- 學位論文
利用線蟲為模型探討精子特異性蛋白質SMZ-1和SMZ-2 PDZ domain的交互蛋白
Identification of cellular factors interact with spermatogenesis-specific PDZ domain proteins SMZ-1/2 in C. elegans
摘要
精子生成(Spermatogenesis)發生在男性生殖系統中，用以產生具有功能的精子，該過程需要雄性特異性調控蛋白(male specific regulators)參與作用。SMZ-1/2蛋白質大量表現於秀麗隱桿線蟲的精子生成期。我們實驗室先前研究顯示SMZ-1/2僅作用於雄性生殖系中，且與雄性生育功能有關。smz-1/2基因缺失會導致雄性減數分裂早期的染色體分離缺陷，使得精子生成失敗。SMZ-1/2具有兩個序列高度相似的PDZ domain，其常見於與其他蛋白質相互作用的支架蛋白(scaffolding protein)。因此我們提出SMZ-1/2作為支架來調控雄性減數分裂必要的細胞內複合物(subcellular complex)或信號的傳導。本研究目的為優化製備富含雄性性腺之線蟲萃取，以利後續利用生化分析SMZ-1/2的交互作用蛋白。 我們利用三個溫度敏感的線蟲性腺突變株，得到只產生精子、只產生卵子、以及不含性腺的蟲株。為優化大規模性腺突變品系同步的培養方法，以得到充沛的蛋白質萃取物，我們評比四個獨立參數：（i）殺死每個成蟲獲得的胚胎數，（ii）L1幼蟲的形態，（iii）L1幼蟲的孵化率，（iv）L1幼蟲的存活率，並獲得大量健康且同步之第一期幼蟲。同步培養之不同突變蟲株經急凍及物理破碎後可重覆地得到與起始蟲數一致的總蛋白質量。三種性腺突變蟲株萃取液以西方點墨法分析後確認SMZ-1/2只能在只含雄性性腺之萃取液中測得。最後，我們執行了小量免疫沉澱測試實驗，內源性SMZ-1/2可成功自線蟲萃取液中以免疫方式分離出。本研究所建立之蛋白質萃取方法可用於後續對SMZ-1/2複合物的質譜儀分析。
並列摘要
Spermatogenesis takes place in male reproductive system to generate functional sperm and requires participation of male specific regulators for the process. SMZ-1/2 are highly spermatogenic enriched proteins expressed during spermatogenesis in Caenorhabditis elegans. Our laboratory had shown SMZ-1/2 are bona fide male fertility factors that only function in male germline previously. Deletion of smz-1 and smz-2 cause defective chromosome segregation in early male meiosis, resulting in failure of sperm generation. SMZ-1/2 have two highly identical PDZ domains, which are protein-protein interaction domains that often found in scaffolding proteins. We thus proposed that SMZ-1/2 function as a scaffold to regulate the mediation of essential subcellular complexes or signaling for male meiotic divisions. The goal of this study is to optimize the procedures of preparing the worm extract that enriched with male germline for applying to analyze the binding partner proteins of SMZ-1/2 biochemically. We used three temperature-sensitive mutant strains that displaying defects in germline and harvested animals which exclusively produce sperm; oocytes or lack the germline. To optimize the procedures of culturing large-scale synchronized germline-defective mutant strains for abundance protein extract, we scored four individual parameters: (i) average embryos obtained per worm, (ii) morphology of L1 larvae, (iii) L1 hatch rate, (iv) L1 survival rate and obtained large quantities of healthy live synchronized L1 larvae. After flash freeze animals and physically fragment the worm pellets, a linear correlation between the total protein amount of worm extracts and the numbers of initial cultured mutants was measured reproducibly. By performing western blot analyses of worm extracts generated from three germline-defective strains, endogenous SMZ-1/2 was confirmed to be detected in lysates prepared from animals that only presenting the male germline. Finally, we used small amounts of the lysate for conducting a test immunoprecipitation experiment and successfully isolated endogenous SMZ-1/2 from the worm extract by immunoprecipitation. The methods of protein extract established in this study can be used for the mass spectrometric analysis of the SMZ-1/2 complexes afterwards.