Identification and immunochemical characterization of spermatogenic cell surface antigens that appear during early meiotic prophase

DEVELOPMENTAL

BIOLOGY

101.307-317

(1984)

Identification and lmmunochemical Characterization of Spermatogenic Cell Surface Antigens That Appear during Early Meiotic Prophase DEBORAH

A. O’BRIEN’

AND CLARKE

F. MILLETTE~

Departmentof Anatomy and Laboratwy

of Human Reproduction and Reproductive Biology, Harvard Medical School, Boston, Massachusetts 02115

Received May

16, 1983;

accepted in revised form .%ptem.ber 14, 1983

Three spermatogenic cell populations isolated from prepuheral mice-type B spermatogonia, preleptotene spermatocytes, and leptotene/zygotene spermatocytes-were used to elicit distinct polyclonal antisera. Surface binding specificities were determined for purified IgGs by indirect immunofluorescence and rosette assays on live cells. Binding activities were assayed both before and after absorptions with a variety of somatic and spermatogenic cells. Each of these antisera binds to surface antigens that are present on germ cells throughout spermatogenesis and are not shared by splenocytes, thymocytes, and erythrocytes. Only the antiserum raised against leptotene and zygotene spermatocytes (ALZ) recognizes a stage-specific subset of surface determinants. After appropriate absorptions, ALZ binds to the surface of early pachytene spermatocytes and germ cells at subsequent stages of differentiation, including vas deferens spermatozoa. Antigens which react with this absorbed IgG are not detected on the surface of spermatogonia or meiotic cells prior to pachynema, including leptotene and zygotene spermatocytes. The observed binding specificities may result from the synthesis of one or more surface molecules during the early meiotic stages, followed by delayed insertion into the plasma membrane during the pachytene stage of meiotic prophase. Stage-specific antigens recognized by ALZ, including both protein and probably lipid, have been localized immunochemically on nitrocellulose blots from onedimensional SDS gels. A dithiothreitol-sensitive constituent (M. - 39,999) recognized by ALZ has been identified as the major protein determinant present in early meiotic cells but absent in %day-old seminiferous cell suspensions containing spermatogonia and Sertoli cells. This determinant is present in populations of preleptotene, leptotene/ zygotene, and early pachytene spermatocytes isolated from Ill-day-old animals, an observation consistent with the hypothesis of delayed insertion into the plasma membrane.

sulfatoxygalactosylacylalkylglycerol, appears on the surface of early primary spermatocytes and is present in subsequent spermatogenic stages in the rat (Kornblatt, 197’9; Shirley and Schachter, 1980; Lingwood and Schachter, 1981). Germ cell specific antigens that appear on the cell surface during the pachytene stage of meiosis have been identified serologically in the mouse (Millette and Bell&, 19’77), rabbit (O’Rand and Romrell, 19’77), and rat (Tung and Fritz, 1978). Although some of these determinants may be transient (Romrell et ah, 1982), many persist on epididymal spermatozoa. RSA-1, a rabbit autoantigen in this class, has been further characterized as a sialoglycoprotein (&& 13,000) that binds Ricinis communis I lectin (O’Rand and Romrell, 1980a, 1981). Additional autoantigenic surface constituents present on mature sperm are first detected during the haploid spermatid stages (Tung et c& 1979; Le Bouteiller et a& 1979; O’Rand and Romrell, 1980b). A third category of surface determinants appears on the spermatozoan surface during epididymal transit (Feuchter et c& 1981). Molecular transitions in germ cell plasma membranes have been detected primarily during the late pachytene or haploid stages of spermatogenesis. This study has focused instead on earlier spermatogenic stages with the hypothesis that important cell surface changes may

INTRODUCTION

Mammalian spermatogenesis is characterized by successive periods of mitotic proliferation, meiosis, and extensive cellular remodeling throughout the haploid spermatid stages. During this developmental sequence regional specialization of the plasma membrane occurs, resulting in a mature gamete with distinctly polarized functional and biochemical properties (for review, see Koehler, 1982; Holt, 1982; Bell& and O’Brien, 1983). Molecular mechanisms underlying these differentiative events have not been well defined. It is clear, however, from a growing number of biochemical and immunological studies that novel surface constituents are inserted into the plasma membrane in a precise temporal sequence during spermatogenesis. Plasma membrane proteins restricted to either pachytene spermatocytes or round spermatids have been identified on two-dimensional polyacrylamide gels (Millette and Moulding, 1981). In addition, a novel glycolipid, ’ Present address: N.I.E.H.S. Research Triangle Park, North Carolina 27709.

sAddress correspondence to Dr. Clarke F. Millet@ LHRRB, Harvard Medical School, 45 Shattuck Street, Boston, Mass. 02115. 307

0012-1606134 Copyright All rights

$3.00

Q 1984 by Academic Press, Inc. of reproduction in any form reserved.

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also occur during the switch from mitosis to meiosis. Type B spermatogonia, preleptotene spermatocytes, and a pooled population of leptotene and zygotene spermatocytes have been utilized in this study to prepare heterologous antisera. One of the antisera (ALZ)3 binds to surface determinants that are first detected during the early pachytene stages of meiotic prophase. Biochemical evidence indicates that at least one of these determinants recognized by ALZ is present within the cell throughout meiosis, prior to detection of surface label by immunofluorescence or rosette assays. MATERIALS

AND

METHODS

Animals and cell preparation. Adult and prepuberal CD-1 mice were obtained from Charles River Breeding Laboratories (Wilmington, Mass.). In some experiments adult Tac:(SW)fBR mice from Taconic Farms, Inc. (Germantown, N. Y.) were used. No differences between these two strains were observed in assays of antibody binding specificities. Mixed cell suspensions from the seminiferous epithelium were prepared from adult and prepuberal animals as described previously (Romrell et & 1976; Bell+ et aL, 19’7’7a,b). Six-day-old testes yield primitive type A spermatogonia and Sertoli cells. Preparations from S-day-old mice contain type A and type B spermatogonia and Sertoli cells. Day 17 or 18 mixed cell suspensions contain predominantly early meiotic germ cells (preleptotene through early pachytene spermatocytes) and Sertoli cells. Late pachytene spermatocytes, spermatids, residual bodies, and testicular sperm are present in adult seminiferous cell suspensions. Initial viabilities of the germ cell suspensions were monitored by trypan blue exclusion and generally exceeded 90%. Purified populations of prepuberal germ cells at defined stages of spermatogenesis were isolated from 17or l&day-old mice by unit gravity sedimentation according to the methods of Bellv6 et al. (1977a,b). Three cell populations-preleptotene, leptotene/zygotene, and early pachytene spermatocytes-were recovered in these separations. Repeated experiments have shown that better yields and purities are obtained when 17-day-old animals weighing 7-10 g are used as testis donors. Mature spermatozoa were isolated from the cauda epididymis and vas deferens of adult mice (at least 10 weeks old) and were washed with either EKRB or PBS ‘Abbreviations used: BSA, bovine serum albumin; DTT, dithiothreitol; EKRB, enriched Krebs-Ringer bicarbonate medium; IgG, immunoglobulin G, NP-40, Nonidet P-40, PBS, phosphate-buffered saline; PMSF, phenylmethylsulfonyl fluoride; SDS, sodium dodecyl sulfate. Antibodies prepared against isolated spermatogenic cells are also abbreviated as follows: ABI and ABc, anti-B spermatogonia; APL, antipreleptotene spermatocytes; ALZ, anti-leptotene and zygotene spermatocytes.

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(Bellve et aL, 1975). In some experiments these cells were fixed with 1% paraformaldehyde (EMS, Ft. Washington, Penn.), 0.2% polyvinylpyrrolidone (PVP-40, Sigma Chemical Co., St. Louis, MO.) in PBS. After a 20min incubation in fixative at 4”C, spermatozoa were pelleted and washed two to three times. Mouse somatic cells including thymocytes, splenocytes, and erythrocytes were obtained from male mice by mincing and gentle homogenization of the appropriate organs in either EKRB or PBS. These cells were washed and filtered through an 80~ Nitex nylon mesh (Tet/Kressilk, Inc., New York, N. Y.) before use. Antibody preparation Heterologous antisera were raised against purified populations of mouse spermatogenie cells in female New Zealand white rabbits (White Pine Rabbitry, Douglas, Mass.) and immunoglobulin G fractions (IgG) were prepared according to the procedures of Millette and Bell& (1977). Antiserum to preleptotene spermatocytes was obtained after 4 injections, averaging 1.7 X lo7 cells per immunization (range, 2.933.8 X lo6 cells) with purities between 74.6 and 81.5%. Leptotene spermatocytes were the principal contaminants in these cell populations. Antiserum to a pooled population of leptotene and zygotene spermatocytes (ALZ) was obtained after five injections, averaging 3.6 X lo7 cells per immunization (range, 7.6-75 X lo6 cells). Purities of these populations ranged from 81.7 to 90.9%, with Sertoli cells as the major contaminant. Surface binding specificities were also determined for two antisera raised against type B spermatogonia isolated from S-day-old mice (Myles and Millette, unpublished observations). Antibody abswptions. Serum and IgG fractions (O.l1 mg/ml in EKRB, 0.5% BSA, 10 mM NaN3) were absorbed repeatedly with thymocytes, splenocytes, and erythrocytes from male mice until surface binding on these cell types was no longer detectable by indirect immunofluorescence. After removing activity shared by these somatic tissues, antibody fractions were further absorbed with mixed seminiferous cell suspensions from either S-day-old or adult mice. All absorptions were conducted at 33-37°C for 1 hr. Indirect immuno~uorescence. Surface binding specificities on both somatic and spermatogenic cells were determined for each antibody by indirect immunofluorescence on live cells. All steps in these assays were conducted at 4°C in the presence of 10 mM NaN3. Cells (3-6 X 106) were first incubated in IgG solutions (0.1-l mg/ml in EKRB, 0.5% BSA) for 30 min. Each sample (0.1-0.2 ml) was then layered over 3 ml 4% BSA in EKRB and centrifuged for 5 min at 2009 in a TJ-6 refrigerated centrifuge (Beckman Instruments, Spinco Division, Palo Alto, Calif.). After carefully aspirating the supernatants, the pelleted cells were resuspended in 0.1-0.2 ml fluo-

O’BRIEN

AND

MILLETTE

Antigens

rescein-conjugated F(ab’), fragment goat anti-rabbit IgG (catalog no. 1312-0081, lots 12368 and 16756, Cappel Laboratories, Cochranville, Penn.) diluted 1:30 with EKRB, 0.5% BSA. Cells were incubated for 30 min, centrifuged through 4% BSA in EKRB as described previously, and were finally resuspended in 0.1 ml EKRB, 0.5% BSA. A Zeiss photomicroscope III equipped with epifluorescence and phase contrast optics was used to evaluate cell surface labeling. Photographs were taken with Kodak TriX film at ASA 1600 using automatic exposure. Rosette assays. Binding of antibodies to the cell surface was also monitored by rosette assays using Immunobeads (Bio-Rad Laboratories, Richmond, Calif.), polyacrylamide beads with covalently attached goat anti-rabbit immunoglobulin. Cells were incubated in appropriate first antibody and washed as described for immunofluorescence assays. Each sample was then resuspended in 0.15 ml EKRB, 0.5% BSA, 10 mM NaN3. Immunobeads (0.15 ml in the same EKRB solution) were added to a final cell to bead ratio of 1:40. Samples were incubated on ice for 30 min with occasional agitation. Rosette formation was monitored with a Zeiss photomicroscope II equipped with Nomarski optics. Only live cells as measured by trypan blue exclusion were scored. Photographs were taken with Kodak Tri-X film at an ASA setting of 400. Polyacrylamide gel electrophoresis. SDS-polyacrylamide gel electrophoresis was conducted according to the basic procedure of Laemmli (1970) except that separating slab gels (0.75 mm thick) were formed with linear 7-12% acrylamide gradients. Stacking gels contained 3.5% acrylamide. Molecular weight standards and reagents for preparing gels were obtained from Bio-Rad Laboratories (Richmond, Calif.). Some gels were fixed and stained with 0.1% Coomassie brilliant blue R (Sigma Chemical Company, St. Louis, MO.) in 45% methanol, 10% acetic acid. Protein samples were prepared in sample buffer containing 0.25% SDS, 20% glycerol, 0.0625 MTris-HCl (pH 6.8). Dithiothreitol (DTT, 40 mM) was included in some samples. Cells were pelleted, suspended in sample buffer, and sonicated (two to three 10 set pulses) using a Braunsonic 1510 (B. Braun Instruments, San Mateo, Calif.). According to the procedure of Schwartz and Nathenson (1971), other samples were prepared by first extracting cells with 0.5% Nonidet P-40 (NP-40, Sigma Chemical Company, St. Louis, MO.) in 0.15 M NaCl, 50 mM TrisHCl (pH 7.0), 0.02% NaN3, 2 mMPMSF (Sigma Chemical Co.). After incubating for 10 min on ice, these samples were centrifuged for 30 set at 1OOOgin a Fisher microcentrifuge (Model 59, Fisher Scientific, Pittsburgh, Penn.) to remove nuclei and cellular aggregates. Supernatants were then mixed with 2X sample buffer and sonicated before loading on gels.

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Chloroform:methanol (2:l v/v) extractions were conducted on Day 17 seminiferous cell samples according to the procedures of Nelson (1975) and Shirley and Schachter (1980). Both the material remaining after extraction and the chloroform:methanol soluble material were solubilized in SDS sample buffer prior to electrophoresis. Immunochemical detection of antigens on SDS-polyacrylamide gels. Proteins were transferred electrophoretically from SDS-polyacrylamide gels to nitrocellulose sheets (BA85, Schleicher & Schuell, Keene, N. H.) according to the basic procedure of Towbin et al (1979). Gels were first incubated in three changes of transfer buffer (25 mM Tris-192 mM glycine, pH 8.3, with 25% (v/v) methanol) for a total of 1 hr. Transfer was conducted overnight (15-17 hr) in an ElectroBlot transfer apparatus (E-C Apparatus Corporation, St. Petersburg, Fl.) at a power supply setting of 15% (-3.9 V). Nitrocellulose blots were then either stained with amido black (0.25% in 45% methanol, 9% acetic acid) or processed for in situ detection of antigens. The method used for antibody staining of these blots was adapted from Hawkes et al. (1982) by E. M. Eddy and associates (personal communication). Blots were first incubated for several hours at 37-40°C in blocking solution containing 0.9% NaCl, 3% BSA, 5% goat serum (GIBCO Laboratories, Grand Island, N. Y.), 10 mMNaN,, 10 mM Tris-HCl (pH 7.4). They were then rinsed briefly with saline (0.9% NaCl, 10 mM Tris-HCl, pH 7.4) and incubated overnight at 4°C in primary antibody diluted in blocking solution. In these experiments the primary antibody was ALZ serum (1:20 dilution) after exhaustive absorptions with splenocytes and thymocytes. The blots were then washed extensively with saline (five changes for a total of 2 hr) at room temperature on a rotary shaker. Incubation in secondary antibody (horseradish peroxidase-conjugated goat anti-rabbit IgG) was carried out at room temperature for 2 hr. This reagent was obtained from Miles Laboratories (Elkhart, Ind.) and was diluted 1:400 (lot S198) or 1:500 (lot S958) with blocking solution. The blots were then washed for an additional 2-hr period with saline. Bound peroxidase was detected by developing the blots with 4-chloro-lnaphthol (Aldrich Chemical Co., Milwaukee, Wise.) and hydrogen peroxide according to the method of Hawkes et al. (1982) except that the final H202 concentration was 0.025% v/v. Positive bands were seen as blue-purple reaction products against a white background. RESULTS

Surface Binding

Spec@city of IgG Preparations

Antibodies were prepared against prepuberal mouse spermatogenic cells to identify novel surface constitu-

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ents that may appear during the transition from mitosis to meiosis. IgG binding specificities were monitored by indirect immunofluorescence and rosette assays on live cells at 4°C in the presence of 10 mM NaN3, conditions which should inhibit endocytosis of surface label. Thus, although the heterologous antibodies may recognize a number of intracellular constituents, cell surface antigens were detected selectively with these techniques. Binding specificities were determined for each antibody before and after absorptions with a variety of somatic cell types. These absorptions were done to remove antibodies that reacted with common mouse cell surface antigens, leaving IgG binding activity restricted primarily to spermatogenic cells. Subsequent absorptions with distinct populations of mouse germ cells were conducted to determine if any of the reactive surface constituents were restricted to specific stages of spermatogenesis. Antibodies to type B sperwmtogonia and preleptotene smtocytes. Antisera raised against these cell populations labeled mouse germ cells at all stages of spermatogenesis, as measured by indirect immunofluorescence. All somatic cells tested were also positive. Following exhaustive absorptions with splenocytes and thymocytes, these antisera still labeled Sertoli cells and both prepuberal and adult spermatogenic cells, although the fluorescent intensity of surface labeling was reduced. Further absorptions with adult spermatogenic cells resulted in the loss of all activity directed against germ cells, including mitotic and early meiotic cells obtained from prepuberal animals. Antibody to leptotene and xygotene spermatocytes. In contrast, the antibody raised against leptotene and zy-

IMMUNOFLUORESCENT

IgG

prepared

against

Leptotene/zygotene Unabsorbed Absorbed-somatic cells Absorbed-somatic and adult germ cellsb

Spermatogonia

BINDING

Preleptotene

101,1984

gotene spermatocytes (ALZ) did exhibit stage-specific surface labeling after appropriate absorptions. Surface binding specificities of ALZ as measured by indirect immunofluorescence are summarized in Table 1. Each germ cell population was assayed at least twice using IgG at a concentration of 0.1 mg/ml. Unabsorbed ALZ bound strongly to all spermatogenic and somatic cells tested, except erythrocytes which were weakly labeled or negative. Like the antibodies previously described, ALZ retained surface binding activity on all spermatogenie and Sertoli cells after somatic cell absorptions (at least 6 X 10’ thymocytes and 19 X 10’ spleen cells, including erythrocytes and splenocytes, per ml containing 1 mg IgG). This activity was completely removed by subsequent absorptions of ALZ with an adult seminiferous cell mixture (al.3 X 10’ cells/O.1 mg IgG). A subset of surface determinants restricted to defined stages of spermatogenesis was recognized by ALZ absorbed first with splenocytes and thymocytes, followed by a Day 8 seminiferous cell mixture. This absorbed antiserum will subsequently be referred to as ALZ8. Absorptions with at least 1.3 X lo* Day 8 cells/O.1 mg IgG were sufficient to remove all surface binding activity on type A spermatogonia, type B spermatogonia, and Sertoli cells. Antigens reactive with ALZ8 were not detected on the surface of early meiotic cells including both leptotene and zygotene spermatocytes. In contrast, early pachytene spermatocytes isolated from l’i-day-old mice and germ cells at subsequent stages of differentiation, including vas deferens spermatozoa, were labeled with ALZ8. As indicated in Table 1, these determinants appear on the surface of spermatogenic cells earlier during the pachytene stage of meiosis than surface con-

TABLE OF ANTI-LZ

1 BEFORE AND AFTER ABSORPTIONS

Leptotene/ zygotene

Early pachytene

Late pachytene

Spermatids

Sperm”

++ +

++ t

++ +

++

+t +

++ +

tt

t

-

-

-

-

-

-

-

Leptotene/zygotene Absorbed-somatic cells and Day 8 cell suspension”

-

-

-

+

t

+

+

Adult

-

-

-

++

++

++

spermatogenic

cellsd

OiSperm isolated from the vas deferens and/or cauda epididymis. b Includes late pachytene spermatocytes, spermatids, residual bodies, testicular sperm. ’ Includes type A and type B spermatogonia and Sertoli cells. d Four antisera prepared against late spermatogenic cell populations including late pachytene sperm, and an adult seminiferous cell suspension (Millette and Bell-&, 1977).

spermatocytes,

round

spermatids,

+

vas deferens

O’BRIEN AND MILLETTE

Antigens

stituents recognized by antisera prepared previously against adult spermatogenic cells (Millette and Bell&, 1977; Millette, 1979). Mature spermatozoa from the cauda epididymis and vas deferens exhibited immunofluorescent surface labeling with ALZ both before and after absorptions (Fig, 1). With unabsorbed ALZ, the convex surface and postacrosomal regions of the sperm head and the midpiece were labeled most prominently. The principal piece of the tail and the lateral surfaces of the head overlying the acrosome were either negative or weakly fluorescent. After absorptions with somatic cells alone or somatic cells plus Day 8 seminiferous cells, ALZ labeled the sperm surface in a similar pattern. However, the intensity of the fluorescent label was reduced, particularly over the midpiece region of the tail. Spermatozoa fixed with 1% paraformaldehyde prior to these assays exhibited similar surface binding patterns with ALZ. Immunofluorescent binding patterns of ALZ8 on Day 17 seminiferous cell suspensions are shown in Fig. 2. Comparison of the same field using phase contrast (Fig. 2a) and fluorescence (Fig. 2b) microscopy illustrates that only the largest cells, identified as early pachytene spermatocytes (Bell& et a& 19’77a,b), were labeled with this absorbed antibody. All smaller cells, including preleptotene, leptotene, and zygotene spermatocytes, were completely negative. The patchy distribution of surface label observed with ALZS was characteristic of all four antisera prepared against prepuberal cells. Positive germ cells at all stages of spermatogenesis, except spermatozoa from the excurrent ducts, consistently exhibited similar nonuniform binding patterns.

FIG. 1. Immunofluorescent surface labeling of unfixed vas deferens spermatozoa with ALZ. X1500. (a) Binding pattern of unabsorbed ALZ. The convex surface and postacrosomal regions of the sperm head and the midpiece region of the tail are labeled most strongly with this antibody. (b) Surface labeling by ALZ after exhaustive absorptions with splenocytes, erythrocytes, and thymocytes. Note that the intensity of fluorescence over the midpiece is reduced in comparison with Fig. la. A similar binding pattern is obtained with ALZS.

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FIG. 2. Surface binding of ALZ8 to Day 17 spermatogenic cells, assayed by indirect immunofluorescence. X480. (a) Seminiferous cell suspension from l%day-old mice containing early pachytene spermatocytes and germ cells at prior stages of differentiation. Phase contrast photomicrograph. (b) Fluorescence photomicrograph of the same field illustrating ALZL) binding to these cells, as detected with fluorescein-conjugated secondary antibody. Assay conditions (live cells, 4°C 10 m&f NaNa) permit selective labeling of the cell surface. Only the largest cells in the field, identifiable as early pachytene spermatocytes, are labeled. Surface binding is not detected on earlier cells, including leptotene and zygotene spermatocytes.

Immunofluorescent results on spermatogenic cells isolated from 8- and 17-day-old animals were verified by Immunobead rosette assays. Figure 3 shows a typical experiment with ALZ8 on seminiferous cells prepared from 17-day-old mice. Early pachytene spermatocytes (average, 93.6% positive) formed rosettes, while spermatocytes at prior stages of differentiation generally did not (average, 89.6% negative). Earlier spermatocytes that were positive tended to be near the large end of their size distribution, and were, therefore, probably at a transition stage between the zygotene and pachytene stages of meiosis. The initial appearance of ALZ8 antigens on early pachytene spermatocytes was unanticipated since the

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FIG. 3. Binding of ALZ8 to Day 17 spermatogenic cells, as detected by a rosette assay using Immunobeads. As in Fig. 2, only the early pachytene spermatocytes (P) exhibit surface labeling. Nomarski photomicrograph, x540.

antiserum was raised against purified populations of leptotene and zygotene spermatocytes. These populations were carefully selected on the basis of size and did not contain significant numbers of the larger pachytene spermatocytes. It is unlikely, therefore, that pachytene-specific antigens in the immunizing population could account for these results. Also, since Day 8 seminiferous cell populations do not contain leptotene and zygotene spermatocytes (Bell& et & 1977a), surface binding activity specific to these meiotic stages could not have been inadvertently removed during absorptions. A possible explanation for the observed binding specificities is that one or more surface molecules are synthesized during the early meiotic stages, but are not actually incorporated into the plasma membrane until the pachytene stage of meiotic prophase. In order to test this hypothesis it was first necessary to identify the ALZS antigens. Immunoch.emical Ident$icaticm of Antigens Recognized b ALZ Immunoblot analyses were conducted to identify biochemically the stage-specific surface antigens recognized

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by ALZS. Since these assays required a minimum of 20 ml absorbed serum, cost considerations made absorptions with Day 8 spermatogenic and Sertoli cells impractical. As an alternative ALZ was absorbed exhaustively with splenocytes and thymocytes and immunoblots of Day 8 and Day 17 seminiferous cells were compared. Antigens shared by these two cell populations could not account for the stage-specific surface reactivity of ALZ8. Only determinants restricted to the Day 1’7 population are potentially responsible for the surface binding of ALZS first seen on early pachytene spermatocytes. Highly purified plasma membrane fractions can be prepared from late spermatogenic cells isolated from adult mouse testes (Millette et al, 1980). These same techniques, however, do not yield suitable plasma membranes from Day 17 seminiferous cell mixtures. Therefore, whole cells from Day 8 and Day 17 seminiferous tubules were solubilized in sample buffer and separated on one-dimensional SDS gels in preparation for immunoblot comparisons. NP-40 extracts prepared from these two seminiferous cell populations were also compared. Coomassie blue stained gels of these samples were qualitatively indistinguishable from amido black stained nitrocellulose blots. In some experiments heavily loaded bands, particularly those migrating in the lowmolecular-weight histone region, were not transferred quantitatively and were still detectable on gels after transfer. After somatic cell absorptions, ALZ still bound to multiple protein constituents in samples prepared from both 8- and 17-day-old testes. Figure 4a illustrates the immunoblot obtained from these two populations when DTT was included in the sample buffer prior to electrophoresis. A corresponding Coomassie blue stained gel run under identical conditions is shown in Fig. 4b. Heavy protein loads were chosen to minimize potential problems of sensitivity in the immunochemical assays. Major antigens that bound to absorbed ALZ were proteins with molecular weights ranging from -42,000 to 90,000. The reactive proteins were very similar in whole cell samples from Day 8 (lane 1) and Day 17 (lane 2) seminiferous mixtures. NP-40 extracts from Day 8 (lane

FIGS. 4 AND 5. Immunoblot analysis comparing antigens recognized by ALZ (after somatic cell absorptions) in cells isolated from Day 8 and Day 17 seminiferous tubules. Prior to electrophoresis samples were solubilized in 0.25% SDS buffer containing DTT (Fig. 4) or in the same buffer without reducing agents (Fig. 5). Proteins were then separated on SDS-polyacrylamide gels, transferred to nitrocellulose, and subjected to sequential incubations in ALZ, horseradish peroxidase conjugated secondary antibody, and 4-chloro-1-naphthol to visualize bound peroxidase. (a) Nitrocellulose blots exhibiting ALZ antigens. The arrow denotes the 39,000 M, protein which is absent in D’IT-treated samples. This constituent is detected only in cell populations from day 17 testes (Fig. 5a, lanes 2 and 4). A presumptive lipid determinant which migrates faster than the protein determinants is also present only in day 17 samples. (b) Corresponding SDS gels stained with Coomassie blue. The following samples were loaded on each gel. (1) Mixed whole cells from Day 8 seminiferous tubules (1.9 X lo6 whole cell equivalents/ lane). (2) Mixed whole cells from Day 17 seminiferous tubules (1.9 X lo6 whole cell equivalents/lane). (3) NP-40 extracts of mixed cells from Day 8 seminiferous tubules (extract from 3.8 X lo6 cells/lane). (4) NP-40 extracts of mixed cells from Day 17 seminiferous tubules (extract from 3.8 X lo6 cells/lane).

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3) and Day 17 (lane 4) samples also exhibited similar binding patterns. The ALZ antigens represent a distinct subset of the protein bands seen in complex Coomassie blue-stained profiles (Fig. 4b). A prominent ALZ antigen restricted to Day 1’7 samples migrated near the bromophenol blue dye front and did not stain with Coomassie blue or Amido black (Fig. 4a, lanes 2 and 4). Furthermore, this material is extractable from Day 17 whole cell samples with chloroform:methanol(2:1 v/v). These features suggest that this constituent may be a lipidcontaining determinant (Larraga and Edidin, 1979; Teuscher et aL, 1982). In preliminary studies a dot-immunobinding assay similar to that described by Hawkes et aL (1982) was used to monitor antigen-antibody interactions in various sample buffers. For several antisera tested, a much stronger reaction product was formed when DTT was omitted from the samples. Therefore, ALZ immunoblot (Fig. 5a) and Coomassie blue (Fig. 5b) comparisons of Day 8 and Day 17 seminiferous cells were also conducted in the absence of this disulfide reducing agent. Protein loads on these gels were identical to those in Fig. 4. As anticipated, the resolution of these gels was diminished compared to those containing DTT. All the antigenic determinants recognized by absorbed ALZ in the presence of DTT (Fig. 4a) were discernible when DTT was omitted from the samples (Fig. 5a). In addition, however, a major antigenic constituent with an apparent molecular weight of -39,000 was present in Day 17 but not Day 8 samples. This determinant was present in both whole cell samples (Fig. 5a, lane 2) and NP-40 extracts (Fig. 5a, lane 4) prepared from 17-day-old seminiferous tubules. These results suggest that a disulfide dependent conformation of the 39,000 J& component is required for recognition by ALZ. Immunoblot controls were conducted on Day 17 seminiferous cell samples (+DTT) with preimmune serum as the first antibody instead of ALZ. These immunoblots were completely negative. In additional controls whole cell samples from spleen and thymus were electrophoresed and transferred to nitrocellulose as described previously. Incubation of these blots with ALZ (absorbed with spleen and thymus) revealed a minor subset of very faint bands, presumably representing intracellular constituents. These bands did not correspond to the major determinants identified in spermatogenic cells. Populations of preleptotene, leptotene/zygotene, and early pachytene spermatocytes were isolated from 17day-old animals to see if the 39,000 M, ALZ determinant was restricted to particular stages of meiotic prophase. Whole cell samples (-+DTT) were electrophoresed on onedimensional SDS gels and were subjected to immunoblot analysis with ALZ after somatic cell absorptions. Major protein determinants recognized by absorbed ALZ were

VOLUME 101, 1934 NO

DTT

+ DTT M,X10-3 t

-66.2

a--

-31

-21.5

PL LZ P PL LZ P FIG. 6. Immunoblots exhibiting ALZ antigens in preleptotene spermatocytes (PL), a pooled population of leptotene and zygotene spermatocytes (LZ), and early pachytene spermatocytes (P) isolated from 17-day-old mice. Samples were prepared with and without DTT and were subjected to identical procedures for immunochemical analysis. The presumptive lipid determinant that migrates near the dye front predominates in the early pachytene spermatocyte samples, with or without DTT. In contrast, the 39,000 M, protein constituent (arrow) is apparent in all three cell populations when samples are prepared without DTT. As in Fig. 4a, this determinant is not detected when reducing agents are present in the sample.

virtually indistinguishable in these three spermatocyte populations, and the DTT-sensitive 39,000 M, determinant was present in each (Fig. 6). Purities of the samples were 80.3 and 1pachytene and leptotene/zygotene 37.3%, respectively, with Sertoli cells constituting the major contaminant in both populations. Purities of the preleptotene spermatocyte fractions used in these experiments averaged m-70%, with both Sertoli cells and leptotene spermatocytes as contaminants. These results indicate that the DTT-sensitive antigen is present in both leptotene/zygotene and pachytene spermatocytes, and may appear even earlier during the preleptotene stage. In contrast, the presumptive lipid-containing determinant(s) recognized by ALZ was restricted primarily to early pachytene spermatocytes (Fig. 6). Leptotene/

O’BRIEN

AND MILLETTE

Antigens

zygotene populations, which were used for immunization, did not exhibit significant antibody staining in this region of the gel. DISCUSSION

Mature mammalian spermatozoa and precursor cells isolated from adult testis have been used in previous studies to elicit a variety of antisera (Millette and Bell&, 1977; Myles et al, 1981; Teuscher et u& 1982; see Introduction). Determinants recognized by these antisera include germ cell-specific surface constituents, some of which are autoantigenic. All of these later spermatogenie stages are localized within the adluminal compartment of the seminiferous tubule, protected from the immune system by the blood testis barrier. Spermatogonia and preleptotene spermatocytes, however, reside in the basal compartment of the seminiferous tubule (Russell, 19’7’7,1978), which communicates with the extracellular fluid compartment of the organism. Characteristics of the three antibodies raised in this study against type B spermatogonia and preleptotene spermatocytes may be related to their position beneath the barrier. In contrast to antibodies raised against later spermatogenic cells (Millette and Bell&, 1977), these IgGs recognize antigens shared with typical somatic cells. In the rat, spermatocytes at the leptotene stage of meiotic prophase are the earliest cells to traverse the Sertoli junctions (Russell, 1977,1978). Perhaps it is not until the spermatogenic cells leave the basal compartment that they begin to express a variety of unique constituents on their surfaces. Unlike antibodies prepared against earlier stages, ALZ8 (raised against a population of leptotene and zygotene spermatocytes) does recognize antigens present on the germ cell surface during restricted periods of spermatogenesis. These determinants are first detected on the surface of early pachytene spermatocytes as measured by both indirect immunofluorescence and rosette assays. Recently, Gaunt (1982) has also identified a surface antigen (& 28,000) that appears on pachytene spermatocytes isolated from prepuberal mice. Since ALZ was raised against leptotene and zygotene spermatocytes, determinants recognized by this antiserum must also be present in these earlier meiotic stages. Immunoblot analyses have identified a DTT-sensitive ALZ determinant (M* - 39,000) in isolated populations of preleptotene, leptotene, and zygotene, as well as early pachytene spermatocytes. Spermatogonia do not exhibit this antigen on immunoblots. This constituent could remain within the cell during the early meiotic stages and then be incorporated into the plasma membrane during pachynema, thus accounting for the stage-specific surface labeling observed with ALZS. De-

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layed insertion of surface constituents does occur during the differentiation of B lymphocytes in both fetal lymphopoietic tissues and adult bone marrow of the mouse (Raff et aH, 1976; Landreth et aL, 1981). In these tissues the p heavy chain of IgM is present within the cytoplasm of B lymphocyte precursors, although no IgM is detectable on the surfaces of these cells. Based on kinetic data in adult bone marrow, Landreth and associates (1981) have proposed a differentiation scheme which includes large and small precursors with this phenotype. They estimate a 72-hr interval between the appearance of ~1chains in the cytoplasm and on the cell surface. In fetal liver cytoplasmic chains also appear several days before surface IgM is detected (Raff et d, 1976). Data presented in this study suggest that delayed insertion of plasma membrane constituents may also occur during mouse spermatogenesis, particularly during the approximate 3 day interval between the early leptotene and early pachytene stages (Oakberg, 1957). Delayed insertion of cell surface constituents during spermatogenesis has been suggested previously for postmeiotic cells (Romrell et a,!, 1982). Verification of this hypothesis would facilitate investigations concerning the significance of delayed insertion of membrane molecules during differentiation, since spermatogenesis is an excellent model system that is accessible for biochemical studies and is well defined both morphologically and kinetically. ALZ also recognizes another stage-specific antigen present in early pachytene spermatocytes but not in mitotic or earlier meiotic stages. This constituent has characteristics which indicate that it may be a lipidcontaining determinant. Recently, a glycolipid autoantigen has been identified in guinea pig spermatozoa and testicular extracts (Teuscher et al, 1982). Furthermore, early primary spermatocytes in the rat display a novel glycolipid on their surfaces as detected by immunofluorescence (Lingwood and Schachter, 1981). The lowmolecular-weight determinant recognized by ALZ may represent a similar constituent in mouse spermatogenic cells. Clearly, further biochemical studies are needed to characterize this antigen. Low-molecular-weight antigens in the presumptive lipid region were not detected with ALZ on immunoblots of isolated populations of preleptotene and leptotene/ zygotene spermatocytes. The absence of this determinant in cells used as the immunogen for this antibody (leptotene and zygotene spermatocytes) suggests that a lipid moiety is probably not responsible for the stage-specific surface labeling observed with ALZ8. However, present data do not rule out the possibility of a common determinant, such as a carbohydrate side chain, shared by protein and lipid. Other carbohydrate determinants, such as those specifying A, B, and H blood groups, have been identified on both glycolipids and glycoproteins

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(for review, see Horowitz, 1978). Similarly, a surface glycolipid that appears at the pachytene stage could display a determinant that is also present on intracellular glycoproteins at earlier stages of meiosis. Both the 39,000 M, protein determinant and the presumptive lipid determinant recognized by absorbed AL,Z are potentially interesting spermatogenic cell constituents since they appear at defined stages during meiosis and are not shared by a number of somatic cell types. Further characterization of these and other stage-specific constituents will contribute to the molecular framework needed for understanding spermatogenesis, particularly during the early stages where little biochemical information is currently available.

The authors gratefully acknowledge Dr. E. M. Eddy for his advice on the immunoblot procedure. Appreciation is also extended to Mr. B. Keyes Scott for excellent technical assistance and to Mr. Steven Borack of the Photographic Unit and Ms. Barbara Lewis for their assistance in preparing the manuscript. Animals used in this study were maintained in accordance with the guidelines of the Committee on Animals at the Harvard Medical School and those prepared by the Committee on Care and Use of Laboratory Animals of the Institute of Laboratory Animal Resources, National Research Council (DHEW publication No. (NIH) 78-23, revised 1978). This research was supported by NICHHD Research Grant HD11267 to C.F.M. and by postdoctoral fellowships to D.A.O. from NSF, NIH, and The Medical Foundation (Boston, Mass.).

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