GnRH Ethylamide Research Articles

Gonadotropin-releasing hormone is prerequisite for the constitutive expression of pituitary annexin A5.

Annexin A5 (ANXA5), a member of the structurally related family of annexin proteins, is expressed in pituitary gonadotropes. We previously reported that ANXA5 expression is stimulated by gonadotropin-releasing hormone (GnRH). In the present study, we investigated ANXA5 expression in the anterior pituitary gland of GnRH-deficient mutant hypogonadal (hpg) mice. RT-PCR demonstrated that luteinizing hormone β subunit (LHβ) and ANXA5 mRNA levels were both lower in the pituitary gland of hpg mice than in wild-type mice. Immunohistochemistry showed that ANXA5 expression throughout the pituitary gland was very low in hpg mice, suggesting that ANXA5 is diminished in gonadotropes and also in other cell types. Subcutaneous administration of a GnRH analogue, des-gly10 (Pro9)-GnRH ethylamide (1 μg/day for 7 days), augmented the expression of LHβ and ANXA5 in the pituitary gland in hpg mice. However, LHβ- and ANXA5-positive cells did not show exactly matched spatial distributions. These findings suggest that GnRH is necessary for constitutive ANXA5 expression in the pituitary gland, not only in gonadotropes but also in other pituitary gland cell types. A close relationship between ANXA5 and LHβ expression was confirmed. It is suggested that a significant role of ANXA5 in the physiologic secretion of LH.

Effects of gonadotropin‐releasing hormone on in vitro gonadotropin release in testosterone‐treated immature rainbow trout

The control mechanism of gonadotropin‐releasing hormone (GnRH) on gonadotropin (GTH) release was studied using cultured pituitary cell or cultured whole pituitary obtained from Testosterone (T) treated and control immature rainbow trout. The release of FSH was not changed by salmon type GnRH (sGnRH), chiken‐II type (cGnRH‐II), GnRH analogue ([des‐Gly10D‐Ala6] GnRH ethylamide) and GnRH antagonist ([Ac‐3, 4‐dehydro‐Pro1, D‐p‐F‐Phe2, D‐Trp3,6] GnRH) in cultured pituitary cells of T‐treated and control fish. Indeed, FSH release was not also altered by sGnRH in cultured whole pituitary. All tested drugs had no effect on the release of LH in both culture systems of control fish. The levels of LH, in contrast, such as the pituitary content, basal release and responsiveness to GnRH were increased by T administration in both culture systems. In addition, the release of LH in response to sGnRH or cGnRH‐ll induced in a dose‐dependent manner from cultured pituitary cells of T‐treated fish, but which is not significantly different between in both GnRH at the concentration examined. Indeed, LH release was also increased by sGnRH in cultured whole pituitary of T‐treated fish. GnRH antagonist suppressed the release of LH by sGnRH (10–8 M) and GnRH analogue (10–8 M) stimulation in a dose‐dependent manner from cultured pituitary cells of T‐treated fish, and which were totally inhibited by 10–7 M GnRH antagonist. These results indicate that the sensitivity of pituitary cells to GnRH is elevated probably through the T treatment, and that GnRH is involved in the regulation of LH release. GnRH‐stimulated LH release is inhibited by GnRH antagonist in a dose‐dependent manner. The effects of gonadal steroids on FSH levels are less clear.

Stimulation of Annexin A5 Expression by Gonadotropin Releasing Hormone (GnRH) in the Leydig Cells of Rats

The distribution and regulation of annexin A5 expression, a gonadotropin releasing hormone (GnRH) receptor regulated protein in gonadotropes and luteal cells, in the testes of rats were examined. Immunocytochemical staining revealed high levels of annexin A5 in the Leydig and endothelial cells and lower levels in the primary spermatocytes and sperm. Hemicastration significantly increased the annexin A5 content of the remaining testis within 24 h. Annexin A5 immunoreactivity was increased mainly in interstitial tissues including the peritubular cells, while some spermatocytes also showed higher intensity of annexin A5 in the remaining testis. Administration of hCG (50 IU) enhanced the testicular content of annexin A5 after 24 h. This treatment expanded the area of interstitial tissue in the testis and increased annexin A5 immunoreactivity, but the area of the endothelial cells was unchanged. Similarly, human chorionic gonadotropin (hCG) enhanced annexin A5 expression in a primary culture of testis cells that consisted of mainly interstitial cells. Because GnRH stimulates the expression of annexin A5 in the gonadotropes and luteal cells, we examined the effect of GnRH on annexin A5 expression in the testes. We found that des-Gly10 [Pro9]-GnRH ethylamide (100 nM), a GnRH agonist, increased annexin A5 expression in cultured testis cells and that Cetrorelix (100 nM), a GnRH antagonist, inhibited the effect of hCG on annexin A5 expression. These results suggest that pituitary luteinizing hormone promotes annexin A5 synthesis in Leydig cells and that this effect could be mediated by local GnRH in the testis.

Gonadotropin Releasing Hormone (GnRH) Enhances Annexin A5 mRNA Expression through Mitogen Activated Protein Kinase (MAPK) in L.BETA.T2 Pituitary Gonadotrope Cells

The mechanism by which GnRH stimulates annexin A5 expression was examined with LbetaT2 gonadotrope cells. Continuous stimulation with GnRH analog (GnRHa, Des-Gly10 [Pro9]-GnRH ethylamide) transiently elevated LHbeta mRNA expression while maintaining annexin A5 mRNA at high levels for 24 h. GnRH antagonist blocked the effect of GnRHa on annexin A5. While 12-O-tetradecanoyl-phorbol-13 acetate, a protein kinase C activator, increased the expression of annexin A5 mRNA, bisindolylmaleimide, an inhibitor of protein kinase C, suppressed GnRHa-stimulated expression of annexin A5 and LHbeta mRNA. GnRHa stimulation of LHbeta mRNA was inhibited to a greater extent than annexin A5 by a calcium chelator BAPTA/AM. Although a calcium ionophore ionomycin stimulated the expression of both genes, only LHbeta was down-regulated. The MAPK kinase inhibitor PD98059 inhibited GnRHa induction of annexin A5 but not LHbeta mRNA. EGF stimulated the expression of annexin A5 mRNA but caused only a transient effect on LHbeta mRNA expression. These results indicate that GnRH stimulation of signaling pathway for annexin A5 mRNA expression is distinct from that of LHbeta mRNA and dependent more on MAPK.

GnRHa-induced spawning of wild-caught spotted rose snapper Lutjanus guttatus

The present study examined des-Gly10, [D-Ala6]-gonadotropin releasing hormone ethylamide (GnRHa) implant-induced spawning of recently caught wild spotted rose snapper (Lutjanus guttatus). The effect of implants containing 0 (control), 25, 50, 75 and 100 μg of GnRHa was assessed in ten females that had an oocyte diameter greater than 430 μm. All fish spawned except for the controls and one 100 μg implanted fish. The 75 μg implant-induced the spawning of eggs with a significantly higher percentage hatch (86.1±7.5 and 87.7±5.0%) and twice as many hatching eggs kg−1 body weight (BW). The 75 μg implant was used to induce the spawning of twelve females in two different tank sizes (700 and 1400 L) and with three different female:male densities (1:1, 1:3, 1:5). There appeared to be no significant effect of tank size or male density on egg quality or quantity. Non-linear 3D regressions for the dependent variables total number of eggs kg−1 BW, number of floating eggs kg−1 BW and number of hatching eggs kg−1 BW against the independent variables oocyte diameter (μm) and GnRHa dose (μg kg−1 BW) were significant (P<0.05) and had respective regression coefficients of r2=0.78, 0.80 and 0.85 (n=22). The models described a narrow range of GnRHa doses (240–280 μg kg−1 BW) and a broader range of oocyte diameters (440–500 μm) that predicted a spawn in excess of 100,000 eggs kg−1 BW. As a result, good quality eggs were obtained that enabled experimentation with larval culture.

Human placental GnRH-like factors: II. Inhibition of enzymatic degradation of GnRH-II and [D-Trp6]GnRH ethylamide tracers by human term placental cytosol fractions reveals the presence of GnRH-binding protein(s).

We describe the preliminary characterization of GnRH-binding protein(s) in human placental cytosol. Samples were analysed by chromatography on Sephadex G25. Radiolabelled GnRH and its analogues elute significantly later than the total column volume (V(t)) on Sephadex G25 column chromatography. However, incubation of GnRH II or GnRH agonist tracers with human placental cytosol reduced the intact tracer peak, with the concomitant appearance of a new peak eluting in the total column volume (V(t)). This peak increased with increasing cytosol concentration and duration of incubation, and probably represented degraded GnRH tracer, since (i) degradation-resistant GnRH agonist tracer, [D-Trp(6)]GnRH EtA, was inactivated more slowly than GnRH II, (ii) boiling of cytosol fractions abolished formation of this peak and (iii) peptidase inhibitors blocked its formation. A second new tracer peak eluted in the column void volume (V(o)) and was largely unaffected by peptidase inhibitor concentrations that blocked tracer degradation. The magnitude of this high molecular weight peak depended on the GnRH tracer employed, cytosol concentration, and the pH, duration and temperature of incubation. Tracer associated with this third peak appeared similar to intact GnRH tracer by TLC. Unlabelled GnRH analogues and isoforms decreased both tracer degradation and formation of the V(o) peak, but their specificity and affinity for the two processes differed. Ligand blots identified several bands that were abolished by inclusion of unlabelled agonist during incubation. Our data indicate the presence of specific GnRH binding protein(s) and GnRH peptidases that may modulate local actions of GnRH in the human placenta.

Paracrine regulation of FSH by follistatin in folliculostellate cell-enriched primate pituitary cell cultures.

Primary pituitary cell cultures are an important tool for understanding pituitary hormone gene expression. In the course of study of pituitary cell cultures from nonhuman adult male primates, pituitary secretory cells were noted to be rapidly overgrown by epithelioid cells with the morphological, immunocytochemical, and proliferative characteristics of folliculostellate cells. Using competitive RT-PCR assays, follistatin mRNA levels were found to increase 4-fold as folliculostellate cells proliferated with time in culture, whereas FSH-beta mRNA and FSH secretion were suppressed. Follistatin gene expression was stimulated by activin-A and pituitary adenylate cyclase-activating polypeptide but not by [D-Trp(6)]-GnRH ethylamide. Testosterone (T) also increased follistatin mRNA levels and follistatin protein secretion. FSH-beta mRNA was stimulated by [D-Trp(6)]-GnRH ethylamide and activin but was suppressed by T. The reciprocal relationship between follistatin and FSH-beta mRNA levels as folliculostellate cells proliferate with time in culture implies a role for folliculostellate cells in the follistatin-activin system in primates. The actions of GnRH and T on follistatin and FSH-beta mRNA levels in these cultures were opposite to effects observed in pituitary cultures from rats and identify species differences in the control of FSH production that may be folliculostellate cell-related.

Annexin 5 Messenger Ribonucleic Acid Expression in Pituitary Gonadotropes Is Induced by Gonadotropin-Releasing Hormone (GnRH) and Modulates GnRH Stimulation of Gonadotropin Release

Our previous studies on annexin 5, a member of the annexin family of proteins, have shown its expression in the anterior pituitary gland, its preferential distribution in gonadotropes, and its increase after ovariectomy. In the present study, we examined (1) whether annexin 5 is synthesized in gonadotropes, (2) whether its expression is under the control of gonadotropin-releasing hormone (GnRH), and (3) the effect of annexin 5 on gonadotropin release. Large cells, also called castration cells, appeared in anterior pituitary tissue 3 weeks after ovariectomy. These cells have been confirmed to be hyperfunctioning gonadotropes and are easily discriminated from other pituitary cells without immunostaining. Using in situ hybridization with a digoxigenin-labeled ribonucleic acid probe, enhanced expression of annexin 5 messenger ribonucleic acid (mRNA) in these gonadotropes was clearly demonstrated. Northern blot analysis showed an increase in the level of annexin 5 mRNA expression 3 weeks after ovariectomy. It was lessened 3 h after the injection of Cetrorelix (GnRH antagonist, 10 µg i.v.). Administration of a GnRH analog [GnRHa; Des-Gly 10 (Pro9) GnRH ethylamide, 0.2 ml of 2.5 µg/ml saline ten times intraperitoneally at 30-min intervals] significantly increased pituitary annexin 5 mRNA. In primary cultures of anterior pituitary cells, recombinant rat annexin 5 stimulated luteinizing hormone (LH) and follicle-stimulating hormone (FSH) release in a dose-dependent manner. Concomitant administration of annexin 5 (1 µg/ml) and GnRHa augmented the LH and FSH release induced by GnRHa. After a 1-hour incubation, cycloheximide (10 µg/ml) apparently inhibited the LH response to GnRHa, while annexin 5 (2 µg/ml) moderated this inhibition. Further, the antisense oligodeoxynucleotide to annexin 5 mRNA blunted the LH response to GnRHa. It is thus concluded that annexin 5 is synthesized in the gonadotropes under the effect of GnRH, and it is suggested that annexin 5 synthesis mediates at least partly GnRH receptor signaling to stimulate gonadotropin secretion.

Effect of Long-Term Treatment with the Antiestrogen EM-652.HCl on Pituitary Estrogen Receptor Alpha and Prolactin mRNA Expression in Intact, Ovariectomized and Gonadotropin- Releasing Hormone-Treated Female Rats

Estrogen receptor α (ERα) is the predominant estrogen receptor subtype in the anterior pituitary gland. In order to assess the influence of the pure antiestrogen EM-652.HCl on ERα gene transcription, we have studied the effect of long-term administration of the antiestrogen in ovariectomized rats as well as in intact female rats treated or not with the GnRH-agonist D-trp⁶, des-Gly-NH₂¹⁰ GnRH ethylamide (GnRH-A), a treatment which induces pharmacological castration. To evaluate the degree of pituitary responsiveness to changes in estrogen exposure, prolactin (PRL) mRNA levels were also measured. ERα and PRL mRNA levels were evaluated by quantitative in situ hybridization. It was found that, 49 weeks after ovariectomy (OVX), pituitary ERα mRNA levels were decreased by 55%. Long-term administration (49 weeks) of EM-652.HCl to OVX animals resulted in a further 41% decrease in ERα mRNA. On the other hand, ovariectomy induced an 82% decrease in PRL mRNA levels while the administration of EM-652.HCl to OVX animals did not further decrease PRL mRNA. The administration of EM.652.HCl or GnRH-A alone to intact rats during 52 weeks did not significantly modify pituitary ERα mRNA levels. Concomitant administration of both GnRH-A and EM-652.HCl induced 41 and 47% decreases in ERα mRNA levels, when compared to the levels measured in vehicle-treated and GnRH-treated animals respectively. Combined administration of EM.652.HCl and GnRH-A induced 56 and 65% decreases in PRL mRNA, respectively. When EM-652.HCl was administered concomitantly with GnRH-A, the inhibitory effect on PRL mRNA levels was more marked than that observed in GnRH-A-treated animals. The present data demonstrate that when circulating estrogens are absent or maintained at very low levels by GnRH administration, EM-652.HCl can still depress ERα gene transcription. It is suggested that estrogens can positively regulate pituitary ERα gene transcription and that the antiestrogen EM-652.HCl can downregulate by itself pituitary ERα gene transcription.

Role of extra-ovarian oestrogens in the regulation of gonadotropin releasing hormone mRNA expression in the rat brain.

To further understand the role of oestrogens in the regulation of gonadotropin releasing hormone (GnRH) mRNA expression in the female rat brain, the effect of EM-652.HCl, a pure anti-oestrogen, was studied in intact and ovariectomized rats as well as in rats chronically treated with a GnRH agonist D-trp6, des-Gly-NH210 GnRH ethylamide (GnRH-A), a treatment which blocks ovarian steroidogenesis. Quantitative in situ hybridization was used to measure GnRH mRNA at the cellular level in the preoptic-anterior hypothalamic area. It was found that, 49 weeks after ovariectomy (OVX), the number of silver grains per cell corresponding to GnRH mRNA was increased by 34%. Long-term administration (49 weeks) of EM-652.HCl to OVX rats resulted in a further increase (11% over the levels measured in OVX rats) in the hybridization signal. By contrast, in intact female rats, treated during 52 weeks with EM-652.HCl, a 49% increase in the GnRH hybridization signal was detected. In rats treated with GnRH-A during the same period, a 20% decrease in GnRH mRNA was observed. When EM-652.HCl was administered concomitantly with GnRH-A, a further 63% increase over the mRNA levels recorded in GnRH-A treated rats was found. Thus, long-term treatment with the anti-oestrogen EM-652.HCl can upregulate GnRH mRNA expression in intact female rats, OVX rats and female rats chronically treated with a GnRH-A. It is suggested that the pure anti-oestrogen EM-652.HCl can exert an influence on the oestrogen feedback mechanism involved in the regulation of GnRH neuronal activity by neutralizing the action of locally produced or low circulating levels of oestrogens remaining after OVX or GnRH-A treatment.

Effect of a single injection of a long-acting gonadotropin-releasing hormone agonist on prepubertal male and female pigs on reproductive organs, growth performance and sensory qualities of pork roasts.

Crossbred pigs (n = 200) were used to study the effects of a long-acting form of gonadotropin-releasing hormone (GnRH) agonist on the reproductive systems of male and female pigs and their growth performance and sensory quality of pork roast. Treatment was a single injection of a controlled release formulation of GnRH agonist [D-Trp6, des-Gly10]-GnRH ethylamide to release 5 micrograms/(kg x day) for 4 months beginning when the pigs were 66 +/- 2 days old. Pigs were allocated to five groups of 40 animals each: males castrated (CM) at 13 +/- 2 days, intact males (IM), treated males (TM), intact females (IF) and treated females (TF). Ovarian and uterine weights at slaughter averaged 3.67 and 79.8 g, respectively, in IF compared with 1.38 and 26.5 g in TF (P < 0.05). Testicular weights were 203 g in IM and 36.8 g in TM (P < 0.05). Microscopic observations of the testes revealed an absence of sperm cells but the presence of germ cells. Steroid concentrations at slaughter from all pigs showed that intact males had significantly more testosterone in their serum (26.36 +/- 9.87 nmol/L) compared with TM, CM, IF or TF groups and that treated males had intermediate concentrations (12.50 +/- 7.44 nmol/L) higher (P < 0.05) than those in CM and TF. Administration of GnRH agonist during the growth period of male pigs had no consistent effect on growth performance, but as compared to IM pigs, some of the carcass characteristics such as meat ratio (49.1 vs 50.2% in TM and IM; P < 0.001), dressing percentage (77.5 vs 76.5% in TM and IM; P < 0.05) and average backfat (20.8 vs 17.6 mm in TM and IM; P < 0.05) were modified by such a treatment. Meat quality, however, as determined by flavor and tenderness evaluations by sensory panelists, were similar (P < 0.05) in all groups and off-flavor scores were lower in TM than in IM (P < 0.001). As for males, backfat and meat ratio were different in TF compared to IF (P < 0.05) and roast juiciness was higher in TF than IF (P < 0.05). These results suggest that GnRH agonist can reduce gonadal secretory activity to castration levels during the growth period of prepubertal male pigs and could be an alternative to surgical castration in the pork industry with no negative effects on growth and meat quality. No advantage to endocrine castration in females was found.

Stimulatory effect of gonadotropin-releasing hormone (GnRH) on in vitro prolactin secretion and presence of GnRH specific receptors in a subset of human prolactinomas.

The purpose of this study was to determine whether gonadotropin-releasing hormone (GnRH) may exert a direct action on human prolactinomas. On a series of 17 adenomas, we studied the effect of GnRH on the in vitro prolactin (PRL) secretion of dispersed and perifused cells of 10 cases and the [125I]GnRH agonist binding on frozen sections of three out of the adenomas studied in perifusion and on the membrane preparations of seven other cases. Two 20-min pulses of GnRH (10(-7) mol/l) stimulated the in vitro PRL secretion of three adenomas out of 10 (increase of 200, 444 and 205%, respectively, above basal levels). The GnRH receptors of three adenomas bound GnRH agonist (Des-Gly10-(D-Ala6)-GnRH ethylamide). The binding was specific, with a high affinity (Kd = 0.60, 0.48 and 0.40 nmol/l) similar to that of two human anterior pituitaries obtained post-mortem (Kd = 0.70 and 0.40 nmol/l). Indirect immunoperoxidase revealed that the majority of the cells (60-90%) in all the adenomas studied contained immunoreactive PRL. Four of them also contained cells immunoreactive to the alpha-subunit of the glycoprotein hormones. In none of the prolactinomas were cells immunoreactive to antiserum of anti-beta-luteinizing hormone, anti-beta-follicle-stimulating hormone or anti-beta-thyrotropin. All the prolactinomas that were responsive to GnRH in perifusion experiments and/or bound specifically to [125I]GnRH agonist were also immunoreactive for alpha-subunit. These results show that GnRH, via GnRH specific receptors, exerts a stimulation on in vitro PRL secretion in a subset of prolactinomas characterized by the presence of alpha-subunit.

Modulation of the steroidogenesis of cultured human granulosa-lutein cells by gonadotropin-releasing hormone analogs.

GnRH analogs are widely used in reproductive medicine to create a hypogonadotropic hypogonadism. However, numerous animal studies have demonstrated a direct regulation of the gonadal function by GnRH. To ascertain this direct ovarian effect in humans, the steroidogenesis of cultured human granulosa cells was studied with or without GnRH and five of its agonists. Buserelin (D-Ser(But)6, desGly10) GnRH ethylamide, leuprorelin (D-Leu6, desGly10) GnRH ethylamide, H4055 (desGly10) GnRH ethylamide, and H4065 (D-Trp6, desGly10) GnRH ethylamide significantly enhanced estradiol secretion. In addition, buserelin induced a significant cell surface decrease that seemed to be mediated by cytoskeleton modifications. The two other molecules, GnRH and triptorelin (D-Trp6) GnRH), had no effect on estrogen secretion at any of the studied concentrations. Thus, despite similar pituitary effects, these agonists did not exhibit the same ovarian action. This may be accounted for by the differences found between pituitary and ovarian receptors. These results suggest that some GnRH analogs can modulate human granulosa cell steroidogenesis at least in the preovulatory period.

Human placental gonadotrophin-releasing hormone (GnRH) binding sites: I. Characterization, properties and ligand specificity

Radioiodinated gonadotrophin-releasing hormone tracers were prepared from mammalian (m GnRH), salmon (s GnRH), lamprey (l GnRH) and the two forms of chicken GnRH (ch GnRH I and ch GnRH II), and also from the GnRH agonist (GnRH A) analogues, Buserelin ([D-Ser(tBu) 6] 1–9 GnRH ethylamide) and Tryptorelin ([D-Trp 6 GnRH]1–9 ethylamide) and a GnRH antagonist (GnRH, ANT [Ac 3, 4-dehydro-Pro 1, D-p-F-Phe 2, D-Trp 3,6 LHRH). Specific binding of hormone tracers was compared in homogenates and membrane fractions from human placenta and rat pituitary. GnRH agonist tracers bound readily to pituitary and placental binding sites. Binding of m GnRH to rat pituitary membranes was low compared to agonist binding, whereas other GnRH iso forms were not bound. Binding of 125I-labelled m GnRH to human placental membranes was also low compared to that of Buserelin, and l GnRH and ch GnRHI tracers bound poorly. However, human placental membranes bounds GnRH and ch GnRH II to the same extent as GnRH A. Studies of the inactivation of GnRH tracers following incubation with rat pituitary and human placental membranes demonstrated that, although GnRH isoforms were degraded at different rates in these tissues, the differential ability of GnRH isoforms to bind to pituitary or placental binding sites was not related to differences in degradation of tracers, but rather to differences in ligand specificity. Specific binding of 125I-labelled GnRH agonists (GnRH A) and mammalian GnRH(m GnRH), s GnRH and ch GnRH II tracers to human placental membrane fractions increased linearly with increasing membrane protein at low concentrations. Binding was dependent on both the duration and temperature of incubation, and pH profiles of 125-labelled GnRHA, s GnRh and ch GnRH II binding to placental membranes were similar. Once bound s GnRH formed a tighter complex with placental receptors than GnRH A, though 125I-labelled s GnRH was inactivated more rapidly than agonist tracer during incubation with placental membranes. Binding of GnRH tracers was specific for molecules with the GnRH structure. Deletions of amino acid residues at positions 1–3 and/or deamidation at Gly 10 reduced binding potencies for both human placental and rat pituitary binding sites, indicating that both ends of the GnRH molecule were required for optimal binding. Modifications which conferred increased agonist activity led to markedly increased receptor binding potency in the rat pituitary, but only slightly increased potency for placental receptors. In contrast, GnRH antagonist analogues had increased potency towards pituitary receptors, but much reduced potency towards human placental binding sites. These studies highlight further the differences between human placental and rat pituitary GnRH receptors.

Conformational and receptor binding studies of gonadotropin releasing hormone and its analogs with respect to their biological activities

The structure of porcine, bovine or ovine hypothalamic gonadotropin releasing hormone (GnRH) as elucidated by Schally and Guillemin and their associates was found to be identical. It is a linear decapeptide with distinctive structural features and blocked N- and C-termini. Chicken has two GnRHs, salmon and mammalian GnRHs are closely related in structure and varied in positions 7 and 8. GnRH of lamprey, the most primitive vertebrate, has 50% of variations in its structure (positions 3,5,6,7, and 8) as compared with others. The synthetic GnRH exhibits potent biological activities among mammalian species via its ability to release the luteinizing hormone (LH) and the follicle-stimulating hormone (FSH) from the anterior pituitary. However&e amount needed to induce carp to spawn was found to be much higher than that required to effect ovulation among mammals (1). It is most probably due to the existence of species variation in the GnRH molecules between mammals and teleosts. We have successfully applied the synthetic analogs, [D-Ala6, des Gly-NH,‘?-GnRH ethylamide (G&H-A), to induce spawning in farm fish in China since 1974 (2). Surprisingly, the synthetic peptide according to structure of salmon GnRH elucidated by Vale et a1.(3) was found to be incapable of inducing carp to spawn more smoothly or in a way better than that obtained with mammalian GnRH. Superactive agonists of GnRH to enhance fertility and exert prolonged pharmacological effects have been synthesized. Most of them are products of GnRH modified in the C-terminal Gly-NH, with ethylamide and in the 6th position Gly, which is involved in the formation of B-bend or U-shaped turn, by D-amino acids having a hydrophobic side chain such as D-Ala, D-Leu, D-Tip, D-Ser(But) and D-Nal. They have much better binding affinity to the GnRH receptor and higher resistance to enzymatic degradation. Conformational studies The conformational study of GnRH and its analogs by fluorescence spectroscopy was described with respect to their biological activities (3). Only the Trp3 fluorescence spectroscopy was observed for GnRH or its analogs (G&H-A) which contains one Trp and one Tyr. The maximal emission wavelength of GnRH in aqueous solution is 352nm, while that of free tryptophane is 360nm (the spectra were not corrected), a slight blue shift of 8nm could be interpreted as being due to the shielding of Trp residue in a certain extent from the aqueous phase by B-bend structure that makes Ser4-Ty?-Gly6-Leu7 to form a reverse turn and the C-terminal region to be in the proximity of the N-terminal region so as to fit well into the recognition site of the receptor molecule.

Pattern of gonadotropin-releasing hormone (GnRH)-like stimuli sufficient to induce follicular growth and ovulation in ewes passively immunized against GnRH.

The pattern of GnRH-like stimuli capable of inducing follicular growth, ovulation, and luteal function was evaluated in ewes passively immunized against GnRH. The estrous cycles of 30 regularly cyclic sheep were synchronized using vaginal pessaries impregnated with a synthetic progestogen. Animals were passively immunized against GnRH (groups 2-5, n = 6) or the carrier protein, keyhole limpet hemocyanin (KLH; group 1, n = 6), at the time of pessary removal (PR). Circhoral delivery of saline (groups 1, 2, and 5) or low amplitude GnRH agonist (des-Gly10 GnRH ethylamide [100 ng/hourly pulse]; groups 3 and 4) was initiated at PR and continued for 3 (groups 4 and 5) or 12 days (groups 1-3). In groups 4 and 5, the amplitude of the GnRH-like stimulus was increased to 800 ng/hourly pulse (stimulus-shift) during the 24-h period beginning 72 h after PR. The amplitude of the hourly stimulus was adjusted to 100 ng/pulse 96 h after PR and continued at that level to Day 12. The endocrine changes associated with follicle growth and maturation (serum concentrations of estradiol [E2] above 10 pg/ml), ovulation (surge-like secretion of LH and FSH), and normal luteal function (serum concentrations of progesterone [P] above 2 ng/ml) were evident in ewes passively immunized against KLH (group 1). In this group, the preovulatory surge of gonadotropins was noted 48.7 +/- 1.2 h after PR. These endocrine events were blocked by passive immunization against GnRH (group 2).(ABSTRACT TRUNCATED AT 250 WORDS)

The gonadotropin-releasing hormone agonist leuprolide affects the thymus and other non-reproductive systems of female rats

To evaluate the effects of a gonadotropin-releasing hormone agonist on non-reproductive systems, we administered [D-Leu6,Des-gly10]-GnRH ethylamide (leuprolide; 5 micrograms/day) for 21 days to female Sprague-Dawley rats. In Experiment 1, continuous infusion (Alzet minipumps sc) was compared to injection. Increased thymus and body weights and decreased estradiol and uterine weights were noted for both administration methods. Spleen weight increased only in rats treated by continuous infusion. Ovary, kidney and liver weights did not change. Only leuprolide-injected rats had elevated LH with decreased corticosterone and ACTH levels, possibly related to the injection process. Glucose, insulin, progesterone, FSH and corticosterone/ACTH were not different. In Experiment 2, intact and ovariectomized rats were implanted with minipumps delivering leuprolide or 0.9% NaCl. Body and thymus weights increased, whereas uterine weight and estradiol declined in both leuprolide-treated and ovariectomized rats. No synergism between leuprolide and ovariectomy was noted. Thymosin alpha 1, but not thymosin beta 4, increased in leuprolide-treated ovariectomized rats. Peripheral white blood cell count was elevated in leuprolide-treated intact rats and ovariectomized rats. In bone marrow, non-nucleated cell count declined in leuprolide-treated intact rats, contributing to the decreased total cell count in this group. Nucleated cell count was unaffected. Therefore, thymus weight gain was accompanied only in some cases by functional changes. Our results demonstrate that leuprolide affects non-reproductive systems, in a similar manner to ovariectomy. We suggest that such alterations may be due to the hypoestrogenic environment produced by leuprolide.

Neuropeptide Y Modulates the Binding of a Gonadotropin-Releasing Hormone (GnRH) Analog to Anterior Pituitary GnRH Receptor Sites*

Neuropeptide Y (NPY) increases LH secretion in part by enhancing the release of LH in response to GnRH. The present studies examined whether NPY influences the binding of GnRH to its receptors and also assessed whether specific binding sites for NPY exist in rat anterior pituitary membranes. In concentrations from 66-200 nM, NPY dose-dependently enhanced the binding of a 125I-labeled GnRH agonist, [D-Ala6, des-Gly10]GnRH ethylamide (GnRHa; 30 pM) to anterior pituitary membranes of chronically ovariectomized rats; higher concentrations of NPY were ineffective. At 200 nM, NPY significantly increased the high affinity binding of the GnRHa to these membranes, without change in the apparent maximum binding capacity. Further, 200 nM NPY significantly increased the binding of [125I]GnRHa when this tracer was used in concentrations of less than 100 pM, but NPY did not increase the binding of higher concentrations of [125I]GnRHa. Peptide YY, a gastrointestinal peptide structurally and functionally related to NPY, and the hypothalamic/pituitary peptide galanin (both at 200 nM) also increased the binding of [125I]GnRHa (30 pM) to anterior pituitary membranes, but to approximately one third of the extent produced by NPY. Salmon calcitonin, which, similar to NPY, is a strongly hydrophobic neuroendocrine peptide, did not alter GnRHa binding. Mg++ ion dose-dependently reduced the affinity of GnRHa binding, without changing the maximum binding capacity, and also attenuated the increase in GnRHa binding produced by NPY. To further characterize the nature of NPY interaction with anterior pituitary membranes, [125I] peptide YY was used to label NPY binding sites in anterior pituitary and hypothalamic membranes from ovariectomized rats. Specific, and predominantly high affinity, NPY binding sites were demonstrated in hypothalamic membranes, whereas NPY binding to anterior pituitary membranes could be resolved into high and low affinity components. These results show that at low nanomolar concentrations, NPY can enhance the association of GnRHa to receptors in anterior pituitary membranes. This demonstration of increased GnRHa binding in the presence of NPY may explain in part the action of this neuropeptide to potentiate GnRH-induced LH release from anterior pituitary cells.

Effects of TAP-144-SR, A Sustained-Release Formulation of a Potent GnRH Agonist, on Experimental Endometriosis in the Rat.

A new, simple experimental endometriosis model was established by auto-transplanting endometrial tissue fragments beneath kidney capsules in female rats. The transplanted endometrial tissue grew well, forming a fluid-filled cyst, which reached maximal size 2 to 3 weeks after transplantation. The growth and maintenance of the transplants was dependent on the ovary: ovariectomy induced regression of well grown transplants. The therapeutic effects of TAP-144-SR (biodegradable microcapsules of copoly (DL-lactic/glycolic acid) copolymer containing a potent GnRH agonist, TAP-144 (D-Leu6-[des-Gly10-NH2]-GnRH ethylamide, leuprolide acetate) were studied with this rat endometriosis model. A single sc injection of TAP-144-SR (corresponding to 1, 10 or 100 micrograms/kg/day of TAP-144), suppressed the growth of the transplanted endometrial tissues and uterine weight in a dose-dependent manner. At 100 micrograms/kg/day, the suppressive effect was more marked in rats given TAP-144-SR than in those given TAP-144 solution. The extent of suppression was comparable to that caused by ovariectomy. Serum and pituitary concentrations of LH and FSH were also reduced more markedly by the administration of TAP-144-SR than by TAP-144 solution. From these results, the present endometriosis model was found to be useful for the evaluation of compounds with potential therapeutic activity. The sustained-release formulation of TAP-144 seems to be beneficial over its solution in terms of both convenience and efficiency for therapy of patients with endometriosis.

Temperature and Protein Kinase C Modulation of Gonadotropin‐Releasing Hormone Receptors in Pituitary Cells from Intact or Castrated Male Rats

Abstract Binding constants of [(125) I]Des-Gly(10)-(D-Ala(6))-gonadotropin-releasing hormone-ethylamide (GnRHa) to dispersed pituitary cells were evaluated in a 4-day culture. Cells were sampled either from intact or from castrated male rats and binding was measured at various temperatures before or after treatment with phorbol-12-myristate-13-acetate (PMA) or 1-5-(isoquinolinyl-sulfoxyl) 2-methylpiperazine (H7), which activate or inhibit, respectively, protein kinase C (PKC). In cells from intact rats incubated with increasing concentrations of ligand at 21 degrees C for 25 min, the Scatchard plot was not linear and calculation of the Hill coefficient (N(H)) was indicative of positive cooperativity (N(H)= 1.26 +/- 0.02). Such non-linearity was not observed when cells were incubated at 0.5 degrees C for 3 h. In that condition the maximal number of binding sites measured at equilibrium (B(max)) increased (15.1 +/- 0.05 versus 9.3 +/- 0.5 fmoles x mg(-1) proteins at 21 degrees C). Two control experiments permitted us to rule out the possibility that lower B(max) at 21 degrees C might reflect internalization: 1) Cells were first incubated with the ligand at 21 degrees C for 25 min and subsequently for 3 additional hours at 0.5 degrees C. Preincubation did not affect the B(max) obtained at 0.5 degrees C; 2) when the radioligand bound to the cell surface was washed out with an acidic buffer, only 13% of the specific radioactivity was retained irrespective of the ligand concentration applied, a much lower value than the 40% binding difference observed between 0.5 degrees C and 21 degrees C. When the cells were incubated with PMA, the Scatchard plot was linearized and the B(max) recorded at 21 degrees C increased by 50% over control cells (13 +/- 0.7 fmoles x mg(-1) proteins). Conversely, inhibition of PKC by H7, a preferential PKC inhibitor, was ineffective. In contrast, cells sampled from castrates exhibited linear and comparable Scatchard plots at either 0.5 degrees or 21 degrees C, with B(max) values of 14.4 +/- 0.3 and 15 +/- 0.34 fmoles x mg(-1) proteins, respectively. PKC activation did not affect binding in that model, but H7 decreased the number of sites (B(max)= 10.7 +/- 0.9) and induced appearance of positive cooperativity (N(H)= 1.36 +/- 0.07). Taken together, these experiments reveal a pool of GnRH receptors in the pituitary of intact rats that recognizes the ligand in a phosphorylation-dependent manner. These binding sites also became evident when biological properties of the membrane were modified by temperature or cell homogenization. After castration, PKC activation was no longer a prerequisite for recruitment of the total population of receptors whereas protein kinase inhibition resulted in a reduction of maximal binding. Finally, our observations demonstrate that GnRH binding can exhibit positive cooperativity, either on normal cells or on cells from castrates after protein kinase inhibition, suggesting that such a cooperativity is not related to a GnRH-dependent phosphorylation.