The role of SPAG6/SPINK2 protein complex in the formation of sperm acrosome in mice / 中华男科学杂志

Objective@#To explore the expression and regulatory function of sperm-associated antigen 6 (SPAG6) in the formation of the sperm acrosome in mice.@*METHODS@#The expression of SPAG6 during the first wave of spermatogenesis on postnatal days (PN) 8, 12, 16, 20, 24, 28, 30 and 35 was examined by Western blot and the localization of SPAG6 in the testicular germ cells was determined by immunofluorescence. The expression plasmids of SPAG6 and serine protease inhibitor Kazal-type 2 (SPINK2) were constructed, the interaction between SPAG6 and SPINK2 in the AH109 and CHO cells examined by yeast two-hybrid and co-localization assays, and the expression and localization of SPINK2 in the testicular germ cells of the SPAG6-knockout (SPAG6 KO) mice detected by immunofluorescence.@*RESULTS@#SPAG6 was highly expressed between PN 16 and 28 and localized in the acrosome of the round spermatids. Yeast two-hybrid assay showed the growth of SPAG6 and SPINK2 in the selective culture medium SD/-Leu/-Trp/-His, and the transfection of the CHO cells revealed the co-localization of SPAG6 and SPINK2 around the nuclei. The expression and acrosomal localization of SPINK2 were not found in the testicular germ cells of the SPAG6-KO mice.@*CONCLUSIONS@#SPAG6 interacts with SPINK2 and probably participates in the formation of the sperm acrosome by stabilizing the expression of SPINK2 during spermatogenesis.

Expressions of SLC22A14 and SPAG6 proteins in the ejaculated sperm of idiopathic asthenozoospermia patients / 中华男科学杂志

Objective@#To investigate the expressions of solute carrier family 22 member 14 (SLC22A14) and sperm-associated antigen 6 (SPAG6) in the sperm of idiopathic asthenospermia men.@*METHODS@#We collected semen samples from 50 idiopathic asthenozoospermia patients and another 50 normal sperm donors, purified the sperm by discontinuous density centrifugation on Percoll gradients, and then determined the mRNA and protein expressions of SLC22A14 and SPAG6 by RT-PCR and Western blot, respectively.@*RESULTS@#Compared with the normal controls, the idiopathic asthenozoospermia patients showed significantly decreased mRNA expressions of SLC22A14 (0.77 ± 0.08 vs 0.53 ± 0.10, P<0.01) and SPAG6 (0.78 ± 0.09 vs0.52 ± 0.10 , P<0.01) and protein expressions of SLC22A14 (0.80 ± 0.09 vs 0.55 ± 0.10 , P<0.01) and SPAG6 (0.78 ± 0.09 vs 0.56 ± 0.09, P<0.01).@*CONCLUSIONS@#T The expressions of SLC22A14 and SPAG6 are reduced in the sperm of the patients with idiopathic asthenospermia, which may be one of the important causes of asthenospermia.

Expression of sperm-associated antigen 6 in liver cancer tissue and its clinical significance / 临床肝胆病杂志

Objective To investigate the expression of sperm-associated antigen 6 (Spag6) in liver cancer tissue and its association with the clinicopathological features and prognosis of liver cancer patients,as well as the effect of Spag6 on the proliferation and migration of HCCLM3 hepatoma cells.Methods Clinical samples were collected from 102 liver cancer patients who were treated in Xiangya Hospital of Central South University from August 2006 to November 2009,and Western blot was used to measure the expression of Spag6 in hepatoma cells,normal liver tissue,tumor tissue,and corresponding adjacent tissue.Immunohistochemistry was used to measure the expression of Spag6 in 102 liver cancer tissue samples,and according to the immunohistochemical scoring criteria,the patients were divided into high Spag6 expression group and low Spag6 expression group.Lentivirus-mediated RNA interference technique was used to silence Spag6 expression in HCCLM3 cells;Western blot was used to analyze silencing effect,wound-healing assay was used to investigate the effect of Spag6 gene silencing on the migration of HCCLM3 cells,and colony formation assay was performed to observe the effect of Spag6 gene silencing on the proliferation of HCCLM3 cells.The chi-square test was used to investigate the association between Spag6 expression and clinicopathological features of liver cancer patients,and the Kaplan-Meier survival analysis and log-rank test were used to analyze the association between Spag6 expression and the prognosis of liver cancer patients.Results Hepatoma cells and liver cancer tissue had significantly higher expression of Spag6 than the normal L02 cells and normal liver tissue.Immunohistochemistry showed that the expression rate of Spag6 was 58.8％ (60/102) in liver cancer tissue samples and 12.7％ (13/102) in adjacent tissue samples (x2 =47.123,P ＜0.001).According to the results of the chi-square test,Spag6 expression was associated with the number of tumor nodules,presence or absence of capsule,vascular invasion,and Edmondson-Steiner classification (x2 =8.360,6.761,4.344,and 7.172,P =0.004,0.009,0.037,and 0.007).Further analysis showed that the high Spag6 expression group had significantly lower 1-,3-,and 5-year survival rates than the low Spag6 expression group (71.5％ vs 90.5％,43.7％ vs 68.8％,19.7％ vs 48.7％,x2 =11.228,P =0.001).Cell assays showed significant reductions in the proliferation and migration of HCCLM3 cells after Spag6 gene silencing (both P ＜ 0.01).Conclusion Spag6 is highly expressed in hepatoma cells and liver cancer tissue,and its high expression is associated with poor clinicopathological features and postoperative survival of liver cancer.Spag6 can promote the proliferation and migration of hepatoma cells,suggesting that Spag6 may be involved in the development and progression of liver cancer.Therefore,it can be used as a reference index for predicting the prognosis of liver cancer patients and a potential target for liver cancer treatment.

Interaction between SPAG6 and TAC1 proteins / 中华男科学杂志

<p><b>Objective</b>To construct eukaryotic expression plasmids of the Tac1 gene and explore the interaction between TAC1 and sperm-associated antigen 6 (SPAG6).</p><p><b>METHODS</b>RNA was extracted from the heart, liver, spleen, lung, kidney, brain, muscle, and testis of 10 Kunming male mice and, after reverse transcription into cDNA, the expression of Tac1 in the above tissues was observed by RT-PCR. Tac1/pEGFP-N2 and Tac1/pGADT7 recombinant plasmids were constructed and Tac1/pEGFP-N2 was transfected into CHO and COS-1 cells, followed by localization and detection of the protein expression of TAC1 by immunofluorescence staining and Western blot. The interaction between TAC1 and SPAG6 was determined by yeast two-hybrid experiment and Western blot.</p><p><b>RESULTS</b>Tac1 was expressed mainly in the testis, brain and heart. The results of restriction enzyme digestion and sequencing indicated successful construction of the recombinant plasmids, with the restriction fragment length of 390 bp. TAC1 was localized in the whole body of the CHO cells when transfected alone, but expressed in the microtubule of the cells when cotransfected with SPAG6, with the molecular weight of 40 000. Yeast two-hybrid experiment showed the colonies of TAC1 and SPAG6 on the culture plate without Leu, Trp and His, both contained in the yeast fusion protein.</p><p><b>CONCLUSIONS</b>The Tac1 recombinant plasmid was constructed successfully and the interaction between TAC1 and SPAG6 was confirmed with the plasmid.</p>