Cloning of gdnf in the mouse testis and its expression in sertoli cells / 中华男科学杂志

<p><b>OBJECTIVE</b>To clone the glial cell line-derived neurotrophic factor (GDNF) from the mouse testis, construct the eukaryotic expression vector and transfect this vector into Sertoli cells in order to use the gdnf-transfected Sertoli cells as the feeder layer to cultivate spermatogonial stem cells (SSCs).</p><p><b>METHODS</b>Total RNA was extracted from the testes of normal mature mice and gdnf was cloned and amplified using RT-PCR, inserted into the eukaryotic expression vector and transfected into sertoli cells (TM4 cell line). Immunofluorescence with anti-GDNF antibodies was performed at 40 h following the transfection.</p><p><b>RESULTS</b>gdnf cDNA was cloned successfully, and GDNF expressed after transfected into Sertoli cells.</p><p><b>CONCLUSION</b>This study provides a basis for culturing SSCs with gdnf-transfected Sertoli cells as the feeder layer.</p>

Infectious bovine rhinotracheitis viral gG expression and gG-ELISA development / 生物工程学报

Taking the genome DNA of Infectious Bovine Rhinotracheitis Virus (IBRV) as the template, the gG gene was amplified with PCR and cloned into the T cloning vector pMD18-T. After being identified by restriction digestion and DNA sequencing, the insert was subcloned into the expression vector pGEX-KG. Sodium docecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot assay showed that this gene was expressed as both soluble form and inclusion body by the transformed E. coli BL21 strain (DE3). The fusion protein was purified and used as the coating antigen to develop the indirect Enzyme-Linked Immunosorbent Assay (ELISA). Comparison between this gG-ELISA and commercial IBRV gB-ELISA Kit (IDEXX) was made in the detection of 380 cow serum samples. The results demonstrated an agreement of 92%. By using this novel gG-ELISA, 1248 cow serum samples were tested and the average positive rate of IBRV antibodies for imported cows is 21.7%, while the positive rate ranged greatly from 0.0%-41.5% for Hubei local Chinese Black and White Dairy Cows.

17beta-estradiol stimulates proliferation of spermatogonia in experimental cryptorchid mice / 亚洲男科学杂志(英文版)

<p><b>AIM</b>To investigate whether estrogen stimulates the proliferation of spermatogonia or induces spermatogenesis in cryptorchid mice.</p><p><b>METHODS</b>Mice were surgically rendered cryptorchid, then treated with different doses of 17beta-estradiol (E2) s.c. once a day. Mice were killed at sexual maturity (45 days of age), and histological analysis and immunofluorescence were performed. Serum follicle stimulating hormone (FSH), estradiol, testosterone and luteinizing hormone (LH) were measured.</p><p><b>RESULTS</b>Low doses of E2 had no notable effect on spermatogonia, but at higher doses, E2 stimulated the proliferation of spermatogonia.</p><p><b>CONCLUSION</b>E2 has a dose-related mitogenic effect on spermatogonia.</p>

Differential analysis of proteomic profiles between cryptorchid and normal mouse testes / 生理学报

To screen factors related to spermatogonial stem cell (SSC) proliferation, and to investigate the mechanism of infertility caused by cryptorchidism, ten-day-old Kunming (KM) mice were used and experimental cryptorchidism was conducted. On the 35th day after cryptorchid operation, the left testes were fixed in Bouin's fluid and used for histological analysis. The testes of 45-day-old mice were subjected to the same histological analysis, and it was found that they contained germ cells at every stage of development, from SSCs to sperm, indicating that the animals were fully sexually mature at this age. While in experimental cryptorchid mice, the spermatogenesis was arrested at the stage of spermatocytes, and only spermatogonia and primary spermatocytes were present in cryptorchid testes. The proportion of spermatogonia to other types of germ cells was much higher than that in sexually mature mice. On the other hand, the right testes were used for proteomic analysis. The total protein in testes was extracted on the 35th day after cryptorchid operation. The differentially expressed proteins in cryptorchid mice and sexually mature mice were screened and compared by the proteomic techniques. Through the separation of two-dimensional gel electrophoresis (2-DE), 20 differential protein spots were found, and 9 of them were digested and identified by the matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrum. In cryptorchid mice, 6 out of 9 proteins were down-regulated, and 3 were up-regulated. Among these proteins, 4 proteins were identified, and they were Stathmin, phosphatidylethanolamine-binding protein1 (PEBP1), HES-related basic helix-loop-helix protein (HERP), and one unnamed protein (we temporarily named it Px). More Stathmin, PEBP1 and Px were expressed in sexually mature mice than in experimental cryptorchid mice. But HERP1 was the other way round. In the present study, we have screened 4 proteins related to cryptorchidism. It is helpful to study the mechanism of SSC proliferation and infertility caused by cryptorchidism.

Cloning of Bmi1 cDNA from mouse testis and its expression in E. coli BL21 / 中华男科学杂志

<p><b>OBJECTIVE</b>To produce BMI1 polyclonal antibody, mouse Bmi1 cDNA was cloned from mouse testis and expressed in E. coli BL21.</p><p><b>METHODS</b>Bmi1 gene was amplified from mouse testis by RT-PCR and inserted into the prokaryotic expression vector pET-28c(+). Subsequently the recombined vector was transformed and expressed in E. coli BL21 (DE3) and the immunogenicity of recombined protein BMI1 (rBMI1) was tested by Western blot.</p><p><b>RESULTS</b>Mouse Bmi1 cDNA of 975 bp was successfully cloned and recombined. E. coli BL21 strains expressed rBMI1 were screened. The expression protein amounted to 12% of the total bacterial protein after induced with IPTG, which included inclusion body and soluble protein. Inclusion body was the major pattern of the expression that amounted to 71% of the insoluble protein. Western blot analysis showed that rBMI1 could be specially recognized by mouse monoclonal IgG1 anti-BMI1 and His-tag antibody.</p><p><b>CONCLUSION</b>There was expression of Bmi1 gene in mouse testis. Mouse Bmi1 cDNA was successfully cloned and expressed prokaryoticly.</p>

Effects of newborn bull serum and vitamins on cryopreservation of mouse seminiferous epithelial cells / 中华男科学杂志

<p><b>OBJECTIVES</b>To investigate the effects of newborn bull serum(NBS), vitamin C and vitamin E on cryopreservation of mouse seminiferous epithelial cells.</p><p><b>METHODS</b>The seminiferous epithelial cells from 7-day-old mice were cryopreserved in different freezing solutions. The cell recoveries were examined by Trypan blue exclusive staining after thawing. The freezing solutions composed of DMEM, 10% dimethylsulphoxide(DMSO), and 0, 5%, 10%, or 20% NBS, respectively, or composed of DMEM, 10% DMSO, 10% NBS, and 150 micrograms/ml vitamin C or 50 micrograms/ml vitamin E, respectively.</p><p><b>RESULTS</b>The cell recoveries in freezing solution containing 0, 5%, 10%, or 20% NBS were 83.4%, 84.7%, 85.7% and 83.6%, respectively. There were no significant differences between them. The cell recoveries in freezing solution containing vitamin C or vitamin E were 88.0% and 82.9%, respectively. There was no significant differences compared with that in freezing solution containing 10% DMSO and 10% NBS.</p><p><b>CONCLUSIONS</b>NBS, vitamin C and vitamin E have no significant protecting effects on mouse seminiferous epithelial cells, and can not significantly improve the cell recoveries.</p>

Effects of SCF, LIF and bFGF on mouse spermatogonial stem cells proliferation in vitro / 生物工程学报

The present study identified the favorable environment conditions for Spermatogomial stem cells in vitro according to their unique biological properties. Three growth factors, stem cell factor (SCF), leukemia inhibitory factor (LIF) and basic fibroblast growth factor (bFGF) were all found to independently contribute to the proliferation of mouse spermatogonial stem cell. The percentage of cell proliferation significantly enhanced by SCF at 30 ng/mL but decreased with heightening its combination after cultured 120 hours. The mice spermatogonial stem cells were significantly proliferated after 120 hours' culture with 10 ng/mL and 20 ng/mL (P < 0.01) of LIF, between 20 ng/mL and 50 ng/mL (P < 0.01) for bFGF. SCF and bFGF were significantly enhanced mice spermatogonial stem cells proliferation after these three factors combination. For LIF, no obvious effect was observed.