Characterization and comparison of testicular LH/hCG receptors of rhesus monkeys (Macaca mulatta) and green monkeys (Cercopithecus aethiops)

Testicular luteinizing hormone (LH/hCG) receptors were characterized in seven green monkeys and compared with those of four rhesus monkeys. Testicular tissue showed high binding affinity for ¹²⁵I-hCG, (0.9–2.5 × 10⁹ M^?1, and 0.7–1.64 × 10⁹ M^?1 respectively, for green and rhesus monkeys) and low binding capacity (0.343–0.682 fmol/mg and 0.198–0.355 fmol/mg testicular homogenate, respectively). There was no difference in binding affinity between the two groups. Testicular LH/hCG receptors in both species bound human LH (hLH) and hCG but did not cross react with ovine LH (oLH). Rat testicular tissue showed similar high binding affinity (6.4 × 10⁹ M^?1) and low binding capacity (1.04 fmol/mg tissue homogenate) for ¹²⁵I-hCG. Rat LH/hCG receptors bound hLH, hCG, and oLH to a similar degree.     

Porcine follicular fluid containing water-soluble LH/hCG receptor

Porcine follicular fluid has been shown to have a specific water-soluble receptor for human chorionic gonadotropin (hCG). The binding of [125I] hCG to follicular fluid is inhibited by unlabelled hCG, LH but not FSH, ACTH and GH. The binding of hormone to the receptor in follicular fluid is a saturable phenomenon and Scatchard analysis suggested that the receptor has high affinity to hCG with no changes as the follicle enlarges. In contrast, follicular fluid from large follicles (6-12 mm) has higher binding capacity (2.04 +/- 0.12 fmol/mg protein) than follicular fluid isolated from medium (3-5 mm) and small (1-2 mm) follicles (0.60 +/- 0.05 and 0.44 +/- 0.04 fmol/mg protein, respectively). With the aid of affinity chromatography on hCG-CNBr-Sepharose 6-B a homogeneous fraction with Mr about 65,000 as estimated by SDS-PAGE was isolated. Treatment of follicular fluid with several protein-modifying reagents changed interactions of [125I] hCG with both soluble receptor and that bound to granulosa cell membrane in the similar manner. The [125I] hCG binding capacity of follicular fluid represents about 9.5% of the total binding capacity of granulosa cells. Finally, soluble LH/hCG receptor is probably secreted actively by follicular cells into follicular fluid. Dead granulosa cells do not release receptor into follicular fluid or incubation medium.     

Follicle stimulating hormone stimulation of 125-I-human chorionic gonadotropin binding in porcine granulosa cell cultures.

In order to see if FSH acts directly upon the granulosa cell to stimulate hCG binding, granulosa cells harvested from small 1-2 mm porcine follicles were grown in 250 ml flasks in chemically defined media containing 0.05 mug/ml highly purified human FSH for 2, 4, and 6 days. The defined medium consisted of culture medium 199 plus 0.4% bovine serum albumin, 0.2% lactalbumin hydrolysate and 10 munit/ml insulin. The cultures were harvested by scraping with a rubber policeman and incubated with 0.1 mug/ml 131-I- or 125-I-hCG. Binding expressed as cpm/culture or per mg protein yielded similar results. In five separate experiments addition of FSH stimulated hCG binding two- to fourfold above control cultures. In a typical experiment after 2 days of culture, the specific binding of control cultures to hCG was 962 plus or minus 45 cpm/culture (-x plus or minus SE; n = 3) and the binding in cultures grown in the presence of 0.05 mug/ml FSH was 3933 plus or minus 1787 (n = 3; P less than 0.01). Granulosa cells harvested from large (8-12 mm) follicles grown under similar conditions bound 29,669 plus or minus 948 cpm/culture (n = 4). These data demonstrate that FSH may have a direct stimulatory role upon induction of granulosa cell LH-hCG receptors in vitro.     

Human chorionic gonadotropin. V. Tissue specificity of binding and partial characterization of soluble human chorionic gonadotropin-receptor complexes.

An in vivo human chorionic gonadotropin (hCG)-receptor complex was solubilized from the subcellular fraction of ovarian and testicular tissues of rats that had been injected with 125-I-labeled hCG. The soluble hCG-receptor complex was partially characterized by Sepharose 6B chromatography in the presence of the nonionic detergent, Emulphogene, and was shown to have a molecular size of about 65 A. By this method it was also shown that the in vivo uptake of radioactivity by rat gonadal tissues represents 125-I-hCG and not the dissociated subunits or degradation products of the hormone. A soluble hCG-receptor complex isolated in vitro in approximately the same yield from both rat testicular and ovarian homogenates was shown to be the same size. The hCG-receptor appears to be specifically located in gonadal tissue; a corresponding hCG-receptor complex was not obtained from liver or kidney that incorporated significant levels of 125-I-hCG administered in vivo. Furthermore, a desialyzed hCG-receptor complex was obtained from rat testis but not liver; desialyzed hCG, like other desialyzed glycoproteins, is nonspecifically bound by rat liver homogenates. The binding of hCG and luteinizing hormone (LH) by rat testis receptor exhibits a high degree of specificity. Other glycoprotein hormones without LH activity, such as follicle-stimulating hormone and thyroid-stimulating hormone, and glycoproteins such as fetuin or alpha1-acid glycoprotein do not bind to the hCG/LH receptors. Desialyzed hCG was 2 times more effective in competing for binding to rat testis receptors than "native" hCG, indicating that caution must be exercised when the radioligand receptor assay is utilized to assay hCG preparations varying in sialic acid content.     

Purification and partial characterization of rat ovarian lutropin receptor

Lutropin (LH) receptor was solubilized from pseudopregnant rat ovaries and purified by two cycles of affinity chromatography on human choriogonadotropin (hCG)-Affi-Gel 10. The purified receptor preparation contained a single class of high-affinity 125I-hCG binding sites with an equilibrium dissociation constant (Kd) of 5.1 X 10(-10) M (at 20 degrees C) and had a specific hormone binding capacity of 7920 pmol/mg of protein. The purified receptor migrated as a single 90-kDa band in sodium dodecyl sulfate-polyacrylamide gel electrophoresis under both nonreducing and reducing conditions. Affinity cross-linking of the purified receptor to 125I-hCG produced a 130-kDa complex. Hormone-binding ability of the purified 90-kDa polypeptide was demonstrated also by ligand blotting. The purified receptor was electroblotted onto nitrocellulose after sodium dodecyl sulfate-polyacrylamide gel electrophoresis under nonreducing conditions followed by incubation with 125I-hCG. Autoradiography revealed labeling of a 90-kDa band. This labeling was displaced by unlabeled hCG and human LH but not by human follitropin or rat prolactin. In addition, LH receptors of bovine corpora lutea and mouse Leydig tumor cells were shown by ligand blotting to contain a 90-kDa hormone binding unit, suggesting that LH receptor structure is well conserved among mammalian species. The purified rat ovarian LH receptor bound to immobilized wheat germ agglutinin, implying that the receptor is a glycoprotein. These results demonstrate that the hormone-binding unit of rat ovarian LH receptor is a 90-kDa membrane glycopolypeptide.     

The porcine LH/hCG receptor. Characterization and purification

Porcine luteal LH/hCG receptor (LH/hCG R) was solubilized with 70-80% recovery from the crude plasma membrane fraction by Triton X-100 in the presence of 25% glycerol and protease inhibitors. The solubilized receptor maintained 90% of original activity at -60 degrees C for 90 days. Equilibrium association constant (Ka) values of 1.92, 2.22, and 2.03 X 10(10) M-1 were observed for the whole homogenate, plasma membrane fraction, and solubilized LH/hCG R preparations, respectively. The specific binding capacity for the same fractions were 49, 70, 55 fmol/mg protein, respectively. Complexes of LH/hCG R and Triton X-100 were resolved into two components with approximate Mr = 2.7 X 10(5) and 5.4 X 10(5) by gel filtration on Sepharose 6B and two glycoprotein components by chromatography on concanavalin A-Sepharose. Solubilized porcine LH/hCG R was purified by two cycles of affinity chromatography on highly purified hCG-Sepharose with an overall recovery of 30-35% of the initial activity in the Triton extract. Purified porcine LH/hCG R had a specific binding capacity of 2300 pmol/mg protein and a Ka = 1.5 X 10(10) M-1. Silver staining of sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels demonstrated that the major protein in porcine LH/hCG R preparations has Mr = 68,000. A weakly staining band at Mr = 45,000 was also observed in the purified receptor preparation. Analysis of iodinated purified LH/hCG R by autoradiography has confirmed these results. Porcine LH/hCG R was purified 40,000-fold by this method.     

Changes in testicular hCG binding and Leydig cell function in rats throughout life

The age dependence of Leydig cell function was investigated in rats from prepuberty (15 days) to senescence (39 months). Serum LH, serum and testicular testosterone were measured by radioimmunoassay. The binding capacity and affinity of LH/hCG receptors were determined by a radioligand receptor assay (hCG/Leydig cells) using 125I-hCG labelled by the lactoperoxidase method. Separation of bound and free 125I and simultaneous concentrations of 125I-hCG was achieved by vacuum ultrafiltration. The biochemical integrity of 125I-hCG tracer was ascertained by various chromatographic procedures. The highest hCG-finding and highest serum LH levels were found during puberty. Serum and testicular testosterone concentrations, however, were maximal in early adulthood. From this period onwards to late senescence hCG-binding changed only slightly, while serum LH and testosterone levels decreased significantly towards late senescence. The study shows that, although hCG binding to the Leydig cell changes characteristically during development, it is minimally affected by aging and cannot therefore be responsible for the reduced androgen biosynthesis in senescence.     

Follitropin receptors in rat testis. Characterization with enzymatically 125I-labeled human follitropin.

The interaction between enzymatically radioiodinated human follitropin and the follitropin receptors in testis homogenate was investigated in immature and adult rats. The 125I-labeled human follitropin exhibited high binding activity with specific binding of up to 17% in the presence of an excess of testis homogenate. Approx. 50% of the bound hormone could be eluted at pH 5, and the receptor purified tracer exhibited a 3.6-fold increase in binding activity when compared with the original tracer preparation. Quantitative analysis of equilibrium binding data was performed with corrections for the measured specific activity and maximum binding activity of the tracer hormone. The equilibrium association constants (Ka) determined 24 degrees C were not significantly different in immature and adult rat testis, and the mean value for Ka was 3.9 . 10(9) M-1. At 37 degrees C, the Ka value obtained using immature rat testis was 1.3 . 10(10) M-1. The association of 125I-labeled human follitropin with immature rat testis homogenate was time and temperature dependent. In the presence of an excess of unlabeled hormone, 30--60% of the preformed hormone . receptor complex was dissociated after 24 h incubation. A specific and sensitive radioligand-receptor assay for follitropin was developed using immature rat testis homogenate. The minimum detectable dose of purified human follitropin was 0.6 ng, and human urinary and pituitary follitropin, ovine follitropin and pregnant mare serum gonadotropin reacted in the assay with equivalent slopes. The potencies of highly purified pregnent mare serum gonadotropin and highly purified human follitropin were similar in the radioligand-receptor assay, consistent with the follitropin bioactivity of the equine gonadotropin.     

Purification, characterization, and amino-terminal sequence of rat ovarian receptor for luteinizing hormone/human choriogonadotropin

The luteinizing hormone (LH)/human choriogonadotropin (hCG) receptor of rat ovary was solubilized with Lubrol PX in the presence of 20% glycerol and protease inhibitors, and purified by one-step affinity chromatography. Purified receptor had a specific hCG binding capacity of 4900 pmol/mg protein, and displayed a single class of high affinity binding sites (Ka = 6.20 X 10(9) M-1). An 11,200-fold purification over the starting crude homogenate was achieved. The purified LH/hCG receptor was identified by sodium dodecyl sulfate-gel electrophoresis and silver staining as a single protein of 92 kDa. The ability of the purified 92-kDa protein to specifically bind hormone was demonstrated by electroblotting onto Immobilon P membrane, incubation with 125I-labeled hCG, and autoradiography of the blot. In addition to a 92-kDa band, ligand blotting also yielded a 170-kDa band representing receptor dimer. Covalent cross-linking of hCG, with isotope in either the alpha- or beta-subunit, to membrane-bound receptor produced complexes that contained a single receptor component of approximately 92 kDa. The cross-linking studies indicated that both subunits interact with receptor and also suggested receptor dimer formation. Following sodium dodecyl sulfate-electrophoresis, purified receptor was electroblotted onto polyethylenimine-treated glass fiber filters for direct microsequencing in a gas-phase sequenator. Eleven cycles of sequence analysis yielded the unique sequence: NH2-Arg-Glu-Leu-Ser-Gly-Ser-Leu-XXX-Pro-Glu-Pro-COOH. These results indicate that the rat ovarian LH/hCG receptor is a protein of 92 kDa which can be easily purified in microgram amounts. This study also describes a relatively simple technique for electroblotting and microsequencing that should be applicable to other membrane-bound hormone receptors.     

Gonadotropin receptor complexes and free receptors in porcine Leydig cell cultures during recovery from hCG stimulation

Free and occupied gonadotropin receptors were studied in vitro in porcine Leydig cells culture maintained in chemically defined medium. Free receptors were evaluated by the binding capacity for 125I-hCG. hCG bound molecules (or hCG receptor complexes) were evaluated using immunocytochemical visualization on fixed cells. Exposure to hCG for 16 hours (.5 to 50 ng/ml) induced the disappearance of free receptors. After removal of the hormone, the return to control levels was observed at 48 and 72 hours. Visualization of hCG bound at the cell surface indicates that, following continuous exposure to gonadotropins for 48 hours, hCG molecules are still present on the cell. Following short-time exposure (1 h) to hCG and 48 hrs washing the number of stained cells is very close to the initial value suggesting that the occupied sites (at 48 hours) represent the initial hormone receptor complexes. These results indicate that, during prolonged incubation, hCG binding is not reversible, that the half-life of some of the complexes at the cell surface is very long and that the receptors recovery is slow and is probably the result of a de novo synthesis.     

An investigation of sites that bind human somatotropin (growth hormone) in the liver of the pregnant rabbit.

The binding of 125I-labelled human somatotropin (growth hormone) to a crude membrane preparation from the liver of pregnant rabbit, and to receptors solubilized from this fraction by Triton X-100, was dependent on time, temperature and receptor concentration. At 4 degrees C a steady state was reached after 20 h, and maximum specific binding (as a percentage of total tracer added) was approx. 50% for both membrane-bound and solubilized receptors. Solubilization did not significantly affect the binding properties of the receptor at low concentrations of Triton X-100 (less than 0.05%, v/v, in the assay tube). However, at higher concentrations (approx. 0.1%, v/v), the detergent lowered the ability of some hormones, for example ovine prolactin, to displace 125I-labelled human somatotropin, but did not affect other hormones such as bovine somatotropin. Some somatogenic hormones, such as bovine somatotropin, and some lactogenic hormones, such as ovine prolactin, displaced 125I-labelled human somatotropin from membrane-bound and solubilized receptor preparations. Furthermore, 85% of 125I-labelled bovine somatotropin was displaced from membrane-bound receptors by ovine prolactin, and 125I-labelled ovine prolactin was almost completely displaced by bovine somatotropin. Scatchard analysis of the binding data for human somatotropin suggested a single class of binding sites in the membrane-bound receptor preparation, with an affinity (Ka) of 1.9 X 10(9) M-1 and a capacity of 1726 fmol/mg of protein; these values were slightly increased by solubilization (Ka = 3.2 X 10(9) M-1, capacity = 2103 fmol/mg of protein). Scatchard analysis of binding to membrane-bound receptors also indicated a single class of high-affinity binding sites for bovine somatotropin (Ka = 4.8 X 10(9) M-1, capacity = 769 fmol/mg) and for ovine prolactin (Ka = 6.1 X 10(9) M-1, capacity = 187 fmol/mg).     

Follitropin receptors in rat testis characterization with enzymatically 125I-labeled human follitropin

The interaction between enzymatically radioiodinated human follitropin and the follitropin receptors in testis homogenate was investigated in immature and adult rats. The ¹²⁵I-labeled human follitropin exhibited high binding activity, with specific binding of up to 17% in the presence of an excess of testis homogenate.Approx. 50% of the bound hormone could be eluted at pH 5, and the receptor purified tracer exhibited a 3.6-fold increase in binding activity when compared with the original tracer preparation. Quantitative analysis of equilibrium binding data was performed with corrections for the measured specific activity and maximum binding activity of the tracer hormone. The equilibrium association constants (K_a) determined at 24°C were not significantly different in immature and adult rat testis, and the mean value for K_a was 3.9 · 10⁹ M^?1. At 37°C, the K_a value obtained using immature rat testis was 1.3 · 10¹⁰ M^?1. The association of ¹²⁵I-labeled human follitropin with immature rat testis homogenate was time and temperature dependent. In the presence of an excess of unlabeled hormone, 30–60% of the preformed hormone · receptor complex was dissociated after 24 h incubation. A specific and sensitive radioligand-receptor assay for follitropin was developed using immature rat testis homogenate. The minimum detectable dose of purified human follitropin was 0.6 ng, and human urinary and pituitary follitropin, ovine follitropin and pregnant mare serum gonadotropin reacted in the assay with equivalent slopes. The potencies of highly purified pregnant mare serum gonadotropin and highly purified human follitropin were similar in the radioligand-receptor assay, consistent with the follitropin bioactivity of the equine gonadotropin.     

Role of gangliosides in gonadotropin and cholera enterotoxin stimulated steroidogenesis in isolated rat ovarian cells

Ovarian cells isolated from 26 day old rats responded to hCG (10 ng/ml) and cholera enterotoxin (100 ng/ml) $\text{in vitro}$ with a forty-five to fifty-fold increase in progesterone production. Both cholera enterotoxin and hCG-stimulated progesterone response was accompanied by a lag period. The duration of the lag period in the production of the progesterone depended on the concentration of gonadotropin or cholera enterotoxin, and with maximally stimulating dose it was 20–30 minutes. Addition of highly purified mixed gangliosides to the incubation medium abolished the stimulatory effect of cholera enterotoxin on progesterone response. In contrast, under identical experimental conditions, ganglioside addition produced no effect on progesterone response elicited by hCG or LH. Similarly mixed gangliosides did not prevent the specific binding of [¹²⁵I]hCG to the ovarian cells or to the membranes isolated from the ovary. In addition preincubation of [¹²⁵I]hCG with ganglioside did not alter the subsequent binding of the hormone to the ovarian cell surface receptor. These findings suggest that gangliosides are not involved in the hormone receptor interactions and subsequent receptor mediated physiological response.     

Solubilization of membrane receptor for epidermal growth factor.

The membrane receptor for epidermal growth factor (EGF) has been solubilized from A-431 tumor cells using Triton X-100. Operational criteria used to define solubilization include failure of the binding activity to be pelleted after centrifugation at 90,000 x g for 1.5 hrs and the requirement for polyethylene glycol precipitation to detect ¹²⁵I-EGF: receptor complexes on membrane filters. Properties of the solubilized EGF are characterized and compared to the properties of the particulate receptor. The specific binding capacity of the solubilized EGF receptor was 8.0 picomoles ¹²⁵I-EGF bound per mg protein--approximately 60% of the binding capacity of particulate receptor preparations. Also, solubilization of the EGF receptor resulted in a 10-fold decrease in the affinity of the receptor for ¹²⁵I-EGF.     

Induction of high-density-lipoprotein receptors in rat corpus luteum by human choriogonadotropin. Evidence of protein synthesis de novo.

The present studies investigated the specific binding of 125I-labelled high-density lipoprotein (125I-HDL) to plasma membranes. Golgi, rough endoplasmic reticulum and mitochondria/lysosomes, prepared from ovaries of rats injected with human choriogonadotropin (hCG) or 0.9% NaCl. Treatment in vivo with hCG resulted in 2-3-fold induction of 125I-HDL binding activity in all the subcellular organelles. The specific binding of HDL to various subcellular organelles was dependent on the amount of protein, lipoprotein concentration and incubation time. Equilibrium-binding studies revealed comparable Kd values (13-22 micrograms of HDL protein/ml) for HDL binding in all the subcellular organelles tested. Treatment with cycloheximide (2.0 mg/kg body wt.) before hCG administration abolished the induction of HDL receptors, suggesting the involvement of a protein-synthesis-dependent process in receptor induction. Analysis of equilibrium dissociation constants (Kd) for 125I-HDL binding in membranes from hCG-, cycloheximide-and saline-treated animals suggests that the increase in binding was due to an increase in the number of binding sites rather than a change in the affinity. Additionally, pretreatment with tunicamycin, an inhibitor of N-linked glycosylation, had no effect on hCG-mediated receptor induction, suggesting that glycosylation of the receptor may not be necessary for the interaction of HDL with its receptors.     

Functional reconstitution of rat ovarian LH/hCG receptor into proteoliposomes

Rat ovarian membrane LH/hCG receptor was solubilized in various detergents and reconstituted into proteoliposomes. Upon removal of sodium cholate by active absorption on Bio-Beads SM-2, the functional interaction between receptor and adenylate cyclase was restored. Adenylate cyclase was stimulated by hCG, HCG+GTP or GppNHp and NaF. Reconstituted proteoliposomes bound more ¹²⁵I-hCG (528 fmol/mg protein) than membrane-bound receptors (384 fmol/mg protein). There was no difference, however, in the relative affinity of reconstituted receptor preparations for hCG.     

Purification and characterization of Leydig cell luteinizing hormone receptor

We have purified the testicular luteinizing hormone (LH/human choriogonadotropin (hCG)) receptor by sequential affinity chromatography on hCG-Sepharose. The purified LH/hCG receptor was identified as a single protein of Mr = 90,000 +/- 2,000 on sodium dodecyl sulfate-gel electrophoresis (SDS-PAGE), showed high affinity binding for hCG, and a binding capacity of 3.8 nmol/mg of protein. Electrophoretically blotted receptor retained the ability to bind 125I-hCG on nitrocellulose membrane, and the Mr of radioactive band was consistent with that revealed by silver staining. Autoradiography after SDS-PAGE analysis of cross-linked purified receptor-hCG complex showed Mr = 145,000 and Mr = 105,000 bands. These results are consistent with a Mr value for the receptor of 90,000 after accounting for contribution by the intact hormone or its alpha-subunit. Analysis of the free receptor by fast protein liquid chromatography on Superose 12 revealed a single peak of binding activity for 125I-hCG which eluted in the position of Mr = 200,000-240,000 in the presence of Triton X-100. Since a single protein species is observed under reducing or nonreducing conditions in SDS-PAGE, the receptor could exist in the membrane as a dimeric form composed of subunits Mr = 90,000 associated through noncovalent interactions. The pure receptor can be phosphorylated in vitro by the catalytic subunit of cAMP-dependent protein kinase (approximately 0.3 mol of phosphate/mol of receptor). This phosphorylation does not affect the binding characteristics of the receptor. The method described is simple and allows rapid purification of microgram amounts of biological active Leydig cell LH/hCG receptor for structural, functional, and immunological studies.     

The pituitary gonadotropin-releasing hormone (GnRH) receptor of the female rabbit: characterization and developmental aspects.

The aim of the present study was to characterize the pituitary gonadotropin-releasing hormone (GnRH) binding site in the rabbit and investigate its possible role in sexual maturation of the female rabbit. A radioligand binding assay was established, and the presence of specific 125I-labelled D-Ala6-des-Gly10-GnRH ethylamide (125I-DAl6EA) binding sites in the anterior pituitary gland of the rabbit was demonstrated. 125I-DAla6EA binding was saturable, specific, displaceable, reversible, correlated with increasing tissue concentrations, and susceptible to physiological manipulation. 125I-DAla6EA binding indicated the presence of two binding sites in the female adult rabbit pituitary: a high affinity, low capacity site (KD = 0.3-0.4 nM; Bmax = 100-200 fmol/mg protein) and a lower affinity, high capacity site (KD = 30 nM; Bmax = 5-8000 fmol/mg protein). Ontogeny of 125I-DAl6EA binding in the female rabbit (40-120 days of age) did not show a correlation between binding site number and serum luteinizing hormone (LH). In addition, the net serum LH response in female rabbits to a subcutaneous injection of DAla6EA (10 ng, 100 ng, and 1 microgram per kilogram body weight) was not significantly different between animals 40, 75, and 120 days of age. This suggests that a decrease in pituitary responsiveness to GnRH is not associated with sexual maturation in the female rabbit. Results indicate that factors other than and (or) in addition to GnRH binding site number, such as postreceptor events, play a role in gonadotropin secretion in the female rabbit.     

Purification and characterization of lutropin receptor from membranes of pig follicular fluid

Membranes derived from free floating granulosa cells in porcine ovarian follicular fluid were used as a starting material for structural characterization of both LH/hCG and FSH receptors. The receptors were highly hormone-specific and showed single classes of high-affinity binding sites (Kd = 19-74 pM). Their molecular weights as determined by affinity cross-linking with their respective 125I-ligands were similarly 70,000. The membrane-localized receptors could be solubilized with reduced Triton X-100 in the presence of 20% glycerol with good retention of hormone binding activity. The Triton extracts of membranes also showed hormone specificity and equilibrium binding constants similar to the membrane receptors (Kd = 32-48 pM). Affinity chromatography on divinylsulfonyl-Sepharose-oLH columns was utilized to purify the solubilized LH/hCG receptor to a specific activity of 2000 pmol/mg of protein. The purified receptor exhibited a high specificity for hCG and hLH but not for hFSH nor bTSH. The purified receptor was iodinated and visualized to be composed of a major protein of Mr approximately 70,000 and other minor proteins of molecular weights ranging from 14,000 to 40,000. Except for the Mr 14,000 protein, all other protein species bound to the concanavalin A-Sepharose column. The data suggest that the ovarian LH/hCG and FSH receptors are structurally similar and consist of a single polypeptide chain, as recently documented for the LH/hCG receptor (Loosefelt et al., 1989; McFarland et al., 1989).     

Interleukin 1: an inhibitor of luteinizing hormone receptor formation in cultured rat granulosa cells

We have shown that interleukin 1 (IL 1) suppresses follicle-stimulating hormone (FSH)-induced progesterone secretion and 125I-labeled human chorionic gonadotropin (hCG) binding (a measurement of LH receptors) in cultured rat granulosa cells. The present study was designed to examine if the reduction of FSH-stimulated 125I-labeled hCG binding by IL 1 was caused by a decline in the binding capacity or by an alteration in the affinity of the LH receptor and, further, to determine the minimum period of exposure of the granulosa cells to IL 1 necessary to suppress 125I-labeled hCG binding. IL 1 produced a dose-dependent inhibition of 125I-labeled hCG binding in FSH-stimulated granulosa cells. Scatchard analysis revealed that this effect resulted from a reduction of the binding capacity of the LH receptor with no change in affinity. Also, a minimum of 12-24 h of exposure to IL 1 is necessary to significantly inhibit FSH-induced LH receptor formation. These results suggest that IL 1 decreases the number of LH receptors and that protein synthesis may be necessary for IL 1's action. However, a physiological/pathological role for IL 1 in ovarian regulation has yet to be established.