p,p'-DDE对离体培养大鼠睾丸支持细胞转铁蛋白表达的影响

目的研究P,P'-DDE对离体培养大鼠睾丸支持细胞转铁蛋白表达的影响.方法对大鼠睾丸支持细胞离体原代培养,运用一步法RT-PCR检测给与P,P'-DDE后24 h支持细胞转铁蛋白mRNA水平.结果睾丸支持细胞转铁蛋白mRNA水平随P,P'-DDE所给剂量的增高而降低,且与对照组差异存在显著性(P＜0.05),呈剂量依赖关系.结论P,P'-DDE 可抑制睾丸支持细胞转铁蛋白的基因转录,从而抑制其合成,导致精子发生障碍和生殖功能紊乱.     

氟对体外大鼠睾丸支持细胞雄激素结合蛋白和抑制素B mRNA表达的影响

目的研究氟对体外培养大鼠睾丸支持细胞雄激素结合蛋白(ABP)和抑制素B(INHB)转录水平的影响。方法建立支持细胞体外培养模型,以浓度分别为2.5、5.0、10.0和20.0mg/L氟化钠溶液染毒细胞,用RT-PCR方法检测ABP和INHB mRNA的相对表达量。结果①ABP mRNA相对表达量在各剂量组与对照组相比,2.5mg/L组高于对照组,且差异有统计学意义(P0.05);5.0mg/L高于对照组,但差异没有统计学意义;其余各组低于对照组,但差异没有统计学意义(P0.05)。②INHB mRNA相对表达量在各剂量组与对照组比较,2.5和5mg/L组均高于对照组,差异具有统计学意义(P0.05);其余两组低于对照组,但是差异均无统计学意义(P0.05)。结论在2.5～20.0mg/L剂量范围内,未观察到氟对体外培养大鼠睾丸支持细胞ABP和INHB mRNA的表达有明显影响。     

p,p’-DDE对离体培养支持细胞DNA损伤与FasL基因表达的影响

目的研究p,p’-DDE对离体培养支持细胞DNA损伤与FasL基因表达的影响。方法从大鼠睾丸组织中分离支持细胞进行离体原代培养3天,加入不同浓度p,p’-DDE继续培养24h,应用单细胞凝胶电泳(SCGE)和反转录聚合链式反应(RT-PCR)研究p,p’-DDE诱导支持细胞DNA损伤和FasL基因表达。结果发现支持细胞DNA迁移度随着p,p’-DDE剂量的增高而增高,同时FasL的基因表达水平也随之增高。结论p,p’-DDE可诱导支持细胞DNA损伤和FasL基因表达,pp’-DDE可能是通过Fas/FasL途径诱导支持细胞损伤,破坏生精过程的动态平衡,最终导致精子减少。     

双酚A对大鼠睾丸支持细胞的功能影响及机制

目的 探讨双酚A(BPA)对雄性SD大鼠睾丸支持细胞功能的影响及损伤机制.方法 将16～22日龄SPF级雄性SD大鼠的睾丸支持细胞制成3.0×10~6～3.5×10~6个/ml的细胞悬液,设立空白对照组、溶剂对照组和1×10~(-7)、1×10~(-6)、1×10~(-5)、1×10~(-4)mol/L BPA染毒组,观察睾丸支持细胞的增殖、细胞周期以及PCNA、Vimentin蛋白表达的情况.结果 各染毒组睾丸支持细胞的存活率明显低于对照组(P<0.05),G_0/G_1期细胞构成比增加,而S期和M期细胞构成比降低,PI下降.免疫细胞化学结果显示,10~(-4)mol/L、10~(-5) mol/L BPA实验组显著抑制大鼠睾丸支持细胞PCNA的表达.BPA在10~(-4) mol/L、10~(-5)mol/L、10~(-6) mol/L剂量组显著抑制大鼠睾丸支持细胞内Vimentin的表达.结论 双酚A可通过影响睾丸支持细胞的细胞增殖、细胞周期和抑制PCNA、Vimentin蛋白表达而表现出生殖毒性.

Abstract：

Objective To investigate the effects of bisphenol A on SD rat Sertoli cell function and the injury mechanism. Methods SD rat Sertoli cells were treated with bisphenol A at different doses,and the control group,solvent control group were set. Sertoli cell proliferation,cell cycle and PCNA.Vimentin protein expression were observed. Results The survival rate of Sertoli cells in the treated groups was significantly lower than the control group (P<0.05), constitution ratio of G_0/G_1 phase cells increased,while for S phase and M-phase, it decreased,and PI decreased. Immunocytochemistry showed PCNA expression in Sertoli cells was significantly inhibited by 10~(-4),10~(-5) mol/L BPA. Vimentin expression in Sertoli cells was significantly inhibited by 10~(-4), 10~(-5),10~(-6) mol/L BPA. Conclusion Bisphenol A can affect Sertoli cell proliferation,cell cycle and inhibit PCNA.Vimentin protein expression in Sertoli cells.     

Chronic alcohol ingestion alters claudin expression in the alveolar epithelium of rats.

Previously we determined that chronic alcohol ingestion (6 weeks) in rats increases lung epithelial permeability in vivo approximately 5-6-fold and promotes flooding of the alveolar airspaces with proteinaceous fluid in response to stresses such as sepsis. In parallel, alveolar epithelial cells isolated from alcohol-fed rats fail to form tight monolayers in vitro, even when cultured for up to 8 days in the absence of alcohol. However, the molecular mechanisms underlying alcohol-induced permeability are unknown. Claudins are key components of tight junctions that restrict the paracellular movement of water, proteins, and solutes across cellular barriers including the alveolar epithelium. In this study, we examined the expression of multiple members of the claudin protein family in the lungs of alcohol-fed versus control-fed rats (Lieber-DeCarli liquid diet with either 36% of calories as alcohol or an isocaloric substitution with maltin-dextrin for 6 weeks). We determined that chronic alcohol ingestion affected the expression of multiple claudins; most striking were decreases in claudin-1 and claudin-7, and an increase in claudin-5, in the whole lung and in alveolar epithelial monolayers derived from alcohol-fed rats. In parallel, immunocytochemistry of alveolar epithelial monolayers from alcohol-fed rats revealed abnormal intracellular accumulation of claudin-7 protein and relatively decreased localization to cell membranes. Claudin-1 and claudin-7 are relatively specific to alveolar epithelial type I pneumocytes that form the vast majority of the alveolar epithelial barrier in vivo, and increases in claudin-5 have been associated with increased epithelial permeability in other systems. Therefore, these findings suggest that changes in claudin expression in the alveolar epithelium produce a "leakier" phenotype that renders the alcoholic lung susceptible to alveolar flooding during acute inflammatory stresses.     

双酚A雄性生殖毒性的体内外实验研究

目的探讨双酚A对雄性动物生殖机能的影响。方法将双酚A混入饲料(0、1和5g／kg)连续饲喂成年32只SD大鼠14d,放免法测定睾酮和雌二醇并进行右睾组织形态分析;对原代培养的支持细胞染毒(0、10^-7、10^-6、10^-5、10^-4moL／L)。结果5g／kg的双酚A组的右睾平均重量1．53g显著低于对照组1．62g,但1g／kg组与对照组差异无统计学意义。形态观察发现,2个双酚A组中的曲细精管基底膜均与生精细胞分离;部分生精细胞和支持细胞发生核固缩和空泡变性;同时,双酚A处理使黏附于支持细胞的生精细胞平均数量由对照组的7．94个分别减少为4．13和3．04个。此外,双酚A在体内外实验中均抑制睾丸支持细胞的波形蛋白表达,阻碍细胞骨架和胞间联结的形成,使支持细胞在体外培养中的形态变得异常细长。但双酚A对血清雌二醇和睾酮浓度的影响没有统计学意义。结论双酚A可能通过破坏支持细胞骨架和改变支持细胞形态而损害雄性生殖功能。     

Increases in apoptosis and declines in Bcl-XL protein characterise testicular regression in American crows (Corvus brachyrhynchos)

The present study investigated the cellular changes observed during testicular regression in American crows. Testes from adults caught during the early (March), progressing (April), peak (early May), transitional (late May), and post- (June) breeding season were examined. Apoptosis was assessed by in situ terminal deoxynucleotidyl transferase-mediated dUTP nick end-labelling (TUNEL) and Bcl-X(L) protein immunolabelling. Testis mass increased two-fold from March to early May (P < 0.05), then declined 19-fold by June (P < 0.001) without corresponding changes in body mass (P > 0.05). Testicular activity, evaluated using a spermatogenic index, increased nearly two-fold from March to early May and declined nine-fold in June (P < 0.001). Seminiferous tubule diameter declined four-fold in June compared with earlier months (P < 0.001). In all testes, TUNEL-positive germ cells were detected at low levels, with the highest levels observed in late May (P < 0.001). In contrast, TUNEL-positive Sertoli cells were maintained at low levels in March-April and increased nine-fold in early May (P < 0.001). The Bcl-X(L) immunostaining was detected in Sertoli cells in March-early May; however, staining was most intense in March-April and substantially weaker by early May. These data suggest that the seasonal rise in testicular competence occurs slowly in American crows; however, testis function is terminated rapidly after the breeding season. Furthermore, it is likely that Sertoli cell apoptosis followed by massive germ cell loss is responsible for the rapid reduction in testis mass.     

Quercetin Decreases Claudin-2 Expression Mediated by Up-Regulation of microRNA miR-16 in Lung Adenocarcinoma A549 Cells

Claudin-2 is highly expressed in human lung adenocarcinoma tissues and cells. Knockdown of claudin-2 decreases cell proliferation and migration. Claudin-2 may be a novel target for lung adenocarcinoma. However, there are no physiologically active substances of foods which decrease claudin-2 expression. We here found that quercetin, a flavonoid present in fruits and vegetables, time- and concentration-dependently decreases claudin-2 expression in lung adenocarcinoma A549 cells. In the present study, we examined what regulatory mechanism is involved in the decrease in claudin-2 expression by quercetin. Claudin-2 expression was decreased by LY-294002, a phosphatidylinositol 3-kinase (PI3-K) inhibitor, and U0126, a MEK inhibitor. These drugs inhibited the phosphorylation of Akt and ERK1/2, which are downstream targets of PI3-K and MEK, respectively. In contrast, quercetin did not inhibit the phosphorylation. Both LY-294002 and U0126 inhibited promoter activity of claudin-2, but quercetin did not. The stability of claudin-2 mRNA was decreased by quercetin. Quercetin increased the expression of microRNA miR-16. An inhibitor of miR-16 rescued quercetin-induced decrease in the claudin-2 expression. These results suggest that quercetin decreases claudin-2 expression mediated by up-regulation of miR-16 expression and instability of claudin-2 mRNA in lung adenocarcinoma cells.     

用睾丸细胞共培养探讨吡哆醇对大鼠睾丸细胞的毒性

目的探讨吡哆醇（ＰＮ）对大鼠睾丸的体外毒性。方法采用在Ｗｉｌｉａｍｓ方法的基础上改进的Ｓｅｒｔｏｌｉｇｅｒｍ细胞共培养系统,观察ＰＮ在不同剂量和接触时间对培养细胞的作用。结果脱落生精细胞数随ＰＮ浓度的增高和接触时间的延长而增加,并有明显的剂量—效应和时间—效应关系。同时,还观察到Ｓｅｒｔｏｌｉ细胞骨架出现松弛、回缩等效应。结论ＰＮ对大鼠生精细胞的体外效应反映了其对Ｓｅｒｔｏｌｉ细胞的损害。睾丸细胞共培养方法对探讨ＰＮ对大鼠睾丸的毒性作用具有实用价值。     

Role of ghrelin on testosterone secretion and the mRNA expression of androgen receptors in adult rat testis

The present study was designed to determine the effects of ghrelin on in vivo and in vitro secretion of testosterone (T) and the expression of androgen receptor (AR) mRNA in the adult rat testis. The distribution of growth hormone secretagogue receptors (GHS-R_1a) in the testis was also investigated. GHS-R_1a immunoreactivity presented mainly in Sertoli and Leydig cells, primary spermatocytes, and secondary spermatocytes. Adult rats that were intracerebroventricularly (i.c.v.) administrated different dosages (1 nmol and 3 nmol) of ghrelin could significantly inhibit the secretion of T. The experession of AR mRNA in the testis was also notably reduced with 3 nmol ghrelin. Additionaly, in vitro exposure of the Leydig cells to increasing concentrations of ghrelin resulted in no obvious changes of T secretion in the culture media and AR mRNA expression of Leydig cells. Overall, our data demonstrate that the i.c.v. injection of ghrelin plays a physiological role in T secretion and AR mRNA expression in the testis, further confirming the reproductive role of ghrelin.     

Role of ghrelin on testosterone secretion and the mRNA expression of androgen receptors in adult rat testis

The present study was designed to determine the effects of ghrelin on in vivo and in vitro secretion of testosterone (T) and the expression of androgen receptor (AR) mRNA in the adult rat testis. The distribution of growth hormone secretagogue receptors (GHS-R(1a)) in the testis was also investigated. GHS-R(1a) immunoreactivity presented mainly in Sertoli and Leydig cells, primary spermatocytes, and secondary spermatocytes. Adult rats that were intracerebroventricularly (i.c.v.) administrated different dosages (1 nmol and 3 nmol) of ghrelin could significantly inhibit the secretion of T. The experession of AR mRNA in the testis was also notably reduced with 3 nmol ghrelin. Additionaly, in vitro exposure of the Leydig cells to increasing concentrations of ghrelin resulted in no obvious changes of T secretion in the culture media and AR mRNA expression of Leydig cells. Overall, our data demonstrate that the i.c.v. injection of ghrelin plays a physiological role in T secretion and AR mRNA expression in the testis, further confirming the reproductive role of ghrelin.     

Role of the Y-located putative gonadoblastoma gene in human spermatogenesis

The gonadoblastoma locus on the human Y chromosome (GBY) is postulated to serve normal functions in spermatogenesis, but could exert oncogenic properties in predisposing susceptible germ cells to tumorigenesis in incompatible niches such as streaked gonads in XY sex reversed patients or dysfunctional testis in males. The testis-specific protein Y-linked (TSPY) repeat gene has recently been demonstrated to be the putative gene for GBY, based on its location on the GBY critical region, expression patterns in early and late stages of gonadoblastoma and ability to induce gonadoblastoma-like structures in the ovaries of transgenic female mice. Over-expression of TSPY accelerates G(2)/M progression in the cell cycle by enhancing the mitotic cyclin B-CDK1 kinase activities. Currently the normal functions of TSPY in spermatogenesis are uncertain. Expression studies of TSPY, and its X-homologue, TSPX, in normal human testis suggest that TSPY is co-expressed with cyclin B1 in spermatogonia and various stages of spermatocytes while TSPX is principally expressed in Sertoli cells in the human testis. The co-expression pattern of TSPY and cyclin B1 in spermatogonia and spermatocytes suggest respectively that 1) TSPY is important for male spermatogonial cell replication and renewal in the testis; and 2) TSPY could be a catalyst/meiotic factor essential for augmenting the activities of cyclin B-cyclin dependent kinases, important for the differentiation of the spermatocytes in prophase I and in preparation for consecutive rounds of meiotic divisions without an intermediate interphase during spermatogenesis.     

Expression patterns and changes of the LCN2 gene in the testes of induced cryptorchidism and busulfan-treated mice

Lipocalin-2 (LCN2) was known to play various roles in different type cells; however, little was known about the effect of LCN2 in male fertility. In this study, we aimed to explore the expression pattern of LCN2 with increasing age in mice, and to obtain insight into the role of LCN2 in mice testes by induced cryptorchidism and busulfan-treated infertility. In situ hybridization showed that LCN2 was localized primarily in Leydig cells, but was absent in Sertoli and germ cells. Its expression in testes exhibited an age-related increase from day 1 to 8 months, then reduced by the twelth month. The mRNA and protein levels of LCN2 in the testes of both infertile models increased as measured by real-time PCR and western blotting, respectively. LCN2 mRNA and protein levels were higher (p<0.05) in busulfan treated mice than that of cryptorchidism. These observations have shown that LCN2 is developmentally regulated and highly expressed in the Leydig cells of mouse testes.     

Health implications of fructose consumption: A review of recent data

Background

An increase in the intestinal permeability is considered to be associated with the inflammatory tone and development in the obesity and diabetes, however, the pathogenesis of the increase in the intestinal permeability is poorly understood. The present study was performed to determine the influence of obesity itself as well as dietary fat on the increase in intestinal permeability.

Methods

An obese rat strain, Otsuka Long Evans Tokushima Fatty (OLETF), and the lean counter strain, Long Evans Tokushima Otsuka (LETO), were fed standard or high fat diets for 16 weeks. Glucose tolerance, intestinal permeability, intestinal tight junction (TJ) proteins expression, plasma bile acids concentration were evaluated. In addition, the effects of rat bile juice and dietary fat, possible mediators of the increase in the intestinal permeability in the obesity, on TJ permeability were explored in human intestinal Caco-2 cells.

Results

The OLETF rats showed higher glucose intolerance than did the LETO rats, which became more marked with the prolonged feeding of the high fat diet. Intestinal permeability in the OLETF rats evaluated by the urinary excretion of intestinal permeability markers (Cr-EDTA and phenolsulfonphthalein) was comparable to that in the LETO rats. Feeding the high fat diet increased intestinal permeability in both the OLETF and LETO rats, and the increases correlated with decreases in TJ proteins (claudin-1, claudin-3, occludin and junctional adhesion molecule-1) expression in the small, but not in the large intestine (cecum or colon). The plasma bile acids concentration was higher in rats fed the high fat diet. Exposure to bile juice and the fat emulsion increased TJ permeability with concomitant reductions in TJ protein expression (claudin-1, claudin-3, and junctional adhesion molecule-1) in the Caco-2 cell monolayers.

Conclusion

Excessive dietary fat and/or increased levels of luminal bile juice, but not genetic obesity, are responsible for the increase in small intestinal permeability resulting from the suppression of TJ protein expression.