人类生精相关基因TSARG4的cDNA克隆

为了探索精子生成的分子机制 ,从人精子外部致密纤维蛋白相关基因SPAG4(spermantigen 4)和小鼠精母细胞中表达的AK0 0 62 2 5基因出发 ,找到两个人类EST ,BG72 0 5 64和AI70 0 45 4,其中BG72 0 5 64在人睾丸中表达。运用“间隙填充法”填平这两个EST之间的间隙 ,从人睾丸文库中快速克隆了同源于SPAG4和AK0 0 62 2 5基因的人类TSARG4基因 (testisandspermatogenesisrelatedgene 4) (GenBank登录号为AF40 13 5 0 ) ,并用RT PCR对该基因阅读框进行验证。TSARG4基因全长 12 5 2bp ,开放阅读框为 94～ 12 3 3bp ,定位于 2 0q11.2 ,推定编码 3 79个氨基酸 ,预计分子量为 43 0 81.45 ,等电点为 8.61,该基因与小鼠精母细胞基因AK0 0 62 2 5编码的氨基酸序列同源性 74% ,与人类SPAG4基因编码的氨基酸序列同源性 45 %。RT PCR表明人类TSARG4基因在多个组织中均有表达 ,而同源的小鼠AK0 0 62 2 5基因仅在睾丸中表达     

Expression of a novel HsMCAK mRNA splice variant,tsMCAK gene,in human testis

Identification of specifically expressed genes in the adult or fetal testis is very important for the study of genes related to the development and function of the testis. In this study, a human adult testis cDNA microarray was constructed and hybridized with 33P-labeled human adult and embryo testis cDNA probes, respectively. After differential display analyzing, a number of new genes related to the development of testis and spermatogenesis had been identified. One of these new genes is tsMCAK. tsMCAK was expressed 2.62 folds more in human adult testis than fetal testis. The full length of tsMCAK is 2401 bp and contains a 2013 bp open reading frame, encoding a 671-amino-acid protein. Sequence analysis showed that it has a central kinesin motor domain and is homologous to HsMCAK gene of the somatic cells. Blasting human genome database localized tsMCAK to human chromosome 1P34 and further investigation showed that it is a splice variant of HsMCAK. The tissue distribution of tsMCAK was determined by RT-PCR and it is expressed highly and specifically in the testis. Southern blot studies of its expression in patients with infertility indicated its specific expression in spermatogenic cells and its correlation with male infertility. The above results suggested that tsMCAK is a candidate gene for the testis-specific KRPs and its specific expression in the testis was correlated with spermatogenesis and may be correlated with male infertility.     

睾丸生精细胞凋亡相关基因TSARG1与Mtsarg1的分子克隆及Mtsarg1基因的表达谱分析

从已获得的在隐睾和正常睾丸对照中表达量有明显差异的EST片段（GenBank登录号：BE644538）出发,利用生物信息学和实验技术,克隆了小鼠睾丸生精细胞凋亡相关新基因Mtsarg1及相应的人类新基因TSARG1,Gen-Bank登录号分别为AF399971和AY032925。小鼠Mtsargl与人类TSARGl基因在氨基酸水平有55％的一致性和61％相似性,与其他已知蛋白质无明显同源性。小鼠10种组织的RT-PCR分析结果表明,Mtsargl基因在睾丸中高表达,在附睾中呈微弱表达,在其他组织不表达,提示Mtsargl和TSARGl基因在生精细胞凋亡或精子发生中具有潜在的重要作用。     

NYD-SP15: a novel gene potentially involved in regulating testicular development and spermatogenesis

By hybridizing human adult testis cDNA microarrays with human adult and embryo testis cDNA probes, we identified a novel human testis gene, NYD-SP15. NYD-SP15 expression was 3.26-fold higher in adult than in fetal testis; however, there was almost no NYD-SP15 expression in the sperm. NYD-SP15 comprises 3364 base pairs, including a 1545 bp open reading frame encoding a 514 amino acid protein possessing 89% sequence identity with the mouse testis homologous protein. NYD-SP15 is located on human chromosome 13q14.2. The deduced structure of the protein contains two dCMP_cyt_deam domains, indicating a potential functional role for zinc ion binding. The gene is expressed variably in a wide range of tissues, with high expression levels in the testis. Sequence analysis revealed that NYD-SP15 is not a highly conserved protein, with its distribution in high-level species such as vertebrates including Homo, Mus, Rattus, and Canis. The results of semiquantitative polymerase chain reaction in mouse testis representing different developmental stages indicate that NYD-SP15 expression was developmentally regulated. These results suggest the putative NYD-SP15 protein may play an important role in testicular development and spermatogenesis and may be an important factor governing male infertility.     

The testis‐specific gene 1700102P08Rik is essential for male fertility

Male infertility is a rising problem around the world. Often the cause of male infertility is unclear, and this hampers diagnosis and treatment. Spermatogenesis is a complex process under sophisticated regulation by many testis‐specific genes. Here, we report the testis‐specific gene 1700102P08Rik is conserved in both the human and mouse and highly expressed in spermatocytes. To investigate the role of 1700102P08Rik in male fertility, knockout mice were generated by CRISPR‐Cas9. 1700102P08Rik knockout male mice were infertile with smaller testis and epididymis, but female knockout mice retained normal fertility. Spermatogenesis in the 1700102P08Rik knockout male mouse was arrested at the spermatocyte stage, and no sperm were found in the epididymis. The deletion of 1700102P08Rik causes apoptosis in the testis but did not affect the serum concentration of testosterone, luteinizing hormone, and follicle‐stimulating hormone or the synapsis and recombination of homologous chromosomes. We also found that 1700102P08Rik is downregulated in spermatocyte arrest in men. Together, these results indicate that the 1700102P08Rik gene is essential for spermatogenesis and its dysfunction leads to male infertility.     

Cloning, characterization, and mapping of the gene encoding the human G protein gamma 2 subunit

G proteins play vital roles in cellular responses to external signals. The specificity of G protein-receptor interaction is mediated mostly by the gamma-subunit and the individual members of the gamma-subunit multigene family would hence be expected to each have a particular expression profile. In an experiment designed to isolate genes expressed predominantly in human testis we identified a cDNA fragment corresponding to the gamma2 gene. Although the protein sequence of the gamma2 subunit has previously been published, the cDNA sequence, expression pattern, genomic structure, and localisation of the human GNG2 gene have not been described. We report the complete sequence of the GNG2 cDNA which is 1066 bp long and contains an open reading frame encoding a protein of 71 amino acids. This protein is 100% homologous to the bovine, mouse, and rat G protein gamma2 subunit. The gene structure is very similar to that of other Ggamma-subunit genes in that there are two introns, one located in the 5' UTR and the other within the ORF. We show that this gene is expressed in a range of foetal tissues as well as adult testis, adrenal gland, brain, white blood cells and lung but not in adult liver, muscle, sperm, prostate gland nor in the testes of two different infertile patients. There is evidence that GNG2 is expressed in malignant tissues. Using two independent methods, we have mapped the human GNG2 gene to chromosome 14q21.     

In‐depth proteomic analysis of whole testis tissue from the adult rhesus macaque

The rhesus macaque is similar to humans both anatomically and physiologically as a primate, and has therefore been used extensively in medical and biological research, including reproductive physiology. Despite sequencing of the macaque genome, limited postgenomic studies have been performed to date. In studies aimed at characterizing spermatogenesis, we successfully identified 9078 macaque testis proteins corresponding to 8662 genes, using advanced MS and an optimized proteomics platform, indicative of complex protein compositions during macaque spermatogenesis. Immunohistochemistry analysis further revealed the presence of proteins from different types of testicular cells, including Sertoli cells, Leydig cells, and various stages of germ cells. Our data provide expression evidence at protein level of 3010 protein‐coding genes in 8662 identified testis genes for the first time. We further identified 421 homologous genes from the proteome already known to be essential for male infertility in mouse. Comparative analysis of the proteome showed high similarity with the published human testis proteome, implying that macaque and human may use similar proteins to regulate spermatogenesis. Our in‐depth analysis of macaque spermatogenesis provides a rich resource for further studies, and supports the utility of macaque as a suitable model for the study of human reproduction.     

大鼠睾丸特异表达基因Ube1的分离鉴定及生物学特征

本研究采用抑制性消减杂交(suppression subtracfive hybridization, SSH)和cDNA快速扩增(rapid amplification of cDNA ends, RACE)技术从大鼠A型精原细胞和粗线期精母细胞中成功克隆出大鼠泛素激活酶(ubiquitin-activating enzyme)基因Ube1 (GenBank登录号EF690356).该基因序列全长3433 bp,其中开放阅读框有3171 bp,编码一个含1057个氨基酸的蛋白质.Blast比对显示,Ube1与小鼠泛素激活酶基因Ubely1的同源性为93%,与人泛素激活酶基因UBE1的同源性为82%.Ube1基因编码的蛋白质含泛素激活酶信号位点和泛素激活酶活化位点,这些位点也存在于人类和小鼠的泛素激活酶1中.RT-PCR分析显示,Ube1在睾丸中大量表达,而在心、肝、脾、肺、肾、肌肉、脑、卵巢中没有表达.荧光定量PCR分析不同生精细胞中Ube1的表达,显示Ube1在A型精原细胞中大量表达,在粗线期精母细胞、圆形精子细胞和支持细胞中微弱表达.以上结果提示,Ube1是大鼠睾丸特异表达基因,可能通过参与泛素/蛋白酶体途径来影响精子发生.     

Spermatogenesis proceeds normally in mice without linker histone H1t

The histone gene H1t is expressed exclusively in pachytene spermatocytes of the testis. In this report we have eliminated the single copy H1t gene by homologous recombination from the mouse genome to analyse the function of the H1t protein during spermatogenesis. Mice homozygous for the mutated H1t gene locus developed normally and showed no anatomic abnormalities until the adult stage. In addition, H1t-deficient mice were fertile and reproduced as wild-type mice. The process of spermatogenesis and the testicular morphology remained unchanged in the absence of H1t. RNase protection analysis demonstrated that H1.1, H1.2 and H1.4 histone gene expression is enhanced during spermatogenesis in H1t-deficient mice.     

Extraordinary sequence divergence at Tsga8, an X-linked gene involved in mouse spermiogenesis

The X chromosome plays an important role in both adaptive evolution and speciation. We used a molecular evolutionary screen of X-linked genes potentially involved in reproductive isolation in mice to identify putative targets of recurrent positive selection. We then sequenced five very rapidly evolving genes within and between several closely related species of mice in the genus Mus. All five genes were involved in male reproduction and four of the genes showed evidence of recurrent positive selection. The most remarkable evolutionary patterns were found at Testis-specific gene a8 (Tsga8), a spermatogenesis-specific gene expressed during postmeiotic chromatin condensation and nuclear transformation. Tsga8 was characterized by extremely high levels of insertion-deletion variation of an alanine-rich repetitive motif in natural populations of Mus domesticus and M. musculus, differing in length from the reference mouse genome by up to 89 amino acids (27% of the total protein length). This population-level variation was coupled with striking divergence in protein sequence and length between closely related mouse species. Although no clear orthologs had previously been described for Tsga8 in other mammalian species, we have identified a highly divergent hypothetical gene on the rat X chromosome that shares clear orthology with the 5' and 3' ends of Tsga8. Further inspection of this ortholog verified that it is expressed in rat testis and shares remarkable similarity with mouse Tsga8 across several general features of the protein sequence despite no conservation of nucleotide sequence across over 60% of the rat-coding domain. Overall, Tsga8 appears to be one of the most rapidly evolving genes to have been described in rodents. We discuss the potential evolutionary causes and functional implications of this extraordinary divergence and the possible contribution of Tsga8 and the other four genes we examined to reproductive isolation in mice.     

DNA methylation and demethylation events during meiotic prophase in the mouse testis.

The genes encoding three different mammalian testis-specific nuclear chromatin proteins, mouse transition protein 1, mouse protamine 1, and mouse protamine 2, all of which are expressed postmeiotically, are marked by methylation early during spermatogenesis in the mouse. Analysis of DNA from the testes of prepubertal mice and isolated testicular cells revealed that transition protein 1 became progressively less methylated during spermatogenesis, while the two protamines became progressively more methylated; in contrast, the methylation of beta-actin, a gene expressed throughout spermatogenesis, did not change. These findings provide evidence that both de novo methylation and demethylation events are occurring after the completion of DNA replication, during meiotic prophase in the mouse testis.     

cDNA structure, alternative splicing and exon-intron organization of the predisposing tuberous sclerosis (Tsc2) gene of the Eker rat model.

The Eker rat hereditary renal carcinoma (RC) is an excellent example of a Mendelian dominant predisposition to a specific cancer in an experimental animal. We recently reported that a germline insertion in the rat homologue of the human tuberous sclerosis gene (TSC2) gives rise to the dominantly inherited cancer in the Eker rat model. We now describe the entire cDNA (5375 bp without exons 25 and 31) and genomic structure of the rat Tsc2 gene. The deduced amino acid sequence (1743 amino acids) shows 92% identity to the human counterpart. Surprisingly, there are a great many (> or = 41) coding exons with small sized introns spanning only approximately 35 kb of genomic DNA. Two alternative splicing events [involving exons 25 (129 bp) and 31 (69 bp)] make for a complex diversity of the Tsc2 product. The present determination of the Tsc2 gene and establishment of strong conservation between the rat and man provide clues for assessing unknown gene functions apart from that already predicted from the GTPase activating proteins (GAP3) homologous domain and for future analysis of intragenic mutations in tumors using methods such as PCR-SSCP and for insights into diverse phenotypes between species.     

Cloning of a novel gene,<Emphasis Type="Italic">Cymg1</Emphasis>, related to family 2 cystatins and expressed at specific stages of mouse testis development

We have cloned a novel gene,Cymg1 (GenBank accession number AY600990), from a mouse testis cDNA library.Cymg1 is located in 2G3 of mouse chromosome 2. The cDNA includes an open reading frame that encodes 141 amino acid residues. The encoded polypeptide has a cysteine protease inhibitor domain found in the family 2 cystatins but lacks critical consensus sites important for cysteine protease inhibition. These characteristics are seen in the proteins of the CRES subfamily of the family 2 cystatins which are expressed specifically in the reproductive tract. CYMG1 protein shows 44% identity with mouse CRES and 30% identity with mouse cystatin C. Northern blot analysis showed that theCymg1 gene was specifically expressed in adult mouse testis. RT-PCR also showed thatCymg1 was expressed in testis and spermatogonial cells.Cymg1 expression level varied in the different developmental stages of mouse testis, and were coincidental with spermatogenesis and sex maturation. These results indicate thatCymg1 may play important roles in mouse spermatogenesis and sex maturation     

The putative peroxisomal gene Pxt1 is exclusively expressed in the testis

Genes reported to be crucial for spermatogenesis are often exclusively expressed in the testis. We have identified a novel male germ cell-specific expressed gene named peroxisomal testis specific 1 (Pxt1) with expression starting at the spermatocyte stage during mouse spermatogenesis. The putative amino acid sequence encoded by the cDNA of the Pxt1 gene contains a conserved Asn-His-Leu (NHL)-motif at its C-terminal end, which is characteristic for peroxisomal proteins. Pxt1-EGFP fusion protein is co-localized with known peroxisomal marker proteins in transfected NIH3T3 cells. In addition, we could demonstrate that the peroxisomal targeting signal NHL is functional and responsible for the correct subcellular localization of the Pxt1-EGFP fusion protein. In male germ cells peroxisomes were reported only in spermatogonia. The Pxt1 gene is so far the first gene coding for a putative peroxisomal protein which is expressed in later steps of spermatogenesis, namely in pachytene spermatocytes.     

Fine mapping of a region of rat chromosome 12 close to the aspermia (as) locus and comparison with the human orthologous regions.

The aspermia mutation of the rat exhibits male sterility caused by arrest of spermatogenesis, which is controlled by an autosomal single recessive gene (as). The as locus has been mapped on rat chromosome 12. We recently identified a causative mutation for the aspermia phenotype of the as homozygous rats in the gene encoding Fkbp6, a member of the immunophilins FK506 binding proteins. In this paper, we report the fine mapping of the as locus by linkage analysis combined with comparative mapping using rat, mouse, and human genomic sequences and expression analysis of genes located in the as region. We constructed a fine linkage map of the region of rat chromosome 12 close to the as locus by using 13 microsatellite markers and localized the as locus to a 1.0-cM interval. Comparison of the linkage map with physical maps of rat, mouse, and human refined the as critical region in a 2.2-Mb segment of the rat physical map between the D12Nas3 and D12Nas8 genes, which includes the Fkbp6 gene. A centromeric part of this segment corresponds to the region commonly deleted in Williams syndrome, a human complex developmental disorder, on human chromosome 7q11.23. The expression analysis of 23 genes located on the 2.2-Mb segments in various mouse tissues identified genes exclusively or strongly expressed in the testis.