以線蟲為模型探討雄性減數分裂特異性CPEB調控蛋白CPB-1在精子生成過程扮演的角色

Investigating the roles of male meiosis-specific CPEB protein CPB-1 during spermatogenesis in Caenorhabditis elegans.

邱雅貞(Ya-Chen Chiu)

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(2022年)

https://doi.org/10.6342/NTU202203909

摘要

精子生成是雄性性腺中產生成熟精子的獨特過程。先前研究發現，在秀麗隱桿線蟲的精母細胞開始減數分裂前，轉錄就停止了，代表該過程主要依賴轉錄後及轉譯調節，這與我們先前研究發現抑制轉譯會阻止減數分裂之進行相互呼應。
本研究探討精子特異性轉譯調節因子CPB-1在秀麗隱桿線蟲精子生成中的作用。CPB-1是一種細胞質聚腺苷酸化元件結合蛋白 (Cytoplasmic polyadenylation element binding, CPEB)，其被認為可以透過與mRNA 3’端非翻譯區中的特定序列結合來調節轉譯。
為了研究CPB-1在精子生成中的作用，我們透過CRISPR技術將帶有AID*標記的序列嵌入cpb-1基因，以利用生長素誘導降解系統 (Auxin-inducible degradation, AID) 使內源性CPB-1降解。
我們發現在雄性的生殖系統中，CPB-1可在幾小時內被誘導降解，且此降解具有可逆性。此外，缺乏CPB-1的雌雄同體線蟲生育力大幅削弱，但其生育能力在提供正常精子後得以恢復，此結果表明CPB-1對雄性生育能力是不可或缺的，但不影響雌性。
利用免疫螢光觀察CPB-1在雄性生殖腺中的分佈，我們發現CPB-1在雄性減數分裂前期 (prophase) 的細胞質中表達，而消失在減數分裂後期和分裂的精母細胞中，此外，缺乏CPB-1的精母細胞染色體無法正確進行染色質濃縮，而且在性腺中堆積未能正常分裂的精母細胞。縮時影像攝影觀察的結果發現缺乏CPB-1的初級精母細胞大量停滯並伴隨嚴重的染色體分離缺陷，且通常含有非溶酶體的細胞質液泡狀結構。綜合以上，我們發現CPB-1在減數分裂前期調控精子第一次減數分裂時所需之蛋白質生成，以協助配對後的同源染色體再次濃縮及促使精子減數分裂。
最後，為了找出由CPB-1調節的目標mRNA及其下游影響的蛋白質，我們比較正常及CPB-1缺乏的雄性線蟲蛋白質萃取物，初步分析顯示，CPB-1缺乏對雄性線蟲整體蛋白質組成沒有顯著影響，但有數個未知蛋白質的表現明顯低下。這些結果將有助於其後蛋白質體分析之研究，以暸解受CPB-1調控之蛋白質如何促進雄性生育能力。

關鍵字

精子生成 ； CPEB ； CPB-1 ； 減數分裂 ； 生長素誘導蛋白質降解

並列摘要

Spermatogenesis is a unique process in the male gonads to produce mature spermatozoa. Previous studies show that transcription ceases before spermatocyte begins meiotic divisions in nematode Caenorhabditis elegans, indicating the process is mainly regulated at the post-transcriptional and translational levels. Consistently, our previous study showed that inhibition of translation prevented the progression of meiotic division.
In this study, we aim to investigate the roles of spermatogenic-specific translation regulator CPB-1 in C. elegans spermatogenesis. CPB-1 is a cytoplasmic polyadenylation element binding (CPEB) protein that is predicted to regulate translation of mRNAs through binding to specific sequences in the 3' untranslated region.
To investigate the roles of CPB-1 in spermatogenesis, we CRISPR-engineered an AID* tag inserted into genomic cpb-1, allowing Auxin-inducible degradation of endogenous CPB-1.
We showed that CPB-1 degradation can be reversibly induced in both male germline within a few hours. CPB-1-depleted hermaphrodites showed significant sterility that could be rescued with wild-type sperm, suggesting that CPB-1 is essential in male but not female fertility.
Results from immunofluorescence staining showed that CPB-1 is expressed in the cytoplasm of male meiotic prophase, and is absent from late meiotic and dividing spermatocytes. Also, paired homologous chromosomes failed to undergo proper condensation stages, leading to accumulation of immature spermatocytes in CPB-1-depleted males. Time-lapse recordings revealed that CPB-1-depleted spermatocytes exhibited severe chromosomal segregation defects and failed to initiate the first meiotic division. Additionally, CPB-1-depleted spermatocytes often contain cytoplasmic vacuole-like structures that were not lysosomal origin. Taken together, we hypothesize that during male meiotic prophase, CPB-1 regulates the production of proteins essential for late meiotic chromosome condensation and initiation of male meiotic division.
To identify the target mRNAs regulated by CPB-1 and their downstream proteins, we compared male worm extracts generated from wild-type and CPB-1-depleted male worms. Preliminary analysis revealed that CPB-1 depletion does not impact global proteome in male worms, however, a few proteins were significantly reduced. These results facilitate further proteomic analyses to elucidate how CPB-1-dependent proteostasis contributes to male fertility.

並列關鍵字

Spermatogenesis ； CPEB ； CPB-1 ； meiosis ； Auxin-induced degradation

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