Anti-FSHB monoclonal antibody, clone F2 (BGN/F63/02)

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 ([Nle(4), D-Phe(7)]-alpha-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.

Background Polycystic ovary syndrome (PCOS), the commonest endocrine disorder affecting young women, appears to be a multigenic trait with contributing genes being unclear. Hence, analysis of polymorphisms in multiple candidate genes is required. Currently available genotyping methods are expensive, time-consuming with limited analytical sensitivity. Aim (i) Develop and validate high resolution melting (HRM) assay and allele-specific real-time quantitative PCR (AS-qPCR) for genotyping selected SNPs associated with PCOS. (ii) Identify selected SNPs and their association with a Sri Lankan cohort of well-characterized PCOS. Methods DNA was extracted from women with well-characterized PCOS from adolescence (n = 55) and ethnically matched controls (n = 110). FTO (Fat mass and obesity associated gene; rs9939609), FSHB (Follicle stimulating hormone beta subunit; rs6169), FSHR (Follicle stimulating hormone receptor; rs6165/rs6166), and INSR (Insulin receptor; rs1799817) genes were genotyped using HRM assay. GnRH1 (Gonadotropin releasing hormone; rs6185), LHB (Luteinizing hormone beta subunit; rs1800447/rs34349826) and LHCGR (Luteinizing hormone/choriogonadotropin receptor; rs2293275) genes were genotyped using AS-qPCR method. Genotyping results were validated using Sanger sequencing. Results A significant association was observed within FTO gene polymorphism (rs9939609) and PCOS. Genotype frequency of FTO gene (rs9939609)-cases versus controls were TT-36.4% vs.65.4% (p< 0.05), AT-23.6% vs. 20.9%, AA-40% vs. 13.6% (p< 0.05). Genotype frequencies of the SNPs GnRH1 (rs6185), FSHB (rs6169), FSHR (rs6165 & rs6166), LHB (rs1800447 & rs34349826), LHCGR (rs2293275) and INSR (rs1799817) were not significantly different between cases and controls (p> 0.05). Only the mutant alleles were observed for LHB rs1800447 and rs34349826 SNPs in both groups. The HRM and AS-qPCR assay results had 100% concordance with sequencing results. Conclusions FTO gene rs9939609 polymorphism is significantly more prevalent among Sri Lankan PCOS subjects while the other selected SNPs of HPG axis genes and INSR gene showed no association. HRM and AS-qPCR assays provide a reliable, fast and user-friendly genotyping method facilitating wider implication in clinical practice.