Expression of 5α－Reductase Type 2 Gene in Human Testis,Epididymis and Vas Deferens

Objectives:To study the expression pattern of 5α-reductase type 2 gene in human male reproductive organs.Methods:The expression level of 5α-reductase type 2 gene inhuman testis,epididymis and vas deferens tissues was determined by in situ hybridization using Digoxin labeled 5α-reductase type 2cRNA probe.Results:The brown granules of hybridizing signals distributed in the cytoplasm of Sertoli and Leydig cells of the testis,the principle cells of epididymis and the epithe-lial cells of vas deferens,but there was no positive signal in the nuclei of above-men-tioned cells.No positive signal was observed in germ cells,basement of the testis,interstium of epididymis and basement,as well as smooth muscle of vas deferens.Conclusion:This study confirmed that the 5α-reductase type 2 gene expressed in Ser-toli,Leydig cells of the testis,and the principle cells of epididymis.The expression pattern of the gene in these cells in human was similar to that of rat and monkey.The presence of 5α-reductase type 2 gene in epithelial cells of the vas deferens suggested it might possess an important physiological role in human reproduction.     

Long-term reproductive consequences of no-scalpel vasectomy in beagles

The effects of vasectomy on the reproductive organs in various species are controversial. This study investigated the morphological change and apoptosis of the testis, epididymis, and vas deferens in beagle dogs 12 months after vasectomy. The male beagles were divided into two groups: vasectomized and sham-operated groups (n=5 in each). Histopathological, ultrastructural, and TUNEL evaluation of the changes in the testis, epididymis, and ductus deferens of each animal were conducted 12 months after surgery. The mean lumen diameter, cellular thickness, mean interstitial distance, and lumen area fraction of each seminiferous tubule and ductus epididymis were measured by stereological analysis. The results showed that, compared with the sham-operated group, the seminiferous tubular epithelial cells of the testes in the vasectomized group were disorderly arranged and scattered. Significant atrophy and apoptosis were found in the endothelial cells, and a range of ultrastructural variations were observed in the cells of testes, epididymis, and vas deferens in vasectomized group. It was concluded that complete obstruction of the vas deferens as a traditional contraception method is not absolutely safe in terms of the reversal of fertility in the long run. Techniques of relieving the inner pressure in the vas deferens while maintaining the efficacy of male contraception need to be explored.     

Effect of Vasocation Intestinal Peptide on Immune Privilege of the Rat Testis

Objective To study the effect of vasocation intestinal peptide （VIP） on immune privilege of the rat testis. Methods The UU infected SD rats and Leydig cells were intervened by VIP, the secretion of TGF-β and the expression of FasL in rat Leydig cells were compared between VIP-intervened group and control group to test the effect of VIP on immune privilege of the rat testis in vitro and in vivo. Results In vitro, the secretion of TGF-β in Leydig cells could be increased by low dosage of VIP while inhibitited by high dosage of VIP; expression of FasL mRNA in Leydig cells could be decreased by VIP In vivo, increased expression of TGF-β mRNA and decreased FasL mRNA were observed in VIP group in 2-3 weeks after infected by UU. In addition, the apoptosis of Jurkat cells mediated by Leydig cells could be prevented by VIP Conclusion When Leydig cells or testis infected by UU, VIP could regulate the immune function of rat Leydig cells and participate in the regulation of immune privilege of testis through the regulation of TGF-β secretion and FasL expression pattern of Leydig cells.     

Immunolocalization of Epidermal Growth Factor in Human Fetal, Adult and Cryptorchid Testes and Seminoma

EGF was localized in human fetal, adult and eryptorchid testis and seminoma using an immunohistoehemical method. Leydig cells of adult testes stained intensely for EGF while those of fetal testes showed weak staining reaction. In addition, some spermatogonia and occasional peritubular myoid cells of adult testis stained positively. In cryptorchid testis, there were clusters of Leydig cells interspersed between seminiferous tubules with varying staining intensities. The number of positiuely stained cells in the cryptorchid testis were fewer than that in adult testes. The decrease in the number of Leydig cells pruducing EGF may account for the spermatogenesis arrest and infertility, associated with this disorder.Intense staining of the cytoplasm of seminoma cells was obserued, suggesting that the high production of EGF may be related to their invasive property. In conclusion, EGF is produced by human testis and Leydig cells are the principal source of this cytokine.     

THE ANTIFERTILITY EFFECT INDUCED BY INJECTION OF SODIUM MORRHUATE VIA VAS DEFFERENS INTO EPIDIDYMIS

2.5% sodium morrhuate (SM) can completely abolish human spermatozoal motility at once. In general azoospermia, oligospermia and asthenospermia were obtained in the cauda epididymis of animals on 3rd to 14th day after a single injection of 0.3 to 2.0 ml SM via the vas deferens into each cauda epididymis. Using the same procedure, a single injection of 1—1.5 ml SM was studied in 1061 fertile male volunteers for 7 to 28 months. The Pearl index was 2.06/100 women years. The effective index was 97.94/100 women years. Blood FSH, LH, T levels and libido were unchanged. After treatment, among the 445 cases of semen analysis, 361 cases had azoospermia, 25 cases had sperm counts lower than 4 million/ml, 20 cases had sperm counts ranging from 4 to 10 million/ml, 39 cases had sperm counts higher than 20 million/ml. The seminiferous tubules of animals on days 3 to 90 exhibited different degrees of damage. Under LM and TEM no discernible abnormality was found in spermatogonia, Sertoli and Leydig cells. On months 15 to 21 spermatogenesis was seen in about 50% of the seminiferous tubules in 6 testicular biopsies of men after treatment, however exfoliated cells were also seen in the tubular lumen. It is evident from our results that injection of SM via the vas into epididymis is a simple procedure for producing birth control in the male.     

Expression of Telomerase Gene mTR in the Testis of SD Rats and Its Significance

Summary:To study the expression of mTR gene in the testis of SD rats with varied ages and its sig-nificance,in situ hybridization(ISH)techniques were applied to detect the expression of telomerasegene mTR mRNA in the testis of SD rats.The expression of mTR was found in testes of different-age male SD rats.There was a positive correlation between the expression of mTR and the location ofgerm cells(spermatogonia,spermatocyte,spermatid).In Setoli cells,leydig cell and spermatozoa,no telomerase mTR was detectable.Type A spermatogonia expressed the highest level of telomerasemTR mRNA.It was suggested that the expression of mTR gene in the testis of SD rats is of lifetimeand coincides with the telomerase activity.     

Molecular cloning and characterization of porcine indoleamine 2, 3-dioxygenase and its expression in various tissues

In order to confirm the existence of indoleamine 2,3-dioxygenase(IDO) gene in swine,and to clone the novel gene followed by the molecule structure properties and expression pattern analysis,the porcine mRNA sequences homologous to human IDO were obtained from GenBank database by bioinformatics method.By using RT-PCR,the IDO gene was cloned from porcine endothelial cell line and the accuracy of the nucleic acid sequence was confirmed,and the expression pattern of the gene was detected.The three-dimensional structure model of porcine IDO was built referring to the tertiary structure of human IDO using biological sequence analysis software and database.The results showed that the porcine IDO was identified by sequencing.The nucleotide sequences were confirmed as a novel gene after submitted to Genbank.Porcine IDO was expressed in the lung,thymus,epididymis and anterior chamber with a basic level,however in peripheral blood mononuclear cells(PBMCs) the IDO gene was highly expressed.The three-dimensional structure model of porcine IDO was similar to that of human IDO.It was suggested that identification of the structure information of porcine IDO is essential to further investigate the immunologic function of the gene.Study of IDO on NK cells-mediated xenograft rejection will be a novel therapeutic target for the development of xenotransplantation.     

EXPRESSION OF HUMAN α-GALACTOSIDASE AND α1,2-FUCOSYLTRANSFERASE GENES MODIFIES THE CELL SURFACE GALα1,3GAL ANTIGEN AND CONFERS RESISTANCE TO HUMAN SERUM-MEDIATED CYTOLYSIS

Objective To explore the strategies which reduce the amount of xenoantigen Galα1, 3 Gal. Methods Human α-galactosidase gene and α1,2-fucosyhransferase gene were transferred into cultured porcine vascular endothelial cells PEDSV. 15 and human α-galactosidase transgenic mice were produced. The Galα1,3Gal on the cell surface and susceptibility of cells to human antibody-mediated lysis were analyzed. Results Human α-galactosidase gene alone reduced 78% of Galα1,3Gal on PEDSV. 15 cell surface while human α-galactosidase combined with α1,2-fucosyltransferase genes removed Galα1,3Gal completely. Decrease of Galα1,3Gal could reduce susceptibility of cells to human antibody-mediated lysis, especially during co-expression of α-galactosidase gene and α1,2-fucosyltransferase gene, RT-PCR indicated positive human α-galactosidase gene expression in all organs of positive human α-galacto-sidase transgenic F1 mice including heart, liver, kidney, lung, and spleen, the amount of Galα1,3Gal antigens on which was reduced largely. 58% of spleen cells from F1 mice were destroyed by complement-mediated lysis compared with 24% of those from normal mice. Conclusions Human α-galactosidase gene and α1,2-fucosyltransferase gene effectively reduce the expression of Galα1,3Gal antigens on endothelial cell surface and confers resistance to human serummediated cytolysis. The expression of human α-galactosidase in mice can also eliminate the Galα1,3Gal antigens in most tissues and decrease the susceptibility of spleen cells to human serum-mediated cytolysis.     

EXPRESSION OF HUMAN α-GALACTOSIDASE AND α1,2-FUCOSYL-TRANSFERASE GENES MODIFIES THE CELL SURFACE GALα1,3GAL ANTIGEN AND CONFERS RESISTANCE TO HUMAN SERUM-MEDIATED CYTOLYSIS

Objective To explore the strategies which reduce the amount of xenoantigen Galα1, 3 Gal. Methods Human α-galactosidase gene and α 1,2-fucosyltransferase gene were transferred into cultured porcine vascular endothelial cells PEDSV.15 and human α-galactosidase transgenic mice were produced. The Galα 1,3Gal on the cell surface and susceptibility of cells to human antibody-mediated lysis were analyzed. Results Human α-galactosidase gene alone reduced 78% of Galα1,3Gal on PEDSV.15 cell surface while human α-galactosidase combined with α 1,2-fucosyltransferase genes removed Galα 1,3Gal completely. Decrease of Galα1,3Gal could reduce susceptibility of cells to human antibody-mediated lysis, especially during co-expression of α-galactosidase gene and α1,2-fucosyltransferase gene. RT-PCR indicated positive human α-galactosidase gene expression in all organs of positive human α-galactosidase transgenic F1 mice including heart, liver, kidney, lung, and spleen, the amount of Galα1,3Gal antigens on which was reduced largely. 58% of spleen cells from F1 mice were destroyed by complement-mediated lysis compared with 24% of those from normal mice. Conclusions Human α-galactosidase gene and α1,2-fucosyltransferase gene effectively reduce the expression of Galα1,3Gal antigens on endothelial cell surface and confers resistance to human serummediated cytolysis. The expression of human α-galactosidase in mice can also eliminate the Galα1,3Gal antigens in most tissues and decrease the susceptibility of spleen cells to human serum-mediated cytolysis.     

Expression profile of a novel germ cell-specific gene,TSCPA, in mice and human

In order to identify novel genes involved in spermatogenesis, testis cDNA samples from Balb/C mice of different postnatal days were hybridized with the whole mouse genome Affymetrix chip to screen the testis-specific genes. The characteristics of the selected genes were analyzed by RT-PCR as well as other bioinformatic tools. A novel differentially expressed testis-specific gene （GenBank Accession No： NM_029042） in the developmental stages of testes was identified, and named TSCPA. Cellular mapping prediction of TSCPA indicated that its protein was probably expressed in nuclei, and one putative domain （aa 332 377） was anchoring domain of cAMP-dependent type Ⅱ PK. The result of subcellular localization of GFP-TSCPA fusion protein in Cos-7 cells showed that TSCPA protein was expressed in nuclei. RT-PCR analysis revealed that TSCPA was expressed specifically in mouse and human testis. TSCPA gene was expressed weakly in 21-day-old mouse testis and the expression was increased gradually from 38th day to 6th month of mouse testes. No expression of hTSCPA was found in cryptorchidism and Sertoli-cell-only syndrome patients. It was concluded that the expression profile of TSCPA in human and mice indicated that TSCPA might play an important role in spermatogenesis.     

Toxicological Evaluation of 504P： An Adhesive Blocking Agent for Vas Deferens

Toxicological studies on an adhesive blocking agent for vas deferens were carried out.It could be excreted from the body soon after some of the agent had been absorbed. No long-term accumulation phenomenon and no obvious effect of acute toxicity on rodent animals have been observed. The parameters of physiology, blood cells and blood biochemistry were not affected apparently. The histomorphological study showed that the blocked part of vas deferens was obliterated with fibrous tissue and sperm deposition was found in the blocked end toward epididymis. The agent had no carcinogenic effect. Moreover, on the basis of mutation assay with Salmonella typhimarium strains, analysis of chromosome aberration, mieronuclei assay in bone marrow cells of mouse and domirrant lethal mutation assay, it was shown that the agent had no mutagenic effect.     

Effect of FasL High Expression on Immune Regulative Function of Sertoli Cell in Transgenic Mouse

Objective To study the immune regulative function of Sertoli cell on testis local infection Methods Ureaplasma urealyticum （UU） was directly injected into bladders of FasL transgenic mice and wild-type mice, which mimicked an ascending infectious way. At week 1, 2 and 3 after injection respectively, the mice were killed to observe the pathological alterations in testis section. And at the same time cytokines was tested by immunohistochemistry. Comparison of levels of FasL, TGF-β, IL-1α and IL-6 between UU-infected and control groups of wild mice and FasL transgenic mice was made respectively. Then the capability of Sertoli cell （FasL^＋） to mediate apoptosis of Fas^＋ cells between wild control and wild UU-infected groups was analyzed. Results The pathological changes of testis in FasL transgenic mice were more seriously compared with wild counterpart and the changing mode of cytokines secreted by Sertoli cells were different between the two kinds of mice. The UU-infected Sertoli cells increased Fas^＋ Jurkat cell apoptosis. Conclusions High expression of FasL in FasL transgenic mice can influence the cytokines secretion during anti-infection, thus affecting the testis immune response to infection and immune balance. The high expression of FasL is not beneficial for body＇s anti-inflection immune response.     

Expression and Bioinformatics Analysis of SPACA4 in Human and Mice

Objective To analyze the expression of SPACA4 in human and mice. Methods Testes cRNA samples from Balb/c mice of different postnatal days were performed with mouse affymetrix chip to screen the expression of SPACA4 in mice. Sub-quantitative RT-PCR and bioinformatic tools were used here to describe the expression profile of SPACA4 in mice and human. Results The results of gene chip analysis indicated that the expression of mSPACA4 began after d 35 of postnatal testis in mice. Sub-quantitative RT-PCR assay showed that SPACA4 gene expressed exclusively in mouse and human testis, and mouse mSPACA4 gene expressed after d 35 of postnatal testis that was consistency with the results of gene chip analysis. By bioinformatics analysis, mSPACA4 is located in cell membrane （34.8%） or plasma membrane （34.8%）, the signal peptide cleavage site between position 19 and 20 amino acids, transmembrane region between 2-20 and 101-126 amino acids, respectively, on mSPACA4 protein. Conclusion mSPACA4 and hSPACA4 were testis-specific genes, and the expression of mSPACA4 begins after d 35 of postnatal testis in mice. SPACA4 is a candidate for targeting in a sperm-based contraceptive vaccine.     

Acceleration of apoptosis by transfection of bak gene in multi-drug resistant (MDR) bladder cancer cellsI

Objective To observe the effects of bak gene on killing MDR bladder cancer cells and to study its mechanisms. Methods Bak gene was transfected into MDR bladder cancer cells by liposome. The mRNA of bak and bcl-2 were detected by in situ hybridization. The protein of bak and bcl-2 were detected by SABC immunohistochemistry. The growth rate of human bladder cancer cells was studied by constructing the growth curve, cell apoptosis being observed by flow cytometry, and the outline of cells observed by fluorescence stain. Results The expression of bak mRNA was positive in EJ/bak cells (64% ,P<0.05).Bak protein expression of EJ/bak cells was positive (60 % ) and bcl-2 protein expression was de creased (P<0.05). The growth of MDR bladder cancer cells was significantly inhibited by 32% after bak gene was transfected (P < 0. 05 ). Apoptosis cells increased significantly. The apoptosis rate was 35 %. Apoptotic bodies can be found in these cells on fluorescence stain. Conclusion Bak gene could inhibit the growth o     

Immunoregulative Mechanism of the Leydig Cell in the Infection by Expression1 of IL-1,IL-6,TGF-β, Fas and FasL mRNA

Objective To investigate the immune regulative mechanism of Leydig cells in the local infection of rat＇s testis. Methods Ureaplasma Urealyticum（UU） was injected into rat＇s bladder, which mimicked an ascending infectious way, and at the same time culture medium was injected into rat＇s bladder as the control. The rats were sacrificed at week 1, 2 and 3 after injection respectively. Then pathological changes in testis were analyzed by histological examination. At the same time Leydig cells were separated from rat＇s testis. The comparasion of levels of IL-1,IL-6, TGF-β, Fas and FasL mRNA expression among the three groups was made by RT-PCR. Results As compared with control group, the levels of IL-1, IL-6, TGF-fl mRNA expression for UU supernatant and living UU groups increased; and levels of Fas and FasL mRNA expression decreased and increased respectively after UU infection. Conclusion During anti-infective immunity, rat＇s Leydig cells may regulate immune function of the testis by changing the levels of IL-1, IL-6, TGF-β, Fas and FasL mRNA expression and may contribute to maintain immune privilege of the testis.     

Identification and expression of a novel human testis-specific gene by digital differential display

Background Evidence for the importance of genetic factors in male infertility is accumulating. This study was designed to identify a novel testis-specific gene related to spermatogenesis by a new strategy of digital differential display (DDD).Methods Based on the generation of expressed sequenced tags (ESTs), comparing the testis libraries with other tissue or cell line libraries by the DDD program, we identified a new contig of the ESTs which were derived from testis libraries and represented a novel gene. Multi-tissue RT-PCR was performed to analyse its tissue-specific expression. The full-length cDNA of the new gene was obtained using the BLAST program. Sequencing was performed and the result was analysed. Semiquantitative RT-PCR and Northern blot analyseis of mRNA from differential normal tissues were performed to clarify the expression pattern of the new gene. The sequence of the opening reading frame was integrated into the pQE-30 vector expressed in Escherichia coil strain M15(pREP4). With IPTG induction, the target protein was detected.Results A full-length cDNA sequence of the new gene named SPATA12 (GeneBank accession number AY221117) in human testis was identified. SPATA12 was 2430 bp in length, located in chromosome 3p21.1-3p21.2. The sequence of the opening reading frame was 676 - 1248 bp, as was confirmed by RT-PCR and sequencing. The cDNA encodes a novel protein of 190 amino acids with a theoretical molecular weight of 20417. 8 and isoelectric point of 5. 23. The sequence has no significant homology with any known protein in databases. Semi-quantitative RT-PCR and Northem-blot analyses of multiple tissues showed that SPATA12 was expressed significantly in normal human testis. The expression recombinant of SPATA12 was constructed and a high level of the histidine-tagged fusion protein was obtained.Conclusions DDD can be confirmed by SPATA12 as a novel computational biology-based approach for identification of the testis-specific expression genes. SPATA12 may function as a testicular germ cell associated gene that plays some roles in spermatogenesis. Moreover, a great amount of SPATA12 protein could be obtained by the gene recombination technique, thus providing a reliable foundation for investigating the biological function of this new protein.     

Epigenetic Regulation of the ERβ Gene on the Estrogen Signal Transfection Pathway in Colon Cancer Cells

We studied the regulatory effects of the estragen receptorβ(ERβ)gene on the downstream estrogen signal transfection pathway in colon cancer cells and the possible mechanisms involved.A human ERβ gene recombinant expression plasmid,pEGFP-C1-ERβ,was constructed and transfected into the Caco-2 colon cancer cell line,a line with low ERβ gene expression.The expression of ERβ mRNA and protein was detected 72h after transfection.RT-PCR was used to examine the expression levels of the progesterone recepror(PR)gene ...     

Activation of protein kinase A alters subnuclear distribution pattern of human steroidogenic factor 1 in living cells

Background The aim of this study was to identify the subnuclear distribution pattern of human orphan nuclear receptor steroidogenic factor 1 (SF-1) in living cells with and without the activation of protein kinase A (PKA) signal pathway, and thus try to explain the unknown mechanism by which PKA potentiates SF-1 transactivation. Methods Full-length cDNAs of wild type and a naturally occurring mutant (G35E) human SF-1 were cloned and fused with green fluorescent protein (GFP). Subcellular distribution pattern of human SF-1 in living cells, whose PKA signaling was either activated or not, was studied by laser confocal microscopy after the validity of the gene sequence was confirmed. Results The transactivation ability of the GFP-SF-1 chimeric protein was highly conserved. Wild type human SF-1 diffused homogeneously within the nuclei of cells when PKA was not active, and converged to clear foci when PKA was activated. Mutant SF-1 diffused within the nuclei even in the presence of PKA activation, surprisingly aggregating as fluorescent dots inside the nucleoli, a phenomenon not altered by PKA. Conclusions Activation of PKA causes wild type, but not mutant SF-1 to alter its subnuclear distribution pattern to a transactivationally active form (foci formation). This finding may throw new light on the mechanism by which PKA activates the orphan nuclear receptor.     

Induction of apoptosis of human colon cancer cells by siRNA recombinant expression vector targeting survivin gene

This study constructed siRNA recombinant expression vector targeting survivin gene and observe the apoptosis induction effect of it in human colon cancer cells, siRNA recombinant expression vector targeting survivin gene was constructed and transfected into human colon cancer cells. The effect of siRNA recombinant expression vector was detected by RT-PCR, Western blot, MTT reduction assay and flow cytometry. It was confirmed by restriction endonuclease and sequence analysis that siRNA recombinant expression vector targeting survivin gene was constructed successfully. Inhibition rate of survivin siRNA at mRNA and protein levels was 36.33% and 44.65% respectively. Growth of cancer cells was inhibited and the apoptosis rate was （17.24±2.13）%. The siRNA recombinant expression vector targeting survivin gene has been constructed successfully. It not only can inhibit the expression of survivin gene, but also can induce apoptosis in human colon cancer cells remarkably.