Heat shock proteins (HSPs) widely exist in all organisms, the structures of which are usually extraordinarily conservative. They are also well-known stress proteins that are involved in response to physical, chemical and biological stresses. HSP70 is an important member of the HSPs family. In order to study the roles of amphibians HSP70 during infection, the cDNA sequence of Rana amurensis hsp70 family genes were cloned by homologous cloning method. The sequence characteristics, three-dimensional structure and genetic relationship of Ra-hsp70s were analyzed by bioinformatics methods. The expression profiles under bacterial infection were also analyzed by real-time quantitative PCR (qRT-PCR). Expression and localization of HSP70 protein were tested by immunohistochemical techniques. The results showed that three conservative tag sequences of HSP70 family, HSPA5, HSPA8 and HSPA13, were found in HSP70. Phylogenetic tree analysis indicated four members are distributed in four different branches, and members with the same subcellular localization motif are distributed in the same branch. The relative expression levels of the mRNA of four members were all significantly upregulated (P < 0.01) upon infection, but the time for up-regulating the expression levels were diverse in different tissues. The immunohistochemical analysis showed that HSP70 was expressed to different degrees in the cytoplasm of liver, kidney, skin and stomach tissue. The four members of Ra-hsp70 family have ability to respond bacterial infection to varying degrees. Therefore, it was proposed that they are involved in biological processes against pathogen and play different biological functions. The study provides a theoretical basis for functional studies of HSP70 gene in amphibians.
Heat-Shock Proteins/genetics* ; Phylogeny ; Amino Acid Sequence ; HSP70 Heat-Shock Proteins/metabolism* ; Stress, Physiological

Objective: To investigate the effect of acetyl-CoA carboxylase 1 (ACC1) knockdown on the migration of esophageal squamous cell carcinoma (ESCC) KYSE-450 cell and underlying mechanism. Methods: Lentiviral transfection was conducted to establish sh-NC control cell and ACC1 knocking down cell (sh-ACC1). Human siRNA HSP27 and control were transfected by Lipo2000 to get si-HSP27 and si-NC. The selective acetyltransferase P300/CBP inhibitor C646 was used to inhibit histone acetylation and DMSO was used as vehicle control. Transwell assay was performed to detect cell migration. The expression of HSP27 mRNA was examined by reverse transcription-quantitative real-time polymerase chain reaction (RT-qPCR) and the expressions of ACC1, H3K9ac, HSP27 and epithelial-mesenchymal transition-related proteins E-cadherin and Vimentin were detected by western blot. Results: The expression level of ACC1 in sh-NC group was higher than that in sh-ACC1 group (P<0.01). The number of cell migration in sh-NC group was (159.00±24.38), lower than (361.80±26.81) in sh-ACC1 group (P<0.01). The protein expression levels of E-cadherin and Vimentin in sh-NC group were statistically significant compared with sh-AAC1 group (P<0.05). The migrated cell number in sh-NC+ si-NC group was (189.20±16.02), lower than (371.60±38.40) in sh-ACC1+ si-NC group (P<0.01). The migrated cell number in sh-NC+ si-NC group was higher than that in sh-NC+ si-HSP27 group (152.40±24.30, P<0.01), and the migrated cell number in sh-ACC1+ si-NC group was higher than that in sh-ACC1+ si-HSP27 group (P<0.01). The protein expression levels of E-cadherin and Vimentin in sh-NC+ si-NC group were significantly different from those in sh-ACC1+ si-NC and sh-NC+ si-HSP27 groups (P<0.01). The protein expression levels of E-cadherin and Vimentin in sh-ACC1+ si-NC group were significantly different from those in sh-ACC1+ si-HSP27 group (P<0.01). After 24 h treatment with C646 at 20 μmmo/L, the migrated cell number in sh-NC+ DMSO group was (190.80±11.95), lower than (395.80±17.10) in sh-ACC1+ DMSO group (P<0.01). The migrated cell number in sh-NC+ DMSO group was lower than that in sh-NC+ C646 group (256.20±23.32, P<0.01). The migrated cell number in sh-ACC1+ DMSO group was higher than that in sh-ACC1+ C646 group (87.80±11.23, P<0.01). The protein expressions of H3K9ac, HSP27, E-cadherin and Vimentin in sh-NC+ DMSO group were significantly different from those in sh-ACC1+ DMSO group and sh-NC+ C646 group (P<0.01). The protein expression levels of H3K9ac, HSP27, E-cadherin and Vimentin in sh-ACC1+ DMSO group were significantly different from those in sh-ACC1+ C646 group (P<0.01). Conclusion: Knockdown of ACC1 promotes the migration of KYSE-450 cell by up-regulating HSP27 and increasing histone acetylation.
Humans ; Esophageal Neoplasms/pathology* ; Esophageal Squamous Cell Carcinoma/genetics* ; Vimentin/metabolism* ; Dimethyl Sulfoxide ; HSP27 Heat-Shock Proteins/metabolism* ; Histones/metabolism* ; Cadherins/metabolism* ; Cell Movement ; Cell Line, Tumor ; Cell Proliferation/genetics* ; Epithelial-Mesenchymal Transition/genetics* ; Gene Expression Regulation, Neoplastic

Heat stress transcription factors (Hsf) family is one of the most important transcription factor families in plants, and plays an important role in the growth and development of plants when encountering abiotic stresses such as heat, drought, and heavy metals. In this study, 20 SpbHsf genes were identified from the full-length transcriptome database of Setcreasea purpurea, and the structure and function of the Hsf gene family were analyzed using bioinformatics tools and qRT-PCR. The results showed that all SpbHsf proteins were hydrophilic. There were 12 SpbHsf proteins located in the nucleus, and the content of α-helix and random coil in the secondary structure of all SpbHsf proteins was high. The SpbHsf genes are divided into three subfamilies, each of which contains unique conserved motifs. All SpbHsf proteins contain DBD and HR-A/B domains. Phylogenetic analysis showed that OsHsf in Oryza sativa protein had the highest homology with SpbHsf protein. All the 20 SpbHsf genes were expressed in the root tissues of S. purpurea. Among them, 8 were significantly up-regulated while 8 were significantly down-regulated under Cu²⁺ stress. This study may help better understand the function and expression pattern of the S. purpurea Hsf gene family.
Droughts ; Gene Expression Regulation, Plant ; Heat Shock Transcription Factors/metabolism* ; Humans ; Phylogeny ; Plant Proteins/metabolism*

OBJECTIVES: The pathogenesis of androgenetic alopecia (AGA) is related to the level of androgen and its metabolic pathways. The binding of androgen and androgen receptor (AR) depends on the assistance of heat shock protein 27 (HSP27). HSP27 combined with microRNAs (miR)-1 can regulate AR levels. However, it is not clear whether HSP27 and miR-1 jointly participate in the pathogenesis of AGA. This study aims to investigate the role of AR up-regulation in the pathogenesis of AGA and underlying mechanisms. METHODS: A total of 46 male AGA patients (AGA group), who admitted to the First Affiliated Hospital of Guangzhou Medical University from September 2019 to February 2020, and 52 healthy controls admitted to the same period were enrolled in this study. Serum levels of dihydrotestosterone (DHT) and HSP27 in patients and healthy controls were measured by ELISA. Western blotting was used to detect the protein expression of HSP27 and AR in scalp tissues of patients and the healthy controls. The levels of HSP27, AR, and miR-1 were analyzed using real-time PCR. Human dermal papilla cells were transfected with HSP27 siRNA to inhibit the expression of HSP27. MiR-1 and miR-1 inhibitors were transfected simultaneously or separately into cells and then the changes in AR protein expression were detected. RESULTS: The levels of DHT and HSP27 in the AGA group were (361.4±187.7) pg/mL and (89.4±21.8) ng/mL, respectively, which were higher than those in the control group [(281.8±176.6) pg/mL and (41.2±13.7) ng/mL, both P<0.05]. However, there was no significant difference in serum HSP27 and AR levels among AGA patients with different degrees of hair loss (P>0.05). Correlation analysis showed that there was a positive correlation between HSP27 level and DHT level in the AGA patients (P<0.05). The level of HSP27 mRNA in scalp tissue was negatively correlated with that of miR-1 mRNA (P<0.05). Compared with the control group, the levels of HSP27 protein, AR protein, HSP27 mRNA, and AR mRNA in scalp tissues of AGA group were significantly increased (P<0.05). The up-regulation of HSP27 in scalp tissues of AGA patients was closely related to the increased levels of AR. However, the level of miR-1 in scalp tissues of AGA patients was significantly down-regulated, contrary to the expression of AR (P<0.05). Further in cell studies showed that inhibition of HSP27 or miR-1 expression in human dermal papilla cells could inhibit the expression of AR, and inhibition of both HSP27 and miR-1 expression was found to have an accumulative effect on AR, with statistically significant differences (all P<0.05). CONCLUSIONS HSP27 could combine with miR-1 to up-regulate AR levels, which is closely related to the development of AGA.
Alopecia/pathology* ; HSP27 Heat-Shock Proteins/metabolism* ; Humans ; Male ; MicroRNAs/genetics* ; RNA, Messenger ; Receptors, Androgen/metabolism* ; Up-Regulation

Objective: To study the effect of anti-fibrotic tetrapeptide N-acetyl-seryl-aspartyl-lysyl-proline (Ac-SDKP) on phosphorylated heat shock protein 27 (P-HSP27) and zinc finger family transcriptional repressor 1 (SNAI1) expression to explore the anti-silicosis fibrosis effect of Ac-SDKP. Methods: In December 2014, the rat silicosis animal model was prepared by one-time bronchial infusion of silicon dioxide (SiO(2)) dust. 80 SPF healthy adult Wistar rats were selected, and the rats were divided into 8 groups according to the random number table method, 10 in each group. Model control group for 4 weeks (feeding for 4 weeks) , model control group for 8 weeks (feeding for 8 weeks) : bronchial perfusion with normal saline 1.0 ml per animal. Silicosis model group for 4 weeks (feeding for 4 weeks) and silicosis model group for 8 weeks (feeding for 8 weeks) : bronchial perfusion of 50 mg/ml SiO(2) suspension 1.0 ml per animal. Ac-SDKP administration group for 4 weeks (feeding for 4 weeks) , Ac-SDKP administration group for 8 weeks (feeding for 8 weeks) : Ac-SDKP 800 μg·kg(-1)·d(-1) was administered by intraperitoneal pump. Ac-SDKP preventive treatment group: 48 h after Ac-SDKP 800 μg·kg(-1)·d(-1) administration, bronchial perfusion of SiO(2) suspension 1.0 ml per animal, raised for 8 weeks. Ac-SDKP anti-fibrosis treatment group: after bronchial perfusion of 1.0 ml of SiO(2) suspension for 4 weeks, Ac-SDKP 800 μg·kg(-1)·d(-1) was administered for 4 weeks. Western blotting was used to detect the expression of P-HSP27, SNAI1, α-smooth muscle actin (α-SMA) , and collage typeⅠ and Ⅲ in each group. The expression of P-HSP27 and SNAI1 was detected by immunohistochemistry, and the co-localized expression of P-HSP27 and α-SMA was detected by laser confocal microscopy. Results: Compared with the model control group, the expressions of P-HSP27, SNAI1, α-SMA, and collage typeⅠ and Ⅲ in the silicosis fibrosis area of the rats in the silicosis model group were enhanced, and the differences were statistically significant (P<0.05) . After Ac-SDKP intervention, compared with silicosis model group for 8 weeks, the expressions of P-HSP27, SNAI1 α-SMA, and collage typeⅠ and Ⅲ in the Ac-SDKP preventive and anti-fibrosis treatment groups were significantly decreased, and the differences were statistically significant (P<0.05) . However, the expressions of P-HSP27 SNAI1, and collage typeⅠ and Ⅲ between the Ac-SDKP administration group and the model control group did not change significantly, and the differences were not statistically significant (P>0.05) . Laser confocal results showed that the positive cells expressing P-HSP27 and α-SMA in the lung tissue of the silicosis model group were more than those in the model control group. Compared with the silicosis model group, the Ac-SDKP prevention and anti-fibrosis treatment groups expressing the positive cells of P-HSP27 and α-SMA decreased. Compared with the model control group for 8 weeks, there were some double-positive cells expressing P-HSP27 and α-SMA in the nodules of the silicosis model group for 8 weeks. Conclusion: Ac-SDKP may play an anti-silicic fibrosis effect by regulating the P-HSP27/SNAI1 pathway.
Animals ; HSP27 Heat-Shock Proteins ; Oligopeptides ; Rats ; Rats, Wistar ; Silicon Dioxide ; Silicosis/metabolism*

Objective: To investigate the effects and mechanism of negative pressure microenvironment on the neogenesis of human umbilical vein endothelial cells (HUVECs). Methods: The experimental research methods were adopted. The third to the fifth passage of HUVECs in the logarithmic growth stage were used for the subsequent experiments. Three batches of cells were taken, with each batch of cells being divided into normal control group and negative pressure treatment alone group (both routinely cultured for 24 h), and 17-allylamino-17-demethoxy-geldanamycin (17-AAG) alone group and 17-AAG+negative pressure treatment group (both cultured with 17-AAG for 24 h). In addition, the intermittent negative pressure suction, with the negative pressure value of -5.33 kPa (suction for 30 s, pause for 10 s) was continuously applied for 8 h on cells in the two negative pressure treatment groups using an automatic three-dimensional cell gradient negative pressure loading device designed and developed by ourselves. After the treatment of the first batch of cells, the cell proliferation level was detected by cell counting kit 8 method at 0 (immediately), 24, 48, and 72 h of culture, with the number of samples being 6. After the treatment of the second batch of cells, the scratch experiment was performed. At 12 h after scratching, the cell migration was observed under an inverted phase contrast microscope and the cell migration rate was calculated, with the number of samples being 3. After the treatment of the third batch of cells, the tubule formation experiment was conducted. After 6 h of culture, the tubulogenesis was observed under an inverted phase contrast microscope and the total tubule length and the number of branch nodes of cells were calculated, with the number of samples being 3. The cells were taken and divided into normal control group, negative pressure treatment alone group, and 17-AAG+negative pressure treatment group. The cells were treated the same as in the previous corresponding group. After the treatment, Western blotting was used to detect the protein expressions of heat shock protein 90 (HSP90), caveolin 1, endothelial nitric oxide synthase (eNOS), and eNOS phosphorylation site 1177 in the cells, and the eNOS phosphorylation site 1177/eNOS ratio was calculated, with the number of samples being 3; co-immunoprecipitation (co-precipitating HSP90 and caveolin 1, caveolin 1 and eNOS) and Western blotting were used to detect the protein expressions of caveolin 1 and eNOS in the cells, with the number of samples being 3; the protein co-localization of HSP90 and caveolin 1 and that of caveolin 1 and eNOS in the cells was assessed by immunofluorescence double staining. The molecular docking prediction of caveolin 1 and eNOS was processed by HADDOCK 2.4 protein-protein docking program. Data were statistically analyzed with analysis of variance for factorial design, one-way analysis of variance, and least significant difference method. Results: Compared with that in normal control group, the cell proliferation level in 17-AAG alone group was significantly decreased at culture hour of 24, 48, and 72 after the treatment (P<0.01), while the cell proliferation level in negative pressure treatment alone group was significantly increased at culture hour of 24, 48, and 72 after the treatment (P<0.01). Compared with that in 17-AAG alone group, the cell proliferation level in 17-AAG+negative pressure treatment group was significantly increased at culture hour of 48 and 72 after the treatment (P<0.05 or P<0.01). Compared with that in negative pressure treatment alone group, the cell proliferation level in 17-AAG+negative pressure treatment group was significantly decreased at culture hour of 24, 48, and 72 after the treatment (P<0.01). At 12 h after scratching, compared with (39.9±2.7)% in normal control group, the cell migration rate in 17-AAG alone group was significantly decreased ((10.7±2.7)%, P<0.01), while the cell migration rate in negative pressure treatment alone group was significantly increased ((61.9±2.4)%, P<0.01). Compared with those in 17-AAG alone group, the cell migration rate in 17-AAG+negative pressure treatment group was significantly increased ((37.7±3.7)%, P<0.01). Compared with that in negative pressure treatment alone group, the cell migration rate in 17-AAG+negative pressure treatment group was significantly decreased (P<0.01). At culture hour of 6 after the treatment, compared with those in normal control group, the total length of the tube formed by the cells in 17-AAG alone group was significantly shortened (P<0.05) and the number of branch nodes was significantly reduced (P<0.05), while the total length of the tube formed by the cells in negative pressure treatment alone group was significantly prolonged (P<0.01) and the number of branch nodes was dramatically increased (P<0.01). Compared with that in 17-AAG alone group, the number of branch nodes of the tube formed by the cells was significantly increased in 17-AAG+negative pressure treatment group (P<0.05). Compared with those in negative pressure treatment alone group, the total length of the tube formed by the cells in 17-AAG+negative pressure treatment group was significantly shortened (P<0.01) and the number of branch nodes was significantly reduced (P<0.01). Western blotting detection showed that after treatment, the overall comparison of eNOS and caveolin 1 protein expressions among the three groups of cells showed no statistically significant differences (P>0.05). The expression of HSP90 protein and the eNOS phosphorylation site 1177/eNOS ratio in the cells of negative pressure treatment alone group were significantly increased (P<0.01) compared with those in normal control group. Compared with those in negative pressure treatment alone group, the HSP90 protein expression and the eNOS phosphorylation site 1177/eNOS ratio in the cells of 17-AAG+negative pressure treatment group were significantly decreased (P<0.01). Co-immunoprecipitation and Western blotting detection after the treatment showed that compared with those in normal control group, the expression of caveolin 1 protein in the cells of negative pressure treatment alone group was significantly increased (P<0.01), while the protein expression of eNOS was significantly decreased (P<0.05). Compared with those in negative pressure treatment alone group, the expression of caveolin 1 protein in the cells of 17-AAG+negative pressure treatment group was significantly decreased (P<0.01), while the protein expression of eNOS was significantly increased (P<0.01). After the treatment, compared with those in normal control group, the co-localization of HSP90 and caveolin 1 protein in the cells of negative pressure treatment alone group was significantly increased, while the co-localization of caveolin 1 and eNOS protein was significantly decreased. Compared with those in negative pressure treatment alone group, the co-localization of HSP90 and caveolin 1 protein in the cells of 17-AAG+negative pressure treatment group was significantly decreased, while the co-localization of caveolin 1 and eNOS protein was significantly increased. Molecular docking prediction suggested that caveolin 1 interacted strongly with eNOS and inhibited the 1177 site phosphorylation of eNOS. Conclusions: The negative pressure microenvironment may inhibit the binding of caveolin 1 to eNOS by promoting the binding of HSP90 to caveolin 1 in HUVECs, so as to relieve the inhibition of 1177 site phosphorylation of eNOS by caveolin 1, thereby promoting the proliferation, migration, and tubulogenesis of HUVECs, and ultimately promoting the neogenesis of HUVECs.
Caveolin 1/metabolism* ; Cells, Cultured ; HSP90 Heat-Shock Proteins/metabolism* ; Human Umbilical Vein Endothelial Cells/metabolism* ; Humans ; Molecular Docking Simulation ; Phosphorylation

With the constant change of global climate, plants are often affected by multiple abiotic stresses such as heat stress, drought stress, cold stress and saline-alkali stress. Heat shock transcription factors (HSFs) are a class of transcription factors widely existing in plants to respond to a variety of abiotic stresses. In this article, we review and summarize the structure, signal regulation mechanism of HSFs and some research in plants like Arabidopsis thaliana, tomato, rice and soybean, to provide reference for further elucidating the role of HSFs in the stress regulation network.
Arabidopsis/metabolism* ; Droughts ; Gene Expression Regulation, Plant ; Heat Shock Transcription Factors/genetics* ; Plant Proteins/genetics* ; Stress, Physiological ; Transcription Factors/metabolism*

N-glycosylation modification, one of the most common protein post-translational modifications, occurs in heat shock protein gp96. The purpose of this study is to investigate the effect of N-glycosylation modification on immunologic function of the recombinant gp96 using the mutant gp96 in N-glycosylation sites. Firstly, wild-type and mutant gp96 proteins were expressed by insect expression system and their glycosylation levels were detected. To determine the effect of N-glycosylation on gp96 antigen presentation function, the IFN-γ+ CD8+ T cells in gp96-immunized mice and secretion level of IFN-γ were examined by flow cytometry and ELISA. The ATPase activity of gp96 was further detected by the ATPase kit. Finally, the effect of N-glycosylation on adjuvant function of gp96 for influenza vaccine was investigated in immunized mice. It was found that total sugar content of mutant recombinant gp96 was reduced by 27.8%. Compared to the wild type recombinant gp96, mutations in N-glycosylation sites resulted in decreased antigen presentation ability and ATPase activity of gp96. Furthermore, influenza vaccine-specific T cell levels induced by mutant gp96 as adjuvant were dramatically reduced compared to those by wild type recombinant gp96. These results demonstrate that N-glycosylation modification is involved in regulation of ATPase activity and antigen presentation function of gp96, thereby affecting its adjuvant function. The results provide the technical bases for development of gp96- adjuvanted vaccines.
Adjuvants, Immunologic ; Animals ; CD8-Positive T-Lymphocytes/metabolism* ; Glycosylation ; Heat-Shock Proteins ; Influenza Vaccines ; Mice

This study was aimed to investigate the regulatory mechanism of heat shock protein 90 (Hsp90) on transcription factor EB (TFEB) during autophagy in liver cancer cells. Human hepatocellular carcinoma cell line HepG2 was treated with Hsp90 N- and C-terminal inhibitors (STA9090 and Novobiocin), respectively. Western blot and RT-PCR were used to detect the expression levels of TFEB and autophagy-related proteins. Chromatin immunoprecipitation (ChIP) assay was used to observe the ability of Hsp90α binding to the TFEB proximal promoter region. The double-luciferase gene reporter experiment was used to determine the activity of TFEB promoter. The results showed that hypoxia induced up-regulation of TFEB protein and mRNA expression levels in the HepG2 cells. The protein expression levels of TFEB, LC3 and P62 were down-regulated significantly by either STA9090 or Novobiocin, under both normoxic and hypoxic conditions. Transfection of Hsp90α-overexpressing plasmids up-regulated TFEB protein levels in either wild-type or Hsp90α knockout HepG2 cells. Hsp90 bound to the TFEB proximal promoter region and was involved in regulating TFEB transcriptional process. Whereas both STA9090 and Novobiocin inhibited Hsp90 to bind to the TFEB proximal promoter region, and decreased the activity of TFEB promoter. These results suggest that Hsp90 promotes TFEB transcription in human hepatocellular carcinoma cells by binding to the proximal promoter region, thereby up-regulating the expression levels of autophagy-related proteins.
Autophagy ; Basic Helix-Loop-Helix Leucine Zipper Transcription Factors ; metabolism ; Carcinoma, Hepatocellular ; metabolism ; pathology ; HSP90 Heat-Shock Proteins ; metabolism ; Hep G2 Cells ; Humans ; Liver Neoplasms ; metabolism ; pathology ; Promoter Regions, Genetic

Testicular cancer seminoma is one of the most common types of cancer among men of reproductive age. Patients with this condition usually present reduced semen quality, even before initiating cancer therapy. However, the underlying mechanisms by which testicular cancer seminoma affects male fertility are largely unknown. The aim of this study was to investigate alterations in the sperm proteome of men with seminoma undergoing sperm banking before starting cancer therapy, in comparison to healthy proven fertile men (control group). A routine semen analysis was conducted before cryopreservation of the samples (n = 15 per group). Men with seminoma showed a decrease in sperm motility (P = 0.019), total motile count (P = 0.001), concentration (P = 0.003), and total sperm count (P = 0.001). Quantitative proteomic analysis identified 393 differentially expressed proteins between the study groups. Ten proteins involved in spermatogenesis, sperm function, binding of sperm to the oocyte, and fertilization were selected for validation by western blot. We confirmed the underexpression of heat shock-related 70 kDa protein 2 (P = 0.041), ubiquinol-cytochrome C reductase core protein 2 (P = 0.026), and testis-specific sodium/potassium-transporting ATPase subunit alpha-4 (P = 0.016), as well as the overexpression of angiotensin I converting enzyme (P = 0.005) in the seminoma group. The altered expression levels of these proteins are associated with spermatogenesis dysfunction, reduced sperm kinematics and motility, failure in capacitation and fertilization. The findings of this study may explain the decrease in the fertilizing ability of men with seminoma before starting cancer therapy.
Acrosin/metabolism* ; Adult ; Case-Control Studies ; Chaperonin Containing TCP-1/metabolism* ; Electron Transport Complex III/metabolism* ; HSP70 Heat-Shock Proteins/metabolism* ; Humans ; Male ; Peptidyl-Dipeptidase A/metabolism* ; Proteasome Endopeptidase Complex/metabolism* ; Proteomics ; Semen Analysis ; Seminoma/metabolism* ; Sodium-Potassium-Exchanging ATPase/metabolism* ; Sperm Count ; Sperm Motility ; Spermatozoa/metabolism* ; Testicular Neoplasms/metabolism*