Melanocortin receptor-4 (MC4R) belongs to the G protein-coupled receptor family, characterized by a classical structure of seven transmembrane domains (7TMD). They play an important role in food intake and weight regulation. In the present study, we identified melanocortin-4-receptor-like (caMC4RL) mutants of goldfish from the Qian River in the Qin Ling region and characterized their functional properties, including the constitutive activities of the mutants, ligand-induced cAMP and ERK1/2 accumulation, and AMPK activation. The results show that six caMC4RL mutants were identified in goldfish from the Qian River in the Qin Ling region, and are located in the conserved position of the Cyprinidae MC4Rs. The mutations (E57K, P296S, and R302T/K) result in the loss of Gs signaling function. The mutations (P296 and R302T/K) exhibited biased signaling in response to ACTH stimulation in the MAPK/ERK pathway. In addition, the E57K mutant may play a role in weight regulation and could serve as molecular markers for molecular breeding. These data will provide fundamental information for functional studies of teleost GPCR mutants and MC4R isoforms.

Super-conserved Receptors Expressed in Brain (SREB) are a highly conserved family of orphan G protein-coupled receptors that consist of three members in most vertebrates: SREB1 (GPR27), SREB2 (GPR85), and SREB3 (GPR173). Each receptor is associated with diverse physiological processes and expressed in both ovaries and testes, but reproductive functions are only beginning to be understood. In addition, some fishes gained a novel fourth gene, SREB3B, which may have unique functions. The purpose of this study was to conduct a spatial and quantitative analysis of SREBs in the gonads of pufferfish (Dichotomyctere nigroviridis), which expresses all four genes. Multiplex RNAscope and absolute qPCR were used to assess gene expression patterns in both ovaries and testes. Expression was detected in early ovaries and dominated by sreb1 (approximately 2500 copies/ng RNA vs. 300 or less for others), with notable expression of all receptors in primary oocytes, granulosa cells, and small numbers of extra-follicular cells. Within primary oocytes, sreb1 and sreb3b exhibited diffuse patterns that may indicate early functions, while sreb2 and sreb3a were granular and may reflect stored mRNA. Early testicular development was dominated by sreb1 and sreb2 (∼5000 copies/ng RNA) in spermatogonia. These patterns were somewhat reduced in late testes (∼1000-2600 copies/ng RNA), but sreb3b exhibited a novel spatial pattern (∼380 copies/ng RNA) within spermatogenic cysts. These results highlight diverse roles for the SREB family, and sreb3b is hypothesized to have unique roles in fish reproduction.

The plains vizcacha is a rodent that shows reactivation of the hypothalamic-pituitary-ovary (HPO) axis activity at mid-gestation. This process is enabled by the secretion of hypothalamic gonadotropin-releasing hormone (GnRH) at mid-gestation, followed by follicle-stimulating hormone (FSH) and luteinizing hormone (LH) secretion. However, a decrease in the pituitary GnRH receptor (GnRHR) expression is concomitantly determined. Moreover, an increment in the pituitary expression of estrogen receptor alpha (ERα) has been determined. This work aimed to study the impact of estradiol (E2) on GnRHR expression, the transcription factors early growth response protein-1 (Egr-1) and steroidogenic factor-1 (Sf-1), as well as on LH secretion. Three experimental approaches were performed: a physiological one with pregnant plains vizcachas, an in vivo approach with ovariectomized (OVX) animals treated with E2 (OVX + E2), and an ex vivo approach using pituitary glands exposed to a combination of GnRH and E2. Significant increased pituitary expression of Sf-1 and Egr-1 was determined at mid-gestation. Ovariectomy significantly increased adenohypophyseal expression levels of GnRHR, Egr-1, and Sf-1, as well as LH secretion. Then, OVX + E2 showed similar levels to SHAM. Adenohypophyses exposed to GnRH showed induced GnRHR, Egr-1, and Sf-1 expression, and LH secretion, while GnRH + E2 reverted these changes. The mid-gestation pituitary GnRHR decrease may result from the combination of increased E2 and GnRH secretion. Nevertheless, the increased expression of Egr-1 and Sf-1 at mid-gestation, together with LH release, suggests the tightly and complex regulatory system that takes place at mid-gestation, enabling a new progesterone surge that successfully carries the pregnancy to term. NEW & NOTEWORTHY: A significant increment of Sf-1 and Egr-1 at the pituitary of mid-gestating plains vizcachas was determined. Moreover, E2 reverted GnRHR, Egr-1, Sf-1, and LH increase in ovariectomized vizcachas' pituitaries and ex vivo pituitaries exposed to GnRH. The decrease of the pituitary GnRHR at mid-gestation may result from the increased E2 and GnRH levels. A tightly and complex regulatory system may take place at mid-gestation enabling a new surge of progesterone that carries pregnancy to term.

Peptide YY (PYY) is an anorectic brain-gut pancreatic peptide that helps in feeding regulation by reducing appetite and is well characterized in mammals. The role of PYY in relation to brain is least studied in mammals as well as in lower vertebrates including fish, however high expression was evident in male reproductive tissue. In this regard, this study attempts to evaluate the significance of PYY in the brain of common carp, Cyprinus carpio. As a first step, the cDNA of PYY from brain of adult male carp was cloned. Following which expression analysis was performed using juvenile and adult fish. The differential distribution pattern in various regions of brain and ontogeny expression analysis indicated that PYY may involve in physiological processes related to brain-pituitary axis. In addition, a significant decrease in neuropeptide Y expression was observed upon PYY- endoribonuclease-prepared small interfering RNA transfection in brain cells, in vitro indicating plausible PYY-NPY interaction in brain-pituitary axis of common carp.

CAPA peptides play diverse roles in insects, modulating muscle contraction, regulating fluid balance, and reproduction. In Rhodnius prolixus, a hematophagous insect and a vector for human Chagas disease, three CAPA peptides are encoded by the capability gene, including RhoprCAPA-1, RhoprCAPA-2, and RhoprCAPA-PK-1. RhoprCAPA-2 is an anti-diuretic hormone in R. prolixus. Here, we explore the involvement of RhoprCAPA-2 in reproduction in adult female R. prolixus. Double-label immunohistochemistry reveals co-localization of RhoprCAPA-2-like and the glycoprotein hormone (GPA2/GPB5) subunit GPB5-like immunoreactivity in neurosecretory cells in the mesothoracic ganglionic mass and in their neurohemal sites, suggesting these peptides can be co-released to regulate physiological processes. qPCR analysis reveals changes in transcript expression levels of the RhoprCAPA receptor (CAPAR) in the fat body and reproductive tissues after feeding in adult female R. prolixus. RNA interference-mediated knockdown of CAPAR transcript decreases egg production and reduces hatching rate and survival rate in female R. prolixus. Downregulation of CAPAR decreases vitellogenin RhoprVg1 transcript expression in the fat body and deceases its receptor RhoprVgR transcript level in the ovaries; accompanied by a reduction in vitellogenin content in the fat body and hemolymph. Incubation of fat body and ovaries in vitro with RhoprCAPA-2 increases RhoprVg1 transcript expression in the fat body, vitellogenin content in the fat body culture medium, and increases RhoprVgR transcript in the ovaries. These findings implicate the CAPA signaling pathway in reproduction, with RhoprCAPA-2 acting as a gonadotropin in adult female R. prolixus.

The present study was aimed at gaining insight into the signalling relationship between glucagon-like peptide-1 (GLP-1) and its receptor (GLP-1R) in the regulation of glucose metabolism. Further, to assess the role of G-protein-coupled receptor 40 (GPR40) and insulin receptor (INSR) in the pancreas of sheep that were supplemented with calcium salts of long-chain fatty acids (CSFAs). An experiment was carried out over a period of 60 days with eighteen sheep, and they were fed with a standard basal diet. The sheep were divided into three groups: CSFA0 (without CSFAs), while CSFA3 and CSFA5 were supplemented with 3 % and 5 % of CSFAs, respectively. Plasma concentrations of GLP-1, insulin, glucagon, and glucose were assessed every two weeks. At the end of the experiment, sheep were slaughtered, and samples of gastrointestinal tract (GIT) epithelial tissues and pancreas were collected to assess the relative expression of mRNA of GPR40, GLP-1R, and INSR. Postprandial GLP-1 and insulin were increased by 3.7-4.1 and 1.45-1.5 times, respectively, in the CSFAs-supplemented groups compared to CSFA0. Post-feeding, glucagon and glucose levels decreased in CSFA3 and CSFA5 compared to CSFA0. The results indicated that the supplementation of LCFAs increased the expression of GLP-1R in the GIT and pancreas, as well as the mRNA of GPR40 and INSR in the pancreas. Chemosensing of LCFAs by GPR40 in the pancreas triggers signalling transduction, and enhanced GLP-1 and GLP-1R resulted in moderately increased insulin secretion and reduced glucagon levels. These combined effects, along with the glucose-lowering effect of GLP-1, effectively lowered glucose levels in normoglycemic sheep.

The vertebrate stress response enables an organism to shift energy towards activities that promote immediate survival when facing a threat to homeostasis, but it can also have detrimental effects on organismal health. Acute and chronic stressors generally have contrasting effects on immune responses, but the timeline of this transition between acute and chronic stressors and their effects on immune responses remains unclear. In this study, we investigate changes in immune markers in captive house sparrows (Passer domesticus) after exposure to normal laboratory conditions, an acute stressor, and chronic stressors for 42 days. Specifically, we examined changes in baseline and stress-induced corticosterone concentrations, body condition, heterophil/lymphocyte (H:L) ratio, hemolysis-hemagglutination, and wound healing. We found that individuals exposed to a single acute stressor had significantly higher stress-induced corticosterone concentrations 24 h after stressor exposure, however this effect was reversed after 48 h. Chronic stressor exposure resulted in generally stronger adaptive immune responses, demonstrated by higher baseline and stress-induced lysis, higher baseline hemagglutination, and slower wound healing. Within-trait correlations also increased with chronic stressor exposure, suggesting limitations on phenotypic plasticity. Most of the effects of chronic stressor exposure on immune markers strengthened over the 42 days of the experiment and differences between captivity-only and treatment groups were not apparent until approximately 20 days of chronic stressor exposure. These results highlight the importance of stressor duration in understanding the effects of chronic stressor exposure on immune responses.

Zebrafish sex chromosomes have been identified in the wild Nadia (NA) strain, and its sex determination belongs to the female-heterogametic ZZ/ZW system. Here, we investigate the correlation between ZZ/ZW sex chromosomes in the NA strain with sex-related factors, and sort out the complicated process of sex determination in zebrafish. Two phases exist during zebrafish sex differentiation. In the first phase, ZW gonads differentiate into juvenile ovary while ZZ gonads remain indifferent. In the second phase, ZW gonads either continue ovary development or undergo female-to-male transition, while ZZ gonads undergo direct male development. The W chromosome may contribute to the first phase while the abundance of germ cells and other factors may be involved in the second phase of sex differentiation in zebrafish.

We analyzed the expression of genes involved in the hypothalamic-pituitary-thyroid axis (HPT-axis) in the longfin yellowtail Seriola rivoliana early larva, including temperature effects (22, 26 and 28 °C) and days of development (day one, day two, and day six after hatching). We aimed to determine if egg and larval incubation at different temperatures could disrupt this critical endocrine axis, which, in an aquaculture context, it could provoke mortality during early metamorphosis. There was a significant interaction between temperature and developmental timing on the relative expression of thyrotropin releasing hormone (trh). Larvae at 22 °C was the longest and increased more trh expression than larvae at higher temperatures. Interestingly, thyrotropin stimulating hormone (tsh) was highly expressed after hatching. Subsequently, it was downregulated at any temperature at least until day four, suggesting a temporal inhibition of the HPT axis. Therefore, we suggest that tsh-binding (tshr) to follicles should have occurred from hatching, creating a further "cascade effect" of upregulation of larval thyroglobulin (tg) from day two in a temperature-dependent manner. Consequently, new thyroid hormones should have been produced after yolk sac absorption. The above may indicate a narrow window of larval survival, where the larval transition from endogenous to exogenous feeding would depend on the correct timing to synthesize tg. Temperature significantly affected the expressions of deiodinase 1 (dio1-downregulated) and deiodinase 2 (dio2-upregulated) after hatching. The expressions of thyroid receptors alpha (trα) and beta (trβ) remained constant after hatching without significant effects of temperature and days of development. Then, the differential expression on day six showed that all HPT-axis transcripts increased their expressions as larvae developed, which suggested a functional HPT. Finally, there was no evidence that any temperature would disrupt the endocrine's larval axis, which indicated that the longfin yellowtail has a wide temperature adaption. Nevertheless, based on tg upregulation, we suggest that larvae should be maintained around 25-26 °C after hatching for a better chance of survival and development.

In Gnathostomes, reproduction is mainly controlled by the hypothalamic-pituitary-gonadal (HPG) axis, with the involvement of the pituitary gonadotropic hormones (GTH), follicle-stimulating hormone (FSH) and luteinizing hormone (LH), which activate their cognate receptors, FSHR and LHR, expressed in gonads. Each GTH consists of a common α subunit and of a specific FSHβ or LHβ subunit. Chondrichthyes (holocephalans and elasmobranchs) is a sister group of bony vertebrates. This position is highly favorable for the understanding of the evolution of endocrine regulations of reproduction among gnathostomes. Surprisingly, the characterization of gonadotropins and their receptors is still limited in chondrichthyes. In the present study, GTH and GTHR sequences have been identified from several chondrichthyan genomes, and their primary structures were analyzed relative to human orthologs. 3D models of GTH/GTHR interaction were built, highlighting the importance of the receptor hinge region for ligand recognition. Functional hormone-receptor interactions have been studied in HEK cells using the small-spotted catshark (Scyliorhinus canicula) recombinant proteins and showed that LHR was specifically activated by LH whereas FSHR was activated by both FSH and LH. Expression profiles of GTHs and their receptors were explored by real-time PCR, in situ hybridization and immunohistochemistry during spermatogenesis, along the male genital tract and other tissues, as well as in some female tissues for comparison. Tissue-expression analyses showed that the highest levels were observed for fshr transcripts in testis and ovary and for lhr in specific extragonadal tissues. The two receptors were expressed at all stages of spermatogenesis by both germ cells and somatic cells, including undifferentiated spermatogonia, spermatocytes, spermatids, somatic precursors and Sertoli cells; differentiated Leydig cells being absent in the testis of S. canicula. Receptors were also expressed by the lymphomyeloid epigonal tissue and the testicular tubules. These results, suggest a wide range of gonadotropin-regulated functions in Elasmobranchs, as well as functional redundancy during spermatogenesis. These extended functions are discussed in an evolutionary context in which the specificity of gonadotropin signaling must have contributed to the evolution of gonadal cells' morphology and function.

Sexual dimorphism in plumage is widespread among avian species. In chickens, adult females exhibit countershading, characterized by dull-colored round feathers lacking fringe on the saddle, while adult males display vibrant plumage with deeply fringed bright feathers. This dimorphism is estrogen-dependent, and administering estrogen to males transforms their showy plumage into cryptic female-like plumage. Extensive studies have shown that estrogen's role in female plumage formation requires thyroid hormone; however, the precise mechanisms of their interaction remain unclear. In this study, we investigated the roles of estrogen and thyroid hormone in creating sexual dimorphism in the structure and coloration of saddle feathers by administering each hormone to adult males and observing the resulting changes in regenerated feathers induced by plucking. RT-PCR analysis revealed that the expression of type 3 deiodinase (DIO3), responsible for thyroid hormone inactivation, correlates with fringing. Estrogen suppressed DIO3 and agouti signaling protein (ASIP) expression while stimulating BlSK1, a marker of barbule cells, resulting in female-like feathers with mottled patterns and lacking fringes. Administration of thyroxine (T4) stimulated BlSK1 and proopiomelanocortin (POMC) expression, with no effect on ASIP, leading to the formation of solid black feathers lacking fringes. Triiodothyronine (T3) significantly increased POMC expression in pulp cells in culture. Taken together, these findings suggest that estrogen promotes the formation of solid vanes by suppressing DIO3 expression, while also inducing the formation of mottled patterns through inhibition of ASIP expression and indirect stimulation of melanocortin expression via changes in local T3 concentration. This is the first report describing molecular mechanism underlying hormonal crosstalk in creating sexual dimorphism in feathers.

Fat mass and obesity associated gene (FTO) has been strongly associated with obesity, and it is functionally linked to the homeobox transcription factor iriquois-3 (IRX3). In mammals, FTO and IRX3 are involved in the regulation of food intake and metabolism. This study aimed to determine whether FTO and IRX3are affected by feeding and food unavailability. FTO and IRX3 mRNA and protein were found widely distributed in all tissues examined, including the brain, muscle, gut, and liver. Postprandial increase in the abundance of FTO and IRX3 mRNAs was observed in metabolic tissues of both male and female zebrafish at 1 h post-feeding. Meanwhile, their expression in the brain and gut decreased at 3 h post-feeding, reaching preprandial levels. Additionally, FTO and IRX3 mRNA abundance in examined tissues increased after 7 days of food deprivation, but substantially decreased after refeeding for 24 h. In summary, we report that both FTO and IRX3 are meal-sensitive genes in zebrafish. The fasting-induced increase suggests a possible appetite regulatory role for FTO and IRX3 in zebrafish. These findings highlight the importance of FTO and IRX3 in appetite and metabolic regulation in zebrafish.