探討Mkx siRNA基因轉殖小鼠生殖異常之可能機制

同源箱基因在胚胎發育、細胞分化及細胞命運的決定上皆扮演很重要的角色，而Mkx基因是同源箱基因家族的一個成員。Mkx轉譯出的同源箱區之胺基酸序列，在不同物種之間的相似度接近百分之百，且小鼠的Mkx幾乎會表現在所有的小鼠成熟個體組織之中，其中以大腦及卵巢的表現量最高。高度的相似性及廣泛的表現位置，暗示著Mkx在小鼠的發育中可能扮演著重要的角色。值得注意的是，Mkx的表現隨著雌性性腺的發育會漸漸的增強，但是在雄性的性腺中則有相反的表現趨勢。為了瞭解Mkx基因在小鼠的發育中扮演的角色，我們製備了表現Mkx siRNA的H1-siMkx構築以抑制Mkx的表現，並製造抑制Mkx表現的基因轉殖小鼠。總共得到三隻siMkx基因轉殖公鼠：TG23，TG43及TG45，在建立個別的基因轉殖鼠line的過程中，發現TG23和TG45基因轉殖小鼠的生育力有異常，TG23完全不孕而TG45只讓母鼠生育過一次。和野生型的小鼠相比較，TG45基因轉殖鼠的Mkx在睪丸之表現量約只有野生型小鼠的20%。為了進一步了解TG45小鼠生殖異常的原因，我們分析TG45基因轉殖小鼠的睪丸組織，發現該小鼠的睪丸明顯的比較破碎，細精管也有明顯的空泡化。此外，TG45基因轉殖小鼠在副睪中所含的成熟精子只有野生型小鼠的6.25%。綜合以上的結果，推論Mkx基因的表現量下降會影響睪丸中的體細胞或生殖細胞的生存。為了證明這個假設，我們利用表現Mkx siRNA來抑制睪丸相關的生殖和體細胞株中的Mkx表現，結果發現抑制Mkx的表現只會讓支持生殖細胞發育的史托利細胞之存活率下降，而對其他的細胞影響則較不顯著。基於這些發現，我們推論在精子生成的過程中，Mkx蛋白扮演著一個重要的角色。

Homeobox gene families play important roles in embryonic development, cellular differentiation and determination of cell fates. Mkx is a novel member of homeobox genes. The homeodomain sequences encoded by Mkx from various vertebrates are almost the same. Mouse Mkx expresses in almost all tested adult mouse tissues and predominately in ovary and brain. The sequence conservation of their homeodomain and expression patterns of Mkx suggest that Mkx proteins play important roles in mouse development. Interestingly, the expression levels of Mkx proteins increase gradually during female gonad development but reversed in male gonad development. To reveal the roles of Mkx in mouse development, we constructed the H1-siMkx DNA, which can express a siRNA RNA that inhibits Mkx expression, and then generated Mkx knockdown transgenic mice. We obtained three siMkx transgenic male mice including TG23, TG43 and TG45. However, two of these transgenic male mice were infertile (TG23) or sexual defect (TG45). TG23 was absolutely infertile, and TG45 could only let a wild-type female pregnant. In comparison with wild-type male mice, the expression level of Mkx in testis is only about 20% of that in wild-type mice. To decipher why TG45 is sexual defect, we analyzed the testis structures of TG45 and wild-type mouse. We found that testis structure of TG45 is more fracture and has obvious vacuolization in its seminiferous tubules than wild-type mouse. Furthermore, TG45 only produced 6.25% mature sperms in comparison with the wild-type mouse. From these results, we hypothesize that down-regulation of Mkx expression may cause germ cell and/or somatic cell death in testis. To confirm this hypothesis, we knocked down Mkx expression in several testis-relative somatic and germ cell lines by expressions of Mkx siRNA. The reductions of Mkx expressions only caused death of Sertoli cells that support germ cell development, but didn’t affect survival of other testis-relative cells. Based on these findings, we recomment that Mkx proteins play an important role in spermatogenesis.