Evidence for the presence of follicle-stimulating hormone receptor antibody in human serum*

Vol. 38, No.3, September 1982

FERTIUTY AND STERILITY Copyright c 1982 The American Fertility Society

Printed in U.8A.

Evidence for the presence of follicle-stimulating hormone receptor antibody in human serum*

James A. Dias, Ph.D.t Sherman A. Gates, M.D.+ Leo E. Reichert, Jr., Ph.D.t§ Albany Medical College of Union University, and Veterans Administration Hospital, Albany, New York

A male patient presented with polyostotic fibrous dysplasia, elevated serum gonadotropin levels, primary gonadal failure, and an immunoglobulin M (lgM) monoclonal gammopathy. When the patient's serum was added to radio iodinated human follicle-stimulating hormone (125I-hFSH)-bovine testis membrane-receptor complex solubilized by detergent, followed by the addition of antihuman immunoglobulin G (anti-hIgG), the preformed complex was precipitated. No such precipitation was seen when normal human or rabbit serum, serum from a patient with polyostotic fibrous dysplasia, or serum from other patients with polyclonal gammopathies were utilized. The patient's serum did not precipitate free 125I_hFSH. Detergent-solubilized testis receptor not complexed to hFSH, as well as highly purified radio iodinated receptor were also precipitated when this serum was added followed by anti-hIgG. Our results indicate that serum from the patient contained antibodies to FSH testis receptor. It is possible that antibodies to gonadal receptors may exist in other patients, although it is not yet possible to assume a causal relationship between the presence of receptor antibodies and gonadal failure. Fertil Steril 38:330, 1982

Antibodies to purified acetylcholine receptors have been demonstrated to elicit experimental myasthenia gravis, l and autoantibodies to insulin receptors have been implicated in insulin-resistant states. 2 Monoclonal antibodies to beta-adrenergic receptor have been generated, and an autoimmune response to beta-adrenergic receptor has been implicated in allergic rhinitis and asthReceived December 9, 1981; revised and accepted June 21, 1982. *Supported by National Institutes of Health Grant HD13938 (LER), and National Research Service Award (JAD) HD-05918. tDepartment of Biochemistry, Albany Medical College of Union University. :j:Department of Medicine, Division of Endocrinology, Veterans Administration Hospital. §Reprint requests: Leo E. Reichert, Jr., Ph.D., Department of Biochemistry, Albany Medical College, 47 New Scotland Avenue, Albany, New York 12208. 330

Dias et aI. Serum antibodies to FSH receptor

ma. 3 Antibodies to receptors for prolactin (PRL)4 and luteinizing hormone (LH)5 have also been reported. However, a search for antibodies to LH receptors in sera of patients with premature ovarian failure was unsuccessful. 6 A prerequisite for detection of receptor antibodies is the availability of a stable, radiolabeled receptor or hormone-receptor complex. We have previously described the properties of a Triton X-IOO solubilized calf testis membrane receptor for follicle-stimulating hormone (FSHf and have succeeded in stabilizing its hormone binding activity through use of glycer0l.8 We undertook the present studies to determine whether receptor autoantibodies could be demonstrated in the serum of a patient with elevated FSH and LH levels accompanied by gonadal failure. Our results show that this patient's serum precipitated free, purified FSH receptor as well as FSH-receptor complexes. The presence of Fertility and Sterility

FSH receptor autoantibodies-in serum could contribute to gonadal dysfunction, although a causal relationship has yet to be established and the mechanism involved remains uncertain.

MATERIALS AND METHODS MATERIALS

Rabbit antisera against immunoglobulin G (lgG) was purchased from Miles-Yeda Ltd., Rehovot, Israel (Lot R-610), Patients' sera were harvested after clotting of blood obtained by cubital venipuncture. Calf testes were obtained from a local abattoir and kept at - 20° C until used. Carrier-free Na 1251 (lMS 300) was provided by Amersham, Chicago, IL; Triton X-100, Tris, polyethylene glycol (PEG) (6000 mol wt), and egg albumin (99% pure) were provided by Sigma Chemical Company, St, Louis, MO; protein A was provided by Calbiochem, Los Angeles, CA; and glycerol was provided by Fisher Scientific Company, Rochester, NY,

PREPARATION OF THE PREFORMED 12'I_hFSH MEMBRANE-RECEPTOR COMPLEX

Calf testes membranes were sedimented at 31,000 x g from supernatants of calf testes parenchyma homogenates initially centrifuged at 1000 x g, Two grams of this membrane fraction was incubated with 100 ng of 1251_hFSH overnight at 25° C. The incubation buffer was 0,05 M Tris, pH 7.5, at 25° C with 5 mM MgCI 2, 0,3 M sucrose, and 0,1% egg albumin, We then washed the membranes twice with assay buffer and counted them to determine the amount of trace bound, The stable preformed hormone-receptor complex was then extracted by incubation of the membranes for 20 minutes either at room temperature or on ice, in detergent buffer (0.05 M Tris, pH 7,5, containing 30% glycerol, [v/v], and 2% Triton X-100 [w/v]), The entire mixture was centrifuged at 100,000 x g for 60 minutes, The straw-colored supernatant contained the preformed hormone-receptor complex. In such experiments, 32% to 38% of the trace added was bound, and 60% to 70% of this bound radioactivity was extracted by detergent. PREPARATION OF SOLUBLE FSH RECEPTOR

IODINATION OF hFSH

Highly purified human FSH (hFSH) (4000 lUI mg)9 was lyophilized in aliquots of 50 J.Lg per vial. All subsequent steps were carried out in an ice bath (4° C). We added 50 J.LI of distilled water to the vial of FSH, followed by 50 J.Ll of 0,5 M phosphate buffer, pH 7.5. After mixing, 2 mCi Na 1251, 10 J.Ll lactoperoxidase (0.5 U), and 5 J.LI H 20 2 (1:30,000 dilution) were added in sequence to the vial containing the hFSH, The reaction proceeded for 4 minutes, with agitation of the vial every 30 seconds, Free iodine and other reagents were separated from labeled hormone by filtration through Sephadex G-25 columns (0.7 cm x 48.5 cm) at room temperature with gravity flow. Specific activity was calculated by the method of Greenwood et al. lO Following iodination, the labeled hFSH was diluted with 0,05 M Tris HCl buffer (pH 7,5, 5 mM MgCl 2 and 0.1% egg albumin) to a final concentration of 5 ng in 50 J.Ll and was stored frozen until used, The specific activity of 1251_hFSH prepared in this fashion was approximately 24 J.LCi/J.Lg. Specific binding of 1251_hFSH to excess calf testis receptors was 28% of total counts added. Vol. 38, No, 3, September 1982

Membrane fractions (31,000 x g) were prepared from calf testes as previously described, 7, 8 The procedure for solubilization of the hormone receptor has been described in detail elsewhere. 8 In brief, 2 gm of membranes was washed twice with rinse buffer (0.05 M Tris, pH 7.5, 5 mM MgCI 2, 0.3 M sucrose). The pellet was then resuspended in 3 ml of detergent buffer (0.05 M Tris, pH 7.5, made 2% with Triton X-100 [w/v] and containing 30% glycerol [v/v]) and incubated on ice for 20 minutes, with occasional aspiration through a 10-ml glass pipette. The entire mixture was then centrifuged at 100,000 x g for 60 minutes. The straw-colored supernatant contained the soluble receptor. 7, 8 IMMUNOPRECIPITATION ASSAY

The immunoprecipitation assay was performed with minor modification as previously described for insulin receptor antibodies l l and consisted of (1) incubation of 100 J.Ll soluble preformed 1251_ hFSH-receptor complex (25,000 to 50,000 CPM) with 100 J.LI or less of serum for 2 hours at 25° C; (2) addition of 100 J.Ll of rabbit antihuman IgG diluted 1:10 in assay buffer, incubated at 4° C for 16 hours; (3) dilution with 1 ml assay buffer folDias et al. Serum antibodies to FSH receptor

331

lowed by centrifugation at 2050 x g for 10 minutes; and (4) counting of the pellets. IMMUNOPRECIPITATION OF FREE 125I_hFSH IN THE PRESENCE OF DETERGENT

In order to ascertain that detergent did not interfere with the antigen-antibody interaction, 125I_hFSH (600 fll) was incubated with 200 fll of a 1:50,000 dilution of rabbit antiserum to hFSH (batch 4, National Institute of Arthritis and Metabolic and Digestive Disease) in a final volume of 800 fll. This initial incubation was performed for 24 hours at 4° C. Following this, 200 fll of 1:10 dilution of goat anti-rabbit gamma globulin was added and allowed to incubate for another 24 hours at 4° C, prior to centrifugation (1250 x g) to separate bound from free hormone. Alternately, 100 fllof a 1:10 dilution of Staphylococcus aureus ~embranes (protein A) was added to each tube of goat anti-rabbit gamma globulin, allowed to incubate at room temperature for 15 minutes and then centrifuged for 10 minutes (1250 x g) to separate bound from free hormone. Triton X-100 at a concentration of 1.0% did not significantly (< 5%) interfere with the binding of antibodies to hFSH; therefore, the failure of the patient J's serum to precipitate 125I_hFSH in the presence of detergent was due to the absence of antibodies to FSH and not due to the presence of detergent. RECEPTOR ASSAYS

Measurement of 125I_hFSH binding to membrane-bound or detergent-solubilized testes receptor was performed as previously described. 7 , 8 SERUM ELECTROPHORESIS

Cellulose acetate electrophoresis and immunoelectrophoresis of serum was performed by the clinical chemistry laboratories at either the Albany Veterans Administration Hospital or Albany Medical College.

urnes of deionized H 20. The ligand solution was added to the moist cake of gel (1.6 mllml of gel bed slurry), and coupling was allowed to proceed overnight at 4° C. Uncoupled groups were blocked by the addition of 500 fll of 1.0 M glycine ethyl ester, followed by incubation for 2 to 3 hours at 4° C. The column was then washed in sequence with 7 M urea, 1 M NaCI, 0.05 M glycine (pH 7.5 containing 0.1% Triton and 30% glycerol), 0.1 N acetate (pH 2.2 containing 0.1 % Triton and 30% glycerol), and the glycine buffer again. Calf testes membranes (31,000 x g) were washed in 0.05 M glycine·HCI, pH 7.5 (25° C) containing 0.05 M MgCl 2 and 30% glycerol (buffer A). Buffer A was made 2% with 10% Triton X-100 (stored with crystals of the antioxidant butylated hydroxytoluene). Two grams of testes membranes was suspended in 3 ml of buffer A made 2% with Triton X-100. After 15 to 20 minutes on ice the suspension was centrifuged at 150,000 x g for 1 hour. The supernatant was diluted to 18 ml with buffer A and immediately added to the affinity gel, followed by rotation on a tube rotator at 25° C for 16 hours. After cooling to 4° C, the gel suspension was poured into a 2.5 x 10-cm BioRad econocolumn and washed with 10-column volumes ofbuffer A. Next 5 ml of buffer A made 30 mM with n-octyl glucoside was added to the column. When 5 ml of 0.1 N acetic acid (pH 2.2) containing 10 mM MgCI 2, 30% glycerol, and 30 mM n-octyl glucoside (buffer B) was added to the gel, the resuspended gel was incubated at 4° C for 5 minutes. After the slurry was poured back into the column, 20 ml of buffer B was added and an equal volume of eluate was collected. This fraction (25 ml) was then precipitated by. addition of an equal volume of 10% trichloroacetic acid in H 20, followed by centrifugation at 200 x g for 20 minutes. The sedimented receptor was redissolved in 0.05 M Tris-30% glycerol (v/v) buffer, pH 7.5, and then utilized for iodination. IODINATION OF THE ACID ELUTED FRACTION

PREPARATION OF THE FSH-AFFINITY COLUMN

Purification of FSH receptor from calf testes was performed as previously described 12 with some modification. The affinity gel was Affigel-15 (BioRad, Richmond, CA). The ligand was hFSH (LER-10065, 800 IU/mg). Eight milligrams of hFSH was dissolved in 3 ml of 0.1 M NaHCOa buffer, pH 8.0. The gel was washed under suction with 3 bed volumes of 2-propanol and 3 bed vol332

Dias et aI. Serum antibodies to FSH receptor

Chloramine T (1.0 mg/1.5 ml) and sodium metabisulfite (3.5 mg/ml) were dissolved in 0,05 M sodium phosphate buffer, pH 7.5. To 100 fllof the receptor solution were added, in sequence, 20 fll of 0.5 M Na2HP04 buffer, pH 7.5, 2 mCi Na 125I, and 10 fll chloramine T. The reaction was stopped after 30 seconds by the addition of 50 fll sodium metabisulfite. After iodination, the labeled receptor was separated from free iodine by filtration through a 0.7 x 50-cm column ofSephFertility and Sterility

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immunoglobulin M (lgM) monoclonal gammopathy, which has progressively increased to where at this time it is classified as Waldenstrom's macroglobulinemia. The patient does not consume alcoholin excess, nor is there any history or clinical evidence of hepatic disease. The liver and the spleen were of normal size, and liver function as measured by serum glutamic oxaloacetic transaminase (SGOT), bilirubin, and cholesterol, were normal.. Alkaline phosphatase was moderately elevated at 163 VII (normal 30 to 97), consistent with his bone disease. The protein profile on J serum as measured by cellulose acetate electrophoresis is shown in Figure 1. The profile of serum from another patient (P) with polyostotic fibrous dysplasia is included for comparison. The quantities

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Figure 1 Cellulose acetate electrophoresis densitometer tracing of serum from patient J and another patient P with polyostotic fibrous dysplasia. The gamma globulin profile illustrates a monoclonal gammopathy for patient J serum. and is compared with a normal serum gamma globulin profile for patient P. The major species of gamma globulin in J serum was determined by immunoelectrophoresis and light scattering to be IgM (kappa type). Protein concentration (gml100 ml) is given above the respective peaks. Total protein for J serum was 9.2 gmldl and for P serum, 6.6 gmldl. For J serum, IgG was 626 mg/dl and IgM was 5330 mg/dl.

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RESULTS

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PATlENTJ 10

The patient is a 57-year-old man with onset, at about age 19, of polyostotic fibrous dysplasia involving primarily the skull. He has no abnormal pigmentation. He was normally developed and the father of two normal children. At about age 47 primary gonadal failure became apparent, with the development ofloss oflibido, decrease in body hair, gynecomastia, testicular atrophy, ejaculatory volume of less than 1 ml, and aspermia. He has a normal sense of smell and normal male karyotype (46 XY). His serum testosterone (T) was low (less than 0.5 ng/ml). He had slightly elevated FSH at 29 mlU/ml (normal 5 to 25 mlVl ml) and LH 23 mlU/ml (normal 3 to 15 mIU/mi). His plasma estradiol (E 2 ) was normal « 10 pg/ mi). There was no history of testicular trauma or inflammation. At age 48 he was found to have an Vol. 38, No.3, September 1982

100

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Figure 2 Precipitation of preformed 125I_hFSH Triton X-100 solubilized receptor complex by J serum. Comparison of normal human serum (line D) and serum from a patient with polyostotic fibrous dysplasia (line C) with patient Jserum (line A). One hundred microliters of detergent solubilized 125I_hFSH receptor complex (50,000 CPM) was added to 12 x 75-mm test tubes, containing various levels of serum in a final volume of 225 ....1. An indication of 10 ....1 serum per tube on the graph reflects a 1:22.5 dilution of serum. Tubes were incubated at 25° C for 2 hours and then cooled on ice. Following this, 100 .....1 of a 1:10 dilution of rabbit antihuman .gamma globulin was added and the tubes were incubated at 4° C. After 16 hours, tubes were centrifuged at 1250 x g for 10 minutes to separate bound from free hormone-receptor complex. Specific precipitation of the 125I-hFSH-soluble receptor complex by J serum (line B) was determined by subtracting. counts precipitated in the presence of normal human serum (line C) from the total counts precipitated in the presence of patient J serum (line A). Dias et aI.

Serum antibodies to FSH receptor

333

Table 1. Precipitation of 1261_hFSH-Detergent-Soluble Receptor Complex by J Serum" Patient

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"Serum from patient J is thought to contain antibodies to testicular FSH receptor. Serum from patients S, L, and E showed gamma globulin levels in excess of the normal range (500 to 1400 mgfd1). One hundred microliters of each serum was incubated with 100 1-'01 of the preformed hormone-receptor complex (26,500 CPM/100 1-'01). After 1 hour at 25° C, 200 1-'01 of a 1:10 dilution of rabbit anti-hIgG was added to half of the tubes while the other half received buffer only. The incubation continued overnight at 25° C. The next day the tubes containing the IgG were diluted with 1 ml PBS and centrifuged at 4000 x g for 10 minutes. The other set of tubes was chilled and 200 1-'01 of 5 mgfml bovine gamma globulin was added, followed by 600 1-'01 of 25% PEG. The tubes with PEG were vortexed and allowed to incubate at 4° C for 10 minutes, followed by centrifugation at 4000 x g for 10 minutes. Percentage of PEG precipitated (column C) = BfA x 100.

of IgG and IgM in patient J serum over a recent 3-year period were as follows: September 1977, 900 and 4600 mg/dl; July 1978, 476 and 1160 mg/dl; July 1979, 636 and 3920 mg/dl; July 1980, 626 and 5330 mg/dl for IgG and IgM, respectively. Normal laboratory values were from 500 to 1400 mg/dl for IgG and 50 to 300 mg/dl for IgM.

ously shown that preformed complexes of 1251_ hFSH and Triton X-100 solubilized calf testis receptor can be quantitatively precipitated by PEG. 7 ,8 Similar amounts of hormone-receptor complex were precipitated by PEG after addition to all sera tested (Table 1, column A). However, anti-hlgG precipitated the 1251-hFSH-receptor complex only from J serum (Table 1, column B), as would be predicted if J serum uniquely contained antibodies to the FSH testes receptor. IMMUNOPRECIPITATION OF FREE SOLUBLE RECEPTOR BYJSERUM

Precipitation of free solubilized receptor by J serum was also studied (Fig. 3). Detergent extracts of 31,000 x g calf testes membranes were prepared as described earlier. To the solubilized FSH receptor preparation were added increasing amounts of J and normal serum, after which the receptor-antibody complex was precipitated by addition of anti-hIgG. The precipitated complex was reconstituted in assay buffer and allowed to incubate with 1251_hFSH. Radioligand bound to the putative receptor-antibody complex was sepa-

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Dias et al. Serum antibodies to FSH receptor

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IMMUNOPRECIPITATION OF PREFORMED HliI-hFSH-

J serum maximally precipitated preformed 1251_hFSH receptor complex (Fig. 2) at a dilution of 1:9 (25 fJ.I of serum per tube in a total volume of 225 fJ.1). This serum (line A, Fig. 2) was compared with that from another patient (P) with polyostotic fibrous dysplasia (line C, Fig. 2). It is evident that ability to precipitate the hormone~ receptor complex is a property of J serum only. Some slight nonspecific precipitation of the complex occurred as the levels of normal human serum increased. Therefore, specific binding (line B, Fig. 2) is determined by subtracting the counts precipitated by normal human serum (line D, Fig. 2) from counts precipitated by J serum (line A,. Fig. 2). Other sera containing elevated gamma globulin levels (polyclonal gammopathies) were also tested for ability to precipitate the preformed hormone-receptor complex. Table 1 summarizes the results of these experiments. We have previ-

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Figure 3 Precipitation of Triton X-100-so1ubilized FSH receptor by J serum. Detergent extract of calf testes membranes was diluted 1:10 in assay buffer containing 30% glycerol. The extract was dispensed (500 1-'01) to each tube followed by 5, 25, or 50 1-'01 of either J serum or normal human serum. The tubes were incubated for 1 hour at 25° C; then 200 1-'01 of rabbit antihuman gamma globulin (1:10) was added, and the tubes were incubated for a further hour at 25° C. Tubes were centrifuged at 1250 x g for 10 minutes and decanted. The pellets were resuspended in 500 1-'01 of assay buffer, and 5 ng of 125I_hFSH was added to each tube. Tubes were incubated for 4 hours at 25° C, and the 125I-hFSH-labeled receptor-antireceptor antibody complex was precipitated by PEG. Nonspecific binding of 125I_hFSH was determined by coincubation with a 1000-fold molar excess of unlabeled hFSH.

Fertility and Sterility

at 4 C after which 100 j.LI of a 1:10 suspension of Staphylococcus aureus cell walls (protein A) was added to precipitate antibody-bound hormone. The tubes were incubated at room temperature for 15 minutes and then centrifuged for 15 minutes. Figure 4 shows that only the rabbit antihFSH serum (line C) bound 125I_hFSH and was precipitated by protein A, indicating that precipitation of the 125I-hFSH-soluble receptor complex by J serum in the earlier study was not due to the presence of antibodies to the hormone. In other . replicate experiments using the double antibody method employing rabbit anti-human gamma globulin as the second antibody, serum from patient J failed to precipitate 125I_hFSH (data not shown). The results also demonstrated that free 125I_hFSH was not precipitated from serum as a consequence of the presence of elevated levels of serum globulin protein (Fig. 4, lines A and B). 0

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Figure 4 Test of 125I_hFSH binding capacity of J serum (line B), normal human serum (line A), or rabbit anti-FSH serum (line C). The horizontal axis represents the reciprocal of the serum dilution. Bound and free 125I_hFSH was separated by incubation with protein A (Cowan type, Staphylococcus aureus cell walls) followed by centrifugation at 1250 x g for 5 minutes. Only rabbit anti-hFSH serum precipitated 125I_hFSH (line C) with no anti-hFSH activity noted with a comparable dilution of normal human serum or serum from patient J.

rated by precipitation with PEG. As can be seen (Fig. 3), 50 j.LI of normal serum did not precipitate detergent-solubilized receptor (Fig. 3B), whereas 50 j.LI of J serum precipitated significant amounts of detergent-solubilized receptor, as measured by precipitation of the 125I-hFSH-Iabeled receptoranti receptor complex by PEG (Fig. 3A). The number of counts precipitated at 0 j.LI of serum reflects precipitation of free 125I_hFSH by the PEG and is routinely included as an assay control. These results suggest that at least some of the sites on the testes receptor that interact with antibody are distinct from those involved in specific binding of the hormone.

INHIBITION OF 126I_hFSH BINDING TO MEMBRANE RECEPTORS BY SERUM GLOBULIN FRACTIONS

Several studies have utilized inhibition of radiolabeled hormone binding to tissue receptors as an indication of the presence of receptor antibody. In this study, a 30% ammonium sulfate fraction was prepared from J serum, as well as from serum 100

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TEST FOR ANTmODIES TO hFSH

Although J serum precipitated free receptor (Fig. 3A), it was necessary to determine whether precipitation of the hormone-receptor complex could be due, in part, to endogenous antibodies to hFSH. In addition, it was necessary to demonstrate the nonspecific co-precipitation of the free or complexed hormone was not occurring due to elevated levels of gamma globulin in J serum. Incremental dilutions of normal human serum (Fig. 4A), serum from patient J (Fig. 4B), or rabbit anti-hFSH serum (batch 4 NIAMDD) (Fig. 4C) were added to 100,000 CPM of 125I_hFSH in a final volume of 300 j.Ll. Incubation was overnight Vol. 38, No.3, September 1982

10

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Figure 5 Inhibition ofFSH binding to FSH receptor by 30% (NH4)2S04 precipitated serum fractions which were desalted, lyophilized, and reconstituted to their original volume prior to assay. Aliquots of serum are therefore expressed as original serum equivalents. Reaction tubes contained 200 ILl of a 1:20 dilution of 31,000 x g calf testes membranes, 5 ng of 125I_hFSH, serum fractions, or unlabeled hFSH (R-1366), and assay buffer to a final volume of 900 ILL Incubation was at 25° C for 16 hours. Bound and free hormones were separated by centrifugation at 1250 x g (10 minutes). Data are expressed as percentage of inhibition of 125I_hFSH binding. Line A = hFSH (LER-1366), 4000 IVlmg; lines Band E = serum from two patients with polyclonal gammopathy; line C = normal rabbit serum; and line D = J serum. Dias et al. Serum antibodies to FSH receptor

335

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Figure 6 Immunoprecipitation of iodinated, purified FSH receptor. Serum was diluted with PBS and 100 fJ.I of 125I-Iabeled purified receptor was added and then incubated for 2 hours at 25° C. One hundred microliters of rabbit antihuman ganIma globulin (1:10 dilution) was added and the mixture incubated at 4° C overnight. One milliliter of PBS was then added to each tube followed by centrifugation at 1250 x g. Counts present in tubes containing normal human serum were subtracted from counts present in tubes containing J serum to give a measure of specific binding of iodinated receptor to receptor antibody.

of patients with polyclonal gammopathies (Fig. 5). As can be seen, all globulin samples tested significantly inhibited binding of 1251_hFSH to testes membrane receptors. However, of these sera, only J serum (Fig. 5, line D) previously gave evidence of the presence of receptor antibody. Further, similarly prepared globulin fraction from normal rabbit serum also inhibited binding of 1251_hFSH to testes membrane receptor. These results indicate a possible caveat in the use of membrane-binding assays as the only criteria for detection of receptor antibody. IMMUNOPRECIPITATION OF PURIFIED FSH RECEPTOR

Details of the procedure for purification of the Triton X-IOO solubilized calf testes receptor are given in Materials and Methods. The receptor was subjected to gel filtration through Sepharose 6 B (Pharmacia, Piscataway, NJ) in 0.05 M Tris buffer (pH 7.5) containing 0.1% Triton. The column size was 1.0 x 90 cm. A single peak of radioactivity with a Stokes radius of approximately 42 to 47 A was observed. Polyacrylamide electrophoresis 13 of the same material under denaturing conditions was performed, followed by autoradiography of the dried slab gels. Several bands of radioactivity were detected in the molecular weight range of 20,000 to 135,000, possibly re336

Dias et aI. Serum antibodies to FSH receptor

flecting the subunit nature of tije iodinated FSH receptor. s The iodinated receptor was incubated with various dilutions of J serum for 2 hours at 25° C, followed by precipitation of the complex with antihuman gamma globulin. As can be seen in Figure 6, maximum counts were precipitated at a 1:250 serum dilution, with fewer counts being precipitated at higher dilutions. Similar dilutions of other human or rabbit serum did not precipitate the radiolabeled purified receptor, and the iodinated receptor exhibited a high level of nonspecific binding to glass tubes. It was necessary to test normal serum at the same levels as J serum in order to account for nonspecific protein effects and to prevent an overestimation of counts precipitated by J serum. The results are interpreted to reflect a complexing of purified iodinated calf testes receptor to receptor antibody present in J serum. DISCUSSION

Several lines of evidence suggest that serum from patient J may contain antibodies to testicular FSH receptor. Preformed complexes of 1251_ hFSH- and Triton X-IOO-solubilized receptor from calf testis 7 were precipitated when J serum was added, followed by anti-hlgG, whereas no precipitation was seen when normal serum, rabbit serum, serum from a patient with polyostotic fibrous dysplasia, or from patients with polyclonal gammopathies were used in place of J serum. Precipitation of the hormone-receptor complex by receptor antibody suggests that some antigenic determinants of the receptor are separate from its hormone binding domain. J serum did not precipitate free 1251_hFSH either in the presence or absence of rabbit anti-hlgG. Free soluble receptor, after addition to all serum samples studied, could be precipitated by PEG, a procedure utilized to quantitatively precipitate detergent solubilized receptor from calf testis. 7 , S However, added free detergent-solubilized receptor could be precipitated only from J serum (and not from other serum tested) upon addition of anti-hlgG, presumably reflecting the presence of receptor antibody in J serum. Anti-hlgG could also precipitate complexes of radioiodinated highly purified calf testis receptor and putative antibodies to testis receptor present in J serum. The demonstration that serum from patient J inhibited FSH binding to its membrane-bound receptor cannot, by itself, be construed as evidence that the antiFertility and Sterility

bodies interact with the hormone binding domain, since ammonium sulfate fractions (30%) of normal human serum as well as normal rabbit serum inhibit FSH binding to its membrane receptors. This may not be surprising, since FSH binding inhibitors have been detected in serum. 14 Thus, a caveat of some concern is that ammonium sulfate precipitation of serum to obtain a gamma globulin fraction does not remove substances that inhibit gonadotropin hormone binding to its receptor. Antireceptor antibodies have been implicated in the pathogenesis of several diseases, such as Graves' disease, with antibodies to the thyroidstimulating hormone (TSH) receptor l5 ; myasthenia gravis, with antibodies to the acetylcholine receptor l6; insulin resistance with acanthosis nigricans, with antibodies to the insulin receptor2; and allergic rhinitis and asthma, with autoantibodies to J32-adrenergic receptors. 3 However, not all patients with these diseases have circulating antireceptor antibodies. Either the development of antibodies is sometimes secondary to target organ failure, or available methods are not sufficiently sensitive to allow antibody detection. The data presented in this study do not demonstrate a causal relationship between FSH receptor antibody and hypogonadism. Indeed, the role of FSH in adult males remains controversial, whereas FSH appears to be essential during the onset17 of spermatogenesis in immature male rats. Although in one study it was not possible to block spermatogenesis in adult male rats by passive immunization with antiserum to FSH,18 in another study active immunization of adult male rats with FSH diminished the numbers of primary spermatocytes and early spermatids. 19 In man, maintenance of normal spermatogenesis and reinitiation of sperm production after short term suppression by exogenous steroids occurred in spite of undetectable « 5 mIU/ml) FSH blood levels. 2o Conversely, men with isolated FSH deficiency « 3 mIU/ml) and normal serum LH (3 to 15 mIU/ml) levels had normal pubertal development but a high incidence of morphologic abnormalities of spermatozoa and were infertile. 21 Finally, two independent reports have demonstrated that, unlike in the rat, passive immunization of adult monkeys with antiserum to FSH results in abnormalities of testicular morphology and function. 22 ,23 Thus, it is not yet clear whether immunoneutralization of FSH or its receptor would necessarily result in infertility of adult males. Recently it has been demonstrated that Vol. 38, No.3, September 1982

two cases of hypergonadotropic amenorrhea were associated with the presence of putative antibodies to the FSH receptor. 24 Patient J exhibited an impaired Leydig cell function, which would be surprising if the antibody were directed to the FSH receptor alone. Although we have not tested for the presence of LH receptor antibody, there are data suggesting some possible explanations for the observed low levels of T. For example, FSH influences the development of interstitial tissue by inducing LH receptors on Leydig cells and increasing Leydig cell ability to secrete T when subsequently stimulated by LH.25 FSH also increases LH-stimulated production of T by adult rat Leydig cells. 26 Thus, there is some experimental evidence suggesting that compromising FSH action may affect Leydig cell function. Likewise, if both FSH and LH are elevated, then desensitization of the target tissue and a loss of tissue responsiveness may occur. The concomitant observation of primary gonadal failure and the presence of putative antibodies to FSH receptors are not sufficient to conclude a causal relationship. However, screening of patients who exhibit gammopathy and gonadal dysfunction for receptor antibody may lead to an acceptable generalization of the autoimmune etiology of some reproductive failures.

REFERENCES 1. Lindstrom J: Immunological studies of acetylcholine re-

ceptors. J Supramol Struct 4:389, 1976 2. Obberghen EV, Kahn CR: Autoantibodies to insulin receptors. Mol Cell Endocrinol 22:227, 1981 3. Venter JC, Fraser CM, Hammon LC: Autoantibodies to J32-adrenergic receptors: a possible cause of adrenergic hyporesponsiveness in allergic rhinitis and asthma. Science 207:1361, 1980 4. Bohnet HG, Shiu RPC, Grinwich D, Friesen HG: In vivo effects of antisera to prolactin receptors in female rats. Endocrinology 102:1657, 1976 5. Luborsky JL, Behrman HR: Antiserum against rat luteinizing hormone (LH) receptors. Biochem Biophys Res Commun 90:1407, 1979 6. Austin GE, Coulam CB, Ryan RJ: A search for antibodies to luteinizing hormone receptors in premature ovarian failure. Mayo Clin Proc 54:394, 1979 7. Abou-Issa H, Reichert LE Jr: Solubilization and some characteristics of the follitropin receptor from calf testes. J BioI Chem 252:4161$, 1977 8. Dias JA, Huston JS, Reichert LE Jr: The interaction of the structure stabilizing solvent glycerol with solubilized FSH receptors from calf testes. Endocrinology 109:736, 1981 9. Steelman S, Pohley FM: Assay of the follicle stimulating hormone, based on augmentation with human chorionic gonadotropin. Endocrinology 53:504, 1953

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10. Greenwood FC, Hunter WM, Glover JS: The preparation of 1311 labeled human growth hormone of high specific radioactivity. Biochem J 89:114, 1963 11. Harrison L, Flier JS, Roth J, Karlsson FA, Kahn CR: Immunoprecipitation of the insulin receptor: assay for receptor antibodies and a specific technique for receptor purification. J Clin Endocrinol Metab 48:59, 1978 12. Reichert LE Jr, Abou-Issa H: Properties of solubilized and purified follitropin receptor from calf testes. Biochim Biophys Acta 631:97, 1980 13. Laemmli UK: Cleavage of structural proteins during the assembly of the head of bacteriophage T4 • Nature 227: 680, 1970 14. Reichert LE Jr, Sanzo MA, Dias JA: Studies on purification and characterization of gonadotropin binding inhibitors and stimulators from human serum and seminal plasma. In Intragonadal Regulation of Reproduction, Edited by P Franchimont, CP Channing. New York, Academic Press, 1981, p 61 15. McKenzie JM, Zakarija M, Sato A: Hormonal immunity in Graves' disease. Clin Endocrinol (Ox!) 7:31, 1978 16. Lindstrom JM, Seybold ME, Lennon VA, Whittingham S, Duane DD: Acetylcholine receptor antibody in myasthenia gravis: incidence, clinical correlates and usefulness as a diagnostic test. Neurology (NY) 26:1054,1976 17. Chemes HE, Dym M, Raj HGM: Hormonal regulation of Sertoli cell differentiation. BioI Reprod 21:251, 1979 18. Dym M, Raj HGM, Lin CY, Chemes HE, Kotite N, Nayfeh SN, French FS: Is FSH required for maintenance of spermatogenesis in adult rats? J Reprod Fertil (SuppD 26: 175, 1979

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19. Lee VQK, Scott RS, Dobos M, Zachariah E, deKretser DM, Burger HG: Inhibin and the biological significance of FSH suppression. In Recent Advances in Reproduction and Regulation of Fertility, Edited by GP Talwar. Amsterdam, Elsevier-North Holland, 1979, p 241 20. Bremner WJ, Matsumoto AM, Sussman AM, Paulsen CA: Follicle stimulating hormone and human spermatogenesis. J Clin Invest 68:1044, 1981 21. Maroulis GB, Parlow AF, Marshall JR: Isolated folliclestimulating hormone deficiency in man. Fertil Steril 28: 818, 1977 22. Rani CSS, Murly GSRC, Moudgal NR: Effect of chronic neutralization of endogenous FSH on testicular function in the adult male Bonnet monkey: assessment using biochemical parameters. Int J Androl 1:489, 1978 23. Wickings EF, Usadel KH, Dathe G, Nieschlag E: The role of follicle stimulating hormone in testicular function of the mature rhesus monkey. Acta Endocrinol (Copenh) 95:117,1980 24. Chiauzzi V, Cigorroga S, Escobar ME, Rivarola MA, Charreau EH: Inhibition of follicle-stimulating hormone receptor binding by circulating immunoglobulins. J Clin Endocrinol Metab 54:1221,1982 25. Odell WD, Swerdloff RS: Etiologies of sexual maturation: a model system based on the sexually maturing rat. Recent Prog Horm Res 32:245, 1976 26. Aznar MA, Diaz GM, Herrera.Justinianno E, Aznar RA: Effect of follicle stimulating hormone on the liberation of testosterone induced by LH in separated Leydig cells. Rev Esp Fisiol 35:9, 1979

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