Giovanna Liguori

Presence, distribution and steroidogenic effect of the peptides orexin A and receptor 1 for orexins in the testis of the South American camelid Alpaca (Vicugna pacos)

The orexins A (oxA) and B are peptides discovered in the rat hypothalamus and successively found in some peripheral organs of the mammalian body. They binds two protein G-coupled receptors defined receptor 1 (ox1r) and 2 for orexins, the first of which is highly specific for oxA while the second binds both the peptides with equal affinity. This work aimed to detect the presence of oxA and ox1r in the testis of the South American camelid alpaca (Vicugna pacos) and investigate the role played by them on Leydig cell steroidogenesis. The species alpaca acquired, in the last years, increasing zootechnical interest for the quality of the wool produced and its breeding spread from the country of origin to USA, Australia and Europe. Immunohistochemistry allowed us to detect oxA in Leydig and Sertoli cells, spermatogonia, resting spermatocytes, round and oval spermatids. Ox1r-immunoreactivity was found in Leydig cells and round, oval and elongated spermatids. The expression of the two peptides in tissue extracts was established by using Western blotting technique. Such results demonstrated that in the alpaca testis exists in a cellular complex able to produce and/or internalize oxA. Finally, the effect of oxA on steroidogenesis was investigated by means of in vitro cultured thin testis slices which were added with oxA or/and Müllerian Inhibiting Substance (MIS), a steroidolitic agent basally produced by the Sertoli cell. OxA evoked increase of testosterone production while MIS a decrease. The consecutive addition of oxA and MIS, or vice versa, highlighted an antagonistic interplay between the two substances which has been thought to be the main molecular event at the basis of the oxA-stimulated steroidogenesis mechanism.

Expression of urocortin and corticotropin-releasing hormone receptors in the bovine adrenal gland.

Urocortin (UCN), a 40 amino acid peptide, is a corticotrophin-releasing hormone (CRH)-related peptide. The biological actions of CRH family peptides are mediated via two types of G protein-coupled receptors, CRH type 1 receptor (CRHR1) and CRH type 2 receptor (CRHR2). The aim of the present study was to investigate the expression of UCN, CRHR1 and CRHR2 by immunohistochemistry, western blot and real-time RT-PCR in the bovine adrenal gland to clarify the mechanisms of the intra-adrenal CRH-based regulatory system. The results showed that UCN, CRHR1 and CRHR2 were expressed in both the cortex and medulla. Specifically, UCN-immunoreactivity (IR) was distributed in the outer part of the zona fasciculata and in the zona reticularis of the cortex and in the medulla. UCN and CRHR2 mRNA expression levels were higher in the cortex than in the medulla, while CRHR1 mRNA levels were undetectable in the cortex. These results suggest that UCN, CRHR1 and CRHR2 are expressed in the bovine adrenal gland and that UCN might play a role in the intra-adrenal CRH-based regulatory system through an autocrine mechanism.

Expression of orexin B and its receptor 2 in rat testis.

The peptides orexin A (OxA) and orexin B (OxB) deriving from a common precursor molecule, prepro-orexin, by proteolytic cleavage, bind the two G-coupled OX1 and OX2 receptors. While OX1 selectively binds OxA, OX2 shows similar affinity for both orexins. Firstly discovered in the hypothalamus, orexins and their receptors have been found in other brain regions as well as in peripheral tissues of mammals, thus resulting involved in the regulation of a broad variety of physiological functions. While the functional localization of OxA and OX1 in the mammalian genital tract has been already described, the expression of OxB and OX2 and their potential role in the reproductive functions remain to be explored. Here, we investigated the presence of OxB and OX2 in the rat testis by immunohistochemical and biochemical analyses. The results definitely demonstrated the localization of OxB and OX2 in pachytene and second spermatocytes as well as in spermatids at all stages of the cycle of the seminiferous epithelium. The expression of both OX2 mRNA and protein in the rat testis was also established by RT-PCR and Western blotting, respectively. The analysis of the molecular mechanism of action of OxB in the rat testis showed that OxB, in contrast with OxA, is unable to promote steroidogenesis. These results translate into the regulation of diverse biological actions by OxA and OxB in the male gonad.

Expression of urocortin and its receptors in the rat epididymis

Urocortin (UCN; 40 aa) is a corticotrophin-releasing hormone (CRH)-related peptide. The biological actions of CRH family peptides are mediated by two types of G-protein-coupled receptors, CRH type 1 receptor (CRHR1) and CRH type 2 receptor (CRHR2). The biological effects of the peptides are mediated and modulated not only by CRH receptors but also by a highly conserved CRH-binding protein (CRHBP). The aim of the present study was to investigate the expression of UCN, CRHR1, CRHR2 and CRHBP by immunohistochemistry, Western blot, RT-PCR and real-time RT-PCR in the rat epididymis. Urocortin, CRHR1 and CRHR2, but not CRHBP, were expressed in all segments of the rat epididymis. Specifically, UCN- and CRHR2-immunoreactivities (IRs) were distributed in epididymal epithelial cells of the caput, corpus and cauda. CRHR1-IR was found in the fibromuscular cells surrounding the epididymal duct and in the smooth musculature of the blood vessels throughout the organ. UCN and CRHR2 mRNA expression levels were higher in the caput and corpus than in the cauda, while CRHR1 mRNA level was higher in the cauda than those in the caput and corpus. In summary, UCN, CRHR1 and CRHR2 are expressed in the rat epididymis. It is suggested that CRH-related peptides might play multiple roles in the maturation and storage of spermatozoa.

Expression of urocortin and corticotropin-releasing hormone receptors in the horse thyroid gland

Urocortin (UCN) is a 40-amino-acid peptide and a member of the corticotropin-releasing hormone (CRH) family, which includes CRH, urotensin I, sauvagine, UCN2 and UCN3. The biological actions of CRH family peptides are mediated via two types of G-protein-coupled receptors, namely CRH type 1 receptor (CRHR1) and CRH type 2 receptor (CRHR2). The biological effects of these peptides are mediated and modulated not only by CRH receptors but also via a highly conserved CRH-binding protein (CRHBP). Our aim was to investigate the expression of UCN, CRHR1, CRHR2 and CRHBP by immunohistochemistry, Western blot and reverse transcription with the polymerase chain reaction (RT-PCR) in the horse thyroid gland. The results showed that UCN, CRHR1 and CRHR2 were expressed in the thyroid gland, whereas CRHBP was not expressed. Specifically, UCN immunoreactivity (-IR) was found in the thyroid follicular cells, CRHR2-IR in the C-cells and CRHR1-IR in blood vessels. Western blot analysis and RT-PCR experiments confirmed the immunohistochemical data. These results suggest that a regulatory system exists in the mammalian thyroid gland based on UCN, CRHR1 and CRHR2 and that UCN plays a role in the regulation of thyroid physiological functions through a paracrine mechanism.

Expression of orexin A and its receptor 1 in the human prostate

The peptides orexin A (OXA) and orexin B, deriving from the cleavage of the precursor molecule prepro‐orexin, bind two G‐coupled transmembrane receptors, named as receptor 1 (OX1R) and receptor 2 for orexin, showing different affinity‐binding properties. First discovered in the rat hypothalamus, orexins and their receptors have been also found in many peripheral tissues where they exert neuroendocrine, autocrine and paracrine functions. Because inconclusive data on their localization in the mammalian prostate are reported, the aim of this study was to investigate the presence of prepro‐orexin, OXA and OX1R in the human normal and hyperplastic gland. Immunohistochemistry revealed the localization of both OXA and OX1R in the cytoplasm of the follicular exocrine epithelium of all tested normal and hyperplastic prostates. Positive immunostaining was mainly observed in the basal cells of the stratified epithelium, and only rarely in the apical cells. The expression of mRNAs coding for prepro‐orexin and OX1R and of proteins in the tissues was also ascertained by polymerase chain reaction and Western blotting analysis, respectively. In order to gain insights into the functional activity of OXA in the prostate, we administered different concentrations of OXA to cultured prostatic epithelial cells PNT1A. We first demonstrated that PNT1A cells express OX1R. The addition of OXA did not affect PNT1A cell proliferation, while it enhanced cAMP synthesis and Ca2+ release from intracellular storage. Overall, our results definitely demonstrate the expression of OXA and OX1R in the human prostate, and suggest an active role for them in the metabolism of the gland.

Expression of orexin A and its receptor 1 in the epididymis of the South American camelid alpaca (Vicugna pacos).

Orexins A (ox A) and B are two peptides originally discovered in neurons of rat hypothalamus, and later found in different cellular types of the gastrointestinal and genital tracts. They arise from the proteolytic cleavage of a common precursor molecule, prepro‐orexin, and bind to two receptors, namely receptor 1 (ox1r) and receptor 2 for orexins, that show different binding affinity. The central role of the two peptides has been extensively studied, whereas their activity in the periphery is still poorly known. Here, we investigated the presence of ox A and ox1r in the epididymis of a South American camelid species, the alpaca, by immunohistochemistry, and we also assessed the expression of prepro‐orexin and ox1r in tissue extracts by Western blotting analysis. Ox A‐ and ox1r‐immunoreactivity was found in the cytoplasm of principal cells of the caput epididymis. A prevalent supranuclear localization of granular‐shaped positive material was observed. No positivity was present in the other cytotypes of epididymis. The expression of two peptides with molecular weight corresponding to those of prepro‐orexin and ox1r, respectively, was detected in the tissue extracts from the organ.

Expression and potential role of the peptide orexin-A in prostate cancer.

The peptides orexin-A and orexin-B and their G protein-coupled OX1 and OX2 receptors are involved in multiple physiological processes in the central nervous system and peripheral organs. Altered expression or signaling dysregulation of orexins and their receptors have been associated with a wide range of human diseases including narcolepsy, obesity, drug addiction, and cancer. Although orexin-A, its precursor molecule prepro-orexin and OX1 receptor have been detected in the human normal and hyperplastic prostate tissues, their expression and function in the prostate cancer (PCa) remains to be addressed. Here, we demonstrate for the first time the immunohistochemical localization of orexin-A in human PCa specimens, and the expression of prepro-orexin and OX1 receptor at both protein and mRNA levels in these tissues. Orexin-A administration to the human androgen-dependent prostate carcinoma cells LNCaP up-regulates OX1 receptor expression resulting in a decrease of cell survival. Noteworthy, nanomolar concentrations of the peptide counteract the testosterone-induced nuclear translocation of the androgen receptor in the cells: the orexin-A action is prevented by the addition of the OX1 receptor antagonist SB-408124 to the test system. These findings indicate that orexin-A/OX1 receptor interaction interferes with the activity of the androgen receptor which regulates PCa onset and progression, thus suggesting that orexin-A and its receptor might represent novel therapeutic targets to challenge this aggressive cancer.

Localization of orexin B and receptor 2 for orexins in testicular cytotypes of the camelid alpaca (Vicugna pacos)

The orexins A (OxA) and B (OxB) are two hypothalamic peptides involved in many physiological functions of the mammalian body. They act through the binding of two G-coupled receptors named receptor 1 (OX1 ) and receptor 2 (OX2 ) for orexins. The first receptor is specific for OxA, while the second binds both the substances with equal affinity. The orexins and the relative receptors have been traced by means of different techniques also at the periphery of the body and particularly in the adrenals, and in gastrointestinal and genital organs. Aim of this work was to investigate the presence of OxB and OX2 by means of immunohistochemistry and Western blotting analysis in the testis of the South American camelid alpaca, a species primarily breed in Chile and Ecuador and recently diffused in Europe where the quality of its wool is particularly appreciated. OxB immunoreactivity (IR) was found in the tubular compartment of the testis where spermatogonia (resting), zygotene and pachytene spermatocytes, and spermatids clearly showed differently sized and shaped cytoplasmic positive structures. OX2 -IR was found both in the interstitial and tubular compartments of the testis and particularly in Leydig cells and round and elongated spermatids. Western blotting analysis of testis lysates showed the presence of a protein band whose molecular weight corresponded to that currently assigned to OX2 . Such findings easily translate the hypothesis that OxB and its receptor 2 play a functional role both in the interstitial and tubular compartments of the alpaca testis.

First study on the peptidergic innervation of the brain superior sagittal sinus in humans

The superior sagittal sinus (SSS) of the mammalian brain is a pain-sensitive intracranial vessel thought to play a role in the pathogenesis of migraine headaches. Here, we aimed to investigate the presence and the potential co-localization of some neurotransmitters in the human SSS. Immunohistochemical and double-labeling immunofluorescence analyses were applied to paraformaldehyde-fixed, paraffin-embedded, coronal sections of the SSS. Protein extraction and Western blotting technique were performed on the same material to confirm the morphological data. Our results showed nerve fibers clustered mainly in large bundles tracking parallel to the longitudinal axis of the sinus, close in proximity to the vascular endothelium. Smaller fascicles of fibers encircled the vascular lumen in a spiral fashion, extending through the subendothelial connective tissue. Isolated nerve fibers were observed around the openings of bridging veins in the sinus or around small vessels extending into the perisinusal dura. The neurotransmitters calcitonin gene related peptide (CGRP), substance P (SP), neuronal nitric oxide synthase (nNOS), vasoactive intestinal polypeptide (VIP), tyrosine hydroxylase (TH), and neuropeptide Y (NPY) were found in parietal nerve structures, distributed all along the length of the SSS. Overall, CGRP- and TH-containing nerve fibers were the most abundant. Neurotransmitters co-localized in the same fibers in the following pairs: CGRP/SP, CGRP/NOS, CGRP/VIP, and TH/NPY. Western blotting analysis confirmed the presence of such neurosubstances in the SSS wall. Overall our data provide the first evidence of the presence and co-localization of critical neurotransmitters in the SSS of the human brain, thus contributing to a better understanding of the sinus functional role.