根據衛生福利部國民健康署的統計數據，台灣的不孕症比率約為10％至15％。導致不孕的夫婦在女性或男性有許多原因，包括年齡，遺傳缺陷，疾病，藥物使用和環境變化。然而，大多數研究集中在女性因素上，對於男性因素的關注較少。值得注意的是，近年來男性不育率正在上升，原因不明的男性不育患者約佔50％。因此，調查男性不育在台灣是一個關鍵問題，因為台灣是世界上生育率最低的國家。無精子症是一種男性不育中最為嚴重的疾病，可分為阻塞性型（OA）或非阻塞性無精子症（NOA）類型。 OA患者可以通過手術獲得體外受精（IVF）的精子，而NOA患者則不能。但是，沒有任何有用的參數來區分OA和NOA患者。因此，了解導致NOA發展的潛在因素並作為NOA患者的生物標誌物是臨床上的關鍵問題。我們與鄭裕生醫師合作於先前文獻表明，MAEL在精子生成中扮演著重要作用。引起逆轉錄轉座子激活的MAEL表達缺失與NOA相關。
為了進一步系統地找尋MAEL的下游目標，對來自MAEL敲低的RNA-seq數據進行了分析，發現丟失MAEL後，一群較長的非編碼RNA（lncRNA）表達水平失調。我們的結果表明，在NOA患者的組織樣本中，LINC02043，LncFAM41C，RPL34-AS1：7，LncZBTB10，LncPXDC1的表達水平上調。為了進一步鑑定具有診斷潛力的LncRNA，從健康患者和NOA患者中分離出精液外泌體，並提取了外泌體的RNA。有趣的是，從NOA患者中分離出的精液外體中僅LncZBTB10表達增加，而其他候選者則沒有。此外，MAEL敲除確實增加了LncZBTB10表達，以進一步證實先前的RNA-seq結果。本文中，我們專注於研究lncZBTB10在NOA中的作用，因為在文獻中目前沒有關於此lncRNA的研究。接下來，分別使用細胞核質分離以及RNA原位雜交（ISH）研究LncZBTB10在體外和在NOA組織中的表達模式。結果表明，LncZBTB10主要表現於細胞核中，並在NOA組織的生殖細胞中過度表達，表示LncZBTB10可能在精子生成過程中發揮重要作用。最後，為了研究LncZBTB10的功能，使用RNA pull down測定結合質譜儀來鑑定與LncZBTB10相互作用的潛在蛋白質。質譜儀的數據通過功能註釋工具DAVID做進一步分析，並與精子生成有關的基因列表進行了交叉比對。更重要的是，許多與LncZBTB10相互作用的蛋白質參與了精子生成並調節了一些關鍵的生物角色。其中，RBMXL1確實通過RNA免疫沉澱（RIP）與LncZBTB10相互作用，而敲低MAEL增加了RBMXL1的表達。綜上所述，我們的研究是第一個研究LncZBTB10在男性不育症中作用的研究團隊。

It is well known that the prevalence rate of infertility in Taiwan is about 10% to 15% according to statistical data from the National Health Administration. Many causes including age, genetic defect, disease, drug usage and environmental change from either female or male gender contributed to infertile couple. However, majority of studies focus on the female factor while male factor is gotten less attentation. It is a remarkable fact that, male infertility rate is increasing in recent years and there is around 50% of male infertile patients with unknown causes. Thus, to investigate male infertility is a critical issue in Taiwan because of its lowest fertility rate in the world. Azoospermia is a serious disease causing male infertility and it can be divided into obstructive (OA) or non-obstructive azoospermia (NOA) type. OA patients can be obtained theirs sperms by surgery for in vitro fertilization (IVF), while NOA patients cannot. However, there is no useful parameter to distinguish OA and NOA patients. Therefore, to identify the potential factor leading to the development of NOA and as a biomarker for NOA patient is a crucial issue in clinical. Previous findings from our cooperators have demonstrated that MAEL plays an important role in spermatogenesis. Loss of MAEL expression causing activation of retrotransposon is associated with NOA.
To further systemically understand the downstream target of MAEL, RNA-seq data from MAEL knockdown was analyzed and found a group of long non-coding RNAs (lncRNA) expression levels were dysregulated after loss of MAEL. Our results showed that expression levels of LINC02043, LncFAM41C, RPL34-AS1:7, LncZBTB10, LncPXDC1 were upregulated in the tissue samples of NOA patients. To further identify LncRNA with diagnostic potential, semen exosome was isolated from healthy donors and NOA patients and extracted exosomal RNA. Interestingly, only LncZBTB10 expression was increased in the semen exosome isolated from NOA patients while others were not. Furthermore, MAEL knockdown indeed increased LncZBTB10 expression to further confirm previous RNA-seq results. Herein, we focus on studying the role of lncZBTB10 in NOA since there is no reference for this novel lncRNA in the literature. Next, nuclear and cytoplasmic extracts and RNA in situ hybridization (ISH) were used to study LncZBTB10 expression pattern in vitro and in NOA tissues, respectively. Results showed that LncZBTB10 was mainly enriched in the nucleus and overexpressed in the germ cells of NOA tissues, suggesting that LncZBTB10 may play an important role during spermatogenesis. Finally, in order to investigate the function of LncZBTB10, RNA pull down assay combined mass spectrometry were used to identify potential proteins interacting with LncZBTB10. Mass data were further analyzed by DAVID, a functional annotation tool, and cross-referenced with a gene list related to spermatogenesis. Crucially, numerous lncZBTB10 interacting proteins were involved in the spermatogenesis and regulated several critical biological events. Among them, RBMXL1 indeed interacted with LncZBTB10 by RNA immunoprecipitation (RIP) and knockdown of MAEL increased RBMXL1 expression. Taken together, our study is the first group to dissect the role of LncZBTB10 in male infertility.

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