苯甲酸雌二醇诱导不育小鼠睾丸中生精细胞增殖紊乱的研究

目的:探讨外源性雌激素苯甲酸雌二醇(EB)对诱导雄性不育小鼠中生精细胞增殖紊乱的影响。方法:60只雄性昆明小鼠随机分为3组,对照组肌肉注射150μl玉米油,EB处理组注射浓度分别为5、10 mg/kg的EB,隔天1次,持续4周。实验结束后取睾丸称其质量并计算睾丸指数;睾丸、附睾尾常规石蜡切片,HE染色;附睾尾制备精子悬液进行精子计数;免疫组化检测睾丸PCNA表达;qRT-PCR分析睾丸细胞周期蛋白A1、细胞周期蛋白B1、VASA、p53的表达变化;Western印迹检测p53及磷酸化p53蛋白表达。结果:与对照组相比,EB处理组小鼠睾丸指数显著下降[(0.56±0.09)%vs(0.43±0.08)%、(0.38±0.10)%,P0.05],精子悬液镜下观察未见精子,HE染色附睾管内未见精子;生精小管中精原细胞、初级精母细胞及支持细胞数量显著下降(P0.05)。免疫组化结果显示,与对照组相比,EB处理组生精小管中细胞PCNA阳性表达细胞数量显著下降(P0.05)。qRTPCR结果表明EB处理组PCNA、细胞周期蛋白A1、细胞周期蛋白B1、VASA mRNA表达与对照组相比显著下降(P0.05);p53表达随EB剂量升高而显著升高(P0.05)。Western印迹结果显示,与对照组相比,EB处理组p53及磷酸化p53蛋白表达水平显著升高,且存在剂量依赖效应,10 mg/kg组显著高于5 mg/kg组(P0.05)。结论:EB以剂量依赖的方式下调细胞周期相关因子表达抑制生精细胞的增殖,是导致雄性不育小鼠睾丸中生精细胞增殖紊乱的重要原因。     

隐睾与不育的关系

隐睾(cryptorchidism)又称睾丸未降,是最为常见的男性生殖系统畸形.隐睾症是导致男性不育的主要原因之一,国外学者对此进行了广泛深入的研究.本文就隐睾与不育症的研究进展综述如下.一、隐睾不育的流行病学隐睾在足月新生儿中的发生率约为4%～5%,在早产儿中的发生率约为9%～30%左右[1],在出生体重低于1800g的婴儿中发生率约为68.5%,体重小于900g的婴儿双侧隐睾发生率为100%.     

隐睾及睾丸固定术后对大鼠生精能力的影响

目的 观察隐睾及睾丸固定术后对睾丸生精能力的影响.方法 通过手术方法对80只SD大鼠制作单侧隐睾模型,随机分为10组,其中4组(每组10只)于隐睾术后7、14 d切取患侧睾丸组织,6组于隐睾术后7、14d行睾丸固定术,分别于术后2、4、6周取材.将所取得的睾丸组织称重后行流式细胞仪检测其细胞凋亡率和各生精细胞百分比以及组织中B淋巴细胞/白血病-2( bcl-2)和bax基因表达量.结果 隐睾睾丸重量明显下降,隐睾组1C细胞(7d组:10.61 ±1.10,14d组:11.79 ±0.91)较对照组降低(16.48±1.60,P＜0.05)、4C细胞也明显降低,而2C细胞(7d组:40.41±2.93,14 d组:51.41±6.45)较对照组增加(30.17±3.24,P＜0.05).隐睾各组生精细胞凋亡率(7d组:14.9±1.26,14 d组:6.90±0.96)高于正常对照组(2.50±0.44,P＜0.01),而睾丸复位固定术后各组生精细胞凋亡率均下降(P＜0.05).隐睾7、14 d睾丸中bc1-2蛋白表达量(7d组:4.68±0.47;14 d组:5.66 ±0.71)较对照组(7.47±1.01)降低而bax蛋白表达量(7d组:8.27±1.08;14 d组:6.26±0.21)较对照组(5.82 ±0.47,P＜0.05)升高,睾丸固定术后各组bcl-2蛋白表达量升高而bax蛋白表达量降低(P＜0.01).结论 隐睾可以使睾丸生精细胞凋亡增加,睾丸复位固定术后,睾丸生精功能可部分或全部恢复,其恢复程度和隐睾时间长短有关;bcl-2和bax表达在生精细胞凋亡的调控中起重要作用.     

中药生精冲剂对生精障碍小鼠治疗作用的实验研究

目的:研究生精冲剂对环磷酰胺所致生精障碍小鼠的治疗作用。方法:46只雄性昆明小鼠随机分为正常组(n=10)、模型组(n=12)、对照组(n=12)和中药组(n=12),后3组腹腔注射环磷酰胺,建立生精障碍模型。造模完成后,中药组和对照组分别灌胃中药生精冲剂[16 g/(kg.d)]和克罗米芬[21.6 mg/(kg.d)],正常组与模型组每天灌胃等量生理盐水,连续15 d。次日,测量睾丸重量,并计算睾丸指数;放射免疫法测定血清FSH、LH、T水平;对睾丸组织进行组织学观察。结果:中药组小鼠血清T[(7.046±0.291)nmol/L]、FSH[(2.947±0.587)mIU/ml]、LH[(3.254±0.492)mIU/ml]、睾丸指数[(3.958±0.342)g/kg]与模型组各检测值相比[(6.231±0.317)nmol/L、(5.428±0.719)mIU/ml、(5.155±0.460)mIU/ml、(3.525±0.462)g/kg]差异有显著性(P<0.05)。组织学观察中药组睾丸生精小管生精细胞层数及管腔内精子数明显高于模型组。结论:生精冲剂治疗小鼠生精障碍有效。     

17β-雌二醇对小鼠生精细胞T型Ca~(2+)通道的作用

目的:研究17β-雌二醇(E2)对小鼠生精细胞T型钙电流(ICaT)的影响。方法:采用膜片钳全细胞记录技术观察E2对急性机械分离的小鼠生精细胞ICaT的影响。结果:E2(1、10、100μmol/L)呈浓度、电压依赖性抑制小鼠生精细胞钙电流,抑制率分别为(13.48±1.86)%、(28.98±2.70)%和(52.93±3.42)%(n=6,P<0.05)。E2对T型钙通道的半数最大抑制浓度(K50)为8.89μmol/L。100μmol/LE2显著改变T型钙通道的激活和失活特性:半数激活电压(Va1/2)和激活斜率因子(κa)分别从(-48.94±0.22)mV、(5.19±0.19)mV变为(-54.34±1.02)mV和(6.02±0.84)mV(n=5,P<0.05);而半数失活电压(Vi1/2)和失活斜率因子(κi)分别从(-56.51±0.13)mV、(3.36±0.11)mV变为(-61.78±0.50)mV、(4.25±0.37)mV(n=5,P<0.05)。结论:E2对生精细胞T型钙通道有抑制作用,呈浓度依赖性。     

睾丸特异表达基因-1在小鼠隐睾模型中的表达及其意义

目的 探讨睾丸特异表达基因-1(TSEG-1)在小鼠隐睾模型中的定位、定量表达变化及其意义.方法 将46只雄性昆明新生小鼠随机分为对照组(n=10)、丙二醇(溶剂)注射组(n=15)、17β-雌二醇注射组(n=21),应用苏术素-伊红(HE)染色观察睾丸组织形态学改变,原位杂交、逆转录-聚合酶链反应(RT-PCR)和实时定量PCR检测TSEG-1 mRNA的定位和定量变化.结果 17β-雌二醇注射组发生隐睾,而对照组和丙二醇注射组睾丸位置正常.与对照组比较,17β-雌二醇组睾丸组织80%曲精小管管腔消失,Ⅰ～Ⅳ级生精细胞数目减少,60%精母细胞或精子细胞核皱缩,90%精子发生障碍.在对照组睾丸组织中,TSEG-1 mRNA表达水平是模型组25倍,主要定位于精母细胞和早期精子细胞,而隐睾组TSEG-1 mRNA表达水平显著下调.结论 17β-雌二醇诱导小鼠隐睾模型是制作隐睾模型的有效方法 ,新基因TSEG-1可能参与雌激素诱导隐睾模型的发生过程.     

在不育合并隐睾的男性中存在AZFc的部分缺失

最近有研究表明有一种特殊的部分性AZFc的缺失即gr／gr缺失，被认为是生精障碍和睾丸肿瘤发生的遗传危险因素。Giachini C等拟证实gr／gr缺失在睾丸发育不全综合征（TDS）的一个主要组成部分即隐睾的发生中的发病遗传机理。Giachini C等选择了146例隐睾患者和140例没有隐睾病史的不育患者，分别采用部分筛选的序列标记位点（STS）的方法并通过CDY1／DAZ的基因含量进行拷贝和分析。     

表观遗传学调控对生精功能障碍性不育C57BL/6J小鼠动物模型影响的实验研究

目的:探讨表观遗传学调控对生精功能障碍性不育C57BL/6J雄性小鼠动物模型的作用及影响.方法:60只C57BL/6J雄性小鼠随机均分为对照组和模型组.应用附睾靶向多肽(CSA)结合吲哚青绿包载游离纳米粒(ICG-NPs)联合白消安和二甲基亚砜构建生精功能障碍性不育动物模型.在附睾中检测精子DNA碎片指数(DFI)、精...     

抗氧化酶对隐睾生精细胞凋亡的影响

目的 :探讨超氧化物歧化酶 (SOD)、过氧化氢酶 (CAT)、谷胱甘肽过氧化物酶 (GSHPx)对隐睾生精细胞凋亡的影响。　方法 :4 8只未成熟SD雄性大鼠随机分为隐睾组和对照组 ,于术后第 1、3、7d采集睾丸。TUNEL法检测其生精细胞凋亡 ;化学比色法测定其SOD、CAT、GSHPx的活性和丙二醛 (MDA)含量。　结果 :术后第 7d ,与对照组相比 ,隐睾重量、SOD活性、CAT活性和SOD/ (CAT +GSHPx)比值均显著降低 (P均 <0 .0 1) ,GSHPx的活性无显著变化 (P >0 .0 5 ) ,生精细胞凋亡指数和MDA含量均显著增加 (P均 <0 .0 1)。　结论 :隐睾生精细胞的凋亡与抗氧化酶活性的降低密切相关     

小鼠隐睾动物模型中CGRP的表达变化

目的 通过氟他胺(Flutamide,FLU)诱导小鼠隐睾动物模型的建立,探索其睾丸组织中降钙素基因相关肽(Calcitonin gene-related peptide,CGRP) mRNA表达的变化.方法 将妊娠BALB/c小鼠随机分成A、B、C、D、E5组;在母鼠孕第12～21d持续10d给予A、B、C、D、E5组氟他胺灌胃,剂量依次为0mg/kg、150mg/kg、300 mg/kg、500 mg/kg、700 mg/kg,A组为空白对照组;在出生后第8周时运用Real-Time PCR技术检测子代小鼠睾丸组织中CGRP mRNA的表达情况及单侧隐睾小鼠中患侧与健侧睾丸CGRP mRNA的表达情况.结果 A组小鼠无隐睾,B、C组有单侧隐睾;D、E组有双侧隐睾的发生.与对照组相比,随着给药剂量的增大,CGRP mRNA的表达强度明显低于对照组(P＜0.01).单侧隐睾小鼠中患侧和对侧睾丸的CGRPmRNA均减少(P＜0.05).结论 剂量为150mg/kg与300 mg/kg的氟他胺能诱导产生单侧隐睾模型,剂量为500与700 mg/kg的氟他胺能诱导产生双侧隐睾模型;单侧隐睾小鼠中双侧睾丸均有损害;CGRP是隐睾产生过程中一个重要的影响因素.     

Pattern of Sertoli cell degeneration in cryptorchid prepubertal tests

Seventy-three testicular biopsies from 54 children (aged 2 months-14 years) with undescended testes were examined by light and electron microscopy. The biopsies included abdominal, inguinally fixed, inguinally moveable, and retractile testes. Alterations in Sertoli cell morphology were found in all biopsies. The alterations included dilated elements of rough endoplasmic reticulum, vacuolization of the cytoplasm, mitochondria with poorly preserved cristae, increase in electron density of the matrix, elongation of the nuclei, and irregularities of the nuclear membrane. According to the numerical appearance of these cells and to the extent of lesions in single Sertoli cells, seven phases in the continuous process of tubular alteration were distinguished. The most severe tubular damaged (phase VII) occurred when the seminiferous epithelium consisted exclusively of necrotic cells. All phases of tubular alterations were seen regularly in each of the biopsies investigated. Germ cells occurred only in phases I-IV and were never observed in tubules in phases V-VII. Significant differences became evident between inguinal and retractile testes by morphometric evaluation. It was demonstrated that the number of germ cells per cross-sectioned tubule (S/T value) correlated negatively with the percentage of tubules in phases V-VII. In contrast to inguinal testes, a complete absence of Sertoli cells and an S/T value less than 0.1 were never found in retractile testes and the percentage of tubules in phases V-VII was reduced significantly compared with inguinal testes. Our findings indicate that (i) maldescended testis in patients between 1 and 15 years-of-age is associated with a special pattern of Sertoli cell degeneration; (ii) Sertoli cell degeneration is a continuous process, which can lead eventually to complete dissolution of the seminiferous epithelium; (iii) total degeneration is not related to age but is dependent on testicular position; (iv) a defined phase of degeneration excludes germ cell development, and therefore enhanced Sertoli cell degeneration in cryptorchid testes must also account for the reduction in germ cell number.     

Assessment of germ cell apoptosis in cryptorchid rats

Aim: To investigate the relationship between germ cell degeneration and apoptosis in cryptorchid rats. Methods: Thirteen 21-day-old Wistar rats were made unilaterally cryptorchid by closing the left inguinal canal. At day 30 (Group 1, n=6) and day 60 (Group 2, n=7) after operation, the testes were removed for histopathological examination. The controls (n=8) were sham operated and were sacrificed at day 60. Germ cell apoptosis was assessed by means of the TUNEL method. Results: Spermatogenesis was arrested and the testicular and seminiferous tubular diameters were significantly reduced In the unilateral undescended testes (UUTs) compared with the contralateral descended testes (CDTs) and the control rats. However, atrophic changes, pathological calcification, necrosis of seminiferous tubule, and absence or sloughing of germ cells were not found in all the animals. The spermatocytes were the main type of germ cells undergoing apoptosis in all the groups. In the UUTs, there was a significant and time-depe     

不同剂量雌激素对裸鼠血管瘤模型的影响

目的：建立人毛细血管瘤裸鼠移植模型,探讨血管瘤裸鼠模型建立的最佳条件。方法：将手术切除的1例雌激素受体阳性的儿童增生期血管瘤组织制成组织块,植入20只裸鼠（BALB/cnudemice）皮下,每只4处,将20只裸鼠分为4个实验组。实验1组在移植后给予普通鼠食喂养;实验2组在1组基础上每周肌注雌二醇0.01mg;实验3组在1组基础上每周肌注雌二醇0.1mg;实验4组在1组基础上每周肌注雌二醇1mg,于移植后第30、60、90天切取移植瘤。移植瘤标本进行病理学光镜检查,用血管内皮细胞单克隆抗体CD31、CD34进行免疫组化染色。结果：移植后早期各组标本内皮细胞大量变性、坏死,30天后,单纯喂养的实验1组及实验2组部分移植瘤开始吸收或形成脓肿及纤维化。实验3、4组移植瘤开始缓慢生长。90天后实验1组、实验2组移植瘤均未成活,实验4组移植瘤部分成活,而实验3组移植瘤全部成活。光镜下成活的移植瘤与原血管瘤组织生物学特点相似。结论：不同剂量的雌激素对血管瘤裸鼠移植模型的建立有一定影响,适量的应用雌激素可建立稳定的人血管瘤裸鼠移植动物模型。该模型可以应用到基础和临床的血管瘤研究。     

Mouse models in male fertility research

Limited knowledge of the genetic causes of male infertility has resulted in few treatment and targeted therapeutic options. Although the ideal approach to identify infertility causing mutations is to conduct studies in the human population, this approach has progressed slowly due to the limitations described herein. Given the complexity of male fertility, the entire process cannot be modeled in vitro. As such, animal models, in particular mouse models, provide a valuable alternative for gene identification and experimentation. Since the introduction of molecular biology and recent advances in animal model production, there has been a substantial acceleration in the identification and characterization of genes associated with many diseases, including infertility. Three major types of mouse models are commonly used in biomedical research, including knockout/knockin/gene-trapped, transgenic and chemical-induced point mutant mice. Using these mouse models, over 400 genes essential for male fertility have been revealed. It has, however, been estimated that thousands of genes are involved in the regulation of the complex process of male fertility, as many such genes remain to be characterized. The current review is by no means a comprehensive list of these mouse models, rather it contains examples of how mouse models have advanced our knowledge of post-natal germ cell development and male fertility regulation.     

Early morphological changes in the abdominal testes in immature unilaterally cryptorchid rats

Testicular descent was prevented unilaterally in newborn rats by cutting the gubernaculum testis. Morphological changes due to non-descent of the testes were studied in 16-, 20-, and 24-day-old rats. Scrotal and abdominal temperatures were measured in control rats, and a difference was noted from 16 days and onwards. At 16 days of age the abdominal testes showed an abnormal accumulation of lipid droplets in the Sertoli cells at certain stages of spermatogenesis, and the number of tubules which had developed a lumen was slightly greater than in the scrotal testes. At 20 days of age, some spermatocytes were degenerating in the abdominal testes, especially in the tubular segments where lipid accumulation had been seen earlier. Lipid accumulation was noted in the Sertoli cells in all stages of spermatogenesis and additional ultrastructural signs of Sertoli cell malfunction such as dilatation of the SER and dilatation of the intercellular space between adjacent Sertoli cells was observed. Also the number of tubules containing a lumen was slightly larger in the abdominal testes. At 24 days of age, the number of spermatocytes was reduced in abdominal testes and the morphological changes seen earlier in the Sertoli cells were more pronounced. The function of the blood-testis barrier was investigated by the ability of the tubules to exclude lanthanum, and no differences were found between scrotal and abdominal testes at 16 and 20 days of age. The present study suggests that the earliest morphological changes in experimentally primary abdominal testes may occur in the Sertoli cells.     

胰岛素样因子3在氟他胺诱导的小鼠隐睾模型中的表达和意义

目的:通过氟他胺诱导小鼠隐睾动物模型的建立,探讨其睾丸组织的胰岛素样因子3(INSL-3)mRNA表达的变化。方法:妊娠BALB/c小鼠随机分成A、B、C、D、E 5组,每组9只(雄鼠3只,雌鼠6只),合笼交配;分别在雌鼠孕第12²1天持续10 d给予氟他胺灌胃,剂量依次为0、150、300、500、700 mg/kg;分别在出生后第4周和第8周时运用荧光定量PCR技术检测子代小鼠睾丸组织中INSL-3 mRNA的表达情况。结果:A组小鼠无隐睾,B、C组有单侧隐睾,发生率分别10.0%和25.0%;D、E组有双侧隐睾的发生,发生率分别为21.1%和40.0%。随着给药剂量的增大,INSL-3 mRNA的表达逐渐降低;与对照组相比,B、C组表达强度无明显变化(P>0.05);D、E组表达强度明显低于A组(P<0.05)。结论:剂量为150与300 mg/kg的氟他胺能诱导单侧隐睾模型,剂量为500与700 mg/kg的氟他胺能诱导双侧隐睾模型;并且隐睾小鼠睾丸的INSL-3表达随氟他胺浓度升高水平明显下降,可能是隐睾发生的机制之一。     

Electron Microscopic Study of Interstitial Cells in Cryptorchid Human Testes. I: Interstitial Cells in Prepubertal Age

In order to establish the ultrastructural features of Leydig cells 50 biopsies or prepubertal human cryptorchid testes have been examined. Light and electron microscopic observations reveal the presence of two types of interstitial cells distinguishable on the basis of the peculiar ultrastructural characteristic of the nucleus and cytoplasm. In our opinion these two types of interstitial cells seem to represent different stages of cell differentiation. There are fibroblast-like cells and cells corresponding to Leydig cells. From our observations it may be inferred that the fibroblast-like cells are the precursor cells of the Leydig cells.     

Spermatogenesis in Snell dwarf, little and congenitally hypothyroid mice

The status of spermatogenesis in Snell dwarf, little and congenitally hypothyroid mice was studied. In all of these mice with a hormone deficiency the seminiferous tubules were smaller in size and contained fewer spermatogonia, spermatocytes, spermatids and spermatozoa than did those of normal control mice. There was no substantial difference in the Johnsen score between the hormone-deficient mice and normal control mice, but the former had underdeveloped seminiferous tubules with a corresponding paucity of germ cells, which may be partly responsible for the infertility of these mice. In the present study, growth hormone and thyroxine were administered separately to growth hormone-deficient and thyroxine-deficient mice, respectively. Such replacement therapy brought about an increase in cell counts of the seminiferous tubules and in sperm counts in both groups.     

Influence for testicular development and histological peculiarity in the testes of flutamide-induced cryptorchid rat model

Objectives: To investigate influence for the testicular development and to assess the usefulness as an animal model, cryptorchid rats were induced by exposure to flutamide during the fetal period and their testes examined histologically. Methods: Flutamide was injected into the abdomen of pregnant rats for 7 days from the 14th to 20th day of gestation. The male offspring in which cryptorchidism was observed at 28 days after birth were defined as the model rats. They were divided into four groups by dosage of flutamide (2.5 mg, 5 mg, 7.5 mg, 15 mg per day), and their testicular weight, spermatogenesis (modified Johnsen score), and germ cell apoptosis were examined histochemically at 10 weeks after birth. Results: The incidence of cryptorchidism including both unilateral and bilateral in the 2.5, 5, 7.5 and 15‐mg flutamide groups was 58.3%, 81.9%, 93.6% and 91.0%, respectively. In the model rats, the undescended testes were located at the caudal end of the abdominal cavity, and these testes weighed less than the contra‐descended testes in each group. Histologically, apoptotic cells were markedly increased, the seminiferous tubules were degenerated and disturbance of spermatid differentiation was observed in the undescended testes compared with the normal or contra‐lateral descended testes. Conclusions: We found out that the incidence of undescended testes increased in a flutamide dose‐dependent manner. The findings of histological examination were independent of the administrated dose of flutamide and it is suggested that exposure of the testes to abdominal temperature causes spermatogenic arrest with germ cell apoptosis. The present animal model indicates high incidence of above 90%, has no surgical stress and dose not require special techniques. We believe that the present model is a useful tool for the understanding of pathogenesis and treatment of cryptorchidism and further biological research into spermatogenesis.     

Regulation of interstitial cell function by seminiferous tubules in intact and cryptorchid rats

The effects of experimental cryptorchidism on seminiferous tubule secretions and interstitial cell testosterone production were studied in vitro. Spent media obtained from incubations of seminiferous tubules (SMST) from cryptorchid rats caused a significant increase in testosterone production when added to interstitial cells isolated from intact rats. The previously noticed inhibitory activity of the SMST from stages VIII–XI of the sperma-togenic epithelial cycle gradually disappeared after the induction of experimental cryptorchidism. SMST obtained from both sham-operated or cryptorchid rats stimulated basal testosterone production when added to interstitial cells from cryptrochid rats. SMST from rats had been cryptorchid for 7, 14 and 28 days stimulated testosterone production when added to interstitial cells prepared from intact animals. Seminiferous tubules from cryptorchid rats therefore appear to be the source of a heat stable, trypsin-resistant factor with an apparent molecular weight of between 5000 and 10 000 daltons which stimulates testosterone production when added to interstitial cells in vitro. Its activity could not be blocked by an LRH antagonist. This factor enhances both basal and LH-stimulated secretion of testosterone in contrast to the inhibitory activity which involves only a partial blockade of LH-dependent steroidogenesis.