A study on the mechanism of the spermatogenic damage after vasectomy in rats

PURPOSE: Vasectomy may result in damage to spermatogenesis. There are several explanations for this damage, including an increase of pressure in the seminiferous tubules and an autoimmune reaction. Recently, vasectomy has been reported to induce germ cell death by apoptosis. However, the exact mechanism of this vasectomy-induced germ cell apoptosis is unclear. To elucidate this mechanism, we designed a vasectomized rat model and examined the testiscular alterations and apoptotic degeneration biochemically and microhistopathologically. Particularly, we analyzed the expression of nitric oxide synthase (NOS) and nuclear factor kappa B (NF kappa B), which plays a critical role in the induction of the iNOS gene, in the testis after vasectomy to gain insight into the association between germ cell apoptosis and these factors. MATERIALS AND METHODS: The testes of 40 Wistar rats (10-weeks old) were studied at 1, 2, 5 and 10 weeks after unilateral (left) vasectomy. Wistar rats weighting 290 to 310 g were divided into 2 groups and subjected to underwent either unilateral vasectomy or sham surgery under ether anesthesia. Bilateral testes were carefully observed biochemically and histopathologically. Apoptosis was detected by an in situ end-labeling technique (detection of cellular DNA fragmentation) and electron microscopy. Neuronal NOS (nNOS), endothelial NOS (eNOS) and inducible NOS (iNOS) protein was detected by Western blotting and immunohistochemical studies using each NOS monoclonal antibody. To confirm the co-localization of cellular DNA fragmentation in germ cells and each NOS, each set of consecutive testis sections (one stained for cellular DNA fragmentation and the others for each NOS) were examined. Expression of NF kappa B proteins was examined immunohistochemically using a NF kappa B p65 polyclonal antibody. RESULTS: At 5 and 10 weeks after vasectomy, the vasectomized left testis was significantly lighter than the unvasectomized right testis and sham-operated testis. At that time, the seminiferous tubules of vasectomized testes were highly damaged, presenting narrow tubular diameter, disorder of cellular arrangement, depletion of the germ cells, and local interstitial fibrosis. Vasectomized testes demonstrated a significantly increased number of apoptotic germ cells per cross-sectional area compared with sham-operated testes at 5 and 10 weeks after operation (p < 0.01). Electron microscopy revealed apoptotic germ cells each with a darkly stained nucleus. Western blotting and immunohistochemical studies demonstrated that iNOS proteins were more strongly expressed on vasectomized testes as time passed after vasectomy. Examination of consecutive sections from the vasectomized testis revealed that visibly apoptotic germ cells that exhibited positive staining for cellular DNA fragmentation were also intensely stained for eNOS and iNOS. NF kappa B p65 proteins were more strongly expressed in the nucleus of germ cells in the vasectomized testis than in the sham-operated testis. CONCLUSIONS: We found that vasectomy results in damage to spermatogenesis in adult rats, that may induce germ cell apoptosis, and that iNOS and NF kappa B may play a critical role in the germ cell apoptosis after vasectomy.     

Morphological alterations in small intestine of rats with myenteric plexus denervation

We aimed to investigate the effect of myenteric denervation by benzalkonium chloride (BAC) on small intestine morphology in the rat, and whether segmental myenteric denervation alters morphology elsewhere in the small intestine. Forty male Sprague-Dawley rats were equally divided into 4 groups: control (0.9% NaCl); denervation (0.062% BAC); chemical inflammation (5% acetic acid), and intraluminal stasis produced by partial obstruction. 28 days after operation tissue samples were taken from the treated segment, 10 cm distal to the treated segment, and 20 cm proximal to the treated segment. Morphological changes and the number of ganglion cells were examined under the light microscope. BAC application reduced the number of myenteric neurons by 85% in the treated segment. Denervation increased villus height and crypt depth in the treated and proximal segments. But changes in muscle thickness were seen throughout the intestine. As a result, although myenteric plexus denervation caused mucosa morphology in the treated and proximal segments, it caused smooth muscle changes throughout the small intestine.     

Effects of hypophysectomy and alterations in spermatogenic function on Leydig cell volume, number, and proliferation in adult rats

The two major objectives of this study were to determine (i) whether the pituitary is required to maintain Leydig cell number per testis, and (ii) whether alterations in spermatogenic function in the absence of LH can affect Leydig cell volume, number, and 3H-thymidine incorporation in vivo. Four experimental treatments tested the combinations of two factors: (i) the intact pituitary vs hypophysectomy (Hypox); and (ii) arrested vs active spermatogenesis. Subdermal Silastic capsules were used to deliver a low dosage of estradiol in addition to a low dosage of testosterone (TE), which arrested spermatogenesis, or a high dosage of testosterone (HTE) which maintained active spermatogenesis. All four treatments (TE, HTE, Hypox and Hypox-HTE) inhibited LH secretion for 16 weeks. Control rats were sham hypophysectomized. Leydig cell volume per testis and the volume of an average Leydig cell decreased 70-85% (P less than 0.01 vs controls) in all treated rats, whether deprived of LH (TE) or of all pituitary secretions (Hypox), and whether spermatogenesis was arrested (TE, Hypox) or maintained by exogenous testosterone (HTE, Hypox-HTE). This result suggested that LH was the only factor required to maintain Leydig cell volume, since the absence of other pituitary factors or alterations in spermatogenic function could not override or modify the effect of LH deprivation. No significant differences were found in Leydig cell number per testis or the proportion of Leydig cells labeled with 3H-thymidine among control and experimentally treated rats. In contrast to Leydig cell volume, which depended on LH, Leydig cell number and Leydig cell division were maintained at control values in the absence of pituitary factors and spermatogenic function for 16 weeks.     

Synergistic action of inflammation and lipid dysmetabolism on kidney damage in rats

In kidney disease, inflammation and lipid dysmetabolism are often associated together, however, the effect and mechanism of inflammatory mediators and lipid dysmetabolism on kidney damage is still unclear. In this study, Wistar rats were randomized into four groups: normal diet?+?saline (Group N), high-fat diet (HF)+?saline (Group HF), normal diet?+?adriamycin (Group ADR), HF?+?adriamycin (Group ADR?+?HF). After 10?weeks of feeding, rats in each group were randomly sacrificed. We found that the protein content of urine in ADR and ADR?+?HF groups were significantly higher than that of group N and HF while the serum levels of total protein and albumin in the ADR and ADR?+?HF groups decreased correspondingly. The serum levels of triglyceride, total cholesterol and low-density lipoprotein in the HF, ADR and ADR?+?HF groups increased. In the treatment groups, mesangial proliferation, matrix accumulation, tubular vacuolization, inflammatory cell infiltration and fat deposition were detected. These pathological changes were the most serious in the ADR?+?HF group. The expression of tumor necrosis factor-α (TNF-α) and transforming growth factor-β1 (TGF-β1) were increased in each treatment group, especially in the ADR?+?HF group. Our results suggested that the inflammatory factors and abnormal lipid levels can activate the inflammatory response in kidney of the Wistar rats, and lead to a series of pathological changes in renal tissue, and inflammatory factors and lipid dysmetabolism can aggravate damage in the kidney.     

长期给予睾酮致大鼠生精小管伴有生精细胞排列疏松

目的:确定睾酮致大鼠睾丸内睾酮抑制因而精子发生障碍是否伴有生精细胞排列疏松。方法:成年雄性SD大鼠肌注十一酸睾酮[19 mg/(kg.15 d)]130 d后取睾丸组织块,作甲基丙烯酸树脂包埋切片,观察睾丸的组织学变化。结果:除精子形成、精子释放障碍等改变以外,11.5%的生精小管轮廓内生精细胞的排列明显较疏松,成串、成束或成团的生精细胞(主要是精母细胞和精子细胞)之间出现朝向小管腔走行的放射状裂隙。结论:生精细胞排列疏松是大鼠睾丸内睾酮抑制所致重要组织学改变之一。     

环磷酰胺对雄性大鼠生精功能的影响

目的 观察环磷酰胺对大鼠生精功能的影响.方法 8周龄成年雄性sD大鼠,分6组,每组16只.前5组为实验组,给药方法分别为每日10、20、40、80、100 mg/kg,另1组为对照组.胃管给药2周和4周后,处死大鼠,取附睾精子行CASA检查;取睾丸组织苏木素.伊红(HE)染色分析睾丸曲细精管结构变化,TUNEL法检测睾丸曲细精管细胞凋亡情况.留取血清标本采用电化学发光法检测性激索水平.结果 环磷酰胺每日40 mg/kg喂养4周,大鼠存活率93.8%,精子数量明显减少(29.36±8.64)X 106个/ml,精子活力降低(22.25±2.03)%,睾丸生精上皮细胞明显损伤变性(58.1±1.2)%,血清睾酮水平下降(0.149±0.020)μg/L.这些指标的变化与环磷酰胺给药剂量及时间旱负相关(P<0.05).结论 通过环磷酰胺诱导可以成功建立大鼠少精/无精症动物模型,环磷酰胺主要通过坏死和凋亡两种途径导致睾丸曲细精管结构变化,导致生精细胞减少并最终引起少精/无精症.     

Co‐administration of ginseng and ciprofloxacin ameliorates epididymo‐orchitis induced alterations in sperm quality and spermatogenic cells apoptosis following infection in rats

Korean red ginseng (KRG) may be a beneficial adjuvant along with ciprofloxacin to ameliorate devastating effects of epididymo‐orchitis (EO) on male fertility. This study intends to assay the effects of KRG and ciprofloxacin on sperm quality and spermatogenic cells apoptosis in EO rats. We divided 54 adult rats into nine groups (n = 6 rats per group): control (CO), sham‐operated (SH), EO (E); ciprofloxacin (C), EO–ciprofloxacin (EC), KRG (G), EO–KRG (EG), ciprofloxacin–KRG (CG) and EO–ciprofloxacin–KRG (ECG). We administered ciprofloxacin and KRG 48 hr after the Escherichia coli (E. coli) injection for 10 days. Bilateral orchiectomy was performed after one sperm cycle (14 days) following the last treatment with ciprofloxacin and KRG. Total and progressive motility of E, C and EC groups decreased. However, motility is improved in CG and ECG in comparison with these groups. The E group induced negative changes in the architecture of testes tissue and dramatic increase in apoptosis indices. Interestingly, co‐administration of ciprofloxacin and KRG has dramatically improved Miller's and Johnsen's scores and decreased the apoptosis indices of animals in the ECG group. Combined treatment of ciprofloxacin and KRG may improve the quality of spermatozoa and attenuated apoptosis indices in the ECG group.     

精索静脉曲张大鼠睾丸自噬对生精细胞的影响

目的:研究精索静脉曲张(VC)大鼠模型中睾丸不同水平自噬对生精细胞凋亡的影响。方法:雄性SD大鼠54只随机分为6组:空白对照组、雷帕霉素对照组、氯喹对照组各6只,VC组、VC+雷帕霉素组、VC+氯喹组各12只。采用HE染色观察睾丸、附睾组织形态学变化,并对睾丸及附睾中精子形成情况进行评分,TUNEL染色检测生精细胞凋亡指数(AI),Western印迹检测LC3、p62、Bax、Bcl-2的表达。结果:空白对照组、雷帕霉素对照组、氯喹对照组大鼠睾丸及附睾组织均未发生明显形态学变化,精子形成情况评分及AI亦无显著差异(P>0.05);VC组大鼠睾丸及附睾组织发生明显病理损伤,精子形成情况评分显著降低(P<0.01),AI显著升高(P<0.01),但VC+雷帕霉素组较VC组明显改善,而VC+氯喹组较VC组轻度加重。此外,与空白对照组比较,VC组自噬相关蛋白LC3(包括LC3-Ⅱ/LC3-Ⅰ的比值)和促凋亡蛋白Bax表达显著增加(P<0.01),抑凋亡蛋白Bcl-2表达则显著降低(P<0.01);且VC+雷帕霉素组LC3与Bcl-2表达显著高于VC组(P<0.01),p62和Bax表达则显著低于VC组(P<0.01);而VC+氯喹组LC3与Bcl-2表达显著低于VC组(P<0.01),p62和Bax的表达则显著高于VC组(P<0.01)。结论:VC可诱导大鼠睾丸自噬和生精细胞凋亡,上调自噬可抑制生精细胞凋亡,阻滞自噬则可促进生精细胞凋亡。     

Sex hormone-induced alterations in the activities of antioxidant enzymes and lipid peroxidation status in the prostate of Noble rats

BACKGROUND: Oxidative stress has been implicated in prostate carcinogenesis. In the Noble rat model, treatment of rats with testosterone (T) plus 17beta-estradiol (E(2)) induced dysplasia and adenocarcinoma. Previous reports from us and other have indicated a linkage between steroid hormones and oxidative status in prostate cells in vivo and in vitro. Here, we provide further evidence that androgens and estrogens could induce a lobe-specific shift of prooxidant-antioxidant balance and alterations in antioxidant enzyme activities, leading to oxidative stress in the rat prostate gland in vivo. METHODS: Male Noble rats were subjected to single (T, E(2), or diethylstilbestrol [DES] alone) or combined (T + E(2) or T + DES) hormone treatments. Lipid peroxidation status and antioxidant enzyme activities of superoxide dismutase (SOD), catalase, glutathione peroxidase (GPx), glutathione reductase (GR), glucose-6-phosphate dehydrogenase (G6PD) were assayed spectrophotometrically. RESULTS: Treatment of rats with T did not alter lipid peroxidation in either the ventral prostate (VP) or the dorsolateral prostate (DLP). In contrast, exposure of rats to DES or E(2) modestly elevated lipid peroxidation in rat VP or DLP, respectively. Of importance, T + DES and T + E(2) treatments of rats induced marked increases in lipid peroxidation in the VP and the DLP, respectively. Antioxidant enzyme activities in the VP and DLP exhibited differential responses to sex hormone challenges. The activities of catalase, GPx, GR, and G6PD were mostly suppressed in either single or dual hormone-treated DLP, whereas there is a general increase of GR and G6PD activities in the VP after hormonal exposures. The changes in T + DES-treated VP were most dramatic with a marked activation of GPx (by one-fold), GR (by one-fold), and G6PD (by five-fold). CONCLUSION: The lobe-specific differential responses of hormone-induced oxidative stress and modulations of antioxidant enzymatic defenses in the rat prostate suggest that reactive oxygen species may play a role in hormone-induced prostate carcinogenesis.     

Association of intratubular seminoma and intratubular embryonal carcinoma with invasive testicular germ cell tumors

The classification of intratubular germ cell neoplasia of the testis includes an unclassified type (IGCNU), in addition to various other intratubular lesions that show specific forms of differentiation, such as intratubular seminoma and intratubular embryonal carcinoma. Although IGCNU is recognized as a precursor lesion for testicular germ cell tumors, the relationship between differentiated types of intratubular germ cell neoplasia and invasive germ cell tumors of the testis is not well established. The aim of the present study was to examine the association between invasive testicular germ cell tumors and intratubular neoplastic lesions, with particular emphasis on differentiated types of intratubular germ cell neoplasia. The seminiferous tubules adjacent to 42 testicular germ cell tumors were evaluated for the presence of various forms of intratubular germ cell neoplasia. IGCNU was observed in 37 (88%) of 42 cases, whereas intratubular seminoma and intratubular embryonal carcinoma were seen in 19% and 7% of the cases, respectively. Intratubular seminoma was associated primarily with seminomas or mixed germ cell tumors with a seminomatous component, but was also present in a case of a nonseminomatous germ cell tumor. Intratubular embryonal carcinoma was associated exclusively with nonseminomatous germ cell tumors. All cases of intratubular embryonal carcinoma were identified morphologically and exhibited histologic features corresponding to traditional definitions of this lesion. No examples of intratubular embryonal carcinoma as defined by CD30 expression alone in the absence of an intratubular proliferation were observed. The presence of intratubular seminoma in a nonseminomatous germ cell tumor suggests that it is a true preinvasive lesion rather than a manifestation of intratubular spread of an established invasive seminoma. The low incidence of intratubular embryonal carcinoma supports the theory that most nonseminomatous germ cell tumors evolve initially as seminomas, rather than directly from a differentiated intratubular neoplastic lesion.     

Skeletal alterations in ovariectomized rats

Summary Female Sprague Dawley rats were subjected to either bilateral ovariectomy or sham surgery. Tetracycline derivatives were administered to each rat on two separate occasions to label sites of bone formation. All rats were sacrificed at 5 weeks postovariectomy and their proximal tibiae were processed undecalcified for quantitative bone histomorphometry. A twofold decrease in trabecular bone volume was noted in the proximal tibial metaphysis of ovariectomized rats. This bone loss was associated with elevated histomorphometric indices of bone resorption and formation. Ovariectomy increased osteoclast surface and numbers as well as osteoblast surface and numbers. Elevations in calcification rate and fractional trabecular bone surface with double tetracycline labels also suggest that bone formation was stimulated in ovariectomized rats. In addition, ovariectomized rats exhibited a greater rate of longitudinal bone growth relative to sham-operated control rats. These histomorphometric data indicate that ovariectomy induces marked bone loss and accelerated skeletal metabolism in rats.     

铅致大鼠睾丸细胞凋亡的实验研究及临床意义

目的探讨铅致大鼠睾丸生精细胞凋亡及其机制。方法40只成年雄性Wistar大鼠随机均分为4组:对照组,低、中、高剂量实验组。实验组分别腹腔注射醋酸铅5、10、20mg/kg体重,对照组注射等体积生理盐水,3周后手术取睾丸,用原位末端脱氧核苷酸转移酶标记(Tunel法)及免疫组化技术检测睾丸组织生精细胞凋亡及凋亡相关蛋白Bcl-2、Bax的表达。结果3周后,中、高剂量组细胞凋亡及Bax基因表达较对照组差异有显著性(P<0.01),低剂量组较对照组无显著性。3个实验组的Bcl-2基因表达较对照组差异有显著性,且各组间亦有显著性(P<0.01)。结论铅负荷至一定程度时可致大鼠生精细胞凋亡,且呈一定量效关系,这种作用可能与Bcl-2基因低表达和Bax基因高表达有关。     

Effect of local testicular heating on spermatogenic cell apoptosis in rats

Aim: To study the effect of testicular local heating on spermatoge-nic cell apoptosis in rats. Methods: Seventy male SD rats were divided into the heat-treated and the control groups. The former was exposed to heat (43 ℃) for 12 hours. Each group was further divided into seven subgroups with respect to the time of observation after heat exposure, i.e., 12 h and 1 days, 3 days, 6 days, 10 days, 50 days and 80 days, respectively. In each subgroup, sper-matogenic cell apoptosis was examined by means of electron microscopy, flow cytometry and terminal deoxynucleotidyl trans-ferase-mediated dUDP-nick end labeling (TUNEL) methods. Results: The percentage of cells with sub-haploid and the percentage of positive TUNEL cells were significantly higher in the heat-treated groups than in the controls (P<0.01). The reaction of cell apoptosis to local heat was highly selective: spermatocytes were the most sensitive, followed by spermatids, spermatozoa and sper-matogonia in a decreasing order. Conclusion: Local testic     

Morphological alterations in dental and periodontal tissues in murine mucopolysaccharidosis type VII

Mucopolysaccharidoses (MPSs) in humans are frequently associated with tooth and periodontal aberrations. Although the cause is known, namely, enzyme deficiency, the pathophysiology of these alterations is not well defined. A murine MPS VII (-glucuronidase deficiency) model has earlier been identified with morphological, genetic, and biochemical characteristics that closely mimic those of human MPS VII. The present investigation describes the histopathological alterations in dental and periodontal tissues from such mutant mice. Homozygous animals were identified by external phenotypical features and as being -glucuronidase deficient by a fluorometric assay of liver samples. In the incisor and the periodontium, abnormalities were evident in both cells and the extracellular matrices. Mesenchyme-derived cells were more aberrant than epithelial cells. Moreover, undifferentiated cells appeared unaffected, whereas actively synthesizing and resorbing cells were distended by virtually empty or granular material-containing vacuoles, the content presumably being glycosaminoglycans. The cells most affected were those in which macromolecular turnover is normally the highest, namely, odontoblasts, postsecretory ameloblasts, and periodontal ligament fibroblasts. Extracellularly, predentin displayed abnormal collagen fibrils, whereas mineralization defects occurred in both dentin and enamel. This murine model of MPS VII provides a good tool for understanding the pathophysiology of this disease in bone, periodontium, and teeth.     

Morphological alterations in dentine after mechanical treatment and ultrashort pulse laser irradiation

The aim of this study was to evaluate and compare the morphological changes that occur in dentine after femtosecond laser irradiation and after mechanical treatment. The duration of the laser pulse is an important parameter, because within the time frame of the pulse heat diffusion plays a very important role in the mechanism of interaction between the light and the tissue. Six totally impacted human third molars were sectioned into sheets approximately 1 mm thick with an Accutom-50 precision cutting machine. The samples were randomly divided into two groups according to their cavity preparation: mechanical cavity preparation and laser cavity preparation. The samples were then examined by light microscopy and scanning electron microscopy. There were clear differences in the results obtained with the two techniques. Cavities prepared with the laser with pulses of <1 ps showed no microcracks, and the treated surface displayed a rough and irregular aspect with no smear layer and exhibited open dentinal tubules. On the contrary, cavities made with a rotatory instrument had a smooth surface and microcracks, a broad area of carbonization and merging, occluded dentinal tubules and a smear layer. This study showed that human dentine can be successfully ablated with the ultrashort pulse laser.     

Study on ethane dimethane sulphonate (EDS)-induced spermatogenic damage and protective drugs against this damage in the rat]

The effects of a single administration of ethane dimethane sulphonate (EDS), which has a direct cytotoxic effect on Leydig cells, was assessed for its spermatogenic damage and intratubular androgen level in SD male adult rats. The protective effect of human chorionic gonadotropins (hCG) (s.c.), testosterone propionate (TP) (s.c.) and intratesticular administration of testosterone microcrystal suspension (Tmcs) against the spermatogenic damage in rats EDS given was also evaluated. EDS caused a decrease of the seminiferous tubular diameter and impaired spermatogenesis remarkably; moreover, it also caused significant decreases in intratubular androgen levels. These results suggest that EDS-treated SD male adult rats may be suitable as a model for hormone dependent infertility. The administration of hCG and intratesticular Tmcs prevented tubular damage and increased the intratubular T level. On the other hand, the administration of TP prevented tubular damage while remarkably decreasing intratubular androgen level. In this connection, it was inferred that priming of rats with TP caused an increase in intratubular androgen binding protein, which would stimulate spermatogenesis. The fact that a single injection of Tmcs caused no tubular damage suggests that intratubular T level is one of the factors playing an important role in spermatogenesis and that an intratesticular injection of Tmcs may be useful for the treatment of some cases of idiopathic male infertility.     

青春期营养性肥胖大鼠睾丸生精细胞周期的变化

目的探讨青春期营养性肥胖大鼠的睾丸生精细胞周期的变化。方法80只新生的雄性清洁级SD大鼠随机分为两组,对照组(n=32只)喂普通饲料,肥胖组(n=48只)喂高脂饲料,分别于喂养后的第3、4、5、6周末观察喂养后体重,计算Lee’s指数,流式细胞分析术检测睾丸生精细胞周期的改变。结果与对照组比较,肥胖组大鼠第3周开始体重有显著性增加(P<0.05),6周内体重持续上升,至第6周末肥胖组大鼠体重超过对照组达26.6%(P<0.01),Lee’s指数随着大鼠肥胖程度的增加而逐渐增高;大鼠G0/G1期细胞在高脂饲料喂养第3周末增多(P<0.05)、肥胖组S期细胞显著下降(P<0.01),此时处于G2/M期细胞的百分数明显增多(P<0.05)。结论青春期时的肥胖可以引起睾丸生精细胞S期细胞百分数逐渐减少,出现G2期细胞阻滞,细胞有丝分裂延迟。