Steroid sulfatase activity in homogenates, microsomes and purified Leydig cells from adult rat testis

Steroid sulfatase (STS) activity was studied in the Long-Evans rat testis. The rate of dehydroepiandrosterone sulfate (DHA-S) hydrolysis determined in whole testis homogenates was low compared to that of the corresponding microsomal fractions, which was, in contrast, as high as that expressed in homogenates from purified Leydig cells. Such an increment in STS activity between total homogenates and the corresponding microsomes was not observed for the seminiferous tubules. The STS affinity reported for total testicular microsomes (Km = 3.47 +/- 0.54 microM; mean +/- SEM) was of the same magnitude as that previously reported for Leydig cells, but was about 3 times higher than that measured for whole testis homogenate (Km = 10.11 +/- 0.92 microM). In vivo hCG treatment decreased the STS affinity in total testicular microsomes without affecting this kinetic parameter in whole testis homogenate. These data suggest that the steroid sulfatase expressed in total testicular microsomes (activity and regulation by hCG) could be considered as a good index of Leydig cell STS activity.     

Oxidation-reduction potentials and spectral properties of some cytochromes from Thiobacillus versutus (A2)

Antibodies raised against rat hepatic epoxide hydrolase (EC 3.3.2.3) and glutathione S-transferases (EC 2.5.1.18) B, C and E were used to determine the presence and localizations of these epoxide-metabolizing enzymes in testes of sexually immature and mature Wistar and Holtzman rats. Unlabeled antibody peroxidase-antiperoxidase staining for each enzyme was readily detected in rat testes at the light microscopic level. Although significant strain-related differences were not apparent, staining intensity for certain enzymes differed markedly between Leydig cells and seminiferous tubules. Leydig cells of immature and mature rats were stained much more intensely for epoxide hydrolase and glutathione S-transferases B and E than were seminiferous tubules, whereas Sertoli cells, spermatogonia, spermatocytes and spermatids, as well as Leydig cells, were stained intensely by the anti-glutathione S-transferase C. Age-related differences in staining for glutathione S-transferase B were not obvious, while the anti-glutathione S-transferase C stained seminiferous tubules more intensely in immature rats, and antibodies to epoxide hydrolase and glutathione S-transferases C and E stained Leydig cells much more intensely in mature rats. These observations thus demonstrate that testes of both sexually immature and mature rats contain epoxide hydrolase and glutathione S-transferases. Except for glutathione S-transferase C in immature rats, Leydig cells appear to contain much higher levels of enzymes than do seminiferous tubules. During sexual maturation, the testicular level of glutathione S-transferase B appears to remain constant, while levels of epoxide hydrolase and glutathione S-transferases C and E increase within Leydig cells and the level of glutathione S-transferase C decreases within seminiferous tubules.     

Ultrastructural localisation of alkaline phosphatase in the intertubular tissue of the testis in the domestic fowl

Alkaline phosphatase activity in the intertubular tissue of the testes of the domestic fowl was examined using an ultracytochemical technique based on the lead capture method. In the interstitial tissue, the Leydig cells, transitional cells and the fibroblasts displayed enzyme activity on their cell membranes. Vacuoles located in the transitional cells were lined by reaction products of enzyme activity, whereas the vacuoles representing extracted lipid droplets and present mainly in the Leydig cells were free of enzyme activity. In the peritubular tissue the cell processes of fibroblasts showed enzyme activity on the cell membranes and in pinocytotic vesicles. Cell processes lying adjacent to blood vessels showed pronounced activity. In the blood vessel itself some activity was present in the basement membrane and the endothelium. The surface of the red blood cell showed moderate activity. The possible role of alkaline phosphatase in the transfer of hormone from the Leydig cells to the seminiferous tubules and from the seminiferous tubules to the interstitium is discussed. The myoid cells and their processes were devoid of enzyme activity.     

Nuclear testicular 1,25-dihydroxyvitamin D3 receptors in Sertoli cells and seminiferous tubules of adult rodents

1,25(OH)2D3 receptors were studied in whole testes, Sertoli cells, seminiferous tubules, Leydig cells and spermatogonia of adult NMRI mice and SD rats. Specific reversible high affinity binding (KD 1.4 x 10(-10)M; Nmax 72 fmol/mg protein) by a 3.5 S macromolecule was demonstrated in whole testes, Sertoli cells and seminiferous tubules. With identical techniques, no receptors were found in Leydig cells despite previous reports of 1,25(OH)2D3 actions on Leydig cell function.     

Stimulation of ornithine decarboxylase activity by luteinizing hormone releasing hormone in the testis of immature rat

The activity of ornithine decarboxylase (ODC) was found to increase in the testis of immature rats following intratesticular injection with luteinizing hormone releasing hormone (LHRH). Maximal stimulation of ODC activity occurred with 1 μg of the hormone at 2 h. The enzyme activity returned to control levels at 4 h. The minimal effective dose was found to be 0.1 μg per testis. The stimulating effect of LHRH was confined to Leydig cells alone. The seminiferous tubules did not show any change in ODC activity following LHRH treatment. These results show that LHRH acts directly on the testis and influences the levels of ODC in the Leydig cells of rat.     

Localization of epoxide-metablozing enzymes in rat testis

Antibodies raised against rat hepatic epoxide hydrolase (EC 3.3.2.3) and glutathione S-transferases (EC 2.5.1.18) B, C and E were used to determine the presence and localizations of these epoxide-metabolizing enzymes in testes of sexually immature and mature Wistar and Holtzman rats. Unlabeled antibody peroxidase-antiperoxidase staining for each enzyme was readily detected in rat testes at the light microscopic level. Although significant strain-related differences were not apparent, staining intensity for certain enzymes differed markedly between Leydig cells and seminiferous tubules. Leydig cells of immature and mature rats were stained much intensely for epoxide hydrolase and glutathione S-transferase B and E than were seminiferous tubules, whereas Sertoli cells, spermatogonia, spermatocytes and spermatids, as well as Leydig cells, were stained intensely by the anti-glutathione S-transferase C. Age-related differences in staining for glutathione S-transferase B were not obvious, while the anti-glutathione S-transferase C stained seminiferous tubules more intensely in immature rats, and antibodies to expoxide hydrolase and glutathione S-transferases C and E stained Leydig cells much more intensely in mature rats. These observations thus demonstrate that testes of both sexually immature and mature rats contain epoxide hydrolase and glutathione S-transferases. Except for glutathione S-transferase C in immature rats, Leydig cells appear to contain much higher levels of enzymes than do seminiferous tubules. During sexual maturation, the testicular level of glutathione S-transferase B appears to remain constant, while levels of epoxide hydrolase and glutathione S-transferases C and E increase within Leydig cells and the level of glutathione S-transferase C decreases within seminiferous tubules.     

Differentiation and development of testis in the oviparous lizard, Calotes versicolor (Daud.)

The differentiation and development of the testis in the lizard Calotes versicolor was studied histologically and histoenzymatically from the day of oviposition (stage 27) to 2 months after hatching. The study reveals the appearance of the gonadal component as a genital ridge at stage 27. The first sign of testis differentiation is observed at stage 33, which displays a well-developed medulla consisting of seminiferous cords comprising Pre-Sertoli cells. The sex differentiation of the embryonic gonads occurs at stage 34. At this stage, seminiferous cords of the testis are prominent and extensive with many pre-Sertoli cells and few spermatogonia. The interstitial space consists of immature fibroblast-type Leydig cells. Pre-Sertoli cells of the seminiferous cords differentiate into Sertoli cells with a triangular nucleus becoming apparent around stages 36-37. The fibroblast-like Leydig cells differentiate into round matured Leydig cells at stage 40. Quantitative estimation of germ cells reveals that the number of germ cells increases in individual gonads, and in 5-day-old hatchling's, this number multiplies by manifold. Spermatogonia show reductional division in the testis of 1-day-old hatchlings.Histochemical localization of Delta5-3beta-HSDH and G-6-PDH activity appears in the seminiferous cords (medulla) of the testis after sexual differentiation (stage 36), indicating that the embryonic medulla is the site of steroidogenesis and not the cortex in C. versicolor. This study also suggests that morphological differentiation of the gonad precedes detectable steroidogenesis in this species. In 10-day-old hatchling's, Delta5-3beta-HSDH activity is seen in the interstitial cells of the testis, which, however, is not detected in the seminiferous tubules. The intensity of the enzyme activity remains more or less the same in the testis up to 10 days after hatching and begins to increase thereafter. The increase in steroidogenesis parallels the progressive post-hatching increase of the interstitial/Leydig cells.     

Immunolocalization and activity of aromatase in the bank vole testes.

A direct approach to identify the cellular source of P450 aromatase in the bank vole testes (seasonally breeding rodents) is the use of immunohistochemistry with a specific antibody that recognizes this enzyme. To confirm the presence of functional aromatase, its activity was measured in microsomal preparations of whole testes and of seminiferous tubules by means of biochemical assay with tritiated androstenedione. The assay was validated using increasing concentrations of both microsomal preparations. Immunoreactive aromatase was found in Leydig cells, Sertoli cells, and germ cells, especially in spermatocytes and spermatids. The aromatase activity was present in microsomal fractions of whole testis and seminiferous tubules. The immunolocalization of P450 aromatase and aromatase activity have been found as photoperiod-dependent.     

Tyrosine protein kinase activity in purified rat Leydig cells

Tyrosine protein kinase activity has been estimated in purified testicular cells with the synthetic peptide substrate NH₂-GLU-ASP-ALA-GLU-TYR-ALA-ALA-ARG-ARG-ARG-GLY-COOH. High levels of enzyme specific activity (56–165 pmol/mg/min) were found in the two populations of Leydig cells isolated by Metrizamide gradient centrifugation. Some activity was also detected in germinal cells, red cells and seminiferous tubules from testis but at levels 6–20 times lower than those found in the Leydig cell fractions. Higher levels of tyrosine protein kinase specific activity were found in population I than in population II Leydig cells.     

Immunocytochemical localization of cytochrome P450 aromatase in the testis of prepubertal, pubertal, and postpubertal horses

The large amount of testicular estrogens produced by the stallion is unique compared to the amounts found in other domestic species. Although the cellular locale of the cytochrome P450 aromatase (P450arom) enzyme that converts C19 androgens to C18 estrogens has been identified in the Leydig cell of adult equine testis, the location in the immature equine testis is not known. The goal of this work was to localize the enzyme in colts and stallions during sexual development. Testes were obtained from prepubertal (n=7), pubertal (n=6), and postpubertal (n=8) colts and stallions during both the breeding and non-breeding seasons. Tissue was fixed and prepared for immunocytochemistry (ICC), carried out with an antiserum against human placental P450arom. In prepubertal colts, there was distinct immunopositive staining of a similar degree within both the Leydig cell and the seminiferous tubule. Horses in the pubertal group had strong Leydig cell immunopositive staining and a slight degree of positive staining within the seminiferous tubules. Postpubertal stallions exhibited definitive immunopositive staining within Leydig cells but not within the seminiferous tubules. Therefore, P450arom is present within the Leydig cell throughout sexual development. In contrast, the presence of P450arom within the seminiferous tubule based upon ICC appeared to be gone by adulthood, suggesting that an age-dependent shift in the locale of this enzyme as the stallion matures.     

Expression of polysialic acid, alpha- and beta-cantenins in adult toad testis in hibernation stage and after gonadotrophin--releasing hormone (GnRH) treatment

The Polysialic Acid (PSA), glycosydic moiety of the Neural Cell Adhesion Molecule (N-CAM), and alpha- and beta-Catenins, which mediate interaction between Cadherins and cytoskeletal proteins, participate in cell adhesion phenomena in numerous organs and tissues. We have performed an immunohistochemical analysis, in hibernating toad testis and in GnRH-reactivated hibernating animals. In hibernating toads we could demonstrate PSA-immunoreactivity (PSA-IR) within the seminiferous tubules, in clusters of primary spermatocytes, spermatids and spermatozoa, in follicular and Sertoli cells. PSA-IR was seen in peritubular, Leydig and efferent duct cells. In GnRH-treated toads PSA-IR persists in primary spermatocyte groups. alpha-Catenin is localized in the basal laminae of seminiferous tubules and in Leydig cells of hibernating toads. This did not change after hormonal treatment. In hibernating toads, beta-Catenin was detected only in Leydig cells and within seminiferous tubules on basal spermatocystes and limiting spermatozoa clusters. In GnRH-treated toads, the beta-Catenin-IR was less intense in Leydig cells and vanished within seminiferous tubules.     

Morphologic study of the testes from spontaneous unilateral and bilateral abdominal cryptorchid boars

Macroscopical and histological characteristics were examined in both testes from three healthy boars, three boars with unilateral abdominal cryptorchidism on the right side, and three boars with bilateral abdominal cryptorchidism. Abdominal cryptorchidism, unilateral and bilateral, provoked a significant decrease of the weight and volume of the ectopic testes. The scrotal testis of the unilateral cryptorchid boars showed an increase in its volume and weight. Cryptorchidism also induced abnormalities in the histological structure of seminiferous tubules, lamina propria, and interstitial tissue of the abdominal testes. The number of seminiferous tubules decreased; the seminiferous epithelium was constituted by few spermatogonia with an atypical pattern and by abnormal Sertoli cells. The lamina propria showed a variable degree of thickening and collagenization. The interstitial tissue was very developed but displayed a decrease in the Leydig cell population. These abnormalities were more critical in bilateral cryptorchidism than in unilateral cryptorchidism. The scrotal testis of the unilateral cryptorchid boars showed normal appearance, but a decrease of the number of seminiferous tubules was observed. Moreover, the seminiferous tubules showed impaired spermatid maturation. The alterations observed in the abdominal testes of the unilateral and bilateral cryptorchid boars were attributed to defective proliferation and differentiation of Sertoli cells and Leydig cells. The anomalies in the scrotal testis of the unilateral cryptorchid boars were due to disturbances in the Sertoli cell activity.     

Changes in Leydig cell activity during the annual testicular cycle of the bat Myotis lucifugus lucifugus: histology and lipid histochemistry

Changes in Leydig cell histology and testicular sudanophilic lipids were examined in relation to spermatogenic activity in the bat Myotis lucifugus lucifugus (Chiroptera: Vespertilionidae) throughout the annual cycle in the northeastern United States. These changes were correlated with annual variations in plasma testosterone concentrations which have recently been described for this species. Gametogenic activity occurred during the months of May-August when bats were metabolically most active. During hibernation (October-April), when sperm are stored in the epididymides, and accessory glands are hypertrophic, the seminiferous tubules were at rest, and the germinal epithelium was reduced to reserve spermatogonia and Sertoli cells. Based on their structure and cyclic pattern of sudanophilic lipids, Leydig cells exhibited a pattern of activity that closely paralleled that of the seminiferous epithelium. On renewal of spermatogenesis in spring, Leydig cells became hypertrophied and accumulated lipid inclusions. These inclusions, seen as vacuoles in plastic sections and sudanophilic droplets in frozen sections, reached maximal accumulations in late June. In late July and during August, when peak testosterone levels occur in blood, lipid droplets were dramatically depleted, and Leydig cells were weakly sudanophilic. In September, when testosterone titers return to low baseline levels, Leydig cells had regressed but exhibited a marked increase in sudanophilic inclusions which appeared to be mostly lipofuscins. During the ensuing mating and hibernation periods, Leydig cells were involuted and filled with lipofuscins. During the periarousal period, however, Leydig cells became weakly Sudan-positive while many large, intensely sudanophilic cells were scattered throughout the interstitium. In electron micrographs these cells were identified as macrophages. They appear to play an important role in the annual testicular cycle by phagocytizing the residues of Leydig cell involution in preparation for a new steroidogenic cycle. Seasonal changes in lipid inclusions were also observed in the seminiferous tubules. In addition, the relationship of the Leydig cell cycle to androgen action and the accessory organs in this bat is discussed.     

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

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

Subcutaneous grafts of fresh or cryopreserved testis in castrated rats and mice: comparison of the histologic aspects of the grafts

In the castrated rat, only testis taken in one to two week-old donors observed three months after sub-cutaneous isograft contain a well developed interstitial tissue and some seminiferous tubules with germinal cells. On the contrary in castrated mice, testicular grafts taken in adult animals show some Leydig cells and degenerating seminiferous tubules. These grafts permit the restoration of androgenic activity in previously castrated recipients.     

Nitric oxide/cGMP pathway components in the Leydig cells of the human testis

In this study we sought to determine whether the main components of the nitric oxide (NO) pathway are localized within the Leydig cells of the human testis and whether the soluble guanylyl cyclase (sGC), the enzyme that accounts for NO effects, is functionally active in these cells. Using an amplified immunocytochemical technique, immunoreactivity for nitric oxide synthase (NOS-I), sGC and cyclic guanosine monophosphate (cGMP) was detected within the cytoplasm of human Leydig cells. Distinct differences in staining intensity were found between individual Leydig cells, between cell groups and between Leydig cells of different patients. By means of a specific cGMP-RIA, a concentration-dependent increase in the quantity of cGMP was measured in primary cultures of human Leydig cells following exposure to the NO donor sodium nitroprusside. In addition, NOS-I immunoreactivity was seen in Sertoli cells, whereas cGMP and sGC immunoreactivity was found in Sertoli cells, some apically situated spermatids and residual bodies of seminiferous tubules. Dual-labelling studies and the staining of consecutive sections showed that there are several populations of Leydig cells in the human testis. Most cells were immunoreactive for NOS-I, sGC and cGMP, but smaller numbers of cells were unlabelled by any of the antibodies used, or labelled for NOS-I or cGMP alone, for sGC and cGMP, or for NOS-I and sGC. These results show that the Leydig cells possess both the enzyme by which NO is produced and the active enzyme which mediates the NO effects. There are different Leydig cell populations that probably reflect variations in their functional (steroidogenic) activity. Received: 27 March 1996 / Accepted: 14 July 1996     

The site of aromatization in the mouse cryptorchid testis

Using the mouse cryptorchid model, degenerations of germ cells were observed as well as a reduced size of seminiferous tubules, while the area of the interstitial tissue increased. Aromatase, the enzyme responsible for the conversion of androgens into oestrogens, was immunolocalized in Leydig cells and in germ cells from both scrotal and abdominal testes, and in Sertoli cells only in a control testis. In the cryptorchid testis, aromatase was strongly expressed in a few tubules, including those spermatids that were still present. Other cells inside the tubules were negative for aromatase. In both testes, oestrogen receptors alpha were expressed only in Leydig cells. Strong aromatase expression in germ cells indicates an additional source of oestrogens in the testis besides the interstitial tissue.     

Cyclic nucleotide phosphodiesterases in somatic and germ cells of mouse seminiferous tubules

The distribution of phosphodiesterase forms in somatic and germ cells, and their variations during testicular development and germ cell differentiation have been investigated. Seminiferous tubules from immature mice and Sertoli cells in culture possessed two enzyme activities which were comparable to forms described for different tissues and species: (a) a calcium-calmodulin-dependent enzyme with high affinity for guanosine 3',5'-(cyclic)-monophosphate (cGMP), and (b) a calcium-calmodulin-independent enzyme with high affinity for adenosine 3',5'-(cyclic)-monophosphate (cAMP) the activity of which increased in cultured Sertoli cells after treatment with FSH or dibutyryl cAMP. Seminiferous tubules from adult animals and germ cells at the meiotic and post-meiotic stage of differentiation possessed two enzyme forms that could be distinguished from those present in somatic cells of the seminiferous tubules: (a) a calcium-calmodulin-dependent form with high affinity for both cAMP and cGMP, similar to forms described in other tissues from different species, and (b) a calcium-calmodulin-independent phosphodiesterase with high affinity for cAMP and present only in post-meiotic cells, previously identified also in germ cells of the rat.     

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.     

Morphometric studies on hamster testes in gonadally active and inactive states: light microscope findings

This study provides quantitative information on the testes of seasonally breeding golden hamsters during active and regressed states of gonadal activity. Seminiferous tubules occupied 92.5% of testis volume in adult gonadally active animals. Leydig cells constituted 1.4% of the testicular volume. The mean volume of an individual Leydig cell was 1092 microns 3, and each testis contained about 25.4 million Leydig cells. The volume of an average Sertoli cell nucleus during stage VII-VIII of the cycle was 502 microns 3. A gram of hamster testis during the active state of gonadal activity contained 44.5 million Sertoli cells, and the entire testis contained approximately 73.8 million Sertoli cells. Testes of the hamsters exposed to short photoperiods for 12-13 wk displayed a 90% reduction in testis volume that was associated with a decrease in the volume of seminiferous tubules (90.8% reduction), tubular lumena (98.8%), interstitium (72.7%), Leydig cell compartment (79.3%), individual Leydig cells (69.7%), Leydig cell nuclei (50.0%), blood vessels (85.5%), macrophages (68.9%), and Sertoli cell nuclei (34.1%). The diameter (61.1%) and the length (36.8%) of the seminiferous tubules were also decreased. Although the number of Leydig cells per testis was significantly lower (p less than 0.02) after short-photoperiod exposure, the number of Sertoli cells per testis remained unchanged. The individual Sertoli cell in gonadally active hamsters accommodated, on the average, 2.27 pre-leptotene spermatocytes, 2.46 pachytene spermatocytes, and 8.17 round spermatids; the corresponding numbers in the regressed testes were 0.96, 0.20, and 0.04, respectively. The striking differences in the testicular structure between the active and regressed states of gonadal activity follow photoperiod-induced changes in endocrine function and suggest that the golden hamster may be used as a model to study structure-function relationships in the testis.