利用轉錄因子 FOXM1 基因剔除小鼠探討其在精子生成之角色

Abstract

轉錄因子 FOXM1 屬於 Forkhead box proteins 之一，已知能調控細胞生長、增生及分化，並高度表達於腫瘤細胞；此外，FOXM1 在哺乳動物的發育過程也扮演了重要角色。本實驗室在初步的研究結果中發現，FOXM1 高度表現於小鼠睪丸組織內的生殖細胞，且小鼠的生殖細胞特異性剔除 Foxm1 基因，便會導致其不孕，顯示 FOXM1 可能也在小鼠的精子生成過程扮演重要的角色。因此本篇研究接續探討 FOXM1 在精子生成過程中所參與的角色以及其對於減數分裂的調節機制。 我們利用重組酶 vasa-Cre 建立條件式剔除生殖細胞之 Foxm1 基因的小鼠模型，在染色體擴散分析的實驗結果中發現，Foxm1 缺失會導致生殖細胞內的性染色體聯會異常 (asynapsis)，且 DNA 延遲修復、精子生成的進程停滯於減數分裂前期 (Meiosis I - Prophase)。而以 Microarray 技術及 RT-qPCR 分析小鼠睪丸組織的基因表現，我們篩選出與精子生成相關的基因群，並透過啟動子序列分析其中的 FOXM1 的核酸結合位，找到了 FOXM1 的潛在調節標的 bromodomain testis-specific protein (Brdt)。接著我們利用基因轉殖技術將標的基因 Brdt 之啟動子序列嵌入螢光報導基因的載體，並透過報導基因的活性分析 (Reporter assay) 結果發現，當 Brdt 啟動子上的 FOXM1 核酸結合序列突變，Brdt 啟動子的轉錄活性便會顯著地下降；此外，以 FOXM1 siRNA 引發細胞內 FOXM1 基因沉默之後，不僅會造成細胞內 BRDT mRNA 的表現量減少，也同樣導致報導基因的轉錄活性下降，顯示 FOXM1 可以調節 BRDT 的轉錄活性。後續我們利用染色質免疫共沉澱技術 (Chromatin immunoprecipitation assay; ChIP) 結合 FOXM1 siRNA 及 FOXM1 的抑制劑 Siomycin A，在人類睪丸癌細胞株 NT2/D1 證明了 FOXM1 可以直接地結合至 Brdt 的啟動子區域，藉此調節 Brdt 的表現。 綜合上述研究結果，我們推論小鼠的 Foxm1 缺失對於生殖細胞在減數分裂過程所產生的不良影響，可能是由於其下游調節標的 Brdt 表現異常所導致的。這項發現或許能為不孕症的診斷或是男性避孕的新藥研發提供一個新穎的方向。

Transcription factor FOXM1 plays important roles in mammalian development. It involves in cell growth, proliferation and differentiation. In addition, it is highly expressed in a variety of human tumor cells. In our previous study, we found that FOXM1 is highly expressed in germ cells of mouse testis, and conditional knockout of Foxm1 in germ cells leads to the absence of sperm and results in infertility. It reveals that FOXM1 may play an important role in mouse spermatogenesis. In this study, we further investigate the molecular functions of FOXM1 during spermatogenesis in mice. We showed that Foxm1 deficiency in germ cells led to abnormal chromosome synapsis, and delayed the DNA repair process. Using gene expression profiles and FOXM1 binding motif analysis, we identified few FOXM1-regulated gene targets, including bromodomain testis-specific protein (brdt). In the reporter assay experiments, deletion or mutation of FOXM1 binding sites on the Brdt promoter significantly reduced its transcriptional activity. Moreover, we further demonstrated that FOXM1 directly bound to Brdt promoter using chromatin immunoprecipitation (ChIP) assay in human testicular embryonal carcinoma cell line NT2/D1. Together, our findings suggest that Brdt may be one of the downstream targets of FOXM1, and that the spermatogenesis defects observed in Foxm1-CKO mice might be due to the reduced expression of Brdt. Our study might provide new insights into male infertility and could lead to novel approaches to make contraception.