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.

Comparative transcriptome analysis of male and female gonads reveals sex-biased genes in spotted scat (Scatophagus argus).

Scatophagus argus is a new emerging aquaculture fish in East and Southeast Asia. To date, research on reproductive development and regulation in S. argus is lacking. Additionally, genetic and genomic information about reproduction, such as gonadal transcriptome data, is also lacking. Herein, we report the first gonadal transcriptomes of S. argus and identify genes potentially involved in reproduction and gonadal development. A total of 136,561 unigenes were obtained by sequencing of testes (n = 3) and ovaries (n = 3) at stage III. Genes upregulated in males and females known to be involved in gonadal development and gametogenesis were identified, including male-biased dmrt1, amh, gsdf, wt1a, sox9b, and nanos2, and female-biased foxl2, gdf9, bmp15, sox3, zar1, and figla. Serum estradiol-17ß and 11-ketotestosterone levels were biased in female and male fish, respectively. Sexual dimorphism of serum steroid hormone levels were interpreted after expression analysis of 20 steroidogenesis-related genes, including cyp19a1a and cyp11b2. This gonadal transcript dataset will help investigate functional genes related to reproduction in S. argus.

Estradiol-17ß regulates the expression of insulin-like growth factors 1 and 2 via estradiol receptors in spotted scat (Scatophagus argus).

Insulin-like growth factors (Igf1 and Igf2) play a key role in growth and development of vertebrates. In mammals, the expression of IGFs is regulated by estradiol-17ß (E2) via estrogen receptors (ESRs). The expression of igfs can also be regulated by E2 in fish, while comparative study of this is still lacking. The present study examined tissue distribution of igfs and hepatic expression of igfs and esrs during gonad development in Scatophagus argus by real-time PCR. Serum E2 concentration was measured by enzyme-linked immunosorbent assay (ELISA). The hepatic expression of igfs and esrs at gonadal phase III, incubated with either E2 (0.1, 1 or 10 µM) alone or in combination with estrogen receptor antagonists-fulvestrant, MPP or PHTPP, was measured. igf1 and igf2 expressed highest in liver of both sexes. Igf1, esr1 and esr2b expressions and serum E2 concentration increased, while igf2 and esr2a expressions decreased, during ovary development. Igfs and esrs expressions increased while serum E2 concentration maintained low during testis development. In females, E2 incubation enhanced the expressions of igf1 and esr1 but inhibited that of igf2 and esr2a. Both fulvestrant and MPP inhibited up-regulation effect of E2 on igf1 and esr1. Fulvestrant enhanced down-regulation effect of E2 on igf2 and esr2a, but MPP conversely. In males, E2 incubation enhanced the expressions of igfs, esr1 and esr2a. Fulvestrant and MPP inhibited up-regulation effect of E2 on igfs and esr1. PHTPP inhibited igf1 and esr2 expressions in both sexes. Our results indicated that the expression of igfs is regulated by E2 via Esrs in S. argus.

Expression and transcriptional regulation of gsdf in spotted scat (Scatophagus argus).

Gonadal soma-derived factor (Gsdf) is critical for testicular differentiation and early germ cell development in teleosts. The spotted scat (Scatophagus argus), with a stable XX-XY sex-determination system and the candidate sex determination gene dmrt1, provides a good model for understanding the mechanism of sex determination and differentiation in teleosts. In this study, we analyzed spotted scat gsdf tissue distribution and gene expression patterns in gonads, as well as further analysis of transcriptional regulation. Tissue distribution analysis showed that gsdf was only expressed in testis and ovary. Real-time PCR showed that both gsdf and dmrt1 were expressed significantly higher in testes at different phases (phase III, IV and V) compared to ovaries at phase II, III and IV, while gsdf was expressed significantly higher in phase II ovaries than those of phase III and IV. Western blot analysis also showed that Gsdf was more highly expressed in the testis than ovary. Immunohistochemistry analysis showed that Gsdf was expressed in Sertoli cells surrounding spermatogonia in the testis, while it was expressed in the somatic cells surrounding the oogonia of the ovary. Approximately 2.7 kb of the 5' upstream region of gsdf was cloned from the spotted scat genomic DNA and in silico promoter analysis revealed the putative transcription factor binding sites of Dmrt1 and Sf1. The luciferase reporter assay, using the human embryonic kidney cells, demonstrated that Dmrt1 activated gsdf expression in a dose-dependent manner in the presence of Sf1 in spotted scat. These results suggest that Gsdf could play a role in regulating the development of spermatogonia and oogonia, and also participate in male sex differentiation by acting as a downstream gene of Dmrt1 in spotted scat.

Phoenixin: Expression at different ovarian development stages and effects on genes ralated to reproduction in spotted scat, Scatophagus argus.

Phoenixin (Pnx), a recently discovered neuropeptide, has been implicated in reproduction. Pnx mainly exists in two active isoforms, phoenixin-14 (Pnx-14) and phoenixin-20 (Pnx-20). However, little is known about the functions of Pnx in teleosts. To determine the roles of Pnx in the regulation of reproduction in Scatophagus argus, the physiological characterization of the Pnx was analyzed. During ovary development, the expression of pnx in phase IV was higher than in phase II and III in the hypothalamus. In the pituitary, pnx expression was highest in phase IV, moderate in phase III, and lowest in phase II. When hypothalamus and pituitary fragments were cultured in vitro with Pnx-14 and Pnx-20 (10 nM and 100 nM) for 6 h, the expression of GnRHR (gonadotropin releasing hormone receptor), lh (luteinizing hormone) and fsh (follicular stimulating hormone) in the pituitary increased significantly, except GnRH (gonadotropin releasing hormone) in the hypothalamus. Similarly, the expression of GnRHR, lh and fsh in the pituitary increased significantly after injecting S. argus with Pnx-14 and Pnx-20 (10 ng/g and 100 ng/g body weight (bw)), except GnRHR and fsh treated with 10 ng/gbw Pnx-20 in the pituitary and GnRHs in the hypothalamus. These results indicate that Pnx may not only stimulate the reproduction of the S. argus through the hypothalamic-pituitary-gonadal (HPG) axis, but also directly through the pituitary.

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.

Cloning, expression and functional characterization on vitellogenesis of estrogen receptors in Scatophagus argus.

Estrogen receptors (Er) play a critical role in vitellogenesis. Three ers (erα, erß1 and erß2) and vitellogenins (vtg-A, vtg-B and vtg-C) subtypes were isolated in various fish species, while the contribution of each Er to the regulation of vtgs expression was not analyzed in detail. Here, erα, erß1 and erß2 were cloned and all were found to be expressed in female liver in Scatophagus argus. During proteic vitellogenesis stage, erα was simultaneously up-regulated, while erß1 and erß2 were not, with three vtgs in female liver. The effects of 17ß-estradiol (E2) alone or combined with Er antagonists on ers, vtgs mRNA expressions and Vtg protein content in incubated male liver were examined by real-time PCR and enzyme-linked immunosorbent assay (ELISA), respectively. The expressions of erα, erß1, vtgs mRNA and Vtg protein increased significantly after 24h incubation with E2 (0.1, 1 and 10µM), while Er nonselective antagonist ICI 182 780 (0.01, 0.1 and 1µM) significantly attenuated the up-regulation effects of E2 on ers, vtgs mRNA and Vtg protein in a dose-dependent manner. Erα selective antagonist Methyl-piperidinopyrazole (MPP) (0.01, 0.1 and 1µM) significantly attenuated the up-regulation effects of E2 on erα, vtg-B, vtg-C mRNA and Vtg protein, while promoted the expression of erß1 and vtg-A. Erß selective antagonist Cyclofenil (0.01, 0.1 and 1µM) attenuated the up-regulation effects of E2 on erß1, erß2, vtg-A, vtg-C mRNA and Vtg protein while promoted the expression of erα and vtg-B. Our results suggest that the regulation of Ers on different vtgs was divergent in S. argus.

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.

Molecular cloning, tissue distribution, and pharmacological characterization of melanocortin-4 receptor in spotted scat, Scatophagus argus.

Melanocortin-4 receptor (MC4R) plays an important role in the regulation of food intake and energy expenditure in mammals. The functions of the MC4R in fish have not been investigated extensively. We herein reported on the cloning, tissue distribution, and pharmacological characterization of spotted scat (Scatophagus argus) MC4R (SAMC4R). It consisted of a 984bp open reading frame predicted to encode a protein of 327 amino acids. Sequence analysis revealed that SAMC4R was highly homologous (>80%) at amino acid levels to several teleost MC4Rs. Phylogenetic analyses showed that SAMC4R was closely related to piscine MC4R. Using RT-PCR, we showed that in addition to brain, pituitary, and gonads, mc4r mRNA was also widely expressed in peripheral tissues of spotted scat in sexually divergent pattern. With human MC4R (hMC4R) as a control, several agonists including α-melanocyte stimulating hormone (α-MSH), [Nle(4), D-Phe(7)]-α-MSH (NDP-MSH), adrenocorticotropic hormone (ACTH) and THIQ (N-[(3R)-1,2,3,4-tetrahydroisoquinolinium3-ylcarbonyl]-(1R)-1-(4-chlorobenzyl)-2-[4-cyclohexyl-4-(1H-1,2,4-triazol-1-ylmethyl)piperidin-1-yl]-2-oxoethylamine), were used to investigate the binding and signaling properties of SAMC4R. The results showed that SAMC4R bound NDP-MSH with the highest affinity followed by ACTH (1-24) and α-MSH. Similar ranking was also found for hMC4R, although SAMC4R had two to five-fold higher affinities for these ligands. THIQ did not displace NDP-MSH from SAMC4R, different from hMC4R. α-MSH, NDP-MSH, and ACTH (1-24) were identified as potent agonists to stimulate cAMP generation followed by THIQ in SAMC4R. The availability of SAMC4R and its pharmacological characteristics will facilitate the investigation of its function in regulating diverse physiological processes in spotted scat.