Inhibition of adenylyl cyclase activity in rat corpora luteal tissue by glycopeptides of human chorionic gonadotropin and the alpha-subunit of human chorionic gonadotropin

Indirect evidence has indicated that the carbohydrate moieties of the glycoprotein hormones are involved in the activation of the receptor-adenylyl cyclase system of reproductive tissues. In the present study, we have isolated the glycopeptides (GP) from human chorionic gonadotropin (hCG), the alpha-subunit of hCG, fetuin, and bovine gamma-globulin (b gamma G). These along with a number of synthetic oligosaccharides were tested for their ability to inhibit adenylyl cyclase (AC). There was less than 0.001% cross-reactivity of the GP from hCG, hCG alpha, fetuin, and b gamma G when tested in a double-antibody hCG radioimmunoassay or rat corpora lutea radioreceptor assay. The GP of fetuin, b gamma G, and the synthetic oligosaccharides did not inhibit AC activity of 2000 g corpora lutea membranes when coincubated with 100 ng of hCG/mL (ED50). However, when the GP of hCG and hCG alpha were included with intact hCG, there was a dose-related inhibition. Inhibition of cyclase activity was enhanced when the hCG GP were desialylated. This occurred without a change in the lag time of hCG activation which was calculated to be 1-1.5 min. Changing the concentration of ATP and Mg2+ did not affect the inhibitory effects of the hCG alpha GP on hCG-stimulated AC activity. Inhibition by hCG GP followed uncompetitive kinetics. The inhibition by the GP of hCG seems to be restricted to the LH/hCG-stimulatable AC system because the same dosage of hCG GP which inhibited the rat luteal AC system did not have any effect on the rat hepatocyte AC system when coincubated with glucagon or on NaF-stimulated activity in luteal membranes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Elution and rebinding of gonadotropins to receptors in corpora lutea: comparisons among treatments and species

Several treatment regimens have been used to dissociate bound gonadotropins from their target tissues to quantify numbers of occupied receptors. To compare the efficacy of these methods, we evaluated the ability of a number of treatments to elute bound gonadotropins from corpora lutea of various species. In addition, we examined the capacity of luteal tissue to rebind gonadotropins after efficacious elution (greater than 90% dissociation of bound gonadotropin). Particulate (20,000 g) preparations of luteal tissue from pseudopregnant rats and from nonpregnant pigs and rhesus monkeys were incubated with 125I-labeled human luteinizing hormone (hLH, NIH-LH-11) or 125I-labelled chorionic gonadotropin (hCG, CR119) for 20 h at 25 degrees C to occupy gonadotropin receptors. Heat treatment (60 degrees C) eluted greater than 80% of bound 125I-hLH from rat tissue within 30 min, but similar treatment dissociated only 35% of 125I-hLH bound to porcine tissue (p less than 0.01). Likewise, heat treatment eluted only 57% of 125I-hLH bound to macaque tissue. At 4 degrees C, the following treatments dissociated greater than 90% of specifically bound 125I-hLH from porcine and rat luteal particulates within 5 min: acetic acid (pH 2.3), formic acid (pH 2.3), propionic acid (pH 2.3), acetic acid: HCl (pH 2.3 and 3.3), and MgCl2 (4 M). After 1-2 h at 4 degrees C, exposure to urea (4 M) or acetic acid:HCl (pH 4.3) also eluted greater than 90% of bound 125I-hLH.(ABSTRACT TRUNCATED AT 250 WORDS)     

125I-luteinizing hormone (LH) binding to soluble receptors from the primate (Macaca mulatta) corpus luteum: effects of ethanol exposure

In vitro exposure to alcohols unmasks additional binding sites for gonadotropin in cell/membrane preparations of the corpus luteum of rhesus monkeys. In the current study, we compared the effects of ethanol on gonadotropin receptors solubilized from macaque luteal membranes to those on receptors associated with the lipid bilayer. Treatment with 1% Triton X-100 for 30 min at 4C, followed by precipitation with polyethylene glycol, resulted in recovery of 50% more binding sites for 125I-human luteinizing hormone (hLH) than were available in particulate preparations (p less than 0.05). However, the soluble receptors displayed a 3-fold lower affinity for 125I-hLH (p less than 0.05). Conditions which enhanced LH binding to particulates, i.e., 1-8% ethanol at 25C, decreased specific 125I-hLH binding to soluble receptors. Steady-state LH binding to soluble receptors during incubation at 4C was half of that observed at 25C. The presence of 8% ethanol at 4C restored LH binding to levels observed in the absence of ethanol at 25C. Thus, LH binding sites in the primate corpus luteum can be effectively solubilized with Triton X-100. The different binding characteristics of particulate and soluble receptors, including the response to ethanol exposure, suggest that the lipid environment in the luteal membrane modulates the availability and affinity of gonadotropin receptors.     

Human chorionic gonadotropin increases chromatin solubility in isolated bovine and human luteal nuclei

We have previously demonstrated that bovine and human luteal nuclei contain human chorionic gonadotropin/luteinizing hormone (hCG/LH) receptors and that these gonadotropins can directly stimulate nuclear membrane enzyme activity (nucleoside triphosphatase) involved in messenger ribonucleic acid (mRNA) transport from the nucleus to the cytoplasm. The present studies were undertaken to investigate the effect or hCG on chromatin solubility, reflecting perhaps synthesis and transport of RNA, in isolated bovine and human luteal nuclei. hCG increased chromatin solubility in a concentration-dependent manner. This hCG effect is either blocked or substantially reduced by the addition of hCG antiserum; denatured hCG had no effect and cyclic adenosine 3',5'-monophosphate could not mimic the hCG response. hCG had no effect on chromatin solubility in bovine liver or kidney nuclei and hormones other than hCG, human LH, or the beta subunit of hCG had no effect on chromatin solubility in bovine luteal nuclei, demonstrating the tissue and hormone specificity of the response. These findings further strengthen the concept of direct gonadotropin regulation of nuclear functions of luteal cells.     

The differential binding affinities of the luteinizing hormone (LH)/choriogonadotropin receptor for LH and choriogonadotropin are dictated by different extracellular domain residues

The high degree of amino acid sequence homology and the divergent ligand binding affinities of the rat (r) and human (h) LH receptors (LHRs) allowed us to identify amino acid residues of their extracellular domain that are responsible for the different binding affinities of bovine (b) and hLH, and human choriogonadotropin (hCG) to the hLHR and rLHR. Because of the proposed importance of the beta-sheets of the leucine-rich repeats (LRRs) of the extracellular domain of the LHR on hormone binding, we examined 10 divergent residues present in these regions by analyzing two complementary sets of mutants in which hLHR residues were substituted with the corresponding rLHR residues and vice versa. These experiments resulted in the identification of a single residue (a Ile or Ser in the C-terminal end of LRR2 of the hLHR or rLHR, respectively) that is important for hLH binding affinity. Surprisingly, however, this residue does not affect hCG or for bLH binding affinity. In fact, the results obtained with bLH and hCG show that several of the divergent residues in the beta-sheets of LRR1-9 affect bLH binding affinity, but none of them affect hCG binding affinity. Importantly, our results also emphasize the involvement of residues outside of the beta-sheets of the LRRs of the LHR in ligand binding affinity. This finding has to be considered in future models of the interaction of LH/CG with the LHR.     

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.     

Site-directed mutagenesis of the human chorionic gonadotropin beta-subunit: bioactivity of a heterologous hormone, bovine alpha-human des-(122-145)beta

Human CG contains an alpha-subunit, common to the pituitary glycoprotein hormones, and a hormone-specific beta-subunit, but unlike the pituitary beta-subunits, hCG beta is characterized by an O-glycosylated carboxy-terminal extension. A mutant beta-subunit, des-(122-145)hCG beta, was prepared using site-directed mutagenesis, and the pRSV expression plasmids were transfected into Chinese hamster ovary cells that produce the bovine alpha-subunit (b alpha). The mutant beta-subunit binds to b alpha, and the heterologous gonadotropin, b alpha-des-(122-145)hCG beta, was capable of stimulating steroidogenesis in cultured Leydig tumor cells (MA-10) to the same extent as standard hCG. When compared with the heterologous gonadotropin, b alpha-hCG beta wild type, the hybrid hormone with the truncated hCG beta exhibited equal potency, within the accuracy of the RIAs used to determine hormone concentrations, and gave a similar time course of steroidogenesis. Interestingly, these transformed Leydig cells do not distinguish between the steroidogenic potencies (as measured by progesterone production) of hCG and human LH (hLH) as do some preparations of normal rodent Leydig cells (as measured by testosterone production). However, the MA-10 cells were able to distinguish hCG from hLH based on their cAMP response; the latter produced a greater response at both maximal and submaximal gonadotropin concentrations. The two expressed heterologous gonadotropins were equipotent in their abilities to stimulate cAMP and gave similar time courses of cAMP accumulation in MA-10 cells. Thus, the carboxy-terminal extension of hCG beta is not required for association with the alpha-subunit nor for functional receptor binding, as judged by cAMP accumulation and progesterone production in MA-10 cells.     

The role of the adenylyl cyclase system in the regulation of corpus luteum function in the human and in nonhuman primates

We have reviewed the properties of luteinizing hormone/human chorionic gonadotropic (LH/hCG)-sensitive adenylyl cyclase (AC) of human corpus luteum (CL) and its regulation by several hormones and nonhormonal activators. We have also described the changes in enzyme activity in membrane preparations of human and cynomolgus monkey CL obtained at various stages of the menstrual cycle and pregnancy. The data have been analyzed with respect to the functional status of the luteal tissue and to the species differences among primate CL. In the menstrual cycle, luteal AC responsiveness to LH/hCG was detectable during the midluteal phase, but not during the late luteal phase or in the follicular phase of the following cycle. In addition, nonhormonal stimulation was high in CL obtained during the midluteal and late luteal phases, but declined drastically by the follicular phase of the next cycle. In early pregnancy, the enzyme was unresponsive to LH/hCG stimulation, yet its sensitivity to nonhormonal stimulation was similar, if not identical, to that of midluteal phase CL. Functional activity was also evident at the end of pregnancy. These results demonstrate that expression of AC activity in primate luteal membrane changes significantly with varying hormonal status under physiologic conditions. It is concluded that the AC system in luteal membranes is an effective model to study the mechanisms that regulate function and life span of the human and nonhuman primate CL.     

Comparison of the steroidogenic response of luteinized granulosa cells from rhesus monkeys to luteinizing hormone and chorionic gonadotropin.

The dynamics of the steroidogenic response of nonprimate gonadal cells to gonadotropins suggests that the biologic action of pituitary LH differs from that of placental CG. To compare the response to LH and CG in primate species, luteinized granulosa cells (LGCs) obtained from rhesus monkeys following follicle stimulation were cultured in vitro. The pattern and levels of progesterone (P) produced during culture was influenced by the concentration (0-10%) and type (fetal bovine or macaque) of serum in the medium and whether LGCs were plated on plastic or extracellular matrix from bovine corneal endothelial cells. After 2-3 days of culture, LGCs were exposed acutely (15-30 min) or chronically (6 h) to 1 or 100 ng/ml human LH (hLH, NIH 1-2) or hCG (CR123), 50 micrograms/ml ovine LH (oLH, NIH-oLH-25), or incubated in the absence of gonadotropins (controls). After the first 15-30 min, the media were changed at 30-min intervals. Both acute and chronic exposure to hLH, hCG, and oLH increased (p less than 0.05) P concentrations above control levels within 15-30 min. There were no differences in the patterns or levels of P elicited by hLH or hCG over time for each treatment condition. Chronic exposure to 1 and 100 ng/ml hLH or hCG and 50 micrograms/ml oLH sustained P levels above that of controls for the 6-h interval. Acute exposure to 1 ng/ml hLH or hCG failed to maintain elevated P levels throughout the experiment.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of luteinizing hormone-releasing hormone agonist and calcium upon adenylyl cyclase activity of human corpus luteum membranes

We have investigated the ability of the agonist analog of luteinizing hormone-releasing hormone (LH-RH), D-Trp6-LH-RH (LH-RHa), and of CaCl2 to inhibit directly gonadotropin stimulation of adenylyl cyclase in a cell-free system prepared from human corpus luteum. In the presence of a submaximally effective concentration of hCG, addition of 10(-5)M final concentration of LH-RHa did not alter the gonadotropin-stimulated enzyme activity, nor did LH-RHa alone show any effect upon basal levels of the enzyme. The failure to inhibit adenylyl cyclase would indicate that the LH-RHa does not affect gonadotropin receptor binding or cAMP synthesis and/or degradation in this membrane system, suggesting that the luteolytic effects of LH-RH are unlikely to involve a direct antigonadotropic activity at the level of the human corpus luteum. In great contrast to LH-RHa, addition of CaCl2 resulted in a dose-dependent inhibition of hCG-stimulable adenylyl cyclase. Thus, in the presence of either a maximally or submaximally effective concentration of hCG, inhibition was significant at 0.5 mM CaCl2 added in excess of ATP (2 mM) and EDTA (1 mM), being about 90% upon addition of 2.5 mM CaCl2. We also found that calcium reduced enzyme stimulation by forskolin and the GTP analog, guanyl 5'-yl imidodiphosphate [GMP-P(NH)P] in a dose-related manner and that activation by NaF was less sensitive to inhibition by calcium. Accordingly, at 2.5 mM CaCl2, guanyl nucleotide and forskolin stimulations were inhibited 96% and 86%, respectively, while NaF stimulation was reduced by 40%. Because previous studies have shown that calcium does not impair gonadotropin binding activity, the calcium-dependent inhibition of gonadotropin responsiveness reported here would imply an alteration in the functional coupling of the components of the luteal adenylyl cyclase system. These data suggest that calcium may play a role in the regulation of gonadotropin action in the human corpus luteum.     

Evidence for dual coupling of the murine luteinizing hormone receptor to adenylyl cyclase and phosphoinositide breakdown and Ca2+ mobilization. Studies with the cloned murine luteinizing hormone receptor expressed in L cells.

The murine receptor for luteinizing hormone (LHR) was cloned and expressed in L cells. This LHR (mature protein of 674 amino acids) is very similar to that of the rat (same length, 36 amino acid differences) but differs significantly more from that of man (673 amino acids, 109 differences). Expression of the murine LHR in L cells led to the appearance of binding sites for human chorionic gonadotropin (hCG) with a Kd of 150 pM and an LH- and hCG-stimulable adenylyl cyclase activity (EC50 = 50-100 pM hCG). Upon labeling pools of phosphoinositides with [3H]myo-inositol, L cells expressing the murine LHR responded to hCG with an increase in their rate of phosphoinositide hydrolysis (EC50 = 2,400 pM hCG). This was accompanied by an increase in intracellular Ca2+ [( Ca2+]i), as determined by the Fura2 method. This increase in [Ca2+]i in response to hCG was dependent on the LHR, for HCG did not affect [Ca2+]i in L cells not expressing the LHR. The effect was not due to the cAMP-forming activity of the LH receptor, for neither forskolin nor prostaglandin E1, which both increase cAMP levels in L cells, had a similar effect in either control or LHR-expressing cells and isoproterenol had no effect in L cells expressing a functionally active hamster beta-adrenergic receptor. The effect was also not due to overexpression of a Gs-coupled receptor, for L cells expressing 8-fold higher levels of the human V2 vasopressin receptor did not mimic the Ca(2+)-mobilizing response of the LH receptor. We conclude that the LH receptor has the capability of activating two intracellular signaling pathways: one leading to stimulation of adenylyl cyclase and resulting in increases in cAMP and a second leading to stimulation of phospholipase C and resulting in formation of inositol phosphates and elevations in [Ca2+]i. These data correlate positively with and provide a mechanistic explanation for previous reports on the ability of hCG to mobilize phosphoinositides and increasing [Ca2+]i in luteal and granulosa cells (e.g. Davis, J. S., West, L. A., and Farese, R. V. (1984) J. Biol. Chem. 259, 15028-15034).     

Activation of the cAMP cascade by steroidogenic hormones and glucagon in the pathogenic fungus Candida albicans

Incubation of Candida albicans yeast cells with human luteinizing hormone (hLH), human chorionic gonadotrophin (hCG) or glucagon produced a significant rise in cAMP total levels. The effect of these hormones in permeabilized cells of the fungus produced a 2-3 fold increase in the Mg2+, GTP-dependent adenylyl cyclase activity as well as full activation of the cAMP-dependent protein kinase (PKA) activity. These results indicate that the interaction of the mammalian hormones with the fungus triggered the cAMP activation cascade in a similar way to that found in higher eukaryotic organisms.     

Application of the immunofluorescence technique and confocal laser scanning microscopy for studying the distribution of the luteinizing hormone/chorionic gonadotropin (LH/CG) receptor on rat luteal cells

We used confocal scanning microscopy to study the semi-quantitative distribution of luteinizing hormone/chorionic gonadotropin (LH/CG) receptors on rat luteal cells at both the two- and the three-dimensional level. The receptors were visualized in 6-microns sections of pseudopregnant rat ovaries using polyclonal rabbit antiserum to hCG-affinity-purified LH/CG receptor in conjunction with rhodamine-conjugated anti-rabbit immunoglobulins. Twenty to 30 optical sections were taken at different focal planes from representative luteal cells with a confocal laser scanning microscope and then processed digitally to two- and three-dimensional pseudocolored images. Distinct differences in fluorescence intensity could be demonstrated at both the two- and the three-dimensional level on the luteal cell surfaces, suggesting an uneven distribution of the LH/CG receptors on the cell membranes. This probably results in the compartmentalization and polarization of luteal cell function.     

Modification of sialyl residues of gonadotropic hormones by reductive amination. Influence on biological activity and circulating half-life

The sialic acid residues of human chorionic gonadotropin, human lutropin and human follitropin were quantitatively modified by introduction of an amino compound. In radioreceptor assays, the modified chorionic gonadotropin, lutropin and follitropin saturated the receptors. However, in the low nanogram range, the gonadotropic binding was higher for the control compared to the modified sample.The hormonal activity of the chorionic gonadotropin was testedin vitro. The modified preparations were four- to thirteen-fold less stimulatory compared to the control but elicited the same maximal response. The biological activity of follitropin was determinedin vivo. In this case, the modified preparations were four- to five-fold less stimulatory than the control. Both the modified chorionic gonadotropin and follitropin preparations were found to act as agonists. Modification of the gonadotropin hormones did not significantly alter the immune recognition of these glycoproteins.The apparent circulating half-life in rats of the modified chorionic gonadotropin and follitropin was increased six- to nine-fold compared to that of native hormones; this might be a consequence of resistance of the modified sialyl residues to sialidases and the resultant slower exposure of terminal galactosyl residues; the plasma half-life of modified lutropin remained the same as that of the native hormone.Abbreviations hCG human chorionic gonadotropin - hLH human lutropin or luteinizing hormone - hFSF human follitropin or follicle stimulating hormone - mala methyl ester of alanine - hCG(ala, mala, etc.) human chorionic gonadotropin modified on sialicacid by reductive amination with alanine, methyl ester of alanine, etc. - IRP-HMG intact rat prostrate-human menopausal gonadotropin     

Effect of human chorionic gonadotropin derivatives on leydig cell function.

BACKGROUND: Several human chorionic gonadotropin (hCG) derivatives have been detected in healthy human subjects, indicating that they may play a role in cell function. These hCG derivatives include deglycosylated hCG, proteolytic digestion products of hCG and free alpha and beta subunits of the hormone. It is well documented that testicular Leydig cells are responsive to luteinising hormone (LH) or its analogue hCG. These hormones have high affinity for LH/hCG receptors on the plasma membrane. METHODS: We designed functional and binding studies to compare the effects of native hCG and several hCG derivatives on a rat Leydig cell system. The molecular weight of the hCG derivatives was determined by SDS-PAGE and the binding affinity to LH/hCG receptors was measured by a radioligand assay. In addition, their ability to produce testosterone, cyclic AMP and arachidonic acid release was also studied. RESULTS: These hCG derivatives, with the exception of the free beta subunit, were able to bind to LH/hCG plasma membrane receptors with different affinities than that of native hCG. In addition, hCG derivatives did not increase intracellular cAMP levels or arachidonic acid release. However, they did increase testosterone production. CONCLUSION: Taken together, the results of this study lead us to suggest that these hCG derivatives may regulate the action of the native hormone in Leydig cells and are, thus, molecules of physiological relevance.     

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).     

Evidence for the existence of gonadotropin receptors in the nuclei isolated from rat ovary

Specific binding of radiolabeled human chorionic gonadotropin (hCG) to nuclei isolated from pseudopregnant rat ovaries was studied. Incubation of cultured luteal cells or isolated nuclei with fluorescein isothiocyanate conjugated hCG showed concentration of fluorescence in the nuclear region. Isolated nuclei exhibited saturable high affinity binding of radiolabeled hCG with an apparent Kd of 3.42 X 10(-10) M. The binding was inhibited by increasing concentrations of unlabeled hCG. Under dissociating conditions, the bound hCG was dissociated from the nuclei. However, unlike the plasma membranes, the hCG bound to nuclei was not degraded before dissociation. Radiolabeled hCG bound to the nuclei could also be dissociated by brief exposure to MgCl2 or acidic incubation medium. The bound hCG was not extractable with 4M KCl or 2% Triton X-100. The available evidence suggest that nuclear receptors are distinct from plasma membrane receptors for hCG.     

Conformational restrictions of the sheep testicular receptor discriminates pituitary lutropin and placental gonadotropins

A membrane preparation from the testis of maturing Dorset-Leicester-Suffolk sheep, capable of discriminating pituitary LH (lutropin) from placental gonadotropins human choriogonadotropin (hCG) and equine choriogonadotropin is described. Maximum binding of 125I-oLH (ovine lutropin) to the testicular receptors occurred at 4 degrees C in a rapid manner, attaining equilibrium in 12-16 h. Under such optimal conditions, only unlabeled ovine LH or the structurally identical bovine LH effectively competed for receptor occupation. Other highly purified pituitary LH preparations from rat and human pituitaries were weakly (4-10%) active in displacement assays. Purified hCG or equine choriogonadotropin, which were highly potent in rat testicular LH receptor assays, could not compete with 125I-oLH for binding to the sheep LH receptor at 4 degrees C. Thus, the sheep testicular LH receptor was highly specific in recognizing pituitary LH conformation. The presence of an ovine/bovine LH alpha- or beta-subunit in recombinants with hCG subunit counterparts was required to generate an effective conformation capable of receptor recognition. Chemically deglycosylated hCG, containing 75% less carbohydrate and which showed greater binding to other LH receptors, failed to recognize sheep LH receptor, suggesting that excess carbohydrate in hCG was not a factor in hindering binding of the native placental hormone. Scatchard analysis using 125I-hCG/125I-oLH revealed that there were separate sites with similar affinities but vastly different capacities. The hCG binding sites, which could also be effectively occupied by oLH, were less than 10% of oLH binding sites. Thus, the Dorset-Leicester-Suffolk sheep testicular receptor provides an important and unique in vitro test system to distinguish pituitary LH from placental LH-like hormones. We infer that temperature-dependent conformational restrictions of the sheep testicular LH receptor are involved in recognizing differences in these highly similar and structurally homologous hormones.     

In vitro and in vivo studies of functional activity of new low molecular weight agonists of the luteinizing hormone receptor

The use of luteinizing hormone (LH) and its structural and functional homolog human chorionic gonadotropin (hCG) leads to hyperstimulation of LH-dependent signalling pathways and to desensitization of LH receptors. Therefore, the development of low molecular weight agonists of the LH receptor without these disadvantages has been carried out in recent years. These agonists, unlike gonadotropins, are active when administered orally. The greatest prospects are associated with the development of the thienopyrimidine derivatives structurally similar to compound Org 43553. The purpose of this work was the synthesis of new thienopyrimidine derivatives TP03 and TP04, the study of the regulation of LH-sensitive adenylyl cyclase signalling system in rat testes, and the evaluation of the ability of TP03 and TP04 to stimulate testosterone synthesis in male rats at different routes of administration. In the concentration range 10^–7–10^–3 M, TP03 and TP04 induced an increase of the basal adenylyl cyclase (AC) activity in rat testicular membranes with the EC₅₀ values of 390 and 759 nM, respectively. At a concentration of 10^–4 M, AC-stimulating effects of TP03 and TP04 were 213 and 122%, respectively, which indicates a higher activity of TP03 under in vitro conditions. At the same time, the AC-stimulating effect of TP03 was approximately 2.5-fold weaker than that of hCG (10^–8 M). Upon simultaneous administration of hCG and thienopyrimidine derivatives, their stimulating effects on the AC activity were additive due to the different localization of their binding sites in the LH receptor. Compound TP03 (25 mg/kg), when administered intraperitoneally to male rats, was more efficient than TP04: the maximal increase in the testosterone level (3 h after administration) was by 60% higher than that induced by TP04. TP03 increased the testosterone level after oral administration (50 mg/kg) as well, but the effect was weaker than in the case of the intraperitoneal injection. Orally administered TP04 exhibited a low activity. The results suggest that 5-amino-N-tert-butyl-2-(methylsulfonyl)-4-(3-(nicotinamide)phenyl) thieno[2,3-d]pyrimidine-6-carboxamide (TP03) can be used for the development of the drugs that stimulate steroidogenesis in Leydig cells.     

Chemical, biological and immunological properties of pituitary gonadotropins from the donkey (Equus asinus): comparison with the horse (Equus caballus)

Donkey gonadotropins (donkey luteinizing hormone, dLH; donkey follicle-stimulating hormone, dFSH) have been isolated in purified form from 191 donkey pituitaries using essentially the same procedures previously employed for the purification of equine gonadotropins. Chemically, dLH and dFSH were observed to be similar to equine LH (eLH) and FSH (eFSH) in fractionation behavior and glycoprotein nature. Two forms of the dFSH molecule were observed, as is the case for eFSH. Donkey LH had significantly less total carbohydrate (13.5%) and sialic acid (1.9%) than eLH (26.7% and 5.8%, respectively). Carbohydrate (17-21%) and sialic acid (2.4%) content of the two dFSH preparations closely resembled that of eFSH. A slightly higher tyrosine content in the donkey gonadotropins was noted in a comparison of amino acid compositions. Immunologically, in a heterologous FSH radioimmunoassay (RIA), dFSH preparations were equal to or twice as active as eFSH preparations. However, in homologous RIAs for equine chorionic gonadotropin (eCG), eFSH and eLH, both the dLH and dFSH preparations were considerably less active than the equine gonadotropins, and their inhibition curves were all nonparallel. Biologically, in the Steelman-Pohley assay both dFSH preparations were equipotent and as potent as eFSH (approximately 40 times NIH-FSH-S12). In the Sertoli cell assay for cAMP (FSH assay) and the Leydig cell assay for testosterone (LH assay), both dFSH and dLH were 2- or 6-fold more active than eFSH and eLH, respectively. In rat and equine testis FSH homologous radioreceptor assays, dFSH preparations were as active and up to 6-fold more active than eFSH. In contrast, dLH was 10-fold less active than eLH in the equine LH homologous radioreceptor assay. Unlike eLH, dLH was found to possess little intrinsic FSH activity or FSH inhibitory activity, and the small amount of FSH activity observed was most likely due to FSH contamination. Therefore, eLH behaves much like eCG (pregnant mare's serum gonadotropin, PMSG) which also possesses both LH and FSH activity. In contrast, dLH behaves more like donkey chorionic gonadotropin (dCG) which possesses only a low degree of FSH activity.