In order to make a series of analyses on the gene expression of Eimeria tenella sensitive strain and anti-maduramycin strain at their different developmental stages, we constructed a mixed cDNA library with unsporulated oocyst, sporulated oocyst, sporozoite and merozoite from Eimeria tenella sensitive strain and antimaduramycin strain (induced by its sensitive strain)respectively. After sequencing reactions, the total 2806 high quality expressed sequence tags (ESTs) of 3' ends were derived from the cDNA library. Results of bioinformatics analysis of all EST data showed that EST sequences assembled 1424 tentative unique transcripts (TUTs) and the redundancy was 49.3%, and that about 83.6% TUTs could not be assigned for functional description. Among the remained annotated genes, infection related proteins and development related proteins such as MIC2 protein, BT1 family protein and some ribosomal protein expressed at high abundant level.
Eimeria tenella ; genetics ; Expressed Sequence Tags ; Gene Library ; Sequence Tagged Sites

OBJECTIVETo evaluate the frequency of microdeletions in the long arm of Y chromosome of idiopathic infertile males with azoospermia and oligospermia in Shaanxi province in China and to investigate the relevance of sperm count to Y microdeletion frequencies.

METHODSAccording to the sequence of sequence-tagged sits (STS) AZFa, AZFb, AZFc and SRY, 4 of the azoospermic factor regions on Y chromosome long-term supplied by GenBank, 5 sets of primers were synthesized. The Y microdeletions in AZF regions were screened by polymerase chain reaction (PCR) in 64 idiopathic cases of azoospermia and oligospermia and 20 men of known fertility.

RESULTSNo microdeletion was detected in the 20 normospermic subjects. Deletion of the AZFc/DAZ was detected in 11 individuals and one patient had both AZFb and AZFc deletion; no deletion of AZFa and SRY region was found. The frequency of Y microdeletions in the subgroups with different sperm count showed the highest value among azoospermic men (3 cases, 21.4%). The percentage progressively decreased with the deletion frequency (20.0%, 17.9% and 8.3%) in the subgroups with sperm counts of < 1 x 10(6)/ml, < (1-5) x 10(6)/ml and < (1 to approximately 10) x 10(6)/ml, respectively.

CONCLUSIONY chromosome microdeletions are specifically associated with severe spermatogenic failure. The rate of deletion involving AZF region of the Y-chromosome is higher in infertile men with azoospermia and oligospermia. PCR amplification of AZF locus is useful for the diagnosis of microdeletions in the Y-chromosome.

Adult ; China ; Chromosome Deletion ; Chromosomes, Human, Y ; Humans ; Male ; Oligospermia ; genetics ; Polymerase Chain Reaction ; Sequence Tagged Sites

OBJECTIVETo investigate the breakpoints of the azoospermia factor c (AZFc) microdeletion in Chinese Han population.

METHODSWe detected 9 sequence tagged sites (sY84, sY86, sY127, sY134, sY152, sY145, sY255, sY254 and sY157) to confirm AZFc microdeletions in the Y chromosome for patients with severe oligozoospermia or non-obstructive azoospermia by multiplex polymerase reaction. To locate the breakpoints of AZFc microdeletions, we analyzed 192 patients with sY255, sY254 and sY157 dele- ted by detecting sY1191, sY1197, sY1054, sY1125 and sY1206, respectively.

RESULTSFive breaking patterns were found in the 192 patients with sY255, sY254 and sY157 deleted, among which the common ones were sY1197(+), sY1191(-), sY1054(-), sY1206(-) and sY1125(+), which accounted fro 54.17% (104/192), sY1197(+), sY1191(+), sY1206(-), sY1054(-) and sY1125(+), which constituted 28.12% (54/192), sY1197(+), sY1191(-), sY1206(-), sY1054(+) and sY1125 (+), which made up 14.58% (28/192). The proximal breakpoint located between sY1197 and sY1191 was 70.83% of AZFc microdeletions, and the distant breakpoint located between sY1054 and sY1125 was 82.29%.

CONCLUSIONThere are 5 breaking patterns of AZFc microdeletions in Chinese Han population, the proximal and distant breakpoints mostly located at the replicons b2 and b4, respectively.

Asian Continental Ancestry Group ; genetics ; Azoospermia ; genetics ; Chromosome Deletion ; Chromosomes, Human, Y ; Humans ; Infertility, Male ; genetics ; Male ; Sequence Tagged Sites

The objective of this study was to elucidate the cause of the spermatogenic defect in idiopathic azoospermia and non-mosaic type of Klinefelter syndrome. Genomic DNAs from 9 cases of Korean idiopathic azoospermia and 6 of Korean non-mosaic type of Klinefelter syndrome were used for the detection of Y chromosome microdeletions by polymerase chain reaction using 60 primers. Microdeletions of the Y chromosome were found in 1 of 9 (11.1%) patients with idiopathic azoospermia, whereas none was deleted in non-mosaic type of Klinefelter syndrome. This result suggests that Y chromosome microdeletions could be one of the etiologic factors in idiopathic azoospermia.
Gene Dosage ; Human ; Klinefelter Syndrome/classification/*genetics ; Male ; Oligospermia/classification/*genetics ; Polymerase Chain Reaction ; *Sequence Deletion ; Sequence Tagged Sites ; Spermatogenesis ; X Chromosome/genetics ; Y Chromosome/*genetics

OBJECTIVETo investigate the genetic causes of azoospermia and severe oligozoospermia.

METHODSCytogenetic analysis and multiplex polymerase chain reaction(PCR) analysis were done on the 148 patients with azoospermia and serious oligozoospermia.

RESULTSEleven of the 148(7.4%) cases showed microdeletion of at least one STS. In fifteen STS of AZFa, AZFb,AZFd, AZFc, thirteen STS, eleven STS,two STS and one STS microdeletion were found in each case respectively, including two with 12 STS, five with 5 STS microdeletion.Seven cases had chromosomal morphologic changes(4.7%),four had deletion and one had deletion with translocation of long arm on Y chromosome. One had enlarged region one band two(q12) on long arm of Y chromosome and one had reciprocal translocation of autosomes.

CONCLUSIONThe findings indicated that AZF microdeletion and chromosomal abnormality should be important causes of male infertility.

Azoospermia ; genetics ; Chromosome Aberrations ; Chromosomes, Human, Y ; genetics ; Genetic Loci ; Humans ; Male ; Oligospermia ; genetics ; Polymerase Chain Reaction ; Seminal Plasma Proteins ; genetics ; Sequence Deletion ; Sequence Tagged Sites

This study examined the gene expression patterns of peripheral blood mononuclear cells (PBMCs) in patients with systemic lupus erythematosus (SLE) by using serial analysis of gene expression (SAGE) technology. Following the construction of serial analysis of gene expression (SAGE) library of PBMCs collected from 3 cases of familial SLE patients, a large scale of tag sequencing was performed. The data extracted from sequencing files was analyzed with SAGE 2000 V 4.5 software. The top 30 expressed genes of SLE patients were uploaded to http://david.niaid.nih.gov/david/ease.htm and the functional classification of genes was obtained. The differences among those expressed gene were analyzed by Chi-square tests. The results showed that a total of 1286 unique SAGE tags were identified from 1814 individual SAGE tags. Among the 1286 unique tags, 86.8% had single copy, and only 0.2% tags had more than 20 copies. And 68.4% of the tags matched known expressed sequences, 41.1% of which matched more than one known expressed sequence. About 31.6% of the tags had no match and could represent potentially novel genes. Approximately one third of the top 30 genes were ribosomal protein, and the rest were genes related to metabolism or with unknown functions. Eight tags were found to express differentially in SAGE library of SLE patients. This study draws a profile of gene expression patterns of PBMCs in patients with SLE. Comparison of SAGE database from PBMCs between normal individuals and SLE patients will help us to better understand the pathogenesis of SLE.
Expressed Sequence Tags ; Gene Expression Profiling/*methods ; Leukocytes, Mononuclear/*metabolism ; Lupus Erythematosus, Systemic/blood ; Lupus Erythematosus, Systemic/*genetics ; Sequence Tagged Sites ; Transcription, Genetic

OBJECTIVETo isolate expressed sequence tags (ESTs) related to K562 cells erythroid differentiation.

METHODSModified differential display reverse transcription polymerase chain reaction (DDRT-PCR) method was applied to identify differential ESTs in uninduced and induced K562 cells by HEMIN for 36 hours. Remarkable differential ESTs were firstly selected for cloning, sequencing and bioinformational analyzing. Several ESTs representing new sequence or providing functional clue were selected for Northern blot analysis.

RESULTSSixty differentially expressed cDNA fragments related to K562 cells inducted into erythroid differentiation by HEMIN were obtained. Among them, 38 were upregulated and 22 downregulated. Among the 40 differential ESTs selected for cloning, sequencing and bioinformationally analyzing, 23 were found to match to known GenBank sequences and 10 represented cDNA sequences with only dbEST database matches and 7 ESTs have no any database matches. The results of 6 in 8 ESTs selected for Northern blot analysis were shown to be consistent with the differential expressions of DDRT-PCR.

CONCLUSIONSThe improved DDRT-PCR method had successfully overcome the problem of false positive. These ESTs provide some clue for studying the molecular mechanisms and regulation network of erythroid differentiation.

Cell Differentiation ; drug effects ; Cell Transformation, Neoplastic ; drug effects ; Erythroid Cells ; cytology ; Expressed Sequence Tags ; Hemin ; pharmacology ; Humans ; K562 Cells ; cytology ; metabolism ; Sequence Tagged Sites

Cytogenetic observations of loss of the distal portion of the Y chromosome long arm were found to be associated with disrupted spermatogenesis. The existence of a gene involved in the regulation of spermatogenesis, the azoospermia factor (AZF), was postulated. In this study, we screened the AZF region including DAZ and DAZH genes and observed the expression pattern of DAZ and DAZH transcript in infertile men with azoospermia and oligospermia by using a sequence-tagged site (STS)-based PCR method. PCR primers were synthesized for 11 STSs that span Yq interval 6, SRY, DAZ, and DAZH, functional DAZ homologue on chromosome 3. Microdeletions were detected in 4/32 (12.5%) azoospermic men and 1/11 (9%) severe oligospermic men. Only 2 of 5 patients had microdeletions of Yq that contained the 342 gene, whereas the other 3 patients had deletions extending from intervals 5L-6F proximal to the DAZ gene on Yq. Testis biopsies of the azoospermic patients revealed a variety from Sertoli cell-only syndrome to testicular maturation arrest. Of 4 men with clinical data available, average testis size was R: 13.8 co, L: 13.8 co, serum T was 4.0+/-1.25 ng/ml, LH was 3.63+/-1.90 mIU/ml, and FSH was 8.85 +/- 5.13 mIU/ml. These values did not differ significantly from the remainder of the patients tested. We could not observed the DAZ transcript in 2 patients, who have no mature spermatozoa. In 11,6% of patients microdeletions of the AZF could be detected. These deletions in the AZF region seem to be involved causing spermatogenic failure. But the frequency of microdeletions proximal to DAZ suggests that DAZ is not the only gene associated with spermatogenic failure.
Arm ; Azoospermia* ; Biopsy ; Chromosomes, Human, Pair 3 ; Cytogenetics ; Genes, vif ; Humans ; Male ; Oligospermia ; Polymerase Chain Reaction ; Sequence Tagged Sites ; Sertoli Cell-Only Syndrome ; Spermatogenesis ; Spermatozoa ; Testis ; Y Chromosome*

Purpose: The prevalence of microdeletion of the Y chromosome is 13% in non-obstructive azoospermic patients. Klinefelter's syndrome may be found in about 11% of azoospermic patients. The prevalence and correlation of microdeletion of the Y chromosome in Klinefelter's syndrome, which is the most common cause of chromosomal disorders in male infertility, were investigated. Materials and Methods: Hormone tests (Testosterone, LH and FSH) were performed and peripheral genomic DNA of 82 patients detected as Klinefelter's syndrome between September 2001 and December 2003. The microdeletion of the Y chromosome was examined by a PCR technique. The primers used for the PCR were Sequence-Tagged sites (STS) of the long arm of the Y chromosome (sY84, sY129, sY134, sY254 and sY255) and SRY (control). Results: The mean age, and values of testosterone, LH and FSH in the 82 Klinefelter's syndrome patients were 32.71 3.13 years, 1.84 1.31ng/ml, 14.88+/-5.38mlU/ml and 38.79 12.40mlU/ml, respectively. No patient in this study was found to have Y chromosomal microdeletion. Conclusions: As the role of the Y chromosome in the spermatogenesis of male is well known, microdeletion of the Y chromosome causes severe damage to the spermatogenesis in infertile males. A microdeletion of the Y chromosome could not be detected in patients with Klinefelter's syndrome. Therefore, multiple factors or other mechanisms that influence the defect of spermatogenesis in Klinefelter's syndrome may exist.
Arm ; Chromosome Deletion ; Chromosome Disorders ; DNA ; Humans ; Infertility, Male ; Klinefelter Syndrome* ; Male ; Polymerase Chain Reaction ; Prevalence ; Sequence Tagged Sites ; Spermatogenesis ; Testosterone ; Y Chromosome*

OBJECTIVES: To estimate the frequency of Y chromosome microdeletions in the Korean population of infertile men and to evaluate the relationship between microdeletion on the Y chromosome and clinical phenotypes of infertile men with idiopathic azoospermia and oligozoospermia. MATERIALS AND METHODS: Genomic DNA was extracted from blood samples collected from 330 infertile men attending the Infertility Clinic at Samsung Cheil Hospital, Korea. Six sequence tagged sites (STSs) spanning the azoospermia factor (AZF) regions of the Y chromosome were amplified by polymerase chain reactions (PCRs). RESULTS: Microdeletions on Y chromosome were detected in 35 (10.6%) of the 330 infertile men. Most of the microdeletions (91.4%) involved AZFb or AZFc. The high incidence of microdeletions were found in AZFc region (57.1%), but the low in AZFa (8.6%) and AZFb (5.7%). Larger microdeletions involving two or three AZF regions were detected in 28.6% of cases. All patients (6 patients) with deletion of AZFa region showed no germ cell phenotypes, Sertoli cell only syndrome or Leydig cell hyperplasia in histopathologic examinations. CONCLUSION: Microdeletions on the Y chromosome, especially, at AZFc/DAZ regions may be the major cause of azoospermia and severe oligozoospermia. We suggest that idiopathic infertile men have genetic counselling and microdeletion analysis on the Y chromosome before IVF-ET and ART program.
Azoospermia ; DNA ; Germ Cells ; Humans ; Hyperplasia ; Incidence ; Infertility ; Korea ; Male ; Oligospermia ; Phenotype ; Polymerase Chain Reaction ; Sequence Tagged Sites ; Sertoli Cell-Only Syndrome ; Spermatogenesis* ; Y Chromosome*