| 摘要: | DAZ家族（Deleted in AZoospermia） 包含兩個體染色體基因，即為BOULE、DAZL（DAZ like）和位於Ｙ染色體的DAZ基因。DAZ是一種RNA binding proteins，其專一的表現在胚胎時期的精細胞。先前研究指出當DAZ家族基因表現異常時將導致造精過程發生障礙進而產生不育限象。本研究主要分析DAZ家族中的BOULE與DAZL蛋白質在睪丸不同細胞株中的表現、蛋白雙聚體及蛋白間相互作用等。在免疫組織切片染色（Immunohistochemistry；IHC）分析發現BOULE主要表現在減數分裂時期的精母細胞（Spermatocyte）時期，DAZL表現在原精母細胞（Spermatogonia）與精母細胞（Spermatocyte）。由免疫螢光分析（Immunofluorescence assay;IFA）結果發現小鼠4N（Leptotene stage）、2N（Spermatogonia、Primary Spermatocyte）、1N（Secondary Spermatocyte、Spermatid）等不同時期的精細胞中，BOULE與DAZL蛋白質表現在相近的位置（colocalization ）。最後利用螢光共振能量轉移技術（Fluorescence Resonance Energy Transfer;FRET）觀察，在不同的細胞株中BOULE與DAZL蛋白質表現位置的相互關係，在GC1細胞(Mouse spermatogonia cell)中，BOULE1、DAZL形成雙聚體（dimer）， BOULE1/DAZL、BOULE2/DAZL 有蛋白質表現在相近的位置，GC2 細胞 (Mouse spermatocyte cell)無雙聚體情形，蛋白質沒有相互關係，TM3 細胞（Mouse Leydig cell）無雙聚體， BOULE1/DAZL。TM4 細胞(Mouse Sertoli)BOULE2 形成雙聚體，BOULE2/DAZL，293T 細胞（Human kidney）BOULE1 、BOULE2 、DAZL 皆有雙聚體，BOULE1/DAZL、BOULE1/BOULE2 蛋白質有相互關係情形。據上述實驗結果，有助於了解DAZ家族中BOULE和DAZL蛋白質表現位置、本身雙聚體及蛋白質表現位置的相互關係。
The DAZ (Deleted in AZoospermia) gene family contains two autosomal genes, BOULE, and DAZL(DAZ like) and the Y chromosome DAZ gene. DAZ gene encoded potential RNA binding protein that is exclusively expressed in the prenatal and the postnatal germ cell lineage. In the previous study, disruption of those genes would contribute to the loss of the differentiation and migration of primordial germ cells then resulted in meiotic arrest and infertility. In this study, we set out to investigate the expression, dimerization, and interaction of these proteins by co-expression of BOULE and DAZL in mouse testis cells. In immunohistochemistry assay, BOULE is expressed in spermatocyte and DAZL is mainly expressed in spermatogonia and spermatocyte. The DAZL and BOULE protein were colocalized in mouse differential germ cells, including 4N（Leptotene stage）、2N（Spermatogonia、Primary Spermatocyte）、1N（Secondary Spermatocyte、Spermatid） cells. The results of Fluorescence Resonance Energy Transfer assay showed that DAZL in GC1, 293T, BOULE2 in TM4、293T, and BOULE1 in GC1, 293T have dimerization of each other. The BOULE1/DAZL, BOULE2/DAZL in GC1, BOULE1/DAZL in TM3, BOULE2/DAZL in TM4, BOULE1/DAZL, and BOULE1/BOULE2 in 293T have colocalized at the sites of cells. The physiological means in cell of dimerization and colocalization among DAZL, BOULE1, and BOULE2 need future investigation. |
