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標題: Hedgehog訊息傳遞在公雞生殖腺發育的研究
A study on hedgehog pathway during male gonad development in chicken
作者: 吳蕙如
Wu, Hui-Ju
關鍵字: Hedgehog signaling pathway
出版社: 生命科學系所
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摘要: Hedgehog (Hh)訊息傳遞路徑對於調控從果蠅至脊椎動物胚胎發育及成體生理機能扮演著不可或缺的角色。Desert hedgehog (Dhh)是哺乳動物三種Hh同源基因中的其中ㄧ員。前人研究指出,Dhh基因剔除會造成公鼠睪丸型態發育不全甚至會導致不孕,進一步結果指出Dhh的基因剔除小鼠中精子生成與Leydig cells的發育有嚴重的缺陷。但是在雞的基因體資料庫中,只能找到兩種Hh基因的存在,分別為Shh及Ihh。而本論文中的Southern blot結果也顯示雞的基因體中除了Shh及Ihh之外沒有第三種Hh基因(Dhh)的存在。雖然雞缺乏Dhh基因的存在,但公雞仍然有生殖能力且具有成熟精子來產生下一代。所以推測公雞性腺中Dhh對於精子發育的功能可能被其他兩種Hh (Shh, Ihh)的其中之ㄧ所取代了。為了確認Hh訊息傳遞途徑在公雞性腺中是否有被活化,我們偵測Hh的下游標的基因Patched1 (Ptch1) 及Gli1在公雞性腺中是否有表現。用取自胚胎發育第6天至20週齡公雞性腺的mRNA作RT-PCR的結果顯示:Ptch1及Gli1在兩種品系的公雞性腺都有持續性的表現。另外,也用免疫染色的方式去偵測所有Hh蛋白在公雞性腺存在的情況,結果顯示Hh蛋白會表現在胚胎發育第3天的生殖脊,此區域是將來會發育成性腺的位置;另外Hh也會表現在胚胎發育第16天Sertoli cells前驅細胞的細胞質中,但在衰退期(46週齡)的公雞性腺中則是無法偵測到明顯的Hh訊號,顯示Hh會表現在發育中的公雞性腺。爲了進一步找出Hh訊息傳遞路徑是由哪一個Hh ligand所活化的,再用RT-PCR去偵測Shh及Ihh mRNA的表現。結果發現不論在胚胎或是成體的公雞性腺中都有Shh表現,據此推論在公雞性腺中Hh訊息傳遞路徑可能是由Shh活化。由以上結果推測,Hh訊息傳遞路徑可能也與公雞的性腺發育有關。因此,最後再嘗試以干擾Hh訊息傳遞路徑的方式去探討Hh訊息傳遞途徑在雞的雄性生殖腺中所扮演的角色。
The Hedgehog (Hh) signaling pathway plays an essential role in regulating embryonic development and adult tissue regeneration from Drosophila to vertebrates. Desert hedgehog (Dhh) is a member of the vertebrate Hh family. It was reported that deletion of Dhh led to male sterility in mice and with hypotrophic testes. Subsequent analysis revealed that Dhh is required for mammalian spermatogenesis and Leydig cells formation. Survey of chicken genomic sequence databases suggested that chicken may possess only two Hh genes (Shh and Ihh). Our Southern hybridization results indicated the absence of Dhh gene in chicken genome. Although chicken lacks Dhh in its genome, the roosters are still fertile with functional sperm. Since lacking Dhh does not affect rooster's fertility, we hypothesized that one of the other two Hh genes (Shh, Ihh) may have substituted the functions of Dhh in spermatogenesis in chicken. To confirm the activation of Hh signaling pathway, we characterized the expression of Hh target genes, Patched1 (Ptch1) and Gli1 in male gonads collected from embryos (Embryo day 6) to adults (week20). RT-PCR analysis showed continuous expression of both Ptch1 and Gli1 in all stages of chicken testes collected from two chicken strains. Furthermore, our immunohistochemistry study with an antibody that recognize all Hh proteins detected Hh expression in genital ridge (Embryo day 3) and Sertoli cell precursor of developing (Embryo day 16) testes, respectively. In order to determine which of the two Hh ligands activates the Hh pathway in chicken testis, we use RT-PCR to identify the expressed Hh mRNA. Shh mRNA was detected in both embryonic and adult testis, suggesting Shh might be responsible for the activation Hh signaling pathway. These results imply that Hh signaling pathway may have a conserved role in chicken gonadogenesis. Therefore, we tried to disrupt the Hh pathway in chicken testes to confirm the functions of Hh pathway in rooster gonad development.
其他識別: U0005-2308200816383000
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