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|Title: ||點帶石斑Epinephelus coioides變性過程生殖細胞與生殖體細胞功能變化之探討|
|Authors: ||Tey, Wei-Guan|
|Contributors: ||NTOU:Department of Aquaculture|
|Issue Date: ||2018-08-22T06:05:01Z
|Abstract: ||點帶石斑(Epinephelus coioides)為先雌後雄之雌雄同體魚類，過去研究顯示石斑魚性別可以雄性素(androgen)誘導提早性轉變(雌→雄)，停止處理會再度性逆轉(雄→雌)。因此本研究將探討性轉變過程中，生殖細胞與生殖體細胞在功能轉換過程中的變化。本研究利用投餵MT(methyltestosterone)與AI(aromatase inhibiter)方式誘導性轉變，並於雄性功能時(spermatids)開始停餵MT或AI，停餵後每階段時間進行BrdU注射追蹤細胞變化並搭配雌雄性生殖相關標誌基因，如雄性：dmrt1、sox9、cyp11b；雌性：figla、foxl2、cyp19a1a進行Q-PCR分析。石斑魚投餵MT或AI三個月後呈雄性功能，停餵MT與AI後兩週開始性逆轉(雄→雌)並於停餵MT後十週與停餵AI後八週呈雌性功能(primary oocyte)。經組織染色(BrdU)顯示AI組性逆轉(雄→雌)速度快於MT組且符合Q-PCR結果，雄性相關基因皆顯著降低。性轉變過程中結果也顯示，雄性新生的生殖細胞(Brdu表現)主要集中在卵巢薄板(ovarian lamella)周圍的結締組織，而雌性新生的生殖細胞，無特定增生位置但大部分主要較集中於精巢組織中央，進行分散增生。生殖細胞在性轉變過程中是先進行細胞凋亡(apoptosis)作用，新的生殖細胞在進行細胞新生(proliferation)。然而生殖體細胞在性轉變過程中是進行功能性轉換(transdifferentiation)作用，來達到性別功能變換。另外為了探討性轉變過程中生殖細胞的靜止作用之原因，本實驗選殖了可能參與生殖細胞增生調控作用相關的amh基因，結果顯示在精巢分化時期amh與其受體amhr2表現量最高，amh基因只表現於精巢精原細胞(spermatogonium)周邊體細胞，並不表現於卵巢卵細胞。隨著生殖細胞發育amh表現量降低，故點帶石斑amh基因可能只參與調控雄性性腺發育並且與雄性早期生殖細胞增生的調節作用有關。|
Orange-spotted grouper (Epinepheluscoioides), a protogynous hermaphroditic fish, could be manipulated their sexuality by exogenous steroid treatment. Previous studies have shown that male fish can induce by androgen treatments. In addition, androgen-induced male are reversible after treatments termination. This study focuses on the germ cells and it surrounding cells functional changes and gene expression during reversible sex change after terminated of MT (methyltestosterone) and AI (aromatase inhibiter) treatments. We performed an immunohisto chemistry analysis of BrdU of these regenerating ovarian tissues to find the sexual fate of BrdU negative cells. Moreover, the male-specific and female-specific markers were be selected to analyze the gene expression pattern during sex reversal (male-specific-dmrt1,sox9,cyp11b;female-specific-figla, foxl2, cyp19a1a).Fish showed male function(spermatids) after three month treatments with MT and AI at the same period. Sex reversal was initiated after terminated treatment in second weeks. However, the female function (primary oocyte) presents two weeks earlier in AI terminated group (eight weeks) than MT terminated group (ten weeks). Regard to gene analysis with Q-PCR, the expression levels of male-related genes was coincident with male gonad development. Male-related genes were reduced following degradation of male gonad development. Sex change process also showed that newborn male germ cells (Brdu express) is mainly concentrated in the connective tissue surrounding the ovarian lamella, and newborn female germ cells, no specific location but most of the major proliferation more concentrated in the central testis tissue. Germ cells undergo apoptosis and it surrounding cells performed transdifferentiation in the process of sex change. In order to investigate the causes of the germ cell quiescence, we choose the Amh gene who participates in regulatory role of cell proliferation. The result shows that amh and its receptor amhrII highest expression in testis differentiation period and is only expressed in spermatogonia surrounding somatic cells, not expressed in ovarian oocytes. Therefore amh gene may be involved in the regulation of male gonadal development and associated with early male reproductive cell proliferation in Orange-spotted grouper.
|Appears in Collections:||[水產養殖學系] 博碩士論文|
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