The insulin signaling pathway is a core and evolutionarily conserved signal transduction network. Here we study hypomorphic mutant conditions in the sole insulin receptor gene (Inr) in the fruit fly Drosophila melanogaster, placed at the beginning of the signaling cascade where it mediates the effects from the seven Drosophila insulin-like peptides. As such, it sits at a nodal point where signaling must pass to ensure and carry out all the functions ascribed to this anabolic pathway. We find that these mutant flies, while viable, exhibit hyperactivity and accumulate glycogen and body lipids. The hyperactivity phenotype was characterized in detail and is significantly different from controls both measuring it in several days' spells, or when done in a more detailed fashion in shorter time periods (twenty-five minutes). The proportion of time spent sleeping is also altered, highlighting the robust role that the insulin pathway has not only regulating metabolism but also behavior. When confronted with mild pro-oxidative conditions, these flies and control wildtype flies have reduced lifespans as well. The metabolic dysregulation is rescued when the Inr mutant flies are also heterozygous for keap1, a negative regulator of the Nrf2 pathway, while the antioxidant response and the activity/sleep levels exhibit only partial and sexually dimorphic improvements. Transcriptomic analyses of Inr mutants show that many metabolism and hormonal signaling genes are dysregulated.

Amphibian organ culture is invaluable for gaining insight into thyroid hormone (TH) action. The olfactory epithelium (OE) of Rana (aka Aquarana or Lithobates) catesbeiana tadpoles is a highly TH-sensitive tissue that regulates essential survival behaviors and undergoes drastic remodeling during TH-dependent metamorphosis. These features make it a valuable bioindicator for normal and disrupted TH action. To our knowledge, we developed and validated the first successful ex vivo culture method for anuran tadpole olfactory epithelium. Pairs of OE-containing principal cavities from premetamorphic tadpoles were excised, each cavity cut in half, and exposed to 10 nM 3,5,3'-triiodothyronine (T3) or solvent control at 24 °C for 24 h. Each half cavity showed consistent enrichment of the olfactory marker omp compared to back skin. Robust and reproducible TH responses were observed in individual, unpooled cultured olfactory epithelium (C-OE) as measured by reverse transcription-quantitative polymerase chain reaction of TH-responsive transcripts thra, thrb, and thibz. No spatial variation in hormone sensitivity within the principal cavity was observed supporting the use of repeated-measures statistics while capturing biological variation between animals. To probe early TH signaling events, C-OE and cultured back skin (C-skin) were treated with T3 in the presence of transcriptional and translational inhibitors at 24 °C or 4 °C. The cold condition represents a metamorphic stasis period during which the TH signal is initiated, but metamorphosis does not proceed. Notably, TH-induction of thibz at 4 °C was unaffected by transcriptional inhibition in both tissues, suggesting that mechanisms other than RNA synthesis may be driving the increase in transcript abundance.

Growth hormone (GH) in mice is primarily expressed in the anterior pituitary, although Gh expression has been reported in extrapituitary tissues, including immune organs. However, the structure of immune-associated Gh transcripts remains poorly characterized. To determine whether splenic Gh transcripts differ from pituitary Gh mRNA, 5'- and 3'-rapid amplification of cDNA ends (RACE) analyses were performed. While 3' RACE showed a shared polyadenylation site, 5' RACE identified a novel exon located approximately 2 kb upstream of the conventional exon 1, generating a transcript (spl-Gh mRNA) with a distinct first exon but shared downstream exons with pituitary Gh mRNA (pit-Gh mRNA). RT-PCR analysis revealed that spl-Gh mRNA is predominantly expressed in immune tissues such as spleen and bone marrow, and its distribution did not correlate with Pit-1 mRNA expression. Quantitative RT-PCR further demonstrated that spl-Gh mRNA was expressed at levels comparable to those of pit-Gh mRNA in the mouse spleen, indicating that spl-Gh is one of the major Gh transcript forms in this tissue. Sequence analysis indicated that spl-Gh mRNA is predicted to retain coding potential for a GH protein. Comparative genomic analyses further demonstrated that genomic features associated with the spl-Gh transcriptional unit are conserved only in a subset of closely related Mus species. In contrast, although a spl-Gh-related transcript was detected in rat spleen, no properly spliced mouse-like transcript was identified under the present experimental conditions. The detected transcript exhibited intron retention and an in-frame stop codon, suggesting that it is unlikely to produce a functional GH protein. These findings identify a distinct immune-associated Gh transcript generated through alternative transcription of the mouse Gh gene and suggest that immune-associated Gh transcriptional mechanisms have undergone species-specific divergence among rodents. Together, these findings reveal previously unrecognized complexity in Gh gene regulation and highlight species-specific differences in immune-associated Gh transcripts.

Relaxin-like gonad-stimulating peptide (RGP) regulates final gamete maturation and spawning in starfish through a specific gonadal receptor, the RGP receptor (RGPR). Homologous RGPs have since been identified in sea cucumbers and shown to induce spawning, supporting a conserved reproductive role across echinoderms. However, the molecular basis of RGP-RGPR signalling in sea cucumbers remained unknown, as the sea cucumber RGPR had not previously been identified. Therefore, we systematically searched for G protein-coupled receptors similar to starfish RGPRs using comparative sequence analysis and identified candidate RGPRs in five sea cucumber species: Holothuria leucospilota, Holothuria glaberrima, Holothuria scabra, Apostichopus japonicus, and Isostichopus badionotus. Receptor sequences from Holothuria species shared 77 to 85% identity, while similarity to Apostichopus and Isostichopus sequences was lower, at about 60%. Structural modelling revealed the expected features of relaxin family peptide receptors, including seven transmembrane helices, a large extracellular domain with multiple leucine-rich repeats, and an N-terminal LDLa module. Functional cell-based assays demonstrated that the candidate H. scabra RGPR responded specifically and dose-dependently to recombinant H. scabra RGP, but not to human H2 relaxin, providing the first functional evidence of an RGP receptor in a sea cucumber. An RGPR homologue was detected in the H. leucospilota ovary across all reproductive stages, although expression did not differ significantly among stages. Together, these findings support the conservation of relaxin-like reproductive signalling in echinoderms, while direct functional characterisation of the H. leucospilota RGP-RGPR pair remains necessary.

Cathepsins (CTS) family of lysosomal proteases is characterized by high expression and proteolytic activity across various species. Notably, cathepsin B (CTSB) and cathepsin L (CTSL) have been implicated in uterine remodeling, follicle atrophy, and granulosa cell apoptosis in various vertebrates. This study investigated the role of the CTS members in post-parturitional ovarian remodeling in ovoviviparous black rockfish (Sebastes schlegelii). We cloned the ctsb (993 bp, encoding 330 amino acids) and ctsl (1011 bp, encoding 336 amino acids) genes from black rockfish. Quantitative Real-time PCR (qPCR) analysis revealed widespread tissue distribution for both genes, with ctsb expression being significantly highest in the ovary and markedly upregulated during the parturition process. In contrast, ctsl exhibited low ovarian expression with no increasing trend. In situ hybridization (ISH) and immunohistochemistry (IHC) localized CTSB to the ovarian stromal periphery and specific oocytes. Functional studies using recombinant mature CTSB demonstrated its proteolytic activity on ovarian tissue, leading to a significant increase in arginine (Arg) content. Post-parturition supplementation with Arg notably restored serum nutritional levels. Furthermore, dual- luciferase assays identified functional binding sites for Estrogen Receptor 1 (ESR1) and Specificity Protein 1 (SP1) within the ctsb promoter, regulating its transcription. Collectively, our findings indicate that CTSB, a member of the CTS family, is likely involved in ovarian remodeling in black rockfish.

Assisted reproduction methodologies are essential to protect species from extinction. But to improve reproductive outcomes, we need to understand gamete quality. Gamete quality assays are powerful functional endpoints for comparative endocrinology, especially when they are integrated with hormone measurements and experimental manipulations. However, the methodologies for assessing amphibian gamete quality do not have published validation or comparisons within the literature. In this study we analysed sperm samples from two bell frog species, looking at morphology (measuring sperm head and sperm tail length) using both wet mounts and dry mounts of three different stains (unstained mounts, Coomassie blue and eosin nigrosin). We also assessed viability in the same sperm samples using three different preparations (eosin nigrosin dry mount, eosin nigrosin wet mount, and fluorescent SYBR-14 propidium iodide). We found that the methods varied in mean morphological measurements. For example, sperm tail lengths were measured longer when the slides were dry mounts, and when the eosin nigrosin stain was used. There were differences among species as well, where, for example, stain type influenced the sperm head length in one species, but not in the other species. Importantly, all methods had similar variance, indicating that all staining techniques are suitable given that the methods used are consistent within a study. For viability assessment, the dried eosin nigrosin staining method showed lower viability, while the wet mount of eosin nigrosin and the fluorescent SYBR-14 propidium iodide stain were statistically equivalent. These results indicate that there are a wide range of methodologies that can be used for sperm quality assessment of amphibian sperm, and researchers must choose the methodology best suited for the study.

Peptide-responsive G protein-coupled receptors (GPCRs) play pivotal roles in a wide variety of physiological regulatory systems in animals. Despite the substantial expansion of the GPCR and endogenous peptide repertoire identified through large-scale genomic and peptidomic studies, a significant number of receptors remain orphan, particularly those for non-homologous or species-specific peptides. Conventional deorphanization approaches based on sequence or ligand similarity, or receptor structure, are frequently ineffective. These current shortcomings in elucidation of peptide-GPCR interactions result in a persistent gap between sequence information and functional characterization. This review consolidates recent advances in computational approaches that address this challenge by focusing on peptide-GPCR pairs rather than on sequences or structures of peptides and receptors. Pair-centric machine learning frameworks, originally developed in the field of chemical genomics, enable systematic prediction of peptide-GPCR interactions without sequence similarity. Our originally developed machine learning system, PD-incorporated SVM, which incorporates peptide-specific descriptors into the frameworks, has enabled the experimentally validated identification of novel peptide-GPCR pairs in a tunicate and a nematode, and has predicted GPCRs for lineage-specific neuropeptides in ctenophore. In addition to interaction prediction, the present study explores the interaction determinant likelihood (IDL) framework, which offers a mechanistic interpretation of predicted interactions. IDL extracts residue-level determinants of ligand recognition directly from trained models, thereby enabling the identification of peptide features and receptor residues that collectively govern interaction specificity. The application of this approach to closely related GPCR paralogs demonstrates the emergence of distinct peptide specificities through limited, context-dependent residue substitutions, without significant alterations in overall receptor structure. Together, these findings highlight interaction determinants as a unifying framework linking deorphanization, molecular mechanism, and evolutionary diversification of peptide-GPCR signaling.

In aquaculture, ovulation is commonly induced using exogenous hormones, which are associated with broodstock stress, variable responses, and environmental concerns. Gap junctions formed by connexin 43 (Cx43) in ovarian granulosa cells play a critical role in maintaining meiotic arrest by facilitating the transfer of meiosis-inhibiting substances from granulosa cells to oocytes. The present study investigated whether pharmacological blockade of Cx43 using the specific inhibitor Gap27 could induce ovulation in zebrafish as a non-hormonal alternative. Sexually mature female zebrafish were divided into four groups: control (saline), low-dose (50 μM Gap27), medium-dose (150 μM Gap27), and high-dose (250 μM Gap27), with injections administered through the genital pore. Ovulation was assessed at 14 days post-injection by gentle abdominal pressure. The high-dose group achieved an ovulation rate of 77.78% (7/9), whereas no ovulation was observed in the control or low-dose groups, and only one female ovulated in the medium-dose group (11.11%). Histological examination revealed that the high-dose group exhibited loose ovarian tissue with abundant stage V (mature) follicles, while the control and low-dose groups were dominated by stage III and IV follicles. Transcriptomic analysis indicated dose-dependent transcriptional remodeling, with the high-dose group displaying a distinct expression profile. Comparative analysis between the medium-dose and high-dose groups identified 255 differentially expressed genes, from which 17 ovulation-related genes were screened, including mmp9, nos2b, fosb, igf3, nppc, il17ra1a, and plk2b, which are involved in meiotic arrest release, extracellular matrix remodeling, inflammatory signaling, and cell cycle regulation. Based on these findings, a "bypass activation" model is proposed, in which Gap27-mediated blockade of Cx43 triggers four coordinated molecular modules-brake release, inflammation and tissue remodeling, stress and proliferation, and alternative growth signals-ultimately leading to ovulation. Collectively, these results suggest that targeted Cx43 blockade by Gap27 effectively induces ovulation in zebrafish, providing a proof-of-concept for non-hormonal ovulation induction strategies in aquaculture.

Stress and reproduction are often linked through interactions between the hypothalamic-pituitary-adrenal (HPA) and hypothalamic-pituitary-gonadal (HPG) axes such that stress can suppress reproduction. It is unclear, however, whether species with enhanced annual fecundity exhibit modifications in this interaction. We addressed this question using a comparative study of two closely related blackbird species that differ markedly in parental care and reproductive output: the brown-headed cowbird (Molothrus ater), an obligate brood parasite that has enhanced annual fecundity without parental care, and the red-winged blackbird (Agelaius phoeniceus), which does not have enhanced annual fecundity and provides parental care. We quantified restraint-induced corticosterone and circulating testosterone, 17β-estradiol, and ovarian follicle size in breeding adult females. We also characterized testosterone and corticosterone profiles in nestlings and adults of both species. Corticosterone responses to restraint did not differ between species or age classes but age-related trajectories diverged as corticosterone declined from nestlings to adults in cowbirds but increased in red-winged blackbirds. On the other hand, testosterone concentrations remained stable across species and ages. In adult red-winged blackbirds, reproductive state positively predicted corticosterone responses to restraint, such that individuals with higher testosterone, estradiol, or larger follicles mounted stronger corticosterone responses. In contrast, cowbirds showed no relationship between reproductive state and stress-induced corticosterone. These findings indicate that cowbirds exhibit an altered relationship between reproductive and stress physiology that suggests a functional decoupling between HPA and HPG activity. Such decoupling may support enhanced fecundity in obligate brood parasites.

The authentic prolactin (PRL) designated PRL1 is a multifunctional hormone secreted by the anterior pituitary gland of vertebrates. The PRL1 gene exists in two forms: fish-type PRL1 gene (PRL1B) and tetrapod-type PRL1 gene (PRL1A). It has been proposed that PRL1B gene underwent duplication during the early amphibian lineage. Subsequently, the ancestral gene (PRL1B) was lost in amniotes, while the newly duplicated gene (PRL1A) was retained. Notably, only amphibians possess both PRL1B and PRL1A genes. In the anuran amphibian bullfrog, PRL1A gene expression is low during the pre- and prometamorphic stages, gradually increasing toward the end of the metamorphic climax stage. In contrast, PRL1B gene expression is relatively high during the early metamorphic stages but significantly declines during the metamorphic climax stage and becomes undetectable after completion of metamorphosis. These differential expression patterns suggest a functional transition of PRL1 paralogs associated with the shift from aquatic to terrestrial life. However, data on PRL1B expression in urodele amphibians remain limited. In this study, we cloned the PRL1B cDNA from the urodele amphibian, the red-bellied newt (Cynops pyrrhogaster) and analyzed its expression in the pituitary gland. Unlike in anuran bullfrog, PRL1B is expressed as the transcript and protein in the adult pituitary of this urodele species, suggesting that the regulatory mechanism controlling PRL1B expression in urodeles differs from that in anurans. This is the first report demonstrating the existence of PRL1B in urodeles in the form of protein as well as mRNA.

In teleosts, gonadal differentiation is a complex and precisely controlled process that requires a proper balance of androgen and estrogen signaling activities. However, the interaction between these steroid signals remains unclear, particularly during the critical stage that determines the sexual fate of the bipotential gonad in zebrafish. In the present study, we investigated the expression of cyp19a1a and foxl2, the amount of primordial germ cells (PGCs), and the status of ovarian differentiation, in response to 17β-estradiol (E2) and 11-ketotestosterone (11-KT) using the Tg(piwil1:EGFP-nanos3'UTR) zebrafish line. The expression of cyp19a1a, foxl2a, foxl2b and foxl2l was significantly higher in the PGC-rich group reared in control medium (CM) at 23 days post-fertilization (dpf) than in the PGC-less group. Treatment with E2 promoted ovarian differentiation in 83% of individuals and increased foxl2 expression. Conversely, 11-KT treatment caused testis-biased differentiation in 86% of individuals and decreased foxl2 and cyp19a1a expression at the same stage. Significant differences were observed when the number of PGCs labeled with green fluorescent protein (GFP) in E2- and 11-KT-treated fish were compared. Furthermore, the inhibitory effect of 11-KT on the expression of foxl2 and ovarian differentiation, which was Androgen receptor (Ar)-dependent, could be completely counteracted in wild-type (WT) fish when E2 was co-administered. In summary, our results showed that estrogen signaling is sufficient to counteract the effect of androgen/Ar signaling on upregulating foxl2 expression and promoting ovarian development and differentiation.

In mammals, insulin-like growth factor (IGF)-1 and IGF-2 regulate postnatal growth and embryonic development, respectively. In fish, as in mammals, IGF-1 promotes somatic growth, whereas evidence suggests the possible involvement of IGF-2 in the regulation of somatic growth. However, the measurement of circulating IGF-2 in fish has been hampered, possibly because of interference from IGF-binding proteins (IGFBPs). Extraction of IGFs from IGFBPs is essential before measuring them using immunoassays. We optimized acid-ethanol (AE) extraction method for salmonid IGF-2 and developed a time-resolved fluoroimmunoassay (TR-FIA) using recombinant IGF-2 as the assay component. In TR-FIA, the standard AE-extraction of IGF-1 was not validated for quantifying serum IGF-2 because of the presence of ethanol. Three treatments-vacuum drying, dialysis, and ultrafiltration-were compared after AE extraction, and ultrafiltration by centrifugation was found to be effective. With this extraction method, circulating IGF-2 levels were measured by TR-FIA in fed, fasted, and refed rainbow trout. Serum IGF-2 levels were similar or slightly lower in fed fish than in fasted fish, and refeeding had no positive effect. Serum IGF-2 levels were not correlated with individual growth rates. GH treatment tended to increase circulating IGF-2 levels albeit not statistically significant. Smolting masu salmon had similar but slightly lower serum IGF-2 levels than non-smolting fish, while exhibiting high serum IGF-1 levels. These results suggest that circulating IGF-2 levels were relatively stable. This newly developed assay should facilitate further exploration of the physiological responses and roles of circulating IGF-2 in salmonids.