Rat Luteinizing Hormone Research Articles

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

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

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.

Regulation of rat luteinizing hormone subunit messenger ribonucleic acids by gonadal steroid hormones.

Little is known about the hormonal regulation of luteinizing hormone (LH) biosynthesis. We have studied the regulation of LH messenger RNA (mRNA) levels by gonadal-steroid hormones in the rat. In one set of experiments, male and female rats were surgically gonadectomized (GDX) and killed 1, 3, 7, 14, 22, and 31 d postoperatively. In another set of experiments, male and female rats were surgically GDX and were injected subcutaneously with testosterone propionate (500 micrograms/100 g body wt per d) or 17 beta-estradiol 3-benzoate (10 micrograms/100 g body wt per d), respectively, beginning 3 wk postoperatively. Levels of serum LH were determined by radioimmunoassay and levels of LH subunit mRNAs in single pituitary glands were determined by blot hybridization analysis using labeled synthetic oligodeoxyribonucleotide probes that correspond to portions of the coding regions of the rat alpha- and LH beta-subunit mRNAs. 4 wk after gonadectomy, serum LH levels rose nine- and 20-fold, while alpha-subunit mRNA levels rose six- and 10-fold, and LH beta-subunit levels rose seven- and 14-fold, compared with controls in males and females, respectively. In gonadal-steroid hormone-treated male and female GDX rats, serum LH levels fell to 8 and 36% of control values, while alpha-subunit mRNA levels declined to 22 and 19%, and LH beta-subunit mRNA levels declined to 6 and 10% of control values, 48 h after injections were initiated, in males and females, respectively. We conclude that gonadal-steroid hormones negatively regulate the levels of both subunit mRNAs in GDX rats in a pattern that parallels the changes in serum LH values. These data suggest that gonadal-steroid hormone regulation of LH biosynthesis occurs, at least in part, at the level of LH subunit mRNAs due to effects at the transcriptional and/or RNA stability levels.

Aluminum alters the permeability of the blood-brain barrier to some non-peptides

The effect of aluminum administered intraperitoneally (i.p.) on the levels of peripherally injected 99mTc labelled red blood cells in brain and on the penetration of the blood-brain barrier by radioiodinated serum albumin (RISA), thyroxine, iodide, cortisol, N-Tyr-delta sleep-inducing peptide ( N-Tyr-DSIP), growth hormone, thyroid stimulating hormone (TSH), prolactin and human and rat luteinizing hormone was examined. Treatment with aluminum did not alter the brain/blood ratio for either 99mTc red blood cells or RISA, although it did increase the blood levels of RISA. These results show that aluminum caused a contraction in the volume of plasma without altering the vascular space of the brain, disrupting the blood-brain barrier, or increasing the “leakiness” of the blood-brain barrier. Aluminum enhanced the permeability of the blood-brain barrier to labelled prolactin, thyroxine, cortisol, growth hormone, N-Tyr-DSIP and rat luteinizing hormone, but not to labelled TSH, iodide, or human luteinizing hormone, a substance with an octanol coefficient markedly different from that of luteinizing hormone from the rat. Incubation of the peptide with aluminum before injection did not increase penetration, demonstrating that aluminum did not increase the permeability of the blood-brain barrier by acting directly on the peptide. Aluminum, administered intraperitoneally, increased the accuracy of lipophilicity as a predictor of penetration of the blood-brain barrier, but the greatest increase in penetration was seen with thyroxine, a substance which crosses the blood-brain barrier by carrier-mediated transport. These results sugest that aluminum acts directly on the blood-brain barrier to enhance the permeability of membranes to lipophilic material and may also enhance carrier-mediated transport mechanisms, but that it does not disrupt the blood-brain barrier or increase its “leakiness”.

Influence of bilateral cryptorchidism on rat pituitary luteinizing hormone charge microheterogeneity.

Chromatofocusing was used to characterize the isohormones of rat luteinizing hormone (rLH) in extracts of pituitaries from intact and bilaterally cryptorchid animals. Pituitary extracts contained at least seven isoelectric variants (isohormones) or rLH: one eluted in the column void volume (Isohormone, I, pI greater than 9.8), five exhibited apparent pI's in the range of 9.25 to 8.97 (designated as Isohormones II-VI) and one bound to the column but eluted with 1.0 M NaCl (Isohormone VII, pI less than 7.0). In both intact and cryptorchid rats, a large percentage of the rLH was present as Isohormone I. Furthermore, Isohormone I was present in greater absolute and relative amounts in cryptorchid rats. All seven rLH isohormones exhibited activity in an in vitro bioassay and the biological to immunological assay (B:I) ratios decreased with their apparent pI's but were not significantly affected by cryptorchidism. When the data were expressed as the product of the amount of rLH and the B:I ratio (termed Bio-Index), Isohormone I was the predominant form of rLH in both cryptorchid and intact rats, with the Bio-Index of Isohormone I being significantly greater for cryptorchid rats. These results suggest that cryptorchidism alters the pattern of rLH isohormones in the pituitary, yielding a greater percentage of the most basic rLH isohormone, which also has the highest B:I ratio. Thus, cryptorchidism significantly alters the qualitative pattern as well as the quantity of rLH in the pituitary.

The gene encoding the beta-subunit of rat luteinizing hormone. Analysis of gene structure and evolution of nucleotide sequence.

The nucleotide sequence of the gene encoding the beta-subunit of rat luteinizing hormone (LH beta) has been determined from a genomic DNA fragment cloned in lambda phage Charon 4A. Blot hybridization of restriction enzyme digests of rat genomic DNA indicates that the gene is present in a single copy. The transcriptional unit is 0.98 kilobase in size and contains three exons interrupted by two introns of 245 and 225 base pairs (bp). The locations of the exon/intron junctions at amino acid codons -16/-15 and +41/+42 have been conserved between the rat LH beta gene and the related genes, human LH beta and human chorionic gonadotropin beta. Using S1 nuclease mapping and oligonucleotide-primed reverse transcription of ovariectomized rat pituitary mRNA, the start of transcription was determined to be 7 bp upstream from the start of translation. Characteristic promoter elements are present in the 5'-flanking region of the gene, including the Goldberg-Hogness sequence, TATAAA, 31 bp, and the consensus CAAT box sequence, 167 bp upstream from the start of transcription, respectively. Within the proximal 200 bp flanking the 5'-region of the transcriptional unit, there is strong homology between the rat and human LH beta genes, suggesting that these regions include sequences which may be important for regulation of gene expression. Isolation and characterization of the rat LH beta gene further defines the evolution of glycoprotein hormone genes and will facilitate the study of cellular and molecular mechanisms which regulate LH beta gene expression.

Monoclonal antibody LICR-LON-E36 recognizes gonadotrophs in female rat pituitaries.

The distribution of the antigen localized by monoclonal antibody LICR-LON-E36 has been studied by means of light and electron microscopical immunocytochemical procedures on resin-embedded pituitaries from male and female rats. Using both immunoperoxidase and immunogold labeling techniques, the localization of LICR-LON-E36 has been compared with that obtained with antibodies against the beta subunit of rat luteinizing hormone (beta LH) and human follicle stimulating hormone (beta FSH), porcine adrenocroticotropic hormone (ACTH) and rat S-100. LICR-LON-E36 is localized in a portion of beta LH-containing cells in female rat pituitaries and where present both antigens are localized within the same storage granules. LICR-LON-E36 is rarely detectable within beta LH-containing cells of male rat pituitaries that also stain positively with anti-beta FSH antibodies. ACTH and S-100 were localized within different cell populations.

Reanalysis of antisera specificities and binding characteristics of rat pituitary hormone assays.

Rat pituitary hormone radioimmunoassays (RIAs) are widely used in reproductive research, yet data on specificity and binding characteristics of many of the antisera are not widely available. This report characterizes one set of rat antisera supplied by the National Institutes of Health (USA). Rat follicle-stimulating hormone (FSH) and thyrotropin-stimulating hormone (TSH) antisera appear specific, but TSH exhibited significant competition in the rat luteinizing hormone (LH) assay. In addition, statistically significant nonparallelism was demonstrable in all three assay systems. This creates further problems in characterizing antisera cross-reactivity and may make potency estimates for pituitary standards inaccurate.

LH responses to LHRH in perifused pituitary cell culture: sex differences in the rat.

The present experiments were designed to study male and female luteinizing hormone (LH) secretory patterns following pulsatile or continuous LH-releasing hormone (LHRH) administration using a perifused dispersed rat anterior pituitary cell culture system. In male cells, consistent LH responses were elicited by hourly LHRH pulses (30 pmol), whereas in the female cells (proestrus or diestrus I), a statistically significant (P less than or equal to 0.01) decrease in the LH secretion occurred with successive LHRH pulses. Proestrous cells secreted significantly (P less than or equal to 0.01) more LH than diestrous cells during the first 24 h but not after 48 h in culture. When exposed to a 6-h continuous infusion of LHRH (10 nM), male cells released LH in a single phase and female cells secreted LH in a biphasic pattern. These data suggest that significant sex differences exist in the rat LH secretory pattern elicited by LHRH in vitro.

Subpopulations of luteinizing hormone (LH) possessing various ratios of bioactivity to immunoreactivity in the female rat pituitary glands and their changes during the estrous cycle.

Twenty-four adult female Sprague-Dawley rats (3 from each of 8 litters), showing 4-day cycles, were used in the present study. Aqueous extracts of pools of 6 pituitary glands in each cycle date were fractionated with a column isoelectrofocusing (IEF) technique, pH range of 3.5-10. Biological and immunological LH activities were determined by an in vitro bioassay and a radioimmunoassay, respectively, in the original aqueous extracts of the pituitary glands and in the fractions separated by IEF. Pituitary content of LH was the highest in the proestrus before the preovulatory LH surge (1243.7 +/- 67.8 micrograms NIAMDD rat LH-RP-1/pituitary gland for the biological activity). In the estrus, after the LH surge, it was reduced to 688.9 +/- 51.2 micrograms/pituitary gland. The decreased pituitary content was recovered to the level in the proestrus during the metestrus and the diestrus (1047.0 +/- 53.8 and 1173.0 +/- 58.5 micrograms/pituitary gland, respectively). Rat LH in the pituitary aqueous extracts was separated into multiple subpopulations in terms of pI values by IEF; i.e. Subpopulations A (pI = 10.3), B (9.3), C (9.0), D (8.7), E (8.3), F (neutral LH), and G (acidic LH). Among them the most predominant one was Subpopulation A throughout the estrous cycle. Subpopulations A, B and C exhibited statistically significant cyclic changes as was observed in the pituitary LH content, whereas the remaining ones stayed at constant levels during the cycle. The highest ratio of biological to immunological LH activities (B/I ratio) was obtained in Subpopulation A (6.41), followed by G, C and B (5.15, 4.24 and 3.99, respectively). Depressed B/I ratios were revealed in D, E and F (2.59, 1.86 and 3.07, respectively). High alkaline LH subpopulations, i.e. A, B and C, preserving high biological potency and showing cyclic changes during the estrous cycle, seem to be the releasable types of the hormone and to be mainly discharged for the preovulatory LH surge. Although characteristic features of other types of the hormone are not known, it is possible that one of them, presumably the acidic LH, might be the newly-synthesized type of the hormone, which might attain releasability by certain molecular modifications involving a shift in the pI value.

Immunofluorescence localization of glycoprotein hormones in the rat anterior pituitary gland using monoclonal antibodies and polyclonal antisera.

A hybridoma-derived monoclonal mouse antibody against bovine luteinizing hormone (LH) cross-reacting with rat LH and conventional polyclonal rabbit antisera against rat follicle-stimulating hormone (FSH) and thyroid-stimulating hormone (TSH) were used for the localization of the cells producing these hormones in rat pituitary glands by the immunofluorescence double staining technique. This approach enables the simultaneous identification of two different hormones on a single tissue section. It was shown that LH and FSH are produced within the same cell type, whereas TSH is synthesized by a different cell.

Further characterization of the molecular species formed during the biosynthesis of rat luteinizing hormone.

During the biosynthesis of rat LH (rLH) there are four molecular species found intracellularly: native rLH, a species which co-elutes with rLHalpha on Sephadex G-100, a species which elutes with an apparent molecular weight of approximately 21 000 and a large molecular weight form (excluded from Sephadex G-100). Significant quantities of native rLH and rLHalpha are secreted. The species of large molecular weight binds to Concanavalin A-Sepharose suggesting that it is glycosylated. The 21 000 molecular weight rLH-like molecule has tentatively been identified as an rLH subunit which has not been completely processed.

Selectivity of melatonin pituitary inhibition for luteinizing hormone-releasing hormone.

The pineal indole melatonin suppresses the neonatal rat luteinizing hormone (LH) and follicle-stimulating hormone (FSH) responses to LH-releasing hormone (LHRH), as shown in previous studies from this laboratory. We show in this study that the melatonin inhibition is a selective effect and is not due to general inhibition of pituitary function. The effects of the indole on the responses to thyrotropin-releasing hormone (TRH) and somatostatin (SRIF) and on basal pituitary hormone secretion were examined with cells in culture. Neonatal rat anterior pituitary cells dissociated with collagenase and hyaluronidase were cultured overnight and distributed to 35-mm dishes at the time of use. For examination of melatonin effects on the response to releasing hormones, the cells were incubated for 3 h in control medium or medium containing LHRH (10-9-10-6 M), TRH (10-10-10-6 M), or SRIF (10-9-10-6 M), either alone or in the presence of melatonin (10-8 or 10-6 M). For examination of basal hormone secretion, the cells were incubated for 1.5, 3, 6, 15, or 24 h in either medium alone or medium containing melatonin (10-6 M). Medium and cell lysate concentrations of LH, FSH, thyroid-stimulating hormone (TSh), prolactin (PRL) and growth hormone (GH) were determined by double antibody RIA. As previously, melatonin (10-8 M) significantly suppressed LH and FSH release by all concentrations of LHRH. This concentration of the indole produced maximal suppression of both LH and FSH responses to LHRH. By contrast, melatonin at a 100-fold greater concentration (10-6 M) had no effect on TRH stimulation of TSH or PRL release or on SRIF inhibition of GH release. Similarly, melatonin had no effect on basal release of TSH, PRL, or GH at the times examined. These findings show that melatonin inhibition of the gonadotroph response to LHRH is a selective effect.

Production and Characterization of Monoclonal Antibodies against Bovine Luteinizing Hormone

Five mouse hybridoma cell lines producing monoclonal antibody against bovine luteinizing hormone (LH) have been established and the respective antibodies characterized by radioimmunoassay, immunofluorescence and immunoelectrophoresis. All antibodies belong to the IgG class and bind to staphylococcus protein A. Intraspecies cross-reactivity studies revealed no reaction with bovine follicle stimulating hormone (FSH). However, all antibodies showed partial cross-reaction with bovine thyroid stimulating hormone (TSH) suggesting a close conformational similarity between bovine LH and TSH. Studies on interspecies cross-reactivity (rat and human) showed that three of these five antibodies strongly react with rat LH but not at all with either rat FSH or rat TSH thus representing monospecific reagents for investigations concerning LH in this species. One of these three antibodies also strongly binds to human LH and to the same extent to human chorionic gonadotropin (CG) but not to human FSH or TSH. It was concluded that at least three different epitopes on the bovine LH molecule are recognized and that they are located on the β-chain of the hormone.

Hypothalamic Luteinizing Hormone (LH): Characteristics and Response to Hypophysectomy

This study describes the finding of an immunoassayable and bioassayable luteinizing hormone (LH) in the rat hypothalamus. Validation of the immunoassay forLH inhypothalamicextractsis presented. Chromatographic patterns of hypothalamic LH were found to be identical with those of rat pituitary LH. Radioimmunoassay values of serial dilutions of hypothalamic extract paralleled dilution values of rat pituitary LH. Furthermore, such homogenates promoted release of testosterone from dispersed rat testis cells in vitro. This effect was largely abolished by prior incubation of homogenate with an antiserum to rat LH. Following removal of the anterior pituitary, LH levels in the hypothalamus persist but drop significantly, signifying that this particular brain-based LH is partially dependent on the presence of a functioning pars distalis. Immunoassayable LH measured in other areas of the rodent central nervous system did not change following hypophysectomy.

Localization of gonadotrophic hormones in the pituitary gland of the California leaf-nosed bat (Macrotus californicus).

Luteinizing hormone (LH) and follicle-stimulating hormone (FSH) cells were immunostained in the anterior pituitary lobe of the California leaf-nosed bat with antisera against the specific beta (beta) subunits of rat LH and rat FSH employing the unlabeled immunoperoxidase technique. Selective immunochemical staining by both antisera was localized primarily in the same cell type. However, some cells consistently reacted solely with antisera to either LH-beta or FSH-beta. Whereas the immunoreactive LH-beta cells, in general, stained more intensely than FSH-beta cells they were usually less numerous. These cells were alcian-blue and PAS-positive, staining pink to purple. The LH/FSH cells were pleomorphic with eccentric nuclei, often with cytoplasmic extensions. They were found throughout the pars distalis.

Cell-free synthesis and processing of the precursors to the subunits of luteinizing hormone.

Polyadenylated RNA prepared from pituitary glands of ovariectomized rats was translated in heterologous cell-free systems derived from wheat germ and rabbit reticulocytes in the absence and in the presence of pancreatic microsomal membranes. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis demonstrated that the major 14,000-dalton product immunoprecipitated by antisera to the alpha subunit of rat and bovine luteinizing hormone from membrane-free translations was processed to a major 21,000-dalton and a minor 17,000-dalton product in the presence of microsomal membranes. These membrane-dependent products were sensitive to cleavage by the glycosidase endo-beta-N-acetylglucosaminidase H, but the 14,000-dalton product was resistant. The 21,000-dalton product was sequestered within the microsomal vesicles as shown by its resistance to limited proteolysis capable of digesting the 14,000-dalton precursor. Glycosylated products with apparent Mr = 18,000 and 17,000 were identified from membrane-supplemented translations by immunoprecipitation using antisera to the beta subunit of rat luteinizing hormone. The immunological identity of the glycosylated subunits of luteinizing hormone was confirmed by showing competition of the immunoprecipitation reactions with unlabeled subunits.

Bacterial peptides with C-terminal similarities to bovine neurotensin

Using radioimmunoassay, peptides resembling the C-terminal region of bovine neurotensin (NT) have been demonstrated in acid/acetone extracts of Rhodopseudomonas palustris, Escherichia coli, and Caulaobacter crescentus. The NT-like bacterial components were shown to behave as peptides of small molecular weight (less than 2000) which were stable to acid and heat but labile to proteolytic digestion. In the radioimmunoassay toward NT they displayed dose-response curves parallel to standard and gave results indicating a competitive type of interaction with NT binding sites on antibody. The bacterial extracts did not register in a control radioimmunoassay toward rat luteinizing hormone. Some of the NT-like immunoreactivity could also be bound to an retrieved from anti-NT-antibody-Sepharose preparations. Since the C-terminal region of NT constitutes its biologically active core, these results suggest that presence of biologically important congeners of NT in bacteria.

23] The Talc-resin-trichloroacetic acid test for screening radioiodinated polypeptide hormones

Publisher Summary This chapter discusses the use of talc-resin trichloroacetic acid test for screening radioiodinated polypeptide hormones. The talc-resin TCA test is a rapid, reliable physicochemical method for predicting the immunologic performance of 125 I-labeled hormones in radioimmunoassays (RIA). With the talc-resin TCA test, it is possible to anticipate the RIA behavior of 125 I -labeled human (h) and rat (r) prolactin (PRL), growth hormone (GH), thyroid-stimulating hormone (TSH), and luteinizing hormone (LH) prepared from more than 100 iodinations. The talc-resin TCA test is based on three different physicochemical properties of iodohormones: adsorption to talc, exclusion from resin, and precipitation by trichloroacetic acid (TCA). The test patterns are distinct for monomeric iodohormone, aggregated iodohormone, and 125 I-labeled salts. Thus, a usable iodohormone may be identified quickly and accurately by its meeting the criteria of greater than 90% talc adsorption, less than 25% bound to resin, and greater than 90% TCA precipitation. In addition, the talc and TCA percentages should agree within 3%. Radioiodinated human and rat PRL, TSH, GH, and LH have been prepared by different iodination procedures. All the purified monomeric iodohormones, producing sensitive and reliable RIA results, showed the characteristic talc-resin TCA test results of >90% talc adsorption, 90% TCA precipitation.

Antiserum against rat luteinizing hormone (LH) receptors

Summary An antiserum to soluble rat luteal LH receptors was produced in rabbits. The presence of LH receptor antibodies was shown by indirect immuno-precipitation of [125]hCG-soluble rat LH receptor-receptor antiserum complexes by antirabbit IgG. The antiserum bound LH receptors from rat ovaries and testes but not those from human or sheep ovaries. Unoccupied LH receptors competed with [125I]hCG occupied receptors in a dose-dependent fashion for antiserum binding. Extracts of rat brain and liver did not appear to bind to the antiserum. In addition, LH-dependent progesterone secretion from isolated rat ovarian cells was reduced by the antiserum although there was no change in hormone binding.