Isolation and expression analysis of the human RNH2 gene encoding ribonuclease inhibitor 2

Ribonuclease inhibitor 1 (RI-1) is a cytoplasmic protein that inhibits a variety of pancreatic-type mammalian Rnases by forming a very tight, reversible 1:1 complex. Recently a novel gene encoding ribonuclease inhibitor 2, Rnh2 has been isolated in mouse. The expression pattern of the mouse Rnh2 is specific to testis, especially in the spermatogonia. Then it has been suggested that the mouse Rnh2 gene may play critical roles in mouse spermatogenesis. In this study, we isolated the human RNH2 cDNA and analyzed its expression pattern. The human RNH2 is also expressed limited to testis, and then it is suggested that the human Rnh2 gene may also play critical roles in human spermatogenesis.     

Isolation of the human <Emphasis Type="Italic">ePAB</Emphasis> and <Emphasis Type="Italic">ePABP2</Emphasis> cDNAs and analysis of the expression patterns

Purpose  Identification of the unique genes playing critical roles in human embryo cleavage. Methods  Isolation of human ePAB cDNA using human ovary cDNA libraries and mouse ePAB amino acid sequences, followed by analysis of its expression pattern in various adult tissues and stages during early oocyte development excluding ePABP2. Results  Human ePAB encodes a 330-aa protein and is located on chromosome 20q12-q13.1. The amino acid sequence is 72% homologous with that of mouse ePab. Human ePAB has only three RRMs and lacks a PABP domain; the expression pattern is nonspecific in adult tissues and detected in all stages, from oocyte to blastocyst. Human ePABP2 encodes a 282-aa protein and is located on chromosome 16q24.3. The amino acid sequence is 68% homologous with mouse ePabp2. Conclusions  We identified human ePAB and ePABP2 cDNA. Human ePAB cDNA is not expressed specific to the ovary. Biological discrepancies exist between the human and the mouse. Capsule   We successfully isolated the human ePAB and ePABP2 cDNAs and analyzed the expression patterns in humans.     

Classical and non-classical Major Histocompatibility Complex class I gene expression in in vitro derived bovine embryos

The role of the Major Histocompatibility Complex class I (MHC-I) genes in human and mouse preimplantation embryo development has received considerable attention. In contrast, information concerning the role of these genes in bovine embryo development is limited. The objective of the current study was to characterize the expression pattern of MHC-I genes during preimplantation embryo development in cattle. To this end, bovine oocytes were harvested from slaughterhouse ovaries, matured, fertilized and cultured in vitro. Samples were collected at immature and mature oocyte, presumptive zygote, 2–4-cell embryo, 8–16-cell embryo, morula, blastocyst and hatched blastocyst stages of development. MHC-I expression was detected using quantitative real-time-PCR, cDNA sequencing, whole mount immunocytochemistry and Western blotting. We report classical and non-classical MHC-I mRNA expression in bovine oocytes and developing embryos. Furthermore, we report that the pattern of MHC-I mRNA expression across development was gene- and stage-specific.     

Isolation of a phylogenetically conserved and testis-specific gene using a monoclonal antibody against the serological H-Y antigen.

Several cDNA clones of a gene termed male-enhanced antigen-2 (Mea-2), have been isolated from a mouse testicular expression cDNA library using a monoclonal histocompatability Y (H-Ys) antibody which detects specific protein(s) present in the mouse testis but not the ovary. The Mea-2 gene is phylogenetically conserved among various mammalian species examined, and is expressed at high levels in adult mouse testis. The expression pattern of Mea-2 is very similar to that of another gene, the male-enhanced antigen-1 (Mea-1), previously isolated using a polyclonal H-Ys antibody. Northern blotting and RT-PCR analyses demonstrated that Mea-2 is also expressed in other adult and fetal mouse organs at low levels. The testis-enhanced expression of this gene is associated with germ cell development at mid- to late-meiotic stages of spermatogenesis. Analysis of an intersubspecies mouse backcross has assigned this gene to chromosome 5, between the loci Gus and Hnf-1.     

Isolation and characterization of novel testis-specific genes from mouse pachytene spermatocyte-enriched cDNA library

Background and Aims:  Isolation and analysis of spermatogenesis-specific genes provide important information for elucidating the mechanisms of human infertility. The aim of the present study was to suggest an effective strategy for the comprehensive isolation of novel genes associated with spermatogenesis in mice.
Methods:  To isolate novel testis-specific genes associated with meiosis in mice, we constructed a mouse pachytene spermatocyte-enriched cDNA library by the centrifugal elutriation method, and sequenced 120 cDNA clones isolated from the cDNA library. A basic local alignment search tool (BLAST) search was carried out on the cDNA clones to find novel genes and then a detailed expression analysis was carried out by Northern blot hybridization and in situ hybridization.
Results:  Of the 120 cDNA clones, 35 clones (29%) were novel and 18 clones (15%) were expressed only in the testis. The expression patterns of seven novel testis-specific clones were examined on the testis sections. Three clones were expressed in spermatocytes and other germ cells, and two clones were exclusively expressed in spermatocytes. Amino acid sequences of seven novel testis-specific clones were deduced from their nucleotide sequences, suggesting that two of them contain known functional repeat structures.
Conclusions:  This method provides a powerful strategy to isolate novel testis-specific genes efficiently. (Reprod Med Biol 2005; 4 : 231–237)     

Pervasiveness of intronless genes expressed in haploid germ cell differentiation

BackgroundcDNA libraries derived from the brain and testis contain genes that encode almost all proteins. The brain is composed of various differentiated cells, and the testis also contains various differentiated cells, such as germ cells, and somatic cells that support germ cell differentiation, such as Sertoli and Leydig cells. Many genes appear to be expressed due to tissue complexity.MethodsThe Genome Project has sequenced the entire genomes of humans and mice. Recent research using new gene analysis technologies has found that many genes are expressed specifically in male germ cells.Main findings (Results)Functional intronless genes are significantly enriched in haploid germ cell‐specific genes.ConclusionFunctional intronless genes associated with fertility are more likely to be inherited in haploid germ cells than in somatic cells.     

小鼠胚胎干细胞体外诱导分化为精原干细胞的研究

目的:初步探讨在体外条件下诱导小鼠胚胎干细胞(EGFP-mESC)分化为精原干细胞(SSC)的条件,建立有效的技术平台。方法:采用部分模拟胚胎早期发育的方法,将EGFP-mESC(XY)经体外悬滴培养,制备拟胚体(EB);使用免疫磁珠分选法从EB中分离出表达原始生殖细胞(PGC)表面标志SSEA-1的细胞;获得的细胞经2μmol/L维甲酸(RA)诱导增殖,使之向雄性生殖细胞分化。对诱导的细胞采用RT-PCR及免疫荧光检测特异性基因及蛋白的碱性磷酸酶。结果:在诱导EGFP-mESC向雄性生殖细胞分化的过程中,采用RT-PCR方法检测到了雄性生殖细胞特异表达基因Sry、fragilis、stella、Tex14等mRNA的表达;利用激光共聚焦方法检测到SSC表面标志蛋白Stra8、integrin-α6及Hsp90α的表达。结论:采用RA体外诱导EGFP-mESC定向分化为SSC是可行的。     

Sex-specific differences in placental global gene expression in pregnancies complicated by asthma

Background

Chronic maternal asthma is associated with reduced growth of the female fetus and normal growth of the male fetus. The mechanisms that control the differential effects of maternal asthma on the fetus have not been fully elucidated but alterations in placental function may play a role. In the current study we have used microarray platform to examine fetal sex-specific global changes in placental gene expression in pregnancies complicated by asthma as compared to non-asthmatic subjects.

Methods

Placental RNA was extracted from 11 control subjects and 38 asthmatic subjects. Labeled cDNA was hybridized to an oligonucleotide chip with 1700 double spotted well-characterized human genes. Global gene expression data analysis and visualization were performed using the Binary Tree-Structured Vector Quantization (BTSVQ) software. Functional relationships of differentially expressed genes were assessed using protein-protein interaction database I2D, network analysis and visualization software NAViGaTOR and Ingenuity Pathway Analysis software.

Results

Overall, 65 genes were found to be altered in placentae of pregnancies complicated by asthma. Of these, only 6 genes were altered in male placentae. There were 59 gene changes in female placentae of asthmatic mothers relative to control placentae. Some of the sex-specific genes were associated with growth, inflammation and immune pathways.

Conclusion

There are sex-specific alterations in placental gene expression in the presence of maternal asthma. Given that many of the identified genes in the female placentae were associated with or involved in cellular growth and tissue development, these may contribute to the sexually dimorphic difference in fetal growth in response to maternal asthma.     

Sequence Analysis of Libraries from Individual Human Blastocysts

Purpose: It has recently become possible to construct cDNA libraries from individual human blastocysts to investigate the expression of embryonic genes in human preimplantation development. We have previously reported the expression of -actin, CD-59, and homeobox OCT-3 and identified almost-complete homology of sequences to human histone 3.1 and human ribosome protein S25. In the present paper, our further sequencing analysis of cDNA libraries from single human blastocysts is described. Methods: cDNA libraries were constructed from 13 blastocysts. Sequence analysis was performed in 120 clones from one of these cDNA libraries with fragments of 50 to 1000 bp. Their sequence identity was analyzed using the expressed sequence tag (EST) database. Results: The presence of two housekeeping genes, hexokinase 1 and serine/threonine phosphorylase, and four other ESTs was demonstrated, the identity of which, with particular gene expression in preimplantation development, has not yet been established. Conclusions: The data demonstrate the usefulness of constructing cDNA libraries from individual human blastocysts and their value in the analysis of genetic expression in human preimplantation development.     

Short Communication: Isolation and Expression Analysis of the Testis-Specific Gene, Human OPPO1

PURPOSE: To investigate human spermatogenesis, we isolated human testis-specific genes. METHODS: Using mouse amino acid sequences, we found the region including homology in amino acid level in the human genome sequences. The primers encompassing introns were made and RT-PCR and RACE were carried out. The resultant PCR products were sequenced. RESULTS: The full-length cDNA of human OPPO1 was isolated. It encodes 257 amino acid residues. The expression of the human OPPO1 was predominantly in the testis. On the other hand, partial cDNAs of ZNF8, GR194, GR219, GR093, GR046, GR163, and GR200 were expressed in the various tissues. CONCLUSIONS: Our data suggests that the human OPPO1 may play important roles in human spermatogenesis.     

Current concepts of human azoospermia and its causes

Infertility is a serious social problem in advanced nations today. One of the most important causes is the male factor. Striking progress has been achieved in recent years in elucidating the mechanisms of spermatogenesis in mice by experimental methods represented by the knockout mouse. Although many factors associated with male infertility are known in mice, the translation of this information to people has been slow. This is because the knockout mouse phenotype cannot necessarily be reproduced faithfully in humans. However, it is known that environmental factors, chromosomal defects and several specific gene mutations result in human male infertility. In this review, we first discuss the environmental factors considered likely to be involved in male infertility, and secondly we describe the Y chromosome and several important genes on the Y chromosome that play critical roles in spermatogenesis in humans. Then, we demonstrate the three critical genes identified in our laboratory in autosomes involved in human spermatogenesis, the SYCP3, MEI1 and PARP-2. Finally, we explain the future directionality and possibilities of research in this field.     

Sperm flagella protein components: Human meichroacidin constructed by the membrane occupation and recognition nexus motif

Background and Aims:   In a previous study, the authors of the present study cloned mouse meichroacidin ( MCA ), which is expressed in stages of spermatogenesis from pachytene spermatocytes through round spermatid germ cells. MCA protein contains the membrane occupation and recognition nexus (MORN) motif and localizes to a male meiotic metaphase chromosome. Recently, a MCA homolog of carp ( Cyprinus carpio ), MORN motif-containing sperm-specific axonemal protein (MSAP), was reportedly identified and localized in sperm flagella. Present knowledge of human spermiogenesis requires the identification of proteins in human sperm. The present study identified the human orthologue of MCA .
Methods:   Colony hybridization using a human testis plasmid cDNA library was carried out to clone human MCA ( h-MCA ) cDNA. Northern blot, Western blot, and immunohistochemical analyses were carried out.
Results:   h-MCA was found to be specifically expressed in the testes. The h-MCA amino acid sequence shared 79.8% identity with mouse MCA and contained MORN motifs. h-MCA localized in the sperm flagellum and basal body, as does MSAP in carp.
Conclusion:   Expression and localization analyses showed that h-MCA is a component of the sperm flagellum and basal body and might play an important role in the development of the sperm flagellum in humans. (Reprod Med Biol 2005; 4: 213–219)     

Co-culture of mouse spermatogonial stem cells with sertoli cell as a feeder layer,stimulates the proliferation and spermatogonial stemness profile

Objectives

Sertoli cells effect the fate map of spermatogonial stem cells (SSCs) to self-renew via providing the special microenvironments. Maintenance of proliferation and self-renewal activity of SSCs may be usable as a therapeutic strategy, leads to increase the recovery of male fertility. This research was aimed to evaluate the effect of mouse sertoli cells on spermatogonia stem cells proliferation and the expression pattern of stemness markers.