Gonadotropin-releasing hormones are composed of 10 amino acids with consensus sequences of pyro-Glu¹-His²-Trp³-Ser⁴ and Pro⁹-Gly¹⁰-amide and play pivotal roles in reproduction as releasing factor of gonadotropins in vertebrates (4). As shown in Table 1, two types of GnRHs (GnRH1 or Type 1 GnRH and GnRH2 or Type 2 GnRH) have been characterized in most vertebrates, whereas the third subtype was found in teleost and lamprey (14–16). Molecular phylogenetic tree and phylogenetic genomic analyses suggest that these subtypes have been generated by gene duplications within the species (15, 16). In other words, teleost GnRH3 and lamprey GnRH-III are specific paralogs to the respective species.

To date, 12 GnRH peptides have been identified in ascidians (Table 1). t-GnRH-1 and -2 were originally identified within the neural extract of an ascidian, Chelyosoma productum (17). Subsequently, ascidian GnRHs were isolated from other ascidians, Ciona intestinalis and Ciona savignyi (18). The former ascidian produces t-GnRH-3 to -8, and the latter generates t-GnRH-5 to -9 (18). In Halocynthia roretzi, t-GnRH-10 and -11 were characterized (19). All of these ascidian GnRHs conserve the consensus sequences of pyro-Glu¹-His²-Trp³-Ser⁴ and Pro⁹-Gly¹⁰-amide of vertebrate GnRHs. Furthermore, a unique GnRH-related peptide, Ci-GnRH-X, was isolated from the neural tissue of C. intestinalis and was found to be composed of 16 amino acids harboring the consensus sequence of pyro-Glu¹-His²-Trp³-Ser⁴ and Pro⁹-Gly¹⁰ and C-terminal Gly-amide (4, 20). The striking feature of ascidian GnRHs is multicopies of GnRH sequences in a single precursor, unlike vertebrate and non-ascidian invertebrate GnRH genes that encode a single GnRH sequence (4, 21). For instance, ci-gnrh-1 encodes t-GnRH-3, -5, and -6, whereas t-GnRH-4, -7, and -8 sequences are found in another gene, ci-gnrh-2 (18). Likewise, the H. roretzi GnRH gene encodes t-GnRH-10 and -11 (19). These findings indicate conservation and species-specific diversification of GnRHs in urochordates.

In the cephalochordate (amphioxus), Branchiostoma floridae, a GnRH-like peptide, Amph.GnRHv (pQEHWQYGHWYa, Table 1) was identified (12). Recently, GnRH-like peptides, SpGnRHP and ArGnRH (Table 1), were identified in the echinoderms, the sea urchin, Strongylocentrotus purpuratus (22) and the starfish, Asterias rubens (8), respectively. Unlike vertebrate and ascidian GnRHs, SpGnRHP and ArGnRH are 12-residue peptides containing a Val²-His³ or Ile²-His³ sequence, respectively (Table 1). These peptides share several amino acids with urochordate and vertebrate GnRHs and protostome GnRH-like peptides, including the N-terminal pGlu, His⁴ (corresponding His² in chordate GnRHs), Gly⁸ (corresponding Gly⁶ in vertebrate GnRHs), Trp⁹ (corresponding Trp⁷ in vertebrate GnRHs), and C-terminal Pro-Gly-amide, whereas the GnRH N-terminal consensus motif displays quite low sequence homology (Table 1). Thus, categorization of the echinoderm peptides as the authentic GnRH family may remain to be concluded.

Over the past 15 years, GnRH-like peptides have been identified in protostomes including mollusks and annelids (4) (Table 1): an octopus, Octopus vulgaris; a cuttlefish, Sepia officinalis; a pacific oyster, Crassostrea gigas; a sea hare, Aplysia californica; a marine worm, Capitella teleta; a leech, Helobdella robusta; a scallop, Patinopecten yessoensis. Noteworthily, two-amino acid insertion after position 1 is found in all protostome GnRH-like peptides (Table 1). Collectively, these GnRH sequences indicate that 10-amino acid sequence length is conserved within ascidians and vertebrates, whereas protostome and non-chordate invertebrate GnRHs are featured by 2-amino acid insertion. In other words, ancestral GnRHs might have harbored such two amino acids after pyro-Glu, which might have been lost during the chordate evolutionary process.

The C-terminal Pro-Gly-amide of ascidian and vertebrate GnRHs is found in oct-GnRH of cephalopods and echinoderms but not in GnRH-like peptides of gastropods, bivalves, and annelids (Table 1), suggesting that cephalopods and echinoderms might have conserved the C-terminal Pro-Gly during their evolutionary processes. Furthermore, all known protostome GnRH-like peptides and SP-GnRHP share the Ser in position 6 or 7, while the Gly⁸-Trp⁹ sequence is conserved in cephalopod GnRH, echinoderm GnRHs, and l-GnRH-II but not in ascidian GnRHs and other vertebrate GnRHs except l-GnRH-II (Table 1). Additionally, substitution of Trp³ in the N-terminal consensus motif with Phe was found in most protostome GnRHs (Table 1). Altogether, these sequences led to the presumption that the ancestral GnRHs might have been composed of pQ-H(F/W)S-GW-PGa or pQ-H(F/W)S-GW-a, and thereafter, chordate GnRHs might have diverged via various substitution and deletion of the two N-terminal amino acids in the evolutionary process of each species.

Gonadotropin-releasing hormone receptors belong to the Class A GPCR family (4, 14). In most vertebrates, two or three molecular forms of GnRHRs are present (14). Molecular phylogenetic analyses have provided evidence that vertebrate GnRHRs are classified into three groups, type-I, -II, and -III. The type-I GnRHRs were characterized from a wide range of vertebrate species such as teleost, amphibians, reptiles, birds, and mammals (23). Mammalian type-I GnRHRs completely lack the C-terminal tail region, which is present in its non-mammalian receptors (14). The type-II gnrhr gene is found in the genome of amphibians, reptiles, aves, and mammals (23). Most mammalian type-II gnrhr is non-functional due to the deletion of functional domains or interruption of full-length translation by the presence of a stop codon. In contrast, type-II GnRHRs of several monkeys, pigs, and other non-mammalian vertebrates were shown to be functional (23). Type-I GnRHRs show high affinity for both GnRH1 and 2, whereas type-II GnRHRs are specifically responsive to GnRH2 (14). Type-III GnRHRs were identified in non-mammalian vertebrates (24). In chicken, type-III GnRHR exhibits a 35-fold higher affinity for GnRH2 than for GnRH1 (24). GnRHRs are in general coupled with Gq protein and activate a typical phospholipase C (PLC)–inositol triphosphate (IP)–intracellular calcium mobilization signaling cascade, occasionally leading to phosphorylation of mitogen-activated protein kinase (MAPK) including ERK1/2 (4, 25, 26), while some GnRHRs are also found to trigger or suppress cAMP production via coupling with Gs or Gi protein (21, 25–28).

Adipokinetic hormone receptors belong to the Class A GPCR family identified in protostomes. Zhu et al. demonstrated that AKH activates both cAMP accumulation and Ca²⁺ mobilization via AKHR of the silkworm moth, Bombix moli (41). Recently, Li et al. demonstrated that AKHR of the oyster Crassostrea gigas was activated by oyster AKH at physiological concentrations (40). Moreover, Nagasawa et al. detected expression of Py-AKHR mRNAs in the nerve ganglia, lip, foot, CPG, mantle, testis, and ovary in Yesso scallop, Patinopecten yessoensis. The differential expression profile of Py-AKHR mRNA in the gonad during gonadal maturation stages suggests their reproductive function (42).

Adipokinetic hormones have so far been shown to stimulate the fat body, resulting in lipid and carbohydrate mobilization into the hemolymph in insects and crustaceans. Furthermore, a homolog of AKH in the northern shrimp, Paudalus borealis, red pigment concentrating hormone, influenced the concentration of pigment chromatophore, causing its body color change (43). Notably, AKHs also showed a reduction in oocyte protein and carbohydrate content in the crickets, Gryllus bimaculatus, and a reduction in vitellogenin of oocytes in L. migratoria (44), indicating a regulatory role for AKHs in insect reproduction. AKH-deficient flies displayed the opposite phenotype in which hemolymph trehalose levels decreased and storage lipid in the fat body accumulated (45). An AKH receptor-deficient strain showed a similar phenotype to AKH-deficient flies (46). In the cricket G. bimaculatus, AKH receptor knockdown by RNAi increased feeding frequency and reduced locomotor activity (47).

Corazonin receptors are class A family GPCRs. The first CRZR was identified in Drosophila melanogaster, and then orthologous receptors have been cloned from moths, mosquitoes, honey bee, and other insects (48) (Figure 3). CRZR of the silkworm moth Bombix mori induces cAMP accumulation, Ca²⁺ mobilization, and ERK1/2 phosphorylation via the Gq- and Gs-coupled signaling pathways in response to CRZ (49). Various molecular phylogenetic analyses indicate that the annelid and molluscan GnRHRs are clustered with the CRZRs and the annelid and molluscan GnRHRs have been recognized as the members of CRZR/GnRHR clade (5, 12, 13). In the starfish, Asterias rubens, CRZ-like peptide (HNTFTMGGQNRWKAG-amide) was identified and also found to activate the cognate receptor (8). Likewise, GnRHR-type receptor was identified and found to be activated specifically by the cognate GnRH-like peptide (pQIHYKNPGWGPG-amide) in a ligand-specific manner (8). Collectively, these results suggest that echinoderms, at least A. rubens, may be endowed with the GnRH- and CRZ-directed signaling systems (8).

Notably, as stated earlier, B. floridae (amphioxus) GnRHR-3 and -4 are highly homologous to the protostome CRZR/GnRHR receptor family and GnRHR-3 was activated by the amphioxus GnRH-like peptide (pQILCARAFTYTHTW-amide), oct-GnRH (pQNYHFSNGWHPG-amide), and AKH (pQLTFTSSW-amide) at physiological concentrations, indicating that B. floridae GnRHR-3 exhibits extensive ligand selectivity for GnRH superfamily peptides. The CRZ/CRZR signaling system has been lost in urochordates, vertebrates, nematodes, and some insects (50).

Corazonins have a number of physiological roles associated with control of heartbeat, ecdysis behavior initiation, and cuticle coloration in the Artholopoda (5, 48). Recently, its regulatory functions on insulin producing cells in the brain of D. melanogaster (51) and on larval–pupal transition and pupariation behavior have been found in the fruit fly, Bactrocera dorsalis (52). Intriguingly, CRZs also show reproductive activities in invertebrates. In male flies, CRZs act on its receptor in a small cluster of posterior serotoninergic neurons to control activity of the accessory glands and sperm ejaculation during mating (53). In the giant freshwater prawn Macrobrachium rosenbergii, CRZs inhibit testicular development and spermatogenesis and androgenic gland secretion (54). Ablation of CRZ-GAL4 neurons increased locomotion and dopamine level in male files, D. melanogaster. Furthermore, silencing of CRZR-GAL4 neurons in male flies elicits infertility and blocks sperm and seminal fluid ejaculation (53). In B. mori, dsRNA-mediated knockdown of BmCrzR indicated a role of CRZ signaling in the regulation of silkworm growth and silk production (49).

Adipokinetic hormone/CRZ-related peptide receptors are Class A family GPCRs identified only in insects (Figure 3). Hansen et al. showed that the A. gambiae ACP receptor transfected into mammalian cells stably expressing the human G-protein G16, a universal G protein adapter, was activated specifically by the cognate ligand (55). Zandawala et al. characterized three splice variants encoding ACP receptors in the kissing bug Rhodnius prolixus; Rhopr-ACPR-A has only five transmembrane (TM) domains, and Rhopr-ACPR-B and C have seven TM domains. All Rhopr-ACPR-A, -B, and -C were activated by Rhopr-ACP but neither Rhopr-AKH nor Rhopr-CRZ with different sensitivities on mammalian cells stably expressing the G-protein G16, whereas Rhopr-ACPR-B and -C indicated coupling with Gq when expressed in CHO-K1-aeq cells (56).

To date, the ACP signaling system has been found only in arthropods and its major biological roles are still unclear. However, recently, ACP was shown to decrease germ cell proliferation and increases in total hemolymph lipids were found by administration of the peptide in female prawn, M. rosenbergii (11). The expression of MroACP mRNA in the eyestalk, central nervous system, thoracic ganglia, and MroACPR mRNA in the neural tissues and the ovary throughout different stages of ovarian maturation indicated a neuronal regulation of ACP signaling in reproduction (11).