Nurit Ben-Aroya

A second isoform of gonadotropin-releasing hormone is present in the brain of human and rodents

Gonadotropin‐releasing hormone‐I (GnRH‐I), present in the mammalian hypothalamus, regulates reproduction. In this study we demonstrate, for the first time, that an additional isoform of GnRH, [His5, Trp7, Tyr8] GnRH‐I (GnRH‐II) is present in the brain of the mouse, rat and human. Human and rat brain extracts contain two isoforms of GnRH, GnRH‐I and GnRH‐II, which exhibited identical chromatographic properties to the respective synthetic peptides, in high performance liquid chromatography. Using immunohistochemical techniques we have found that GnRH‐II is present in neuronal cells that are localized mainly in the periaqueductal area as well as in the oculomotor and red nuclei of the midbrain. It is of interest to note that in the hypogonadal mouse, although the GnRH‐I gene is deleted, GnRH‐II is present. Substantial concentrations of GnRH‐II are also present in the hypothalamus and stored in the human pituitary stalk or in the mouse median eminence. By using reverse transcription (RT)‐PCR we have also found that while GnRH‐II is not expressed in the cerebellum, it is expressed in all three structures of the brain stem: midbrain, pons and medulla oblongata.

The neuropeptides GnRH-II and GnRH-I are produced by human T cells and trigger laminin receptor gene expression, adhesion, chemotaxis and homing to specific organs

Can T cells be directly activated to de novo gene expression by gonadotropin-releasing hormone-II (GnRH-II), a unique 10-amino-acid neuropeptide conserved through 500 million years of evolution? GnRH-II, which has been identified in mammals, shares 70% homology with the mammalian hypothalamic neurohormone GnRH (GnRH-I), the primary regulator of reproduction, but is encoded by a different gene. Although both neuropeptides are produced mainly in brain, their localization and promoter regulation differ, suggestive of distinct functions. Indeed, GnRH-II barely affects reproduction and its role in mammalian physiology is unknown. We find here that human normal and leukemic T cells produce GnRH-II and GnRH-I. Further, exposure of normal or cancerous human or mouse T cells to GnRH-II or GnRH-I triggered de novo gene transcription and cell-surface expression of a 67-kD non-integrin laminin receptor that is involved in cellular adhesion and migration and in tumor invasion and metastasis. GnRH-II or GnRH-I also induced adhesion to laminin and chemotaxis toward SDF-1α, and augmented entry in vivo of metastatic T-lymphoma into the spleen and bone marrow. Homing of normal T cells into specific organs was reduced in mice lacking GnRH-I. A specific GnRH-I-receptor antagonist blocked GnRH-I- but not GnRH-II-induced effects, which is suggestive of signaling through distinct receptors. We suggest that GnRH-II and GnRH-I, secreted from nerves or autocrine or paracrine sources, interact directly with T cells and trigger gene transcription, adhesion, chemotaxis and homing to specific organs, which may be of clinical relevance.

The gonadotropin‐releasing hormone family of neuropeptides in the brain of human, bovine and rat: identification of a third isoform

The mammalian gonadotropin‐releasing hormone (GnRH‐I), which regulates reproduction, was the first isoform of GnRH that was identified in mammals. Recently, we and others have demonstrated the existence of a second isoform of GnRH in the brain of mammals. The presence of a third isoform of GnRH, GnRH‐III, in the brain of mammals is reported herein. GnRH‐III, extracted from the brain of bovine and human, was purified by high performance liquid chromatography, using two distinct elution programs. In both, GnRH‐III was eluted at the same positions as synthetic salmon GnRH, as demonstrated by radioimmunoassay. The luteinizing hormone‐releasing activity of purified GnRH‐III, using dispersed rat pituitary cells, was found to be similar to that of synthetic salmon GnRH. The total amount of GnRH‐III, determined by radioimmunoassay, in the hypothalamus and midbrain of humans and calves is similar to that of GnRH‐I. Immunohistochemical studies demonstrated GnRH‐III‐containing neurons in the hypothalamus and midbrain of human and GnRH‐III fibers in the median eminence of rats. The distribution of GnRH‐III in the brain suggests that in addition to a putative function as a neurohormone at the hypothalamic–pituitary axis, GnRH‐III may have other functions. Our present results suggest that multiple isoforms of GnRH are present in the brain of mammals, and further studies are required in order to elucidate their biological functions.

Two Isoforms of Gonadotropin-Releasing Hormone Are Coexpressed in Neuronal Cell Lines1

GnRH-I serves as the neuropeptide that regulates mammalian reproduction. Recently, several groups have identified in the brain of rodents, monkeys, and humans a second isoform of GnRH (GnRH-II) whose structure is 70% identical to that of GnRH-I. In this study we demonstrate for the first time human and mouse neuronal cell lines that express both GnRH-I and GnRH-II. Following the screening of several human neuronal cell lines by RT-PCR and Southern hybridization, we demonstrated that two cell lines, TE-671 medulloblastoma and LAN-1 neuroblastoma cells, coexpress messenger RNA encoding the two isoforms of GnRH. Nucleotide sequencing indicated that the complementary DNA fragments are identical to those of the known human GnRH-I and GnRH-II sequences. Extracts obtained from the TE-671 and LAN-1 cell lines as well as from the immortalized mouse hypothalamic GT1–7 neuronal cell line were found to contain the two isoforms of GnRH, which exhibited identical chromatographic properties as synthetic GnRH-I and GnRH-...

Daily GnRH and GnRH-receptor mRNA expression in the ovariectomized and intact rat.

We recently described patterns of GnRH and GnRH receptor (GnRH-R) expression in the hypothalamus, pituitary and ovary throughout the rat estrus cycle. Here, we wished to distinguish between regulatory effects of ovarian factors and underlying circadian rhythmicity. We quantified GnRH and GnRH-R mRNA in the pituitary and hypothalamus of long-term ovariectomized (OVX) rats, at different times of day, using real-time PCR. Furthermore, we expanded our previous study of hypothalamic and pituitary GnRH and GnRH-R expression in intact rats by including more time points throughout the estrus cycle. We found different daily patterns of GnRH and GnRH-R expression in intact versus OVX rats, in both tissues. In the hypothalamus of OVX rats, GnRH mRNA peaked at 12, 16 and 20 h, whereas in the hypothalamus of intact rats we observed somewhat higher GnRH mRNA concentrations at 19 h on every day of the estrus cycle except proestrus, when the peak occurred at 17 h. In this tissue, GnRH-R fluctuated less significantly and peaked at 16 h in OVX rats. During the estrus cycle, we observed higher levels in the afternoon of each day except on estrus. In OVX rats, pituitary GnRH mRNA rose sharply at 9 h, with low levels thereafter. In these animals, pituitary GnRH-R also peaked at 9h followed by a second rise at 22 h. In intact rats pituitary GnRH was high at noon of diestrus-II and on estrus, whereas GnRH-R mRNA was highest in the evening of diestrus-II. This is the first demonstration of daily GnRH and GnRH-R mRNA expression patterns in castrated animals. The observed daily fluctuations hint at underlying tissue-specific circadian rhythms. Ovarian factors probably modulate these rhythms, yielding the observed estrus cycle patterns.

Conjugates of gonadotropin releasing hormone (GnRH) with carminic acid : Synthesis, generation of reactive oxygen species (ROS) and biological evaluation

We synthesized two carminic acid (7-alpha-d-glucopyranosyl-9,10-dihydro-3,5,6,8-tetrahydroxy-1-methyl-9,10-dioxo-2-anthracene carboxlic acid, CA)-GnRH conjugates to be used as a model for potential photoactive targeted compounds. CA was conjugated to the epsilon-amino group of [d-Lys(6)]GnRH through its carboxylic moiety or via a beta-alanine spacer (beta-ala). Redox potentials of CA and its conjugates were determined. We used electron spin resonance (ESR) and spin trapping techniques to study the light-stimulated redox properties of CA and its CA-GnRH conjugates. Upon irradiation, the compounds stimulated the formation of reactive oxygen species (ROS), that is, singlet oxygen ((1)O(2)) and oxygen radicals (O(2)(-*) and OH(*)). Both conjugates exhibited higher ROS production than the non-conjugated CA. The bioactivity properties of the CA conjugates and the parent peptide, [d-Lys(6)]GnRH, were tested on primary rat pituitary cells. We found that the conjugates preserved the bioactivity of GnRH as illustrated by their capability to induce ERK phosphorylation and LH release.

SYNTHESIS AND BIOACTIVITY OF FATTY ACID-CONJUGATED GNRH DERIVATIVES

Transdermal delivery of peptidic drugs is usually inefficient, notably due to their hydrophilic character which makes it difficult to cross the hydrophobic layers of the skin. In order to obtain transdermally deliverable analogs of gonadotropin releasing hormone (GnRH), we have synthesized related hydrophobic derivatives by attaching various aliphatic acids to the N(epsilon)-amino side chain of [D-Lys]6GnRH, a superactive GnRH agonist. It was found that the affinity toward the GnRH receptor gradually decrease with increased hydrophobicity, i.e. increase in chain length of the attached aliphatic acid. Nevertheless, analogs with 12-carbon or shorter aliphatic acids were shown to be GnRH superagonists, with in vitro and in vivo potencies similar to that of [D-Lys]6GnRH. [D-Lys-lauryl]6GnRH was shown to have a longer duration of action in vivo, as compared to [D-Lys]6GnRH. The transdermal penetration of the peptides was evaluated by in vivo functional experiments in rats. According to these studies the efficiency of penetration is gradually lowered in increasingly hydrophobic analogs. These results are discussed with respect to the circular dichroism spectra of the peptides in trifluoroethanol. The spectra of the aliphatic acid-conjugated superagonists examined do not express a significant tendency towards a beta-turn conformation, typical of GnRH and its agonists. This finding contradict previous publications which suggested a correlation between the conformations of GnRH analogs in trifluoroethanol and their biological activities.

Expression of the gene for the receptor of gonadotropin-releasing hormone in the rat mammary gland

Recent findings have demonstrated that the GnRH gene is expressed in the mammary gland of pregnant and lactating rats but not of virgin rats. Indeed, significant concentrations of biologically active GnRH have been found in milk of human, cow, sheep and rat. We have, therefore, looked for expression of the GnRH receptor in the rat mammary gland. By reverse transcription (RT)‐PCR amplification, we have demonstrated the presence of GnRH receptor mRNA in mammary gland samples derived from virgin, pregnant and lactating rats. The GnRH receptor transcript cloned from the mammary gland was sequenced and found to have an identical coding region to the one cloned from the pituitary gland. In addition, we have found that the mammary gland, as the pituitary gland, contains at least two transcripts having the same coding region but different 5′ non‐coding regions. Binding studies, however, could demonstrate only low‐affinity binding sites. These results, therefore, suggest that the regulation of the GnRH receptor occurs posttranscriptionally rather than at the level of transcription.