Porcine Luteinizing Hormone Research Articles

Chemical cross-linking of porcine luteinizing hormone: location of the cross-link and consequences for stability and biological activity

To study the interaction between the subunits of LH and determine which amino acid residues are involved in this interaction, porcine and ovine LH (pLH and oLH) were cross-linked with 0.02 M 1-ethyl-(3-(3-dimethylaminopropyl)carbodiimide to generate one specific intersubunit cross-link. The cross-linked hormone was separated from the noncross-linked dissociated subunits by gel filtration on a Sephadex G-75 column. The position of the cross-link in cross-linked pLH (X-pLH) was determined by sequencing peptide fragments that were generated by digestion with endoproteinase Arg-C. In accordance with previous data for bovine LH, the position of the cross-link was between alpha-Lys49 and beta-Asp111, indicating that these residues are at the subunit-subunit interface. The biological activity of cross-linked hormone was tested by radioreceptor binding assay. The receptor-binding activity of X-pLH was slightly reduced to 84%, suggesting that the conformational stability of X-pLH is similar to that of pLH as a result of the introduction of the covalent cross-link. The receptor-binding activity of X-oLH was decreased by approximately 30%, which we attribute to the formation of multiple cross-links within the ovine molecule, making the molecule more rigid, and to oligomeric forms, resulting from multiple intermolecular cross-links, that are not able to bind to the testicular LH receptor. This observation implies that the oLH has more amino-carboxyl functional groups that are sterically susceptible to carbodiimide cross-linking than does pLH under the reaction conditions used.

Expression of Human Luteinizing Hormone (LH) Receptor: Interaction with LH and Chorionic Gonadotropin from Human but not Equine, Rat, and Ovine Species

Studies on human LH receptors are difficult due to the limited availability of clinical samples. Recent cloning of rat and porcine LH receptor cDNAs indicated that these binding sites are single polypeptides of the G-protein-coupled receptor family with seven transmembrane domains. Based on the conserved sequences of rat and porcine receptors, we performed reverse transcription polymerase chain reaction, using human ovarian mRNA as template and obtained partial human LH receptor cDNA clones. Further screening of a human ovary cDNA library and subsequent ligation of individual cDNA clones generated a human LH receptor cDNA containing the entire amino acid-coding region. Sequence analysis indicated that the human receptor cDNA displays 89% and 82% homology at the nucleotide level with its porcine and rat counterparts, respectively. A region spanning the second extracellular and third transmembrane domains is highly conserved among the human LH, FSH, and TSH receptors. The ovarian LH receptor clone is, however, significantly different from an incompletely spliced LH receptor cDNA recently obtained from a human thyroid library. Unlike the thyroid clone, the ovarian LH receptor cDNA could be expressed in the human fetal kidney cell line (293), and radioligand receptor assay identified high affinity (Kd, 1.2 x 10(-10) M) LH/hCG-binding sites on the plasma membrane. Binding specificity of the human LH receptor was studied using recombinant human CG, LH, and FSH secreted by CHO cells transfected with the respective genes. Human CG and LH displaced [125I]hCG binding with an ED50 of 4.3 and 4.8 ng/ml, respectively. In contrast, recombinant FSH was not effective. Treatment of transfected cells with recombinant gonadotropins also induced dose-dependent increases in extracellular cAMP production (hCG = LH much greater than FSH; ED50 25, 10, and greater than 3000 ng/ml). Although equine, rat, and ovine LH as well as equine CG competed effectively for rat testicular LH receptor binding, these hormones were unable to displace [125I]hCG binding to the human receptor, suggesting evolutionary changes in receptor binding specificity and the importance of using human receptors for clinical studies. Thus, the cloning and expression of the human LH receptor cDNA allowed analysis of interactions between human LH receptor and gonadotropins from diverse species. The present work should provide the basis for future design of therapeutic agents capable of interacting with the human receptor and for understanding the structural basis for LH receptor binding to different gonadotropins.

Endocytosis of the lutropin receptor is mediated by a low affinity binding site

Porcine Leydig cells in primary culture were incubated to equilibrium with increasing doses of either porcine luteinizing hormone (pLH) or human chorionic gonadotropin (hCG). Then, free and total high affinity gonadotropin receptors on the cell surface were dosed with [ 125I]hCG as tracer. In isotonic high salt medium, the pLH concentration for half receptor occupancy ( K occ) and for half receptor endocytosis ( K endo) were nearly indistinguishable (1−3×10 −7 M). But, when the medium was changed to an isotonic low salt buffer, K occ shifted to 1.2×10 −9 M and K endo to 1.5×10 −8 M. However, with hCG, both values were largely independent of the ionic strength ( K occ = 10 −10 M and K endo = 10 −8 M). The fact that K endo is higher than K occ suggests that the endocytosis of the high affinity gonadotropin receptor is controlled by the hormone binding to another lower affinity site.

Downward Regulation of Plasma LH by LHRH Agonist, Leuprolide Acetate, Resulting in Inhibited Renal Growth and Function in the Castrated Male Rat.

We previously reported that ovine and porcine luteinizing hormone (LH) stimulated kidney growth in castrated hypophysectomized rats. Our present study focuses on the physiological role of the renotropic activity of LH isoforms. Plasma LH levels were decreased to 10% of that of castrated control rats by injections of a slow-releasing LHRH agonist, leuprolide acetate, from microcapsules. Compared to controls, which were injected with microcapsules only, the kidney weight in leuprolide-treated castrated rats decreased 12%. Renal protein and DNA contents decreased significantly. Body, liver and spleen weights were not changed by the treatment, however. This effect on the kidney was not observed in castrated hypophysectomized rats, suggesting that leuprolide affected the kidneys indirectly, rather than directly, by suppressing LH secretion. In leuprolide-treated castrated rats, urinary fractional excretion of sodium (FENa) increased, indicating suppressed renal function at the proximal tubules. We concluded that the secretion of renotropically active LH isoforms was regulated at least partially by LHRH and played a physiological role in growth and the function of the proximal tubules.

Potassium and chloride conductances in rat Leydig cells: effects of gonadotrophins and cyclic adenosine monophosphate.

1. The effects of gonadotrophins (luteinizing hormone and human chorionic gonadotrophin) and cyclic AMP on ionic conductances were investigated using the tight-seal whole-cell recording technique in Leydig cells freshly isolated from nature rat testis by enzymatic treatment. 2. In resting cells, the predominant ionic conductance is a voltage-dependent K+ conductance resembling the delayed rectifier K+ conductance of T-lymphocytes. This conductance is characterized by: (1) a time-dependent inactivation for potentials more positive than +20 mV, (2) a reversal potential near -65 mV, (3) a sensitivity to intracellular Cs+, and (4) a sensitivity to extracellular TEA and 4-aminopyridine. 3. A Cl- conductance is also present resembling the Cl- background conductance in squid axons and heart cells. In resting cells, this conductance contributes only a small component of the total outward current obtained with depolarizing pulses. 4. Gonadotrophins (human chorionic gonadotrophin, porcine luteinizing hormone and ovine luteinizing hormone) have little effect on the K+ conductance. They transiently increase a Cl- conductance after a delay of up to 30 s. This response does not occur if the hormones are applied late in the whole-cell recording. Gonadoliberine (GnRH) does not affect the Cl- or K+ conductance. 5. Internal cyclic AMP (100 microM) mimics all these effects while internal application of a GTP-ATP mixture induces a similar response, which is, however, sustained rather than transient. 6. The Cl- conductance was studied quantitatively with a GTP-ATP internal solution. This conductance is activated by depolarizing voltage steps to test potentials of -40 mV or more. Under these conditions, the instantaneous current observed as soon as the depolarizing pulse is applied displays outward rectification and reverses near ECl. During the pulses, a strong inactivation is observed for potentials greater than +40 mV. This conductance is independent of external and internal calcium. 7. It is concluded that the gonadotrophins act through a cyclic AMP-dependent process to activate a Cl- conductance. This conductance is different to the hyperpolarization-activated Cl- conductance and the calcium-activated Cl-conductance also present in the membrane of resting cells.

Cloning of the LH/CG receptor implications for a unique G-protein coupled receptor

The proteins encoded by the rat and porcine luteinizing hormone (LH)lchorionic gonadotropin (CG) receptor cDNAs appear to be unique members of the G -protein-coupled receptor family. Although they have the characteristic seven transmembrane domains, the LH/CG receptors have relatively low homology to other members o f this family, do not have a G-protein-coupling domain corresponding to that of the a-adrenergic receptor, and contain an unusual long extracellular domain. Experience in the molecular dissection of this receptor family will help guide investigation of the LHICG receptor's functional domains. Further studies are needed to clarify the origin and significance of the various mRNA species hybridizing on Northern blot analyses. Finally, the rat and porcine receptor cDNAs should permit cloning of the human LH/CG receptor, as well as cloning of the thyroid-stimulating hormone and follicle-stimulating hormone receptors.

Localization of the human luteinizing hormone/choriogonadotropin receptor gene (LHCGR) to chromosome 2p21

Probes corresponding to human and porcine LH (luteinizing hormone) receptor cDNA were used for in situ hybridization to human chromosomes. This allowed us to assign the LH receptor gene to chromosome 2p21.

Cloning and DNA sequence analysis of the cDNA for the precursor of porcine luteinizing hormone (LH) β subunit

The cDNAs encoding the porcine luteinizing hormone (LH) β subunit (pLHβ) were isolated from a cDNA library of porcine anterior pituitary constructed in an expression vector λgt11 using anti-pituitary glycoprotein hormone antisera. The nucleotide sequence of pLHβ subunit cDNA clone (543 bases) was determined. The cDNA encodes a signal sequence (20 amino acids) and a further 121 amino acids corresponding to the mature LHβ molecule. The predicted protein sequence differs from that determined by direct peptide sequencing at 6 residues and additionally shows an extended carboxyl terminus. The 5′ untranslated sequence shows a low homology and is extremely long when compared with other mammalian LHβ subunit cDNAs. Northern analysis showed that the length of the pLHβ subunit mRNA is about 0.85 kb.

Determination of porcine plasma follitropin levels during superovulation treatment in cows

Porcine follicle stimulating hormone (pFSH) and porcine luteinizing hormone (pLH), are widely used to induce superovulation in cows. An advantage of this treatment is that the LH:FSH ratio can be varied to optimize the growth of the ovarian follicles. However, due to the relatively short half-life of FSH, the superovulatory treatment requires numerous injections. A performant radioimmunoassay system (sensitivity=0.2 ng/ml plasma) was used to determine plasma pFSH levels in cows that were superovulated with 2 daily injections of 4 Armour Units (A.U.) of pFSH for 4 d. From plasma profiles, the half-life and the disappearance of pFSH were estimated at 5 h and at 10 to 12 h, respectively, confirming the necessity of using two daily injections.

The effect of estradiol with Synchro-Mate-B in the superovulation of beef heifers with FSH-P

A study was conducted to determine whether luteal tissue formed after administration of exogenous luteinizing hormone (LH) was capable of maintaining pregnancy in the absence of the original corpus luteum (CL) of pregnancy in heifers. Nineteen beef heifers, confirmed pregnant by ultrasonography approximately 28 days after breeding, were randomly assigned to one of two groups. Nine heifers remained as untreated controls to determine the incidence of spontaneous embryonic or fetal loss. Each of the remaining ten heifers was given two Syncro-Mate-B (SMB) ear implants (12 mg of norgestomet) between 30 and 35 days after breeding (day of SMB implant was Day 1). An additional SMB was implanted on Day 8 and another on Day 15. All implants were removed on Day 20. On Day 2, SMB-implanted heifers were given an intramuscular (i.m.) injection of 500 μg of cloprostenol to induce luteal regression. On Day 5, regression of the CL was confirmed by ultrasonography and these heifers received a highly purified porcine LH preparation (50 mg Armour, i.m.); an additional 25 mg was injected i.m. on Day 6. Ovulation occurred in all ten LH-treated heifers and was detected on Day 7.2±0.1 (mean±SEM). In five of ten LH-treated heifers, the induced CL regressed, and was last detected by ultrasonography on Day 15.8±0.4 (range, Day 15–17). Pregnancy was not maintained in these heifers after SMB implants were removed. Pregnancy was maintained until Day 40 (end of experiment) in the remaining five LH-treated heifers and in all nine control heifers. In all five heifers in which the induced CL regressed, this CL had formed in the ovary contralateral to the original CL of pregnancy. In contrast, in four of five heifers in which the induced CL was maintained, this CL had formed in the ovary ipsilateral to the original CL of pregnancy. Although LH induced ovulation and CL formation in the absence of the CL of pregnancy and in the presence of continuous progestagen support, short-lived CL were formed in five of ten heifers.

Purification and characterization of luteinizing hormone from the dromedary ( Camelus dromedarius)

Luteinizing hormone (LH) has been purified from 150 dromedary pituitaries and its partial physicochemical, biological and immunological characterization has been achieved. Purification of the hormone was monitored by a porcine LH radioreceptor assay (RPA). In this system, the final camLH preparation exhibited an activity 0.6-fold that of highly purified porcine LH. The acid half-dissociation of camLH at equilibrium was observed at pH 4.2. A homologous camLH RRA was developed using the testicular plasma membrane fraction from prepubertal camels and radioiodinated, highly-purified camLH. Pituitary and chorionic gonadotropins (CG) from several mammalian species were compared to camLH in this system. The equine gonadotropins eLH and eCG were shown to be 6 times less potent in the camel RRA than in the porcine RRA, whereas the LH from other species exhibited similar activities in both systems. This particularity of camel LH receptors offers a new tool for the study of structural features of gonatropin interactions with their receptors.

Functional states of the luteinizing hormone/choriogonadotropin-receptor complex in rat Leydig cells.

Three classes of gonadotropins with different ratios of stimulating to binding activities (S/B ratio) in rat Leydig cells have been identified. An S/B ratio of 1 was observed for rat luteinizing hormone (LH), porcine LH, and equine choriogonadotropin (CG) (class I), whereas ovine and equine LH exhibited and S/B ratio of 10-20 (class II) and human CG (hCG) (class III) an S/B ratio of 60. We coined the term "superactivity" to designate this particular behavior. This phenomenon was further studied by comparing the competitive activities of porcine LH (pLH) and hCG in radioreceptor assays using rat Leydig cell membranes and either radiolabeled oLH or hCG as the tracer, in the presence or absence of 150 mM NaCl. At equilibrium, both native hormones were equipotent in competing with 125I-oLH binding, but hCG was 4-fold more potent than pLH when 125I-hCG was used. Moreover, the binding rates of both hormones were considerably diminished in the presence of NaCl, but hCG binding at equilibrium was not affected, whereas that of oLH was almost completely abolished. From these results and previous data on the binding and internalization of these hormones, we suggest the existence of two interconvertible functional states of the hormone-receptor complex: (formula; see text). The equilibrium constant k3/k4 would be extremely high for hCG and lower and lower for the hormones in class II and class I, respectively. The equilibrium constant k1/k2 would be the one affected by the presence of NaCl and seems to be similar for all the hormones tested. The normal activity or superactivity of gonadotropins would thus be primarily dependent on the equilibrium between HR1 and HR2.

Rapid in vitro desensitization of the testosterone response in rat Ley dig cells by sub-active concentrations of porcine luteinizing hormone

We have studied in rat Leydig cells, the effect of sub-active concentrations of porcine LH on the subsequent stimulation of the cAMP and testosterone production by a sub-maximal concentration of pLH or hCG. We found that extremely low concentrations of pLH (0.01–2.0 ng ml ) were able to induce rapidly a partial but highly significative desensitization of the testosterone response without affecting the cyclic AMP response. These data indicate that desensitization of the steroidogenic response might be due to some lesion beyond cAMP formation or at the level of one discrete compartment of cyclic AMP, directly involved in the control of steroidogenesis. Moreover, our data strongly suggest that the basal circulating concentrations of LH can exert an inhibitory control on the testosterone response to LH pulses in vivo.

Action of nitroguanidinated luteinizing hormone on different rat gonadal tissues

The biological activities of nitroguanidinated derivatives prepared from ovine or porcine luteinizing hormone were investigated using rat Leydig cells and pseudopregnant rat ovaries. In these tissues nitroguanidyl ovine luteinizing hormone (NGoLH) or nitroguanidyl porcine luteinizing hormone (NGpLH) were unable to stimulate adenylate cyclase or steroidogenesis but were able to inhibit the binding of ovine or porcine native LH to their specific receptors. When added to incubations of isolated Leydig cells or pseudopregnant ovary slices, NGoLH as well as NGpLH inhibited the stimulating action of native LH on adenylate cyclase or steroidogenesis. However, these derivatives had no inhibiting action on the stimulation of adenylate cyclase and steroidogenesis induced in the Leydig cells by choleratoxin or on the stimulation of testosterone production induced by 8-bromo-cyclic AMP. Since NGpLH (which does not contain lysine residues or free α-amino groups in the β-subunit) exhibits the same antagonist action as NGoLH, we conclude that the nitroguanidination of the α-subunit is sufficient to endow the derivative with antihormone properties.

Purification of equine gonadotropins and comparative study of their acid-dissociation and receptor-binding specificity

A method for the simultaneous purification of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) from equine pituitaries is briefly described. Different forms of each hormone were obtained. The total yield of LH was 24.2 mg·kg −1 with a recovery of 22% and the yield of FSH was 26 mg·kg −1 with a recovery of 34%. The specific activities of both hormones, measured in homologous equine radio-receptor assays are equal to or higher than those of the preparations described so far. In all species studied so far the acid-dissociation curves of LH and FSH are similar; this is an agreement with the view that the binding of the common α-subunit and the specific β-subunits involves polypeptide regions which are identical in both hormones. In contrast, the acid-dissociation p K a of equine LH was found to be considerably lower (3.9) than that of equine FSH (5.8). The equine gonadotropins exhibit a much lower specificity with receptors of a porcine testicular fraction compared with an equine fraction. Equine LH exhibited a binding activity on FSH receptors from a porcine testicular fraction equal to 20% that of equine FSH instead of only 1% for an equine binding fraction. Similarly, all the equine FSH preparations tested exhibited a five-fold higher binding-activity on porcine LH receptors than on equine LH receptors. In the porcine system, pregnant mare serum gonadotropin behaved like equine LH towards LH and FSH receptors. In contrast, on equine binding fraction, pregnant mare serum gonadotropin was only 4% as active as equine LH and was devoid of FSH activity. All the data we have obtained are consistent with the ‘negative specificity’ model we proposed recently.

Porcine LH levels during the estrous cycle, gestation, parturition and early lactation.

Thirty-five sows were used in a study of plasma luteinizing hormone (LH) concentrations during the estrous cycle, including first and second estrus postweaning, during gestation, at parturition and during the first 5 d of lactation. After cannulation of the anterior vena cava via the cephalic vein, blood samples were collected four times daily for 2 d before estrus, during estrus and for 3 d after estrus and once daily at 1100 h throughout the rest of the estrous cycle. One sample was collected from each sow on alternate days (1100 h) during the gestation period. Four daily samples were obtained on d 60 through 68 of gestation and for 5 d after parturition. Preovulatory surges of LH occurred 8 to 32 h before standing estrus in four of seven sows during first estrus and in three of seven sows during second estrus. The mean preovulatory LH surge during the first estrus postweaning (3.00 +/- .46 ng/ml) was lower than that during second estrus (4.24 +/- .60 ng/ml). Throughout the first 12 d of gestation, LH concentrations were relatively high and variable, ranging from 1.2 to 2 ng/ml. They then decreased to a range of .42 to .62 ng/ml after d 24. Similar LH concentrations were noted throughout the remainder of gestation except for some slight fluctuations. Higher concentrations (P less than .05) were noted with evening collections (1900 h, 1:17 +/- .09 ng/ml) than in daytime collections (0700 h, .64 +/- .08: 1100 h, .61 +/- .06; 1500 h, .72 +/- .06 ng/ml) during d 60 to 68 of gestation. Low plasma concentrations of LH (.3 to .5 ng/ml) were observed for the 5 d before and during parturition. After parturition, considerable variation of LH occurred, with rising concentrations observed on d 5 postpartum. Results of this study illustrate the occurrence of LH surges before behavioral estrus with increased plasma LH during implantation.

DEVELOPMENT AND APPLICATION OF HOMOLOGOUS RADIOIMMUNOASSAYS FOR PORCINE GONADOTROPHINS

Antisera were raised against highly purified preparations of porcine luteinizing hormone (pLH) and follicle-stimulating hormone (pFSH). Highly specific and sensitive radioimmunoassay systems were developed. The antisera to LH and FSH were used at working dilutions of 1:500,000 and 1:200,000 respectively and the sensitivities of the assays were 0.1 ng LH/ml serum (3 x 10(-12) mol/l) and 0.5 ng FSH/ml serum (1.5 x 10(-11) mol/l). The LH and FSH preparations used as standards were 1.2 and 81 times as potent as NIH-LH-S15 and NIH-FSH-P1 respectively. Both assays were validated and adapted for the measurement of the gonadotrophin content of porcine serum. The concentrations of LH and FSH in blood were measured simultaneously in prepubertal sows throughout a 24 h period, in adult sows during the oestrous cycle and in both prepubertal and adult animals after treatment with LH releasing hormone.

SOLID PHASE SYNTHESIS OF PORCINE LH‐RELEASING HORMONE WITH HYDROGEN CHLORIDE IN FORMIC ACID AS THE DEBLOCKING REAGENT*

The decapeptide corresponding to the amino acid sequence of porcine luteinizing hormone-releasing hormone (LH-RH) which involves 1 mol of tryptophan was synthesized via solid phase synthesis with two different deblocking procedures which used hydrogen chloride in formic acid and hydrogen chloride in acetic acid containing 1% 2-mercaptoethanol. After some fundamental studies on the former reagent with respect to deblocking efficiency toward the Boc group, 0.5 M hydrogen chloride (a 10-fold molar excess with respect to the N-terminal Boc group) in formic acid was used in the present synthesis. The two synthetic products exhibited the same chemical and biological properties as an authentic LH-RH. Hydrogen chloride in formic acid has proved effective without a scavenger although loss of peptide from the resin occurred to a somewhat greater extent than that with hydrogen chloride in acetic acid. A derivative of the synthetic LH-RH formylated at the indole nitrogen had a greatly diminished biological activity, indicating that the intact indole side chain is essential for the activity.

The Specificity of Gonadotropin Binding by the Human Corpus Luteum

The specificity of gonadotropin binding was studied in fresh and frozen human corpora lutea. Ovine, bovine, and porcine luteinizing hormone (LH) competed with 125I-labeled human LH (125I-hLH) and 125I-labeled human chorionic gonadotropin (125I-hCG) for binding to tissue receptors in homogenates of human corpora lutea frozen for 3 to 12 months. In contrast, oLH, bLH, and pLH competed minimally for 125I-hLH and 125I-hCG binding sites in homogenates of fresh human corpora lutea. Ovine follicle-stimulating hormone (FSH) and thyroid-stimulating hormone (TSH) did not compete in homogenates of fresh or frozen tissue. Competition of oLH and hCG for 125I-hCG binding sites at several dose levels in a homogenate of a fresh corpus luteum was studied. One hundred micrograms of oLH and ten nanograms of hCG gave an equivalent competition--a 10,000-fold difference in competitive potency. Only hCG competed with 125I-hCG for binding when the competition of oLH, bLH, pLH, oFSH, oTSH, hCG and hCG subunits, and hCG were compared at the 10-mug level in a homogenate of fresh human corpus luteum. The binding of 125I-labeled homologous human hormones by the corpus luteum was examined in a limited fashion. 125I-Prolactin did not bind to preparations of fresh stroma from a patient with polycystic ovaries nor did it bind to three separate preparations of fresh corpora luteum which did bind 125I-hCG. 125I-hTSH did not show significant binding to a fresh human corpus luteum preparation which did bind 125I-hCG. These studies indicate that the gonadotropin receptor of the fresh human corpus luteum possesses a unique species specificity and illustrate the importance of working with human corpora lutea in their most native state.

Porcine thyrotropin. The amino-acid sequence of the alpha and beta subunits.

The amino acid sequences of both the alpha and beta subunits of porcine thyrotropin have been studied. Bovine thyrotropin primary structure was taken as a model for ordering the tryptic peptides of porcine thyrotropin. The amino acid sequence of the alpha subunit is identical to that of porcine luteinizing hormone, while oligosaccharide side-chains differ in composition. The primary structure of the beta subunit differs from that of bovine thyrotropin by six amino acid replacements, in positions 22, 24, 26, 36, 62 and 69, and by the absence of a methionyl residue at the carboxy terminus. Chemical evolutions of thyrotropin and luteinizing hormone are compared.