Identification and expression profiling of 10 novel spermatid expressed CYPT genes

To identify candidate genes for poor sperm morphology, we have screened for genes expressed during spermiogenesis. We identified 10 new members of the cysteine-rich perinuclear theca (CYPT) family showing that this family contains at least 15 members, which also includes the casein kinase II target genes. Based on similarity the CYPT sequences could be divided into two groups, Cypt1-10 and the novel members Cypt12-15. The 5'-end of the CYPT family is highly similar to exon1A and part of the first intron of Zfy2. Seven CYPT genes mapped to the X chromosome; six contained an intron and one was intron-less. One CYPT gene mapped to chromosome 3 and one mapped to chromosome 9 which were both intron-less. The upstream region of the CYPT family and Zfy2 genes is conserved. For some the conservation extended over a large region, however, only about 150 nucleotides is conserved among all CYPT members and Zfy2. Nevertheless, the short conserved promoter leads to essentially identical expression profiles for the CYPT family members and Zfy2, which was clearly different from the profile of Zfy1. Expression of the CYPT family and Zfy2 preceded the expression of other spermatid-specific genes such as the transition proteins and the protamines. In situ hybridization revealed a low expression in pachytene spermatocytes from stages IX-X followed by a strong upregulation in spermatids from stage VI with maximum expression in spermatids in stages VII-VIII. The CYPT family may function in the remodeling of the spermatid nucleus before condensation of the DNA.     

Prioritization of differentially expressed genes through integrating public expression data

Differentially expressed gene (DEG) analysis is a major approach for interpreting phenotype differences and produces a large number of candidate genes. Given that it is burdensome to validate too many genes through benchwork, an urgent need exists for DEG prioritization. Here, a novel method is proposed for prioritizing bona fide DEGs by constructing the normal range of gene expression through integrating public expression data. Prioritization was performed by ranking the differences in cumulative probability for genes in case and control groups. DEGs from a study on pig muscle tissue were used to evaluate the prioritization accuracy. The results showed that the method reached an area under the receiver operating characteristic curve of 96.42% and can effectively shorten the list of candidate genes from a differential expression experiment to find novel causal genes. Our method can be easily extended to other tissues or species to promote functional research in broad applications.     

Identification of a novel gene SRG4 expressed at specific stages of mouse spermatogenesis

Spermatogenesis is a complex process. Duringspermatogenesis, the production of sperm occurs withinthe testicular seminiferous tubules through three separatedphases. First of all, diploid germ cells, primitivespermatogonia, will self renew to amplify and producetypes A and B spermatogonia. Type B spermatogonia willdifferentiate into primary spermatocytes. Then, meioticdivisions of spermatocytes will produce round spermatids.Finally, after a series of biochemical and morphologicalchanges, sper…     

Low rates of expression profile divergence in highly expressed genes and tissue-specific genes during mammalian evolution

Evolutionary rates provide important information about the pattern and mechanism of evolution. Although the rate of gene sequence evolution has been well studied, the rate of gene expression evolution is poorly understood. In particular, it is unclear whether the gene expression level and tissue specificity influence the divergence of expression profiles between orthologous genes. Here we address this question using a microarray data set comprising the expression signals of 10,607 pairs of orthologous human and mouse genes from over 60 tissues per species. We show that the level of gene expression and the degree of tissue specificity are generally conserved between the human and mouse orthologs. The rate of gene expression profile change during evolution is negatively correlated with the level of gene expression, measured by either the average or the highest level among all tissues examined. This is analogous to the observation that the rate of gene (or protein) sequence evolution is negatively correlated with the gene expression level. The impacts of the degree of tissue specificity on the evolutionary rate of gene sequence and that of expression profile, however, are opposite. Highly tissue-specific genes tend to evolve rapidly at the gene sequence level but slowly at the expression profile level. Thus, different forces and selective constraints must underlie the evolution of gene sequence and that of gene expression.     

人体肝癌细胞急性低氧及低氧习服差异表达基因分析

本文分析了人体肝癌细胞(HepG2)急性低氧处理以及低氧习服处理后基因表达谱的改变。急性低氧处理为细胞在1％氧气中培养48h,低氧习服处理为细胞在1％氧气中培养24h,常氧培养24h,以此作为一个周期,重复6个周期。联合应用抑制消减杂交技术和cDNA芯片技术,筛选HepG2细胞经急性低氧处理与正常培养细胞相比差异表达的基因,以及经低氧习服处理细胞与正常培养细胞相比差异表达的基因。结果显示,HepG2细胞经急性低氧处理与在常氧条件下培养相比,差异表达的基因有37个,表达水平全部表现为下调,其中包括参与细胞周期、细胞应激、细胞信号转导、细胞骨架形成、转录相关蛋白及细胞代谢相关蛋白的基因,1个未知基因序列、4个EST序列、5个线粒体蛋白基因,另外有功能不明的蛋白质基因12个。低氧习服处理的细胞与常氧条件下培养的细胞相比,差异表达的基因有6个,其中包括两个线粒体蛋白基因、金属蛋白酶1基因、转铁蛋白基因、Thymosin ．beta-4和TPT1基因。其中线粒体蛋白ND4、转铁蛋白、Thymosin.beta-4和TPT1基因的表达呈上调,线粒体NDl及金属蛋白酶1基因的表达水平呈下调。经低氧习服处理后,细胞低氧耐受力提高,低氧习服处理细胞基因的表达与急性低氧处理细胞和正常培养细胞的基因表达不同,这种变化可能与低氧习服细胞低氧耐受力的增强有关。     

兔精子发生过程中cyclin B1基因表达和蛋白定位的发育阶段依赖性

利用原位杂交和免疫组化等方法,研究兔精子发生过程中生精细胞cyclin B1 mRNA的表达和蛋白定位特点,结果显示,兔生精上皮中Cyclin B1 mRNA的主要分布在初级精母细胞中,直至圆形精子细胞仍然存在,于精子细胞的变态过程中逐渐消失,在伸长的精子细胞和精子中未检测出cyclin B1 mRNA,Cyclin B1蛋白在进入分裂期的精原细胞和精母细胞中表达,在圆形精子细胞和伸长的精子细胞中呈现大量的cyclin B1蛋白,上述结果表明,在兔精子发生过程中,cyclin B1 mRNA表达和蛋白定位具有发育阶段依赖性的特征。     

Up-regulation of Arl4a gene expression by broccoli aqueous extract is associated with improved spermatogenesis in mouse testes

Introduction: Broccoli (Brassica oleracea) is well known for its properties as an anticancer, antioxidant, and scavenger of free radicals. However, its benefits in enhancing spermatogenesis have not been well established.Objective: To study broccoli aqueous extract effects on sperm factors and the expression of genes Catsper1, Catsper2, Arl4a, Sox5, and Sox9 in sperm factors in mice.Material and methods: Male mice were divided randomly into six groups: (1) Control; (2) cadmium (3 mg/kg of mouse body weight); (3) orally treated with 200 µl broccoli aqueous extract (1 g ml^-1); (4) orally treated with 400 µl of broccoli aqueous extract; (5) orally treated with 200 broccoli aqueous extract plus cadmium, and (6) orally treated with 400 µl of broccoli aqueous extract plus cadmium. We analyzed the sperms factors and Catsper1, Catsper2, Arl4a, Sox5, and Sox9 gene expression.Results: An obvious improvement in sperm count and a slight enhancement in sperm motility were observed in mice treated with broccoli extract alone or with cadmium. Sperm viability was reduced by broccoli extract except for the 200 µl dose with cadmium, which significantly increased it. Interestingly, Arl4a gene expression increased in the 400 µl broccoli- treated group. Likewise, the Arl4a mRNA level in mice treated with cadmium and 200 µl of broccoli extract was higher than in the cadmium-treated mice. Furthermore, broccoli extract enhanced the mRNA level of Catsper2 and Sox5 genes in mice treated with 200 µl and 400 µl broccoli extract plus cadmium compared with the group treated solely with cadmium.Conclusion: The higher sperm count in broccoli-treated mice opens the way for the development of pharmaceutical products for infertile men.     

Investigation of differentially expressed genes during the development of mouse cerebellum

Before the discovery of DNA microarray and DNA chip technology, the expression of only a small number of genes could be analyzed at a time. Currently, such technology allows us the simultaneous analysis of a large number of genes to systematically monitor their expression patterns that may be associated with various biological phenomena. We utilized the Affymetrix GeneChip Mu11K to analyze the gene expression profile in developing mouse cerebellum to assist in the understanding of the genetic basis of cerebellar development in mice. Our analysis showed 81.6% (10,321/12,654) of the genes represented on the GeneChip were expressed in the postnatal cerebellum, and among those, 8.7% (897/10,321) were differentially expressed with more than a two-fold change in their maximum and minimum expression levels during the developmental time course. Further analysis of the differentially expressed genes that were clustered in terms of their expression patterns and the function of their encoded products revealed an aspect of the genetic foundation that lies beneath the cellular events and neural network formation that takes place during the development of the mouse cerebellum.     

Enhanced expression of various exogenous genes in recombinant Chinese hamster ovary cells in presence of dimethyl sulfoxide

Dimethyl sulfoxide (DMSO) (1% v/v) stimulated stable transformed Chinese hamster ovary (CHO) cells to synthesize different recombinant proteins and repress their proliferation rate. The expressions of a fusion protein and -galactosidase were increased 1.6- and 1.4-fold after adding DMSO. The expression of fusion protein was increased by up to 2.8-fold that of uninduced control by the simultaneous addition of DMSO and pentanoic acid. However, DMSO did not increase the production of the monoclonal antibody (immunoglobulin G) of three hybridoma cell lines (OKM1, OKT4 and HyGPD-YK-1-1), although it could inhibit the growth rates of the hybridoma.     

Gene expression profiling revealed specific spermatogonial stem cell genes in mouse

Mammalian spermatogenesis originates from spermatogonial stem cells (SSCs), which undergo mitosis, meiosis and spermiogenesis in order to generate mature spermatozoa. SSCs are adult stem cells that can both self‐renew and differentiate. To maintain pluripotency, SSCs are regulated by both extrinsic factors secreted from surrounding somatic cells and intrinsic factors including specific gene expression programs. Using fluorescent labeled germ line stem cells, mouse gonocytes and SSCs were purified up to 97% by improved FACS method. Through microarray analyses, global gene expression profiles of gonocytes, SSCs, and differentiated cells were compared. A large number of distinctive genes were found to be enriched in respective cell populations, indicating different functional requirements of each cell type. Functional clustering analyses revealed that while gonocytes and SSCs preferentially express genes implicated in gene expression regulation and epigenetic modifications, differentiated cells including somatic cells are enriched with genes encoding proteins involved in various cellular activities. Further in situ hybridization and RT‐PCR experiments confirmed SSC specific expression of several genes of which functions have not been characterized in SSCs. The comparative gene expression profiling provides a useful resource for gene discovery in relation to SSC regulation and opens new avenues for the study of molecular mechanisms underlying SSC self‐renewal and differentiation. genesis 51:83–96, 2013. © 2012 Wiley Periodicals, Inc.     

The differential expression of lamin epitopes during mouse spermatogenesis

The presence of lamin proteins in mouse spermatogenic cells has been examined by using an anti-lamin AC and an anti-lamin B antisera which recognize somatic lamins A and C, and somatic lamin B, respectively. Anti-lamin B binds to the nuclear periphery of all cell types examined, including Sertoli cells, primitive type A spermatogonia, preleptotene, leptotene, zygotene and pachytene spermatocytes, and round spermatids. In sperm nuclei, the antigenic determinants are localized to a narrow domain of the nucleus. However, after removing the perinuclear theca, anti-lamin B localizes to the entire nuclear periphery in a punctate pattern, suggesting that it is binding to determinants previously covered by the theca constituents. On immunoblots anti-lamin B reacts with a ～ 68 kD polypeptide in all germ cells and, to a lesser extent, with four additional polypeptides present only in meiotic and post-meiotic nuclear matrices. Anti-lamin AC also reacts with the perinuclear region of the somatic cells in the testes, in particular, those of the interstitium and also the Sertoli cells of the seminiferous epithelium. In contrast to anti-lamin B, anti-lamin AC does not bind to the germ cells at any stage of spermatogenesis. In addition, nuclear matrix proteins from isolated spermatogenic cells do not bind anti-lamin AC on immunoblots, suggesting the lack of reactivity is not due to the masking of any antigenic sites. These data demonstrate that germ cells contain lamin B throughout spermatogenesis, even during meiosis and spermiogenesis when the nuclear periphery lacks a distinct fibrous lamina. © 1993 Wiley-Liss, Inc.     

筛选差异表达基因方法的新进展

了解不同细胞或同类细胞在不同发育阶段、不同生理状态下的基因表达状况,可以为研究生命活动过程提供重要信息。以差别筛选、削减杂交等基本方法为出发点,研究基因表达差异的方法不断完善,先后出现了DDRT—PCR、RDA、SSH、cDNA微阵列(基因芯片)、基因表达的系统分析(SAGE)等技术。本着重对这些方法的优缺点及改进进行论述和评介,并对技术的发展趋势进行了分析。     

Patatin and four serine proteinase inhibitor genes are differentially expressed during potato tuber development

A highly efficient and synchronousin vitro tuberization system is described. One-node stem pieces from potato (Solanum tuberosum cv. Bintje) plants grown under short day-light conditions containing an axillary bud were cultured in the dark on a tuber-inducing medium. After 5 or 6 days all axillary buds started to develop tubers. To study gene expression during tuber development, RNA isolated from tuberizing axillary buds was used for bothin vitro translation and northern blot hybridizations. The genes encoding the proteinase inhibitors I and II (PI-I and PI-II), a Kunitz-and a Bowman-Birk-type proteinase inhibitor were already expressed in uninduced axillary buds. The length of the day-light conditions differently influenced the expression level of the individual genes. In addition, the expression of each of these genes changed specifically during the development of the axillary bud to tuber. In contrast to the expression of these proteinase inhibitor genes, patatin gene expression was only detectable from the day tuberization was manifested as a radial expansion of the axillary bud.These results are discussed with respect to the regulation of the expression of the genes studied in relation to the regulation of tuber development.     

Newt RAD51: Cloning of cDNA and analysis of gene expression during spermatogenesis

A cDNA encoding a newt homolog of Escherichia coli RecA and yeast RAD51 from a testis cDNA library was isolated. The newt RAD51 (nRAD51) cDNA predicted a 337 amino acid protein with a 95-96% amino acid identity to Xenopus and mammalian RAD51. Northern blot analysis showed that nRAD51 mRNA, 1.7 kb in length, was expressed strongly in the testis and ovary, but weakly in the liver, kidney and brain. In situ hybridization revealed that expression of nRAD51 mRNA was barely observed in primary spermatogonia (one cell in a cyst) and early secondary spermatogonia (two to four cells in a cyst), but increased in late secondary spermatogonia (> or =eight cells in a cyst), reaching a maximum level in leptotene-zygotene spermatocytes, and thereafter declined. These results suggest that nRAD51 is involved in mitotic recombination in spermatogonia as well as in meiotic recombination in spermatocytes.