The partial purification and characterization of GnRH-like activity from prostatic biopsy specimens and prostatic cancer cell lines

We have investigated the possibility of the secretion of gonadotrophin-releasing-hormone (GnRH)-like peptides by prostatic cancer cells in culture and their presence in cytosolic preparations from human prostatic biopsy specimens. A GnRH-specific radioimmunoassay showed GnRH-like activity in concentrated cytosolic preparations and conditioned media from DU 145, an androgen-insensitive human prostatic cell line and from LNCaP, an androgen-responsive prostatic cancer cell line. GnRH immunoreactivity in culture media correlated directly with cell numbers. HPLC demonstrated that this GnRH-like material co-migrated with synthetic GnRH. This homology between synthetic GnRH and partially purified prostatic GnRH was confirmed following V8 protease and trypsin digestion which resulted in similar alterations in HPLC characteristics. The mean content of GnRH-like activity/g specimen tissue was significantly more in malignant tissue (88.5 ± 80.5 fmol) than in benign (29.6 ± 22 fmol), though more specimens of benign tissue were positive (37/54) than malignant tissue (6/22). This observation, taken with an earlier finding of GnRH-specific receptors in a hormone-sensitive cell line and human cancer specimens provides supportive evidence for the autocrine hypothesis of cell regulation.     

GnRH-like proteins in cows: concentrations during corpora lutea development and selective localization in granulosal cells

Gonadotropin-releasing hormone (GnRH)-like proteins with anti-gonadotropic properties were recently discovered in the ovaries of several species, including humans. Since neither GnRH receptors nor GnRH are in bovine ovarian tissue, we examined, in the present studies, whether concentrations of GnRH-like proteins varied during development of the corpus luteum (CL) and whether GnRH-like proteins were selectively localized in ovarian cells of cows. For these studies, GnRH-like proteins were extracted from various ovarian and nonovarian tissues and fluids and fractionated for hydrophobic interaction chromatography. A highly specific and sensitive radioreceptor assay (RRA) was used to quantify concentrations of GnRH-like proteins. The major findings of these studies demonstrated that 1) the amount of GnRH-like proteins in the corpus luteum (CL) was proportional to the weight of the CL; 2) the concentration of GnRH-like proteins in luteal tissue decreased during development of the CL; 3) GnRH-like proteins were in ovarian and numerous nonovarian tissues, but were not in the heart, plasma, or follicular fluid; 4) the retention time for GnRH-like proteins following high-pressure liquid chromatography (HPLC) varied with the tissue source; and 5) compared with all other tissues, the greatest concentration of GnRH-like proteins was in granulosal cells. We concluded that the concentration of GnRH-like proteins in luteal cells decreased during development of the CL, and that a specific GnRH-like protein was selectively localized in bovine granulosal cells.     

Identification of a gonadotropin-releasing hormone-like factor in the rabbit fetal placenta

The biological and immunological gonadotropin-releasing hormone (GnRH)-like activities in rabbit fetal placentas collected at Day 18 of gestation were investigated. Both crude and partially purified acid extracts of placental tissue were tested. A similarly prepared liver extract served as a control. Immunological GnRH-like activity, determined through a GnRH radioimmunoassay was 1.3-2.0 pg/mg protein for the crude placental extract, 7.1-9.2 pg/mg protein for the partially purified placental extract and was nondetectable for liver extract. Both the crude and partially purified placental extracts increased (P less than 0.01) luteinizing hormone (LH) release by dispersed rabbit pituitary cells, whereas the liver extract had no effect. The (Ac-D-p-Cl-Phe1,2, D-Trp3, D-Arg6, D-Ala10)-GnRH antagonist was used to determine whether the biological GnRH-like activity in the placental extract was mediated through GnRH receptors. All three doses of antagonist (10, 100 and 1000 ng) suppressed the biological GnRH-like activity in the placental extracts. Molecular sieve chromatography of the partially purified placental extract showed that the immunoreactive GnRH-like factor eluted in the same fractions as the GnRH standard. These data indicate that the rabbit fetal placenta has both immunological and biological GnRH-like activity.     

Identification and partial characterization of gonadotropin-releasing hormone-like factors in human seminal plasma

Gonadotropin-releasing hormone (GnRH)-like material was measured by radioimmunoassay in acid-ethanol-extracted human seminal plasma using radiolabeled D-[Leu6] GnRH ethylamide as labeled ligand, authentic GnRH as standard, and antibody raised against D-[Lys6] GnRH analog. The mean amount of GnRH-like material measured in the seminal plasma of semen samples with sperm counts greater than 20 X 10(6)/ml was 229.0 +/- 66 pg/ml, with sperm counts less than 20 X 10(6)/ml was 213 +/- 42 pg/ml, and from vasectomized samples was 252 +/- 36 pg/ml. There was no significant difference among the three groups. Scatchard analysis of radioreceptor binding data demonstrated significant displacement of GnRH agonist ligand from castrated male rat pituitary membrane preparations. Ultrafiltration and gel column chromatography of pooled extracted seminal plasma identified two compounds with apparent molecular weights of 2600 and 5000 that differ chemically and immunologically from native GnRH. Further characterization using affinity column chromatography suggests that at least one of these GnRH-like factors is a glycosylated protein.     

Bioassayable gonadotropin releasing hormone-like activity in the spent nutrient medium of rat sertoli cells in primary cultures

Investigations were undertaken to demonstrate bioassayable GnRH-like activity in the spent nutrient medium of Sertoli Cells in Primary Cell Cultures (SCCM) using an in vitro bioassay procedure. The bioassay involved the use of intact pituitaries from day-12 male rats. Amounts of LH and FSH released into the incubation medium in response to varying doses of synthetic GnRH were measured by radio-immunoassays. SCCM was extracted with methanol and diethylether. The extracted SCCM was evaporated under a stream of nitrogen at 50 degrees C. The dry residue was re-suspended in the assay medium and centrifuged. The supernatant was used as the source of GnRH-like activity. The treated SCCM gave a dose response parallel to that given by synthetic GnRH. Boiling and trypsinization of extracted SCCM destroyed the GnRH-like activity. Addition of synthetic GnRH to different doses of extracted SCCM gave additive response.     

Evolution of a Neuropeptide Family: Gonadotropin-Releasing Hormone

SYNOPSIS. Gonadotropin-releasing hormone (GnRH), a small peptidein the brain, is essential for reproduction. It is now clearthat GnRH is part of a family of closely related molecules.The primary structure has been identified for 4 GnRH molecules:mammalian, chicken I, chicken II and salmon. During evolutionthe molecule has been conserved in length, terminal amino acidstructure, 70–90% of amino acid sequence and the His²Trp³residues,residues, which are important in the release of gonadotropin.Alterations have occurred in positions 5, 7 and 8, regions thoughtto be involved in receptor binding. The receptors for GnRH haveapparently evolved also in that the mammalian and avian receptorsvary considerably in their ability to bind different GnRH molecules.Other GnRH family members have been distinguished indirectlyby chromatographic or immunological means; 3 different GnRH-likemolecules are present, respectively, in lamprey, sturgeon andsalmon (a second form). Several GnRH-like molecules includingthose in chondrichthyes have not yet been distinguished fromthe proposed salmon II molecule. The lamprey GnRH-like moleculemay be a nodal point inthe analysis of the ancestral molecule;hagfish do not contain a detectable GnRH molecule. The elucidationof the GnRH precursor molecule in human placenta showed thepresence of a 53-amino-acid gene-related peptide of unknownfunction, but did not reveal the basis for expression of multipleGnRH forms in many nonmammalian species. GnRH has a varietyof novel functions in addition to release of gonadotropin fromthe pituitary. During evolution certain functions such as thosein the retina and sympathetic ganglia have apparently disappearedin amniotes, but GnRH placental functions have appeared in mammals.     

Novel cyclic azo-bridged analogs of gonadotropin-releasing hormone.

Five linear analogs of GnRH containing a p-aminophenylalanine (Pap) residue in their sequence and their six corresponding azo-bridged cyclic derivatives were synthesized. The precyclic peptides were prepared on solid-support, while azo-cyclization was performed in solution by diazotization of the p-aminophenylalanine residue followed by intramolecular coupling of the formed diazo salt with either tyrosine or histidine side chains present in the sequence. All peptides were examined for their binding ability to the GnRH receptor expressed on rat pituitary membranes and for their LH-release activity from dispersed rat pituitary cells. Linear analogs 1 i.e [Pap(5)] GnRH and 3, i.e. [Tyr(3), Pap(5)] GnRH, were found to bind to the GnRH receptors only slightly less avidly than native GnRH. Their cyclization, however, led to a marked reduction in the binding capacity, i.e. from IC(50) of 10(-9) M to the 10(-7) M range, and in biopotency, i.e. LH-release. All other linear and cyclic peptides were found to bind selectively to the GnRH receptor only in the low microM range. Only peptide 1 was found comparable to native GnRH in respect to LH-release activity and thus may potentially be a good agonist of the parent peptide. Peptides 1-4, the most potent GnRH receptor binders, were examined for their conformational properties using CD. Cyclic-azo peptides 2 and 4 were further evaluated by NMR spectroscopy in solution combined with molecular modeling. The structural information obtained explains in part the GnRH-like biological activity observed.     

GnRH在文昌鱼脑和哈氏窝的分布

用免疫细胞化学方法和抗哺乳动物ＧｎＲＨ抗体研究了ＧｎＲＨ免疫活性细胞在文昌鱼脑和哈氏窝的分布。ＡＰＡ免疫染色法证明了哺乳动物ＧｎＲＨ免疫活性结构分布在文昌鱼端脑前端和靠近中脑的背部,中脑中部,神经管和哈氏窝中。ＧｎＲＨ－ｉｒ胞体有多种类型,一种是大脑胞体,可见在端脑和中脑,另一种为中等和小型胞体,这些胞体聚集在中脑中部（相当于鱼类下丘脑）以及端脑。长的念珠状ＧｎＲＨ－ｉｒ神经纤维从端脑腹外侧面延伸     

Identification of gonadotropin-releasing hormone and associated binding substances in the blood serum of a holocephalan (Hydrolagus colliei)

The identity of the gonadotropin-releasing hormone (GnRH) form and the presence of GnRH-binding substances in the blood serum of the holocephalan, spotted ratfish (Hydrolagus colliei), were investigated. The GnRH-like peptides in the serum were identified on the basis of relative hydrophobicity using reverse-phase HPLC. [His⁵,Trp⁷,Tyr⁸]GnRH (chicken GnRH-II) was the only GnRH form detected in the serum. It has been previously shown to be the only GnRH form in the brain of this species. The presence of GnRH-binding substances was inferred by anomalous HPLC elution of GnRH, ultrafiltration behavior, and by the direct binding of iodinated GnRH analogues by blood serum components. The mean GnRH concentration in the extracted blood serum was 125 ± 11 pg ml⁻¹ (n = 5) in males and 64 ± 48 pg ml⁻¹ (n = 4) and 155 ± 26 (n = 4) in two separate groups of females. Measurement of GnRH in the blood serum is complicated by the presence of GnRH-binding substances, which may cause the coprecipitation of GnRH during extraction with organic solvents. The high concentration of GnRH and the presence of GnRH-binding substances suggest that systemic blood is the route by which GnRH reaches the gonadotropes and/or that GnRH may have a hormonal role in H. colliei.     

Testicular GnRH-like factors: characterization of biologic activity

Observations that gonadotropin releasing hormone and its agonists directly inhibit gonadal function by binding to receptors on the Leydig cells had led to search for testicular GnRH-like peptide(s). This communication presents evidence that GnRH-like factors isolated from rat testis by immunoaffinity chromatography and previously characterized by radioimmunoassay and radioreceptor assay possess biologic activity. The partially purified material led to dose dependent inhibition of oLH stimulated testosterone production in a mixed Sertoli-Leydig cell monolayer culture. Pre-incubation of the cells with a potent GnRH antagonist prevented the inhibitory effects of the partially purified material suggesting that inhibition of oLH stimulated testosterone production may be receptor mediated.     

排卵,排精前后文昌鱼体内GnRH的研究

利用放射免疫分析法测定了排卵、排精前后青岛文昌鱼体内促性腺激素释放激素（ＧｎＲＨ）的含量变化,并通过高效液相色谱比较了雌、雄文昌鱼性腺及除性腺外体部ＧｎＲＨ的种类和含量的异同。结果表明：１）生殖过程中雌、雄文昌鱼体内ＧｎＲＨ含量的变化规律不同;雌性文昌鱼体内ＧｎＲＨ总含量在排卵时有所下降,排后１２小时政策最为明显,以后逐渐上升到排前水平;雄性文昌鱼仅在排精时有所下降,２小时后即稳定在排水平。２）文     

D-Ala6-des-Gly10-gonadotropin-releasing hormone ethylamide: absence of binding sites and lack of a direct effect in rabbit corpora lutea

Rat ovarian tissue has been shown to contain high-affinity gonadotropin-releasing hormone (GnRH) receptors, and synthetic GnRH analogues have been shown to inhibit steroid production by rat corpora lutea in vivo and in vitro. These results raise the possibility that an ovarian GnRH-like peptide may be involved in normal luteal regression. We have examined binding of D-Ala6-des-Gly10-GnRH ethylamide (D-Ala) to rabbit corpora lutea, and have investigated the luteolytic activity of this analogue in hypophysectomized, pseudopregnant rabbits. Three hypophysectomized estrogen-treated rabbits were injected with 0.25 mg D-Ala s.c. every 6 h for 48 h during mid-pseudopregnancy, and three were injected with vehicle only. Treatment with D-Ala produced no acute changes in serum progesterone, nor was the time of luteal regression altered. Rabbit anterior pituitary tissue was found to contain high-affinity GnRH receptors (Ka = 7.0 X 10(9) M-1; 188.2 +/- 35.6 fmol/mg protein). However, no similar high-affinity GnRH receptors were detected in rabbit luteal tissue from any stage of pseudopregnancy. Some apparent low-affinity binding was observed, but this displaceable binding was subsequently observed in all control tissues tested. Thus, a potent GnRH analogue does not have any detectable direct effect on steroidogenesis in the rabbit corpus luteum, nor are high-affinity GnRH binding sites present in rabbit luteal tissue.     

Antigonadotropic effects of the bovine ovarian gonadotropin-releasing hormone-binding inhibitor/histone H2A in rat luteal and granulosal cells

A gonadotropin-releasing hormone (GnRH)-binding inhibitor (GnRH-BI) was purified from bovine ovaries and identified as histone H2A. In the present studies, the biological effects of partially purified and purified ovarian GnRH-BI, as well as calf thymus histone H2A, were examined in rat ovarian cells. Since GnRH has direct antigonadotropic actions on these cells, the effects on luteinizing hormone-stimulated cAMP accumulation in luteal cells and follicle stimulating hormone-induced cAMP and progesterone production in granulosal cells were evaluated. Antigonadotropic activity in both luteal and granulosal cells coeluted directly with GnRH-BI activity during purification from bovine ovaries, and the antigonadotropic effects were dose dependent and reversible. In contrast to GnRH, GnRH-BI maximally inhibited gonadotropin responses and the effects of GnRH-BI were not blocked by a GnRH antagonist. The purified ovarian GnRH-BI and calf thymus histone H2A had identical antigonadotropic properties, and the half-maximal concentrations for inhibiting the gonadotropin responses of granulosal and luteal cells was 2 and 5 microM, respectively. In conclusion, the ovarian GnRH-binding inhibitor, identified as histone H2A, not only inhibits the high affinity binding of GnRH to rat ovarian membranes but also evokes GnRH-like antigonadotropic responses in rat ovarian cells that do not appear to be mediated by binding to GnRH receptors.     

Detection and localization of gonadotrophin-releasing hormone (GnRH)-like material in the frog,Rana esculenta,ovary

GnRH-like material has been identified using HPLC followed by RIA in the ovary of Rana esculenta. During the reproductive cycle three immunoreactive GnRH peaks were eluted. One of them coeluted with s-GnRH, the other two forms between GnRH and cII-GnRH. During the recovery phase s-GnRH immunoreactivity disappears. By immunocytochemistry, cII-GnRH immunostaining was localized to granulosa cells while s-GnRH was present in the perinuclear zone of the oocytes.     

A comparative study of the mechanism of action of luteinizing hormone and a gonadotropin releasing hormone analog on the ovary

The mechanism of action of a gonadotropin releasing hormone (GnRH) agonistic analog ([D-Ala6]GnRH) on the rat ovary has been studied in comparison to similar effects of luteinizing hormone (LH). Stimulation of meiosis resumption in vitro in follicle-enclosed oocytes by both LH and [D-Ala6] GnRH, was blocked by elevated levels of cAMP as demonstrated when either dibutyryl cAMP or the phosphodiesterase inhibitor methylisobutylxanthine was present in the culture medium. In vivo, the prostaglandin synthase inhibitor indomethacin, which blocks LH-induced ovulation, also inhibited ovulation induced by the GnRH analog in hypophysectomized rats. On the other hand, the potent GnRH-antagonist [D-pGlu1, pClPhe2, D-Trp3,6] GnRH which blocked the stimulatory effect of the agonist on oocyte maturation and ovulation had no effect on LH action. It is concluded that while a GnRH-like peptide does not seem to mediate LH action on the ovarian follicles, both LH and GnRH agonist share some common mechanistic pathways at a post-receptor locus.     

Characterization of Leydig cell gonadotropin-releasing hormone binding sites utilizing radiolabeled agonist and antagonist

Gonadotropin-releasing hormone (GnRH) binding sites in intact Leydig cells and in membrane preparations were investigated using 125I-labeled GnRH agonist and antagonist. Binding was saturable and involved a single class of high affinity sites. Intact Leydig cells and a membrane preparation had a higher affinity for GnRH agonist (Kd 3.0 +/- 1.7 X 10(-10) M) than for GnRH antagonist (Kd 10.0 +/- 1.8 X 10(-10) M). With anterior pituitary membranes the Kd was 2.8 +/- 0.7 X 10(-10) M for the agonist and 2.4 +/- 1.4 X 10(-10) M for the antagonist. The Kd for GnRH was similar for Leydig cells and the anterior pituitary. Chymotrypsin and trypsin digestion decreased receptor binding, but neuraminidase increased Leydig cell binding in contrast to the decrease in binding observed with pituitary receptors. The results suggest that the Leydig cell GnRH binding sites may differ from the pituitary receptor which may be related to structural differences in GnRH-like peptides recently described in extracts of rat testis.     

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)     

Presence of two neuropeptides in the fusiform ganglion and reproductive ducts of Octopus vulgaris: FMRFamide and gonadotropin-releasing hormone (GnRH)

We have found evidence of FMRFamide-like and cGnRH-I-like immunoreactivity in the central nervous system (CNS) and in the reproductive ducts of both female and male cephalopod Octopus vulgaris. Cell bodies and fibers were immunolocalized in the fusiform ganglion from which the nerves that reach the female and male reproductive ducts arise. FMRFamide-like and cGnRH-I-like immunoreactive nerve endings were present in the oviduct, and in the oviducal gland of the female and in the seminal vesicle of the male. The GnRH-like peptide from the reproductive ducts has been partially characterized by HPLC. The retention time of the Octopus vulgaris GnRH-like peptide was similar to the retention time of cGnRH-I. Based on these observations we suggest that FMRFamide-like and a novel GnRH-like peptide are involved in the control of reproductive ducts of Octopus vulgaris. One possibility is that the peptides affect gamete transport. Another possibility is that they regulate secretory products such as mucus and mucilaginous substances from the oviducal gland and the seminal vesicle. Our data provide further evidence to support the hypothesis of the existence of a central and peripheral peptidergic control of reproduction of Octopus vulgaris.