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.     

Multiple rearrangements of mitochondrial DNA in unfertilized human oocytes

OBJECTIVE: To determine the rearrangement of mitochondrial DNA (mtDNA) in unfertilized human oocytes and compromised embryos to evaluate the fertilization capacity of oocytes. DESIGN: Prospective laboratory research. SETTING: IVF laboratory in a university hospital. PATIENT(S): One hundred twenty-four unfertilized oocytes, 98 arrested embryos, and 45 tripronucleate (3PN) embryos from 65 female patients undergoing in vitro fertilization (IVF). INTERVENTION(S): Unfertilized oocytes and poor quality embryos were collected 48 hours after IVF. MAIN OUTCOME MEASURE(S): Comparison of the frequency of mtDNA deletions and fertilization rates of oocytes. RESULT(S): Multiple deletions of mtDNA were found in unfertilized oocytes and arrested embryos obtained from IVF patients. A 4977-bp deletion was the most frequent deletion in human oocytes and embryos. About 66.1% of the unfertilized oocytes, 34.8% of the arrested or fragmented embryos, and 21.1% of the 3PN embryos harbored the 4977-bp deletion of mtDNA. There was a significant increase in the proportion of deleted mtDNA in unfertilized oocytes. CONCLUSION(S): Accumulation of mtDNA deletions may contribute to mitochondrial dysfunction and impaired ATP production. We conclude that the accumulation of rearranged mtDNA may interfere with fertilization of human oocytes and further embryonic development.     

Problems in the cryopreservation of unfertilized eggs by slow cooling in dimethyl sulfoxide

The survival, fertilization, development, and viability in vitro and in vivo of unfertilized mouse eggs frozen by slow cooling to -36 degrees C or -80 degrees C in 1.5 M dimethyl sulphoxide (DMSO) was examined in a series of experiments which explored some of the problems in freezing the egg. DMSO was added to the eggs at either room temperature or at 0 degrees C. Maximum success rate (42% of frozen eggs developing to two cells) was obtained when DMSO was added at 0 degrees C and the eggs slow cooled to -80 degrees C. Removal of cumulus failed to improve freezing success rates. Addition of DMSO at temperatures above 0 degrees C significantly reduced the fertilizing capacity of eggs. Excessive exposure of eggs to temperatures around 15 degrees C also caused a significant reduction in fertilization rates. The effects of DMSO and cooling on fertilization are likely to be due to zona hardening by cortical granule release and to disorganization of the egg cytoskeleton and plasma membrane. These problems will be difficult to overcome if cryopreservation of the unfertilized human egg is preferred to the fertilized egg or early cleavage stage embryo in clinical in vitro fertilization.     

Long-term survival of human spermatogonial stem cells in mouse testes

OBJECTIVE: To evaluate colonizing ability of human spermatogonial stem cells in mouse testes. DESIGN: Transplantation of human testis cells into the seminiferous tubules of immunodeficient mice. SETTING: University hospital and academic laboratory. PATIENT(S): Men with obstructive azoospermia or maturation arrest of spermatogenesis.Analyzed up to 6 months after transplantation. Also analyzed: cryopreservation of donor cells, donor cell concentrations, and leuprolide treatment of recipients. MAIN OUTCOME MEASURE(S): Detection of human donor cells in recipient testes using whole-mount immunohistochemistry with antibodies that react with human germ cells. RESULT(S): Mouse testes were colonized by human testis cells obtained from each of 6 patients; overall, human spermatogonia were found in 16 of 22 (73%) recipient testes. Human spermatogonial stem cells survived in mouse testes for at least 6 months and proliferated during the first month after transplantation. No human-differentiating spermatogonia were identified, and meiotic differentiation did not occur in mouse testes. In this initial study, human stem cell colonization was not influenced by cryopreservation of donor cells, donor cell concentration, or leuprolide treatment of recipient mice. CONCLUSION(S): Xenogeneic transplantation of human germ cells using mice as recipients is feasible and could be used as a biological assay system to further characterize human spermatogonial stem cells. This study might provide a mechanism to evaluate the status of the stem cell population in selected infertile male patients.     

小鼠胚泡着床相关基因的差减筛选

目的:筛选新的小鼠胚泡着床相关基因。方法:对孕4.5 d小鼠着床和非着床位点的子宫内膜组织进行PCR差减。获得了2个新的在小鼠胚泡着床位点高表达的EST(EST8和EST81)。结果:EST8在孕4.5 d小鼠着床位点、肝脏中表达较高,在非着床位点和卵巢中亦有微量表达。EST81主要在孕4.5 d小鼠子宫着床组织和卵巢中表达,其它各种组织中也有微量表达。用PCR方法获得了相应的全长。cDNA,长度分别为1665bp和1364bp。结论:用差减筛选获得的两种cDNA是孕小鼠着床相关基因,其功能有待进一步研究。     

Circulating IL-8 and anti-IL-8 autoantibody in patients with ovarian cancer

OBJECTIVES: In an ongoing effort to identify diagnostic ovarian cancer biomarkers, SEREX (serological analysis of recombinant cDNA expression libraries) technique was employed resulting in detection of 20 known genes, nine ESTs and one novel sequence. Interleukin-8 (IL-8) was one of ovarian cancer-associated antigens identified by SEREX screening. The objective of this study was, therefore, to evaluate the potential importance of circulating anti-IL-8 antibody as ovarian cancer biomarker. METHODS: We developed and optimized a new immunofluorescent bead-based assay for detection of anti-IL-8 antibody in blood serum. Circulating IL-8 and anti-IL-8 IgG concentrations were measured in blood sera from 44 patients with early stage (I-II) ovarian cancer, 50 patients with late stage (III-IV) ovarian cancer, 37 patients with benign pelvic masses, and 80 healthy women using the bead-based assay. RESULTS: Our data indicate that serum contains IL-8 cytokine, anti-IL-8 antibody, and IL-8:anti-IL-8 complexes. We found that concentrations of IL-8 and anti-IL-8 antibody were elevated in sera of patients with ovarian cancer as compared with healthy controls. Logistic regression analysis of circulating concentrations of anti-IL-8 IgG in patients with stages I-II ovarian cancer versus healthy controls allowed for prediction of early ovarian cancer with 98% specificity, 65.5% sensitivity, 80.3% of patients correctly classified. Combining IL-8 and anti-IL-8 IgG with CA 125 resulted in increased classification power as compared to individual markers analyzed separately. CONCLUSION: Thus, IL-8 and anti-IL-8 autoantibody might potentially serve as additional biomarkers for ovarian cancer.     

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.     

Isolation and expression analysis of the testis-specific gene,STRA8, stimulated by retinoic acid gene 8

Retinoids are known to be required for vertebrate reproduction, and in the male, for the maintenance of normal testicular structure and function. Previously several novel retinoic acid responsive genes, collectively designated as the Stra genes, had been isolated in the mouse. The Stra8 gene encodes a cytoplasmic protein and is expressed specific to the developing male gonad during mouse embryogenesis. In adult mouse, its expression is restricted to the premeiotic germ cells. Thus it has been suggested that the mouse Stra8 protein may play a role in the premeiotic phase of spermatogenesis.Recently a lot of genes that are expressed only in male germ cells have been isolated in the mouse. The mouse Stra8, Rnh2, Piwil2, Tex17, and Tuba7 were identified as testis-specific expressed genes. In addition, the Figla was known to be a testis- and ovary-specific gene. Recently we had reported the isolation of the human RNH2 cDNA and its expression, which is limited to the human testis. In the present study, we have isolated full-length cDNA of STRA8 and partial cDNAs of PIWIL2, FIGLA, TEX17, and TUBA7, and analyzed their expression patterns in human tissues.     

Cyclin-dependent kinase 5 is expressed in both Sertoli cells and metaphase spermatocytes

OBJECTIVE: To gain insight into the function of cyclin-dependent kinase 5 (Cdk5) in spermatogenesis. DESIGN: The expression of the Cdk5 protein was determined with the use of immunohistochemical and immunoblot analysis. SETTING: Academic research laboratory. ANIMAL(S): Adult mouse and archival human testicular tissue were used for the immunohistochemical analysis. Adult mice were used as the source of tissues for the immunoblot analysis. INTERVENTION(S): The immunohistochemical analysis was performed with an anti-Cdk5 antibody. The double immunohistochemical analysis was performed with anti-Cdk5 and alpha-tubulin antibodies. Immunoblotting was used to examine multiple mouse tissues for Cdk5 expression. MAIN OUTCOME MEASURE(S): Analysis of Cdk5 protein distribution. RESULT(S): Cdk5 was localized specifically within the cytoplasm of Sertoli cells and meiotic metaphase germ cells. The double immunohistochemistry analysis demonstrated the co-localization of Cdk5 and alpha-tubulin within the Sertoli cells. Western blot analysis revealed a high level of expression of Cdk5 in the testicular lysate. CONCLUSION(S): The cyclin-dependent kinases are known regulators of the cell cycle; however, Cdk5 expression previously has been described in terminally differentiated cells of the brain. The present evidence of an association between Cdk5 and microfilaments of Sertoli cells and meiotic metaphase germ cells suggests a role of Cdk5 in both seminiferous tubule function and meiosis.