Neuropeptide expression and action in the reproductive system of the starfish Asterias rubens

Reproductive processes are regulated by a variety of neuropeptides in vertebrates and invertebrates. In starfish (phylum Echinodermata), relaxin‐like gonad‐stimulating peptide triggers oocyte maturation and spawning. However, little is known about other neuropeptides as potential regulators of reproduction in starfish. To address this issue, here, we used histology and immunohistochemistry to analyze the reproductive system of the starfish Asterias rubens at four stages of the seasonal reproductive cycle in male and female animals, investigating the expression of eight neuropeptides: the corticotropin‐releasing hormone‐type neuropeptide ArCRH, the calcitonin‐type neuropeptide ArCT, the pedal peptide‐type neuropeptides ArPPLN1b and ArPPLN2h, the vasopressin/ocytocin‐type neuropeptide asterotocin, the gonadotropin‐releasing hormone‐type neuropeptide ArGnRH, and the somatostatin/allatostatin‐C‐type neuropeptides ArSS1 and ArSS2. The expression of five neuropeptides, ArCRH, ArCT, ArPPLN1b, ArPPLN2h, and asterotocin, was detected in the gonoducts and/or gonads. For example, extensive ArPPLN2h expression was revealed in the coelomic epithelial layer of the gonads throughout the seasonal reproductive cycle in both males and females. However, seasonal and/or sexual differences in the patterns of neuropeptide expression were also observed. Informed by these findings, the in vitro pharmacological effects of neuropeptides on gonad preparations from male and female starfish were investigated. This revealed that ArSS1 causes gonadal contraction and that ArPPLN2h causes gonadal relaxation, with both neuropeptides being more effective on ovaries than testes. Collectively, these findings indicate that multiple neuropeptide signaling systems are involved in the regulation of reproductive function in starfish, with some neuropeptides exerting excitatory or inhibitory effects on gonad contractility that may be physiologically relevant when gametes are expelled during spawning.


INTRODUCTION
The maturation and function of the reproductive system in animals are regulated by a variety of neuropeptides and peptide hormones.
In mammals, reproductive maturation and function are initiated and maintained by pulsatile hypothalamic release of the neuropeptide gonadotropin-releasing hormone (GnRH) (Amoss et al., 1971;Schally et al., 1971), with GnRH release regulated by other neuropeptides such as kisspeptins and tachykinins (Terasawa, 2022).GnRH stimulates the pituitary synthesis and release of the gonadotropic glycoprotein-type hormones follicle-stimulating hormone (FSH) and luteinizing hormone (LH), which then act on the gonads to regulate steroid hormone synthesis and gamete production (Pierce & Parsons, 1981).Accordingly, research on non-mammalian vertebrates has revealed conserved roles for GnRH-type neuropeptides and glycoprotein-type hormones as regulators of reproduction (Dufour et al., 2020).Furthermore, evidence that GnRH-type neuropeptides are evolutionarily ancient regulators of reproductive processes has been obtained with the discovery that GnRH-type neuropeptides trigger spawning in the invertebrate chordate Ciona intestinalis (sub-phylum Urochordata) (Adams et al., 2003;Terakado, 2001).However, other neuropeptides have also been identified as regulators of reproductive processes in C. intestinalis.Thus, tachykinin-type neuropeptides induce oocyte growth and transition from the vitellogenic stage to the post-vitellogenic stage (Aoyama et al., 2008;Satake et al., 2019), and the cholecystokinin/gastrin-type neuropeptide cionin triggers germinal vesicle breakdown in mature eggs (Osugi et al., 2021).
In 1959, it was reported that extracts of radial nerve cords trigger spawning in the starfish Asterias forbesi (Phylum Echinodermata) (Chaet & McConnaughy, 1959), providing the first evidence for the existence of gonadotropic neurohormones in invertebrates.Subsequently, the active constituent known as gonad-stimulating substance or gamete-shedding substance (GSS) was found to be a peptide.Furthermore, it was discovered that GSS exerts its effects on female starfish by stimulating follicle cells that surround oocytes to produce 1-methyladenine (Chaet, 1966a(Chaet, , 1966b;;Kanatani et al., 1969).
1-Methyladenine then triggers oocyte maturation by germinal vesicle breakdown, which is followed by contractions of the gonadal wall that expel gametes into the surrounding seawater where external fertilization occurs (Mita, 2019).Importantly, the molecular identity of GSS was determined in 2009 when it was isolated from the starfish Patiria pectinifera and identified as a heterodimer that shares sequence similarity with relaxins, hormones that have important roles in the regulation of reproductive physiology in mammals (Mita et al., 2009).
Because RGP was first detected in and then isolated from extracts of starfish radial nerve cords, it has generally been assumed that these are the physiological sources for its role as a gonadotropin.However, the molecular identification of RGP has enabled detailed anatomi-cal analysis of its expression in the starfish A. rubens using mRNA in situ hybridization and immunohistochemistry.This has revealed that RGP is not only expressed in the radial nerve cords, but it is also expressed in the digestive system, tube feet (locomotory organs), arm tips, associated sensory organs, and the body wall.Furthermore, and importantly from a reproductive perspective, RGP expression was also revealed in the gonoducts, tubular-shaped organs that link the gonads to gonopores in the body wall and that convey gametes to the external environment when starfish spawn (Feng et al., 2023;Lin, Mita, et al., 2017).Because RGP expression in the gonoducts is proximal to its site of action in the gonads, it is postulated that the gonoducts may be the physiological source of RGP that triggers spawning in starfish and that RGP produced in other parts of the body may be involved in the regulation of other processes (Feng et al., 2023).
With the discovery that RGP is expressed in the gonoducts of starfish, it is of interest to determine if other neuropeptide types are expressed in the starfish reproductive system.Currently, there are very few reports of studies that have investigated neuropeptide expression in the starfish reproductive system.An immunohistochemical study using antibodies to the SALMFamide-type neuropeptide S1 revealed immunoreactive nerve fibers in the wall of the gonad in the starfish P. pectinifera (Mita et al., 2004).More recently, the expression of the calcitonin-type neuropeptide ArCT in the gonoducts of the starfish A. rubens was revealed using mRNA in situ hybridization (Cai et al., 2018).Opportunities for a more comprehensive investigation of neuropeptide expression in the starfish reproductive system have been provided by the development of antibodies to a variety of neuropeptide types in A. rubens.Informed by the analysis of transcriptome sequence data, over 40 candidate neuropeptide precursors have been identified in A. rubens (Semmens et al., 2016).Subsequently, detailed investigations of the expression and actions of neuropeptides derived from some of these precursor proteins have been reported.
To facilitate the anatomical interpretation of patterns of neuropeptide expression investigated here in the reproductive system of A.
rubens, histological analysis of the reproductive system was also performed using Masson's trichrome staining method.Previously, Walker (1974Walker ( , 1975) ) described the morphology and histology of the reproductive system in the starfish A. rubens, but referring to it as Asterias vulgaris, which is no longer an accepted name (Clark & Downey, 1991).
A detailed description of the morphology and histology of the gonads (ovaries and testes) was presented based on the analysis of starfish specimens collected near to Portsmouth, New Hampshire, USA, at four stages of the seasonal reproductive cycle in October and December 1970and March and July 1971(Walker, 1974).However, a limitation of this study was the use of black and white photographs in the published study.Furthermore, when investigating the morphology and histology of the gonoducts in A. rubens, sexually mature and sexually immature specimens were analyzed but without any reported investigation of seasonal changes in gonoduct structure (Walker, 1975).Therefore, to complement and extend the findings of Walker (1974Walker ( , 1975), here we used Masson's trichrome staining method to analyze the structure of the gonoducts and gonads (ovary and testis) from specimens of A.
rubens collected at all four stages of the reproductive cycle in which neuropeptide expression was also examined: early stage growth (October), mid-stage growth (December), late stage growth (March), and after spawning (May).
Previous studies have reported the in vitro effects of neurotransmitters on starfish gonads, revealing that glutamate inhibits spawning induced by 1-methyladenine and acetylcholine induces Ca 2+dependent contraction of muscle in the gonad wall (Mita, 2017;Mita et al., 2020).However, with the exception of RGP, nothing is known about the effects of other neuropeptides on the reproductive system in starfish.Therefore, informed by our immunohistochemical analysis of neuropeptide expression in the reproductive system of A. rubens, here we also investigated the in vitro pharmacological effects of neuropeptides on gonad preparations.

Animals
Specimens of A. rubens were obtained from a fisherman based at Whitstable (Kent, UK) and transported to Queen Mary University of London.
The animals were then maintained in an aquarium with circulating artificial seawater at ∼12 • C. Starfish were fed on mussels (Mytilus edulis), which were collected near Margate (Kent, UK).To enable immunohistochemical investigation of any sex-specific differences in neuropeptide expression in the reproductive system and/or changes in neuropeptide expression in the reproductive system over the seasonal reproductive cycle, both male (n = 3) and female (n = 3) specimens of A. rubens were analyzed at four times of the year: October 2020 (early gametogenic stage), March 2021 (late gametogenic stage), May 2021 (spawning stage), and December 2021 (mid-gametogenic stage).
It was not possible to collect starfish during the mid-gametogenic stage in December 2020 because of restrictions to research activity in the United Kingdom caused by the COVID-19 pandemic, and for this reason, starfish were collected in December 2021 instead.The mean (± standard deviation) diameter and mass of the 24 animals analyzed were 15.2 ± 1.32 cm and 73.6 ± 16 g, respectively.The reproductive system was dissected from starfish no longer than 1 day after collection from the sea and transportation to the aquarium so as to minimize any potential effects of the non-natural aquarium environment on neuropeptide expression.For investigation of the in vitro pharmacological effects of neuropeptides on gonad preparations, experiments were performed on gonads dissected from starfish collected in April 2022 (mean ± standard deviation diameter = 16.8 ± 3.0 cm; mean ± standard deviation mass = 95.1 ± 34.6 g).

Fixation and decalcification of body regions containing the reproductive system
Starfish were narcotized in artificial seawater containing 3.5% of MgCl 2 for 30 min and then were dissected.Regions of the body at the junction between adjacent arms were dissected and trimmed so that a pair of adjacent gonads and their associated gonoduct from each arm were retained (Figure 1a,b).These dissected regions of the body were fixed in Bouins solution (75% saturated picric acid in seawater, 25% formalin, and 5% acetic acid) for 2 days at 4 • C.Then, the fixed body parts were decalcified in 2% ascorbic acid/0.15M sodium chloride for 2 weeks at 4 • C, with fresh decalcification fluid used every 2 days.Finally, following dehydration through an ethanol series and transfer to xylene, the body parts were embedded in paraffin wax, sectioned at 10 µm with a microtome (Leica, RM 2145), and mounted on chrome alum/gelatin-coated microscope slides.

Masson's trichrome staining
To facilitate the interpretation of immunostaining observed with antibodies to neuropeptides (see below), sections of the A. rubens reproductive system were also stained using Masson's trichrome method, which differentiates collagenous tissue (blue) from muscle, cytoplasm, keratin (red), and nuclei (black) (Blowes et al., 2017).Sections of the reproductive system from both male and female starfish previously mounted on slides were dewaxed in xylene and rehydrated through an ethanol series (100%, 90%, 70%, and 50%; 10 min for each step) and transferred to distilled water (2 × 10 min).Then, the slides were placed in Weigerts iron hematoxylin solution (50% of a solution of hematoxylin [1 g] in 100 mL of ethanol and 50% of a solution of 4 mL of ferric chloride at 29% in water plus 95 mL distilled water and 1 mL concentrated hydrochloric acid) for 10 min, rinsed in warm running tap water (10 min), and then washed in distilled water (2 × 10 min).
Following staining in Biebrich scarlet-acid fuchsin solution (90 mL of Biebrich scarlet, 1% aqueous, 10 mL acid fuchsin, 1% aqueous, and 1 mL glacial acetic acid) and washing in distilled water (3 × 5 min), slides were placed in a solution of phosphomolybdic-phosphotungstic acid (25 mL Photographs showing how gonad preparations from specimens of A. rubens were isolated and prepared for histological/immunohistochemical analysis and for in vitro pharmacological experiments.(a) Mature specimen of A. rubens viewed aborally, with the white trapezium showing the region of the body that was dissected so that a pair of gonads and their associated gonoducts were removed from two adjacent arms.(b) Dissected region highlighted in (a) but with the aboral body wall removed, which was then fixed, decalcified, and sectioned for histological/immunohistochemical analysis.The boxed region shows where the gonads are connected to the body wall via the gonoducts.(c) Isolated gonad in an organ bath with cotton ligatures attaching the gonad to a hook at the base and to an isotonic force transducer above (not shown).Am, ampullae; AR, ambulacral ridge; Gd, gonad.
of phosphomolybdic acid at 5% and 25 mL of phosphotungstic acid at 5%) for 10 min or until collagenous tissue no longer appeared red.Then, slides were transferred directly (without rinsing) into an aniline blue solution (2.5 g of aniline blue, 2 mL of glacial acetic acid, and 100 mL of distilled water) for 5 min, rinsed briefly in distilled water, differentiated in 1% acetic acid (2 min), and washed in distilled water (3 × 5min).
Finally, the slides were placed in 95% ethanol followed by two changes of 100% ethanol to dehydrate sections, and then xylene (2 × 10 min) was used to clear sections before mounting with coverslips over DPX (Thermo Scientific).

Imaging
Images

In vitro pharmacology
To investigate if neuropeptides affect the contractility of gonads in A. rubens, pieces of gonads were dissected from starfish arms and cut into segments ∼2 cm in length, and cotton ligatures were tied at each end.The gonad preparations were linked to an isotonic force transducer and maintained in a 20-mL aerated organ bath containing circulating artificial sea water at 12 • C until a stable baseline tone was reached (Figure 1c).Then, in a series of preliminary experiments, the following neuropeptides (synthesized by PPR Ltd.) were tested on gonad preparations at a concentration of 10 −6 M: ArCRH, ArCT, ArGnRH, ArPPLN1b, ArPPLN2h, ArSS1, ArSS2, and asterotocin.No effects were observed with ArCRH, ArCT, ArGnRH, ArPPLN1b, ArSS2, and asterotocin.However, ArPPLN2h and ArSS1 caused relaxation and contraction, respectively, of gonad preparations.To facilitate the detection and measurement of the relaxing effect of ArPPLN2h, additional experiments were performed in which gonad preparations were first induced to contract using acetylcholine (ACh; 10 −6 M) before the application of ArPPLN2h (10 −6 M).Likewise, to enable comparative quantification of the contracting effect of ArSS1 on gonad preparations, 10 −6 M ACh was also tested on gonad preparations after testing of ArSS1.The relaxing effect of ArPPLN2h was quantified as the percentage reversal of the maximum contraction induced by 10 −6 M ACh.For ArSS1, the contracting effect was expressed as a percentage relative to the maximal contraction induced by 10 −6 M ACh.
These experiments were performed on gonads from 10 male starfish and 10 female starfish.Effects of ArPPLN2h were observed and measured with gonad preparations from six males and nine females, and effects of ArSS1 were observed and measured with gonad preparations from seven males and nine females.To enable comparative quantification of the effects of the neuropeptides on male and female gonads following normalization to the effects of ACh, means and standard errors of responses were plotted in bar graphs and analyzed using t-tests.Generation and editing of graphs were performed using R Stu-dio (version 2022.02.3) and the packages ggplot2 (Wickham, 2009), ggrepel (Slowikowski, 2023), and egg (Auguie, 2019), with significance values added using ggsignif (Ahlmann-Eltze & Patil, 2021).Statistical analysis of data was done using the base R packages (R-CoreTeam, 2022).

Histology of the reproductive system in A. rubens
Seasonal growth and maturation of the reproductive system of A.
rubens have been reported previously, highlighting the dramatic change in the size of the gonads from tiny organs after spawning in May/June into large organs that fill the coelomic cavity of each arm prior to spawning in March/April of the following year (Barker & Nichols, 1983;Schoenmakers et al., 1981).Seasonal changes in the histology of the gonads have also been described previously, although only with the accompaniment of black and white photographs (Walker, 1974).Here, we performed an analysis of the histology of the reproductive system in A. rubens at different stages of the seasonal reproductive cycle using Masson's trichrome staining method.However, to avoid repetitious reporting of features that have been described in detail before, we focused our attention on the gonoducts, the structure of which has been analyzed previously (Walker, 1975) but not over the seasonal reproductive cycle.Furthermore, the use of color photographs enabled the visualization of histological features of the gonoducts and proximal regions of the gonads that have not, to the best of our knowledge, been shown previously.
Analysis of the size of the gonoducts revealed an ∼2-fold increase in length from the early stage of gonad growth in October (mean ± standard error = 258.4± 22.5 µm in width, 483.5 ± 50.1 µm in length; n = 3) to the final stages of gonad growth just prior to spawning in March (mean ± standard error = 276.2± 54.2 µm in width, 1055 ± 219.2 µm in length; n = 3) (Figure 2a-h).However, no major changes in the histological structure of the gonoducts were observed from October to March (Figure 2a-c, e-g).During this period, the histology of the gonoduct in both male and female animals was found to be consistent with a previous report (Walker, 1975).The outermost layer of the gonoduct is formed by a coelomic epithelium, which is continuous with the coelomic epithelial lining of the body wall and the coelomic epithelium of the gonad, as illustrated in high magnification images of the gonoducts (Figure 2i,m) and gonads (Figure 2l,p).Beneath the coelomic epithelium is a layer of collagenous tissue which is continuous with collagenous tissue of the body wall and a thin layer of collagenous tissue underlying the coelomic epithelium of the gonads.The collagenous tissue layer is largely composed of collagen fibrils, but it also contains a sparse population of cells and their processes (Figure 2i,m,n).Beneath the collagenous tissue layer of the gonoducts is an inner epithelial layer that lines the lumen.However, an unusual feature of this lumenal epithelium is that it is highly folded to form cross-ridges that span across the lumen of the duct and intertwine to partially block the lumen  F I G U R E 3 Diagram summarizing seasonal expression patterns of five neuropeptides in the reproductive system of A. rubens.Colors represent expression in the different tissue layers of the reproductive system: blue, coelomic epithelial layer of the gonoduct (CE(Gt)); orange, lumenal epithelial layer of the gonoduct (LE); gray, cross-ridges of the gonoduct (CR); yellow, coelomic epithelial layer of the gonad (CE(Gd)); white, no expression detected.In the colored boxes without numbers, expression was detected in three out of three animals analyzed.In colored boxes with 1 or 2, expression was detected in one or two of the three animals analyzed.Both female (F) and male (M) animals were analyzed at four stages of gonad growth: early stage (October), mid-stage (December), late stage (March), and after spawning (May), as shown at the top of the diagram.The expression of five neuropeptides was detected: ArCT, calcitonin-type; ArPPLN1b, pedal peptide-like neuropeptide 1b; ArCRH, corticotropin releasing hormone-type; ArPPLN2h, pedal peptide-like neuropeptide 2h; Ast, vasopressin/oxytocin-type neuropeptide asterotocin.
(Figure 2j,k,n,o).The lumenal epithelial layer comprises both cells and fibers, but the density of cells is higher in the cross-ridges than in the inner region adjacent to the collagenous tissue (Figure 2j,k,n,o).
Following spawning in May, changes in the structure of the gonoducts were observed in comparison with gonoducts prior to spawning.
The most striking feature was a widening of the lumen of the gonoduct, which could also be seen to extend through the body wall toward the gonopores (Figure 2d,h).Furthermore, in female starfish, several oocytes and other material that is probably cell debris could be seen in the lumen of the gonoducts at the position where it passes through the body wall.Accordingly, prior to spawning in March, the regions of the gonads proximal to the gonoducts could be seen to be packed full of unreleased gametes (Figure 2c,g), whereas after spawning in May, the gonads were shrunken but with the initial stages of gametogenesis occurring in preparation for spawning in the following year (Figure 2d,h,l,p).

Neuropeptide expression in the reproductive system of A. rubens
Immunohistochemical analysis of neuropeptide expression in the reproductive system of A. rubens revealed immunostaining in the gonads and/or gonoducts with antisera/antibodies to ArCRH, ArCT, ArPPLN1b, ArPPLN2h, and asterotocin, as described in detail below, but not with antisera/antibodies to ArGnRH, ArSS1, and ArSS2 (Figure 3).The validity of the results of our immunohistochemical analysis in the reproductive system was also investigated by analysis of the radial nerve cord tissue in sections, which served as positive control experiments.Importantly, immunostaining was observed in the radial nerve cords with all eight antisera/antibodies tested (Figure S1), consistent with our previously published findings.Therefore, the absence of immunostaining in the reproductive system with antisera/antibodies to ArGnRH, ArSS1, and ArSS2 indicates that these neuropeptides are either not expressed in the gonoducts/gonads or are expressed at low levels that are beyond the detection threshold of the immunohistochemical methods employed.Hereafter, we describe in detail the patterns of immunostaining observed in the reproductive system of A. rubens with antisera/antibodies to ArCRH, ArCT, ArPPLN1b, ArPPLN2h, and asterotocin, including seasonal changes in expression revealed by our analysis of tissue obtained from animals collected at four different times: October 2020 (early gametogenic stage), March 2021 (late gametogenic stage), May 2021 (spawning stage), and December 2021 (mid-gametogenic stage).The order in which we describe the results obtained with different antibodies/antisera follows that shown in Figure 3, where neuropeptides sharing some similarities in their expression patterns are paired sequentially.

3.2.1
ArCT expression in the reproductive system of A. rubens ArCT-immunoreactivity was detected in the coelomic epithelial layer of the gonoducts in both male and female starfish at all four stages of the seasonal reproductive cycle and in all 24 of the animals analyzed, highlighting the consistency of this immunostain-ing (Figure 4a,b,e,f,i,j,m,n).Immunostaining was localized in both cells and/or fibers in the coelomic epithelial layer of the gonoducts, with fiber staining in some cases coalescing into a strongly immunoreactive sub-epithelial nerve plexus (Figure 4j).ArCT-immunoreactivity was also consistently observed in the sub-epithelial nerve plexus underlying the lumenal epithelium of the gonoducts during all four stages of the seasonal reproductive cycle analyzed and in all 24 of the animals analyzed (Figure 4n).Furthermore, ArCT-immunoreactivity was also observed in cells and/or processes in the cross-ridges of the gonoducts (Figure 4c,d,j), although this was observed less consistently (see Figure 4n) than the immunostaining in the coelomic epithelial layer of the gonoducts and in the sub-epithelial nerve plexus underlying the lumenal epithelium of the gonoducts.Thus, ArCT immunostaining in the cross-ridges of the gonoducts was observed in only one of three females at the mid-stage of gonad growth but in none of the three males analyzed (Figure 3).Furthermore, at the late stage of gonad growth and after spawning, only one of three males analyzed exhibited ArCT immunoreactivity in the cross-ridges of the gonoducts (Figure 3).ArCT-immunoreactivity was not observed in the gonads in any of the 24 animals analyzed (Figure 3).

3.2.2
ArPPLN1b expression in the reproductive system of A. rubens ArPPLN1b-immunoreactivity was detected in the coelomic epithelial layer of the gonoducts in both male and female starfish and in all four stages of the seasonal reproductive cycle analyzed (Figure 3).Furthermore, all 24 of the animals analyzed exhibited immunostaining in the coelomic epithelial layer of the gonoducts, highlighting the consistency of this immunostaining (Figure 3; Figure 5a,b,e,f,i,j,k,m,n,o,p).Immunostaining was localized in both cells and fibers in the coelomic epithelial layer of the gonoducts, with fiber staining coalescing into a strongly immunoreactive sub-epithelial nerve plexus (Figure 5n,p).In female animals, ArPPLN1b-immunoreactivity was consistently observed in the sub-epithelial nerve plexus underlying the lumenal epithelium of the gonoducts during all four stages of the seasonal reproductive cycle analyzed (Figures 3 and 5b).Furthermore, during the early and late stages of gonad growth in females, ArPPLN1b-immunoreactivity was also observed in cells and/or processes in the cross-ridges of the gonoducts (Figure 5c,d).In contrast, in male animals, ArPPLN1bimmunoreactivity was observed less consistently in the sub-epithelial nerve plexus underlying the lumenal epithelium of the gonoducts, with no animals exhibiting staining after spawning (Figures 3 and 5m,o), only one out of the three animals exhibiting staining during the early stage of gonad growth (Figure 3), two out of the three animals exhibiting staining during the late stage of gonad growth (Figures 3 and 5g,h) Only in the mid-stage of gonad growth was immunostaining observed in the sub-epithelial nerve plexus underlying the lumenal epithelium of the gonoducts in all three of the animals analysed (Figure 3).Furthermore, in males, ArPPLN1b-immunoreactivity was not observed in the cross-ridges of the gonoducts in any of the animals analyzed (Figures 3   and 5g,o).Lastly, no ArPPLN1b-immunoreactivity was observed in the gonads in any of the 24 animals analyzed (Figure 3).

3.2.3
ArCRH expression in the reproductive system of A. rubens ArCRH-immunoreactivity was detected in the coelomic epithelial layer of the gonoducts in both male and female starfish at all four stages of the seasonal reproductive cycle and in all 24 of the animals analyzed, highlighting the consistency of this immunostaining (Figures 3 and 6a,b,e,f,i,k,m,n,o).Immunostaining was localized in cells and/or fibers in the coelomic epithelial layer of the gonoducts, with fiber staining in some cases coalescing into a strongly immunoreactive sub-epithelial nerve plexus (Figure 6k).ArCRH-immunoreactivity was also observed in the sub-epithelial nerve plexus underlying the lumenal epithelium of the gonoducts, but this was only observed after spawning in females (n = 3) (Figures 3 and 6j,k).Furthermore, in one female, immunostaining in the lumenal epithelium of the gonoducts also extended into the cross-ridges (Figure 6k).
ArCRH-immunoreactivity was also detected in the coelomic epithelial layer of the gonads in both male and female starfish, and consistent with immunostaining observed in the coelomic epithelial layer of the gonoducts, immunostaining was observed in both cells and fibers (Figure 6c,d,e,g,h,i,l).However, this was only observed in a subset of the 24 animals analyzed (Figure 3).During the early stage of gonad growth, ArCRH-immunoreactivity in the coelomic epithelium of the gonad was observed in two male and two female individuals, whereas in the midstage of gonad growth, ArCRH-immunoreactivity was present in just one male and one female (Figures 3 and 5c,d,e,g,h).At the late stage of gonad growth, ArCRH-immunoreactivity in the coelomic epithelium of the gonad was only observed in one of the three females analyzed and not in any of the three males analyzed (Figure 3), and after spawning, it was observed in two of the three females analyzed but not in any of the three males analyzed (Figures 3 and 6i,l,n,p).

3.2.4
ArPPLN2h expression in the reproductive system of A. rubens ArPPLN2h-immunoreactivity was detected in the coelomic epithelial layer of the gonoducts in both male and female starfish, with cells and fibers exhibiting immunostaining (Figure 3).However, while this immunostaining was observed during the late stage of gonad growth in both males and females and after spawning in females (Figures 3   and 7g,i), during the early and mid-stages of gonad growth, it was only observed in one (male) of the six animals analyzed (Figures 3 and 7d,e).
In all 24 animals analyzed, no immunostaining was observed in the lumenal epithelial layer of the gonoducts.
ArPPLN2h-immunoreactivity was also detected in the coelomic epithelial layer of the gonads in both male and female starfish, and this was consistently observed in all four stages of the seasonal reproductive cycle analyzed and in all 24 of the animals analyzed (Figure 3).ArPPLN2h-immunoreactivity in the coelomic epithelial layer of the gonads was more extensive than for any other neuropeptide analyzed here.Furthermore, consistent with immunostaining observed in the coelomic epithelial layer of the gonoducts, immunostaining was observed in both cells and fibers in the coelomic epithelial layer of the gonads (Figure 7a,b,c,d,e,f,h,j,l,m).

3.2.5
Asterotocin expression in the reproductive system of A. rubens Asterotocin-immunoreactivity was only detected in the coelomic epithelial layer of the gonoducts (Figure 3).In female starfish, this was detected in all four stages of the seasonal reproductive cycle analyzed (Figures 3 and 8a,b,e,f), whereas, in male starfish, it was only detected in the early and mid-stages of gonad growth (Figures 3 and 8c,d,g,h).Furthermore, by comparison with the expression of other neuropeptides in the coelomic epithelial layer, as described above, asterotocin-immunoreactivity was relatively sparse.Thus, as shown in Figure 8b,d,f, in sections of the gonoduct, only a few immunostained cells were observed in the coelomic epithelial layer, and no immunostaining was observed in nerve fibers.

In vitro pharmacological effects of neuropeptides on gonad preparations from A. rubens
ArCT, ArCRH, ArGnRH, ArPPLN1b, ArSS2, and asterotocin had no observable effects on the contractile state of gonad preparations from A. rubens when tested at a concentration of 1 µM (n = 6-9 for ArSS1 caused contraction of gonad preparations from both male and female specimens of A. rubens (Figure 9a-c).When tested at a concentration of 1 µM, the mean (±standard error) magnitude of contraction induced by ArSS1 in ovary preparations was 29.9 ± 3.3% (n = 9) relative to the contracting effect of 1 µM ACh (Figure 9c).In contrast, when tested at a concentration of 1 µM, the mean (±standard error) magnitude of contraction induced by ArSS1 in testis preparations was 6.6 ± 1.2% (n = 7) relative to the contracting effect of 1 µM ACh (Figure 9c).Furthermore, statistical analysis revealed there to be a statistically significant difference in the effect of ArSS1 on ovary and testis preparations (p ≤ .001;t-test).
ArPPLN2h caused relaxation of gonad preparations from both male and female specimens of A. rubens, with ArPPLN2h partially reversing the contracting effect of ACh (Figure 10a,c).When tested at a concentration of 1 µM on ovary preparations, the mean (±standard error) reversal of the contracting effect of 1 µM ACh induced by ArPPLN2h was 29.2 ± 4.5% (n = 9) (Figure 10c).In contrast, when tested at a concentration of 1 µM on testis preparations, the mean (±standard error) reversal of the contracting effect of 1 µM ACh induced by ArPPLN2h was 14.7 ± 3.2% (n = 6) (Figure 10c).Furthermore, statistical analysis revealed there to be a statistically significant difference in the effect of ArPPLN2h on ovary and testis preparations (p ≤ .05;t-test).

DISCUSSION
Spawning in starfish is triggered by RGP, a neuropeptide that was first isolated from radial nerve cord extracts (Chaet & McConnaughy, 1959;Mita, 2019;Mita et al., 2009).It was recently reported that in the starfish A. rubens, RGP is expressed in the gonoducts that link the gonads to the gonopores, where gametes are expelled into the external environment.Furthermore, it was hypothesized that the gonoducts may be the physiological source of the RGP that triggers spawning in starfish, with RGP expressed in other parts of the body being involved in the regulation of other processes (Feng et al., 2023).Evidence that other neuropeptides are expressed in the starfish reproductive system, including the calcitonin-type neuropeptide ArCT and the SALMFamide neuropeptide S1, has been reported previously (Cai et al., 2018;Mita et al., 2004).In this study, we report a more comprehensive immunohistochemical analysis of the expression of a variety of neuropeptides in the reproductive system of the starfish A. rubens.Of the eight neuropeptides analyzed, the expression of five neuropeptides (ArCRH, ArCT, ArPPLN1b, ArPPLN2h, and asterotocin) was detected in cells and/or fibers located in the gonoducts and/or gonads of A. rubens.However, no immunostaining was observed in the reproductive system of A. rubens with antibodies/antisera to ArGnRH, ArSS1, or ArSS2.It is noteworthy that for two of these neuropeptides (ArGnRH and ArSS1), affinity-purification of antibodies was required for the immunohistochemical visualization of their expression in A. rubens (Tian et al., 2017; Y. Zhang, Yañez Guerra, et al., 2022).Furthermore, while immunohistochemical analysis of the expression of ArCRH, ArCT, ArPPLN1b, and ArPPLN2h has been accomplished with antisera at dilutions ranging from 1:10,000 to 1:100,000 (Cai et al., 2023(Cai et al., , 2018;;Lin, Egertovà, et al., 2017;Lin et al., 2018), the visualization of ArSS2 expression in A. rubens has been accomplished with antiserum at a lower dilution (1:5,000) (Y.Zhang et al., 2020).Therefore, although, in this study, no immunostaining was observed with ArGnRH, ArSS1, and ArSS2 antisera/antibodies in the reproductive system of A. rubens, we cannot rule out the possibility that these neuropeptides are expressed in the reproductive system F I G U R E 1 0 The pedal peptide-type neuropeptide ArPPLN2h causes relaxation of gonad preparations from A. rubens.(a) After inducing contraction of an ovary preparation with acetylcholine (ACh; 10 −6 M), ArPPLN2h (10 −6 M) induced relaxation can be observed, causing partial reversal of the effect of ACh.Washing with artificial sea water (ASW) causes complete reversal of the effect of ACh.(b) After inducing contraction of testis preparation with acetylcholine (ACh; 10 −6 M), ArPPLN2h (10 −6 M) induced relaxation can be observed, causing partial reversal of the effect of ACh.Washing with ASW causes complete reversal of the effect of ACh.(c) Graph comparing mean (±SEM) relaxing effect of ArPPLN2h (10 −6 M) on gonads from female starfish (ovary; n = 9) and male starfish (testis; n = 6), with relaxation quantified as percentage reversal of the contracting effect ACh (10 −6 M).The magnitude of the effects ArPPLN2h on ovaries and testes is significantly different (p ≤ .05;t-test).
of A. rubens but at low levels of expression beyond the detection threshold of the immunohistochemical methods employed.It should be noted, however, that because sections of gonoduct and gonad tissue tested in this study also contained radial nerve cord tissue, this provided material for positive control tests, and immunostaining was observed in the radial nerve cords with all the antisera/antibodies tested, including the ArGnRH, ArSS1, and ArSS2 antisera/antibodies, consistent with our previous reports (Tian et al., 2017;Y. Zhang et al., 2020;Y. Zhang, Yañez Guerra, et al., 2022).Therefore, the absence of staining in the reproductive system with the ArGnRH, ArSS1, and ArSS2 antisera/antibodies was validated by accompanying positive controls (Figure S1).

Masson's trichrome staining reveals changes in the structure of the gonoducts in A. rubens in association with spawning
As a prelude to immunohistochemical analysis of neuropeptide expression, Masson's trichrome staining was employed to investigate seasonal changes in the histology of the reproductive system in A. rubens.
The first detailed morphological and histological analysis of the gonads and gonoducts in immature and mature specimens of male and female starfish was reported by Walker (1974Walker ( , 1975)).Using the common starfish A. vulgaris (synonym A. rubens) as an experimental animal, changes in the structure of the gonads during a single seasonal reproductive cycle were analyzed (Walker, 1974).However, when analyzing the gonoducts, seasonal changes in their structure were not examined (Walker, 1975).
Here, we used Masson's trichrome staining to analyze the histology of the reproductive system of A. rubens at four stages of the seasonal reproductive cycle, focusing on the gonoducts.During the period of gonad growth from October to March, the structure and histology of the gonoducts were consistent with that reported previously (Walker, 1975).However, in May, after spawning had occurred, the structure of the gonoducts was different, exhibiting morphological changes that appear to be associated with facilitating the expulsion of gametes during spawning.These changes include (i) enlargement of the lumen of the gonoducts and widening of channels that link the gonoducts to the gonopores; (ii) elongation and narrowing of the collagenous tissue layer of the gonoducts; and (iii) flattening of the lumenal epithelium of the gonoduct, which appears to be associated with at least partial loss of the cross-ridges.Thus, after spawning, in some individuals, the cross-ridges appear to be shorter and less numerous than in individuals prior to spawning.These findings suggest that the cross-ridges undergo a process of flattening and/or loss in association with the widening and elongation of the lumen of the gonoduct prior to and/or during spawning, which is presumably an adaptation to facilitate the ejection of gametes.Furthermore, these changes in the structure of the reproductive system of A. rubens in association with spawning are consistent with previous reports for other echinoderms (Hamel & Mercier, 2007;McEuen, 1988).

Physiological significance of neuropeptide expression in the gonoducts of A. rubens
In interpreting the results obtained with the five antisera/antibodies for which immunostaining was observed in the reproductive system of A. rubens, we will focus first on the gonoducts.As summarized in Figure 3, the expression of all five neuropeptides (ArCRH, ArCT, ArP-PLN1b, ArPPLN2h, and asterotocin) was detected in the coelomic epithelial layer of the gonoducts, and for three of the neuropeptides (ArCT, ArPPLN1b, and ArCRH), the expression was detected in both males and females at all four stages of the seasonal reproductive cycle analyzed.In contrast, ArPPLN2h expression in the coelomic epithelial layer was only consistently detected in both males and females in March, just prior to spawning, whereas after spawning in May, ArPPLN2h expression was only detected in females.Similarly, a sexspecific difference in asterotocin expression was also observed, with asterotocin-immunoreactivity observed in the coelomic epithelial layer of the gonoducts both prior to spawning (March) and after spawning (May) in females, but not in males.The functional significance of these seasonal/sexual differences in neuropeptide expression in the coelomic epithelial layer of the gonoducts remains to be determined, but they seem to be indicative of regulatory roles associated with preparation for and/or initiation of spawning.With this in mind, it is worthwhile to consider in more detail the anatomy and physiological relevance of neuropeptide expression in the coelomic epithelial layer of the gonoducts.This layer is contiguous with the coelomic epithelial layer of the adjacent body wall and the coelomic epithelial layer of the gonads.To the best of our knowledge, the gonoducts of A. rubens have not been examined using electron microscopy, and therefore the ultrastructure of the coelomic epithelial layer is not known.However, the coelomic epithelium of the gonoducts is continuous with the coelomic epithelium of the gonads, which comprises myoepithelial cells, coelomocytes, and neurons (Chia & Koss, 1994;Schoenmakers et al., 1981).Accordingly, the immunohistochemical analysis reported here has revealed the presence of neuropeptide-immunoreactivity in a nerve plexus located beneath the coelomic epithelium of the gonoducts.Furthermore, within the coelomic epithelium there are immunoreactive cells that may be neuronal cell bodies.For example, see Figure 4b,f for ArCT; Figure 6b,f for ArCRH; Figure 5k,n for ArPPLN1b; Figure 7i for ArPPLN2h; and Figure 8b,d,f for asterotocin.Thus, at least some of the immunoreactive fibers in the sub-epithelial nerve plexus of the coelomic epithelial layer of the gonoducts are likely to be derived from these cells.However, since the coelomic epithelial layer of the gonoducts it contiguous with the coelomic lining of the body wall, some of the immunoreactive fibers in the sub-epithelial nerve plexus of the coelomic epithelial layer of the gonoducts could be derived from cell bodies situated outside the gonoducts.For example, neurons are located in the aboral coelomic epithelium of the body wall, which include neurons associated with the apical muscle that runs longitudinally along the aboral coelomic lining of the body wall in each arm (Blowes et al., 2017).Consistent with this hypothesis, all of five neuropeptides detected in the coelomic epithelial layer of the gonoducts are also expressed by cells in the aboral coelomic epithelium of the body wall in A. rubens.Thus, for ArCT, see figs.What then might be the physiological role of neuropeptides in the coelomic epithelial layer of the gonoducts?In the body wall, beneath the coelomic epithelial layer, there are both circularly and longitudinally orientated layers of muscle fibers that are separated by a thin layer of collagenous tissue (Blowes et al., 2017).In the gonoducts, there is a prominent layer of collagenous tissue beneath the coelomic epithelial layer, and muscle fibers may also be present but if they are, they are not prominent enough to form a clearly visible layer.Nevertheless, we speculate that neuropeptides expressed in the coelomic epithelial layer of the gonoducts may regulate the mechanical/contractile state of the gonoducts through effects on collagenous tissue and/or muscle.Furthermore, such effects could be important in adaptive changes in the mechanical/contractile state of the gonoducts in preparation for and during spawning.Some, but not all, of the five neuropeptides detected in the gonoducts were found to be expressed in the lumenal epithelial layer of the gonoducts.Importantly, although the ultrastructure of the gonoduct in A. rubens is not known, electron microscopic analysis of the gonoduct in the starfish Asterina gibbosa has been reported.This revealed that between the lumenal epithelium and a thick layer of collagenous tissue, there is a layer of tissue that comprises nerve fibers and smooth muscle fibers (Bruslé, 1969).Here, in our study, we refer to this layer of tissue, together with lumenal epithelium, as the lumenal epithelial layer and, in accordance with the gonoduct ultrastructure of A. gibbosa (Bruslé, 1969), we observe neuropeptide immunoreactivity in this layer in A. rubens, as discussed in more detail below.
ArCT expression was detected in the lumenal epithelial layer of the gonoducts over all four stages of the seasonal reproductive cycle in A. rubens in both males and females and in all animals analyzed (Figure 3).
In contrast, ArPPLN1b expression was detected in all females and over all four stages of the seasonal reproductive cycle, whereas in males, ArPPLN1b expression in the lumenal epithelial layer of the gonoducts was much more variable.Thus, none and one or two out of the three males analyzed after spawning, at the early stage of gonad growth and at the late stage of gonad growth, respectively, exhibited ArP-PLN1b expression in the lumenal epithelial layer of the gonoducts.
Lastly, ArCRH expression in the lumenal epithelial layer of the gonoducts was only observed in females, including one out of three females prior to spawning and all three females analyzed after spawning.Thus, as with the expression of some neuropeptides in the coelomic epithelial layer, there appear to be dynamic seasonal differences and/or sexual differences in the expression of some neuropeptides in the lumenal epithelial layer of the gonoducts.Furthermore, this also extends to the cross-ridges of the lumenal epithelium, with expression of some neuropeptides such as ArCT generally detected in both males and females, albeit with some seasonal variation in consistency of expression, and others such as ArPPLN1b and ArCRH only expressed in the crossridges in females, before (March) or after (May) spawning, respectively.Interpretation of immunostaining in the lumenal epithelial layer of the gonoduct is challenging because of the lack of information on gonoduct ultrastructure in A. rubens.Nevertheless, we infer that immunostained fibers located beneath the lumenal epithelium are likely to be of neuronal origin, consistent with the presence of axon-like structures in this position in the gonoducts of A. gibbosa (Bruslé, 1969).However, the identity of immunostained cells and/or processes located in the crossridges of the gonoducts remains to be determined, with possible cell types including neurons, neuron-like cells (e.g., juxtaligamental cells), or non-neuronal cells (e.g., endocrine-type cells).
As with neuropeptide expression in the coelomic epithelial layer, our findings from the lumenal epithelial layer and the associated crossridges invite functional explanations.It is noteworthy in this regard that the expression of the gonadotropic neuropeptide RGP is only detected in the lumenal epithelial layer of the gonoducts, and not in the coelomic epithelial layer (Feng et al., 2023).Furthermore, it is hypothesized that it is the release of RGP from the lumenal epithelial layer of the gonoducts that triggers spawning in starfish, with RGP presumed to diffuse into the lumen of the gonoduct and then into the lumen of the gonads (Feng et al., 2023), where it could bind to receptors located on follicle cells that surround oocytes in ovaries or interstitial cells associated with sperm in testes (Kubota et al., 1977;Mita, 2019).
Accordingly, we hypothesize that the expression of other neuropeptides in the lumenal epithelial layer of the gonoducts may likewise be associated with roles in the regulation of gonad growth and/or maturation, with neuropeptides gaining access to the gonad via the lumen of gonoducts and gonads.Furthermore, neuropeptide expression in the lumenal epithelial layer and cross-ridges of the gonoducts may also be associated with dynamic changes in the morphology of the gonoducts that occur before, during, and after spawning, and neuropeptide release could be either a cause and/or a consequence of changes in gonoduct morphology.However, clearly further experimental studies will be needed to test such hypotheses.

Physiological significance of neuropeptide expression and action in the gonads of A. rubens
As summarized in Figure 3, of the eight neuropeptides analyzed, only the expression of ArCRH and ArPPLN2h was detected in the gonads, with immunoreactivity localized in the coelomic epithelial layer.The expression of ArPPLN2h was detected in the coelomic epithelial layer of the gonads in both males and females at all four stages of the seasonal reproductive cycle and in all animals analyzed.In contrast, the expression of ArCRH in the gonad was found to be less consistent, with only one or two out of the three females analyzed exhibiting ArCRH-immunoreactivity in each of the four stages of the seasonal reproductive cycle.Furthermore, ArCRH-expression in the coelomic epithelial layer of the gonad was only observed in one or two out of three males analyzed during early (October) and mid (December) stages of gonad growth but not in any animals just prior to (March) or after (May) spawning.However, the functional significance of such sexdependent differences in ArCRH expression in the gonad remains to be determined.
Having established that ArPPLN2h is expressed in the coelomic epithelial layer of the gonad throughout the seasonal reproductive cycle, we sought to investigate the effects of this peptide on gonad function.Analysis of the ultrastructure of the gonads in A. rubens and in the closely related species A. amurensis has revealed that beneath the coelomic epithelium, there is a nerve plexus and a layer of muscle (Kalachev & Dyachuk, 2022;Walker, 1974).Accordingly, ArPPLN2himmunoreactivity can be seen in the sub-epithelial nerve plexus of the coelomic epithelial layer, and ArPPLN2h-immunoreactive cells in the coelomic epithelium of the gonad are therefore inferred to be neuronal cell bodies with axonal processes that contribute to this nerve plexus.
Informed by these observations, we hypothesized that ArPPLN2-type neuropeptides might affect the contractile state of muscle in the gonads.Previous studies have revealed that ArPPLN2h causes relaxation of in vitro preparations of the cardiac stomach of A. rubens, but this peptide has no effect on in vitro preparations of the tube feet and apical muscle of A. rubens (Lin et al., 2018).Therefore, we tested the effects of ArPPLN2h on in vitro preparations of gonads from both female (ovary) and male (testis) starfish.Importantly, and consistent with its effects on the cardiac stomach, ArPPLN2h caused relaxation of ovary and testis preparations, partially reversing the contracting effect of acetycholine.However, the relaxing effect of ArPPLN2h on ovary preparations was stronger than its effect on testis preparations.Having discovered that ArCRH is also expressed in the coelomic epithelial layer of the gonad in A. rubens, we also tested this peptide on in vitro preparations of ovaries and testes, but no effects on contractility were observed.However, it should be noted that when ArCRH was tested on in vitro preparations of other organs from A. rubens (cardiac stomach, tube feet, and apical muscle), it also had no effect on contractility (Cai et al., 2023).Therefore, ArCRH may not act as a myoregulatory substance in starfish, and instead it may have other physiological roles.
Although the expression of other neuropeptides (ArCT, ArPPLN1b, asterotocin, ArGnRH, ArSS1, and ArSS2) was not detected in the coelomic epithelial layer of the gonads, we nevertheless tested them to determine if they affect gonad contractility.No effects were observed with all but one of the neuropeptides tested.Thus, ArSS1 caused transient contraction of gonad preparations and, consistent with the bioactivity of ArPPLN2h as a relaxant, ArSS1 was more effective as a contractant of ovaries than as a contractant of testes.Furthermore, the myoexcitatory effect of ArSS1 on the gonads is consistent with its effects on other starfish organs because a previous study has revealed that ArSS1 also causes contraction of cardiac stomach, tube foot, and apical muscle preparations from A. rubens (Y.Zhang, Yañez Guerra, et al., 2022).How can the effect of ArSS1 on the gonads be explained, given that expression of ArSS1 was not detected in the gonads?As highlighted above, it may simply reflect a technical failure to detect the low-level expression of ArSS1 in the gonad wall with the affinitypurified antibodies generated previously (Y.Zhang, Yañnez Guerra, et al., 2022) and used in this study.Alternatively, it is possible that ArSS1 acts as a neurohormone in starfish and is released elsewhere in the body and gains access to the gonad wall via the coelomic fluid that surrounds the gonads.
What could be the physiological relevance of the pharmacological effects of ArPPLN2h and ArSS1 on gonad contractility in A. rubens?When spawning occurs in starfish, the expulsion of gametes from the gonads into the gonoducts and out of the gonopores into the surrounding seawater requires muscular contraction of the gonad wall.Previous studies have revealed that RGP-induced oocyte maturation and spawning in starfish is mediated via the production of 1-methyladenine by follicle cells that surround oocytes.Furthermore, 1-methyladenine causes contraction of starfish ovaries containing oocytes, but it does not cause the contraction of isolated ovarian walls.Thus, it is inferred that 1-methyladenine does not act directly on gonad muscle but acts indirectly via other molecules, including an egg-derived jelly substance (Shirai et al., 1981).Recently, evidence that the neurotransmitter acetylcholine mediates the effect of 1-methyladenine in triggering contraction of the gonad wall has been reported (Mita et al., 2020).However, there remains the possibility that other neurotransmitters/neuromodulators are involved in the control of gonad wall contraction during spawning in starfish.As ArSS1 causes the contraction of gonad preparations in vitro, it may also be involved in the mechanisms of neural and/or hormonal control of spawning in starfish.Furthermore, if rhythmic contractions are required for the expulsion of gametes from the gonads, alternating contraction and relaxation of the gonad wall may occur.Therefore, ArPPLN2-type neuropeptides may also be involved as myorelaxants in facilitating gamete expulsion during spawning.However, taking into account that the expression of ArPPLN2h is detected in the coelomic epithelial layer of the gonads throughout the seasonal reproductive cycle, it is possible that ArPPLN2-type neuropeptides are involved in maintaining the gonad wall in a relaxed state throughout the different stages of gonad growth.Thus, neuropeptide-induced relaxation of the gonad wall through effects on muscle and/or collagenous tissue may be needed to accommodate the growing number and volume of gametes in the lumen as the gonads grow.

Comparative physiology of neuropeptides as regulators of reproduction
It is clear from the findings of this study that the reproductive system of starfish is regulated by many neuropeptide types.In addition to the known role of RGP as a neuropeptide that triggers oocyte maturation and spawning, we have discovered other neuropeptides that affect gonad activity.In particular, we have identified a neuropeptide that causes gonadal contraction (ArSS1) and a neuropeptide that causes gonadal relaxation (ArPPLN2h) and we speculate, as discussed above, that these neuropeptides may be involved in the regulation of gonad contractility during spawning.Other neuropeptides with as yet unknown functions were also shown to be expressed in the gonoducts of A. rubens (ArCT, ArCRH, ArPPLN1b, and asterotocin).
Furthermore, given the large number of neuropeptides (>40) that have been identified in A. rubens (Semmens et al., 2016), it is likely that other neuropeptides that remain to be investigated are also expressed in the gonoducts and/or gonads.Consistent with this hypothesis, transcriptomic and mass spectroscopic analysis of the starfish Acanthaster planci has revealed a variety of neuropeptides that are expressed in the gonads, including orthologs of neuropeptides analyzed in this study (SS1-type, PPLN1-type, PPLN2-type, and GnRH-type), and others (e.g., kisspeptin-type, luqin-type, orexin-type, SALMFamide-type, and NGFFYamide) (Jonsson et al., 2022;Smith et al., 2017).
Currently, little is known about the roles of neuropeptides as regulators of reproductive processes in other echinoderms.Consistent with the role of RGP as a regulator of spawning in starfish, it has recently been reported that a relaxin-type neuropeptide also triggers headwaving behavior followed by spawning in mature individuals of the sea cucumber species Holothuria fuscogilva and Holothuria scabra (Chieu et al., 2019).Furthermore, it has been discovered that another neuropeptide type known as cubifrin, which is identical to the previously identified myotropic neuropeptide NGIWYamide (Inoue et al., 1999;Iwakoshi et al., 1995), triggers head-waving behavior, germinal vesicle breakdown, and spawning in the sea cucumber Apostichopus japonicus (Fujiwara et al., 2010;Kato et al., 2009).NGIWYamide and the related peptide NGIWFamide also trigger spawning in the sea cucumber H. scabra, and transcripts encoding the precursor of these peptides are detected in the wall of the ovary, suggesting that these peptides may be produced and released locally to trigger spawning (Chaiyamoon et al., 2020).Thus, in sea cucumbers, at least two types of neuropeptides (relaxin-type and NGIWYamide-type) appear to be involved in the regulation of spawning, and others may remain to be discovered.Clearly, there is scope for further investigation of neuropeptide expression and action in sea cucumbers and in other echinoderms, and the findings of this study provide a basis and a rationale for this.
While a detailed comparative analysis of neuropeptides as regulators of reproductive processes in other invertebrates is beyond the scope of this study, it is of interest here to highlight some examples relevant to the findings of this study.Thus, in accordance with the expression and/or action of pedal peptide-type neuropeptides (ArP-PLN1b, ArPPLN2h) in the A. rubens reproductive system, homologous orcokinin-type neuropeptides regulate reproductive processes in the insect Drosophila melanogaster, including effects on courtship behavior in males and egg production in females (Silva et al., 2021).Consistent with the expression of ArCRH in the A. rubens reproductive system, a CRH-related peptide known as egg-laying hormone induces spawning in the mollusk Aplysia californica (Chiu et al., 1979;DesGroseillers, 1990), and the CRH-related diuretic hormone DH44 controls sperm ejection and storage in D. melanogaster (Lee et al., 2015) and inhibits oocyte growth in the locust Schistocerca gregaria (Van Wielendaele et al., 2012).With regard to the expression of ArCT in the A. rubens reproductive system, it is noteworthy that the calcitoninrelated diuretic hormone DH31 regulates reproductive dormancy in D. melanogaster (Kurogi et al., 2023).In accordance with the expression and action of the allatostatin-C (ASTC)-type neuropeptide ArSS1 in the A. rubens reproductive system, ASTC is involved in the regulation of a variety of reproduction-associated processes in D. melanogaster, including roles in the generation of the circadian rhythm for oogenesis (C.Zhang et al., 2021), recovery from cold-induced reproductive dormancy (Meiselman et al., 2022), and control of vitellogenesis during reproductive maturation (C.Zhang, Kim, et al., 2022).Lastly, consistent with the expression of asterotocin in the A. rubens reproductive system, there is extensive evidence that vasopressin/oxytocin-type neuropep-tides regulate reproduction in a variety of invertebrates, including roles in the control of egg-laying in annelids (Oumi et al., 1996), sexual behavior in the hermaphrodite mollusk Lymnaea stagnalis (Van Kesteren et al., 1995), and mating behavior in the nematode Caenorhabditis elegans (Garrison et al., 2012).
In conclusion, the findings of this study combined with those of previous studies on a variety of protostome invertebrates, as discussed above, indicate that the evolutionary origin of a variety of neuropeptides as regulators of reproductive processes can be traced back to the urbilaterian common ancestor of deuterostomes and protostomes.
However, in accordance with the variety in structure and mechanisms of reproductive systems in invertebrates, specific reproductive roles of different neuropeptide types may vary between taxa.Furthermore, knowledge of the evolutionary conservation and diversification of neuropeptide function in reproduction will clearly require many more experimental studies on a variety of invertebrate taxa, including starfish and other echinoderms.

F
I G U R E 2 Histology of the gonoducts and gonads in A. rubens at different stages of seasonal gonad growth.Longitudinally orientated sections of gonoducts were stained using Masson's trichrome method (cytoplasm is red/pink, nuclei are dark purple/black, and connective tissue is blue).(a) Gonoduct and ovary at the early stage of gonad growth (October).(b) Gonoduct and ovary at the mid-stage of gonad growth (December).(c) Gonoduct and ovary at the late stage of gonad growth (March).(d) Gonoduct and ovary after spawning (May).(e) Gonoduct and testis at the early stage of gonad growth (October).(f) Gonoduct and testis at the mid-stage of gonad growth (December).(g) Gonoduct and testis at the late stage of gonad growth (March).(h) Gonoduct and testis after spawning (May) (i).High magnification image of right-side boxed region in (b), showing coelomic epithelial layer, collagenous tissue layer, and lumenal epithelial layer of the gonoduct in a female animal.(j) High magnification image of right-side boxed region in (c), showing the bases of the cross-ridges in the gonoduct of a female animal.(k) High magnification image of left-side boxed region in (c), showing how cross-ridges occlude the lumen of the gonoduct in a female animal.(l) High magnification image of left-side boxed region in (b), showing coelomic epithelium, germinal epithelium, and mature oocytes in the ovary.(m).High magnification image of lower boxed region in (g), showing coelomic epithelium and collagenous tissue layer of the gonoduct in a male animal.(n) High magnification image of left-side boxed region in (f), showing the collagenous tissue layer and bases of the cross-ridges in the gonoduct of a male animal.(o) High magnification image of right-side boxed region in (f), showing cross-ridges extending into the lumen of the gonoduct in a male animal.(p) High magnification image of upper boxed region in (g), showing coelomic epithelium, germinal epithelium, and mature sperm in the testis.BW, body wall; CE(Gd), coelomic epithelium of the gonad; CE(Gt), coelomic epithelium of the gonoduct; CR, cross-ridges; CT, collagenous tissue; GE, germinal epithelium; LE, lumenal epithelium of the gonoduct; LG, lumen of the gonoduct; MO, mature oocyte; MS, mature sperm; Ov, ovary; Te, testis.Scale bars: (a-h) = 500 µm; (i-p) = 25 µm.

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I G U R E 4 ArCT-immunoreactivity in the reproductive system of A. rubens, showing expression at two stages of the seasonal reproductive cycle.(a-d) Females at the mid-stage of gonad growth; (e-h) males at the mid-stage of gonad growth; (i-l) females after spawning; (m-p) males after spawning.(a) Immunostaining in the gonoduct.(b) High magnification image of the upper boxed region in (a), showing immunostaining in cells (blue arrowhead) located in the coelomic epithelial layer.(c) High magnification image of the lower boxed region in (a), showing immunostaining in cells located in the lumenal epithelial layer (orange arrowhead) and in cells located in the cross-ridges (gray arrowhead).(d) High magnification image of the boxed region in (c), showing immunostaining in cells located in the cross-ridges (gray arrowhead).(e) Immunostaining in the gonoduct.(f) High magnification image of the right-side boxed region in (e), showing immunostaining in cells (blue arrowhead) located in the coelomic epithelial layer.(g) High magnification image of the left-side boxed region in (e), showing immunostaining in cells located in the lumenal epithelial layer (orange arrowhead).(h) High magnification image of the boxed region in (g).(i) Immunostaining in the gonoduct.(j) High magnification image of the boxed region in (i), showing immunostaining in cells (blue arrowhead) located in the coelomic epithelium and in the underlying nerve plexus (blue arrow).Extensive immunostaining of cells and/or fibers can also be seen here in the lumenal epithelial layer (orange arrowhead) and in the cross-ridges (gray arrowhead) (k).The expanded lumen of the gonoduct can be seen here, with immunostaining in the lumenal epithelial layer extending to the gonopore.(l) High magnification image of the boxed region in (k), showing immunostained cells in the lumenal epithelial layer and in cellular material located within the gonopore.(m) Immunostaining in the gonoduct.(n) High magnification image of the boxed region in (m), showing immunostaining in the coelomic epithelial layer (blue arrowhead) and immunostained cells (orange arrowhead) and fibers (orange arrow) in the lumenal epithelial layer.Also note the absence of immunostaining in the cross-ridges.(o) Immunostaining in the lumenal epithelial layer of the gonoduct that extends to the gonopore.(p) High magnification image of the boxed region in (o), showing immunostaining in the lumenal epithelial layer (orange arrowheads) and in tissue that forms the lining of the gonopore.BW, body wall; CE(Gt), coelomic epithelium of the gonoduct; CR, cross-ridges; CT, collagenous tissue; GP, gonopore; LE, lumenal epithelium of the gonoduct; LG, lumen of the gonoduct; Ov, ovary; Te, testis.Scale bars: (e), (i), (m), (k), and (o) = 500 µm.(a) Scale bar = 250 µm.(c), (g), (p), and (j) = 50 µm.(b), (d), (f), (h), (n), and (l) = 25 µm.F I G U R E 5 ArPPLN1b-immunoreactivity in the reproductive system of A. rubens, showing expression at two stages of the seasonal reproductive cycle.(a-d) Females at the late stage of gonad growth; (e-h) males at the late stage of gonad growth; (i-l) females after spawning; (m-p) males after spawning.(a) Immunostaining in the gonoduct.(b) High magnification image of the upper boxed region in (a), showing immunostaining in cells/fibers (blue arrowhead) located in the coelomic epithelial layer and in cells (orange arrowhead) and fibers (orange arrow) in the lumenal epithelial layer.(c) High magnification image of the lower boxed region in (a), showing immunostaining in cells located in the lumenal epithelial layer (orange arrowhead) and in cells located in the cross-ridges (gray arrowhead).(d) High magnification image of the boxed region in (c), showing stained cells (gray arrowheads) in the cross-ridges.(e) Immunostaining in the gonoduct.(f) High magnification image of the upper boxed region in (e), showing immunostaining in cells (blue arrowheads) located in the coelomic epithelium.(g) High magnification image of the lower boxed region in (e), showing immunostaining in cells (orange arrowhead) located in the lumenal epithelial layer.(h) High magnification image of the middle boxed region in (e), showing immunostained cells in the lumenal epithelium (orange arrowheads).(i) Immunostaining in the gonoduct.(j) High magnification image of the lower boxed region in (i), showing immunostaining in cells (blue arrowhead) located in the coelomic epithelium.Immunostaining can also be seen here in the lumenal epithelial layer, including stained cells (orange arrowhead).(k) High magnification image of the boxed region in (j), showing immunostained cells (arrowhead) in the coelomic epithelium (l).High magnification image of the upper boxed region in (i), showing immunostaining in the lumenal epithelial layer, including stained cells (orange arrowheads); note that the cross-ridges are unstained.(m) Immunostaining in the gonoduct.(n) High magnification image of the upper boxed region in (m), showing immunostained cells (blue arrowheads) and fibers (blue arrow) in the coelomic epithelial layer.(o) High magnification image of the lower boxed region in (m), showing immunostaining in the coelomic epithelial layer.Also note the absence of staining in the cross-ridges.(p) High magnification image of the boxed region in (o), showing immunostained cells (blue arrowhead) and fibers (blue arrow) in the coelomic epithelial layer.BW, body wall; CE(Gt), coelomic epithelium of the gonoduct; CR, cross-ridges; CT, collagenous tissue; LE, lumenal epithelium of the gonoduct; LG, lumen of the gonoduct; Ov, ovary; Te, testis.Scale bars: (a), (e), (i), and (m) = 500 µm.(b), (c), (j), (l), and (o) = 50 µm.(d), (f), (g), (h), (k), (n), and (p) = 25 µm.F I G U R E 6 ArCRH-immunoreactivity in the reproductive system of A. rubens, showing expression at two stages of the seasonal reproductive cycle.(a-d) Females at the mid-stage of gonad growth; (e-h) males at the mid-stage of gonad growth; (i-l) females after spawning; (m-p) males after spawning.(a) Immunostaining in the gonoduct.(b) High magnification image of the boxed region in (a), showing immunostaining in cells (blue arrowhead) located in the coelomic epithelium.(c) Immunostaining in the gonad.(d) High magnification image of the boxed region in (c), showing immunostaining in cells (yellow arrowhead) and fibers (yellow arrow) in the coelomic epithelial layer.(e) Immunostaining in the gonoduct.(f) High magnification image of the boxed region in (e), showing immunostaining in cells (blue arrowhead) located in the coelomic epithelial layer.(g) Immunostaining in the gonad, localized in the coelomic epithelial layer.(h) High magnification image of the boxed region in (g), showing immunostaining in cells (yellow arrowheads) in the coelomic epithelium.(i) Immunostaining in the gonoduct.(j) High magnification image of the middle boxed region in (i), showing immunostained cells (blue arrowhead) and fibers (blue arrow) in the coelomic epithelial layer of the gonoduct.(k) High magnification image of the left boxed region in (i), showing immunostaining in cells (orange arrowhead) and fibers (orange arrows) in the lumenal epithelial layer and in cells (gray arrows) in the cross-ridges.(l) High magnification image of the right boxed region in (i), showing immunostained cells (yellow arrowheads) in the coelomic epithelial layer of the gonad (ovary).(m) Immunostaining in the gonoduct.(n) High magnification image of the boxed region in (m), showing immunostaining in the coelomic epithelial layer.Note the absence of staining the cross-ridges (o).High magnification image of the left boxed region in (n), showing immunostained cells (blue arrowheads) in the coelomic epithelial layer.(p) High magnification image of the right boxed region in (n), showing the absence of immunostaining in the coelomic epithelial layer of the gonad, contrasting with immunostaining seen in (l).BW, body wall; CE(Gd), coelomic epithelium of the gonad; CE(Gt), coelomic epithelium of the gonoduct; CR, cross-ridges; CT, collagenous tissue; LE, lumenal epithelium of the gonoduct; LG, lumen of the gonoduct; Ov, ovary; Te, testis.Scale bars: (a), (e), (i), and (m) = 500 µm; (c) = 250 µm; (n) = 100 µm; (g), (j) = 50 µm.(b), (f), (d), (h), (k), (l), (o), and (p) = 25 µm.F I G U R E 7 ArPPLN2h-immunoreactivity in the reproductive system of A. rubens, showing expression at two stages of the seasonal reproductive cycle.(a-c) Females at the early stage of gonad growth; (d-f) males at the early stage of gonad growth; (g-j) females after spawning; (k-m) males after spawning.(a) Immunostaining in the gonad (ovary).(b) High magnification image of the lower boxed region in (a), showing immunostaining in cells (yellow arrowhead) and fibers (yellow arrow) in the coelomic epithelial layer in a region of the gonad (ovary) proximal to the gonoduct.(c) High magnification image of the right boxed region in (a), showing immunostaining in cells (yellow arrowhead) in the coelomic epithelial layer of the gonad (ovary).(d) Immunostaining in the gonoduct and gonad (testis).(e) High magnification image of the upper boxed region in (d), showing immunostained cells in the coelomic epithelial layer of the gonoduct (blue arrowhead) and testis (yellow arrowhead).(f) High magnification image of the lower boxed region in (d), showing immunostained cells (yellow arrowhead) and fibers (yellow arrow) in the coelomic epithelial layer of the testis.(g) Immunostaining in the gonoduct and proximal region of the gonad (ovary).(h) Immunostaining in the gonad (ovary).(i) High magnification image of the boxed region in (g), showing immunostaining in cells (blue arrowhead) and fibers (blue arrow) in the coelomic epithelial layer of the gonoduct.(j) High magnification image of the boxed region in (h), showing immunostained cells (yellow arrowheads) in the coelomic epithelial layer of the ovary.(k) Note here the absence of immunostaining in the gonoduct.(l) Immunostaining in the gonad (testis), localized in the coelomic epithelial layer.(m) High magnification image of the boxed region in (l), showing immunostained cells (yellow arrowhead) in the coelomic epithelial layer of the gonad (testis).BW, body wall; CE(Gd), coelomic epithelium of the gonad; CE(Gt), coelomic epithelium of the gonoduct; CR, cross-ridges; CT, collagenous tissue; Ov, ovary; Te, testis.Scale bars: (a), (d), (g), (k), and (h) = 500 µm.(l) = 250 µm.(b), (c), (e), (f), (i), (j), and (m) = 25 µm.

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I G U R E 8 Asterotocin-immunoreactivity in the reproductive system of A. rubens, showing expression at two stages of the seasonal reproductive cycle.(a and b) Females at the early stage of gonad growth; (c and d) males at the early stage of gonad growth; (e and f) females at the late stage of gonad growth; (g and h) males at the late stage of gonad growth.(a) Immunostaining in the gonoduct.(b) High magnification image of the boxed region in (a), showing immunostaining in cells (blue arrowhead) located in the coelomic epithelial layer of the gonoduct.(c) Immunostaining in the coelomic epithelial layer of the gonoduct.(d) High magnification image of the boxed region in (c), showing immunostaining in cells (blue arrowhead) located in the coelomic epithelial layer of the gonoduct.(e) Immunostaining in the gonoduct.(f) High magnification image of the boxed region in (e), showing immunostaining in cells (blue arrowhead) located in the coelomic epithelial layer.(g) No immunostaining is present in the gonoduct, contrasting with (a-f).(h) High magnification image of the boxed region in (g).CR, cross-ridges; CT, collagenous tissue; Ov, ovary; Te, testis.Scale bars: (g) = 500 µm, (e) = 250 µm, (a) = 100 µm, (c) = 50 µm, (b), (d), (f), and (h) = 25 µm.each neuropeptide tested) (Figure S2).However, ArSS1 and ArPPLN2h caused contraction and relaxation, respectively, of gonad preparations (Figures 9 and 10).
The allatostatin-C type neuropeptide ArSS1 causes contraction of gonad preparations from A. rubens.(a) ArSS1 (10 −6 M) induces transient contraction of an ovary preparation and after washing with artificial sea water (ASW).The contracting effect of acetylcholine (ACh; 10 −6 M) can be observed for comparison.(b) ArSS1 (10 −6 M) induces transient contraction of a testis preparation and after washing with ASW.The contracting effect of ACh (10 −6 M) can be observed for comparison.(c) Graph comparing mean (±SEM) contracting effect of ArSS1 (10 −6 M) relative to the effect of ACh (10 −6 M) on gonads from female starfish (ovary; n = 9) and male starfish (testis; n = 7).The magnitude of the effects ArSS1 on ovaries and testes is significantly different (p ≤ .001;t-test).