cm3-7227/92/1313-1212$03.ca/0 Endocrinology Copyright 0 1992 by The Endocrine Society

Vol. 131, No. 3 Printed in U.S.A.

Biological Mimicry of Gonadotropin Action by Antiidiotypic Antibodies to Luteinizing Hormone: Characterization and Biological Properties* M. R. SAIRAM,

G. N. BHARGAVI,

AND

B. R. DOWNEY

Reproduction Research Laboratory, Clinical Research Institute of Montreal, Montreal, Quebec, Canada HZ W lR7; and the Department of Animal Science, Macdonuld Campus of McGill University (B.R.D.), Ste-Annede-Belkvue, Quebec, Canada H9X 1CO ABSTRACT High affinity polyclonal rabbit antibodies to ovine (0) pituitary LH [anti-oLH-immunoglobulin G (IgG) Abl] were used to immunize young Southdown lambs, Their semm samples as well as those from controls receiving normal rabbit IgG were studied for the presence of anti-Abl antibodies. In RIAs using [‘?]oLH and affinity-purified Abl, sera from experimental sheep showed high activity, as expressed in oLH equivalents. These sera also showed ability to compete with [“‘I]oLH for binding to receptor on pig ovarian and testicular membranes. The antiidiotypic antibodies (Ab2) in experimental sheep sera were purified by successive affinity chromatography on immobilized rabbit normal IgG and immobilized oLH-IgG columns. Ab2-IgG eluted from the latter mimicked oLH in RIAs and RFUs. These purified Ab2 antibodies were also of a stimulatory type, because they elicited progesterone production in rat granulosa cells and collagenase-dispersed rat Leydig cells. This

stimulatory action was counteracted by coincubation with anti-oLHIgG, which would also terminate (oLG) hormone action in a similar manner. The Ab2 antibodies had no effect on oFSH RIA or on the binding of [‘*61]oFSH to pig ovarian receptors, indicating specificity with respect to LH antigenic structure and function. As can be expected from the choice of the immunogen (polyclonal anti-oLH-IgG), only a small percentage of the true Ab2 population could display biological mimicry of the original antigen (oLH). Their presence in circulation during 6-8 months had no effect on testicular size or body growth. The formation of Ab2 antibodies to rabbit anti-oLH-IgG was also demonstrated in male rats, but these were not purified. In this instance also there was no effect on testicular weight after 6 months of immunization. These results show the feasibility of producing antiidiotypic antibodies that stimulate gonadal function in a manner much like the pituitary gonadotropin (oLH). (Endocrinology 131: 1212-1222,1992)

T

albeit weakly. This has been subsequently confirmed (4) and extended to include other hormones, such as TSH (5), PRL (6), fl-adrenergic ligands (7), GH (a), and tachykinins, such as substance-P (9). The receptors for many hormones, neurotransmitters, neuropeptides, and growth factors (5, 10, 11) were studied by using antiidiotypic antibodies as probes. Even with the advent of cloning of several receptors, antiidiotypic antibodies against hormones will have potential as receptor probes because of the ease with which they can be purified in a stable form and studied by many techniques, including x-ray crystallography. The high antigenicity of purified gonadotropins, such as ovine LH (oLH), has been known for over 3 decades (12), and such antisera have been widely used to understand the physiological role of gonadotropins (13-l 6) in many species. The availability of affinity-purified highly active anti-oLHIgG from rabbit antisera (17) prompted us to investigate the possibility of developing antiidiotypic antibodies and evaluate their biological properties. In characterizing such antisera produced in sheep, we have found that they are capable of recognizing the LH receptor in rodents and stimulate ovarian and testicular cells in a manner similar to the original hormone. Thus, these antibodies can mimic the action of oLH. Portions of this investigation have been reported in preliminary form (18).

HE VERSATILE immune system produces an endless variety of antibodies (Abl) directed against different epitopes of an antigen. Among these different antibodies raised against a protein hormone, for example, there could be some that recognize the hormone in a manner much like a functional receptor does for that hormone. The structural features of these select antibodies might resemble those of the hormone-binding domains on the receptor. Subsequent immunization of experimental animals with some of these selected immunoglobulins (Igs) could produce a second set of antibodies (Ab2) directed against the combining site of the receptor-like molecules. These combining sites are called the idiotopes, and antibodies specific to these domains may in their variable region be similar to those features of the hormone in its binding to the cellular receptor. These second antibodies, categorized under the term antiidiotypic antibodies, are thought to possess an #internal image” of the original immunizing antigen, and this concept became part of the immune network hypothesis (1, 2). Applying this idea to hormones, Sege and Peterson (3) first demonstrated that antiidiotypic antibodies against insulin could recognize the insulin receptor and mimic some of its biological effects, Received March 24, 1992. Address all correspondence and requests for reprints to: Dr. M. R. Sairam, Reproduction Research Laboratory, Clinical Research Institute of Montreal, 110 Pine Avenue West, Montreal, Quebec, Canada H2W lR7. * This investigation was supported by the Medical Research Council of Canada (to M.R.S.) and the Natural Sciences and Engineering Research Council of Canada (to B.R.D.).

Materials

and Methods

Materials oLH and oFSH were isolated in our laboratory starting from frozen or Iyophilized pituitary glands of New Zealand origin. Sepharose-4B 1212

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ANTIIDIOTYPIC

ANTIBODIES

was obtained from Pharmacia (Dorval, Quebec, Canada). Purified rabbit IgG fraction, normal sheep y-globulin, BSA, adjuvants, and lactoperoxidase were purchased from Sigma Chemical Co. (St. Louis, MO). Protein-A and Iodogen were purchased from Pierce Chemical Co. (Rockford, IL). Antibodies to progresterone and testosterone were kindly donated by Dr. B. E. P. Murphy (Montreal, Quebec, Canada), and the dog second antibody used in oLH RIA was a gift from Dr. F. Labrie (Ste-Foy, Quebec, Canada). All other reagents were of the highest purity available from commercial suppliers. Affinity-purified

antibodies

to oLH

Antisera produced in adult male rabbits to our purified oLH (2-3 X NIH LH-Sl) were subjected to affinity chromatography on columns of hormone immobilized on divinylsulfonyl (DVS)-Sepharose. This was performed according to a previously described procedure (17) with slight modifications. Briefly, 10 mg of the hormone powder dissolved in 6 ml 0.25 M carbonate-bicarbonate buffer, pH 9.0, containing 0.5 M NaCl were mixed with 5 ml activated DVS-Sepharose 4B and shaken gently for 6 h at room temperature. The extent of coupling, determined by absorbance at 280 nm, was over 80%. After this, the addition of 1 g alvcine along with fresh buffer, pH 9.0, terminated the coupling and &&ved to b&k the remaining a&e groups on the matrix. The ga was then exhaustivelv washed with 0.1 M Tris-HCl, oH 7.6, containinp; 0.3 M NaCl and kept at 4 C until use. Such columns have been used o;er 3 yr without deterioration. Routinely, columns were washed with 0.1 M NI&OH for 30-60 min at room temperature and reequilibrated with buffer (see below) on the day of use. Two milliliters of rabbit antiserum to oLH were diluted 5-fold with RIA buffer (0.05 M PO4 buffer, pH 7.5; 0.1 M EDTA; 0.9% NaCl; and 0.05% sodium azide) and circulated through a 1.5-ml DVS-Sepharose-oLH column for 3 h at room temperature and then overnight at 4 C. The next day, the unadsorbed fraction was collected, and the column was extensively washed successively with 0.1 M Tris-HCl-NaCl buffer, pH 7.6, and 0.05 M NH*HCOa at room temperature. The latter served to remove all nonvolatile salts, after which the adsorbed antibody (IgG) was eluted by 0.2 M NHnOH. Eluted fractions were pooled, diluted with water, and lyophilized directly to provide highly purified anti-oLH-IgG. After the elution, columns were reequilibrated with RIA buffer for storage or subsequent fractionations. For the present series of experiments, several batches of the IgG preparation made in 1976 and stored at 4 C for 10 yr were pooled. They were found to retain all of their original characteristics (see Results). In more recent purifications, we have used larger columns containing 10 ml DVS-oLH to fractionate up to 10 ml LH antiserum/run. RIA RIA using [‘r51]oLH was performed in 12 X 75-mm plastic disposable tubes accordine to the method of Sineh et al. (19). using doa second antibody to rabvbit IgG for separation lf bound and. free horm&e. The first antibody used was either the diluted antiserum against oLH or the affinity-purified anti-oLH-IgG, weighed on a electrobalance and dissolved in RIA buffer. Purified oLH and its subunits (u and p), separated by reverse phase HPLC (Xl), were used for displacement/competition studies. In some experiments sheep antisera (preimmune/postimmune; see below), their affinity-purified IgGs, as well as normal sheep IgG (Sigma) were included (see figure legends). Immunization

of animals

against IgG

In November 1986, young male rats (Charles River, CR-CD, St. Constant, Quebec, Canada), weighing approximately 150 g, and 6month old Southdown rams were used for immunization using rabbit IgG (Sigma) for controls or anti-oLH-IgG for experimental groups (n = 6 rats for each; n = 2 rams in control and 3 rams in experimental groups). Each rat was injected with 100 rg of the antigen dissolved in 0.25 ml saline, emulsified with an equal volume of Freund’s complete adjuvant (Sigma) and administered SCat 2 sites. Injections were repeated every 2-3 weeks for a total of 13 immunizations. About 1 ml blood was withdrawn from rats by cardiac puncture under anaesthesia after the sixth injection and at the termination of the study (6 months). The rams received an injection of 1 mg control IgG or anti-oLH-IgG dissolved in

RECOGNIZING

LH

RECEPTOR

1213

1 ml saline and emulsified with 1 ml Freund’s complete adjuvant. The emulsion was injected SC or intradermally at a minimum of 8 sites/ animal. Injections were repeated about every 2 weeks for the first 6 injections, after which they were boosted monthly. Freund’s incomplete adjuvant was used from the fourth immunization onward in rams. A preimmune blood sample was collected from each ram before the start of the immunization and at monthly intervals after the first injection until termination at the end of September 1987. Before each booster injection, about 150-200 ml blood were collected via jugular vein and allowed to clot, and serum was separated. Serum samples were stored at -20 C until analysis. All rats and rams were weighed at monthly intervals. A monthly measurement of scrotal circumference in rams was also performed according to the method of Sanford and Yamey (21). The average of three values for each testis was recorded. Affinity

purification

of sheep antiidiotypic

antibodies

(Ab2)

In addition to testing the unfractionated antisera derived from immunized sheep, they were further purified by affinity chromatography in the following manner. In the first series of trials, selected serum (10 ml filtered through 5-pm membranes) from each of the immunized sheep was fractionated on a DVS-Sepharose column containing immobilized normal rabbit IeG (&ma). One hundred millierams of this protein were coupled tothe‘m&ix (10 ml) by the procedures outlined above (17), and the adsorbed anti-IgG was eluted with 0.2 M NHIOH and lyophilized. Double diffusion tests in agar plates indicated complete adsorption of the anti-IgG from sheep used for control immunization with normal rabbit IgG when 10 ml sheep serum were used for fractionation. It should be noted that this fractionation removes isotypic- as well as allotype-specific antibodies in the antisera. When antisera from the experimental group immunized with rabbit anti-oLH-IgG were processed, the unadsorbed fraction containing potential antiidiotypic antibodies was saved for fractionation on DVS-Sepharose containing immobilized anti-oLH-IgG. For this purpose, purified anti-oLH-IgG was also coupled to the matrix and handled by the same procedure outlined above (17), except that a smaller column (1.5 ml) containing 5 mg antioLH-IgG was used. The unadsorbed fraction from the first column was circulated through the second, and the adsorbed Ab2 was eluted and lyophilized. When control sheep sera immunized with normal rabbit IgG were processed on the fit column, there was no antibody remaining in the unadsorbed reaction to react with columns containing immobilized anti-oLH-IgG. In some experiments we processed the sera from experimental rams directly on DVS-anti-oLH-IgG columns to recover the Ab2 population. However, it should be noted that this preparation is likely to be contaminated with isotypic antibodies against irrelevant portions of rabbit anti-oLH-IgG. Solid phase detection

of Ab2

Besides using the qualitative double diffusion test for checking the success of sheep immunizations, we also performed experiments using anti-oLH-IgG coated on plastic wells. For this purpose, Costar 96-well plastic strips (Costar, Cambridge, MA) were coated with 200 pl antioLH-IgG solution (2 pg/ml) prepared in 0.05 M glycine buffer, pH 9.2, during an overnight incubation at room temperature. After removing the unbound

anti-oLH-IgG

(for subsequent

use), the remaining

available

sites were saturated with 200 ~1 1 mg/ml BSA or 3% norma rabbit serum in RIA buffer. Sera (200 ~1; 1:lOO dihrtion) from immunized sheep (control and experimental) were allowed to react with immobilized Abl for 3 h at 37 C, after which they were removed and washed. Detection was possible using i’sI-labeled anti-oLH-IgG (Iodogen method; SA, 5080 &i/pg), because antibody molecules are bimodal and flexible. About 100,000 cpm/well [iz51]oLH-IgG were added, and after l-h incubation at 37 C, the wells were washed twice, and radioactivity bound to the plastic wells was counted. To a parallel series of wells, 1 rg purified oLH was added along with the sheep sera to verify competition (if any) between Ab2 and hormone for binding to wells with the coated Abl antibody. This experiment was repeated as described above, except for the use of iZ51-labeled oLH in the last step for detection.

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ANTIIDIOTYPIC

1214 In vitro effects of antiidiotypic

antisera

ANTIBODIES

and purified

IgGs

RRA for oLH. Assessment of binding of [‘251]oLH labeled by the Iodogen method or the lactoperoxidase method (19) to pig ovarian (22) or testicular membranes (23) was performed in 12 x 75mm plastic tubes. Unlabeled hormone at different concentrations, preimmune sheep serum, or immune serum containing the test antibodies or the affinitypurified fractions were incubated with the membranes and 11251]oLH i50,000-75,000 cpm) in a total volume of 500 ~1 assay buffer (0.65 M Tris-HCl, pH 7.5, containing 10 mu MgC12 and 0.1% BSA (Sigma). After a 3-h incubation at 37 C or overnight at room temperature, the reaction was terminated by the addition of 2 ml assay buffer and centrifuged. Radioactivity in the pellet was determined in an LKB-7 counter (LKB, Rockville, MD). Activify in steroidogenic cells. The steroidogenic activity of the test samples, including the oLH standard, was determined in rat granulosa cells and collagenase-dispersed rat Leydig cells in suspension.

Rat grunulosn cells. Immature female Sprague-Dawley rats, 24 days of age, were primed with 15 IU eqine CG (Ayerst Laboratories, Montreal, Quebec, Canada) (19) and killed 2 days later. The ovaries were removed and cleaned of adherent tissue, and the follicles were punctured under the microscope by gentle pressure to squeeze out the granulosa cells. Approximately 400,000 cells were distributed into disposable glass tubes (12 x 75 mm) in Dulbecco’s Modified Eagle’s Medium (Gibco, Grand Island, NY) containing 20 mu HEPES, pH 7.5, and 0.1% BSA. The test substances were prepared in the same solution, and the cells were incubated in a total volume of 0.5 ml for 4 h at 37 C under 95% COZ5% Oz. Progesterone in the medium was estimated by RIA, using [3H] progesterone and a specific antibody (19). Steroidogenic activity was also evaluated using collagenase-dispersed rat Leydig cells (24) by measuring testosterone accumulated in the medium after a 4-h exposure, as described above. Mouse Leydig tumor cells. These cells (MA-10 cell line) provided by Dr. M. Ascoli (Iowa City, IA) were grown and maintained as previously described (25) with some modifications (19). They were grown in 24well plates for hormone binding assays as well as for the measurement of steroidogenic responses. Analytical experiments. Purified IgG samples were evaluated by sodium dodecyl sulfate-pol~acrylamide gel electrophoresis and Western blotting (19), using either [’ 51]protein-A (Pierce) or [‘251]oLH. Statistical analysis, where necessary, was performed using Student’s t test. Checks for parallelism in displacement assays or bioassays were performed on a program adapted for the IBM computer.

RECOGNIZING

LH RECEPTOR

Endo * 1992 Vol131. No 3

[‘251]oFSH (this laboratory) or [‘*‘I]bovine TSH (courtesy of Dr. J. Pierce, University of California-Los Angeles; data not shown). These data reveal that the polyclonal antibodies studied here are specific to the native hormone (oLH) conformation. Both the immune serum from rabbits immunized with oLH and their affinity-purified antibodies (Fig. 1) effectively inhibited the binding of [‘*‘I]oLH to MA-10 cells. In these experiments, the MA-10 cells were cultured in 24-well plates according to previously described procedures (19, 25). Control preimmune serum from the same rabbits had no effect on hormone binding during the 3-h incubation at 37 C. The rat granulosa cells obtained from eCG primed rats were highly responsive to the stimulatory action of pituitary oLH (Fig. 2). The simultaneous presence of 10 ng purified anti-oLH-IgG blocked LH action by more than 90%, and at 30 or 100 rig/tube, the hormone response was completely neutralized. Because the hormone and anti-oLH-IgG were added to the cells at the same time, these data imply effective competition between the receptor and antibody for the hormone. As expected, 100 ng anti-oLH-IgG by itself had no effect on the basal low production of progesterone during the 4-h incubation. Similar inhibitory effects of anti-oLHIgG on LH action in MA-10 cells were observed (not shown). We also assessed the in uivo neutralizing potential of these purified antibodies by testing their effect on pregnant rats. When injected before day 12 (26) of pregnancy, there was complete resorption (data not shown) of the implantation sites. These tests suggested that the anti-oLH-IgG preparation contained some antibodies directed against the receptorbinding sites of the hormone. Alternatively, their binding at sites in this vicinity was adequate to neutralize hormone action. The preparation derived from an antiserum pool of 100

-

80

Results Characteristics

of affinity-purified

rabbit anti-oLH-IgG

(Abl)

The testes of male rabbits that were used to produce antibodies to oLH became completely atrophic after four or five immunizations. Before using their affinity-purified antioLH-IgG for immunizing the rats and rams, it was desirable to establish their binding characteristics and neutralizing potential. Lyophilized preparations of anti-oLH-IgG, kept at 4 C for over 10 yr, were remarkably stable, showing high reactivity with [1251]oLH in RIAs. In an assay using only 510 ng purified oLH antibody/tube, the reaction was conformation specific, because only the native oLH could compete effectively (data not shown). The two isolated subunits were very weakly active, showing less than 1% cross-reaction (20). When these subunits were recombined, full immunological reactivity was regenerated. In addition to this preparation used for immunization, antibody purified from four other rabbits were similar in these tests. All of these were also highly specific to [1251]oLH, showing little or no binding of

0 I

-

-

CONTROL PRE-IMMUNE SERUM (1:lOO

oLH a/s

- oLH IgGlpg+ #1 #2

Diln)

1. Effect of oLH antiserum and affinity-purified oLH-IgG on the binding of ‘l-labeled oLH to receptors on MA-10 cells. MA-10 cells were grown to confluence in 24-well plates, and just before the experiment, cells were washed with serum-free medium and incubated for 3 h at 37 C with [iz51]oLH (220,000 cpm/well) and additions as shown (oLH-IgG = anti-oLH-IgG). After incubation, the medium was removed,cells were washed with medium (twice) and lysed with 0.1 M NaOH for counting. The radioactivitv found in control wells and representing specific binding (determined in the presence of 1 pg oLH) of 2219 + 116 cpm is shown as 100%. FIG.

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ANTIIDIOTYPIC

oLH

ANTIBODIES

oLH, ng Ab, ng

FIG. 2. Effect of purified anti-oLH-IgG on LH action. Rat granulosa cells (see Mater& and Methods) were incubated with hormone alone or an affinity-purified preparation of rabbit anti-oLH-IgG. Progesterone (mean k SEM; n = 3) was estimated by RIA of the medium after a 3-h incubation at 37 C.

polyclonal nature could also have IgGs directed against other determinants present elsewhere on the oLH, which may be remote from hormone-binding domains. To examine if any of these would still be available subsequent to binding of the hormone to the receptor, the following experiment was performed. A pig ovarian receptor preparation (22) was incubated with increasing concentrations of unlabeled oLH (0.151000 rig/tube) at pH 7.5 for 16 h at room temperature to allow binding and stabilization of the hormone-receptor complex. After this, 2 ml cold assay buffer were added and centrifuged, and the supematant was carefully siphoned, removing the unbound oLH. To the resuspended pellet, lz51labeled anti-oLH-IgG (100,000 cpm) was added in 500 ~1 assay buffer and incubated for an additional 3 h at 37 C. It was then diluted with 2 ml assay buffer and centrifuged, and radioactivity associated with the pellet was determined. As indicated in Fig. 3, the amount of labeled anti-oLH-IgG bound to the membrane pellet was proportional to the concentration of unlabeled oLH added in the first incubation for binding to the receptor. Virtually all of the radioactivity in the tube was associated with the pellet and specifically bound, because less than l-4% remained after dissociation of the receptor-hormone (R-H)-oLH-IgG complex by a change in pH (see Fig. 3). The binding of other hormones, such as 200 ng purified hCG or 1 pg purified oFSH (which also binds to its own receptor in this membrane preparation), was not detected by the added ‘251-labeled LH-IgG because of its hormone (oLH) specificity. These data reveal the presence of antibodies directed against oLH epitopes that are away from the regions responsible for binding of hormone to the receptor. Inhibition of r251]oLH antiidiotypic antibodies

binding (Ab2)

to anti-oLH-IgG

(Abl)

by sheep

The affinity-purified idiotype antibody (Abl) was used to immunize sheep to induce potential antiidiotypes (Ab2) that might be able to mimic LH in its action on the receptor and cells. Before conducting these detailed tests, it was necessary

RECOGNIZING

LH RECEPTOR

1215

to verify the presence of anti-oLH antibodies in the sheep sera. This may have arisen from the oLH leaking from the affinity columns and inducing anti-oLH formation. Although this possibility is highly unlikely in sheep, as the antigen (oLH) itself is from the same species, confirmation was provided in the following way. Binding assays using [lz51] oLH and immunized sheep sera were negative. Using 12.5% polyethylene glycol for separation of bound and free hormone, no binding of [‘z51]oLH to immunized sheep sera could be demonstrated. No signal could be detected in Western blots performed with unfractionated sheep sera or affinitypurified Ab2 preparations and [‘251]oLH for detection (not shown). The antiidiotypic antibodies in sheep sera became detectable by about 3 months, the earliest time at which the following analyses were performed. In sandwich assays designed to detect their presence (Table l), serum of sheep immunized with Abl showed significantly greater binding of [‘251]anti-oLH-IgG (23%) added during the second incubation than that in wells in which serum of sheep receiving irrelevant normal rabbit IgG as antigen was added. Epitopes common to both antigens and present in the Fc region or elsewhere in the variable domains would be detected by the added ‘251-labeled Abl. The greater binding seen when sera from sheep immunized with Abl were used in the first incubation must have been due to determinants specific to idiotypes recognizing oLH. This was confirmed by the data which showed reduction (41%) of the counts bound when unlabeled oLH was also present in the first incubation (Table 1, see treatment 3). Such a reduction was not seen in treatment 2, in which the antiidiotypic antibodies were not specific (to oLH-IgG). Similarly, in another series of incubations in which [l*‘I] oLH was used for detection in anti-oLH-IgG-coated wells, there was complete inhibition (data not shown) by sheep antibodies specific to Abl (treatment 3). We were able to quantitate such antibodies by performing direct RIAs. Antiidiotypic antibodies (Ab2) against rabbit anti-oLH-IgG (Abl) in three sheep inhibited the specific binding of [‘251]oLH to affinity-purified Abl (Fig. 4). Preimmune sera from the same animals or normal sheep IgG (commercial, Sigma) at the dilutions or concentrations employed had no effect on hormone binding to Abl. The postimmune serum taken from sheep 3-6 months after immunization inhibited hormone binding to Abl. All sera (control and experimental groups) showed good competition, and the displacement curves were parallel to that shown by purified oLH (antigen). Because of this feature we could calculate the relative hormone content (oLH equivalent) in each of these immunized sheep sera. The two animals in the control group that received normal rabbit IgG for immunization showed oLH equivalents of l-4 pg/ ml serum. The values for three animals immunized with antioLH-IgG ranged from 40-480 ccg/ml (272 + 90.6; n = 5), representing an increase of 40-120 X over control values. Such levels persisted in two animals that were kept for 18 months. As these animals were bled before the booster injections each time, these estimates could not have been influenced by the circulating anti-oLH-IgG in the serum left

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ANTIIDIOTYPIC

1216

FIG. 3. Binding of anti-oLH-IgG to preformed oLH-pig receptor complex. Ovarian membranes were incubated with medium alone or with increasing concentrations of unlabeled oLH for 16 h at room temperature to form a tight H-R complex. The tubes were diluted with assay buffer (2 ml) and centrifuged, and supernatant was removed completely. The pellet was resuspended in 0.5 ml assay buffer containing 100,000 cpm 12’I-labeled rabbit anti-oLH-IgG (Abl) and incubated for 3 h at 37 C. After dilution and wash, the radioactivity in the pellet (R-H-Abl*) was determined and expressed as a percentage of the total counts per min added. Duplicate tubes showed minimal variation (-1%). AntioLH-IgG* binding increases with increasing concentration of hormone in the first incubation (0). Note that although unlabeled hCG (200 ng) bound to the same receptor, it was not detected by the antibody in the second incubation. Likewise, oFSH (1 pg), which also binds to its own receptor in this membrane preparation (22), was not detected by this specific labeled oLH antibody. A, Radioactivity remaining in the pellet after a change in pH (0.1 M NHdOH; 5 min; 22 C). R-H, Receptor-hormone.

TABLE 1. Recognition by sheep antiidiotypic oLH-binding sites on immobilized anti-oLH-IgG

ANTIBODIES

1. Buffer 2. Sheep antiserum to normal rabbit r-globulin 3. Sheep antiserum to anti-oLH-IgG

Endo. 1992 Vol131. No 3

2

oLH(ng)

antibody (Abz) of

1,160 & 51 22,627 f 1,286 (6)”

In presence of 1 pg oLH 1,171 f 44 22,468 zk 1,591 (6)O

27,997 f 2,668 (9)*

15,877 2~ 627 (9)”

In absenceof oLH

LH RECEPTOR

12

cpm/well Treatment

RECOGNIZING

Affinity-purified rabbit anti-oHL-IgG (2 rg/ml; 200 pi/well) was coated in 96-well plates. After blocking with 200 ~1 3% normal rabbit serum, wells were treated as indicated with 200 ~1 of a 1:200 dilution of sheep sera for 3 h at 37 C. They were then removed and allowed to react with 100,000 cpm iz51-labeled anti-oLH-IgG for 1 h at 37 C. Wells were washed and counted. Different superscripts indicate differences (P < 0.01) in binding. In each treatment there were three to nine wells, indicated in parentheses.

over from the immunization performed 30 days previously. As little as 40 ng of each purified Ab2 inhibited [“‘I]oLH binding to Abl by about 50%. In a RIA employing [“‘I]oFSH and its rabbit antibody (27), the samples tested in Fig. 4 had no effect (data not shown), indicating that the antiidiotypic antibodies examined were specific in mimicking LH. Like the sheep antibodies, rats immunized with anti-oLH-IgG had high hormone-like activity in the circulation, as revealed by RIAs. Six months after immunization, equivalents of 165 + 25 ng oLH/ml were found in these rats.

Inhibition of f2’I]oLH antiidiotypic antibodies

binding

to membrane

receptors by

As preliminary experiments revealed that sheep sera immunized with anti-oLH-IgG, but not normal rabbit IgG or preimmune serum, were able to compete in receptor binding assays for LH, we focussed our attention on analyzing them further. These sera from three sheep taken from 3 months to 1.5 yr after immunization competed effectively in the RRA for LH using [‘251]oLH and pig testicular membranes (Fig. 5). Displacement was virtually complete at the highest concentration tested for all sera, and the curves were parallel to that of purified oLH. All of these sera contained approximately 30-50 ng/mleq purified oLH. These values are much lower than estimates provided by RIA (Fig. 4) and show that in the population of Ab2 antibodies produced against anti-oLHIgG, only selected molecules have a configuration (mirror image of hormone) capable of recognizing the LH receptor. These sera had no effect in a RRA for oFSH using the same membranes and [‘251]oFSH (data not shown). The affinity-purified Ab2 preparations also were active in competing with [‘251]oLH for binding to the LH receptor. Of the two preparations tested in Fig. 6, one showed almost complete displacement at 100 fig/tube. Such displacement was obtained with 10 ng purified hormone in this assay. In an attempt to demonstrate direct binding of the purified sheep Ab2 IgG to the membrane LH receptor, the preparation that showed the most potent activity in Fig. 6 was labeled with 12’1by the Iodogen method to a specific activity of 65 &i/pg. This labeled preparation showed excellent binding to Abl (oLH antiserum) when it was sandwiched to oLH in

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ANTIIDIOTYPIC

ANTIBODIES

RECOGNIZING

LH RECEPTOR

1217

SERUM DILUTION 'ho6 I FIG. 4. Effect of sheep sera on the recognition of ‘x61-labeled oLH by antioLH-IgG. The RIA was performed using 10 rig/tube affinity-purified anti-oLHIgG (same preparation as the immunogen for sheep) and [‘x’?]oLH. The various serum samples were diluted with RIA buffer and added as shown. Preimmune serum shows pooled line for three animals. X, Sera of two sheep immunized with normal rabbit IgG. A, n , and 0, Sera from selected bleeding of three animals immunized with rabbit anti- # oLH-IgG (Abl). AbZ-IgG, Aftinity-puriSed antiidiotypic antibody (note additions in micrograms). The radioactivity bound (32%) by Abl in the absence of unlabeled oLH was set at 100%.

'/'OS I

'/'Ok I

'ho0 I SHEEP

IgG

oLH

100

80

40

20 0

~1 SERUM 10

0.01

0.1 ng oLH/pg

1.0

10

100

IgG

IO0

0.1

ng oLH

FIG. 5. RRA using [‘261]oLH and pig testicular membranes. The specific binding (9.5%) in a 3-h 37 C assay was set at 100% for calculations. Serum from three experimental animals immunized with anti-oLHIgG was tested (0).

wells (Fig. 7A), revealing its high immunoreactivity. However, its binding to the pig ovarian LH receptor was low (Fig. 7B). Only about 2.2% of the total counts added were specifically bound, i.e. displaceable by 10 pg unlabeled Ab2 preparation. The high nonspecific binding (80%) precluded further direct analysis. Purified oLH (1 pg) competed with labeled Ab2 to the extent of 50%, and this inhibition was significant. By employing a different detection strategy, similar to that shown in Fig. 3, an improved signal for binding of relevant Ab2 to receptor could be demonstrated (Fig. 7C). The pig testicular membrane containing the LH receptor was incubated with diluted sheep sera in the presence of a proteolytic enzyme inhibitor (phenylmethylsulfonylfluoride). After washing off unbound proteins, 1251-labeled anti-oLHIgG was added for a second incubation. In this case, the second arm of the receptor-bound antibody (if any) Ab2 plastic

'/to3 I

1

10

100ng oLH 119 Ab

FIG. 6. Competition for receptor binding by affinity-purified sheep antiidiotype preparations. A RRA was performed as described in Fig. 3, using [1261]oLH (specific binding, 15% of the total counts added and shown as 100). V, Ab2 from sheep (control) immunized with normal rabbit IgG. A and n , Two AbP’s from experimental sheep (anti-oLHIgG immunogen).

would be recognized by the [1251]anti-oLH-IgG and be counted in the pellet. Sera from both sheep immunized with normal rabbit IgG and containing irrelevant second antibodies were not bound by the receptor, as there was no net radioactivity bound. However, samples from sheep containing Ab2 antibodies mimicking LH were detected by the added [‘251]anti-oLH-IgG. The radioactivity was higher than that found for separate incubation tubes receiving unlabeled oLH (1 pg), reflecting the fact that the variable region of the free arm of the receptor-bound Ab2 could recognize more of the added [‘251]anti-oLH-IgG (Abl). Steroidogenic activity of affinity-purified

sheep antiidiotypes

The data in Figs. 4-7 suggested that the relevant sheep antiidiotypic Igs had some structural similarity to 0LI-I with respect to both their antigenicity and specific recognition by

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ANTIIDIOTYPIC

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ANTIBODIES

RECOGNIZING

Endo l 1992 Voll31. No 3

LH RECEPTOR

2500 r t3 - 2000 52 g 1500 0 w” 1000 c-4 2 zA 500 None

Normal OLH rabbit AntIserum serum < I : 50,000>

‘cg oLH

‘P!3 oLH

Ab2 Control serum

Ab2 Experlmentol serum

7. Ab2 recognition of Abl by immunoassay and RRA. A, oLH-coated plastic wells were first incubated with a 1:50,000 dilution (200 pl) of normal rabbit serum or oLH antiserum (used for affinity purification of anti-oLH-IgG) overnight at room temperature. After a wash, 50,000 cpm 1261-labeled affinity-purified sheep Ab2 were added and incubated for 4 h at 37 C. Wells were washed and counted. B, The 1261-labeled sheep Ab2 used in A was incubated overnight at room temperature with pig ovarian membranes. “None” indicates specific binding calculated from counts per min found in the absence and presence of 10 pg unlabeled Ab2; 1 pg oLH decreased binding by 53%. C, Detection of binding of Ab2 to pig testicular LH receptor by the use of ‘Y-labeled anti-oLH-IgG (Abl IgG). In the first part the added samples shown were incubated for 2 h at 37 C in assay buffer containing 100 PM phenylmethylsulfonylfluoride. After dilution and wash, the pellets containing bound materials were suspended and incubated for 2 h at 37 C with 100,000 cpm 1261-labeled anti-oLH-IgG. Membranes were washed, and radioactivity in the pellet was determined. Ab2 control serum, Sheep immunized with normal rabbit IgG. Ab2 experimental, Sheep immunized with anti-oLH-IgG. Both of these were used at a 1:lO dilution, 100 pi/tube. All panels show the mean + SD (n = 6-9). FIG.

the receptor. Thus, it was of interest to further examine if steroidogenically active gonadal cells would be stimulated by these preparations. Three affinity-purified sheep antiidiotypic antibodies (IgGs) enhanced progesterone production by rat ovarian granulosa cells, and this was clearly concentration dependent (Fig. 8). However, compared to purified oLH, higher concentrations (microgram) of relevant sheep anti-IgGs were required for stimulation. These patterns are also consistent with their weaker receptor-binding ability shown in Fig. 6. Purified nonspecific sheep second antibodies, which could not bind to the LH receptor, showed no stimulation of progesterone production. Preparations of antiidiotypic IgGs that were active in the

,-.

oLH

rat granulosa cells were also effective in stimulating rat Leydig cells to cause testosterone production (Table 2). Here again, irrelevant sheep Ab2’s were not active in this regard. When combinations of oLH and a relevant Ab2 preparation were exposed to rat granulosa cells, there was a suggestion of an additive effect in the presence of low concentrations (0.1 ng for oLH and 1 pg for Ab2; see Table 3). However, when these constituents were present at maximal or near-maximal stimulatory concentrations, the progesterone produced by the cells remained the same. These data imply that the hormone and the Ab2 with a mirror image of the hormone most likely acted on the cells via a common pathway, including binding to the same receptor(s) and at a similar site(s). From the data in Fig. 2, it was evident that the highly TABLE cells

125

2. Effect of sheep antiidiotypic Treatment

nonspecific -2nd Ab

.-.-.-.-

25

lk

4

4

0.010

0.100 Concentration(

1.000 ( Hormone ng) Antibody Pg)

10.000

8. Steroidogenic activity of affinity-purified sheep antiidiotypic antibodies in rat granulosa cells. Experimental conditions are similar to those described in Table 3. Nonspecific second Ab, From sheep immunized with normal rabbit IgG. n and A, Three preparations from sera of animals immunized with anti-oLH-IgG. FIG.

antibodies on rat Leydig Testosterone (P&a

Control 1,680 f 116 oLH 1w 10,566 + 528 10 ng 18,572 f 742 Experimental Ab:! 10 rg 2,699 f 189 30 a! 7,500 f 452 Control Abz 36 wg 1,800 -e 98 About 300,000 collagenase-dispersed testicular Leydig cells from young rats were incubated with Dulbecco’s Modified Eagle’s MediumBSA/trypsin inhibitor and samples for 4 h at 37 C, and testosterone in the medium was measured by RIA. Values are the mean f SEM.

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ANTIIDIOTYPIC

ANTIBODIES

TABLE 3. Combined effects of oLH and experimental on rat granulosa cells

sheep Abs

Progesterone (pg/tube)

oLH (w)

0a 520 643 1445

0

0.1 3

Ah + 32” + 80” f 50”

1 /a Ah 585 f 35” 805 k 96b

30pgAbz

1298 + 41’ 1424 f 22’

Rat granulosa cells were incubated with medium or with hormone or affinity-purified Abz alone, as indicated, and in combination. Progesterone was estimated by RIA after a 4-h incubation at 37 C (n = 3). Different letters in superscript indicate a significant difference (P < 0.05). Values are the mean + SEM. TABLE 4. Neutralization by rabbit anti-oLH-IgG

of stimulatory

Treatment

effect of experimental

Abs

Progesterone (Pidwell)

166 -c 18.5

Control 0.3 ng oLH

1582 f 40

10 ag Abn prep. 1 10 pg Abn prep. 2

1708 f 24 1880 f 51

0.3 ng oLH + 30 ng anti-oLH-IgG 0.3 ng oLH + 1 pg anti-oLH-IgG

192.5 f 16 90 f 8

10 pg Abl prep. 1 + 1 pg antioLH-IgG 10 pg Abs prep. 2 + 1 rg antioLH-IgG

106 zk 13 91 f 17

Steroidogenesis was assessed in rat granulosa cells as described for the experiment shown in Table 3. Abl (anti-oLH-IgG) and hormone or Abs where present were added together. Values are the mean f SEM.

potent polyclonal Abl preparation neutralized hormone (oLH) action. If some of the antibodies in the sera of the experimental sheep also contained a structure that stimulated granulosa cells, such an action might be subject to inhibition by Abl. The action of affinity-purified Ab2 preparation from two sheep (Table 4) was completely blocked by the simultaneous addition of Abl. Discussion

This report documents the production of antiidiotypic antibodies to oLH which can mimic the hormone in some of its epitopes, receptor binding, and function. These antibodies have been fractionated and characterized. We used the affinity-purified polyclonal rabbit anti-oLH-IgG to prepare the antiidiotype in the sheep, demonstrating that a mirror image of its hormone could be created by immunization. It is worth noting that the affinity-purified anti-oLH IgGs were highly stable and active after storage for more than 10 yr, indicating a long shelf-life. These polyclonal antibodies were highly conformation specific, requiring the union of (Y- and @subunits of oLH, a structural requirement that is also essential for efficient receptor recognition by the hormone. In addition to being specific, their affinity to oLH (2 X 10” liters/mol) was high. These are also features necessary for efficient hormone-receptor interaction. The preservation of these ele-

RECOGNIZING

LH

RECEPTOR

1219

ments perhaps contributed in part to the formation of useful antiidiotypic IgGs upon subsequent immunization of the sheep. We believe that both the polyclonal first antibody (rabbit) and the second antibody (sheep) were IgG, based on the 150,000 mol wt found in sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. While the purification of the polyclonal Abl preparation by affinity chromatography using the antigen is not absolutely necessary, its avaiIability enhances the opportunity to generate potentially useful antiidiotypes by eliminating unwanted Igs. This became very evident in the data from our study. Although it was not essential for us to immunize control sheep with normal rabbit IgG, we elected to do so to demonstrate that these irrelevant Ab2 would not mimic hormone action. Similar studies describing the formation of antiidiotypic antibodies have been reported for many hormones, such as insulin (3, 4), PRL (6), ,&adrenergics (7), pituitary GH (B), and TSH (5, 28). For the pituitary glycoprotein hormones, only TSH has been studied in some detail with respect to the production of antiidiotypic antibodies, which were shown to mimic the hormone both structurally and functionally (5, 28). Besides our own preliminary report (18) and that of Kotagi et al. (29) for FSH, there have been no studies on the generation of antiidiotypic antibodies for gonadotropins. This latter report only mentioned that the antiidiotypic FSH antibodies could bind to the testicular FSH receptor, but no other functional tests were performed. From the data presented herein, we have established that the sheep injected with affinity-purified rabbit anti-oLH-IgG produced antiidiotypes that not only resembled oLH in antigenic properties, but could also bind to both ovarian and testicular receptors and further stimulate both of these target cells to produce steroids, a response typical of pituitary LH action. Antibodies capable of altering receptor (cell) function in some manner have been reported for many hormone receptors, including those for gonadotropins and TSH. A polyclonal antibody to the FSH receptor could prevent hormone binding to testicular cells and also stimulate estradiol production (30). Polyclonal antibodies against the purified LH/ hCG receptor (31,32) and various peptides deduced from its sequence (33) recognize the cellular receptor in its native form or upon denaturation. Among monoclonal antibodies reported for the LH/hCG receptor, some are known to be of a stimulatory type (34), while others inhibit function, either partially or completely (35). These are true antibodies to the receptor, because the antigen used was the purified receptor or one of its peptidic segments. However, the antiidiotypic antibodies that we studied would be different from that described above. Although both kinds of antibodies bind to the cellular LH receptor, their direct comparison is precluded, as the responsible binding domains are not defined. It is unlikely that the oLH-like hormone activity detected in the sera of immunized sheep (experimental group) by both RIA and RRA could be due to some contaminating anti-oLHIgG immunogen from the affinity column and sequestering the tracer [‘251]oLH used in such experiments. Control runs with buffers and eIuents from these columns showed no

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1220

ANTIIDIOTYPIC

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RECOGNIZING

LH RECEPTOR

Endo. 1992 Vol131.

leakage of either hormone or antibody (36). In addition, there was no evidence of such a presence in Western blotting performed with affinity-purified sheep Ab2 preparations. In such tests less than 1 ng anti-oLH-IgG, if any, could be detected by [“‘I]oLH treatment of the proteins transferred to nitrocellulose (data not shown). Secondly, as the sheep were bled before each booster injection, it might be expected that the oLH-IgG immunogen from the preceding booster would have disappeared from circulation during the 30-day interval allowed between bleedings. We also believe that an argument to explain the steroidogenic ability of sheep antiidiotypic IgG as being due to the concentration of oLH in the circulation of the sheep by the affinity chromatographic procedures used is untenable, because purified sheep IgGs from control animals receiving commercial normal rabbit y-globulins did not show any such activity in rat granulosa and mouse Leydig cells. The conditions required for classifying the second antibodies in sheep as antiidiotypic antibodies were satisfied. The binding of Ab2 to Abl was effectively inhibited by the antigen oLH in sandwich assays, and likewise, the recognition of labeled antigen by Abl was competed by Ab2 present in the serum or affinity-purified form. In addition, it is important to note that these same Ab2 populations from all experimental animals were specific because they were without effect on a structurally analogous hormone, oFSH, in its interaction with its own antibody or receptor. The high hormone-like activity of Ab2 assessed by the oLH RIA was not matched by equivalent high activity in either RRA or steroidogenic assays, in which they were quite effective nevertheless. This is most likely due to the fact that among the variety of Ab2’s generated in response to the specific anti-oLH-IgG, which itself was directed against numerous epitopes of the antigen oLH, only a few could be expected to bear a mirror image of the receptor-binding domain of the hormone. Even in this specific Ab2 population, other antibodies, although detectable in RIAs of the type shown in Figs. 4 or 7, B or C, would not be relevant as far as receptor recognition and cell stimulation are concerned. As we used affinity-purified polyclonal antibody, it was not possible to select only those Abl populations that were directed solely against the receptor-binding domain(s) of the oLH. For this purpose, one would have to rely on highly specific monoclonal antibodies specific to the receptor-binding regions of the hormone to increase the chances of producing Ab2 capable of biological mimicry and pronounced activity. Even in this case, it is essential to establish if the Ab2’s so generated would be of the stimulatory or inhibitory type. It appears that the polyclonal Ab2’s that were produced in the present study by all three sheep were of the stimulatory type. If there were any of an inhibitory nature as far as cell function is concerned, they were either not predominant or were masked by the others. As no success has been indicated in producing antiidiotypic antibodies starting from hCG monoclonal antibodies (5), the search for suitable ones as potential antigens may have to be widened. Numerous studies on the structure-function relationships of gonadotropins and TSH (37-39), including synthetic pep-

No 3

tides (39), have suggested the presence of multiple sites in the hormone for binding the receptor(s). The long extracellular domain of these receptors predicted from cDNA cloning studies (40-43) is thought to accommodate one or more of these sites in the hormone to effect signal transduction. In the light of this knowledge and the present work, occupation of one or a few such sites on the cell by an antiidiotypic antibody may give rise to partial stimulation. Thus, if selected monoclonal antibodies to hormones representing different receptor-binding domains were chosen as immunogens, improvements in response may be possible by combined immunizations. Because we employed a polyclonal Abl as antigen, it is most likely that the idiotopes were against more than one receptor-binding site. This would be an explanation for attaining a stimulation equivalent to the hormone by the purified Ab2’s in steroidogenic assays. It may also be possible to enhance relevant Ab2 formation by choosing either the Fab or (Fab)2 fragment of Abl for immunizations. As this study was not originally designed to examine the effect of immunization on the androgenic status of the sheep, we did not measure testosterone levels in the circulation of the animals before and after the start of the injections. In these young and growing animals, a visual record of scrotal circumference was made as an indirect assessment, because any sustained and drastic reduction in androgen levels would be reflected in testis size. From the data indicated in Fig. 9, we can infer that the formation of irrelevant antibodies in control animals as well as the stimulatory antibody to antioLH-IgG (experimental) during 6-8 months of study showed no marked differences. The changes seen were deemed to be within the experimental error typical for these recordings. In two experimental sheep that were killed after 18 months, testis size was normal. All animals sustained a uniform increase in body weight (+231.8 + 10.7%) during the course of the immunizations, indicating no deleterious effects on 37 35

-E sY z

r-

3331-

29-

E

g 270’ fbi 2526

23

t

II

Nou

I

b

II

Dot

Jon

Feb

I,,

Mar

Apr

May

June

FIG. 9. Testicular growth in control and experimental sheep. The scrotal circumferenceof eachtestis was measured,and averagevalues are indicated. Variations are within tti point. 1 and 2, Control immunized with normal rabbit IgG; 3 and 4, immunized with rabbit antioLH-IgG. Arrows indicate the start of immunization in 1986.

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ANTIIDIOTYPIC

ANTIBODIES

growth. Similarly, there were no decreases in either the final body weights or testicular weights (1.549 f 0.07 VS.1.663 f 0.037 g) of the control and experimental group of male rats. These animals are sensitive to androgen deprivation, because testicular regression occurs shortly after active immunization with LH (44) or passive immunization using LH antiserum (45). In conclusion, the present study has documented the potential for generation of antiidiotypic polyclonal antisera in sheep that can mimic pituitary LH action. It would be of interest to explore the potential benefit of such an approach as a vaccine, particularly with FSH, for improving and enhancing the fertility of farm animals. In addition, these antibodies may serve as useful surrogates to study receptor structure and function, Acknowledgments

Ann

1974 Towards Immunol125C:373-389

a network

theory

of the immune

internal

images.

EMBO

17.

idiotypes

19. 20.

21.

6.

system.

27.

7. Schreiber AB, Couraud PO, Andre C, Vray B, Strosberg AD 1980

8.

Anti-alprenolol anti-idiotypic antibodies bind to fi-adrenergic receptors and modulate catecholamine sensitive adenylate cyclase. Proc Nat1 Acad Sci USA 77~7385-7389 Garder MJ, Morrison CA, Stevenson CQ, Flint DJ 1990 Production of anti-idiotypic antisera to rat GH antibodies capable of binding to GH receptors and increasing body weight gain in hypophysectomized rats. 1 Endocrinol 125:53-59

9. Couraud

Jk, Maillet

S, Grassi J, Frobert

Y, Pradelles

95:93-98 13. Young WP, Nasser R, Hayashida

28.

T 1963 Antiserum inhibition of the oestrous cycle in normal rats. Nature 197:1117 14. Bourdel G, Li CH 1961 Effect of rabbit antiserum to sheep pituitary interstitial cell stimulating hormone in adult female rats. Acta Endocrinol (Copenh) 42:473-479 15. Jagannadha Rao A, Moudgal NR, Madhwa Raj HG, Lipner H, Greep RO 1974 The role of FSH and LH in the initiation of ovulation in rats and hamsters. A study using rabbit antisera to ovine FSH

Eur J Biochem

BR,

86:121-131

of hormone induced cyclic AMP production and steroidogenesis in interstitial cells by deglycosylated lutropin. Mol Cell Endocrinol22:251-264 Ascoli M 1981 Characterization of several clonal lines of cultured Leydig tumor cells: gonadotropin receptors and steroidogenic responses. Endocrinology 108:88-95 Madhwa Raj HG, Moudgal NR 1970 Hormonal control of gestation in the intact rat. Endocrinology 86:874-889 Sairam MR 1978 Studies on pituitary follitropin. IV. A conformation specific radioimmunoassay for the ovine hormone. Endocr Res Commun 5:279-291 Hill BL, Erlanger BF 1988 Monoclonal antibodies to the thyrotropin receptor raised by an autoantiidiotypic protocol and their relationship to monoclonal autoantibodies from Graves’ patients. Endocrinology 122:2840-2850

29. Kotagi SG, Nath N, Raj SG, Singh KB, MadhwaRaj Development of ovine antiidiotypic antibodies Acad Sci 513:516-519

HG 1987

follicle stimulating hormone (FSH) and its as vaccine for male contraception. Ann NY

30. Dattatreyamurthy

B, Zhang SB, Reichert Jr LE 1990 Polyclonal antibodies against follitropin (FSH) receptor Interfere with hormone binding, but mimic the effects of oFSH. Endocrinology 126:1318-

1326 31. Metsikko

L, Rajaniemi

hormone receptor 109:1399-1403

32. Rosemblit

P 1989

Characterization and properties of antisubstance P anti-idiotypic antibodies. Methods Enzymol178:275-300 10. Gaulton GN, Greene MI 1986 Idiotypic mimicry of biological receptors. Annu Rev Immunol4:253-280 11. Schick MR, Kennedy RC 1989 Production and characterization of anti-idiotypic antibody reagents. Methods Enzymol 178:36-48 12. Moudgal NR, Li CH 1961 An immunochemical study of sheep pituitary interstitial cell stimulating hormone. Arch Biochem Biophys

GN, Downey

24. Sairam MR, Fleshner P 1981 Inhibition

Sege K, Peterson PA 1978 Use of anti-idiotvnic

5.

RG, Bhargavi

recognize the luteinizing hormone 21st Annual Meeting of the Society Seattle WA, 1988 (Abstract 214) Singh V, Sairam MR, Bhargavi GH, Akhras RG 1989 Hormonotoxins. Preparation and characterization of ovine luteinizing hormone-gelonin conjugate. J Biol Chem 264:3089-3095 Sairam MR 1991 Facile separation of the subunits of ovine and bovine gonadotrophins by high-performance liquid chromatography: microheterogeneity and biological activity. J Endocrinol 130:415-424 Sanford LM, Yarney TA 1983 Clrcannual changes in serum levels of pituitary hormones and testosterone and in testis size of sexually active and inactive adult rams. Can J Anim Sci 63:811-821 Sebok K, De Lean A, Sairam MR 1987 Analysis of the kinetics of ovine follitropin agonist-antagonist interactions with pig ovarian membranes. Biochemistry 263650-3658

23. Rogister GM, Closset J, Combarnous G, Dechenne C, Ketelslegers JM 1978 Study of follitropin receptors in testis using a homologous

and

antibodies as cell suaace.receptor probes. Proc Nat1 Acad Sci USA 75:2443-2447 Shechter Y. Maron R. Elias D. Cohen IR 1982 Autoantibodies to insulin receptor spontaneouslv develop as anti-idiotvpes in mice Iimmunized *with insulin. Science 216:542-545 Farid NR, Lo TCY 1985 Anti-idiotvuic antibodies as urobes for I receptor &ucture and function. End&r Rev 6:1-23 Amit T, Barkey RJ, Gavish M, Youdim MBH 1986 Anti-idiotypic antibodies raised against anti-prolactin (PRL) antibodies recognize the PRL receptor. Endocrinology 118:835-843

GJ, Greep RO 1971 Role of endogenous hormone (LH) in maintaining corpus luteum of Endocrinol Metab 35:113-l 16 Sairam MR, Porath J 1976 Isolation of antibodies to protein hormones by bioaffinity chromatography on divinylsulfonyl Sepharose. Biochem Biophys Res Commun 69:190-196

Anti-idiotypic antibodies that receptor: biological properties. for the Study of Reproduction,

system.

J 1:243-247

Fertil37:323-330

NR, Macdonald

18. Sairam MR, Sairam J, Akhras

26.

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1221

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We thank J. Sairam, Mira Dobias-Goff, S. Payne, and R. Akhras for assistance in the various phases of this investigation. The help of Drs. B. E. P. Murphy and F. Labrie in providing some of the antisera is gratefully acknowledged. Mrs. Francine De Coste helped in the preparation of the manuscript.

Jerne NK

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antiserum receptor.

33. Rodriguez

localize

H 1981 Antibodies receptor

to purified luteinizing at the cell surface. Endocrinology

N, Ascoli M, Segaloff DL 1988 Characterization

of an luteinizing hormone/chorionic gonadotropin 123:2284-2290 Segaloff DL 1990 The orientation of lutropin/ receptor in rat luteal cells as revealed by site Endocrinology 127~674-681

to rat luteal Endocrinology

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choriogonadotropin specific antibodies.

34. Podesta EJ, Solano AR, Attar R, Sanchez ML, Vedia LMY 1983 Receptor biological

aggregation induced by antilutropin response in rat testis Leydig cells.

receptor antibody and Proc Nat1 Acad Sci USA

80:3986-3990 35. Indrapichate K, Meehan D, Lane TA, Chu SY, Rao ChV, Johnson D, Chen TT, Wimalasena T 1992 Biological actions of monoclonal 36.

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luteinizing hormone/human chorionic gonadotropin receptor antibodies. Biol Reprod 46:265-278 Sairam MR 1988 Affinity chromatography of hormone receptors and antibodies on divinylsulfonyl Sepharose. In: Hutchens TW (ed) UCLA Svmuosia on Molecular and Cellular Bioloev.v, Liss. New York. New Series’80:303-314 Gordon WG, Ward DN 1985 Structural aspects of luteinizing

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RECOGNIZING

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Endo. 1992 Vol131. No 3

Cloning and sequencing of porcine LH-hCG receptor cDNA: variants lacking transmembrane domain. Science 245:525-528 Sprengel R, Braun T, Nikolics K, Segaloff DL, Seeburg PH 1990 The testicular receptor for follicle stimulating hormone: structure and functional expression of cloned cDNA. Mol Endocrinol4:525530 Parmentier M, Libert F, Maenhaut C, Lefort A, Gerard C, Perret J, VanSande J, Dumont JF& Vassart G 1989 Molecular cloning of the thyrotropin receptor. Science 246:1620-1622 Monastirsky R, Laurence KA, Tovar E 1971 The effects of gonadotropin immunization of prepubertal rabbits on gonadal development. Fertil Steril22:318-324 MadhwaRaj HG, Dym M 1976 The effects of selective withdraw1 of FSH or LH on spermatogenesis in the immature rat. Biol Reprod 14:489-494

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