運動神經元存活蛋白於精原幹細胞潛能與精子發育功能角色之研究

Abstract

運動神經元存活蛋白（Survival motor neuron, SMN）廣泛表現於多種細胞間，其編碼基因SMN並高度保留於跨物種間，主要功能為參與RNA剪接體（RNA spliceosome）重要組成之一，涉及細胞轉錄調控及胞內運輸。脊髓性肌肉萎縮症（spinal muscular atrophy, SMA）是由於SMN1基因發生缺失或突變的疾病，患者發生漸進性地運動神經元萎縮、肌肉退化，嚴重者甚至死亡。為探究運動神經元存活蛋白缺失對於病程演進是基於肌肉退化抑或是發育受影響，近年與合作團隊的共同研究發現，運動神經元存活蛋白表現在果蠅的神經幹細胞與生殖幹細胞中，子細胞分化程度愈高，運動神經元存活蛋白表現量愈低；而運動神經元存活蛋白在小鼠胚胎發育、胚胎幹細胞潛能中亦扮演重要角色，並發現運動神經元存活蛋白影響生殖細胞的生長。此等研究結果重複驗證運動神經元存活蛋白維持著多種幹細胞之潛能，並調控其增生與分化，且與生殖系統高度相關。故本研究進而提出運動神經元存活蛋白在生殖幹細胞中扮演著重要角色。本研究係透過以活體外及活體內兩層面，探討運動神經元存活蛋白對於精原幹細胞（spermatogonial stem cells, SSCs）潛能維持及其分化生成精子（spermatogenesis）能力之影響。試驗結果顯示，在脊髓性肌肉萎縮症疾病模式的精原幹細胞試驗中，低量表現的運動神經元存活蛋白影響了精原幹細胞的生長及潛能的維持；而在以慢病毒（lentivirus）轉導高量表現運動神經元存活蛋白之試驗組發現，精原幹細胞於體外培養的生長情形獲得改善，包括細胞增生情形、精原幹細胞特異標誌蛋白PLZF表現比例（潛能維持）等方面。進一步在體內移植試驗中，可觀察到高量表現運動神經元存活蛋白後的精原幹細胞顯著表現了更好的歸航能力（homing）及更完整的分化表現。本試驗結果不但證明運動神經元存活蛋白有利於小鼠精原幹細胞的體外培養，將有助於穩定精原幹細胞體外培養之研究與應用，並為該蛋白有效提升精原幹細胞於活體內之分化能力表現提供直接證據，闡明運動神經元存活蛋白參與生殖幹細胞之生長調控。

Survival motor neuron (SMN) is ubiquitously expressed in numerous cell types and its encoding gene SMN is highly conserved in various species. SMN is involved in the assembly of RNA spliceosomes which are important for pre-mRNA splicing. One related neurogenic disease, spinal muscular atrophy (SMA), is caused by the loss or mutation of the survival motor neuron 1 gene (SMN1). Patients with this disease gradually lose their spinal motor neurons. It is unknown how exactly the reduction of a ubiquitously functional protein results in tissue-specific impairment. Recently, several studies sought to determine whether this disease is caused by developmental or degenerative defects. In Drosophila germline stem cells, SMN is expressed in a striking concentration gradient in the differentiating progeny. In mice, SMN has been proven to maintain stemness and regulate the proliferation and differentiation of several types of stem cells, as well as being highly related with germline systems. The purpose of this study is to determine if SMN plays any role in maintaining spermatogonial stem cells (SSCs) potency and in the spermatogenesis process. In in vitro culture, SSCs obtained from SMA model mice, comparing to those from wild-type (WT) mice, showed slow cell growth accompanied by significantly reduced expression of spermatogonia marker PLZF (Promyelocytic leukemia zinc finger). SMA SSCs failed to proliferate and quickly lost SSC characteristics. In contrast, SMN overexpressed-SSCs showed enhanced cell proliferation and improved potency maintenance capacity than WT ones. Moreover, the transplantation experiments revealed that SMN overexpressed-SSCs demonstrate superior ability of homing in host seminiferous tubule and more complete performance in differentiating progeny compared to control groups. These findings strengthen our earlier findings that SMN is involved in germline stem cell development.