Immunocytochemical localization of aromatase in rat testis

Summary The immunocytochemical localization of armatase in the testes of young and adult rats was investigated by an indirect-immunofluorescent method using antihuman placental aromatase-II cytochrome P-450 antibody. In both young (1 and 2 weeks old) and adult rats, only the Leydig cells in the interstitial tissue showed a positive immunoreaction for aromatase, while the germ cells and Sertoli cells in the seminiferous tubule were entirely negative. In addition, electron microscopy revealed that the Leydig cells in the testes of young as well as adult rats have a well-developed smooth endoplasmic reticulum, mitochondria with tubulovesicular cristae, and a few lipid droplets; these structures being characteristic of steroid secretory cells. On the basis of these results, we suggest that estrogens are mainly synthesized in Leydig cells of the testes.Supported by grants from the Ministry of Education, Science, and Culture, Japan, and USPHS HD 04945     

Cytochrome P450 aromatase in the testis of immature and mature pigs

Aromatization of androgens into estrogens is performed by a microsomal enzyme, the cytochrome P450 aromatase. A direct approach for identifying the cellular source of aromatase is the use of immunohistochemistry with a specific antibody that recognizes aromatase. The pig presents some unusual features with regard to the synthesis of testosterone and estrogens in the male gonads. In testes from prepubertal males, testosterone level measured radioimmunologically, was lower than in testes from adult pig, while estrogen secretion was relatively high and comparable to that of mature porcine gonads. Immunolocalization of aromatase in testes from both immature and mature pigs was confined to the Leydig cell cytoplasm. The intensity of immunohistochemical staining indicated the presence of unsynchronous Leydig cell population. Other somatic cells and germ cells were negative for aromatase. In control tissue sections, incubated in the absence of the primary antibody or in the presence of normal rabbit serum, no positive staining was observed. Western blot analysis revealed one major band of aromatase about 50-52 kDa in testes from both immature and mature pigs.     

Testicular morphology and expression of aromatase in testes of mice with the mosaic mutation (Atp7a mo-ms)

The aim of this study was to determine whether testicular cells of mice with the mosaic mutation, associated with abnormal copper metabolism, are able to aromatize androgens to estrogens, and what is the putative role of estrogens in the gonad of the mutant male. Mosaic is a lethal mutation; affected males usually die on about day 16. Those, which survive to reach sexual maturity, are valuable research subjects. In testes of young and adult mutants, histological analysis revealed the presence of many degenerating seminiferous tubules besides normal-looking ones. Additionally, high numbers of apoptotic germ cells were observed, especially in young mutants when compared with the controls. Positive immunostaining for aromatase was found in cultured Leydig cells and testicular sections of both control and mutant males. The intensity of immunostaining was always stronger in the mosaic mice. In both groups, Western-blot analysis revealed the presence of aromatase protein as a single band of approximately 55 kDa. In the mosaic males, levels of testosterone in cultured Leydig cells, whole testes, and in blood plasma were lower than in those of the respective controls. On the contrary, estradiol concentrations were always higher in the mutants. Both in vivo and in vitro studies indicate that morphological and functional changes in the testes of the mosaic mice mainly result from defective copper metabolism. The higher level of endogenous estrogens can additionally enhance morphological alterations within the testes. It seems also likely that excess estrogens may affect the survival rate of the mosaic males.     

Aromatase in the human testis

Low levels of testicular estrogen synthesis have been reported in a number of species, but the cellular localization has not been unequivocally established. To study aromatase in the human testis, we have combined immunocytochemistry with direct measurement of enzyme activity in the testicular 6μm cryosections. Thus, the functionality of the immunoreaction and its sensitivity can be assessed in quantitative terms. Testes were obtained from immediate autopsy from men aged 18–53 years, from surgery from two patients with prostatic cancer (67 and 74 years) and from two normal children aged 8 months and 3 years at autopsy. Benign testicular sex cord tumors were also examined from two unrelated patients aged 5 and 8 years with gynecomastia and diagnosed with Peutz-Jeghers syndrome. Our results consistently showed low to moderate staining intensity of immunoreactive aromatase in comparison to that of normal human placental cryosections. Immunoreactive aromatase was only present in the interstitial Leydig cells and absent from the Sertoli cells of all normal adult testes showing spermatogenesis. Aromatase activity correlated well with the intensity of the immunostain. However, there was no obvious relationship between the level of aromatase activity and increasing age. Generally higher levels were present in testes of young men (18–22 years). No immunostain in any cell type was detected in one 33-year-old patient with testicular cancer. In the testes of the two normal prepubertal boys, no immunostaining was observed. However, intensely stained Sertoli cells as well as high aromatase activity were observed in the testicular tumors of the patients with Peutz-Jeghers syndrome. Our results suggest that Leydig cells are the source of aromatase in normal men but that Sertoli cells may express this enzyme under abnormal conditions. The combined methods for measuring enzyme activity and immunoreactive aromatase are suitable for application to tissues expressing low levels of aromatase.     

Differential effects of the destruction of Leydig cells by administration of ethane dimethane sulphonate to postnatal rats

Ethane dimethane sulphonate (EDS) is a cytotoxic drug that selectively destroys Leydig cells in adult testes. This study has examined the effect of a single injection of EDS on the Leydig cell populations present in the testes of rats aged 5, 10, or 20 days. Microscopic examination of the tissue demonstrated that the fetal Leydig cell population was destroyed at all ages, but that subsequent development of the adult population of Leydig cells was not affected. Whilst the destruction of the fetal Leydig cells in this acute phase of EDS on 5-day-old rats was accompanied by a decline in serum testosterone levels, there was no apparent effect on this hormone when EDS administered at 10 or 20 days of age, despite the destruction of fetal Leydig cells in these rats. The long-term effects of EDS on Day 5 of age resulted in proliferation of the intertubular tissue in which more Leydig cells were observed, but serum testosterone and testosterone levels in response to human chorionic gonadotropin stimulation in vitro were normal despite moderate or severe disruption of the seminiferous epithelium. These data show that the fetal Leydig cells of immature testes are sensitive to the cytotoxic effects of EDS in the adult, but the response of the testes differs depending on the age at which the drug is administered.     

Structural and functional abnormality of ectopic testes in rats

Some males of a mutant strain of King-Holtzman rats exhibit an anomalous heritable defect manifested as either unilateral or bilateral ectopic testes. In the adult, these testes contain seemingly immature Sertoli and Leydig cells, seminiferous tubules greatly reduced in diameter, and exhibit arrested spermatogenesis. Thus, the affected testis is essentially sterile. An inability to produce normal amounts of testosterone and androstenedione by these gonads is probably a reflection of changes that have been effected in their Leydig cells. Thus, this study suggests that abnormal function of the Leydig and Sertoli cells and seminiferous tubule failure in these mutant animals result from the physiologically cryptorchid condition.     

Cyclic AMP phosphodiesterase in Leydig cell preparations of the rat testis

Enzymatic digestion of the interstitial tissue of early juvenile and adult rat testes resulted in an enrichment of the Leydig cell population. The cells of the intertubular preparation from adult testes were separated by centrifugal elutriation, according to differences in sedimentation velocity, a counter-flow centrifugation technique leading to 70% Leydig cell purity. Using this approach, it was possible to demonstrate that Leydig cells from adult testes contain only low affinity isoenzymes of cyclic AMP phosphodiesterase (PDE; E.C.: 3.1.4.17), an intracellular regulator of cAMP. Starch gel electrophoresis showed that the isozyme of cAMP PDe of Leydig cells is masked in crude testis homogenates due to the relatively low level of these cells in the total population. In Leydig cells, there are two different electrophoretic forms expressed which resemble two of eleven different molecular forms of cAMP PDE demonstrated for comparison in 21 different organs of the adult rat. An interstitial cell preparation from early juvenile testes, with a Leydig cell content of up to 20%, was also investigated electrophoretically with regard to molecular forms of cAMP PDE, the properties of which were characterized by kinetics analysis of cAMP hydrolysis. The results presented are discussed in relation to the onset of testosterone synthesis in Leydig cells of prepubertal rats leading to the initiation of male puberty.     

Ultrastructure and morphometry of testicular Leydig cells and the interstitial components correlated with testosterone in aging rats

The ultrastructure of testicular interstitium in young and aged adult rats was analysed using morphometric methods, and the plasma testosterone concentration was measured. With increasing age there was an augumentation in the volume of collagen fibrils in the intercellular matrix and in blood vessels. During the aging process (approximately two years) the average volume of the Leydig cell decreased from 1364 m³ to 637 m³, but the number of Leydig cells in paired testes increased from 53x10⁶ to 113x10⁶. The absolute volume of smooth surfaced endoplasmic reticulum (SER) per Leydig cell amounted in aged rats to 78% of that in young adult rats. The total amount of SER in paired testes increased by 62% with aging. The present analysis suggests that the ability of SER to maintain peripheral testosterone concentration decreases with age. In young adult rats the absolute volume of peroxisomes per Leydig cell correlated significantly with the concentration of testosterone in blood and also with the absolute volume of SER per Leydig cell. These results combined with ultrastructural observations of close apposition of peroxisomes and SER suggest that peroxisomes have a role in testosterone secretion by Leydig cells.Visiting scientist to Laboratory of Electron Microscopy (Director: Prof. L.J. Pelliniemi)     

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.     

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.     

Cyclic AMP Phosphodiesterase in Leydig Cell Preparations of the Rat Testis

Enzymatic digestion of the interstitial tissue of early juvenile and adult rat testes resulted in an enrichment of the Leydig cell population. The cells of the intertubular preparation from adult testes were separated by centrifugal elutriation, according to differences in sedimentation velocity, a counter-flow centrifugation technique leading to 70% Leydig cell purity. Using this approach, it was possible to demonstrate that Leydig cells from adult testes contain only low affinity isoenzymes of cyclic AMP phosphodiesterase (PDE; E.C.: 3.1.4.17), an intracellular regulator of cAMP. Starch gel electrophoresis showed that the isozyme of cAMP PDE of Leydig cells is masked in crude testis homogenates due to the relatively low level of these cells in the total population. In Leydig cells, there are two different electrophoretic forms expressed which resemble two of eleven different molecular forms of cAMP PDE demonstrated for comparison in 21 different organs of the adult rat.
An interstitial cell preparation from early juvenile testes, with a Leydig cell content of up to 20%, was also investigated electrophoretically with regard to molecular forms of cAMP PDE, the properties of which were characterized by kinetic analysis of cAMP hydrolysis. The results presented are discussed in relation to the onset of testosterone synthesis in Leydig cells of prepubertal rats leading to the initiation of male puberty.     

Immunolocalization of aromatase in stallion Leydig cells and seminiferous tubules.

High levels of plasma estrogens constitute an endocrine peculiarity of the adult stallion. This is mostly due to testicular cytochrome p450 aromatase, the only irreversible enzyme responsible for the bioconversion of androgens into estrogens. To identify more precisely the testicular aromatase synthesis sites in the stallion, testes from nine horses (2-5 years) were obtained during winter or spring. Paraplast-embedded sections were processed using rabbit anti-equine aromatase, followed by biotinylated goat anti-rabbit antibodies, and amplified with a streptavidin-peroxidase complex. Immunoreactivity was detected with diaminobenzidine. Immunofluorescence detection, using fluoroisothiocyanate-conjugated goat anti-rabbit antibodies, was also applied. Specific aromatase immunoreactivity was observed intensely in Leydig cells but also for the first time, to a lesser extent, in the cytoplasm surrounding germ cells at the junction with Sertoli cells. Interestingly, the immunoreactivity in Sertoli cells appears to vary with the spermatogenic stages in the basal compartment (with spermatogonia) as well as in the adluminal one (with spermatids). Relative staining intensity in Leydig and Sertoli cells and testicular microsomal aromatase activity increased with age. The present study in stallions indicates that in addition to Leydig cells, Sertoli cells also appear to participate in estrogen synthesis, and this could play a paracrine role in the regulation of spermatogenesis.     

Müllerian-inhibiting substance inhibits rat Leydig cell regeneration after ethylene dimethanesulphonate ablation

The postnatal development of Leydig cell precursors is postulated to be controlled by Sertoli cell secreted factors, which may have a determinative influence on Leydig cell number and function in sexually mature animals. One such hormone, Mullerian inhibiting substance (MIS), has been shown to inhibit DNA synthesis and steroidogenesis in primary Leydig cells and Leydig cell tumor lines. To further delineate the effects of MIS on Leydig cell proliferation and steroidogenesis, we employed the established ethylene dimethanesulphonate (EDS) model of Leydig cell regeneration. Following EDS ablation of differentiated Leydig cells in young adult rats, recombinant MIS or vehicle was delivered by intratesticular injection for 4 days (Days 11-14 after EDS). On Days 15 and 35 after EDS (1 and 21 days post-MIS injections), endocrine function was assessed and testes were collected for stereology, immunohistochemistry, and assessment of proliferation and steroidogenesis. Although serum testosterone and luteinizing hormone (LH) were no different, intratesticular testosterone was higher on Day 35 in MIS-treated animals. At both time points, intratesticular 5alpha-androstan-3alpha,17beta-diol concentrations were much higher than that of testosterone. MIS-treated animals had fewer mesenchymal precursors on Day 15 and fewer differentiated Leydig cells on Day 35 with decreased numbers of BrdU+ nuclei. Apoptotic interstitial cells were observed only in the MIS-treated testes, not in the vehicle-treated group on Day 15. These data suggest that MIS inhibits regeneration of Leydig cells in EDS-treated rats by enhancing apoptotic cell death as well as by decreasing proliferative capacity.     

Hormonal regulation of lh stimulation of testosterone production in isolated leydig cells of immature rats: The effect of hypophysectomy,fsh, and estradiol-17β

Testosterone production in isolated Leydig cells from testes of immature and adult rats was stimulated by addition of LH in a dose dependent way. Hypophysectomy of adult rats had no influence on LH-stimulated testosterone production in isolated Leydig cells after 5 days. In contrast hypophysectomy of immature rats resulted after 5 days in an almost complete loss of LH sensitivity of isolated Leydig cells. Daily administration of FSH during 5 days starting immediately after hypophysectomy maintained LH responsiveness of isolated Leydig cells of immature rats. Also FSH administration starting on day 5 after hypophysectomy resulted in a restoration of LH responsiveness. Estradiol benzoate, injected simultaneously with FSH, abolished the FSH-induced LH responsiveness.     

Cell-specific knockout of steroidogenic factor 1 reveals its essential roles in gonadal function

Knockout (KO) mice lacking the orphan nuclear receptor steroidogenic factor 1 (SF-1, officially designated Nr5a1) have a compound endocrine phenotype that includes adrenal and gonadal agenesis, impaired expression of pituitary gonadotropins, and structural abnormalities of the ventromedial hypothalamic nucleus. To inactivate a conditional SF-1 allele in the gonads, we targeted the expression of Cre recombinase with a knock-in allele of the anti-Müllerian hormone type 2 receptor locus. In testes, Cre was expressed in Leydig cells. The testes of adult gonad-specific SF-1 KO mice remained at the level of the bladder and were markedly hypoplastic, due at least partly to impaired spermatogenesis. Histological abnormalities of the testes were seen from early developmental stages and were associated with markedly decreased Leydig cell expression of two essential components of testosterone biosynthesis, Cyp11a and the steroidogenic acute regulatory protein. In females, the anti-Müllerian hormone type 2 receptor-Cre allele directed Cre expression to granulosa cells. Although wild-type and SF-1 KO ovaries were indistinguishable during embryogenesis and at birth, adult females were sterile and their ovaries lacked corpora lutea and contained hemorrhagic cysts resembling those in estrogen receptor alpha and aromatase KO mice. Collectively, these studies establish definitively that SF-1 expression in the gonads is essential for normal reproductive development and function.     

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.     

Aromatase messenger RNA is derived from the proximal promoter of the aromatase gene in Leydig, Sertoli, and germ cells of the rat testis

It has long been recognized that individual cell types within the testes possess the capacity to synthesize estrogen. A number of studies on different species have demonstrated that the levels of aromatase expression and the patterns of regulation are distinct between the different cell types of the testes. Whereas a variety of promoters have been shown to contribute to the patterns of aromatase expression in different cell lineages, studies using ovarian RNA, testis RNA, and Leydig cell tumor lines have demonstrated that the same promoter (promoter II) was used in each. Recent experiments using potent aromatase inhibitors or analysis of animals in which the genes encoding the estrogen receptor-alpha (ER-alpha) or the aromatase, P450, are defective, have confirmed the importance of local estrogen formation in normal testicular function. In order to permit experiments to identify the elements controlling aromatase expression in the individual cell compartments of the testes, we prepared RNA from purified preparations of Leydig, Sertoli, and germ cells. Using specific oligonucleotide primers, the sites of initiation of the aromatase mRNA were determined using rapid amplification of cDNA ends (RACE) and nucleotide sequence analysis of the resulting cDNA fragments. Our results indicate that aromatase mRNA is derived from the proximal promoter (PII) of the aromatase gene in each of the major cell types of the rat testes.     

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.