Invertebrate Gonadotropin-Releasing Hormone Receptor Signaling and Its Relevant Biological Actions

Gonadotropin-releasing hormones (GnRHs) play pivotal roles in reproduction via the hypothalamus-pituitary-gonad axis (HPG axis) in vertebrates. GnRHs and their receptors (GnRHRs) are also conserved in invertebrates lacking the HPG axis, indicating that invertebrate GnRHs do not serve as “gonadotropin-releasing factors” but, rather, function as neuropeptides that directly regulate target tissues. All vertebrate and urochordate GnRHs comprise 10 amino acids, whereas amphioxus, echinoderm, and protostome GnRH-like peptides are 11- or 12-residue peptides. Intracellular calcium mobilization is the major second messenger for GnRH signaling in cephalochordates, echinoderms, and protostomes, while urochordate GnRHRs also stimulate cAMP production pathways. Moreover, the ligand-specific modulation of signal transduction via heterodimerization between GnRHR paralogs indicates species-specific evolution in Ciona intestinalis. The characterization of authentic or putative invertebrate GnRHRs in various tissues and their in vitro and in vivo activities indicate that invertebrate GnRHs are responsible for the regulation of both reproductive and nonreproductive functions. In this review, we examine our current understanding of and perspectives on the primary sequences, tissue distribution of mRNA expression, signal transduction, and biological functions of invertebrate GnRHs and their receptors.

Over the past quarter-century, GnRH-GnRHR signaling systems have also been identified by molecular and in silico cloning or transcriptome analysis in invertebrate deuterostomes and protosomes: urochordates, cephalochordates, echinoderms, crustaceans, mollusks, and annelids (Table 1). These species lack a hypothalamus and pituitary. Absence of the HPG axis and gonadotropins in invertebrates indicates that biological functions of GnRHergic signaling in these species do not include the release of gonadotropins. The evolutionary relationships of the GnRH-related peptides corazonins (CRZs) and adipokinetic hormones (AKHs) have been identified, and the cognate receptors of these structurally related peptides have been documented based on GnRHR, CRZ receptor, and AKH receptor molecular phylogenetic trees [9][10][11][12][13]. There is a recent growing body of reports describing GnRH-GnRHR signaling and the resultant biological effects in invertebrates. These insights will aid investigations into the biological roles of GnRHs in invertebrates and into evolutionary aspects of GnRH-related peptides.
In this review, we summarize our current understanding of the primary sequences, tissue distribution, signaling cascades, and biological functions of invertebrate GnRHs and their receptors.
Recently, the precise microscopic observation of the nervous system of transgenic adult C. intestinalis, which expresses the Kaede reporter gene driven by a protein convertase 2 gene promoter, suggested that neuropeptides produced in the cerebral ganglion are secreted to the ovary via neurons of the dorsal strand plexus and/or visceral nerves and regulate the gonads [20]. This observation is consistent with reports on the distribution of GnRH immunoreactive neurons of the dorsal strand plexus from the cerebral ganglion to the gonads along the dorsal strand of C. intestinalis [17,21].
The induction of calcium mobilization by tGnRH-6 via Ci-GnRHR-1, expressed in HEK293MSR cells, was shown to lead to the translocation of a typical calcium ion-dependent protein kinase, PKCα, from the cytoplasm to the plasma membrane. This is a canonical activation process of PKC [25]. The tGnRH-6-Ci-GnRHR-1 interaction was also found to enhance the translocation of a calcium ion-independent PKC, PKCζ [25], indicating that the tGnRH-6-Ci-GnRHR-1 interaction activates different signaling pathways ( Figure 1). Furthermore, tGnRH-5 was shown to enhance the translocation of the calcium ion-independent PKC, PKCζ but not PKCα, although tGnRH-5 fails to elevate the intracellular calcium or cAMP production [25]. We also demonstrated that the activation of both PKCα and PKCζ triggered a canonical signaling process-the phosphorylation of ERK1/2, which belongs to the MAPK family [25]. In combination, tGnRH-5 and -6 regulate multiple signaling cascades via calcium ion-dependent and independent pathways, leading to the phosphorylation of ERK1/2 ( Figure 1). Heterodimerization between Ci-GnRHR-1 and -4 results in a 10-fold more potent intracellular calcium ion mobilization response to tGnRH-6 compared with the Ci-GnRHR-1 monomer/homodimer. Heterodimerization between Ci-GnRHR-2 and -4 decreases cAMP production by 50% in response to tGnRH-6, 7, and 8 compared with the Ci-GnRHR-1 monomer/homodimer.

Int
The induction of calcium mobilization by tGnRH-6 via Ci-GnRHR-1, expressed in HEK293MSR cells, was shown to lead to the translocation of a typical calcium ion-dependent protein kinase, PKCα, from the cytoplasm to the plasma membrane. This is a canonical activation process of PKC [25]. The tGnRH-6-Ci-GnRHR-1 interaction was also found to enhance the translocation of a calcium ion-independent PKC, PKCζ [25], indicating that the tGnRH-6-Ci-GnRHR-1 interaction activates different signaling pathways ( Figure 1). Furthermore, tGnRH-5 was shown to enhance the translocation of the calcium ion-independent PKC, PKCζ but not PKCα, although tGnRH-5 fails to elevate the intracellular calcium or cAMP production [25]. We also demonstrated that the activation of both PKCα and PKCζ triggered a canonical signaling process-the phosphorylation of ERK1/2, which belongs to the MAPK family [25]. In combination, tGnRH-5 and -6 regulate multiple signaling cascades via calcium ion-dependent and independent pathways, leading to the phosphorylation of ERK1/2 ( Figure 1).
These findings verify that Ci-GnRHR-4 serves as a protomer of Ci-GnRHR heterodimers rather than as a ligand-unidentified orphan GPCR (Figure 2) [7,15,27]. Taken together, these findings indicate urochordate-specific molecular and functional diversity in GnRH signaling systems.

Reproductive Effects
Ascidians are not endowed with a hypothalamus or a pituitary in the central nervous system, or with gonadotropin genes, and thus, tGnRHs cannot serve as "gonadotropin-releasing hormones" in the HPG axis but, rather, function as neuropeptides that directly regulate target tissues. Tissue distribution of the gnrh genes Ci-gnrh-1 and -2 and their receptor genes Ci-gnrhr-1 to -4 in adult C. intestinalis strongly support this notion. RT-PCR analysis of total RNA extracted from the neural complex, testis, ovary, heart, and hepatic organ of adult C. intestinalis, and in situ hybridization, revealed that the two gnrh genes are exclusively expressed in the neural complex, especially in the cerebral ganglion [28]. In contrast, the four gnrhr genes are expressed in various tissues, such as the neural complex, gonad, heart, intestine, endostyle, and branchial sac in adult Ciona [23]. These gene expression profiles of tGnRHs and Ci-GnRHRs suggest that urochordate GnRH signaling regulates reproductive and nonreproductive activities. Notably, several studies demonstrated that the administration of tGnRHs to adult urochordates influences the reproductive activities of the ascidian. In C. intestinalis, tGnRHs were found to increase the water flow by body contraction and then induce the release of eggs and sperm by the injection of water into the gonaducts, ovary, stomach, and posterior body cavity [17,21]. Furthermore, the colocalization of Ci-GnRHR-4 and Ci-GnRHR-1 or R-2 was detected exclusively in test cells inside the inner follicles of oocytes in the vitellogenic follicle, indicating the involvement of GnRH signaling in oocyte growth via GPCR heterodimerization [25][26][27].

Nonreproductive Effects
Expression of the GnRH genes Ci-gnrh-1 encoding tGnRH-3, 5, and 6; Ci-gnrh-2 encoding tGnRH-4, 7, and 8; and four GnRH receptor genes have been reported in the nonreproductive stage of C. intestinalis [17,24,29,30]. Ci-gnrh1 and Ci-gnrh2 are expressed at the four-cell stage and gastrulation, tailbud, and tail absorption stages of larvae during development [17]. Furthermore, these genes are expressed in the motor ganglion and central nervous systems of larvae, including in papillar neurons, and ascidian metamorphosis is triggered upon adhesion to a substrate [24,30]. All four Ci-gnrhr genes are also expressed in the central nervous system of the larva [24,30]. Moreover, Ci-gnrhr-1 and -2 are expressed in the adhesive papillae and tail muscle, whereas Ci-gnrhr-3 is expressed in notochord cells in the larval tail, which is rapidly absorbed during metamorphosis [24]. Such a distribution of gene expression suggests that GnRH signaling regulates the sensory reception, neuronal processing, and muscle functions in Ciona larvae [24]. Importantly, our group demonstrated that tGnRH-3 and -5 induce tail absorption and inhibit the growth of adult organs in the larval trunk by arresting cell cycle progression [29]. These findings indicate that tGnRHs play a pivotal role in metamorphosis. Intriguingly, expression of the Ci-GnRH-X gene was detected specifically in the larva, as well as in the adult neural complex [19]. The specific interactions of tGnRH-3 and -5 with Ci-GnRHR-3, and a partial inhibitory effect of Ci-GnRH-X on Ci-GnRHR-3, suggested that the antagonistic effect of Ci-GnRH-X on Ci-GnRHR-3 is likely indicative of a role in the metamorphosis of the larva. Quite recently, gene functional analyses and pharmacological assays demonstrated that gamma-butyric acid (GABA) induces the secretion of GnRH through the metabotropic GABA receptor to initiate Ciona metamorphosis [30]. Ci-gnrh2-knockout larvae produced by genome editing exhibited failed tail regression, while Ci-gnrh1-knockout counterparts exhibited a moderate reduction of tail regression. This study indicated that the GABA-GnRH axis participates in postembryonic development in ascidians [30].

Cephalochordate GnRHs
A hidden Markov model for a variety of vertebrate and invertebrate GnRH, AKH, and CRZ propeptides in the cephalochordate amphioxus Branchiostoma floridae identified two putative GnRH-like peptides: amphioxus GnRHv (pQEHWQYGHWYa, Table 1) and the invertebrate GnRH-and CRZ-like peptide amphioxus GnRH-like peptide (pQILCARAFTYTHTWa, Table 1) [31]. Synteny analysis of the amphioxus GnRH-like peptides with human GnRH paralogons and limpet AKH indicated the lineage of the ancestral-type GnRH superfamily peptides and its evolutionary relevance to vertebrate GnRHs [31].

Cephalochordate Receptors
Four putative GnRHRs have been cloned from the amphioxus B. floridae [32]. Two putative GnRH-like peptides and several peptides were tested for bioactivity in vitro with the four receptors. Amphioxus GnRHR-1 and -2, expressed in COS-7 cells, were activated by vertebrate GnRH1 (pQHWSYGLRPGa) but not by Amph.GnRHv and Amph.GnRH-like peptides. Amphioxus GnRHR-3 was activated exclusively by the Amph.GnRH-like peptide oct-GnRH (pQNYHFSNGWPGa) and by silkworm AKH (pQLTFTSSWa) at physiological concentrations [31,32]. In contrast, no ligands induced IP 3 accumulation or cAMP stimulation via amphioxus GnRHR-4 [31,32]. This functional assay indicated that Amph.GnRHv, which has a higher sequence similarity to other species' GnRHs than the AmphGnRH-like peptide, does not interact with any hereto known putative amphioxus GnRHRs [31]. The response of amphioxus GnRHR-4 remains to be investigated, but mismatches between putative amphioxus GnRHRs and cultured cells used for heterologous functional analysis may cause unsuccessful functional expression of the receptor mRNA or degradation of the receptor protein in heterologous expression systems, as seen with other invertebrate GPCRs [33]. Molecular phylogenetic analysis demonstrated that amphioxus GnRHR-1 and -2 are members of the vertebrate GnRHR clade, while amphioxus GnRHR-3 and -4 belong to the CRZ receptor/invertebrate GnRHR clade. Collectively, the sequences of Amph.GnRH-like peptides might reflect the ancestral sequence of CRZ/GnRH or the transition state between CRZ and GnRH [31].
To our knowledge, the detailed tissue distribution of amphioxus GnRHs and their receptors has not been published. Thus, the biological functions of GnRHergic signaling in the amphioxus remain unclear. The identification of authentic ligands for amphioxus GnRHR-1 and -2 and the tissue distribution of amphioxus GnRH and receptor pairs await further investigation.

Echinoderm GnRHs
To date, two echinoderm GnRH peptides have been identified. The GnRH-like peptide SpGnRHP (Table 1) was identified in the sea urchin Strongylocentrotus purpuratus as the first echinoderm GnRH-related peptide [34], followed by the identification of ArGnRH (Table 1) in the starfish Asterias rubens [35]. SpGnRHP and ArGnRH comprise 12 amino acids, with Val 2 -His 3 and Ile 2 -His 3 inserted in the N-terminus of these peptides, respectively (Table 1). Furthermore, the echinoderm GnRHs share several amino acids with urochordate and vertebrate GnRHs and protostome GnRH-like peptides (see Section 4.2), including the N-terminal pGlu, His 4 (corresponding to His 2 in chordate GnRHs), Gly 8 (corresponding to Gly 6 in vertebrate GnRHs), Trp 9 (corresponding to Trp 7 in vertebrate GnRHs), and C-terminal Pro-Gly-amide (Table 1). SpGnRHP and ArGnRH were recently shown to have weak sequence similarity with CRZ (pQTF(Q/H)YS(R/Q)GW(T/Q)N-amide), an arthropod neuropeptide [9][10][11]. Consequently, it has been presumed that echinoderm GnRHs are included in the GnRH/CRZ family [9][10][11]. In addition, ArGnRH was shown to bind to copper (II) ion and nickel (II) ion with much higher affinity compared with human GnRH [36]. Although the physiological significance of this high-affinity binding remains to be investigated, the N-terminus of ArGnRH forms an ATCUN motif copper binding site, in which the third histidine is responsible for the high-affinity binding of copper and nickel [36].

Echinoderm GnRH Receptors
Genome surveys suggested that four GnRH/CRZ-type receptors are encoded in the sea urchin S. purpuratus [9], although the functions of these receptors remain to be investigated. These four receptors belong to a separate cluster in the molecular phylogenetic tree of GnRH receptors and CRZ receptors, indicating that these receptors were generated in the sea urchin lineage [9]. The cognate receptor for ArGnRH, ArGnRHR, was identified in the starfish A. rubens [35], and ArGnRH was shown to specifically activate intracellular calcium mobilization via ArGnRHR expressed in CHO-K1 cells [35]. Molecular phylogenetic tree analysis demonstrated that ArGnRHR forms a cluster with the aforementioned four sea urchin putative GnRHRs, suggesting that echinoderm GnRHRs were generated in common ancestors of echinoderms [9]. In addition, ligand-receptor signaling for CRZ was also identified in A. rubens [35]. In combination, these findings indicate that GnRH-type and CRZ-type signaling pathways function in A. rubens [10,11] and represent the first functional characterization of GnRH-type and CRZ-type signaling pathways in an organism.

Biological Effects of Echinoderm GnRH
Of the echinoderm GnRHs identified to date, only the biological effects of ArGnRH have been characterized. ArGnRH was shown to stimulate the contraction of cardiac, stomach, and apical muscles and tube foot preparations in vitro [37], suggesting that ArGnRH participates in regulating locomotion and food digestion. In contrast, no reproductive functions of this neuropeptide have been examined. Investigation of the tissue distribution of ArGnRHR and ArGnRHergic innervation would provide insight into the reproductive roles of ArGnRH.

Protostome GnRHs
GnRH-like peptides have been identified over the past fifteen years in protostomes, including mollusks, annelids, and crustaceans ( Table 1). The first protostome GnRH, oct-GnRH, was isolated from the central nervous system of the octopus Octopus vulgaris [38], followed by the identification of oct-GnRH in other cephalopods such as cuttlefish Sepia officinalis and swordtip squid Loligo edulis [7,9,39]. Oct-GnRH, like echinoderm GnRHs, is composed of 12 amino acids and conserves amino acids corresponding to pyro-Glu, His 2 , Ser 4 , Gly 6 , Pro 9 , and C-terminally amidated Gly 10 in typical vertebrate GnRHs and tunicate t-GnRH6, -10, and -11 (Table 1) This amino acid sequence identity demonstrates that oct-GnRH shares more common amino acids with vertebrate and tunicate GnRHs compared with the echinoderm GnRHs SpGnRHP and ArGnRH. Oct-GnRH-like peptides [9,[40][41][42][43][44] have also been identified in other protostomes, including bivalves, gastropods, annelids, and crustaceans (Table 1). In contrast, GnRH-like peptides have not been identified in insects or nematodes, although CRZs and AKHs, which are believed to have originated from common ancestors of GnRHs, are found in these organisms [7,9,11]. Unlike deuterostome and cephalopod GnRHs, these non-cephalopod protostome GnRHs, except Has-GnRH [40], comprise 11 amino acids and lack the C-terminal Gly-amide (Table 1). However, most protostome GnRHs also conserve amino acid residues corresponding to pyro-Glu, His 2 , Ser 4 , Gly 6 , Trp 7 , Pro 9 , and C-terminal amidation in vertebrate GnRHs and tunicate tGnRH6, -10, and -11 (Table 1). Such sequence identity and diversity suggest that common ancestral GnRHs might have harbored C-terminal Pro-Gly-amide, which is conserved in cephalopods and echinoderms but was lost during the evolutionary processes of protostomes. Consistent with this, the sequence diversity of GnRH showed that ancestral GnRHs might have comprised pQ-H(F/W)S-GW-PGa or pQ-H(F/W)S-GW-a. Subsequent vertebrate and tunicate GnRHs could have arisen via deletion of the two N-terminal amino acids and several substitutions of amino acids during the evolutionary process of each species.

Protostome GnRH Receptors
As shown in Table 1, putative protostome GnRHR sequences were detected in several species [9,11,43,45,46], but only three protostome GnRHRs have been shown to interact with the cognate GnRH ligands. The first protostome GnRHR identified was oct-GnRHR in the octopus O. vulgaris [47]. Oct-GnRHR expressed in Xenopus oocyte activates intracellular calcium mobilization in response to oct-GnRH but not to vertebrate GnRHs. Moreover, an oct-GnRH synthetic analog lacking Asn 2 -Tyr 3 failed to activate calcium mobilization via oct-GnRHR, whereas a chicken GnRH-II synthetic analog with an Asn-Tyr insertion after position 1 exhibited weak activity [47]. These findings revealed that Asn 2 -Tyr 3 is a requisite for signaling by oct-GnRHR, suggesting that the two amino acids after position 1 in non-chordate GnRHs are responsible for activating protostome GnRHRs. In the gastropod sea hare (Aplysia californica), ap-GnRH was shown to increase IP 3 accumulation but not cAMP production in ap-GnRHR-expressing Drosophila S2 cells [48]. Recently, my-invGnRHR was identified in the bivalve yesso scallop (Mizuhopecten (Patinopecten) yessoensis) [49]. This receptor, transfected into HEK293MSR cells, was shown to trigger intracellular calcium mobilization, but not cAMP production, in response to the cognate ligand py-GnRH [49]. Molecular phylogenetic trees demonstrated that these molluscan GnRHRs are clustered in a separate clade and suggested that protostome GnRHRs might have originated from common ancestors of deuterostome GnRHRs, CRZ receptors, and AKH receptors [9,11,49]. Taken together, these findings support the evolutionary view that the original GnRHRs (or their common ancestors) might have only been capable of activating the PLC-IP 3 -intracellular calcium mobilization signaling cascade, whereas ascidian and vertebrate GnRHRs both conserved the PLC-IP 3 -intracellular calcium mobilization signaling cascade and acquired cAMP production signaling during their evolutionary processes.

Biological Effects of Protostome GnRHs
In vitro experiments showed that oct-GnRH stimulates the contractions of the heart and oviduct, and secretion of testosterone-, progesterone-, and 17β-estradiol-immunoreactive steroids, from the follicle and spermatozoa in O. vulgaris [47,50]. These effects are compatible with the expression of oct-gnrhr in the heart, oviduct, testis, and ovary [47,50]. The localization of oct-gnrhr in these organs will stimulate investigation of the underlying molecular mechanisms. Furthermore, expression in the salivary gland, branchia, and radula retractor muscle [47] suggest nonreproductive roles of oct-GnRHs.
The injection of ap-GnRH into sexually mature and immature sea hare (A. californica) for 10-14 days resulted in stimulation of the parapodial opening, inhibition of feeding, and promotion of substrate attachment but no reproduction-related functions. Specifically, there was no alteration in ovotestis mass, reproductive tract mass, egg-laying, penile eversion, oocyte growth, or egg-laying hormone accumulation and secretion [51]. These biological effects are at least partially in accordance with the expression of ap-GnRHR in the abdominal, cerebral, and buccal ganglia of the central nervous system, chemosensory organ, small hermaphroditic duct, and ovotestis [48,52]. Consequently, these findings suggest that ap-GnRH is not involved in reproductive functions in the sea hare, although the possibility cannot be excluded that the ap-GnRH injection experiment failed to reproduce in vivo functions in the genital organs.
In another gastropod, the abalone Haliotis asinina, treatment with 250 and 500-ng/g body weight (BW) synthetic Has-GnRH and buserelin (a synthetic mammalian GnRH analog) in vitro and in vivo enhanced oogonia and oocyte proliferation in one-year-old female H. asinina [40]. In addition, the injection of 1000-ng/g BW Has-GnRH and buserelin was less effective in stimulating oocyte cell proliferation [40], suggesting its involvement in feedback-like systems in abalone. Quite recently, Sharker et al. [53] verified the in vivo effects of Hdh-GnRH1 and -2 in the abalone Haliotis discus hannai. The injection of 250 and 500 ng/g BW of Hdh-GnRH1, -2, and buserelin resulted in the enhancement of spermatogonial cell and oogonial cell proliferation. Moreover, the expression of GnRH, GnRHR, and the serotonin receptor in the cognate male and female gonads were shown to be upregulated by the treatment with 250-ng/g BW Hdh-GnRH and buserelin [53]. These biological effects are compatible with the expression of the putative cognate receptor Hdh-GnRHR [46]. Similar to Has-GnRH in Haliotis asinine [40], the injection of 1000-ng/g BW Hdh-GnRH and buserelin was less effective than the aforementioned gonadal cell proliferation and gene expression effects in Haliotis discus hannai [53].
In the bivalve Mizuhopecten (Patinopecten) yessoensis, py-GnRH was found to enhance spermatogonial cell division in cultured testis [54]. Furthermore, a six-week administration of py-GnRH to yesso scallops using an original slow-release delivery system resulted in the acceleration of spermatogenesis and suppression of oocyte growth [55]. These biological functions are compatible with the high-level expression of the cognate receptor my-invGnRHR in the gonads during maturation [56]. Intriguingly, py-GnRH is highly likely to regulate masculinization. The administration of py-GnRH for six weeks to the scallop gonads altered the sex ratio (male:female = 1:1) to 100% male scallops by stimulating the generation of unnatural hermaphrodites (8.3% in week two and 33.3% in week four), whereas the sex ratio of the untreated group was unaffected [55]. These findings strongly suggest that py-GnRH is responsible for sexual differentiation or male gonad maturation. In particular, an enhancement of spermatogonial cells has been observed in mollusks but not in vertebrates, suggesting that this biological role is specific to mollusks or possibly other phyla of invertebrates. In combination, molluscan GnRHs play various unique roles in both reproductive and nonreproductive systems, with abalone and scallop GnRHs likely playing pivotal common roles in the proliferation and development of male germ cells. Exploration of the biological roles of GnRHs in various species will aid in verifying the functional evolutionary origin of ancestral GnRHs.

Conclusions and Perspectives
As stated above, a number of GnRH-GnRHR-signaling cascades remain to be investigated in invertebrates, despite a growing body of reports on the molecular cloning of invertebrate GnRHs and GnRHRs via omics analyses and in silico homology-searching. Nevertheless, the hitherto-elucidated GnRH-GnRHR signaling pathways in urochordates, echinoderms, and mollusks confirm that intracellular calcium mobilization is a major second messenger. In other words, the GnRHergic PLC-IP 3 intracellular calcium ion pathway is conserved in invertebrates. This is in agreement with the view that GnRH-GnRHR signaling and CRZ-CRZ receptor signaling might have originated from common ancestors, given that, in addition to the moderate sequence similarity of CRZs and CRZ receptors to GnRHs and GnRHRs, respectively, intracellular calcium ion is also the second messenger in CRZ-CRZ receptor signaling pathways [7,[9][10][11]. On the other hand, the functional characterization of GnRHRs has revealed that intracellular calcium mobilization is the major signaling pathway for GnRHRs in cephalochordates, echinoderms, and protostomes, whereas all Ciona GnRHR1 to -3 regulate the cAMP production pathways [9,12,13]. Notably, multiple GnRHs and GnRHRs have emerged in cephalochordates, urochordates, and vertebrates, whereas only a single GnRH and GnRHR have been identified in each echinoderm or protostome species (Table 1). Moreover, neither Gs nor Gi-coupled signaling has been detected in invertebrate GnRH-GnRHR signaling pathways, except for Ciona paralogs (see Section 1). These findings suggest that some invertebrate GnRHRs may also participate in the signaling of cAMP regulation. Elucidation of the functional interactions between more GnRHs and the cognate receptors in a wide range of invertebrate species will likely provide crucial clues to understanding molecular and functional divergence and conservation in the animal kingdom. Combined with the tissue distribution of mRNAs, the identification of ligand-receptor pairs is a crucial step in elucidating the endogenous roles of GnRHs. Of particular interest are the biological roles of GnRHs in protostomes, echinoderms, cephalochordates, and urochordates, all of which lack the HPG axis. As illustrated in Figure 3, GnRHs exhibit both common and species-specific reproductive and nonreproductive functions. Invertebrate GnRHs are highly likely to act directly on the genital organs in a neuropeptidergic manner, as supported by the expression of cognate GnRHRs in the ovary, follicles, and testis. In particular, GnRHs were shown to induce the proliferation of spermatogonial cells in several mollusks (Figure 3), suggesting a central role of GnRHs in the growth and maturation of the male gonads. Figure 3, GnRHs exhibit both common and species-specific reproductive and nonreproductive functions. Invertebrate GnRHs are highly likely to act directly on the genital organs in a neuropeptidergic manner, as supported by the expression of cognate GnRHRs in the ovary, follicles, and testis. In particular, GnRHs were shown to induce the proliferation of spermatogonial cells in several mollusks (Figure 3), suggesting a central role of GnRHs in the growth and maturation of the male gonads. In addition to the elucidation of ligand-receptor pairs, two research strategies will prompt advances in verifying GnRH signaling and its relevant biological functions. First is the development of artificial agonists and antagonists specific to GnRHRs in each species. In general, the exploration of a specific agonist and antagonist for a receptor is costly, time-consuming, and serendipitous. However, recently, Shiraishi et al. efficiently and systematically identified multiple species-specific novel neuropeptide-GPCR pairs of C. intestinalis Type A by a combination of machine learning-based prediction and cell-based functional characterization. This strategy can also be applied to the prediction of artificial peptidic agonist and antagonist candidates in various organisms [57], likely supporting the systematic and efficient development of a species-specific agonist and antagonist for invertebrate GnRHRs. Second is the development of genome editing for invertebrates. Invertebrate genome editing experimental techniques, to date, have been developed only in insects, nematodes, and Ciona [58]. Thus, the development of genome editing in other invertebrates-in particular, starfish and mollusks-is highly likely to aid in the elucidation of the biological roles of GnRHs in invertebrates, given that the cognate GnRH-GnRHR pairs have been elucidated (Table 1) and several in vivo biological functions have been observed in these species (Figure 3). The identification of authentic GnRH-GnRHR pairs, combined with the development of species-specific GnRH agonists and antagonists and gene editing techniques for each invertebrate species, will pave the way for exploring the biological significance of invertebrate GnRHs, the underlying signaling mechanisms, and the evolutionary processes of biological functions that cannot be verified by a simple comparison of the sequence alignments and analyses of molecular phylogenetic trees and gene syntenies of GnRHs or GnRHRs. In addition to the elucidation of ligand-receptor pairs, two research strategies will prompt advances in verifying GnRH signaling and its relevant biological functions. First is the development of artificial agonists and antagonists specific to GnRHRs in each species. In general, the exploration of a specific agonist and antagonist for a receptor is costly, time-consuming, and serendipitous. However, recently, Shiraishi et al. efficiently and systematically identified multiple species-specific novel neuropeptide-GPCR pairs of C. intestinalis Type A by a combination of machine learning-based prediction and cell-based functional characterization. This strategy can also be applied to the prediction of artificial peptidic agonist and antagonist candidates in various organisms [57], likely supporting the systematic and efficient development of a species-specific agonist and antagonist for invertebrate GnRHRs. Second is the development of genome editing for invertebrates. Invertebrate genome editing experimental techniques, to date, have been developed only in insects, nematodes, and Ciona [58]. Thus, the development of genome editing in other invertebrates-in particular, starfish and mollusks-is highly likely to aid in the elucidation of the biological roles of GnRHs in invertebrates, given that the cognate GnRH-GnRHR pairs have been elucidated (Table 1) and several in vivo biological functions have been observed in these species (Figure 3). The identification of authentic GnRH-GnRHR pairs, combined with the development of species-specific GnRH agonists and antagonists and gene editing techniques for each invertebrate species, will pave the way for exploring the biological significance of invertebrate GnRHs, the underlying signaling mechanisms, and the evolutionary processes of biological functions that cannot be verified by a simple comparison of the sequence alignments and analyses of molecular phylogenetic trees and gene syntenies of GnRHs or GnRHRs.