Testicular involution following optic enucleation

Summary The testes of adult male Syrian hamsters underwent involution within six weeks after optic enucleation. The diameter of the seminiferous tubules was 39% less than controls. Sertoli cells, spermatogonia, and primary spermatocytes were still present, but all steps of spermatids were completely absent from the involuted testes. Lipid droplets filled the Sertoli cell cytoplasm and often encroached upon the nucleus. Sertoli cells had sparse mitochondria and smooth endoplasmic reticulum, but Golgi cisternae were abundant. Typical SertoliSertoli junctions attached contiguous Sertoli cells. With lanthanum tracers it was demonstrated that these junctions were impenetrable; therefore, the bloodtestis barrier was deemed intact. Irregularly shaped protrusions often arose from the peritubular tissue and extended inward toward the seminiferous epithelium, often displacing the cytoplasm of the Sertoli cells and spermatogonia. The core of these protrusions consisted of irregular extensions of myoid cell cytoplasm surrounded by the myoid cells' basal lamina. External to the myoid cell basal lamina were bundles of collagen filaments with the basal lamina of the seminiferous epithelium forming the outermost layer of these protrusions. The apices of the Sertoli cells gave rise to numerous leaf-like processes that extended into and obliterated the lumen of the tubules. The Sertoli cell basal cytoplasm often contained phagocytized degenerating germ cells that appeared to give rise to the lipid droplets that filled the Sertoli cell cytoplasm. Acid phosphatase rich lysosome-like organelles were seen fusing with the degenerating germ cells and lipid droplets. The degenerating germ cells also were shown to contain acid phosphatase activity.     

A dense-cored filamentous body in Leydig cells of the golden hamster

Summary A unique cytoplasmic structure has been observed in Leydig cells of the golden hamster. It consists of a laminar core made up of electron dense material surrounded by a filamentous matrix of lower density, and is tentatively called a dense-cored filamentous body (DCFB). DCFBs vary in overall size and in configuration of the centrally disposed dense lamina. They are typically located in the vicinity of the centrosome and the Golgi complex. The body has no limiting membrane, and may be in contact with virtually every type of organelle. The DCFB is well developed in active Leydig cells, whereas it is small in the quiescent stage of the secretory cell. It is likely that the DCFB is a constant organelle in the hamster Leydig cell and may be involved in the physiological function of the Leydig cell, which remains to be specified.This work was supported in part by a grant from the National Science Council, the Republic of China (NSC-66B-0412-02-13)     

睾丸间质细胞瘤3例报告及文献复习

目的：探讨睾丸间质细胞瘤的临床病理特点及诊疗方法。方法：分析并总结3例睾丸间质细胞瘤患者的,J盏床病理资料并文献复习。结果：1例术中冰冻切片诊断为睾丸间质细胞瘤,2例术前细针穿刺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。2例患者行单侧睾丸切除,1例行睾丸肿瘤剜除术,术后分别随访24、15、10个月未见复发。结论：睾丸间质细胞瘤发病率低,临床表现缺乏特异性,易误诊,确诊需依赖病理组织学检查,细针穿刺病理可明确诊断并有助于手术的选择及手术范围的确定。     

Ultrastructural changes induced by HCG in the Leydig cell of the adult mouse testis

Summary Adult mice were treated daily with HCG (human chorionic gonadotropin). The most notable changes were found 24 hours after the second injection of 100 i.u. Nearly all lipid droplets had disappeared. Mitochondria showed a constriction linking the two portions of the organelle with widened intracristal spaces. Doughnut-shaped, bifurcated, and elongated mitochondria occurred commonly; their intracristal spaces were consistently widened. The Golgi complex was well developed. Dense bodies and phagolysosomes seemed to disappear. The results indicate that the mitochondrion is the first and most responsive organelle affected by HCG treatment. A hypothesis is advanced that the mitochondria of the Leydig cell provide the energy required for the lipolytic effect of HCG.     

Characterization of Nestin-positive stem Leydig cells as a potential source for the treatment of testicular Leydig cell dysfunction

The ability to identify and isolate lineage-specific stem cells from adult tissues could facilitate cell replacement therapy. Leydig cells (LCs) are the primary source of androgen in the mammalian testis, and the prospective identification of stem Leydig cells (SLCs) may offer new opportunities for treating testosterone deficiency. Here, in a transgenic mouse model expressing GFP driven by the Nestin (Nes) promoter, we observed Nes-GFP⁺ cells located in the testicular interstitial compartment where SLCs normally reside. We showed that these Nes-GFP⁺ cells expressed LIFR and PDGFR-α, but not LC lineage markers. We further observed that these cells were capable of clonogenic self-renewal and extensive proliferation in vitro and could differentiate into neural or mesenchymal cell lineages, as well as LCs, with the ability to produce testosterone, under defined conditions. Moreover, when transplanted into the testes of LC-disrupted or aging models, the Nes-GFP⁺ cells colonized the interstitium and partially increased testosterone production, and then accelerated meiotic and post-meiotic germ cell recovery. In addition, we further demonstrated that CD51 might be a putative cell surface marker for SLCs, similar with Nestin. Taken together, these results suggest that Nes-GFP⁺ cells from the testis have the characteristics of SLCs, and our study would shed new light on developing stem cell replacement therapy for testosterone deficiency.     

Stimulatory effect of follicle-stimulating hormone on rat Leydig cells

Summary The effects of FSH on the testicular interstitial tissue of immature hypophysectomized rats were studied by comparing morphological changes in Leydig cells with quantitative changes in interstitial tissue histology using morphometric analysis. Three groups of rats received subcutaneous injections of 0.5 ml saline vehicle or 10 g rFSH or 20 ng oLH (equivalent to the amount of LH known to contaminate the FSH), twice daily for 7 days. Administration of FSH significantly increased testis weight and stimulated more advanced spermatogenesis compared to saline or LH. Morphometric analysis of testes of LH-treated rats showed a small but significant increase in total interstitial cell volume compared to saline treatment. FSH caused much greater increases in the total volume of interstitial tissue and interstitial cells than either saline or LH and significantly increased the total volume of interstitial fluid by comparison with the other groups. FSH but not saline or LH treatment resulted in a striking hypertrophy of Leydig cells, to produce cells ultrastructurally identical to Leydig cells from adults. Since the target tissue of FSH is the seminiferous epithelium, the observed effects on Leydig cells by FSH treatment suggest that the secretion of factors by the seminiferous tubules may mediate the maturation of Leydig cells.     

Effects of scrotal hyperthermia on Leydig cells in long-term: a histological,immunohistochemical and ultrastructural study in rats

We have previously demonstrated that scrotal hyperthermia induce Leydig cell (LC) damage in short-term. The objectives of this pilot study were to investigate morphological changes and regulation of steroidogenesis on LC in long-term and the time of observation were extended to investigate whether the LC would eventually make a recovery after scrotal hyperthermia. The rats were randomly allotted into one of four groups: A (control), B (70 days after scrotal hyperthermia), C (105 days after scrotal hyperthermia), D (140 days after scrotal hyperthermia); each group contain seven animals. Scrotal hyperthermia was carried out in a thermostatically controlled water bath at 43°C for 30 min once daily for six consecutive days. Control rats were treated in the same way, except the testes were immersed in a water bath maintained at 22°C. Hyperthermia applied rats were sacrificed under 50 mg/kg ketamine anaesthesia after 70, 105 and 140 days, and biopsy materials of testes were obtained for light and electron microscopic examinations. Morphologically normal and the number of testosterone positive LC was significantly higher in 140 days after last heat than all other heat treatment groups. In heat treated groups, a dilated smooth endoplasmic reticulum, swollen mitochondria, and vanished mitochondrial cristae were observed. In the 140 days after scrotal hyperthermia, the severities of degenerative changes of LC were less than that observed in the other heat treated groups. We conclude that, scrotal hyperthermia cause morphological damaging and impaired steroidogenesis in LC and recovery of these findings were noted first time in 140 days after the last heat treatment.     

A morphological study on Leydig cells of scrotal hyperthermia applied rats in short-term

Testicular function is highly dependent on temperature control. The aim of this study was designed to investigate the morphological changes and regulation of steroidogenesis by light and electron microscopic level in Leydig cells (LC) after scrotal hyperthermia in rats. The rats were randomly allotted into one of four experimental groups: A (Control), B (1 day after scrotal hyperthermia), C (14 days after scrotal hyperthermia), D (35 days after scrotal hyperthermia); each group contain seven animals. Scrotal hyperthermia was carried out in a thermostatically controlled water bath at 43°C for 30 min once daily for 6 consecutive days. Control rats were treated in the same way, except the testes were immersed in a water bath maintained at 22°C. Hyperthermia applied rats were sacrificed under 50 mg/kg ketamine anaesthesia after 1, 14 and 35 days, and biopsy materials of testis were obtained for light and electron microscopic examinations. To date, no histopathological changes of LC injury after scrotal hyperthermia in rats have been reported. Light microscopic examinations indicated increase degenerative LC, decrease in number of testosterone positive LC in interstitial area after scrotal hyperthermia in short-term. In scrotal hyperthermia, a dilated smooth endoplasmic reticulum, swollen mitochondria, and vanished mitochondrial cristae were observed. The nuclei of some LC displayed deep invaginations and irregular outlines. The number of lipid droplets was very considerably increased in most LC when compared to control group. As a conclusion, we claim that temperatures higher than the body temperature may cause infertility by damaging LC.     

Leydig cell mitoses in human testes bearing early germ cell tumors

Summary Numerous mitoses were noted in testicular tissue from adult men with early germ cell tumors. More than 15 Leydig cells undergoing mitosis were found in the interstitial compartment. The presence of specific crystalline intracytoplasmatic inclusions demonstrated for the first time that differentiated Leydig cells are capable of proliferation. Occasionally cells are difficult to discriminate during mitosis. To establish reference criteria, the light- and electron-microscopic features of the following mitotic cells were examined: Leydig cells, fibroblasts, perivascular cells, peritubular cells, and lymphocytes. Supplementary mitoses in germ cell tumors and in a case of Leydig cell tumor were investigated. In the literature, only single reports of mitoses in Leydig cells are available. The frequent incidence of Leydig cell mitosis in early germ cell tumors may be due to the presence of growth-promoting factors in the testicular tissue.     

Paracrine effect of seminiferous tubule factors on rat Leydig cell testosterone production: Role of cytoskeleton

In Percoll purified Leydig cells from mature rat we have demonstrated that the basal testosterone production (9.5 ng/10⁶ Leydig cells/24 h) is increased 10-fold in presence of a saturating amount of hCG (1 IU/mL) and diminished in a dose-related manner when larger concentrations of gonadotropin are used to reach 14 ng/10⁶ Leydig cells for 50 IU of hCG. If 40% (v/v) seminiferous tubule medium (STM) is added together with hCG (1 IU/mL) to the incubation medium, a further increase (62%) of testosterone output is noticed. Obviously, when the testosterone production is low as a consequence of a higher dose of hCG (50 IU/mL), the STM (80%) improves the steroid synthesis five-fold (67.4 ng). Concerning the cytoskeletal components (microtubules, intermediate filaments and microfilaments) which have been examined in presence or absence of hCG and STM, we have found a rearrangement of cytoskeletal elements as well as cell-shape changes in relation with hormonal activity of the cells. The most prominent alterations of cytoskeletal elements have been observed after 24 h of incubation with 1 IU/mL of hCG added together with 80% of STM. The obtained results suggest that paracrine factor(s) presents in STM and acting in synergy with LH/hCG generate(s) the rearrangement of cytoskeletal structures which, in turn, facilitates the availability of cholesterol for the mitochondria and finally enhances the testosterone production in the rat Leydig cells.     

Glycogen content during the postnatal differentiation of the Leydig cell in the mouse testis

Summary The concentration and distribution of glycogen in relation to postnatal differentiation of the mouse Leydig cell are studied by biochemical and ultrastructural methods. Glycogen decreases to less than one third in the first twelve days after birth. This decrease is accompanied by modifications of its distribution in the cytoplasm. In the newborn it is abundant and arranged in clusters of beta particles; in the mature Leydig cell, glycogen is found scattered in extremely low concentration interspersed among elements of the endoplasmic reticulum.The role of glycogen during Leydig cell differentiation can be interpreted as a source of energy and/or as a source of building material in the biogenesis of membranous components.This work was supported by Grant M 63,121 from the Population Council, U.S.A.Fellow Consejo Nacional de Investigaciones Cientificas y Técnicas, Argentina.     

Leydig cell loss and spermatogenic arrest in platelet-derived growth factor (PDGF)-A-deficient mice

Platelet-derived growth factor (PDGF)- A-deficient male mice were found to develop progressive reduction of testicular size, Leydig cells loss, and spermatogenic arrest. In normal mice, the PDGF-A and PDGF-Ralpha expression pattern showed positive cells in the seminiferous epithelium and in interstitial mesenchymal cells, respectively. The testicular defects seen in PDGF-A-/- mice, combined with the normal developmental expression of PDGF-A and PDGF-Ralpha, indicate that through an epithelial-mesenchymal signaling, the PDGF-A gene is essential for the development of the Leydig cell lineage. These findings suggest that PDGF-A may play a role in the cascade of genes involved in male gonad differentiation. The Leydig cell loss and the spermatogenic impairment in the mutant mice are reminiscent of cases of testicular failure in man.     

H2O2 at physiological concentrations modulates Leydig cell function inducing oxidative stress and apoptosis

H₂O₂ is one of the active reactive oxygen species secreted by macrophages that are seen closely aligned with Leydig cells in the testicular interstitium. The present study was initiated to investigate the role of H₂O₂ on Leydig cell function in vitro at physiological concentrations. Significant decrease in both testosterone production (p < 0.05) and 3 β-hydroxysteroid dehydrogenase activity (p < 0.05) in adult Leydig cells were observed even with H₂O₂ at low concentrations (30 – 50 μM). H₂O₂ exposure increased oxidative stress in Leydig cells with the rise in lipid peroxidation and fall in the activities of the antioxidant enzymes; superoxide dismutase (SOD), catalase (CAT) & glutathione-s-transferase (GST). There was also a marginal increase (∼8%) in cell apoptosis accompanied by rise in FasL expression and caspase-3 activation. The above findings indicate that H₂O₂ as a bio-molecule modulates Leydig cell function at or below physiological concentrations through a variety of actions like decrease in steroidogenic enzyme activity and increase in oxidative stress and apoptosis.     

Ultrastructure of germ cells, the Leydig cells, and Sertoli cells during spermatogenesis in Boleophthalmus pectinirostris (Teleostei, Perciformes, Gobiidae)

The ultrastructures of germ cells, Leydig cells, and Sertoli cells during spermatogenesis in male Boleophthalmus pectinirostris were investigated by electron microscopic observations. During the period of maturation divisions, well-developed Leydig cells have three major morphological characteristics: a vesicular nucleus, mitochondria with tubular cristae, and a number of smooth endoplasmic reticulum. Based on cytoplasmic features, it appears that Leydig cells are responsible for the synthesis of male sex steroids. Although no clear evidence of steroidogenesis was found in the Sertoli cells, they were found to perform a phagocytic function in the seminiferous lobules. Most Sertoli cells contain granules thought to represent deposited glycogen or lipid but there is no indication of a transfer of nutrients to the spermatids. During the period of germ cell degeneration, several characteristics of phagocytosis appear in the cytoplasm of the Sertoli cells. In particular, it is assumed that the Sertoli cells are involved in the degeneration and resorption of undischarged spermatids after spermiation. No acrosome of the sperm is formed. The structure of the spermatozoon in B. pectinirostris is very similar and closely resembles to those of suborder Gobioidei (perciform type teleosts). The flagellum or sperm tail shows the typical 9+2 array of microtubules.     

Ultrastructure and morphometry of testicular Leydig cells and the interstitial components correlated with testosterone in aging rats

The ultrastructure of testicular interstitium in young and aged adult rats was analysed using morphometric methods, and the plasma testosterone concentration was measured. With increasing age there was an augumentation in the volume of collagen fibrils in the intercellular matrix and in blood vessels. During the aging process (approximately two years) the average volume of the Leydig cell decreased from 1364 m³ to 637 m³, but the number of Leydig cells in paired testes increased from 53x10⁶ to 113x10⁶. The absolute volume of smooth surfaced endoplasmic reticulum (SER) per Leydig cell amounted in aged rats to 78% of that in young adult rats. The total amount of SER in paired testes increased by 62% with aging. The present analysis suggests that the ability of SER to maintain peripheral testosterone concentration decreases with age. In young adult rats the absolute volume of peroxisomes per Leydig cell correlated significantly with the concentration of testosterone in blood and also with the absolute volume of SER per Leydig cell. These results combined with ultrastructural observations of close apposition of peroxisomes and SER suggest that peroxisomes have a role in testosterone secretion by Leydig cells.Visiting scientist to Laboratory of Electron Microscopy (Director: Prof. L.J. Pelliniemi)     

Two distinct origins for Leydig cell progenitors in the fetal testis

During the differentiation of the mammalian embryonic testis, two compartments are defined: the testis cords and the interstitium. The testis cords give rise to the adult seminiferous tubules, whereas steroidogenic Leydig cells and other less well characterized cell types differentiate in the interstitium (the space between testis cords). Although the process of testis cord formation is essential for male development, it is not entirely understood. It has been viewed as a Sertoli-cell driven process, but growing evidence suggests that interstitial cells play an essential role during testis formation. However, little is known about the origin of the interstitium or the molecular and cellular diversity within this early stromal compartment. To better understand the process of mammalian gonad differentiation, we have undertaken an analysis of developing interstitial/stromal cells in the early mouse testis and ovary. We have discovered molecular heterogeneity in the interstitium and have characterized new markers of distinct cell types in the gonad: MAFB, C-MAF, and VCAM1. Our results show that at least two distinct progenitor lineages give rise to the interstitial/stromal compartment of the gonad: the coelomic epithelium and specialized cells along the gonad–mesonephros border. We demonstrate that both these populations give rise to interstitial precursors that can differentiate into fetal Leydig cells. Our analysis also reveals that perivascular cells migrate into the gonad from the mesonephric border along with endothelial cells and that these vessel-associated cells likely represent an interstitial precursor lineage. This study highlights the cellular diversity of the interstitial cell population and suggests that complex cell–cell interactions among cells in the interstitium are involved in testis morphogenesis.