Proc. Natl. Acad. Sci. USA Vol. 77, No. 6, pp. 3440-3444, June 1980 Cell Biology Aggregation of receptors in granulosa cells: A possible mechanism of desensitization to the hormone (adenylate cyclase/antibodies to /immunofluorescence/receptor internalization/radioautography) ABRAHAM AMSTERDAM*, ARIE BERKOWITZ, ABRAHAM NIMROD, AND FORTUNA KOHEN Department of Hormone Research, The Weizmann Institute of Science, Rehovot, Israel Communicated by Roy Hertz, February 26, 1980

ABSTRACT The temporal relationship between redistri- ceptor sites, referred to as "down regulation" (for review, see bution of receptors to lutropin (luteinizing hormone)/human ref. 19). chorionic in cultured rat ovarian granulosa cells We have previously demonstrated that internalization of the and the cellular response to hormonal challenge were studied. Visualization of receptor-bound human chorionic gonadotropin receptor-bound hCG in cultured rat granulosa cells into lyso- by indirect immunofluorescence, with hormone-specific anti- somes occurs after hormone binding, but that this process bodies after fixation with 2% formaldehyde, revealed the ex- cannot account for the loss of the cellular response to the hor- istence of small clusters around the entire cell circumference mone because desensitization of the hormone-stimulated ade- 5-20 min after exposure to the hormone at 370C. Such small nylate cyclase preceded the massive loss of specific receptors receptor aggregates were also evident if hormone incubation (10). In the present study we have examined whether desensi- was at 40C or if cells were fixed with 2% formaldehyde before incubation. Larger clusters were evident after prolonged incu- tization of granulosa cells to LH or hCG can be due to rear- bation with the hormone (2-4 hr) at 370C. The later change rangement or clustering of these receptors, reflecting their coincided with diminished cyclic AMP accumulation in re- lateral mobility and the efficiency of their coupling to adenylate sponse to challenge with fresh hormone. When the fixation step cyclase. was omitted and antibodies to human chorionic gonadotropin were applied after hormonal binding, acceleration of both re- ceptor clustering and the desensitization process was observed. MATERIALS AND METHODS This maneuver also induced capping of the hormone receptors. Materials. Highly purified hCG [CR-119; 12,800 interna- In contrast, monovalent Fab' fragments of the antibodies were tional units (IU)/mg], rat FSH, and pregnant mare serum go- without effect. Internalization of the bound hormone in lyso- somes, and subsequent degradation, was evident 8 hr after nadotropin were kindly provided by the National Institutes of hormonal application and was not accelerated by the anti- Health (Bethesda, MD). Goat anti-rabbit IgG conjugated to bodies. It is suggested that clustering of the luteinizing hormone fluorescein was obtained from Hyland (Costa Mesa, CA). receptors may play a role in cellular responsiveness to the hor- Photographic emulsion L4 was from Ilford (Essex, England). mone. Massive aggregation of the receptors may desensitize the 3-Isobutylmethylxanthine (IBMX) was from Aldrich (Mil- cell by interfering with coupling to adenylate cyclase. waukee, WI). Granulosa . Granulosa cells were collected from Since the observation by Frye and Edidin (1) of lateral mobility rat preovulatory follicles on the morning of proestrus by of membrane proteins, movement and clustering have been puncture and gentle expression into McCoy's 5a (modified) demonstrated for receptors to immunoglobulins (2) and lectins medium containing 2 mM glutamine, 100 units of penicillin (3) as well as receptors to hormones and neurotransmitters (4-7). per ml, 100,tg of streptomycin per ml, and 20% (vol/vol) fetal Moreover, it was recently suggested that receptors associated calf serum. Cells (106 cells per ml) were introduced into Falcon with the plasma membrane can be internalized after binding Optilux tissue culture dishes (35 X 10 mm) and cultured for 24 specific ligands (8-10). However, the biological significance hr as described (20). Alternatively, granulosa cells were obtained of such a receptor redistribution is not fully understood. from 26-day-old immature rats treated with pregnant mare Hertz and Hisaw demonstrated (11) that pituitary extracts, serum gonadotropin (15 IU for 48 hr; see ref. 21) and cultured containing gonadotropic hormones such as follitropin (folli- as described above. In some control experiments, cells were cle-stimulating hormone; FSH), play an important role in the obtained from immature rats treated with diethylstilbestrol. regulation of ovarian development. It is now known that go- These cells contain FSH but not LH receptors (22). nadotropic hormones have receptors on the plasma membrane Preparation of Specific Antibodies to hCG. Antibodies to of ovarian target cells (12) and that they exert their effect via hCG were raised in rabbits with the highly purified hormone activation of a hormone-sensitive adenylate cyclase (13, 14). as the antigen. Immunoglobulin (IgG) fractions were prepared Prolonged exposure of ovarian tissue to lutropin (luteinizing from normal and immune sera by ammonium sulfate precipi- hormone; LH) or human chorionic gonadotropin (hCG), which tation followed by DEAE-52 column chromatography (23). The bind to the same receptor, causes prompt stimulation of ade- divalent fragment F(ab')2 was prepared by pepsin digestion of nylate cyclase followed by desensitization of the enzyme to the IgG fraction (24) followed by removal of the Fc fragment renewed challenge with the hormone in vmvo and in vitro by staphylococcal protein A absorption. The monomer, Fab', (13-16). A similar phenomenon was also described in other was prepared by reductive alkylation of the divalent fragment systems where hormones exert their effects via activation of The the divalent and adenylate cyclase (17, 18). The mechanism responsible for this (25). immunoglobulin fraction, fragment, refractoriness is not clear. Several explanations have been Abbreviations: FSH, follitropin (follicle-stimulating hormone); LH, suggested, among them an agonist-induced depletion of re- lutropin (luteinizing hormone); hCG, human chorionic gonadotropin; 125I-hCG, l25I-labeled hCG; IBMX, 3-isobutyl-1-methylxanthine; IU, The publication costs of this article were defrayed in part by page international units. charge payment. This article must therefore be hereby marked "ad- * Present address: and Reproduction Research Branch, vertisement" in accordance with 18 U. S. C. §1734 solely to indicate National Institute of Child Health and Human Development, Na- this fact. tional Institutes of Health, Bethesda, MD 20205. 3440 Downloaded by guest on September 26, 2021 Cell Biology: Amsterdam et al. Proc. Natl. Acad. Sci. USA 77 (1980) 3441

the monovalent fragment were checked for purity by Na- Aggregation of hCG Receptors. The effect of incubation DodSO4 gel electrophoresis. The titer of the specific -ntibodies of granulosa cells with hCG on receptor distribution was studied and their fragments was checked by a double-antibody radio- in detail. Fixation of the cells with 2% formaldehyde after immunoassay procedure (26) with '25I-labeled hCG (125I- hormone binding and before the application of the specific hCG). antibodies to the hormone allowed us to differentiate between Immunofluorescent Staining of Cells. Cell layers were in- the effect of the hormone itself and that of the antibodies. cubated with hCG (20-200 IU/ml for 5-240 min) at 37 or 40C, Moreover, fixation of the cells prior to hormone binding allowed followed by incubation either with antibodies to hCG [IgG, us to visualize the receptor distribution before they were oc- Fab', or F(ab')2 fragments; 1-2 mg/ml] or with whole antise- cupied. When the cells were incubated for 5 min with 200 IU rum (diluted 1:10-1:20) for 30-90 min and finally with goat of hCG or for 20 min with 20 IU at 37"C, followed by formal- anti-rabbit IgG conjugated to fluorescein (1 mg/ml for 30-45 dehyde fixation and incubation with hormone-specific anti- min). Concentrations of antisera, IgG fraction, and antibody bodies, small clusters of fluorescent particles were evident at fragments were chosen to give a similar titer in radioimmu- the circumference of granulosa cells after the application of noassay (1:8000) with 125I-hCG. In some experiments, cells were fluorescein-labeled goat anti-rabbit IgG (Fig. 1A). Essentially, fixed with 2% formaldehyde (30 min at room temperature or thisame pattern of fluorescence was obtained when cells were 40C) before or after incubation with the hormone and rinsed. incubated with 20-200 IU of the hormone per ml at 4VC or Remaining aldehyde groups were blocked with 0.2 M Tris were fixed with the aldehyde prior to incubation with hormone. buffer (pH 7.4) containing 1 mg of bovine serum albumin per This suggests that small clusters of receptors exist before ap- ml before hormones or antibodies were applied. Such a fixation plication of the hormone. procedure did not alter the specific binding of the hormone to Significantly, larger clusters were observed when cells were cells, unlike glutaraldehyde which completely destroyed spe- incubated with 20 IU of hCG per ml for 2 hr at 370C and fixed cific binding even at concentrations as low as 1.0%. with formaldehyde before the application of the antibodies Light Microscopy. Cultures were inspected and photo- (Fig. 1B). When the fixation step was omitted and antibodies graphed with a photomicroscope III (Zeiss, West Germany) were applied, bulky fluorescent agglomerates appeared (Fig. equipped for fluorescent and phase-contrast microscopy. 1C), followed by cap formation after an additional 1-2 hr of Iodination of hCG and Fragments of Antibodies. hCG was incubation with antibody-free medium (Fig. 1D). Prolonged iodinated by the lactoperoxidase method (27). The specific incubation with the hormone itself even up to 5 hr at 370C did activity of the iodinated hormone was 30,Ci/mg (1 Ci = 3.7 not result in formation of these bulky agglomerates or cap X 1010 becquerels). Iodination of Fab' and F(ab')2 fragments formation. of immunoglobulins was as described (28). The integrity and ultrastructural characteristics of the cells Autoradiography. Cells were processed for high-resolution were well preserved during incubation of the cultures with the hormones as well autoradiography after binding of '25I-hCG as described (10). as with the antibodies (Figs. 1E and 2). Radioautograms were examined by electron microscopy (JEOL Internalization and Degradation of Receptor-Bound Hormone After Application of Antibodies to hCG. We have 100-B). shown that Assays. Production of cyclic AMP in cells in the presence of already 125I-hCG is internalized in lysosomes, where it is to a inhibitor was measured by the probably degraded single amino acids (10). We found phosphodiesterase (IBMX) that the extent of method of Gilman (29) and as described earlier (10). In some hormone degradation after internalization is not the experiments the assay was performed according to Humes et significantly changed by external application of al. (30). The content of trichloroacetic acid-soluble and -in- specific antiserum. After a 3-hr incubation with 125I-hCG fol- soluble radioactive material in the culture incubation media, lowed by a 1-hr incubation with the antibodies and an addi- tional 4 hr with antibody-free medium, about 20% of the ra- after binding of 125I-hCG to cells, was measured as described elsewhere dioactivity primarily associated with the cells was recovered (10). as trichloroacetic acid-soluble material in the incubation me- dium. At this time, half of the radioactivity associated with the RESULTS cells was found in lysosome-like structures, as revealed by Specificity of hCG Antiserum to Stain Cell-Bound Hor- high-resolution radioautography (Fig. 2). Similar data were mone by Indirect Immunofluorescence Technique. When obtained after cells were incubated with 125I-hCG for the same 24- to 48-hr cultured granulosa cells, obtained from preovula- time and subsequently with hormone-free medium without tory follicles, were incubated with a saturating concentration antibodies (see also ref. 10). These data suggest that the for- of hCG followed by anti-hCG IgG and stained with fluores- mation of bulky aggregates of receptors, subsequent to hor- cein-conjugated goat anti-rabbit IgG, bright fluorescence was mone-induced formation of clusters of receptors, does not ac- evident at the circumference of the cells (Fig. 1 A-D). Because celerate the rate of internalization and degradation of the re- the hormone served as a specific marker for the LH/hCG re- ceptor-bound hormone. ceptor, it was essential to determine that the antibodies bound Hormone-Induced Formation of Cyclic AMP. Granulosa exclusively to the hormone molecule on the cell. Replacement cell cultures not previously exposed to hCG responded to the of the specific antibodies with nonspecific ones abolished the hormone with approximately a 70-fold rise in cyclic AMP ac- fluorescent staining of the cells, whereas elimination of the cumulation over basal levels (Tables 1 and 2). Previous studies hormone from the incubation resulted in only weak staining in our laboratory indicated that incubation of such cells with of the cells. This might be due to binding of some LH contained hCG for 2-3 hr results in partial desensitization to the hormone, in the fetal calf serum of the culture medium (see Materials and so that after rinsing and challenge with fresh hormone in the Methods). However, granulosa cell cultures that had already presence of a phosphodiesterase inhibitor (IBMX), cyclic AMP lost their LH receptors (12 days in culture) or that contained accumulation is reduced to about 40% of the initial response only FSH receptors, did not show any staining. Moreover, no (ref. 10 and Table 1). When anti-hCG serum was added for 30 staining was obtained if cells from diethylstilbestrol-treated mn afteran initial incubation for 90 mn with the hormone and animals were incubated with a saturating concentration of rat excess anti-hCG was then removed (see Table 1), this desensi- FSH and subsequently with antibodies to hCG. tization was accelerated, so that only 17% of the initial cyclic Downloaded by guest on September 26, 2021 3442 Cell Biology: Amsterdam et al. Proc. Nati. Acad. Sci. USA 77 (1980) I

FIG. 1. Localization ofreceptor-bound hCG in granulosa cell cultures by indirect immunofluorescence. Incubations with hormone and antibodies were at 370C. Calibration = 30 ,sm. (A) Cells were incu- bated with the hormone (200 IU/ml) for 5 min and then fixed with formaldehyde. Tiny specks of fluorescence (arrowheads) are visible around the entire circumference of an elongated granulosa cell (g). (B) Cells were incubated for 120 min with hCG (20 *.9 IU/ml) and fixed with formaldehyde. ,. 2~ Clusters of fluorescence are visible (ar- If rowheads) over three granulosa cells (g). The clusters are larger than those obtained ik" after a 5-min incubation with the hormone. (C) Cells were incubated for 90 min with the .i. It,s * hormone and subsequently with anti-hCG without prior fixation of the cell. Large .i clusters are seen at the circumference of several granulosa cells (g). (D) Treatment as in C, but the cells were incubated for an additional 1 hr after removal of excess an- tibodies. Note large clusters and caps (ar- rowheads) over granulosa cells (g). (E) The same field as in D, through phase-contrast ...... optics, indicating the integrity of the E cells.

AMP response was observed at the end of a .150-min incubation. Table 2 shows that hormone-induced cyclic AMP accumu- Addition of the antiserum prior to hormonal challenge did not lation was suppressed by the whole antiserum, the IgG fraction, significantly affect the cyclic AMP response. Application of and the divalent F(ab')2 fragments, but not by the monovalent second antibody (by itself, or after the first antibody) did not Fab' fragment. Introduction of excess hCG in the challenge change the degree of desensitization (Table 1, Exp. II). incubation increased cyclic AMP accumulation somewhat in To study the effect of antibodies to the hormone in more cells preincubated with the divalent antibody. However, cyclic detail, we preincubated cells with hCG for only 20 min to AMP formation in these systems did not exceed 25-50% that minimize the degree of cell desensitization due to the hormone produced by cultures treated with the Fab' fragments of the alone. When rates of cyclic AMP accumulation in cells prein- specific antiserum or with F(ab')2 fragments obtained from cubated with the hormone and subsequently incubated in the nonimmune serum. Cells preincubated with the hormone presence or absence of antibodies were compared, 65% of in- bound '25I-Fab' or '25I-F(ab')2 fragments of the specific anti- hibition in cyclic AMP formation was achieved after 1 hr of bodies to the same extent. incubation with the antibodies and 90% of inhibition was seen after 90 min (Fig. 3). DISCUSSION Incubation of the cells with the antibodies for 30 min and Loss of receptors is one explanation for the loss of cellular re- subsequently with antibody-free medium for an additional 60 sponse to hormones. However, we have demonstrated earlier min gave essentially the same depression of cyclic AMP accu- (10), as well as in this work, that refractoriness precedes massive mulation as did continuous incubation with antiserum. internalization of the receptor-bound hormone and coincides When cells were preincubated with 125I-hCG (2 IU of hor- with receptor aggregation. This is also consistent with the ob- mone per ml), rinsed, and subsequently incubated for 90 min servations of Harden et al. (17) and Hoffman et al. (18), who with the antiserum, no increase in the release of the labeled found no change in 3-adrenergic receptor number during de- hormone was detected compared to control cultures incubated sensitization of astrocytoma cells and turkey erythrocytes, re- with antibody-free medium (data not shown). spectively, to catecholamines. Downloaded by guest on September 26, 2021 Cell Biology: Amsterdam et al. Proc. Natl. Acad. Sci. USA 77 (1980) 3443

Table 1. Effect of attachment of antibodies to receptor-bound hCG on adenylate cyclase activity in cultured granulosa cells Challenge Preincubation, min incubation* No Exp. 0-90 91-120 121-150 hormone +hCGt I -hCG NRS NRS <5 197 I 3 -hCG Anti-hCG NRS 8 + 1 189 + 14 +hCG NRS NRS 87 4 90 11 +hCG Anti-hCG NRS 31 2 55 + 5 II -hCG NRS FITC-aRIgG <5 231 L 13 +hCG NRS FITC-aRIgG 138 + 24 134 4 6 +hCG Anti-hCG FITC-aRIgG 53 I 3 70 ± 10 Normal rabbit serum (NRS) and anti-hCG serum were used at 1:10 dilution. Cultures were thoroughly rinsed after each incubation (at 371C). Concentration of hCG was 20 IU/ml at the preincubation step. Fluorescein-conjugated goat anti-rabbit IgG (FITC-aRIgG) was used at 1 mg/ml. The challenge medium contained 10 ,g of IBMX per ml. * Cyclic AMP formation, given as pmol per dish per 20 min. Results are expressed as mean :1 SEM (n = 3). t 10 IU/ml.

to be elucidated. Schlessinger et al. recently reported (33) that the clustering of receptors for insulin and epidermal growth factor that occurs after ligand binding results in a decrease of the lateral mobility of these membrane proteins. We suggest that massive aggregation of LH/hCG receptors induced by the hormone in granulosa cells may restrict the mobility of these receptors in the plane of the membrane. This may result in a decrease in efficiency of coupling between the receptor and the adenylate cyclase system. Alteration of the lipid composition of cell membranes con- FIG. 2. Electron microscope autoradiograph of thin section of taining f,-adrenergic receptors, which probably increase cultured granulosa cell incubated for 3 hr with 125I-hCG (2 IU/ml) and membrane fluidity, results in increased adenylate cyclase ac- subsequently for 1 hr with antiserum to the hormone (1:10 dilution) tivity in response to catecholamines (346). This is consistent and an additional 4 hr with antibody-free medium at 37°C. Note silver with the hypothesis that increased membrane fluidity and grains associated with lysosome-like structure (L) and small, smooth increase membrane vesicle (V) whereas the plasma membrane (M) is essen- lateral mobility of membrane proteins the coupling tially free of radioactive material. Calibration = 0.5 ,m. between hormone receptors and the cyclase system, whereas restriction of receptor mobility leads to a refractory state. The ovarian follicular cells respond to three different agonists, LH (hCG), FSH, and prostaglandin E2, with increased cyclic AMP state is restricted accumulation. However, the refractory Table 2. Effect of attachment of mono- and divalent fragments to the homologous hormone. Thus, cells that are refractory to of antibodies to receptor-bound hCG on adenylate cyclase activity LH are responsive to FSH and prostaglandin E2 (31). Therefore, in cultured granulosa cells it seems unlikely that the adenylate cyclase per se is affected during the desensitization period. On the other hand, the recent Incubation with Challenge incubation (20 min)* report (19) that fluoride-sensitive cyclase activity in luteal tissue antibodies (90 min) No hormone +hCGf is temporarily and partially blocked during the period of de- None$ 71.0 L 3.0 71.0 + 4.3 sensitization does not exclude the possibility that the enzyme NRS 18.21I1.7 20.01±2.3 might be directly affected in the refractory state. Anti-hCG serum 2.4 4 0.5 8.4 L 3.0 We present evidence that failure of granulosa cells to respond Anti-hCG IgG 3.7 + 0.3 5.3 + 0.4 to hormonal challenge after prolonged incubation with the Anti-hCG F(ab')2 2.5 ± 1.2 9.7 + 2.9 hormone is associated with massive aggregation of the LH/hCG Anti-hCG Fab' 22.4 k 1.9 19.7 + 1.7 receptors on the cell membrane. Moreover, divalent antibodies Nonspecific F(ab')2 18.0 ± 2.4 18.0 k 1.1 to hCG, which enhance receptor aggregation, also accelerate All cultures were preincubated with hCG (20 IU/ml) for 20 min and the rate of desensitization in these cells. Preliminary data were thoroughly rinsed after each incubation (at 370C). Normal rabbit suggest that receptors to hCG are more aggregated in Leydig serum (NRS) and anti-hCG serum were used at 1:10 dilution. IgG, cells obtained from refractory testis than in cells obtained from F(ab')2, and Fab' fractions were used at concentrations of 2.0 mg/ml. the unstimulated gland (32). Therefore, clustering of receptors The challenge medium contained IBMX (10 ,g/ml). * AMP as relative to the basal level. Re- may a in the mechanism of refractoriness both in vitro Cyclic accumulation, given play role sults are expressed as mean I SEM (n = 3). and in vivo. t 10 IU/ml. The process by which massive receptor aggregation may lead t Immediate challenge incubation after a 20-min preincubation with to inability of the cell to respond to hormonal challenge remains hormone. Downloaded by guest on September 26, 2021 3444 Cell Biology: Amsterdam et al. Proc. Natl. Acad. Sci. USA 77 (1980)

1. Frye, L. D. & Edidin, M. (1970) J. Cell Sci. 7,319-35. 2. de Petris, S. & Raff, M. (1972) Eur. J. Immunol. 2,523-535. 3. Edelman, G. M. (1976) Science 192,218-226. 1.0 4. Orly, J. & Schramm, M. (1976) Proc. NatI. Acad. Sci. USA 73, 4410-4414. _ 5. Privas, J., Silman, I. & Amsterdam, A. (1976) Cell 7,543-550. * 0.8 6. Amsterdam, A., Hollander, Z., Nimrod, A., Reisel, R. & Kohen, F. (1977) J. Cell Biol. 75, 222a (abstr.). 0.6 7. Amsterdam, A., Kohen, F., Nimrod, A. & Lindner, H. R. (1979) 0 in Ovarian Follicular and Function, eds. Channing, C. P., Marsh, J. M. & Sadler, W. A. (Plenum, New 0.4 _ York), pp. 69-75. 0. 8. Carpenter, G. & Cohen, S. (1976) J. Cell Biol. 71, 159-171. 9. Brown, M. S. & Goldstein, J. L. (1976) Science 191, 150-154. g 0.2 10. Amsterdam, A., Nimrod, A., Lamprecht, S. A., Burstein, Y. & Lindner, H. R. (1979) Am. J. Physiol. 5, E129-E138. 11. Hertz, R. & Hisaw, F. L. (1934) Am. J. Physiol. 108, 1-13. 30 60 75 90 12. Han, S. S., Rajaniemi, H. J., Cho, M. I., Hirshfield, A. N. & Time of incubation, min Midgley, A. R., Jr. (1974) Endocrinology 95,589-598. 13. Lamprecht, S. A., Zor, U., Tsafriri, A. & Lindner, H. R. (1973) FIG. 3. Effect of specific antiserum to hCG on cyclic AMP J. Endocrinol. 57,217-233. (cAMP) accumulation in granulosa cell cultures preincubated with 14. Marsh, J. M., Mills, T. M. & Lemarie, W. J. (1973) Biochim. the hormone; cells were incubated with hCG (20 IU/ml) followed by Biophys. Acta 304, 197-202. incubation with or without 1:20 dilution of the specific antisera and 15. Selstam, G., Rosberg, S., Liljekvist, J., Gronquist, L., Perklev, T. finally with IBMX (10 ,g/ml) for 20 min at 370C. Cultures were & Ahren, K. (1976) Acta Endocrinol. 81, 150-164. thoroughly washed after each incubation. Cyclic AMP production was measured during the last 20 min of incubation in triplicate culture 16. Lamprecht, S. A., Zor, U., Salomon, Y., Koch, Y., Ahren, K. & H. R. (1977) J. Cyclic Nucleotide Res. 3,69-83. dishes where the variation from the mean did not exceed 10%. Data Lindner, 17. Harden, T. K., Su, Y. F. & Perkins, J. P. (1979) J. Cyclic Nucle- are expressed as the ratio between the amount of cyclic AMP pro- duced in the presence of anti-hCG serum to that produced in its ab- otide Res. 5, 99-106. 18. Hoffman, B. B., Kilpatrick, D. M. & Lefkowitz, R. J. (1979) J. sence. Cyclic Nucleotide Res. 5, 355-366. 19. Harwood, J. P., Conti, M., Conn, P. M., Dufau, M. L. & Catt, K. Schlegel et al. (37) reported that glucagon receptors in he- J. (1978) Mol. Cell. Endocrinol. 11, 121-135. patic membranes may be present in small aggregates and that 20. Nimrod, A. & Lindner, H. R. (1976) Mol. Cell. Endocrinol. 5, these aggregates must break down to their monomeric form in 315-320. the process of combining with the catalytic unit of the cyclase. 21. Salomon, Y., Yanovsky, A., Mintz, Y., Amir, Y. & Lindner, H. R. Whether this mechanism also applies to the LH/hCG-sensitive (1977) J. Cyclic Nucleotide Res. 3, 163-176. 22. Nimrod, A., Erickson, G. F. & Ryan, K. (1976) Endocrinology adenylate cyclase remains to be seen. 98,56-64. By using antibodies to insulin receptors and to receptor- 23. Fahey, J. L. & Terry, E. W. (1967) in Handbook ofExperimental bound insulin, Kahn et al. (38) indicated that aggregation of Immunology, ed. Weir, D. M., (Davis, Philadelphia), p. 19. insulin receptors results in enhancement of the biological re- 24. Stanworth, D. R. & Turner, M. W. (1973) in Immunochemistry, sponse and, thus, may be important for hormone action. ed. Weir, D. M. (Blackwell, Oxford, England), pp. 10.0-10.97. However, when we used nonsaturating concentrations of hCG 25. Nisonoff, A., Markus, G. & Wissler, F. C. (1961) Nature (London) followed by incubation of granulosa cells with the specific an- 189,293-295. 26. Moudgal, N. R. & Madhwa Raj, H. G. (1974) in Methods of tibodies to the hormone, we failed to enhance the cellular re- Hormone Radiommunoassay, ed. Behrman, J. (Academic, New sponse to the hormone (unpublished observations). York), pp. 57-85. The existence of small clusters of LH receptors on granulosa 27. Marchalonis, J. J. (1969) Biochem. J. 113, 299-305. cells prior to hormonal incubation revealed by immunofluo- 28. Fraker, P. J. & Speck, J. C. (1978) Biochem. Biophys. Res. rescence may be of biological significance. However, the pos- Commun. 80, 849-857. sibility that they were formed mainly after application of the 29. Gilman, A. G. (1970) Proc. Natl. Acad. Sci. USA 67,305-312. 30. Humes, J. H., Rounbehler, M. & Kuehl, F. A., Jr. (1968) Anal. antibodies to the fixed cells cannot be completely excluded Biochem. 32, 210-217. because formaldehyde fixation might not completely prevent 31. Zor, U., Lamprecht, S. A., Mislovin, Z., Koch, Y. & Lindner, H. translocation of proteins within biological specimens (39). In R. (1976) Biochim. Biophys. Acta 428,761-765. addition, some of these aggregates might be produced after 32. Brandtzaeg, P., Purvis, K. & Hansson, V. (1978) lnt. J. Androl. partial occupancy of the receptors by some hormone (LH) Suppl. 2, 287-302. present in the serum of the culture medium (see Materials and 33. Schlessinger, J., Schechter, Y., Cuatrecasas, P., Willingham, M. 5353- Methods). Nonetheless, our observations clearly indicate that C. & Pastan, I. (1978) Proc. Natl. Acad. Sci. USA 75, 5357. desensitization of granulosa cells to hormone challenge is as- 34 Schramm, M. & Orly, J. (1975) Proc. Natl. Acad. Sci. USA 72, sociated with pronounced aggregation of the LH/hCG recep- 3433-3437. tors. Whether such a phenomenon also occurs in other de- 35. Hirata, F., Strittmatter, W. J. & Axelrod, J. (1979) Proc. Natl. sensitized cells, in which various hormones exert their effect Acad. Sci. USA 76,368-372. via activation of adenylate cyclase, remains to be seen. 36. Hanski, E., Rimon, E. & Levitzki, A. (1979) Biochemistry 18, 846-853. 37. Schlegel, W., Kempner, E. S. & Rodbell, M. (1979) J. Biol. Chem. We thank Drs. G. T. Ross and H. R. Lindner for helpful discussion 254,5168-5176. and Mrs. A. Azrad, Mrs. Y. Osher, and Mr. S. Gordon for skillful 38. Kahn, C. R., Baird, K. L., Jarrett, D. B. & Flier, J. S. (1978) Proc. technical assistance. The work was supported by grants from the United Natl. Acad. Sci. USA 75,4209-4213. States-Israel Binational Foundation (to A.A.) and from the Ford 39. Tokuyasu, K. T. & Singer, S. J. (1976) J. Cell Biol. 71, 894- Foundation and Population Council of New York (to H.R.L.). 906. Downloaded by guest on September 26, 2021