Synthesis and degradation of cyclic nucleotides in the pituitary gland,ovary and testis of rats treated with d-Trp6-luteinizing hormone-releasing hormone

The effect of administration of d-Trp⁶-Luteininzing Hormone-Releasing Hormone (LH-RH) on synthesis and degradation of cyclic nucleotides was studied in the rat. There were no significant changes in the rate of synthesis and degradation of cyclic AMP in the ovary, testis and pituitary gland of d-Trp⁶ LH-RH-treated rats as compared to controls. On the other hand, the levels of cyclic GMP and activity of guanylate cyclase were significantly higher in the ovary and testis as well as in the pituitary gland of animals which received the analog. The rate of hydrolysis of cyclic GMP was unchanged by the administration of d-Trp⁶-LH=RH. Interestingly, the cyclic CMP phosphodiesterase seemed to be activated in animals treated with d-Trp⁶-LH-RH.     

Changes in circulating levels of immunoreactive follicle stimulating hormone, luteinizing hormone, and testosterone during sexual development in the rhesus monkey, Macaca mulatta

Basal serum levels of follicle stimulating hormone (FSH), luteinizing hormone (LH), and testosterone (T) and the responsiveness of these hormones to a challenge dose of luteinizing hormone releasing hormone (LHRH), were determined in juvenile, pubertal, and adult rhesus monkeys. The monkey gonadotrophins were analyzed using RIA reagents supplied by the World Health Organization (WHO) Special Programme of Human Reproduction. The FSH levels which were near the assay sensitivity in immature monkeys (2.4 +/- 0.8 ng/ml) showed a discernible increase in pubertal animals (6.4 +/- 1.8 ng/ml). Compared to other two age groups, the serum FSH concentration was markedly higher (16.1 +/- 1.8 ng/ml) in adults. Serum LH levels were below the detectable limits of the assay in juvenile monkeys but rose to 16.2 +/- 3.1 ng/ml in pubertal animals. When compared to pubertal animals, a two-fold increase in LH levels paralleled changes in serum LH during the three developmental stages. Response of serum gonadotrophins and T levels to a challenge dose of LHRH (2.5 micrograms; i.v.) was variable in the different age groups. The present data suggest: an asynchronous rise of FSH and LH during the pubertal period and a temporal correlation between the testicular size and FSH concentrations; the challenge dose of LHRH, which induces a significant rise in serum LH and T levels, fails to elicit an FSH response in all the three age groups; and the pubertal as compared to adult monkeys release significantly larger quantities of LH in response to exogenous LHRH.     

Higher molecular weight immunoreactive species of luteinizing hormone releasing hormone: possible precursors of the hormone.

Separation of extracts of sheep hypothalami on Sephadex G-25 gave three peaks exhibiting luteinizing hormone releasing hormone immunoreactivity. One peak corresponded in elution volume with luteinizing hormone releasing hormone but the others (I and II) eluted earlier, indicating that they are of higher molecular weight. Elution volumes were unaffected by 8 M urea treatment. Incubation of I and II with hypothalamic peptidases produced a small quantity of immunoreactive material eluting in the luteinizing hormone releasing hormone region. Digestion of I with trypsin resulted in a marked increase in total immunoreactivity and the production of material with the same elution volume as II. Tryptic digestion of II gave rise to a small quantity of immunoreactive peptide eluting in the luteinizing hormone releasing hormone region. The amount of I and II relative to luteinizing hormone releasing hormone was lower in the median eminence than in the supra optic chiasmatic and basal hypothalamic regions.     

Ontention of antisera against a hypothalamic decapeptide (luteinizing hormone/follicle stimulating hormone releasing hormone) which stimulates the release of pituitary gonadotropins and development of its radioimmunoassay

Using the classical approach, a decapeptide was synthesized with the structure of porcine luteinizing hormone/follicle stimulating hormone releasing hormone reported by Matsuo, H., Baba, Y., Nair, R. M. G., Arimura, A. and Schally, A. V. (1971) Biochem. Biophys. Res. Commun. 43, 1393–1399. As already reported, this peptide was capable of inducing in vitro the release of luteinizing hormone and follicle stimulating hormone from rat pituitary glands. A specific antiserum against luteinizing hormone/follicle stimulating hormone releasing hormone has been generated in the guinea pig and this allowed the development of a radioimmunoassay for this peptide. The antisera, at a final dilution of to depending on the antiserum used, were able to bind 35% of the ¹³¹I-labelled antigen. The sensitivity of this assay method was 50 pg of luteinizing hormone/follicle stimulating hormone releasing hormone. The following substances did not cross-react: oxytocin, lysine-vasopressin, synthetic thyroid stimulating hormone releasing hormone, ovine luteinizing hormone, follicle stimulating hormone and prolactin. Des-Trp³ luteinizing hormone/follicle stimulating hormone releasing hormone, pyroglutamyl-histidyl-tryptophan and seryl-tyrosyl-glycyl-leucyl-arginyl-prolyl-glycinamide, exhibited flatter curves than luteinizing hormone/follicle stimulating hormone releasing hormone with a cross-reactivity of about . Using this method, luteinizing hormone/follicle stimulating hormone releasing hormone was assayed in extracts of the sheep stalk-median eminence and of the hypothalamus and in jugular vein blood from a normal ram and from normal male rats, from cyclic ewe and from hypophysectomized ram and rats. It was concluded that luteinizing hormone/follicle stimulating hormone releasing hormone is present in hypothalamic extracts and in plasma of sheep and rat.     

Dynamic alterations in luteinizing hormone-releasing hormone (LHRH) neuronal cell bodies and terminals of adult rats

Summary 1. The decapeptide lueteinizing hormone-releasing hormone (LHRH) is synthesized in neuronal cell bodies diffusely distributed across the basal forebrain and is secreted from neuronal terminals in the median eminence. Once secreted, LHRH enters the portal vessels and is then transported to the anterior pituitary, where it modulates the synthesis and secretion of gonadotropins, which are essential to gonadal function and reproduction.2. Because of the difficulties encountered in studying these diffusely distributed neurons, we have developed strategies which combine immunocytochemistry and computer-assisted techniques to examine individual LHRH neuronal cell bodies, as well as the entire population of LHRH neurons from the diagonal band of Broca to the mammillary bodies. In addition, we have examined LHRH neuronal terminals in the median eminence using computer-assisted imaging techniques to examine individual terminals by electron microscopy or across all rostral-caudal regions of the median eminence by light microscopy. In our most recent studies using confocal microscopy, we have examined the relationships of LHRH terminals to glial processes.3. These studies reveal a very dynamic system of LHRH neuronal cell bodies and terminals. The population of neurons in which LHRH can be detected varies as a function of time after gonadectomy, during the estrous cycle, and during the preovulatory surge of LH during the afternoon of proestrus. Dynamic changes are also observed in LHRH terminals in the median eminence as a function of time after gonadectomy and in specific rostral-caudal regions of the median eminence during the preovulatory surge of LH. Finally, confocal microscopy reveals that LHRH terminals are prevented from contacting the basal lamina of the brain by glial end-feet.4. We are currently examining the hypothesis that these relationships change as a function of endocrine milieu and, therefore, participate in the modulation of LHRH secretion. Ongoing studies focus on defining the sites of action and synergy of multiple sources of regulation of LHRH secretion and their relative importance to ensuring reproductive success.     

Amplitude and frequency modulation of pulsatile luteinizing hormone-releasing hormone release

Summary 1. A variety of neuroendocrine approaches has been used to characterize cellular mechanisms governing luteinizing hormone-releasing hormone (LHRH) pulse generation. We review recentin vivo microdialysis,in vitro superfusion, andin situ hybridization experiments in which we tested the hypothesis that the amplitude and frequency of LHRH pulses are subject to independent regulation via distinct and identifiable cellular pathways.2. Augmentation of LHRH pulse amplitude is proposed as a central feature of preovulatory LHRH surges. Three mechanisms are described which may contribute to this increase in LHRH pulse amplitude: (a) increased LHRH gene expression, (b) augmentation of facilitatory neurotransmission, and (c) increased responsiveness of LHRH neurons to afferent synaptic signals. Neuropeptide Y (NPY) is examined as a prototypical afferent transmitter regulating the generation of LHRH surges through the latter two mechanisms.3. Retardation of LHRH pulse generator frequency is postulated to mediate negative feedback actions of gonadal hormones. Evidence supporting this hypothesis is reviewed, including results ofin vivo monitoring experiments in which LHRH pulse frequency, but not amplitude, is shown to be increased following castration. A role for noradrenergic neurons as intervening targets of gonadal hormone negative feedback actions is discussed.4. Future directions for study of the LHRH pulse generator are suggested.     

Steroid imprinting and modulation of sexual dimorphism in the luteinizing hormone-releasing hormone neuronal system

Summary 1. Sex differences in the control of gonadotropin secretion and reproductive functions are a distinct characteristic in all mammalian species, including humans. Ovulation and cyclicity are among the most distinct neuroendocrine markers of female brain differentiation, along with sex behavioral traits that are also evident in different species.2. The luteinizing hormone-releasing hormone (LHRH) neuronal system is the prime regulator of neuroendocrine events leading to ovulation and hormonal changes during the menstrual cycle and, as such, is the potential site where many of these sex differences may be expressed or, at the very least, integrated. However, until recently, no significant differences were seen in LHRH neurons between male and female brains, including cell number, pattern of distribution, and expression of message or peptide (LHRH) levels.3. Recently, we reported that galanin (GAL), a brain-gut peptide, is coexpressed in LHRH neurons and that this coexpression is sexually dimorphic. When GAL is used as a marker for this neuronal system, it is clear that estradiol as well as progesterone profoundly affects the message and expression of the peptide and that this regulation, at least in rodents, is neonatally predetermined by gonadal steroid imprinting.4. Changes in GAL expression and message can also be seen at puberty, during pregnancy and lactation, and in aging, all situations that affect the function of the LHRH neuronal system. Using an immortalized LHRH neuronal cell line (GT1) we have recently observed that these neurons express estrogen receptor (ER) and GAL and that estradiol can increase the expression of GAL, indicating functional activation of the endogenous ER.     

Administration of human pancreatic growth hormone-releasing factor (GRF) analogs enhances responsiveness of cultured rat pituitary cells to GRF

The aim of this study was to investigate whether anterior pituitary responsiveness to human pancreatic growth hormone-releasing factor containing 29 amino acids (GRF-29) can be modulated by GRF-29 itself. Male rats were injected (sc) daily for 3 days with 50 ug of GRF-29, or were treated twice daily for 14 days with 5 ug of [D-Ala-2]-GRF-29 (a potent GRF agonist). Control animals were injected with saline. After the last injection, pituitaries were removed, dispersed, cultured for 96 h and then challenged with either GRF-29 or [D-Trp-6]-LHRH (a LHRH agonist). Cultured cells from analog-treated rats were more responsive to GRF-29 stimulation than were cells obtained from controls. In contrast, neither treatment altered the response to [D-Trp-6]-LHRH. These studies indicate that periodic administration of GRF analogs can increase hypophyseal GRF responsiveness. Such control may be an important component in the physiological regulation of GH secretion and has important implications for potential therapeutic uses of GRF analogs.     

Control of luteinizing hormone-releasing hormone pulse generation in nonhuman primates

Summary 1. The pulsatile release of luteinizing hormone-releasing hormone (LHRH) is critical for reproductive function. However, the exact mechanism of LHRH pulse generation is unclear. The purpose of this article is to review the current knowledge on LHRH pulse generation and to discuss a series of studies in our laboratory.2. Using push-pull perfusion in the stalk-median eminence of the rhesus monkey several important facts have been revealed. There is evidence indicating that LHRH neurons themselves have endogenous pulse-generating mechanisms but that the pulsatility of LHRH release is also modulated by input from neuropeptide Y (NPY) and norepinephrine (NE) neurons. The release of NPY and NE is pulsatile, with their pulses preceding or occurring simultaneously with LHRH pulses, and the neuroligands NPY and NE and their agonists stimulate LHRH pulses, while the antagonists of the ligands suppress LHRH pulses.3. The pulsatile release of LHRH increases during the estrogen-induced LH surge as well as the progesterone-induced LH surge. These increases are partly due to the stimulatory effects of estrogen and progesterone on NPY neurons.4. An increase in pulsatile LHRH release occurs at the onset of puberty. This pubertal increase in LHRH release appears to be due to the removal of tonic inhibition from aminobutyric acid (GABA) neurons and a subsequent increase in the inputs of NPY and NE neurons to LHRH neurons.5. There are indications that additional neuromodulators are involved in the control of the LHRH pulse generation and that glia may play a role in coordinating pulses of the release of LHRH and neuromodulators.6. It is concluded that the mechanism generating LHRH pulses appears to comprise highly complex cellular elements in the hypothalamus. The study of neuronal and nonneuronal elements of LHRH pulse generation may serve as a model to study the oscillatory behavior of neurosecretion.     

The structure and function of glycoprotein hormone receptors: ganglioside interactions with luteinizing hormone.

A minor glycopeptide was newly isolated from the exhaustive pronase digest of crystalline ovalbumin by Dowex-50w column chromatography, and its structure was determined as Manα1→3Manα1→6 (Manα1→3) Manβ1→4GlcNAcβ1→4GlcNAc→Asn. This glycopeptide (GP-VI) has the smallest carbohydrate unit among the ovalbumin glycopeptides so far reported, and is also the smallest glycopeptide of all which are susceptible to endo-β-N-acetylglucosaminidases C_II and H. This finding, together with the already reported data of the action of both enzymes to glycopeptides of known structures, elucidates that the structural requirement of C_II enzyme for its substrate is R→2Manα1→3 (R→6) Manα1→6 (R→2Manα1→3) (R→4) Manβ1→4GlcNAcβ1→4GlcNAc→Asn, in which R represents either hydrogen or sugars, and that of H enzyme is R→2Manα1→3 (R→6) Manα1→6 (R→4) Manβ1→4GlcNAcβ1→4GlcNAc→Asn.     

Binding of luteinizing hormone-releasing hormone analogues to dispersed rat pituitary cells

The binding of luteinizing hormone-releasing hormone (LHRH) to dispersed rat pituitary cells was studied by using ¹²⁵I-labeled analogues of the neurohormone: a superactive agonist [D Ser (Bu^t)⁶]LHRH(1–9) ethylamide and an antagonist DpGlu¹, DPhe², DTrp^3,6-LHRH. Although these cells were exposed to proteolytic enzymes, their ability to respond to LHRH stimulation by gonadotropin release, is preserved. The time course of binding of the two analogues at different temperatures has demonstrated that highest specific binding is evident at 4°C and that equilibrium is reached after 90 min of incubation at this temperature. Incubation of pituitary cells with the labeled analogues together with increasing concentrations of LHRH or unlabeled analogues exhibited parallel competition curves, suggesting binding to the same receptor sites but with different affinities. Biologically inactive analogues of LHRH or unrelated peptides such as TRH did not compete for binding sites. K_a values for the agonist, LHRH and the antagonist were 2.1 × 10⁹M^?1, 0.92 × 10⁸M^?1 and 0.76 × 10⁹M^?1, respectively, and the binding capacity was 116 fmoles/10⁶ pituitary cells.     

A model system for the biochemical study of luteinizing hormone/chorionic gonadotropin receptor synthesis

A model system for the biochemical study of LH/CG receptor synthesis has been developed. Culture conditions for porcine granulosa cells were adapted that maximized the selective induction of LH/CG receptors by cAMP-inducing stimuli with an elimination of background LH/CG receptor appearance. It was found that the addition of FSH (1.5 μg/ml) or cholera toxin (10 ng/ml) 1 day after plating resulted in optimal induction of the LH/CG receptor (20–60 pg [¹²⁵I]CG bound/μg DNA 72 h after addition) with virtually no LH/CG receptor appearance in the absence of added stimuli. Later additions of FSH or cholera toxin required insulin (1.0 μg/ml) which alone caused background LH/CG receptor appearance in the absence of any additional stimuli. Furthermore, insulin increased the general rate of cellular protein synthesis, whereas FSH or cholera toxin each decreased it. Thus, the use of FSH or cholera toxin, without insulin, may enable one to detect the synthesis of the LH/CG receptor by metabolic labeling techniques where background protein synthesis has been lowered.     

Homeodomain transcription factor Hesx1/Rpx occupies Prop-1 activation sites in porcine follicle stimulating hormone (FSH) beta subunit promoter

Homeodomain repressor factor Hesx1/Rpx plays a crucial role in the formation of Rathke's pouch at the start of pituitary organogenesis and represses the Prop-1-dependent expression of Pit-1 gene, which promotes the differentiation of Pit-1-dependent hormone producing cells. Recently, we discovered a novel function of Prop-1 by which it activates the porcine follicle stimulating hormone beta subunit (FSHbeta) gene through Fd2 region (-852/-746). The present study aimed to determine whether Hesx1 exerts its role in the Prop-1-dependent activation of FSHbeta gene. Transient transfection assay for the porcine FSHbeta promoter -985/+10, electrophoretic mobility shift assay (EMSA) and DNase I footprinting analysis for Fd2 region were carried out. Transfection assay in GH3 cells demonstrated that expression of Hesx1 alone does not change the promoter activity but the coexpression with Prop-1 represses the Prop-1-dependent activation of FSHbeta promoter. Similar results were obtained for the mutant reporter vector deleting the region -745/-104 indicating that Fd2 region is a target site of Hesx1 as well as Prop-1. EMSA and DNase I footprinting analysis using recombinant Hesx1 and Prop-1 protein demonstrated that Hesx1 and Prop-1 certainly bind to the AT-rich regions in a different manner. These results suggest that Hesx1 blocks the advanced expression of FSHbeta gene in the early stage of pituitary development, and Prop-1 thereafter appears and activates this gene.     

Implication of microtubules and microfilaments in the response of the ovarian adenylate cyclase-cyclic AMP system to gonadotropins and prostaglandin E2.

Addition of anti-actin serum or cytochalasin B (3 μg/ml) to the medium abolished the stimulatory effect of LH and of choleragen, and inhibited the action of FSH, but not of PGE₂, on cyclic AMP production in cultured rat Graafian follicles. Colchicine and anti-sera to BSA, tubulin or smooth-muscle myosin, as well as anti-actin serum absorbed with actin, had no effect on the follicular response to LH, but anti-tubulin serum and colchicine inhibited the response to FSH and PGE₂. The inhibitory effect of cytochalasin B on LH-action was fully reversed 24 h after transfer of the follicles to drug-free medium. Neither anti-actin serum nor cytochalasin B had any effect on the binding of ¹²⁵I-hCG by the follicular cell membrane. The results suggest that microfilaments, but not microtubules, are intimately involved in the process of LH- and choleragen-stimulated ovarian adenylate cyclase activity. By contrast, the action of PGE₂ is dependent on microtubule assembly, while the action of FSH seems to depend on both these components of the cytoskeleton.     

Expression of luteinizing hormone and follicle-stimulating hormone receptor in the dog prostate

A possible role for gonadotrophins luteinizing hormone (LH) and follicle-stimulating hormone (FSH) in the prostate physiology has been suggested in humans and rats. This study aimed at investigating the presence of receptors for LH and FSH (LHR and FSHR) in the canine prostate. Prostates were collected at post mortem from 6 clinically healthy, sexually intact beagles free from any prostatic disorder. Tissue was sampled from dorsal, middle and ventral regions of each prostate. Immunohistochemical localization was performed on wax-embedded sections using polyclonal antibodies for LHR or FSHR. The pattern and intensity of staining in the parenchyma (glandular epithelium) and stroma were determined using a semiquantitative histologic assessment. Receptors for LH and FSH were consistently present in both the glandular epithelium and the stroma in all tissue samples examined. Expression for both receptors was higher in the glandular epithelium than the stroma of all prostatic regions (P < 0.001). In the glandular epithelium, LHR (P < 0.01) and FSHR (P < 0.05) expression was lower in the lateral than the other regions, and there was no difference between dorsal and ventral regions. However, variations in the expression for LHR and FSHR among prostatic regions were not found in the stroma. These findings have demonstrated that LHR and FSHR are expressed in the dog prostate, and the variation observed in their levels of expression among its regions and tissue layers suggests a potential role of gonadotrophins LH and FSH in the regulation of the prostate physiology, particularly the glandular epithelium.     

Leydig cell receptors for luteinizing hormone releasing hormone and its agonists and their modulation by administration or deprivation of the releasing hormone

Leydig cells isolated from adult rat testes bound ¹²⁵I-labelled luteinizing hormone releasing hormone (LHRH) agonist with high affinity (K_A=1.2 × 10⁹M) and specificity. LHRH and the 3–9 and 4–9 fragments of LHRH agonist competed for binding sites with ¹²⁵I-LHRH agonist but with reduced affinities, whereas fragments of LHRH, and oxytocin and TRH were largely inactive. Somatostatin inhibited binding at high (10^?4M) concentrations but was inactive at 10^?6M and less. Pretreatment of rats for 7 days with 5 μg/day of LHRH agonist reduced binding of ¹²⁵I-LHRH agonist to Leydig cells $\text{in vitro}$ by 25%, whilst inhibition of endogenous LHRH by antibodies for 7 days caused a 40% decrease.     

Comparative structure-activity studies on mammalian [Arg8] LH-RH and chicken [Gln8] LH-RH by fluorimetric titration

Fluorimetric titrations of mammalian [Arg8] LH-RH, chicken [Gln8] LH-RH and an analogue [Lys8] LH-RH revealed pK values of 5.80, 6.22 and 6.01 for His2, and 9.65, 9.88 and 9.88 for Tyr5. The titration ranges for His2 were 1.72, 2.03 and 1.71 while the range for Tyr5 was rather similar (approximately 1.7) for all three peptides. Biological activity and receptor binding in the mammalian system for chicken LH-RH was 1% relative to mammalian LH-RH while [Lys8] LH-RH had a relative activity of approximately 10%. In contrast, mammalian and chicken LH-RH were equipotent in stimulating LH release from chicken pituitary cells. The results indicate differences in the receptors related to the conformations of LH-RH and position 8-substituted analogues.     

Characterization of a rat anterior pituitary cell bioassay

Summary We have described the protocols and characterization of a pituicyte culture, which became established as a reliable and reproducible bioassay for the secretion of follicle-stimulating hormone (FSH) and luteinizing hormone (LH). The bioassay was used to measure the bioactivity of factors that inhibit and stimulate gonadotrophin secretion. The protocol that was used involved the culling of female Wistar rats (200 to 250 g weight), at random stages of their cycle, and dispersal of their pituicytes in a concentration of 0.4 × 10⁶ cells · ml⁻¹ · well⁻¹ in serum-free medium (Dulbecco’s modified Eagle’s medium/Ham’s F12 mixture, supplemented with insulin and transferrin) in Falcon 3047 24-well culture plates. After 24 h of pre-culture, the medium was changed and the cells cultured for a further 48 h. The supernatant was removed and assayed for basal secretion of FSH and LH. The cells were then stimulated with 10⁻⁸ M GnRH for 4 h and the supernatant assayed for gonadotrophin-releasing hormone (GnRH)-stimulated FSH and LH secretion. All samples were assayed as pairs of duplicates (i.e. quadruplicate samples) which were randomly added to the plates to minimize plate effects. Random number tables were used to achieve this randomization.     

Direct tritium-labelling into histidine of luteinizing hormone-releasing hormone agonists and use of the tracers in studies on proteolytic breakdown

Analogs of luteinizing hormone- releasing hormone (LHRH) having higher biological activity than LHRH itself are being mainly used to study the biological effects and the mechanism of action of LHRH. In the present study, conditions for the direct ³H-labelling at the histidine residue of analogs of LHRH were worked out, circumventing the synthesis of precursor peptides for labelling. [D-Phe⁶,desGly¹⁰]-LHRH ethylamide and [D-Ser(Bu^t)⁶,desGly¹⁰]-LHRH ethylamide were tritiated by tritium gas and a 10% Pd/Al₂O₃ catalyst to high specific radioactives. The labelled peptides are sufficiently stable to be used in biochemical studies. The degradability of the analogs by homogenates of various of rats was compared with that of the native LHRH. The analogs were shown to be distinctly degradable, but to a lower extent. The kidney homogenate degrades the analogs [D-Phe⁶,desGly¹⁰]- and [D-Ser(Bu^t)⁶, desGly¹⁰]-LHRH ethylamide with 35 and 50%, respectively, of the velocity observed with LHRH, whereas the degradation velocity of the analogs by a homogenate of the hypothalamus and pituitary is only 10% of that of LHRH. It is suggested that the lower degradability of tha analogs at peripheral sites and target sites (pituitary, ovary) explains partly their higher biological activity.