Polymorphism of Usp26 correlates with idiopathic male infertility / 中华男科学杂志

<p><b>OBJECTIVE</b>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.</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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.</p><p><b>CONCLUSION</b>The nucleotide polymorphisms of the Usp26 gene might be closely related with idiopathic male infertility, and exert negative effect on the testis function.</p>

Recombinant lentivirus-mediated gene transfer of NT4-p53(N15)-Ant inhibits the growth of hepatocellular carcinoma cells in vitro / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To construct a recombinant lentivirus vector containing fusion gene NT4-p53(N15)-Ant and transfer it into HepG2 cancer cells for gene therapy.</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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.</p><p><b>CONCLUSION</b>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.</p>

Effects of progesterone on intracellular free Ca2+ concentration in the spermatozoa of fertile men and patients with unexplained infertility / 中华男科学杂志

<p><b>OBJECTIVE</b>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.</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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).</p><p><b>CONCLUSION</b>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.</p>

Experimental varicocele affects VEGF and Flt-1 protein expressions in the testis and epididymis of adolescent rats / 中华男科学杂志

<p><b>OBJECTIVE</b>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.</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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).</p><p><b>CONCLUSION</b>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.</p>

Effects of experimental left varicocele on SPAG11 mRNA and SPAG11E in the testis and epididymis of adolescent rats / 中华男科学杂志

<p><b>OBJECTIVE</b>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.</p><p><b>METHODS</b>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).</p><p><b>RESULTS</b>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.</p><p><b>CONCLUSION</b>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.</p>

Effects of prepubertal exposure to diethylstilbestrol on testicular development and function of SD rats / 中华男科学杂志

<p><b>OBJECTIVE</b>To investigate the effects of prepubertal exposure to diethylstilbestrol (DES) on the testicular development and function of Sprague-Dawley (SD) rats.</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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).</p><p><b>CONCLUSION</b>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.</p>

Expressions of VEGF and Flt-1 in the testis, epididymis and epididymal sperm of adolescent rats / 中华男科学杂志

<p><b>OBJECTIVE</b>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.</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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.</p><p><b>CONCLUSION</b>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</p>

Adenovirus-mediated killing of hepatocellular carcinoma HepG2 cells by heterogeneous fusion gene NT4p53(N15)Ant / 南方医科大学学报

<p><b>UNLABELLED</b>OBJECTIVE To construct a recombinant adenovirus Ad.NT4p53(N15)Ant and explore its cytotoxic effect against hepatocellular carcinoma HepG2 cells in vitro.</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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.</p><p><b>CONCLUSION</b>The recombinant adenovirus containing NT4p53(N15)Ant fusion gene can inhibit the growth the of HepG2 cells in vitro partially by inducing cell apoptosis.</p>

Radioligand binding assay of progesterone receptors on normal fertile human sperm membrane / 中华男科学杂志

<p><b>OBJECTIVE</b>To investigate the progesterone-binding site on the normal fertile human sperm membrane after 2 hours of in vitro capacitation.</p><p><b>METHODS</b>Viable spermatozoa were selected by a swim-up method. After 2 hours of in vitro capacitation, multipoint saturation binding experiments were performed. Sperm suspension and increasing concentrations of progesterone-11alpha-glucuronide-[125I] iodotyramine (125I-P) were added to 7 total binding tubes respectively, and equal amounts of sperm suspension and 125I-P were added to another 7 corresponding non-specific binding tubes in the presence of 10 micromol/L progesterone. After incubation for 1 hour at 4 degrees C, the radioactivity of both the tubes and the pellets after centrifugation was measured respectively. The equilibrium dissociation constant (Kd) and maximum binding capacity (Bmax) were calculated using the mathematical model of single site multi-point saturation method of Scatchard function and least-squares regression.</p><p><b>RESULTS</b>Kd was (0.61 +/- 0.04) nmol/L and Bmax was (830 +/- 344) sites/cell. The significance test of the regression equation indicated that r = -0.980, P < 0.01.</p><p><b>CONCLUSION</b>There is a high affinity and low capacity binding site for the progesterone (progesterone receptor) on the normal fertile human sperm membrane.</p>

Novel mutations in ubiquitin-specific protease 26 gene might cause spermatogenesis impairment and male infertility / 亚洲男科学杂志(英文版)

<p><b>AIM</b>To study the incidence of single nucleotide polymorphisms in ubiquitin-specific protease 26 (USP26) gene and its involvement in idiopathic male infertility in China.</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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.</p><p><b>CONCLUSION</b>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.</p>

Effect of Astragalus polysaccharide on pancreatic cell mass in type 1 diabetic mice / 中国中药杂志

<p><b>OBJECTIVE</b>To study the effect of Astragalus polysaccharide (APS) on pancreatic beta cell mass in type 1 diabetic mice.</p><p><b>METHOD</b>Diabetic mice induced by multiple low dose streptozotocin (MLD-STZ) were administered either APS (100, 200, 400 mg x kg(-1) body weight) or saline intraperitoneally daily, and sacrificed after 15 or 30 days of treatment. Streptavidin-peroxidase immunohistochemical method with counterstain was performed to determine the effect of APS on insulitis. Indirect double immunofluorescence for Insulin/Ki67 (counterstained by Hoechst33258) and Insulin/Cleaved caspase-3 was used to evaluate pancreatic cell (besides beta cell) proliferation, beta cell neogenesis, beta cell apoptosis and beta cell mass. Semi-quantitative RT-PCR was utilized to characterize pancreatic regenerating protein 1 mRNA levels, and ELISA method was performed to measure the levels of cytokine IFN-gamma and IL-4 secreted by splenocytes.</p><p><b>RESULT</b>Attenuated insulitis, upregulated beta cell mass, increased number of neogenetic pancreas islets, decreased number of apoptosis beta cells and downregulation of Th1/Th2 cytokine ratio were significantly time-and dose-dependent on APS treatment, when compared to saline controls. However, no significant differences of the number of pancreatic proliferative cells or replicative cells and pancreatic regenerating protein 1 mRNA levels were demonstrated between APS (APS100, APS200 and APS400) and saline vehicle group on day 15 and 30 with APS treatment.</p><p><b>CONCLUSION</b>APS can upregulate pancreatic beta cell mass in type 1 diabetic mice, strongly associated with improved autoimmunity.</p>

Effect of tail-suspension on the reproduction of adult male rats / 中华男科学杂志

<p><b>OBJECTIVE</b>To study the effects on the male reproduction in adult male rats and its mechanisms through simulated weightlessness using tail-suspension, in order to do a basic works of exploring the effects on human being's reproduction in outer space.</p><p><b>METHODS</b>Forty Spraque-Dawley adult male rats were randomly divided into four groups, two experimental groups and two control groups. Rats in the two experimental groups were tail-suspended for 14 d and 28 d respectively, then we examined the weight and morphology of testis, the quality and amount of sperm, also tested the serum hormone by radioimmunoassay and analyzed apoptosis rate of testicular cells by TUNEL in the experimental rats and control rats.</p><p><b>RESULTS</b>After tail-suspension, the weight of testis, the sperm count and sperm motility significantly decreased (P <0.05), while the apoptosis rate of testicular cells and the amount of abnormal sperm markedly increased (P <0.05). The content of testosterone significantly decreased (P <0.05), but the contents of FSH and LH mildly increased (P > 0.05). These changes were not significant between two experimental groups (P > 0.05). In addition, the seminiferous tubules became atrophy with the reduction of the layers of seminiferous epithelium, and sperm amount in lumens of seminiferous tubules decreased in experimental groups. The above were more remarkable in the 28 d experimental group.</p><p><b>CONCLUSION</b>Simulating weightlessness has a harmful effect on reproduction of adult male rats. These may be caused by inducing apoptosis. The blocking apoptosis of testicular cells may be useful in improving the harmful effect.</p>

Effects of ketamine-midazolam anesthesia on the expression of NMDA and AMPA receptor subunit in the peri-infarction of rat brain / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Activation of N-methyl-D-aspartate (NMDA) receptors and alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA) receptors play an important role in the neurons death induced by ischemia. The mitigating effect of intravenous anesthetics on ischemic neuron injury is related to their influence on NMDA receptors. This study was performed to investigate the effect of ketamine-midazolam anesthesia on the NMDA and AMPA receptor subunits expression in the peri-infarction of ischemic rat brain and explore its potential mechanism of neuroprotection.</p><p><b>METHODS</b>Thirty Sprague Dawley (SD) rats were subjected to permanent middle cerebral artery occlusion under ketamine/atropine (100/0.05 mg/kg) or ketamine-midazolam/atropine (60/50/0.05 mg/kg) intraperitoneal anesthesia (n=15 each). Twenty-four hours after ischemia, five rats in each group were killed by injecting the above dosage of ketamine or ketamine-midazolam intraperitoneally and infarct size was measured. Twenty-four and 72 hours after ischemia, four rats in each group were killed by injecting the above dosage of ketamine or ketamine-midazolam intraperitoneally. After staining the brain tissue slices with toluidine blue, the survived neurons in the peri-infarction were observed. Also, the expression level of NMDA receptors 1 (NR1), NMDA receptors 2A (NR2A), NMDA receptors 2B (NR2B) and AMPA (GluR1 subunit) were determined by grayscale analysis in immunohistochemical stained slices.</p><p><b>RESULTS</b>Compared with ketamine anesthesia, ketamine-midazolam anesthesia produced not only smaller infarct size [(24.1+/-4.6)% vs (38.4+/-4.2)%, P<0.05], but also higher neuron density (24 hours: 846+/-16 vs 756+/-24, P<0.05; 72 hours: 882+/-22 vs 785+/-18, P<0.05) and lower NR2A (24 hours: 123.0+/-4.9 vs 95.0+/-2.5, P<0.05; 72 hours: 77.8+/-4.1 vs 54.2+/-3.9, P<0.05) and NR2B (24 hours: 98.5+/-2.7 vs 76.3+/-2.4, P<0.05; 72 hours: 67.2 +/-7.5 vs 22.2+/-2.6, P<0.05) expression level in the peri-infarction following ischemia.</p><p><b>CONCLUSION</b>The protective effects of ketamine-midazolam anesthesia on ischemic brain injury may related to decreasing NR2A and NR2B expression.</p>

Expression of human epididymis protein 2beta1 in the testis and epididymis of adolescent male rats / 中华男科学杂志

<p><b>OBJECTIVE</b>To investigate the expression of human epididymal secretary protein 2 isoform human epididymal protein 2beta1(HE2beta1) in the testis and epididymis of adolescent male rats along with its significance.</p><p><b>METHODS</b>Immunohistochemical staining was used to detect the expression and localization of HE2beta1 in the testis and epididymis of 15 adolescent SD rats.</p><p><b>RESULTS</b>HE2beta1 immunoreactive staining was detected in the testis and epididymis. In the epithelia of the epididymal duct, HE2beta1 expressed mainly in the supranuclear region of the principle cells and the basement membrane of some epithelial cells; there were no immunostaining in the n clear cells, halo cells and basal cells. The immunopositive reaction was detected, weak in the distal caput, strong in the proximal, middle corpus and the cauda, but negative in the initial segment. Immunopositive results of HE2beta1 were also observed in some of the nuclei of spermatogonia and Sertoli cells with negatively-stained cytoplasm.</p><p><b>CONCLUSION</b>Immunohistochemical staining is a fairly sensitive method for detecting HE2beta1 expression. The localization and expression level of HE2beta1 in the genital duct of adolescent male rats exhibited a region- and cell-specific expression pattern, which suggests that HE2beta1 may play an important role in spermatogenesis, maturation and epididymal epithelial innate defense mechanisms.</p>

A preliminary study on the effect of prepubertal exposure of male rats to diethylstilbestrol on the apoptosis of spermatogenic cells after sexual maturation and its mechanism / 中华男科学杂志

<p><b>OBJECTIVE</b>To preliminarily study the effect of prepubertal exposure of male SD (Sprague-Dawley) rats to diethylstilbestrol (DES) on the apoptosis of spermatogenic cells after sexual maturation and its mechanism.</p><p><b>METHODS</b>Thirty 21-day-old male SD rats were randomly divided into 4 experimental groups, DES 0.01, 0.1, 1.0 and 10.0 microg/(kg x d) and 1 control group. The experimental groups were injected (s.c.) with different doses of DES (dissolved in corn oil) during prepuberty [from postnatal day (PND) 22 to PND 35] and the control group with medium only. The apoptosis and related proteins Bcl-2 and Bax expressions of testicular spermatogenic cells were studied with TUNEL and immunohistochemistry after the rats sexual maturation (at PND 64).</p><p><b>RESULTS</b>Compared with the control group, the apoptosis of testicular spermatogenic cells in the DES 0.01 microg/kg group had no difference, but significantly increased in the DES 0.1, 1.0 and 10.0 microg/kg groups and the apoptosis increased with the increase of DES dose. In the control and DES 0.01 microg/kg groups, Bax protein expressed weakly but Bcl-2 protein strongly in spermatogenic cells. With the increase of DES exposure, Bax protein expression in spermatogenic cells increased but Bcl-2 protein expression decreased.</p><p><b>CONCLUSION</b>Prepubertal exposure of SD rats to inappropriate dose of DES can make the apoptosis of spermatogenic cells increase after sexual maturation. Bax and Bcl-2 proteins participate in the apoptotic course caused by prepubertal DES exposure.</p>

The protective effect of vitamin E against oxidative damage caused by formaldehyde in the testes of adult rats / 亚洲男科学杂志(英文版)

<p><b>AIM</b>To investigate the effect of formaldehyde (FA) on testes and the protective effect of vitamin E (VE) against oxidative damage by FA in the testes of adult rats.</p><p><b>METHODS</b>Thirty rats were randomly divided into three groups: (1) control; (2) FA treatment group (FAt); and (3) FAt + VE group. FAt and FAt + VE groups were exposed to FA by inhalation at a concentration of 10 mg/m(3) for 2 weeks. In addition, FAt + VE group were orally administered VE during the 2-week FA treatment. After the treatment, the histopathological and biochemical changes in testes, as well as the quantity and quality of sperm, were observed.</p><p><b>RESULTS</b>The testicular weight, the quantity and quality of sperm, the activities of superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) and glutathione (GSH) were significantly decreased whereas the level of malondialdehyde (MDA) was significantly increased in testes of rats in FAt group compared with those in the control group. VE treatment restored these parameters in FAt + VE group. In addition, microscopy with hematoxylin-eosin (HE) staining showed that seminiferous tubules atrophied, seminiferous epithelial cells disintegrated and shed in rats in FAt group and VE treatment significantly improved the testicular structure in FAt + VE group.</p><p><b>CONCLUSION</b>FA destroys the testicular structure and function in adult rats by inducing oxidative stress, and this damage could be partially reversed by VE.</p>

Effects of experimental varicocele on CRES protein in the testis and epididymis of adolescent rats / 中华男科学杂志

<p><b>OBJECTIVE</b>To investigate the effects of experimental left varicocele (ELV) on the cystatin-related epididymal spermatogenic (CRES) protein in the testis and epididymis of adolescent rats.</p><p><b>METHODS</b>The ELV model of Sprague-Dawley (SD) male adolescent rats was established, and the expression of CRES protein in the testis and epididymis was detected by immunohistochemistry and Western-blot at 2 and 4 weeks after surgery.</p><p><b>RESULTS</b>Immunohistochemistry and Western-blot detected CRES protein in both the testis and the epididymis of the ELV rats and the control rats. Immunohistochemistry showed that within the testis, CRES protein was expressed mainly in the cytoplasm of round spermatids and elongating spermatids, sperm acrosomes and residual bodies. The expression was most intensive at Stages I-III and IX-XIV, and then decreased gradually at Stages VII-VII and IV-VI. Within the epididymis, CRES protein was expressed mainly in the cytoplasm of the principal cells of epididymal epithelia. Western-blot detected CRES protein in Mr 19,000 and 14,000, stronger in the former than in the latter. Image and statistical analyses showed that the expression of CRES protein in the 2-week and 4-week ELV groups was significantly higher than in the control group (P < 0.05, or P < 0.01).</p><p><b>CONCLUSION</b>CRES protein expressed in both the testis and epididymis of adolescent rats and the expression is stage-specific and cell-specific in the testis and segment-specific and cell-specific in the epididymis. The expression of CRES protein in the ELV rats is much stronger than in their corresponding controls. It is suggested that CRES protein may be significantly involved in the regulation of spermatogenesis and sperm maturation, and possibly associated with varicocele-related male infertility or subfertility.</p>

Effect of secretory human calcitonin gene-related peptide recombinant AAV on penile erection in streptozotocin-induced diabetic rat / 中华男科学杂志

<p><b>OBJECTIVE</b>To determine whether recombinant adeno-associated virus-mediated overexpression of hCGRP in the corpus cavernosum can affect the continuous production of hCGRP in the penile tissue and enhance erectile responses in STZ-induced diabetic rats.</p><p><b>METHODS</b>Diabetes mellitus was induced by a single intraperitoneal injection of 60 mg/kg streptozotocin in male SD rats. VssHGCMV-hCGRP, VssCMV-GFP and rAAV solution were injected into the corporal cavernosum of STZ-induced diabetic rats, respectively. The corporal tissue was obtained from groups of 8 rats on day 5 post-injection, and the expressions of CGRP and GFP in cavernosal tissue were detected using immunohistochemistry and laser scanning confocal microscopy, respectively. Cavemosal tissue cAMP and cGMP levels were measured using radioimmunoassay. On day 5 post-injection, intracavernous pressure induced by electrostimulation of penile dorsal nerves was measured and recorded with a biological signal processing system in each group rat.</p><p><b>RESULTS</b>rAAV transduction efficiency of GFP reporter gene was measured by laser scanning confocal microscopy and was observed in the penile tissue, especially in the corporal cavernosum and the vessel 5 days after transfection with VssCMV-GFP. Immunohistochemistry showed that the CGRP increased in the corporal cavernosum. In addition, both cAMP and cGMP levels in the corpora cavernosa transfected with VssHGCMV-hCGRP were significantly increased, compared with controls [(48.4 +/- 6.5) nmol/L and (21.2 +/- 13.6) nmol/L vs (16.7 +/- 2.5) nmol/L and (0.42 +/- 0.12) nmol/L, respectively]. More importantly, 5 days after administration of VssHGCMV-hCGRP,a significant increase was observed in the erectile response to penile dorsal nerve stimulation in the diabetic rat [(60.5 +/- 4.5) mm Hg vs (22. 3 +/- 1.3) mm Hg].</p><p><b>CONCLUSIONS</b>This results demonstrate that rAAV-mediated transfer of the CGRP gene can increase production of endogenous CGRP, cAMP and cGMP in corpora cavernosa of STZ-induced diabetic rats. Moreover, overexpression of CGRP enhances ICP and the erectile response to penile dorsal nerve stimulation in the diabetic rat.</p>

Expression and effect of hCGRP recombinant adeno-associated virus in primary cultured corporal cavernosum smooth muscle cells of the rat in vitro / 中华男科学杂志

<p><b>OBJECTIVE</b>To observe the expression and effect of human calcitonin gene-related peptide (hCGRP) gene mediated by recombinant adeno-associated virus (rAAV) in primary cultured corporal cavernosum smooth muscle cells of the rat and explore the possibility of using CGRP gene for gene therapy in erectile dysfunction.</p><p><b>METHODS</b>The primary cultured corporal cavernosum smooth muscle cells of the rat were randomly divided into 4 groups and infected with recombinant virus VssHGCMV-hCGRP, VssHGCMV, VssC-MV-GFP and the untreated, respectively. CGRP-like immunoreactivity was measured by protein dot blot assay in the 24 h-culture medium, and intracellular cAMP and cGMP levels in the cultured cells were also determined using radioimmunoassay to ascertain bioactivity of transduced CGRP.</p><p><b>RESULTS</b>The exogenous gene was transferred into primary corporal cavernosum smooth muscle cells by VssHGCMV-hCGRP infection and efficiently expressed. Compared with the control group, intracellular cAMP level in the cell infected by VssHGCMV-hCGRP was significantly increased (48.7 +/- 1.1 nmol/L vs 7.8 +/- 1.4 nmol/L, P < 0.01), whereas cGMP level remained unchanged in two groups, and CGRP-like immunoreactivity was also detected in the culture medium infected by VssHGCMV- hCGRP.</p><p><b>CONCLUSION</b>The system of secretory expressing bioactive peptide rAAV mediated gene transfer may be used to express efficiently exogenous gene in corporal cavernosum smooth muscle cells and affect cAMP level in the corporal cavernosum smooth muscle cells of the rat.</p>

Intracavernous pressure responses induced by electrical stimulation of penile dorsal nerves and intracavernosal drug injection in rats / 中华男科学杂志

<p><b>OBJECTIVE</b>To evaluate the application of intracavernous pressure (ICP) monitoring in the electrophysiologic and pharmacologic induction of penile erection.</p><p><b>METHODS</b>The penile dorsal nerves (DN) of 8 anesthetized adult male rats were isolated and the corpora cavernosa exposed. A heparinized 25-gauge angiocath (intravenous catheter) was inserted into the right corpus cavernosum to monitor the ICP and a 30-gauge needle was inserted into the left corpus cavernosum for intracavernosal drug administration. Penile erection was induced by electrical stimulation of the dorsal nerve (16 Hz frequency, 0.5 ms pulse width, 20 s duration, 4 volts) and intracavernous papaverine hydrochloride injection (0.4 mg). ICP was recorded with the SMUP-PC biological signal process system.</p><p><b>RESULTS</b>In the anesthetized rats, the baseline level of ICP was (12.3 +/- 3.1) mm Hg and the electrical stimulation of the DN significantly increased ICP[(36.4 +/- 2.3) mm Hg, P < 0.05], which slowly returned to baseline pressure after termination of the electrical stimulation. The intravavernosal administration of papaverine also induced a significant increase in ICP [(28.4 +/- 6.1) mm Hg, P < 0.05].</p><p><b>CONCLUSION</b>ICP monitoring in rats provides a useful scientific tool for in vivo studies of penile erection in experimental rat models. It is of great significance for further studying the physiology of penile erection and the pathogenesis of erectile dysfunction as well as for evaluating the efficacy of novel therapies for erectile dysfunction.</p>