ECB-ART-51410
J Comp Neurol
2023 Sep 01;53113:1299-1316. doi: 10.1002/cne.25496.
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Localization of relaxin-like gonad-stimulating peptide expression in starfish reveals the gonoducts as a source for its role as a regulator of spawning.
Feng Y
,
Piñon Gonzalez VM
,
Lin M
,
Egertová M
,
Mita M
,
Elphick MR
.
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Oocyte maturation and gamete release (spawning) in starfish are triggered by relaxin-like gonad-stimulating peptide (RGP), a neuropeptide that was first isolated from the radial nerve cords of these animals. Hitherto, it has generally been assumed that the radial nerve cords are the source of RGP that triggers spawning physiologically. To investigate other sources of RGP, here we report the first comprehensive anatomical analysis of its expression, using both in situ hybridization and immunohistochemistry to map RGP precursor transcripts and RGP, respectively, in the starfish Asterias rubens. Cells expressing RGP precursor transcripts were revealed in the ectoneural epithelium of the radial nerve cords and circumoral nerve ring, arm tips, tube feet, cardiac stomach, pyloric stomach, and, most notably, gonoducts. Using specific antibodies to A. rubens RGP, immunostaining was revealed in cells and/or fibers in the ectoneural region of the radial nerve cords and circumoral nerve ring, tube feet, terminal tentacle and other arm tip-associated structures, body wall, peristomial membrane, esophagus, cardiac stomach, pyloric stomach, pyloric caeca, and gonoducts. Our discovery that RGP is expressed in the gonoducts of A. rubens proximal to its gonadotropic site of action in the gonads is important because it provides a new perspective on how RGP may act as a gonadotropin in starfish. Thus, we hypothesize that it is the release of RGP from the gonoducts that triggers gamete maturation and spawning in starfish, while RGP produced in other parts of the body may regulate other physiological/behavioral processes.
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???displayArticle.link??? J Comp Neurol
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FIGURE 1. Starfish anatomy. (a) Photograph of a specimen of Asterias rubens. The lines labeled b, c, and d show the position and orientation of the diagrams in panels b, c, and d, respectively. (b) Schematic diagram of the central disc and proximal region of an arm. (c) Schematic diagram of a transverse section of an arm. (d) Schematic diagram of a sagittal section through the tip of an arm. AHR, aboral hemal ring; AL, aboral lappet; AO, ambulacral ossicles; CS, cardiac stomach; CONR, circumoral nerve ring; CC, coelomic cavity; G, gonad; Gd, gonoduct; HR, hemal ring; md, madreporite; MN, marginal nerve; O, ossicle; OC, optic cushion; PC, pyloric caecum; PS, pyloric stomach; ped, pedicellaria; RNC, radial nerve cord; RWV, radial water vessel; TF, tube foot; TO, terminal ossicle; TT, terminal tentacle. The photograph in Figure 1a was taken using an iPhone13 pro with a 12‐megapixel sensor camera (Apple, USA) and then background removal and labeling were accomplished using Photoshop CC2020 (San Jose, CA, USA). Images b and c were adapted from Yanez‐Guerra et al. (2018) and image d was adapted from Smith (1937). Images b–d were drawn in Photoshop CC2020 (San Jose, CA) using the digital pen of a creative drawing tablet (Wacom, Tokyo, Japan), which was connected to a laptop (MacBook Pro, Apple, USA). |
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FIGURE 2. Localization of AruRGP precursor transcripts in radial nerve cord, tube foot, and arm tip of Asterias rubens using in situ hybridization. (a) Transverse section of a radial nerve cord incubated with antisense probes, revealing a bilaterally symmetrical pattern of staining, with two or three stained cells located in the epithelium of the ectoneural region on both sides. The inset shows absence of staining in a radial nerve cord incubated with AruRGP precursor sense probes (see also Figure S1a). (b) A higher magnification image of the boxed region in panel a. (c) Longitudinal section of a tube foot showing stained cells (arrowheads) in the subepithelial nerve plexus of the podium. (d) Transverse section of an arm tip showing strongly stained cells (arrowhead) in the body wall epithelium lining the cavity that surrounds the terminal tentacle. The inset of panel d shows absence of staining in a transverse section of an arm tip incubated with sense probes (see also Figure S1b). Ec, ectoneural region; Hy, hyponeural region; RHS, radial hemal strand or sinus; RNC, radial nerve cord; TT, terminal tentacle. Scale bars: 50 μm in panels a, c, and d and in insets of panels a and d; 30 μm in panel b. |
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FIGURE 3. Localization of AruRGP precursor transcripts in the digestive system of Asterias rubens using in situ hybridization. (a) Staining (boxed region) in a section of cardiac stomach incubated with AruRGP precursor antisense probes. The inset shows absence of staining in an adjacent section of cardiac stomach incubated with AruRGP precursor sense probes (see also Figure S1c). (b). A higher magnification image of the boxed region in panel a, showing a stained cell (arrowhead). (c) Staining (boxed region) in a section of pyloric stomach incubated with AruRGP precursor antisense probes. The inset shows absence of staining in an adjacent section of pyloric stomach incubated with AruRGP precursor sense probes (see also Figure S1d). (d) A higher magnification image of the boxed region in panel c, showing a stained cell (arrowhead). CS, cardiac stomach; Lu, lumen of gut; PS, pyloric stomach. Scale bar: 100 μm in panel c and in insets of panels a and c; 8 μm in panels b and d. |
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FIGURE 4. Localization of AruRGP precursor transcripts in the reproductive system of Asterias rubens (female) using in situ hybridization. (a) Longitudinal section of a gonoduct incubated with AruRGP antisense probes showing stained cells at the base of the cross‐ridge epithelium, proximal to the junction between the gonoduct and the body wall. The inset shows absence of staining in an adjacent section of gonoduct incubated with AruRGP precursor sense probes (see also Figure S1e). (b) Higher magnification image of boxed region in panel a. (c) Higher magnification image of boxed region in panel b; one of the stained cells (arrowhead) clearly has a stained process emanating from it. (d) Staining in an adjacent section to that shown in panels a–c. (e) Higher magnification image of the boxed region in panel d. BW, body wall; Cr, cross‐ridge epithelium. Scale bar: 100 μm in panels a and d and in inset in panel a; 50 μm in panel b; 8 μm in panels c and e. |
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FIGURE 5. Immunohistochemical localization of AruRGP in the nervous system of Asterias rubens. (a, b) AruRGP‐immunoreactivity in transverse sections of radial nerve cord from the regions of an arm proximal to the arm tip (a) or proximal to the circumoral nerve ring (b). The inset of panel a shows absence of immunostaining in a transverse section of the radial nerve cord incubated with AruRGP antiserum that had been preabsorbed with AruRGP (see also Figure S1f). (c) Higher magnification image of the boxed region on the left side of panel a, showing a cluster (two or three) of immunostained cells in the subcuticular epithelium of the ectoneural region (arrowhead). (d) Higher magnification image of the boxed region on the right side of panel a, showing punctate staining of axonal processes in the neuropile of the ectoneural region. Absence of staining in the hyponeural region is also seen clearly here. (e) Higher magnification image of the boxed region in panel b, showing staining in the ectoneural neuropile. Note that there is a band of strongly stained large fibers at the boundary between the largely unstained oral region of neuropile and the immunostained aboral region of neuropile (bracket). Note also the columnar organization of the neuropile, with columns of stained axonal processes separated by the unstained processes of supporting cells (radial glial‐like cells; arrow). (f) AruRGP‐immunoreactivity in the marginal nerve. (g) Parasagittal section of a radial nerve cord showing AruRGP‐immunoreactivity in the ectoneural region, but not in hyponeural region. Note also the presence of an immunostained cell (arrowhead) in the ectoneural epithelium. (h) Higher magnification image of the boxed region in panel g. Note the banded appearance of the immunostaining, with two clusters of longitudinally projecting immunostained axons (asterisks) that are separated and bounded by three less intensely stained regions of neuropile (triangles). (i) Horizontal section of a juvenile starfish showing AruRGP‐immunoreactive fibers in the ectoneural neuropile at the junction between a radial nerve cord and the circumoral nerve ring. (j) Transverse section of circumoral nerve ring showing AruRGP‐immunoreactivity in the ectoneural region but not in the hyponeural region, which has become detached from the ectoneural region during processing of this tissue section. AruRGP‐immunoreactivity in the circumoral nerve ring is continuous with immunostaining in the subepithelial nerve plexus of the peristomial membrane. (k) Higher magnification image of lower boxed region in panel j, showing stained axonal processes in the ectoneural neuropile proximal to where the hyponeural region is located naturally, but which has become detached during tissue processing (as seen in panel j). (l) Higher magnification image of the upper boxed region of panel j showing intense AruRGP‐immunoreactivity at the junction between the circumoral nerve ring and the peristomial membrane. BW, body wall; CONR, circumoral nerve ring; Ec, ectoneural region; Hy, hyponeural region; MN, marginal nerve; OHR, oral hemal ring; PM, peristomial membrane; RHS, radial hemal strand or sinus; RNC, radial nerve cord; TF, tube foot. Scale bar: 40 μm in panels a, b, f, g, i, and j; 10 μm in panels c, d, e, h, k, and l. |
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FIGURE 6. Immunohistochemical localization of AruRGP in tube feet, arm tip, and body wall of Asterias rubens. (a) Longitudinal section of a tube foot showing AruRGP‐immunoreactivity in the subepithelial nerve plexus of the podium (boxed region) and in the basal nerve ring (arrowheads) proximal to the disc region. (b) Higher magnification image of boxed region in panel a, showing immunostained fibers in the subepithelial nerve plexus. (c) Sagittal section of an arm tip showing AruRGP‐immunoreactivity in the body wall subepithelial nerve plexus, terminal tentacle, optic cushion, and radial nerve cord. The margins of the coelomic cavity and radial water vessel of the terminal tentacle are labeled with dotted lines to highlight the presence of tissue that separates the coelomic cavity and radial water vessel. (d) Higher magnification image of boxed region in panel c, showing immunostained cells and/or processes in the aboral body wall epithelium above the terminal tentacle, in the aboral lappet, and in the terminal tentacle. (e) Higher magnification image of boxed region in panel d, showing a strongly immunostained bipolar shaped cell body in the body wall epithelium, with a stained neurite projecting toward the external surface of the epithelium (black arrowhead) and a stained axonal process projecting into the subepithelial nerve plexus (white arrowhead). (f) Transverse section of an arm tip showing AruRGP‐immunoreactivity in the terminal tentacle, lateral lappet, and body wall epithelium surrounding the cavity containing the terminal tentacle. (g) Higher magnification image of the right boxed region of panel f showing immunostained cells (asterisks) in the body wall epithelium. A stained neurite of one of the cells can be seen projecting to the external surface of the epithelium (black arrowhead). (h) Higher magnification image of left boxed region of panel f showing a bipolar shaped immunostained cell in the epithelial layer of the terminal tentacle (asterisk) with an axonal process projecting into the underlying subepithelial nerve plexus. (i) AruRGP‐immunoreactivity in the aboral body wall epithelium and subepithelial nerve plexus of an arm, proximal to a cluster of pedicellariae. (j) Higher magnification image of the boxed region in panel i showing AruRGP‐immunoreactivity in the subepithelial nerve plexus (asterisk). AL, aboral lappet; AO, ambulacral ossicles; BNR, basal nerve ring; BW, body wall; CC, coelomic cavity; Di, disk; Ep, epithelium; LL, lateral lappet; ML, muscle layer; OC, optic cushion; Pe, pedicellaria; RWV, radial water vessel; SNP, subepithelial nerve plexus; TO, terminal ossicle; TT, terminal tentacle. Scale bar: 100 μm in panels a, b, and i; 50 μm in panel e; 20 μm in panels c, d, g, and h; 6 μm in panels f and j. |
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FIGURE 7. Immunohistochemical localization of AruRGP in the digestive system of Asterias rubens. (a–c) Transverse section of the central disc region showing AruRGP‐immunoreactivity in the subepithelial nerve plexus of the peristomial membrane, which is contiguous with immunostaining in the adjacent ectoneural region of the circumoral nerve ring. (d) Transverse section of a central disc showing AruRGP‐immunoreactivity in the subepithelial nerve plexus of the peristomial membrane and esophagus. (e) Higher magnification image of the boxed region of panel d showing immunostained fibers in the subepithelial nerve plexus at the junction between the peristomial membrane and the esophagus. (f) Transverse section of a central disc showing AruRGP‐immunoreactivity in the highly folded cardiac stomach. (g) Higher magnification image of the boxed region in panel f showing AruRGP‐immunoreactivity in the basiepithelial nerve plexus (arrowhead) of cardiac stomach. (h) Transverse section of a central disc showing sparse AruRGP‐immunoreactivity in the pyloric stomach. (i) Higher magnification image of boxed region in panel h showing immunostaining in the basiepithelial nerve plexus (arrowhead). (j) AruRGP‐immunoreactivity in a transverse section of a pyloric caecum. (k) Higher magnification image of the boxed region in panel j showing immunostaining in the basiepithelial nerve plexus (arrowhead). CONR, circumoral nerve ring; CS, cardiac stomach; Es, esophagus; Lu, lumen of gut; Mu, mucosa; PM, peristomial membrane; PS, pyloric stomach. Scale bar: 200 μm in panel h; 80 μm in panels d, f, and j; 60 μm in panels a–c; 20 μm in panels e and i; 10 μm in panels i and k. |
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FIGURE 8. Immunohistochemical localization of AruRGP in the reproductive system of Asterias rubens. (a–d) AruRGP‐immunoreactivity in longitudinal (a, b) and transverse (c, d) sections of the gonoduct in male (a, c) and female (b, d) starfish. Note the absence of immunostaining in the testes (a, c) and ovaries (O). (e) Higher magnification image of the boxed region in panel a showing AruRGP‐immunoreactivity in a nerve plexus (arrowhead) located at the base of the cross‐ridged luminal epithelial layer of the gonoduct (male). (f) Higher magnification image of the boxed region in panel c showing AruRGP‐immunoreactivity in a nerve plexus (arrowhead) located at the base of the cross‐ridged luminal epithelial layer of the gonoduct (male). (g) Higher magnification image of the boxed region in panel b showing AruRGP‐immunoreactivity in a nerve plexus (arrowhead) located at the base of the cross‐ridged luminal epithelial layer of the gonoduct (female). (f) Higher magnification image of the boxed region in panel d showing AruRGP‐immunoreactivity in a nerve plexus (arrowhead) located at the base of the cross‐ridged luminal epithelial layer of the gonoduct (female). CR, cross‐ridge epithelium; Gd, gonoduct; O, ovary; T, testis. Scale bar: 150 μm in panels a–d; 20 μm in panels e–h. |
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FIGURE 9. Schematic diagrams showing the anatomy of RGP signaling in starfish, with the gonoduct as a source of RGP and the gonad as a site of action. (a) Diagram showing a model of our hypothesis that prior to spawning in starfish, RGP molecules released from nerve processes in the gonoduct diffuse into lumen of the gonoduct and then into the lumen of the adjoining gonad (ovary), where they can bind to cell surface receptor proteins on follicle cells surrounding immature oocytes. (b) Diagram showing how binding of RGP to RGP receptors on follicle cells stimulates formation of 1‐methyladenine, which then binds to receptors on oocytes to trigger oocyte maturation (germinal vesicle breakdown). (c) Detail of signal transduction pathway in follicle cells, in which binding of RGP to a G‐protein‐coupled receptor triggers G‐protein (Gs)‐mediated stimulation of adenylyl cyclase activity and elevation of intracellular cyclic adenosine monophosphate, which then triggers formation of 1‐methyladenine via as yet unknown molecular mechanisms. α, alpha subunit of G‐protein; ATP, adenosine triphosphate; β, beta subunit of G‐protein; BW, body wall; Cr, cross‐ridged epithelium of gonoduct; γ, gamma subunit of G‐protein; Gd, gonoduct; GDP, guanosine diphosphate; GTP, guanosine triphosphate; 1‐MeAde, 1‐methyladenine; O, ovary; RGP, relaxin‐like gonad‐stimulating peptide; RGPR, RGP receptor. The diagram in panel a is adapted from figure 1 in Walker (1975) and was drawn in Photoshop CC2020 (San Jose, CA) using the digital pen of a creative drawing tablet (Wacom, Tokyo, Japan), which was connected to a laptop (MacBook Pro, Apple, USA). The diagrams in panels b and c are based upon figure 2 in Mita (2019) and were created using BioRender (https://www.biorender.com/). |
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