小鼠睾丸移植物中3种生精阶段特异性基因的表达

目的 采用逆转录-聚合酶链反应，探讨新生小鼠睾丸组织移植到裸鼠体内后，3种阶段特异性基因，缺失型无精子基因（Daz1）、磷酸甘油激酶2（Pgk2）和鱼精蛋白-2（Prm2）在不同发育阶段移植物中的表达情况。方法 将180只出生1-2d昆明小鼠睾丸移植到60只7-12周去势雄性免疫缺陷小鼠背部；在移植后不同时间段（分为3d、1周-8周和12周10个组）取出移植物，对在发育不同阶段表达的3种基因出现的时间及表达情况进行分析测定，并与相应各年龄段正常小鼠睾丸中的基因表达相比较。同时进行组织形态学观察，对比各种基因出现的时间与小鼠睾丸发育周期的吻合性。结果 在10个时间段取出的移植物中，所测定的3种基因表达趋势与在正常昆明小鼠睾丸中所见基本相同，并与小鼠发育周期基本吻合。结论 新生小鼠睾丸组织移植到免疫缺陷小鼠体内后，生精细胞的发育在形态学和几种受试基因出现的时间上与在正常小鼠中表现均相似。从而对该睾丸组织移植模型用于生精细胞异体异位生长发育研究及基因调控机制研究的可行性提供了进一步的理论依据。     

受体激素水平对未成熟睾丸移植物中生精细胞的生长发育影响

目的 采用已建立的未成熟睾丸组织异体异位移植模型,研究受体小鼠激素水平变化对未成熟睾丸组织移植物生长发育的影响.方法 以免疫缺陷小鼠为受体,将新生1～2d龄昆明小鼠睾丸移植到受体小鼠背部皮下.在移植后不同阶段取出移植物,进行组织形态学观察,分析移植物中生精细胞的发育情况,同时采用放射免疫分析法检测受体动物血清中睾酮(T)、促卵泡刺激素(FSH)和促黄体生成素(LH)水平,并以同龄正常昆明小鼠作对照.结果 受体小鼠血清中LH水平没有明显波动,而T和FSH浓度在移植的前8周呈逐渐上升趋势,从9周以后开始下降,且显著低于正常受体内睾酮和FSH浓度,与组织学中所观察到的生精上皮受损程度的增加是同步的.结论 以上结果提示,应注意获取移植物中精子的最佳时间.对于小鼠来说,从新生小鼠睾丸移植物中获取精子的最佳时间是术后5-8周.     

小鼠睾丸组织移植物二次移植后的生长发育研究

目的 通过将第一次移植后的移植物进行再移植,探讨二次移植的睾丸组织生长发育情况,为今后采用睾丸组织裸鼠移植模型探讨性成熟期较长动物(包括人类)睾丸组织的体外成熟提供实验依据.方法 将1～2 d龄昆明小鼠睾丸组织植入去势裸鼠背部,于移植后2周取出移植物,并将其立即移植到另一只去势裸鼠背部,并在二次移植4周后取出移植物,制作HE染色切片,在光镜下观察二次移植后睾丸组织的生长发育情况;同时采用检测鱼精蛋白-2 mRNA的方法对二次移植物的生长发育程度进行鉴定.结果 移植2周后的小鼠睾丸组织再次移植后,仍可观察到移植物中的血管组织再生以及生精小管的继续发育;二次植入的44块组织经4周生长发育后收集到22个二次移植物,回收率为50%;在仅含有精原细胞和精母细胞的移植物二次移植后收集到的移植物中,观察到了圆形精子细胞甚至长形精子细胞,并在所有的二次移植物中检测到鱼精蛋白-2 mRNA的表达.结论 新生小鼠睾丸组织移植物转移到另一只受体中后可继续存活并生长发育.     

异体异位发育小鼠睾丸组织中精子功能相关基因和蛋白的表达研究

目的:采用已建立的未成熟睾丸组织移植模型,研究异体异位生长发育的小鼠睾丸组织中精子功能相关基因Asrgl1,Gpx4,Asp,Prdx4和Spa17及其相关蛋白GPX-4和PRX-Ⅳ的表达,以迸一步评估该移植模型在生殖医学中应用的可行性.方法:以1～2 d小鼠睾丸组织为供体,7～12周雄性免疫缺陷小鼠为受体进行组织移植;移植8周后收取移植物组织,采用荧光定量PCR法检测移植物中Asrgl1,Opx4,Asp,Prdx4和Spa17的表达情况,并与正常小鼠睾丸组织进行对比;同时采用Western blot检测GPX-4和PRX-Ⅳ 2种蛋白的表达情况.结果:基因检测显示,5种受试基因在移植物中的表达与8周正常小鼠睾丸组织相比均有下调;蛋白分析提示,GPX-4和PRX-Ⅳ 2种蛋白在8周正常小鼠睾丸组织中表达而在小鼠睾丸组织移植物中表达缺陷.结论:几种主要在精子运动、精子顶体形成及精子的受精功能中发挥作用的基因和蛋白在移植物中表达下调及缺失提示,异体异位睾丸组织移植在临床应用的可行性还需进一步评估     

冷冻保存未成熟睾丸组织移植后生精细胞的生长发育木

目的采用睾丸组织移植模型,探讨通过简单易行的冷冻保存程序保存未成熟睾丸组织,并使冷冻后的组织恢复生精过程的可能性,为该动物模型应用在保存雄性生殖细胞、帮助癌症患者保存生殖能力以及保存濒危物种等方面提供进一步的实验依据。方法用DMEM培养基加入二甲基亚砜配制冷冻保存液,将新生1～2d昆明小鼠睾丸组织按分步冷冻的方法进行冷冻,最终放入-80℃超低温冰箱中保存起来。2周后取出冻存的睾丸组织,移植到雄性去势免疫缺陷小鼠背部皮下,分别于移植后4周、5周和8周取材,制作HE染色切片,观察移植物中生精小管结构和生精细胞的组成,并与新鲜移植的睾丸组织进行对比分析。结果采用实验中的冷冻程序保存的新生小鼠睾丸组织,在冷冻保存一段时间后再移植,其表现与新鲜睾丸组织移植相同,其中末成熟的生精细胞可以在受体中继续生长发育,并完成整个生精过程,发育成为精子。结论本实验中所采用的睾丸组织冷冻保存方法具有不需特殊设备、简便易行、适用范围广泛等优点,适用于多种场合中生精细胞的冷冻保存。     

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…     

雄,雌激素对睾丸表皮生长因子及受体表达的影响

运用＾１２５Ｉ－ＥＧＦ放射受体法，探讨对不同周龄Ｗｉｓｔａｒ鼠使用雄、雄激素过程中及停药后睾丸表皮生长因子与ＥＧＦ受体结合情况，结果表明：ＥＧＦ与睾丸细胞膜在２７℃孵育７０分钟结合最大，年龄增长与睾丸重量呈正比，与ＥＧＦ呈反比；雄激素治疗中，睾丸重量、ＥＧＦ均上升，且大于正常（Ｐ＜０．０１），ＲＴ骤各；停药时，ＥＧＦ降至正常；雌激素治疗时，ＥＧＦ亦高于正常（Ｐ＜０．０１），ＫＤ微上升，停药时，ＥＧ     

睾丸组织异体异位移植研究进展

自从2002年《自然》杂志发表了第1篇用同种或异种睾丸组织移植进行的生精细胞体外发育研究以来,该技术被广泛应用于不同种类动物(包括人类)睾丸组织的体外成熟的探讨。这种采用免疫缺陷动物作为受体,同种或异种睾丸组织为供体的技术与其他生精细胞体外成熟方法相比具有环境条件较易控制、重复性较好等优点。迄今为止,在用小鼠、仓鼠、猫、兔、猪、山羊、牛和恒河猴等动物睾丸组织进行的移植研究中,未成熟生精细胞在作为受体的裸鼠体内均发育到精子阶段,并通过卵细胞胞质内单精子注射(ICSI)技术,用发育成的小鼠、兔精子分别产生了下一代。本文总结了供体组织的种类和年龄,受体的性别、完整性、免疫状况和移植部位以及移植时间对移植物发育的影响,概述了近几年来采用该技术所进行的睾丸组织移植研究发展现状,并对该技术的应用以及存在的问题进行了讨论。     

Age-specific effect of phthalate ester on testicular development in rats

Purpose

Phthalate esters have been shown to induce testicular damage in both adult and immature rats; however, there have been, so far, few reports describing the age-specific effects of phthalate esters on testicular function. The aim of this study is therefore to investigate the age-specific effects of mono-n-butyl phthalate (MBP) on the testes in both prepubertal and mature adult rats.

Materials and methods

Both prepubertal male rats and adult mature male rats were fed special rat chow containing 1% MBP for 10 days. Control prepubertal and adult rats were fed standard commercial rat chow during the same period as the MBP-treated rats. After 10 days of feeding, all rats were killed, and the testes were removed. The weight of the testis was measured, and histological examination of the testis was performed. In addition, the frequency of an apoptotic cell appearance in the seminiferous tubules was determined in both MBP-treated and control groups.

Results

In the prepubertal rats, the mean weight of the testes was significantly lower in the MBP-treated rats than in the control rats. A histological examination of the MBP-treated testes showed a decreased seminiferous tubular diameter and an inhibited maturation of germ cells in comparison to those of the control testes. Furthermore, apoptotic cells appeared more frequently in the MBP-treated testes than in the control testes. Although in adult mature rats, no significant difference was observed in either the testicular weight or the histological findings between the MBP-treated and control rats.

Conclusions

The oral administration of MBP to male rats was observed to produce more pronounced testicular damage in prepubertal rats than in adult mature rats. Immature testes may thus be more sensitive to MBP, which induces the germ cell apoptosis in seminiferous tubules and testicular atrophy in prepubertal young rats.     

Effect of cryptorchidism on testicular and Leydig cell androgen production in the mouse

Unilateral cryptorchidism was induced surgically in adult mice and the effects on testicular and Leydig cell steroidogenesis were studied after 7 weeks. There was a 60% reduction in weight of the cryptorchid testis and this was associated with a significant reduction in intratesticular androgen content, both under basal conditions and following an injection of hCG. Testicular androgen production in vitro was also significantly lower in the cryptorchid testis compared to the scrotal testis, again under both basal conditions (29 +/- 6% of control) and in the presence of hCG (46 +/- 9% of control). Scrotal testes from the unilaterally cryptorchid animals did not show any significant difference in steroidogenic capacity compared to testes from untreated control animals. The decrease in steroidogenic capacity of the cryptorchid testis was due, at least in part, to a reduction in activity for each Leydig cell. In four experiments, androgen production by Leydig cells isolated from cryptorchid testes was 48 +/- 9% of cells from scrotal testes in the presence of a saturating dose of hCG. Under basal conditions the effect was more variable between experiments with steroid secretion by Leydig cells from cryptorchid testes being 58 +/- 32% of that for cells from scrotal testes. Leydig cell steroidogenesis in the scrotal testes of unilaterally cryptorchid animals did not differ significantly from untreated controls. These results show that induced cryptorchidism in the mouse causes a significant reduction in Leydig cell activity. This is apparently different from the effects of this procedure on the rat and raises the possibility that intratesticular regulation differs between the two species.     

The effect of Chrysophyllum albidum fruit on testicular functions in rats

The prevalence of male infertility is a well‐known public health issue with majority of cases due to deficient sperm production of unknown origin. Studies have associated dietary habits with male factor infertility. Chrysophyllum albidum is a common plant that produces a popular fruit, widely consumed for its nutritional and medicinal values. This study investigates the effects of C. albidum fruit methanol extract on the reproductive functions of male Wistar rats. Ripe C. albidum fruit was extracted using methanol and subjected to phytochemical screening. Fifteen male Wistar rats (100–120 g) divided into three (n = 5) received distilled water (control), 1.0 and 6.4 g kg^?1 day^?1 extract, respectively, for 28 days via oral gavage. The sperm count, motility, percentage sperm aberration, histology of testes and epididymides were examined by microscopy. Serum levels of luteinising hormone (LH), follicle‐stimulating hormone (FSH) and testosterone were quantified using ELISA. Data were analysed using ANOVA at p < 0.05 significance. Sperm count significantly increased in 6.4 g kg^?1 day^?1 extract. Serum testosterone level decreased in 1.0 and 6.4 g kg^?1 day^?1 extract. The architecture of sections of testes and epididymides showed anomalies. C. albidum fruit adversely altered reproductive functions of male Wistar rat.     

The effect of varicocele repair on testicular volume in children and adolescents with varicocele

PURPOSE: We investigated the effect of varicocele repair on testicular volume according to age in children and adolescents and review the long-term results of varicocele surgery. MATERIALS AND METHODS: The study included 39 boys 11 to 19 years old with clinical palpable varicocele who underwent varicocele surgery with at least 1 year of postoperative followup. Preoperative and postoperative testicular volumes were monitored and measured with an ellipsoid Prader orchidometer. Physical examination findings (testicular volumes and testicular consistency) in all boys, and serum hormone values and semen parameters in 16 adolescents were recorded and compared before and after surgery. RESULTS: Left unilateral varicocelectomy was done in 29 boys (74%) and bilateral varicocelectomy in 10 (26%). While no postoperative hematoma, infection or testicular atrophy was observed, 1 boy (2.5%) had varicocele recurrence and 2 boys (5.1%) had minimal hydroceles that required no intervention. Significant increases were observed in postoperative sperm concentration (p = 0.01), total motile sperm count (p = 0.009), testis volume (p = 0.000) and serum testosterone level (p = 0.014). All 15 boys with preoperative soft testis had normal testicular consistency postoperatively. Of the 19 boys with preoperative testicular atrophy 10 (53%) did regain normal testicular growth, while 9 (47%) retained testicular volume loss after surgery. When comparing preoperative to postoperative increase in testicular volume according to age in all boys, the mean was statistically significantly higher in boys younger than 14 years (left testis p = 0.037, right testis p = 0.000). CONCLUSIONS: Testicular consistency achieved normal firmness after varicocelectomy in all boys with preoperative soft testis. While there was catch-up growth in comparison to the contralateral testis, testicular consistency improved but testicular volumes may not increase significantly after varicocele repair at ages older than 14 years. However, in these adolescents postoperative semen parameters and serum hormone values may significantly improve regardless of testicular volume. Therefore, boys with varicocele and their families should be fully informed in light of these findings.     

Effect of bilateral testicular resection on thymocyte and its microenvironment in aged mice

Aim: To observe the changes in thymocyte and its microenvironment in aged mice after bilateral testicular resection. Methods: In male old mice, at the 25th day after testicular resection, the peripheral blood and thymus were collected. Blood and thymus suspension smears were prepared for quantitative histochemistry and immunohistochemistry study under light and electron microscopes. Results: In testes resected mice the size and the weight of thymus were markedly increased. The demarcation between cortex and medulla was clear. The cortex was thickened and the cell density was increased. The ratio of cortex/medulla stereometry was increased. The total cell count, thymocyte count, the percentage of acid α-naphthyl acetate esterase (ANAE) positive thymocytes, nonlymphocytes and the rosette formation of macrophages and thymocytes were all increased. The thymocytes surrounded closely to the light thymic epithelial cells, dendritic cells or macrophages. The lymphocytes, particularly the ANAE positive lymphocytes of peripheral blood were increased. Conclusion: After bilateral testicular resection, the thymus of aged male mice showed morphological regeneration and the thymocytes and its microenvironment appeared to be definitely improved. It is suggested that testicular resection may improve immune function.     

糖尿病对小鼠睾丸组织中PCNA表达的影响

为研究糖尿病对睾丸生殖细胞DNA合成的影响，应用免疫组化ABC法检测增殖细胞核抗原（proliferation cell nuleus antigen PCNA)在睾丸组织的表达。结果显示免疫阳性细胞包括精原细胞和初级精母细胞，少数支持细胞和肌样细胞胞核也有着色。PCNA阳性率随糖尿病病程进展呈明显下降趋势，而且每年龄段糖尿病组PCNA阳性率均明显低于相应正常对照组。由此得出结论，糖尿病对PCNA在睾丸组织中表达起抑制作用，且与糖尿病病程呈正相关，即糖尿病时生精过程受到明显抑制，这种抑制作用可能与糖尿病所导致的雄性生殖能力下降有关。     

The protective effect of darbepoetin alfa on experimental testicular torsion and detorsion injury

AIM: Testicular torsion is a serious urological emergency, usually involving newborns, children, and adolescents which can lead to subfertility and infertility. Prevention of testicular damage caused by torsion is still a clinical and experimental problem. So far many chemicals and drugs have been investigated for decreasing ischemia/reperfusion (I/R) injury in experimental animals. The possible protective effect of darbepoetin alfa, a novel erythropoietic protein, on testicular tissue after I/R injury was examined in this study. METHODS: Thirty rats were divided into three groups: sham operation, torsion/detorsion, and torsion/detorsion plus darbepoetin alfa groups. After torsion (2 hours) and detorsion (4 hours), bilateral orchiectomy was performed. Malondialdehyde, nitric oxide and glutathione levels were determined in testicular tissue. RESULTS: Administration of darbepoetin alfa caused a decrease of malondialdehyde and nitric oxide levels and an increase in glutathione levels compared with the torsion/detorsion group. In addition, histological injury scores were significantly decreased in the treatment group more than the torsion/detorsion group. CONCLUSION: The results suggest that darbepoetin alfa may be a potential protective agent for preventing testicular injury caused by testis torsion.     

The effect of norepinephrine, isoprenaline and acetylcholine on the testicular and epididymal circulation in the pig

Blood flow to the testis and epididymis of 7 anaesthetized male pigs was measured using electromagnetic blood flow sensors. An average blood flow to the testis of 12 ml/min/100 g and in the epididymal arteries of 19 ml/min/100 g was measured. The effect of intravenously administered norepinephrine, isoprenaline and acetylcholine on α and β₂ adrenergic and cholinergic receptor sites, respectively, was investigated. As neither norepinephrine nor isoprenaline, nor acetylcholine changed vascular conductance of the testis, we could not demonstrate adrenergic and cholinergic receptor activity. The possibility of autonomic regulation by the nonvascular smooth muscles of the spermatic cord and the inner layer of the tunica albuginea has been discussed.     

The effect of environmental contaminants on testicular function

Male reproductive health has deteriorated considerably in the last few decades. Nutritional, socioeconomic, lifestyle and environmental factors (among others) have been attributed to compromising male reproductive health. In recent years, a large volume of evidence has accumulated that suggests that the trend of decreasing male fertility (in terms of sperm count, quality and other changes in male reproductive health) might be due to exposure to environmental toxicants. These environmental contaminants can mimic natural oestrogens and target testicular spermatogenesis, steroidogenesis, and the function of both Sertoli and Leydig cells. Most environmental toxicants have been shown to induce reactive oxygen species, thereby causing a state of oxidative stress in various compartments of the testes. However, the molecular mechanism(s) of action of the environmental toxicants on the testis have yet to be elucidated. This review discusses the effects of some of the more commonly used environmental contaminants on testicular function through the induction of oxidative stress and apoptosis.