The reproductive regulation of LPXRFa and its receptor in the hypothalamo-pituitary-gonadal axis of the spotted scat (Scatophagus argus).

Gonadotropin-inhibitory hormone (GnIH) plays a critical role in regulating gonadotropin-releasing hormone (GnRH), gonadotropin hormone (GtH), and steroidogenesis. The Lpxrfa (the piscine ortholog of GnIH) system has been found to regulate fish reproduction. To gain insight into the role of Lpxrfa in the regulation of spotted scat (Scatophagus argus) reproduction, spotted scat Lpxrfa (ssLpxrfa), and its receptor (ssLpxrfa-r) were cloned and analyzed. Tissue distribution and expression patterns at the hypothalamo-pituitary-gonadal axis (HPG axis) of sslpxrfa and sslpxrfa-r mRNA were also investigated during gonadal development of spotted scat. The open reading frame (ORF) of the sslpxrfa was 606 bp encoding 201 amino acids and includes a putative signal peptide and two mature ssLpxrfa peptides with LPXRFamide motif at their C-terminus. The sslpxrfa-r ORF was 1449 bp encoding 482 amino acids and contracted a seven-hydrophobic transmembrane (TM) domain structure. The tissue distribution showe d that the sslpxrfa was highly expressed in hypothalami, gill, and the gonads. In addition, sslpxrfa-r was highly expressed in hypothalami, pituitaries, and the gonads. Quantitative real-time polymerase chain reaction (qPCR) revealed that sslpxrfa had the highest expression in the hypothalami and pituitaries, and the lowest expression in the gonads in stage V. During gonadal development, the expression of sslpxrfa-r was gradually increased in the hypothalami but reduced in the gonads. However, no obvious trend was observed in the pituitaries. The expression of sslpxrfa and sslpxrfa-r decreased significantly after injection with 17ß-estradiol (E2). However, the expression of both sslpxrfa and sslpxrfa-r was not changed after injection with 17α-methyltestosterone(17α-MT) in the hypothalami. In addition, no changes were observed in the expression of fshß and lhß in the pituitaries after injecting ssLpxrfa-1. However, ssLpxrfa-2 could downregulate the expression of sbgnrh and fshß in the hypothalami and pituitaries, respectively. Taken together, these findings suggested that ssLpxrfa may participate in E2 feedback in reproduction and regulate the reproductive axis of spotted scat.

Identification, functional characterization, and estrogen regulation on gonadotropin-releasing hormone in the spotted scat, Scatophagus argus.

Gonadotropin-releasing hormone (GnRH) is a key neuropeptide of the reproductive system. However, little is known about the role of GnRH in the spotted scat (Scatophagus argus). Here, three GnRH subtypes (cGnRH-II, sGnRH, and sbGnRH) were identified in the spotted scat. cGnRH-II and sGnRH were only expressed in the brains and gonads of both male and female fish, exhibiting a tissue-specific expression pattern, while sbGnRH was expressed at different transcription levels in all examined tissues. During ovarian maturation, hypothalamus-associated sbGnRH was upregulated, while the expression of sGnRH was variable and cGnRH-II first increased and then decreased. In vivo experiments showed that sbGnRH significantly promoted the expression of fsh and lh genes in a dose-dependent manner and exhibited a desensitization effect on lh expression at high concentrations. For sGnRH and cGnRH-II, only high concentrations could induce fsh and lh expression. Furthermore, treatment with highly concentrated sbGnRH peptide also induced fsh and lh expression, whereas the sGnRH and cGnRH-II peptides only induced fsh expression in vitro. 17ß-Estradiol (E2) significantly inhibited the expression of sbGnRH mRNA in a dose-dependent manner and did not impact sGnRH and cGnRH-II mRNA levels in vivo or in vitro. The inhibitory effect of E2 on sbGnRH expression was attenuated by the estrogen receptor (ER) broad-spectrum antagonist (fulvestrant) and the ERα-specific antagonist (methyl-piperidinopyrazole), respectively, implying that the feedback regulation on sbGnRH is mediated via ERα. This study provides a theoretical basis for the reproductive endocrinology of the spotted scat by studying GnRH.

Phoenixin participated in regulation of food intake and growth in spotted scat, Scatophagus argus.

Phoenixin (Pnx) is an endogenous peptide known to be involved in reproduction and food intake in rats, with two active isoforms, phoenixin-14 (Pnx-14) and phoenixin-20 (Pnx-20). However, little is known about the functions of Pnx in teleost. Here, pnx was cloned and was detected in all tissues of both male and female in spotted scat (Scatophagus argus), including growth axis, hypothalamus, pituitary, and liver. Real-time PCR analysis showed that pnx in the hypothalamus increased significantly after 2 d and 7 d fasting, while reduced significantly after re-feeding (P < 0.05). When pituitary and liver fragments were cultured in vitro with Pnx-14 and Pnx-20 (10 nM and 100 nM) for 6 h, the expression of ghrhr (growth hormone-releasing hormone receptor) and gh (growth hormone) in the pituitary, and ghr1 (growth hormone receptor 1) in the liver increased significantly, except ghr2 (growth hormone receptor 2) incubated with 10 nM and 100 nM Pnx-20 and ghr1 incubated with 10 nM Pnx-20. Similarly, the expression of ghrhr and gh in the pituitary, as well as ghr1 and ghr2 in the liver, increased significantly after injecting S. argus with Pnx-14 and Pnx-20 (10 ng/g and 100 ng/g body weight). These results indicate that Pnx is likely to be involved in the regulation of food intake, and also regulates the growth of S. argus by increasing ghrhr and gh expression in the pituitary, ghr1 and ghr2 in the liver, and ghr1 directly in the liver.

Molecular cloning, characterization and expression analysis of spexin in spotted scat (Scatophagus argus).

Spexin (Spx), a novel neuropeptide, composed of 14 amino acid residues, is evolutionally conserved from fish to mammals. It has been suggested that Spx has pleiotropic functions in mammals. However, reports about Spx are very limited. To clarify the roles of Spx in the regulation of reproduction and food-intake in the spotted scat, the spx (ssspx) gene was cloned and analyzed. Analysis of the tissue distribution by RT-PCR showed that ssspx expression was widespread. During ovary development, expression of ssspx was found to be highest in phase II, moderate in phase III, and at its lowest level in phase IV. Ssspx expression was significantly down-regulated in the hypothalamus after treatment with E2 both in vitro and in vivo. A significant increase of ssspx was observed after 2 and 7 days of food deprivation. However, the ssspx transcript levels in the 7 day fasting group decreased significantly after refeeding 3 h after the scheduled feeding time. This suggests that ssSpx may be involved in the regulation of reproduction and food-intake in the spotted scat.

Effects of melanocortin-4 receptor agonists and antagonists on expression of genes related to reproduction in spotted scat, Scatophagus argus.

Melanocortin-4 receptor (Mc4r) function related to reproduction in fish has not been extensively investigated. Here, we report on gene expression changes by real-time PCR following treatment with Mc4r agonists and antagonists in the spotted scat (Scatophagus argus). Using in vitro incubated hypothalamus, the Mc4r nonselective agonist NDP-MSH ([Nle4, D-Phe7]-α-melanocyte stimulating hormone; 10-6 M) and selective agonist THIQ (N-[(3R)-1, 2, 3, 4-Tetrahydroisoquinolinium-3-ylcarbonyl]- (1R)-1-(4-chlorobenzyl)-2-[4-cyclohexyl-4-(1H-1,2,4-triazol-1-ylmethyl) piperidin-1-yl]-2-oxoethylamine; 10-7 M) significantly increased the expression of gnrh (Gonadotropin releasing hormone), while the Mc4r nonselective antagonist SHU9119 (Ac-Nle-[Asp-His-DPhe/DNal(2')-Arg-Trp-Lys]-NH2; 10-6 M) and selective antagonist Ipsen 5i (compound 5i synthesized in Ipsen Research Laboratories; 10-6 M) significantly inhibited gnrh expression after 3 h of incubation. In incubated pituitary tissue, NDP-MSH and THIQ significantly increased the expression of fshb (Follicle-stimulating hormone beta subunit) and lhb (Luteinizing hormone beta subunit), while SHU9119 and Ipsen 5i significantly decreased fshb and lhb expression after 3 h of incubation. During the in vivo experiment, THIQ (1 mg/kg bw) significantly increased gnrh expression in hypothalamic tissue, as well as the fshb and lhb expression in pituitary tissue 12 h after abdominal injection. Furthermore, Ipsen 5i (1 mg/kg bw) significantly inhibited gnrh expression in hypothalamic tissue, as well as fshb and lhb gene expression in pituitary tissue 12 h after abdominal injection. In summary, Mc4r singling appears to stimulate gnrh expression in the hypothalamus, thereby modulating the synthesis of Fsh and Lh in the pituitary. In addition, Mc4r also appears to directly regulate fshb and lhb levels in the pituitary in spotted scat. Our study suggests that Mc4r, through the hypothalamus and pituitary, participates in reproductive regulation in fish.

Effects of fish oil on ovarian development in spotted scat (Scatophagus argus).

The effects of different concentrations of dietary fish oil (0, 2%, or 6%) on ovarian development in 2-year-old female Scatophagus argus were investigated. The levels of serum sex steroid hormones (estradiol-17ß, E2; testosterone, T), protein phosphorus (SPP), and protein calcium (SPC), as well as vitellogenin (vtg) mRNA expression in livers and ovaries were measured. Over the eight week experimental period, oocytes did not develop further and remained at phase III in fish fed with the control diet with no supplement of fish oil. Fish fed with 2% fish oil supplement had oocytes at transition phase from III to IV. Fish fed with 6% fish oil supplement had oocytes at late phase IV. Higher gonadosmatic index, serum E2, SPP, SPC, and liver vtg expression were found in 6% fish oil group compared to that in the 2% fish oil group (except E2) and the control group (P<0.05). In addition, vtg expression in livers was 600-1000 times higher than that in the ovaries. Gonadosmatic index, E2, and SPP, as well as liver vtg expression increased during the experiment and peaked at the end of experiment. However, hepatosomatic index, serum T, and ovarian vtg expression peaked at 4 weeks, and then decreased at 8 weeks, with no significant difference among the 3 groups. In summary, we showed that 6% fish oil supplementation in S. argus could effectively promote ovarian development, with associated increases in E2 secretion and increased liver vtg mRNA expression.

Curcumin alleviates dystrophic muscle pathology in mdx mice.

Abnormal activation of nuclear factor kappa B (NF-kappaB) probably plays an important role in the pathogenesis of Duchenne's muscular dystrophy (DMD). In this report, we evaluated the efficacy of curcumin, a potent NF-kappaB inhibitor, in mdx mice, a mouse model of DMD. We found that it improved sarcolemmic integrity and enhanced muscle strength after intraperitoneal (i.p.) injection. Histological analysis revealed that the structural defects of myofibrils were reduced, and biochemical analysis showed that creatine kinase (CK) activity was decreased. We also found that levels of tumor necrosis factor alpha (TNF-alpha), interleukin-1 beta (IL-1beta) and inducible nitric oxide synthase (iNOS) in the mdx mice were decreased by curcumin administration. EMSA analysis showed that NF-kappaB activity was also inhibited. We thus conclude that curcumin is effective in the therapy of muscular dystrophy in mdx mice, and that the mechanism may involve inhibition of NF-kappaB activity. Since curcumin is a non-toxic compound derived from plants, we propose that it may be useful for DMD therapy.