Regulation of type II luteinizing hormone-releasing hormone (LHRH-II) gene expression by the processed peptide of LHRH-I, LHRH-(1-5) in endometrial cells

Luteinizing hormone-releasing hormone (LHRH) was first isolated in the mammalian hypothalamus and shown to be the primary regulator of the reproductive system through its initiation of pituitary gonadotropin release. Since its discovery, this form of LHRH (LHRH-I) has been shown to be one of many structural variants with a variety of roles in both the brain and peripheral tissues. Enormous interest has been focused on LHRH-I, LHRH-II, and their cognate receptors as targets for designing therapies to treat cancers of the reproductive system. LHRH-I is processed by a zinc metalloendopeptidase EC 3.4.24.15 (EP24.15) that cleaves the hormone at the Tyr(5)-Gly(6) bond. We have previously reported that the autoregulation of LHRH gene expression can also be mediated by its processed peptide, LHRH-(1-5). Given its importance in the brain, we have investigated the role of the specific processed peptide of LHRH-I, LHRH-(1-5), within Ishikawa cells, a human endometrial cell line. Using real-time polymerase chain reaction, we observed that LHRH-(1-5) upregulates LHRH-II mRNA expression in Ishikawa cells but does not exert any influence on LHRH-I mRNA levels. This is in contrast to the effects of LHRH-I, which affects the expression of LHRH-I mRNA. Our findings support a potential role for LHRH-(1-5) as a processed metabolite in the endometrium. Further investigations are needed to determine the role of this processed metabolite and to identify specific pathways involved in LHRH-(1-5) signaling.     

Presence and distribution of LHRH- and alpha-MSH-like immunoreactive nerve fibers in the epithalamus of the sheep

The presence of luteinizing-hormone-releasing hormone (LHRH)-like and alpha-melanocyte-stimulating hormone (alpha-MSH)-like immunoreactive fibers, nerve terminals or cellular elements in the pineal gland of the sheep has been investigated by immunohistochemistry. No LHRH-or alpha-MSH-like immunoreactive fiber, nerve terminals or cellular elements have been demonstrated in the pineal organ of the sheep. However, LHRH- and alpha-MSH-like immunoreactive fibers are present in the posterior commissure. Immunoreactive LHRH and alpha-MSH nerve endings are evident in the medial and lateral habenular nuclei. Discrepancies with the results obtained in other mammals are indicative of species differences in the distribution of LHRH- and alpha-MSH-like immunoreactive material in the pineal region.     

Production of monoclonal antibodies against avian LHRH-I

Summary Production of antibodies against peptides or poorly antigenic proteins by conventional methods often requires either large quantities of the native immunogen or some chemical modification to increase their antigenicity. In this study an in vivo and in vitro immunization protocol has been used to generate monoclonal antibodies against the decapeptide luteinizing hormone-releasing hormone (LHRH). Two injections of 100 μg of avian LHRH-I into BALB/c mice were given 7 d apart. Dissociated splenocytes were collected under sterile conditions. They were incubated with 100 μg of the immunogen in 75-cm² tissue culture flasks in thymocyte-conditioned media. After 5 to 8 d exposure to the antigen, splenocytes were fused with SP2/O myeloma cells by polyethylene glycol. The cells were plated into 24 wells and then incubated in hypoxanthine aminopterin and thymidine selective media. After 14 d an initial screening was done by enzyme immunoassay. The positive wells (6/24) were expanded into 96-well plates and rescreened. Selected lines were cloned out 3 times by limiting dilution and the most positive expanded for ascites production. The antibody was affinity purified in a protein A column. The antibody cross-reacted with LHRH-I and II but preferentially to LHRH-I, as shown by competitive assay. A hypothalamic extract from a mature chick showed a higher response than preparations from whole brain explants of 1- to 3-d posthatched chicks, mature quail, and mature mouse. This work was funded by the Maryland Agricultural Experiment Station artical no. A4975, contribution no. 8019.     

Postmenopausal increase in KiSS-1, GPR54, and luteinizing hormone releasing hormone (LHRH-1) mRNA in the basal hypothalamus of female rhesus monkeys

The G-protein coupled receptor, GPR54, and its ligand, kisspeptin-54 (a KiSS-1 derived peptide) have been reported to be important players in control of LHRH-1 release. However, the role of the GPR54 signaling in primate reproductive senescence is still unclear. In the present study we investigated whether KiSS-1, GPR54, and LHRH-1 mRNA in the brain change after menopause in female rhesus monkeys using quantitative real-time PCR. Results indicate that KiSS-1, GPR54, and LHRH-1 mRNA levels in the medial basal hypothalamus (MBH) in postmenopausal females (28.3+/-1.1 years of age, n=5) were all significantly higher than that in eugonadal adult females (14.7+/-2.1 years of age, n=9), whereas KiSS-1, GPR54, and LHRH-1 mRNA levels in the preoptic area (POA) did not have any significant changes between the two age groups. To further determine the potential contribution by the absence of ovarian steroids, we compared the changes in KiSS-1, GPR54, and LHRH-1 mRNA levels in young adult ovarian intact vs. young ovariectomized females. Results indicate that KiSS-1 and LHRH-1 mRNA levels in the MBH, not POA, in ovariectomized females were significantly higher than those in ovarian intact females, whereas GPR54 mRNA levels in ovariectomized females had a tendency to be elevated in the MBH, although the values were not quite statistically significant. Collectively, in the primate the reduction in the negative feedback control by ovarian steroids appears to be responsible for the aging changes in kisspeptin-GPR54 signaling and the elevated state of the LHRH-1 neuronal system.     

Ovulation inhibition in the pregnant mare's serum gonadotropin-treated immature rat: a bioassay for luteinizing hormone-releasing hormone antagonists

A convenient method for evaluating the biological activity of luteinizing hormone-releasing hormone (LHRH) antagonists was devised. Pregnant mare's serum gonadotropin (PMSG) treatment of immature rats is known to stimulate follicular growth and estrogen production, that in turn stimulates the release of LHRH which triggers an ovulatory discharge of luteinizing hormone (LH) from the pituitary. The present bioassay of the antagonists is based on the inhibition of ovulation in the PMSG-treated rats. Twenty-eight-day-old Sprague Dawley rats maintained under a light period of 12 h/day (lights on at 0630 h) were given 10 IU of PMSG s.c. at 0930 h. On Day 30 of age the antagonist was given s.c. at 1430 h. The rats were killed on the following morning and the oviducts examined for the presence of ova. In addition, the antagonists were compared in their ability to inhibit serum testosterone levels in adult male rats. In the PMSG-treated rats the order of ovulation-inhibiting potency of the following antagonists was: [Ac-D-NAL(2)1,4FD-Phe2,D-Trp3,D-Arg6]-LHRH (LHRH-1) greater than [Ac-delta 3 Pro1,4FD-Phe2,D-NAL(2)3.6]-LHRH (LHRH-2) greater than [Ac-delta 3 Pro1,4FD-Phe2,D-Trp3,6]-LHRH (LHRH-3). The order of potency was confirmed by their antitesticular effects in adult male rats.     

Inhibition of melatonin-induced ascorbic acid and LHRH release by a nitric oxide synthase and cyclic GMP inhibitor

Melatonin (MEL), the principle secretory product of the pineal gland, has been shown to function as an antioxidant and free-radical scavenger. We previously showed that the release of ascorbic acid (AA) and luteinizing hormone releasing hormone (LHRH) from medial basal hypothalamus (MBH) was mediated by nitric oxide (NO) that released cyclic guanosine 3'5'-mono-phosphate (cGMP). Therefore, it was of interest to evaluate the effect of MEL on AA and LHRH release and study the effect of a nitric oxide synthase (NOS) inhibitor, 6-anilino-5,8-quinoline-dione (LY 83583), and a guanylyl cyclase (GC) inhibitor, 1H-[1,2,4] oxadiazolo [4,3-a] quinoxalin-1-one (O.D.Q.), on the release process. Because NO has been shown to activate soluble guanylyl cyclase that elicited an elevation of cGMP in target cells, in the current investigation LY 83583, O.D.Q., or N(G)-monomethyl-l-arginine (NMMA), a competitive inhibitor of NOS, were used to evaluate their effects on MEL-induced AA and LHRH release. Medial basal hypothalami were incubated in 0.5 ml of Krebs-Ringer bicarbonate (KRB) buffer for 1 hr. Subsequently, the tissues were incubated with graded concentrations of MEL (10(-8) to 10(-4) M), MEL + NMMA (3 x 10(-4) M), MEL + LY 83583 (10(-6) M), or MEL + O.D.Q. (10(-5) M) for 1 hr. Ascorbic acid and LHRH released into the medium were measured by high-performance liquid chromatography (HPLC) and radio-immunoassay (RIA), respectively. Melatonin (10(-6) and 10(-5) M) significantly stimulated both AA and LHRH release, but the lower and the highest concentrations were ineffective. A combination of MEL + NMMA completely blocked both AA and LHRH release, supporting a role for NO in the releasing action. Both LY 83583 and O.D.Q. significantly suppressed MEL-induced AA and LHRH release, emphasizing the role of NOS, GC, and cGMP in mediating the action of MEL. The data of these in vitro experiments support a role for MEL in the hypothalamic control of AA and LHRH release.     

Compromised reproductive function in adult female mice selectively expressing mutant ErbB-1 tyrosine kinase receptors in astroglia

The ErbB-1 tyrosine kinase receptor plays critical roles in regulating physiological functions. This receptor-mediated signaling in astroglia has been implicated in controlling female sexual development via activating neurons that release LH-releasing hormone (LHRH), the neuropeptide required for the secretion of LH. It remains unknown whether astroglial ErbB-1 receptors are necessary for maintaining normal adult reproductive function. Here we provide genetic evidence that astroglia-specific and time-controlled disruption of ErbB-1 receptor signaling by expressing mutant ErbB-1 receptors leads to compromised reproduction due to alteration in LHRH neuron-controlled secretion of LH in adult female mice. Therefore, astroglial ErbB-1 receptors are required for controlling LHRH neuronal function and thus maintaining adult reproduction, suggesting that compromised astroglial ErbB-1 signaling may also contribute to reproductive abnormalities in aging females.     

诱导大鳍鱯和长吻鮠排卵过程中血清GTH水平的变化

繁殖期从嘉陵江收集性成熟的大鳍■ 和长吻 野生亲鱼,用Ｌｉｎｐｅ方法（即ＬＨＲＨ－Ａ加多巴胺Ｄ２受体拮抗剂地欧酮）或传统的ＬＨＲＨ—Ａ加脑垂体的方法进行催产,定时取血样,用放射免疫方法测定催产过程中血清ＧＴＨ水平的变化,进一步证实鲇形目鱼类ＧＴＨ的分泌受到下丘脑分泌的促性腺激素释放激素ＧｎＲＨ和多巴胺的双重调节;排卵和产卵也是以血清ＧＴＨ的急剧升高为先导的,而最终能否排卵还有赖于血清ＧＴＨ峰是否超过“排卵阈值”。尽管催产后的大鳍 和长吻 雄鱼血清ＧＴＨ水平也有一个高峰出现,但血清ＧＴＨ水平升高幅度都大大低于雌鱼,这种现象在硬骨鱼类可能具有普遍性。     

Immunization of male mice with luteinizing hormone-releasing hormone fusion proteins reduces testicular and accessory sex gland function

Genes for ovalbumin-luteinizing hormone-releasing hormone 7 (LHRH-7) and thioredoxin-LHRH-7 fusion proteins (containing seven LHRH inserts) were constructed by cassette and mismatch mutagenesis and expressed in Escherichia coli. In experiment 1, 10 microgram of either ovalbumin-LHRH-7 or thioredoxin-LHRH-7 were suspended in Z-max adjuvant and injected three times at 4-wk intervals into postpubertal male BALB/c mice. In experiment 2, the fusion proteins were suspended in Immumax adjuvant and administered in equimolar quantities (0.4 nmol per injection) to postpubertal male BALB/c mice. In addition to injection of these two proteins alone, the proteins were also administered in different sequences or together in a mixture. Both LHRH fusion proteins induced significant antibody titers, which resulted in a significant decrease in vesicular gland and anterior prostate weight (measure of biological response) in both experiments. Vesicular gland and anterior prostate weight and LHRH antibody titers were significantly correlated in experiments 1 (r = -0.64) and 2 (r = -0.53). Percentage of animals responding to treatment varied from 40-60% in experiment 1 and from 11-89% in experiment 2, with the highest responses in treatments that used a combination of both fusion proteins. The variation in responders and nonresponders was evaluated by estimating antibody K(D) from displacement curves. Part, but not all, of the high antibody nonresponders can be explained by antibody affinity.     

Isocratic liquid chromatographic assay for monitoring the degradation of luteinizing hormone releasing hormone by extracts from the gastrointestinal tract of possums

A simple HPLC method to separate human luteinizing hormone releasing hormone (LHRH) from its metabolites using an isocratic elution is described. Intact LHRH and five metabolites were separated in 11.4 min. The calibration curve (peak area versus concentration) was linear over the concentration range 1.25-35 microg/ml (r(2)=0.99) with the intercept not significantly different from zero (P>0.05). Intra-day and inter-day variability of the assay was less than 5% for repeat injections of 5, 14.5 and 29 microg/ml. The method was applied to evaluate the susceptibility of LHRH to enzymes present in the lumen and mucosal extracts of the gastrointestinal tract of possums. The major degradation products of LHRH were identified by HPLC separation, amino acid analysis and mass spectrometry as LHRH (1-5), LHRH (1-4), LHRH (1-3) and LHRH (3-4).     

Chromatographic and biologic analysis of ME and OVLT LHRH

The luteinizing hormone (LH)-releasing activities a pooled rat organum vasculosum lamina terminalis (OVLT) and median eminence (ME) tissues were evaluated for chromatographic and biologic similarity and compared to those of synthetic decapeptide LH-releasing hormone (LHRH). The LHRH detected in these extracts appeared similar chromatographically (Sephadex G-25) to synthetic LHRH. These extracts, as well as synthetic LHRH, were also capable of stimulating dose dependent gonadotropin release form cultures rat gonadotrophs. These findings suggest a physiological role of the LHRH present in the rat OVLT in the control of gonadotropin secretion.     

Benzimidazoles as non-peptide luteinizing hormone-releasing hormone (LHRH) antagonists. Part 3: Discovery of 1-(1H-benzimidazol-5-yl)-3-tert-butylurea derivatives

1-(1H-Benzimidazol-5-yl)-3-tert-butylurea derivatives have been identified as a novel class of non-peptide luteinizing hormone-releasing hormone (LHRH) antagonists. Herein, we disclose the synthesis and structure-activity relationships (SAR) of this class resulting in the identification of compound 12c, with dual functional activity on human and rat receptors (rat LHRH: IC50=120 nM; human LHRH: IC50=18 nM). These SAR studies suggest that 1-(1H-benzimidazol-5-yl)-3-tert-butylurea is a new pharmacophore for small molecule LHRH antagonists.     

Chitosan-Nanoconjugated Hormone Nanoparticles for Sustained Surge of Gonadotropins and Enhanced Reproductive Output in Female Fish

A controlled release delivery system helps to overcome the problem of short life of the leutinizing hormone releasing hormone (LHRH) in blood and avoids use of multiple injections to enhance reproductive efficacy. Chitosan- and chitosan-gold nanoconjugates of salmon LHRH of desired size, dispersity and zeta potential were synthesized and evaluated at half the dose rate against full dose of bare LHRH for their reproductive efficacy in the female fish, Cyprinus carpio. Whereas injections of both the nanoconjugates induced controlled and sustained surge of the hormones with peak (P<0.01) at 24 hrs, surge due to bare LHRH reached its peak at 7 hrs and either remained at plateau or sharply declined thereafter. While the percentage of relative total eggs produced by fish were 130 and 67 per cent higher, that of fertilised eggs were 171 and 88 per cent higher on chitosan- and chitosan-gold nanoconjugates than bare LHRH. Chitosan nanoconjugates had a 13 per cent higher and chitosan gold preparation had a 9 per cent higher fertilization rate than bare LHRH. Histology of the ovaries also attested the pronounced effect of nanoparticles on reproductive output. This is the first report on use of chitosan-conjugated nanodelivery of gonadotropic hormone in fish.     

Endopeptidase-24.15 Is the Primary Enzyme that Degrades Luteinizing Hormone Releasing Hormone Both In Vitro and In Vivo

The concentration of luteinizing hormone releasing hormone (LHRH) (pGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH2), which reaches the anterior pituitary via the hypothalamo-hypophyseal portal system, appears to be controlled in part by the rate of LHRH degradation within the hypothalamus and/or pituitary. Specific, active site-directed endopeptidase inhibitors synthesized in our laboratory were used to identify the enzyme(s) involved in LHRH degradation by hypothalamic and pituitary membrane preparations, and by an intact anterior pituitary tumor cell line (AtT20). Incubation of LHRH with pituitary and hypothalamic membrane preparations led to the formation of pGlu-His-Trp (LHRH1-3) as the main reaction product. Under the same conditions, addition to the incubation mixtures of captopril, an inhibitor of the angiotensin converting enzyme, led to accumulation of pGlu-His-Trp-Ser-Tyr (LHRH1-5) and, to a lesser extent, pGlu-His-Trp-Ser-Tyr (LHRH1-6). The degradation of LHRH and the formation of the N-terminal tri- and pentapeptides was blocked by N-[1-(R,S)-carboxy-3-phenylpropyl]-Ala-Ala-Phe-p-aminobenzoate (cFP-AAF-pAB), a specific, active site directed inhibitor of endopeptidase-24.15. Some inhibition of LHRH degradation and formation of the N-terminal hexapeptide was also obtained in the presence of N-[1-carboxy-2-phenylethyl]-Phe-p-aminobenzoate (cFE-F-pAB), an inhibitor of endopeptidase-24.11. Similar results were obtained with AtT20 cell membranes and with intact AtT20 cells in monolayer culture. Following cleavage by endopeptidases the C-terminal part of LHRH was rapidly degraded by aminopeptidases. Superactive analogs of LHRH in which Gly6 was replaced by a D-amino acid are resistant to degradation by both endopeptidase-24.11 and -24.15. In vivo, when LHRH was injected directly into the third ventricle of rats, the presence of cFP-AAF-pAB inhibited LHRH degradation. It is concluded that LHRH degradation is primarily initiated by the membrane-bound form of endopeptidase-24.15 to yield pGlu-His-Trp-Ser-Tyr and to a lesser extent by endopeptidase-24.11 to yield pGlu-His-Trp-Ser-Tyr-Gly.     

Secretion-stimulating and secretion-inhibiting hormones stimulate high-affinity pertussis-toxin-sensitive GTPases in membranes of a pituitary cell line

Different peptide hormones influence hormone secretion in pituitary cells by diverse second messenger systems. Recent data indicate that luteinizing-hormone-releasing hormone (LHRH) stimulates and somatostatin inhibits voltage-dependent Ca2+ channels of GH3 cells via pertussis-toxin-sensitive mechanisms [Rosenthal et al. (1988) EMBO J. 7, 1627-1633]. In other pituitary cell lines, somatostatin has been shown to cause a pertussis-toxin-sensitive decrease in adenylate cyclase activity, and LHRH and thyrotropin-releasing hormone (TRH) stimulate phosphoinositol lipid hydrolysis in a pertussis-toxin-independent manner. Whether stimulation of Ca2+ influx by TRH is affected by pertussis toxin is not known. In order to elucidate which of the hormone receptors interact with pertussis-toxin-sensitive and -insensitive G-proteins, we measured the effects of LHRH, somatostatin and TRH on high-affinity GTPases in membranes of GH3 cells. In control membranes, both LHRH and TRH stimulated the high-affinity GTPase by 20%, somatostatin by 25%. Maximal hormone effects were observed at a concentration of about 1 microM. Pretreatment of cells with pertussis toxin abolished pertussis-toxin-catalyzed [32P]ADP-ribosylation of 39-40-kDa proteins in subsequently prepared membranes and reduced basal GTPase activity. The toxin also reduced by more than half the increases in GTPase activity induced by LHRH and TRH; stimulation of GTPase by somatostatin was completely suppressed. Stimulation of adenylate cyclase by vasoactive intestinal peptide (VIP) was not impaired by pretreatment of cells with pertussis toxin. Somatostatin but not LHRH and TRH decreased forskolin-stimulated adenylate cyclase activity. The results suggest that the activated receptors for LHRH and TRH act via pertussis-toxin-sensitive and -insensitive G-proteins, whereas effects of somatostatin are exclusively mediated by pertussis-toxin-sensitive G-proteins.     

Precursor and deaminated forms of both luteinizing hormone-releasing hormone (LHRH) and (hydroxyproline9)LHRH are present in the rat hypothalamus.

A naturally occurring analog of the decapeptide luteinizing hormone-releasing hormone ([Hyp9]LHRH) has been described previously in the hypothalamus of several mammals. It derives from post-translational hydroxylation of the LHRH proline9 residue. In the present work, intermediate LHRH precursors exhibiting both Pro9 or Hyp9 residues in the LHRH sequence were characterized in the rat hypothalamus. Hydroxylation of the Pro9 residue can thus be assumed to occur at an early stage of post-translational maturation. Deaminated, free acid forms of both native decapeptides were also detected. They correspond most likely to catabolites from incompletely processed precursors.     

Vitamin E stimulates luteinizing hormone-releasing hormone and ascorbic acid release from medial basal hypothalami of adult male rats

Vitamin E, a dietary factor, is essential for reproduction in animals. It is an antioxidant present in all mammalian cells. Previously, we showed that ascorbic acid (AA) acted as an inhibitory neurotransmitter in the hypothalamus by scavenging nitric oxide (NO). Earlier studies have shown the antioxidant synergism between vitamin E and ascorbic acid (AA). Therefore, it was of interest to evaluate the effect of vitamin E on luteinizing hormone-releasing hormone (LHRH) and AA release. Medial basal hypothalami from adult male rats of the Sprague Dawley strain were incubated with Krebs-Ringer bicarbonate buffer or graded concentrations of a water soluble form of vitamin E, tocopheryl succinate polyethylene glycol 1000 (TPGS, 22-176 microM) for 1 hr. Subsequently, the tissues were incubated with vitamin E or combinations of vitamin. E + N-methyl-D-aspartic acid (NMDA), an excitatory amino acid for 30 min to study the effect of prior and continued exposure to vitamin E on NMDA-induced LHRH release. AA and LHRH released into the incubation media were determined by high-performance liquid chromatography and radioimmunoassay, respectively. Vitamin E stimulated both LHRH and AA release. The minimal effective concentrations were 22 and 88 microM, respectively. NMDA stimulated LHRH release as previously shown and this effect was not altered in the combined presence of vitamin E plus NMDA. However, AA release was significantly reduced in the combined presence of vitamin E plus NMDA. To evaluate the role of NO in vitamin E-induced LHRH and AA release, the tissues were incubated with vitamin E or combinations of vitamin E + NG-monomethyl-L-arginine (NMMA), a competitive inhibitor of NO synthase. NMMA significantly suppressed vitamin E-induced LHRH and AA release indicating a role of NO in the release of both LHRH and AA. The data suggest that vitamin E plays a role in the hypothalamic control of LHRH and AA release and that the release is mediated by NO.     

Interactions between type 1 astrocytes and LHRH-secreting neurons (GT1-1 cells): modification of steroid metabolism and possible role of TGFbeta1

The hypothesis that type 1 astrocytes (A1) might modify the activities of the enzymes 5alpha-reductase (5alpha-R) and 3alpha-hydroxysteroid dehydrogenase (3alpha-HSD) present in the GT1-1 cells has been tested. The data obtained indicate that, utilizing a co-culture technique, A1 are able to: (1) decrease the formation of dihydrotestosterone (DHT) from testosterone (T); (2) increase the formation of dihydroprogesterone (DHP) from progesterone (P); (3) decrease the conversion of DHP into tetrahydroprogesterone (THP) in GT1-1 cells. Moreover, GT1-1 cells are able to increase the formation of DHP in A1; that of DHT was unchanged. The present data might suggest the possible existence of a third isoform of the enzyme 5alpha-R; details on this hypothesis are provided in the text. Interestingly, the inhibitory effect exerted by A1 on the formation of DHT in GT1-1 cells can be mimicked by transforming growth factor beta1 (TGFbeta1). Since TGFbeta1 had been previously shown to be directly involved in the stimulatory control of LHRH secretion by GT1-1 cells, acting both on LHRH release [R.C. Melcangi, M. Galbiati, E. Messi, F. Piva, L. Martini, M. Motta, Type 1 astrocytes influence luteinizing hormone-releasing hormone release from the hypothalamic cell line GT1-1: is transforming growth factor-beta the principle involved? Endocrinology 136 (1995) 679-686.] and gene expression [M. Galbiati, M. Zanisi, E. Messi, I. Cavarretta, L. Martini, R.C. Melcangi, Transforming growth factor-beta and astrocytic conditioned medium influence LHRH gene expression in the hypothalamic cell line GT1, Endocrinology 137 (1996) 5605-5609], the present data also show that TGFbeta1 might intervene in modulating feedback signals reaching hypothalamic LHRH producing neurons. The present findings underline once more the importance of the physiological cross-talk between A1 and neurons.     

The prolonged action of the LHRH agonist buserelin (HOE 766) may be due to prolonged binding to the LHRH receptor

Hemi-pituitary glands of ovariectomized rats were superfused for 4 h with either LHRH or the analog buserelin (HOE 766) at several concentrations, and thereafter with medium only for another 1.5 h. In a further experiment glands were exposed for 2.5 h to LHRH or buserelin at a single concentration (5 ng/ml) and subsequently for another 2.5 h to either the same agonist (LHRH or buserelin) alone (5 ng/ml), the agonist plus an LHRH-antagonist (ORG 30093, 1000 ng/ml), the LHRH- antagonist alone, or medium alone. LHRH and buserelin stimulated gonadotropin release equally well. After cessation of this stimulation, the gonadotropin release by the buserelin-treated pituitary glands and the glands, treated with the highest dose of LHRH (1000 ng/ml), continued, while the release by the glands, treated with the lower doses of LHRH, declined. The LHRH-antagonist completely blocked the release of LH, stimulated by buserelin or LHRH, as well as the prolonged activation of the release, caused by buserelin pre-treatment. In a superfusion experiment with pituitary cell aggregates of 14-day-old intact female rats, buserelin stimulated the release of LH much more effectively than LHRH itself. Moreover, the release caused by buserelin declined more slowly after cessation of the stimulation. Finally, in a pituitary cell monolayer culture the Kd's of LHRH, buserelin and the antagonist were determined as 4.7 X 10(-9) M, 2.4 X 10(-10) M and 4.6 X 10(-9) respectively. It was concluded that the estimates of the potencies of LHRH and buserelin depend on the choice of the test-system. It is suggested that the long duration of action of buserelin is at least partly due to prolonged binding to the LHRH-receptor.     

Delay in the age of balano-preputial skinfold cleavage and alterations in serum profiles of testosterone, 5 alpha-androstane-3 alpha,17 beta-diol, and gonadotropins in adult rats treated during puberty with luteinizing hormone releasing hormone

Previous work has shown that chronic treatment of intact, immature male rats with luteinizing hormone releasing hormone (LHRH) decreases sex accessory gland weights and results in retardation of the normal developmental increase in the ratio of serum testosterone (T)/5 alpha-androstane-3 alpha,17 beta-diol (3 alpha-Diol) via an apparent enhancement of testicular 5 alpha-reductase or 3 alpha-hydroxysteroid oxidoreductase activities. In the present work, androgen dependent balano-preputial skinfold cleavage was significantly delayed by approximately one week in intact, immature male rats which were treated daily for two weeks with either 1.0 micrograms, 2.5 micrograms or 5.0 micrograms of LHRH during a discrete phase of pubertal development (28-41 days of age). In intact, adult (62 day old) animals which received LHRH treatments during pubertal development, serum T concentrations and sex accessory gland weights were reduced compared to control animal values. Serum 3 alpha-Diol content in the adult rats was either unaltered or increased significantly depending on the LHRH dosage employed during sexual development. Serum luteinizing hormone concentrations were not different between control and LHRH-pretreated adult rats whereas the highest dosage of LHRH employed (5.0 micrograms) during puberty resulted in a significant elevation of adult serum follicle stimulating hormone levels. It is suggested that chronic LHRH treatment of the male rat during puberty results in a perturbation in testicular androgen biosynthetic activities and an impairment of pituitary-testicular hormone feedback mechanisms which persist at least through early adulthood.