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標題: | 利用線蟲為模型探討PP1去磷酸酶GSP-1與GSP-2在雄性減數分裂中染色體分離的角色 Investigate the role of PP1 phosphatases GSP-1 and GSP-2 in male meiotic chromosome segregation in C. elegans |
作者: | Yi-Hsiu Lin 林怡秀 |
指導教授: | 吳瑞菁(Jui-Ching Wu) |
關鍵字: | 雄性減數分裂,染色體分離,PP1去磷酸?,染色體轉向,黏連蛋白, male meiosis,chromosome segregation,PP1 phosphatase,chromosome orientation,cohesin, |
出版年 : | 2016 |
學位: | 碩士 |
摘要: | 精確的染色體分離對於生物的正常發育十分重要。不正確的染色體分配會導致細胞癌化、不孕以及子代死亡率上升等問題,因此探討細胞是如何調節染色體分離的過程是非常重要的。本論文以線蟲(C. elegans)為模型探討染色體分離的調節機制。已知在有絲分裂(mitosis)與雌性減數分裂(female meiosis)中,PP1去磷酸酶(Protein Phosphatase 1) GSP-1與GSP-2會調控染色體正確地轉向要分開的方向;另一方面也調節染色體間的黏連蛋白(cohesin)在染色體要分離時才被移除。但是目前仍不清楚雄性減數分裂是否也受相似的機制調控。
首先,為了探討GSP-1與GSP-2在雄性減數分裂中的角色,我們觀察PP1去磷酸酶基因缺陷的公蟲與只產生卵子的母蟲交配後對子代存活率的影響,發現gsp-1基因缺陷的公蟲對子代存活率沒有影響;gsp-2基因缺陷的公蟲會使子代存活率下降,根據此結果推測只有GSP-2對正常的精子生成是必要的。我們推測gsp-2突變的子代存活率下降可能與產生染色體數目異常的精子相關,使用DAPI染色法以及染色體帶有紅色螢光的活體精子進行精子細胞核的觀察,發現gsp-2突變會導致精子的染色體數目異常。進一步使用免疫染色法與活體影像攝影法探討gsp-2基因突變會使精子形成過程的哪些部份產生缺陷,從染色結果發現gsp-2突變的公蟲性腺中出現異常的染色體分離型態,包括染色體分不開的chromatid bridges以及染色體未在同時間點分開的lagging chromosome;從活體影像攝影發現gsp-2突變的精原細胞在染色體排列上出現異常,我們推測此現象可能是導致染色體分離異常的原因。透過測量染色體分離前的染色體寬度以及使用活體影像的方式以BUB-1::GFP觀察染色體的轉向(chromosome orientation),此兩項結果皆支持gsp-2突變會導致chromosome orientation出現異常。除了染色體轉向上的異常,我們同時也從染色與活體影像攝影的結果觀察到gsp-2突變在第一次染色體分離時出現數目較多的染色體,此結果顯示GSP-2在雄性減數分裂可能也具有避免cohesin被提前移除的功能。進一步藉由DAPI染色法計數第一次染色體分離之前與之後的染色體數目,觀察到gsp-2突變相較於野生型,在第一次染色體分離時會產生較多數目的染色體。同時,我們也觀察到gsp-2突變並不會改變cohesin REC-8在第一次染色體分離之前的分布,但是會使促進cohesin移除的磷酸酶AIR-2在染色體上的位置不再侷限於要分離的染色體之間,根據此結果推測GSP-2會透過拮抗AIR-2調節cohesin的移除。 儘管gsp-1突變在本論文的實驗中皆未觀察到明顯的缺陷,但是從定量的PCR結果發現雄性性腺中的gsp-1基因呈現高表現,因此我們不排除GSP-1可能在雄性減數分裂中扮演輔助的角色。綜合所有結果,我們假設GSP-2在雄性減數分裂中具有調節染色體bi-orientation以及避免cohesin提前被移除的功能。 Accurate chromosome segregation is critical for normal development of organism. Defective chromosome partition induces tumorigenesis, infertility and progeny lethality. In both mitotic and female meiotic cells, PP1 phosphatases GSP-1 and GSP-2 have been shown to regulate multiple aspects in chromosome segregation, including chromosome bi-orientation and cohesion removal. However, whether sperm meiotic chromosome segregation events are regulated under similar mechanisms remains unclear. To investigate the roles of GSP-1 and GSP-2 in male meiotic divisions, we examined the fertility of male worms defective of either gsp-1 or gsp-2. Surprisingly, gsp-1 mutant males were able to sire viable progeny when mated with wild type females. Contrarily, female worms mated with gsp-2 mutant males produced mostly dead embryos. This suggests GSP-2, but not GSP-1, is essential to normal sperm production. By examination of DAPI-stained gonads as well as live sperm nuclei, we found that gsp-2 mutant males generate aneuploid sperm. By immunofluorescence staining, gsp-2 mutant spermatocytes exhibit abnormal chromosome segregation defects, including lagging chromosome and chromosome bridges. Time lapse recording of the male meiotic divisions revealed failure of chromosome alignment in gsp-2 mutant spermatocytes, resulting in lagging chromosome and chromosome bridges during divisions. We measured the width of aligned chromosomes before anaphase onset and used BUB-1::GFP as a marker to observe the orientation of chromosomes via live imaging. The results showed that gsp-2 mutant is defective in chromosome bi-orientation. In addition to chromosome bi-orientation, we also observed increased number of chromosome mass in staining and live imaging in gsp-2 mutant, indicating GSP-2 also prevents the premature cohesin removal in male meiosis. By counting the chromosome number in DAPI-stained male gonads, we found gsp-2 mutant induced increased chromosome number after anaphase I. Furthermore, while cohesin REC-8 appears to be normally localized in gsp-2 mutant spermatocytes, AIR-2, a kinase promoting the removal of cohesin, was mis-localized on chromosomes, suggesting GSP-2 plays a role in counteracting AIR-2 in timely cohesin removal. Despite gsp-1 does not exhibit specific defect in any of the analyses we conducted, quantitative RT-PCR results showed that gsp-1 is highly expressed in male gonads, suggesting that GSP-1 might play supportive roles in sperm meiotic chromosome segregation. Taken together, our results suggest that GSP-2 is required for both proper chromosome bi-orientation and timely cohesin removal during male meiotic divisions. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/50223 |
DOI: | 10.6342/NTU201601820 |
全文授權: | 有償授權 |
顯示於系所單位: | 醫學檢驗暨生物技術學系 |
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