Zinc alpha 2 glycoprotein as an early biomarker of diabetic nephropathy in patients with type 2 diabetes mellitus / Zinco-alfa2-glicoproteina (ZAG) como biomarcador precoce de nefropatia diabética em pacientes com diabetes mellitus tipo 2

Abstract Introduction: Although microalbuminuria remains the gold standard for early detection of diabetic nephropathy (DN), it is not a sufficiently accurate predictor of DN risk. Thus, new biomarkers that would help to predict DN risk earlier and possibly prevent the occurrence of end-stage kidney disease are being investigated. Objective: To investigate the role of zinc-alpha-2-glycoprotein (ZAG) as an early marker of DN in type 2 diabetic (T2DM) patients. Methods: 88 persons were included and classified into 4 groups: Control group (group I), composed of normal healthy volunteers, and three patient groups with type 2 diabetes mellitus divided into: normo-albuminuria group (group II), subdivided into normal eGFR subgroup and increased eGFR subgroup > 120 mL/min/1.73m2), microalbuminuria group (group III), and macroalbuminuria group (group IV). All subjects were submitted to urine analysis, blood glucose levels, HbA1c, liver function tests, serum creatinine, uric acid, lipid profile and calculation of eGFR, urinary albumin creatinine ratio (UACR), and measurement of urinary and serum ZAG. Results: The levels of serum and urine ZAG were higher in patients with T2DM compared to control subjects and a statistically significant difference among studied groups regarding serum and urinary ZAG was found. Urine ZAG levels were positively correlated with UACR. Both ZAG levels were negatively correlated with eGFR. Urine ZAG levels in the eGFR ˃ 120 mL/min/1.73m2 subgroup were higher than that in the normal eGFR subgroup. Conclusion: These findings suggest that urine and serum ZAG might be useful as early biomarkers for detection of DN in T2DM patients, detectable earlier than microalbuminuria.

Resumo Introdução: Embora a microalbuminúria continue sendo o padrão ouro para a detecção precoce da nefropatia diabética (ND), ela não é um preditor suficientemente preciso do risco de ND. Assim, novos biomarcadores para prever mais precocemente o risco de ND e possivelmente evitar a ocorrência de doença renal terminal estão sendo investigados. Objetivo: Investigar a zinco-alfa2-glicoproteína (ZAG) como marcador precoce de ND em pacientes com debates mellitus tipo 2 (DM2). Métodos: Os 88 indivíduos incluídos foram divididos em quatro grupos: grupo controle (Grupo I), composto por voluntários saudáveis normais; e três grupos de pacientes com DM2 assim divididos: grupo normoalbuminúria (Grupo II), subdivididos em TFG normal e TFG > 120 mL/min/1,73 m2), grupo microalbuminúria (Grupo III) e grupo macroalbuminúria (Grupo IV). Todos foram submetidos a urinálise e exames para determinar glicemia, HbA1c, função hepática, creatinina sérica, ácido úrico, perfil lipídico, cálculo da TFG, relação albumina/creatinina (RAC) e dosagem urinária e sérica de ZAG. Resultados: Os níveis séricos e urinários de ZAG foram mais elevados nos pacientes com DM2 em comparação aos controles. Foi identificada diferença estatisticamente significativa entre os grupos estudados em relação aos níveis séricos e urinários de ZAG. Os níveis urinários de ZAG foram positivamente correlacionados com a RAC. Ambos os níveis de ZAG foram negativamente correlacionados com TFG. Os níveis urinários de ZAG no subgrupo com TFG ˃ 120 mL/min/1,73m2 foram maiores do que no subgrupo com TFG normal. Conclusão: Constatamos que a ZAG sérica e urinária pode ser um útil biomarcador precoce para detecção de ND em pacientes com DM2, sendo detectável mais precocemente que microalbuminúria.

ODF4, MAGEA3, and MAGEB4: potential biomarkers in patients with transitional cell carcinoma

Background: This study aimed to evaluate the diagnostic value of outer dense fiber 4 [ODF4], melanoma associated antigen A3 [MAGEA3], and MAGEAB4 mRNAs in transitional cell carcinoma [TCC], using a small amount of cell reverse transcriptase-polymerase chain reaction [RT-PCR] on urinary exfoliated cells

Methods: We recruited a total of 105 suspected TCC patients and 54 sex- and age-matched non-TCC controls. The candidates' genetic expression patterns were investigated with RT-PCR, while reverse transcription quantitative PCR was applied to quantify and compare each mRNA level between cases and control groups

Results: The sensitivity of ODF4, MAGEA3, and MAGEAB4 RT-PCR was 54.8%, 63%, and 53.4%, whereas the specificity was 73.7%, 86%, and 94.7%, respectively. Combining ODF4, MAGEA3, and MAGEAB4 RT-PCR offered a relatively higher sensitivity [83.6%]

Conclusion: RT-PCR with ODF4, MAGEA3, and MAGEAB4 on urinary exfoliated cells could provide clinicians with a promising method to improve TCC diagnosis, especially in the case of gross hematuria and catheterization. The method used here is non-invasive, simple and convenient, and unlike cytology, it does not rely directly on expert professional opinions. These features can be of particular importance to the management of TCC patients in whom regular and lifelong surveillance is required

Correlation of seminal plasma zinc alpha-2 glycoprotein with semen quality in obese males / 中华男科学杂志

<p><b>Objective</b>To investigate the relationship between seminal plasma zinc alpha-2 glycoprotein (ZAG) and semen quality in obese males.</p><p><b>METHODS</b>This study included 130 obese male patients with idiopathic infertility Based on the concentration of seminal plasma ZAG, we divided the patients into three tertile groups: tertile 1 (T1, 73.45－97.15 μg/ml, n = 43), T2 (97.16－115.46 μg/ml, n = 44), and T3 (115.47－220.11 μg/ml, n = 43). We measured the concentrations of seminal plasma zinc (SPZ) and ZAG of the patients by ELISA, obtained the semen parameters, and analyzed the correlation of semen quality with the levels of SPZ and ZAG and the influence of obesity on SPZ, ZAG and semen quality.</p><p><b>RESULTS</b>The mean level of seminal plasma ZAG in the 130 obese male patients was (111.29 ± 26.50) μg/ml. There were statistically significant differences in sperm concentration and total sperm count among the three tertile groups (P < 0.05). The level of seminal plasma ZAG was correlated negatively with the body mass index (BMI), waist circumference (WC), sperm concentration and sperm count (P < 0.01), that of SPZ positively with BMI and WC (P < 0.05) but negatively with semen volume and the percentage of progressively motile sperm (P < 0.05). The level of serum ZAG, however, exhibited no correlation with SPZ, seminal plasma ZAG or semen quality. Obesity was found to be associated with significantly decreased concentration of seminal plasma ZAG and percentage of progressively motile sperm but remarkably increased level of SPZ (P < 0.05).</p><p><b>CONCLUSIONS</b>Obesity may induce the metabolic disorder of SPZ and ZAG, change the microenvironment of seminal plasma, and consequently affect semen quality.</p>

Correlation of serum zinc alpha 2 glycoprotein with blood lipid and reproductive hormone levels in men / 中华男科学杂志

Objective@#To study the changes of the serum zinc alpha 2 glycoprotein (ZAG) level in men and its relationship with blood lipid male reproductive hormones.@*METHODS@#We enrolled 297 men aged 25－ 65 years in this study, 152 with hyperlipemia (HL) and the other 145 with normal blood lipid (normal control). We divided them into four age groups (25－35 yr, 36－45 yr, 46－55 yr, and 56－65 yr) and three tertile groups (Q1, Q2, and Q3) according to the tertiles of the serum ZAG level, and examined their blood lipid, blood glucose, serum ZAG, and reproductive hormones.@*RESULTS@#The serum ZAG level was decreased gradually with the increase of age in both the HL patients and normal controls, significantly in the 36－45 and 56－65 yr age groups (P <0.05), and markedly lower in the HL than in the control men in the 25－35 and 36－45 yr groups (P <0.05). The levels of follicle-stimulating hormone (FSH) and total testosterone (TT) changed significantly with the ZAG level. The level of serum ZAG was correlated negatively with age (r = －0.58, P<0.05), waist circumference (r = －0.21, P <0.05), body mass index (BMI) (r = －0.22, P <0.05), fasting blood glucose (r = －0.16, P <0.05) , and triglyceride (TG) (r = －0.27, P <0.05) but positively with TT (r = 0.36, P <0.05). Age, BMI and TG were independent factors influencing the serum ZAG level.@*CONCLUSIONS@#The serum ZAG level is decreased with the increase of age and associated with lipid metabolism, abdominal obesity, and reproductive hormone levels in males.

Effect of curcumin on visfatin and zinc-α2-glycoprotein in a rat model of non-alcoholic fatty liver disease

ABSTRACT PURPOSE: To investigate the effect of curcumin on visfatin and zinc-α2-glycoprotein (ZAG) expression levels in rats with non-alcoholic fatty liver disease (NAFLD). METHODS: Fifty-six male rats were randomly divided into a control group (n=16) and model group (n=40) and were fed on a normal diet or a high-fat diet, respectively. Equal volumes of sodium carboxymethyl cellulose (CMC) were intragastrically administered to the control group for 4 weeks. At the end of the 12th week, visfatin and ZAG protein expression levels were examined by immunohistochemistry. Visfatin mRNA levels were measured by semi-quantitative reverse transcription polymerase chain reaction. RESULTS: Compared with the control group, the model group showed significantly increased expression of visfatin in liver tissue (P < 0.01) and significantly decreased expression of ZAG (P < 0.01). These effects were ameliorated by curcumin treatment. CONCLUSIONS: Visfatin and zinc-α2-glycoprotein may be involved in the pathogenesis of NAFLD. Treatment of NAFLD in rats by curcumin may be mediated by the decrease of visfatin and the increase of non-alcoholic fatty liver disease.

Recombinant expression and purification of the bovine acidic Seminal Fluid Protein

The acidic Seminal Fluid Protein (aSFP), a 12.9 kDa protein is a maker for bovine semen freezability possibly due to its antioxidant activity and effect on sperm mitochondrial function. However, its precise function on sperm preservation during freezing thaw is poorly understood. The use of recombinant DNA technology allows new approaches on the study of function and structure of proteins, and its production in procaryote systems offers several advantages. The present work describes the recombinant expression of the bovine aSFP and its binding properties. A cDNA library from the bovine seminal vesicle was used as template for amplification of the aSFP coding region. The amplicon was cloned into a pET23a (+) vector and transformed into E.coli BL21 pLysS strain. The recombinant expression was obtained in E coli. One step ion immobilized affinity chromatography was performed, resulting in high yield of purified protein. To determine the bioactivity of the r aSFP, the protein was incubated in different concentrations with 10 7 spermtozoa at 37°C for 5 h. Western blotting and fluorescence microscopy analyses showed the ability of the recombinant aSFP to attach to the spermatozoa. Based on our results, the described method can be used to obtain mg levels of recombinant aSFP.

Proteins in sperm and seminal plasma associated with human sperm resistance to cryopreservation / 中华男科学杂志

<p><b>OBJECTIVE</b>To identify the proteins that could improve the resistance of human sperm to cryopreservation using comparative proteomics.</p><p><b>METHODS</b>A total of 31 semen samples from 10 donors were divided into a high recovery and a low recovery group. Differentially expressed proteins in sperm and seminal plasma were detected and compared between the two groups by two-dimensional differential gel electrophoresis and mass spectrometry.</p><p><b>RESULTS</b>Totally, 22 differentially expressed proteins were found in the two groups, 12 seminal plasma proteins, 9 sperm proteins, and 1 belonging to both. These identified proteins were involved in the maturation, movement, energy metabolism, DNA repair and other activities of spermatozoa.</p><p><b>CONCLUSION</b>Many proteins were identified in sperm and seminal plasma that might influence the resistance of human sperm to cryopreservation.</p>

Abnormal semen liquefaction and seminal plasma lipoprotein (a) / 中华男科学杂志

<p><b>OBJECTIVE</b>To explore the effect of seminal plasma lipoprotein (a) in abnormal semen liquefaction and its clinical significance.</p><p><b>METHODS</b>According to The WHO Laboratory Manual for the Examination and Processing of Human Semen, we conducted semen routine analyses of 101 patients with abnormal semen liquefaction and 26 normal healthy controls. We added chymotrypsin to the semen for 30 minutes of incubation at 37 degrees C. When there were filaments, we centrifuged the semen and obtained the upper seminal plasma to determine the level of lipoprotein (a).</p><p><b>RESULTS</b>The level of lipoprotein (a) was significantly higher in the 101 patients ([526.2 +/- 243.5] mg/L) than in the 26 normal controls ([296.9 +/- 105.2] mg/L) (P < 0.01) .</p><p><b>CONCLUSION</b>Lipoprotein (a) can inhibit fibrin dissolution, and delayed fibrin dissolution in semen liquefaction may be related to the increased level of seminal plasma lipoprotein (a). The seminal plasma lipoprotein (a) level should be taken into account in the clinical diagnosis of male infertility caused by abnormal semen liquefaction.</p>

Expression of testis-specific genes, TEX101 and ODF4, in chronic myeloid leukemia and evaluation of TEX101 immunogenicity

Cancer-testis [CT] antigens are a group of antigens with a restricted expression in normal tissues, except testis, and they have aberrant expression in different tumors. This pattern of expression has made them promising targets for immunotherapy and cancer detection. Our aim was to find new members of this group that might be useful as markers in the detection of cancer and immunotherapy. A descriptive study conducted in referral centers of Tehran University of Medical Science from january 2008 to January 2009. We analyzed the expression of two testis-specific genes named ODF4 [outer dense fiber of sperm tails 4] and TEX101 [testis expressed 101] in 20 chronic myeloid leukemia [CML] and 20 normal samples by reverse transcription-polymerase chain reaction and sequencing. Immunogenicity of TEX101 was evaluated by means of enzyme-linked immunosorbent assay. These two genes were expressed in 30% of CML patients but not in any of the healthy donors. Humoral response against TEX101 was not detected in any samples. TEX101 and ODF4 are CT genes useful for detection of CML. Unlike many CT genes, overexpression of TEX101 was not shown to induce immunologic responses in these samples. According to the previous studies, overexpression of TEX101 leads to suppression of cancer invasion and metastasis; thus, the induction of the expression of TEX101 in cancer by epigenetic mechanisms may be a treatment strategy

Multiplex ligation-dependent probe amplification for detecting AZF microdeletions on the Y chromosome in infertile men with azoospermia or severe oligozoospermia / 中华男科学杂志

<p><b>OBJECTIVE</b>To 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.</p><p><b>METHODS</b>DNA 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.</p><p><b>RESULTS</b>STS 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.</p><p><b>CONCLUSION</b>Compared 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.</p>

Presencia de epimastigotes de Trypanosoma cruzi en el plasma seminal de ratones con infección aguda / Trypanosoma cruzi epimastigotes in seminal plasma in acute infected mice

Se reporta la presencia de formas evolutivas de Trypanosoma cruzi en el plasma seminal (PS) de ratones NMRI, inoculados por vía subcutánea con 2x104 tripomastigotes metacíclicos cepa P6 obtenidos de Rhodnius prolixus. Al separar las muestras de sangre a los 15 días pos-infección, un ratón eyaculó espontáneamente y el examen directo del PS reveló la presencia de formas epimastigotes de T. cruzi en activo movimiento mezclados con los espermatozoides. Las preparaciones del PS coloreadas con Giemsa, mostraron formas epimastigotes libres y en división, tripomastigotes y amastigotes extracelulares y dentro de células fagocíticas. Los resultados de este estudio revelaron los diferentes estadios de T. cruzi en el PS de ratón, con morfogénesis similar a como ocurre en el insecto vector. El parasitismo encontrado en el PS del ratón con infección aguda, aporta importante información epidemiológica sobre la vía de transmisión sexual de T. cruzi, principalmente entre la población de reservorios silvestres que se encuentran en áreas endémicas y no endémicas para la enfermedad de Chagas.

We report the presence of evolving forms of Trypanosoma cruzi in the seminal plasma (SP) of NMRI mice subcutaneously inoculated with 2x104 metacyclic trypomastigotes obtained from P6 strain Rhodnius prolixus. When taking blood samples at 15 days post-infection, the mouse spontaneously ejaculated and the direct SP exam revealed the presence of active epimastigotes of T. cruzi mixed with spermatozoids. SP preparations stained with Giemsa showed free and dividing epimastigotes, extracellular trypomastigotes and amastigotes, as well as, within phagocytic cells. The results showed the presence of T. cruzi at the different stages of its life cycle in the mouse PS, observing similar morphogenesis in the PS to the one known in the insect vector. The parasitism found in the SP of this mouse with acute infection, provides important epidemiological information about the T. cruzi pathway of sexual transmission, mainly among the population of wild reservoirs found in endemic and non-endemic areas for Chagas`disease.

Topografia de ligação de proteínas do plasma seminal à membrana de espermatozoides bovinos epididimários e ejaculados / Binding patterns of seminal plasma plasma proteins on bovine epididymal and ejaculated sperm membrane

The present study was designed to investigate the topographical distribution of seminal plasma (SP) proteins on epididymal and ejaculated bovine sperm. Using immunocytochemistry and confocal microscopy the binding patterns of bovine SP proteins BSP-A3, albumin, transferrin, prostaglandin D-synthase (PGDS) and nucleobindin in ejaculated and cauda epididymal sperm from adult bulls were evaluated. Experiments were performed using sperm from 5 males. Data showed a positive signal, only detected for anti-PGDS, in the acrosomal cap of epididymal and ejaculated sperm. In ejaculated sperm, a very weak signal for nucleobindin 2 in the midpiece and equatorial regions was detected, using the anti-rat nucleobindin. BSP-A3 was detected on all sperm regions studied, with a more evidenced signal in acrosome and midpiece. However, no binding was detected for albumin or transferrin in neither epididymal nor ejaculated sperm. In conclusion, PGDS, BSP-A3 and nucleobindin interact directly with bovine sperm, with specific topographic distribution. These findings may add to the knowledge of how these proteins modulate sperm functions, thus providing fundamental support for studies designed to evaluate how they influence sperm functions.

Investigou-se a distribuição topográfica da ligação de proteínas seminais à membrana de espermatozoides bovinos epididimários e ejaculados. Utilizando imunocitoquímica e microscopia confocal, avaliaram-se a topografia de ligação das proteínas BSP-A3, albumina, transferrina, prostaglandina D sintetase (PGDS) e nucleobindina 2 (NUC2) à membrana espermática. Os experimentos foram realizados utilizando espermatozoides de cinco touros. Os resultados mostraram que, para espermatozoides epididimários, somente detectou-se a PGDS na crista do acrossomo. Nos espermatozoides ejaculados, a PGDS ligou-se de forma mais intensa à crista acrossômica, enquanto a NUC2 apresentou sinal bastante fraco na peça intermediária e região equatorial. A BSP-A3 ligou-se a todas as regiões estudadas, de forma mais intensa na peça intermediária e acrossomo. Nenhum sinal foi detectado para albumina ou transferrina, seja em espermatozoides epididimários ou ejaculados. Concluiu-se que PGDS, BSP-A3 e NUC2 interagem diretamente com espermatozoides bovinos, e mostrou distribuição topográfica específica. Estes achados permitem melhor compreensão sobre o papel desempenhado por essas proteínas na regulação da função espermática e da fertilidade.

Porcine oviduct sperm binding glycoprotein and its deleterious effect on sperm: a mechanism for negative selection of sperm?

In their journey through the oviduct some subpopulations of sperm are preserved in a reservoir, while others are negatively selected. Sperm binding glycoprotein (SBG) is a pig oviductal epithelial cell glycoprotein that produces, under capacitating conditions, acrosome alteration, p97 tyrosine-phosphorylation and reduction of the motility of sperm. In this paper, we show that SBG is accessible at the extracellular surface of the oviductal epithelial cells, supporting a sperm interaction biological role in situ. We analyze the possible dependence of the tyrosine-phosphorylation of p97 on the PKA mechanism, finding that apparently it is not PKA dependent. Also, after SBG treatment the phosphorylated proteins locate mainly at the detached periacrosomal region and at the tail of sperm; the latter may be related to SBG's motility reduction effect. The study of the time course effect of SBG on sperm as detected by chlortetracycline (CTC) staining and of its binding to sperm by immunodetection in conjunction with CTC, shows results in agreement with the hypothesis that this glycoprotein is involved in the alteration of acrosomes in a specific sperm subpopulation. The results suggest that SBG may be part of a mechanism for negative selection of sperm.

High prevalence of AZFb microdeletion in Iranian patients with idiopathic non-obstructive azoospermia.

Background & objectives: Genetic factors contribute about 10 per cent of male infertility. Among these, genes in azoospermia factor (AZF) region including AZFa, AZFb, AZFc and AZFd on the long arm of Y chromosome are considered most important for spermatogenesis. Deletions in these regions are thought to be involved in some cases of male infertility associated with azoospermia or oligozoospermia. We studied the incidence of AZF deletions among Iranian infertile men with idiopathic non-obstructive azoospermia. Methods: A total of 100 Iranian azoospermic infertile men were selected for the molecular study of Y chromosome microdeletions. The presence of 13 sequence tagged site (STS) markers from AZF region was investigated using multiplex polymerase chain reaction (M-PCR). One hundred fertile men were also studied as control group. Results: Twelve (12%) patients showed Y chromosome microdeletions and among these, deletion in AZFb region was the most frequent (66.67%) followed by AZFc (41.67%), AZFd (33.33%) and AZFa (8.33%), respectively. Interpretation & conclusions: Because of relatively high incidence of Y chromosome microdeletions among Iranian azoospermic patients, molecular screening may be advised to infertile men before using assisted reproductive treatments.

Carnitine supplement: Does it worth trial to improve semen quality among infertile Sudanese men?

Carnitine supplement proves to upgrade the quality of semen by increasing sperm count and motility. In this study we have determined the level of L - carnitine in the seminal plasma of men with normal and abnormal seminal analysis. L - carnitine levels among the normal group was significantly higher than the abnormal group. We recommend trials of carnitine supplements to evaluate its usefulness in correcting some infertility cases. Subjects and methods: A total of 52 men; recruited from fertility centers in Khartoum ;were included in this study. Colorimetric carnitine determination kits were used for estimation of L - carnitine in seminal plasma. Results: Collectively; men with normal values of semen analysis had significantly higher mean seminal plasma carnitine levels compared to abnormal values (p = 0.028). Oligospermic men had significantly lower levels of carnitine compared to normal (p = 0.046). Conclusion: Seminal plasma carnitine level seems to correlate with seminal quality and its deficiency may be a reason for infertility among some Sudanese men

Evaluation of a Multiplex PCR Kit Used for Detecting Y Chromosome Microdeletions / 대한진단검사의학회지

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.

Construction of Zinc-alpha2-glycoprotein expression plasmid and its expression in 3t3-l1 preadipocytes / 中国医学科学院学报

<p><b>OBJECTIVE</b>To construct mouse Zinc-alpha2-glycoprotein (mZAG) eucaryotic expression plasmid and identify its expression in 3T3-L1 preadipocytes.</p><p><b>METHODS</b>The 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.</p><p><b>RESULTS</b>DNA 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).</p><p><b>CONCLUSIONS</b>mZAG 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.</p>

Chromosome abnormalities and Y chromosome microdeletions in patients with the azoospermia and cryptozoospermia / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To study the incidence of the chromosome abnormalities and Y chromosome microdeletions in Chinese patients with azoospermia and cryptozoospermia.</p><p><b>METHODS</b>Conventional 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.</p><p><b>RESULTS</b>The 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).</p><p><b>CONCLUSION</b>Chromosome 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.</p>

Identification of asthenozoospermia-associated proteins in human seminal plasma by shotgun proteomic strategy / 中华男科学杂志

<p><b>OBJECTIVE</b>To identify asthenozoospermia-associated proteins in seminal plasma by the shotgun proteomic strategy.</p><p><b>METHODS</b>Six seminal plasma samples were collected by Percoll respectively from healthy fertile and asthenozoospermia volunteers, balanced, mixed, and then the mixture was separated by SDS-PAGE. The proteins in the gel were enzymolyzed, extracted and identified by the shotgun proteomic strategy. The identified proteins with the unique peptide count > or =2 or the unique peptide count=1 but the total count > or =4 were compared between the two groups.</p><p><b>RESULTS</b>A total of 172 differential proteins were identified, of which, 89 were exclusively from the asthenozoospermia and 83 exclusively from the healthy fertile men. According to the molecular function, these differential proteins were mainly the types of signal transduction and catalytic activity.</p><p><b>CONCLUSION</b>Functionally, 10 of the proteins are particularly important, which include annexin VI isoform 2, isoform 1 of interleukin-6 receptor subunit beta precursor, Mr 400,000 protein, cytosolic dynein heavy chain, alpha-actinin-4, receptor-type tyrosine-protein phosphatase eta precursor, vitamin D-binding protein precursor, protein S100-A11, protein S100-A9 and ANXA4.</p>