Neuroendocrine marker substances in human Leydig cells — changes by disturbances of testicular function

Summary. A number of neuroendocrine and neuronal markers were demonstrated in Leydig cells of the testes of 18 men aged between 20 and 81 years. Tissue sections were divided into five groups, i.e. carcinoma of the prostate (control cases; n = 4), seminoma ( n = 8), anti-androgen therapy ( n = 3), oestradiol therapy ( n = 2) and cryptorchidism ( n = 1). The following substances were immunocytochemically tested: the monoamine synthesizing enzymes tyrosine hydroxylase, aromatic L-amino acid decarboxylase, dopamine-β-hydroxylase and phenylethanolamine-N-methyltransferase, the indolamine serotonin, the calcium-binding proteins parvalbumin, calbindin and S-100 protein, the microtubule associated protein-2, as well as neurofilament protein 200, synaptophysin, neuron specific enolase, substance P and chromogranin A + B. All these substances were found in Leydig cells of all sections independently of the pathological changes of the testes. Compared with the control cases, all the other groups showed a significantly weaker immunore-activity for all markers. The uniformity of staining among the different antibodies allows the deduction that these neuroactive peptides may belong to a basic equipment of Leydig cells probably stabilizing their function in an autocrine manner. On the other hand, Leydig cells themselves seem to be a stable structural component of the testis, which are not essentially involved in the pathogenesis of the disturbances mentioned above.     

Non-tumoural parenchyma in Leydig cell tumours: pathogenetic considerations

Little is known about the pathogenesis of Leydig cell tumours (LCTs) of the testis. The observation of several associated dysgenetic features in the non-tumoural parenchyma and in the contralateral testes of men with testicular germ cell neoplasms has served as the basis to propose that there may be a common mechanism for different male reproductive disorders. However, the possible relationship between LCTs and other testicular lesions has not been explored. Here we describe the presence of primary lesions in the non-tumoural parenchyma of testes with LCT, from which we try to establish possible pathogenetic associations. We studied the non-tumoural parenchyma adjacent to 16 LCT specimens. Parameters as Leydig cell hyperplasia (LCHY), qualitative evaluation of the germinal epithelium and spermatogenesis, the presence of Sertoli cell-only tubules (SCOT), and the Sertoli cell nuclear morphology were consistently assessed in all cases. SCOT associated with Sertoli cell dysgenetic morphology was the most frequent finding, present in 50% of the cases. Another interesting finding was the presence of LCHY in four cases (25%). Abnormal spermatogenesis was found in 81.25% of the cases, and it consisted of lesions of the adluminal or basal compartments of seminiferous tubules. The occurrence of either dysgenetic Sertoli cells or LCHY adjacent to LCTs could represent primary anomalies, resulting from a common insult also involved in tumourigenesis. The abnormalities in spermatogenesis observed here are likely to represent consequences of either tumour compression or abnormal hormonal production. The significance of these associations merits further investigation regarding a common pathogenesis.     

Is testosterone involved in the initiation of spermatogenesis in humans? A clinicopathological presentation and physiological considerations in four patients with Leydig cell tumours of the testis or secondary Leydig cell hyperplasia

The purpose of this study was to evaluate the role of testosterone on the puberal development of spermatogenesis and to present additional clinicopathological data which bring about new information to this controversial subject. Four pre-pubertal patients are presented, 2 of them bearing Leydig cell tumours of the testis in the form of nodular masses. In both cases seminiferous tubules in the immediate vecinity to the tumours showed complete development of spermatogenesis, while those located away from the tumours were infantile in nature. Gonadotrophic levels were within the normal pre-pubertal range in these 2 cases. In one of the patients, testosterone concentration in the testis showed higher values than normal, and a concentration gradient was detected between the tumoral nodule and non-tumoral parenchyma. The 3rd patient had a pineal choriocarcinoma producing high amounts of hCG and consequently a diffuse hyperplasia of Leydig cells with high levels of plasma testosterone. Seminiferous tubules showed development up to pachytene spermatocytes. The last case was a precocious puberty in a boy with a tumour of the 3rd ventricle area. He had elevated levels of testosterone in the testis and plasma. In the testicular biopsy, stimulation of Leydig cells was detected. The seminiferous tubules showed mature Sertoli cells and pachytene spermatocytes. FSH levels were abnormally low. These 4 cases present in common different situations in which abnormally high amounts of testos-happens in the immature rat, the interaction between testosterone and gonadotrophins is essential for the normal initiation of spermatogenesis in normal puberty. Considerations are discussed on the possible synergistic role of gonadotrophins or other factors in relation with stimulation of seminiferous tubules by testosterone.     

睾丸间质细胞瘤二例报告并文献复习

目的探讨睾丸间质细胞瘤的临床病理特点及鉴别诊断方法。方法总结2例睾丸间质细胞瘤患者的临床病理资料并复习文献。2例患者年龄均为27岁。例1因左侧睾丸胀痛不适4个月余,例2因婚后3年不育、查体发现右侧睾丸占位1个月入院。B超分别显示睾丸1.0 cm×1.3 cm、0.8 cm×0.6 cm强回声光团,边界锐利。2例均行睾丸肿瘤切除术。结果术中快速冰冻病理诊断为睾丸间质细胞瘤。病理组织学表现为瘤细胞呈团、条索或弥漫分布,体积较大,呈多角形,胞质丰富嗜酸性,边界清楚。免疫组化:波形蛋白,钙结合蛋白和抑制素均阳性。2例术后病理诊断均为睾丸间质细胞瘤。2例术后分别随访17、9个月未见复发。结论睾丸间质细胞瘤发病率低,临床易误诊,确诊需依赖病理组织学检查,尤其对年轻未育者,术中快速冰冻检查有助于手术范围的选择。     

睾丸间质细胞瘤一例报告（附文献复习）

报告双侧睾丸间质细胞瘤并性早熟1例,复习文献并讨论了睾丸间质细胞瘤的发生率、病理、临床表现、治疗和预后。     

Neuron-specific enolase-like immunoreactivity in human Leydig cells

Using the peroxidase anti-peroxidase immunocytochemical technique, neuron-specific enolase (NSE)-like immunoreactivity (NSE-LI) was revealed in Leydig cells of adult human testes at the light microscopic level. Differences in the NSE staining intensity were observed between the individual Leydig cells, separate cell groups within a testis and between the testes of individual patients. Together with the already established substance P-like immunoreactivity (SP-LI), the results obtained provided further evidence for the possible neuroectodermal origin of human Leydig cells and their presumable relation to the APUD- or the Diffuse Neuroendocrine System (DNES).     

睾丸间质细胞凋亡及调控

睾丸间质细胞 (Leydigcells,LC)的发生与成熟过程都和细胞凋亡相关 ,一定范围内的凋亡对机体具有积极的生理意义 ,但过度凋亡会使睾酮 (T)分泌明显减少 ,导致生精细胞凋亡增加 ,甚至不育。二甲磺基乙烷 (ethanedimethane sulphonate,EDS) ,糖皮质激素 ,下丘脑 垂体 睾丸轴分泌的促性腺激素释放激素 (GnRH)、卵泡刺激素 (FSH)、黄体生成素 (LH) /绒毛膜促性腺激素 (hCG)、T等激素 ,LC发育阶段及其他一些因素都会影响其凋亡。多个基因参与LC凋亡的调控 ,SCF/c kit、Bcl 2、Bcl xl可抑制其凋亡 ,caspase 3、Fas、Bax和clusterin可促进其凋亡     

A malignant Leydig cell tumor of the testis

A 40-year-old man was referred to our hospital with gynecomastia and painless swelling of the right scrotum. Ultrasonography revealed a 15×10 mm mass with low echogenicity of the right testis. We performed right high orchiectomy. Histologically, Reinke’s crystals and capsular invasion by tumor cells were found. Final diagnosis, the tumor was a malignant Leydig cell tumor of the testis.     

睾丸间质干细胞分化和移植

睾丸间质细胞是男性体内合成雄激素的主要细胞,胚胎发育期中肾胚的间质细胞及生精小管周成纤维样细胞可能是睾丸间质细胞的干细胞。在胚胎期间质干细胞分化为胎儿型间质细胞;出生后间质干细胞经间质祖细胞、未成熟间质细胞分化为成熟间质细胞。老年期间质细胞数量可能不变,但雄激素合成下降。间充质干细胞及脂肪干细胞等干细胞经诱导可分化为分泌雄激素的睾丸间质细胞,因此,间质干细胞移植可望成为治疗男性性腺功能不全和中老年雄激素缺乏的创新方法,本文对睾丸间质干细胞的分化及移植方面研究进行综述。     

Response of testicular macrophages to EDS-induced Leydig cell death

Summary.  The response of testicular macrophages to massive Leydig cell death was studied by the administration of the specific Leydig cell cytotoxic ethylene dimethane sulphonate (EDS) to sham-operated (SO), short-term (STHX), and long-term (LTHX) hypophysectomized rats. EDS-killed Leydig cells showed the morphological features of the programmed cell death or apoptosis. A 2-fold increase in the number of macrophages was found on days 1–2 after treatment in both SO and STHX rats, and dead Leydig cells were completely eliminated by day 3 after treatment. Otherwise, in LTHX rats, there was a delay in the increase in the number of macrophages, and EDS-killed Leydig cells remained in the testicular interstitium for several days. These results indicate that the phagocytic capacity of the macrophage population was diminished in hypophysectomized rats, and particularly after long-term hypophysectomy.     

Ectopic Leydig Cells in Seminiferous Tubules of an Infertile Human Male with a Chromosomal Aberration

A case of a human male infertility with chromosomal aberration is reported. The patient showed neither mental retardation nor physical abnormalities except that the testes were somewhat small and soft. Plasma follicle stimulating hormone and luteinizing hormone were 49.0 and 19.0 mIU/ml. Plasma testosterone was 2.6 ng/ml. Karyotype was considered to be 46 XY q-, long arms of the Y chromosome being deleted. Histological features of the testis were peculiar. Seminiferous tubules were small and devoid of spermatogenic cells, consisting only of Sertoli cells. Peritubular boundary layer of the tubules showed a marked increase in width due to the increase of collagen fibers. The base of some Sertoli cells was seen to protrude into the thickened peritubular boundary layer or, though rare, into the interstitial space. Unusual cells which had a round vesicular nucleus and abundant, dense cytoplasms also occurred in the boundary layer of most tubules. These cells were identified as Leydig cells because of an extensively developed smooth endoplasmic reticulum in their cytoplasm, although they lacked Reinke's crystals. These ectopic Leydig cells sometimes lay in direct contact with Sertoli cells in the seminiferous tubule.     

Long-term followup and clinical characteristics of testicular Leydig cell tumor: experience with 24 cases

PURPOSE: The natural history of Leydig cell testicular tumors is not well known, and differentiation between malignant and benign forms is not easy. We performed a retrospective multicenter evaluation of clinical and histological characteristics as well as followup of Leydig cell tumors. MATERIALS AND METHODS: From 1990 to August 2004 surgery was performed on 24 Leydig cell tumors. Before surgery all patients underwent clinical examination, tumor markers (alpha-fetoprotein, beta-human chorionic gonadotropin, lactate dehydrogenase), scrotal ultrasound and chest x-ray. Surgery was performed under ultrasound or palpation guidance via inguinotomy with clamping of the spermatic cord. Lesions less than 3 cm in diameter were resected and sent for frozen section examination. Histological criteria were considered. Abdominal computerized tomography or ultrasound and chest x-ray were performed postoperatively every 3 months for the first 2 years and every 6 months thereafter. RESULTS: Patient age range was 22 to 61 years (mean 37.75). In 7 patients (29.2%) the lesion was palpable and incidental diagnosis was made in 10 patients (41.7%). In the other patients diagnosis was made by ultrasound performed for testicular pain (4 patients, 16.6%) or the appearance of gynecomastia (3 patients, 12.5%). Frozen section examination was done in 20 of 24 patients (83%). In 17 of 20 patients (85%) Leydig cell tumor was diagnosed on frozen section examination. All patients underwent radical orchiectomy. All definitive diagnoses interpreted the neoplasia as benign. Average followup was 117 months (range 11 to 241). There was no disease recurrence or progression and all patients are currently disease-free. CONCLUSIONS: In our case study all Leydig cell tumors were interpreted as benign and long-term followup was negative.     

The Ultrastructural Response of Human Leydig Cells to Exogenous Estrogens

The effect of long term treatment with estrogens alone or along with medroxyprogesterone acetate on the Leydig cell ultrastructure was studied in testes from males undergoing surgery for sexual reassignment. The testes were fixed for electron microscopy by a perfusion method to insure uniform preservation. The morphological features were not the same in all the treated testes. Therefore, the cells found in the intertubular region were classified into three groups: (A) Leydig cells very similar to controls; (B) Absence of typical Leydig cells, but with cells having increased microfilaments, abundant smooth endoplasmic reticulum and some lipid droplets; (C) Absence of any cell type possessing abundant smooth endoplasmic reticulum, but having varying amounts of microfilaments and pigmentation. It is suggested that some of the cell types found in the intertubular region are dedifferentiated Leydig cells. This study indicates that the human testis from transsexuals of reproductive age is an appropriate model to study the indirect and direct effects of estrogens on the ultrastructure of cell types found in the human testes.     

Cytogenetic studies of human testicular germ cell tumours

In a search for consistent cytogenetic alterations in testicular germ cell cancers we have thus far studied some twenty surgical specimens of seminomas and nonseminomatous tumours. From the literature and our results it is now clear that such testicular tumours generally have a hyperdiploid to hypotriploid chromosomal content, and frequently possess a possibly site-specific chromosomal marker, an isochromosome 12p. A significant correlation between the presence of the i(12p) and advanced clinical stages has been revealed in our study. Several other chromosomal regions are consistently involved in cytogenetic changes: 1p and 1q, 6q, 7p, 9q, 12p, 17q, and 22q. Although there is little doubt that characteristic chromosomal lesions exist in testicular germ cell tumours, the impact which specific lesions may have on tumour progression is still unclear.     

Increased incidence of Leydig cell tumours of the testis in the era of improved imaging techniques

Study Type – Diagnostic (exploratory cohort) Level of Evidence 2b What’s known on the subject? and What does the study add? Leydig cell tumours (LCTs) of the testis are rare tumours, accounting for 1–3% of all testicular neoplasms. Our data indicate that using scrotal ultrasound with high resolution imaging in routine checkups leads to an earlier detection of LCTs. Most patients underwent an organ‐sparing surgery and no androgen deprivation was observed.

OBJECTIVE

? To report an observed high frequency of Leydig cell tumours (LCTs) diagnosed at our centre.

PATIENTS AND METHODS

? Charts of all patients who underwent surgery for a testicular tumour between 1999 and 2008 at our department were searched and data from patients with LCT were collected. ? Before surgery all patients underwent ultrasound and complete staging. In all but two patients with LCT an organ‐sparing surgery was performed. Surgery was performed under ultrasound or palpation guidance. ? All patients underwent postoperative follow‐up. We retrospectively reviewed surgical technique, histology, epidemiology and outcome in all LCT patients.

RESULTS

? In the study period, 197 testicular tumours were surgically removed of which 29 were diagnosed as LCT (14.7% of 197; further study group) in 25 patients. Mean age of patients with LCT was 45 years (range 21–68 years). ? Tumour size ranged from 1.2 to 80 mm (mean 10.23 mm). In two patients (8%) the lesion was palpable whereas incidental diagnosis was made in seven patients (28%). ? In the remaining patients diagnosis was made by ultrasound performed for testicular pain (six patients, 24%) or during infertility or erectile dysfunction evaluation (10 patients, 40%). ? Definitive histology reported no malignant histopathological features in all but one patient; this particular patient experienced tumour progression after 2 months and died from advanced disease 1 year later. All other patients are free of disease after a mean follow up of 56 months (range 7–93 months). ? During this period one patient developed a second LCT on the contralateral side; another patient had a recurrence within the same testicle, but on the opposite pole. Both underwent a subsequent organ‐sparing tumour resection.

CONCLUSION

? The percentage of LCT (14.7% of all testicular tumours removed) was significantly higher than expected from the literature. One possible explanation for this phenomenon is the increasing use of better ultrasound technology and the subsequent increased detection of small nodules that have not been found in historical series. Use of ‘observation‐only’ for very small lesions detected at infertility clinics is under debate.     

体外诱导人脂肪干细胞向Leydig细胞定向分化的研究

目的:探讨体外诱导脂肪干细胞(ADSCs)向Leydig细胞分化的可能性和条件。方法:应用I型胶原酶消化法分离人皮下脂肪组织中ADSCs,原代培养,适时传代。免疫组化方法检测波形蛋白的表达。以人绒毛膜促性腺激素(hCG)不同浓度及不同作用时间进行诱导后,实时荧光PCR检测类固醇激素合成急性调节蛋白(StAR)基因的表达。MTT法测定hCG诱导下的细胞增殖情况。在hCG、DMSO诱导1周后行3β-HSD免疫组化染色,并采用放射免疫法检测其培养上清及细胞裂解液的睾酮水平。结果:ADSCs增殖迅速,ADSCs波形蛋白呈黄褐色阳性表达;ADSCs中StAR基因的表达在一定范围内与hCG诱导浓度的增加成正相关,hCG 10 U/ml达到高峰,该浓度诱导1周内StAR表达随时间延长而增加;hCG诱导增加ADSCs细胞增殖;hCG 10 U/ml、DMSO 3.2×10-6mol/L条件下诱导1周后细胞中3β-HSD免疫组化染色可见胞质呈浅褐色弱阳性,且该条件下细胞裂解液睾酮测定值明显高于其它组。结论:①hCG在一定范围内促进ADSCs的StAR基因表达;②hCG可以促进ADSCs增殖;③在hCG 10 U/ml、DMSO 3.2×10-6mol/L诱导下,人ADSCs有向Leydig细胞分化的可能。     

Processing of Testicular Biopsies — Fixed in Stieve''s Solution — for Visualization of Substance P- and Methionine-Enkephalin-Like Immunoreactivity in Leydig Cells

In the present report a schedule for processing testicular biopsies fixed in Stieve's solution is described which is suitable for modern immunocytochemical techniques. By means of this procedure the existence of immunoreactivity in Leydig cells can be demonstrated using antisera against substance P and methionine-enkephalin.