Lactational exposure of polychlorinated biphenyls downregulates critical genes in Leydig cells of F1 male progeny (PND21)

Polychlorinated biphenyls (PCBs) are environmental contaminants. The present study was aimed to test the effect of lactational exposure of PCBs on Leydig cellular mRNA and protein expressions of 5α‐reductase, aromatase and androgen receptor (AR) in F1 male offspring. Lactating dams were orally gavaged with different doses of PCBs (Aroclor 1254) 0, 1, 2 and 5 mg kg b.wt^?1 day^?1, respectively, from PND1 to PND21. Male offsprings were sacrificed at PND21. Testes were used to isolate Leydig cells. Blood was collected. Serum testosterone (T) and oestradiol (E₂) were measured. Anogenital distance was measured. Dams’ milk lipid and serum lipids of male pups were estimated. PCB (Aroclor 1254) concentration of dams’ milk and serum of male pups were analysed by GC‐ECD. Leydig cellular mRNA and protein expressions of 5α‐reductase, aromatase and AR were significantly decreased. Our data suggest that lactational exposure of PCBs downregulates selected genes in Leydig cells of F1 generation on post‐natal day 21.     

Brief maternal exposure of rats to the xenobiotics dibutyl phthalate or diethylstilbestrol alters adult-type Leydig cell development in male offspring

Maternal exposure to estrogenic xenobiotics or phthalates has been implicated in the distortion of early male reproductive development, referred to in humans as the testicular dysgenesis syndrome. It is not known, however, whether such early gestational and/or lactational exposure can influence the later adult-type Leydig cell phenotype. In this study, Sprague–Dawley rats were exposed to dibutyl phthalate (DBP; from gestational day (GD) 14.5 to postnatal day (PND) 6) or diethylstilbestrol (DES; from GD14.5 to GD16.5) during a short gestational/lactational window, and male offspring subsequently analysed for various postnatal testicular parameters. All offspring remained in good health throughout the study. Maternal xenobiotic treatment appeared to modify specific Leydig cell gene expression in male offspring, particularly during the dynamic phase of mid-puberty, with serum INSL3 concentrations showing that these compounds led to a faster attainment of peak values, and a modest acceleration of the pubertal trajectory. Part of this effect appeared to be due to a treatment-specific impact on Leydig cell proliferation during puberty for both xenobiotics. Taken together, these results support the notion that maternal exposure to certain xenobiotics can also influence the development of the adult-type Leydig cell population, possibly through an effect on the Leydig stem cell population.     

Effects of maternal exposure to 3,3',4,4',5-pentachlorobiphenyl (PCB126) or 3,3',4,4',5,5'-hexachlorobiphenyl (PCB169) on testicular steroidogenesis and spermatogenesis in male offspring rats

On days 7-21 of gestation, Sprague-Dawley rats were orally administered 3 or 30 mug/kg/d of 3,3',4,4',5-pentachlorobiphenyl (PCB126) or 3,3',4,4',5,5'-hexachlorobiphenyl (PCB169) daily. Their male offspring were autopsied at 3, 6, and 15 weeks after birth to investigate the effects of the 2 polychlorinated biphenyls (PCBs) on spermatogenesis and steroidogenesis in their testes. PCB treatment caused a decrease in the area ratio of 3beta-hydroxysteroid dehydrogenase (HSD)-expressing cells (Leydig cells)/testis at 3 weeks after birth. When PCB126 was administered to pregnant rats, the plasma testosterone levels in their offspring were decreased at 3 weeks. The expression levels of P450scc, 3beta-HSD, and P450(17alpha) mitochondrial RNAs (mRNAs) were unchanged, although the StAR (steroidogenic acute regulatory protein) mRNA expression level was increased at 6 weeks. On the other hand, when PCB169 was administered, plasma testosterone levels were decreased at 3 and 6 weeks and were increased at 15 weeks. Plasma luteinizing hormone (LH) levels were decreased at 6 weeks, and plasma follicle-stimulating hormone (FSH) levels were increased at 15 weeks. The expression levels of 3beta-HSD and P450(17alpha) were increased, and the mRNA level of 5alpha-reductase 1 was decreased at 15 weeks. PCB169 treatment suppressed the conversion of round spermatids between stages VII and VIII. These results indicate that in utero and lactational exposure to PCB126 or PCB169 decreases plasma testosterone levels in 3-week-old rats, with no change in the expression levels of the mRNAs of enzymes, and that PCB169 inhibits testicular steroid synthesis more strongly than PCB126. PCB169 greatly altered the concentration of testosterone, indicating a stronger inhibitory effect on spermatogenesis. Low testosterone and LH levels in prenatally PCB169-exposed rats until 6 weeks after birth presumably retard the functional differentiation of testicular Leydig cells; however, the increased testosterone levels at 15 weeks suggest that Leydig cells in PCB-exposed rats are virtually mature by the 15th week.     

The effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin on foetal male rat steroidogenesis

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is the most toxic and widely investigated dioxin congener. In utero and lactational exposure to TCDD results in developmental and reproductive defects that are the most sensitive endpoints for TCDD toxicity. TCDD has a potential to interfere with steroid metabolism, but the mechanisms by which this occurs are not well understood. In this study, we investigated the effects of TCDD on prenatal rat steroidogenesis. Pregnant Sprague-Dawley female rats were treated once with TCDD (0, 0.3 or 1 microg/kg) by gavage on embryonic day (ED) 11 and the expression levels of androgen (AR) and estrogen receptors (ER), steroidogenic enzymes (P450scc and 3beta-HSD) and four regulatory factors (StAR, SF-1, GATA-4 and Insl-3) involved in foetal Leydig cell and adrenal function were analysed on ED 19.5. Hormonal status of male foetuses was determined by measuring testicular testosterone (T) levels, plasma luteinizing hormone (LH) and corticosterone concentrations. In utero exposure to TCDD reduced intratesticular T of foetal males (significant at 0.3 microg/kg TCDD) and tended to reduce the protein expression of ERalpha and AR of foetal male rat testis. Foetal male rat plasma LH levels were significantly reduced at the dose of 1 microg/kg TCDD, while corticosterone levels tended to be increased possibly because of the TCDD-induced stress. Only minor alterations in steroidogenesis were observed in rat adrenal. mRNA expression of developmental regulatory factors was not influenced by foetal TCDD exposure, except for significantly reduced adrenal SF-1. The results demonstrate that maternal exposure to TCDD suppressed testicular steroidogenesis of 19.5-day-old foetal male Sprague-Dawley rat. The highest dose of TCDD (1 microg/kg) had also an effect on pituitary LH secretion. Our data implicate that TCDD has direct testicular and pituitary effects on foetal male rat but with different dose-responses. These changes can lead to impaired steroidogenesis and it may result in the maldevelopment of the testis and weaken masculinization.     

乳头状甲状腺癌中谷胱甘肽过氧化物酶3启动子区高甲基化与表达缺失

目的 探讨乳头状甲状腺癌(PTC)中谷胱甘肽过氧化物酶3(GPX3)基因启动子区域甲基化状态,分析GPX3甲基化与其基凶表达的关系.方法 应用甲基化特异性聚合酶链式反应(MSP)检测47例PTC、10例非癌组织标本和乳头状甲状腺癌细胞株TPC-1中GPX3的甲基化状态,并用逆转录-聚合酶链式反应(RT-PCR)检测TPC-1细胞中GPX3的mRNA表达.结果 48.9％( 23/47) PTC发生GPX3基因启动子区域甲基化,而10例非癌组织均未发生甲基化;TPC-1中GPX3启动子区甲基化,而且GPX3的mRNA不表达,经5-aza-2’-deoxycytidine干预96h后GPX3重新表达.结论 GPX3基因启动子区域频繁发生甲基化.,可能作为PTC的诊断标志物;GPX3启动子区的甲基化是其基因表达的重要调节机制.     

Epigenetic regulation of human bone morphogenetic protein 6 gene expression in prostate cancer.

Bone morphogenetic proteins (BMPs), belonging to the transforming growth factor-beta (TGF-beta) superfamily, are multifunctional molecules that regulate bone induction and organ development. Among BMPs, BMP-6 has been shown to be overexpressed in prostate cancer and is speculated to be associated with bone-forming skeletal metastasis. We investigated the regulatory mechanism of the BMP-6 gene expression in prostate cancer cell lines DU-145, LNCaP, PC-3, and PC-3M with regard to the methylation status of the CpG island in the 5' flanking region of the human BMP-6 gene. By sequence-specific analysis of methylated cytosines, we show here that the methylation status of the CpG loci around the Sp1 site of the BMP-6 promoter is related to its steady-state expression and an alternative splicing of messenger RNA (mRNA) in prostate cancer cell lines. Furthermore, a study of clinical cases of benign and malignant prostate lesion by in situ hybridization showed that BMP-6 expression was high at both primary and secondary sites in cases of advanced cancer with metastasis. Demethylation of the CpG loci around the Spl binding site was shown in cases with high BMP-6 expression by sequencing analysis of the methylated cytosine from paraffin-embedded materials. Our results suggested that during cancer progression, besides inactivation of tumor suppressor genes by hypermethylation, activation of certain genes like BMP-6 by selective demethylation was a common epigenetic event giving a variable character to the invading and metastasizing cancer cells.     

Suppression of androgen action and the induction of gross abnormalities of the reproductive tract in male rats treated neonatally with diethylstilbestrol

This study evaluated whether androgen action is altered in rats treated neonatally with diethylstilbestrol (DES) at a dose that induced reproductive tract abnormalities. Rats were treated on alternate days 2-12 with 10 microg DES and studied on Day 18. DES-induced abnormalities included a 70% reduction in testis weight, distension and overgrowth of the rete, distension and reduction in epithelial height of the efferent ducts, underdevelopment of the epididymal duct epithelium, reduction in epithelial height in the vas deferens, and convolution of the extra-epididymal vas. In DES-treated rats, androgen receptor (AR) immunoexpression was virtually absent from all affected tissues and the testis, whereas AR expression in controls was intense in epithelial and stromal cells. The DES-induced change in AR immunoexpression was confirmed by Western analysis for the testis. In rats treated neonatally with 1 microg DES, reproductive abnormalities were absent or minor, except for a 38% reduction in testis weight; loss of AR immunoexpression also did not occur in these rats. Treatment-induced changes in AR expression were paralleled by changes in Leydig cell volume per testis (91% reduction in the 10-microg DES group; no change in the 1-microg DES group). To test whether suppression of androgen production or action alone could induce comparable reproductive abnormalities to 10 microg DES, rats were treated neonatally with either a potent gonadotropin-releasing hormone antagonist (GnRHa) or with flutamide (50 mg/kg/day). These treatments reduced testis weight (68% for GnRHa, 40% for flutamide), and generally retarded development of the reproductive tract but failed to induce the abnormalities induced by 10 microg DES. GnRHa and flutamide caused no detectable change in AR immunoexpression in target tissues, with the exception of minor changes in the testes of flutamide-treated males. GnRHa treatment caused a reduction (83%) in Leydig cell volume comparable to that caused by 10 microg DES. Immunoexpression of estrogen receptor alpha (ER alpha) in the efferent ducts and of ER beta in all tissues studied were unaffected by any of the above treatments. Neonatal coadministration of testosterone esters (TE; 200 microg) with 10 microg DES prevented most of the morphological abnormalities induced by 10 microg DES treatment alone, though testis weight was still subnormal (46% reduction in DES + TE vs 72% in DES alone and 49% with TE alone) and some lumenal distension was still evident in the efferent ducts. Coadministration of TE with DES prevented DES-induced loss of AR immunoexpression (confirmed for testis by Western blot analysis). It is concluded that 1) reproductive tract abnormalities induced in the neonatal male rat by a high (10 microg) dose of DES are associated with reduced AR expression and Leydig cell volume; 2) these changes are largely absent with a lower dose of DES (1 microg); 3) treatments that interfere with androgen production (GnRHa) or action (flutamide) alone failed to induce reproductive tract abnormalities or alter AR expression as did 10 microg DES; 4) a grossly altered androgen:estrogen balance (low androgen + high estrogen) may underlie the reproductive tract abnormalities, other than reduced testis weight, induced by high doses of DES.     

转化生长因子β1对系膜细胞内1型纤溶酶原激活物抑制物基因组蛋白乙酰化修饰的影响

目的 探讨转化生长因子β1(TGF-β1)对1型纤溶酶原激活物抑制物(PAI-1)基因启动子及转录区域内组蛋白尾部乙酰化修饰的影响.方法 采用染色质共免疫沉淀与实时定量PCR技术,观察高糖及TGF-β1对PAI-1基因启动子及转录区组蛋白H3赖氨酸残基9位乙酰化(H3K9Ac)修饰的影响;并应用共免疫沉淀方法探讨转录因子CREB结合蛋白(CBP)与Smad3、Sp1蛋白之间的相互作用.结果 在PAI-1基因启动子的4个区域内,TGF-β1(10 μg/L)刺激显著增加了P1、P2、P3区域的H3K9Ac修饰(均P＜0.05),而在距离转录起始点较远的P4区域,H3K9Ac修饰水平没有明显改变;而在PAI-1基因的转录区,TGF-β1刺激显著增加了T1区域的H3K9Ac修饰(P＜0.05),而T2区没有明显改变.高糖刺激引起系膜细胞内PAI-1基因的表达水平及其启动子P1区域H3K9Ac修饰显著增加(P＜0.05),应用TGF-β1中和性抗体预处理显著抑制了高糖引起的PAI-1基因启动子区H3K9Ac修饰(P＜0.01).TGF-β1刺激能够显著诱导Smad3、CBP蛋白结合在P1、P2、P3区域(均P＜0.05);同时,TGF-β1刺激能够诱导CBP、Sp1与Smad3蛋白的结合,进而引起H3K9Ac修饰.结论 TGF-β1能够诱导PAI-1基因启动子与转录起始区发生H3K9Ac修饰,促进Smad3与Sp1及CBP蛋白相互结合,促进PAI-1基因的转录表达.     

FasL启动子调控的报告基因表达质粒的构建及活性分析

目的扩增FasL启动子并构建带有FasL启动子的荧光素酶报告质粒,并评价在皮质酮(啮齿类动物体内的糖皮质激素)作用的睾丸Leydig细胞中,由FasL启动子调控的荧光素酶的表达活性。本研究将为进一步分析Leydig细胞中FasL启动子转录调控的作用机制奠定基础。方法采用DNA重组技术构建由FasL启动子调控的荧光素酶报告基因的表达质粒。此重组质粒经阳离子脂质体LipofectAMINE介导,瞬时转染入Leydig细胞中,36h后细胞经皮质酮处理,并于48h时收集细胞。测定荧光素酶的相对表达活性。结果(1)酶切及测序证实成功扩增FasL基因启动子并构建了带有FasL基因启动子的报告基因表达质粒；(2)皮质酮处理的Leydig细胞中FasL启动子调控的荧光素酶表达质粒的表达活性显著增高。结论构建成功的带有FasL启动子的荧光素酶报告质粒可准确地评价FasL启动子在凋亡过程中的作用。     

Hypermethylation of the thrombospondin-1 gene is associated with poor prognosis in penile squamous cell carcinoma

OBJECTIVE

To evaluate the presence of human papillomavirus (HPV) infection, the methylation status in the promoter region of thrombospondin‐1 (TSP‐1), RAS association domain family 1A (RASSF1‐A) and p16 genes, and the expression of TSP‐1, CD31, p16 and p53 proteins in patients diagnosed with penile cancer, and the possible associations between these variables and clinical and pathological features.

PATIENTS AND METHODS

HPV types, gene promoter hypermethylation and protein expression were analysed by reverse line blot, methylation‐specific polymerase chain reaction, and immunohistochemistry, respectively, in 24 penile squamous cell carcinomas.

RESULTS

HPV infection was detected in 11 of 24 cases (46%), and TSP‐1, RASSF1‐A and p16 genes were hypermethylated in 46%, 42% and 38% of the tumours, respectively. TSP‐1 hypermethylation was associated with unfavourable histological grade (grade 3; P = 0.033), vascular invasion (P = 0.023), weak expression of TSP‐1 protein (P = 0.041), and shorter overall survival (P = 0.04). TSP‐1 expression was not associated with microvessel density. However, RASSF1‐A hypermethylation was more frequent in T1 tumours (P = 0.01), and p16 hypermethylation was not associated with any of the tested variables except for absence of p16 expression (P = 0.022).

CONCLUSION

In summary, the epigenetic inactivation of TSP‐1 and RASSF1‐A genes is associated with pathological variables and seems to be of prognostic significance in penile cancer.     

邻苯二甲酸二乙基己酯对新生小鼠睾丸及Leydig细胞影响的实验研究

目的:探讨邻苯二甲酸二乙基己酯(DEHP)对新生小鼠睾丸及Leyd ig细胞形态结构及功能的影响。方法:DEHP分别以低、中、高3组剂量[100、200、500 mg/(kg.d)]灌胃作用于怀孕12 d到产后3 d(GD12~PND3)的KM母鼠,观察DEHP对新生雄性仔鼠体重、睾丸重量、Leyd ig细胞形态结构和3β-羟基类固醇脱氢酶(3-βHSD)活性、酶反应面积的影响。结果:DEHP作用于母鼠后,其雄性子代幼鼠体重和睾丸重量减轻,睾丸Leyd ig细胞形态、超微结构发生改变;高剂量组Leyd ig细胞数量明显增多;低、中剂量组睾酮合成关键酶3β-HSD酶活性下降,酶反应面积减小,但高剂量组在仔鼠出生后15 d时酶活性降低[(吸光度值(0.154±0.011)vs空白对照组(0.222±0.013),P<0.01],而酶反应面积增大[(6 303.0±745.6)μm2vs空白对照组(5 091.4±214.4)μm2,P<0.01)]。结论:DEHP能影响新生雄性小鼠体重、睾丸重量、Leyd ig细胞的形态结构和3β-HSD活性,具有抗雄激素效应。