A quantitative morphological study of human Leydig cells from birth to adulthood

Summary Human testicular specimens were obtained from biopsies and autopsies covering the period from birth to adulthood. The number of testosterone-containing Leydig cells was determined using the peroxidase-anti-peroxidase method. This number decreased markedly from 3–6 months of age to the end of the first year of life and, up to 6 years of age, only a small number of testosterone-containing cells was found. From 6 years onwards the number of Leydig cells progressively increased. Ultrastructural examination revealed four types of Leydig cells: (1) fetal-type Leydig cells (from birth to 1 year of age) with round nuclei, abundant smooth endoplasmic reticulum and mitochondria with tubular cristae; (2) infantile-type Leydig cells (from birth to 8–10 years of age), showing a multilobated nucleus, moderately abundant smooth endoplasmic reticulum, some lipid droplets and mitochondria with parallel cristae; (3) prepubertal, partially differentiated Leydig cells (from 6 years of age onwards) with regularly-outlined round nuclei, abundant smooth endoplasmic reticulum, mitochondria with tubular cristae, and some lipid droplets and lipofuscin granules; and (4) mature adult Leydig cells (from 8–10 years of age onwards). The ultrastructure of the infantile-type Leydig cells and the lack of delay between the disappearance of the fetal-type Leydig cells and the appearance of infantile-type Leydig cells suggest that fetal-type Leydig cells give rise to the infantile-type Leydig cells. Before puberty, myofibroblast-like precursor cells differentiate into the prepubertal, partially differentiated Leydig cells, which complete their differentiation into the adult Leydig cells.This work was supported by grants from the Comisión Asesora de Investigation Científica y Técnica, and the Fondo de Investigaciones Sanitarias de la Seguridad Social, Madrid, Spain     

Mitosis in adult human Leydig cells

Summary The ultrastructural study of testicular biopsies from 87 adult men revealed mitosis in two mature Leydig cells, each from a different man. The men showed normal hormone levels and had received no previous chemotherapy or hormone treatment, nor had they been exposed to known toxic agents. The presence of mitotic Leydig cells suggests that differentiated Leydig cells may divide and contribute either to the increase in the number of Leydig cells or to the formation of multinucleate Leydig cells.This work was partially supported by a grant from the Comisión Asesora de Investigación Científica y Técnica, Madrid, Spain     

Seasonal changes in the seminiferous epithelium of rhesus and bonnet monkeys

With a view to elucidate seasonal variations in testicular spermatogenesis, quantitative analysis of spermatogenic cells was carried out in non-human primate species viz. rhesus (Macaca mulatta) and bonnet (M. radiata) monkeys during breeding (October-December) and non-breeding (May-June) seasons. The results revealed significant inhibition of testicular germ cell population during non-breeding compared with the breeding period in both the species. Quantitative determination of Sertoli cell-germ cell ratio showed a marked decrease in the number of type A-spermatogonia, spermatocytes (non-pachytene and pachytene) and spermatids (in steps 1-12 of spermiogenesis) in rhesus monkey during the non-breeding period. Bonnet monkeys exhibited the significant decline in the number of primary spermatocytes and spermatids during the non-breeding phase. In addition, average diameter of round seminiferous tubules and nuclear diameter of Leydig cells also decreased significantly in rhesus monkeys. However, bonnet monkeys did not show any significant change in nuclear diameter/morphology of Leydig cells, testicular tubular diameter and number of type A-spermatogoniae. Sertoli cell number did not show any significant change during both breeding and non-breeding periods in both the species. The results of this study indicate a prominent seasonal variation in testicular spermatogenic/Leydig cells in rhesus monkeys than those observed in bonnet monkeys.     

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.     

The expression profiles of fibroblast growth factor 9 and its receptors in developing mice testes

An expressional lack of fibroblast growth factor 9 (FGF9) would cause male-to-female sex reversal in the mouse, implying the essential role of FGF9 in testicular organogenesis and maturation. However, the temporal expression of FGF9 and its receptors during testicular development remains elusive. In this study, immunohistochemistry was used to identify the localization of FGF9 and its receptors at different embryonic and postnatal stages in mice testes. Results showed that FGF9 continuously expressed in the testis during development. FGF9 had highest expression in the interstitial region at 17–18 d post coitum (dpc) and in the spermatocytes, spermatids and Leydig cell on postnatal days (pnd) 35–65. Regarding receptor expression, FGFR1 and FGFR4 were evenly expressed in the whole testis during the embryonic and postnatal stages. However, FGFR2 and FGFR3 were widely expressed during the embryonic testis development with higher FGFR2 expression in seminiferous tubules at 16–18 dpc and higher FGFR3 expression in interstitial region at 17–18 dpc. In postnatal stage, FGFR2 extensively expressed with higher expression at spermatids and Leydig cells on 35–65 pnd and FGFR3 widely expressed in the whole testis. Taken together, these results strongly suggest that FGF9 is correlated with the temporal expression profiles of FGFR2 and FGFR3 and possibly associated with testis development.     

Postnatal somatic cell proliferation and seminiferous tubule maturation in pigs: A non-random event

Although seminiferous tubule maturation in horses begins in the central area of the testis, this process is thought to occur randomly throughout the testis in most mammals. Studies in our laboratory revealed that the establishment of spermatogenesis may not be a synchronous event in the testicular parenchyma of pigs. The objectives of the present study were to evaluate the pattern of seminiferous cord/tubule maturation and the morphological and functional characteristics of testicular somatic cells during postnatal development in three regions of the pig testis: a) near the tunica albuginea (TA); b) in the transitional area between the seminiferous tubules and mediastinum (TR); and c) in the intermediate area (ID) between the TA and TR. Based on the diameter of seminiferous cords/tubules, nucleus size of Sertoli cells and fluid secretion, mainly at 90 and 120 d of age, seminiferous tubule maturation was more advanced in the ID and TR. The mitotic activity of Sertoli cells was higher (P < 0.05) in the TR than the ID and TA at 7 and 120 d. Except for the mitotic index of the Leydig cells, which was lower (P < 0.05) in the ID at 7, 30, and 180 d than in the TA and TR, other Leydig cell ebd points, e.g., individual cell size, nuclear volume, and cytoplasmic volume, were consistently higher (P < 0.05) in the ID, suggesting that steroidogenesis was more active in this region during the period investigated. Overall, we inferred that Leydig cells in the ID may play a pivotal role in postnatal testis development in pigs and this type of cell is likely related to asynchronous testicular parenchyma development, with the transitional area providing the primary zone for growth of seminiferous tubules.     

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.     

Galectin-1, a cell adhesion modulator, induces apoptosis of rat Leydig cells in vitro

Galectin-1 (Gal-1), a beta galactoside-binding lectin, is involved in multiple biological functions, such as cell adhesion, apoptosis, and metastasis. On the basis of its ability to interact with extracellular matrix (ECM) glycoproteins, we investigated the Gal-1 effect on Leydig cells, which express and are influenced by ECM proteins. In this study, Gal-1 was identified in Leydig cell cultures by immunofluorescence. To gain insight into its biological role, Gal-1 was added to purified rat Leydig cells, under both basal and human chorionic gonadotrophin-stimulated conditions. Substantial morphological changes were observed, and cell viability showed an 80% decrease after 24 h culture. As a functional consequence of Gal-1 addition, testosterone production was reduced in a dose-dependent fashion, reaching a minimum of 26% after 24 h compared with basal values. cAMP showed a similar variation after 3 h. Assessment of DNA hypodiploidy and caspase activity determinations indicated that the reduction in viability and in steroidogenesis was caused by apoptosis induced by Gal-1. Besides, addition of Gal-1 caused Leydig cell detachment. Presence of laminin-1 or lactose prevented the effect of Gal-1, suggesting that the carbohydrate recognition domain is involved in inducing apoptosis. These findings demonstrate a novel mechanism, based on Gal-1 and laminin-1 interaction, which could help us better understand the molecular basis of Leydig cell function and survival control.     

The expression of neurotrophins and their receptors in the prenatal and adult human testis: evidence for functions in Leydig cells

Previous studies have demonstrated local functions for neurotrophins in the developing and mature testis of rodents. To examine whether these signaling molecules are present and also potentially active in the human testis, we characterized immunohistochemically the expression and cellular localization of the known neurotrophins and their receptors during prenatal testicular development as well as in the adult human testis. Results obtained revealed the presence of nerve growth factor (NGF), brain-derived neurotrophic factor, neurotrophin-3 and 4, as well as neurotrophin receptors p75^NTR, TrkA, TrkB, and TrkC during testis morphogenesis. These proteins were also detectable in the adult human testis, and their local expression could be confirmed largely by immunoblot and RT-PCR analyses. Remarkably, the Leydig cells were found to represent the predominant neurotrophin/receptor expression sites within both fetal and adult human testes. Functional assays performed with a mouse tumor Leydig cell line revealed that NGF exposure increases cellular steroid production, indicating a role in differentiation processes. These findings support previously-recognized neuronal characteristics of Leydig cells, provide additional evidence for potential roles of neurotrophins during testis morphogenesis and in the mature testis, and demonstrate for the first time a neurotrophin-induced functional activity in Leydig cells.     

大鼠睾丸中S-100蛋白生后发育变化的免疫细胞化学研究

本文应用免疫细胞方法,研究大鼠生后４天、７天、１４天、３０天、２个月和３个月睾丸中Ｓ－１００蛋白的分布和变化规律。结果表明：Ｓ－１００蛋白染色反应位于睾丸间质细胞,直到生后３０天才出现阳性细胞,数量少,着色浅,而２月龄和３月龄大鼠睾丸Ｓ－１００蛋白阳性细胞数量多,着色深。提示Ｓ－１００蛋白可能参与间质细胞的合成和分泌睾酮过程。     

Ultrastructural and biochemical characteristics of leydig cells from newborn androgen-insensitive rats

Summary Leydig cells of the testis of newborn pseudohermaphrodite (tfm) rats have an ultrastructure similar to that of the normal, containing well developed organelles and inclusions. The cytoplasm is filled with smooth endoplasmic reticulum forming a network of interconnected tubules. Lipid droplets are surrounded by cisternae of smooth endoplasmic reticulum and are in close association with pleomorphic mitochondria. Many of the latter are cup-shaped and have tubular cristae and intramitochondrial dense bodies.Essentially, these are characteristics of normal Leydig cells. Accordingly, the production of testosterone by testes from newborn tfm rats is the same as that by testes from normal newborns and adults. However, it is significantly higher than that by testes of tfm adults. Also, the plasma testosterone levels of newborn tfm rats are the same as in the normal newborn, but lower than in normal adults and much lower than in adult tfm animals.Thus, since in the tfm rat the morphology of Leydig cells, androgen production, and maintenance of plasma levels of testosterone are normal in the newborn, but become abnormal with advancing age, it appears that defective androgen action rather than insufficient androgen production is the cause of male pseudohermaphroditism.     

Postnatal differentiation of Leydig cells in the African green monkey

At two years of age the interstitial tissue of Cercopithecus aethiops is composed principally of undifferentiated, fibroblast-like cells. Also present during this time are scattered differentiating Leydig cells, which are characterized by a large nucleus, numerous mitochondria, elements of smooth reticulum, and small cisternae of rough reticulum. A mean level of 1.69 ± 0.66 ng/ml of testosterone was found. At three years Leydig cells are much more numerous and developed; since all the elements of steroid secreting cells are present, even their morphology differs from that observed in mature cells. Lipid accumulation is characteristic during this period. A mean testosterone level of 2.28 ± 0.47 ng/ml was found. Mature Leydig cells are basically similar to that of other mammals, while they differ significantly from that of human Leydig cells.     

Fate determination of fetal Leydig cells

Leydig cell (LC) is one of the most important somatic cell types in testis, which localized in the interstitium between seminiferous tubules. The major function of Leydig cells is to produce steroid hormone, androgens. LC differentiation exhibits a biphasic pattern in rodent testes, which are divided into two different temporal mature populations, fetal Leydig cells (FLCs) and adult Leydig cells (ALCs). FLCs are transiently present in fetal testes and undergo involution or degeneration after birth. FLCs are completely devoid and replaced by ALCs in adult testes. Comparing to ALCs, FLCs display unique morphology, ultrastructure and functions. The origin of FLCs has been debated for many years, but it is still a mystery. Many factors have been reported regulating the specification, proliferation and differentiation of FLCs. FLCs degenerate in a few weeks postnatally, however, the underlying mechanism is still unknown. In this review, we will focus on the fate determination of FLCs, and summarize the resent progress on the morphology, ultrastructure, function, origin and involution of FLCs.