Exogenous Hemin enhances the antioxidant defense system of rice by regulating the AsA-GSH cycle under NaCl stress.

Abiotic stress caused by soil salinization remains a major global challenge that threatens and severely impacts crop growth, causing yield reduction worldwide. In this study, we aim to investigate the damage of salt stress on the leaf physiology of two varieties of rice (Huanghuazhan, HHZ, and Xiangliangyou900, XLY900) and the regulatory mechanism of Hemin to maintain seedling growth under the imposed stress. Rice leaves were sprayed with 5.0 µmol·L-1 Hemin or 25.0 µmol·L-1 ZnPP (Zinc protoporphyrin IX) at the three leaf and one heart stage, followed by an imposed salt stress treatment regime (50.0 mmol·L-1 sodium chloride (NaCl)). The findings revealed that NaCl stress increased antioxidant enzymes activities and decreased the content of nonenzymatic antioxidants such as ascorbate (AsA) and glutathione (GSH). Furthermore, the content of osmoregulatory substances like soluble proteins and proline was raised. Moreover, salt stress increased reactive oxygen species (ROS) content in the leaves of the two varieties. However, spraying with Hemin increased the activities of antioxidants such as superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) and accelerated AsA-GSH cycling to remove excess ROS. In summary, Hemin reduced the effect of salt stress on the physiological characteristics of rice leaves due to improved antioxidant defense mechanisms that impeded lipid peroxidation. Thus, Hemin was demonstrated to lessen the damage caused by salt stress.

TAC3 regulates GnRH/gonadotropin synthesis in female chickens.

Neurokinin B (NKB), a peptide encoded by the tachykinin 3 (TAC3), is critical for reproduction in all studied species. However, its potential roles in birds are less clear. Using the female chicken (c-) as a model, we showed that cTAC3 is composed of five exons with a full-length cDNA of 787 bp, which was predicted to generate the mature NKB peptide containing 10 amino acids. Using cell-based luciferase reporter assays, we demonstrated that cNKB could effectively and specifically activate tachykinin receptor 3 (TACR3) in HEK293 cells, suggesting its physiological function is likely achieved via activating cTACR3 signaling. Notably, cTAC3 and cTACR3 were predominantly and abundantly expressed in the hypothalamus of hens and meanwhile the mRNA expression of cTAC3 was continuously increased during development, suggesting that NKB-TACR3 may emerge as important components of the neuroendocrine reproductive axis. In support, intraperitoneal injection of cNKB could significantly promote hypothalamic cGnRH-Ι, and pituitary cFSHß and cLHß expression in female chickens. Surprisingly, cTAC3 and cTACR3 were also expressed in the pituitary gland, and cNKB treatment significantly increased cLHß and cFSHß expression in cultured primary pituitary cells, suggesting cNKB can also act directly at the pituitary level to stimulate gonadotropin synthesis. Collectively, our results reveal that cNKB functionally regulate GnRH/gonadotropin synthesis in female chickens.

Hepatic-Specific FGF21 Knockout Abrogates Ovariectomy-Induced Obesity by Reversing Corticosterone Production.

Increased glucocorticoid (GC) levels act as a master contributor to central obesity in estrogen-depleted females; however, what factors cause their increased GC production is unclear. Given (1) liver fibroblast growth factor 21 (FGF21) and GCs regulate each other's production in a feed-forward loop, and (2) circulating FGF21 and GCs are parallelly increased in menopausal women and ovariectomized mice, we thus hypothesized that elevation of hepatic FGF21 secretion causes increased GGs production in estrogen-depleted females. Using the ovariectomized mice as a model for menopausal women, we found that ovariectomy (OVX) increased circulating corticosterone levels, which in turn increased visceral adipose Hsd11b1 expression, thus causing visceral obesity in females. In contrast, liver-specific FGF21 knockout (FGF21 LKO) completely reversed OVX-induced high GCs and high visceral adipose Hsd11b1 expression, thus abrogating OVX-induced obesity in females. Even though FGF21 LKO failed to rescue OVX-induced dyslipidemia, hepatic steatosis, and insulin resistance. What's worse, FGF21 LKO even further exacerbated whole-body glucose metabolic dysfunction as evidenced by more impaired glucose and pyruvate tolerance and worsened insulin resistance. Mechanically, we found that FGF21 LKO reduced circulating insulin levels, thus causing the dissociation between decreased central obesity and the improvement of obesity-related metabolic syndromes in OVX mice. Collectively, our results suggest that liver FGF21 plays an essential role in mediating OVX-induced central obesity by promoting GC production. However, lack of liver FGF21 signaling reduces insulin production and in turn causes the dissociation between decreased central obesity and the improvement of obesity-related metabolic syndromes, highlighting a detrimental role for hepatic FGF21 signals in mediating the development of central obesity but a beneficial role in preventing metabolic abnormality from further exacerbation in estrogen-depleted females.

Active immunization against gonadotropin-releasing hormone affects thymic T cell production, migration, and colonization in male rat lymphoid tissue.

Active immunization against gonadotropin-releasing hormone (GnRH) inhibits animal reproduction and has become a friendly alternative to surgical castration, which has been reported to affect the proportion of thymic T cell subpopulations. The effects of active immunization against GnRH on T cell migration from the thymus to the periphery and T cell distribution in lymphoid tissues remain unclear. Here, we showed that active immunization against GnRH increased thymic size and weight, enlarged the number of thymocytes, and enhanced CD4+ recent thymic emigrants (RTEs) and CD8+ RTEs migration to the blood and spleen. Active immunization against GnRH had no significant effect on naïve CD4+, naïve CD8+, CD4+ memory/activated, or CD8+ memory/activated T cells. In addition, active immunization against GnRH increased the proportion of CD3+ T cells in the spleen and lymph nodes. The percentages of CD3+CD4+ and CD3+CD8+ T cells in the blood, spleen, and lymph nodes were not significantly affected by GnRH immunization. Overall, these results enhance our understanding of thymic T cell production, migration, and colonization in rat lymphoid tissues affected by GnRH immunization.

Efficacy of Immunization against a Novel Synthetic 13-Amino Acid Betaglycan-Binding Peptide Sequence of Inhibin α Subunit on Promoting Fertility in Female Rats.

Inhibins suppress the FSH production in pituitary gonadotrope cells by robustly antagonizing activin signaling by competitively binding to activin type II receptors (ACTR II). The binding of inhibin A to ACTR II requires the presence of its co-receptor, namely, betaglycan. In humans, the critical binding site for betaglycan to inhibin A was identified on the inhibin α subunit. Through conservation analysis, we found that a core 13-amino-acid peptide sequence within the betaglycan-binding epitope on human inhibin α subunit is highly conserved across species. Based on the tandem sequence of such a conserved 13-amino-acid betaglycan-binding epitope (INHα13AA-T), we developed a novel inhibin vaccine and tested its efficacy in promoting female fertility using the female rat as a model. Compared with placebo-immunized controls, INHα13AA-T immunization induced a marked (p < 0.05) antibody generation, enhanced (p < 0.05) ovarian follicle development, and increased ovulation rate and litter sizes. Mechanistically, INHα13AA-T immunization promoted (p < 0.05) pituitary Fshb transcription and increased (p < 0.05) serum FSH and 17ß-estradiol concentrations. In summary, active immunization against INHα13AA-T potently increased FSH levels, ovarian follicle development, ovulation rate and litter sizes, thus causing super-fertility in females. Therefore, immunization against INHα13AA is a promising alternative to the conventional approach of multiple ovulation and super-fertility in mammals.

Spexin2 Is a Novel Food Regulator in Gallus gallus.

Spexin2 (SPX2), a paralog of SPX1, is a newly identified gene in non-mammalian vertebrates. Limited studies in fish have evidenced its important role in food intake and energy balance modulation. However, little is known about its biological functions in birds. Using the chicken (c-) as a model, we cloned the full-length cDNA of SPX2 by using RACE-PCR. It is 1189 base pair (bp) in length and predicted to generate a protein of 75 amino acids that contains a 14 amino acids mature peptide. Tissue distribution analysis showed that cSPX2 transcripts were detected in a wide array of tissues, with abundant expression in the pituitary, testis, and adrenal gland. cSPX2 was also observed to be ubiquitously expressed in chicken brain regions, with the highest expression in the hypothalamus. Its expression was significantly upregulated in the hypothalamus after 24 or 36 h of food deprivation, and the feeding behavior of chicks was obviously suppressed after peripheral injection with cSPX2. Mechanistically, further studies evidenced that cSPX2 acts as a satiety factor via upregulating cocaine and amphetamine regulated transcript (CART) and downregulating agouti-related neuropeptide (AGRP) in hypothalamus. Using a pGL4-SRE-luciferase reporter system, cSPX2 was demonstrated to effectively activate a chicken galanin II type receptor (cGALR2), a cGALR2-like receptor (cGALR2L), and a galanin III type receptor (cGALR3), with the highest binding affinity for cGALR2L. Collectively, we firstly identified that cSPX2 serves as a novel appetite monitor in chicken. Our findings will help clarify the physiological functions of SPX2 in birds as well as its functional evolution in vertebrates.

Corticosterone stage-dependently inhibits progesterone production presumably via impeding the cAMP-StAR cascade in granulosa cells of chicken preovulatory follicles.

Stress can suppress reproduction capacity in either wild or domestic animals, but the exact mechanism behind it, especially in terms of steroidogenesis, remains under-investigated so far. Considering the important roles of progesterone in avian breeding, we investigated the modulation of corticosterone on progesterone production in cultured granulosa cells of chicken follicles at different developmental stages. Using enzyme immunoassays, our study showed that corticosterone could only inhibit progesterone synthesis in granulosa cells from F5-6, F4, and F3 follicles, but not F2 and F1 follicles. Coincidentally, both quantitative real-time PCR and western blotting revealed that corticosterone could downregulate steroidogenic acute regulatory protein (StAR) expression, suggesting the importance of StAR in corticosterone-related actions. Using the dual-luciferase reporter system, we found that corticosterone can potentially enhance, rather than inhibit, the activity of StAR promoter. Of note, combining high-throughput transcriptomic analysis and quantitative real-time PCR, phosphodiesterase 10A (PDE10A), protein kinase cAMP-dependent type II regulatory subunit alpha (PRKAR2A) and cAMP responsive element modulator (CREM) were identified to exhibit the differential expression patterns consistent with cAMP blocking in granulosa cells from F5-6, F4, and F3, but not F2 and F1 follicles. Afterward, the expression profiles of these genes in granulosa cells of distinct developmental-stage follicles were examined by quantitative real-time PCR, in which all of them expressed correspondingly with progesterone levels of granulosa cells during development. Collectively, these findings indicate that corticosterone can stage-dependently inhibit progesterone production in granulosa cells of chicken preovulatory follicles, through impeding cAMP-induced StAR activity presumptively.

Evidence for progesterone acting as an inhibitor of stress axis via stimulating pituitary neuropeptide B/W receptor 2 (NPBWR2) expression in chickens.

In vertebrates, the hypothalamus-pituitary-adrenal gland (HPA) axis is the main endocrine pathway regulating the stress response, thus also called the stress axis. It has been well-accepted that the stress axis is tightly controlled by both hypothalamic stimulators and inhibitors [e.g. corticotropin (ACTH)-releasing inhibitory factor (CRIF)]. However, the identity of authentic CRIF remains unclear for decades. Recently, neuropeptide W (NPW) was proved to be the physiological CRIF in chickens. Together with its functional receptor (NPBWR2), they play critical roles in attenuating the activity of the chicken stress axis. Because increasing pieces of evidence suggested that sex steroids could regulate the stress axis, using chicken as a model, we investigated whether the newly identified CRIF and its receptor are under the control of sex steroids in this study. Our results showed that: (1) expression of NPW-NPBWR2 in the hypothalamus-pituitary axis was sexually dimorphic and developmental stage-dependent; (2) progesterone (P4), rather than 17ß-estradiol (E2) and dihydrotestosterone (DHT), could dose- and time-dependently upregulate NPBWR2 expression, which was accompanied with the decrease of ACTH synthesis and secretion, in cultured pituitary cells; (3) intraperitoneal injection of P4 could elevate the mRNA level of pituitary NPBWR2; (4) P4-stimulated NPBWR2 expression was relevant to both nPR-mediated genomic action and mPRs-triggered nongenomic route associated with MEK/ERK, PI3K/AKT cascade, and calcium influx. To our knowledge, our results discover a novel route of sex steroids in modulating the stress axis of chickens, which lays a foundation to reveal the complicated interaction network between reproduction and stress axes in chickens.

Two Synthetic Peptides Corresponding to the Human Follicle-Stimulating Hormone ß-Subunit Promoted Reproductive Functions in Mice.

A follicle stimulating hormone (FSH) is widely used in the assisted reproduction and a synthetic peptide corresponding to a receptor binding region of the human (h) FSH-ß-(34−37) (TRDL) modulated reproduction. Furthermore, a 13-amino acid sequence corresponding to hFSH-ß-(37−49) (LVYKDPARPKIQK) was recently identified as the receptor binding site. We hypothesized that the synthetic peptides corresponding to hFSH-ß-(37−49) and hFSH-ß-(34−49), created by merging hFSH-ß-(34−37) and hFSH-ß-(37−49), modulate the reproductive functions, with the longer peptide being more biologically active. In male or female prepubertal mice, a single injection of 200 µg/g BW ip of hFSH-ß-(37−49) or hFSH-ß-(34−49) hastened (p < 0.05) puberty, whereas the same treatments given daily for 4 d promoted (p < 0.05) the gonadal steroidogenesis and gamete formation. In addition of either peptide to the in vitro cell cultures, promoted (p < 0.05) the proliferation of primary murine granulosa cells and the estradiol production by upregulating the expression of Ccnd2 and Cyp19a1, respectively. In adult female mice, 200 µg/g BW ip of either peptide during diestrus antagonized the FSH-stimulated estradiol increase and uterine weight gain during proestrus. Furthermore, hFSH-ß-(34−49) was a more potent (p < 0.05) reproductive modulator than hFSH-ß-(37−49), both in vivo and in vitro. We concluded that hFSH-ß-(37−49) and especially hFSH-ß-(34−49), have the potential for reproductive modulation.

Effects of active immunization against a 13-amino acid receptor-binding epitope of FSHß on fertility regulation in female mice.

Follicle-stimulating hormone (FSH) is crucial for ovarian folliculogenesis and thus essential for female fertility. Here, we developed a novel FSH vaccine based on the tandem of a 13-amino acid receptor-binding epitope of FSHß (FSHß13AA-T) and used a mouse model to test its efficacy in female fertility regulation. Compared to placebo-immunized controls, FSHß13AA-T vaccination: induced a marked (P < 0.05) antibody generation; reduced (P < 0.05) serum concentrations of FSH, inhibin B and 17ß-estradiol; disrupted (P < 0.05) normal estrous cyclicity; delayed (P = 0.08) establishment of pregnancy; blocked (P < 0.05) folliculogenesis; and reduced (P < 0.05) litter size. Mechanistically, FSH vaccination reduced (P < 0.05) ovarian estrogen production by decreasing Lhcgr, Cyp19a1 and HSD3ß1 expression, and suppressed ovarian follicular development by decreasing ovarian Fshr, Inhα, Foxo3a, Bmp15 and Cdh1 expression. Overall, vaccination of female mice with FSHß13AA-T substantially disrupted FSH-dependent ovarian steroidogenesis and folliculogenesis, and caused subfertility. Therefore, vaccines based on FSHß13AA-T have potential as anti-fertility/contraceptive agents in females.

Physiological mechanisms of ABA-induced salinity tolerance in leaves and roots of rice.

Abscisic acid (ABA) plays a crucial role in response to abiotic stress as important small molecules in regulating metabolism. This study aimed to evaluate the ability of foliar spraying ABA to regulate growth quality at rice seedling stage under salt stress. Results demonstrated that salt stress strongly reduced all the growth parameters of two rice seedlings ('Chaoyouqianhao' and 'Huanghuazhan'), caused prominent decrease in the levels of photosynthetic pigments (mainly in Huanghuazhan), photosynthesis and fluorescence parameters. Salinity treatment increased the concentration of malondialdehyde (MDA) and hydrogen peroxide (H2O2) in roots, whereas significant decreased H2O2 was found in leaves of Huanghuazhan. Additionally, salinity triggered high Na+ content particularly in leaves and enhanced catalase (CAT) activities, ascorbate peroxidase (APX) and peroxidase (POD) activities of the two rice seedlings. Nevertheless, salinity-induced increased root ascorbic acid (AsA) and glutathione (GSH) levels while decreased in leaves, which depended on treatment time. Conversely, ABA application partially or completely mitigated salinity toxicity on the seedlings. ABA could reverse most of the changed physiological parameters triggered by salt stress. Specially, ABA treatment improved antioxidant enzyme levels and significantly reduced the Na+ content of two varieties as well as increased the K+, Mg2+ and Ca2+ content in leaves and roots. ABA treatment increased the hormone contents of 1-aminocclopropane carboxylic acid (ACC), trans-zeatin (TZ), N6-isopentyladenosine (IPA), Indole-3-acetic acid (IAA), and ABA in leaves of two rice varieties under salt stress. It is suggested that ABA was beneficial to protect membrane lipid peroxidation, the modulation of antioxidant defense systems and endogenous hormonal balance with imposition to salt stress.

A novel follicle-stimulating hormone vaccine for controlling fat accumulation.

Follicle-stimulating hormone (FSH) has been newly demonstrated to play a great role in promoting fat accumulation, providing a potential to target FSH for controlling fat accumulation and treating obesity. A short, 13-amino acid of FSHß (FSHß13AA) was indentified to be the FSH receptor-binding epitope in both humans and mice. By conservation analysis, we found such FSHß13AA is highly conserved across species. Accordingly, we designed a new FSH antigen by synthesizing a tandem of FSHß13AA (LVYKDPARPNIQK) and then conjugating it to ovalbumin (FSHß13AA-T-OVA). Then, we tested its efficacy in suppressing fat accumulation in both ovariectomized and intact mouse models. Vaccination with this novel antigen emulsified in mild adjuvant, Specol, was highly effective in preventing ovariectomy-induced body weight gain and fat accumulation in mice (P < 0.01). Mechanistically, FSH vaccination treatment inhibited lipid biosynthesis by inactivating PPARγ adipogenic signaling pathway and simultaneously enhanced adipocyte themogenesis via upregulating UCP1 expression in both visceral and subcutaneous adipose tissues. Moreover, injection of this novel FSH vaccine also substantially reduced (P < 0.05) fat accumulation in both intact male and female mice. These actions result from the specific binding of the generated antibody to the ß-subunit to block its action, rather than lowering the circulating levels of FSH, as evidenced by nearly no alterations in serum FSH levels in mice following FSH vaccination. Overall, we developed a novel FSH antigen and vaccine, and demonstrated it is highly efficacious in suppressing fat accumulation.

Oral oyster polypeptides protect ovary against d-galactose-induced premature ovarian failure in C57BL/6 mice.

BACKGROUND: Oyster polypeptides have various biofunctions, such as anti-cancer and anti-oxidative stress, but whether it has the protective effects to primary ovarian failure (POF) remains poorly understand. To address this issue, daily gavage of oyster polypeptides was performed to investigate their protective effect, basing on d-galactose-induced POF model in C57BL/6 female mice. RESULTS: Oyster polypeptides restored the irregular estrous cycles and the abnormal serum follicle stimulating hormone (FSH), luteinizing hormone (LH) and progesterone (P) levels as well as the decreased mRNA expression level of Amh that were induced by d-galactose. The follicle development of POF mice was improved by increasing the primordial follicle ratio and decreasing the atretic follicle number after oral administration of oyster polypeptides. Moreover, in the oyster polypeptides treated mice, the total superoxide dismutase (T-SOD) activity was significantly increased, while the malondialdehyde levels were significantly decreased. The mRNA expression levels of stress-related genes (SOD2, SIRT1 and FOXO3a) were remarkably up-regulated after d-galactose induction, but the up-regulation was weakened or disappeared by the gavage of oyster polypeptides. In addition, oyster polypeptides treatment also reduced the apoptosis of the ovarian granulosa cells and down-regulated the mRNA expression levels of apoptosis-related genes (p53 and Bad but not Bcl-2). CONCLUSION: This study reveals that oyster polypeptides may protect ovary against d-galactose-induced POF by their anti-oxidative stress activity to rescue d-galactose-induced ovarian oxidative damage and therefore to prevent ovarian cells apoptosis, thereby tipping the abnormality trigged by POF to get close to the normal levels. © 2019 Society of Chemical Industry.

Mechanistic insight into the role of immunocastration on eliminating skatole in boars.

The accumulation of skatole in fat tissue is one of the predominant factors, causing boar taint. The present study was aimed to understand the mechanism whereby active immunization against GnRH (immunocastration) eliminates skatole in boars. Thirty-six boars were assigned within litter into three groups (n = 12): control, surgically castrated, or immunized against GnRH at 10 wk of age (with a booster 8 wk later). Faecal and blood samples (for skatole and skatole-regulatory hormone profiles) were collected at 4-wk intervals until boars were slaughtered (26 weeks). Immunocastration reduced (P < 0.05) serum levels of androstenone, 17ß-estradiol and IGF1 especially after the booster immunization, and down-regulated (P < 0.05) mRNA expressions of both IGF1 and IGF1receptor (IGF1R) in mucosa of ileum as well as colon at slaughter. Compared to intact controls, immunocastration substantially decreased (P < 0.05) faecal skatole contents subsequent to the decrease of serum IGF1 levels, which persisted in boars after surgical castration. In parallel with the decreased formation of skatole in the intestine, levels of skatole in serum and then in fat tissue were also decreased (P < 0.05). On the other hand, deprivation of testicular steroids, especially androstenone and 17ß-estradiol accelerated skatole degradation metabolism in the liver by increasing (P < 0.05) hepatic CYP2E1, CYP2A, CYP2C49 and CYB5A expressions. Collectively, our results suggested that immunocastration decreased skatole formation in the intestine and meanwhile accelerated skatole degradation metabolism in the liver, resultantly eliminating skatole accumulation in male pigs. Decreased intestinal skatole formation by immunocastration appeared to be associated with the attenuated actions of IGF1 on the turnover of both ileal and colon mucosa.

Peptides from Colochirus robustus Enhance Immune Function via Activating CD3ζ- and ZAP-70-Mediated Signaling in C57BL/6 Mice.

Colochirus robustus, a species of sea cucumber, has long been used in East and Southeast Asia as nutritious food as well as for certain medicinal purpose. Studies have shown a number of biological functions associated with consumption of sea cucumber, many of which are attributed to its major component, sea cucumber peptides (SCP). However, how SCP impacts immune system, which is critical for host defense, has not been defined. To address this issue, in the present study, we conducted comprehensive analysis of immune function after oral administration of SCP (0, 25, 50, and 75 mg/kg body weigh) for eight weeks in C57BL/6 mice. We found that SCP treatment significantly enhanced lymphocyte proliferation, serum albumin (ALB) levels, and the natural killer (NK) cell activity. Moreover, SCP promoted functions of helper T cells (Th) as indicated by increased production of Th1 type cytokines of Interleukin (IL)-1ß, IL-2, Interferon (IFN)-γ and TNF-α and Th2 type cytokines (IL-4, IL-6, and IL-10). To determine the effective components, SCP was hydrolyzed into 16 types of constituent amino acids in simulated gastrointestinal digestion and these hydrolytic amino acids (HAA) were used for the mechanistic studies in the in vitro models. Results showed that HAA enhanced lymphocyte proliferation and production of IL-2, IL-10 and IFN-γ. Furthermore, CD3ζ (CD3ζ) and ζ-chain-associated protein kinase 70 (ZAP-70), the signaling molecules essential for activating T lymphocytes, were significantly up-regulated after HAA treatment. In summary, our results suggest that SCP is effective in enhancing immune function by activating T cells via impacting CD3ζ- and ZAP-70-mediated signaling pathway.

Effects of active immunization against GnRH versus surgical castration on hypothalamic-pituitary function in boars.

The objective was to compare effects of anti-GnRH immunization (immunocastration) versus surgical castration on hypothalamic-pituitary function in boars. Thirty-six boars were randomly divided into three groups (n = 12/group): control, surgically castrated, or immunized against GnRH at 10 wk of age (boostered 8 wk later). Compared to intact boars, immunocastration reduced (P < 0.05) serum concentrations of LH, FSH, testosterone and inhibin B and caused severe testicular atrophy, whereas surgical castration increased (P < 0.05) serum concentrations of LH and FSH. Both immunocastration and surgical castration consistently reduced hypothalamic GnRH synthesis, with decreased (P < 0.05) mRNA expressions of GnRH, GnRH up-stream gatekeeper genes kiss1 and its receptor (GPR54), and androgen receptor in the hypothalamic arcuate nucleus (ARC) and anteroventral periventricular nucleus (AVPV), as well as GnRH content in the median eminence. Inconsistently, mRNA expressions of gonadotropin-inhibitory hormone (GnIH) in ARC and AVPV as well as its receptor (GPR147) in pituitary were selectively reduced (P < 0.05), but mRNA expressions of estrogen receptor alpha and aromatase (CPY17A1) in pituitary were selectively increased (P < 0.05) in surgical castrates. In response to selectively attenuated suppressive signaling from GnIH and testosterone, mRNA expressions of GnRH receptor (GnRHR), LH-ß and FSH-ß in pituitary were increased (P < 0.05) in surgical castrates, whereas these pituitary gene expressions were decreased (P < 0.05) in immunocastrates, due to loss of hypothalamic GnRH signaling. We concluded that immunocastration and surgical castration consistently reduced hypothalamic GnRH synthesis due to a testosterone deficiency disrupting testosterone-Kisspeptin-GPR54-GnRH signaling pathways. Furthermore, selectively attenuated GnIH and testosterone signaling in the pituitary increased gonadotropin production in surgical castrates.

Molecular characterization of neuropeptide Y (NPY) receptors (Y1, Y4 and Y6) and investigation of the tissue expression of their ligands (NPY, PYY and PP) in chickens.

Neuropeptide Y (NPY) receptors and its ligands, NPY, peptide YY (PYY) and pancreatic polypeptide (PP), are suggested to regulate many physiological processes including food intake in birds. However, our knowledge regarding this avian NPY system remains rather limited. Here, we examined the tissue expression of NPY, PYY and PP and the gene structure, expression and signaling of three NPY receptors (cY1, cY4 and cY6) in chickens. The results showed that 1) NPY is widely expressed in chicken tissues with abundance noted in the hypothalamus via quantitative real-time PCR, whereas PYY is highly expressed in the pancreas, gastrointestinal tract and various brain regions, and PP is expressed almost exclusively in the pancreas; 2) cY1, cY4 and cY6 contain novel non-coding exon(s) at their 5'-UTR; 3) The wide tissue distribution of cY1 and cY4 and cY6 were detected in chickens by quantitative real-time PCR and their expression is controlled by the promoter near exon 1, which displays strong promoter activity in DF-1 cells as demonstrated by Dual-luciferase reporter assay; 4) Monitored by luciferase reporter assays, activation of cY1 and cY4 expressed in HEK293 cells by chicken NPY1-36, PYY1-37, and PP1-36 treatment inhibits cAMP/PKA and activates MAPK/ERK signaling pathways, while cY6-expressing cells show little response to peptide treatment, indicating that cY1 and cY4, and not cY6, can transmit signals in vitro. Taken together, our study offers novel information about the expression and functionality of cY1, cY4, cY6 and their ligands in birds, and helps to decipher their conserved roles in vertebrates.

Physiological interactions between the hypothalamic-pituitary-gonadal axis and spleen in rams actively immunized against GnRH.

Hypothalamic-pituitary-gonadal (HPG) axis is strongly implicated in the regulation of immune system. The objective was to determine the effects of immunocastration on splenic reproduction- and immunity-related gene expressions, and serum cytokine profiles in rams. Forty rams were randomly allocated into three groups: control (n=14); surgically castrated (n=13); or immunized (n=13) against 100µg D-Lys6-GnRH-tandem-dimer peptide conjugated to ovalbumin in Specol adjuvant at 6months of age (with a booster 2months later). Blood samples (for hormone and immune cytokine profiles) were collected at 1-month intervals until rams were slaughtered (10months). Compared to intact controls, anti-GnRH immunization reduced (P<0.05) serum concentrations of LH, FSH, and testosterone. Reduced testosterone abrogated its inhibitor feedback effect on the synthesis of GnRH in spleen, as evidenced by increased (P<0.05) protein content and mRNA expressions of GnRH, and simultaneously decreased (P<0.05) mRNA expressions of androgen receptor in spleen. In parallel with the increased GnRH production in spleen, the mRNA expressions of interleukin (IL)-2, IL-4, IL-6 and tumor necrosis factor alpha (TNF-α) as well as lymphocyte marker CD4, CD8 and CD19 molecules were increased (P<0.05) in spleen. Consistently, serum concentrations of IL-2, IL-4, IL-6, TNF-α were increased (P<0.05) in rams following immunization. Similarly, deprivation of testosterone by surgical castration also increased (P<0.05) GnRH and thus immune cytokine expressions in spleen. Collectively, our data suggested that immunocastration increased GnRH production in spleen by abrogating the inhibitory feedback effects from testosterone, consequently improving the immune markers of spleen and serum immune cytokines in rams.

Molecular characterization of three NPY receptors (Y2, Y5 and Y7) in chickens: Gene structure, tissue expression, promoter identification, and functional analysis.

Six neuropeptide Y (NPY) receptors are suggested to mediate the biological actions of NPY, peptide YY (PYY), and pancreatic polypeptide (PP), such as food intake in birds, however, information regarding the structure and signaling of avian NPY receptors are rather limited. In this study, we investigated the gene structure, tissue expression and signaling property of three NPY receptors (cY2, cY5 and cY7) in chickens. The results showed that 1) cY2, cY5 and cY7 contain novel non-coding exons upstream of their start codon and alternative mRNA splicing in their 5'-UTR results in the formation of multiple transcript variants; 2) cY2, cY5 and cY7 transcripts were detected to be widely expressed in adult chicken tissues including various brain regions by RT-PCR, and their expression is controlled by a promoter(s) near exon 1, which display promoter activity in DF-1 cells as demonstrated by Dual-luciferase reporter assay; 3) cY2, cY5 and cY7 expressed in HEK293 cells were preferentially (or potently) activated by cNPY1-36 and cPYY1-37, but not by cPP1-36, and their activation led to the inhibition of cAMP/PKA signaling pathway and activation of MAPK/ERK signaling pathway, monitored by the cell-based luciferase reporter systems or western blots, indicating that the three NPY receptors are functional and capable of transmitting signals effectively. On the whole, our data establishes a molecular basis to elucidate the actions of three functional NPY receptors (cY2, cY5 and cY7) and their ligands in birds, which helps to uncover the conserved roles of these ligand-receptor pairs in vertebrates.

Glucagon-like peptide (GCGL) is a novel potential TSH-releasing factor (TRF) in Chickens: I) Evidence for its potent and specific action on stimulating TSH mRNA expression and secretion in the pituitary.

Our recent study proposed that the novel glucagon-like peptide (GCGL), encoded by a glucagon-like gene identified in chickens and other lower vertebrates, is likely a hypophysiotropic factor in nonmammalian vertebrates. To test this hypothesis, in this study, we investigated the GCGL action on chicken pituitaries. The results showed that: 1) GCGL, but not TRH, potently and specifically stimulates TSH secretion in intact pituitaries incubated in vitro or in cultured pituitary cells monitored by Western blotting or a cell-based luciferase reporter assay; 2) GCGL (0.1nM-10nM) dose dependently induces the mRNA expression of TSHß but not 5 other hormone genes in cultured pituitary cells examined by quantitative real-time RT-PCR, an action likely mediated by intracellular adenylate cyclase/cAMP/protein kinase A and phospholipase C/inositol 1,4,5-trisphosphate/Ca(2+) signaling pathways coupled to GCGL receptor (GCGLR); 3) GCGLR mRNA is mainly localized in pituitary cephalic lobe demonstrated by in situ hybridization, where TSH-cells reside, further supporting a direct action of GCGL on thyrotrophs. The potent and specific action of GCGL on pituitary TSH expression and secretion, together with the partial accordance shown among the temporal expression profiles of GCGL in the hypothalamus and GCGLR and TSHß in the pituitary, provides the first collective evidence that hypothalamic GCGL is most likely to be a novel TSH-releasing factor functioning in chickens. The discovery of this novel potential TSH-releasing factor (GCGL) in a nonmammalian vertebrate species, ie, chickens, would facilitate our comprehensive understanding of the hypothalamic control of pituitary-thyroid axis across vertebrates.