Hormonotoxins: synthesis, characterization and bioefficacy of some defined disulfide linked conjugates of ovine LH with a ribosome inactivating protein, gelonin.

In order to synthesise a bioeffective hormonotoxin for selective targeting to specific cells in the gonads, gelonin, a single chain RIP obtained from an Indian plant, Gelonium multiflorum of Euphorbeaceae family was covalently linked to oLH with the use of N-succinimidyl-3-(2-pyridyldithio)propionate, generating a linkage containing a disulfide bond and a amide bond. The hormonotoxins were separated according to their molecular weight (indirectly according to oLH:gelonin molar ratio) and a complete biochemical analysis was performed. The linkage occurred through the epsilon-NH2 group of alpha oLH as judged from RP-HPLC analysis. The conjugates were devoid of ingredients as determined by SDS-PAGE and RP-HPLC analysis. The hormonotoxins retained substantial receptor binding, steroidogenic activity and immunoreactivity of oLH and gelonin to their antibodies. Hormonotoxins bind to the Leydig tumour cells via oLH part leaving gelonin free as judged by competitive displacement analysis. The hormonotoxin was internalized to the sufficient degree to effectively inhibit protein synthesis. The cytotoxicity of 1:1 molar ratio conjugate was relatively higher than that of others. The cytotoxicity of presently described more defined hormonotoxins exhibited higher receptor binding and cytotoxicity than the hormonotoxins reported earlier [Singh, et al., J Biol Chem, 264 (1989) 3089].     

Hormonotoxins. Preparation and characterization of ovine luteinizing hormone-gelonin conjugate

With the aim of targeting toxins to selected cells in the gonad, we have prepared conjugates of ovine luteinizing hormone (oLH) with a single chain ribosome-inactivating protein called gelonin. The two proteins were thiolated by using N-succinimidyl-3-(2-pyridyldithio)propionate and subsequently reacted under appropriate conditions to form oLH-S-S-gelonin complex. A complete biochemical analysis of thiolated oLH and oLH-gelonin conjugates has been performed. The linkage of the hormone to the toxin probably occurred through a single amino group in the alpha-subunit, with the beta-subunit remaining free. Modification of a single amino group on the alpha-subunit reduced receptor binding and immunological reactivity of the thiolated oLH, but subsequent complexing with the toxin-gelonin did not seriously compromise these activities. oLH and gelonin were calculated to be present in a 1:1 ratio in the hormonotoxin preparation. The conjugate retained significant steroidogenic activity in rat granulosa cells. Upon reaction with mouse tumor Leydig cells (MA-10 cells), the toxin component of the complex became internalized to a sufficient degree to effectively inhibit protein synthesis. The studies provide a rational basis for the design and study of large hormonotoxins.     

Hormonotoxins: the role of positive charge of lysine residue on the immunological,biological and cytotoxic properties of ovine lutropin-S-S-gelonin conjugates

Since the positive charge on the lysine residues plays an important role in the receptor recognition ability of oLH, the hormonotoxin has been synthesised with the use of 2-iminothiolane HC1 (2IT) and N-Succinimidyl-3-(2-pyridyldithio)-propionate (SPDP). The oLH activated with 2IT (oLH-10) was then mixed with SPDP activated gelonin (gelonin-30) in order to obtain a oLH-S-S-gelonin hormonotoxin. The conjugation mixture containing hormonotoxin was purified by gel-filtration chromatography according to the molecular weight and a complete physico-chemical, immunochemical and biochemical analysis were performed. The linkage occured through the -NH₂ groups of -subunit of oLH as judged from RP-HPLC analysis. A 11 (oLH:gelonin) molar ratio was obtained when determined with the use of several techniques. The hormonotoxins retained substantial receptor binding, steroidogenic activity and immunoreactivity. The competitive displacement analysis indicate that the binding occurs via the hormone part leaving the gelonin free which was probed with the gelonin antibodies. The presently described (C150A-02, C160A-02 and C170A-02) hormonotoxins exhibited higher receptor binding and toxicity to the target cells than the hormonotoxins prepared with the use of SPDP only. Therefore it is concluded that higher receptor binding and cytotoxicity may be due to the retention of positive charge on the lysine residues of oLH which was preserved during the conjugation process.Abbreviations BSA Bovine Serum Albumin - CMC Carboxy methyl Cellulose - DTT Dithiothreitol - DMEM Dulbeco's Modified Eagle's Medium - DTNB Ellman's reagent [5,5-dithio-bis-(2-nitrobenzoic acid)] - EDTA Ethylenediaminetetraacetic acid - FPLC Fast Protein Liquid Chromatography - FCA Freund's Complete Adjuvant - FCS Fetal Calf Serum - Gelonin-30 Gelonin modified by SPDP - GnRH Gonadotropin-Releasing Hormone - Gelonin-SPDP SPDP modified derivative of gelonin - HEPES (N-[2-hydroxyethyl] piperazine-N-[-2-ethanesulphonic acid]) - IFA Incomplete Freund's Adjuvant - 2IT 2-Iminothiolane - IODOGEN 1,3,4,6-tetrachloro 3,6-diphenylglycouril - oLH Ovine Luteinizing Hormone - oLH-SPDP SPDP modified derivative of oLH - oLH-10 oLH modified by 2IT - oLH2IT Molar ratio of oLH and 2IT - PDP 2-Pyridyl-dithiopropionate - PAP Pokeweed Antiviral Protein - RIP Ribosome Inactivating Protein - RP-HPLC Reverse-Phase High Performance Liquid Chromatography - RPMI Roswell Park Memorial Institute - RIA Radioimmunoassay - RRA Radioreceptor Assay - SPDP N-Succinimidyl-3(2-pyridyldithio)propionate - SDS-PAGE Sodium Dodecyl Sulphate-Polyacrylamide Gel Electrophoresis - TCA Trichloroacetic acid - TFA Trifluroacetic acid     

Hormonotoxins: abrogation of ribosome inactivating property of gelonin in the disulfide linked ovine luteinizing hormone-gelonin conjugates.

In order to synthesize a bioeffective hormonotoxin for selective targeting to specific cells in the gonads, gelonin, a single chain ribosome-inactivating protein obtained from an Indian plant called Gelonium multiflorum was covalently linked to ovine luteinizing hormone (oLH) by a disulfide bond. Ovine LH-S-S-gelonin conjugates of different molar ratios were subjected to determine the ribosome-inactivating property in a cell-free translation assay using rabbit reticulocyte lysate system. A single amino group modification with N-succinimidyl-3-(2-pyridyldithio)propionate resulted in a loss of 90% protein synthesis inhibition activity. Upon conjugation of gelonin to oLH, the activity was further inhibited ranging from 2.5-6.4%. A 1:1 to 1:1.5 molar ratio (oLH-S-S-gelonin) conjugates showed 2.5-4.6% activity while 1:2.8 to 1:2.2 molar ratio exhibited 5.5-6.4% inhibition ability.     

Lutropin receptors from male and female tissues: different responses to a lutropin receptor binding inhibitor.

An inhibitor for lutropin receptor site binding (LH-RBI), which strongly inhibited the binding of 125I-labeled ovine lutropin ([125I]oLH) to ovarian LH receptors, did not inhibit the [125I]oLH binding to testicular LH receptors. Preincubation of the LH-RBI with [125I]oLH did not affect the binding of preincubated ]125I]oLH to ovarian LH receptors. No inhibition of [125I]oLH binding to testicular LH receptors was observed even uhen the concentration of LH-RBI was significantly increased or when the testicular LH receptors uere first incubated with LH-RBI prior to the addition of [125I]oLH and a second incubation. Scatchard analysis revealed that the dissociation constant of [125I]oLH binding was essentially the same in the presence or absence of LH-RBI. The results suggest that: (i) the lutropin receptor of ovaries, but not of testes, has a specific LH-RBI binding site in addition to the lutropin binding site, and (ii) the binding of the LH-RBI produces an "allosteric" type of inhibition to the binding of lutropin at the lutropin binding site.     

Effect of lysine residue modification of ovine luteinizing hormone by heterobifunctional crosslinking reagent SPDP on subunit-subunit association, receptor binding and biological activity.

The increasing use of heterobifunctional crosslinking agent in the design of hormone-carrier conjugates for selective targeting or inducing immune response against the hormone has prompted us to study the effect of epsilon-NH2 group modification of oLH-subunit, their recombination, immunoreactivity, receptor binding and biological activity. The epsilon-NH2 groups of oLH alpha and oLH beta subunits were modified by using SPDP. The SPDP modified oLH alpha derivatives hybridize to native OLH beta as judged by RP-HPLC analysis. The sequential modification of alpha and beta subunits led to progressive reduction in immunoreactivity and receptor binding activities. The steroidogenic potential of oLH beta.SPDP.alpha oLH recombinant was relatively comparable. The modification of six or more epsilon-NH2 groups in oLH alpha although recombine fully with native oLH beta but failed to react to anti-oLH antibody. Moreover, steroidogenic activity was also abolished. Introduction up to four SPDP groups in oLH alpha compromised immunological and biological activities but further addition of two more SPDP groups completely abolished antibody reactivity, receptor binding and steroidogenic activity indicating the importance of later two -NH2 groups in the receptor recognition and steroidogenic potential.     

Modification of ovine luteinizing hormone subunits with SMPT and its effect on subunit recombination, immunological activity, receptor binding and steroidogenic activity

The increasing use of heterobifunctional cross-linking agents in the design of defined conjugates for selective targeting and inducing immune response has prompted us to study the role of epsilon-NH2 group modification of oLH subunits, their recombination and effect on immunoreactivity, receptor binding and biological activity. The epsilon-NH2 groups of alpha oLH and beta oLH subunits were separately modified by using SMPT. The alpha oLH-SMPT modified derivatives hybridize to beta oLH. Similarly, the beta oLH-SMPT derivatives recombined with alpha oLH. The recombination was judged by gel filtration chromatography and RP-HPLC analysis. The sequential modification of subunits led to progressive reduction in immunoreactivity and receptor binding activity. The modification of six or more epsilon-NH2 groups in alpha oLH although recombine fully with native beta oLH but failed to react to anti-oLH antibody. Moreover, the steroidogenic activity was also abolished. Introduction upto four SMPT groups in alpha oLH compromised immunological and biological activities but further addition of two or more SMPT groups completely abolished antibody reactivity, receptor binding and steroidogenic activity indicating the importance of later two amino groups in the receptor binding and steroidogenic activity. The present investigation clearly demonstrate that only 1:2-3 molar ratio of oLH subunits:SMPT could generate the site(s) in the subunits of the oLH that retained reasonable immunological, receptor binding and biological activity of the hormone. Therefore, this molar ratio may be used in future for the design and synthesis of bioeffective hormonotoxins.     

A superactive hormonotoxin prepared with truncated diphtheria toxin

A chemically truncated form of diphtheria toxin, DT51, which lacks the cell-binding site but retains the membrane-translocating function, was covalently linked to luteinizing hormone (LH) and compared to similar conjugates containing diphtheria toxin (DT) or diphtheria toxin A-chain (DTA). The DT51 hormonotoxin killed cells possessing an LH receptor at concentrations similar to that of DT hormonotoxin and orders of magnitude lower than DTA hormonotoxin. The DTA hormonotoxin exhibited an LD-50 similar to that of previously reported hormonotoxins which employed DTA, ricin A-chain, or gelonin as toxic moieties.     

Testicular GnRH-like factors: characterization of biologic activity

Observations that gonadotropin releasing hormone and its agonists directly inhibit gonadal function by binding to receptors on the Leydig cells had led to search for testicular GnRH-like peptide(s). This communication presents evidence that GnRH-like factors isolated from rat testis by immunoaffinity chromatography and previously characterized by radioimmunoassay and radioreceptor assay possess biologic activity. The partially purified material led to dose dependent inhibition of oLH stimulated testosterone production in a mixed Sertoli-Leydig cell monolayer culture. Pre-incubation of the cells with a potent GnRH antagonist prevented the inhibitory effects of the partially purified material suggesting that inhibition of oLH stimulated testosterone production may be receptor mediated.     

Significance of charge on lysine residue of ovine luteinizing hormone on immunological and biological properties of the hormone

In order to understand the significance of positive charge of lysine residues of ovine luteinizing hormone (oLH) on immunological and biological activity, the epsilon-NH2 group(s) of ovine LH were sequentially modified with 2-iminothiolane (2IT) that preserves the positive charge of the lysine while the overall charge of the hormone remains unchanged. These studies have also been compared with the oLH modified by N-succinimidyl 3-(2 pyridyldithio) propionate (SPDP) and succinimidyl 6-[3-(2-pyridyldithio)propionamido]hexanoate (LC-SPDP) that abolish positive charge of lysine residues. The modification primarily occurs in the alpha-subunit. Sequential modification led to progressive reduction in receptor binding and immunological activities. However, the steroidogenic activity was substantially retained. The immunoreactivity and receptor binding properties of 2IT modified oLH (oLH-2IT) were less affected when compared to SPDP (oLH-SPDP) or LC-SPDP (oLH-LC-SPDP) modified derivatives suggesting that increase in hydrophobic carbon chain in oLH-LC-SPDP molecule resulted in drastic inhibition in immunological and biological properties. But the steroidogenic potential of oLH-2IT, oLH-LC-SPDP or oLH-SPDP was relatively comparable. This suggests that a single -NH2 group modification with 2IT would generate the site in the hormone for conjugation to the toxin/carrier proteins that may retain better immunological and biological activity compared to that of SPDP or LC-SPDP modified oLH.     

In vitro studies on steroidogenesis in the presence of pregnenolone as precursors by the follicular tissue of the domestic fowl (Gallus domesticus)

Chicken granulosa and theca cells were isolated from F1 and F4-6 follicles 2-4 h before ovulation, and the amounts of progesterone, testosterone and oestradiol released in the medium during incubation for 3 h, in the presence or absence of pregnenolone as a percursor and stimulatory drugs or inhibitory drugs, were measured. Progesterone synthesis by granulosa cells was stimulated with oLH or theophylline. Much more progesterone was synthesized when pregnenolone was added to the medium. The amount of testosterone produced by the granulosa cells was similar to that produced by the theca cells. The production of testosterone was increased by the addition of oLH or theophylline. Oestradiol synthesis by F4-6 follicles was higher than by F1 follicles, and it was higher in the theca cells than in the granulosa cells. The addition of oLH or theophylline increased oestradiol synthesis in the theca cells and the granulosa cells of F4-6 follicles. The results indicate that oestradiol can be produced from pregnenolone by the theca cells alone. It is possible, however, that the theca cells also take in the precursors for the production of oestradiol from the granulosa cells.     

Disulfide linked αoLH-gelonin conjugate failed to recombine with βoLH subunit to generate bioeffective hormonotoxin

Since, linking of ovine luteinizing hormone (oLH) to ribosome inactivating protein gelonin (in oLH-gelonin conjugate) occur via the alpha-subunit, oLH, an attempt has been made to develop a universal hormonotoxin for selective targeting to specific cells in the gonads. Four different molar ratios of oLH and N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP) were used to activate the epsilon amino (-NH₂) groups of oLH. The oLH-SPDP derivatives recombine to native beta subunit of oLH (oLH) and the purified recombinants retained substantial receptor binding, steroidogenic activity and immunoreactivity to native oLH. The disulfide linked oLH-S-S-gelonin conjugates prepared by SPDP method were purified by gel filtration chromatography and analysed by reverse-phase high performance liquid chromatography (RP-HPLC). In order to obtain specificity and bioeffectivity, the oLH-S-S-gelonin conjugates were allowed to recombine to native oLH and the recombination mixture was further purified by gel-filtration chromatography. The RP-HPLC analysis of these recombinants indicated that oLH-S-S-gelonin did not recombine to oLH. The failure of recombination may be due to the reasons. (i) The site of -NH₂ activation by SPDP may be different in the oLH than the native oLH. (ii) The activation site may be in close proximity to the annealing site which facilitates the recombination of -subunit but failured to reassociate to oLH-S-S-gelonin conjugate. (iii) The introduction of gelonin (30 kDa basic protein) might have induced some steric hinderence for oLH to recombine to the oLH site which might have been masked in oLH-S-S-gelonin conjugates. (Mol Cell Biochem120: 95–102, 1993)Abbreviations oLH ovine Luteinizing Hormone - oLH alpha subunit of oLH - oLH beta subunit of oLH - BSA Bovine Serum Albumin - DTT Dithiothreitol - RP-HPLC Reverse Phase High Performance Liquid Chromatography - TSH Thyroid Stimulating Hormone - FSH Follicle Stimulating Hormone - LH Luteinizing Hormone - eCG equine Chorionic Gonadotropin - DMEM Dulbecco's Modified Eagles Medium - HEPES 4-(2-Hydroxyethyl)-1 Piperazine Ethane Sulfonic acid - PAP Pokeweed Antiviral Protein - RIA Radioimmunoassay - hCG human Chorionic Gonadotropin - TRH Thyrotropin Releasing Hormone - CRF Corticotropin Releasing Factor - hPL human Placental Lactogen - TFA Trifluroacetic Acid - oLH-SPDP SPDP activated derivative of oLH     

Effects of photoperiod, hypophysectomy, and follicle-stimulating hormone on testicular follicle-stimulating hormone binding sites in golden hamsters

Experiments were conducted to partially characterize and to examine the regulation of unoccupied testicular follicle-stimulating hormone (FSH) binding sites in adult golden hamsters. Testicular FSH binding sites were measured in the 1800 X gav fraction of whole testicular homogenates using iodinated bovine FSH. Binding of FSH was highly specific for FSH, located primarily in the testes, was time- and temperature-dependent, initially reversible, saturable, and consistent with a model consisting of a single class of high-affinity binding sites (range of equilibrium association constants (Ka) 2-12 X 10(10) M-1). Exposure of hamsters to a short photoperiod consisting of 5L:19D was associated with an increase in concentration (fmol/mg protein), but a reduction in total content (fmol/testes) of testicular FSH binding sites. There was no appreciable 5L:19D-associated alteration in receptor affinity (average Ka = 7.83 X 10(10) M-1). Injections of ovine prolactin (oPRL), ovine luteinizing hormone (oLH), or ovine FSH (oFSH) for 3 days into hamsters housed in 5L:19D for 12 wk had no effect on photoperiod-induced changes in testicular FSH binding sites. On Days 5 and 6 post hypophysectomy, a dramatic increase in FSH binding site concentration occurred, with but marginal effects on binding site affinity. Injections of 5 micrograms oFSH on Days 2, 3, and 4 after hypophysectomy prevented the increase in binding site concentrations measured on Day 5. Injection of a combination of 5 micrograms oFSH, 50 micrograms oPRL, and 25 micrograms oLH also reduced testicular FSH binding site concentrations in hypophysectomized hamsters, but oPRL or oLH by themselves were ineffective. The data indicate a homologous down-regulation of testicular FSH binding sites, but do not exclude the involvement of other hormones.     

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.     

Inhibition of adenylate cyclase from luteinized rat ovary by monovalent cations: roles of the stimulatory guanine nucleotide-binding regulatory component and stimulatory hormone receptor

Sodium and other monovalent cations (added as chloride salts) inhibited adenylate cyclase of luteinized rat ovary. Sodium chloride (150 mM) inhibited basal enzyme activity by 20%. Sodium chloride inhibition was enhanced to 34-54% under conditions of enzyme stimulation by guanine nucleotides (GTP and its nonhydrolyzable analog 5'-guanylyl imidodiphosphate), fluoride anion, and agonists (ovine luteinizing hormone (oLH) and the beta-adrenergic catecholamine isoproterenol) acting at stimulatory receptors linked to adenylate cyclase. Sodium chloride inhibition was dependent on salt concentration over a wide range (25-800 mM) as well as the concentrations of GTP and oLH. Inhibition by NaCl was of rapid onset and appeared to be reversible. The order of inhibitory potency of monovalent cations was Li+ greater than Na+ greater than K+. The role of individual components of adenylate cyclase in the inhibitory action of monovalent cations was examined. Exotoxins of Vibrio cholerae and Bordetella pertussis were used to determine respectively the involvement of the stimulatory and inhibitory guanine nucleotide-binding regulatory components (Ns and Ni) in NaCl inhibition. Sodium chloride inhibited cholera toxin-activated adenylate cyclase activity by 29%. Ni did not appear to mediate cation inhibition of adenylate cyclase because pertussis toxin did not attenuate inhibition by NaCl. Enzyme stimulation by agents (forskolin and Mn2+) thought to activate the catalytic component directly was not inhibited by NaCl but was instead significantly enhanced. Sodium chloride (150 mM) increased both the Kd for high-affinity binding of oLH to 125I-human chorionic gonadotropin binding sites and the Kact for oLH stimulation of adenylate cyclase by sevenfold. In contrast, NaCl had no appreciable effect on either isoproterenol binding to (-)-[125I]iodopindolol binding sites or the Kact for isoproterenol stimulation of adenylate cyclase. The results suggest that in luteinized rat ovary monovalent cations uncouple, or dissociate, Ns from the catalytic component and, in a distinct action, reduce gonadotropin receptor affinity for hormone. Dissociation of the inhibitory influence of Ni from direct catalytic activation could account for NaCl enhancement of forskolin- and Mn2+-associated activities. On the basis of these results, the spectrum of divergent stimulatory and inhibitory effects of monovalent cations on adenylate cyclase activities in a variety of tissues may be interpreted in terms of differential enzyme susceptibilities to cation-induced uncoupling of N and catalytic component functions.     

Radioimmunoassay of Bovine,Ovine and Porcine Luteinizing Hormone with a Monoclonal Antibody and a Human Tracer

A radioimmunoassay for bovine (bLH), ovine (oLH) and porcine (pLH) luteinizing hormone was developed using a human 125 ILH tracer from a commercial kit and a monoclonal antibody (518B7) specific for LH but with low species specificity. Standard curves demonstrated similar binding kinetics when bLH, oLH and pLH were incubated with tracer and antibody for 2 h at room temperature. A 30-min delay in the addition of the tracer gave sufficient sensitivity when analysing pLH. Separation of antibody-bound LH from free hormone was achieved by using second antibody-coated micro Sepharose beads. The assay was validated and the performance compared with that of an RIA currently in use for determination of bLH and oLH (coefficient of correlation: 0.99 and 0.98). Regardless of the standards used, intra-assay coefficients of variation were <10% for LH concentrations exceeding 1 µg/L. The inter-assay coefficients of variation were <15%. The assay was used for clinical evaluation demonstrating the pre-ovulatory LH surge in two cyclic cows, LH pulsatility in an oophorectomized ewe and LH response to GnRH injection in a boar.     

Differential steroidogenic responses of ovine luteal cells to ovine luteinizing hormone and human chorionic gonadotropin

Experiments were conducted in vitro on ovine small luteal cells to evaluate their steroidogenic response to ovine luteinizing hormone (oLH) and human chorionic gonadotropin (hCG) administered continuously throughout the experimental period or as a 15-min pulse. Both oLH and hCG stimulated a significant increase in progesterone secretion (P less than 0.001) by small luteal cells. Human chorionic gonadotropin administered continuously or as a pulse maintained progesterone secretion at 40-55% of experimental maximum at least 6 hr while oLH-stimulated progesterone secretion declined to basal levels by 4 hr after a 15-min pulse or declined to 25% of the experimental maximum within 6 hr under constant stimulation. The responses of small luteal cells to oLH and hCG were found to differ (P less than 0.001). The sustained progesterone secretion of luteal cells in response to a pulse of hCG may be due to longer residence of occupied receptor complex on the cell membrane. In contrast, the decline in oLH stimulated progesterone secretion, even when hormone is continuously present in the medium, may be related to a rapid internalization of receptor-hormone complexes and down-regulation of receptors.     

Selective proteolysis of ovine lutropin or its beta subunit by endoproteinase Arg-C. Properties of the Arg beta 43 cleaved hormone

Ovine lutropin (oLH) and its beta subunit (oLH beta) were nicked by short-term incubations with endoproteinase Arg-C. Isolated oLH beta was rapidly nicked and converted from an Mr 18,000 band on sodium dodecyl sulfate-polyacrylamide gels to an Mr 13,000 band. Partial nicking of only the beta subunit in intact oLH was also observed as indicated by the appearance of small amounts of the Mr 13,000 band detected in Arg-C-treated oLH samples. The alpha subunit was protected by association with the beta subunit, but free alpha subunit was rapidly degraded. Sequence analysis of nicked oLH beta indicated that one of the peptide bonds on either side of Arg43 was cleaved by the protease, with a slight preference for the amino side of this residue. Nicked oLH beta was reassociated with oLH alpha, and the resulting dimer was separated from unrecombined subunits. The biologic activity of nicked oLH beta + oLH alpha in an LH radioligand assay was only 2% that of intact oLH.     

Effects of the transmethylation inhibitor S-adenosyl-homocysteine and of the methyl donor S-adenosyl-methionine on rat Leydig cell function in vitro

In purified rat Leydig cells, the methyl donor S-adenosyl-methionine (SAM), increases significantly in a dose dependent manner the [125I]hCG binding as well as the productions of cAMP and of testosterone; the competitive inhibitor of methylations S-adenosyl-homocysteine (SAH), has an opposite effect. Associated to oLH, SAM further enhances the cAMP synthesis while SAH inhibits significantly the adenylate cyclase activity. With regard to testosterone synthesis, SAM potentiates the stimulating roles of oLH and dbcAMP (27 and 38% increases, respectively) although SAH diminishes testosterone productions (48 and 35%, respectively under oLH and dbcAMP stimulations). Scatchard analysis has shown that SAM (1.4 mM) increases the number of LH/hCG binding sites on Leydig cells while SAH (1.4 mM) decreases it; LH/hCG Ka values are not modified neither by SAM nor by SAH. These data suggest that the in vitro regulation of steroidogenesis in purified rat Leydig cells may involve methylation processes (presumably phospholipids are the potential substrates of these reactions) which modulates the transmission of the hormonal signal through the membrane and affects the testosterone synthesis at a step beyond the adenylate cyclase.     

Activin A and gonadotropin regulation of follicle-stimulating hormone and luteinizing hormone receptor messenger RNA in avian granulosa cells.

Activin A regulation of the expression of mRNA for the LH receptor, FSH receptor, and the inhibin alpha subunit as well as the effect of activin A on the secretion of progesterone were investigated in chicken granulosa cell cultures. Granulosa layers were isolated from the F(1) and F(3) + F(4) follicles from five hens, pooled according to size, dispersed, and cultured for 48 h. In experiment 1 (n = 3 replications), granulosa cells were cultured with or without highly purified ovine (o) FSH at 50 ng/ml and in the presence of 0, 10, or 50 ng/ml of recombinant chicken activin A. Experiment 2 (n = 4 replications) followed the same protocol as experiment 1, except that oFSH was replaced with oLH. Results from these experiments showed that addition of activin A to the granulosa cell cultures had no effect on the expression of mRNA for the inhibin alpha subunit or the FSH receptor, but it did affect the expression of mRNA for the LH receptor. Treatment of F(3) + F(4) granulosa cells with LH stimulated the expression of mRNA for the LH receptor; however, when LH was combined with either dose of activin A, this induction was prevented. The highest dose of activin A with or without LH resulted in decreased expression of the LH receptor compared to the untreated controls in the F(3) + F(4) cell cultures. Progesterone secretion by the granulosa cells from both follicle sizes was not altered by activin A. In experiment 3 (n = 3 replications), the effect of activin A on the growth of granulosa cells was examined with the following treatments: 0, 10, or 50 ng/ml of activin A; 50 ng/ml of either oLH or oFSH; and oLH or oFSH combined with 10 ng/ml of activin A. The highest dose of activin reduced the rate of granulosa cell proliferation in both follicle types. Growth of F(1) and F(3) + F(4) granulosa cells was stimulated by the addition of either gonadotropin, and the presence of 10 ng/ml of activin A with either gonadotropin did not alter this proliferation, except for the LH-treated F(3) + F(4) granulosa cells, in which the increase in proliferation was prevented. The results suggest that activin A could act as a local factor that regulates follicular maturation by preventing excessive or untimely LH receptor expression.