Nestin, a neuroectodermal stem cell marker molecule, is expressed in Leydig cells of the human testis and in some specific cell types from human testicular tumours

The intermediate filament protein nestin is predominantly expressed in some stem/progenitor cells and appears to be a useful molecular tool to characterise tumours originating from precursor cells of neuroectodermal and mesenchymal lineages. Leydig cells originate in the adult testis by differentiation from stem cells and express a variety of neural and neuroendocrine markers. The possible expression of the neural stem cell marker nestin in Leydig cells and testicular tumour cells was determined by analysing the patterns of nestin expression in normal and pathological human testes by Western blot and immunohistochemical methods. In normal testis, nestin was found in some vascular endothelial cells, a subset of peritubular spindle-shaped cells and some Leydig cells; spermatogenic and Sertoli cells were unstained. In normal Leydig cells, nestin was distributed in the perinuclear cytoplasm and accumulated in the crystalloids of Reinke with ageing. In non-tumour pathologies (cryptorchidism, impaired spermatogenesis), the seminiferous tubules were immunonegative, whereas hyperplastic Leydig cells showed cytoplasmic immunolabelling. In testicular malignancies, nestin was localised in the Sertoli cells of the seminiferous tubules affected with intratubular germ cell neoplasia, in the hyperplastic Leydig cells associated with these tumours and in some components (mesenchymal and neuroepithelial cells) of teratomas; spermatocytic and non-spermatocytic seminomas were unstained. Some vascular endothelial cells were immunolabelled in all tumour samples. Thus, nestin is expressed in a population of normal and hyperplastic Leydig cells and in Sertoli cells in the presence of intratubular germ-cell neoplasia. Nestin may be a good marker for identifying components of testicular teratomas.The two first authors participated equally in this workThis work was supported by a grant from the Fondo de Investigaciones Sanitarias (FIS 02/3003 to M.V.T. Lobo)     

Selective destruction and regeneration of rat Leydig cells in vivo

Summary The effect of a single i.p. administration of ethane dimethanesulphonate (EDS) upon rat testicular histology was studied by light microscopy and morphometry up to 4 weeks after treatment. One day after injection the interstitial tissue exhibited degenerating Leydig cells, abundant pyknotic interstitial cells, deposition of cellular debris and extensive networks of fibrillar material. Macrophages contained greatly increased numbers of cytoplasmic inclusion bodies. From 3 to 7 days morphometric analysis showed that Leydig cells and cellular debris had disappeared from the interstitial tissue, leaving only macrophages, fibroblasts and lymphatic endothelial tissue. A very small number of new Leydig cells were seen on day 14, often located in peritubular or perivascular positions. Regeneration of foetal-like Leydig cells occurred by 4 weeks, their cytoplasm containing large lipid inclusions and, numerous Leydig cells were often observed closely applied to the walls of the seminiferous tubules. The observations suggest that, after experimental destruction and depletion of Leydig cells, an interstitial precursor cell, as yet unidentified, gives rise to a new Leydig cell population. EDS thus offers a valuable opportunity to study further the interactions between the seminiferous tubules and the interstitial tissue following the destruction and subsequent regeneration of the Leydig cells.     

Deprival of testicular innervation induces apoptosis of Leydig cells via caspase-8-dependent signaling: A novel survival pathway revealed

Leydig cells are the primary source of testosterone in adult males. Recently, a growing body of evidence has shown that testicular innervation functions as a major regulator in Leydig cell steroidogenesis. The question then arises whether this novel regulatory pathway also plays an important role in other biological behaviors of this cell type. In the present study, we selectively resected the superior spermatic nerves (SSNs) or the inferior spermatic nerves (ISNs) to investigate the effects of testicular denervation on survival of Leydig cells. After testicular denervation, Leydig cells displayed morphological characteristics of apoptosis, such as chromatin condensation, cell shrinkage and apoptotic body formation. Flow cytometry combined with TUNEL labeling demonstrated dramatic and persistent apoptosis of Leydig cells in the denervated testes 14 and 21 days after operation. Meanwhile, serum T concentrations in the SSN- or ISN-denervated rats dramatically decreased on day 14 and declined further on day 21. Plasma LH levels underwent a remarkable rise, while serum FSH levels remained unchanged. Immunofluorescent staining and flow cytometry further demonstrated that testicular denervation activated caspase-3 and caspase-8, but not caspase-9 in Leydig cells. Our data indicate that testicular innervation functions as an important survival factor for Leydig cells in vivo.     

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.     

Autoradiographical localization of luteinizing hormone releasing hormone (LHRH) receptors on rat testicular intertubular cells fractionated on Percoll density gradients

The specific binding of 125I-labelled [D-Ser(tBu)6,des-GlyNH2(10)] LHRH ethylamide (LHRH-A) to testicular intertubular cells fractionated on Percoll density gradients was investigated. The greatest binding per cell occurred in the density region which contained the largest proportion of Leydig cells (sp. gr. 1.0820-1.0585). Autoradiographs of the cells from this region confirmed that silver stains were predominantly located over the Leydig cell, significantly (P less than 0.01) more grains were observed over this cell type in the total binding fractions than in the non-specific binding fractions. However, 5.9% of cells other than Leydig cells (testicular macrophages and indeterminate connective tissue cells) from this region also displayed significant displaceable binding (P less than 0.01). The location of [125I]LHRH-A binding to cells in other density regions, which did not contain identifiable Leydig cells, could not be established by autoradiography. These results confirm that the Leydig cell possesses LHRH receptors, but also indicate that other testicular cells have specific, high-affinity binding sites for LHRH-A, and may either be responsive to direct stimulation by LHRH, or may partially mediate the effects of LHRH and its agonists on Leydig cell function.     

The germ cell development strategy and seasonal changes in spermatogenesis and Leydig cell morphologies of the spiny lizard Sceloporus mucronatus (Squamata: Phrynosomatidae)

The viviparous lizards of the Sceloporus genus exhibit both seasonal and continuous spermatogenesis. The viviparous lizard Sceloporus mucronatus from Tecocomulco, Hidalgo, México, exhibits seasonal spermatogenesis. This study demonstrates the relationship between changes in testis volume, spermatogenesis activity, and Leydig cells during the male reproductive cycle of S. mucronatus. A recrudescence period is evident, which starts in the winter when testicular volume is reduced and climaxes in February, when the greatest mitotic activity of spermatogonia occurs. The testicular volume and Leydig cell index increase gradually during the spring with primary spermatocytes being the most abundant cell type observed within the germinal epithelium. In the summer, the secondary spermatocytes and undifferentiated round spermatids are the most abundant germinal cells. The breeding season coincides with spermiogenesis and spermiation; testicular volume also increases significantly as does the Leydig cell index where these cells increase in both cytoplasmic and nuclear volume. During fall, testicular regression begins with a significant decrease in testicular volume and germinal epithelium height, although there are remnant spermatozoa left within the lumen of the seminiferous tubules. During this time, the Leydig cell index is also reduced, and there is a decrease in cellular and nuclear volumes within these interstitial cells. Finally, during quiescence in late fall, there is reduced testicular volume smaller than during regression, and only spermatogonia and Sertoli cells are present within the seminiferous tubules. Leydig cells exhibit a low index number, their cellular and nuclear volumes are reduced, and there is a depletion in lipid inclusion cytoplasmically.     

Multiple regulation of testicular steroidogenesis

One single injection of ethylene dimethane sulfonate (EDS) to mature rats causes specific degeneration of testicular Leydig cells which is complete after 3 days. At this time no steroidogenic activities can be detected, indicating that Leydig cells are the source of steroids. The mechanism of this cytotoxic effect of EDS has been investigated with isolated cells. Extensive protein alkylation has been shown to occur in Leydig cells, Sertoli cells and hepatocytes. Steroid production by Leydig cells is always inhibited by EDS, but cytotoxic effects of EDS could only be demonstrated in Leydig cells from mature rats or tumour tissue and not in Leydig cells from immature rats. A new population of Leydig cells develops during the next 2-5 weeks after EDS treatment. In hypophysectomized rats this repopulation only occurs when hCG is given daily. FSH has no effects. The proliferative activity in the interstitial tissue increases within 2 days after administration of hCG or EDS and there are indications that LH and locally produced factors are involved in the proliferation of Leydig cells or Leydig cell precursor cells. Inhibition of cAMP production with inhibitors of adenylate cyclase results in an enhancement of the LH-stimulated steroid production similar to that observed with an LHRH agonist and phospholipase C (PLC). Since the effects of LHRH and PLC on protein phosphorylation and steroid production are similar and different from LH or active phorbol esters, it is proposed that LHRH and PLC may stimulate steroid production via liberation of calcium from a specific intracellular pool. Sterol carrier protein2 (SCP2) which is specifically localized in Leydig cells and regulated by LH probably plays a role in the delivery of cholesterol to the mitochondria although the mechanism of this carrier function is not clear. The results indicate that regulation of Leydig cell development and the steroidogenic activities by gonadotrophins and locally produced factors occur via different transducing systems and regulatory pathways.     

Paracrine regulation of testicular function

Data from several experimental approaches have been reviewed and the findings clearly indicate the existence of multiple interactions between testicular cells and the potential role of these interactions in the paracrine control of testicular functions. Both testicular interstitial fluid and spent media from cultured Sertoli cells had an acute steroidogenic effect on Leydig cells, and this effect is not species specific. The secretion of this steroidogenic factor(s), which is probably a protein, is enhanced by previous FSH treatment of Sertoli cells. Coculture for 2-3 days of pig Leydig cells with homologous or heterologous Sertoli cells enhances Leydig cell specific functions (hCG receptor number and hCG responsiveness) and induces Leydig cell hypertrophy. A similar but less pronounced trophic effect is seen when Leydig cells are cultured with spent media from Sertoli cells cultured in the presence of FSH and high concentrations of insulin, but the spent media from Sertoli cells cultured in the absence of these two hormones inhibits Leydig cell specific functions. Somatomedin-C might play an important role in the positive trophic effect of Sertoli cells on Leydig cells, since this peptide is secreted by Sertoli cells and it has trophic effects on the specific function of Leydig cells. Moreover, Sertoli cells, probably through a diffusible factor and cell-to-cell contacts, control the multiplication, meiotic reduction and maturation of germ cells. In turn, the activity of Sertoli cells is modulated by the stage of neighbouring germ cells. Thus, if a normal Sertoli cell function (which depends not only on FSH but also on Leydig and myoid cell secretory products) is an absolute requirement for germ cell multiplication and maturation, these cells, in turn, cyclically regulate Sertoli cell function and through these cells the size and probably the function of Leydig cells.     

Immunocytochemical localization of 17 beta-hydroxysteroid dehydrogenase in porcine testis

The immunocytochemical localization of 17 beta-hydroxysteroid dehydrogenase (17 beta-HSD) in porcine testes was examined by applying an indirect-immunofluorescence method using an antiporcine testicular 17 beta-HSD antibody. Only the Leydig cells located in the interstitial tissue exhibited a positive immunoreaction for 17 beta-HSD: the germ cells and Sertoli cells located in the seminiferous tubules were entirely negative. These results suggest that, in porcine testis, the biosynthesis of testicular testosterone, the final step of which is the conversion of androstenedione to testosterone, takes place in the Leydig cells.     

Over expression of interleukin-1alpha,interleukin-1beta and interleukin-1 receptor antagonist in testicular tissues from sexually immature mice as compared to adult mice

The levels of IL-1alpha, IL-1beta and IL-1Ra were higher in homogenates of testicular tissue from sexually immature than those from mature mice. Immunohistochemical staining of testicular tissues from sexually immature and adult mice show that differentiated germ cells express higher levels of IL-1alpha compared to Sertoli cells and Leydig cells/interstitial cells. Peritubular cells of sexually immature and adult mice did not express IL-1alpha. Testicular tissue cells of adult mice showed high levels of expression of IL-1beta, mainly in the cytoplasm and nucleus of the spermatogonia and in spermatocytes. Sertoli cells and Leydig/interstitial cells were also highly stained for IL-1beta. However, peritubular cells did not express IL-1beta. On the other hand, testicular tissue cells from sexually immature mice, showed high levels of IL-1beta, mainly in spermatocytes. Spermatogonia showed low levels of IL-1beta expression. Also, high levels of IL-1beta expression were detected in Leydig/interstitial cells. Peritubular cells clearly showed IL-1beta expression. Testicular tissue cells from adult mice, showed IL-1Ra expression in spermatogonia, Sertoli and Leydig/interstitial cells. IL-1Ra expression was clearly present in the Golgi apparatus of spermatogonia and Sertoli cells. However, peritubular cells did not show IL-1Ra expression. Testicular tissue cells from sexually immature mice, also showed high levels of IL-1Ra expression mainly in the cytoplasm and nucleus of the spermatogonia and Sertoli cells. In addition, Leydig/interstitial cells and peritubular cells also expressed IL-1Ra. Our results demonstrate, for the first time, the expression of IL-1beta in germ and Sertoli cells, and IL-1Ra in Leydig/interstitial cells of testicular tissues from adult and sexually immature mice, under in vivo conditions. In addition, the relative elevated levels of the IL-1 system in the testis of immature mice compared to mature mice may indicate its involvement in the spermatogenesis.     

Insulin-like growth factor-I (IGF-I) protects cultured equine Leydig cells from undergoing apoptosis

Leydig cells located in the interstitial space of the testicular parenchyma produce testosterone which plays a critical role in the maintenance and restoration of spermatogenesis in many species, including horses. For normal spermatogenesis, maintaining Leydig cells is critical to provide an optimal and constant level of testosterone. Recently, an anti-apoptotic effect of IGF-I in testicular cells in rats has been reported, but a similar effect of IGF-I on equine Leydig cells remains to be elucidated. If IGF-I also protects stallion testicular cells from undergoing apoptosis, then IGF-I may have potential as a treatment regime to prevent testicular degeneration. The present study was designed to evaluate the anti-apoptotic effect of IGF-I on cultured equine Leydig cells. Testes were collected from 5 post-pubertal stallions (2-4 years old) during routine castrations. A highly purified preparation of equine Leydig cells was obtained from a discontinuous Percoll gradient. Purity of equine Leydig cells was assessed using histochemical 3β-HSD staining. Equine Leydig cells and selected doses of recombinant human IGF-1 (rhIGF-I; Parlow A.F., National Hormone and Peptide Program, Harbor-UCLA Medical Center) were added to wells of 24 or 96 well culture plates in triplicate and cultured for 24 or 48 h under 95% air:5% CO(2) at 34°C. After 24 or 48 h incubation, apoptotic rate was assessed using a Cell Death Detection ELISA kit. Significantly lower apoptotic rates were observed in equine Leydig cells cultured with 5, 10, or 50ng/ml of rhIGF-I compared with control cells cultured without rhIGF-I for 24h. Exposure to 1, 5, 10 or 50 ng/ml of rhIGF-I significantly decreased apoptotic rate in equine Leydig cells cultured for 48 h. After 48 h incubation, cells were labeled with Annexin V and propodium iodine to determine the populations of healthy, apoptotic, and necrotic cells by counting stained cells using a Nikon Eclipse inverted fluorescence microscope. As a percentage of the total cells counted, significantly lower numbers of apoptotic cells were observed in cells treated with 10 (9%) or 50 ng/ml (10%) of rhIGF-I compared with cells cultured without rhIGF-I (control, 22%). In this study, the results from the two assays indicated that rhIGF-I protected equine Leydig cells from undergoing apoptosis during cell culture for 24h or 48 h. In conclusion, IGF-I may be an important paracrine/autocrine factor in protecting equine Leydig cells from undergoing apoptosis.     

Development of cytoplasmic digitations between Leydig cells and testicular macrophages of the rat

Summary Testicular macrophages and Leydig cells from adult animals are known to be functionally coupled. For example, secreted products from macrophages stimulate testosterone secretion by Leydig cells. In adult rat testes, structural coupling also exists between these cells. This coupling consists of cytoplasmic projections from Leydig cells located within cytoplasmic invaginations of macrophages. Although macrophages are known to exist in the testis in immature animals, it is not known when these digitations develop. The purpose of the present study was to determine whether the time of their development coincides with known maturational events that occur in Leydig cells, particularly during the peripubertal period. Testes from rats at 20, 30 and 40-days-of-age as well as testes from mature rats weighing more than 500 gm were prepared for ultrastructural analysis. It was found that digitations form between 20 and 30-days-of-age. These structures varied from simple tubular projections to complicated branched structures, suggesting that digitations are more than simple invaginations of microvilli into coated vesicles as previously described. Subplasmalemmal linear densities were also observed within macrophages juxtaposed to Leydig cells. Collagen was commonly observed between macrophages and Leydig cells in animals 20 days old. These studies demonstrate that although macrophages are present in the testis in maximal numbers at 20 days-of-age, they do not form junctions with Leydig cells until day 30. This is just prior to the major increase in secretory activity of rat Leydig cells that occurs during puberty.     

Testicular interstitial cells, and steroidogenic detection in the protogynous fish, Synbranchus marmoratus (Teleostei, Synbranchidae)

The swamp eel, Synbranchus marmoratus, is a freshwater protogynic diandric species. Primary males develop directly as males while secondary males arise from the sex reversal of females. Fishes from Argentine and Brazil inland waters were collected, examined and compared for this study. In order to characterize the interstitial testicular compartment, light and electron microscopy techniques and an enzyme histochemical examination for steroidogenic cells detection were used. The interstitial compartment of S. marmoratus is composed of Leydig and myoid cells, collagen fibers, blood cells, macrophages,and amyelinic nerves. At the ultrastructural level, no differences were observed in the interstitial tissue, either between specimens from the different sampling sites or between primary and secondary males. Leydig cells are present in all testes examined throughout the year. A cytoplasmatic reaction of 3beta-HSD was detected only in Leydig cells during sex reversal and in both type of males, mainly during the regressed and early maturation classes (autumn and winter). Leydig cells possess the typical fine structural characteristics associated with steroidogenesis. Furthermore, in both type of males, during sex reversal and after the spawning period, the number of granulocytes and macrophages present in the testes increased, suggesting that they could be involved in phagocytosis and resorption of damaged cells.     

Les endozépines, facteurs locaux de régulation de la stéroïdogenèse testiculaire

Testicular endocrine and exocrine functions are controlled by multiple signals including circulating gonadotropins and locally produced factors. Among these factors, endozepines (EZ), which are the endogenous ligands for benzodiazepine receptors, seem to exert an intracrine, autocrine and/or paracrine stimulatory effect on Leydig cell testosterone production. Benzodiazepine effects are mediated by two types of receptors, i.e. the central-type benzodiazepine receptor (CBR) associated with the GABAA-receptor complex, and the peripheral-type benzodiazepine receptor (PBR) principally located on the mitochondrial membrane and extremely abundant in steroidogenic cells. All EZ characterized to date are derived from an 86 amino acid polypeptide called diazepam binding inhibitor (DBI) that generates, via proteolytic cleavage, several biologically active peptides including the triakontatetraneuropeptide DBI17-50 (TTN) and the octadecaneuropeptide DBI33-50 (ODN). EZ are widely distributed in the brain and various peripheral organs, particularly in steroidogenic glands. A number of data suggest that, in rats, EZ could regulate testicular steroidogenesis. Firstly, DBI gene expression and the presence of DBI-like peptides have been shown in Sertoli cells, Leydig cells and in late-differentiated germ cells. Moreover, EZ are able to stimulate progesterone and testosterone production by rat Leydig cells and by MA-10 or R2C Leydig tumor cells. Finally, pharmacological studies have shown that EZ stimulate rat testicular steroidogenesis via activation of PBR. PBR appears to be an important component of a dynamic multistep process involving protein-protein interactions, to promote cholesterol translocation in the mitochondria, where it is converted into pregnenolone by cytochrome P450scc.     

Studies on the onset of Leydig precursor cell differentiation in the prepubertal rat testis

Leydig cells of the adult rat testis differentiate postnatally from spindle-shaped cells in the testis interstitium during the neonatal-prepubertal period. Which spindle-shaped cell types are the precursor for Leydig cells and the stimulus for initiation of their differentiation are, however, two unresolved issues. In the present study, our objectives were to identify unequivocally which spindle-shaped cells are the precursors to Leydig cells and to test whether the initiation of their differentiation into Leydig cells depends on LH. Testes from fifteen groups of Sprague-Dawley rats (n = 4 per group) from 7-21 days of age were fixed in Bouin solution and embedded in paraffin. Immunoexpression of 3beta-hydroxysteroid dehydrogenase (3betaHSD), cytochrome P450 side-chain cleavage (P450(scc)), 17alpha-hydroxylase cytochrome P450 (P450(c17)), and LH receptors (LHR) in interstitial cells (other than fetal Leydig cells) was observed using the avidin biotin method. Of all spindle-shaped cell types in the testis interstitium, only the peritubular mesenchymal cells showed positive immunolabeling for all three steroidogenic enzymes, beginning from the 11th postnatal day. All three enzymes were expressed simultaneously in these cells, and their numbers increased significantly thereafter. Immunoexpression of LHR in a few of these cells was just evident for the first time on postnatal Day 12 (i.e., after acquiring the steroidogenic enzyme activity). Their numbers gradually increased with time. The number of immunolabeled cells per 1000 interstitial cells (excluding fetal Leydig cells and capillary endothelial cells) was not significantly different for the three steroidogenic enzymes tested at all ages; however, a lower value was observed for LHR at each time-point. Based on these observations, we suggest that 1) the precursor cell type for the adult generation of Leydig cells in the postnatal rat testis is the peritubular mesenchymal cells, 2) precursor cells acquire 3beta-HSD, P450(scc), and P450(c17) enzyme activity simultaneously during Leydig cell differentiation, and 3) onset of precursor cell differentiation during Leydig cell development does not depend on LH.     

Leydig cell hyperplasia in fetal mice treated transplacentally with ethinyl estradiol

Pregnant female mice were given ethinyl estradiol on days 11 through 17 of gestation. On day 18 the dams were killed and the male fetuses were examined for testicular differentiation. Three of 12 males from dams treated with the highest dose of ethinyl estradiol showed cryptorchid testes with uterine tubes. Light and electron microscopic evaluation of the testes, both cryptorchid and normal, demonstrated foci of hyperplasia of Leydig cells showing cytoplasmic and nuclear pleomorphism, increase in lipid droplets, and decrease in smooth endoplasmic reticulum and ribosomes when compared to testes from control fetal mice. Morphometric determinations of the testes indicated that the number of Leydig cells in a unit area (mm2) in the interstitial tissue showed a dose-response relationship to ethinyl estradiol in the normal testes. The number of Leydig cells in the testes exposed to the highest dose of estrogen showed a significant difference between cryptorchid and normal testes: the former had fewer Leydig cells than the latter. These morphological observations indicate that hyperplasia of Leydig cells of fetal mouse testis at term can be induced by transplacental treatment with ethinyl estradiol and suggest that a malignant transformation into a Leydig cell tumor is possible.     

Meiosis in autologous ectopic transplants of immature testicular tissue grafted to Callithrix jacchus

Grafting of immature testicular tissue provides a tool to examine testicular development and may offer a perspective for preservation of fertility in prepubertal patients. Successful xenografting in mice, resulting in mature spermatids, has been performed in several species but has failed with testicular tissues from the common marmoset, Callithrix jacchus. Previous data indicate that the hormonal milieu provided by the mouse host might cause this failure. We conducted autologous ectopic transplantation of testicular fragments under the back skin in newborn marmoset monkeys. Seventeen months after transplantation, we found viable transplants in 2 out of the 4 grafted animals. In the transplants, tubules developed up to a state intermediate between the pregraft situation and adult controls. Dividing spermatogonia and primary spermatocytes were present. Boule-like positivity and CDC25A negativity indicated that spermatogenesis was arrested at early meiosis. Immunohistochemistry revealed normal maturation of Sertoli cells, Leydig cells, and peritubular cells. Serum testosterone values were not restored to the normal range and bioactive chorionic gonadotropin levels increased to castrate levels. Meiotic arrest could have occurred in the grafts because of lack of sufficient testosterone or because of hyperthermia caused by the ectopic position of the grafts. We conclude that autologous transplants of immature testicular tissues in the marmoset can mature up to meiosis but that normal serum testosterone levels are not restored. Further studies have to be performed to overcome the meiotic arrest to explore the model further and to develop therapeutic options.     

Functional properties of developing rat Leydig cells after treatment with ethylene dimethanesulphonate (EDS)

The development of a new population of Leydig cells after specific elimination of existing Leydig cells in mature rats by ethylene dimethanesulphonate (EDS) was characterized by investigating the testicular activities of 5 alpha-reductase and non-specific esterase, the serum concentrations of 3 alpha-androstanediol and testosterone and the Leydig cell morphology. Plasma concentrations of both androgens were strongly reduced up to 15 days after administration of EDS. Thereafter, in contrast to the gradual and continuous increase of serum testosterone values, the changes in serum 3 alpha-androstanediol were transient, with the highest level on Day 35. The temporal pattern of testicular 5 alpha-reductase activity was almost similar to that of serum 3 alpha-androstanediol. The testicular esterase activity increased gradually from Day 25 until Day 76. The temporal changes in steroid concentrations and enzyme activities after EDS administration indicate that the development of the Leydig cells in EDS-treated rats occurs in a fashion similar to that in pubertal rats. However, the numerous lipid droplets and large nuclei in these Leydig cells indicate that these cells may also be classified as fetal cells. It is concluded that, after treatment with EDS, fetal and pubertal characteristics are present in Leydig cells. It is, however, unknown whether both characteristics are present in one or in two distinct cell populations.     

Immunoexpression of androgen receptors and aromatase in testes of patient with Klinefelter's syndrome

Klinefelter's syndrome (47, XXY) is the most common chromosome aneuploidy in men and is usually characterized by underdeveloped testes and sterility. The aim of the present study was to detect cellular distribution of androgen receptors (AR) and aromatase in testes of patient with KS. The tissue sections were processed for morphological and immunohistochemical staining. Additionally, levels of FSH, LH, PRL, estradiol, and testosterone were measured in the plasma. Morphological analysis revealed a complete absence of spermatogenesis. No germ cells were present in seminiferous tubules. In some tubules, nests of apparently degenerating Sertoli cells were found. In the interstitium, Leydig cell hyperplasia was observed. Using immunohistochemistry, nuclear AR staining was detected in Sertoli cells and peritubular cells, whereas in Leydig cells the staining was exclusively cytoplasmic. The immunostaining of aromatase was detected in the cytoplasm of Sertoli cells and Leydig cells. Increased levels of gonadotropins and decreased level of testosterone concomitantly with the cytoplasmic localization of AR in Leydig cells might contribute to the impaired testicular function in patient with KS.