SAGE标签3’端长片段克隆方法的建立和精子中的新基因QSA克隆及功能初探
|
摘要
传统观念认为，精子是个高度分化的终末细胞，仅仅将父本基因组传递给卵细胞。但近年来的研究证实哺乳动物精子中存在有复杂的mRNA,基因芯片方法在人的成熟精子中检测出大约3500-5500种的mRNA。我们实验室用基因序列分析（SAGE）的方法也对人成熟精子中的mRNA进行了聚类和功能分析。其中在389高丰度表达的基因中，最突出的是一组核蛋白基因，占全部聚类分析基因的四分之一(96/389)，主要参与DNA依赖的基因转录和翻译调节。此外，还包括蛋白多肽溶解相关者、蛋白激酶、免疫相关者、蛋白转运和定位相关者、翻译后修饰相关者、线粒体电子传递相关者、ATP合成传递相关者ATP、ATPase活性相关者、蛋白质向核内运输传递相关者等。另外，还发现54个重叠性标签没有基因匹配，属于未知的新基因。
为何转录静止的精子细胞有选择性地保留种类繁多的mRNA，这些mRNA又执行怎样的功能呢？研究人员发现精子中的mRNA在受精过程中会传递给卵子且持续存在，从而推测其可能在受精和胚胎早期发育中具有重要作用。而以往的母系效应观点认为，在胚胎基因组激活前，合子发育由卵子发生过程中储存在成熟卵母细胞质中的母源因子（包括mRNA和蛋白质）所调控。在卵子受精或核移植的胚胎激活后，这些母源因子启动早期胚胎发育，基因组开始转录，表达新的基因和合成新的因子，并发生级联反应，合成更多的细胞因子，保持早期胚胎继续发育。精子mRNA群进入合子中是否也同样起着类似的功能，起着调控胚胎的早期发育作用？这种父源因子的调控效应是否可能在胚胎发育中与母系效应互相补充，融为一体共同调控胚胎早期发育？为了探究精子中的mRNA的功能，本研究在已有人的精子mRNASAGE数据库基础上，首先试图从54个未匹配SAGE标签克隆出所对应的新基因，进而研究这些新基因的功能，分析这些功能在精子发生及胚胎早期发育过程中的作用。本研究可分为两个部分：
第一部分研究，为了便于从SAGE标签克隆出对应新基因序列，我们研究创建了一种新的克隆方法。基因序列分析（SAGE）技术是一种高通量研究转录本的方法，通过定义每种mRNA特定位置的14bp长度的核苷酸片段（SAGE标签）可以系统，综合全面的构建出整个的表达谱。但同时由于SAGE标签仅有14bp碱基，承载有限的遗传信息，因此需要克隆标签所代表的基因全长序列，以便研究其功能。常规的3’和5’RACE方法都无法从14bp的短标签克隆其cDNA的全长，因而出现了一些特殊的方法如，RAST-PCR、GLGI、rSAGE等方法。这些方法各有所长，但都存在费时费力，克隆效率低，易产生非特异性条带，尤其需要很高的初始mRNA模板量。本研究中，精子中的mRNA含量非常少，仅有体细胞的1/600，因此很难用上述方法克隆其标签。为此，我们创建了新两步克隆方法，第一步骤是模板cDNAs的富集。利用夹在所有cDNAs两端序列为引物，经过PCR可以富集扩增出总cDNAs模板；第二步骤利用上述模板进行含tag的特异性片段克隆。根据半巢式PCR原理设计成2条下游套式引物，结合上游含标签特异性引物采用两次半巢氏PCR扩增出目的片度。与同类方法比较而言，本研究方法扩增特异性高，所需初始mRNA模板量少，尤其克隆一些低丰度表达的基因效果好。用此方法，我们对54个中11未知标签进行其3’cDNA端克隆，并成功得到了11个标签对应的3’cDNA长片段，便于后续基因全长的克隆及分析。
第二部分研究，利用上述创新的方法并结合5’RACE等方法，从54个未知标签中成功克隆出一个基因。该序列在NCBI人的refseq_rna库未见匹配的同源序列，根据人类基因命名的规则初步命名该新基因为QSA。通过生物信息学分析，我们发现该基因由人第19号染色体上三个外显子拼接而成，物种之间同源性较低，但编码28aa蛋白质序列在物种之间具有非常高的保守性，且含有一段signalanchor信号序列。利用RT-PCR及原位杂交分析基因QSA的mRNA时空表达分布发现，该基因在24种人组织切片中部分腺体组织（如：甲状旁腺、垂体、前列腺等）中具有明显的表达；另外，睾丸、精子、受精卵中也检测出基因QSA的表达，而在卵细胞及囊胚均未见基因QSA的mRNA。同时，我们原核表达了抗原多肽QSA，并以此免疫兔产生抗多肽QSA的抗体。根据原位杂交结果，我们选择了部分组织利用Western-blot，免疫组化及间接免疫荧光实验检测了基因QSA的表达蛋白分布。该蛋白的分布情况与原位杂交结果一致，特别在睾丸组织中，主要分布在各级生精细胞中，而其他支持细胞、间质细胞中均未见。为了研究与蛋白QSA发生结合作用的蛋白，我们做了人的16,000种类蛋白点阵的蛋白芯片分析，发现核糖核酸酶P/MRP的可变亚基蛋白前体4(POP4)与待测蛋白QSA具有很强的结合信号。
结合上述实验结果，我们初步得出如下结论：我们改进了一种新的分子克隆方法，可以便捷、特异性从SAGE标签克隆出基因全长序列，尤其克隆一些低丰度表达的标签特异性较同类方法高。我们用此方法成功地从人精子mRNASAGE库中克隆出新基因QSA。该基因QSA的核酸和蛋白在进化过程中保守性的差异，说明基因QSA在分子进化水平上突变属于无害突变，不影响蛋白序生物功能性质；遵循分子中性进化理论，即在生物分子层次上的进化改变不是由自然选择作用于有利突变而引起的，而是在连续的突变压之下由选择中性或非常接近中性的突变的随机固定造成的（这里所谓选择中性的突变是指对当前适应度无影响的突变）。由于含有signalanchor信号序列，蛋白QSA可能为膜蛋白。同时，新基因QSA表达分布于部分腺体组织细胞，以及睾丸、精子、受精卵中。蛋白芯片实验揭示蛋白前体4(POP4)与蛋白QSA发生结合作用。POP4为核糖核酸酶P/MRP一个可变亚基。核糖核酸酶P对于正常并有效地转录多种非编码RN（A包括tRNA、5SrRNA、SRPRNA和U6snRNA）的基因是必要的。
我们根据上述结论做出推论，基因QSA在部分腺体组织和睾丸各级生精细胞及受精卵中表达，推测基因QSA可能具体某种激素产生、分泌等有关且可能参与精子发生及胚胎的早期发育过程。潜在的途径可能是蛋白QSA可能位于核膜上，介导POP4组装成核糖核酸酶参与激素的合成、分泌等过程，从而通过激素发挥在精子发生及胚胎发育过程中的作用。因而，推测精子的mRNA在胚胎的早期发育过程中可能产生父系效应。
In the conservative perception the spermatozoon is a highly differentiated and specialized cell, which until recently was thought only to transport the paternal genome to the oocyte. However, recently more and more evidences have been accumulating that human ejaculate spermatozoa convincingly retain a complex and yet specific population of RNAs. For example, by using a complementary DNA (cDNA) microarray, about3500-5500mRNA species were detected in human spermatozoa. Also, in our lab we have already clustered and analyzed function of the complex population of RNAs by using an alternative approach of serial analysis of gene expression (SAGE). Among389SAGE tags of high abundance genes, the most obvious group claimed one fourth (96/389) of the general genes analyzed in spermatozoa as nucleus proteins related to transcription and transcription regulation (DNA-dependent). The rest included:ribosomal subunit (84/389) involving protein biosynthesis; the genes related to spermiogenesis process; proteolysis and peptidolysis; protein kinase (24/389); antigen presentation and cell adhere and immune (22/389); protein transportation and localization (18/389) and posttranslational modification (16/389), etc. Furthermore,54SAGE tags had no matches on the SAGEmap and therefore representing potential novel gene.
Thus, it raised a puzzle why these mRNAs were reserved in such a 'quiescent' specialized cell and what possible functions of those mRNAs could perform. And now, many evidences indicate that the spermatozoa RNA penetrates the oocyte during fertility and remains therein during the early embryonic development, it therefore appears that spermatozoa RNA are not merely non-functional remnants during spermatogenesis, but that they may play an important role in fertilization and early embryo development. As an accepted dogma of maternal effect, the maternal factors (including mRNAs and proteins) derived from oocytes regulate zygotic development before activation of zygotic genome. After fertilized by a sperm, in zygote the maternal factors initiate early embryonic development. Its approach is a cascade reaction process, in which maternal factors firstly activate zygotic genome transcription, expression of new genes and translating new factors, thus more cellular factors are generated to keep early embryonic development. Resembling the manner of maternal factors, whether does spermatozoon RNA penetrating the oocyte during fertility possible regulate early embryonic development? And whether is paternal effect possible necessary complementary with maternal effect during the early embryonic development? To clarify the function of spermatozoa RNA and validate the hypothesis about paternal effect, we want to identify and character the possible new genes from the54unmatched SAGE tags based on our SAGE library of spermatozoa mRNA, and then analyze their functions during the spermatogenesis and the early embryonic development. Through elucidating the function of these potential new genes, we will get more insight into the transcript complexity of human ejaculated spermatozoa.
Part Ⅰ-Establishment of innovative method of3'-end amplification from SAGE tags (Semi-nested PCR analysis of unknown tags on serial analysis of gene expression).
The serial analysis of gene expression (SAGE) technique is a high-throughput method, which can allows the construction of a comprehensive expression profile, in which each mRNA is defined by a specific14-mer. However, we could not get more genetic information from the tag than from its full-length gene because of short SAGE tag (14bp). Also it was difficult to indentify the full-length cDNA of gene by3'and5' RACE technique due to its short tag. Hence, the special clone methods were developed to overcome the shortage of SAGE tag, such as RAST-PCR、GLGI、rSAGE. There methods share different characteristics and advantages. Yet there are many flaws about these methods, intensive labor; low clone efficiency; nonspecific amplification; what more, large amount requirement of initial mRNA, which limit the widespread use of them. In our study, it is hard to identify gene using the methods above because each human spermatozoon is estimated to contain just0.015pg of total RNA, only1/600of the amount of somatic total RNA. Therefore, we have developed a technique called the two-step analysis of unknown SAGE tags (TSAT-PCR) to generate the3'-longer cDNA ends. The first step is enrichment of cDNA template using PCR method with a pair of primes added to the5'and3'ends of cDNAs. The second step is to clone the3'-longer cDNA ends corresponding to SAGE tags based on semi-nested PCR with two downstream nested primers. Compared to analogous methods mentioned above, the TSAT-PCR method is significantly superior to them in the follow aspect, low amount of initial mRNA, high specific amplification, especially better effect for clone of low abundance genes.11of54unmatched tags were tested for the the TSAT-PCR method, and3'-longer cDNA ends corresponding to11tags were successfully amplified and cloned for following experiment.
Part II-accomplishment of clone and functional study of a new gene from54unmatched SAGE tags using our new method above.
Through the combined application of TSAT-PCR and5'-RACE, we have successfully amplied and identified a new gene QSA from54unmatched SAGE tags. BLAST result in NCBI web showed the new gene encoding28aa peptide, which we called QSA gene, consisted of three exons spliced from human chromosome19. Homology analysis of QSA gene disclosed there was no homologous nucleotide sequence blasting among other species, yet the28aa peptide encoding by QSA gene displayed high conservation during five species (pan troglodytes, dog, human, rattus norve, mus musculus). Besides homology analysis, we also had predicted the cellular localization of the28aa peptide was plasma membrane by online prediction software (website, http://www.cbs.dtu.dk/services/SignalP/). To investigate tissue distribution of QSA gene mRNA,24human tissues were tested and compared each other through RT-PCR and in situ hybridization. Among24tissues, its mRNA exists in great quantity at several glands, such as, parathyroid gland, pituitary gland, and prostate. In addition, the mRNA was detected at testicle, spermatozoon and zygote, while no QSA gene mRNA was detected at oocyte and blastula. Then we constructed prokaryotic expression plasmid (pET-28a-QSA) of QSA gene and it was expressed in E. coli BL21cells, the recombinant QSA fusion protein was purified and immunized in rabbit. After immunizing, polyclonal antibodies against recombinant protein QSA were generated in rabbit serum. According to result of in situ hybridization, several representative human tissues (parathyroid gland, pituitary gland, prostate, testicle, spermatozoon and ovary) were selected to screen tissue distribution of the peptide QSA using western-blot, immunohistochemistry, and indirect immunofluorescent technique. The case of peptide QSA distribution was in accordance with the result of in situ hybridization. And there was a significant case that peptide QSA was detected in human testicular spermatogenic cells at different stages, yet it was undetectable in other testicular cell, e.g., leydig cell and sertoli cell. To initially explore molecular function of peptide QSA, we tried to screen interacting protein of peptide QSA among16,000human proteins using protein chip technique. The result revealed Homo sapiens processing of precursor4(POP4), ribonuclease P/MRP variant subunit, interacted strongly with peptide QSA.
In sum, the conclusion can be drawn from above two parts of studies:
We developed a innovative method of3'-end amplification from SAGE tags, which possesses the advantages of being simple, rapid, low in cost, and highly efficient, a low amount of mRNA requirement, and what more, amplifying target PCR products from low-abundance transcripts. Using our new method, a new gene QSA was cloned and sequenced successfully. Molecular evolution analysis of QSA gene followed the neutral theory of molecular evolution, which if a population carries several different versions of a gene; odds are that each of those versions is equally good at performing its job-in other words, that variation is neutral. High expression of gene QSA was detected at several kinds of glands cell, such as, thyroid gland, pituitary gland, prostate, testicle, and spermatozoon. Result of protein chip showed peptide QSA was possibly associated with POP4, a variant subunit of ribonuclease P/MRP. Ribonuclease P participates in transcription of non-coding RNA genes including tRNA,5S rRNA, SRP-RNA and U6snRNA.
Summarily, some inferences were drawn According to the results above.
The case of peptide QSA distribution at several glands and testicle cell revealed that QSA gene possibly was related to synthesis and/or secretion of a hormone and played an important role during the early embryonic development. It is a speculated pathway that peptide QSA located at nuclear membranes mediates assembly of ribonuclease P/MRP through POP4; then, it would regulate synthesis and/or secretion of a hormone; last, the hormone acts on the early embryonic development. In conclusion, spermatozoa mRNA, a kind of paternal factors, may play a significant role in the early embryonic development, it is a obvious case of paternal effect.
为何转录静止的精子细胞有选择性地保留种类繁多的mRNA，这些mRNA又执行怎样的功能呢？研究人员发现精子中的mRNA在受精过程中会传递给卵子且持续存在，从而推测其可能在受精和胚胎早期发育中具有重要作用。而以往的母系效应观点认为，在胚胎基因组激活前，合子发育由卵子发生过程中储存在成熟卵母细胞质中的母源因子（包括mRNA和蛋白质）所调控。在卵子受精或核移植的胚胎激活后，这些母源因子启动早期胚胎发育，基因组开始转录，表达新的基因和合成新的因子，并发生级联反应，合成更多的细胞因子，保持早期胚胎继续发育。精子mRNA群进入合子中是否也同样起着类似的功能，起着调控胚胎的早期发育作用？这种父源因子的调控效应是否可能在胚胎发育中与母系效应互相补充，融为一体共同调控胚胎早期发育？为了探究精子中的mRNA的功能，本研究在已有人的精子mRNASAGE数据库基础上，首先试图从54个未匹配SAGE标签克隆出所对应的新基因，进而研究这些新基因的功能，分析这些功能在精子发生及胚胎早期发育过程中的作用。本研究可分为两个部分：
第一部分研究，为了便于从SAGE标签克隆出对应新基因序列，我们研究创建了一种新的克隆方法。基因序列分析（SAGE）技术是一种高通量研究转录本的方法，通过定义每种mRNA特定位置的14bp长度的核苷酸片段（SAGE标签）可以系统，综合全面的构建出整个的表达谱。但同时由于SAGE标签仅有14bp碱基，承载有限的遗传信息，因此需要克隆标签所代表的基因全长序列，以便研究其功能。常规的3’和5’RACE方法都无法从14bp的短标签克隆其cDNA的全长，因而出现了一些特殊的方法如，RAST-PCR、GLGI、rSAGE等方法。这些方法各有所长，但都存在费时费力，克隆效率低，易产生非特异性条带，尤其需要很高的初始mRNA模板量。本研究中，精子中的mRNA含量非常少，仅有体细胞的1/600，因此很难用上述方法克隆其标签。为此，我们创建了新两步克隆方法，第一步骤是模板cDNAs的富集。利用夹在所有cDNAs两端序列为引物，经过PCR可以富集扩增出总cDNAs模板；第二步骤利用上述模板进行含tag的特异性片段克隆。根据半巢式PCR原理设计成2条下游套式引物，结合上游含标签特异性引物采用两次半巢氏PCR扩增出目的片度。与同类方法比较而言，本研究方法扩增特异性高，所需初始mRNA模板量少，尤其克隆一些低丰度表达的基因效果好。用此方法，我们对54个中11未知标签进行其3’cDNA端克隆，并成功得到了11个标签对应的3’cDNA长片段，便于后续基因全长的克隆及分析。
第二部分研究，利用上述创新的方法并结合5’RACE等方法，从54个未知标签中成功克隆出一个基因。该序列在NCBI人的refseq_rna库未见匹配的同源序列，根据人类基因命名的规则初步命名该新基因为QSA。通过生物信息学分析，我们发现该基因由人第19号染色体上三个外显子拼接而成，物种之间同源性较低，但编码28aa蛋白质序列在物种之间具有非常高的保守性，且含有一段signalanchor信号序列。利用RT-PCR及原位杂交分析基因QSA的mRNA时空表达分布发现，该基因在24种人组织切片中部分腺体组织（如：甲状旁腺、垂体、前列腺等）中具有明显的表达；另外，睾丸、精子、受精卵中也检测出基因QSA的表达，而在卵细胞及囊胚均未见基因QSA的mRNA。同时，我们原核表达了抗原多肽QSA，并以此免疫兔产生抗多肽QSA的抗体。根据原位杂交结果，我们选择了部分组织利用Western-blot，免疫组化及间接免疫荧光实验检测了基因QSA的表达蛋白分布。该蛋白的分布情况与原位杂交结果一致，特别在睾丸组织中，主要分布在各级生精细胞中，而其他支持细胞、间质细胞中均未见。为了研究与蛋白QSA发生结合作用的蛋白，我们做了人的16,000种类蛋白点阵的蛋白芯片分析，发现核糖核酸酶P/MRP的可变亚基蛋白前体4(POP4)与待测蛋白QSA具有很强的结合信号。
结合上述实验结果，我们初步得出如下结论：我们改进了一种新的分子克隆方法，可以便捷、特异性从SAGE标签克隆出基因全长序列，尤其克隆一些低丰度表达的标签特异性较同类方法高。我们用此方法成功地从人精子mRNASAGE库中克隆出新基因QSA。该基因QSA的核酸和蛋白在进化过程中保守性的差异，说明基因QSA在分子进化水平上突变属于无害突变，不影响蛋白序生物功能性质；遵循分子中性进化理论，即在生物分子层次上的进化改变不是由自然选择作用于有利突变而引起的，而是在连续的突变压之下由选择中性或非常接近中性的突变的随机固定造成的（这里所谓选择中性的突变是指对当前适应度无影响的突变）。由于含有signalanchor信号序列，蛋白QSA可能为膜蛋白。同时，新基因QSA表达分布于部分腺体组织细胞，以及睾丸、精子、受精卵中。蛋白芯片实验揭示蛋白前体4(POP4)与蛋白QSA发生结合作用。POP4为核糖核酸酶P/MRP一个可变亚基。核糖核酸酶P对于正常并有效地转录多种非编码RN（A包括tRNA、5SrRNA、SRPRNA和U6snRNA）的基因是必要的。
我们根据上述结论做出推论，基因QSA在部分腺体组织和睾丸各级生精细胞及受精卵中表达，推测基因QSA可能具体某种激素产生、分泌等有关且可能参与精子发生及胚胎的早期发育过程。潜在的途径可能是蛋白QSA可能位于核膜上，介导POP4组装成核糖核酸酶参与激素的合成、分泌等过程，从而通过激素发挥在精子发生及胚胎发育过程中的作用。因而，推测精子的mRNA在胚胎的早期发育过程中可能产生父系效应。
In the conservative perception the spermatozoon is a highly differentiated and specialized cell, which until recently was thought only to transport the paternal genome to the oocyte. However, recently more and more evidences have been accumulating that human ejaculate spermatozoa convincingly retain a complex and yet specific population of RNAs. For example, by using a complementary DNA (cDNA) microarray, about3500-5500mRNA species were detected in human spermatozoa. Also, in our lab we have already clustered and analyzed function of the complex population of RNAs by using an alternative approach of serial analysis of gene expression (SAGE). Among389SAGE tags of high abundance genes, the most obvious group claimed one fourth (96/389) of the general genes analyzed in spermatozoa as nucleus proteins related to transcription and transcription regulation (DNA-dependent). The rest included:ribosomal subunit (84/389) involving protein biosynthesis; the genes related to spermiogenesis process; proteolysis and peptidolysis; protein kinase (24/389); antigen presentation and cell adhere and immune (22/389); protein transportation and localization (18/389) and posttranslational modification (16/389), etc. Furthermore,54SAGE tags had no matches on the SAGEmap and therefore representing potential novel gene.
Thus, it raised a puzzle why these mRNAs were reserved in such a 'quiescent' specialized cell and what possible functions of those mRNAs could perform. And now, many evidences indicate that the spermatozoa RNA penetrates the oocyte during fertility and remains therein during the early embryonic development, it therefore appears that spermatozoa RNA are not merely non-functional remnants during spermatogenesis, but that they may play an important role in fertilization and early embryo development. As an accepted dogma of maternal effect, the maternal factors (including mRNAs and proteins) derived from oocytes regulate zygotic development before activation of zygotic genome. After fertilized by a sperm, in zygote the maternal factors initiate early embryonic development. Its approach is a cascade reaction process, in which maternal factors firstly activate zygotic genome transcription, expression of new genes and translating new factors, thus more cellular factors are generated to keep early embryonic development. Resembling the manner of maternal factors, whether does spermatozoon RNA penetrating the oocyte during fertility possible regulate early embryonic development? And whether is paternal effect possible necessary complementary with maternal effect during the early embryonic development? To clarify the function of spermatozoa RNA and validate the hypothesis about paternal effect, we want to identify and character the possible new genes from the54unmatched SAGE tags based on our SAGE library of spermatozoa mRNA, and then analyze their functions during the spermatogenesis and the early embryonic development. Through elucidating the function of these potential new genes, we will get more insight into the transcript complexity of human ejaculated spermatozoa.
Part Ⅰ-Establishment of innovative method of3'-end amplification from SAGE tags (Semi-nested PCR analysis of unknown tags on serial analysis of gene expression).
The serial analysis of gene expression (SAGE) technique is a high-throughput method, which can allows the construction of a comprehensive expression profile, in which each mRNA is defined by a specific14-mer. However, we could not get more genetic information from the tag than from its full-length gene because of short SAGE tag (14bp). Also it was difficult to indentify the full-length cDNA of gene by3'and5' RACE technique due to its short tag. Hence, the special clone methods were developed to overcome the shortage of SAGE tag, such as RAST-PCR、GLGI、rSAGE. There methods share different characteristics and advantages. Yet there are many flaws about these methods, intensive labor; low clone efficiency; nonspecific amplification; what more, large amount requirement of initial mRNA, which limit the widespread use of them. In our study, it is hard to identify gene using the methods above because each human spermatozoon is estimated to contain just0.015pg of total RNA, only1/600of the amount of somatic total RNA. Therefore, we have developed a technique called the two-step analysis of unknown SAGE tags (TSAT-PCR) to generate the3'-longer cDNA ends. The first step is enrichment of cDNA template using PCR method with a pair of primes added to the5'and3'ends of cDNAs. The second step is to clone the3'-longer cDNA ends corresponding to SAGE tags based on semi-nested PCR with two downstream nested primers. Compared to analogous methods mentioned above, the TSAT-PCR method is significantly superior to them in the follow aspect, low amount of initial mRNA, high specific amplification, especially better effect for clone of low abundance genes.11of54unmatched tags were tested for the the TSAT-PCR method, and3'-longer cDNA ends corresponding to11tags were successfully amplified and cloned for following experiment.
Part II-accomplishment of clone and functional study of a new gene from54unmatched SAGE tags using our new method above.
Through the combined application of TSAT-PCR and5'-RACE, we have successfully amplied and identified a new gene QSA from54unmatched SAGE tags. BLAST result in NCBI web showed the new gene encoding28aa peptide, which we called QSA gene, consisted of three exons spliced from human chromosome19. Homology analysis of QSA gene disclosed there was no homologous nucleotide sequence blasting among other species, yet the28aa peptide encoding by QSA gene displayed high conservation during five species (pan troglodytes, dog, human, rattus norve, mus musculus). Besides homology analysis, we also had predicted the cellular localization of the28aa peptide was plasma membrane by online prediction software (website, http://www.cbs.dtu.dk/services/SignalP/). To investigate tissue distribution of QSA gene mRNA,24human tissues were tested and compared each other through RT-PCR and in situ hybridization. Among24tissues, its mRNA exists in great quantity at several glands, such as, parathyroid gland, pituitary gland, and prostate. In addition, the mRNA was detected at testicle, spermatozoon and zygote, while no QSA gene mRNA was detected at oocyte and blastula. Then we constructed prokaryotic expression plasmid (pET-28a-QSA) of QSA gene and it was expressed in E. coli BL21cells, the recombinant QSA fusion protein was purified and immunized in rabbit. After immunizing, polyclonal antibodies against recombinant protein QSA were generated in rabbit serum. According to result of in situ hybridization, several representative human tissues (parathyroid gland, pituitary gland, prostate, testicle, spermatozoon and ovary) were selected to screen tissue distribution of the peptide QSA using western-blot, immunohistochemistry, and indirect immunofluorescent technique. The case of peptide QSA distribution was in accordance with the result of in situ hybridization. And there was a significant case that peptide QSA was detected in human testicular spermatogenic cells at different stages, yet it was undetectable in other testicular cell, e.g., leydig cell and sertoli cell. To initially explore molecular function of peptide QSA, we tried to screen interacting protein of peptide QSA among16,000human proteins using protein chip technique. The result revealed Homo sapiens processing of precursor4(POP4), ribonuclease P/MRP variant subunit, interacted strongly with peptide QSA.
In sum, the conclusion can be drawn from above two parts of studies:
We developed a innovative method of3'-end amplification from SAGE tags, which possesses the advantages of being simple, rapid, low in cost, and highly efficient, a low amount of mRNA requirement, and what more, amplifying target PCR products from low-abundance transcripts. Using our new method, a new gene QSA was cloned and sequenced successfully. Molecular evolution analysis of QSA gene followed the neutral theory of molecular evolution, which if a population carries several different versions of a gene; odds are that each of those versions is equally good at performing its job-in other words, that variation is neutral. High expression of gene QSA was detected at several kinds of glands cell, such as, thyroid gland, pituitary gland, prostate, testicle, and spermatozoon. Result of protein chip showed peptide QSA was possibly associated with POP4, a variant subunit of ribonuclease P/MRP. Ribonuclease P participates in transcription of non-coding RNA genes including tRNA,5S rRNA, SRP-RNA and U6snRNA.
Summarily, some inferences were drawn According to the results above.
The case of peptide QSA distribution at several glands and testicle cell revealed that QSA gene possibly was related to synthesis and/or secretion of a hormone and played an important role during the early embryonic development. It is a speculated pathway that peptide QSA located at nuclear membranes mediates assembly of ribonuclease P/MRP through POP4; then, it would regulate synthesis and/or secretion of a hormone; last, the hormone acts on the early embryonic development. In conclusion, spermatozoa mRNA, a kind of paternal factors, may play a significant role in the early embryonic development, it is a obvious case of paternal effect.
引文
1.阮杰刘新光梁念慈蛋白激酶CK2与精子发生关系的研究进展生殖与避孕2006vol.26,165-174
2. Salisbury GW, Hart RG, Lodge, JR. The spermatozoan genome and fertility. American Journal of Obstetrics and Gynecology.1997,128(93):342-350
3. Clermont Y. Quantitative analysis of spermatogenesis of the rat:A revised model for the renewal of spermatogonia. AmJ Anat.1962,111:111-129
4. Clermont Y. The cycle of the seminiferous epithelium in man. AmJ Anat1963,112:35-51
5. Forti G, Vannelli GB, Barni T, Orlando C, Balboni GC, Serio M. Androgen-binding protein and other Sertoli cell proteins in human testis. Annals of the New York Academy of Sciences Volume1988,538:167-172
6. Vogl AW. Distribution and function of organized concentrations of actin filaments in mammalian spermatogenic cells and Sertoli cells. International Review of Cytology1989,119:1-56
7. Fraser L R, Dudley K. New insights into the t-complex and control of sperm function. Bioessays,1999,21(4):304-312
8. Aranha I P, Martin DeLeon P A. Mouse chromosome6in Rb translocations: consequences in singly and doubly heterozygous males. Cytogenet Cell Genet,1995,69(34):253-259
9. Conway S J, Mahadevaiah S K, Darling S M, et al. Y353/B:a candidate multiple copy spermiogenesis gene on the mouse Y chromosome. Mamm Genome,1994,5(4):203-210
10. Kimmins S, Kotaja N, Davidson I, Sassone-Corsi P. Testis-specific transcription mechanisms promoting male germ-cell differentiation. Reproduction.2004,128(1):5-12
11. Ellis PJI, Furlong RA, Wilson A, et al. Modulation of the mouse testis transcriptome during postnatal development and in selected models of male infertility. Molecular Human Reproduction2004,10(4):271-281
12. Prasanth SG, Giran HM, Ali S. Biology of protooncogene c-kit receptor and spermatogenesis. Current Pharma cogenomics2004,2(1):47-60
13. Miller, D. et al. Differential RNA fingerprinting as a tool in the analysis of spermatozoal gene expression. Hum. Reprod.1994,9,864-869
14. Lambard, S. et al. Analysis and significance of mRNA in human ejaculated sperm from normozoospermic donors:relationship to sperm motility and capacitation. Mol. Hum. Reprod.2004,10,535-541
15. Ostermeier, G.C. et al. Spermatozoal RNA profiles of normal fertile men. Lancet2002,360,772-777
16. Hecht, N.B. Molecular mechanisms of male germ cell differentiation. Bioessays1998,20:555-561
17. Pessot, C.A. et al. Presence of RNA in the sperm nucleus. Biochem. Biophys. Res. Commun.1989,158,272-278
18. Kumar, G. et al. c-MYC mRNA is present in human sperm cells. Cell. Mol. Biol. Res.1993,39,111-117
19. Wykes, S.M. et al. Haploid transcripts persist in mature human spermatozoa. Mol. Hum. Reprod.1997,3,15-19
20. Chiang, M.H. et al. Detection of human leukocyte antigen class I messenger ribonucleic acid transcripts in human spermatozoa via reverse transcription-polymerase chain reaction. Fertil. Steril.1994,61,276-280
21. Goodwin, L.O. et al. L-type voltage-dependent calcium channel a-1C subunit mRNA is present in ejaculated human spermatozoa. Mol. Hum. Reprod.2000,6,127-136
22. Goodwin, L.O. et al. Presence of N-cadherin transcripts in mature spermatozoa. Mol. Hum. Reprod.2000,6,487-497
23. Durkee, T.J. et al. Identification of estrogen receptor protein and messenger ribonucleic acid in human spermatozoa. Am. J. Obstet. Gynecol.1998,178,1288-1295
24. Richter, W. et al. Detection of mRNA transcripts of cyclic nucleotide phosphodiesterase subtypes in ejaculated human spermatozoa. Mol. Hum. Reprod.1999,5,732-736
25. Rohwedder, A. et al. Detection of mRNA transcripts of b1integrins in ejaculated human spermatozoa by nested reverse transcription-polymerase chain reaction. Mol. Hum. Reprod.1996,2,499-505
26. Lambard, S. et al. Human immature germ cells and ejaculated spermatozoa contain aromatase and oestrogen receptors. J. Mol. Endocrinol.2004,32,279-289
27. Miller, D. et al. A complex population of RNAs exists in human ejaculate spermatozoa: implications for understanding molecular aspects of spermiogenesis. Gene1999,237,385-392
28. Zhao Y, Li Q, Yao C, Wang Z, Zhou Y, Wang Y, et al. Characterization and quantification of mRNA transcripts in ejaculated spermatozoa of fertile men by serial analysis of gene expression. Hum Reprod2006;21:1583-1590
29. Kumar G, Patel D, Naz RK. C-Myc Messenger-RNA Is Present in Human Sperm Cells. Cell Mol Biol Res1993;39:111-7
30. Ostermeier GC, Dix DJ, Krawetz SA. A bioinformatic strategy to rapidly characterize cDNA libraries. Bioinformatics2002;18:949-52.
31. Balhorn R, Cosman M, Thornton KH, Krishnan VV, Corzett M, Bench G, et al. Protamine mediated condensation of DNA in mammalian sperm. In:Gagnon C, editor. The Male Gamete:From Basic Science to Clinical Applications. St. Louis, MO:Cache River Science;1999. p55-70
32. Dix DJ, Garges JB, Hong RL. Inhibition of hsp70-1and hsp70-3expression disrupts preimplantation embryogenesis and heightens embryo sensitivity to arsenic. Mol Reprod Dev1998,51:373-80.
33. Ostermeier, G.C. et al. Reproductive biology:delivering spermatozoan RNA to the oocyte. Nature2004,429,154
34. Ostermeier, G.C. et al. A suite of novel human spermatozoal RNAs. J. Androl.2005,26,70-74
35. Lee RC, Feinbaum RL, Ambros V. The C. elegans heterochronic gene lin-4encodes small RNAs with antisense complementarity to lin-14. Cell1993;75:843-54
36. Wightman B, Ha I, Ruvkun G. Posttranscriptional regulation of the heterochronic gene lin-14by lin-4mediates temporal pattern formation in C. elegans. Cell1993;75:855-62
37. Reinhart BJ, Slack FJ, Basson M, Pasquinelli AE, Bettinger JC, Rougvie AE, et al. The21-nucleotide let-7RNA regulates developmental timing in Caenorhabditis elegans. Nature2000,403:901-6.
38. Slack FJ, Basson M, Liu Z, Ambros V, Horvitz HR, Ruvkun G. The lin-41RBCC gene acts in the C. elegans heterochronic pathway between the let-7regulatory RNA and the LIN-29transcription factor. Mol Cell2000;5:659-69
39. Mao B, Niehrs C. Kremen2modulates Dickkopf2activity during Wnt/LRP6signaling. Gene2003;302:179-83
40. Davidson G, Mao B, del Barco Barrantes I, Niehrs C. Kremen proteins interact with Dickkopfl to regulate anteroposterior CNS patterning. Development2002;129:5587-96
41. Mao B, Wu W, Davidson G, Marhold J, Li M, Mechler BM, et al. Kremen proteins are Dickkopf receptors that regulate Wnt/beta-catenin signalling. Nature2002;417:664-7
42. Mao B, Wu W, Li Y, Hoppe D, Stannek P, Glinka A, et al. LDL-receptor-related protein6is a receptor for Dickkopf proteins. Nature2001;411:321-15
43. Mitsui K, Nakajima D, Ohara O, Nakayama M. Mammalian fat3:a large protein tha contains multiple cadherin and EGFlike motifs. Biochem Biophys Res Commun2002;290:1260-6
44. Watts JL, Phillips E, Griffing KR, Browse J. Deficiencies in C20polyunsaturated fatty acids cause behavioral and developmental defects in Caenorhabditis elegans fat-3mutants. Genetics2003;163:581-9
45. Morisato D, Anderson KV. Signaling pathways that establish the dorsal-ventral pattern of the Drosophila embryo. Annu Rev Genet1995;29:371-99
46. Wang Q, Latham KE. Translation of maternal messenger ribonucleic acids encoding transcription factors during genome activation in early mouse embryos. Biol Reprod2000; 62:969-78
47. Tong ZB, Gold L, Pfeifer KE, Dorward H, Lee E, Bondy CA, et al. Mater, a maternal effect gene required for early embryonic development in mice. Nature Genet2000;26:267-8
48. Gardner RL. Can developmentally significant spatial patterning of the egg be discounted in mammals? Hum Reprod Update1996;2:3-27
49. David Miller and G.Charles Ostermeier Towards a better understanding of RNA carriage by ejaculate spermatozoa Human Reproduction Update, Vol.12, No.6pp.757-767,2006
50. Barone, J.G. et al. DNA organization in human spermatozoa. J. Androl.1994,15,139-144
51. Kramer, J.A. and Krawetz, S.A. Nuclear matrix interactions within the sperm genome.J. Biol. Chem.1996,271,11619-11622
52. Jackson, D.A. Chromatin domains and nuclear compartments:establishing sites of gene expression in eukaryotic nuclei. Mol. Biol. Rep.1997,24,209-220
53. Pederson, T. Half a century of 'the nuclear matrix'. Mol. Biol. Cell11,2000,799-805
54. Ward,W.S.and Coffey,D.S. DNA packaging and organization in mammalian spermatozoa: comparison with somatic cells. Biol. Reprod.1991,44,569-574
55. Pombo, A. et al. Specialized transcription factories within mammalian nuclei. Crit. Rev. Eukaryot. Gene Expr.2000,10,21-29
56. Kramer, J.A. et al. Human spermatogenesis as a model to examine gene potentiation. Mol. Reprod.2000, Dev.56(Suppl.),254-258
57. Ward, W.S. et al. An intact sperm nuclear matrix might be necessary for the mouse paternal genome to participate in embryonic development. Biol. Reprod.1999,60, 702-706
58. Cho, C. et al. Protamine-2deficiency leads to sperm DNA damage and embryo death in mice. Biol. Reprod.2003,69,211-217
59. Escalier, D. Failure of differentiation of the nuclear-perinuclear skeletal complex in the round-headed human spermatozoa, hit. J. Dev. Biol.1990,34,287-297
60. Schatten, G. et al. Nuclear lamins and peripheral nuclear antigens during fertilization and embryogenesis in mice and sea urchins. Proc. Natl. Acad. Sci. U. S. A.1985,82,4727-4731.
61. Kono, T. et al. Birth of parthenogenetic mice that can develop to adulthood. Nature2004,428,860-864
62. Trasler, J.M. Origin and roles of genomic methylation patterns in male germ cells. Semin. Cell Dev. Biol.1998,9,467-474
63. Kierszenbaum, A.L. Genomic imprinting and epigenetic reprogramming:unearthing the garden of forking paths. Mol. Reprod. Dev.2002,63,269-272
64. Kerjean, A. et al. Establishment of the paternal methylation imprint of the human H19and MEST/PEG1genes during spermatogenesis. Hum. Mol. Genet.2000,9,2183-2187
65. Wykes, S.M. and Krawetz, S.A. The structural organization of sperm chromatin. J. Biol. Chem.2003,278,29471-29477
66. Rassoulzadegan M, Grandjean V, Gounon P, et al. RNA-mediated non-mendelian inheritance of an epigenetic change in the mouse. Nature2006;441(7092):469-474
67.王玲基于知识发现的生物信息学.生物工程进展,2000,20(3)：27-29
68. Lamar EE, Palmer E. Y-encoded, spcies-specific DNA in mice:evidence that the chromo some exists in two polymorphic forms in inbred strains [J]. Cell,1984,37:171-177
69. Liang P et al. Differential display of eukaryotic messenger RNA by means of the polymerase chain reaction [J]. Science,1992Aug14;257(5072):967-71
70. Lisitsyn N, Wigler M. Cloning the differences between two complex genomes [J]. Sience,1993,259:946-951
71. Liang P et al. Distribution and cloning of eukaryotic mRNAs by means of differential display:refinements and optimization [J]. Nucleic Acids Res,1993Jul11;21(14):3269-75
72. Velculescu VE, Zhang L, Vogelstein B&Kinzler KW (1995) Serial analysis of gene expression. Science270,484-487.
73. Velculescu V E et al. Tantalizing transcriptomes2SA GE and its use in global gene expression analysis [J]. Science,1999,286:1491-1492
74.何志巍,姚开泰DNA微阵列(或芯片)技术原理及应用.生物化学与生物物理进展,1999,26：507
75. Schena M, Shalon D, Davis RW et al. Quantitative monitoring of gene expression patterns with a complementary DNA microarry. Science,1995,270:467-470
76. Etienne W, Meyer MH et al. Comparison of mRNA gene expression by RT-PCR and DNA microarray [J]. Biotechniques.2004, Apr;36(4):618-20,622,624-6
77.陈杰大规模平行测序技术(MPSS)研究进展.生物化学与生物物理进展2004;31(8)：761-765
78. Schena M, Shalon D, Davis RW et al. Quantitative monitoring of gene expression patterns with a complementary DNA microarry. Science,1995,270:467-470
79. Hubank M, Chatz DG. Identifying differences in mRNA expression by representation ddifference analysis of cDNA. Nucleic Acids Res.1994,22(25):5640-5648
80. Adams MD, Kelley JM, Gocayne JD et al. Complementary DNA sequencing:expressed sequence tags and the human genome project. Science,1991,252(5013):1651-1656
81. Velculescu VE, Zhang L, Zhou W, et al. Characterization of the yeast transcriptome. Cell.1997,88(2):243-251
82. Yamamoto M, Wakatsuki T, Hada A, et al. Use of serial analysis of gene expression (SAGE) technology. J Immunol Methods,2001,250(1-2):45-66
83. Huibc, Bareravan schaik, Merlijnvander Mee et al. The human transcriptome map:clustering of highly expressed genesin chromosomal domains. Science,2001,291(5507):1289-1292
84. Welles, Bhattk, Thorntonc A. High abundance mRNA in human muscle:comparison between young and old. Journal of applied physiology,2000,89(1):297-304
85. Welles, Brooks A, Thornton C A. Senescence related changes in gene expression in muscle:similarities and differences between mice and man. Physiology Genomics,2001,5(2)67-73
86. Inadera H, Hashimoto S, Dongl H Y et al. WISP2as a novel estrogen responsive gene in human breast cancer cells. Biochemical and Biophysical Research Communcation,2000,275(1):108-114
87. Xu L L, Shanmugam N, Sesterhenn I A et al. A novel androgen regulated gene, PMEPAI, located on chromosome20113exhibit high level expression in protstate. Genomics,2000,66(3):257-263
88.吴志革,邹方东强大的广谱基因表达分析技术——基因表达系列分析法四川动物2006年第25卷第3期
89.谢超,刘荷中,裴雪涛.基因表达连续分析技术及应用研究的进展[J].国外医学-分子生物学分册,2002,24(6)：369-373
90. Chen JJ, Janet DR, Wang SM. Generation of longer cDNA fragments from serial analysis of gene expression tags for gene identification. Proc Natl Acad Sci USA,2000,97(1):349-353
91. Anke van den Berg, Judith van der Lei, Sibrang P. Serial analysis of gene expression: rapid RT-PCR analysis of unknown SAGE tags. Nucleic Acids Research.1999,27(17):17
92. Wang SM, Janet DR. A strategy for genome-wide gene analysis:Intrgrated procedure for gene identification. Proc Natl Acad Sci USA,1998,95(20):11900-11910
93. Frohman, M.K. Dush and G.R. Martin, Rapid production of full-length cDNA from rare transcripts:amplification using a single gene-specific oligonucleotide primer, Proc. Natl. Acad. Sci. USA.85(1988), pp.8998-9002
94. Ohara O, Dorit R, Gilbert W. One-sided polymerase chain reaction:the amplification of cDNA[J]. Proc Natl Acad Sci USA,1989,86:5673-5677
95. Borson, ND, Salo, W L, and Drewes, L R A lock-docking oligo(dT) primer for5'and3' RACE PCR. PCR Methods Appl.1992,2,144-148
96. Troutt AB, Mcheyzer-Williams MG, Pulendran B, et al. Ligation-anchored PCR:a simple amplification technique with singled-sided specificity [J]. Proc Nail Acad Sci USA,1992,89:9823-9825.
97. Bertling WM, Beier F, and Reichenberger E. Determination of5'Ends of Specific mRNAs by DNA Ligase-dependent Amplification. PCR Method App1,1993,3:95-99
98. Huang X, Yuan Z, Chen G, et al. Cloning and characterization of a novel ITIM containing Iectin2like immunoreceptor LL IR and its two transmembrane region deletion variants [J]. Biochem Biophys Res Commun,2001,281:131-140
99. Van den Berg A, van der Leij J&Poppema S. Serial analysis of gene expression:rapid RT-PCR analysis of unknown SAGE tags. Nucleic Acids Res,1999,27, e17.
100. Chen JJ, Rowley JD&Wang SM. Generation of longer cDNA fragments from serial analysis of gene expression tags for gene identification. Proc Natl Acad Sci USA,2000,97,349-353
101. Richards M, Tan SP, Chan WK&Bongso A. Reverse serial analysis of gene expression (SAGE)characterization of orphan SAGE tags from human embryonic stem cells identifies the presence of novel transcripts and antisense transcription of key pluripotency genes. Stem Cells,2006.24,1162-1173
102. Xu WJ, Li QL, Yao CJ, Wang ZX, Zhao YX&Qiao ZD. Semi-nested PCR analysis of unknown tags on serial analysis of gene expression. FEBS J.2008.275,5422-5428.
103. Wahl MB, Heinzmann U&Imai K (2005) LongSAGE analysis significantly improves genome annotation:identifications of novel genes and alternative transcripts in the mouse. Bioinformatics21,1393-1400
104. Yamashita T, Hashimoto S, Kaneko S, Nagai S,Toyoda N, Suzuki T, Kobayashi K&Matsushima K(2000) Comprehensive gene expression profile of a normal human liver. Biochem Biophys Res Commun269,110-116.
105. Matsumura H, Reich S, Ito A, Saitoh H, Kamoun S,Winter P, Kahl G, Reuter M, Kruger DH&Terauchi R(2003) Gene expression analysis of plant host-pathogen interactions by SuperSAGE. Proc Natl Acad Sci USA100,15718-15723.
106. Boon WM, Beissbarth T, Hyde L, Smyth G, Gunnersen J, Denton DA, Scott H&Tan SS (2004) A comparative analysis of transcribed genes in the mouse hypothalamus and neocortex reveals chromosomal clustering.Proc Natl Acad Sci USA101,14942-14977.
107. Halaschek-Wiener J, Khattra JS, McKay S, Pouzyrev A, Stott JM, Yang GS, Holt RA, Jones SJ, Marra MA,Brooks-Wilson AR et al.(2005) Analysis of long-lived C. elegans daf-2mutants using serial analysis of gene expression. Genome Res15,603-615.
108. Ryu EJ, Angelastro JM&Greene LA (2005) Analysis of gene expression changes in a cellular model of Parkinson disease. Neurobiol Dis18,54-75.
109. Li QL, Zhao YX, Ni B, Yao CJ, Zhou Y, Xu WJ, Wang ZX&Qiao ZD (2008) Comparison of the expression profiles of promastigotes and axenic amastigotes in Leishmania donovani using serial analysis of gene expression. Parasitol Res103,821-828.
110. Matsumura H, Reuter M, Kru" ger DH, Winter P, KahlG&Terauchi R (2008) SuperSAGE. Methods Mol Biol387,55-70.
111. Wei CL, Ng P, Chiu KP, Wong CH, Ang CC, Lipovich L, Liu ET&Ruan Y (2004)5' Long serial analysis of gene expression (LongSAGE) and3'LongSAGE for transcriptome characterization and genome annotation. Proc Natl Acad Sci USA101,11701-11706.
112. Peters DG, Kassam AB, Yonas H, O'Hare EH, Ferrell RE&Brufsky AM (1999) Comprehensive transcript analysis in small quantities of mRNA by SAGE-lite. Nucleic Acids Res27, e39
113. Saha S, Sparks AB, Rago C, Akmaev V, Wang CJ, Vogelstein B, Kinzler KW&Velculescu VE (2002) Using the transcriptome to annotate the genome. Nat Biotechnol20,508-512.
114. Gowda M, Jantasuriyarat C, Dean RA&Wang GL (2004) Robust-Long SAGE (RL-SAGE):a substantially improved LongSAGE method for gene discovery and transcriptome analysis. Plant Physiol134,890-897.
115. Heidenblut AM, Lu" ttges J, Buchholz M, Heinitz C, Emmersen J, Nielsen KL, Schreiter P, Souquet M, Nowacki S, Herbrand U et al.(2004) a RNA-long-SAGE:a new approach to generate SAGE libraries from micro dissected cells. Nucleic Acids Res32, e131
116. Kodzius R, Kojima M, Nishiyori H, Nakamura M, Fukuda S, Tagami M, Sasaki D, Imamura K, Kai C, Harbers M et al.(2006) CAGE:cap analysis of gene expression. Nat Methods3,211-222.
117. Neilson L, Andalibi A, Kang D, Coutifaris C, Strauss JF Ⅲ, Stanton JA&Green DP (2000) Molecular phenotype of the human oocyte by PCR-SAGE. Genomics63,13-24.
118. Zhang L, Zhou W, Velculescu VE, Kern SE, Hruban RH, Hamilton SR, Vogelstein B&Kinzler KW (1997)Gene expression profiles in normal and cancer cells. Science276,1268-1272.
119. Lee S, Chen J, Zhou G&Wang SM (2001) Generation of high quality and quantity of tag/ditag for SAGE analysis. BioTechniques31,348-354.
120. Wahl M, Shukunami C, Heinzmann U, Hamajima K, Hiraki Y&Imai K (2004) Transcriptome analysis of early chondrogenesis in ATDC5cells induced by bone morphogenetic protein4. Genomics83,45-58.
121. Siddiqui AS, Khattra J, Delaney AD, Zhao Y, Astell C,Asano J, Babakaiff R, Barber S, Beland J, Bohacec S et al.(2005) Large-scale digital gene-expression profiles from precisely defined developing C57BL/6J mouse tissues and cells. Proc Natl Acad Sci USA102,18485-18490.
122. Chen J, Lee S, Zhou G&Wang SM (2002) Highthroughput GLGI procedure for converting a large number of serial analysis of gene expression tag sequences into3' complementary DNAs. Genes Chromosomes Cancer33,252-261.
123. Miller D, Ostermeier GC and Krawetz SA. The controversy, potential and roles of spermatozoal RNA. Trends Mol. Med.2005,11,156-63.
124. Li HG, Liao AH, Ding XF, et al. The expression and significance of CATSPER1in human testis and ejaculated spermatozoa [J]. Asian J Androl,2006,8(3):301-306.
125. Li HG, Ding XF, Liao AH, et al. Expression of CatSper family t ranscripts in the mouse testis during post2natal development and human ejaculated spermatozoa: relationship to Sperm motility [J]. Mol Hum Reprod,2007,13(5):299-306.
126Hargreaves CA, Rogers S, Hills F, Howell RJ and Homa ST. Effects of co-trimoxazole, erythromycin, amoxycillin, tetracycline and chloroquine on sperm function in vitro.Hum. Reprod.1998,13,1878-86.
127Hargreaves TB. Genetics and male infertility. Curr Opin Obstet Gynecol.2000,12(3):207-219.
128Kimura, M.(1968)."Evolutionary rate at the molecular level". Nature217:624-626.
129Kimura, M.(1983). The Neutral Theory of Molecular Evolution. Cambridge University Press, Cambridge. ISBN0-521-23109-4.
130Themmen APN, Huhtaniemi IT. Mutations of gonadotropins and gonadotropin receptors:elucidating the physiology and pathophysiology of pituitary-gonadal function [J]. Endocr Rev,2000,21(5):551-583.
131Layman LC. Mutations in the follicle-stimulating hormone-beta (FSH beta) and FSH receptor genes in mice and humans [J]. Semin Reprod Med,2000,18(1):5-10.
132Ramaswamy S, Plant TM. Operation of the follicle-stimulating hormone (FSH)-inhibin B feedback loop in the control of primate spermatogenesis [J]. Mol Cell Endocrinol,2001,180(1-2):93-101.
133Kinniburgh D, Anderson RA, Baird DT. Suppression of spermatogenesis with desogestrel and testosterone pellets is not enhanced by addition of finasteride [J]. J Androl,2001,22(1):88-95.
134Plant TM, Marshall GR. The functional significance of FSH in spermatogenesis and the control of its secretion in male primates [J]. Endocr Rev,2001,22(6):764-786.
135O'Donnell L, Narula A, Balourdos G, et al. Impairment of spermatogonial development and spermiation after testosterone-induced gonadotropin suppression in adult monkeys (Macaca fascicularis)[J]. J Clin Endocrinol Metab,2001,86(4):1814-1822.
136葛秦生主编.临床生殖内分泌学——男性与女性[M].北京：科学技术文献出版社,2001.762-800.
137Wang C, Swerdloff RS. Male contraception [J]. Best Pract Res Clin Obstet Gynaecol,2002,16(2):193-203.
138Seshagiri PB, Terasawa E, Hearn JP. The secretion of gonadotrophin-releasing hormone by peri-implantation embryos of the rhesus monkey:comparison with the secretion of chorionic gonadotrophin. Hum Reprod,1994,9(7):1300-7.
139Casan EM, Raga F, Polan ML. GnRH mRNA and protein expression in human preimplantation embryos. Mol Hum Reprod,1999,5(3):234-9.
140Francisco R, Eva MC, Jan K, et al. The role of gonadotropin releasing hormone in murine preimplantation embryonic development. Endocrinology,1999,140(8):3602-8.
141Patsoula E, Loutradis D, Drakakis P, et al. Expression of mRNA for the LH and FSH receptors in mouse oocytes and preimplantation embryos. Reproduction,2001,121(3):455-61.
142Hou Q, Paria BC, Mui C, et al. Immunolocalization of estrogen receptor protein in the mouse blastocyst during normal and delayed implantation. PNAS,1996,93(6):2376-81.
143Pantaleon M, Whiteside EJ, Harvey MB, et al. Functional growth hormone (GH) receptors and GH are expressed by preimplantation mouse embryos:a role for GH in early embryogenesis? ProcNatl Acad Sci USA,1997,94(10):5125-30.
144Welting, T.J., Kikkert, B.J., van Venrooij, W.J. and Pruijn, G.J. Differential association of protein subunits with the human RNase MRP and RNase P complexes RNA12(7),1373-1382(2006)
145Sharin,E., Schein,A., Mann,H., Ben-Asouli,Y. and Jarrous,N. RNase P:role of distinct protein cofactors in tRNA substrate recognition and RNA-based catalysis Nucleic Acids Res.33(16),5120-5132(2005)
146van Eenennaam,H., Pruijn,G.J. and van Venrooij,W.J. hPop4:a new protein subunit of the human RNase MRP and RNase P ribonucleoprotein complexes Nucleic Acids Res.27(12),2465-2472(1999)
147Welting,T.J., van Venrooij,W.J. and Pruijn,G.J.Mutual interactions between subunits of the human RNase MRP ribonucleoprotein complex Nucleic Acids Res.32(7),2138-2146(2004)
148Jarrous N, Reiner R (2007), Human RNase P:a tRNA-processing enzyme and transcription factor, Nucleic Acids Res.35(11):3519-24, PMID17483522, DOI:10.1093/nar/gkm071
149Reiner R, Ben-Asouli Y, Krilovetzky I, Jarrous N (2006), A role for the catalytic ribonucleoprotein RNase P in RNA polymerase Ⅲ transcription, Genes Dev.20(12):1621-35, PMID16778078, DOI:10.1101/gad.386706
150Monika Martick, Lucas H. Horan, Harry F. Noller and William G. Scott, A Discontinuous Hammerhead Ribozyme Embedded in a Mammalian mRNA. Nature454:899-902(2008).
151赵春丽,焦丽红,李雪梅,陈新,郝艳红,孙悍军,王柳小鼠母源因子对早期胚胎发育的影响遗传28(5)：601-605,2006
2. Salisbury GW, Hart RG, Lodge, JR. The spermatozoan genome and fertility. American Journal of Obstetrics and Gynecology.1997,128(93):342-350
3. Clermont Y. Quantitative analysis of spermatogenesis of the rat:A revised model for the renewal of spermatogonia. AmJ Anat.1962,111:111-129
4. Clermont Y. The cycle of the seminiferous epithelium in man. AmJ Anat1963,112:35-51
5. Forti G, Vannelli GB, Barni T, Orlando C, Balboni GC, Serio M. Androgen-binding protein and other Sertoli cell proteins in human testis. Annals of the New York Academy of Sciences Volume1988,538:167-172
6. Vogl AW. Distribution and function of organized concentrations of actin filaments in mammalian spermatogenic cells and Sertoli cells. International Review of Cytology1989,119:1-56
7. Fraser L R, Dudley K. New insights into the t-complex and control of sperm function. Bioessays,1999,21(4):304-312
8. Aranha I P, Martin DeLeon P A. Mouse chromosome6in Rb translocations: consequences in singly and doubly heterozygous males. Cytogenet Cell Genet,1995,69(34):253-259
9. Conway S J, Mahadevaiah S K, Darling S M, et al. Y353/B:a candidate multiple copy spermiogenesis gene on the mouse Y chromosome. Mamm Genome,1994,5(4):203-210
10. Kimmins S, Kotaja N, Davidson I, Sassone-Corsi P. Testis-specific transcription mechanisms promoting male germ-cell differentiation. Reproduction.2004,128(1):5-12
11. Ellis PJI, Furlong RA, Wilson A, et al. Modulation of the mouse testis transcriptome during postnatal development and in selected models of male infertility. Molecular Human Reproduction2004,10(4):271-281
12. Prasanth SG, Giran HM, Ali S. Biology of protooncogene c-kit receptor and spermatogenesis. Current Pharma cogenomics2004,2(1):47-60
13. Miller, D. et al. Differential RNA fingerprinting as a tool in the analysis of spermatozoal gene expression. Hum. Reprod.1994,9,864-869
14. Lambard, S. et al. Analysis and significance of mRNA in human ejaculated sperm from normozoospermic donors:relationship to sperm motility and capacitation. Mol. Hum. Reprod.2004,10,535-541
15. Ostermeier, G.C. et al. Spermatozoal RNA profiles of normal fertile men. Lancet2002,360,772-777
16. Hecht, N.B. Molecular mechanisms of male germ cell differentiation. Bioessays1998,20:555-561
17. Pessot, C.A. et al. Presence of RNA in the sperm nucleus. Biochem. Biophys. Res. Commun.1989,158,272-278
18. Kumar, G. et al. c-MYC mRNA is present in human sperm cells. Cell. Mol. Biol. Res.1993,39,111-117
19. Wykes, S.M. et al. Haploid transcripts persist in mature human spermatozoa. Mol. Hum. Reprod.1997,3,15-19
20. Chiang, M.H. et al. Detection of human leukocyte antigen class I messenger ribonucleic acid transcripts in human spermatozoa via reverse transcription-polymerase chain reaction. Fertil. Steril.1994,61,276-280
21. Goodwin, L.O. et al. L-type voltage-dependent calcium channel a-1C subunit mRNA is present in ejaculated human spermatozoa. Mol. Hum. Reprod.2000,6,127-136
22. Goodwin, L.O. et al. Presence of N-cadherin transcripts in mature spermatozoa. Mol. Hum. Reprod.2000,6,487-497
23. Durkee, T.J. et al. Identification of estrogen receptor protein and messenger ribonucleic acid in human spermatozoa. Am. J. Obstet. Gynecol.1998,178,1288-1295
24. Richter, W. et al. Detection of mRNA transcripts of cyclic nucleotide phosphodiesterase subtypes in ejaculated human spermatozoa. Mol. Hum. Reprod.1999,5,732-736
25. Rohwedder, A. et al. Detection of mRNA transcripts of b1integrins in ejaculated human spermatozoa by nested reverse transcription-polymerase chain reaction. Mol. Hum. Reprod.1996,2,499-505
26. Lambard, S. et al. Human immature germ cells and ejaculated spermatozoa contain aromatase and oestrogen receptors. J. Mol. Endocrinol.2004,32,279-289
27. Miller, D. et al. A complex population of RNAs exists in human ejaculate spermatozoa: implications for understanding molecular aspects of spermiogenesis. Gene1999,237,385-392
28. Zhao Y, Li Q, Yao C, Wang Z, Zhou Y, Wang Y, et al. Characterization and quantification of mRNA transcripts in ejaculated spermatozoa of fertile men by serial analysis of gene expression. Hum Reprod2006;21:1583-1590
29. Kumar G, Patel D, Naz RK. C-Myc Messenger-RNA Is Present in Human Sperm Cells. Cell Mol Biol Res1993;39:111-7
30. Ostermeier GC, Dix DJ, Krawetz SA. A bioinformatic strategy to rapidly characterize cDNA libraries. Bioinformatics2002;18:949-52.
31. Balhorn R, Cosman M, Thornton KH, Krishnan VV, Corzett M, Bench G, et al. Protamine mediated condensation of DNA in mammalian sperm. In:Gagnon C, editor. The Male Gamete:From Basic Science to Clinical Applications. St. Louis, MO:Cache River Science;1999. p55-70
32. Dix DJ, Garges JB, Hong RL. Inhibition of hsp70-1and hsp70-3expression disrupts preimplantation embryogenesis and heightens embryo sensitivity to arsenic. Mol Reprod Dev1998,51:373-80.
33. Ostermeier, G.C. et al. Reproductive biology:delivering spermatozoan RNA to the oocyte. Nature2004,429,154
34. Ostermeier, G.C. et al. A suite of novel human spermatozoal RNAs. J. Androl.2005,26,70-74
35. Lee RC, Feinbaum RL, Ambros V. The C. elegans heterochronic gene lin-4encodes small RNAs with antisense complementarity to lin-14. Cell1993;75:843-54
36. Wightman B, Ha I, Ruvkun G. Posttranscriptional regulation of the heterochronic gene lin-14by lin-4mediates temporal pattern formation in C. elegans. Cell1993;75:855-62
37. Reinhart BJ, Slack FJ, Basson M, Pasquinelli AE, Bettinger JC, Rougvie AE, et al. The21-nucleotide let-7RNA regulates developmental timing in Caenorhabditis elegans. Nature2000,403:901-6.
38. Slack FJ, Basson M, Liu Z, Ambros V, Horvitz HR, Ruvkun G. The lin-41RBCC gene acts in the C. elegans heterochronic pathway between the let-7regulatory RNA and the LIN-29transcription factor. Mol Cell2000;5:659-69
39. Mao B, Niehrs C. Kremen2modulates Dickkopf2activity during Wnt/LRP6signaling. Gene2003;302:179-83
40. Davidson G, Mao B, del Barco Barrantes I, Niehrs C. Kremen proteins interact with Dickkopfl to regulate anteroposterior CNS patterning. Development2002;129:5587-96
41. Mao B, Wu W, Davidson G, Marhold J, Li M, Mechler BM, et al. Kremen proteins are Dickkopf receptors that regulate Wnt/beta-catenin signalling. Nature2002;417:664-7
42. Mao B, Wu W, Li Y, Hoppe D, Stannek P, Glinka A, et al. LDL-receptor-related protein6is a receptor for Dickkopf proteins. Nature2001;411:321-15
43. Mitsui K, Nakajima D, Ohara O, Nakayama M. Mammalian fat3:a large protein tha contains multiple cadherin and EGFlike motifs. Biochem Biophys Res Commun2002;290:1260-6
44. Watts JL, Phillips E, Griffing KR, Browse J. Deficiencies in C20polyunsaturated fatty acids cause behavioral and developmental defects in Caenorhabditis elegans fat-3mutants. Genetics2003;163:581-9
45. Morisato D, Anderson KV. Signaling pathways that establish the dorsal-ventral pattern of the Drosophila embryo. Annu Rev Genet1995;29:371-99
46. Wang Q, Latham KE. Translation of maternal messenger ribonucleic acids encoding transcription factors during genome activation in early mouse embryos. Biol Reprod2000; 62:969-78
47. Tong ZB, Gold L, Pfeifer KE, Dorward H, Lee E, Bondy CA, et al. Mater, a maternal effect gene required for early embryonic development in mice. Nature Genet2000;26:267-8
48. Gardner RL. Can developmentally significant spatial patterning of the egg be discounted in mammals? Hum Reprod Update1996;2:3-27
49. David Miller and G.Charles Ostermeier Towards a better understanding of RNA carriage by ejaculate spermatozoa Human Reproduction Update, Vol.12, No.6pp.757-767,2006
50. Barone, J.G. et al. DNA organization in human spermatozoa. J. Androl.1994,15,139-144
51. Kramer, J.A. and Krawetz, S.A. Nuclear matrix interactions within the sperm genome.J. Biol. Chem.1996,271,11619-11622
52. Jackson, D.A. Chromatin domains and nuclear compartments:establishing sites of gene expression in eukaryotic nuclei. Mol. Biol. Rep.1997,24,209-220
53. Pederson, T. Half a century of 'the nuclear matrix'. Mol. Biol. Cell11,2000,799-805
54. Ward,W.S.and Coffey,D.S. DNA packaging and organization in mammalian spermatozoa: comparison with somatic cells. Biol. Reprod.1991,44,569-574
55. Pombo, A. et al. Specialized transcription factories within mammalian nuclei. Crit. Rev. Eukaryot. Gene Expr.2000,10,21-29
56. Kramer, J.A. et al. Human spermatogenesis as a model to examine gene potentiation. Mol. Reprod.2000, Dev.56(Suppl.),254-258
57. Ward, W.S. et al. An intact sperm nuclear matrix might be necessary for the mouse paternal genome to participate in embryonic development. Biol. Reprod.1999,60, 702-706
58. Cho, C. et al. Protamine-2deficiency leads to sperm DNA damage and embryo death in mice. Biol. Reprod.2003,69,211-217
59. Escalier, D. Failure of differentiation of the nuclear-perinuclear skeletal complex in the round-headed human spermatozoa, hit. J. Dev. Biol.1990,34,287-297
60. Schatten, G. et al. Nuclear lamins and peripheral nuclear antigens during fertilization and embryogenesis in mice and sea urchins. Proc. Natl. Acad. Sci. U. S. A.1985,82,4727-4731.
61. Kono, T. et al. Birth of parthenogenetic mice that can develop to adulthood. Nature2004,428,860-864
62. Trasler, J.M. Origin and roles of genomic methylation patterns in male germ cells. Semin. Cell Dev. Biol.1998,9,467-474
63. Kierszenbaum, A.L. Genomic imprinting and epigenetic reprogramming:unearthing the garden of forking paths. Mol. Reprod. Dev.2002,63,269-272
64. Kerjean, A. et al. Establishment of the paternal methylation imprint of the human H19and MEST/PEG1genes during spermatogenesis. Hum. Mol. Genet.2000,9,2183-2187
65. Wykes, S.M. and Krawetz, S.A. The structural organization of sperm chromatin. J. Biol. Chem.2003,278,29471-29477
66. Rassoulzadegan M, Grandjean V, Gounon P, et al. RNA-mediated non-mendelian inheritance of an epigenetic change in the mouse. Nature2006;441(7092):469-474
67.王玲基于知识发现的生物信息学.生物工程进展,2000,20(3)：27-29
68. Lamar EE, Palmer E. Y-encoded, spcies-specific DNA in mice:evidence that the chromo some exists in two polymorphic forms in inbred strains [J]. Cell,1984,37:171-177
69. Liang P et al. Differential display of eukaryotic messenger RNA by means of the polymerase chain reaction [J]. Science,1992Aug14;257(5072):967-71
70. Lisitsyn N, Wigler M. Cloning the differences between two complex genomes [J]. Sience,1993,259:946-951
71. Liang P et al. Distribution and cloning of eukaryotic mRNAs by means of differential display:refinements and optimization [J]. Nucleic Acids Res,1993Jul11;21(14):3269-75
72. Velculescu VE, Zhang L, Vogelstein B&Kinzler KW (1995) Serial analysis of gene expression. Science270,484-487.
73. Velculescu V E et al. Tantalizing transcriptomes2SA GE and its use in global gene expression analysis [J]. Science,1999,286:1491-1492
74.何志巍,姚开泰DNA微阵列(或芯片)技术原理及应用.生物化学与生物物理进展,1999,26：507
75. Schena M, Shalon D, Davis RW et al. Quantitative monitoring of gene expression patterns with a complementary DNA microarry. Science,1995,270:467-470
76. Etienne W, Meyer MH et al. Comparison of mRNA gene expression by RT-PCR and DNA microarray [J]. Biotechniques.2004, Apr;36(4):618-20,622,624-6
77.陈杰大规模平行测序技术(MPSS)研究进展.生物化学与生物物理进展2004;31(8)：761-765
78. Schena M, Shalon D, Davis RW et al. Quantitative monitoring of gene expression patterns with a complementary DNA microarry. Science,1995,270:467-470
79. Hubank M, Chatz DG. Identifying differences in mRNA expression by representation ddifference analysis of cDNA. Nucleic Acids Res.1994,22(25):5640-5648
80. Adams MD, Kelley JM, Gocayne JD et al. Complementary DNA sequencing:expressed sequence tags and the human genome project. Science,1991,252(5013):1651-1656
81. Velculescu VE, Zhang L, Zhou W, et al. Characterization of the yeast transcriptome. Cell.1997,88(2):243-251
82. Yamamoto M, Wakatsuki T, Hada A, et al. Use of serial analysis of gene expression (SAGE) technology. J Immunol Methods,2001,250(1-2):45-66
83. Huibc, Bareravan schaik, Merlijnvander Mee et al. The human transcriptome map:clustering of highly expressed genesin chromosomal domains. Science,2001,291(5507):1289-1292
84. Welles, Bhattk, Thorntonc A. High abundance mRNA in human muscle:comparison between young and old. Journal of applied physiology,2000,89(1):297-304
85. Welles, Brooks A, Thornton C A. Senescence related changes in gene expression in muscle:similarities and differences between mice and man. Physiology Genomics,2001,5(2)67-73
86. Inadera H, Hashimoto S, Dongl H Y et al. WISP2as a novel estrogen responsive gene in human breast cancer cells. Biochemical and Biophysical Research Communcation,2000,275(1):108-114
87. Xu L L, Shanmugam N, Sesterhenn I A et al. A novel androgen regulated gene, PMEPAI, located on chromosome20113exhibit high level expression in protstate. Genomics,2000,66(3):257-263
88.吴志革,邹方东强大的广谱基因表达分析技术——基因表达系列分析法四川动物2006年第25卷第3期
89.谢超,刘荷中,裴雪涛.基因表达连续分析技术及应用研究的进展[J].国外医学-分子生物学分册,2002,24(6)：369-373
90. Chen JJ, Janet DR, Wang SM. Generation of longer cDNA fragments from serial analysis of gene expression tags for gene identification. Proc Natl Acad Sci USA,2000,97(1):349-353
91. Anke van den Berg, Judith van der Lei, Sibrang P. Serial analysis of gene expression: rapid RT-PCR analysis of unknown SAGE tags. Nucleic Acids Research.1999,27(17):17
92. Wang SM, Janet DR. A strategy for genome-wide gene analysis:Intrgrated procedure for gene identification. Proc Natl Acad Sci USA,1998,95(20):11900-11910
93. Frohman, M.K. Dush and G.R. Martin, Rapid production of full-length cDNA from rare transcripts:amplification using a single gene-specific oligonucleotide primer, Proc. Natl. Acad. Sci. USA.85(1988), pp.8998-9002
94. Ohara O, Dorit R, Gilbert W. One-sided polymerase chain reaction:the amplification of cDNA[J]. Proc Natl Acad Sci USA,1989,86:5673-5677
95. Borson, ND, Salo, W L, and Drewes, L R A lock-docking oligo(dT) primer for5'and3' RACE PCR. PCR Methods Appl.1992,2,144-148
96. Troutt AB, Mcheyzer-Williams MG, Pulendran B, et al. Ligation-anchored PCR:a simple amplification technique with singled-sided specificity [J]. Proc Nail Acad Sci USA,1992,89:9823-9825.
97. Bertling WM, Beier F, and Reichenberger E. Determination of5'Ends of Specific mRNAs by DNA Ligase-dependent Amplification. PCR Method App1,1993,3:95-99
98. Huang X, Yuan Z, Chen G, et al. Cloning and characterization of a novel ITIM containing Iectin2like immunoreceptor LL IR and its two transmembrane region deletion variants [J]. Biochem Biophys Res Commun,2001,281:131-140
99. Van den Berg A, van der Leij J&Poppema S. Serial analysis of gene expression:rapid RT-PCR analysis of unknown SAGE tags. Nucleic Acids Res,1999,27, e17.
100. Chen JJ, Rowley JD&Wang SM. Generation of longer cDNA fragments from serial analysis of gene expression tags for gene identification. Proc Natl Acad Sci USA,2000,97,349-353
101. Richards M, Tan SP, Chan WK&Bongso A. Reverse serial analysis of gene expression (SAGE)characterization of orphan SAGE tags from human embryonic stem cells identifies the presence of novel transcripts and antisense transcription of key pluripotency genes. Stem Cells,2006.24,1162-1173
102. Xu WJ, Li QL, Yao CJ, Wang ZX, Zhao YX&Qiao ZD. Semi-nested PCR analysis of unknown tags on serial analysis of gene expression. FEBS J.2008.275,5422-5428.
103. Wahl MB, Heinzmann U&Imai K (2005) LongSAGE analysis significantly improves genome annotation:identifications of novel genes and alternative transcripts in the mouse. Bioinformatics21,1393-1400
104. Yamashita T, Hashimoto S, Kaneko S, Nagai S,Toyoda N, Suzuki T, Kobayashi K&Matsushima K(2000) Comprehensive gene expression profile of a normal human liver. Biochem Biophys Res Commun269,110-116.
105. Matsumura H, Reich S, Ito A, Saitoh H, Kamoun S,Winter P, Kahl G, Reuter M, Kruger DH&Terauchi R(2003) Gene expression analysis of plant host-pathogen interactions by SuperSAGE. Proc Natl Acad Sci USA100,15718-15723.
106. Boon WM, Beissbarth T, Hyde L, Smyth G, Gunnersen J, Denton DA, Scott H&Tan SS (2004) A comparative analysis of transcribed genes in the mouse hypothalamus and neocortex reveals chromosomal clustering.Proc Natl Acad Sci USA101,14942-14977.
107. Halaschek-Wiener J, Khattra JS, McKay S, Pouzyrev A, Stott JM, Yang GS, Holt RA, Jones SJ, Marra MA,Brooks-Wilson AR et al.(2005) Analysis of long-lived C. elegans daf-2mutants using serial analysis of gene expression. Genome Res15,603-615.
108. Ryu EJ, Angelastro JM&Greene LA (2005) Analysis of gene expression changes in a cellular model of Parkinson disease. Neurobiol Dis18,54-75.
109. Li QL, Zhao YX, Ni B, Yao CJ, Zhou Y, Xu WJ, Wang ZX&Qiao ZD (2008) Comparison of the expression profiles of promastigotes and axenic amastigotes in Leishmania donovani using serial analysis of gene expression. Parasitol Res103,821-828.
110. Matsumura H, Reuter M, Kru" ger DH, Winter P, KahlG&Terauchi R (2008) SuperSAGE. Methods Mol Biol387,55-70.
111. Wei CL, Ng P, Chiu KP, Wong CH, Ang CC, Lipovich L, Liu ET&Ruan Y (2004)5' Long serial analysis of gene expression (LongSAGE) and3'LongSAGE for transcriptome characterization and genome annotation. Proc Natl Acad Sci USA101,11701-11706.
112. Peters DG, Kassam AB, Yonas H, O'Hare EH, Ferrell RE&Brufsky AM (1999) Comprehensive transcript analysis in small quantities of mRNA by SAGE-lite. Nucleic Acids Res27, e39
113. Saha S, Sparks AB, Rago C, Akmaev V, Wang CJ, Vogelstein B, Kinzler KW&Velculescu VE (2002) Using the transcriptome to annotate the genome. Nat Biotechnol20,508-512.
114. Gowda M, Jantasuriyarat C, Dean RA&Wang GL (2004) Robust-Long SAGE (RL-SAGE):a substantially improved LongSAGE method for gene discovery and transcriptome analysis. Plant Physiol134,890-897.
115. Heidenblut AM, Lu" ttges J, Buchholz M, Heinitz C, Emmersen J, Nielsen KL, Schreiter P, Souquet M, Nowacki S, Herbrand U et al.(2004) a RNA-long-SAGE:a new approach to generate SAGE libraries from micro dissected cells. Nucleic Acids Res32, e131
116. Kodzius R, Kojima M, Nishiyori H, Nakamura M, Fukuda S, Tagami M, Sasaki D, Imamura K, Kai C, Harbers M et al.(2006) CAGE:cap analysis of gene expression. Nat Methods3,211-222.
117. Neilson L, Andalibi A, Kang D, Coutifaris C, Strauss JF Ⅲ, Stanton JA&Green DP (2000) Molecular phenotype of the human oocyte by PCR-SAGE. Genomics63,13-24.
118. Zhang L, Zhou W, Velculescu VE, Kern SE, Hruban RH, Hamilton SR, Vogelstein B&Kinzler KW (1997)Gene expression profiles in normal and cancer cells. Science276,1268-1272.
119. Lee S, Chen J, Zhou G&Wang SM (2001) Generation of high quality and quantity of tag/ditag for SAGE analysis. BioTechniques31,348-354.
120. Wahl M, Shukunami C, Heinzmann U, Hamajima K, Hiraki Y&Imai K (2004) Transcriptome analysis of early chondrogenesis in ATDC5cells induced by bone morphogenetic protein4. Genomics83,45-58.
121. Siddiqui AS, Khattra J, Delaney AD, Zhao Y, Astell C,Asano J, Babakaiff R, Barber S, Beland J, Bohacec S et al.(2005) Large-scale digital gene-expression profiles from precisely defined developing C57BL/6J mouse tissues and cells. Proc Natl Acad Sci USA102,18485-18490.
122. Chen J, Lee S, Zhou G&Wang SM (2002) Highthroughput GLGI procedure for converting a large number of serial analysis of gene expression tag sequences into3' complementary DNAs. Genes Chromosomes Cancer33,252-261.
123. Miller D, Ostermeier GC and Krawetz SA. The controversy, potential and roles of spermatozoal RNA. Trends Mol. Med.2005,11,156-63.
124. Li HG, Liao AH, Ding XF, et al. The expression and significance of CATSPER1in human testis and ejaculated spermatozoa [J]. Asian J Androl,2006,8(3):301-306.
125. Li HG, Ding XF, Liao AH, et al. Expression of CatSper family t ranscripts in the mouse testis during post2natal development and human ejaculated spermatozoa: relationship to Sperm motility [J]. Mol Hum Reprod,2007,13(5):299-306.
126Hargreaves CA, Rogers S, Hills F, Howell RJ and Homa ST. Effects of co-trimoxazole, erythromycin, amoxycillin, tetracycline and chloroquine on sperm function in vitro.Hum. Reprod.1998,13,1878-86.
127Hargreaves TB. Genetics and male infertility. Curr Opin Obstet Gynecol.2000,12(3):207-219.
128Kimura, M.(1968)."Evolutionary rate at the molecular level". Nature217:624-626.
129Kimura, M.(1983). The Neutral Theory of Molecular Evolution. Cambridge University Press, Cambridge. ISBN0-521-23109-4.
130Themmen APN, Huhtaniemi IT. Mutations of gonadotropins and gonadotropin receptors:elucidating the physiology and pathophysiology of pituitary-gonadal function [J]. Endocr Rev,2000,21(5):551-583.
131Layman LC. Mutations in the follicle-stimulating hormone-beta (FSH beta) and FSH receptor genes in mice and humans [J]. Semin Reprod Med,2000,18(1):5-10.
132Ramaswamy S, Plant TM. Operation of the follicle-stimulating hormone (FSH)-inhibin B feedback loop in the control of primate spermatogenesis [J]. Mol Cell Endocrinol,2001,180(1-2):93-101.
133Kinniburgh D, Anderson RA, Baird DT. Suppression of spermatogenesis with desogestrel and testosterone pellets is not enhanced by addition of finasteride [J]. J Androl,2001,22(1):88-95.
134Plant TM, Marshall GR. The functional significance of FSH in spermatogenesis and the control of its secretion in male primates [J]. Endocr Rev,2001,22(6):764-786.
135O'Donnell L, Narula A, Balourdos G, et al. Impairment of spermatogonial development and spermiation after testosterone-induced gonadotropin suppression in adult monkeys (Macaca fascicularis)[J]. J Clin Endocrinol Metab,2001,86(4):1814-1822.
136葛秦生主编.临床生殖内分泌学——男性与女性[M].北京：科学技术文献出版社,2001.762-800.
137Wang C, Swerdloff RS. Male contraception [J]. Best Pract Res Clin Obstet Gynaecol,2002,16(2):193-203.
138Seshagiri PB, Terasawa E, Hearn JP. The secretion of gonadotrophin-releasing hormone by peri-implantation embryos of the rhesus monkey:comparison with the secretion of chorionic gonadotrophin. Hum Reprod,1994,9(7):1300-7.
139Casan EM, Raga F, Polan ML. GnRH mRNA and protein expression in human preimplantation embryos. Mol Hum Reprod,1999,5(3):234-9.
140Francisco R, Eva MC, Jan K, et al. The role of gonadotropin releasing hormone in murine preimplantation embryonic development. Endocrinology,1999,140(8):3602-8.
141Patsoula E, Loutradis D, Drakakis P, et al. Expression of mRNA for the LH and FSH receptors in mouse oocytes and preimplantation embryos. Reproduction,2001,121(3):455-61.
142Hou Q, Paria BC, Mui C, et al. Immunolocalization of estrogen receptor protein in the mouse blastocyst during normal and delayed implantation. PNAS,1996,93(6):2376-81.
143Pantaleon M, Whiteside EJ, Harvey MB, et al. Functional growth hormone (GH) receptors and GH are expressed by preimplantation mouse embryos:a role for GH in early embryogenesis? ProcNatl Acad Sci USA,1997,94(10):5125-30.
144Welting, T.J., Kikkert, B.J., van Venrooij, W.J. and Pruijn, G.J. Differential association of protein subunits with the human RNase MRP and RNase P complexes RNA12(7),1373-1382(2006)
145Sharin,E., Schein,A., Mann,H., Ben-Asouli,Y. and Jarrous,N. RNase P:role of distinct protein cofactors in tRNA substrate recognition and RNA-based catalysis Nucleic Acids Res.33(16),5120-5132(2005)
146van Eenennaam,H., Pruijn,G.J. and van Venrooij,W.J. hPop4:a new protein subunit of the human RNase MRP and RNase P ribonucleoprotein complexes Nucleic Acids Res.27(12),2465-2472(1999)
147Welting,T.J., van Venrooij,W.J. and Pruijn,G.J.Mutual interactions between subunits of the human RNase MRP ribonucleoprotein complex Nucleic Acids Res.32(7),2138-2146(2004)
148Jarrous N, Reiner R (2007), Human RNase P:a tRNA-processing enzyme and transcription factor, Nucleic Acids Res.35(11):3519-24, PMID17483522, DOI:10.1093/nar/gkm071
149Reiner R, Ben-Asouli Y, Krilovetzky I, Jarrous N (2006), A role for the catalytic ribonucleoprotein RNase P in RNA polymerase Ⅲ transcription, Genes Dev.20(12):1621-35, PMID16778078, DOI:10.1101/gad.386706
150Monika Martick, Lucas H. Horan, Harry F. Noller and William G. Scott, A Discontinuous Hammerhead Ribozyme Embedded in a Mammalian mRNA. Nature454:899-902(2008).
151赵春丽,焦丽红,李雪梅,陈新,郝艳红,孙悍军,王柳小鼠母源因子对早期胚胎发育的影响遗传28(5)：601-605,2006