Vzpostavitev celične kulture spermatogonijev in oogonijev potočne postrvi.

Zabret, Nika (author), Sušnik Bajec, Simona (mentor), Djurdjevič, Ida (comentor)

Linija zarodnih celic vključuje vse razvojne stadije celic, ki vodijo v nastanek spermijev in jajčec. Od somatskih celic se izvorne zarodne celice ločijo že v zgodnjem embrionalnem razvoju, nato migrirajo do mesta gonad in tam diferencirajo v spermatogonije in oogonije, ki predstavljajo zarodne matične celice. Zaradi zmožnosti samoobnavljanja in diferenciacije v gamete so spermatogoniji in oogoniji primerni za transplantacijo ali krioprezervacijo. Za povečanje uspešnosti transplantacije in krioprezervacije je potrebno željene celice predhodno namnožiti. Cilj našega dela je bil vzpostaviti celično kulturo spermatogonijev in oogonijev potočne postrvi. Celice smo po izolaciji iz gonad očistili z gradientnim centrifugiranjem ter jih nasadili v osnovno in bogato gojišče. Dodatno stopnjo čistosti smo dosegli z diferencialnim presajanjem. Po šestnajstih dneh gojenja smo delitve celic preverili z detekcijo z EdU. Z merjenjem fluorescence smo dokazali, da so se spermatogoniji in oogoniji v celični kulturi delili. Na podlagi rezultatov lahko zaključimo, da sta gradientno centrifugiranje in diferencialno presajanje učinkoviti metodi za čiščenje spermatogonijev in oogonijev. Prav tako pa obogatitev osnovnega gojišča ugodno vpliva na rast celic. Z vzpostavitvijo celične kulture smo uspešno namnožili spermatogonije in oogonije ter jih tako pripravili za nadaljnjo uporabo.

• Language: Slovenian
• Keywords: oogonij, spermatogonij, zarodne celice, celična kultura, potočna postrv, transplantacija
• Work type: Bachelor thesis/paper
• Typology: 2.11 - Undergraduate Thesis
• Organization: BF - Biotechnical Faculty
• Publisher: [N. Zabret]
• Year: 2020
• PID: 20.500.12556/RUL-119714
• UDC: 597.552.56:601.2:575.853:602.9:591.81:57.086(043.2)
• COBISS.SI-ID: 31142403
• Publication date in RUL: 11.09.2020

Secondary language

• Language: English
• Title: Establishment of spermatogonial and oogonial cell culture in brown trout

Abstract

The germ cell line includes all cell types that lead to the development of eggs and sperm. The somatic cells already separate from the primordial germs cells in the early stage of embryonic development. Afterwards they migrate to the gonads where they differentiate into spermatogonia and oogonia which represent germ stem cells. The ability of self-renewal and differentiation into gametes gives the spermatogonia and oogonia potential use for transplantation and cryopreservation. To improve the success of transplantation and cryopreservation isolated cells need to be multiplied beforehand. The objective of our study was to establish spermatogonial and oogonial cell culture in brown trout. After isolating the cells from gonads we enriched these cells with gradient centrifugation and then seeded them in a basic and enriched medium. Additional enrichment of spermatogonia and oogonia was achieved by differential plating. After sixteen days of cultivation we used EdU detection to determine cell proliferation. The observed fluorescence shows evidence of spermatogonia and oogonia proliferation. Based on our results we can conclude that gradient centrifugation and differential plating are efficient methods for cell enrichment. We also concluded that a positive effect on cell growth is shown in cells seeded in the enriched medium. With cell culturing we achieved the proliferation of spermatogonia and oogonia and prepared them for further use.

• Keywords: oogonium, spermatogonium, germ cells, cell culture, brown trout, transplantation