The fate of fetal Leydig cells during the development of the fetal and postnatal rat testis

The ultrastructure and developmental fate of the fetal generation of Leydig cells of the rat testis was studied from the 17th day of fetal life up to 100 days after birth. The number of fetal Leydig cells per testis was determined by light microscopic morphometric analysis of semithin plastic sections. In fetal testes (days 17-22 postconception), Leydig cells exhibited a characteristic ultrastructure, containing smooth endoplasmic reticulum, many lipid inclusions and glycogen. Testes of 17-day-old fetuses contained about 25 x 10(3) fetal Leydig cells, rapidly increasing to 90 x 10(3) per testis in 21-day-old fetuses. After birth, fetal Leydig cells per testis remained relatively constant up to 2 weeks (80-90 x 10(3) per testis) and were identified by light and electron microscopy which showed their numerous lipid inclusions, their tendency for clustering and their association with interstitial tissue fibroblasts which partly encapsulated the fetal Leydig cells. From 21-100 days after birth, fetal Leydig cell numbers were quite variable with a mean of 45-60 x 10(3) per testis. These results are the first to show that the fetal generation of Leydig cells persist in the adult testis and do not undergo early postnatal degeneration or dedifferentiation into other interstitial cells. The simultaneous occurrence of the fetal Leydig cells and the adult population of Leydig cells indicates that these cells are distinct cell generations which are developmentally unrelated.     

Changes of the lingual epithelium in Ambystoma mexicanum

Changes in the lingual epithelium during ontogenesis and after induced metamorphosis in Ambystoma mexicanum are described as observed by light microscopy and scanning electron microscopy. The epithelium of the tongue is always multilayered in the larva as well as in the adult. It consists of a stratum germinativum with little differentiated basal cells and a stratum superficiale (superficial layer) with specialized superficial cells and goblet cells. Usually, there are more than two layers because of a stratum intermedium consisting of replacement cells. The apical cell membrane of the superficial cells is perforated by fine pores. Its most typical feature are microridges. Maturing superficial cells possess microvilli. Goblet cells occur in early larvae primarily in the centre of the tongue. They spread throughout the dorsal face of the tongue as their numbers increase during ontogenesis. The small apices of the goblet cells are intercalated in the wedges between the superficial cells. Leydig cells are not found on the larval tongue but on that of adults. Due to metamorphosis, the epithelium of the tongue changes. It is furrowed in its anterior part. The furrows house the openings of the lingual glands. The surface is further modulated by ridges which are densely coated by microvilli and which bear the taste buds. The villi of the tongue which lack extrusion pores show cilia and microvilli but lack microridges. The Leydig cells disappear during metamorphosis. In addition to the two types of goblet cells found in different regions of the glandular tubules, goblet cells occur in the caudal part. They secrete directly into the cavity of the mouth. The posterior part is characterised by a dense coat of cilia.     

Developmental Changes in the Epidermal Surface Cells of Salamander Larva

Epidermal surface cells were studied ultrastructurally and histochemically at various larval stages in Salamandra salamandra. The most outstanding cellular structures undergoing changes were found to be secretory vesicles, mitochondria and tonofilaments. The highest density of secretory vesicles was noticed immediately after birth, declining in number at later stages and almost disappearing at metamorphosis. The mitochondria, whose appearance at early stages indicates a high metabolic activity, hypertrophy and degenerate close to metamorphosis. Simultaneously, there is an increase in the cellular tonofilaments' density reaching its climax with keratinization.     

Postnatal differentiation of the gametogenic and endocrine functions of the testis in the tree-shrew (Tupaia belangeri)

Summary Testicular development was studied in Tupaia belangeri (tree-shrew) from birth to sexual maturity. At birth the seminiferous cords contained peripheral supporting cells and centrally located gonocytes. Large foetal Leydig cells were prominent in the interstitium. The mitotic index of the gonocytes was low at birth and rose to peak levels at Day 20, following the regression of the foetal generation of Leydig cells, and during the nadir in circulating testosterone concentrations. Mitotic activity returned to low levels at Day 30 in association with the reappearance of differentiated Leydig cells and the first signs of increased androgenesis. The negative temporal relationship between mitogenesis and androgenic function suggests that the proliferation of the gonocytes does not require, and may be inhibited by, high titres of androgens. Post-mitotic development of the gonocytes occurred during a period of rising testosterone levels, and the first appearance of spermatogonia coincided with peak testosterone levels. This indicates that androgens may be specifically involved in the initiation of spermatogenesis. Spermatogenesis progressed to completion during a phase of declining testosterone levels. The precise temporal correlations established during post-natal development suggest that the tree-shrew is a suitable animal model for studies on the endocrine control of the initiation of spermatogenesis in primates.     

Cell proliferation and hormonal changes during postnatal development of the testis in the pig

Histometrical evaluation of the testis was performed in 36 Piau pigs from birth to 16 mo of age to investigate Sertoli cell, Leydig cell, and germ cell proliferation. In addition, blood samples were taken in seven animals from 1 wk of age to adulthood to measure plasma levels of FSH and testosterone. Sertoli cell proliferation in pigs shows two distinct phases. The first occurs between birth and 1 mo of age, when the number of Sertoli cells per testis increases approximately sixfold. The second occurs between 3 and 4 mo of age, or just before puberty, which occurs between 4 to 5 mo of age, when Sertoli cells almost double their numbers per testis. The periods of Sertoli cell proliferation were concomitant with high FSH plasma levels and prominent elongation in the length of seminiferous cord/tubule per testis. Leydig cell volume increased markedly from birth to 1 mo of age and just before puberty. In general, during the first 5 mo after birth, Leydig cell volume growth showed a similar pattern as that observed for testosterone plasma levels. Also, the proliferation of Leydig cells per testis before puberty showed a pattern similar to that observed for Sertoli cells. However, Leydig cell number per testis increased up to 16 mo of age. Substantial changes in Leydig cell size were also observed after the pubertal period. From birth to 4 mo of age, germ cells proliferated continuously, increasing their number approximately two- to fourfold at each monthly interval. A dramatic increase in germ cells per cross-section of seminiferous tubule was observed from 4 to 5 mo of age; their number per tubule cross-section stabilized after 8 mo. To our knowledge, this is the first longitudinal study reporting the pattern of Sertoli cell, germ cell, and Leydig cell proliferative activity in pigs from birth to adulthood and the first study to correlate these events with plasma levels of FSH and testosterone.     

Basement membrane and epithelial features of fetal-type Leydig cells in rat and human testis

The basement membranes of developing Leydig cells in fetal and newborn testis of rat were studied by ultrastructural and immunocytochemical methods. Fetal-type Leydig cells in prenatal rats were organized in irregularly outlined groups in the interstitium and were extensively surrounded by ultrastructurally identifiable basement membranes and immunocytochemically localized laminin and collagen type IV. Prenatal Leydig cell precursors had small patches of laminin and collagen type IV on their surfaces, which indicated that changes in extracellular matrix took place during their differentiation to mature fetal-type Leydig cells. Additionally, ultrastructural evidence was obtained for a basement membrane surrounding the fetal human Leydig cells similar to that in fetal rats. Soon after birth the rat fetal-type cells gathered into distinct clusters surrounded by delicate envelope cells and a discontinuous basement membrane. Basement-membrane structures, laminin, and collagen type IV were observed between the clustered cells as well. The basement membranes covering large cell surface areas of the fetal-type Leydig cells in fetal and newborn rats differed from those of the adult-type cells, which, according to our earlier study, are covered only by small patches of basement membrane. The difference between the basement membranes of the fetal- and adult-type rat Leydig cells further supports the concept of two different Leydig cell populations. The earlier findings of the epithelial nature of the Leydig cells agree with the observation of basement membranes in the Leydig cells.     

Metamorphosis of the central nervous system of Drosophila

The study of the metamorphosis of the central nervous system of Drosophila focused on the ventral CNS. Many larval neurons are conserved through metamorphosis but they show pronounced remodeling of both central and peripheral processes. In general, transmitter expression appears to be conserved through metamorphosis but there are some examples of possible changes. Large numbers of new, adult-specific neurons are added to this basic complement of persisting larval cells. These cells are produced during larval life by embryonic neuroblasts that had persisted into the larval stage. These new neurons arrest their development soon after their birth but then mature into functional neurons during metamorphosis. Programmed cell death is also important for sculpting the adult CNS. One round of cell death occurs shortly after pupariation and a second one after the emergence of the adult fly.     

Abnormal development of the testis after administration of the Leydig cell cytotoxic ethylene-1,2-dimethanesulphonate to the immature rat

Male rats were injected with 50 mg ethylene-1,2-dimethanesulphonate/kg from Day 5 to Day 16 after birth and control rats received injections of the same volume of vehicle. Testes were studied at various times from Day 6 to Day 108 using histochemistry, light and electron microscopy. Fine structural degenerative changes were observed in the Leydig cells and seminiferous tubules of EDS-treated animals as early as Day 6. By Day 11 no Leydig cells could be detected and the interstitia of EDS-treated testes contained large numbers of fibroblast-like cells which formed peritubular collars 3-5 cells thick; the tubules contained Sertoli cells with heterogeneous inclusions and large numbers of lipid droplets. A small number of Leydig cells was found at Day 14 and their numbers increased so that, in animals of 28 days and older, large clusters of Leydig cells were present between severely atrophic tubules. These tubules contained Sertoli cells with few organelles; germinal cells were not observed after 28 days in EDS-treated animals. These results show that EDS destroys the fetal population of Leydig cells postnatally and this mimics the well documented effect of EDS on adult Leydig cells. The seminiferous tubules were permanently damaged by EDS in the present experiments. Tubular damage could have been due to a direct cytotoxic effect of multiple injections of EDS on the tubule before the blood-testis barrier develops or due to withdrawal of androgen support secondary to Leydig cell destruction.     

Developmental stages of fetal-type Leydig cells in prepubertal rats

Fetal Leydig cells were studied in rats during and after the perinatal-neonatal period by comparing changes in morphology, number and volume with changes in testicular steroids and serum luteinizing hormone (LH) concentration. Stereologic examination indicated regression of fetal Leydig cells in testis by showing that their total volume as well as the average cell volume decreased between prenatal day 20 and postnatal day 3. The total number and total volume of cells both increased between postnatal days 3 and 11 but the average cell volume did not change during the same time period. Determination of serum LH showed a close correlation between an increase in LH concentration and increases in total number and volume of cells. The combined number of fetal- and adult-type Leydig cells on day 20 was more than 20 times the number of fetal cells at 3 days of age. Electron microscopic analysis showed that fetal Leydig cells after birth formed conspicuous clusters, which were surrounded by a layer of envelope cells and extracellular material. Occasional dividing fetal Leydig cells and possible precursors of fetal or adult Leydig cells were observed. Mitoses of spindle-shaped pericordal cells were frequent during the neonatal period. During and after the second postnatal week fetal Leydig cells again showed signs of regression, indicated by disintegration of the cell clusters, a decrease in cell size, accumulation of collagen between the cells and a decrease in steroid content per cell.(ABSTRACT TRUNCATED AT 250 WORDS)     

Oncostatin M inhibits differentiation of rat stem Leydig cells in vivo and in vitro

Oncostatin M (OSM) is a pleiotropic cytokine within the interleukin six family of cytokines, which regulate cell growth and differentiation in a wide variety of biological systems. However, its action and underlying mechanisms on stem Leydig cell development are unclear. The objective of the present study was to investigate whether OSM affects the proliferation and differentiation of rat stem Leydig cells. We used a Leydig cell regeneration model in rat testis and a unique seminiferous tubule culture system after ethane dimethane sulfonate (EDS) treatment to assess the ability of OSM in the regulation of proliferation and differentiation of rat stem Leydig cells. Intratesticular injection of OSM (10 and 100 ng/testis) from post‐EDS day 14 to 28 blocked the regeneration of Leydig cells by reducing serum testosterone levels without affecting serum luteinizing hormone and follicle‐stimulating hormone levels. It also decreased the levels of Leydig cell‐specific mRNAs (Lhcgr, Star, Cyp11a1, Hsd3b1, Cyp17a1 and Hsd11b1) and their proteins by the RNA‐Seq and Western blotting analysis. OSM had no effect on the proliferative capacity of Leydig cells in vivo. In the seminiferous tubule culture system, OSM (0.1, 1, 10 and 100 ng/mL) inhibited the differentiation of stem Leydig cells by reducing medium testosterone levels and downregulating the expression of Leydig cell‐specific genes (Lhcgr, Star, Cyp11a1, Hsd3b1, Cyp17a1 and Hsd11b1) and their proteins. OSM‐mediated action was reversed by S3I‐201 (a STAT3 antagonist) or filgotinib (a JAK1 inhibitor). These data suggest that OSM is an inhibitory factor of rat stem Leydig cell development.     

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     

Organization of interstitial tissue in the testis of the salamander Necturus maculosus (Caudata: Proteidae)

In Necturus maculosus the organization of the interstitial tissue varies according to the stage of spermatogenesis. Leydig cells at various stages of differentiation and myoid cells are always present in this tissue. The Leydig cells are undifferentiated at all phases of germ cell activity and only hypertrophy following spermiation and degeneration of Sertoli cells. These Leydig cells are structurally analogous to mammalian Leydig cells. They do not form part of the lamina propria of the seminiferous lobules and hence cannot be referred to as lobule-boundary cells previously described in the urodele testis (Lofts, '74). When the Leydig cells hypertrophy, numerous unmyelinated axons appear in the interstitial tissue. These axons, often devoid of Schwann-cell cytoplasm, occur in close proximity to Leydig cells. Because the levels of both Substance P and neurotensin increased in the testis of Necturus maculosus as Leydig cells differentiated, we concluded that these neural elements may regulate Leydig-cell function locally, through the release of neuropeptides.     

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.     

Pathogenesis of cryptorchidism.

Cryptorchidism was induced experimentally by treating pregnant mice on the 14th day of pregnancy with 5 mg estrogen. Testes from cryptorchid and control newborn and adult mice were investigated with radioimmunoassay and electron microscopy. It was concluded that a normal Leydig cell function plays a decisive role in testicular descent. In cryptorchidism, Leydig cells at birth are atrophic. Testicular testosterone content is diminished as compared to controls. Ultrastructural alterations of Leydig cells observed in our experiments closely resemble those found in biopsies of cryptorchid patients. In male mouse offspring, prenatal estrogen injection induced not only a cryptorchidism but also Leydig cell atrophy and a permanently impaired function. Testosterone content is still significantly diminished after puberty. It is proposed therefore that an insufficiency of endocrine gonadal function of hypothalamo-pituitary origin occurring during intrauterine development is one of the main causes of cryptorchidism. An appropriate long-term therapy could diminish the high sterility rate.     

Fetal Leydig cells: cellular origin, morphology, life span, and special functional features.

The Leydig cells, responsible for testicular androgen production, have two growth phases during the life-span of mammals. The fetal population appears during fetal life and is responsible for the androgen-induced differentiation of the male genitalia. The fetal Leydig cells disappear after birth, and the other population, the adult Leydig cells, appears during puberty and persists for the whole adult life. The fetal Leydig cells, evidently due to the intrauterine endocrine milieu and their special functional requirements in genital differentiation, differ both morphologically and functionally from the adult population. The purpose of this review is to elucidate the special features of the mammalian fetal Leydig cell population, which presents an intriguing experimental model for studies of function and regulation of steroidogenic cells.     

Developmental fates of larval tissues after metamorphosis in the ascidian, Halocynthia roretzi

Cell lineages during ascidian embryogenesis are invariant. Developmental fates of larval mesodermal cells after metamorphosis are also invariant with regard to cell type of descendants. The present study traced developmental fates of larval endodermal cells after metamorphosis in Halocynthia roretzi by labeling each endodermal precursor blastomere of larval endoderm. Larval endodermal cells gave rise to various endodermal organs of juveniles: endostyle, branchial sac, peribranchial epithelium, digestive organs, peripharyngeal band, and dorsal tubercle. The boundaries between clones descended from early blastomeres did not correspond to the boundaries between adult endodermal organs. Although there is a regular projection from cleavage stage and larval stage to juvenile stage, this varies to some extent between individuals. This indicates that ascidian development is not entirely deterministic. We composed a fate map of adult endodermal organs in larval endoderm based on a statistical analysis of many individual cases. Interestingly, the topographic position of each prospective region in the fate map was similar to that of the adult organ, indicating that marked rearrangement of the positions of endodermal cells does not occur during metamorphosis. These findings suggest that fate specification in endoderm cells during metamorphosis is likely to be a position-dependent rather than a deterministic and lineage-based process. Received: 16 June 1999 / Accepted: 16 August 1999     

Physiological and anatomical properties of Leydig cells in the segmental nervous system of the leech

Summary Each of the 21 segmental ganglia in the American leechMacrobdella decora contains a pair of Leydig cells (ca. 45 m) each of which is located in a posteriolateral glial packet. Leydig cells exhibit spontaneous action potentials (1–10/s) whose duration and undershoot depend upon membrane polarization. The two Leydig cells within each ganglion are bidirectionally-coupled (V ₂/V ₁0.3). Pairs of ipsilateral Leydig cells within adjacent ganglia are mutually excitatory such that an impulse in one generates an impulse in the other. The interganglionic latency for any cell pair is constant regardless of the direction of impulse conduction and is unchanged by 20 mM Mg²⁺ saline. These data indicate that the interactions are not mediated by chemical synapses. Additionally, the results of collision experiments lead us to infer that ipsilateral Leydig cell pairs utilize common axonal pathways for interganglionic interactions. If Leydig cells are driven by current injection to fire impulses at frequencies of six to ten per s, cells in adjacent ganglia exhibit impulse failures. The combination of spontaneous activity, intraganglionic coupling and interganglionic interactions results in the generation of constant, low frequency impulse activity and can cause impulse reverberations.The branching pattern of Leydig cells filled with HRP is consistent with their functional properties and connectivity. Each cell sends axons to both adjacent ganglia through the ipsilateral connectives and projects to the periphery only by the lateral roots of these adjacent ganglia. This unusual morphology was verified Lucifer Yellow CH.In addition to intraganglionic dye-coupling, dye coupling was occasionally evident between ipsilateral cells in adjacent ganglia. Electron microscopy of Leydig cells depicts abundant 100 nm granules in both their somata and neuropilar processes. Although this fine structure suggests a neurosecretory role, we were unable to discern a peripheral function for these neurons.Abbreviation H R P horseradish peroxidase     

Histological, histochemical, enzyme histochemical and ultrastructural investigations of the testis of Padogobius martensi between annual breeding seasons

From July to March, the testis of the spring‐spawning freshwater goby Padogobius martensi is characterized by spermatogonial proliferation. A close correlation exists among type of proliferating spermatogonia, gonado‐somatic (I_G) profiles and morphological and functional variations of the Leydig cells. The I_G reach their minimal levels by the end of summer and increase progressively but modestly during autumn and winter. Declining I_G levels are associated with proliferation of primary spermatogonia only, whereas increasing I_G levels are associated with predominant proliferation of secondary spermatogonia. Minimal I_G levels are reached when the germinal epithelium is formed by a continuum of primary spermatogonia and associated Sertoli cells. The proliferation of secondary spermatogonia begins only at this time. Spermatogenesis in autumn occurs when spermatogonial cysts contain at the most 16 cells and it rarely results in the maturation of several cysts so that the amount of sperm cells produced is either negligible or scarce. A number of degenerating cells are usually present within the spermatogonial and meiotic cysts. Leydig cells are the unique cells that display features of steroidogenic cells: mitochondria with tubular cristae, extensive smooth endoplasmic reticulum (SER), 3β‐hydroxysteroid dehydrogenase (3β‐HSD) and glucose‐6‐phosphate dehydrogenase (G6PD) activity and sudanophilia. Light and dark Leydig cell varieties are always present. During regression, Leydig cells undergo a marked decrease in SER amount, mitochondrial sizes and number of mitochondrial cristae. In parallel, the 3β‐HSD and G6PD activities and sudanophilia decrease progressively until they become undetectable by the end of regression. In autumn, mitochondria increase in size, reaching sizes similar to those observed at the end of the spawning season in the light cells, but not in the dark cells. The SER, on the contrary, undergoes a modest and irregular increase only in a part of the Leydig cells, mostly of the light type. In parallel, the 3β‐HSD and G6PD activities increase until they become moderately intense by the end of autumn. At the end of winter, the SER is extensive and regularly dilated in both Leydig cell types, whereas mitochondria still have sizes similar to those observed in December. The 3β‐HSD and G6PD activities are strong and sudanophilia is again detectable. Sertoli cells undergo changes in shape and position in relation to the proliferation of primary spermatogonia and the development of cysts. A junction modulation occurs in association with these changes. Sertoli cells also undergo changes indicative of a decrease in activity immediately after spawning (loss of mitochondrial cristae and clarification of the mitochondrial matrix) and of an increase in activity by the end of the regressing phase (darkening of the mitochondrial matrix and increase in mitochondrial cristae, rough endoplasmic reticulum (RER) and free ribosomes). In addition, they are involved in the phagocytosis of degenerating germ cells at all stages of their development. Macrophages are found in the testis interstitium only, where they are usually adjacent to Leydig cells, myoid cells and blood capillaries and do not participate in the phagocytosis of degenerating germ cells. Myoid cells do not undergo ultrastructural changes except for an increase in the amount of heterochromatin by the end of spawning. The meaning of the autumnal spermatogenic wave and the relationships between the development of the germinal epithelium and the changes of the Leydig and Sertoli cells are discussed.     

Epidermal growth factor regulates the development of stem and progenitor Leydig cells in rats

Epidermal growth factor (EGF) has many physiological roles. However, its effects on stem and progenitor Leydig cell development remain unclear. Rat stem and progenitor Leydig cells were cultured with different concentrations of EGF alone or in combination with EGF antagonist, erlotinib or cetuximab. EGF (1 and 10 ng/mL) stimulated the proliferation of stem Leydig cells on the surface of seminiferous tubules and isolated CD90⁺ stem Leydig cells and progenitor Leydig cells but it blocked their differentiation. EGF also exerted anti‐apoptotic effects of progenitor Leydig cells. Erlotinib and cetuximab are able to reverse EGF‐mediated action. Gene microarray and qPCR of EGF‐treated progenitor Leydig cells revealed that the down‐regulation of steroidogenesis‐related proteins (Star and Hsd3b1) and antioxidative genes. It was found that EGF acted as a proliferative agent via increasing phosphorylation of AKT1. In conclusion, EGF stimulates the proliferation of rat stem and progenitor Leydig cells but blocks their differentiation.