Ontogeny of the specificity of gonadotropin receptors and gene expression in carp

Abstract

The pituitary gonadotropins, luteinizing hormone (LH) and follicle-stimulating hormone (FSH), are the principle endocrine drivers of reproductive processes in the gonads of jawed vertebrates. Canonically, FSH recruits and maintains selected ovarian follicles for maturation and LH induces the stages of germinal vesicle breakdown and ovulation. In mammals, LH and FSH specifically activate cognate G-protein-coupled receptors that affect the proteins involved in steroidogenesis, protein hormone synthesis, and gametogenesis. This dual-gonadotropin model also exists in some fish species, but not in all. In fact, due to their diverse number of species, extended number of ecological niches, and remarkably flexible reproductive strategies, fish are appropriate as models to understand the co-evolution of gonadotropins and their receptors. In this study, we cloned and characterized the expression profile over the final stages of ovarian maturation of carp (Cyprinus carpio) LHCGR and FSHR. Expression of both gonadotropin receptors increased in the later stage of early vitellogenesis, suggesting that both LH and FSH play a role in the development of mature follicles. We additionally tested the activation of cLHCGR and cFSHR using homologous and heterologous recombinant gonadotropins in order to gain insight into an evolutionary model of permissive gonadotropin receptor function. These data suggest that carp (Cyprinus carpio) gonad development and maturation depends on a specific gonadotropin profile that does not reflect the temporally distinct dual-gonadotropin model observed in salmonids or mammals, and that permissive gonadotropin receptor activation is a specific feature of Ostariophysi, not all teleosts.

Differential Regulation of Gonadotropins as Revealed by Transcriptomes of Distinct LH and FSH Cells of Fish Pituitary

“…cDNA preparation was accomplished with 2 ng of the FACS cells mRNA using the Verso cDNA Synthesis Kit (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s instruction. Real-time PCR validations were performed as described previously [ 154 ]. cDNA (3 μL) was used as template in a 20 μL final reaction volume with Platinum SYBR Green qPCR SuperMix (Invitrogen).…”

“…To assess the mRNA expression of lhβ, fshβ, and gh in each fraction, mRNA levels (the averaged of two technical replicates) were normalized against the reference gene, EF1α, using the comparative threshold cycle method (−∆CT). Ef1α was previously shown to be a suitable reference gene for gene expression in several fish species including tilapia [ 14 , 154 ]. Primer sequences are detailed in Supplementary Table S1 .…”

Identifying the Interaction of the Brain and the Pituitary in Social – and Reproductive – State of Tilapia by Transcriptome Analyses

“…mRNA levels were normalized against the reference genes, EF1α , using the comparative threshold cycle method (−∆CT). Ef1α was previously shown to be a suitable reference gene for tissue analysis in fish [32, 33]. Primer sequences are detailed in online supplementary Table 4 (for all online suppl.…”

“…For the cDNA preparation, 1-µg mRNA from each sample (from the three pairs used for the RNAseq and four new pairs of fish, in total seven pairs of fish) was used in the Verso cDNA Synthesis Kit (Thermo Fisher), according to the manufacturer's instructions. Real-time PCR validations were 1240 DOI: 10.1159/000524437 performed as described previously [32]. In short, cDNA (3 μL) was used as a template in a 20-μL reaction volume of Platinum SYBR Green qPCR SuperMix (Invitrogen).…”

Characteristics of Neurokinin-3 Receptor and Its Binding Sites by Mutational Analysis

“…We previously demonstrated that cAMP response element (CRE-LUC; Invitrogen, Massachusetts, United States) reporter systems are a useful tool for discriminating cAMP/PKA signaling pathways [47], and that PKA is the dominant pathway for tiTac3Ra [20,22]. Transient transfection, cell procedures and stimulation protocols followed the procedures previously described [48]. Briefly, 3 µg of each construct, together with 3 µg of a luciferase reporter plasmid were transiently transfected into COS-7 cells (American Type Culture Collection, Manassas, VA, USA).…”