JAK inhibitors attenuate hyperactivation of nonswitched memory B cells in rheumatoid arthritis patients in remission.

OBJECTIVE: To investigate the distribution and activation of B-cell subpopulations in rheumatoid arthritis (RA) patients treated with Janus kinase inhibitors (JAKis) and to analyze their correlation with disease remission. METHODS: Peripheral blood samples were collected from 23 adult healthy controls and 58 RA patients, 31 of whom were treated with JAKis and assessed during a 24-month follow-up. The number of peripheral B-cell subpopulations (including naive B cells, nonswitched memory B (NSMB) cells, switched memory B cells, and double-negative B cells), their activation, and phosphorylation of SYK and AKT upon B-cell receptor (BCR) stimulation in each population were analyzed by flow cytometry. RESULTS: Compared with that in healthy controls, the frequency of NSMB cells was significantly lower in new-onset untreated RA patients. However, expression of CD40, CD80, CD95, CD21low and pAKT significantly increased in these NSMB cells. Additionally, the number of NSMB cells correlated negatively with DAS28-ESR and IgG and IgA levels in these patients; expression of CD80, CD95 and CD21low on NSMB cells correlated positively with DAS28-ESR and IgG and IgA levels. After treatment with JAKis, the serum IgG concentration significantly decreased in RA patients in remission, but CD40, CD95 and pAKT levels in NSMB cells significantly decreased. CONCLUSION: RA patients present different B-cell subpopulations, in which the frequency of NSMB cells is negatively associated with disease activity. However, treatment with JAKis can inhibit activation of NSMB cells, restore the balance of kinase phosphorylation, and facilitate disease remission in RA patients.

A recombinant IL-1ß vaccine attenuates bleomycin-induced pulmonary fibrosis in mice.

Interleukin-1ß (IL-1ß) contributes to interstitial lung disease (ILD) and pulmonary fibrosis (PF), thus representing a potential therapeutic target for PF. In this study, we first verified the increased expression of IL-1ß in human fibrotic lung specimens and mouse lung tissues after intratracheal (i.t.) instillation of bleomycin (BLM), after which the pro-inflammatory and pro-fibrotic effects of recombinant IL-1ß were tested in mice. The results above suggested that vaccination against IL-1ß could be an effective strategy for managing PF. An anti-IL-1ß vaccine (PfTrx-IL-1ß) was designed by incorporating two IL-1ß-derived polypeptides, which have been verified as the key domains that mediate the binding of IL-1ß to its type I receptor, into Pyrococcus furiosus thioredoxin (PfTrx). The fusion protein PfTrx-IL-1ß was prepared by using E. coli expression system. The vaccine was well tolerated; it induced robust and long-lasting antibody responses in mice and neutralized the biological activity of IL-1ß, as shown in cellular assays. Pre-immunization with PfTrx-IL-1ß effectively protected mice from BLM-induced lung injury, inflammation, and fibrosis. In vitro experiments further showed that anti-PfTrx-IL-1ß antibodies counteracted the effects of IL-1ß concerning pro-inflammatory and pro-fibrotic cytokine production by primary mouse lung fibroblast, macrophages (RAW264.7), and type II alveolar epithelial cell (A549), primary mouse lung fibroblast activation and epithelial-mesenchymal transition (EMT) of alveolar epithelial cells. In addition, the vaccination did not compromise the anti-infection immunity in mice, as validated by a sepsis model. Our preliminary study suggests that the anti-IL-1ß vaccine we prepared has the potential to be developed as a therapeutic measure for PF. Further experiments are warranted to evaluate whether IL-1ß vaccination has the capacity of inhibiting chronic progressive PF and reversing established PF.

Effect of moxa smoke on sperm parameters and oxidative stress in rats with asthenozoospermia.

Asthenozoospermia is a leading cause of male infertility, characterized by reduced sperm motility. In this study, we determined sperm motility and the activities of antioxidant enzymes and oxidation products in the testis of rats with ornidazole (ORN)-induced asthenozoospermia and further examined and compared the differential effects of moxa smoke (MS) and cigarette smoke (CS) on sperm motility and oxidative stress (OS) of asthenozoospermic rats. The smoke intervention was initiated 11 days after intragastric administration of ORN, followed by the examination of testis index, sperm parameters, OS-related gene levels, and testicular histopathology. Sperm motility and antioxidant enzyme activities, as well as oxidation products significantly decreased in ORN-induced rats compared with MS-treated rats (p < .05-.001). MS treatment restored the reduced sperm motility and activities of glutathione peroxidase, superoxide dismutase, and catalase, but increased the malondialdehyde and nitric oxide synthetase levels in ORN-induced rats (p < .05-.001). Also, the histopathological changes in the testis of ORN-induced rats were improved by MS treatment. The study highlighted that MS was an effective factor in moxibustion therapy, which notably improved the sperm motility of asthenozoospermic rats by inhibiting OS in the reproductive system.

The proportion and phenotypic changes of CD4+CD25-Foxp3+ T cells in patients with untreated rheumatoid arthritis.

OBJECTIVE: CD4+CD25+Foxp3+ regulatory T (Treg) cell-mediated immunosuppression is an essential mechanism of rheumatoid arthritis (RA). However, little is known regarding the specific role of CD4+CD25-Foxp3+ Treg cells in RA. This study aimed to investigate the frequency of circulating CD4+CD25-Foxp3+ Treg cells and their role in RA. METHODS: Sixty-one untreated RA patients and 40 healthy controls (HCs) were enrolled in this study. The proportion of CD4+CD25-Foxp3+ T cells and CD4+CD25+Foxp3+ Tregs; the levels of CTLA4, GITR, Helios, and ICOS; and the production of IL-17A, IFN-γ, and IL-10 were assessed by flow cytometry. The correlation of CD4+CD25-Foxp3+ T cells and CD4+CD25+Foxp3+ Tregs with the clinical indicators was conducted by Spearman correlation analysis. RESULTS: The proportion of CD4+CD25-Foxp3+ T cells was elevated in RA and positively correlated with disease activity. CD4+CD25-Foxp3+ T cells expressed less Helios and produced more IFN-γ than conventional Tregs in RA. Additionally, the proportion of CD4+CD25-Foxp3+ T cells was positively correlated with DAS28 score, IgG titer, and anti-CCP titer. CONCLUSIONS: These data indicate that CD4+CD25-Foxp3+ T cells in RA exhibit several different functional properties from conventional Tregs and are correlated with RA disease activity.

HIP/PAP protects against bleomycin-induced lung injury and inflammation and subsequent fibrosis in mice.

Hepatocarcinoma-intestine-pancreas/pancreatitis-associated protein (HIP/PAP), a C-type lectin, exerts anti-oxidative, anti-inflammatory, bactericidal, anti-apoptotic, and mitogenic functions in several cell types and tissues. In this study, we explored the role of HIP/PAP in pulmonary fibrosis (PF). Expression of HIP/PAP and its murine counterpart, Reg3B, was markedly increased in fibrotic human and mouse lung tissues. Adenovirus-mediated HIP/PAP expression markedly alleviated bleomycin (BLM)-induced lung injury, inflammation, and fibrosis in mice. Adenovirus-mediated HIP/PAP expression alleviated oxidative injury and lessened the decrease in pulmonary superoxide dismutase (SOD) activity in BLM-treated mice, increased pulmonary SOD expression in normal mice, and HIP/PAP upregulated SOD expression in cultured human alveolar epithelial cells (A549) and human lung fibroblasts (HLF-1). Moreover, in vitro experiments showed that HIP/PAP suppressed the growth of HLF-1 and ameliorated the H2 O2 -induced apoptosis of human alveolar epithelial cells (A549 and HPAEpiC) and human pulmonary microvascular endothelial cells (HPMVEC). In HLF-1, A549, HPAEpiC, and HPMVEC cells, HIP/PAP did not affect the basal levels, but alleviated the TGF-ß1-induced down-regulation of the epithelial/endothelial markers E-cadherin and vE-cadherin and the over-expression of mesenchymal markers, such as α-SMA and vimentin. In conclusion, HIP/PAP was found to serve as a potent protective factor in lung injury, inflammation, and fibrosis by attenuating oxidative injury, promoting the regeneration of alveolar epithelial cells, and antagonizing the pro-fibrotic actions of the TGF-ß1/Smad signaling pathway.

Disruption of ubiquitin specific protease 26 gene causes male subfertility associated with spermatogenesis defects in mice†.

Ubiquitin-specific protease 26 (USP26) is an X-linked gene exclusively expressed in the testis and codes for the USP26, a peptidase enzyme that belongs to the deubiquitinating enzyme family. Recent studies have indicated that mutations in USP26 affect spermatogenesis and are associated with male infertility in humans and mice. However, the exact role of USP26 in spermatogenesis and how it affects male reproduction remains unknown. In this study, we generated a conventional Usp26 knockout mouse model and found that deletion of Usp26 in male mice (Usp26-/Y) leads to significantly reduced pup numbers per litter and significantly increased intervals between two consecutive offspring. We also found that the serum follicle stimulating hormone and testosterone levels of adult Usp26-/Y mice were significantly decreased compared to those of Usp26+/Y mice. Histological examination results showed that Usp26-/Y mice had significantly increased percentage of abnormal seminiferous tubules at different ages. Flow cytometry results exhibited that Usp26-/Y mice had significantly reduced percentage of mature haploid cells in the testes compared to Usp26+/Y mice. Sperm counts in epididymis were also significantly declined in Usp26-/Y mice compared to those in Usp26+/Y mice. Immunohistochemistry and immunofluorescence staining and immunoprecipitation analysis results showed that USP26 and androgen receptor were co-localized in mouse testicular cells at different ages and they both had physiological interactions. All these results demonstrated that the loss of Usp26 affects spermatogenesis and hormone secretion and causes male subfertility. Our study also provides the evidence on the interactions between USP26 and androgen receptor in mouse testis, whereby pointing to a potential mechanism.

Moderate Dose of Trolox Preventing the Deleterious Effects of Wi-Fi Radiation on Spermatozoa In vitro through Reduction of Oxidative Stress Damage.

BACKGROUND: The worsening of semen quality, due to the application of Wi-Fi, can be ameliorated by Vitamin E. This study aimed to demonstrate whether a moderate dose of trolox, a new Vitamin E, inhibits oxidative damage on sperms in vitro after exposure to Wi-Fi radiation. METHODS: Each of the twenty qualified semen, gathered from June to October 2014 in eugenics clinic, was separated into four aliquots, including sham, Wi-Fi-exposed, Wi-Fi plus 5 mmol/L trolox, and Wi-Fi plus 10 mmol/L trolox groups. At 0 min, all baseline parameters of the 20 samples were measured in sequence. Reactive oxygen species, glutathione, and superoxide dismutase were evaluated in the four aliquots at 45 and 90 min, as were sperm DNA fragments, sperm mitochondrial potential, relative amplification of sperm mitochondrial DNA, sperm vitality, and progressive and immotility sperm. The parameters were analyzed by one-way analysis of variance and Tukey's posttest. RESULTS: Among Wi-Fi plus 5 mmol/L trolox, Wi-Fi-exposed and Wi-Fi plus 10 mmol/L trolox groups, reactive oxygen species levels (45 min: 3.80 ± 0.41 RLU·10-6·ml-1 vs. 7.50 ± 0.35 RLU·10-6·ml-1 vs. 6.70 ± 0.47 RLU·10-6·ml-1, P < 0.001; 90 min: 5.40 ± 0.21 RLU·10-6·ml-1 vs. 10.10 ± 0.31 RLU·10-6·ml-1 vs. 7.00 ± 0.42 RLU·10-6·ml-1, P < 0.001, respectively), percentages of tail DNA (45 min: 16.8 ± 2.0% vs. 31.9 ± 2.5% vs. 61.3 ± 1.6%, P < 0.001; 90 min: 19.7 ± 1.5% vs. 73.7 ± 1.3% vs. 73.1 ± 1.1%, P < 0.001, respectively), 8-hydroxy-2'-deoxyguanosine (45 min: 51.89 ± 1.46 pg/ml vs. 104.89 ± 2.19 pg/ml vs. 106.11 ± 1.81 pg/ml , P = 0.012; 90 min: 79.96 ± 1.73 pg/ml vs. 141.73 ± 2.90 pg/ml vs. 139.06 ± 2.79 pg/ml; P < 0.001), and percentages of immotility sperm (45 min: 27.7 ± 2.7% vs. 41.7 ± 2.2% vs. 41.7 ± 2.5%; 90 min: 29.9 ± 3.3% vs. 58.9 ± 4.0% vs. 63.1 ± 4.0%; all P < 0.001) were lowest, and glutathione peroxidase (45 min: 60.50 ± 1.54 U/ml vs. 37.09 ± 1.77 U/ml vs. 28.18 ± 1.06 U/ml; 90 min: 44.61 ± 1.23 U/ml vs. 16.86 ± 0.93 U/ml vs. 29.94 ± 1.56 U/ml; all P < 0.001), percentages of head DNA (45 min: 83.2 ± 2.0% vs. 68.2 ± 2.5% vs. 38.8 ± 1.6%; 90 min: 80.3 ± 1.5% vs. 26.3 ± 1.3% vs. 26.9 ± 1.1%; all P < 0.001), percentages of sperm vitality (45 min: 89.5 ± 1.6% vs. 70.7 ± 3.1% vs. 57.7 ± 2.4%; 90 min: 80.8 ± 2.2% vs. 40.4 ± 4.0% vs. 34.7 ± 3.9%; all P < 0.001), and progressive sperm (45 min: 69.3 ± 2.7% vs. 55.8 ± 2.2% vs. 55.4 ± 2.5%; 90 min: 67.2 ± 3.3% vs. 38.2 ± 4.0% vs. 33.9 ± 4.0%; all P < 0.001) were highest in Wi-Fi plus 5 mmol/L trolox group at 45 and 90 min, respectively. Other parameters were not affected, while the sham group maintained the baseline. CONCLUSION: This study found that 5 mmol/L trolox protected the Wi-Fi-exposed semen in vitro from the damage of electromagnetic radiation-induced oxidative stress.

Generation and Applications of a DNA Aptamer against Gremlin-1.

Gremlin-1, a highly conserved glycosylated and phosphorylated secretory protein, plays important roles in diverse biological processes including early embryonic development, fibrosis, tumorigenesis, and renal pathophysiology. Aptamers, which are RNA or DNA single-stranded oligonucleotides capable of binding specifically to different targets ranging from small organics to whole cells, have potential applications in targeted imaging, diagnosis and therapy. In this study, we obtained a DNA aptamer against Gremlin-1 (G-ap49) using in vitro Systematic Evolution of Ligands by Exponential Enrichment (SELEX). Binding assay and dot-blot showed that G-ap49 had high affinity for Gremlin-1. Further experiments indicated that G-ap49 was quite stable in a cell culture system and could be used in South-Western blot analysis, enzyme-linked aptamer sorbent assay (ELASA), and aptamer-based cytochemistry and histochemistry staining to detect Gremlin-1. Moreover, our study demonstrated that G-ap49 is capable of revealing the subcellular localization of Gremlin-1. These data indicate that G-ap49 can be used as an alternative to antibodies in detecting Gremlin-1.

In vitro selection and characterization of deoxyribonucleic acid aptamers against connective tissue growth factor.

Connective tissue growth factor (CTGF) is a secreted matricellular protein possessing complex biological functions. CTGF modulates a number of signaling pathways that are involved in cell adhesion, migration, angiogenesis, myofibroblast activation, extracellular matrix deposition and tissue remodeling. Aptamers are oligonucleic acid chains or polypeptides that bind with specific target molecules hence have the potential to be used in the detection and blockade of the targets. In this study, we selected CTGF-targeting DNA aptamers by using systematic evolution of ligands by exponential enrichment (SELEX). After 8 iterative rounds of selection, cloning, DNA sequencing and affinity determination, six aptamers with high affinities to CTGF were obtained. Among them, one (C-ap17P) binds with the N-terminal region (aa 1-190) and the other five (C-ap11, 12, 14, 15 and 18) bind with the C-terminal region (aa 191-350) of hCTGF specifically. The biological stability assay indicated that a representative aptamer, C-ap17P, could keep its integrity at a rather high level for at least 24 h in complete DMEM cell culture medium. These CTGF aptamers might be used as a easy and fast detection tool for CTGF and be developed as CTGF-specific inhibitors for both research works and clinical applications.

Effects of low-dose, long-term formaldehyde exposure on the structure and functions of the ovary in rats.

Formaldehyde (FA) is a ubiquitous environmental pollutant. However, the effects of FA exposure on reproduction are still a matter of scientific controversy. In this study, we assessed the ovarian toxicity of long-term, low-dose FA exposure in rats and explored the potential oxidative stress mechanisms. A total of 30 Sprague-Dawley female rats were randomly allotted to three groups, in which rats were exposed to FA at a dose of 0 mg/m(3) (control), 0.5 mg/m(3) and 2.46 mg/m(3), respectively, by inhalation consecutively for 60 days. The results showed that the ovarian toxicity of FA is dose dependent. Ovarian structure and function in the group of rats exposed to 0.5 mg/m(3) FA showed no obvious difference when compared with those in the control group. However, the activity of superoxide dismutase was significantly decreased, whereas the level of malondialdehyde was significantly increased in ovaries of rats exposed to 2.46 mg/m(3) FA. Moreover, histopathological results demonstrated that the number and size of mature follicles significantly decreased, vascular congestion and interstitial edema in the ovaries of rats exposed to 2.46 mg/m(3) FA. In conclusion, this study may suggest that the FA level of 0.5 mg/m(3) can be considered as a safe level for FA exposure, but long-term FA exposure at a dose of 2.46 mg/m(3) has a harmful effect on ovary by inducing oxidative stress.

Association between XRCC1 single-nucleotide polymorphisms and infertility with idiopathic azoospermia in northern Chinese Han males.

X-ray repair cross-complementing group 1 (XRCC1) is a scaffold protein that plays a critical role in DNA base excision repair. To explore the association between XRCC1 single-nucleotide polymorphisms and infertility with idiopathic azoospermia in a northern Chinese Han population, PCR restriction fragment length polymorphism was used to genotype a SNP locus (rs25487) of XRCC1 in 112 patients with idiopathic azoospermia and 156 healthy controls. Furthermore, nucleotide sequences were sequenced. The results showed that, compared with GG genotype, the GA and GA+AA genotypes showed a significant association with an increased risk of idiopathic azoospermia (OR 2.119, 95% CI 1.245-3.606, P=0.005), (OR 2.052, 95% CI 1.227-3.431, P=0.006) respectively. Meanwhile, the A allele frequency was significantly higher in azoospermic patients than that in controls (OR 1.472, 95% CI 1.029-2.105, P=0.034). The substitutions bring about an amino acid alteration: G→A changes the arginine residue into glutamine. In conclusion, the SNP locus rs25487 of XRCC1 could be a marker for genetic susceptibility to idiopathic azoospermia and the A allele might be a risk gene of idiopathic azoospermia in the northern Chinese Han population.

Effects of paternal occupation exposure to formaldehyde on reproductive outcomes.

OBJECTIVE: To investigate whether paternal occupational exposure to formaldehyde (FA) affects the reproductive outcomes. METHODS: Data were collected from 302 male workers occupationally exposed to FA and 305 referent controls through interview questionnaires. Formaldehyde exposure level was measured and calculated for every subject. Different reproductive outcomes were compared for two groups by logistic regression analyses. RESULTS: A significant increased risk of prolonged time to pregnancy (P = 0.034; odds ratio, 2.828; 95% confidence interval, 1.081 to 7.406) and significant elevated risk of spontaneous abortion (P = 0.021; odds ratio, 1.916; 95% confidence interval, 1.103 to 3.329) were observed in wives of male workers occupationally exposed to FA after correction for confounding factors. Moreover, reproductive toxicity due to FA exposure is dose dependent. CONCLUSIONS: This epidemiological study adds some evidence for the hypothesis that paternal FA occupation exposure has adverse effects on reproductive outcomes.

Adenovirus-mediated hepatocarcinoma-intestine-pancreas/pancreatitis-associated protein suppresses dextran sulfate sodium-induced acute ulcerative colitis in rats.

BACKGROUND: Although increased expression of hepatocarcinoma-intestine-pancreas/pancreatitis-associated protein (HIP/PAP) has been reported in ulcerative colitis (UC), its role in UC remains unclear. This study was designed to assess the function of HIP/PAP in experimental UC and further to explore its underlying mechanisms. METHODS: Recombinant adenovirus was prepared to mediate ectopic expression of HIP/PAP in the colon of rats. The effect of HIP/PAP on dextran sodium sulfate (DSS)-induced colitis was assessed by disease activity index (DAI), macroscopic, and histological evaluations. Superoxide dismutase (SOD) and myeloperoxidase (MPO) activities, malondialdehyde (MDA) content, and tumor necrosis factor α (TNF-α) and interleukin-6 (IL-6) production were determined in colonic mucosa. Proliferation cell nuclear antigen (PCNA) was immunostained to reflect the proliferation of colonic epithelia. The effects of HIP/PAP on proliferation and H(2)O(2) -induced apoptosis of SW480 and LoVo colonic adenocarcinoma cells were also determined. Gene expression profiles in SW480 after HIP/PAP overexpression were analyzed by microarray analysis. RESULTS: The protective effect of HIP/PAP against DSS-induced colitis in rats was confirmed. Ectopic expression of HIP/PAP resulted in attenuation of oxidative damage, reduction of TNF-α and IL-6 expression, and elevation of epithelial proliferation in colonic mucosa and led to decreased apoptosis and increased proliferation in colonic adenocarcinoma cells. Microarray analysis revealed altered expression of inflammation-related molecules, growth factors, proliferation-related molecules, and antioxidant enzymes under overexpression of HIP/PAP. CONCLUSIONS: HIP/PAP has a protective effect against DSS-induced colitis in rats via inhibiting inflammation, alleviating oxidative damage, and promoting colonic epithelium regeneration. HIP/PAP might represent a new promising therapeutic strategy in UC.

Correlation of Sperm Associated Antigen 11 (SPAG11) and its Isoforms with Varicocele in Rats.

BACKGROUND: We undertook this study to investigate the variation relationship of sperm associated antigen 11 (Spag11) mRNA expression and SPAG11E protein in the epididymis and spermatozoa of experimental left varicocele (ELV) rats. These findings could contribute to the understanding of the role of epididymal proteins in sperm functions and the mechanism of male infertility induced by varicocele. METHODS: The ELV model was established in adolescent male Sprague-Dawley rats. Four weeks after the operation, tissue distribution and changes in the expressions of Spag11 mRNA and SPAG11E protein caused by ELV in the whole of left epididymis and spermatozoa were studied using quantitative reverse transcription-polymerase chain reaction (RT-QPCR), immunohistochemistry and immunofluorescence. Significant differences were identified using one-way ANOVA followed by Student-Newman-Keuls test. Significance level (p) was fixed at 0.05. RESULTS: The expected product of Spag11 was 96 bp that amplified by RT-QPCR was detected in the epididymal tissue and spermatozoa. SPAG11E protein was confined mainly to the supranuclear region of the principal cells and the stereocilium of the epididymal epithelium, it was concentrated on the acrosome and the tail of spermatozoa except the terminal piece. Statistical analyses of the images and the data indicated that Spag11 mRNA and SPAG11E protein expressions in the left epididymis and spermatozoa of ELV rats presented a considerable decrease (p<0.001) compared with that of the corresponding control group. CONCLUSION: The expressions of Spag11 mRNA and SPAG11E protein declined markedly in ELV rats, which suggest that SPAG11E may not only play an important role in sperm maturation, but it may also be influenced by varicocele.

Association study of HLA-B alleles with idiopathic male infertility in Han population of China.

PURPOSE: To investigate the distributions of HLA-B alleles and estimate their associations with idiopathic male infertility in Chinese Han population. METHODS: Polymerase chain reaction-sequence-based typing (PCR-SBT) method was used for DNA typing at HLA-B locus in 109 patients with idiopathic male infertility and 152 healthy controls in male Han population of Shaanxi Province, situated in northwestern China. RESULTS: In total, we detected 45 HLA-B alleles in idiopathic infertile patients, 48 HLA-B alleles in control subjects. However, no significant differences of these allelic frequencies were found between the infertile patients and the controls. CONCLUSION: HLA-B gene was unlikely a major risk factor of idiopathic male infertility in this sample population. As different populations have different HLA polymorphisms, investigation of the relationship of other HLA genes and idiopathic male infertility with larger sample size, is warranted in the future.

[Experimental varicocele affects VEGF and Flt-1 protein expressions in the testis and epididymis of adolescent rats].

OBJECTIVE: To investigate the effects of experimental left varicocele (ELV) on the vascular endothelial growth factor (VEGF) and its receptor fms-like tyrosine kinase-1 (Flt-1) proteins in the testis and epididymis of adolescent rats, and to find out the correlation of the two proteins with varicocele-induced male infertility. METHODS: We established the ELV model in adolescent male SD rats, and detected the expressions of VEGF and Flt-1 proteins in the testis and epididymis by immunohistochemistry at 2 and 4 weeks after surgery. RESULTS: Cell- and region-specific expressions of VEGF and Flt-1 were observed in the testis and epididymis of the ELV and control groups. Statistical analysis showed that, in comparison with the corresponding control groups, the 2- and 4-week ELV groups exhibited a notable increase in the VEGF protein expression in the hibateral testis and epididymis (P < 0.01, P < 0.05); the Flt-1 expression was obviously upregulated in the hibateral testis and epididymis of the 2-week ELV group (P < 0.01, P < 0.01), but remarkably reduced in the hibateral testis and left epididymis of the 4-week ELV group (P < 0.01, P < 0.05), with no statistic difference in the right epididymis (P > 0.05). CONCLUSION: ELV can cause changes in the expressions of VEGF and Flt-1 proteins in the testis and epididymis of adolescent rats, and consequently affect spermatogenesis and spermiotelcosis, which may be one of the causes of varicocele-induced male infertility or subfertility.

The expression of Usp26 gene in mouse testis and brain.

Deubiquitinating enzymes (DUBs) play an important role in ubiquitin-dependent processes as negative regulators of protein ubiquitination. Ubiquitin-specific protease 26 (USP26) is a member of this family. The expression of Usp26 in mammalian testis and in other tissues has yet to be fully elucidated. To study the expression of Usp26 mRNA and protein in various murine tissues, reverse transcription (RT)-PCR and immunohistochemistry analyses were carried out. The RT-PCR analysis showed that the Usp26 transcript was expressed in all of the tested tissues. USP26 protein localization was examined by immunohistochemistry, and it was shown that USP26 was not detectable at 20 days postpartum, with the expression restricted to the cytoplasm of condensing spermatids (steps 9-16), Leydig cells and nerve fibers in the brain. In addition, the USP26 protein was detected at moderate levels in myocardial cells, the corpus of epidydimis, epithelium of the renal tubules and the seminal gland of postnatal day 35 mice. Its spatial and temporal expression pattern suggests that Usp26 may play an important role in development or function of the testis and brain. Further research into these possibilities is in progress.

[Effects of progesterone on intracellular free Ca2+ concentration in the spermatozoa of fertile men and patients with unexplained infertility].

OBJECTIVE: To investigate the difference in the responsiveness of intracellular free Ca2+ concentration ([Ca2+]i) to progesterone in the spermatozoa of normal fertile men and patients with unexplained infertility. METHODS: Nine normal fertile men and 10 patients with unexplained infertility were selected in this study. After swim-up separation of the motile fraction and 2-hour in vitro capacitation, the spermatozoa were loaded with the fluorescent calcium indicator Fluo-3/AM (8.85 micromol/L) for 40 minutes away from the light, and then the sperm suspension was mixed with equal amount of 20% gelatin to immobilize the spermatozoa. The basal intracellular free [Ca2+]i and that induced by 10 micromol/L progesterone in the individual sperm were assessed by laser scanning confocal microscopy. RESULTS: The infertile patients had a significantly lower basal level of [Ca2+]i in the capacitated sperm than the fertile men (P < 0.01). The sperm from the normal controls responded to progesterone by exhibiting a rapid but transient rise in [Ca2+]i, with the peak level significantly higher than the basal level (P < 0.05), while those from the infertile patients by showing a slight increase, with no significant difference between the peak and basal levels (P > 0.05). Both the peak of the progesterone-induced [Ca2+]i and its increase amplitude expressed as the difference between the peak and basal levels were significantly higher in the normal fertile group than in the infertile patients (P < 0.01). CONCLUSION: The responsiveness of [Ca2+]i to progesterone is reduced in the spermatozoa of patients with unexplained infertility, which suggests a functional defect in the non-genomic sperm membrane progesterone receptor responsible for calcium influx.

Androgen receptor: a new player associated with apoptosis and proliferation of pancreatic beta-cell in type 1 diabetes mellitus.

Androgen receptor (AR) mediates a wide range of cellular processes, such as proliferation, differentiation and apoptosis. Here we sought to identify whether AR was located in pancreatic beta-cells and investigate its functions in type 1 diabetes induced by multiple low doses of streptozotocin. Double/triple immunofluorescence, Western blot and semi-quantitative RT-PCR were carried out to determine variances of AR expression in beta-cells and correlation between AR and apoptosis/proliferation of beta-cells with progress of diabetes. In addition, in vitro primary beta-cells from control mice were cultured for 3 days or 6 days with compound stimulation in order to further identify effect of AR on beta-cell apoptosis and proliferation. AR expression in beta-cells peaked in control and 1-day diabetic mice, gradually and significantly decreased, even disappeared in diabetic mice with progress of diabetes. TUNEL-positive beta-cells were concomitant with overexpression of AR, and Ki67-positive beta-cells showed extremely weak, even negative AR staining. In vitro, AR could mediate beta-cell apoptosis, and AR antagonist flutamide contributed to beta-cell proliferation. In conclusion, AR is abundantly expressed in pancreatic beta-cell cytoplasm of control mice. With progress of type 1 diabetes, decrement of AR expression in diabetic mice contributes to prohibit beta-cells from apoptosis, and is strongly associated with beta-cell proliferation.

[Expressions of VEGF and Flt-1 in the testis, epididymis and epididymal sperm of adolescent rats].

OBJECTIVE: To study the expressions of the vascular endothelial growth factor (VEGF) and its receptor fms-like tyrosine kinase-1 (Flt-1) in the testis, epididymis and epididymal sperm of adolescent rats and explore the functions of both the proteins in the male reproductive system. METHODS: The expressions of VEGF and Flt-1 were detected in 20 adolescent SD rats, immunohistochemical staining used for both the testis and the epididymis and immunofluorescent staining for sperm. RESULTS: VEGF and Flt-1 proteins were specifically present in the testis, epididymis and sperm. In the testis, VEGF immunoreactive particles were localized in the cytoplasm of spermatogenic cells, the developing acrosome of spermatids, Sertoli cells and Leydig cells, while Flt-1 expressed mainly in the developing acrosome of spermatids and Leydig cells. In the epididymis, the cell-specific and region-specific expressions of VEGF and Flt-1 proteins were observed in the principal cells of epididymal epithelia, VEGF in the whole epididymis, while Flt-1 only in the caput and cauda segments. Both VEGF and Flt-1 were localized in the acrosome of the sperm head as well as in the neck, middle and principal segments of the sperm tail. CONCLUSION: The specific expression patterns of VEGF and Flt-1 in the rat testis, epididymis and sperm indicate that they may independently or collectively affect spermatogenesis and spermiotelcosis in either an autocrinological or a