OBJECTIVETo investigate the possibility of applying multiplex ligation-dependent probe amplification (MLPA) to the detection of azoospermia factor (AZF) microdeletion on the Y chromosome in infertile men with azoospermia or severe oligozoospermia.

METHODSDNA samples were obtained from 147 azoospermia or severe oligozoospermia patients and 154 normal controls. After denatured at 95 degrees C, the samples were hybridized to the specific probes designed for the AZF region. With the ligase, the hybrid products were amplified by a pair of universal primers labeled with FAM fluorescence, and then separated by capillary electrophoresis for data analysis. Meanwhile all the samples were subjected to multiplex-PCR (mPCR) analysis for sequence-tagged sites (STS) in the AZF region.

RESULTSSTS deletion was detected in 22 (15.0%) of the 147 patients but not in the normal controls. By MLPA, 40 (27.2%) of the patients were found with specific probe omission in the AZF region, as compared with 20 cases in the control group.

CONCLUSIONCompared with mPCR, MLPA has a better sensitivity in detecting AZF microdeletions, and it provides more precise genetic information on the AZF regions, which may contribute to in-depth exploration into the etiological mechanism of impaired spermatogenesis.

Adult ; Azoospermia ; genetics ; Case-Control Studies ; Chromosome Deletion ; Chromosomes, Human, Y ; genetics ; DNA Probes ; Genetic Loci ; Humans ; Infertility, Male ; Male ; Nucleic Acid Amplification Techniques ; methods ; Oligospermia ; genetics ; Polymerase Chain Reaction ; methods ; Seminal Plasma Proteins ; genetics ; Sequence Tagged Sites ; Sex Chromosome Aberrations ; Sex Chromosome Disorders of Sex Development ; genetics ; Young Adult

BACKGROUND: In addition to Klinefelter's syndrome, microdeletion of Yq is the most common genetic cause of male infertility; 15% of azoospermic or 5-10% of oligozoospermic males have Yq deletions. We evaluated a Yq microdeletion kit (LG Life Sciences, Korea) for identifying microdeletions in the azoospermic factor (AZF) regions of the Yq. METHODS: The kit was designed to amplify 3 regions of the AZF gene (AZFa, AZFb, and AZFc) using 15 sequence-tagged sites. We evaluated the preclinical performance of the kit. For clinical validation, 58 patients including 25 idiopathic azoospermic or oligozoospermic patients were examined. RESULTS: We observed clear bands on electrophoresis of DNA, up to a DNA concentration of 3.12 ng/microliter; the known microdeletion regions of all 6 reference cell-lines (Coriell, USA) were accurately detected and no false positive/negative results showed with normal female (n=11) and fertile male (n=15) specimens. This kit could identify the same microdeletions in the common regions, similar to another commercial kit. Among the 58 male infertile patients, 7 (12.1%) had microdeletions of the Yq. Among the idiopathic azoospermic (n=22) and oligozoospermic (n=3) patients, 3 (12.0%) had microdeletions. Further, 2 of 21 varicocele patients (9.5%), 1 of 4 patients with testicular failure, and 1 patient with a 45,X/46,XY mosaic had microdeletions. CONCLUSIONS: The kit was effective for detecting microdeletions of the Yq. We identified microdeletions in 12% of the infertile patients. This Y chromosome microdeletion detection kit is useful for screening Yq microdeletions in infertile patients.
Azoospermia/genetics ; *Chromosome Deletion ; *Chromosomes, Human, Y ; Electrophoresis, Agar Gel/methods ; Female ; Humans ; Infertility, Male/genetics ; Male ; Oligospermia/genetics ; Polymerase Chain Reaction/*methods ; Reagent Kits, Diagnostic ; Reproducibility of Results ; Seminal Plasma Proteins/*genetics ; Sensitivity and Specificity ; Varicocele/genetics

OBJECTIVETo investigate the relationship between the partial deletions in the azzospermia factor(AZFc) region of Y chromosome and male infertility.

METHODSMultiplex PCR technology was performed to screen the partial deletions in the AZFc region in 158 azoospermia, 160 severe oligozoospermia and 135 oligozoospermia patients and 236 men with normal spermatogenesis.For samples with gr/gr, b2/b3 recombinogenic deletions, author applied RFLP method to identify which DAZ gene doublet deletion was involved.

RESULTSThe gr/gr and b2/b3 were two types of common deletions detected. There were significant differences in the b2/b3 deletion in patients with oligozoospermia and severe oligozoospermia compared to the controls (both P< 0.05). However, there was no difference for the gr/gr deletion between the patients and controls.

CONCLUSIONThe results suggested that the b2/b3 deletion might be a risk factor to spermatogenic impairment and might lead to male infertility.

Chromosomes, Human, Y ; genetics ; Genetic Loci ; Humans ; Infertility, Male ; genetics ; Male ; Polymerase Chain Reaction ; Seminal Plasma Proteins ; genetics ; Sequence Deletion ; genetics

OBJECTIVETo study the incidence of the chromosome abnormalities and Y chromosome microdeletions in Chinese patients with azoospermia and cryptozoospermia.

METHODSConventional chromosomal karyotyping was used to analyze the chromosome abnormalities. Genomic DNA was extracted from peripheral blood samples and multiplex polymerase chain reactions (PCR) analyses were performed using specific primers to confirm the presence or absence of Y chromosome microdeletions. A total of 997 patients with azoospermia and cryptozoospermia were enrolled in the study.

RESULTSThe incidence of chromosome abnormalities in the patient with azoospermia and cryptozoospermia was 28.4%. The major abnormal karyotypes included 47,XXY, 46,XY (Y>G), 46,XX, chimera and translocations. The incidence of the Y chromosome microdeletions was 17.4%. They were mainly found in the karyotypes of 46,XY and 46,XY (Y>G).

CONCLUSIONChromosome abnormalities were the most common hereditary causes of the patients with azoospermia and cryptozoospermia. The incidence of Y chromosome microdeletion was higher in the patients with karyotype of 46,XY and 46,XY (Y>G). Therefore, detection of the AZF microdeletion in these patients is helpful to determine the etiology and avoid the unnecessary treatment and vertical transmission of the genetic defects.

Azoospermia ; genetics ; Chromosome Deletion ; Chromosomes, Human, Y ; genetics ; Female ; Genetic Testing ; Humans ; Infertility, Male ; genetics ; Male ; Middle Aged ; Oligospermia ; genetics ; Seminal Plasma Proteins ; genetics

OBJECTIVETo construct mouse Zinc-alpha2-glycoprotein (mZAG) eucaryotic expression plasmid and identify its expression in 3T3-L1 preadipocytes.

METHODSThe total RNA from mouse liver tissue was extracted. The reverse-transcript(RT)-PCR method was used to amplify the complete domain sequence of mZAG, and the confirmed PCR products was inserted into expression plasmid by DNA ligation. The mZAG expression plasmids with various concentrations (0, 0.4, 0.8, and 1.6 microg) were transfected into 3T3-L1 preadipocytes, and ZAG expression in mRNA and protein level was determined by real-time fluorescence quantitative PCR and Western blot, respectively.

RESULTSDNA sequencing confirmed the right sequence of mZAG expression plasmid pcDNA3.1(-)-mZAG. After the mZAG expression plasmid with different concentrations were transfected into 3T3-L1 preadipocytes, mZAG mRNA level significantly increased and reached 2.58 folds (P=0.002), 3.67 folds (P=0.000 and 5.19 folds (P=0.001) of that in the control group (no mZAG transfection). mZAG protein level also significantly increased and reached 2.75 folds of that in the control group (P=0.017). Treating 3T3-L1 cells with small interfering RNA (siRNA) sequence siRNA 1 and siRNA 4 resulted in a decrease of mZAG mRNA to 49% and 41% of those in the control group(no siRNA sequence transfection) (P=0.002P=0.000)and a decrease of mZAG protein to 55% and 62% of that in the control group (P=0.004,P=0.025).

CONCLUSIONSmZAG expression plasmid pcDNA3.1(-)-mZAG was successfully established in this study. This plasmid can be well expressed in 3T3-L1 preadipocytes. siRNA 1 and siRNA 4 can effectively inhibit the expression of mZAG in these cells.

3T3-L1 Cells ; Adipocytes ; metabolism ; Animals ; Genetic Vectors ; Mice ; Plasmids ; genetics ; RNA, Small Interfering ; genetics ; Seminal Plasma Proteins ; genetics ; metabolism ; Transfection

OBJECTIVEThe past few years have seen great progress in the studies of the relationship between AZF microdeletions and male infertility. However, some molecular and clinical concerns are not supported by definitive data. The aim of this study was to investigate the prevalence and types of AZF microdeletions in infertile Chinese men, and the indications for genotype-phenotype correlation.

METHODSWe retrospectively analyzed Y chromosome AZF microdeletions among 502 patients with nonobstructive azoospermia and 306 with severe oligozoospermia received in our hospital during the past five years.

RESULTSMicrodeletions were detected in 7.80% of the patients (63/808), 9.16% in the men with nonobstructive azoospermia (46/502) and 5.56% in those with severe oligozoospermia (17/306). Complete AZFa and AZFb (P5/Proximal P1) deletions were associated with azoospermia, whereas AZFc deletion with variable spermatogenic phenotypes. A mild decline in sperm concentration was found in one male with partial AZFb deletion. The most frequent deletion was the AZFc b2/b4 subtype (60.32%, 38/63), and 39.47% of the cases (15/38) had sperm in the ejaculate. Of the 63 deletions, only one case of the AZFc b2/b4 type had a sperm concentration of over 2 million sperm/ml.

CONCLUSIONAZF microdeletions play a significant role in the diagnosis and evaluation of spermatogenic defects. Larger-scale clinical researches on Y chromosome microdeletions may give us a deeper insight into their mechanism and the genotype-phenotype relationship.

Adult ; Asian Continental Ancestry Group ; genetics ; Azoospermia ; genetics ; Chromosome Deletion ; Chromosomes, Human, Y ; Genetic Loci ; Humans ; Male ; Middle Aged ; Oligospermia ; genetics ; Retrospective Studies ; Seminal Plasma Proteins ; genetics ; Sequence Deletion ; Young Adult

OBJECTIVETo explore the effects of zinc-alpha2-glycoprotein (ZAG) on body weight and body fat in high-fat-diet (HFD)-induced obesity in mice and the possible mechanism.

METHODSThirty-six male mice were fed with standard food (SF) (n = 9) and HFD (n = 27), respectively. Five weeks later, 9 mice fed with HFD were subjected to ZAG expression plasmid DNA transfection by liposome transfection method, and another 9 mice to negative control plasmid transfection. Two weeks later, serum ZAG level in the mice was assayed by Western blot, and the effects of ZAG over-expression on body weight, body fat, serum biochemical indexes, and adipose tissue of obese mice were evaluated. The mRNA expressions of fatty acid synthase (FAS) and hormone-sensitive lipase (HSL) in liver tissue were determined by reverse transcription-polymerase chain reaction.

RESULTSSerum ZAG level significantly lowered in simple HFD-fed mice in comparison to SF-fed mice (0.51 +/- 0.10 AU vs. 0.75 +/- 0.07 AU, P < 0.01). Further statistical analysis demonstrated that ZAG level was negatively correlated with body weight (r = -0.56, P < 0.001), epididymal fat mass (r = -0.67, P < 0.001), percentage of epididymal fat (r = -0.65, P < 0.001), and increased weight (r = -0.57, P < 0.001) in simple SF- and HFD-fed mice. ZAG over-expression in obese mice reduced body weight and the percentage of epididymal fat. Furthermore, FAS mRNA expression decreased (P < 0.01) and HSL mRNA expression increased (P < 0.001) in the liver in ZAG over-expressing mice.

CONCLUSIONSZAG is closely related to obesity. Serum ZAG level is inversely correlated with body weight and percentage of body fat. The action of ZAG is associated with reduced FAS expression and increased HSL expression in the liver of obese mice.

Adipose Tissue ; metabolism ; Animals ; Fatty Acid Synthases ; genetics ; physiology ; Liver ; enzymology ; Male ; Mice ; Mice, Obese ; Seminal Plasma Proteins ; blood ; physiology ; Sterol Esterase ; genetics ; physiology ; Weight Loss

OBJECTIVETo investigate the incidence of abnormal karyotypes and Y chromosome microdeletion in Chinese men with azoospermia, and the relationship with reproductive hormones.

METHODSFour hundred and eighty nine cases of azoospermic patients and 20 fertile men were studied. Karyotypes and Y chromosome microdeletion were analyzed by G-banding and mutiplex polymerase chain reaction, respectively. Chemiluminescene immunoassay technique was applied to measure the serum levels of follicle-stimulating hormone (FSH), luteinizing hormone (LH), testosterone (T), and prolactine (PRL).

RESULTSChromosome abnormalities were found in 102 out of 489 azoospermic patients (20.86%), among them 86 (84.31%) cases had sex chromosome abnormalities, with 73 cases being Klinefelter syndrome. Y chromosome microdeletions were detected in 58 (11.86%) cases out of the 489 patients, and deletion of the AZFc region was the leading group (63.8% of all deletions), followed by AZFbc (19.0%), AZFabc (10.3%), AZFb or AZFa (3.4%). FSH, LH levels were significantly increased and T level was decreased in azoospermic patients compared with the fertile men group (P<0.01). Furthermore, in the azoospermic patients with Klinefelter syndrome or AZFabc microdeletions, FSH and LH levels were increased more significantly, and were statistically different from azoospermic patients with normal karotype or without Y chromosome microdeletion (P<0.05).

CONCLUSIONIn the Chinese men with azoospermia, the incidence of abnormal karyotype and Y chromosome microdeletion were similar to those described previously in other populations. In azoospermia with Klinefelter syndrome or AZFabc microdeletions, FSH and LH levels increased markedly indicating the protracted stimulation of gonadotrophs due to lack of androgen feedback.

Adult ; Azoospermia ; blood ; genetics ; Case-Control Studies ; Chromosomes, Human, Y ; genetics ; Follicle Stimulating Hormone ; blood ; Genetic Association Studies ; Genetic Loci ; Humans ; Karyotyping ; Luteinizing Hormone ; blood ; Male ; Prolactin-Releasing Hormone ; blood ; Seminal Plasma Proteins ; genetics ; Sequence Deletion ; Testosterone ; blood

OBJECTIVETo analyze the characteristics of azoospermia factor(AZF) deletions in Y-chromosome.

METHODSBased on the AZF microdeletion screening on 272 cases of azoospermia and 240 cases of severe oligozoo spermia, 49 cases were investigated using 23 sequence-tagged sites (STS) in AZFa, AZFb and AZFc. For some cases, single nucleotide rarians (SNV) method was applied to identify the single nucleotide polymorphism (SNPs) in four DAZ gene copies and to determine the copy number of the DAZ gene.

RESULTSIn 6 cases with deletions of AZFb+c, there was 1 case with sY98/sY1206 deletion, 4 cases with P5/distal-P1 recombination and 1 with P4/distal-P1 recombination. In 3 cases with deletions in AZFb, 1 case showed P5/P3 deletion and 2 cases showed P5/proximal-P1 recombination with DAZ1 and DAZ2 deletions. b2/b4 recombination was observed in all the 40 cases with deletions in AZFc. A fraction of patients with AZFb and AZFb+c deletions showed oligospermia and spermatogenic failure by testicular biopsy.

CONCLUSIONBreakpoint localization of deletions in AZF regions may help elucidating the mechanisms of microdeletions, and analysis of the characteristics and quantity of deleted genes essential for normal spermatogenesis may evaluate the association of phenotype with spermatogenic failure.

Chromosome Deletion ; Chromosomes, Human, Y ; genetics ; Deleted in Azoospermia 1 Protein ; Gene Dosage ; Genetic Loci ; Humans ; Male ; Oligospermia ; genetics ; physiopathology ; RNA-Binding Proteins ; genetics ; Seminal Plasma Proteins ; genetics ; Spermatogenesis

Fertilization is a complex process involving multiple steps, of which sperm-egg fusion is most important. This article presents a detailed review of some of the key sperm membrane proteins closely related with fertilization, such as the Izumo, the ADAMs gene family and the Crisp gene family proteins, which is of practical significance for deeper insights into the mechanisms of sperm-egg fusion, as well as for the improvement of clinical diagnosis of male infertility and development of novel contraceptive drugs.
Animals ; Cell Fusion ; Gene Expression ; Humans ; Male ; Membrane Proteins ; genetics ; metabolism ; Oocytes ; cytology ; metabolism ; Seminal Plasma Proteins ; genetics ; metabolism ; Sperm-Ovum Interactions ; Spermatozoa ; cytology ; metabolism