Laser capture microdissection of gonads from juvenile zebrafish

Anne Jørgensen, John E. Nielsen, Jane Ebsen Morthorst, Poul Bjerregaard, Henrik Leffers

    Publikation: Bidrag til tidsskriftTidsskriftartikelForskningpeer review

    Resumé

    Background: Investigating gonadal gene expression is important in attempting to elucidate the molecular mechanism of sex determination and differentiation in the model species zebrafish. However, the small size of juvenile zebrafish and correspondingly their gonads complicates this type of investigation. Furthermore, the lack of a genetic sex marker in juvenile zebrafish prevents pooling gonads from several individuals. The aim of this study was to establish a method to isolate the gonads from individual juvenile zebrafish allowing future investigations of gonadal gene expression during sex determination and differentiation.

    Methods: The laser capture microdissection technique enables isolation of specific cells and tissues and thereby removes the noise of gene expression from other cells or tissues in the gene expression profile. A protocol developed for laser microdissection of human gonocytes was adjusted and optimised to isolate juvenile zebrafish gonads.

    Results: The juvenile zebrafish gonad is not morphologically distinguishable when using dehydrated cryosections on membrane slides and a specific staining method is necessary to identify the gonads. The protocol setup in this study allows staining, identification, isolation and subsequent RNA purification and amplification of gonads from individual juvenile zebrafish thereby enabling gonadal gene expression profiling.

    Conclusion: The study presents a protocol for isolation of individual juvenile zebrafish gonads, which will enable future investigations of gonadal gene expression during the critical period of sex differentiation. Furthermore, the presented staining method is applicable to other species as it is directed towards alkaline phosphatase that is expressed in gonocytes and embryonic stem cells, which is conserved among vertebrate species.

    OriginalsprogEngelsk
    TidsskriftReproductive Biology and Endocrinology
    Vol/bind7
    Sider (fra-til)97
    ISSN1477-7827
    DOI
    StatusUdgivet - 2009

    Bibliografisk note

    Art. no. 1477

    Citer dette

    Jørgensen, A., Nielsen, J. E., Morthorst, J. E., Bjerregaard, P., & Leffers, H. (2009). Laser capture microdissection of gonads from juvenile zebrafish. Reproductive Biology and Endocrinology, 7, 97. https://doi.org/10.1186/1477-7827-7-97
    Jørgensen, Anne ; Nielsen, John E. ; Morthorst, Jane Ebsen ; Bjerregaard, Poul ; Leffers, Henrik. / Laser capture microdissection of gonads from juvenile zebrafish. I: Reproductive Biology and Endocrinology. 2009 ; Bind 7. s. 97.
    @article{2dda8350f54411dea1c9000ea68e967b,
    title = "Laser capture microdissection of gonads from juvenile zebrafish",
    abstract = "Background: Investigating gonadal gene expression is important in attempting to elucidate the molecular mechanism of sex determination and differentiation in the model species zebrafish. However, the small size of juvenile zebrafish and correspondingly their gonads complicates this type of investigation. Furthermore, the lack of a genetic sex marker in juvenile zebrafish prevents pooling gonads from several individuals. The aim of this study was to establish a method to isolate the gonads from individual juvenile zebrafish allowing future investigations of gonadal gene expression during sex determination and differentiation. Methods: The laser capture microdissection technique enables isolation of specific cells and tissues and thereby removes the noise of gene expression from other cells or tissues in the gene expression profile. A protocol developed for laser microdissection of human gonocytes was adjusted and optimised to isolate juvenile zebrafish gonads.Results: The juvenile zebrafish gonad is not morphologically distinguishable when using dehydrated cryosections on membrane slides and a specific staining method is necessary to identify the gonads. The protocol setup in this study allows staining, identification, isolation and subsequent RNA purification and amplification of gonads from individual juvenile zebrafish thereby enabling gonadal gene expression profiling.Conclusion: The study presents a protocol for isolation of individual juvenile zebrafish gonads, which will enable future investigations of gonadal gene expression during the critical period of sex differentiation. Furthermore, the presented staining method is applicable to other species as it is directed towards alkaline phosphatase that is expressed in gonocytes and embryonic stem cells, which is conserved among vertebrate species.",
    author = "Anne J{\o}rgensen and Nielsen, {John E.} and Morthorst, {Jane Ebsen} and Poul Bjerregaard and Henrik Leffers",
    note = "Art. no. 1477",
    year = "2009",
    doi = "10.1186/1477-7827-7-97",
    language = "English",
    volume = "7",
    pages = "97",
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    Jørgensen, A, Nielsen, JE, Morthorst, JE, Bjerregaard, P & Leffers, H 2009, 'Laser capture microdissection of gonads from juvenile zebrafish', Reproductive Biology and Endocrinology, bind 7, s. 97. https://doi.org/10.1186/1477-7827-7-97

    Laser capture microdissection of gonads from juvenile zebrafish. / Jørgensen, Anne; Nielsen, John E.; Morthorst, Jane Ebsen; Bjerregaard, Poul; Leffers, Henrik.

    I: Reproductive Biology and Endocrinology, Bind 7, 2009, s. 97.

    Publikation: Bidrag til tidsskriftTidsskriftartikelForskningpeer review

    TY - JOUR

    T1 - Laser capture microdissection of gonads from juvenile zebrafish

    AU - Jørgensen, Anne

    AU - Nielsen, John E.

    AU - Morthorst, Jane Ebsen

    AU - Bjerregaard, Poul

    AU - Leffers, Henrik

    N1 - Art. no. 1477

    PY - 2009

    Y1 - 2009

    N2 - Background: Investigating gonadal gene expression is important in attempting to elucidate the molecular mechanism of sex determination and differentiation in the model species zebrafish. However, the small size of juvenile zebrafish and correspondingly their gonads complicates this type of investigation. Furthermore, the lack of a genetic sex marker in juvenile zebrafish prevents pooling gonads from several individuals. The aim of this study was to establish a method to isolate the gonads from individual juvenile zebrafish allowing future investigations of gonadal gene expression during sex determination and differentiation. Methods: The laser capture microdissection technique enables isolation of specific cells and tissues and thereby removes the noise of gene expression from other cells or tissues in the gene expression profile. A protocol developed for laser microdissection of human gonocytes was adjusted and optimised to isolate juvenile zebrafish gonads.Results: The juvenile zebrafish gonad is not morphologically distinguishable when using dehydrated cryosections on membrane slides and a specific staining method is necessary to identify the gonads. The protocol setup in this study allows staining, identification, isolation and subsequent RNA purification and amplification of gonads from individual juvenile zebrafish thereby enabling gonadal gene expression profiling.Conclusion: The study presents a protocol for isolation of individual juvenile zebrafish gonads, which will enable future investigations of gonadal gene expression during the critical period of sex differentiation. Furthermore, the presented staining method is applicable to other species as it is directed towards alkaline phosphatase that is expressed in gonocytes and embryonic stem cells, which is conserved among vertebrate species.

    AB - Background: Investigating gonadal gene expression is important in attempting to elucidate the molecular mechanism of sex determination and differentiation in the model species zebrafish. However, the small size of juvenile zebrafish and correspondingly their gonads complicates this type of investigation. Furthermore, the lack of a genetic sex marker in juvenile zebrafish prevents pooling gonads from several individuals. The aim of this study was to establish a method to isolate the gonads from individual juvenile zebrafish allowing future investigations of gonadal gene expression during sex determination and differentiation. Methods: The laser capture microdissection technique enables isolation of specific cells and tissues and thereby removes the noise of gene expression from other cells or tissues in the gene expression profile. A protocol developed for laser microdissection of human gonocytes was adjusted and optimised to isolate juvenile zebrafish gonads.Results: The juvenile zebrafish gonad is not morphologically distinguishable when using dehydrated cryosections on membrane slides and a specific staining method is necessary to identify the gonads. The protocol setup in this study allows staining, identification, isolation and subsequent RNA purification and amplification of gonads from individual juvenile zebrafish thereby enabling gonadal gene expression profiling.Conclusion: The study presents a protocol for isolation of individual juvenile zebrafish gonads, which will enable future investigations of gonadal gene expression during the critical period of sex differentiation. Furthermore, the presented staining method is applicable to other species as it is directed towards alkaline phosphatase that is expressed in gonocytes and embryonic stem cells, which is conserved among vertebrate species.

    U2 - 10.1186/1477-7827-7-97

    DO - 10.1186/1477-7827-7-97

    M3 - Journal article

    VL - 7

    SP - 97

    JO - Reproductive Biology and Endocrinology

    JF - Reproductive Biology and Endocrinology

    SN - 1477-7827

    ER -