Mammalian Function Research Articles

A saposin domain-containing protein of tongue sole Cynoglossus semilaevis: Antimicrobial activity and mechanism

Prosaposin is a precursor that can be processed into four different saposins, designated as A, B, C, and D, which have multiple functions in mammals, including neuroprotection and immune modulation. The immune function of saposin in teleost remains largely unknown. In the present study, a saposin (SAP) domain-containing protein was identified in half-smooth tongue sole Cynoglossus semilaevis and named CsSDP. CsSDP harbors one SAP A domain and two SAP B domains. When expressed in HEK293T cells, CsSDP was specifically localized in the lysosome. When overexpressed in Escherichia coli, CsSDP markedly inhibited bacterial growth, and the inhibitory effect depended on two specific regions in the SAP A and SAP B domains. Two polypeptides (P32 and P30) derived from the above SAP A and B domains could bind to and inhibit the growth of both Gram-negative and Gram-positive bacteria. The ultrastructural analysis revealed that P32 and P30 killed target bacteria by disrupting the bacterial cell wall and inducing substantial release of cytoplasmic contents. These results shed new lights on the immune function of saposin domain-containing protein in teleost.

Evolutionary conservation of Zinc-Activated Channel (ZAC) functionality in mammals: a range of mammalian ZACs assemble into cell surface-expressed functional receptors.

In contrast to the other pentameric ligand-gated ion channels in the Cys-loop receptor superfamily, the ZACN gene encoding for the Zinc-Activated Channel (ZAC) is exclusively found in the mammalian genome. Human ZAC assembles into homomeric cation-selective channels gated by Zn2+, Cu2+ and H+, but the function of the receptor in human physiology is presently poorly understood. In this study, the degree of evolutionary conservation of a functional ZAC in mammals was probed by investigating the abilities of a selection of ZACs from 10 other mammalian species than human to be expressed at the protein level and assemble into cell surface-expressed functional receptors in mammalian cells and in Xenopus oocytes. In an enzyme-linked immunosorbent assay, transient transfections of tsA201 cells with cDNAs of hemagglutinin (HA)-epitope-tagged versions of these 10ZACs resulted in robust total expression and cell surface expression levels of all proteins. Moreover, injection of cRNAs for 6 of these ZACs in oocytes resulted in the formation of functional receptors in two-electrode voltage-clamp recordings. The ZACs exhibited robust current amplitudes in response to Zn2+ (10mM) and H+ (pH 4.0), and the concentration-response relationships displayed by Zn2+ at these channels were largely comparable to that at human ZAC. In conclusion, the findings suggest that the functionality of ZAC at the molecular level may be conserved throughout mammalian species, and that the channel thus may govern physiological functions in mammals, including humans.

Editing the Genome of the Golden Hamster (Mesocricetus auratus).

The golden (Syrian) hamster (Mesocricetus auratus) is a small rodent belonging to the Cricetidae family. Golden hamsters have several unique characteristics that are advantageous in the study of reproductive and developmental biology: a highly stable 4-day estrous cycle, a high responsiveness to conventional superovulation methods, and a shortest gestation period (16days) known among eutherian mammals. Besides these advantages, the technical ease of in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) in this species has contributed much to our understanding of the basic mechanisms of mammalian fertilization. However, the exceptionally strong in vitro developmental block of hamster embryos, especially at the two-cell stage, has hampered the production of genetically modified hamsters, which has resulted in limited use of this species for biomedical research. However, the recently developed in vivo genome editing method (improved genome editing via oviductal nucleic acid delivery, i-GONAD) has overcome this shortcoming and made production of gene-edited hamsters much easier than before. This method has the potential to provide a means of reexamining genes whose functions cannot be identified using mouse models, thus leading to the better understanding of gene functions in mammals. In this chapter, we present our procedure for editing the genome of the golden hamster using i-GONAD.

Cisplatin attenuates taste cell homeostasis and induces inflammatory activation in the circumvallate papilla.

Rationale: Gustation is important to several biological functions in mammals. However, chemotherapy drugs often harm taste perception in cancer patients, while the underlying mechanism is still unclear for most drugs and there is no effective way to restore taste function. This study investigated the effects of cisplatin on the taste cell homeostasis and gustatory function. Methods: We used both mice and taste organoid models to study the effect of cisplatin on taste buds. Gustometer assay, gustatory nerve recording, RNA-Sequencing, quantitative PCR, and immunohistochemistry was performed to analyze the cisplatin-induced alteration in taste behavior and function, transcriptome, apoptosis, cell proliferation and taste cell generation. Results: Cisplatin inhibited proliferation and promoted apoptosis in the circumvallate papilla, leading to significant impairment in taste function and receptor cell generation. The transcriptional profile of genes associated with cell cycle, metabolic process and inflammatory response was significantly altered after cisplatin treatment. Cisplatin inhibited growth, promoted apoptosis, and deferred taste receptor cell differentiation in taste organoids. LY411575, a γ-secretase inhibitor, reduced the number of apoptotic cells and increased the number of proliferative cells and taste receptor cells, potentially suggesting as a taste tissue protective agent against chemotherapy. LY411575 treatment could offset the increased number of Pax1+ or Pycr1+ cells induced by cisplatin in the circumvallate papilla and taste organoids. Conclusion: This study highlights the inhibitory effects of cisplatin on taste cell homeostasis and function, identifies critical genes and biological processes regulated by chemotherapy, and proposes potential therapeutic targets and strategy for taste dysfunction in cancer patients.

Polymyxin B neutralizes endotoxin and improves the quality of chicken semen during liquid storage

Despite its importance in gamete utilization for livestock production, poultry semen cryopreservation in a liquid state, is limited in the poultry industry due to a significant decline in sperm viability and functionality during liquid storage. Lipopolysaccharide (LPS) is released from gram-negative bacteria and impairs sperm function in mammals. Using exogeneous LPS, we show this endotoxin compromises sperm viability and function, including motility and penetrability to the inner peri-vitelline layer (IPVL) during liquid storage at 4 °C. This outcome was supported by LPS quantification showing an extreme increase in the first 24 h of storage. Polymyxin B (PMB) is an LPS neutralizer previously shown to improve fertility in boar semen, thus we explored the effect of PMB on chicken semen quality during liquid storage. Sperm viability and penetrability tests showed that PMB completely abolishes the deleterious effect by LPS. However, co-addition of PMB with penicillin G (PenG), an antibiotic against gram positive bacteria, reduces IPVL-penetrability while improving sperm viability post-storage. Furthermore, artificial insemination trials showed that PMB addition improves semen fertility at the post liquid storage. Our results show that chicken semen quality during liquid storage is impaired by bacterial LPS, but improved by PMB addition due to cancelled endotoxic effects, which offers a new approach for prolonged fertility of poultry semen storage in a liquid state.

In-silico identification of novel inhibitors for human Aurora kinase B form the ZINC database using molecular docking-based virtual screening

Introduction: Aurora kinase enzymes play critical functions in mammals. Aurora kinases are mitotic regulators that are involved in a variety of processes during cell division. The overexpression of these enzymes is associated with tumour formation and is symptomatic of clinical circumstances in cancer patients who have been diagnosed.
 Materials and methods: The current study reports an in-silico virtual screening (VS) and molecular docking analysis of 2500 compounds retrieved from the ZINC database and five current clinical trial compounds against Aurora Kinase B using AutoDock Vina to identify potential inhibitors.
 Results and discussion: The top six compounds that resulted from the screening were ZINC00190959, ZINC07889110, ZINC0088285, ZINC01404326, ZINC00882846 and ZINC08813187, which showed lower free energy of binding (FEB) against the target protein binding pocket. The FEB were as follows: -11.92, -11.85, -11.46, -11.33, -11.21 and -11.1 kcal/mol, using AutoDock, and -11.7, -11.5, -11.2, -11.0, -10.8 and -10.6 kcal/mol for AutoDock Vina, respectively. These findings were superior to those obtained with the co-crystallized ligand VX-680, with a -7.5 kcal/mol and the current clinical trial drug. Finally, using a VS and molecular docking approach, novel Aurora kinase B inhibitors were effectively identified from the ZINC database fulfilling the Lipinski rule of five with low FEB and functional molecular interactions with the target protein.
 Conclusion: The findings suggest that the six compounds could be used as a potential agent for cancer treatments.
 Graphical abstract:

Sulphide donors affect the expression of mucin and sulphide detoxification genes in the mucosal organs of Atlantic salmon (Salmo salar).

Hydrogen sulphide (H2S) is a gas that affects mucosal functions in mammals. However, its detrimental effects are less understood in fish despite being known to cause mass mortality. Here we used explant models to demonstrate the transcriptional responses of Atlantic salmon (Salmo salar) mucosa to the sulphide donor sodium hydrosulphide (NaHS). The study focused on two groups of genes: those encoding for sulphide detoxification and those for mucins. Moreover, we performed pharmacological studies by exposing the organ explants to mucus-interfering compounds and consequently exposed them to a sulphide donor. Exposure to NaHS significantly affected the expression of sulphide:quinone oxidoreductase (sqor1, sqor2) and mucin-encoding genes (muc5ac, muc5b). The general profile indicated that NaHS upregulated the expression of sulphide detoxification genes while a significant downregulation was observed with mucins. These expression profiles were seen in both organ explant models. Pharmacological stimulation and inhibition of mucus production used acetylcholine (ACh) and niflumic acid (NFA), respectively. This led to a significant regulation of the two groups of marker genes in the gills and olfactory rosette explants. Treatment of the mucosal organ explants with the mucus-interfering compounds showed that low dose NFA triggered more substantial changes while a dose-dependent response could not be established with ACh. Pharmacological interference demonstrated that mucins played a crucial role in mucosal protection against H2S toxicity. These results offer insights into how a sulphide donor interfered with mucosal responses of Atlantic salmon and are expected to contribute to our understanding of the least explored H2S-fish interactions-particularly at the mucosa.

Selenium deficiency increased duodenal permeability and decreased expression of antimicrobial peptides by activating ROS/NF-κB signal pathway in chickens.

Selenium (Se) is an essential trace element for the body. Various organs of the body, including the intestine, are affected by its deficiency. Se deficiency can induce oxidative stress and inflammatory responses in the intestine. It can also increase intestinal permeability and decrease intestinal immune function in mammals. However, the detailed studies, conducted on the intestinal molecular mechanisms of Se deficiency-induced injury in poultry, are limited. This study explored the adverse effects of Se deficiency on intestinal permeability and its mechanism. A Se-deficient chicken model was established, and the morphological changes in the chicken duodenum tissues were observed using a light microscope and transmission electron microscope (TEM). Western blotting, qRT-PCR, and other methods were used to detect the expression levels of selenoproteins, oxidative stress indicators, inflammatory factors, tight junction (TJ) proteins, antimicrobial peptides, and other related indicators in intestinal tissues. The results showed that Se deficiency could decrease the expression levels of selenoproteins and antioxidant capacity, activate the nuclear factor kappa-B (NF-κB) pathway, cause inflammation, and decrease the expression levels of TJ proteins and antimicrobial peptides in the duodenum tissues. The study also demonstrated that Se deficiency could increase intestinal permeability and decrease antimicrobial peptides via reactive oxygen species (ROS)/NF-κB. This study provided a theoretical basis for the scientific prevention and control of Se deficiency in poultry. Se deficiency decreased the expression levels of selenoproteins and increased ROS levels to activate the NF-κB pathway, resulting in the production of pro-inflammatory cytokines, reducing the expression levels of TJ protein, and weakening the expression of antimicrobial peptides, which contributed to the higher intestinal permeability. Oxidative stress weakened the expression of antimicrobial peptides.

Mammalian CDC14 phosphatases control exit from stemness in pluripotent cells.

Maintenance of stemness is tightly linked to cell cycle regulation through protein phosphorylation by cyclin-dependent kinases (CDKs). However, how this process is reversed during differentiation is unknown. We report here that exit from stemness and differentiation of pluripotent cells along the neural lineage are controlled by CDC14, a CDK-counteracting phosphatase whose function in mammals remains obscure. Lack of the two CDC14 family members, CDC14A and CDC14B, results in deficient development of the neural system in the mouse and impairs neural differentiation from embryonic stem cells (ESCs). Mechanistically, CDC14 directly dephosphorylates specific proline-directed Ser/Thr residues of undifferentiated embryonic transcription Factor 1 (UTF1) during the exit from stemness, triggering its proteasome-dependent degradation. Multiomic single-cell analysis of transcription and chromatin accessibility in differentiating ESCs suggests that increased UTF1 levels in the absence of CDC14 prevent the proper firing of bivalent promoters required for differentiation. CDC14 phosphatases are dispensable for mitotic exit, suggesting that CDC14 phosphatases have evolved to control stemness rather than cell cycle exit and establish the CDK-CDC14 axis as a critical molecular switch for linking cell cycle regulation and self-renewal.

Targeting Abnormal PI3K/AKT/mTOR Signaling in Intracerebral Hemorrhage: A Systematic Review on Potential Drug Targets and Influences of Signaling Modulators on Other Neurological Disorders.

PI3K/AKT/mTOR (phosphoinositide 3-kinase/protein kinase B/mammalian target of rapamycin) signaling pathway is an important signal transduction pathway mediated by enzyme-linked receptors with many biological functions in mammals. This pathway modulates the epigenetic modification of DNA and target gene histones and plays a significant role in regulating biological activity, disease progression, oncogenesis, and cancer progression. PI3K/AKT/mTOR signaling pathway involves and mediates many cellular processes such as nutrient uptake, proliferation, anabolic reactions, and cell survival. Several studies have shown that PI3K/AKT/mTOR has been a promising therapeutic approach to intracerebral hemorrhage (ICH). ICH is characterized by the progressive development of hematoma, which leads to the structural destabilization of the neurons and glial cells, leading to neuronal deformation, further contributing to mitochondrial dysfunction, membrane depolarization, oligaemia, and neurotransmitter imbalance. Partial suppression of cell metabolism and necrosis can occur, depending on the degree of mitochondrial dysfunction. Therefore in the following review, we discuss whether or not the activation of the PI3K/AKT/mTOR signaling pathway could minimize neuronal dysfunction following ICH. We further elaborate the review by discussing the updated pathophysiology of brain hemorrhage and the role of molecular targets in other neurodegenerative diseases. This review provides current approachable disease treatment in various disease states, single and dual PI3K/AKT/mTOR signaling pathway modulators.

The cold-sensing ion channel TRPM8 regulates central and peripheral clockwork and the circadian oscillations of body temperature.

Physiological functions in mammals show circadian oscillations, synchronized by daily cycles of light and temperature. Central and peripheral clocks participate in this regulation. Since the ion channel TRPM8 is a critical cold sensor, we investigated its role in circadian function. We used TRPM8 reporter mouse lines and TRPM8-deficient mice. mRNA levels were determined by in situ hybridization or RT-qPCR and protein levels by immunofluorescence. A telemetry system was used to measure core body temperature (Tc). TRPM8 is expressed in the retina, specifically in cholinergic amacrine interneurons and in a subset of melanopsin-positive ganglion cells which project to the central pacemaker, the suprachiasmatic nucleus (SCN) of the hypothalamus. TRPM8-positive fibres were also found innervating choroid and ciliary body vasculature, with a putative function in intraocular temperature, as shown in TRPM8-deficient mice. Interestingly, Trpm8-/- animals displayed increased expression of the clock gene Per2 and vasopressin (AVP) in the SCN, suggesting a regulatory role of TRPM8 on the central oscillator. Since SCN AVP neurons control body temperature, we studied Tc in driven and free-running conditions. TRPM8-deficiency increased the amplitude of Tc oscillations and, under dim constant light, induced a greater phase delay and instability of Tc rhythmicity. Finally, TRPM8-positive fibres innervate peripheral organs, like liver and white adipose tissue. Notably, Trpm8-/- mice displayed a dysregulated expression of Per2 mRNA in these metabolic tissues. Our findings support a function of TRPM8 as a temperature sensor involved in the regulation of central and peripheral clocks and the circadian control of Tc.

Plant N-acylethanolamines play a crucial role in defense and its variation in response to elevated CO2 and temperature in tomato.

The ubiquitous lipid-derived molecules N-acylethanolamines (NAEs) have multiple immune functions in mammals, but their roles and mechanisms in plant defense response during changing environment remain largely unclear. Here, we found that exogenous NAE18:0 and NAE18:2 promoted defense against the necrotrophic pathogen Botrytis cinerea but suppressed defense to the hemi-biotrophic pathogen Pseudomonas syringae pv. tomato (Pst) DC3000 in tomato. The knocking-down and overexpression function analysis of the pathogen-responsive NAE synthetic gene PHOSPHOLIPASE Dγ (PLDγ) and hydrolytic gene FATTY ACID AMID HYDROLASE 1 (FAAH1) revealed that the NAE pathway is crucial for plant defense response. Using exogenous applications and SA-abolished NahG plants, we unveiled the antagonistic relationship between NAE and SA in plant defense response. Elevated CO2 and temperature significantly changed the NAE pathway in response to pathogens, while inhibition of the NAE pathway led to the alternation of environment-mediated defense variations against Pst DC3000 in tomato, indicating that NAE pathway is associated with plant defense variations in response to elevated CO2 and temperature. The results herein reveal a new function of NAE in plant defense, and its involvement in environment-mediated defense variation in tomato. These findings shed light on the NAE-based plant defense, which may have relevance to crop disease management in future changing climate.

CDK8 and CDK19 regulate intestinal differentiation and homeostasis via the chromatin remodeling complex SWI/SNF.

Initiation and maintenance of transcriptional states are critical for controlling normal tissue homeostasis and differentiation. The cyclin dependent kinases CDK8 and CDK19 (Mediator kinases) are regulatory components of Mediator, a highly conserved complex that orchestrates enhancer-mediated transcriptional output. While Mediator kinases have been implicated in the transcription of genes necessary for development and growth, its function in mammals has not been well defined. Using genetically defined models and pharmacological inhibitors, we showed that CDK8 and CDK19 function in a redundant manner to regulate intestinal lineage specification in humans and mice. The Mediator kinase module bound and phosphorylated key components of the chromatin remodeling complex switch/sucrose non-fermentable (SWI/SNF) in intestinal epithelial cells. Concomitantly, SWI/SNF and MED12-Mediator colocalized at distinct lineage-specifying enhancers in a CDK8/19–dependent manner. Thus, these studies reveal a transcriptional mechanism of intestinal cell specification, coordinated by the interaction between the chromatin remodeling complex SWI/SNF and Mediator kinase.

Hydrogen Sulfide and Its Donors: Keys to Unlock the Chains of Nonalcoholic Fatty Liver Disease.

Hydrogen sulfide (H2S) has emerged as the third “gasotransmitters” and has a crucial function in the diversity of physiological functions in mammals. In particular, H2S is considered indispensable in preventing the development of liver inflammation in the case of excessive caloric ingestion. Note that the concentration of endogenous H2S was usually low, making it difficult to discern the precise biological functions. Therefore, exogenous delivery of H2S is conducive to probe the physiological and pathological roles of this gas in cellular and animal studies. In this review, the production and metabolic pathways of H2S in vivo, the types of donors currently used for H2S release, and study evidence of H2S improvement effects on nonalcoholic fatty liver disease are systematically introduced.

Single-cell epigenome analysis reveals age-associated decay of heterochromatin domains in excitatory neurons in the mouse brain

Loss of heterochromatin has been implicated as a cause of pre-mature aging and age-associated decline in organ functions in mammals; however, the specific cell types and gene loci affected by this type of epigenetic change have remained unclear. To address this knowledge gap, we probed chromatin accessibility at single-cell resolution in the brains, hearts, skeletal muscles, and bone marrows from young, middle-aged, and old mice, and assessed age-associated changes at 353,126 candidate cis-regulatory elements (cCREs) across 32 major cell types. Unexpectedly, we detected increased chromatin accessibility within specific heterochromatin domains in old mouse excitatory neurons. The gain of chromatin accessibility at these genomic loci was accompanied by the cell-type-specific loss of heterochromatin and activation of LINE1 elements. Immunostaining further confirmed the loss of the heterochromatin mark H3K9me3 in the excitatory neurons but not in inhibitory neurons or glial cells. Our results reveal the cell-type-specific changes in chromatin landscapes in old mice and shed light on the scope of heterochromatin loss in mammalian aging.

New roles for GAPDH, Hsp90, and NO in regulating heme allocation and hemeprotein function in mammals.

The intracellular trafficking of mitochondrial heme presents a fundamental challenge to animal cells. This article provides some background on heme allocation, discusses some of the concepts, and then reviews research done over the last decade, much in the author's laboratory, that is uncovering unexpected and important roles for glyceraldehyde 3-phosphate dehydrogenase (GAPDH), heat shock protein 90 (hsp90), and nitric oxide (NO) in enabling and regulating the allocation of mitochondrial heme to hemeproteins that mature and function outside ofthe mitochondria. A model for how hemeprotein functions can be regulated in cells through the coordinate participation of GAPDH, hsp90, and NO in allocating cellular heme is presented.

The Role of Plant Progesterone in Regulating Growth, Development, and Biotic/Abiotic Stress Responses.

Progesterone is a steroid hormone that performs important functions in mammals. However, studies on its physiological functions in plants have gradually increased in recent years. Therefore, this review summarizes the regulatory functions of progesterone on plant growth and development, as well as its response to stress. Moreover, the plant metabolic processes of progesterone are also discussed. Overall, progesterone is ubiquitous in plants and can regulate numerous plant physiological processes at low concentrations. Since progesterone shares similar characteristics with plant hormones, it is expected to become a candidate for plant hormone. However, most of the current research on progesterone in plants is limited to the physiological level, and more molecular level research is needed to clarify progesterone signaling pathways.

Cumulative risk assessment of phthalates exposure for recurrent pregnancy loss in reproductive-aged women population using multiple hazard indices approaches

Background and aim: Phthalates, which are commonly used in flexible plastics and consumer products, have been reported to be toxic to reproductive and developmental function in mammals. Past studies have focused on the toxic effects on male reproduction, with only a few studies conducted on the risks that cumulative exposure to phthalates have on the female reproductive system. Methods: We recruited 260 patients with recurrent pregnancy loss (RPL) of unknown etiology and 203 controls from the clinics of Obstetrics and Gynecology at a medical center in southern Taiwan from 2013 to 2020. The daily intake of phthalates was estimated from urine samples using the back-calculation method, after which the cumulative risk was determined using multiple hazard indices, including a dose-addition model, a receptor effect model, and a hazard index approach. Results: The patients with RPL had a significantly higher cumulative exposure to phthalates (p < 0.05) than did the controls with a hazard index above one. After adjusted logistic regression analysis, we found that the risk of RPL was strongly related to the higher quartiles of DEHP, the DEHPTEQ for the antiandrogenic effect and adverse effects of the female reproductive system and the ERα binding effect (p < 0.05). Conclusions: Our work suggests that more attentions should be paid to the adverse effects induced by phthalates on female reproduction, especially the effects caused by the cumulative exposure to phthalates in women of reproductive age. Keywords: Phthalates; Cumulative risk assessment; Relative potency factor; Recurrent pregnancy loss

Evolution of calcitonin/calcitonin gene-related peptide family in chordates: Identification of CT/CGRP family peptides in cartilaginous fish genome

The calcitonin (CT)/CT gene-related peptide (CGRP) family is a peptide gene family that is widely found in bilaterians. CT, CGRP, adrenomedullin (AM), amylin (AMY), and CT receptor-stimulating peptide (CRSP) are members of the CT/CGRP family. In mammals, CT is involved in calcium homeostasis, while CGRP and AM primarily function in vasodilation. AMY and CRSP are associated with anorectic effects. Diversification of the molecular features and physiological functions of the CT/CGRP family in vertebrate lineages have been extensively reported. However, the origin and diversification mechanisms of the vertebrate CT/CGRP family of peptides remain unclear. In this review, the molecular characteristics of CT/CGRP family peptides and their receptors, along with their major physiological functions in mammals and teleosts, are introduced. Furthermore, novel candidates of the CT/CGRP family in cartilaginous fish are presented based on genomic information. The CT/CGRP family peptides and receptors in urochordates and cephalochordates, which are closely related to vertebrates, are also described. Finally, a putative evolutionary scenario of the CT/CGRP family peptides and receptors in chordates is discussed.

Spexin mRNA profile and its response to different photoperiods in Chinese Yangzhou geese (Anas cygnoides).

Spexin (SPX, NPQ), a novel neuropeptide composed of 14 amino acid residues, is evolutionarily conserved among different species. Spexin has been suggested to have pleiotropic functions in mammals. However, reports on spexin in birds are limited. To clarify the role of spexin in goose reproduction, the spexin gene was cloned and analyzed. Analysis of tissue distribution by RT-PCR showed that the expression of spexin and its two receptors was widespread. During the long photoperiod, the expression levels of spexin in the pituitary and hypothalamus and of GALR2/3 in the pituitary decreased, and the GnRH, LHβ, and FSHβ expression levels increased significantly. This suggests that a long photoperiod regulates reproductive activities by activating the gonadotrope-axis, which is modulated by decreased spexin levels.