., ACTON, R. T., and WILLIAMS, A. F. (1974). Preliminary characterization of Thy-l.1 and Ag-B antigens from rat tissues solubilized in detergents. B&hem J. 143, 51-61. LITTLEFIELD, J. W. (1964). Selections of hybrids from matings of fibroblasts in vitro and their presumed recombinanta. Science 145, 709-710. MCMASTER, W. R., and WILLIAMS, A. F. (1979). Monoclonal antibodies to Ia antigens from rat thymus: Cross reactions with mouse and human and use in purification of rat Ia glycoproteins. Immunol Rev. 47,117-137.
VOLUME 89, 1982
MILLETTE, C. F. (1977). Distribution and mobility of lectin binding sites on mammalian spermatozoa. In “Immunobiology of Gametes” (M. Edidin and M. H. Johnson, eds.), Clincial and Experimental Immunoreproduction Series No. 4. pp. 51-64.. Cambridge Univ. Press, London/New York. MILLETTE, C. F., and BELLV~~, A. R. (1977). Temporal expression of membrane antigens during mouse spermatogenesis. J. Cell BioL 74, 86-97. MINTZ, B. (1967). Mammalian embryo culture. In “Methods in Developmental Biology” (F. H. Wilt and N. K. Wessels, eds.), pp. 379401, Y. Y. Crowell, New York. MIYAMOTO, H., and CHANG, M. C. (1973). The importance of serum albumen and metabolic intermediates for capacitation of spermatozoa and fertilization of mouse eggs in vitro. J Reprod. Fed 32, 193-205. MORRIS, R. J., and WILLIAMS, A. F. (1975). Antigens on mouse and rat lymphocytes recognized by rabbit antiserum against rat brain: The quantitative analysis of a xenogeneic antiserum. Eur. J. ImmunoL 5, 274-281. OLIPHANT, G., and BRACKETT, B. G. (1973). Immunological assessment of surface changes of rabbit sperm undergoing capacitation. BioL Reprod 9,404-414. O’RAND, M. G., and PORTER, J. P. (1979). Isolation of a sperm membrane silaoglycoprotein from rabbit testes. J ImmunoL 122, 12481254. O’RAND, M. G., and ROMRELL, L. J. (1977). Appearance of cell surface auto and isoantigens during spermatogenesis in the rabbit. Develop. BioL 55, 347-358. O’RAND, M. G., and ROMRELL, L. J. (1980). Appearance of regional surface autoantigens during spermatogenesis: Comparison of antitestis and anti-sperm autoantisera. Develop. BioL 75,431-441. REINHERZ, E. L., HUSSEY, R. E., and SCHLOSSMAN, S. F. (1980). A monoclonal antibody blocking human T cell function. Eur. J. Immunol 10, 758-762. ROMRELL, L. J., and O’RAND, M. G. (1978). Capping and ultrastructural localization of sperm surface isoantigens during spermatogenesis. Devebp. Biol. 63, 76-93. SETCHELL, B. P. (1967). The blood-testicular fluid barrier in sheep. J. PhysioL 189, 63-65. SHEN, T. F., and MENGE, A. C. (1971). Separation of testis-specific antigens of the rabbit. BioL Reprod 5, 96 (Abstract). STEINBERGER, E., STEINBERGER, A., and FICHER, M. (1970). Study of spermatogenesis and steroid metabolism in cultures of mammalian testes. Recent Progr. Harm. Res. 26, 547-588. SUNDERLAND, C. A., MCMASTER, W. R., and WILLIAMS, A. F. (1979). Purification with monoclonal antibody of a predominant leukocytecommon antigen and glycoprotein from rat thymocytes. Eur. J. ImmunoL 9,155-159. TOULLET, F., VOISIN, G. A., and NEMIROVSKY, M. (1973). Histochemical localization of three guinea pig spermatozoa1 autoantigens. Immunology 24,635-653. TUNG, P. S., and FRITZ, I. B. (1978). Specific surface antigens on rat pachytene spermatocytes and successive classes of germinal cells. Develop. BioL 64, 297-315. TUNG, K. S. K., HAN, L. B., and EVAN, A. P. (1979). Differentiation autoantigen of testicular cells and spermatozoa in the guinea pig. Develop. BioL 68, 224-238. WEBB, M., MASON, D. W., and WILLIAMS, A. F. (1979). Inhibition of mixed lymphocyte response by monoclonal antibody specific for a rat T lymphocyte subset. Nature (&m&m) 282, 841-843. ZIPARO, E., GEREMIA, R., Russo, M. A., and STEFANINI, M. (1980). An in vitro study of surface mediated interactions between germ cells and Sertoli cells. Eur. J Cell BioL 22, 236 (Abstract).
BIOLOGY
89,
(1982)
92-1~
A 28K-Dalton Cell Surface Autoantigen of Spermatogenesis: Characterization Using a Monoclonal Antibody STEPHEN J. GAUNT’ Department
of
Zoology, Received
University January
of
Oxford,
South
30, 1981; accepted
Parks
Road,
in revised
Oxford, form
OX1
August
3PS, United
Kingdom
5, 1981
In mammals, some differentiation antigens of spermatogenesis are autoantigens. One of these autoantigens has now been identified and characterized using a monoclonal antibody, lB3. The antibody-producing cells were prepared using the spleen cells of a mouse which had been immunized syngeneically with vas deferens spermatozoa. Evidence is presented that the lB3 antigen resides on a surface protein or glycoprotein of molecular weight 28K daltons. In absorption assays using a variety of different mouse tissues, the lB3 antigen was detected only on testicular cells and spermatozoa. The antigen appeared on the cell surface at a specific stage in spermatogenesis: it was not found on cells of the prepuberal, 8-day mouse testis, but was first seen on pachytene primary spermatocytes arising at 14 days. lB3 antigen was then retained on all postpachytene cells within the testis. By immunofluorescence, lB3 antigen was seen to be present over the entire cell surface of spermatozoa within the testis and caput epididymis, but cauda epididymal and vas deferens spermatozoa showed fluorescent lahelling only upon the anterior tip of the head. Although tissue specific, lB3 antigen is not species specific. It was found on testicular cells of rat, rabbit, vole, and guinea pig, but not of the rhesus monkey. Some possible applications of monoclonal antibodies to the study of spermatogenesis are discussed.
INTRODUCTION
Romrell, 1980). Thus, the differentiation antigens of spermatogenesis are autoantigens. This is a consequence of the development of spermatozoa within the “adlumenal compartment” of the testicular seminiferous tubules (Dym and Fawcett, 1970), a site which is isolated from the immune system by the blood-testis barrier (Kormano, 1967; Setchell, 1967), and which is therefore immunologically privileged (Johnson, 1973). Unfortunately, each autoantiserum may recognize several different antigenic determinants (O’Rand and Porter, 1979). Such antisera do not therefore allow the characterization of single antigens. Use of monoclonal antibodies (Kohler and Milstein, 1976) does, however, permit analysis of individual antigens, and the feasibility of preparing monoclonal antibodies to the differentiation antigens on spermatogenic cells has already been demonstrated by Bechtol et al., (1979). This paper describes a monoclonal antibody, lB3, which recognizes an autoantigen on spermatogenic cells. In the mouse, the lB3 antigen is specific to testicular cells, first appears on pachytene-stage primary spermatocytes, and resides on a 28K-dalton polypeptide.
During spermatogenesis, germ cells of the male, the spermatogonia, differentiate to form spermatozoa. Cell surface molecules specific to the process of spermatogenesis arise during this differentiation, and these are of interest for several reasons. First, we may gain an understanding at the molecular level of the cell surface phenomena which are peculiar to spermatogenic cells. These include interaction between germinal cells and Sertoli cells (Ziparo et al., 1980), capacitation of mature spermatozoa (Oliphant and Brackett, 19’73), fertilization of the egg, and a process whereby surface molecules on spermatozoa become extensively immobilized (Millette, 19’7’7; Millette and Bell&, 1977). Second, they provide markers to assess differentiation during the in vitro culture of germinal cells (Steinberger et al., 1970). Third, they provide some of the antigens which are responsible for the diseases of experimental allergic orchitis in animals, and autoimmune infertility in man (reviewed in Johnson, 1973; Johnson et al., 1975). Use of antisera to cell surface antigens has been a popular approach in the study of the cell surface during spermatogenesis. Antisera specific to the differentiation antigens of spermatogenesis may be prepared simply by immunizing an animal with its own testicular cells or spermatozoa (Tung et al., 1979; O’Rand and ’ Present address: A.R.C. tingdon Road, Cambridge,
Institute of Animal CB3 OJQ, U.K.
0012-1606/82/010092-09$02.00/O Copyright All rights
Q 1982 by Academic Press, Inc. of reproduction in any form reserved.
Physiology,
MATERIALS
AND
METHODS
Immunixation and cell fusion. A female C3H mouse was immunized with spermatozoa from C3H males. Immunizations were at 2-week intervals, and each immunizing dose consisted of the entire contents extruded
307 Hun-
9.2
STEPHEN
J. GAUNT
28K Autoantigen
from the vasa deferentia of a single male mouse. The first four injections were given intraperitoneally in Freunds complete adjuvant (GIBCO). The final injection was given intravenously in Alpha medium (Flow Laboratories Ltd.). Four days later, the mouse was killed and 5 X lo7 spleen cells, purified on Ficoll-Paque (Pharmacia), were mixed with 5 X lo6 NSl nonsecretor myeloma cells (Kohler and Milstein, 1976). The cells were washed in serum-free Alpha medium, centrifuged (100~ for 2 min) and then resuspended, for fusion, in 100 ~1 of 50% (w/v) polyethylene glycol 1500 (BDH Ltd.). After 1 min at room temperature, the mixture was diluted over a further 2 min with 10 ml Alpha medium. The fused cells were centrifuged, resuspended in Alpha medium with 10% (v/v) foetal calf serum and HAT supplement (Littlefield, 1964), and then dispensed as 96 separate cultures in four multiwell dishes (Flow Laboratories, Ltd.). At 2-day intervals, half of the medium in each well was replaced with fresh HAT medium. After 2-weeks, the supernatants of 50 wells which contained colonies were tested by indirect immunofluorescence for binding to vas deferens spermatozoa. Only one of the supernatants was positive. Cells from this well were plated sparsely (500 cells per 60-mm dish) into 0.25% (w/v) agarose (LGT agarose, Miles Ltd.) and allowed to grow as colonies. Of 10 colonies picked, 9 produced antibody. Repeating this procedure, one positive clone was recloned to obtain 12 subclones, all of which produced antibody. One subclone, designated lB3, was used in further investigations. Indirect immunojluorescence. For use in indirect immunofluorescence, lB3 culture supernatant was used either without prior concentration, or after fivefold concentration using an Amicon ultrafiltration device. A suspension of testicular cells was obtained by gentle disruption of mouse testes using a loose-fitting homogenizer. The cells (approximately 20 mg wet wt) were incubated in lB3 antibody (250 ~1) for 20 min at 4°C washed twice in PBS/BSA (phosphate-buffered saline plus 0.5% bovine serum albumen), incubated for 20 min at 4°C in fluorescein-conjugated rabbit anti-mouse IgG antiserum (Miles Ltd.), and then washed a further three times in PBEVBSA. All incubations were carried out in the presence of 0.1% azide. Cells were examined using a Zeiss RA microscope fitted with epifluorescent illumination. As judged by the proportion of testicular cells labelled, and their intensity of fluorescence, the lB3 antibody was always present in saturating concentrations. Dissociated ovarian cells and preimplantation mouse embryos were examined as above. Prior to immunofluorescent labelling, zonae were removed from embryos using pronase (Mintz, 1967) and the embryos were then
of Spermatogenesis
93
cultured for 12 hr in an attempt to permit recovery of the cell surface. Absorption assays. The amounts of lB3 antigen on different tissues were compared by absorption assays. For this, 280-~1 samples of a constant dilution of lB3 culture supernatant were incubated with serial dilutions of each of the tissues under test. After 1 hr at 4°C the absorption mixtures were centrifuged (5000~ for 20 min) and the supernatants collected. Duplicate, loo-p.1 aliquots of each supernatant were assayed for remaining lB3 activity by an indirect radioactive binding assay (Morris and Williams, 1975). For this assay, the aliquots were incubated for 30 min at 4°C with 3mg (wet weight) samples of testicular cells in roundbottom wells of a microtiter plate. Cells were washed twice in PBS/BSA, and then the cells of each well were incubated for a further 30 min in 30 ~1 of PBS/BSA containing lo5 cpm of ‘251-labelled F(ab’)z rabbit antimouse IgG (Jensenius and Williams, 1974). After three further washes in PBS/BSA solution, cells were dissolved in 2 M sodium hydroxide (100 ~1 per well) and then transferred to tubes for counting. The dilution of the lB3 antibody used in absorptions was derived by titration of the batch of supernatant against 3 mg of testicular cells and lo5 cpm ‘251-labelled second-layer antibody. The dilution chosen (l/32) gave 80% of the maximal binding activity. The conditions used in the assays therefore allowed quantitation of relative binding activity (Morris and Williams, 1975). Solubilixation of the lB3 antigen. Using the procedures described by Letarte-Muirhead et al. (1974), a variety of detergents were tested for their ability to solubilize the lB3 antigen. The antigen was solubilized either directly from testicular cells or, when using deoxycholate, from a crude testicular cell membrane fraction. To prepare the membrane fraction, testicular cells were first allowed to swell for 20 min at 4°C in a hypotonic buffer (10 mMTris-HCl, pH 8.0). The cells were then disrupted by means of a mechanically driven homogenizer, and the suspension was gently centrifuged (1OOg for 2 min) in order to pellet nuclei and unbroken cells. These procedures were repeated until such time as the supernatant appeared to be clear of nuclei and whole cells by phase-contrast microscopy. A pellet of crude membranes was then obtained by centrifugation of this supernatant for 30 min at 50,OOOg. Fifty milligrams (wet weight) of either testicular cells or their crude membranes were incubated at 0°C for 1 hr in 100 ~1 of 2% detergent in either PBS or, for deoxycholate, 0.9% NaCl, 10 mM Tris-HCl, pH 8.0 buffer. Alpha medium (100 ~1) was then added. For each detergent, duplicate samples of these mixtures were prepared. One sample was centrifuged (150,OOOg for 1
94
DEVELOPMENTALBIOLOGY
hr) and the supernatant, containing the detergent-solubilized material, was collected. lB3 culture supernatant and Alpha medium were then added to both samples of each duplicate set in order to obtain solutions which contained 0.25% detergent and a l/8 dilution of lB3 antibody. All samples were then centrifuged (50009 for 20 min) and the supernatants collected. Duplicate lOO-~1 aliquots of each of the supernatants were assayed for lB3 antibody activity by indirect radioactive binding assay as described above, but using testicular cells (3 mg/well) which had been fixed in 0.1% glutaraldehyde. The concentration of the lB3 antibody used in the absorption was found in a preliminary experiment to be a limiting concentration when working with glutaraldehyde-fixed target cells. Preparation
of mono&ma1 antibody affinity
column.
lB3 antibody was found to be an IgM when tested by immunodiffusion against immunoglobulin class- and subclass-specific antisera (Miles Ltd.). Ascites fluid, obtained from mice bearing lB3 cell tumours, was used as the source of lB3 antibody for the preparation of affinity columns. The mice were C3H X Balb/c F1 hybrids. Ascites fluid was clarified by centrifugation, and the IgM fraction, containing lB3 activity, was isolated by gel filtration as the first protein peak to elute from a Sephacryl S-300 (Pharmacia) column (80.0 X 4.0 cm), using 0.5 M NaCl, 0.1 M Tris-HCl, pH 8.0, buffer. The IgM preparation was dialysed against carbonate buffer (0.5 M NaCl, 0.1 M carbonate, pH 8.3). Sepharose 4B (Sigma) was activated by cyanogen bromide using the procedures developed by McMaster and Williams (1979). After activation, the Sepharose was washed with water, then carbonate buffer. For coupling to Sepharose beads, IgM was gently stirred with the activated Sepharose (2 mg protein/ml of beads) in carbonate buffer for 12 hr at 4°C. The beads were washed, then stirred for a further 2 hr in 1 M ethanolamineHCl, pH 9.0, in order to block any remaining active sites. After washing, conjugated beads were stored in 0.5 MNaCl, 0.1% azide, 10 mMTris-HCl, pH 8.0, buffer. A 2C5 monoclonal antibody column served as a control during affinity purification of the lB3 antigen. 2C5, a gift from B. J. Randle, is a monoclonal mouse IgM which binds to mouse teratocarcinoma cells, but not to testicular cells (unpublished). Preparation of 1B3 antigen by affinity chromatography. A solution of the lB3 antigen was prepared, as described above, by solubilizing the crude membrane pellet obtained from 20 mouse testes in 1 ml of deoxycholate solution (2% sodium deoxycholate, 0.9% NaCl, 10 mM Tris-HCl, pH 8.0). Material which remained insoluble was then pelleted (150,OOOg for 1 hr) and the clear supernatant was reduced to 1% deoxycholate so-
VOLUME 89,1982
lution by addition of an equal volume of NaCl/Tris buffer (0.9% NaCl, 10 mM Tris-HCl, pH 8.0). Phenylmethylsulphonyl fluoride (Sigma) was added to a concentration of 2 mM as a protease inhibitor. The affinity column was set up using 200 ~1 of antibody-conjugated beads. The beads were washed in 1% DOC/NaCl/Tris buffer (1% sodium deoxycholate, 0.9% NaCl, 10 mMTris-HCl, pH 8,O) prior to passage through the column of the detergent-solubilized material. The column was again washed by passing approximately 2 ml of 1% DOC/NaCl/Tris buffer prior to the elution of bound antigen by a high-pH buffer (1% deoxycholate, 0.9% NaCl, 0.05 M diethylamine-HCl, pH 11.5). After initial entry of elution buffer into the column, the first 50 ~1 of solution to be voided was discarded, and the following 300 ~1 was collected as the lB3 antigen preparation. This was immediately neutralised by addition of 50 ~1 of 1 M Tris-HCl, pH 8.0, and then dialysed against 0.5% DOC/NaCl/Tris buffer. Affinity-purified antigen was radiolabelled with ‘%I using the chloramine T method (Jensenius and Williams, 1974). The reaction was carried out in the presence of 0.5% deoxycholate. Labeled protein was separated from nonincorporated ‘%I by gel filtration in G25 Sephadex (Pharmacia) using 0.5% DOC/NaCl/Tris buffer. In some preparations, the labelled antigen was subjected to a second affinity-column purification step. Prior to electrophoresis, labelled antigen preparations were precipitated in 15% TCA at 4°C and precipitates were washed twice with cold acetone as described by Sunderland et al. (1979). Air-dried precipitates were then solubilized in SDS denaturing buffer, containing 1% P-mercaptoethanol, prior to electrophoresis in 15% polyacrylamide slab gels (Laemmli, 1970). After drying, gels were exposed to X-ray film to detect radioactivity. RESULTS Binding of lB3 Antibody Testicular Cells
to Mouse Spermatozoa and
Of the 50 independent hybrid cultures which were tested, only one produced antibody which bound to vas deferens spermatozoa. This culture, and its subclone lB3, made antibody which gave fluorescent labelling of most (approximately 70%), but not all, spermatozoa at the anterior tip of the head. These labelled spermatozoa (Figs. 2, 3) were seen to bear material, which was irregular in its form, along the acrosomal border. Spermatozoa not showing this material were never labelled (Fig. 1). The attached material showed speckled fluorescence (Fig. 3) but, in general, fluorescent labelling was mostly confined to the associated surface of the spermatozoon head.
STEPHEN
J. GAUNT
.%K Autoantigenof Spermatogenesis
AS judged by the pattern of fluorescent labelling, cauda epididymal spermatozoa were identical to vas deferens spermatozoa in their surface distribution of lB3 antigen. In contrast, all spermatozoa obtained from the caput epididymis bound lB3 antibody over their entire surface (Fig. 4). Forty percent of cells from the testis bound lB3 antibody when tested by indirect immunofluorescence. These cells included spermatozoa, spermatids (recognised by presence of the developing sperm head-Fig. 5), and round cells. All spermatozoa and spermatids were seen to be labelled. Spermatozoa from the testis, like those from the caput epididymis, were strongly fluorescent over their entire surface. Vas deferens spermatozoa were also tested by immunofluorescence after their in vitro incubation for 2 hr in Krebs-Ringer solution plus 10% BSA. These conditions, developed by Miyamoto and Chang (1973) to permit capacitation of mouse spermatozoa, did not result in any change in the distribution of the lB3 antigen on the cell surface. Tissue Distribution
95
Leydig cells) which are present at this time (Bellve et al., 19’77) do not bear surface lB3 antigen. The first cells to bind lB3 antibody were seen at 14 days (Fig. 6). These were among the largest cells present in the 14-day testis and they comprised about 15% of the total cell population. The time of appearance, and the size of these labelled cells suggest that they are pachytene-stage primary spermatocytes (Bell& et al., 1977), although these criteria cannot exclude the possibility that the antigen may first appear at a slightly earlier stage than true pachytene. Since all spermatids and spermatozoa of the adult testis were also found to be fluorescence positive, it seemed likely that the lB3 antigen, after first appearing on pachytene cells, was retained on all postpachytene stages of spermatogenesis within the testis. The foetal gonads of 15-day, female mouse embryos contain the pachytene stage of primary oocyte (Borum, 1961). None of the cells dissociated from these foetal gonads was seen, by immunofluorescence, to bear lB3 antigen. The antigen thus seems to be restricted to gametogenesis in the male.
of the lB3 Antigen
The distribution of the lB3 antigen throughout a variety of different mouse tissues was examined by absorption assays (Fig. 7). Of the tissues tested, only testicular cells and spermatozoa extruded from the vas deferens were found to absorb lB3 antibody activity. For 50% absorption, the weight of spermatozoa needed was approximately 25 times greater than that of testicular cells. Thus, the lB3 antigen in the spermatozoa preparations was only 1/25th as abundant as in the testis. Absorption of lB3 antibody by testicular cells was examined for several different strains of adult mice: 129, A2G, Balb/c, AKR, CFLP, and C57Bl. For each of these strains, the absorption was similar to that of C3H mice (data not shown). Ovarian cells from adult mice, mouse teratocarcinoma cells, and cells of the preimplantation mouse embryo were all negative for lB3 antigen as judged by indirect immunofluorescence. Time of Appearance of the lB3 Antigen During Spermatogenesis
In the mouse, spermatogenesis begins at 8 days of age and successive stages in meiosis appear at predictable times thereafter. Testes from mice of ages 8, 10, 12, and 14 days were examined for the presence of lB3 antigen-positive cells. Up to 12 days there were no cells positive for lB3 antigen as judged by immunofluorescence. It was therefore concluded that spermatogonia, and the somatic cells of the testis (including Sertoli and
Species Distribution
of the lB3 Antigen
The lB3 antigen was present, but at different concentrations, on testicular cells of the rabbit, rat, vole, and guinea pig. As judged by the weights of testicular cells required for 50% absorption of antibody (Fig. 8a), the concentration of lB3 antigen in the rat testis resembled that in mouse testis. The antigen concentrations in guinea pig and rabbit testis were, respectively, 36-fold less and 6-fold greater than in mouse testis. After indirect immunofluorescent labelling of cells dissociated from the guinea pig testis, lB3 antigen was not detectable on the majority of spermatids or spermatozoa. Patches of fluorescence were, however, seen on a few spermatozoa. In this species, round cells, which accounted for approximately 10% of the total cell population, were the only cell type seen to be clearly labelled. Like the mouse testis, the rabbit testis contained approximately 40% of cells which bore lB3 antigen. Rabbit spermatozoa, like mouse spermatozoa, retained lB3 antigen over the entire cell surface when in the testis and the caput epididymis (Fig. 9), but fluorescent labelling was confined to a small patch on the head of spermatozoa which had been taken from the cauda epididymis or vas deferens (Fig. 10). This fluorescent patch, present on approximately 80% of the cells, resembled that found on mouse spermatozoa: labelling was restricted to the anterior tip of the sperm head and, in general, was not detected unless fragments of material were seen to be attached to this region of the spermatozoon.
FIGS. contrast; FIGS. FIG. FIG. FIG.
l-6. Indirect immunofluorescent labelling of mouse spermatozoa and testicular cells using lB3 monoclonal right: uv illumination. 1-3. Spermatozoa from the vas deferens (arrows indicate material attached to the acrosomal region). 4. Spermatozoa from the &put epididymis. 5. A cluster of spermatids from the adult testis (arrow indicates the developing sperm heads). 6. Cells dissociated from the testes of 14-day-old mice. Bars, 10 pm. 96
antibody.
Left:
phase
STEPHEN
J. GAUNT
28K
Autoantigen
0
50
100 E fi
a -
i OS s
50
100
b. IL 625
125
25
5
1 IBoWl
Reciprocal
Dilution
of Tissue
FIG. ‘7. Tissue distribution of lB3 antigen in the mouse. A constant dilution of lB3 antibody was absorbed with serial dilutions of testis (a), kidney (A), spleen (A), heart (D), lung (e), liver (D), brain (0), or cells extruded from the vas deferens (0). In each case, the maximum weight of tissue used was 30 mg. Antibody remaining unabsorbed was then assayed, by an indirect binding assay, using testicular cells as targets. Percentage absorption values give the results of indirect binding assays using lB3 antibody without prior absorption (OS),
or using
no lB3
antibody
(100%).
In absorption assays, the lB3 antigen was not detected on testicular cells of the rhesus monkey (Fig. 8b). Characterization
of the lB3 Antigen
Incubation of testicular cells with proteases greatly reduced their ability to absorb activity of lB3 monoclonal antibody (Fig. 11). Therefore, it was considered most likely that the antigenic activity was carried by a protein or glycoprotein molecule. As a first step towards isolation of the molecule bearing the antigen, a variety of detergents were tested for their ability to solubilize the molecule with optimal retention of its antigenic activity (Table 1). As tested in absorption assays, the activity on cells or membrane fractions was retained in each of the detergents tested. Detergents differed, however, in their ability to solubilize the antigen. Solubilities varied from 4% in Tween 40 to 55% in deoxycholate. A 2% deoxycholate solution was chosen as the best solvent for the antigen prior to its purification. The lB3 antigen was solubilized, purified using a monoclonal antibody affinity column, and then radiolabelled using the procedures described under Materials and Methods. Of the total radioactivity incorporated, 90% was recovered after precipitation in 15% TCA and then washing of the precipitate in acetone to remove
of Sperma@mis
97
deoxycholate. After electrophoresis in SDS-polyacrylamide gels, most of the radioactivity present was found to run as a single band of molecular weight 28K daltons, but several less abundant molecules were also present (Fig. 12, Tracks B). As controls on this experiment, the procedure was repeated using columns of Sepharose 4B beads which either had no bound antibody, or had bound 2C5 monoclonal antibody. 2C5 is a monoclonal mouse IgM which has no binding to testicular cells. Neither of these two columns gave any purification of the 28K molecule (Fig. 12, Tracks A, C). These controls were carried out in order to ensure that the 28K product had not simply bound nonspecifically to either Sepharose beads or IgM. Incubation of ‘%I-labelled, affinity-purified antigen with proteases (0.5% papain or Pronase for 2 hr at 3’7°C) prior to acrylamide gel electrophoresis left only a trace of the labelled 28K molecule (data not shown). The most likely explanation for this finding is that the 28K molecule is a protein or glycoprotein. DISCUSSION
In recent years, several authors have used antisera, prepared by immunizing animals with testicular cells or spermatozoa, to examine the appearance of cell surface antigens during spermatogenesis (Toullet, et al., 1973; Millette and Bellve, 1977; O’Rand and Romrell, 1977, 1980; Tung and Fritz, 1978; Tung et al., 1979). Antisera prepared in this way have been found to recognize a multiplicity of antigens (Shen and Menge, 1971; O’Rand and Porter, 1979), and it remains unclear as to whether the same groups of cell surface molecules are being characterized by the different antisera used. Use of monoclonal antibodies will permit a very systematic analysis of the differentiation antigens of spermatogenesis, since for each antibody there is a single antigenie determinant, and since the molecule which bears this determinant can be identified. The present study describes the characterization of a differentiation antigen of spermatogenesis using a monoclonal antibody, lB3. The lB3 antigen is present on the germinal cells of male mice, rats, rabbits, guinea pigs, and voles. It appears to be specific to the germinal cells and tests upon adult mice showed that it was not detectable in somatic tissues. The antigen, solubilized in deoxycholate, was purified from mouse testes by use of an antibody affinity column. The only major component eluting from the column was a 28K-dalton polypeptide, and so it is probable that this molecule bears the lB3 antigenic site. Two pieces of evidence suggest that the lB3 differentiation antigen is an autoantigen. First, the antibody was
raised
following
a syngeneic
dure and, second, the antigen
immunization
proce-
did not appear on the
98
DEVELOPMENTAL
BIOLOGY
germinal cells of mice until they had reached the pachytene stage of primary spermatocyte, a cell type concealed from the immune response of the host by the blood-testis barrier (Dym and Fawcett, 1970). That the autoantigens of spermatogenesis first appear on pachytene cells is already known from previous studies using conventional antisera (O’Rand and Romrell, 1977,198O; Romrell and O’Rand, 1978; Tung and Fritz, 1978). Pachytene antigens, such as lB3 antigen, have been classified as “early autoantigens” (O’Rand and Romrell, 1980), distinguishing them from “late autoantigens” which first appear on spermatids (Toullet et al., 1973; Tung et al., 1979; O’Rand and Romrell, 1980). Like lB3 antibody, the monoclonal antibody XT-l, described by Bechtol et al. (1979), may recognize a pachytene-stage autoantigen of spermatogenesis. Thus, XT-l antigen was first detectable in mouse testes of 13 days of age, the approximate time of first appearance of pachytene cells (Bell+ et al, 1977). Since the authors did not identify the XT-l antigen, it is not known whether or not it is the 28K polypeptide now described. The lB3 antigen remained detectable on postpachytene stages of mouse germinal cells. With the maturation of spermatozoa within the epididymis, however, there was a marked change in the pattern of immunofluorescent cell surface labelling. Thus, while mouse spermatozoa within the testis and caput epididymis were labelled over their entire surface, cauda epididyma1 and vas deferens spermatozoa only showed labelling of the anterior head region of most, but not all, of the cells. Rabbit spermatozoa showed a similar change. It was obvious, from immunofluorescence studies, that there was an overall loss of lB3 antigen from spermatozoa within the epididymis. Two different expla-
VOLUME 89, 1982
0
b
7776
12%
216
36
6
1
K375mgl
Reciprocal
Dilution
of Tissue
8. Species distribution of lB3 antigen. lB3 antibody was absorbed with serial dilutions of testicular tissue taken from mouse (o), rat (0), rabbit (A), vole (W), guinea pig (a), or rhesus monkey (0). In each case, the maximum wet weight of tissue used was 375 mg. Unabsorbed antibody was then assayed, by indirect binding, using mouse testicular cells as targets. Values for 0 and 100% absorption were obtained as described in the legend to Fig. 7. FIG.
nations may account for this loss. First, it may be directly associated with the process of maturation within the epididymis. For example, adsorption of epididymal proteins onto the spermatozoon surface (Killian and Amann, 1973) may mask the antigen. A second explanation, however, is that loss of the antigen is merely due to shedding from the membrane, a phenomenon simply associated with aging of the cells. Loss of lB3
FIGS. 9, 10. Indirect immunofluorescent labelling of rabbit spermatozoa using lB3 monoclonal antibody. Left: phase contrast; right: uv illumination. The cells were obtained from the caput epididymis (Fig. 9), and the cauda epididymis (Fig. 10). Arrow indicates material attached to the acrosomal region of a cauda epididymal spermatozoon. Bars, 10 pm.
STEPHEN
J. GAUNT
28K Autoantigen
of Spermatogwis
99
FIG. 11. Sensitivity of lB3 antigen to proteases. Mouse testicular cells (300 mg in 10 ml of solution) were incubated for 1 hr at 37°C in PBS (a), 0.5% trypsin (A), or 0.5% papain (0). The cells were then washed extensively in foetal calf serum prior to their serial dilution for absorption assays. In each case, the maximum wet weight of tissue used was 100 mg. Unabsorbed antibody was assayed, by indirect binding, using mouse testicular cells as targets. Values for 0 and 100% absorption were obtained as described in the legend to Fig. 7.
antigen from germinal cells of the guinea pig was already evident by the time of spermatid and spermatozoon formation within the testis. It seems plausible that the rate of loss of lB3 antigen from the germinal cells might be approximately the same for all species, and that the essential difference lies in the amount of antigen which appears on the cell surface during pachytene. The fluorescent labelling at the anterior tip of mature spermatozoa, seen in both mouse and rabbit, occurred in association with attachment of debris in this region. The debris itself showed only speckled fluorescence, while the underlying spermatozoon surface was more intensely labelled. One possible explanation for this finding is that antigen-positive material present in the epididymal tract may have adsorbed on to the surface of the spermatozoon. It is unclear, however, as to why such adsorption should be limited to the head region of only some of the spermatozoa. As a second TABLE ACTIVITY
1
AND SOLUBILITY OF lB3 ANTIGEN OF DETERGENTS
IN A VARIETY
lB3 activity (W) Detergent
Fraction
Tween 40 Triton X-100 Berol Brij 96 Lubrol-PX Deoxycholate
C C C C C M
Detergent/ buffer 114 92 102 114 127 92
Soluble/ buffer 4 12 26 30 47 55
Note. Detergents were used in extractions at a concentration of 2%. Detergent/buffer: antigenic activity of cells (C) or membranes (M) in detergent expressed as a percentage of activity in buffer only. Soluble/buffer: activity in 150,OOOgdetergent supernatant expressed as a percentage of activity of cells or membranes in buffer only.
FIG. 12. Purification of lB3 antigen from a deoxycholate extract of mouse testes. SDS-polyacrylamide gel electrophoresis of ‘%I-labelled eluates from: Tracks A, control column of Sepharose beads without conjugated antibody; Tracks B, lB3 antibody column; Tracks C, 2C5 antibody column. In A, B, and C, there were threefold differences in the amount of radioactivity loaded onto adjacent lanes, and the maximum radioactivity loaded was the same in each case. The figure shows an autoradiograph of the dried gel.
explanation, the debris may result from detachment of the outer acrosomal membrane, such as that which occurs during acrosome vesiculation or vacuolation (Jones, 1975). Fluorescent labelling of the anterior sperm head would then indicate that the lB3 antigen is present within the acrosomal contents, or upon the inner acrosomal membrane. Support for this interpretation comes from the work of Toullet et al. (1973). Using an antiserum to guinea pig spermatozoa, these authors described “T antigen” which is present in both the plasma membrane and the acrosomal membrane. Within the acrosome, the lB3 antigen might be protected from whatever mechanism causes its loss from the surface plasma membrane during maturation of spermatozoa in the epididymis. The experiments described in this paper do not indicate the function of the 28K molecule which bears the lB3 antigenic site, However, the specificity of the antigen to the testis, and its presence on testicular cells of a variety of different mammalian species, suggest that it may play a role in a cell surface phenomenon which is peculiar to spermatogenic cells. Availability of a monoclonal antibody to a cell surface molecule provides an opportunity to examine the molecule’s function. This opportunity has been exploited in the field
DEVELOPMENTAL BIOLO~SY
100
of cellular immunology where monoclonal antibodies have been identified which block in vitro cell-to-cell interactions between lymphocytes (Webb et al., 1979; Reinherz, et al., 1980). Two in vitro systems of cellular interaction have so far been developed using spermatogenie cells: fertilization, and a specificity in adhesion to Sertoli cells shown by pachytene-stage primary spermatocytes (Ziparo et al., 1980). It will be of interest to test whether lB3 antibody can inhibit either of these two cell-to-cell interactions. I thank K. Chada, B. J. Randle, J. K. Heath, and C. F. Graham for their valuable comments. The work was supported by grants from the Medical Research Council and the Cancer Research Campaign. REFERENCES BECHTOL, K. B., BROWN, S. C., and KENNE’IT, R. H. (1979). Recognition of differentiation antigens of spermatogenesis in the mouse by using antibodies from spleen cell-myeloma hybrids after syngeneic immunization. Proc. Nut. Acad. Sci. USA 76, 363-367. BELLV~, A. R., CAVICCHIA, J. C., MILLETTE, C. F., O’BRIEN, D. A., BHATNAGAR, Y. M., and DYM, M. (197’7). Spermatogenic cells of the prepuberal mouse: Isolation and morphological characterization. J. Cell. BioL 74, 68-85. BORUM, K. (1961). Oogenesis in the mouse. Exp. Cell Res. 24.495-507. DYM, M., and FAWCET~, D. W. (1970). The blood-testis barrier in the rat and the physiological compartmentation of the seminiferous epithelium Biol. Repro& 3, 308-326. JENSENIUS, J. C., and WILLIAMS, A. F. (1974). The binding of antiimmunoglobulin antibodies to rat thymocytes and thoracic duct lymphocytes. Eur. J. Immunol. 4, 91-97. JOHNSON, M. H. (1973). Physiological mechanisms for the immunological isolation of spermatozoa. Advan Reprod Physiol6,279-324. JOHNSON, M. H., HEKMAN, A., and R~~MKE, P. H. (1975). The male and female genital tracts in allergic disease. In “Clinical Aspects of Immunology” (P. G. H. Gell, R. R. A. Coombs, and P. J. Lachmann, eds.), 3rd ed., pp. 1509-1544. Blackwell, London. JONES, R. C. (1975). Fertility and infertility in mammals in relation to sperm structure. In “The Biology of the Male Gamete” (J. G. Duckett and P. A. Racey, eds.), pp. 343-365. Academic Press, New York. KILLIAN, G. J., and AMANN, R. P. (1973). Immunoelectrophoretic characterization of fluid and sperm entering and leaving the bovine epididymis. Biol. Reprod 9,489-499. KOHLER, G., and MILSTEIN, C. (1976). Derivation of specific antibodyproducing tissue culture and tumour lines by cell fusion. Eur. J Immunol. 6.511-519. KORMANO, M. (1967). Dye permeability and alkaline phosphatase activity of testicular capillaries in the postnatal rat. Histochemie 9, 327-338. LAEMMLI, II. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London) 227, 680-685. LETARTE-MUIRHEAD, M., ACTON, R. T., and WILLIAMS, A. F. (1974). Preliminary characterization of Thy-l.1 and Ag-B antigens from rat tissues solubilized in detergents. B&hem J. 143, 51-61. LITTLEFIELD, J. W. (1964). Selections of hybrids from matings of fibroblasts in vitro and their presumed recombinanta. Science 145, 709-710. MCMASTER, W. R., and WILLIAMS, A. F. (1979). Monoclonal antibodies to Ia antigens from rat thymus: Cross reactions with mouse and human and use in purification of rat Ia glycoproteins. Immunol Rev. 47,117-137.
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MILLETTE, C. F. (1977). Distribution and mobility of lectin binding sites on mammalian spermatozoa. In “Immunobiology of Gametes” (M. Edidin and M. H. Johnson, eds.), Clincial and Experimental Immunoreproduction Series No. 4. pp. 51-64.. Cambridge Univ. Press, London/New York. MILLETTE, C. F., and BELLV~~, A. R. (1977). Temporal expression of membrane antigens during mouse spermatogenesis. J. Cell BioL 74, 86-97. MINTZ, B. (1967). Mammalian embryo culture. In “Methods in Developmental Biology” (F. H. Wilt and N. K. Wessels, eds.), pp. 379401, Y. Y. Crowell, New York. MIYAMOTO, H., and CHANG, M. C. (1973). The importance of serum albumen and metabolic intermediates for capacitation of spermatozoa and fertilization of mouse eggs in vitro. J Reprod. Fed 32, 193-205. MORRIS, R. J., and WILLIAMS, A. F. (1975). Antigens on mouse and rat lymphocytes recognized by rabbit antiserum against rat brain: The quantitative analysis of a xenogeneic antiserum. Eur. J. ImmunoL 5, 274-281. OLIPHANT, G., and BRACKETT, B. G. (1973). Immunological assessment of surface changes of rabbit sperm undergoing capacitation. BioL Reprod 9,404-414. O’RAND, M. G., and PORTER, J. P. (1979). Isolation of a sperm membrane silaoglycoprotein from rabbit testes. J ImmunoL 122, 12481254. O’RAND, M. G., and ROMRELL, L. J. (1977). Appearance of cell surface auto and isoantigens during spermatogenesis in the rabbit. Develop. BioL 55, 347-358. O’RAND, M. G., and ROMRELL, L. J. (1980). Appearance of regional surface autoantigens during spermatogenesis: Comparison of antitestis and anti-sperm autoantisera. Develop. BioL 75,431-441. REINHERZ, E. L., HUSSEY, R. E., and SCHLOSSMAN, S. F. (1980). A monoclonal antibody blocking human T cell function. Eur. J. Immunol 10, 758-762. ROMRELL, L. J., and O’RAND, M. G. (1978). Capping and ultrastructural localization of sperm surface isoantigens during spermatogenesis. Devebp. Biol. 63, 76-93. SETCHELL, B. P. (1967). The blood-testicular fluid barrier in sheep. J. PhysioL 189, 63-65. SHEN, T. F., and MENGE, A. C. (1971). Separation of testis-specific antigens of the rabbit. BioL Reprod 5, 96 (Abstract). STEINBERGER, E., STEINBERGER, A., and FICHER, M. (1970). Study of spermatogenesis and steroid metabolism in cultures of mammalian testes. Recent Progr. Harm. Res. 26, 547-588. SUNDERLAND, C. A., MCMASTER, W. R., and WILLIAMS, A. F. (1979). Purification with monoclonal antibody of a predominant leukocytecommon antigen and glycoprotein from rat thymocytes. Eur. J. ImmunoL 9,155-159. TOULLET, F., VOISIN, G. A., and NEMIROVSKY, M. (1973). Histochemical localization of three guinea pig spermatozoa1 autoantigens. Immunology 24,635-653. TUNG, P. S., and FRITZ, I. B. (1978). Specific surface antigens on rat pachytene spermatocytes and successive classes of germinal cells. Develop. BioL 64, 297-315. TUNG, K. S. K., HAN, L. B., and EVAN, A. P. (1979). Differentiation autoantigen of testicular cells and spermatozoa in the guinea pig. Develop. BioL 68, 224-238. WEBB, M., MASON, D. W., and WILLIAMS, A. F. (1979). Inhibition of mixed lymphocyte response by monoclonal antibody specific for a rat T lymphocyte subset. Nature (&m&m) 282, 841-843. ZIPARO, E., GEREMIA, R., Russo, M. A., and STEFANINI, M. (1980). An in vitro study of surface mediated interactions between germ cells and Sertoli cells. Eur. J Cell BioL 22, 236 (Abstract).