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Please use this identifier to cite or link to this item: http://ntour.ntou.edu.tw:8080/ir/handle/987654321/30929

Title: 點帶石斑生殖腺性別分化與性轉變探討
The gonadal sex differentiation and sex change of orange-spotted grouper, Epinephelus coioides
Authors: Ya-Ju Tsai
蔡雅如
Contributors: NTOU:Department of Aquaculture
國立臺灣海洋大學:水產養殖學系
Keywords: 點帶石斑;生殖腺性別分化;性轉變;免疫組織染色
Epinephelus coioides;gonadal sex differentiation;sex change;immunohistochemistry stain
Date: 2012
Issue Date: 2012-04-16T01:53:43Z
Abstract: 點帶石斑 (Epinephelus coioides) 生殖腺先發育為雌性,孵化後四月齡兩葉生殖腺的兩股分支逐漸併合形成卵巢空腔 (ovarian cavity, OC),於四至五月齡卵巢空腔 (OC) 的形成作為雌魚性別分化的依據。隨著雌性生殖配子發育、增生,六個半月至七月齡出現初級卵母細胞 (primary oocyte, PO),與卵原細胞 (oogonium, OG) 共同夾雜於卵巢薄板 (OL) 內,一直到一年七月齡出現皮質小粒卵細胞 (cortical alveolar, CA)。最早在兩年又二月齡 (2+2 -age) 出現內生性卵黃堆積的成熟卵細胞 (vitellogenic oocyte, VO)。實驗以環化酵素抑制物 (aromatase inhibiter, AI) 或雄性素 (methyltestosterone, MT) 投餵或埋植處理性別分化期間 (3~5個月齡)、1~2年齡與四年齡成魚誘導性別轉換。利用生殖腺環化酵素 (cyp19a1a)、dmrt1 (doublesex and mab-3-related transcription factor, number 1) 與性別分化相關基因 (sf1、dax1、foxL2、11β) 從基因表現,以及P450arom、Dmrt1、Pcna、Vasa組織免疫染色 (immunohistochemical stain, IHC) 切入探討點帶石斑的性別分化與性轉變。 點帶石斑diandric type雄魚的來源大部分自functional female性轉變而來 (secondary male),亦有少部分的自然性轉變由孵化後直接分化發育產生 (primary male),兩者皆可產生不同性轉變程度的精巢組織:a single spermatogenic cyst (SSC)、various stages of spermatogenic cyst (VSCs) 與mature stage of cysts (AMC)。自然性轉變 (primary male) 的精子生成包囊 (SSC, VSCs) 最早於一年半月齡出現。 點帶石斑cyp19a1a專一表現於7月齡生殖腺,cyp19a1a於性別分化期間至雌性發育階段其表現逐漸上升,至一年齡有一表現高峰,隨後下降,至兩~三年齡皆呈現生理低表現,於3+7 -age再度有一cyp19a1a表現高峰。由IHC染色結果,P450arom於性別分化後的雌性germ cell (OG, CA, VO) 外之濾泡層表現,尤其在ZR層明顯增厚階段卵細胞,P450arom只在濾泡層之granulose cells表現。無論任何階段雌性卵巢P450arom皆不會在初級卵母細胞 (PO) 表現。人工性轉變後P450arom微弱表現於SSC/VSCs階段生殖腺之精原細胞 (spermatogonium, SG) 外體細胞,與內含精母細胞 (spermatocyte, SC)、精細胞 (spermatid, SD) 包囊之間質細胞表現,以及退化卵細胞 (atretic oocyte) 外圍濾泡層細胞。 點帶石斑dmrt1專一表現於7月齡點帶石斑生殖腺,dmrt1於生殖腺性別分化過程逐漸上升,並高度表現於性轉變後精巢 (IHC結果Dmrt1強烈表現於SG細胞質);由in situ與IHC結果顯示dmrt1 (Dmrt1) 於性別分化期間與早期雌性發育卵巢之germ cells細胞質表現,且同時表現於卵巢與精巢之germ cells,因此認為點帶石斑的Dmrt1不止參與精巢的發育,亦對於早期雌性的分化 (卵細胞的生成、卵巢空腔的維持) 相當重要。由cyp19a1a與dmrt1基因表現與IHC結果,推測secondary male的發生可能在2+3 齡之後,2+3 齡亦是區別primary male與secondary male關鍵點。 性別分化及內分泌相關等基因,於性別分化期間 (4-5個月齡) 皆會有上升之表現,而在MT或AI投餵處理後,抑制生殖腺雌性生殖配子 (OG) 生成與發育、卵巢空腔 (OC) 萎縮或形成VSCs精巢結構,造成生殖腺dax1、foxL2、cyp19a1a、vasa、與pcna等基因於性別分化期間表現顯著下降,sf1與dmrt1基因則表現顯著上升,其結果亦反應在P450arom、Dmrt1、Pcna與Vasa免疫組織染色上,故認為dax1、foxL2、cyp19a1a等基因有助於點帶石斑早期雌性分化與發育,sf1與dmrt1對點帶石斑雄性性別分化上扮演重要角色。
The sex of Orange-Spotted grouper gonad (Epinephelus coioides) firstly differentiated into female, each lobe of the paired gonads began to form an ovarian cavity at 4 mo of age. The formation of the ovarian cavity is an early characteristic of female differentiation for orange-spotted grouper. The newly budded ovarian lamella around the inside of the ovarian cavity in the gonad mainly contained oogonia and a few primary oocytes in the fish at 6.5-7 mo of age. Cortical alveoli (CA) appeared untill 1yr7mo age,and vitellogenic oocytes were present within the mature ovarian lamellae.Treament with aromatase inhibiter (AI) or methyltestosterone (MT) by feed and implantation in sex differentiation period (3-5mo)、1-2yr and 4yr adult fish inducesd sex change. To investigate gene expression of cyp19a1a、dmrt1 and sex-differentiation related genes ex. sf1、dax1、foxL2、11β, and localization of P450arom、Dmrt1、Pcna、Vasa during sex differentiation and sex change by immunohistochemistry (IHC). The protogynous orange-spotted grouper E. coioides is a diandric type of hermaphrodite. Most secondary males occur through a sex change in adult female fish and few primary males develop directly from juveniles. primary males and secondary males have varying degree sex change testis tissue:a single spermatogenic cyst (SSC)、various stages of spermatogenic cyst (VSCs) and mature stage of cysts (AMC). Primary males (sex change naturally) with single spermatogenic cyst and various stages of spermatogenic cyst firstly occur in 1.5 yr. The expressions of cyp19a1a is single-mindedly in 7 mo orange-spotted grouper gonad. The gene expression of cyp19a1a expressed increasingly from sex differentiation to female adult fish. There are two gene expression peaks of cyp19a1a in 1 and 3+7 -age. From IHC results, P450arom appeared in follicular layers of oogonia, cortical alveolar oocytes, and vitellogenic oocytes but not in primary oocytes. It was interesting to observe that testicular tissues in the sex-changing male grouper (such as cysts,spermatids, and spermatogonia) showed positive aromatase staining. The expressions of dmrt1 is only in 7 mo orange-spotted grouper gonad. The gene expression is gradually expressed increasingly during sex differentiation and highly expressed in testis after sex change (From IHC results, Dmrt1 express strongly in cytoplasm of SG);From IHC and in situ results, Dmrt1 (dmrt1) expresses in cytoplasm of germ cells during sex differentiation or early female gonad development stage, and also expresses in the germ cells of ovary or testis. So dmrt1 is vary important not only involved in testis growth but also in early female differentiation (including the production of oocytes and maintain of ovarian cavity). Based on the gene expression of cyp19a1a and dmrt1 and IHC results, primary male first appears at 1+5 years. The expression peak of dmrt1 indicates that 2+3 years is critical time point to distinguish primary and secondary males. Sex-differentiation and endocrine related genes expressed increasingly during sex differentiation period, but treatment with MT or AI, it inhibit the female gonad germ cell production and growth、the atrophy ovarian cavity or formation of various stages of spermatogenic cyst (VSCs). MT induces musculinization which causes the decreased expression of dax1, foxL2, cyp19a1a, vasa, pcna and the increased expression of sf1 and dmrt1. Therefore, dax1, foxL2, cyp19a1a might be related with early sex differentiation and development. sf1 and dmrt1 might play an important role in male sex differentiation of E. coioides.
URI: http://ethesys.lib.ntou.edu.tw/cdrfb3/record/#G0D91330005
http://ntour.ntou.edu.tw/handle/987654321/30929
Appears in Collections:[水產養殖學系] 博碩士論文

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