Male germ cell transplantation: an experimental approach with a clinical perspective

Germ cell transplantation was developed in strains of mice. The infusion of germ cell preparations into the seminiferous tubules of infertile hosts led to repopulation of the testis with donor germ cells and restored fertility. Meanwhile, this technique has become a powerful tool to study the expansion of the testicular stem cell population and the kinetics of spermatogonial proliferation and re-induction of spermatogenesis. Further approaches, such as the transfer of rat/hamster/rabbit germ cells into mouse testes or cryopreservation or culture of spermatogonia, have widened the spectrum of applications associated with germ cell transplantation. Since the devastating effects of chemo- or radiotherapy on spermatogonia are the cause of infertility in oncological patients, germ cell transplantation might become an alternative approach for gonadal protection in prepubertal and postpubertal male tumour patients. This review focusses on recent developments with respect to germ cell transplantation and highlights the problems and perspectives of future clinical applications.     

OCT4 和P27kip1 蛋白在肾透明细胞癌中的表达及其相关性

目的研究OCT4 和P27kip1 蛋白在肾透明细胞癌中的表达及其相关性。方法采用免疫组织化学方法对 24 例肾透明细胞癌标本OCT4 和P27kip1 的表达进行检测。观察OCT4 和P27kip1 在肾癌组织中的阳性表达百分比及细胞分布情况。考察OCT4 和P27kip1 在性别、年龄（＜60 岁和≥60 岁）及癌细胞有无转移间的表达水平差异。对两者进行相关性分析。结果肾透明细胞癌标本OCT4 和P27kip1 蛋白阳性表达分别为16 例（66.7%）和18 例（75%）, OCT4 蛋白高、中及低表达分别为8 例（33.3%）、5 例（20.5%）及3 例（12.5%）,主要在细胞质和细胞核中表达,另有少部分在胞膜中表达。P27kip1 高、中及低表达分别为3 例（12.5%）、6 例（25%）、9 例（37.5%）,阳性着色在胞浆和胞膜,少部分细胞核可表达。OCT4 和P27kip1 的表达水平在年龄、性别间差异无统计学意义,在肿瘤有无转移间差异有统计学意义（P < 0.05)。OCT4 和P27kip1 在肾癌中的表达呈负相关(rs=-0.408,P < 0.05)。结论OCT4 和P27kip1 呈负相关,采用抑制OCT4 表达或P27kip1 的降解,尤其是降低胞质中P27kip1 的表达,可作为基因治疗肾癌的一个新的的作用靶点。     

Comparative activity of four anthracyclines against heterotransplanted germ cell tumor lines

Though the majority of patients with metastatic nonseminomatous germ cell tumors can be cured by modern combination chemotherapy, for those patients who do not respond to standard therapy additional drugs are needed. The activity of three new anthracycline derivatives, 4-epidoxorubicin, THP-doxorubicin and mitoxantrone against two established human testicular cancer cell lines in comparison to doxorubicin and to cisplatin, vinblastine, bleomycin and ifosfamide was studied in a xenograft model. All drugs were given at equitoxic doses. There were no differences in antitumor activity between the four anthracycline derivatives. In line H 12.1, which is very sensitive to the standard drugs cisplatin, vinblastine, bleomycin and ifosfamide, all four anthracycline derivatives were inferior to these agents. In contrast, in line H 23.1, where all four standard agents showed a significant lower antitumor activity when compared to line H 12.1, the anthracyclines preserved their activity, indicating a lack of cross resistance. Thus the anthracycline derivatives seem to be inferior to the standard drugs as first line treatment but because of apparent lack of cross resistance they deserve further evaluation in refractory germ cell tumors.     

Advances in the treatment of testicular cancer

Testicular cancer is the most common solid tumour in young men, and the treatment of testicular germ cell tumours (TGCT) has been called a success story of medical oncology, germ cell cancer being regarded as the "model of a curable neoplasm". Even with metastatic disease, high cure rates can be achieved: the overall 5-year survival for all stages of TGCT is approximately 80%. Today, elaborate systems for prognostic evaluation for gonadal and extragonadal germ cell tumours facilitate the choice of the most appropriate therapy for individual patients. In doing so, the ultimate goal of treatment is tumour-free survival for any patient with TGCT.This goal has already been reached for >99% of the patients with early-stage tumours, as well as for the majority of patients with advanced disease (56% of patients with metastases are considered to have a good prognosis at the time of diagnosis; the 5-year survival rate for this group is 90%). However, patients with 'intermediate' or 'poor' prognosis at the time of diagnosis, as well as patients with relapsed disease after cisplatin-containing therapy, still have an unsatisfactorily low 5-year survival rate after standard therapy with PEB (cisplatin, etoposide, bleomycin) of only 80%, 45-55% and 20-25%, respectively.Therefore, our goals must be (i) to limit acute and chronic toxicity by avoiding overtreatment for patients with localised disease and/or good prognosis with advanced disease; and (ii) to identify patients with poor prognosis and treat them in specialised centres, where not only is optimal interdisciplinary care available but new treatment strategies are being applied. For example, tandem high-dose chemotherapy regimens might be effective in achieving higher cure rates in these patients.     

The Effects of Prenatal Administration of Azo Dyes on Testicular Development in the Mouse: A Structure Activity Profile of Dyes Derived from Benzidine, Dimethylbenzidine, or Dimethoxybenzidine

In mice and rats, prenatal exposure to the dye Congo red permanentlyreduces the number of germ cells in male and female offspring.In the current investigation, nine other dyes structurally relatedto Congo red were examined for developmental testicular toxicity.In this study, the structural component of the dyes responsiblefor the prenatal induction of germ cell aplasia was identified.We found that only benzidine-based dyes altered testicular developmentand caused hypospermatogenesis in mice during adulthood. Dimethyl-anddimethoxybenzidine-based dyes were without effect. Pregnantmice were dosed orally on Days 8–12 of gestation witha benzidine-, dimethlybenzidine-, or a dimethoxybenzidine-baseddye and the testes of 45- to 50-day-old male offspring wereexamined. The testes of postpubertal male offspring exposedto the benzidine-based dyes, Congo red, diamine blue, and ChlorazolBlack E, were small and contained some tubules completely devoidof germ cells, but the dimethlybenzidine-based dyes, trypanblue, Evans blue, and benzopurpurin 4B, and the dimethoxybenzidine-baseddye, Chicago sky blue, did not alter testicular developmentin this manner. Azoic diazo component 48, a dimeth-oxybenzidinecongener, and two other diazo dyes, naphthol blue black andSudan III, were also without effect on the germ cells. Experimentswith Chlorazol Black E (CBE) indicate that the period of susceptibilityin the male fetus is limited to the period of primordial germcell migration and division. When CBE was administered on Days8–10 of gestation it reduced testis weight after pubertyby 30%, while treatment after Day 13 did not affect testicularfunction. It is interesting to note that the structure—activityrelationship of the dyes for developmental toxicity followingoral administration differs considerably from that producedby maternal ip administration.     

Immunohistochemical detection of melphalan-DNA adducts in colon cancer cells in vitro and human colorectal liver tumours in vivo

Melphalan is a chemotherapeutic drug that exerts its cytotoxic effect mainly through the formation of DNA adducts. We report the specific immunohistochemical detection and visualisation of melphalan-DNA adducts using the monoclonal antibody MP5/73 in cultured tumour cells and solid tumour tissue from colorectal liver metastases from patients treated with melphalan. The human colon cancer cell lines HT29, SW480 and SW1116, and the rat colon cancer cell line CC531 were exposed to different concentrations of melphalan. In addition, tumour samples from 17 patients with colorectal liver metastases treated by isolated hepatic perfusion with high dose melphalan (200mg) were collected. Cell lines and tumour samples were stained with the MP5/73 antibody against melphalan-DNA adducts and cell viability was determined by an MTT assay. Melphalan-DNA adducts could be visualised by immunohistochemistry in both cultured cells and solid tumour tissue. A correlation between melphalan exposure concentration, the subsequent melphalan-DNA adduct staining intensity, and melphalan cytotoxicity existed for each individual cell line, but the level of both parameters independently differed between cell lines. Specific staining for melphalan-DNA adducts also was feasible in the human solid tumour tissue. There was considerable variation in melphalan-DNA adduct staining, staining intensity, and distribution in the tumour stroma and the tumour epithelium among the different patients. Melphalan-DNA adducts appeared to be more intense in tumour cells at the border of the tumour nodules than in tumour cells in the centre. Thus, visualisation of melphalan-DNA adducts by immunohistochemistry allows the study of distribution of melphalan-DNA adducts in solid tumours.     

Bis(4,7-dimethyl and 5-dinitro-1,10-phenanthroline) sulfato-oxovanadium(IV)-mediated in vivo male germ cell apoptosis

Oxovanadium(IV) [VO] complexes of 1,10-phenanthroline are a new class of potent apoptosis-inducing cytotoxic agents against human testicular cancer cells in vitro. The present study investigated the in vivo ability of four(bis)-chelated 1,10-phenanthroline [phen] complexes of sulfato-oxovanadium(IV)-VO(phen)(2), VO(Cl-phen)(2), VO(Me(2)-phen)(2) and VO(NO(2)-phen)(2)-with and without substitutions, to induce testicular germ cell apoptosis. Male germ cell loss in mice was measured by determining the epididymal sperm count, testicular weight and histological evaluation of the testes. Repetitive intratesticular injection (7.5 mg kg(-1) testis(-1)) of bis-chelated 1,10-phenanthroline complexes of oxovanadium(IV) with 4,7-dimethyl [VO(Me(2)-phen)(2)] and 5-dinitro [VO(NO(2)-phen)(2)] substitution led to decreased sperm counts and reduced testicular weights. Histopathological examination of testicular sections from VO(Me(2)-phen)(2)- and VO(NO(2)-phen)(2)-treated mice revealed a marked inhibition of spermatogenesis and preferential loss of maturing, as well as elongated spermatids. In situ evaluation of seminiferous tubule cross-sections by terminal deoxynucleotidyl transferase-mediated FITC-deoxyuridine triphosphate nick end-labeling (TUNEL) and laser scanning confocal microscopy showed characteristic apoptotic germ cells delineating the periphery of the seminiferous tubules. The ability of bis-chelated 4,7-dimethyl- and 5-dinitro-substituted 1,10-phenanthroline complexes of oxovanadium(IV) to induce germ cell apoptosis in vivo may have potential utility in the treatment of human testicular germ cell tumors.     

Acute testicular toxicity induced by melamine alone or a mixture of melamine and cyanuric acid in mice

Eight-week-old male Kunming mice were administered either melamine (MA, 30, 140, or 700 mg/kg/day), a melamine and cyanuric acid mixture (MC, each at 15, 70, or 350 mg/kg/day), or vehicle (control) for 3 consecutive days. Testicular toxicity was evaluated on days 1 and 5 after the final exposure. The testicular and epididymal weights and serum testosterone level were significantly decreased in the highest MC group (350 mg/kg/day). Histopathologically, both MA and MC caused obvious lesions in the testis and epididymis, with significant increases in sperm abnormalities. By TEM, the blood–testis barrier was damaged dose dependently. TUNEL staining showed that both MA and MC induced increases in germ cell apoptosis. The Sertoli cell vimentin was collapsed in the treated animals as detected by immunohistochemical staining and Western blotting. This study demonstrated that both MA and MC treatments could disrupt the blood–testis barrier and cause a clear testicular toxicity.     

川芎嗪对隐睾固定术大鼠生精细胞影响的实验研究

目的：观察睾丸固定术前使用盐酸川芎嗪（Ligustrazine Hydrochloride）是否对大鼠隐睾所致的生精细胞凋亡有协同保护作用。方法：22日龄雄性SD大鼠40只，随机分成4组，假手术组（A组）、隐睾组（B组）、睾丸固定术组（C组）、固定术＋川芎嗪组（D组），制作大鼠隐睾模型，D组注射盐酸川芎嗪。14d后，C组和D组行睾丸下降固定术。28d后测定睾丸组织中超氧化物歧化酶（sod）、丙二醛（MDA）的含量，TUNEL法测定生精细胞的凋亡指数。结果：固定术＋川芎嗪组隐睾睾丸内SOD活性增强，MDA含量明显下降，生精细胞凋亡指数进一步减低。结论：睾丸下降固定术能够减轻大鼠睾丸生精细胞的凋亡，而固定术前使用川芎嗪对隐睾所致的睾丸生精细胞凋亡有协同保护作用。     

Quantitative immunohistochemical analysis of the glutathione S-transferase GSTM1: in situ phenotyping in archival material.

1. GSTM1 is present in only approximately 50% of Caucasian individuals and deficiency of GSTM1 is associated with susceptibility to a growing number of diseases, especially cancer. Thus, a method that would allow accurate, retrospective determination of the GSTM1 phenotype in different patient populations would have many applications. 2. Developed, therefore, is a quantitative, image-analysis-based immunohistochemical technique for the analysis of GSTM1 protein in paraffin-embedded tissue samples. It was applied to the determination of the GSTM1 phenotype using liver biopsies taken from 70 patients. 3. Of the 70 cases (depending on the cut-off point), 51-54% were deficient in GSTM1. A single 27 kD band characteristic for GSTM1 was found in seven of 16 cases analysed by Western blotting using the same GSTM1 antibody as in the immunohistochemical analysis. There was a good correlation (r = 0.87) between the staining intensity of the GSTM1 band and the staining intensity evaluated by immunohistochemistry. 4. It is concluded that this quantitative immunohistochemical method permits accurate determination of the GSTM1 phenotype and is well suited for retrospective analysis of GSTM1 expression in specific tissues in situ.     

Mode of action: impaired fetal leydig cell function--effects on male reproductive development produced by certain phthalate esters

Certain phthalate esters (di-2-ethylhexyl; di-n-butyl and butyl benzyl) have profound effects on the developing male reproductive system when administered orally to pregnant experimental animals during a critical window of development. These esters produce a syndrome of adverse effects that are characteristic of a disturbance in androgen-mediated development and include a variety of reproductive tract malformations and effects on developmental phenotypic markers. A testicular dysgenesis syndrome has been proposed to explain the secular increases in a number of human male reproductive deficits, including decreased semen parameters, increased incidence of cryptorchidism and hypospadias (two of the most common human birth defects), and increased incidence of testicular (germ cell derived) cancer. The rodent phthalate data lend support to the hypothesis. This example illustrates a number of points in the use of the Human Relevance Framework. First, chemical agents may have more than one mode of action (MOA): for example, phthalate-induced peroxisome proliferation leading to hepatocarcinogensis, compared with the induction of developmental effects via effects on androgen signaling. Second, the case demonstrates the life-stage sensitivity of the response to these compounds. Third, because humans may be exposed to multiple phthalate esters producing adverse effects on reproductive development, these compounds may be useful in testing the utility of the Human Relevance Framework (HRF) approach for evaluating cumulative and aggregate risk.     

Vanadocene-mediated in vivo male germ cell apoptosis

Vanadocenes are potent apoptosis-inducing cytotoxic agents against human testicular cancer cells in vitro. The present study investigated the ability of four vanadocenes-vanadocene diazide (VDA), vanadocene dicyanate (VDCN), vanadocene dioxycyanate (VDOCN), and vanadocene monochloro oxycyanate (VDCO)-to induce male germ cell apoptosis in vivo in mouse testes by repetitive intratesticular injection of vanadocenes (7.5 mg/kg/testis) for 28 days. Germ cell loss in vivo was measured by epididymal sperm count, testes weights, and histologic evaluation of the testes. Repetitive intratesticular injection of vanadocenes led to decreased sperm counts and reduced testicular weights. Histopathological examination revealed seminiferous tubular atrophy, inhibition of spermatogenesis, and the preferential loss of maturing and elongated spermatids. In situ evaluation by the terminal deoxynucleotidyl transferase-mediated FITC-deoxyuridine triphosphate nick-end labeling (TUNEL) of seminiferous tubule cross sections and laser confocal microscopy showed characteristic apoptotic cells identified primarily as pachytene spermatocytes delineating the periphery of the seminiferous tubules. The ability of vanadocenes to induce germ cell apoptosis in vivo may have potential utility in the treatment of testicular seminomas in humans.     

Hesperetin protects testicular toxicity of doxorubicin in rat: Role of NFκB, p38 and caspase-3

Doxorubicin is a widely used chemotherapeutic agent causing serious dose-dependent toxicity to non-target tissues such as testis. Its testicular toxicity is mainly due to the induction of oxidative stress. Hesperetin exerts its beneficial effects against oxidative stress-induced cellular damage. In the present investigation, doxorubicin was administered intraperitoneally at the dose of 4 mg/kg bw/week for a period of 5 consecutive weeks. Hesperetin was administered at the doses of 25, 50 and 100 mg/kg bw per oral by gavage for 5 consecutive days in a week for 5 weeks. Animals were sacrificed 1 week after the last injection of doxorubicin. The results of the present study clearly indicate the prevention of oxidative stress, DNA damage and the cellular toxicity by hesperetin treatment as evident from the analysis of biochemical parameters, comet assay, halo assay, Terminal Deoxynucleotidyl Transferase-mediated dUTP Nick End Labeling assay, immunohistochemistry and histology. Hesperetin protection against doxorubicin-induced germ cell toxicity was further evident from the sperm count and sperm head morphological evaluation. Moreover, the role of nuclear factor-kappa B, p38 and caspase-3 on hesperetin-mediated protection against doxorubicin-induced testicular toxicity was also investigated. The present study clearly revealed the amelioration of doxorubicin-induced testicular toxicity by the intervention with hesperetin.     

Mode of Action: Impaired Fetal Leydig Cell Function—Effects on Male Reproductive Development Produced by Certain Phthalate Esters

Certain phthalate esters (di-2-ethylhexyl; di-n-butyl and butyl benzyl) have profound effects on the developing male reproductive system when administered orally to pregnant experimental animals during a critical window of development. These esters produce a syndrome of adverse effects that are characteristic of a disturbance in androgen-mediated development and include a variety of reproductive tract malformations and effects on developmental phenotypic markers. A testicular dysgenesis syndrome has been proposed to explain the secular increases in a number of human male reproductive deficits, including decreased semen parameters, increased incidence of cryptorchidism and hypospadias (two of the most common human birth defects), and increased incidence of testicular (germ cell derived) cancer. The rodent phthalate data lend support to the hypothesis. This example illustrates a number of points in the use of the Human Relevance Framework. First, chemical agents may have more than one mode of action (MOA): for example, phthalate-induced peroxisome proliferation leading to hepatocarcinogensis, compared with the induction of developmental effects via effects on androgen signaling. Second, the case demonstrates the life-stage sensitivity of the response to these compounds. Third, because humans may be exposed to multiple phthalate esters producing adverse effects on reproductive development, these compounds may be useful in testing the utility of the Human Relevance Framework (HRF) approach for evaluating cumulative and aggregate risk.     

睾丸精原细胞瘤的CT诊断

目的探讨睾丸精原细胞瘤的CT特点,以期提高对本病的认识。方法总结分析经手术病理证实的8例精原细胞瘤（其中3例为隐睾合并精原细胞瘤）,对其图像进行回顾性分析.结果5例均表现为单侧睾丸肿块,内见多发小片状低密度坏死灶;3例隐睾合并精原细胞瘤,均位于腹膜后或盆腔腹膜外,肿块长轴与睾丸下行路径一致。结论睾丸精原细胞瘤的CT征象具有相对的特征性,密切结合临床综合分析,对于本病的诊断有重要的价值。     

Rat testis during 2,5-hexanedione intoxication and recovery. I. Dose response and the reversibility of germ cell loss

The histopathology of the testicular injury induced by 2,5-hexanedione (2,5-HD) exposure was examined in the rat. Charles River CD rats (200 g) were intoxicated by consuming 1% 2,5-HD in the drinking water or by intraperitoneal injection of the toxicant. Both neurotoxic and subneurotoxic exposures were studied, the total dose ranging from 40 to 211 mmol/kg. The following results were obtained: (1) there was a time delay between administration of the toxicant and development of the testicular injury, (2) Sertoli cell vacuolation in stages associated with the meiotic metaphase was the first histological sign of cellular injury at all doses, (3) subneurotoxic doses produced selective defects in germ cells in stages I-VIII of the spermatogenic cycle, (4) both subneurotoxic and neurotoxic doses produced germ cell necrosis and generalized sloughing of germ cells, and (5) intensive intoxication followed by a 17-week recovery period resulted in an absence of all postspermatogonial germ cells from the seminiferous epithelium of three of five treated rats. These data demonstrate that 2,5-hexanedione-induced testicular atrophy occurs at exposure levels below those producing clinical neurotoxicity and that, within the time frame of this study, the testicular injury is at least partially irreversible.     

Epigenetic alterations in sperm DNA associated with testicular cancer treatment

DNA methylation, a key component of the epigenome involved in regulating gene expression, is initially acquired in the germ line at millions of sites across the genome. Altered sperm methylation patterns are associated with infertility and transgenerational effects in humans and rodents. Testicular cancer is the most common form of cancer among men of reproductive age and has a high cure rate associated with chemotherapy treatment with bleomycin, etoposide, and cis-platinum (BEP). Although these drugs result in improved survival, they also affect the number and quality of germ cells. Our goal was to assess germ cell methylation patterns in a rodent model emulating the BEP treatment regimens used in human testicular cancer treatment. Animals were treated with control, or 0.3× (low) or 0.6× (high) dose of BEP, where a 1× dose is equivalent to human treatment regimens. Both dose-dependent and germ cell-dependent DNA methylation alterations were found at numerous loci throughout the genome. Of about 3000 loci tested, 42 loci were affected by BEP at the round spermatid stage of germ cell development, whereas 101 loci were affected in spermatozoa; 15 loci were consistently altered in spermatozoa of all high dose-treated rats. Both hyper- and hypomethylation were detected, suggesting either an interference with normal methylation patterning or abnormal repair of damaged patterns during spermatogenesis. The results indicate that a combination chemotherapy regimen used for testicular cancer treatment can result in altered DNA methylation patterns in spermatozoa and that some loci are more susceptible to damage than others.     

Gonadal function in young adults after surgical treatment of cryptorchidism.

In a follow-up study of 48 young men who had been surgically treated for cryptorchidism before puberty testicular function was assessed by examining the genitalia, testicular volume, secondary sex characteristics, semen, plasma luteinising hormone (LH) and follicle-stimulating hormone (FSH) concentrations after luteinising hormone-releasing hormone stimulation, and plasma testosterone concentrations. Clinical androgen effects were normal. The mean testicular volume of both testes was in the low normal range in those who had had unilateral cryptorchidism and below normal in those who had had bilateral cryptorchidism. Of 37 patients whose sperm counts were recorded (14 bilateral) six showed azoospermia (all bilateral), five had severe oligospermia (four bilateral), and 10 had moderate oligospermia (one bilateral). In nearly all those who had had bilateral cryptorchidism and most of those who had had unilateral cryptorchidism plasma gonadotrophin levels were increased. Four cases of possible partial LH deficiency were identified. Plasma testosterone concentrations were normal in all except two patients.     

干细胞标记物OCT4在膀胱癌中的表达及其临床意义

目的：探讨干细胞关键转录因子OCT4在膀胱癌患者组织中的表达与临床病理因素及预后的相关性。方法选取河北大学附属医院病理科收集的73例经泌尿外科手术切除的膀胱癌组织(膀胱癌组)及30例癌旁组织(癌旁组)作为标本进行免疫组化染色观察,对比两种OCT4蛋白的表达程度,并分析其与患者的临床病理特征及远期预后的关系。结果膀胱癌组织中OCT4蛋白的阳性表达率(71.23%)显著高于癌旁组织(10.00%)(P<0．05);膀胱癌组织中OCT4蛋白的阳性表达与膀胱癌分化程度、发生血管淋巴结转移、浸润深度有关(P<0．05),与年龄、性别、病理分期无关(P>0．05);膀胱癌组织中OCT4蛋白的阳性表达3年生存率显著低于阴性表达(P<0．05),3年生存中位时间显著短于阴性表达(P<0．05)。结论 OCT4表达与膀胱癌转移有关,对远期预后具有不良影响。