Biomedical Advances, Editors’ Picks, August 2017

Comprehensive Histological and Immunological Studies Reveal a Novel Glycoprotein Hormone and Thyrostimulin Expressing Proto-Glycotrope in the Sea Lamprey Pituitary Timothy J. Marquis1,2, Masumi Nozaki3, Wayne Fagerberg2 and Stacia A. Sower1,2* 1Center for Molecular and Comparative Endocrinology, University of New Hampshire, Durham, NH 03824 2Department of Molecular, Cellular and Biomedical Sciences, University of New Hampshire, Durham, NH 03824 3Sado Marine Biological Station, Niigata University, Tassha, Sado, Niigata 952-2135, Japan *Corresponding Author: Dr. Stacia A. Sower University of New Hampshire Center for Molecular & Comparative Endocrinology, Department of Molecular, Cellular & Biomedical Sciences 46 College Road, Durham, NH 03824-3544 E-mail: sasower@unh.edu

Cell Tissue Res (2017) 367: 311-338 | PMID: 27771775

ABSTRACT: In the adenohypophysis (anterior pituitary) of all gnathostomes, there are six tropic cell types: corticotropes, melanotropes, somatotropes, lactotropes, gonadotropes, and thyrotropes; each cell type produces specific tropic hormones. In contrast, we report in this study that there are only four tropic cell types in the sea lamprey (Petromyzon marinus) adenohypophysis. We specifically focused on the cell types that produce the glycoprotein hormones (GpHs). The gnathostome adenohypophyseal GpHs are follicle-stimulating hormone (FSH), luteinizing hormone (LH), thyroid-stimulating hormone (TSH), and thyrostimulin. However, lampreys only have two heterodimeric adenohypophyseal GpHs consisting of unique α and β subunits, lamprey GpH (lGpH) (lGpA2/lGpHβ) and thyrostimulin (lGpA2/lGpB5). We used an array of histological techniques to determine the (co)-localization and (co)-expression of the lGpH and thyrostimulin subunits in the lamprey adenohypophysis at different life stages (larval, parasitic, adult) and to identify their synthesizing cell(s). The thyrostimulin subunits (lGpA2/lGpB5) were co-expressed throughout the adenohypophysis (larval, parasitic, adult), while GpH β-subunit (lGpHβ) exhibited localized distribution (adult); all three subunits were co-localized and co-expressed, suggesting both GpHs are synthesized in the same cells, novel proto-glycotropes, in specific adenohypophyseal regions at different life stages. In summary, we provide the first comprehensive study using histology, transmission electron microscopy, in-situ hybridization, and immunohistochemistry that strongly supports further evidence for four definitive adenohypophyseal cell types in the lamprey, including: corticotropes, somatotropes, melanotropes, and the first identification of a novel proto-glycotrope. In addition, our studies show that there is developmental/region-specific co-localization and co-expression of lGpH and thyrostimulin in the lamprey adenohypophysis. KEYWORDS: glycoprotein hormones; proto-glycotrope; sea lamprey pituitary; thyrostimulin; ISH, FISH, IHC, histology and TEM of lamprey pituitary cell types

Biomedical Advances, Editors’ Picks, August 2017 SUPPLEMENT: Lampreys – A Reference Species for Biomedical & Pharmacological Research & Development Sea lampreys (Petromyzon marinus), along with hagfish, represent the oldest lineage of vertebrates, agnathans (jawless fish), which likely arose around 530 million years ago following the Cambrian explosion (Forey and Janvier, 1993). All other vertebrates have jaws and are called gnathostomes. Vertebrate reproduction is regulated by a hierarchically organized endocrine system, known as the hypothalamic-pituitary-gonadal (HPG) axis, which is highly conserved across all vertebrates, from lampreys to humans (Sower et al., 2009, Sower et al., 2006). Comparative endocrinology is one excellent approach for biomedical researchers and physician-scientists to foster a complete understanding and appreciation of the mammalian pituitary. Central to this approach are lampreys, as a reference species, whose remarkable long-term existence on earth transcends the age of the dinosaurs. By studying lampreys, we can unlock the secrets they hold to understanding normal human neuroendocrinology, physiology, pathophysiology, and developing breakthrough medical treatments for diseases, such as cancer (Lajko et al., 2013). (Figure 1)

Figure 1. Lampreys as a Reference Species. Lampreys, along with hagfish, represent the oldest lineage of vertebrates, Agnathans (jawless fish), which likely arose around 530 million years ago following the Cambrian explosion, before the age of the dinosaurs. A comparative endocrinology approach for biomedical and pharmacological research, can help to unlock the secrets to normal human physiology, disease, and treatment.

Biomedical Advances, Editors’ Picks, August 2017 The Pituitary – From Lampreys to Humans – A Brief Introduction The pituitary gland (hypophysis) is present in all vertebrates, from agnathans to mammals, but it is absent in all invertebrates (Gorbman, 1983, Green and Maxwell, 1958). The vertebrate pituitary consists of two main regions, the anterior pituitary (adenohypophysis, AH) and the posterior pituitary (neurohypophysis, NH). In most mammalian vertebrates, the anterior pituitary comprises three distinct regions, the pars distalis (PD), the pars tuberalis (PT), and the pars intermedia (PI); while in most non-mammalian vertebrates, the anterior pituitary consists of slightly different regions: the rostral pars distalis (RPD), the proximal pars distalis (PPD), and the pars intermedia (PI). In gnathostomes, six major anterior pituitary tropic cell types have been identified: corticotropes, melanotropes, somatotropes, lactotropes, gonadotropes and thyrotropes. These cells synthesize the following specific tropic hormones: adrenocorticotropin (ACTH) (corticotropes), melanocyte-stimulating hormone (MSH) (melanotropes), growth hormone (GH) (somatotropes), prolactin (PRL) (lactotropes), follicle-stimulating hormone (FSH) and luteinizing hormone (LH) (gonadotropes), and thyroid-stimulating hormone (TSH) (thyrotropes). One family of the pituitary tropic hormones is the glycoprotein hormones (GpHs). In gnathostomes, the classical GpH family consists of one thyrotropin, TSH, and two gonadotropins, FSH and LH (Gorbman et al., 1983), as well as a relatively new member, thyrostimulin (Nakabayashi et al., 2002). In contrast to gnathostomes and mammals, lampreys have only been definitively shown to have one pituitary GpH, lamprey (l) GpH (lGpH) (Sower et al., 2015), as well as a putative pituitary thyrostimulin hormone (Dos Santos et al., 2009). (Figure 2)

Figure 2. Evolution of Pituitary Cell Types. The pituitary was acquired during evolution following the protochordates, which have no pituitary and thus no pituitary hormones. Lampreys have a pituitary that consists of four main cell types that produce five hormones, including the novel proto-glycotrope. Teleosts (bony fish) acquired the more specialized cell types, gonadotropes, which produce the gonadotropins (FSH and LH); the thyrotropes, which produce TSH; and the lactotropes, which produce PRL. Therefore, teleosts have a pituitary that consists of seven main cell types that produc produce seven hormones. Mammals have a pituitary that consists of six main cell types that produce eight hormones. GpH,

glycoprotein hormone [family]; GH/PRL, growth hormone / prolactin family; POMC, pro-opiomelanocortin family; POM/POC, pro-opiomelanotropin (POM) / pro-opiocortin (POC) family; ACTH, adrenocorticotropic hormone; GH, growth hormone; LH, luteinizing hormone; MSH, melanocyte-stimulating hormone; PRL, prolactin; TSH, thyroid stimulating hormone.

Biomedical Advances, Editors’ Picks, August 2017 Discovery of a Novel Pituitary Cell Type – New Insights into Neuroendocrinology This report is the first comprehensive study using specialized histology, transmission electron microscopy (TEM), fluorescent in situ hybridization (FISH), and immunohistochemistry (IHC), to reveal the different tropic cell types of the lamprey anterior pituitary at all three major life stages (larval, parasitic, and adult). We discovered a novel pituitary tropic cell type that we have named the proto-glycotrope, which is differentially expressing the two lamprey pituitary glycoprotein hormones, lGpH and thyrostimulin, in overlapping and unique regions of the anterior pituitary in different stages of development. This is in contrast to one previous study that showed thyrostimulin is expressed in the corticotropes of the human anterior pituitary (Okada et al., 2006). Therefore, it is possible that the distinct spatial organization of the proto-glycotropes and corticotropes in lampreys may indicate paracrine and/or juxtacrine signaling mechanisms between the two cell types as a means of intercellular regulation of thyrostimulin and/or ACTH production and secretion. Our results suggest that as lampreys mature, along with other developmental changes in the body, the pituitary undergoes dramatic development as well, leading to highly organized arrangements of tropic cells with unique ultrastructural cellular characteristics. Our study provides important new insights into the neuroendocrine regulation of reproduction in lampreys relative to other vertebrates, based on the adenohypophyseal cell types that are present, including the novel proto-glycotropes, which supports our working hypothesis that lampreys are in an evolutionary intermediate stage of pituitary development, leading to the differentiation of the more highly specialized tropic hormones and synthesizing tropic cells found in gnathostomes (Sower, et al., 2015, Sower, et al., 2009). (Figure 3)

Figure 3. Comparative Schematic of Lamprey and Mammalian Pituitaries. The pituitary gland consists of two main regions the AH and the NH; in most mammalian vertebrates the AH is further divided into the PD, PT and PI, while in lampreys, the AH is divided into the RPD, PPD, and PI. In lampreys, the corticotropes produce ACTH and are located in the RPD, the somatotropes produce GH and are located in the dorsal PPD, the melanotropes produce MSH and are located in the PI, and the novel proto-glycotropes produce GpH and thyrostimulin and are located in the RPD, PPD, and PI. In mammals, the corticotropes produce ACTH and thyrostimulin and are located in the PD, the somatotropes produce GH and are located in the PD, the melanotropes produce MSH and are located in the PI, the thyrotropes produce TSH and are located in the PD, and the gonadotropes produce FSH and LH and are located in the PD. ACTH, adrenocorticotropic hormone; FSH, follicle-stimulating hormone; GH, growth hormone; GpH, lamprey (l) glycoprotein hormone (lGpH); LH, luteinizing hormone; MSH, melanocyte-stimulating hormone; PRL, prolactin; TSH, thyroid-stimulating hormone; AH, adenohypophysis (anterior pituitary); PD, pars distalis; RPD, rostral pars distalis; PPD, proximal pars distalis; PI, pars intermedia; NH, neurohypophysis (posterior pituitary); III, third ventricle. The pituitaries are not drawn to scale.

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