Membrane methylation in isolated rat testis interstitial cells unmasks functional luteinizing hormone receptors.

Treatment of intact isolated rat testis interstitial cells with S-adenosylmethionine as methyl donor, increases substantially the number of LH human CG receptors (100-200%) without modifying the equilibrium dissociation constant. The increase in binding capacity was associated with an augmentation in the sensitivity of the rat testis interstitial cells to produce testosterone in response to LH, suggesting a functional role of the unmasked receptors. The amount of S-adenosylmethionine necessary to obtain an increase in LH binding capacity and preserve cell viability was 25-50 micrograms/ml per 1.6 X 10(7) cells. 10 mM MgCl2 in addition to the Mg2+ present in the medium was necessary to maintain cell viability. 3H-labelled methyl groups were incorporated mainly into the lipid fraction (208 fmol/10(6) cells) when 3H-S-adenosylmethionine was incubated with the cells for 2 h at 30 degrees C. Our results are consistent with the conclusion that early action of LH may involve an activation of methyltransferase activity, phospholipid methylation, an increase in LH binding capacity and an increase in receptor function.

Penetrance and clinical manifestations of non-hotspot germline RET mutation, C630R, in a family with medullary thyroid carcinoma.

Germline mutations in specific hot spot-codons of the RET proto-oncogene are associated with multiple endocrine neoplasia type 2 (MEN 2). Clinical RET gene testing has been routine for the last 10 years in some countries. In Argentina, RET testing excluding MEN 2B was always reported with a mutation at codon 634, with one exception: we described a novel mutation T > C transition at codon 630 (C630R), the family to which we extend the study in the present report. This family comprised 29 members in four generations including 6 individuals affected with medullary thyroid cancer (MTC), positive for the C630R mutation and normal adrenaline/ noradrenaline and ionized calcium/parathyroid hormone levels. Two asymptomatic mutation carriers aged 5 and 11 years underwent total thyroidectomy. The histopathologic examination showed C-cell hyperplasia and microcarcinoma foci, while preoperative basal calcitonins were normal for both. Our report emphasizes the importance of testing for non-hot spot RET mutations in apparently mutation negative MEN 2 families. Furthermore, it would appear that C630R mirrors C634R in penetrance (100% in this family) and in early age of onset of MTC, although paradoxically, no pheochromocytomas and hyperparathyroidism have developed. In addition to recommending RET testing before 5 years of age; we also can postulate that codon 630 may be the key point along the extracellular domain, important in the tissue-specific penetrance.

Luteinizing hormone triggers two opposite regulatory pathways through an initial common event, receptor aggregation.

LH receptor internalization was studied with an antireceptor monoclonal antibody (aLHR) which induces Leydig cells to produce testosterone. To follow receptor-mediated aLHR internalization, cells were incubated with aLHR at 10 C for 3 h to generate an aLHR complex; this was followed by a second incubation with fluorescent labeled antimouse immunoglobulin at 34 C, a temperature which allows internalization. Within 15 min at 34 C, cytoplasmic fluorescent staining was detectable; this staining was strongly visible after 60 min. At no time was nuclear staining observable. Employing such an approach, it has also been possible to follow the fate of unoccupied receptors when cells are stimulated with a submaximal dose of LH. The results show that LH interactions with 20% of its receptors produces microaggregation, patching, capping, and internalization of free receptor sites. The results further demonstrate that cells with receptors in the state of capping are less sensitive to a second LH stimulation, suggesting that in this state receptors are no longer coupled to the adenylate cyclase system.

Bioassay and radioimmunoassay of serum luteinizing hormone in the male rat.

Measurements of immunoactive and bioactive serum LH in the rat were performed after optimization of both RIA and rat interstitial cell-testosterone (RICT) assay to measure the low circulating LH levels of intact male rats. The effective sensitivity of the RIA was increased 3- to 5-fold to 6.2 or 4.7 ng RP1/ml by extraction of 1--1.5 ml serum, respectively. Serum LH levels of adult male rats were 48.5 +/- 16.3 ng RP-1/ml (n = 50), became undetectable after hypophysectomy, and were suppressed 50% after estrogen treatment. For RICT assay of serum rat LH, sensitivity was increased 5-fold by reduction of the incubation volume to 0.35 ml containing 2--4 x 10(6) interstitial cells/tube, with a detection limit of 3 ng RP-1 or 0.1 ng pure LH. The within-assay coefficient of variation for measurement of a pool of control male rat serum (126 +/- 19l6 ng RP-1) WAS +/- 12.5% and the between-assay variation was +/- 15%. Bioactive serum LH levels in adult male rats were 124.8 +/- 32.3 ng RP-1/ml or 2.74 +/- 0.71 ng rat LH/ml, with biopotency to immunopotency (B:I) ratios of 2.8 +/- 0.2 and 1.1 +/- 0.1, respectively, that were unchanged during elevations of serum LH by LHRH administration. Pituitary LH content was 726 +/- 183 micrograms RP-1 or 16.4 micrograms rat LH/gland, with B:I ratios of 2.3 +/- 0.4 and 1.0 +/- 0.2, respectively. The difference between B:I ratios in assays employing pure rat LH and assays using the RP-1 preparation was attributable to the presence of alpha-subunit and biologically inactive material in the RP-1 standard. Precise measurements of immunoactive and bioactive rat LH in male rat plasma can not be performed by these modified RICT and RIA procedures. The sensitive RICT assay can also be applied to the measurement of low levels of LH in the serum of primates and other species.

Luteinizing hormone triggers a molecular association between its receptor and the major histocompatibility complex class I antigen to produce cell activation.

We studied the involvement of major histocompatibility (MHC) class I antigens on the mechanism of LH/hCG receptor activation. For this purpose we investigated the effects of anti-MHC class I antibodies on hormone-receptor interaction, signal transduction, and MHC class I antigen-receptor interaction. Monoclonal antibodies against MHC class I antigen were able to stimulate testosterone production in mouse Leydig cells with the same potency as LH. This biological effect depends on the concentration of antibody used and could be abolished by a LH antagonist. There is a perfect parallelism, for each monoclonal antibody, between the specificity for a particular haplotype and the response of the target cells from the strains carrying such a haplotype. The same antibodies were able to precipitate the soluble LH/hCG receptors, as both a hormone-receptor complex and a free receptor. The results suggest that bound hormone triggers an association of the MHC class I antigen with the LH/hCG receptor, resulting in activation of the target cell.

Modulation of serum and pituitary luteinizing hormone bioactivity by androgen in the rat.

The biological and immunological activities of serum and pituitary LH were analyzed in normal and castrated rats using the rat interstitial cell testosterone assay and RIAs for intact rat LH (rLH), rLH alpha, and rLH beta. After castration, the bioactivity to immunoactivity ratio of both serum and pituitary LH decreased by about 50% between the 5th and 25th days and returned to normal by 60 days. This decrease in the bioactivity to immunoreactivity ratio of LH in castrated males was not due to increased subunit production and was prevented by the administration of testosterone propionate (50 micrograms/day) from the time of castration. These observations indicate that androgens of their metabolites modify the bioactivity of the circulating and stored forms of the LH molecule.

Regulation of pituitary receptors for gonadotropin-releasing hormone during the rat estrous cycle.

A radioiodinated superagonist analog of gonadotropin-releasing hormone (GnRH:[D-Ser(TBu)6]des-GLy10-GnRH N-ethylamide) was used to quantitate the GnRH receptor content of single pituitary glands. This ligand binds with high affinity (Ka = 4.9 X 10(9) M-1) to a single class of sites in pituitary homogenates, without significant tracer degradation, during equilibration for 80 min at 4 C. The GnRH receptor content of adult male rat pituitaries [112 +/- 6.4 (SE) fmol/gland; n = 9] was similar to that of adult female rat pituitaries obtained at estrus or metestrus (104.8 +/- 6.4 fmol/gland, n = 13). Between 1800 h on metestrus and 1800 h on diestrus, the pituitary content of GnRH receptors increased to 200 fmol/gland or greater in each of three separate experiments. The pituitary content of GnRH receptors remained elevated until the time of the serum LH surge, then consistently fell sharply to metesterus levels. These data imply a physiological relevance for the GgRH-binding sites measured by radioassay and demonstrate their relationship to the events of the estrous cycle. The increased GnRH receptor content from the evening of diestrus until late in the afternoon of proestrus may contribute to the enhanced sensitivity of the pituitary to GnRH during proestrus. The close temporal relationship between rising blood estrogen levels and increasing pituitary GnRH receptors suggests that steroid-mediated receptor induction occurs before proestrus. Such an effect of estrogen could be exerted directly upon the pituitary gland or indirectly via the hypothalamus to increase the release of endogenous GnRH, which subsequently induces its own receptors in pituitary gonadotrophs.

Lipoxygenase products as common intermediates in cyclic AMP-dependent and -independent adrenal steroidogenesis in rats.

Aldosterone secretion from adrenal glomerulosa cells can be stimulated by angiotensin II (AII), extracellular potassium and ACTH. Mitochondria from these cells respond to intracellular factors generated by AII (cyclic AMP (cAMP)-independent steroidogenesis) and ACTH (cAMP-dependent steroidogenesis), suggesting that the two-signal-transduction mechanisms are linked by a common intermediate. We have evaluated this hypothesis by stimulating mitochondria from the unstimulated zona glomerulosa with a subcellular post-mitochondrial fraction (PMF) obtained from the zona glomerulosa after stimulation with AII or from the fasciculata gland after stimulation with ACTH; the subcellular fractions were also tested on mitochondria from fasciculata cells. PMFs obtained after incubation of adrenal zona glomerulosa with or without AII (0.1 microM) or ACTH (0.1 nM) were able to increase net progesterone synthesis 4.5-fold in mitochondria isolated from unstimulated rat zona glomerulosa. AII-pretreated PMFs from the zona glomerulosa also stimulated steroidogenesis by mitochondria from zona fasciculata cells. Separate experiments showed that inhibitors of arachidonic acid release and metabolism (bromophenacyl bromide, nordihydroguaiaretic acid, caffeic acid or esculetin) blocked corticosterone production in fasciculata cells stimulated with ACTH, suggesting that arachidonic acid could be the common intermediate in the actions of AII and ACTH on steroid synthesis. Evidence to support this concept was obtained from experiments in which the formation of an activated PMF by treatment of zona fasciculata with ACTH was blocked by the presence of the same inhibitors. Moreover, the inhibitory effects of these substances on PMF activation by ACTH were overcome by exogenous arachidonic acid and, in addition, arachidonic acid release was stimulated by ACTH.(ABSTRACT TRUNCATED AT 250 WORDS)

Stimulation of an individual cell with peptide hormone in a prescribed region of its plasma membrane results in a compartmentalized cyclic AMP-dependent protein kinase response.

This work describes the stimulation by a peptide hormone of an individual cell in a prescribed region of its plasma membrane. When Leydig cells were stimulated via a section of membrane tightly sealed to an electrode containing LH, a very localized area exhibited the morphological change known as 'rounding up', which is a cyclic AMP-dependent protein kinase-mediated response. This localized stimulation did not produce a wider response through intracellular, intermembranous or extracellular signals. Each individual cell responded to peptide stimulation gradually, with an increase over time and with dose. In contrast, when the stimulation was accomplished using a non-hydrolysable cyclic AMP analogue in the patch electrode, a general response throughout an individual cell was produced. Locally stimulated peptide hormone receptors, adenylate cyclases and cyclic AMP-dependent protein kinases appear to be closely associated so that second messenger production and the effects it mediates are compartmentalized.

The cytosol as site of phosphorylation of the cyclic AMP-dependent protein kinase in adrenal steroidogenesis.

The mitochondria, the microsomes and the cytosol have been described as possible sites of cAMP-dependent phosphorylation. However, there has been no direct demonstration of a cAMP-dependent kinase associated with the activation of the side-chain cleavage of cholesterol. We have investigated the site of action of the cAMP-dependent kinase using a sensitive cell-free assay. Cytosol derived from cells stimulated with ACTH or cAMP was capable of increasing progesterone synthesis in isolated mitochondria when combined with the microsomal fraction. Cytosol derived from cyclase or kinase of negative mutant cells did not. Cyclic AMP and cAMP-dependent protein kinase stimulated in vitro a cytosol derived from unstimulated adrenal cells. This cytosol was capable of stimulating progesterone synthesis in isolated mitochondria. Inhibitor of cAMP-dependent protein kinase abolished the effect of the cAMP. ACTH stimulation of cytosol factors is a rapid process observable with a half maximal stimulation at about 3 pM ACTH. The effect was also abolished by inhibitor of arachidonic acid release. The function of cytosolic phosphorylation is still unclear. The effect of inhibitors of arachidonic acid release, and the necessity for the microsomal compartment in order to stimulate mitochondrial steroidogenesis, suggest that the factor in the cytosol may play a role in arachidonic acid release.

The action of luteinizing hormone on the testis.

Luteinizing hormone (LH) and human chorionic gonadotrophin (hCG) receptors are coupled to intracellular effector systems, most notably adenylate cyclase, through guanyl nucleotide-binding proteins or G-proteins. The molecular mechanism involved in the dynamic coupling of the LH/hCG receptor however, are not known. It has been postulated that receptor aggregation at the molecular level plays a critical role in this process. There have been attempts to understand the receptor association and dissociation phenomena at the molecular level. One of them involves the participation of the major histocompatibility complex (MHC) class I antigen in the mechanism of receptor activation and/or expression. One molecular basis for these mechanisms consists of a physical interaction between MHC proteins and receptors to form "compound receptors" able to transfer a hormonal signal to the cell. Using a photo-reactive probe we demonstrated that the LH/hCG receptors and the class I antigens are closely associated in the membrane. Thus, it is possible to form covalent complexes of hCG and class I antigens through the binding of the hormone to specific receptors. These findings imply that LH/hCG receptors and the MHC class I antigens may interact at the level of the plasma membrane in the mechanism of LH action. We also performed experiments using a single cell and limiting stimulation to a patch of membrane. The results stimulating the cell in a localized area suggested that even if all components are entirely free to float there is a constraint in the localization of the receptor, G-protein, and/or the effector, supporting the constraint dissociation model. Within a limited area subunits could dissociate, but they would not be free to diffuse throughout the membrane. Moreover the concept of compartmentalization that has been utilized to explain some inconsistencies in second-messenger action now can be proved by experimental design.

The Human Variome Project country node of Argentina in the first two years of activity: past, present and future / El nodo Argentino del Proyecto Varioma Humano en los primeros dos años de actividad: pasado, presente y futuro

The Human Variome Project (HVP) is an international effort aiming systematically to collect and share information on all human genetic variants. It has been working for years in collaboration with local scientific societies by establishing systems to collect every genetic variant reported in a country and to store these variants within a database repository: LOVD (Argentinian chapter: ar.lovd.org). Formally established in 2017 in the Argentinian Node, up to June 2019 we collected more than 25,000 genetic variants deposited by 17 different laboratories. Nowadays the HVP country nodes represent more than 30 countries. In Latin America there are four country nodes: Argentina, Brazil, Mexico and Venezuela; the first two interacted recently launching the LatinGen database. In the present work we want to share our experience in applying the HVP project focusing on its organization, rules and nomenclature to reach the goal of sharing genetic variants and depositing them in the Leiden Open Variation Database. Contributing laboratories are seeking to share variant data to gain access all over the country. It is one of our goals to stimulate the highest quality by organizing courses, applying current nomenclature rules, sponsoring lectures in national congresses, distributing newsletter to serve the Argentinian genomics community and to stimulate the interaction among Latin America countries.

El Proyecto Varioma Humano (HVP) es un esfuerzo internacional que tiene como objetivo recopilar y compartir sistemáticamente información sobre todas las variantes genéticas humanas. Hemos estado trabajando durante tres años en colaboración con sociedades científicas locales, mediante el establecimiento de sistemas para recolectar todas las variantes genéticas reportadas en el país y almacenarlas dentro de la base de datos LOVD (capítulo argentino: ar.lovd.org). En el año 2017 fue establecido formalmente el Nodo Argentino del HVP, habiéndose recolectado más de 25.000 variantes genéticas depositadas por 17 laboratorios diferentes hasta junio de 2019. Hoy en día existen al menos 30 nodos del HVP, correspondientes a diferentes países. En América Latina hay cuatro nodos: Argentina, Brasil, México y Venezuela; Los dos primeros interactuaron recientemente lanzando la base de datos LatinGen. En el presente trabajo queremos compartir nuestra experiencia en la aplicación del proyecto HVP centrándonos en su organización, reglas y nomenclatura para alcanzar el objetivo de compartir variantes genéticas y depositarlas en la base de datos de variaciones abiertas de Leiden (LOVD). Es uno de nuestros objetivos estimular la más alta calidad mediante la organización de cursos, aplicación de las reglas de nomenclatura actuales, patrocinio de conferencias en congresos nacionales, distribución de boletines informativos para la comunidad de genómica argentina, y estimulación de la interacción entre los países de América Latina.

Rat adrenal cycloheximide-sensitive factors and phospholipids in the control of acute steroidogenesis.

ACTH in vivo induces the formation of several steroidogenic factors in both cytosol and extramitochondrial particulate fractions of rat adrenal. Cycloheximide prevents the formation of these factors. Here we show the presence of a cytosolic steroidogenic component (C1) which is cycloheximide-sensitive and not ACTH-dependent. C1 is able to solubilize an ACTH-dependent steroidogenic factor (C2) from particulate fractions resulting in the release of the rat-limiting constraint of mitochondrial steroidogenesis. The thermolabile and trypsin-resistant factor C1 has an apparent mol.wt of 28,000 Daltons. In contrast, the cycloheximide-sensitive factor C2 from extra-mitochondrial fractions of ACTH-treated rats comigrates on Sephadex G-10 with phospholipids. Endogenous phospholipids isolated from particulate adrenal fractions of ACTH-treated rats or exogenous phospholipids will also stimulate steroidogenesis in vitro. Indeed, cytosolic solubilizing factor C1 enhances the exogenous phospholipid effect 3-4-fold. The results taken together suggest that C1 may be very similar to a well defined phospholipid exchange protein and C2 is itself a phospholipid. Both factors seem to be obligatory for the ACTH-induced steroidogenesis.

Gonadotropin-releasing hormone action upon luteinizing hormone bioactivity in pituitary gland: role of sulfation.

The regulation of rat luteinizing hormone (rLH) bioactivity was studied in an in vitro system using isolated pituitaries from male rats. Stored and released rLH was evaluated in terms of mass (I-LH), bioactivity (B-LH), mobility in nonequilibrium pH gradient electrophoresis, and mannose and sulfate incorporation either in the presence or absence of gonadotropin-releasing hormone (GnRH). GnRH increased the biological potency of stored and released rLH. The pituitary content revealed seven I-LH species (pH 7.2, 7.8, 8.5, 9.0, 9.1, 9.3, and 9.7) and five B-LH species (pH 8.5, 9.0, 9.2, 9.4, and 9.7). The major I-LH and B-LH peaks were at pH 9.0 and 9.2, respectively. I-LH peaks at pH 7.2 and 7.8 are devoid of bioactivity; at these pH values, free rLH subunits are detectable. GnRH increases the amount of both I-LH and B-LH material secreted into the medium, and the major component migrates at pH 8.5 and is probably the alpha beta dimer. [3H]Mannose and [35S]sulfate can be incorporated into stored and released rLH (pH 7.2, 7.8, 9.0, 9.1, and 9.3 and 7.2, 7.8, 8.5, and 9.0, respectively). GnRH decreases [2-3H]mannose incorporation into secreted rLH. [35S]Sulfate was incorporated into I-LH released spontaneously into the medium; the form at pH 7.2 has no biological activity and is probably the free alpha subunit. GnRH decreases the [35S]sulfate-labeled rLH content of the pituitary concomitantly with a 500% increase in [35S]sulfate-labeled released rLH, suggesting that, soon after [35S]sulfate is incorporated, sulfated rLH is released. Sulfatase action on released rLH reveals that sulfation may be related to release of rLH but that sulfate residues are not involved in the expression of rLH bioactivity. In conclusion, GnRH stimulates carbohydrate incorporation and processing of the oligosaccharide residues giving the highest biological potent rLH molecule and also increases sulfation; this step is closely related to the step limiting the appearance of LH in the medium in the absence of GnRH.

Compartmentalization of corticotropin-dependent steroidogenic factors in adrenal cortex: evidence for a post-translational cascade in stimulation of the cholesterol side-chain split.

A sensitive cell-free assay was developed for the analysis of corticotropin-dependent factors that stimulate the rate-limiting step of adrenal steroidogenesis. In this assay adrenal post-mitochondrial supernates from corticotropin-stimulated rats caused a 10- to 100-fold increase in the de novo synthesis of pregnenolone and progesterone. A similar stimulation was observed by corresponding fractions from Leydig cells and mouse Y-1 adrenal tumor cells, but not from rat liver. Subcellular fractionation of rat adrenal tissue showed several steroidogenic factors to be present in various compartments. Recombination of them produced highly synergistic effects. The activation of some components could also be demonstrated in vitro, suggesting a cascade of events possibly linking the cAMP-dependent phosphorylation pathway with the rate-limiting step. Cycloheximide prevented the production of these steroidogenic factors in vivo upon stimulation but had no effect in vitro, suggesting a post-translational cascade involved in the activation of the cholesterol side-chain split.

Production of steroid hormone and cyclic AMP in hybrids of adrenal and Leydig cells generated by electrofusion.

Electrofusion of rat adrenal and Leydig cells generated hybrids capable of synthesizing simultaneously both testosterone and corticosterone, under stimulation of lutropin or adrenocorticotropin. Evidence was obtained indicating that under such circumstances, heterologous lutropin receptor--adrenal adenylate cyclase complexes were formed.

Molecular and biological interaction between major histocompatibility complex class I antigens and luteinizing hormone receptors or beta-adrenergic receptors triggers cellular response in mice.

Purified IgG from BALB/c mouse anti-C3H serum exerts positive inotropic and chronotropic effects in C3H mouse atria and induces testosterone synthesis in C3H mouse Leydig cells. The effect depends on IgG concentration and can be abolished by beta-adrenergic-receptor and luteinizing hormone-receptor antagonists. IgG interferes with the binding of dihydroalprenolol and luteinizing hormone. Monoclonal antibodies against major histocompatibility complex class I antigens were active on the Leydig cells of C3H and BALB/c mice. There was a parallelism between the effect of each individual monoclonal antibody with specificity for a particular haplotype and the response of the target cell from the strains carrying such haplotypes. These antibodies could precipitate the soluble luteinizing hormone-receptor complex. The results suggested that bound hormone triggers the association of major histocompatibility class I antigen with the receptor, thereby activating the respective target cells.

Effect of the antiandrogen flutamide on pituitary LH content and release.

Flutamide is a nonsteroidal antiandrogen that blocks androgen receptors, with a consequent increase in serum immunoreactive LH (I-LH) in the presence of high testosterone concentrations. Several studies suggested that the gonadal steroids also play an important role in the regulation of LH bioactivity (B-LH). Therefore, it seems difficult to understand how the blockade of pituitary androgen receptors leads to the increase in testosterone levels. The present study was designed to elucidate the effect of flutamide on serum I-LH, B-LH and testosterone, as well as on in vitro stimulation of pituitaries by gonadotropin-releasing hormone (GnRH), in intact and androgen-treated castrated rats. In intact animals, a dose of flutamide as low as 0.5 mg/day provoked a 7- to 8-fold increase in serum I-LH levels over the vehicle-injected controls, whereas B-LH and testosterone were unaffected. However, higher doses significantly increased serum B-LH to values similar to those obtained in vehicle-injected castrated animals, resulting in high testosterone levels. Flutamide treatment provoked a decrease in I-LH and B-LH pituitary content; this effect was significantly higher under in vitro GnRH stimulation. The releasable I-LH under GnRH stimulation was not affected by flutamide treatment; however, a marked decrease was observed in B-LH.(ABSTRACT TRUNCATED AT 250 WORDS)

Receptor aggregation induced by antilutropin receptor antibody and biological response in rat testis Leydig cells.

Antibodies against the lutropin receptor have been obtained by the monoclonal antibody technique. Mice were immunized with luteal membrane from ovaries from pseudopregnant rats, containing high lutropin receptor concentration. Hybridoma cells were obtained by fusing mouse myeloma cells with spleen cells from the immunized animal. Five clones were produced that secreted monoclonal antibodies that specifically inhibited lutropin binding to its receptor in a competitive fashion. Antibodies from three clones were capable of blocking biological response to lutropin (e.g., testosterone production by isolated rat Leydig cells). Antibodies secreted by two other clones, however, were capable of acting as Leydig cell stimulators. Immunofluorescence studies demonstrated the presence of receptor capping which may be associated with receptor-mediated testosterone production. Antagonist antibodies could be transformed into agonist by the addition of a second crosslinking anti-mouse IgG. The discovery of agonist antibodies against the receptor molecule proves that the biological information of the lutropin-receptor complex resides in the receptor and not in the hormone.