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     

单侧隐睾大鼠对侧睾丸的损害与Bcl-2和Bax基因表达

目的:探讨单侧隐睾大鼠对侧睾丸生精细胞凋亡与Bcl-2/Bax基因表达的关系。方法:20只健康SD雄性大鼠(22日龄)随机分成隐睾组和对照组,每组10只。通过手术建立单侧隐睾动物模型。术后90 d取对侧睾丸,采用原位缺口末端标记(TUNEL)法检测生精细胞凋亡,免疫组化SP法检测Bcl-2/Bax基因表达。结果:与对照组相比,隐睾组对侧睾丸生精细胞凋亡显著增多(P<0.01),重量显著减轻(P<0.01),Bax表达显著升高(P<0.01),Bcl-2表达显著降低(P<0.01)。凋亡细胞主要是初级精母细胞和圆形精子细胞。结论:单侧隐睾大鼠对侧睾丸的生精细胞凋亡增多与Bcl-2基因表达降低、Bax基因表达升高密切相关。细胞内Bc l-2/Bax比值是影响生精细胞凋亡的重要因素之一。     

诱生型一氧化氮合成酶基因在隐睾中的表达及意义

目的：探讨隐睾中诱生型一氧化氮合成酶（ｉＮＯＳ）基因表达与生殖细胞凋亡的关系。方法：选取日龄为２２ｄ的ＳＤ雄性大鼠２０只，建立单侧隐睾模型，用生物素－ｄＵＴＰ／酶标亲和素测定法检测睾丸生殖细胞凋亡，用免疫组织化学ＳＰ法检测ｉＮＯＳ基因表达。结果：隐睾生殖细胞凋亡指数在术后７ｄ内随时间的延长而增加；从术后第４天起，与对侧正常睾刃相比，隐睾发生凋亡的生殖细胞显著增加（Ｐ〈０．０１）；在正常的睾丸组织中     

一氧化氮合酶基因表达对实验性大鼠隐睾生殖细胞凋亡的影响

目的　研究诱生型一氧化氮合酶 (iNOS)及其mRNA表达与实验性大鼠隐睾生殖细胞发育、凋亡的关系。方法　 (1)采用SD雄性健康大鼠 16只 ,日龄 2 2天时复制单侧隐睾模型。 (2 )采用生物素 dUTP/酶标亲和素测定法检测睾丸生殖细胞凋亡。 (3)采用免疫组织化学方法检测大鼠睾丸生殖细胞中iNOS基因表达。 (4 )采用原位杂交法检测大鼠生殖细胞中iNOSmRNA的表达。结果　 (1)术后第 7天 ,与自身对侧正常睾丸相比 ,隐睾侧睾丸发生凋亡的生殖细胞数显著增加 (P <0 .0 1)。 (2 )单侧隐睾模型建立术后第 7天 ,在双侧睾丸的间质细胞、支持细胞和初级精母细胞中均可见iNOS蛋白及iNOSmRNA的弱阳性表达 ,在隐睾侧睾丸曲细精管中脱落的生殖细胞中可见iNOS蛋白及iNOSmRNA的强阳性表达。术后第 7天 ,与自身对侧正常睾丸相比 ,隐睾侧睾丸生殖细胞中iNOSmRNA表达显著增加 (P <0 .0 1)。结论　 (1)实验性大鼠隐睾可以导致睾丸生殖细胞凋亡增加。 (2 )iNOS蛋白及其mRNA的表达增加是隐睾生殖细胞凋亡增加的分子机制之一     

Bcl-2/Bax基因表达对隐睾生殖细胞凋亡的影响

为探讨Ｂｃｌ－２／Ｂａｘ基因表达对隐睾生殖细胞凋亡的影响。采用ＳＤ雄性大鼠日龄２２天时复制单侧隐睾模型;用生物素－ｄＴＵＰ／酣标亲和素测定法检测睾丸生殖细胞凋亡;用免疫组织化学ＳＰ法检测Ｂｃｌ－２／Ｂａｘ基因表达。结果发现术后第７天,与对侧正常睾丸相比,隐睾侧睾丸发生凋亡的生殖细胞显著增加（Ｐ〈０．０１）;Ｂｃｌ－２、Ｂａｘ表达均有显著差异（Ｐ〈０．０１）。提示手术诱导的隐睾生殖细胞凋亡增加;Ｂｃ     

Overexpression of endothelial nitric oxide synthase in transgenic mice accelerates testicular germ cell apoptosis induced by experimental cryptorchidism

Surgical induction of cryptorchidism in experimental animals causes testicular germ cell apoptosis and infertility. The mechanisms of germ cell apoptosis have been associated with oxidative stress or testicular exposure to elevated temperature. Nitric oxide (NO) has been associated with apoptosis in a number of cell types. The objective of this study was to investigate whether overexpression of endothelial NO synthase (eNOS) could accelerate apoptosis of germ cells in the testes of transgenic mice. There are 3 NOS isoforms, and we restricted the analysis to eNOS at this time. For the colocalization of eNOS, staining in degenerating germ cells that were apoptotic cells suggested that eNOS may be related to germ cell apoptosis. eNOS overexpression in the testes of eNOS transgenic (eNOS-Tg) mice was examined using Western blot analysis. Unilateral cryptorchidism was surgically induced in both eNOS-Tg and wild-type (WT) adult mice. The testes were evaluated 1, 3, 5, 7, and 14 days after the operation by weighing the testes and examining histopathologic features and cell apoptosis using in situ microscopic analysis of DNA fragmentation. Immunoblotting for eNOS protein demonstrated increases in eNOS protein expression in testes, as well as the lung and aorta. In eNOS-Tg mice, weight reduction of cryptorchid testis was significantly increased on days 3, 5, and 7 (P = .02, .02, and .04, respectively). The numbers of spermatocytes and spermatids of eNOS-Tg cryptorchid testis significantly decreased compared with those of WT cryptorchid testis from day 3 (spermatocytes: P = .04; spermatids: P = .02). Moreover, terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling demonstrated that eNOS-Tg mice significantly accelerate germ cell apoptotic changes induced by experimental cryptorchidism compared with WT mice from day 3 (P = .03). We have provided evidence that eNOS plays a functional role in mouse spermatogenesis in cryptorchidism-induced apoptosis.     

Reproductive aging in the Brown Norway rat is characterized by accelerated germ cell apoptosis and is not altered by luteinizing hormone replacement.

Reproductive aging in the male Brown Norway (BN) rat is characterized by decreased Leydig cell steroidogenesis associated with seminiferous tubule dysfunction. This could be a result of a combination of a primary testicular defect and a secondary hypothalamic pituitary dysfunction. In the present study, we determined in the BN rat whether germ cell loss occurred via apoptosis. We then defined the age of onset of Leydig cell dysfunction and germ cell loss and examined whether chronic luteinizing hormone (LH) replacement would delay or prevent reproductive aging. Plasma hormone levels, testicular sperm concentrations, and germ cell apoptosis were studied in 6, 9, 12, 15, 18, and 21-month-old BN rats. Beginning at 15 months, testicular weight, sperm concentration, total sperm counts, plasma testosterone, LH, and inhibin decreased, whereas the proportion of regressed testes and plasma follicle-stimulating hormone (FSH) levels increased with aging. Accelerated germ cell apoptosis involving spermatogonia, preleptotene and pachytene spermatocytes, and spermatids was evident in some tubules of the relatively normal testes from 21-month-old rats. In the regressed testes, complete cessation of spermatogenesis occurred. The apoptotic index was higher in the testes of old (21-month-old) rats in particular at stages XII-XIV when compared with younger animals. Chronic LH replacement (0.5 microg i.p. twice per day) administered to 15-month-old BN rats for 6 months did not alter plasma hormone levels, testes weight, sperm concentration or content, or the germ cell apoptotic index. In the control group, 3 out of 10 testes were regressed, whereas in the LH-replaced group, only 1 out of 12 testes was regressed. We show in this study that early reproductive aging in the BN rat began at around 15 months. Germ cell loss associated with aging occurs via apoptosis. Replacement therapy with LH for 6 months does not decrease or delay the testicular dysfunction associated with aging. It is unlikely that hypothalamic-pituitary dysregulation is the major cause of testicular aging.     

Cisplatin-induced germ cell apoptosis in mouse testes

The purpose of this study was to investigate whether exposure of male mice to cisplatin induces apoptosis in male germ cells and the possible role of apoptosis in cisplatin-induced testicular damage. Forty-eight male BALB/c mice were divided into cisplatin and control groups. The mice from the cisplatin group received a single intraperitoneal injection of cisplatin of either 1, 5, or 10 mg/kg. The control group received a single intraperitoneal injection of saline alone. The testes were removed on days 1, 3, and 7 after cisplatin administration, respectively. Following histological examination, apoptotic indices (AIs) were measured within seminiferous tubules of the mouse testes by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling assay. A low incidence of spontaneous apoptosis was observed in controls, particularly in spermatogonia and spermatocytes of the mouse testes. After cisplatin administration, both increased Als and decreased spermatozoa and spermatids were found in the seminiferous tubules of the mouse testes. Cisplatin-induced apoptosis was found in spermatogonia, spermatocytes, and spermatids of the mouse testes. In comparison to the control values, AIs increased 2.6- to 6.8-fold in cisplatin-treated mouse testes. AIs reached the highest level on day 1 following 1 mg/ kg, on day 3 following 5 mg/kg, and on day 7 following treatment of 10 mg/kg cisplatin. The study showed that cisplatin-induced germ cell apoptosis in the mouse testes was related to both the dose response and the time course of response. It is suggested that cisplatin-induced germ cell apoptosis may result in decreased spermatogenesis, and the higher dose of cisplatin may delay the occurrence of apoptosis in the mouse testes.     

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.     

血管内皮细胞生长因子和内皮型一氧化氮合酶在大鼠单侧隐睾对侧睾丸损害中的表达

目的 探讨大鼠隐睾中内皮型一氧化氮合酶(eNOS)和血管内皮细胞生长因子(VEGF)的表达与单侧隐睾对侧睾丸损害的关系.方法 将45只雄性SD大鼠(22 日龄)随机分成单侧隐睾并生殖股神经离断组(A组)、单侧隐睾组(B组)和假手术组(C组),每组15只.动物模型建立后(65日龄),苏木素-伊红(HE)染色观察睾丸生精上皮形态,原位缺口末端标记法(TUNEL)检测对侧睾丸生精细胞的凋亡,免疫组织化学和Western blot方法检测对侧睾丸组织中eNOS和VEGF基因表达的变化.结果 B组相对于A组和C组对侧睾丸组织中生精细胞的凋亡率最高(t1=3.04,t2=3.94,t1,t2＞t28,P＜0.01),Western blot和免疫组织化学方法检测结果也显示B组eNOS和VEGF的表达含量较A组和C组都显著升高,差异有统计学意义(P＜0.01),而A组和C组的各指标差异均无统计学意义(P＞0.05).结论 eNOS和VEGF的高表达和生殖股神经在单侧隐睾对侧睾丸的损害中起重要作用,而切断生殖股神经可以阻断这一损害过程.

Abstract：

Objective To study the relationship between the expression of endothelial nitric oxide synthase (eNOS) and vascular endothelial growth factor (VEGF) with the damage of contralateral testis of unilateral cryptorchidism in the experimental rats. Methods Forty-five immature male Sprague-Dawley rats (aged 22 days) were randomly divided into the group A (unilateral cryptorchidism and the ipsilateral genitofemoral nerve division), group B (unilateral cryptorchidism), group C (sham operation), n = 15 in each group. When the rats were aged 65 days, all the rats were sacrificed and the testes were obtained. The morphological changes of the spermatogenic epithelium in the testes were observed, and the germ cell apoptosis was detected by the TdT-mediated dUTP-biotin nick end labeling (TUNEL) technique. The expression of eNOS and VEGF was detected by using Western blotting and immunohistochemistry in the testicular tissues.Results The germ cell apoptosis was increased significantly ( t1 = 3. 04, t2 = 3. 94, t1 ,t2 ＞ t28 , P ＜0. 01) , and the levels of eNOS and VEGF in the contralateral testes were also increased obviously in group B as compared with groups A and C ( P ＜ 0. 01) , but the entire indexes in groups A and C had no significant difference. Conclusion eNOS, VEGF and genitofemoral nerve play a important role in the damage of the contralateral testes of unilateral cryptorchidism, and the damage can be prevented by genitofemoral nerve division.     

A new experimental model for cryptorchidism: inguinoscrotal approach

We investigated the effect of inguinal canal closure as a new mechanically induced cryptorchid rat model. The effectiveness of this new model was evaluated by histopathological examination. Thirty-one 21-day-old Wistar rats were divided into four groups. In groups 1 (n=6), 2 (n=6) and 3 (n=7), unilateral undescended testis was created by performing inguinal canal closure with inguinoscrotal approach. Sham-operated rats were used as controls in group 4 (n=12). The rats were killed on day 30 after surgery in group 1, day 45 in group 2 and day 60 in group 3. The seminiferous tubular diameter, number of tubules with mature germ cell and Leydig cell clusters were evaluated. None of the rats were lost during the study period. Signs of infection were not detected in operation site although antibiotics were not used. Overall only three (16%) testes descended into scrotum in study groups. The operation time was 3–4 min for each rat. Histopathological examination revealed detrimental effects of cryptorchidism on testicular growth in study groups. In all groups, except the sham group, the mean tubular diameter and the number of tubules with mature germ cells in the left testicle were significantly decreased compared to the right ones. Our findings were in correlation with other experimental studies using different rat models of cryptorchidism. This new model of cryptorchidism is considered to provide a simple and effective technique for investigating the impaired development of the testes in cryptorchidism. Received: 26 September 2000 / Accepted: 27 December 2000     

一氧化氮、总抗氧化能力对大鼠隐睾生殖细胞凋亡的影响

目的 :探讨一氧化氮 (nitricoxide,NO)、总抗氧化能力 (totalantioxidecapacity ,T AOC)对大鼠隐睾生殖细胞凋亡的影响。　方法 :健康SD雄性大鼠 2 0只 ,日龄 2 2d时复制单侧隐睾模型。生物素 dUTP/酶标亲和素测定法检测睾丸生殖细胞凋亡 ,采用硝酸还原酶法测定睾丸组织中NO含量 ,化学比色法测定睾丸组织中T AOC。　结果 :术后第 7d ,与对侧正常睾丸相比 ,隐睾侧睾丸发生凋亡的生殖细胞数显著增加 (P <0 .0 1) ;T AOC显著下降 ;NO含量显著上升 (P <0 .0 1)。　结论 :实验性大鼠隐睾可导致睾丸生殖细胞凋亡增加 ,且与睾丸组织中NO升高及T AOC下降密切相关     

Influence of a leptin deficiency on testicular morphology, germ cell apoptosis, and expression levels of apoptosis-related genes in the mouse

Leptin-deficient (ob/ob) male mice are morbidly obese and exhibit impaired reproductive function. The objective of this study was to assess the effect of a leptin deficiency on testicular morphology, germ cell development, apoptotic activity within germ cells, and expression levels of apoptosis-related genes in the testis. Sixteen week-old ob/ob male mice (n = 8) and controls (n = 8) were killed, and their reproductive organs were weighed. Testes were processed for either histomorphological analysis (hematoxylin and eosin [H&E] staining), germ cell apoptosis assessment (deoxy-UTP-digoxigenin nick end labeling [TUNEL] method), or apoptosis-related gene expression analysis (microarray). Cross sections of the testes of leptin-deficient animals showed reduced seminiferous tubule area, fewer pachytene spermatocytes, and fewer tubules exhibiting elongated spermatids/mature spermatozoa. Condensation of germ cell nuclei and Sertoli cell vacuolization were evident in the testes of some ob/ob animals. Overall there was an elevation of apoptotic activity in the germ cells of ob/ob mice, particularly within the pachytene spermatocytes. With microarray technology, we identified 9 proapoptosis-related genes that were expressed at a significantly higher level in the testes of ob/ob mice than in the testes of the controls. Among these were members of the tumor necrosis factor receptor super family 1A and 5 (TNFR1 and 5) and peptidoglycan recognition proteins (associated with the extrinsic apoptotic pathway), and granzymes A and B, growth arrest and DNA damage inducible 45 gamma, sphingosine phosphate lyase 1, and caspase 9 (associated with the intrinsic apoptotic pathway). The results of the current study show that a leptin deficiency in mice is associated with impaired spermatogenesis, increased germ cell apoptosis, and up-regulated expression of proapoptotic genes within the testes.     

大鼠睾丸扭转后生殖细胞凋亡与Bcl-2和Bax基因表达

目的 :研究睾丸扭转复位后生殖细胞凋亡与Bcl 2 /Bax基因表达的关系。　方法 :30只成年健康SD雄性大鼠随机分成扭转组 (n =15 )和对照组 (n =15 )。建立单侧睾丸扭转复位动物模型 (72 0° 2h)。术后 3d取手术侧睾丸 ,采用原位缺口末端标记法 (TUNEL)检测生殖细胞凋亡 ,免疫组化SP法检测Bcl 2 /Bax基因表达。　结果 :与对照组相比 ,扭转组手术侧睾丸生殖细胞凋亡显著增多 (P <0 .0 1) ,Bax表达显著升高 (P <0 .0 1) ,Bcl 2表达显著降低 (P <0 .0 1)。凋亡细胞主要是初级精母细胞和圆形精子细胞。　结论 :睾丸扭转复位后 ,生殖细胞凋亡与Bcl 2 /Bax基因表达密切相关。细胞内Bcl 2 /Bax比值是影响生殖细胞凋亡的重要因素之一。     

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     

The effect of varicocele repair on experimental varicocele-induced testicular germ cell apoptosis

The purpose of this study was to evaluate the variance of apoptosis in rats in which experimental varicocele was induced and then treated by varicocelectomy. Forty adult male Wistar albino rats were used in this experimental study. Experimental varicocele was created in 30 rats. A total of 5 rats underwent a sham operation, and the remaining 5 rats were the control group. A total of 5 rats from the varicocele group were sacrificed on the 14th postoperative day, and 5 more were sacrificed on the 28th postoperative day to document the level of apoptosis due to varicocele. Varicocelectomy was performed on 20 rats with varicocele on the 14th postoperative day. These 20 rats were divided into 4 groups to evaluate the level of apoptosis in their testis after varicocelectomy. They were sacrificed on days 7, 14, 21, and 28 after varicocelectomy. The testes were fixated by perfusion with 10% formaldehyde and then placed in paraffin blocks. From each testis, 2 samples were stained with hematoxylin and eosin, and 2 samples were stained using the TUNEL method. In each specimen, apoptotic germ cells stained by TUNEL were counted in the cross section of 100 seminiferous tubules. The apoptotic index was defined by calculating the number of apoptotic cells per seminiferous tubule. Apoptotic index = total apoptotic germ cell count/100. In the adult rats on which experimental varicocele was performed, both in the second and fourth week, apoptosis in both left and right testes were significantly higher compared with the control group (with varicocele day 14: 0.25-0.26, with varicocele day 28: 0.28-0.32, control: 0.11-0.13). After varicocelectomy on the 7th and 14th days, the slight increase in the level of apoptosis continued (day 7 left testis: 0.30, day 7 right testis: 0.28; day 14 left testis: 0.25, day 14 right testis: 0.31). After varicocelectomy, apoptosis decreased significantly on day 21 (left testis: 0.16, right testis: 0,22), and on day 28 it was almost equal to the level of the control group (left testis: 0.14, right testis: 0.16). After the creation of unilateral varicocele, the level of apoptosis increased in both the left and right testes. Apoptosis in both testes decreased after surgical treatment.     

COCAINE INDUCED APOPTOSIS IN RAT TESTES

PURPOSE: Exposure of rats to chronic cocaine results in disruption of spermatogenesis including reduction of germ cells. However, the cellular mechanism responsible for the testicular damage in testes is still unknown. We have studied the role of apoptosis in cocaine induced testicular damage. MATERIALS AND METHODS: Thirty-day-old male Sprague-Dawley rats were given cocaine hydrochloride (15 mg./kg. body weight) subcutaneously daily for 90 days. Control animals received equal volumes of normal saline daily for 90 days. Testes were removed at 15, 30, 60, and 90 days of cocaine administration. In situ detection of germ cells with DNA strand breaks in paraffin-embedded testicular section (5 microm.) was achieved by the terminal deoxynucleotidyl transferase (TdT)-mediated dUTP in situ nick end-labeling (TUNEL) method. DNA fragmentation was also determined by gel electrophoresis. RESULTS: Apoptotic cells were found in the spermatocytes and spermatogonia of germinal epithelium. Less than 7% of seminiferous tubule cross sections showed a high level of apoptosis (> or =3 apoptotic cells per tubule) in control animals compared with experimental group where 25% of the tubules showed a high level of apoptosis (p<0.05). The number of apoptotic cells was significantly increased by 15 days, peaked at 30 days and persisted up to 90 days of cocaine exposure when compared with controls (p<0.05). DNA isolated from the cocaine treated testes displayed a clear ladder pattern whereas the DNA from controls did not. CONCLUSIONS: The experimental results presented here suggest that cocaine exposure leads to significant apoptosis in rat testes and the mechanism of cocaine induced testicular injury may be related to the induction of apoptosis.     

Altered distribution of Sertoli cell vimentin and increased apoptosis in cryptorchid rats

Background/Purpose: Sertoli cell cytoskeletons play an important role in the process of spermatogenesis. The authors investigated the effects of cryptorchidism in immature rats on the distribution of Sertoli cell cytoskeletons and the incidence of testis germ cell apoptosis. Methods: Immature 3-week-old rats were made cryptorchid unilaterally, and the distribution of Sertoli cell cytoskeletons by immunohistochemistry and germ cell apoptosis by TUNEL technique were evaluated at different day intervals. Results: Immunohistochemical staining from control rats showed that the distribution of vimentin in Sertoli cells changes in developing rats. Basally, it is localized in the perinuclear region of the cell as well as in filament networks that project into the apical Sertoli cell cytoplasm in a [ldquo ]spokelike[rdquo ] pattern. In cryptorchidism, there was an intense vimentin immunoreactivity surrounding Sertoli cell nuclei along with the collapse of the apical extensions. This became significantly evident by 7 days. Microtubules occur in Sertoli cell cytoplasm apical to the nucleus. Cryptorchidism did not alter their distribution. Spontaneous apoptosis of germ cells occurs in the control testis, and a markedly increased incidence of apoptosis was observed in cryptorchidism by 7 days with decreased testis weight. Conclusions: Taken together, the authors propose that Sertoli cells are affected in cryptorchidism, and altered distribution of Sertoli cells vimentin filaments correlates with the increased germ cell apoptosis. Sertoli cell vimentin filaments are important for maintaining the structural integrity of the seminiferous epithelium.     

Immunohistochemical study of glutathione S-transferase in normal and experimental cryptorchid testes rats]

Using the antibody for glutathione S-transferase (GST) purified from human kidney, normal testes and experimental cryptorchid testes from newborn to 20-week-old rats were immunohistochemically stained by the peroxidase antiperoxidase (PAP) method. The cryptorchidism was surgically created at 1 week of age. The localization of GST was particularly examined by light microscopy, and the amount of Leydig cells was measured by a stereological method. 1. Leydig cells in the normal and cryptorchid testes showed strong GST activity at all ages. The amount of these cells in normal testes increased from 4 to 8 weeks of age and then slightly decreased, whereas in cryptorchid testes it was significantly larger than in the normal testes at 20 weeks of age, indicating hyperplasia of Leydig cells. 2. In the normal and cryptorchid testes, degenerating primary spermatocytes with GST activity appeared in the seminiferous tubules at 2 to 4 weeks of age. In the cryptorchid testis, degenerating germ cells with GST activity were also found in the regressing seminiferous tubules after 4 weeks of age. It is possible that GST acts as a detoxification system in the degenerating germ cells. 3. The PAP staining of GST in the rat testes is considered to be useful method for evaluating metabolic function of the spermatogenic cells and the distribution and amount of Leydig cells. 4. Experimental cryptorchidism showed that germ cells become sensitive after 4 weeks of age.     

Evolution of somatic and germ cell populations after busulfan treatment in utero or neonatal cryptorchidism in the rat

Summary.  In order to elucidate the respective effects of depletion of germ cells and of increase in testicular temperature, rats of the same Wistar strain were rendered experimentally bicryptorchid or sterilized by a busulfan injection in utero and compared to control animals. In both models, germ cells were depleted but numeric evolution and functions of somatic cells differed. The aim of that work was to compare the numeric evolutions of testicular somatic and germ cells to their respective functions in each model before puberty and in adult rats of the same strain. Serum concentrations of FSH, LH and testosterone were compared at 20, 40 and 110 days of age. Histological analyses of Sertoli and germ cells in the seminiferous tubules and of Leydig cells in the intertubular tissue were performed before puberty and at adulthood. Testosterone serum concentrations were depleted in both models starting at 40 days of age and more in busulfan-treated rats. Both FSH and LH levels were increased from 20 days onwards in experimental rats. Additional cryptorchidism in busulfan-treated rats depressed the serum testosterone concentration. At 17 days of age, the cryptorchidism do not modify somatic or germ cell populations while busulfan treatment has induced a decrease of both these populations. Conversely, the cross sectional area of the somatic testicular cells was not affected whatever the treatment. In adult testes of busulfan-treated and cryptorchid rats, the total numbers of Sertoli and Leydig cells and of germ cells per testis were decreased. The cellular size of the perivascular Leydig cells was not modified by any of the treatments whereas the size of the Sertoli cells was reduced.
In conclusion, in both models the absence of germ cells induces a decrease in Sertoli cell function, while the increase in testicular temperature provokes degeneracies of Sertoli and germ cells in the seminiferous tubules of the rat.