利用線蟲為模型探討ZYG-1及兩溫度敏感性突變株tpe1和tpe2在精子減數分裂之角色

Abstract

第一部分:中心體對於一般細胞分裂形成雙極的微管絲構造是重要的。而在每一次的有絲分裂裡，染色體複製一次後會進行一次的細胞分裂，微管絲則是扮演把染色體拉開角色，因此中心體在有絲分裂只需要複製一次。而在精子減數分裂裡，染色體在進行一次的複製之後會進行兩次的細胞分裂，暗示著中心體在精子減數分裂的過程中有多一次的複製。至於在精子減數分裂裡這個多一次的中心體複製是如何被調控的是我們所感興趣的。過去已有報導指出在線蟲胚胎發育中，中心體的複製會經由ZYG-1來調控，對於溫度感受性強的zyg-1(b1)突變株在形成精子時只帶有一個中心粒，在受精後無法形成雙極的紡錘體構造，由此暗示著在精子減數分裂的兩次中心體複製，ZYG-1應該至少有參與一次的複製過程。在核染色裡，我們觀察zyg-1(b1)突變株精母細胞和精子的染色體，發現在精子減數分裂過程中zyg-1(b1)突變株形成了不正常形態的核，觀察活體精子時卻又沒有看到與核染色形態相似的細胞核。進一步的經由活體影像攝影觀察精子減數分裂過程後發現zyg-1(b1)突變株在第二次減數分裂時形成了單極的紡錘體構造，此結果指出了ZYG-1的確有參與第二次的中心體複製。為了探討在精子減數分裂中中心體的數目，我們將公蟲性腺做了γ-tubulin和SAS-4的螢光免疫染色，發現γ-tubulin在進入分裂期之前的訊號是散佈在細胞質，相反的SAS-4的訊號在性腺中則是從有絲分裂期就可以明顯的在每個染色體旁觀察到，也因此SAS-4對於要計數中心體數目來說是較為有效的。綜合了計數帶有SAS-4訊號數目和量化SAS-4螢光強度後可以藉由趨勢來推測中心粒的分開和成長過程，這對於未來要藉由計數量化zyg-1(b1)突變株的中心粒數目而進一步的探討ZYG-1在精子減數分裂的角色來說是重要的。 第二部分:在很多物種中，對溫度感受性高的突變株被廣泛地用來探討必要性基因的功能。在研究精子減數分裂ZYG-1所扮演角色的過程中，我們實驗室發現了tpe1和tpe2這兩個對溫度感受性高的新突變株。將這兩個突變株放到高溫後，雌雄同體的蟲全部都是不孕的。而造成不孕的原因有可能是精子或卵子有問題，將tpe1的公蟲和能夠產生正常卵子的蟲交配後發現胚胎有很低的孵化率，顯示出tpe1的精子是有問題的。觀察活細胞的精子後發現tpe1和tpe2的精子都沒沒有核，而造成產生沒有核精子的可能原因是染色體在分離的過程中發生了問題，因此我們將tpe1和tpe2的公蟲性腺做了核的染色，發現tpe1和tpe2都產生了不正常型態的染色體和數目較少的精子，在tpe1突變株更是觀察到了較多位於細胞分裂中期型態的染色體。我們想知道tpe1和tpe2中這些可能是染色體分離異常的表現形是不是由於紡錘絲在形成的過程中受到破壞，對中心體做了染色後卻發現tpe1和tpe2都形成了雙極構造的紡錘體。而進一步的藉由活體影像攝影的方式觀察tpe1和tpe2的精子減數分裂發現這兩個突變株在第一次減數分裂中期都有停滯的現象，說明了tpe1和tpe2可能使得紡錘體組成檢查點(spindle assembly checkpoint)活化。

Chapter1: Centrosomes are crucial for assembly bipolar microtubule spindle during normal cell division. Like chromosome duplication, centrosome duplication is limited to once per cell cycle in mitotic cells. During male meiosis, however, two consecutive divisions occur after one round of chromosome duplication, indicating an additional centrosome duplication event in male meiosis. I am interested in how this extra centrosome duplication is regulated in normal male meiosis. It has been reported that Polo-like kinase ZYG-1 is an essential regulator of centrosome duplication during embryogenesis in the nematode C. elegans. Previous studies show that zyg-1(b1) mutant generates sperm with only one centriole, resulting in failure of bipolar spindle assembly in zygote after fertilization. To examine if ZYG-1 regulates both centrosome duplication events in male meiosis, I examined the process of sperm production in zyg-1(b1) male worms. DAPI-stained male germline and live sperm cells of zyg-1(b1) showed atypical morphology nuclei of division zone chromosome and sperm. Results from live imaging of zyg-1(b1) spermatocytes showed monopolar spindle phenotype in meiosis I and/or meiosis II, indicating one centrosome duplication event occur before meiosis I and the other occur between meiosis I and meiosis II. The result also shows ZYG-1 participate in both centrosome duplication events of spermatogenesis. Next, I stained γ-tubulin and SAS-4 for probing the centrosome duplication events during male meiosis and found SAS-4 foci stably associated with nuclei. Investigating morphological and quantity changes of centrosome in male meiotic stages suggest that centrioles separate at least from pachytene. Quantitative analysis of centrosome duplication during male meiosis shows centrosome elongation lasting from mitotic zone to diakinesis. However, there is no increasing intensity of SAS-4 signal between anaphase I to metaphase II. Taken together, my results indicate ZYG-1 have roles in both centrosome duplication events in C. elegans spermatogenesis. Chapter2: Temperature sensitive mutants have been used extensively in various organisms for investigating the functions of essential gene. In the process of investigating the role of ZYG-1 in spermatogenesis, our lab identified two new temperature-sensitive mutant, tpe1 and tep2, that show full penetrant sterility when grown at non-permissive temperature. Sperm generated by tpe1 males could not support normal development of embryos, suggesting the mutants are defective in sperm generation. Examination of mature sperm showed that both tpe1 and tpe2 generate anucleate sperm. One explanation of anucleate sperm production is that chromosome segregation errors occur during division. Therefore, DAPI-stained tpe1 and tpe2 male germline showed atypical morphology of chromosome and less number of sperm in both mutant, also an elevation number of metaphase-like nuclei in tpe1. One possibility caused chromosome segregation defect is the defective of spindle organization, however both tpe1 and tpe2 showed bipolar spindles in γ-tubulin stained spermatocytes. Live image the spermatogenesis of tpe1 and tpe2 observed a prolonged metaphase, suggesting the activation of spindle assembly checkpoint.