LGR8基因与隐睾相关性的研究进展

LGR8(leucine rich repeat containing G protein coupled receptor 8),中文名称为富含亮氨酸的G蛋白偶联受体8,是体内胰岛素样因子3(INSL3)的特异性受体,属于G蛋白偶联受体(GPCR)家族.LGR8能与INSL3结合形成受体配体复合物,参与睾丸下降的第一过程(经腹下降阶段),被认为是导致人类隐睾症的一个重要因素.本文就近年来对LGR8基因及其突变与隐睾相关性的研究作一综述.     

胰岛素样激素3与睾丸引带发育和睾丸下降

睾丸下降是生殖系统发育过程中的重要阶段,其机制尚未阐明。睾丸引带发育与睾丸下降关系密切。睾丸下降不全常常伴随睾丸引带发育异常,它们除了可引起隐睾和睾丸发育不良等疾病外,还是生殖系统先天畸形以及以后生殖功能、性功能异常和高比例生殖系统肿瘤发生的原因。胰岛素样激素3是近年来倍受关注的影响睾丸引带发育和睾丸下降的重要因素,本文就相关进展进行综述。     

小鼠睾丸引带细胞的培养及己烯雌酚对培养睾丸引带细胞的影响

目的:探讨小鼠睾丸引带细胞体外培养的有效方法,并进行形态学观察。研究经典的外源性雌激素己烯雌酚(DES)体外对小鼠睾丸引带发育的影响。方法:手术放大镜解剖出3日龄雄性昆明小鼠的睾丸引带组织并进行细胞培养。台盼蓝染色检测细胞存活率,HE染色观察细胞形态。传代培养并随机分为空白对照组、溶剂对照组(DMSO组)和实验组(DES 1~4组)共6组。溶剂对照组加入DMSO,实验组分别加入0.01、0.10、1.00以及10.00μg/ml DES。分别于培养12、24、48 h后进行细胞形态学观察,CCK-8法检测睾丸引带细胞的生长情况。结果:培养睾丸引带细胞大部分为成纤维细胞型,有少数的上皮样细胞。原代细胞的存活率为85%~90%。在不同剂量DES作用后的3个不同时间段里,细胞增殖性的检测结果存在时间-剂量效应的显著差异(P<0.01)。结论:睾丸引带细胞可经一定的方法进行体外培养,外源性雌激素对睾丸引带细胞的生长有抑制作用,且呈现一定的时间-剂量效应。对培养睾丸引带细胞影响的研究是外源性雌激素影响生殖系统发育研究的一条有效的睾丸外途径。     

己烯雌酚影响新生小鼠睾丸引带相关基因表达的初步研究

目的:通过不同剂量己烯雌酚(DES)孕期染毒,了解其对新生仔鼠睾丸引带中雄激素受体(AR)、雌激素受体α(ERα)、增殖细胞核抗原(PCNA)和肌动蛋白α1(ACTα1)mRNA表达的影响,以期窥探DES影响睾丸引带的作用途径。方法:将发现雌性昆明小鼠阴道栓当天记为孕第0天(GD0),在GD9~GD17分别给予不同剂量DES[0.02、0.1、0.5、10、50μg/(kg·d)]作为实验组,给予等体积二甲基亚砜及生理盐水分别作为溶剂和空白对照组。生后第1天处死雄性仔鼠获取睾丸引带,通过RT-PCR检测AR、ERα、PCNA和ACTα1 mRNA的表达量。结果:随着DES浓度的增加,ERαmRNA的表达逐渐增加,各实验组与溶剂对照组比较差异有统计学意义(RE~2=0.825,P0.05);AR、PCNA和ACTα1 mRNA的表达都呈下降趋势,高剂量组[10、50μg/(kg·d)DES组]与溶剂对照组比较差异有统计学意义(RA~2=0.713,RP~2=0.946,RT2=0.960,P0.01)。结论:正常新生昆明小鼠睾丸引带中存在ERα、AR、PCNA及ACTα1 mRNA的表达。孕期不同剂量DES染毒对新生小鼠睾丸引带细胞受体蛋白(ERα和AR)及功能蛋白(PCNA和ACTα1)mRNA表达的影响不同。外源性雌激素可能通过影响ER和或AR的代谢而影响睾丸引带细胞的增殖和收缩等功能,从而影响睾丸甚至整个雄性生殖系统的正常发育。     

影响睾丸引带发育的内分泌因素研究进展

睾丸引带与睾丸下降关系密切,它的分化发育和多种因素有关,特别是雄激素、降钙素基因相关肽、胰岛素样因子3、苗勒管抑制物质、表皮生长因子以及环境雌激素(EEs)等,其中降钙素基因相关肽、胰岛素样因子3和EEs对生殖系统的影响是目前研究的热点。从影响睾丸引带发育的角度探讨EEs对睾丸及生殖发育的影响很有意义。     

RXFP2在己烯雌酚染毒小鼠睾丸引带细胞中的作用机制研究

目的通过检测己烯雌酚(DES)对小鼠睾丸引带细胞形态与增殖性影响过程中松弛素家族肽受体2(RXFP2)的表达水平,探讨RXFP2在外源性雌激素影响小鼠睾丸引带细胞中的可能作用机制。方法小鼠睾丸引带组织进行细胞培养,传代后随机分为正常组、溶剂对照组及实验组,实验组分别加入DES终浓度为0.01、0.10、1.00、10.00μg/m L作用48 h。观察细胞形态变化,CCK-8检测其增殖情况,应用免疫荧光和Western Blot对小鼠睾丸引带细胞中RXFP2进行定位和蛋白定量分析。结果正常组细胞呈成纤维细胞型生长,同源性好。各实验组随DES剂量增加细胞数及细胞生存率均下降,细胞增殖明显受抑制,失去正常形态,胞浆收缩,胞体形态不规则。免疫荧光显示RXFP2高表达于细胞膜上。蛋白定量分析发现各实验组RXFP2水平与正常组相比表达降低,且差异有统计学意义。结论DES可影响小鼠睾丸引带细胞形态与增殖性以及RXFP2蛋白的表达,推测DES可能通过影响RXFP2信号系统介导睾丸引带的发育,进而影响睾丸的下降。     

己烯雌酚对大鼠睾丸组织干细胞因子表达水平的影响

雌激素是精子发生过程中关键激素之一 ,其具体作用机制尚待探讨[1] 。c kit受体与其配体干细胞因子 (SCF)的相互作用 (SCF/c kit系统 )在精子生成中有重要的作用。在成年雄性大鼠睾丸中 ,编码c kit的mRNA存在于精原细胞、初级精母细胞、圆形精子细胞及睾丸间质细胞 ,但表达SCF的细胞只有支持细胞[2 ] 。所以目前认为支持细胞损伤及恢复与SCF表达水平有重要联系。本实验拟应用己烯雌酚 (DES)对 2 ,5 己二酮(支持细胞毒物 )导致的大鼠生精障碍模型进行干预 ,以探索DES与睾丸内SCF表达水平的关系。一、材料和方法1.实验动物 :健康雄…     

不同剂量己烯雌酚对新生小鼠睾丸表皮生长因子及受体的影响

目的 观察不同剂量己烯雌酚(DES)对新生小鼠睾丸组织中表皮生长因子(EGF)及其受体(EGFR)的影响,探讨DES对睾丸发育影响的机制.方法 建立小鼠DES模型.将72只怀孕的雌性昆明小鼠随机分成3组:正常组、对照组及实验组1～4(DES 10、25、50、100 μg/kg).采用免疫组织化学方法检测各组新生小鼠睾丸组织中EGF、EGFR的表达.结果 EGF和EGFR主要表达在新生小鼠睾丸的间质细胞.实验组1～4中EGF阳性细胞的累积吸光度值(IA)分别为75.43±1. 42、52.22±5.67、13.75±3.14、6.38±3.20,显著低于正常组及对照组中阳性细胞的IA值433.88±11.64、23.44±4.70;实验组EGFR阳性细胞的IA值分别为198.16±34.35、138.00±12.04、46.03±6.74、27.22±5.52,显著低于正常组及对照组中阳性细胞的IA值804.74±22.52、800.03±21.96.两者在正常、不同剂量DES实验组的两两比较差异有统计学意义(P＜0.05).随DES剂量增加,EGF和EGFR表达减弱,各组间差异有统计学意义(P＜0.05).EGF与EGFR呈强正线性相关(r=0.750,P＜0.01).结论 不同剂量DES对新生小鼠睾丸组织中EGF和EGFR表达强度均有影响,可能是影响睾丸发育机制之一.

Abstract：

Objective To investigate the effects of prenatal exposure to diethylstilbestrol (DES)with different dosages on epidermal growth factor (EGF) and epidermal growth factor receptor (EGFR) in offspring mice testis, and the possible mechanism. Methods DES model was induced in mice by DES.Female Kungmiag mice were randomly divided into normal group, control group and experimental groups 1-4 ( DES 10, 25, 50, 100 μg/kg). Immunohistochemistry was applied to detect the expression of EGF and EGFR in the testicular tissue in each group. Results EGF and EGFR were expressed mainly in sffspring mice testis Leydig cells. The cumulative absorbance ( IA ) values of EGF positive cells in experimental groups 1-4 were 75. 43 ± 1.42, 52. 22 ± 5.67, 13.75 ± 3. 14, and 6. 38 ± 3.20 respectively, which were significantly lower than in normal and control groups (433.88 ± 11.64,423.44 ±4. 70 respectively).The IA values of EGFR positive cells in experimental groups 1-4 were 198. 16 ± 34. 35, 138.00 ± 12.04,46.03 ± 6. 74, 27.22 ± 5.52 respectively, which were significantly lower than in normal and control groups (804. 74 ± 22. 52,800. 03 ± 21.96 respectively). The expression levels of EGF and EGFR could be detected in each subgroup and statistically significant differences existed in the expression of EGF and EGFR between any two groups ( P ＜ 0. 05 ). With the increase of DES dosage, the expression of EGF and EGFR was decreased, with the difference being significant amond the groups ( P ＜ 0. 05 ). Conclusion DES can influence the expression of EGF and EGFR in mice testis, which might be one of possible mechanisms effecting the development of testis.     

青春期己烯雌酚暴露对大鼠睾丸发育及功能的影响

目的 探讨青春期己烯雌酚(diethylstilbestrol,DES)暴露对SD(Sprague-Dawley)大鼠睾丸发育及功能的影响.方法 35日龄雄性SD大鼠90只,随机分为DES 0.01、0.1、1.0、10.0 μg·kg-1·d-14个实验组和1个对照组(编码为Bda、BDb、BDc、BDd和BC组,每组18只).于青春期,即出生后第36天(postnatal day 36,PND 36)至PND 49,实验组每日皮下注射相应剂量的DES,共14 d,对照组仅注射溶媒.于青春期晚期(PND 50)、性成熟后(PND 64)和成年期(PND 130)分3批(每批6只)处死各组大鼠取材,测定睾丸重量,观察比较睾丸组织形态学变化,分析PND 130大鼠附睾尾精子质量.结果 PND 50时,BC、Bda、BDb、BDc和BDd组单侧睾丸重量分别为(1.26±0.13)、(1.23±0.20)、(0.99±0.15)、(0.85±0.23)和(0.60±0.04)g,其中BDb、BDc和BDd组均较BC组减轻(P＜0.05);与BC组比较,BDb组仅有少数生精小管生精上皮中的细胞数目稍减少,BDc和BDd组生精小管发育较差、生精上皮中细胞数目减少、精子发生阻滞、间质细胞发育幼稚,其程度随DES暴露剂量增加而加重.PND 64时,BC、Bda、BDb、BDc和BDd组单侧睾丸重量分别为(1.54±0.14)、(1.55±0.17)、(1.52±0.11)、(1.37±0.14)和(0.88±0.15)g,其中BDc和BDd组均较BC组减轻(P＜0.05);BDc和BDd组睾丸组织形态学改变与PND 50时类似,但较PND 50时有所改善.PND 130时,各实验组与对照组比较,单侧睾丸重量差异无统计学意义(P＞0.05),睾丸组织形态学改变未见明显差异;BC、Bda、BDb、BDc和BDd组大鼠附睾尾精子密度分别为(71.00±14.85)、(69.00±23.98)、(67.00±13.52)、(31.67±12.94)和(18.83±6.68)×106/ml,其中BDc和BDd组精子密度均较BC组明显降低(P＜0.01);与BC组比较,BDd组精子活动率下降(P＜0.01),BDb、BDc和BDd组A级精子比例降低(P＜0.05),BDd组B级精子比例降低(P＜0.01).结论 青春期小剂量DES(0.01μg·kg-1·d-1×14 d)暴露对SD大鼠睾丸发育及功能无明显影响,大剂量DES(1.0～10.0 μg·kg-1·d-1×14 d)暴露对大鼠睾丸发育及功能具有明显的近期(PND 50和PND 64)和较远期(PND 130)毒性作用,该毒性作用随DES暴露剂量增加而加重,随鼠龄增长而逐渐减退,其机制可能与间质细胞和支持细胞的发育及功能受损密切相关.     

新生小鼠睾丸引带形态结构及其异常的显微连续研究

目的:应用新生小鼠生殖系统整体原位固定、横断面连续组织切片显微观察的研究方法,整体展现正常及异常状态下新生小鼠睾丸引带的形态结构,以探讨应用整块组织固定、连续切片显微研究的方法在判定生殖系统形态异常中的适用性。方法:正常及孕期(9~17d)接触不同剂量外源性雌激素己烯雌酚(DES)的新生雄性昆明小鼠,切取其腹部(自膈下)固定、包埋,作连续切片、染色。显微镜下顺序摄像,分析睾丸引带的形态结构,并测算其相应位置和大小。结果:显微连续观察显示新生小鼠睾丸引带的结构清晰,形态结构各段变化较大且左右不对称,DES对睾丸引带形态结构发育的影响各段不尽相同,而且明显影响整个引带的长度。结论:孕期接触DES对新生小鼠睾丸引带的发育有明显影响,其影响是整体性的。整块组织切片显微连续研究对睾丸引带或及其它相关生殖系统器官的形态结构评价能够比较全面和精确。     

A novel technique for reinforcing the neourethra in hypospadias associated with bilateral undescended testis: use of the gubernaculum

The authors present 3 cases of proximal hypospadias associated with bilateral undescended testes (UDT) in which the gubernaculum was used to reinforce the neourethra to prevent urethrocutaneous fistula. This is the first report of the gubernaculum being used to reinforce the neourethra. The technique is simple and should be applicable to any hypospadias patient with concomitant UDT, especially in severe hypospadias. Good outcome is likely because of the excellent blood supply of the gubernaculum.     

Insulin-like 3 signalling in testicular descent

Undescended testis is one of the most common congenital defects in the newborn boys and the common cause of cryptorchidism. If left untreated, this condition is strongly associated with infertility and drastically increased risk of testicular cancer in adulthood. Testis position in developing males is defined by sexual dimorphic differentiation of two gonadal ligaments, gubernaculum and cranial suspensory ligament. Recent transgenic mouse studies identified testicular hormone insulin-like 3 (INSL3), and its receptor, GREAT/LGR8, as the critical regulators of the gubernacular differentiation. Mutation analysis of the two genes in patients with undescended testis revealed functionally deleterious mutations, which may be responsible for the abnormal phenotype in some of the patients.     

隐睾症睾丸引带异常的临床意义

目的探讨睾丸引带异常的临床意义。方法1994年1月至2003年12月对169例接受手术的隐睾症患者进行术中直接观察,记录隐睾位置、睾丸引带长度和附着点。结果169例212枚隐睾中,腹腔内睾丸42枚(19.8%),腹股沟管睾丸134枚(63.2%),阴囊上部睾丸36枚(17.0%)。腹腔内睾丸引带附着点位于腹股沟管外环周围、阴囊上部和其他位置者分别占73.8%(31)、21.4%(9)和4.8%(2);腹股沟管睾丸引带附着点位于腹股沟管外环周围者64.9%(87),阴囊上部者26.1%(35),阴囊底部者6.7%(9);阴囊上部睾丸引带附着点位于腹股沟管外环周围者25.0%(9),阴囊上部者52.8%(35),阴囊底部者22.2%(8)。腹腔内、腹股沟管和阴囊上部睾丸引带长度分别为(3.1±1.1)、(3.2±0.7)和(2.5±0.4)cm,阴囊上部睾丸引带长度明显短于其他2型隐睾(P<0.01)。结论大多数隐睾有引带异常如迁移停止或未退化或异常附着,这可能是隐睾症激素治疗难以奏效的原因。保留引带睾丸固定术对需要切断精索血管的高位隐睾和精索血供不良的隐睾的血供具有重要价值。     

保留睾丸引带在腹腔镜小儿腹腔型隐睾手术中的应用

目的:探讨保留睾丸引带在腹腔镜小儿腹腔型隐睾手术中的疗效.方法:回顾分析2007年6月至2010年8月为24例腹腔型隐睾患儿行腹腔镜睾丸阴囊肉膜囊固定术的临床资料,术中保留睾丸引带和精索双向血供.结果:24例均成功完成腹腔镜Ⅰ期睾丸下降固定术,无一例中转手术,手术时间45～60 min,平均55 min,无并发症发生....     

Effect of neonatal exposure to diethylstilbestrol on testicular gene expression in adult mouse: comprehensive analysis with cDNA subtraction method

Summary In utero or neonatal exposure to high levels of exogenous steroid hormones, such as the potent synthetic diethylstilbestrol (DES), incurs an increased risk of malfunctional male reproduction. In this study, we investigated whether neonatal exposure to DES induces the alteration of mRNA expression in adult mouse testis. Using a cDNA subtraction method, we isolated seven gene clones whose expression was changed in neonatally DES-treated mouse testis. Northern blot analysis revealed that five up-regulated genes (AF326230, AF356521, AK004975, AK006136 and BM237156) and two down-regulated genes (AK017044, AK017130) were predominantly expressed in testes of 8-week-old mice. Moreover, we confirmed that the expression of these seven genes was altered by neonatal DES-exposure using Northern blot analysis. Our results suggest that neonatal exposure to DES leads to the alteration of gene expression in the testis in the long term. These genes might be useful as biological markers of foetal or neonatal exposure to exogenous steroid hormones, such as DES.     

趋化素样因子超家族在小鼠睾丸中的基因发育表达调控和蛋白表达定位

目的研究CKLFSF2小鼠同源序列cklfsf26基因在睾丸组织发育中的表达和定位,为其功能研究做出提示。方法利用RT-PCR方法检测cklfsf26基因在不同周龄小鼠睾丸组织中的表达情况；制备cklfsf2b多克隆抗体,利用免疫组织化学方法,检测cklfsf2b蛋白在小鼠睾丸组织中的表达定位。结果cklfsf2bmRNA在出生后第2周内出现,表达量逐渐增加,在成年前即达高峰并持续表达。cklfsf2b蛋白特异性的表达于睾丸Leydig细胞和Sertoli细胞,阳性信号均位于胞质内。结论cklfsf2b基因存在发育的表达调控,随着睾丸发育的日趋成熟,表达量逐渐增加：在Leydig细胞和Sertoli细胞的特异性表达表明其在睾酮合成和精子发生中均可能发挥作用,但其具体功能还需深入研究。     

Testicular migration: remodeling of connective tissue and muscle cells in human gubernaculum testis

PURPOSE: We present the main morphological modifications in the human gubernaculum during testicular migration in humans. MATERIALS AND METHODS: We obtained 12 gubernacula from fresh, macroscopically normal human fetuses at 15 to 29 weeks of gestation. Collagen was evidenced using trichrome and Sirius red staining procedures, while Weigert's resorcinol-fuchsin and anti-human elastin antibody were used to reveal elastic system fibers. Smooth muscle cells were detected by anti-human smooth muscle alpha-actin antibody. RESULTS: When the testes were still located in the abdomen at 15 to 16 weeks of gestation, collagen fibers were sparse and embedded in a loose extracellular matrix. The amount of fibers then gradually increased with age and at 28 weeks of gestation the gubernaculum was mostly collagenous in composition. Elastic fibers had a similar growth pattern, although they were located mainly at the distal end of the gubernaculum. Fibroblasts largely predominated over other cell types and decreased in number with gestational age, whereas smooth muscle cells were restricted to the walls of blood vessels. Striated muscle cells were detected at the scrotal end of the gubernaculum, where they were disposed as isolated and scattered bundles running in various directions. Like fibroblasts, their number also decreased with age. CONCLUSIONS: During testicular migration gubernacular connective tissue undergoes extensive remodeling and ultimately becomes an essentially fibrous structure rich in collagen and elastic fibers. Such changes should decrease the size of the gubernaculum and, thus, contribute to other forces that cause the testes to move toward the scrotum. In fact, because of the lack of smooth muscle cells, and the amount and organization of striated muscle cells, active contraction of the gubernaculum is less likely to be an important factor in testicular descent.