Lehrstuhl für Physiologische Chemie

    Sex determination and reproductive development in fish

    (Dr. Verena Kottler, Dr. Álvaro S. Roco, MSc. Mateus C. Adolfi, MSc. Anabel Martinez B., Ruiqi Liu)

    In fish, mechanisms of sex determination are very diversified: even within groups of closely related species a wide spectrum of different systems can be found. We are studying the molecular mechanisms of sex determination and the genetic organization of sex chromosomes in selected fish species to generate a database that will allow a better understanding of the evolutionary processes generating such apparent diversity.

    Evolution of sex determination and sex chromosomes

    Evolution of sex determination and sex chromosomes

    Poeciliids are a suitable animal model to study the evolution of sex determination and sex chromosomes. Species with either male (XX/XY) or female (WZ/ZZ) heterogamety exist within this group and in some cases even three types of sex chromosomes (W, Y, X) have been reported, e.g., in the platyfish (Xiphophorus maculatus).

    Sex-linked inheritance of traits has been demonstrated; for instance, many of the highly polymorphic color patterns of the guppy (Poecilia reticulata) and platyfish are linked to the respective male sex-determination locus on the Y chromosome. Nevertheless, the presence of large amounts of repeats, caused by the suppression of recombination around the sex-determining gene, makes it difficult to identify the genes located in these regions.

    In order to characterize the sex-determining region in the guppy, platyfish, and other poeciliids, we utilize Next Generation Sequencing (NGS) (whole genome sequencing, RNA-seq, and RAD-seq) as well as cytogenetic and molecular biology techniques, for instance, FISH, cloning of candidate genes, in situ hybridization and BAC and fosmid libraries. Identification of the genes in these regions will help to understand how the high degree of color polymorphism is generated and maintained in these species and will reveal the evolutionary relationships of these sex chromosomes.

    Xiphophorus maculatus male and female
    Xiphophorus maculatus male and female
    Xiphophorus maculatus chromosomes. FISH with BACs probes for the sex chromosomes
    Xiphophorus maculatus chromosomes.
    FISH with BACs probes for the sex chromosomes
    Endler guppy male
    Endler guppy male
    Mechanisms of sex determination in the genus Oryzias

    Mechanisms of sex determination in the genus Oryzias

    The diversity of sex determination mechanisms is particularly evident in species of the genus Oryzias, which includes the medaka, O. latipes. In this fish the master male sex determination gene (dmrt1bY) is known and it is present in the sister species O. curvinotus but absent in the other closely related species. Despite the huge diversity of upstream and downstream factors and networks involved in the process of sex determination, some phenomena still appear to be highly conserved throughout evolution. A common feature found in most studied vertebrates is an early morphological difference between male and female that becomes apparent on the germ cell level. The germ cells start to proliferate and enter meiosis first in female compared to male. Thus, main questions of current interest in our group are: How similar are the downstream gene networks that respond to these triggers and actually how do they work? Did the diversity of sex determination mechanism converge to conserved key effectors, like germ cells proliferation and meiosis entry? We use techniques like gene expression by qRT-PCR and in situ hybridization, transgenic reporter lines, genome editing by CRISPR/Cas9, immunohistochemistry and confocal microscope imaging.

    Medaka fish Oryzias latipes
    Medaka fish Oryzias latipes
    Fluorescent in situ hybridization in medaka ovary
    Fluorescent in situ hybridization in medaka ovary
    Medaka transgenic GFP line
    Medaka transgenic GFP line
    Molecular dynamics of Mc4r in the Xiphophorus fish reproduction

    Molecular dynamics of Mc4r in the Xiphophorus fish reproduction

    Reproduction is a fundamental feature of life. Puberty is an important physiological process in the reproduction, and timing of puberty is a polymorphic traits within species affected by genetic polymorphism.

    As male fish in the Xiphophorus genus cease to grow when they reach puberty, the size of the fish correlates with the timing of the puberty. The wild population contains naturally “early-matured” small males and “late-matured” large males. They also apply different reproduction strategies, in which the latter ones court, and the former ones do sneak mating. The puberty timing of these fish is determined by the mc4r gene on the Puberty locus. In platyfish and swordtails, the P locus encodes wild type and non-functional versions of Mc4r and the accessory protein MRAP2a is acting on the Mc4r system. This system is involved in energy balance in vertebrate and puberty in Xiphophorus fish. The melanocortin 4 receptor belongs to melanocortin receptor family. The Mcrs affects distinct physiological processes, such as pigmentation, steroidogenesis, and energy homeostasis.

    Our goal is to investigate the molecular basis of the dynamic interaction of Mc4r alleles and Mc4r with MRAP2a, as well as functional consequences of such interactions. The main techniques used include molecular cloning, transgenic reporter line, luciferase assay, fluorescence resonance energy transfer techniques, confocal or other microscopy imaging.

    Wild type Xiphophorus nigresis male fish showing body length polymorphism (A) and genetic polymophism (B) (Source: modified from Maderspacher F. 2010, Curr Biol and Lampert KP et al. 2010, Curr Biol)
    Wild type Xiphophorus nigresis male fish showing body length polymorphism (A) and genetic polymophism (B) (Source: modified from Maderspacher F. 2010, Curr Biol and Lampert KP et al. 2010, Curr Biol)
    Mc4r may form homodimers or heterodimers, which can be measured by FRET technique
    Mc4r may form homodimers or heterodimers, which can be measured by FRET technique
    Dimerization of Mc4r (a Class A GPCR) may have distinct functions
    Dimerization of Mc4r (a Class A GPCR) may have distinct functions

    Selected references (Sex determination and reproductive development in fish)

    EMBO Rep. 2015 Oct;16(10):1260-74. Plasticity of gene-regulatory networks controlling sex determination: of masters, slaves, usual suspects, newcomers, and usurpators. Herpin A, Schartl M.

    Genetics. 2014 Jun;197(2):625-41. A RAD-tag genetic map for the platyfish (Xiphophorus maculatus) reveals mechanisms of karyotype evolution among teleost fish. Amores A, Catchen J, Nanda I, Warren W, Walter R, Schartl M, Postlethwait JH.

    Mol Biol Evol. 2013 Oct;30(10):2328-46. Divergent expression regulation of gonad development genes in medaka shows incomplete conservation of the downstream regulatory network of vertebrate sex determination. Herpin A, Adolfi MC, Nicol B, Hinzmann M, Schmidt C, Klughammer J, Engel M, Tanaka M, Guiguen Y, Schartl M.

    Nat Genet. 2013 May;45(5):567-72. The genome of the platyfish, Xiphophorus maculatus, provides insights into evolutionary adaptation and several complex traits. Schartl M, Walter RB, Shen Y, Garcia T, Catchen J, Amores A, Braasch I, Chalopin D, Volff JN, Lesch KP, Bisazza A, Minx P, Hillier L, Wilson RK, Fuerstenberg S, Boore J, Searle S, Postlethwait JH, Warren WC.

    Chromosoma 123(4):373-383. Sex chromosome polymorphism in guppies. Nanda I, Schories S, Tripathi N, Dreyer C, Haaf T, Schmid M, Schartl M.

    Genetics (2013) 195: 1337-1352 Gene amplification and functional diversification of melanocortin 4 receptor at an extremely polymorphic locus controlling sexual maturation in the platyfish. Volff J-N, Selz Y, Hoffmann C, Froschauer A, Schultheis C, Schmidt C, Zhou Q, Bernhardt W, Hanel R, Böhne A et al.

    Current Biology (2010) 20: 1729-1734. Determination of onset of sexual maturation and mating behavior by melanocortin receptor 4 polymorphisms. Lampert KP, Schmidt C, Fischer P, Volff J-N, Hoffmann C, Muck J, Lohse MJ, Ryan MJ, Schartl M.

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