Differentiation of adult Leydig cells

Adult Leydig cells originate within the testis postnatally. Their formation is a continuous process involving gradual transformation of progenitors into the mature cell type. Despite the gradual nature of these changes, studies of proliferation, differentiation and steroidogenic function in the rat Leydig cell led to the recognition of three distinct developmental stages in the adult Leydig cell lineage: Leydig cell progenitors, immature Leydig cells and adult Leydig cells. In the first stage, Leydig cell progenitors arise from active proliferation of mesenchymal-like stem cells in the testicular interstitium during the third week of postnatal life and are recognizable by the presence of Leydig cell markers such as histochemical staining for 3β-hydroxysteroid dehydrogenase (3β-HSD) and the present of luteinizing hormone (LH) receptors. They proliferate actively and by day 28 postpartum differentiate into immature Leydig cells. In the second stage, immature Leydig cells are morphologically recognizable as Leydig cells. They have an abundant smooth endoplasmic reticulum and are steroidogenically active, but primarily produce 5-reduced androgens rather than testosterone. Immature Leydig cells divide only once, giving rise to the total adult Leydig cell population. In the third and final stage, adult Leydig cells are fully differentiated, primarily produce testosterone and rarely divide. LH and androgen act together to stimulate differentiation of Leydig cell progenitors into immature Leydig cells. Preliminary data indicate that insulin like growth factor-1 (IGF-1) acts subsequently in the transformation of immature Leydig cells into adult Leydig cells.     

Differentiation of the adult Leydig cell population in the postnatal testis

Five main cell types are present in the Leydig cell lineage, namely the mesenchymal precursor cells, progenitor cells, newly formed adult Leydig cells, immature Leydig cells, and mature Leydig cells. Peritubular mesenchymal cells are the precursors to Leydig cells at the onset of Leydig cell differentiation in the prepubertal rat as well as in the adult rat during repopulation of the testis interstitium after ethane dimethane sulfonate (EDS) treatment. Leydig cell differentiation cannot be viewed as a simple process with two distinct phases as previously reported, simply because precursor cell differentiation and Leydig cell mitosis occur concurrently. During development, mesenchymal and Leydig cell numbers increase linearly with an approximate ratio of 1:2, respectively. The onset of precursor cell differentiation into progenitor cells is independent of LH; however, LH is essential for the later stages in the Leydig cell lineage to induce cell proliferation, hypertrophy, and establish the full organelle complement required for the steroidogenic function. Testosterone and estrogen are inhibitory to the onset of precursor cell differentiation, and these hormones produced by the mature Leydig cells may be of importance to inhibit further differentiation of precursor cells to Leydig cells in the adult testis to maintain a constant number of Leydig cells. Once the progenitor cells are formed, androgens are essential for the progenitor cells to differentiate into mature adult Leydig cells. Although early studies have suggested that FSH is required for the differentiation of Leydig cells, more recent studies have shown that FSH is not required in this process. Anti-Müllerian hormone has been suggested as a negative regulator in Leydig cell differentiation, and this concept needs to be further explored to confirm its validity. Insulin-like growth factor I (IGF-I) induces proliferation of immature Leydig cells and is associated with the promotion of the maturation of the immature Leydig cells into mature adult Leydig cells. Transforming growth factor alpha (TGFalpha) is a mitogen for mesenchymal precursor cells. Moreover, both TGFalpha and TGFbeta (to a lesser extent than TGFalpha) stimulate mitosis in Leydig cells in the presence of LH (or hCG). Platelet-derived growth factor-A is an essential factor for the differentiation of adult Leydig cells; however, details of its participation are still not known. Some cytokines secreted by the testicular macrophages are mitogenic to Leydig cells. Moreover, retarded or absence of Leydig cell development has been observed in experimental models with impaired macrophage function. Thyroid hormone is critical to trigger the onset of mesenchymal precursor cell differentiation into Leydig progenitor cells, proliferation of mesenchymal precursors, acceleration of the differentiation of mesenchymal cells into Leydig cell progenitors, and enhance the proliferation of newly formed Leydig cells in the neonatal and EDS-treated adult rat testes.     

The intertubular compartment morphometry in capybaras (Hydrochoerus hydrochaeris) testis

The aim of the present study was to characterize the intertubule element volume density, individual and total Leydig cells volume, Leydig cell number per testis and per gram of testis, and leydigosomatic index in adult capybaras. Eight capybaras from a commercial abattoir were utilized. The intertubular compartment volume density and the Leydig cells were 45.2 and 31.13%, respectively. The individual and total Leydig cell volumes were 8.51 and 2169.41 x 10(-12) mL, respectively. The Leydig cell number per testis was 3.8 billion and the Leydig cell number per gram of testis was 126 million. The leydigosomatic index was 0.037%. In conclusion, this study shows that capybaras have one of the greatest individual and total Leydig cell volume and Leydig cell volume density, and that the Leydig cell number per gram of testis is at least double the mean for mammals previously investigated in its order.     

Leydig cell development

This review is about the study of the testis Leydig cells formation and development in prenatal and postnatal periods. Leydig cells of testis are the main place of synthesis and secretion of androgens including testosterone--the main male sexual hormone. Testosterone plays an important role in male reproduction regulation. There are two types (two populations) of Leydig cells during ontogenesis. The first type is fetal Leydig cells, which appear and function in the prenatal masculinization period of the male urogenital system. Another type is adult Leydig cells, which originate during sexual maturation postnatally. Fetal and adult Leydig cells pass the same stages both in the prenatal and postnatal periods. They are Leydig cell progenitors, immature Leydig cells and adult Leydig cells.     

Seasonal variation in the total volume of Leydig cells in stallions is explained by variation in cell number rather than cell size

Stereological methods were employed in two studies with stallions 1) to determine if seasonal variation in the total volume of Leydig cells is a function of cell number or cell size and 2) to characterize the annual cycle of the Leydig cell population. In the first study, numbers of Leydig cells were calculated for 28 adult (4-20 yr) stallions in the breeding or nonbreeding seasons from nuclear volume density (percentage of the decapsulated testicular volume), parenchymal volume (decapsulated testicular volume), and the volume of individual Leydig cell nuclei. The average volume of the individual Leydig cells was calculated as the total Leydig cell volume/testis (volume density of Leydig cells in the parenchymal volume times parenchymal volume) divided by the number of Leydig cells. The average volume of an individual Leydig cell varied within each season, but means were almost identical for the nonbreeding (6.94 +/- 0.61 picoliter) and breeding (6.91 +/- 0.45 picoliter) seasons. However, Leydig cell numbers per testis were 57% higher in the breeding season, which also had a 58% higher total volume of Leydig cells per testis. In the second study, the numbers of Leydig cells were determined for 43-48 adult horses in each 3-mo period for 12 mo. The number of Leydig cells per testis in May-July was higher (p less than 0.05) than in August-October or February-April, and higher (p less than 0.01) than in November-January. Thus, seasonal fluctuations in the total volume of Leydig cells in adult stallions is a function of the number of Leydig cells that cycle annually.     

Effects of thyroid hormones on Leydig cells in the postnatal testis

Thyroid hormones (TH) stimulate oxidative metabolism in many tissues in the body, but testis is not one of them. Therefore, in this sense, testis is not considered as a target organ for TH. However, recent findings clearly show that TH have significant functions on the testis in general, and Leydig cells in particular; this begins from the onset of their differentiation through aging. Some of these functions include triggering the Leydig stem cells to differentiate, producing increased numbers of Leydig cells during differentiation by causing proliferation of Leydig stem cells and progenitors, stimulation of the Leydig cell steroidogenic function and cellular maintenance. The mechanism of action of TH on Leydig cell differentiation is still not clear and needs to be determined in future studies. However, some information on the mechanisms of TH action on Leydig cell steroidogenesis is available. TH acutely stimulate testosterone production by the Leydig cells in vitro via stimulating the production of steroidogenic acute regulatory protein (StAR) and StAR mRNA in Leydig cells; StAR is associated with intracellular trafficking of cholesterol into the mitochondria during steroid hormone synthesis. However, the presence and/or the types of TH receptors in Leydig cells and other cell types of the Leydig cell lineage is still to be resolved. Additionally, it has been shown that thyrotropin-releasing hormone (TRH), TRH receptor and TRH mRNA in the testis in many mammalian species are seen exclusively in Leydig cells. Although the significance of the latter observations are yet to be determined, these findings prompt whether hypothalamo-pituitary-thyroid axis and hypothalamo-pituitary-testis axis are short-looped through Leydig cells.     

Effect of luteinizing hormone deprivation in situ on steroidogenesis of rat Leydig cells purified by a multistep procedure

Depriving rats of luteinizing hormone (LH) causes Leydig cells to lose smooth endoplasmic reticulum and diminishes their P450 C17-hydroxylase/C17,20-lyase activity (Wing et al., 1984). LH administration to hypophysectomized rats prevents these changes in Leydig cell structure and function (Ewing and Zirkin, 1983). We adopted a multistep procedure of rat Leydig cell isolation to study the trophic effects of LH on steroidogenesis in the Leydig cell. Our method employs vascular perfusion, enzymatic dissociation, centrifugal elutriation, and Percoll gradient centrifugation. The purified Leydig cell fraction obtained after Percoll density-gradient centrifugation contains 95% well-preserved 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD)-staining cells with ultrastructural characteristics of Leydig cells. These Leydig cells produced 248 and 29 ng of testosterone/10(6) Leydig cells when incubated for 3 h with and without a maximally stimulating concentration of ovine LH. Purified Leydig cells obtained from control rats and rats treated with testosterone-estradiol (T-E) implants for 4 days to inhibit LH production were incubated with a saturating concentration (2 microns) of pregnenolone. Leydig cells from control and T-E-implanted rats produced 537 and 200 ng of testosterone/10(6) Leydig cells X 3 h, respectively, suggesting a defect in the steroidogenic reactions converting pregnenolone to testosterone in Leydig cells from T-E-implanted rats. By using rabbit antibodies to the P450 C17-hydroxylase/C17,20-lyase pig microsomal enzyme, immunoblots of one-dimensional sodium dodecyl sulfate polyacrylamide gels of Leydig cell microsomal protein from control and 4- and 12-day T-E implanted rats revealed a continued loss of enzyme as the period of LH withdrawal continues. These results show that Leydig cells from animals deprived of LH had diminished capacity to convert pregnenolone to testosterone and reduced P450 C17-hydroxylase/C17,20-lyase content.     

Seminiferous tubules and daily sperm production in older adult men with varied numbers of Leydig cells

Previous studies of adult men have failed to reveal a relationship between numbers of Leydig cells in the testes and rates of sperm production, perhaps because of a functional excess of these cells in younger men. Hence, a possible relationship between Leydig cell numbers and sperm production was sought in 50 older men, aged 50-90 years, in whom the Leydig cell population had been depleted by age-related attrition. When these men were sorted by increasing numbers of Leydig cells per man into two, three, or five groups, no difference could be found between or within these groups when daily sperm production per man (DSP); seminiferous tubular volume, diameter, or length; or seminiferous epithelial volume was examined. Furthermore, no significant correlation could be detected between Leydig cell numbers and DSP in these 50 men. The only relationship between numbers of Leydig cells and spermatogenesis appeared to be a threshold effect, in that men with fewer than 60 million Leydig cells (4 in this study) had drastically reduced DSP. Men with few Leydig cells tended to have larger Leydig cells, and the increased size was due to more cytoplasm instead of nucleoplasm. There were weak but significant positive correlations between total Leydig cell cytoplasm per man and DSP and between average size of a Leydig cell and DSP. These findings suggest that a relationship may exist between sperm production and the amount of cytoplasm containing testosterone-producing organelles in surviving Leydig cells of older men.     

Leydig cells, thyroid hormones and steroidogenesis

Leydig cells are the primary source of androgens in the mammalian testis. It is established that the luteinizing hormone (LH) produced by the anterior pituitary is required to maintain the structure and function of the Leydig cells in the postnatal testis. Until recent years, a role by the thyroid hormones on Leydig cells was not documented. It is evident now that thyroid hormones perform many functions in Leydig cells. For the process of postnatal Leydig cell differentiation, thyroid hormones are crucial. Thyroid hormones acutely stimulate Leydig cell steroidogenesis. Thyroid hormones cause proliferation of the cytoplasmic organelle peroxisome and stimulate the production of steroidogenic acute regulatory protein (StAR) and StAR mRNA in Leydig cells; both peroxisomes and StAR are linked with the transport of cholesterol, the obligatory intermediate in steroid hormone biosynthesis, into mitochondria. The presence of thyroid hormone receptors in Leydig cells and other cell types of the Leydig lineage is an issue that needs to be fully addressed in future studies. As thyroid hormones regulate many functions of Sertoli cells and the Sertoli cells regulate certain functions of Leydig cells, effects of thyroid hormones on Leydig cells mediated via the Sertoli cells are also reviewed in this paper. Additionally, out of all cell types in the testis, the thyrotropin releasing hormone (TRH), TRH mRNA and TRH receptor are present exclusively in Leydig cells. However, whether Leydig cells have a regulatory role on the hypothalamo-pituitary-thyroid axis is currently unknown.     

Inhibitory effect of Sertoli cell-cultured media on LH binding to mouse Leydig cells in culture

The aim of this study is to examine the influence of Sertoli cells on LH binding to Leydig cells in culture in immature mice. Leydig cells and Sertoli cells were obtained from the testes of immature C57BL/6Ncrj mice and were cultured in serum-free medium for 7 days. The LH binding to Leydig cells and the FSH binding to Sertoli cells were dependent on incubation time, the number of cells, and the amount of labelled hormone added. The dissociation constant for LH binding to Leydig cells was 7.3 x 10(-10) M. Co-culture of Leydig cells with Sertoli cells for 7 days decreased LH binding to Leydig cells. The binding was 34.9% of that to Leydig cells cultured alone. After cultivation of Leydig cells with spent Sertoli cell-cultured medium (SM) for the last 4 days of the 7-day culture period, LH binding to Leydig cells decreased to as low as 17.4% of that of the controls. For the controls, LH binding was measured in Leydig cells cultured in spent Leydig cell-cultured medium (LM). There was no difference between SM- and LM-cultures in the final survival rate or the percentage of cells showing histochemically demonstrated 3 beta-hydroxysteroid dehydrogenase activity. These data suggest that some factor or factors are secreted from the cultured Sertoli cells and inhibit the binding of LH to Leydig cells in culture.     

Interactions between immature porcine Leydig and Sertoli cells in vitro

Summary Interactions between Leydig and Sertoli cells, as well as a stimulatory effect of FSH on Leydig cell activity, have been reported in many studies. In order to investigate these interactions, the ultrastructure of immature pig Leydig cells under different culture conditions has been studied. When cultured alone in a chemically defined medium, there is a marked regression of the Leydig cell smooth endoplasmic reticulum and a swelling of the mitochondria. Addition of FSH or hCG does not prevent these phenomena. Co-culturing of Leydig cells with Sertoli cells from the same animal maintains the smooth endoplasmic reticulum at the level seen in vivo and in freshly isolated Leydig cells. The addition of FSH to the co-culture stimulates its development and increases Leydig cell activity, as assessed by an increase in hCG binding sites and an increased steroidogenic response to hCG. These results suggest that Sertoli cells exert a trophic effect on Leydig cells, and that the stimulatory effect of FSH on Leydig cell function is mediated via the Sertoli cells. These results reinforce the concept of a local regulatory control of Leydig cell steroidogenesis.Post-Doctoral fellow supported by CIRIT, Generalitat de Catalunya, Spain     

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.     

Changes in Leydig cell ultrastructure and function during pubertal development in the boar

Changes in the ultrastructure of Leydig cells during pubertal development in the boar (40 to 250 days of age) were assessed using quantitative morphometric procedures, and the results were compared to the in vitro steroid-producing capacity and gonadotropin sensitivity of testicular tissue obtained from the same boars. Volume of individual Leydig cells declined through 100 days of age, increased rapidly to a peak at 130-160 days (i.e., puberty), and then declined to intermediate levels by 220-250 days of age. The pattern of change in the number of intracellular organelles per Leydig cell was very similar to the change that occurred in Leydig cell volume. Changes in the total intracellular volume occupied by each type of organelle were highly correlated with changes in Leydig cell volume (r = 0.40-0.99, p less than 0.01), and this was particularly true for the nucleus (r = 0.63), mitochondria (r = 0.88), smooth endoplasmic reticulum (SER; r = 0.97), and total cytoplasm (r = 0.99) of the boar Leydig cell. In vitro production of testosterone and estradiol, expressed per Leydig cell, also peaked at 130-160 days, and was highly correlated to average Leydig cell volume, volume of SER, and number and total volume of mitochondria (r = 0.63-0.84; p less than 0.01). Observations in the present study indicated that onset of puberty in boars coincides with a dramatic increase in average Leydig cell size and SER volume per Leydig cell, accompanied by an increase in number of other intracellular organelles, including mitochondria, lysosomes, and lipid droplets, and a peak in the steroid-producing capacity per Leydig cell. A decline in Leydig cell size, intracellular organelles, and sensitivity to gonadotropin stimulation occurred postpubertally.     

Comparison of components of the testis interstitium with testosterone secretion in hamster, rat, and guinea pig testes perfused in vitro

Components of the testis and cytoplasmic organelles in Leydig cells were quantified with morphometric techniques in hamster, rat, and guinea pig. Testosterone secretory capacity per gram of testis and per Leydig cell in response to luteinizing hormone (LH) (100 ng/ml) stimulation was determined in these three species from testes perfused in vitro. Numerous correlations were measured among structures, and between structures and testosterone secretion, to provide structural evidence of intratesticular control of Leydig cell function. Testosterone secretion per gm testis and per Leydig cell was significantly different in the three species: highest in the guinea pig, intermediate in the rat, and lowest in the hamster. The volume of seminiferous tubules per gm testis was negatively correlated, and the volumes of interstitium, Leydig cells, and lymphatic space per gm testis were positively correlated with testosterone secretion. No correlations were observed between volumes of blood vessels, elongated spindleshaped cells, or macrophages per gm testes and testosterone secretion. The average volume of a Leydig cell and the volume and surface area of smooth endoplasmic reticulum (SER) and peroxisomes per Leydig cell were positively correlated, and the volume of lysosomes and surface area of inner mitochondrial membrane per Leydig cell were negatively correlated with testosterone secretion. No correlations were observed between volume and surface area of rough endoplasmic reticulum (RER), Golgi apparatus, and lipid, and volume of ribosomes, cytoplasmic matrix, and the nucleus with testosterone secretion per Leydig cell. These results suggest that Leydig cell size is more important than number of Leydig cells in explaining the difference in testosterone-secreting capacity among the three species, and that this increase in average volume of a Leydig cell is associated specifically with increased volume and surface area of SER and peroxisomes. An important unresolved question is what is the role of peroxisomes in Leydig cell steroidogenesis.     

Functional and physical characteristics of rat Leydig cell populations isolated by metrizamide and Percoll gradient centrifugation

The physical and functional properties of Leydig cell populations obtained by centrifugation of testicular cells in two different density gradient media, Percoll and Metrizamide, were compared. Percoll-gradient centrifugation yielded two Leydig cell bands (Peak I and Peak II) that were comparable, as to their density and testosterone-producing capacity, to the respective Leydig cell bands, Population I and Population II, isolated in a Metrizamide gradient. The denser Leydig cell band (II) had a greater capacity for testosterone production than the less dense band (I), regardless of the type of gradient used for its isolation. Metrizamide gradient centrifugation separated the majority of germ cells from the "light" (Population I) Leydig cells, whereas in the Percoll gradient, germ cells comigrated with Peak I Leydig cells. Leydig cell separation by Percoll gradients was highly dependent on the presence of Ca2+ and Mg2+ in the medium, while these cations had no effect on the separation of Leydig cells by Metrizamide. In conclusion, Metrizamide gradient centrifugation yielded two Leydig cell populations of similar functional and physical properties to the respective populations isolated in Percoll gradients.     

Changes of oestrogen receptor levels in Leydig cells from mice and rats during culture

Two functional properties of Leydig cells in culture, i.e. LH-stimulated steroidogenesis and nuclear oestrogen receptor levels have been investigated. Leydig cells isolated from testes of immature rats and mature mice maintained their responsiveness to LH during 48-72 h of cell culture, although the mouse Leydig cells appeared to be less responsive to LH after 72 h of culture. In contrast, nuclear oestrogen receptor levels in both types of Leydig cells declined to 10-20% of the initial value after 24 h in culture. In the 48-72 h culture period nuclear oestrogen receptor levels recovered to 75% of the initial value only in Leydig cells from immature rats, whereas the nuclear oestrogen receptor levels in Leydig cells from mature mice remained low. These data demonstrate that during in vitro culture of Leydig cells, preservation of LH responsiveness does not necessarily warrant that other Leydig cell parameters e.g. nuclear oestrogen receptors also remain unaltered.     

The regenerative capacity of testicular interstitial tissue

Summary A single intraperitoneal injection of ethane dimethanesulphonate (EDS) destroys all Leydig cells in the adult rat testis but 1–2 weeks later new foetal-type Leydig cells begin to regenerate within the interstitial tissue. A further EDS treatment at 4 weeks failed to kill the new population of foetal-type Leydig cells. Between 10–20 weeks, the new Leydig cells exhibited the characteristics of adult-type Leydig cells. These cells responded to another EDS treatment by exhibiting a second phase of complete degeneration followed by regeneration of a foetal-type and subsequently an adult-type cell population. The results indicate that the testis retains the ability to replenish its supply of Leydig cells despite successive phases of total degradation of Leydig cells.     

Effect of prostaglandins on hormonal function of cultured rat Leydig cells

The effect of PGF2 alpha and its analogues on androgen production and activity of delta 5,3 beta-hydroxysteroid dehydrogenase in rat Leydig cells in vitro was investigated. Prostaglandin of the F type inhibit the enzyme activity and hormone secretion by cultured Leydig cells. This effect was considerably stronger in Leydig cells isolated from mature rats, than by Leydig cells from immature animals.