Testosterone regulates thymic remodeling by activating glucocorticoid receptor signaling pathway to accelerate thymocyte apoptosis in male rats.

Thymic atrophy affects T cell generation and migration to the periphery, thereby affecting T cell pool diversity. However, the mechanisms underlying thymic atrophy have not been fully elucidated. Here, gonadotropin-releasing hormone (GnRH) immunization and surgical castration did not affect thymocyte proliferation, but significantly reduced the apoptosis and increased the survival rate of CD4-CD8-, CD4+CD8+, CD4+CD8-, and CD4-CD8+ thymocytes. Following testosterone supplementation in rats subjected to GnRH immunization and surgical castration, thymocyte proliferation remained unchange, but the apoptosis of CD4-CD8-, CD4+CD8+, CD4+CD8-, and CD4-CD8+ thymocytes significantly increased. Transcriptome analyses of the thymus after GnRH immunization and surgical castration showed a significant reduction in the thymus's response to corticosterone. Cholesterol metabolism and the synthesis and secretion of corticosterone were significantly reduced. Analysis of the enzyme levels involved in the corticosterone synthesis pathway revealed that corticosterone synthesis in thymocytes was significantly reduced after GnRH immunization and surgical castration, whereas exogenous testosterone supplementation relieved this process. Testosterone promoted thymocyte apoptosis in a concentration-dependent manner, and induced corticosterone secretion in vitro. Blocking the intracellular androgen receptor (AR) signaling pathway did not significantly affect thymocyte apoptosis, but blocking the glucocorticoid receptor (GR) signaling pathway significantly reduced it. Our findings indicate that testosterone regulates thymus remodeling by affecting corticosterone synthesis in thymocytes, which activates GR signal transduction and promotes thymocyte apoptosis.

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.

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.

Novel insights into the roles of RNA N6-methyladenosine modification in regulating gene expression during environmental exposures.

N6-methyladenosine (m6A) is one of the most common RNA modifications in eukaryotes involved in the regulation of post-transcriptional gene expression, as well as the occurrence and development of diseases related to environmental exposures. Adverse factors produced by environmental exposures, such as reactive oxygen species, inflammation, and cyclobutane pyrimidine dimers, mediate m6A modification, thereby regulating downstream gene and protein expression, and signaling pathways, such as FTO/m6A RNA/p53 axis, PI3K/AKT/mTOR pathway, and PARP/METTL3/m6A RNA/Pol κ pathway. Moreover, an imbalance in m6A methylation levels directly mediates disease pathogenesis. To date, some studies have detailed the mechanisms underlying environmental exposure-mediated global changes in RNA m6A methylation. Based on our current understanding, we aimed to elaborate on the molecular mechanisms through which RNA m6A methylation regulates gene expression under environmental exposures. In this review, we outline the biogenesis and functions of RNA m6A modification. Furthermore, we focus on the effects of environmental exposures on m6A levels and highlight the relationships between environmental exposures (doses and time) and m6A levels. Although the molecular mechanisms regulating gene expression remains to be elucidated, m6A has potential applications as a disease biomarker.

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.

Opioid Peptides and Their Receptors in Chickens: Structure, Functionality, and Tissue Distribution.

Opioid peptides, derived from PENK, POMC, PDYN and PNOC precursors, together with their receptors (DOR, MOR, KOR and ORL1), constitute the opioid system and are suggested to participate in multiple physiological/pathological processes in vertebrates. However, the question whether an opioid system exists and functions in non-mammalian vertebrates including birds remains largely unknown. Here, we cloned genes encoding opioid system from the chicken brain and examined their functionality and tissue expression. As in mammals, 6 opioid peptides encoded by PENK (Met-enkephalin and Leu-enkephalin), POMC (ß-endorphin), PDYN (dynorphin-A and dynorphin-B) and PNOC (nociceptin) precursors and four opioid receptors were found to be highly conserved in chickens. Using pGL3-CRE-luciferase and pGL4-SRE-luciferase reporter systems, we demonstrated that chicken opioid receptors (cDOR, cMOR, cKOR and cORL1) expressed in CHO cells, could be differentially activated by chicken opioid peptides, and resulted in the inhibition of cAMP/PKA and activation of MAPK/ERK signaling pathways. cDOR is potently activated by Met-enkephalin and Leu-enkephalin, and cKOR is potently activated by dynorphin-A, dynorphin-B and nociceptin, whereas cORL1 is specifically activated by nociceptin. Unlike cDOR, cKOR and cORL1, cMOR is moderately/weakly activated by enkephalins and other opioid peptides. These findings suggest the ligand-receptor pair in chicken opioid system is similar, but not identical to, that in mammals. Quantitative real-time PCR revealed that the opioid system is mainly expressed in chicken central nervous system including the hypothalamus. Collectively, our data will help to facilitate the better understanding of the conserved roles of opioid system across vertebrates.

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.

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.

Insulin Stimulates Goose Liver Cell Growth by Activating PI3K-AKT-mTOR Signal Pathway.

BACKGROUND/AIMS: Recent studies have suggested a crucial role for PI3K-Akt-mTOR pathway in regulating cell proliferation, so we hypothesize that insulin acts goose hepatocellular growth by PI3K-Akt-mTOR signal pathway. Because the physiological status of liver cells in vitro is different from that in vivo, a simplified cell model in vitro was established. METHODS: Goose primary hepatocytes were isolated and incubated in either no addition as a control or insulin or PI3K-Akt-mTOR pathway inhibitors or co-treatment with glucose and PI3K-Akt-mTOR pathway inhibitors; Then, cell DNA synthesis and cell cycle analysis were detected by BrdU-incorporation Assay and Flow cytometric analysis; the mRNA expression and protein expression of factors involved in the cell cycle were determined by Real-Time RT-PCR, ELISA, and western blot. RESULTS: Here we first showed that insulin evidently increased the cell DNA synthesis, the mRNA level and protein content of factors involved in the cell proliferation of goose primary hepatocytes. Meanwhile, insulin evidently increased the mRNA level and protein content of factors involved in PI3K-Akt-mTOR pathway. However, the up-regulation of insulin on cell proliferation was decreased significantly by the inhibitors of PBK-Akt-mTOR pathway, LY294002, rapamycin or NVP-BEZ235. CONCLUSION: These findings suggest that PI3K-Akt-mTOR pathway plays an essential role in insulin-regulated cell proliferation of goose hepatocyte.

Active immunization with GnRH-tandem-dimer peptide in young male rats reduces serum reproductive hormone concentrations, testicular development and spermatogenesis.

GnRH sterilization vaccines have been developed for various practical and clinical reasons. However, conjugation of GnRH peptide to carrier protein has many drawbacks, hampering the further commercialization of GnRH vaccines. In this study, a new nonconjugated GnRH vaccine, D-Lys6-GnRH-tandem-dimer peptide (TDK), emulsified in Specol adjuvant was investigated for its immunocastration efficacy in young male rats. Prepubertal male rats were randomly allocated into three groups (n = 12): control (no treatment), surgically castrated or immunized against 100 µg TDK in Specol adjuvant at 6 weeks of age (with a booster 8 weeks later). Blood samples (for antibody titers and hormone concentrations) were collected at 2-week intervals until rats were killed (18 weeks of age). Compared to intact controls, active immunization against TDK reduced (P < 0.05) serum concentrations of testosterone, inhibin B, LH and FSH, prevented the onset of spermatogenesis at puberty. Furthermore, mRNA expressions of GnRH receptor, LH-ß and FSH-ß in the pituitary, LH receptor, FSH receptor, inhibin α, ßA and ßB subunit in the testes were decreased in immunocastrated rats compared to intact controls (P < 0.05). These results demonstrate for the first time that GnRH-tandem-dimer peptide emulsified in Specol is a promising veterinary sterilization medicine.

Effect of immunization against GnRH on hypothalamic and testicular function in rams.

The objective was to determine effects of active immunization against GnRH on reproductive function in Tibetan rams. Peripubertal Tibetan rams (n = 30) were randomly and equally allocated into three groups: control (no treatment); surgically castrated; or immunized against 100-µg d-Lys6-GnRH-tandem-dimer peptide conjugated to ovalbumin in Specol adjuvant at 24 weeks of age (with a booster 8 weeks later). Blood samples (for antibody titers and hormone concentrations) were collected at 4-week intervals until rams were killed (40 weeks). Immunization triggered a good antibody response in all immunized rams (P < 0.01). Compared with intact controls, anti-GnRH immunization reduced (P < 0.01) serum concentrations of testosterone, inhibin A, LH, and FSH, and it induced testicular atrophy (suppression of spermatogenesis). Androstenone concentrations in fat tissues of GnRH-immunized rams were also rendered nondetectable (P < 0.001). Furthermore, mRNA expressions of GnRH receptor, LH-ß, and FSH-ß in the pituitary and of LH receptor, FSH receptor, and inhibin α and ßA subunits in the testes were decreased in immunized rams compared with intact controls (P < 0.05). This was apparently the first report that active immunization against GnRH-tandem-dimer-ovalbumin conjugate in Specol adjuvant was an effective alternative to surgical castration for Tibetan rams under practical Tibetan plateau conditions.