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Comprehensive histological and immunological studies reveal a novel glycoprotein hormone and thyrostimulin expressing proto-glycotrope in the sea lamprey pituitary

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, and adult), while the GpH β-subunit (lGpHβ) exhibited localized distribution (adult); all three subunits were co-localized and co-expressed, suggesting that 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 and region-specific co-localization and co-expression of lGpH and thyrostimulin in the lamprey adenohypophysis.

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Acknowledgments

We would like to sincerely thank the members of the Sower Laboratory for their assistance with tissue collection, including: Shannon Barton, Christopher Cape, James Gargan, Krist Hausken, Katie Hayes, Eric MacDonald, Rebecca Scialabba, Daniel Turner, Sabah Ul-Hasan and Emily Van Gulick. In particular, we thank Dr. Jeffrey Hall and Wayne Decatur, from the Sower Laboratory, for their help in training on in situ hybridization; and Dr. Mark Townley and Ms. Nancy Cherim, from the University of New Hampshire University Instrumentation Center, for their help in training on the respective confocal and electron microscopy techniques; and additionally, Dr. Steve McCormick, from the U.S. Geological Survey, Conte Anadromous Fish Research Center, who provided larval and parasitic phase lampreys. This work was supported by the National Science Foundation: NSF IOS-1257476 (to S.A.S.) and the University of New Hampshire Hamel Center for Undergraduate Research: URA-2014 (to T.J.M.). Partial funding was provided by the New Hampshire Agricultural Experiment Station. This is Scientific Contribution Number 2671. This work was supported by the USDA National Institute of Food and Agriculture HATCH Project (AES NH00571 and AES NH00624) (to S.A.S.)

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Correspondence to Stacia A. Sower.

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Fig. S1
figure 15

Dot blot analysis to test the specificity of GpA2, GpHβ and GpB5 riboprobes for in situ hybridization. There was no expression observed for the no-probe controls for GpA2 (GpA2: C1), GpHβ (GpHβ: C1), or GpB5 (GpB5: C1). There was specific expression for the antisense (AS) probes for GpA2, GpHβ and GpB5, against the respective complementary sense DNA strands (GpA2: AS; GpHβ: AS; GpB5: AS), respectively; and sense (S) probes for GpA2, GpHβ and GpB5, against the respective complementary antisense DNA strands (GpA2: S; GpHβ: S; GpB5: S). There was no cross-reactivity between: GpA2 AS probes and GpHβ DNA (GpA2: C2) or GpB5 DNA (GpA2: C3); GpHβ AS probes and GpA2 DNA (GpHβ: C2) or GpB5 DNA (GpHβ: C3); or GpB5 AS probes and GpA2 DNA (GpB5: C2) or GpHβ DNA (GpB5: C3). Black arrowheads indicate the expression. White asterisks indicate pencil-mark reference points. (GpA2 shared lamprey thyrostimulin and GpH alpha (α) subunit (lGpA2), GpB5 hormone-specific lamprey thyrostimulin beta (β) subunit (lGpB5), GpHβ hormone-specific lamprey GpH beta (β) subunit (lGpHβ), AS antisense, S sense). (GIF 34 kb)

Fig. S2
figure 16

ISH: GpA2. Single-labeled chromogenic in situ hybridization for the common alpha (α) subunit, GpA2, of lamprey (l) adenohypophyseal glycoprotein hormone (GpH) (lGpH) and thyrostimulin in female ammocoete, female parasitic phase and adult female and male lamprey adenohypophyses. GpA2 was minimally expressed in the rostral pars distalis (RPD) (a, b), proximal pars distalis (PPD) (a, c) and pars intermedia (PI) (a, d) of female ammocoete lampreys; moderately expressed in the RPD (e, f), PPD (e, g) and PI (e, h) of female parasitic phase lampreys; and highly expressed in the RPD (i, j), dorsal (i, k) and ventral (i, m) PPD and PI (i, l) of adult female lampreys; and highly expressed in the RPD (n, o), dorsal (n, p) and ventral (n, r) PPD and PI (n, q) of adult male lampreys. Arrowheads indicate the regions of expression. Scale bars (a) 100 μm, (bd) 20 μm. l lamprey, GpH lamprey glycoprotein hormone (lGpH), GpA2 shared lamprey thyrostimulin and GpH alpha (α) subunit (lGpA2), GpB5 hormone-specific lamprey thyrostimulin beta (β) subunit (lGpB5), GpHβ hormone-specific lamprey GpH beta (β) subunit (lGpHβ), AH adenohypophysis (anterior pituitary), RPD rostral pars distalis (region of AH), PPD proximal pars distalis (region of AH), PI pars intermedia (region of AH), NH neurohypophysis (posterior pituitary), III third ventricle. (GIF 242 kb)

Fig. S3
figure 17

ISH: GpHβ. Single-labeled chromogenic in situ hybridization for the hormone specific beta (β) subunit, GpHβ, of lamprey (l) adenohypophyseal glycoprotein hormone (GpH) (lGpH) in female ammocoete, female parasitic phase and adult female and male lamprey adenohypophyses. GpHβ was not expressed in female ammocoete lampreys (ad); was moderately expressed in the RPD (e, f), PPD (e, g) and PI (e, h) of female parasitic phase lampreys; minimally expressed in the RPD (i, j), moderately expressed in the dorsal (i, k) and ventral (i, m) PPD and PI (i, l) of adult female lampreys; and minimally expressed in the RPD (n, o) and dorsal PPD (n, p) and highly expressed in the ventral PPD (n, r) and PI (n, q) of adult male lampreys. Arrowheads indicate the regions of expression. Scale bars (a) 100 μm, (bd) 20 μm. For abbreviations, see Fig. S2. (GIF 209 kb)

Fig. S4
figure 18

ISH: GpB5. Single-labeled chromogenic in situ hybridization for the hormone specific beta (β) subunit, GpB5, of thyrostimulin in female ammocoete, female parasitic phase and adult female and male lamprey adenohypophyses. GpB5 was minimally to moderately expressed in the RPD (a, b), PPD (a, c) and PI (a, d) of female ammocoete lampreys; moderately expressed in the RPD (e, f), PPD (e, g) and PI (e, h) of female parasitic phase lampreys; and highly expressed in the RPD (i, j), dorsal (i, k) and ventral (i, m) PPD and PI (i, l) of adult female and male lampreys. Arrowheads indicate the regions of expression. Scale bars (a) 100 μm, (bd) 20 μm. For abbreviations, see Fig. S2. (GIF 251 kb)

Fig. S5
figure 19

FISH: GpA2/GpHβ. Dual-labeled fluorescent in situ hybridization of the alpha (α) subunit, GpA2 and beta (β) subunit, GpHβ, of lamprey (l) adenohypophyseal glycoprotein hormone (GpH) (lGpH) in female ammocoete, female parasitic phase and adult female and male lamprey adenohypophyses. GpA2 and GpHβ transcripts were not co-expressed in female ammocoete lampreys (a, b: b2, c: c2, d: d2); were highly co-expressed in the RPD (e, f: f2), moderately co-expressed in the PPD (e, g: g2) and highly co-expressed in the PI (e, h: h2) of female parasitic phase lampreys; moderately to highly co-expressed in the dorsal PPD (i, k: k2), moderately co-expressed in the ventral PPD (i, l: l2) and minimally co-expressed in the PI (i, m: m2) of adult female lampreys; and moderately co-expressed in the dorsal PPD (n, p: p2), moderately to highly co-expressed in the ventral PPD (n, q: q2) and highly co-expressed in the PI (n, r: r2) of adult male lampreys. Arrowheads indicate the regions of expression. Scale bars (a) 100 μm, (bd) 20 μm. For abbreviations, see Fig. S2. (GIF 472 kb)

Fig. S6
figure 20

FISH: GpA2/GpB5). Dual-labeled fluorescent in situ hybridization of the alpha (α) subunit, GpA2 and beta (β) subunit, GpB5, of thyrostimulin in female ammocoete, female parasitic phase and adult female and male lamprey adenohypophyses. GpA2 and GpB5 transcripts were moderately co-expressed in the PI of female ammocoete lampreys (a, d: d2); highly co-expressed in the RPD (e, f: f2), PPD (e, g: g2) and PI (e, h: h2) of female parasitic phase lampreys; highly co-expressed in the RPD (i, j: j2), dorsal (i, k: k2) and ventral PPD (i, l: l2) and PI (i, m: m2) of adult female lampreys; and highly co-expressed in the RPD (n, o: o2), dorsal (n, p: p2) and ventral PPD (n, q: q2) and PI (n, r: r2) of adult male lampreys. Arrowheads indicate the regions of expression. Scale bars (a) 100 μm, (bd) 20 μm. For abbreviations, see Fig. S2. (GIF 465 kb)

Fig. S7
figure 21

FISH/IHC: triple co-expression of GpA2, GpHβ and GpB5 in adult female and male. Triple-labeled fluorescent in situ hybridization (FISH) and immunohistochemistry (IHC) of thyrostimulin subunits (α: GpA2, β: GpB5) and glycoprotein hormone subunits (α: GpA2, β: GpHβ) in adult female and male lamprey adenohypophyses. By FISH, all of the thyrostimulin and GpH subunits, GpA2, GpB5 and GpHβ were triple co-expressed (light teal) only in the ventral PPD of the AH in adult female (a) and male (b) lampreys, magnified from adult female (Fig. 3 e: e2) and male (Fig. 4 e: e2) AH, respectively. By IHC, all of the thyrostimulin and GpH subunits, GpA2, GpB5 and GpHβ were triple co-expressed (dark gray-magenta) only in the ventral PPD of the AH in adult female (c) and male (d) lampreys, magnified from adult female (Fig. 7 e: e2) and male (Fig. 8 e: e2) AH, respectively. Arrowheads in merged and individual images indicate the regions of triple co-expression of the lGpH and thyrostimulin subunits, GpA2, GpHβ and GpB5. Scale bars (a) 100 μm, (bd) 20 μm. l lamprey, GpH lamprey glycoprotein hormone (lGpH), GpA2 shared lamprey thyrostimulin and GpH alpha (α) subunit (lGpA2), GpB5 hormone specific lamprey thyrostimulin beta (β) subunit (lGpB5), GpHβ hormone specific lamprey GpH beta (β) subunit (lGpHβ), AH adenohypophysis (anterior pituitary), RPD rostral pars distalis (region of AH), PPD proximal pars distalis (region of AH), PI pars intermedia (region of AH), NH neurohypophysis (posterior pituitary), III third ventricle. (GIF 496 kb)

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Table S1

Summary table of the individual expression of GpA2, GpHβ and GpB5 by single-label chromogenic ISH and co-expression of lGpH subunits, GpA2/GpHβ and thyrostimulin subunits, GpA2/GpB5, by dual-label FISH in female ammocoete, female parasitic phase and adult female and male lampreys. l lamprey, GpH lamprey glycoprotein hormone (lGpH), GpA2 shared lamprey thyrostimulin and GpH alpha (α) subunit (lGpA2), GpB5 hormone specific lamprey thyrostimulin beta (β) subunit (lGpB5), GpHβ hormone specific lamprey GpH beta (β) subunit (lGpHβ), AH adenohypophysis (anterior pituitary), RPD rostral pars distalis (region of AH), PPD proximal pars distalis (region of AH), D dorsal PPD, V ventral PPD, PI pars intermedia (region of AH), + minimal expression, ++ moderate expression, +++ high expression, — no expression. (DOCX 32 kb)

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Marquis, T.J., Nozaki, M., Fagerberg, W. et al. Comprehensive histological and immunological studies reveal a novel glycoprotein hormone and thyrostimulin expressing proto-glycotrope in the sea lamprey pituitary. Cell Tissue Res 367, 311–338 (2017). https://doi.org/10.1007/s00441-016-2502-y

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Keywords

  • Glycoprotein hormones
  • Proto-glycotrope
  • Sea lamprey pituitary
  • Thyrostimulin
  • ISH, FISH, IHC and TEM of lamprey pituitary cell types