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.

PNX14 but not PNX20 as a novel regulator of preadipocyte differentiation via activating Epac-ERK signaling pathway in Gallus gallus.

Small integral membrane protein 20 (SMIM20) could generate two main peptides, PNX14 and PNX20, which participate in multiple biological roles such as reproduction, inflammation and energy metabolism in mammals. However, little is known about their physiological functions in non-mammalian vertebrates. Using chicken (c-) as an animal model, we found cSMIM20 was moderately expressed in adipose tissues, and its expression was gradually increased during the differentiation of chicken preadipocytes, suggesting that it may play an important role in chicken adipogenesis. Further research showed cPNX14 could facilitate the differentiation of chicken preadipocytes into mature adipocytes by enhancing expression of adipogenic genes including PPARγ, CEBPα and FABP4, and promoting the formation of lipid droplets. This pro-adipogenic effect of cPNX14 was completely attenuated by Epac-specific and ERK inhibitor. Interestingly, cPNX20 failed to regulate the adipogenic genes and lipid droplet content. Collectively, our findings reveal that cPNX14 but not cPNX20 can serve as a novel adipogenesis mediator by activating the Epac-ERK signaling pathway in chickens.

Mechanic Insight into the Distinct and Common Roles of Ovariectomy Versus Adrenalectomy on Adipose Tissue Remodeling in Female Mice.

Dysfunctions of the ovaries and adrenal glands are both evidenced to cause aberrant adipose tissue (AT) remodeling and resultant metabolic disorders, but their distinct and common roles are poorly understood. In this study, through biochemical, histological and RNA-seq analyses, we comprehensively explored the mechanisms underpinning subcutaneous (SAT) and visceral adipose tissue (VAT) remodeling, in response to ovariectomy (OVX) versus adrenalectomy (ADX) in female mice. OVX promoted adipocyte differentiation and fat accumulation in both SAT and VAT, by potentiating the Pparg signaling, while ADX universally prevented the cell proliferation and extracellular matrix organization in both SAT and VAT, likely by inactivating the Nr3c1 signaling, thus causing lipoatrophy in females. ADX, but not OVX, exerted great effects on the intrinsic difference between SAT and VAT. Specifically, ADX reversed a large cluster of genes differentially expressed between SAT and VAT, by activating 12 key transcription factors, and thereby caused senescent cell accumulation, massive B cell infiltration and the development of selective inflammatory response in SAT. Commonly, both OVX and ADX enhance circadian rhythmicity in VAT, and impair cell proliferation, neurogenesis, tissue morphogenesis, as well as extracellular matrix organization in SAT, thus causing dysfunction of adipose tissues and concomitant metabolic disorders.

UNC93B1 facilitates TLR18-mediated NF-κB signal activation in Schizothorax prenanti.

Toll-like receptor 18 (TLR18), a non-mammalian TLR, has been believed to play an important role in anti-bacterial immunity of teleost fishes. UNC93B1 is a classical molecular chaperone that mediates TLRs transport from endoplasmic reticulum to the located membrane. However, TLR18-mediated signal transduction mechanism and the regulatory effect of UNC93B1 to TLR18 are still unclear in teleost fishes. In this study, the coding sequences of TLR18 and UNC93B1 were cloned from Schizothorax prenanti, named spTLR18 and spUNC93B1, respectively. The spTLR18 and spUNC93B1 are 2583 bp and 1878 bp in length, encode 860 and 625 amino acids, respectively. The spTLR18 widely expressed in various tissues with the highest expression level in liver. After stimulation of Aeromonas hydrophila, lipopolysaccharide (LPS) and Poly(I:C), the expression levels of spTLR18 were significantly increased in spleen and head kidney. The spTLR18 located in the cell membrane, while spUNC93B1 located in the cytoplasm. Luciferase and overexpression analysis showed that spTLR18 activated NF-κB and type I IFN signal pathways, and spTLR18-mediated NF-κB activation might depend on the adaptor molecule MyD88. Besides, spUNC93B1 positively regulates spTLR18-mediated NF-κB signal. Our study first uncovers TLR18-UNC93B1-mediated signal transduction mechanism, which contributes to the understanding of TLR signaling pathway in teleost fishes.

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.

Characterization of spexin (SPX) in chickens: molecular cloning, functional analysis, tissue expression and its involvement in appetite regulation.

Spexin (SPX) is a conservative tetradecapeptide which has been proven to participate in multiple physiological processes, including anxiety, feed intake, and energy metabolism in fish and mammals. However, whether SPX exists and functions in birds remain largely unknown. Using chicken (c-) as a model, the full-length cDNA encoding cSPX precursor was cloned, and it was predicted to generate a mature peptide with 14 amino acids conserved across vertebrates. The pGL4-SRE-luciferase reporter system-based functional analysis demonstrated that cSPX was effective in activating chicken galanin type Ⅱ receptor (cGALR2), cGALR2-like receptor (cGALR2L) and galanin type Ⅲ receptor (cGALR3), thus to stimulate intracellular MAPK/ERK signaling pathway. Quantitative real-time PCR revealed that SPX was widely expressed in chicken tissues, especially abundant in the central nervous system, pituitary, testes, and pancreas. Interestingly, it was noted that chicken hypothalamic SPX mRNA could be up-regulated by 24-h and 36-h fasting, heralding its latent capacity in appetite regulation. In accordance with this speculation, peripheral injection of cSPX was proved to be functional in reducing feed intake of 3-wk-old chicks. Furthermore, we found that cSPX could reduce the expression of AgRP and MCH, with a concurrent rise in CART1 mRNA level in the hypothalamic of chicks. Collectively, our findings not only provide the evidences that SPX can act as a satiety factor by orchestrating the expression of key feeding regulators in the chicken hypothalamus but also help to facilitate a better understanding of its functional evolution across vertebrates.

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.

Research Note: The gut microbiota varies with dietary fiber levels in broilers.

It is generally accepted the gut microbiota have a profound effect on the nutrition, health, and production in poultry. To deeply understand the gut microbiota composition with the dietary fiber level in broilers, we evaluated the cecal microbiota profiles feeding on different dietary fiber level with alfalfa as additive in Dahen broilers based on 16S rRNA gene sequencing and gas chromatography. As a result, the gut microbiota diversity was greatly accelerated with the dietary fiber level. The dietary fiber stimulated the growth of many intestinal communities such as Rikenellaceae RC9 gut group, Faecalibacterium, Prevotellaceae UCG 001 and Ruminococcaceae UCG 014, and led to an altered microbial function such as Carbohydrate metabolism and Genetic information processing. Meanwhile, we found the genera Anaerofilum and Dielma were significantly correlated with the production of short chain fatty acids (SCFAs). All these results provide a reference for the broilers gut microbiota changes with different dietary fiber level. The key role of the altered microbiota with the dietary fiber may mediate beneficial effects in broiler production, which also reflect the substantial potential of dietary fiber level in poultry.

Evidence for Neuropeptide W Acting as a Physiological Corticotropin-releasing Inhibitory Factor in Male Chickens.

In vertebrates, adrenocorticotropin (ACTH), released by the pituitary gland, is a critical part of the stress axis and stress response. Generally, the biosynthesis and secretion of ACTH are controlled by both hypothalamic stimulatory factors and inhibitory factors [eg, ACTH-releasing inhibitory factor (CRIF)], but the identity of this CRIF remains unrevealed. We characterized the neuropeptide B (NPB)/neuropeptide W (NPW) system in chickens and found that NPW could directly target the pituitary to inhibit growth hormone (GH) and prolactin (PRL) secretion via neuropeptide B/W receptor 2 (NPBWR2), which is completely different from the mechanism in mammals. The present study first carried out a series of assays to investigate the possibility that NPW acts as a physiological CRIF in chickens. The results showed that (1) NPW could inhibit ACTH synthesis and secretion by inhibiting the 3',5'-cyclic adenosine 5'-monophosphate/protein kinase A signaling cascade in vitro and in vivo; (2) NPBWR2 was expressed abundantly in corticotrophs (ACTH-producing cells), which are located mainly in cephalic lobe of chicken pituitary, as demonstrated by single-cell RNA-sequencing, immunofluorescent staining, and fluorescence in situ hybridization; (3) dexamethasone could stimulate pituitary NPBWR2 and hypothalamic NPW expression in chicks, which was accompanied by the decease of POMC messenger RNA levels, as revealed by in vitro and subcutaneous injection assays; and (4) the temporal expression profiles of NPW-NPBWR2 pair in hypothalamus-pituitary axis and POMC in pituitary were almost unanimous in chicken. Collectively, these findings provide comprehensive evidence for the first time that NPW is a potent physiological CRIF in chickens that plays a core role in suppressing the activity of the stress axis.

Corticosterone triggers anti-proliferative and apoptotic effects, and downregulates the ACVR1-SMAD1-ID3 cascade in chicken ovarian prehierarchical, but not preovulatory granulosa cells.

The coordinated proliferation and apoptosis of granulosa cells plays a critical role in follicular development. To identify the exact mechanisms of how stress-driven glucocorticoid production suppresses reproduction, granulosa cells were isolated from chicken follicles at different developmental stages and then treated with corticosterone. Using CCK-8, EDU and TUNEL assays, we showed that corticosterone could trigger both anti-proliferative and pro-apoptotic effects in granulosa cells from 6 to 8 mm follicles only, while depicting no influence on granulosa cells from any preovulatory follicles. High-throughput transcriptomic analysis identified 1362 transcripts showing differential expression profiles in granulosa cells from 6 to 8 mm follicles after corticosterone treatment. Interestingly, Kyoto Encyclopedia of Genes and Genomes enrichment analysis revealed that 17 genes were enriched in the TGF-ß signaling pathway, and 13 showed differential expression patterns consistent with corticosterone-induced effects. The differential expression profiles of these 13 genes were examined by quantitative real-time PCR in cultured chicken ovarian granulosa cells at diverse developmental stages following corticosterone challenge for a short (8 h) or long period (24 h). After 24 h of treatment, INHBB, FST, FMOD, NOG, ACVR1, SMAD1 and ID3 were the genes that responded consistently with corticosterone-induced proliferative and apoptotic events in all granulosa cells detected. However, only ACVR1, SMAD1 and ID3 could initiate coincident expression patterns after being treated for 8 h, suggesting their significance in corticosterone-mediated actions. Collectively, these findings indicate that corticosterone can inhibit proliferation and cause apoptosis in chicken ovarian prehierarchical, but not preovulatory granulosa cells, through impeding ACVR1-SMAD1-ID3 signaling presumptively.

RNA-seq coupling two different methods of castration reveals new insights into androgen deficiency-caused degeneration of submaxillary gland in male Sprague Dawley rats.

BACKGROUND: Salivary gland (SMG) degeneration and dysfunction are common symptoms that occur after sex hormone deprivation, but the underlying mechanisms remain largely unknown. Additionally, immunocastration, which causes drop of sex hormones, has been developed as an alternative to surgical castration, however whether it exerts similar effects as surgical castration on the salivary glands is unknown. Through histological and RNA-seq analysis, we assessed changes in morphology and transcriptome of SMG in response to immunocastration (IM) versus surgical castration (bilateral orchiectomy, ORC). RESULTS: Compared to entire males (EM), ORC caused severe degeneration of SMG in rats, as evidenced by both decreased (P < 0.01) SMG weight and organ index, and by decreased (P < 0.01) quantity of SMG acini and ducts. IM had minimal effects (P > 0.05) on SMG weight and organ index, but it still caused degeneration (P < 0.05) of the acini and ducts. Even though, the quantity of both SMG acini and ducts was much higher (P < 0.001) in IM than in ORC. Functional enrichment analysis of the common regulated genes by ORC/IM revealed disrupted epithelial cell development, angiogenesis, anatomical structure morphogenesis and enhanced cell death are associated with SMG degeneration in deprivation of androgens. Integrated data analysis shown that there existed a selective hyperfunction of SMG ribosome and mitochondrion in ORC but not in IM, which might be associated with more severe degeneration of SMG in ORC than in IM. CONCLUSIONS: Our findings suggested that both surgical castration and immunocastration caused SMG degeneration by disrupting epithelial cell development, angiogenesis, anatomical structure morphogenesis and enhancing cell death. But, surgical castration selectively induced hyperfunction of SMG ribosome and mitochondrion, thus causing more severe degeneration of SMG than immunocastration.

Characterization of prolactin (PRL) and PRL receptor (PRLR) in Chinese soft-shelled turtle: Molecular identification, ligand-receptor interaction and tissue distribution.

Prolactin (PRL) plays crucial roles in many physiological and pathological processes through activating its specific membrane-anchored receptor (PRLR). Although this ligand-receptor pair has been extensively studied in mammals, birds and fishes, researches examining their significance is rather scarce in reptiles. Additionally, the interaction mechanism of PRL-PRLR has abortively understood across vertebrates, since two tandem repeated ligand-binding domains of PRLR have been identified in birds and few reptiles. To lay the foundation to clarify their roles and ligand-receptor interaction in reptiles, using Chinese soft-shelled turtle as model, the cDNAs containing open reading frame of PRL and PRLR were cloned. The cloned PRL consisted of 710 bp and encoded a precursor of 228 amino acid (-aa), while PRLR was 2658 bp in length and predicted to generate a 828-aa precursor. Furthermore, the recombinant PRL protein with high-purity was prepared from Escherichia coli (E. coli) strain Rosetta gamiB (DE3) by using cobalt resin. Using the 5 × STAT5-Luciferase reporter system, we found PRLR and PRLR-M2 (the PRLR-mutant lacking membrane-distal ligand-binding domain) could be dose-dependently activated by recombinant PRL, thereby triggering the intracellular JAK2-STAT5 signaling cascade, suggesting PRL-PRLR is functional in Chinese soft-shelled turtle, and the membrane-proximal ligand-binding domain of PRLR is the critical domain involving in PRL-binding. Quantitative real-time PCR revealed that PRL was predominantly and abundantly expressed in pituitary, while PRLR exhibited ubiquitous expression in all of the tissues examined. Collectively, our data indicate the PRL-PRLR pair may function in reptiles including Chinese soft-shelled turtle, in a way similar to that in birds.

Identification and functional analysis of NOD2 and its two splicing variants associated with a novel pattern of signal regulation in teleost fishes.

The nucleotide-binding oligomerization domain 2 (NOD2) has been identified as an important sensor for microorganic invasion in both mammals and teleost fishes. In this study, two splicing variants of NOD2 (NOD2-v1 and NOD2-v2) were identified as truncating the functional domains of wild-type NOD2 in the teleost fish Schizothorax prenanti. NOD2-v1 included an intron sequence that terminated within the third leucine-rich repeat (LRR) domain, while NOD2-v2 incorporated an insertion of one and half intron sequences and truncated within the second caspase activation and recruitment domain (CARD). NOD2, NOD2-v1 and NOD2-v2 genes were ubiquitously expressed. All three genes positively responded to exposure of Aeromonas hydrophila and lipopolysaccharide stimulation in varying degrees. Using luciferase activity assays in HEK293T cells, our results revealed that NOD2 activated the NF-κB signal and recognized muramyl dipeptide (MDP). NOD2-v1 exhibited deficiency in the LRR domains and could not sense MDP, but maintained the ability to activate NF-κB and enhanced NOD2-mediated MDP recognition. Given the significant change to the functional structure, NOD2-v2 lost its capacity for NF-κB activation, but interestingly repressed NOD2-mediated MDP sensing and NF-κB activation, and even NOD2-v1-induced NF-κB activation. Altogether, our study reveals a novel pattern of signal regulation by splicing variants in teleost fishes.

FSH promotes fat accumulation by activating PPARγ signaling in surgically castrated, but not immunocastrated, male pigs.

Follicle-stimulating hormone (FSH) was recently implicated as a novel regulator of fat accumulation. Surgical castration causes high FSH concentrations and increases fat accumulation, whereas immunocastration results in low FSH concentrations and less fat in immunocastrated boars versus barrows. However, detailed information regarding the role of FSH in regulation of fat accumulation in male pigs is unclear. First, expression of FSH receptor was confirmed (real-time quantitative PCR) in subcutaneous and visceral adipose tissues (SAT and VAT, respectively) of boars. Then, surgical castration (high FSH model) was compared to immunocastration (low FSH model) to investigate potential roles of FSH in adipogenesis and fat accumulation. High FSH concentrations after surgical castration activated PPARγ signaling by upregulating expression of CREB (P < 0.05), and then recruited an array of PPARγ target adipogenic genes, including transcription factor (C/EBPα), long-chain fatty acid uptake (LPL), fatty acid de novo synthesis (FASN, ACACA) and lipid droplet formation (PLIN1) in both SAT and VAT, promoting fat accumulation in barrows. In contrast, much lower serum FSH concentrations in immunocastrates attenuated (P < 0.05) expressions of PPARγ and PPARγ target genes in both SAT and VAT, resulting in less fat accumulation in immunocastrated boars versus barrows. We concluded that the substantially elevated FSH concentrations in barrows promoted fat accumulation by activating the PPARγ signaling pathway in adipose tissues, whereas immunocastrates accumulated less fat due to low FSH.