以線蟲為模型探討在精子生成過程中兩次減數分裂之間過渡時期的表徵

Characterization of the transition between meiosis I and meiosis II during spermatogenesis in Caenorhabditis elegans

陳宇豪(Yu-Hao Chen)

指導教授 ： 吳瑞菁

國立臺灣大學/醫學院/醫學檢驗暨生物技術學研究所/碩士(2021年)

https://doi.org/10.6342/NTU202104310

摘要

細胞分裂對於生物體的生長、繁殖是一個重要的過程。在細胞分裂期間，染色體會分離並且受到紡錘體檢查點(spindle assembly checkpoint, SAC)調控以確保準確的染色體分離。在雄性減數分裂過程中會牽涉到兩次連續的細胞分裂階段，目前尚不清楚在兩次分裂之間的調控機制。先前本實驗室發現在給予轉譯抑制劑時雖然精母細胞可以成功完成第一次的染色體分離，但細胞遂即停滯而無法進行第二次染色體分離。這顯示在第一次減數分裂過程中製造的新合成蛋白質是為了提供給第二次的減數分裂。因此本篇研究想要探討這些參與在兩次減數分裂之間的重要分子。
如同實驗室先前的研究成果，位在kinetochore外側的蛋白質BUB-1在第一次染色體開始分離後會快速的掉下來，降至低點後會以較慢的速率回升訊號並進入metaphase II，顯示有部分kinetochore蛋白需要被拆解下來並重新組裝回去以利進入第二次的減數分裂。我們發現在兩次細胞分裂期間有兩階段的染色體分離。在BUB-1訊號回升時期，染色體分開速率下降。在抑制轉譯時，會使anaphase I的染色體分開速率以及BUB-1訊號下降變慢；此外，當BUB-1訊號降至低點後不再回升且染色體分離也停滯。我們發現藉著BUB-1而能連接上kinetochore 的CENP-F蛋白HCP-1也有相同的訊號變化。因此我們推測在兩次染色體分離之間部分kinetochore勢必要重新建構。我們進一步檢視其他outer kinetochore是否也需被拆解再重組。我們發現NDC-80和HIM-10，兩個主要連結紡錘絲的Ndc80 complex成員，在兩次減數分裂過程中維持在染色體上，且至少NDC-80在染色體上的訊號不受轉譯抑制的影響。此外，和SAC調控的相關蛋白RZZ complex (ROD-1)兩次減數分裂過程中維持在染色體上。綜上所述，在transition中只有部分outer kinetochore需要被拆解再重組，轉譯對於outer kinetochore的重組及能否進到第二次減數分裂是必須的。
由於主要連接microtubules的Ndc80 complex成員在兩次減數分裂過程中依然維持在染色體上，我們進一步檢視spindle microtubules在兩次分裂期間的動態變化。我們發現spindle microtubules在anaphase I開始後有變少再變多的現象。同時，我們發現spindle pole成員γ-tubulins的訊號在兩次減數分裂期間會分裂並且也有訊號變弱再回升現象，當抑制轉譯時，訊號下降後不再回升，我們認為轉譯對於spindle pole的成熟並長出spindle microtubules是必須的。
最後，根據我們先前在抑制轉譯後對於精母細胞的觀察，我們認為在meiosis I期間的新合成蛋白質對於精母細胞能否過渡到第二次的減數分裂是重要的。雖然抑制轉譯不太影響第一次減數分裂的完成，但在第一次減數分裂中勢必會有新製造出來的蛋白質提供給第二次減數分裂。因此，我們也利用了代謝性蛋白質標定(metabolic labeling)技術來找出新合成蛋白質在初級精母細胞中的分布。

關鍵字

雄性減數分裂 ； 染色體分離 ； 紡錘體檢查點 ； 動粒 ； 微管 ； 中心體 ； 代謝性蛋白質標定 ； 轉譯

並列摘要

Cell division is an important process for growth and reproduction in organisms. During the cell division, chromosomes separate to the dividing cells and the spindle assembly checkpoint (SAC) ensures the accurately chromosome segregation. In the male meiosis, there are two consecutive cell divisions in the same M phase. It is unclear the regulations between the two cell divisions. In our lab’s preliminary results, we found that translation inhibition did not affect the progression of the first division but stall the cells after completion of meiosis I. This indicates new factors are synthesized during meiosis I for the progression of meiosis II. The goal of this study is to characterize the dynamics of key division-related molecules between meiosis I and meiosis II.
According to our preliminary results, the levels of the outer kinetochore protein BUB-1 dropped rapidly after anaphase I and the signals recovered slowly to reach metaphase II. This indicates that part of the kinetochore is disassembled and then is reassembled on chromosomes for the second meiosis. We also found the two phases of chromosomes separation between the two cell divisions. The chromosomes separation rate was reduced during BUB-1 recovery phase. Inhibition of translation slightly reduced the rates of initial anaphase I chromosome separation and the departure of BUB-1. Furthermore, BUB-1 levels did not recover when translation was inhibited and the chromosomes ceased to separate when BUB-1 levels dropped to the lowest levels. HCP-1CENP-F, which depends on BUB-1 for kinetochore localization, exhibits similar kinetics with BUB-1. We thus propose that part of the kinetochore should re-build between the two divisions. We further examined whether other outer kinetochore proteins were required to be disassembled and reassembled on chromosomes or not. We found NDC-80 and HIM-10, the two major binding components with microtubules in Ndc80 complex, maintained on chromosomes between the two meiotic divisions. We also found translation inhibition did not affect the NDC-80 signals on chromosomes. Furthermore, the SAC related proteins in the outer kinetochore region, RZZ complex (ROD-1) appeared to be associated with chromosomes between the two meiotic divisions. Taken together, only part of outer kinetochore had to be disassembly and reassembled on chromosomes. Translation was required for the reassembly of outer kinetochore and the transit into the second meiosis.
Because the major binding components with spindle microtubules, Ndc80 complex maintained on chromosomes between the two meiosis, we therefore examined the kinetics of spindle microtubules. We found the intensities of spindle microtubules decreased and recovered after anaphase I onset. Simultaneously, we found the signals of γ-tubulins split and also decreased and recovered between the two meiotic divisions. Translation inhibition affected the recovery of γ-tubulins levels, translation was required for spindle pole maturation to grow spindle microtubules.
Finally, according to our observations in spermatocytes with translation inhibition, we considered the newly synthesized proteins during meiosis I were important for spermatocytes to transit into second meiosis. Although translation inhibition did not absolutely affect the completion of meiosis I, it was required to generate new proteins for the second meiosis during meiosis I. Therefore, we also use the metabolic labeling techniques to find the localization of newly synthesized proteins in the primary spermatocytes.

並列關鍵字

male meiotic divisions ； chromosome segregation ； spindle assembly checkpoint ； kinetochore ； microtubules ； centrosome ； metabolic labeling ； translation

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