DNA replication and germ cell apoptosis during spermatogenesis in the cat

Stages at which DNA synthesis and germ cell death take place have recently been found to be equivalent in rabbits and rats. Preservation of the timing of these processes in different orders of mammals indicates that this timing may be crucial for testis cell biology. Since there is no previous study on either germ cell proliferation or apoptosis in upper mammals, an analysis of DNA replication (by bromodeoxyuridine labeling) and of the location of apoptotic germ cells (by the TUNEL assay) has been performed in 3 young adult cats. Our observations indicated that in this animal, spermatogonial DNA synthesis occurs at stages V (at which point the first generation of replicating spermatogonia appears, together with replicating preleptotene spermatocytes), early VII, VIII, and early I, II, and IV. Apoptosis of both spermatogonia and spermatocytes was located mostly at stages early I, early VI, early VII, and VIII. Interestingly, DNA synthesis and germ cell death were found to occur at the same stages of the spermatogenic cycle (that is, to occur at the same specific stages of development) as those reported for the rabbit and small rodents.     

Decrease in apoptosis of germ cells in the testes of infertile men with varicocele

To determine whether the regulation of apoptosis in the testes of patients with varicocele testes was abnormal, affecting germ-cell differentiation and sperm production, we studied apoptosis in the testes of normal men and infertile men with varicocele. In all, 56 testicular biopsy specimens were collected from 28 varicocele patients. The specimens from the testes of five normal volunteers with informed consent were used as controls. In situ end-labeled cells were counted with a CAS 200 image analyzer, and an apoptotic index (AI) was calculated by division of the number of labeled cells by the total number of spermatocytes and spermatogonia in over 20 seminiferous tubules. The apoptosis was also examined by electron microscope. The mean AI was 9.67 ± 0.93% in normal testes (n = 5). In contrast, the mean AIs determined in the right and left testes of varicocele patients (n = 28) were 3.90 ± 2.28% and 3.78 ± 2.87%, respectively. The AIs recorded for the testes of varicocele patients were significantly lower than those noted for normal men (P < 0.05). In varicocele patients the AI obtained in the right testis was not statistically significantly different from that found in the left testis. The numbers of apoptotic cells per Sertoli cell also decreased in the testes of varicocele patients as compared with normal men (P < 0.01). Evaluation of all specimens, including the normal controls, revealed no significant correlation either between the AI and the sperm concentration on the seminogram or between the AI and Johnsen's mean score. There was also no relationship between the AI and the serum level of follicle-stimulating hormone, lutenizing hormone, testosterone, or estradiol. In conclusion, apoptosis is decreased in germ cells in the testes of infertile men with varicocele as compared with normal men.     

Expression of BCL-2 family genes in germ cells undergoing apoptosis during the first wave of spermatogenesis in the rat

Apoptosis is a key event controlling sperm output both in normal and pathological conditions. However, the mechanisms involving germ cell apoptosis is far from being understood. In this work, we have immunoisolated germ cells undergoing apoptosis by taking advantage of the up-regulation of Fas, a dead receptor involved in apoptosis induction in these cells. Analysis of specific markers showed that this cell population is composed of spermatogonia and meiotic spermatocytes. We measured the mRNA levels of several apoptosis-inducing proteins belonging to the BCL-2 family (BAX, BAD, PUMA, BOK and BAK) as well as those that prevent apoptosis (BCL-2, BCL-W and BCL-XL). Results showed that apoptotic germ cells have elevated mRNA levels of all studied genes (both pro and anti-apoptotic) compared with non-apoptotic cells. Our data would help to define the molecular mechanisms involving germ cell apoptosis under physiological conditions.     

Intercellular bridges and apoptosis in clones of male germ cells

When an As spermatogonium divides to form a pair of Apr spermatogonia the two daughter cells stay interconnected by an intercellular bridge. These cytoplasmic bridges form after every subsequent division leading to large clones of interconnected germ cells. Cohorts of spermatogonia maintain synchronous development throughout spermatogenesis, which has been attributed to the presence of these intercellular bridges. To examine whether apoptotic signals are transduced through the intercellular bridges we studied germ cell apoptosis in whole mounts of seminiferous tubules from non-irradiated and irradiated mouse testes, using whole mount seminiferous tubules and confocal microscopy. This allowed us to use TUNEL staining of apoptotic germ cells and at the same time to study these apoptotic germ cells in their topographical context. Our results show that in response to ionizing radiation single spermatogonia within a clone can undergo apoptosis without affecting their neighboring cells. Additionally, also early spermatocytes were shown to undergo apoptosis individually. Both radiation-induced spermatogonial apoptosis and spontaneous apoptosis of spermatocytes are caused by DNA damage of individual cells. Degeneration of healthy spermatogonia because of regulatory signals, however, follows other death inducing mechanisms, which lead to apoptosis of chains of interconnected spermatogonia.     

Vasectomy impairs spermatogenesis through germ cell apoptosis mediated by the p53-Bax pathway in rats

PURPOSE: The pathophysiology of impaired spermatogenesis after vasectomy has not been completely investigated. We examined the role of p53 protein in cell cycle arrest and apoptosis of germ cells after vasectomy in the rat. MATERIALS AND METHODS: Eight-week old rats underwent bilateral vasectomy and the testes were harvested 1, 4, 8, 12 and 24 weeks after surgery. Germ cell apoptosis was evaluated by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end-labeling (TUNEL) and electrophoresis assay of DNA fragmentation. Western blot analysis and immunohistochemistry were performed to examine the expression of p53, proliferating cell nuclear antigen, Bax, Bcl-2, p21WAF1/Cip1 and Gadd45. To evaluate spermatogenesis, testicular weight and percent of haploid cells flow cytometry was done. RESULTS: Spermatogenesis impairment was associated with increased p53 and decreased proliferating cell nuclear antigen expression at the delayed phase more than 8 weeks after vasectomy. The number of TUNEL positive germ cells was increased at the early 1-week and delayed phases after vasectomy. Bax but not p21WAF1/Cip1 or Gadd45 expression was increased. p53, Bax and TUNEL positive cells were co-localized in the seminiferous tubules. CONCLUSIONS: Spermatogenesis was impaired after vasectomy by apoptosis but not by cell cycle arrest. The p53-Bax pathway effects apoptosis in the delayed phase after vasectomy in some seminiferous tubules.     

Prepubertal exposure to 4-tert-octylphenol induces apoptosis of testicular germ cells in adult rat

The effects of chronic exposure of 4-tert-octylphenol (OP) on the testicular development of prepubertal male rats were evaluated. 4 weeks old rats were injected with 0.8 microg of estradiol valerate (EV) or 20, 40, or 80mg of OP three times a week for one month. A marked reduction of the size and weight of the testis, epididymis, and seminal vesicle was observed in all the three dosages. Serum testosterone concentration was dramatically decreased while serum LH concentration was increased. Seminiferous tubules were reduced in size and showed no mature spermatozoa or late-stage developing spermatids. In addition, testicular germ cells undergoing apoptosis were obviously increased in all the treated groups. The expression of bcl-xL mRNA was significantly decreased in the OP treated groups, whereas the expressions of bcl-2 and bax mRNA were not significantly changed. In conclusion, these results demonstrate that OP severely reduce the size and/or function of the male reproductive organs due to increased apoptosis of testicular germ cells and the decreased biosynthesis of testosterone.     

Retarded differentiation of Leydig cells and increased apoptosis of germ cells in the initial round of spermatogenesis of rats with lethal dwarf and epilepsy (lde/lde) phenotypes

The lde/lde rats show a severe dwarf phenotype with early postnatal lethality and a high incidence of epileptic seizure. Seizures are first detected in this model between 16 and 63 days of age, and mostly begin as wild running and progress to generalized tonic-clonic convulsions. Because our histological examination detected many extracellular vacuoles in the hippocampus and amygdaloid bodies of these animals at 28 days of age, these pathological alterations may be related to the epileptogenesis in lde/lde rats. In addition to these defects, male lde/lde rats have apparently smaller testes with reduced number of germ cells and poorly matured adult-type Leydig cells in comparison with wild-type controls. In the present study, we performed anatomical, histological, and endocrinologic examinations to characterize the testicular phenotype of lde/lde rats at 21, 28, 35, and 56 days of age. Male lde/lde rats showed severely retarded growth of the testes and accessory sex organs. Their seminiferous tubules were significantly smaller and contained markedly fewer germ cells at all time points examined as compared with controls. Significantly fewer Sertoli cells at 21 and 28 days of age, markedly decreased spermatocyte number at 28 days of age, and delayed appearance of spermatids at 56 days of age were observed in the testes of lde/lde rats. More TUNEL (T&T-mediated duTP-biotin nick-end labeling)-positive cells were detected in lde/lde seminiferous tubules, and the largest number of apoptotic cells was recorded at 28 days of age. The increases in 3beta-hydroxysteroid dehydrogenase-positive adult-type Leydig cells and 11beta-hydroxysteroid dehydrogenase-positive mature adult-type Leydig cells were also severely retarded in the testes of lde/lde rats. Consistent with these defects, significantly lower plasma follicle-stimulating hormone (FSH), luteinizing hormone (LH), and testosterone concentrations were detected in lde/lde males at 28 days of age, and weak immunostaining for FSH and smaller cytoplasm of LH-positive cells were detected in the anterior pituitary lobes of lde/lde males. Despite a normal level of plasma LH after 35 days of age, a significantly lower level of plasma testosterone was detected at 56 days of age. These results indicate that the normal lde allele is related to prepubertal elevations of gonadotropins and normal development of adult-type Leydig cells. Because lde/lde rats experience epileptic seizures during the period when the hypothalamus-pituitary-testicular axis is established, lde/lde rats would be useful as a model for reproductive disorder with pediatric epilepsy.     

Irradiated mouse testes efficiently support spermatogenesis derived from donor germ cells of mice and rats

Testicular cell transplantation has been widely used to investigate the biology of spermatogonial stem cells, production of transgenic animals, and restoration of fertility in rodent models. One critical step in successful transplantation is the preparation of the recipient testes. Busulfan has been widely used, but irradiation has been often suggested as an alternative. There have only been limited reports of the use of irradiated animals as transplant recipients for studying differentiation of transplanted cells, and there has been no direct comparison of irradiation and busulfan as preparation methods. Mouse testes treated with local fractionated irradiation (1.5 + 12 Gy) were compared with busulfan-treated testes as recipients using mouse-to-mouse and rat-to-mouse germ cell transplantation. The fractionated irradiation schedule resulted in depletion of endogenous spermatogenesis similar to that produced by busulfan doses of 50-55 mg/kg. When immature mouse or rat testicular germ cells were transplanted into the irradiated testes, donor cells derived from either rat or mouse spermatogonial stem cells were able to form colonies of differentiated spermatogenic cells 10-13 weeks after transplantation with similar efficiencies as in busulfan-treated testes. Locally irradiated testes could be considered as an alternative to busulfan treatment for animal recipients of germ cell transplants that cannot endure the systemic toxicity of busulfan.     

Autophagic deficiency is related to steroidogenic decline in aged rat Leydig cells

Late-onset hypogonadism (LOH) is closely related to secondary androgen deficiency in aged males, but the mechanism remains unclear. In this study, we found that reduced testosterone production in aged rat Leydig cells is associated with decreased autophagic activity. Primary rat Leydig cells and the TM3 mouse Leydig cell line were used to study the effect of autophagic deficiency on Leydig cell testosterone production. In Leydig cells from young and aged rats, treatment with wortmannin, an autophagy inhibitor, inhibited luteinising hormone (LH)-stimulated steroidogenic acute regulatory (StAR) protein expression and decreased testosterone production. In contrast, treatment with rapamycin, an autophagy activator, enhanced LH-stimulated steroidogenesis in Leydig cells from aged, but not young, rats. Intracellular reactive oxygen species (ROS) levels were increased in both young and aged Leydig cells treated with wortmannin but decreased only in aged Leydig cells treated with rapamycin. Furthermore, an increased level of ROS, induced by H(2)O(2), resulted in LH-stimulated steroidogenic inhibition. Finally, knockdown of Beclin 1 decreased LH-stimulated StAR expression and testosterone production in TM3 mouse Leydig cells, which were associated with increased intracellular ROS level. These results suggested that autophagic deficiency is related to steroidogenic decline in aged rat Leydig cells, which might be influenced by intracellular ROS levels.     

蒲葵子提取物对膀胱癌细胞增殖凋亡的影响及其相关机制

目的:探讨蒲葵子提取物对膀胱癌细胞增殖、凋亡的影响及其相关机制。方法:将T24细胞分别用含蒲葵子提取物且终浓度分别为0、25、50、100 mg/L的培养液培养,分别记为对照组和蒲葵子低、中、高剂量组。用细胞计数试剂盒8(CCK-8)检测细胞存活率;克隆形成实验检测细胞克隆形成数量;流式细胞术检测细胞凋亡;蛋白质印迹法...     

老年男性骨密度与IGF-1及骨代谢相关激素的关系研究

目的：探讨老年男性骨密度（BMD）与胰岛素样生长因子-1（IGF－1）及骨代谢相关影响激素的关系。方法：采用双能X线骨密度仪测量120例正常老年男性骨密度（BMD）、血IGF－1及生长激素（GH）、雌二醇（E2）、血睾酮（T）、甲状旁腺激素（PTH）等指标，并与青中年男性对照，进行统计分析。结果：老年男性胰素样生长因子－1、雌二醇（E2）及血睾酮（T）呈现随着年龄增长而降低的趋势，并且在骨质疏松组均显低于非骨质疏松组（P＜0．01），IGF－1与骨密度（P＜0．01）、E2（P＜0．005）、T（P＜0．05）呈正相关。结论：IGF－1的增龄性减少同时伴雌激素、雄激素水平的降低可能是老年男性骨质疏松发生的重要机制。     

探索中老年男性骨量减少和骨质疏松的相关因素

目的探索中老年男性骨量减少和骨质疏松的相关因素,为防治骨量减少发展为骨质疏松提供参考。方法选取2017年1月1日至2020年8月31日在某三甲医院健康体检的中老年男性为研究对象。测量空腹血糖、高密度脂蛋白等生化指标,用双能X线骨密度仪测量腰椎和髋关节骨密度。结果单因素Logistic回归分析发现,BMI、腰围、血糖和高密度脂蛋白有统计学意义(P 0.05)。偏瘦、血糖为疾病进展的危险因素;适度肥胖和高密度脂蛋白为疾病进展的保护因素。多因素Logistic回归发现,BMI、血糖为疾病进展的相关因素,且差异有统计学意义。与正常BMI相比,偏瘦为疾病进展的危险因素,适度肥胖为该病进展的保护因素;血糖控制较差的患者疾病进展风险是血糖正常人群的1.4倍,且差异有统计学意义。结论年龄、BMI、腰围、血糖和高密度脂蛋白为骨量减少进展为骨质疏松的相关因素,其中BMI和血糖可能为疾病进展的综合因素。在预防骨量减少进展为骨质疏松时,需综合考虑各种因素。     

紫草素抑制胰腺癌细胞增殖并诱导凋亡的研究

胰腺癌是一种恶性肿瘤。预计到2030年,胰腺癌将上升为第二大致死性肿瘤[1]。本研究旨在观察中药紫草素抗胰腺癌细胞的具体机制。一、材料与方法1.材料:PATU8988胰腺癌细胞、紫草素、膜联蛋白V-异硫氰酸荧光素(Annexin V-fluoresceine isothiocyanate,Annexin V-FITC)凋亡试剂盒、锥虫蓝、BALB/c-nude小鼠。2.方法:Alamar Blue检测细胞活力;锥虫蓝染色计数死亡细胞;Annexin V-FITC细胞凋亡法检测凋亡;蛋白质印迹法(Western blot)检测Notch1、聚腺苷二磷酸核糖聚合酶(poly adenosine diphosphate-ribose polymerase,PARP)表达;人源肿瘤细胞系异种移植模型(CDX)检测紫草素对体内胰腺癌细胞的影响,应用Graphpad prism 7.0统计软件,One-way分析方法,组间比较采用t检验,以P<0.05为差异有统计学意义。     

YAP 基因干扰对乳腺癌细胞增殖和凋亡的影响

目的：探讨干扰YAP基因的表达对乳腺癌细胞增殖和凋亡的影响。 方法：通过逆转录病毒介导的方法,分别用YAP shRNA（实验组）或阴性对照shRNA（对照组）转染MCF-7乳腺癌细胞,用qRT-PCR和Western blot方法检测干扰效率;溴脱氧核苷尿嘧啶（BrdU）结合实验和MTT法检测细胞增殖活性;流式细胞术和DAPI染色检测细胞凋亡情况。 结果：干扰效率检测显示,转染72 h后,实验组MCF-7细胞YAP mRNA表达量明显降低（P<0.05）,同时YAP蛋白表达也明显下调;细胞增殖检测显示,与对照组比较,实验组MCF-7细胞BrdU结合与OD值明显降低（P<0.05）,转染后24、48、72 h增殖抑制率分别为19.1%、38.5%、53.5%;凋亡检测显示,实验组细胞凋亡率与凋亡细胞数较对照组明显增加（均P<0.05）。 结论：干扰YAP基因的表达能有效抑制乳腺癌细胞的增殖并促进凋亡,YAP可能是乳腺癌潜在治疗靶点。     

内质网应激在高脂血症大鼠睾丸生殖细胞凋亡中的作用

目的:探讨内质网应激途径在高脂血症大鼠睾丸生殖细胞损伤中的作用。方法:42只雄性Wistar大鼠于鼠龄4周末随机化分为两组:对照组(12只)和高脂组(30只),分别给予普通饲料和高脂高热量饲料喂养建立高脂血症大鼠模型。第10周末(即鼠龄14周)全自动生化分析仪检测外周血甘油三酯(TG)和总胆固醇(TC)含量,TUNEL法检测睾丸组织中凋亡生殖细胞,免疫组化法检测睾丸组织中葡萄糖调节蛋白78(GRP78)及caspase-12蛋白表达,RT-PCR法检测睾丸组织GRP78及caspase-12 mRNA的表达。结果:高脂组大鼠血清TG、TC[(3.00±0.92)mmol/L、(3.04±0.39)mmol/L]较对照组[(1.43±0.41)mmol/L、(1.55±0.23)mmol/L]显著升高(P0.01),睾丸生殖细胞凋亡指数[(37.17±2.74)%]较对照组[(5.16±0.81)%]显著升高(P0.01);以精原细胞和精母细胞凋亡为主。高脂组睾丸组织中GRP78蛋白(0.32±0.03)及caspase-12蛋白(0.34±0.02)表达较对照组(0.19±0.01、0.12±0.01)明显升高(P0.01),GRP78 mRNA及caspase-12 mRNA(0.86±0.05、0.87±0.01)较对照组(0.37±0.03、0.34±0.03)明显增加(P0.01)。结论:高脂血症大鼠睾丸生殖细胞凋亡增多;内质网应激可能是高脂血症大鼠睾丸生殖细胞凋亡的主要途径之一。     

干细胞向生殖细胞分化的研究进展

干细胞(SCs)具有在体外分化为生殖细胞的潜能,为研究生殖细胞(GCs)早期发育提供了良好的模型,并将为干细胞移植修复生殖功能提供细胞资源。本文综述了胚胎干细胞/诱导多能干细胞(ESCs/iPSCs)、新生儿附属物来源干细胞(NDSCs)以及成体干细胞(ASCs)向生殖细胞分化所取得的研究进展,同时总结了各类干细胞向生殖细胞分化时所遇到的障碍及所面临的挑战,为干细胞在生殖医学领域的应用提供理论依据。     

Effects of triptolide on proliferation and apoptosis in rat mesangial cells induced by transforming growth factor β1 and related mechanisms

Objective To investigate the effects of triptolide on proliferation, apoptosis and the changes of Ski, Smad3, Smad7 and collagen type Ι (ColΙ) in cultured rat mesangial cells induced by transforming growth factor (TGF)?β1. Methods Cultured HBZY?1 rat mesangial cells were divided into 5 groups: (1)normal control group; (2)TGF?β1 group (10 μg/L); (3)-(5)triptolide (0.4, 2, 10 μg/L)+TGF?β1 (10 μg/L) groups. The cell proliferation was detected by MTT. Apoptosis of mesangial cells was detected by TUNEL assay. The expressions of Ski, Smad3, Smad7 mRNA were examined by real?time quantitative PCR. The expressions of Ski, Smad3, Smad7 and ColΙ protein were detected by Western blotting. The localizations of Ski and Smad3 protein were detected by laser confocal fluorescence microscope. Results Compared with the normal control, TGF?β1 (10 μg/L) significantly stimulated mesangial cells proliferation, while decreased apoptosis. The mRNA and protein expressions of Ski, Smad7, Smad3 and ColΙ protein expression in TGF?β1 group were increased (P＞0.05). In comparison with TGF?β1 group, triptolide could significantly inhibit TGF?β1?induced mesangial cells proliferation in dose?dependent manner, and promote the apoptosis of mesangial cells. In TGF?β1 group, mRNA and protein expresscons of Ski and Smad7 were increased (P<0.05), Smad3 mRNA and protein were decreased (P＞0.05), and ColΙ protein was decreased (P<0.01). In comparison with TGF?β1 group, fluorescence intensity of Ski, Smad3 proteins was significantly increased in cytoplasm, while decreased in nucleus. Conclusions Triptolide can inhibit TGF?β1?induced mesangial cells proliferation through regulating the expressions of Ski, Smad7 mRNA and protein, inhibiting Ski. Smad7 translocation to the nucleus, and down?regulating Smad3 mRNA and protein expression. Triptolide can promote apoptosis of mesangial cells.     

苯肾上腺素对人前列腺上皮细胞增殖和凋亡的作用

目的　探讨交感神经递质对人前列腺上皮细胞增殖和凋亡的作用。方法　在不同浓度的苯肾上腺素下培养人良性前列腺增生 (BPH)上皮细胞系BPH1,观察苯肾上腺素对前列腺上皮细胞增殖和凋亡的影响及其能否被盐酸特拉唑嗪阻断。结果　苯肾上腺素能够刺激体外培养的人前列腺上皮细胞数量增加 ,r1=0 .82 1,P <0 .0 5 ) ;能够增加体外培养的人前列腺上皮细胞上清液A值的增加 ,r2 =0 .85 4,P <0 .0 5 ;能够增加体外培养的人前列腺上皮细胞S期细胞所占的比例(r3 =0 .85 0 ,P <0 .0 5 ) ,这种作用能被盐酸特拉唑嗪阻断。苯肾上腺素不能诱导或抑制体外培养的前列腺上皮细胞发生凋亡。结论　苯肾上腺素能通过α1受体途径刺激前列腺上皮细胞增殖。