Associations between occupation exposure to Formaldehyde and semen quality, a primary study.

Formaldehyde (FA), a ubiquitous environmental pollutant, has long been suspected of having male reproductive toxicity. However, FA male reproductive toxicity was inconclusive due to dearth of human studies. Therefore, we sought to investigate whether occupational exposure to FA affects semen quality. Semen quality including five conventional parameters and seven kinematics parameters were compared between 114 male workers occupationally exposed to FA and 76 referents. FA exposure index (FEI) was measured and calculated. Our results showed that sperm progressive motility, total sperm motility, VCL, VSL and VAP were statistically significant decreased in FA exposure workers compared with the referents. Moreover, FEI was significantly negative associated with sperm progressive motility (ß = -0.19, P = 0.01) and total sperm motility (ß = -0.23, P = 0.004). In addition, a significant elevated risk of abnormal sperm progressive motility were observed in both low- (OR = 2.58; 95% CI: 1.11-5.97) and high-FA-exposed group (OR = 3.41; 95% CI: 1.45-7.92) respectively. Furthermore, a significant increased risk was also estimated for abnormal total sperm motility in both low- (OR = 3.21; 95% CI: 1.24-8.28) and high-FA-exposed group (OR = 4.84; 95% CI: 1.83-12.81) respectively. In conclusion, our study revealed the adverse effects of FA occupation exposure on semen quality, especially on sperm motion parameters.

[Diabetes induced by multiple low doses of STZ can be spontaneously recovered in adult mice].

In a previous study, we observed that multiple low doses of streptozotocin (STZ) can induce spontaneous restoration of normoglycemia in adult mouse diabetes models. In the present study, we sought to identify when spontaneous recovery from diabetes occurs and to disclose the changes in the diabetic data of diabetic mice induced by multiple low doses of streptozotocin (MLD-STZ mice). After inducing type 1 diabetes mellitus, radioimmunoassay, indirect immunofluorescence and hematoxylin-eosin staining were used to determine the diabetic data of MLD-STZ mice. In MLD-STZ mice, the diabetic indicators, including food intake, water consumption, body weight, blood glucose level and serum insulin concentration, developed gradually until week 20. Thereafter, the symptoms of diabetes gradually improved. By the week 36, although body weight and ß cell masses remained significantly different between the MLD-STZ mice and the age-matched control animals, food intake, water consumption, blood glucose levels and serum insulin concentrations had all returned to normal levels, and no lymphocytic infiltrations were observed in pancreatic islets. This data demonstrates that MLD-STZ can induce spontaneous recovery from diabetes mellitus in adult mice, suggesting further research into the processes by which normoglycemia is recovered.

[The change of alpha cell mass and its mechanism with diabetic progress].

OBJECTIVE: To explore the change of a cell mass and its mechanism with diabetic progress. METHODS: Diabetic mice were killed by exsanguinations after 4, 12 and 20 weeks of diabetes, respectively. Indirect double immunofluorescences for Insulin/Ki67, or BrdU, Cleaved-Caspase 3, TUNEL were used to evaluate pancreatic alpha cell mass, regeneration and apoptosis of a cells. Indirect triple immunofluorescences for Glucagon/ Neurogenin 3/MafA and Western blot analysis for Neurogenin 3 were used to determine neogenesis of pancreatic alpha cells. RESULTS: Pancreatic alpha cell mass was gradually increased with diabetic progress. It was significantly different from that of controls. There weren't any proliferation and apoptosis of alpha cell during diabetes, however, many Neurogenin 3+ cells appeared in the pancreatic islets of diabetic mice, and most of them were co-stained with MafA and Glucagon. CONCLUSION: Pancreatic alpha cell mass is gradually increased with diabetic progress. It seems to be strongly associated with neogenesis of pancreatic alpha cells.

[Polymorphism of Usp26 correlates with idiopathic male infertility].

OBJECTIVE: To study the correlation of nucleotide polymorphism in the ubiquitin-specific protease 26 (Usp26) gene with idiopathic male infertility and its action mechanism in spermatogenesis. METHODS: Based on the WHO criteria (4th ed.), we selected 41 patients with idiopathic infertility from 150 infertile males, and enlisted 50 normal fertile men as controls. We examined the selected patients for mutations using polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) analysis, and determined how and where the mutations occurred by gene sequencing. RESULTS: Low sperm concentration and poor sperm motility were found in the 41 men with idiopathic infertility. Nine (22.0%) of them exhibited changes in the Usp26 gene (P = 0.01), including compound mutations of 364insACA and 460G > A in 8 (19.5%, P = 0.01) and 1 044T > A substitution in 1 (2.4%, P > 0.05). The above three variations led to changes in the coding amino acids. No other changes were found in the remaining patients and normal fertile controls. CONCLUSION: The nucleotide polymorphisms of the Usp26 gene might be closely related with idiopathic male infertility, and exert negative effect on the testis function.

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.

Vasoactive intestinal peptide protects against ischemic brain damage induced by focal cerebral ischemia in rats.

Vasoactive intestinal peptide (VIP) exerts neuroprotective effects under various neurotoxic conditions in vitro. In the present study, we investigated the effects of VIP on transient ischemic brain damage. Focal cerebral ischemia was induced using middle cerebral artery occlusion (MCAO) for 120 min in the adult rat brain. Either a single intracerebroventricular injection of VIP or saline was given at the beginning of reperfusion. Forty-eight hours after MCAO, the rats were sacrificed for evaluation of the infarct volume and histological analysis. ELISA was performed to assay levels of serum S100B before being sacrificed. We also evaluated the blood-brain barrier (BBB) permeability using Evans blue dye injection method. In contrast to the cases treated with vehicle, the infarct volume was significantly (P<0.05) reduced, and terminal deoxynucleotidyl transferase-mediated dUTP-nick end labeling (TUNEL) staining and immunoreactivity for S100B were also significantly (P<0.05) decreased in the ischemic hemisphere with VIP treatment. In addition, the elevations of serum S100B were significantly (P<0.01) attenuated in VIP-treated rats compared with those of control rats. Treatment with VIP did not result in a significant reduction of Evans blue leakage, although it tended to be lower than that in the control rats. Our data suggest that treatment with VIP reduces brain damage in ischemic rats, and this effect may be associated with the attenuation of apoptosis and S100B expression.

[Recombinant lentivirus-mediated gene transfer of NT4-p53(N15)-Ant inhibits the growth of hepatocellular carcinoma cells in vitro].

OBJECTIVE: To construct a recombinant lentivirus vector containing fusion gene NT4-p53(N15)-Ant and transfer it into HepG2 cancer cells for gene therapy. METHODS: The gene of p53(N15)-Ant was obtained by T-vector method. After restriction enzyme digestion, the interest gene of p53(N15)-Ant was inserted in pBV220/NT4 vector and fusion gene of NT4-p53(N15)-Ant was subcloned into the plasmid of lentivirus and cotransferred into HEK-293 cells with helper plasmid. The recombinant lentivirus was produced by homologous recombination of the above mentioned two plasmids in HEK-293 cells and its titer was measured by plaque-forming. The expression of LV. NT4-p53-Ant in transfected HepG2 cells was finally confirmed by reverse transcription polymerase chain reaction (RT-PCR) procedure. The effect of LV. NT4-p53(N15)-Ant on HepG2 cells was measured by a colorimetric 3-[4,5-dimethyl thiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay. The inhibition effect on HepG2 cells of LV. NT4-p53(N15)-Ant and its potential mechanism was detected by light microscopy, electron microscopy, MTT, LDH-release assay and annexin V-PI double staining. RESULTS: The gene of p53(N15)-Ant was confirmed by restriction enzyme digestion and DNA sequencing. High titer of the recombinant lentivirus was obtained by homologous recombination in HEK-293 cell lines (1 x 10(11) pfu/ml), and the expression of NT4-p53(N15)-Ant gene in HepG2 cells was confirmed by RT-PCR. The viability of HepG2 cells was decreased to 83.4%, 46.9% and 33.9%, at 24 h, 48 h and 72 h, respectively, after infection by LV. NT4-p53(N15)-Ant. Compared with the LV. EGFP control group, there were significant differences (P < 0.01). The LDH level in HepG2 cells infected by LV. NT4-p53(N15)-Ant at 48 h, 72 h and 96 h after infection was 682 IU/L, 815 IU/L and 979 IU/L, respectively, significantly increased than that in the LV. EGFP group (P < 0.01), indicating the cell membrane destruction. CONCLUSION: The recombinant lentivirus vector encoding gene NT4-p53(N15)-Ant is successfully constructed in this experiment by molecular cloning and recombination in vitro techniques, and the results suggested that this fusion gene has an anti-tumor effect, which provides the basis for further research on recombinant adenovirus for cancer gene therapy.

NT4(Si)-p53(N15)-antennapedia induces cell death in a human hepatocellular carcinoma cell line.

AIM: To construct the recombinant lentivirus expression plasmid, pLenti6/V5-NT4 p53(N15)-antennapedia (Ant), and study its effect on HepG2 cells. METHODS: Plasmid pLenti6/V5-NT4 p53(N15)-Ant was constructed incorporating the following functional regions, including signal peptide sequence and pro-region of neurotrophin 4, N-terminal residues 12-26 of p53 and 17 amino acid drosophila carrier protein, Ant. Hepatocellular carcinoma (HepG2) cells were used for transfection. 3-[4,5-dimethyl-thiazol-2yl]-2,5 diphenyl tetrazolium bromide (MTT) assay, lactate dehydrogenase (LDH) release assay, transmission electron microscopy (TEM) and flow cytometric analysis (FCM) were employed to investigate the effects of LV-NT4(Si)-p53(N15)-Ant in vitro on HepG2 cells. In vivo experiment was also performed to investigate the inhibitory effect of LV-NT4(Si)-p53(N15)-Ant on tumor growth in nude mice. RESULTS: LV-NT4(Si)-p53(N15)-Ant significantly suppressed the growth of HepG2 cells. MTT assay showed that the growth of HepG2 cells was mucj more significantly inhibited by LV-NT4(Si)-p53(N15)-Ant than by LV-EGFP. The inhibition rate for HepG2 cell growth in the two groups was 46.9% and 94.5%, respectively, 48 h after infection with LV-NT4(Si)-p53(N15)-Ant, and was 33.9% and 95.8%, respectively, 72 h after infection with LV-NT4(Si)-p53(N15)-Ant (P < 0.01). Light microscopy and TEM showed morphological changes in HepG2 cells infected with LV-NT4(Si)-p53(N15)-Ant, but no significant changes in HepG2 cells infected with LV-EGFP. Changes were observed in ultra-structure of HepG2 cells infected with LV-NT4(Si)-p53(N15)-Ant, with degraded membranes, resulting in necrosis. LDH release from HepG2 cells was analyzed at 24, 48, 72 and 96 h after infection with LV-NT4(Si)-p53(N15)-Ant and LV-EGFP, which showed that LDH release was significantly higher in LV-NT4(Si)-p53(N15)-Ant treatment group (682 IU/L) than in control group (45 IU/L, P < 0.01). The longer the time was after infection, the bigger the difference was in LDH release. FCM analysis showed that LV-NT4(Si)-p53(N15)-Ant could induce two different kinds of cell death: necrosis and apoptosis, with apoptosis being the minor type and necrosis being the main type, suggesting that LV-NT4(Si)-p53(N15)-Ant exerts its anticancer effect on HepG2 cells by inducing necrosis. The in vivo study showed that LV-NT4(Si)-p53(N15)-Ant significantly inhibited tumor growth with an inhibition rate of 66.14% in terms of tumor size and weight. CONCLUSION: LV-NT4(Si)-p53(N15)-Ant is a novel recombinant lentivirus expression plasmid and can be used in gene therapy for cancer.

[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 prepubertal exposure to estradiol benzoate on testicular development and function of Sprague-Dawley rats].

OBJECTIVE: To investigate the effects of prepubertal exposure to estradiol benzoate (EB) on the testicular development and function of SD rats. METHODS: Fifty four 21-day-old male SD rats were randomly and equally divided into 2 experimental groups (AEa and AEb, n = 18 each) and one control group (AC, n = 18). The rats in the experimental groups were injected (s. c.) with EB dissolved in corn oil at a dose of 0.1 (AEa) and 100.0 microg/(kg x d) (AEb) respectively during prepuberty for 14 d [from postnatal day (PND) 22 to PND 35]. The rats in the control group received vehicle injections only. The testicular development of the rats was observed. The testes were harvested at the stages of late puberty (PND 50), sexual maturity (PND 64) and adulthood (PND 130) (n = 6 at each stage). The serum testosterone, the weights and histology of the testes, and the quality of sperms in the epididymal cauda were examined. RESULTS: The testes of rats in group AEb descended at day 43.17 +/- 1.72, later than the rats in the AC group (27.00 +/- 0.89, P < 0.01). At the stage of late puberty, the AEb group had lower serum testosterone than the AC group (P < 0.05) and had less unilateral testes weights than the AC group (P < 0.01). Compared with the AC group, the histological alteration of the testes of rats in the AEb group included seminiferous tubules maldevelopment, decreased cell numbers of seminiferous epithelia, spermatogenesis blocking and Leydig cells aplasia. At the stage of sexual maturity, the AEb group had less unilateral testes weight than the AC group (P < 0.01) and maintained similar histological alterations of testes as at the stage of late puberty. At the stage of adulthood, no differences were observed among the three groups in the unilateral testes weights and the histological features of the testes (P > 0.05). However, the AEb group had significant lower density and motility of sperms and percentage of grade a+b sperms in the epididymal cauda than the AC group (P < 0.05). CONCLUSION: Prepubertal exposure to low dose of EB does not affect the testicular development and function of SD rats, while high dose [100 microg/(kg x d) x 14 d] exposure has significant short (PND 50 and 64) and long-term (PND 130) toxic effects. The toxic effects are associated with the damage of Leydig and Sertoli cells.

[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.

[Effects of experimental left varicocele on SPAG11 mRNA and SPAG11E in the testis and epididymis of adolescent rats].

OBJECTIVE: To investigate the effects of experimental left varicocele (ELV) on the expression of sperm associated antigen 11 (SPAG11) mRNA and its protein isomer SPAG11E in the testis and epididymis of adolescent rats, and to explore the mechanism of infertility caused by varicocele. METHODS: The experimental left varicocele model was established in the adolescent male Sprague-Dawley rats. Two and 4 weeks after the operation, the changes of SPAG11 mRNA and SPAG11E expression in the testis and epididymis were detected using immunohistochemistry and reverse transcription-polymerase chain reaction (RT-PCR). RESULTS: The expected product of SPAG11 367 bp amplified by RT-PCR was detected only in the epididymis. SPAG11E protein was observed mainly in the acrosomal vesicles and acrosome of round and elongating spermatids of the seminiferous epithelium, in the cytoplasm of Leydig cells, and in the supranuclear region of principle cells and stereocilia of the epididymal epithelium. Imaging and statistical analysis showed that SPAG11 mRNA and SPAG11E protein expressions in the left epididymis of the 2- and 4-week ELV groups presented a remarkable decrease (P < 0.05 or P < 0.01) compared with the right side and the corresponding control group, and the same decreased change in the left epididymis (P < 0.05 or P < 0.01) and an obvious reduction of SPAG11E immunopositive reaction in the right epididymis (P < 0.01) were noted in the 4-week group as compared with the 2-week group. No statistical difference of SPAG11E expression in the bilateral testes was found (P > 0.05) between the ELV group and the control, as well as between the 2- and 4-week ELV groups. CONCLUSION: SPAG11 is a specific gene expressed in the epididymis. The localization and expression of SPAG11E exhibited a region- and cell-specific pattern in both the testis and epididymis of adolescent rats. The expression levels of both SPAG11 mRNA and SPAG11E protein altered obviously in ELV rats. The results suggest that SPAG11 may not only play an important role in spermatogenesis and sperm maturation, but also be associated with varicocele-induced male infertility or subfertility.

[Effects of prepubertal exposure to diethylstilbestrol on testicular development and function of SD rats].

OBJECTIVE: To investigate the effects of prepubertal exposure to diethylstilbestrol (DES) on the testicular development and function of Sprague-Dawley (SD) rats. METHODS: Ninety 21-day-old male SD rats were randomly and equally divided into 4 experimental groups (Da, Db, Dc and Dd), which were injected with DES dissolved in corn oil at the dose of 0.01, 0.1, 1.0 and 10.0 microg/(kg x d) from postnatal day (PND) 22 to 35, and a control group (C), which received vehicle only. The testicular development of all the rats was observed, and their testes were harvested in the stages of late puberty (PND 50), sexual maturity (PND 64) and adulthood (PND 130) respectively to determine the weight and histological features of the testis and examine the quality of the sperm in the epididymal cauda of the PND 130 rats. RESULTS: The testis descent in the C, Da, Db, Dc and Dd groups occurred on PND 26.17 +/- 1.94, 26.83 +/- 1.47, 28.68 +/- 1.03, 33.50 +/- 1.87 and 41.50 +/- 2.74 respectively, significantly delayed in the Db, Dc and Dd groups compared with the C group (P < 0.05 or P < 0.01). On PND 50, the unilateral testis weights in the C, Da, Db, Dc and Dd groups were (1.38 +/- 0.01) g, (1.38 +/- 0.12) g, (1.30 +/- 0.14) g, (0.86 +/- 0.18) g and (0.73 +/- 0.27) g respectively, significantly less in the Dc and Dd groups than in the C group (P < 0.01). Compared with the C group, there was a slight decrease in the number of the cells in the epithelia of a few seminiferous tubules in the Db group on PND 50, maldevelopment of seminiferous tubules, reduced cell number in seminiferous epithelia, blocked spermatogenesis and aplasia of Leydig cells in the Dc and Dd groups in a dose-dependent manner. On PND 64, the unilateral testis weights in the C, Da, Db, Dc and Dd groups were (1.60 +/- 0. 06) g, (1.62 +/- 0.11) g, (1.58 +/- 0.08) g, (1.47 +/- 0.10) g and (0.99 +/- 0.37) g respectively, significantly less in the Dc and Dd groups than in the C group (P < 0.05 or P < 0.01), and the histological alteration of the testis in the Dc and Dd groups was similar to or less than that on PND 50. On PND 130, no statistic difference was observed either in unilateral testis weight or in the histological features of the testis between any experimental group and the control (P > 0.05). The sperm concentration in the epididymal cauda in the C, Da, Db, Dc and Dd groups were (73.00 +/- 16.90) x 10(6)/ml, (68.00 +/- 19.67) x 10(6)/ml, (68.67 +/- 12.15) x 10(6)/ml, (35.17 +/- 15.64) x 10(6)/ml and (19.13 +/- 5.17) x 10(6)/ml, significantly lower in the Dc and Dd groups than in the C group (P < 0.01). There was a significant decrease in sperm motility in the Dd group (P < 0.01), the percentage of grade a sperm in the Db, Dc and Dd groups (P < 0.05) and the percentage of grade b sperm in the Dd group (P < 0.01). CONCLUSION: Prepubertal exposure to low dose of DES (0.01 microg/[kg x d] x 14 d) does not significantly affect the testicular development and function of SD rats, while high dose (1.0-10.0 microg/[kg x d] x 14 d) has significant short- (PND 50 and 64) or long-term (PND 130) toxic effect, which increases with dose and decreases with age. The mechanism of the toxic effect involves the insults to the development and function of Leydig and Sertoli cells.

[Immunomodulatory effects of Astragalus polysaccharide in diabetic mice].

OBJECTIVE: To study the immunomodulatory effects of Astragalus polysaccharide (APS) in type 1 diabetic mice. METHODS: A mouse model of type 1 diabetes mellitus was established by intraperitoneal injection of multiple low dose streptozotocin (MLD-STZ). The diabetic mice were intraperitoneally administered 100, 200, 400 mg/kg APS or 1 ml normal saline (NS) every day respectively, then the diabetic mice were sacrificed after 15 or 30 days of treatment. The effect of APS on insulitis was determined via pancreatic histological analysis. Serum insulin autoantibody (IAA) levels were measured by radio-immunoassay (RIA). Proliferation ability of splenocytes to concanavalin A was tested by using [(3)H] thymidine incorporation assay. The levels of cytokine interferon-gamma (IFN-gamma) and interleukin-4 (IL-4) secreted by splenocytes were determined by enzyme linked immunosorbent assay (ELISA) method, and the expression of peroxisome proliferator-activated receptor gamma (PPARgamma) in spleens was characterized using Western-blot analysis. RESULTS: Attenuated insulitis, down-regulation of the serum IAA levels and Th1/Th2 cytokine ratio, decrease of the proliferation ability of splenocytes to concanavalin A, and up-regulation of the PPARgamma levels in spleens showed a significant time- and dose-dependent response to APS treatment as compared with the NS-treated group. CONCLUSION: APS possesses immunotherapeutic effects on mice with type 1 diabetes mellitus through improving the cell- and humoral-mediated immunity.

[Construction and antitumor effect of recombinant adenovirus vector containing heterofusion gene NT4p53(N15)Ant].

BACKGROUND & OBJECTIVE: Peptide p53(N15)Ant from the amino-terminal of p53 fused with cell penetrating peptide antennapedia (Ant) can induce rapid cell death resembling necrosis in breast cancer and pancreatic cancer, whereas it is low cytotoxic to normal cells. This study was to construct an recombinant adenovirus vector containing NT4p53(N15)Ant gene and investigate its antitumor effects. METHODS: Adenovirus packaging system was used to construct Ad-NT4p53(N15)Ant by recombinant technique in vitro. The recombinant adenovirus was packaged in HEK-293 cells and identified by reverse transcription-polymerase chain reaction (RT-PCR). Human hepatocellular carcinoma (HCC) cell line HepG2 were infected with Ad-NT4p53(N15)Ant or parallel control recombinant adenovirus Ad-GFP. After infection of Ad-NT4p53(N15)Ant, cell morphology was observed under inverted phase contrast microscope and transmission electron microscope. The effect of Ad-NT4p53(N15)Ant on HepG2 cells was detected by MTT assay and lactate dehydrogenase (LDH) release assay. RESULTS: NT4p53(N15)Ant significantly inhibited the proliferation of HepG2 cells. The survival rates of HepG2 cells in NT4p53(N15)Ant group were 36.67% at 48 h, 20.47% at 72 h and 17.82% at 96 h after infection, which were significantly lower than those in Ad-GFP group (P<0.05). Distinct cell membrane disruption, pore-like structures in cytoplasm, and chromatin condensation were observed in Ad-NT4p53(N15)Ant group. LDH release in supernatant of HepG2 cells were 94 U/L at 24 h, 236.3 U/L at 48 h, 267 U/L at 72 h and 313 U/L at 96 h in Ad-NT4p53(N15)Ant group, which were significantly higher than those in Ad-GFP group (P<0.05). CONCLUSION: NT4p53(N15)Ant could significantly inhibit the proliferation of HepG2 cells through necrosis pathway.

[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

Novel mutations in ubiquitin-specific protease 26 gene might cause spermatogenesis impairment and male infertility.

AIM: To study the incidence of single nucleotide polymorphisms in ubiquitin-specific protease 26 (USP26) gene and its involvement in idiopathic male infertility in China. METHODS: Routine semen analysis was performed. Infertility factors such as immunological, infectious and biochemical disorders were examined to select patients with idiopathic infertility. DNA was isolated from peripheral blood of the selected patients and control population, which were examined for mutations using polymerase chain reaction-single strand conformation polymorphism analysis. Furthermore, nucleotide sequences were sequenced in some patients and controls. RESULTS: Of 41 infertile men, 9 (22.0%, P = 0.01) had changes in USP26 gene on the X chromosome. A compound mutation (364insACA; 460G right triple arrow A) was detected in 8 patients (19.5%, P = 0.01) and a 1044T right triple arrow A substitution was found in 1 patient (2.4%, P > 0.05). All three variations led to changes in the coding amino acids. Two substitutions predict some changes: 460G right triple arrow A changes a valine into an isoleucine, and 1044T right triple arrow A substitutes a leucine for a phenylalanine. Another insertion of three nucleotides ACA causes an insertion of threonine. No other changes were found in the remaining patients and fertile controls. CONCLUSION: The USP26 gene might be of importance in male reproduction. Mutations in this gene might be associated with male infertility, and might negatively affect testicular function. Further research on this issue is in progress.

[Adenovirus-mediated killing of hepatocellular carcinoma HepG2 cells by heterogeneous fusion gene NT4p53(N15)Ant].

UNLABELLED: OBJECTIVE To construct a recombinant adenovirus Ad.NT4p53(N15)Ant and explore its cytotoxic effect against hepatocellular carcinoma HepG2 cells in vitro. METHODS: The recombinant adenovirus containing the fusion gene of neurotrophin 4 (NT4)signal peptide, N-terminal residues (12-26) of p53 and 17 amino acid Drosophila homeobox protein Antennapedia (Ant) was constructed by gene cloning protocol. The effect of this fusion gene on HepG2 cells was evaluated by MTT assay, PI staining and flow cytometry. RESULTS: The fusion gene Ad.NT4p53(N15)Ant was successfully constructed, as verified by restriction endonuclease digestion and PCR. Ad.NT4p53(N15)Ant could strongly suppress the growth of HepG2 cells (with a growth inhibition rate of 63.3% 48 h after infection) without affecting NIH-3T3 cells. Flow cytometry showed that Ad.NT4p53(N15)Ant could induce obvious apoptosis of HepG2 cells. CONCLUSION: The recombinant adenovirus containing NT4p53(N15)Ant fusion gene can inhibit the growth the of HepG2 cells in vitro partially by inducing cell apoptosis.