长期给予睾酮致大鼠生精小管伴有生精细胞排列疏松

目的:确定睾酮致大鼠睾丸内睾酮抑制因而精子发生障碍是否伴有生精细胞排列疏松。方法:成年雄性SD大鼠肌注十一酸睾酮[19 mg/(kg.15 d)]130 d后取睾丸组织块,作甲基丙烯酸树脂包埋切片,观察睾丸的组织学变化。结果:除精子形成、精子释放障碍等改变以外,11.5%的生精小管轮廓内生精细胞的排列明显较疏松,成串、成束或成团的生精细胞(主要是精母细胞和精子细胞)之间出现朝向小管腔走行的放射状裂隙。结论:生精细胞排列疏松是大鼠睾丸内睾酮抑制所致重要组织学改变之一。     

Role of glyceraldehyde 3-phosphate dehydrogenase in the development and progression of diabetic retinopathy

OBJECTIVE—Mitochondrial superoxide levels are elevated in the retina in diabetes, and manganese superoxide dismutase overexpression prevents the development of retinopathy. Superoxide inhibits glyceraldehyde-3-phosphate dehydrogenase (GAPDH), which activates major pathways implicated in diabetic complications, including advanced glycation end products (AGEs), protein kinase C, and hexosamine pathway. Our aim is to investigate the role of GAPDH in the development and progression of diabetic retinopathy and to elucidate the mechanism.RESEARCH DESIGN AND METHODS—Rats with streptozotocin-induced diabetes were in a state of poor control (GHb >11%) for 12 months, good control (GHb <7) soon after induction of diabetes, or poor control for 6 months with 6 months’ good control. Retinal GAPDH, its ribosylation and nitration, AGEs, and PKC activation were determined and correlated with microvascular histopathology.RESULTS—In rats with poor control, retinal GAPDH activity and expressions were subnormal with increased ribosylation and nitration (25–30%). GAPDH activity was subnormal in both cytosol and nuclear fractions, but its protein expression and nitration were significantly elevated in nuclear fraction. Reinstitution of good control failed to protect inactivation of GAPDH, its covalent modification, and translocation to the nucleus. PKC, AGEs, and hexosamine pathways remained activated, and microvascular histopathology was unchanged. However, GAPDH and its translocation in good control rats were similar to those in normal rats.CONCLUSIONS—GAPDH plays a significant role in the development of diabetic retinopathy and its progression after cessation of hyperglycemia. Thus, therapies targeted toward preventing its inhibition may inhibit development of diabetic retinopathy and arrest its progression.Retinopathy is a multifactorial sight-threatening complication of diabetes. It is a progressive disease associated with chronic hyperglycemia (1). Although many glucose-induced retinal metabolic abnormalities are postulated to contribute to its development, the exact mechanism remains elusive (2–5). We have shown that in diabetes, retinal mitochondria experience increased oxidative damage and the mitochondrial enzyme that scavenges superoxide (manganese superoxide dismutase [MnSOD]) prevents vascular histopathology that is characteristic of diabetic retinopathy (6–8). Increased mitochondrial superoxide production inactivates glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in vascular endothelial cells, and inhibition of GAPDH is postulated to activate some of the key pathways that are associated with the development of diabetes complications, including increased formation of advanced glycation end products (AGEs) and activation of protein kinase C (PKC) and hexosamine pathway (9,10).GAPDH is a glycolytic enzyme that catalyzes the conversion of glyceraldehyde-3-phosphate to 1,3-bis-phosphoglycerate. Recent studies have shown that GAPDH is a protein with multiple cytoplasmic, membrane, and nuclear functions and is a major intracellular messenger mediating apoptosis of cells (11,12). GAPDH translocation to the nucleus is considered an important step in glucose-induced apoptosis of retinal Muller cells (13). The mechanism that initiates its translocation is not well understood; covalent modification by nitration/ribosylation is considered a likely possibility (14–16). How GAPDH contributes to the pathogenesis of diabetic retinopathy remains to be established.Good glycemic control attenuates the development/progression of retinopathy in diabetic patients, but its effects on the progression of retinopathy are not immediate, and it takes years for retinopathy to halt progression after the reestablishment of good control. The imprinted effects of prior glycemic control either produce the long-lasting benefits of good control or resist the arrest of progression of diabetic retinopathy after reinstitution of good control. Reinstitution of good control after a profound period of poor glycemic control does not immediately benefit the progression of retinopathy. This suggests a “metabolic memory” phenomenon (17–20). Metabolic memory phenomenon is observed also in animal models of diabetic retinopathy (21–26); the formation of acellular capillaries, characteristic of early signs of diabetic retinopathy, does not stop for at least 6 months when good control is initiated 6 months after induction of diabetes in rats, and nitrotyrosine levels and oxidative stress remain elevated (24,26). These abnormalities are, however, partially inhibited if the duration of poor control is reduced to 2 months, suggesting the role of oxidative stress in the metabolic memory phenomenon (24). The role of GAPDH in metabolic imprinting remains to be elucidated.In the present study, we investigated how GAPDH inhibition contributes to the development of retinopathy in diabetes and the mechanism(s) that could result in its inactivation. We have also explored the role of GAPDH in the metabolic memory phenomenon in diabetic rats by maintaining them in a state of poor control before initiation of a state of good control.     

Human ribonuclease 9, a member of ribonuclease A superfamily,specifically expressed in epididymis,is a novel sperm-binding protein

To explore the functions of human ribonuclease 9 (RNase 9), we constructed a mammalian fusion expression vector pcDNA-hRNase9, prepared recombinant human RNase 9-His fusion protein from HEK293T cells and determined its N-terminal amino acid sequences. According to the determined mature protein, recombinant human RNase 9 was prepared in E. coli. Ribonucleolytic activity and antibacterial activity of recombinant human RNase 9 were detected, and the distribution of human RNase 9 on tissues and ejaculated spermatozoa and in vitro capacitated spermatozoa were analyzed via indirect immunofluorescence assay. The results showed that recombinant human RNase 9 did not exhibit detectable ribonucleolytic activity against yeast tRNA, but exhibited antibacterial activity, in a concentration/time dependent manner, against E. coli. Immunofluorescent analyses showed that the predicted human RNase 9 was present throughout the epididymis, but not present in other tissues examined, and human RNase 9 was also present on the entire head and neck regions of human ejaculated spermatozoa and in vitro capacitated spermatozoa. These results suggest that human RNase 9 may play roles in host defense of male reproductive tract.     

Human T cells specifically activated against autologous malignant melanoma

Lymphocytes from ten patients with melanoma were specifically stimulated in vitro with autologous melanoma cells and expanded in interleukin 2. Significant lysis of autologous melanoma cells was demonstrated in T cells derived from six of these patients. The mean percent of lysis of autologous tumor cells at an effector-target ratio of 20:1 was 46% among these six patients. The T cells derived from two patients developed specificity in lysing autologous melanoma cells. In both cases, specificity was enhanced by the in vitro stimulation with autologous tumor cells. Restimulation with autologous melanoma cells was associated with increasing specificity over time. Whether derived from peripheral blood lymphocytes or from lymph node cells, T cells from one patient lysed fresh autologous melanoma cells more potently than K562, allogeneic melanoma cells, and nonmelanoma cells. On day 38, at an effector-target ratio of 10:1, cell lysis of K562, an osteosarcoma, a pancreatic cancer, and three allogeneic melanomas was 3%, 4%, 7%, 8%, 7%, and 2%, respectively, while lysis of autologous melanoma cells was 47%. Specificity was maintained beyond day 60. The T cells could be expanded over 50-fold within one month.     

饮酒对大鼠生精细胞iNOS,Bcl-2基因表达的影响

目的 探讨酒精对大鼠睾丸诱导型一氧化氮合酶（iNOS）、Bcl-2基因表达和生精细胞凋亡的影响。方法30只成年健康SD雄性大鼠随机均分为对照组、低剂量组和高剂量组，用不同剂量的酒精灌胃成年大鼠26d（两个生精周期）后，免疫组织化学法（SP法）检测睾丸iNOS、BCl-2基因表达的变化；原位缺口末端标记法（TUNEL法）检测细胞凋亡指数（A1）的变化。结果 与对照组相比，低剂量组大鼠睾丸iNOS、Bcl-2基闪表达强度和细胞凋亡指数（A1）无明显变化（P〉0．05）：而高剂量组与对照组和低剂量组相比，iNOS表达显著增强（P〈0．01），Bcl-2基因表达明显减弱（P〈0．01，P〈0．05），细胞凋亡指数则增加（P〈0.01）。结论 长期大量饮酒可以诱导睾丸生精细胞凋亡增加，iNOS与Bcl-2基因表达的改变是重要原因之一。     

Gene functional research using polyethylenimine-mediated in vivo gene transfection into mouse spermatogenic cells

Aim:To study polyethylenimine(PEI)-mediated in vivo gene transfection into testis cells and preliminary functionalresearch of spermatogenic cell-specific gene NYD-SP12 using this method.Methods:PEI/DNA complexes wereintroduced into the seminiferous tubules of mouse testes using intratesticular injection.Transfection efficiency andspeciality were analyzed on the third day of transfection with fluorescent microscopy and hematoxylin staining.Thelong-lasting expression of the GFP-NYD-SP12 fusion protein and its subcelluar localization in spermatogenic cells atdifferent stages were analyzed with fluorescent microscopy and propidium iodide staining.Results:With the media-tion of PEI,the GFP-NYD-SP12 fusion gene was efficiently transferred and expressed in the germ cells(especially inprimary spermatocytes).Transfection into Sertoli cells was not observed.The subcellular localization of the GFP-NYD-SP2 fusion protein showed dynamic shifts in spermatogenic cells at different stages during spermatogenesis.Conclusion:PEI can efficiently mediate gene transfer into spermatocytes.Thus,it might be useful for the functionalresearch of spermatogenic-cell specific genes such as the NYD-SP12 gene.In our study,the NYD-SP12 protein wasvisualized and was involved in the formation of acrosome during spermatogenesis.Our research will continue into thedetailed function of NYD-SP12 in spermatocytes.(Asian J Androl 2006 Jan;8:53-59)     

Urine glyceraldehyde excretion is elevated in the renal Fanconi syndrome

We analyzed urinary constituents using GC/MS in 16 children with the renal Fanconi syndrome and 13 normal individuals. Urine glyceraldehyde levels were strikingly elevated in the renal Fanconi syndrome group (mean 5.1 +/- 4.8 mg/mg creatinine) compared to levels in the normal group (mean 0.04 +/- 0.04 mg/mg creatinine, P less than 0.001). Urine lactate levels were also elevated in the renal Fanconi syndrome group (mean 2.3 +/- 2.6 mg/mg creatinine) compared to normals (mean 0.01 +/- 0.01 mg/mg creatinine, P less than 0.003). Only small elevations of glyceraldehyde and lactate were found in urine from children with other renal disorders. Serum levels of glyceraldehyde and lactate were no greater in individuals with the Fanconi syndrome than in the normals. The fractional reabsorption of both glyceraldehyde and lactate was virtually complete in the normals, but was markedly impaired in the Fanconi syndrome patients where, in some cases, glyceraldehyde excretion greatly exceeded the excretion of creatinine. We conclude that marked glyceraldehyde excretion is a previously unrecognized feature of the renal Fanconi syndrome which may result from disordered proximal tubular glycolytic metabolism. Further studies will be required to determine the role of glyceraldehyde loss in the pathogenesis of this generalized disturbance of proximal tubular function.     

Follicle-stimulating hormone autoantibody is involved in idiopathic spermatogenic dysfunction

Aim： To detect the anti-follicle-stimulating hormone （FSH） antibody in idiopathic infertile patients and fertile subjects in order to determine the role of this antibody in patients with spermatogenic dysfunction. Methods： The anti-FSH antibody in serum was detected by an enzyme-linked immunosorbent assay （ELISA）. The functional and structural integrity of the sperm membrane was evaluated with hypo-osmotic swelling （HOS） test and the ultrastructure of the spermatozoa was investigated by transmission electron microscopy （TEM）. Results： The extent of positive FSH antibody in the patients with oligozoospermia and/or asthenozoospermia was significantly higher than that in the fertile subjects and infertile patients with normal sperm concentration and motility, but it was significantly lower than that in the patients with azoospermia. The extent of anti-FSH antibody in the patients with azoospermia was significantly greater than that in patients with oligospermia and/or asthenospermia, infertile people with normal sperm density and motility and fertile people. The hypo-osmotic swelling test showed that the percentage of HOS-positive spermatozoa （swollen） was 45.1% ±3.5% in the FSH antibody-positive group and 59.1% ± 6.2% in the FSH antibody-negative control group. The percentage of functional membrane damage to spermatozoa was significantly higher in the anti- FSH antibody-positive group than in the control group. TEM showed that the outer acrosomal membrane was located far from the nucleus, and detachment of the acrosome was found in the FSH autoantibody-positive group. Conclusion： These data suggest that the presence of anti-FSH antibody is strongly correlated with the sperm quantity and quality in idiopathic male infertility. Anti-FSH antibody may be an important factor causing spermatogenic dysfunction and infertility.     

Biologically active ADAMTS13 is expressed in renal tubular epithelial cells

ADAMTS13 mRNA, which encodes the von Willebrand factor-cleaving protease, has been detected in a variety of tissues, including the kidney. The aim of our study was to characterize tubular expression and bioactivity of ADAMTS13. ADAMTS13 mRNA was detected in cultured primary human renal tubular epithelial cells (HRTEC) and in A498 cells, a human renal carcinoma cell line, by real-time PCR. Protein was detected using immunofluorescence and immunoblotting. Immunoblots demonstrated that the protein was secreted. The protease was proteolytically active in both cell lysates and cleaved the FRETS–VWF73 substrate. ADAMTS13 was demonstrated in situ in the renal cortex by immunohistochemistry. Protease was detected in both the proximal and distal renal tubules in normal renal tissue (n = 3) as well as in patients with tubular disorders (n = 3). Immunoblotting revealed that ADAMTS13 was present in the urine of patients with tubulopathy (n = 5) but not in normal urine. ADAMTS13 in urine had a molecular size similar to that in plasma, which indicates that the protease originates in the tubuli because such large proteins do not normally pass the glomerular filter. In conclusion, human renal tubular epithelial cells synthesize biologically active ADAMTS13 which may, after release from tubuli, regulate hemostasis in the local microenvironment.     

Human kallikrein-2 gene polymorphism is associated with the occurrence of prostate cancer

PURPOSE: Human glandular kallikrein, which is encoded by the human kallikrein-2 (KLK2) gene, is significantly associated with the occurrence of prostate cancer (PCa). We tested the association between a functional C748T polymorphism of the KLK2 gene and the occurrence of PCa. MATERIALS AND METHODS: Peripheral venous blood samples were obtained from 254 patients with PCa and 168 controls with benign prostatic hyperplasia. All control subjects had normal serum prostate specific antigen and proved to be free from malignancy on pathological examination of resected prostatic tissues. Serum prostate specific antigen, testosterone, Gleason score, clinical and pathological stage, tumor and prostate volume of the patients were investigated. The KLK2 gene polymorphism was determined by the polymerase chain reaction based restriction fragment length polymorphism method. RESULTS: The PCa group had a younger mean age +/- SEM (73.0 +/- 0.5 vs 74.7 +/- 0.5 years, p = 0.010) and higher C allele frequency (82.1% vs 74.7%, p = 0.010) than the control group. The frequency of the CC, CT and TT genotypes was 65.7%, 32.7% and 1.6% in patients with PCa, and 56.0%, 37.5% and 6.5%, respectively, in controls (p = 0.010). C allele carriers (CC and CT genotypes) were at significantly higher risk for PCa than TT homozygous subjects (p = 0.002). CC homozygous subjects were at significantly higher risk for PCa than T allele carriers (CT and TT genotypes) (p = 0.043). The PCa subgroup of patients with pathologically proved, localized PCa also had a higher frequency of the C allele (87.5% vs 74.7%, p = 0.026) and CC genotypes (78.7% vs 56.0%, p = 0.019) than controls. CONCLUSIONS: Our results suggest that the C allele of the functional C748T polymorphism of KLK2 may increase the risk of PCa.     

精液生精细胞检查的临床意义

本文报告了精液生精细胞检查与疾病的关系。化疗、放疗可引起生精细胞形态异常，精液中以多核精子细胞为主；长期服用雷公藤药物生精停滞于初级精母细胞阶段；长期接触高温、肾功能不全而行血透者生精细胞停滞于初级精母细胞及精子细胞阶段；双侧隐睾及克氏综合征患者精液中无精子及生精细胞；阴囊鞘膜积液患者约20％生精停滞于精子细胞阶段；精索静脉曲张（Ⅱ～Ⅲ°）精液中以精子细胞为主；睾丸及附睾炎症患者精液中除出现次级精母细胞、精子细胞外，以中性粒细胞、淋巴细胞、巨噬单核细胞多见。本研究为精液常规检查增加了新内容，为诊断与治疗不育症及环境遗传因素引起的生精障碍提供了客观指标。     

母源性印记基因3抑癌作用机制的研究进展

随着长链非编码RNA的研究热潮,母源性印记基因3(MEG3)作为长链非编码RNA家庭的一员也被广泛关注.MEG3不仅在良性疾病中有所研究,而且在恶性疾病中研究的更多且更为深入.MEG3因其抑癌的独特性在肿瘤研究中备受关注,但其抑癌的具体作用机制及相关途径还未完全明确,仍有待深入的研究与阐明.现本文将MEG3的抑癌作用及相关作用通路归纳总结.