【作者】 刘美玲；

【导师】 王介东； 贾孟春；

【作者基本信息】 中国协和医科大学 ， 细胞生物学， 2010， 博士

【摘要】 哺乳动物的精子发生是一个复杂而独特的细胞增殖与分化的过程,其中包括精原细胞的有丝分裂增殖,精母细胞的减数分裂和精子细胞的变态过程,最终形成成熟的精子。精子发生过程涉及复杂的形态变化、遗传重组及染色体倍数的减半。这一过程是一个受到精细调节的众多基因参与的复杂而有序的过程。因此,筛选和研究精子发生过程中起重要作用的特异表达基因,对研究精子发生的分子机制,探讨男性不育的分子机理,寻找新的诊断和治疗措施及研究新的节育手段具有重要意义。LM23基因是本实验室克隆出的一条新基因,前期的初步研究提示,它是一个新的大鼠精子发生相关基因。BLAST工具对LM23基因的同源基因进行比对分析发现,LM23基因与小鼠、人类的speedy homolog A(Spdya, Speedy)基因具有同源性。本研究对该基因在精子发生过程中的明确作用及其机制进行了探讨。首先我们对该基因的表达特性进行了进一步的鉴定。利用RT-PCR检测了LM23基因在正常大鼠的心、肝、脾、肺、肾、脑、卵巢、睾丸、肌肉组织中的表达,结果显示睾丸组织有较高水平的LM23基因表达,其余组织均为阴性,从而提示LM23基因的表达具有睾丸特异性。分离9天龄雄性SD大鼠A型精原细胞及成年大鼠粗线期精母细胞和圆形精子细胞进行荧光定量PCR检测,结果显示该基因在精母细胞中表达量最高,显著高于精原和精子细胞的表达量；原位杂交检测结果也提示LM23基因主要在精母细胞中表达。这些结果说明LM23基因的表达具有组织特异性和阶段特异性的特点。为了探讨LM23基因在精子发生中的功能,本研究针对LM23基因序列,设计了4个RNA干扰靶点序列,克隆到慢病毒载体上,筛选出含目的序列的慢病毒载体,包装成病毒颗粒。选5周龄的雄性SD大鼠为研究对象,经睾丸输出管注射法将上述包装好的病毒颗粒注射到睾丸。实验组注射液中含干扰片段病毒,对照组含无关片段病毒,单侧注射,未注射侧睾丸作为空白对照。注射后4周,拉颈处死大鼠,开腹取出睾丸。结果发现,注射干扰病毒的睾丸比对照侧右侧睾丸略小,且附睾也没有右侧附睾饱满;而注射对照病毒的睾丸与对侧睾丸比较无明显差异。在体式荧光显微镜下观察,可见已经在大鼠睾丸表达慢病毒载体的绿色荧光蛋白。在荧光显微镜下观察睾丸冰冻切片,显示生精上皮细胞中有绿色荧光表达,说明慢病毒颗粒已成功转染生精细胞。Real-time PCR检测注射后2周和4周的实验组、对照组和空白对照组睾丸LM23的表达,结果显示实验组LM23的表达显著降低,与对照组比较,2周和4周表达量分别减低了69%和87%。通过Western blot方法检测干扰侧大鼠组睾丸中LM23蛋白的表达情况,结果显示,发现RNA干扰大鼠睾丸未LM23蛋白表达。这些结果提示成功建立LM23基因沉默大鼠模型。睾丸组织切片HE染色检查,结果显示干扰侧睾丸的精曲小管组织紊乱,有断裂和生精细胞脱落至管腔的现象,生精细胞发育阻滞在精母细胞阶段。镜下大约可以观察到三种类型的精曲小管,在Ⅰ型管中含有3-4层的精母细胞；在Ⅱ型管中含更多的精母细胞,并且细胞有向管腔中脱落的现象,管腔中有伊红染色很重的细胞；在Ⅲ型管腔中仅有1-2层精原细胞和支持细胞,管腔空洞。对照侧附睾中充满成熟精子,而干扰侧附睾管的管腔缩小,未见成熟精子。TUNEL检测LM23基因沉默后睾丸显示,在一些精曲小管中有大量的凋亡细胞存在,而这些精曲小管与上述提到的Ⅱ型管基本对应,而在对应的Ⅰ型管腔和Ⅲ型管腔中存有少量的凋亡细胞,而在对照侧睾丸则几乎未检测到凋亡细胞。根据这些结果推测,LM23基因沉默通过某种机制引起精子发育到精母细胞阶段发生阻滞,造成了精母细胞在精曲小管中堆积,出现了Ⅰ型管中所观察到的现象。由于这些精母细胞不能继续分化可能启动了凋亡通路而发生凋亡,出现了Ⅱ型管中所观察到的现象；最后由于凋亡细胞被清除而出现了Ⅲ型管中所观察到的现象。为了探讨LM23基因的作用机制,本研究利用大鼠全基因组芯片筛查LM23基因沉默引起的睾丸表达谱的改变状况,并对所得数据进行验证和分析。基因芯片结果分析显示,LM23基因沉默大鼠睾丸发生大量的基因表达改变,其中包括多个精子发生、减数分裂及细胞周期和凋亡相关的基因。综上所述,LM23基因的具有睾丸特异表达的特点,且主要在精母细胞表达,该基因在精子发生中起重要的作用,抑制其作用造成精子发生阻滞在精母细胞阶段,大量生精细胞凋亡,许多与精子发生、减数分裂和凋亡相关基因的表达发生改变。更多还原

【Abstract】 Spermatogenesis is a complicated and specific process of cell proliferation and differentiation including mitotic division of spermatogonia, meiotic division of spermatocytes and morphological transformation of spermatids.It is a complex process involving cell division, diffentiation and interactions between cells in the microenviroment of the seminiferous tubule. It is regulated by lots of genes; generally, these kinds of regulation genes are expressed under precise temporal and spatial regulation in sperm cells specifically. The separating and identifying of genes related to spermatogenesis and the study of their molecular regulating mechanism at proteome level are very important for clinical diganosis and treatment of male infertility.LM23 (AF492385) is a gene specifically expressed in the testes of Rattus norvegicus previously reported by our laboratory. A BLAST homology search against the NCBI non-redundant database and an Ambystoma EST database revealed that LM23 is a R. norvegicus homologue of Speedy A (Spdya). In this study, we clarified the majoy role and mechanism of LM23 in the proeess of spemratogenesis.Firstly, RT-PCR analysis of RNA from the nine different tissues including testis and ovary showed that LM23 RNA was only present in testis. Real-time PCR analysis showed that the expression level of LM23 was highest in spermatocytes and very low in spermatogonia and spermatids. In situ hybridization revealed a strong positive signal in the cytoplasm of spermatocytes and a weak signal in spermatids and spermatogonia. This testis-specific and stage-specific expression pattern suggested that LM23 might be involved in R. norvegicus spermatogenesis.To reveal the function of LM23 in the testis, we used lentivirus-mediated RNA interference (RNAi) to knock down LM23 expression in a tissue-specific manner in vivo. A lentiviral vector expressing a short hairpin RNA (shRNA) targeting LM23 was microinjected into the efferent ducts of R. norvegicus testes. The infectious lentivirus was microinjected into testes of 5-week-old R. norvegicus just completing the first wave of spermatogenesis. The enhanced green fluorescent protein (EGFP) signal in about 75% of whole testes of R. norvegicus at four weeks post-transfection is shown in a stereomicroscope view. Next, to examine the efficiency of LM23 RNAi, we analyzed the expression levels of LM23 mRNA in testes by real-time PCR at two weeks and four weeks post-transfection. Compared with scrambled RNAi-transfected testes, LM23 mRNA expression was significantly reduced (69% and 87%, respectively). There was no difference in LM23 mRNA level between scrambled RNAi-transfected testes and wild type testes. Western blot analysis showed LM23 protein expression was not detected in testis after LM23 RNAi. These data showed that the specific in vivo knockdown of LM23 in testes of R. norvegicus via lentivirus-mediated RNAi was effective and stable.The size and weight of LM23-shRNA testes had no significant differences from the controls. Seminiferous tubules of control testes were well organized and contained a full spectrum of spermatogenic cells, including spermatogonia, spermatocytes, spermatids and spermatozoa. In contrast, seminiferous tubules of LM23-shRNA testes appeared disorganized, disrupted, and shedding germ cells into the lumina; the germ cells exhibited complete meiotic arrest in spermatogenesis. Spermatocytes were accumulated, round spermatids were few and elongating spermatids, spermatozoa were absent in certain LM23-shRNA seminiferous tubules. Three major types of seminiferous tubules were observed in LM23-shRNA testes. TypeⅠtubules contained 3-4 layers of spermatocytes. In typeⅡtubules, there were more layers of spermatocytes and many heavily eosin-stained cells, which might be apoptotic cells. TypeⅢtubules were characterized by a few layers of spermatogenic cells/Sertoli cells and big empty lumina. The epididymal tubules of control R. norvegicus were filled with spermatozoa, whereas those of LM23-shRNA testes R. norvegicus were empty. A TUNEL assay showed the presence of many apoptotic cells in certain tubules, which were likely typeⅡtubules. In contrast, few apoptotic cells were present in typeⅠor typeⅢtubules. Few apoptotic cells were detected in tubules of control testes. One possible explanation for the presence of three types of tubules in LM23-knockdown testes might be coordinated differentiation of the germ cells in a given tubule. In LM23-knockdown testes, spermatogenesis proceeded from spermatogonia to spermatocytes, but further differentiation was blocked, resulting in the accumulation of spermatocytes in type I tubules. Subsequently, these spermatocytes failed to further differentiate and underwent apoptosis in typeⅡtubules. Eventually, most apoptotic spermatocytes were eliminated in typeⅢtubules.Microarray analyses of the transcriptomes of the LM23-shRNA and control testes were performed to screen for genes regulated by LM23. The results revealed that the expression of some genes related to spermatogenesis, meiosis, the cell cycle, and apoptosis were significantly changed after LM23 knockdown. Real-time PCR analysis confirmed that some meiotic genes involved in synapsis, recombination (Sycp1, Sycp2, Sycp3, Msh5) and meiotic sister-chromatid cohesion (Stag3, rec8Ll) had lower expression. Many pro-apoptotic genes were up regulated such as Bcl-2 family members including Bax, Bid3, Bak1 et al. And many anti-apoptotic genes were down regulated such as Fafl and Zfp9.Collectively, these studies demonstrate that LM23 is required for meiosis in spermatogenesis.更多还原