Hormonal secretion and enzyme activity of cultured roe-deer (Capreolus capreolus L.) Leydig cells, as measured by radio-immunological and histochemical assays

Leydig cells from roe-deer collected according to Steinberger's (1975) technique were cultured as monolayers in Leighton tubes for 10 days. Cultures were grown in medium 199 supplemented with 10% calf serum. Androgen and oestrogen secretion by Leydig cells into the culture medium was measured using appropriate radio-immunoassays. Using histochemical tests the activity of the following oxydoreductive enzymes in cultured Leydig cells was shown: delta 5, 3 beta-hydroxysteroid dehydrogenase (delta 5, 3 beta-HSD), 17 beta-hydroxysteroid dehydrogenase (17 beta-HSD), succinate and lactate dehydrogenases (SDH and LDH). Strong activity of the enzymes investigated during the first 4 days of culture was observed. The androgen level was high throughout the second and fourth day of culture. A decrease in hormone secretion after day 4 occurred, and this was closely correlated with enzyme activity. The oestrogen level was very low during culture. The direct effect of the luteinizing hormone (LH) added into the culture medium caused an increase in not only enzyme activity but also androgen and oestrogen levels.     

Interaction between Leydig and Sertoli cells in vitro

The interaction between Leydig and Sertoli cells grown in co-culture was studied. After 3 to 4 days in culture, Leydig and Sertoli cells formed monolayers. To distinguish functional Leydig cells from Sertoli cells, a histochemical test for delta 5,3 beta-HSD activity was performed, and cells which showed a positive reaction were defined as Leydig cells, in contrast to Sertoli cells which did not manifest enzyme activity. Testosterone and oestradiol levels in culture media were determined by radioimmunological assays. Sertoli cells in co-culture showed a tendency to organize themselves as in vivo, forming a kind of pseudo-wall of the tubule. This process becomes more evident with the time of culture. Co-cultures secreted more androgens than Leydig cells alone and more oestradiol than Sertoli cells alone. This influence was strengthened by the presence of follicle stimulating hormone (FSH) in the culture medium, which was not the case in cultures of Leydig and Sertoli cells cultured separately.     

Correlation between NADPH-diaphorase and iNOS in bank vole Leydig cells in vitro and in testicular sections with the use of histochemistry and immunocytochemistry

Histochemistry for NADPH-diaphorase detects an enzymatic activity associated with nitric oxide synthase while immunohistochemistry detects the nitric oxide synthase molecule. NADPH-diaphorase and inducible isoform of nitric oxide synthase in Leydig cells in vitro and in testis sections of the bank vole were demonstrated histochemically and immunocytochemically. Histochemical studies revealed localization of NADPH-diaphorase reaction product in the cytoplasm of cultured Leydig cells as well as in the interstitial area, mainly in Leydig cells and in vascular endothelium. Distribution pattern of NADPH-diaphorase was different in Leydig cell cytoplasm of individual cells. Using immunocytochemistry, the immunoreactivity for nitric oxide synthase was observed both in cultured Leydig cells and testis sections. Moreover, a co-localization of positively immunostained cells with those histochemically detected was noticed. Addition of hCG to the cultured medium or injections in vivo resulted in a small decrease in reaction intensity in Leydig cells. Treatment with N omega-nitro-L-arginine methyl ester resulted in distinctly weaker reactivity of the enzymes studied which was correlated with a higher testosterone and estradiol levels in Leydig cells measured radioimmunologically. The results have indicated that nitric oxide synthase is able to act directly within the male gonad regulating androgen secretion by Leydig cells.     

Catecholamine stimulation of androgen production by rat Leydig cells. Interactions with luteinizing hormone and luteinizing hormone-releasing hormone

The mechanism(s) of the development of response to catecholamines (CA) by Leydig cells in culture was investigated with the use of primary culture of purified Leydig cells of adult rats. The interactions of a CA agonist, isoproterenol (ISOP), with luteinizing hormone (LH) and a luteinizing hormone-releasing hormone agonist analog (LHRHa) on production of androgen by the Leydig cells were also studied. Cells incubated with ISOP for 3 h increased release of cyclic adenosine 3',5'-monophosphate (cAMP) to similar extents at 0, 3, and 24 h of culture. The beta-agonist did not increase androgen release at 0 h but had a concentration-dependent effect at 3, 24, and 48 h of culture, with maximal effects at 24 h. LH stimulated high increases in production of cAMP and androgen by the cells at 0-24 h of culture. Leydig cell beta-receptors decreased with culture time. Low concentrations but not high levels of LH had additive effects with ISOP on androgen release. ISOP showed a complex interaction with LHRHa on androgen release. Chronic exposure of Leydig cells to LHRHa reduced basal androgen release as well as release of androgen stimulated by ISOP, forskolin, and LH. These studies suggest that the development of response to CA by rat Leydig cells is a postreceptor, postcAMP event and showed that CA can interact with LH or LHRH to regulate Leydig cell function.     

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.     

Possible involvement of microtubules and microfilaments of the epididymal epithelial cells in 17beta-estradiol synthesis

The rat epididymal epithelial cells revealed features of steroidogenic cells and released 17beta-estradiol (E2) into the culture medium. In steroidogenic cells, elements of the cytoskeleton due to their influence on organelle distribution are implicated in the regulation of steroidogenesis. In the present study, the morphology of cultured epididymal epithelial cells in light, scanning and transmission electron microscopes was evaluated. The organization of microtubules and microfilaments revealed by fluorescence microscopy, and the concentration of E2 in cultured medium were also studied. The epididymal epithelial cells were cultured in different conditions: in the medium with or without exogenous testosterone (T) and in the co-culture with Leydig cells as a source of androgens. The cells in co-culture located close to Leydig cells were rich in glycogen, PAS-positive substances and lipid droplets, in higher amount than the cells cultured with addition of exogenous testosterone. Stress fibers and microtubules of epididymal epithelial cells cultured with exogenous T and in co-culture with Leydig cells presented typical structure, and numerous granular protrusions appeared on the surface of the cells. Disorganization of microtubules and shortening of stress fibers as well as the smooth cell surface deprived of granular protrusions were observed in the epididymal epithelial cells cultured without T. Change of the cytoskeleton organization caused by the absence of androgen in culture medium resulted in an increased E2 secretion.     

Regulation of 3 beta-hydroxysteroid dehydrogenase activity by human chorionic gonadotropin, androgens, and anti-androgens in cultured testicular cells

delta 5-3 beta-Hydroxysteroid dehydrogenase is a key enzyme for testicular androgen biosynthesis and a marker for the Leydig cells. The hormonal regulation of this enzyme was studied in cultured rat testicular cells. Human chorionic gonadotropin (hCG) increased testosterone production in vitro while time course studies indicated a biphasic action of the gonadotropin on 3 beta-hydroxysteroid dehydrogenase activity. An initial stimulation (51%) of the enzyme was detected between 3 and 12 h of culture when medium testosterone was low. This is followed by an inhibition of 3 beta-hydroxysteroid dehydrogenase activity on days 2 and 3 of culture when medium testosterone was elevated. Concomitant treatment with a synthetic androgen (R1881) inhibited 3 beta-hydroxysteroid dehydrogenase activity and testosterone production in hCG-treated cultures while an anti-androgen (cyproterone acetate) increased 3 beta-hydroxysteroid dehydrogenase activity and testosterone biosynthesis. Addition of 10(-5) M spironolactone, an inhibitor of 17 alpha-hydroxylase, blocked the hCG stimulation of testosterone production but increased medium progesterone. In the absence of the secreted androgen, hCG stimulated 3 beta-hydroxysteroid dehydrogenase activity in a time- and dose-related manner. Furthermore, hCG stimulation of 3 beta-hydroxysteroid dehydrogenase activity and progesterone accumulation in spironolactone-supplemented cultures was decreased by concomitant treatment with R1881 but was not affected by cyproterone acetate. The inhibitory effect of R1881 was blocked by the anti-androgen. In the absence of hCG, treatment with testosterone, dihydrotestosterone, or R1881, but not promegestone, alone also inhibited 3 beta-hydroxysteroid dehydrogenase activity while the inhibitory effect of testosterone was blocked by cyproterone acetate. Thus, hCG stimulates 3 beta-hydroxysteroid dehydrogenase activity in cultured testicular cells. The androgenic steroidogenic end products, in turn, inhibit this enzyme. The hormonal regulation of 3 beta-hydroxysteroid dehydrogenase activity may be important in the ultrashort loop autoregulation of androgen biosynthesis.     

Effect of in vitro administration of LH, prolactin separately, and LH and prolactin in mixture on cultured Leydig cells from mouse testes: II. Age-related alterations of androgen secretion

The effect of gonadotropic hormones on androgen level in media of cultured Leydig cells isolated from mouse testes of various age was investigated. Changes of androgen level were connected with the age of mice. The highest secretory level of androgen was found in cultures of Leydig cells from mature 60 day old animals, which then decreased in 7 month old mice. Stimulating effect of LH and PRL on hormonal secretion by cultured Leydig cells was observed.     

Prolactin augments luteinizing hormone binding to rat Leydig cells in serum-free culture

Effect of prolactin on the testicular luteinizing hormone binding was studied in a serum-free culture system. By the collagenase digestion of decapsulated testes taken out from 25-day-old rats, Leydig cells were isolated and cultured for 7 days in DME/F12 (1:1) medium supplemented with insulin, transferrin, epidermal growth factor, and gentamicin. The cultured cells exhibited the 3β-hydroxysteroid dehydrogenase activity. Hill plots constructed from the data of competition experiment showed that the dissociation constant (K_d) was 0.33 × 10^–10M. The K_d value was approximately the same as the known value for the rat testicular homogenates. When the Leydig cells were cultured with ovine prolactin for the last 3 days of 7-day culture period, the binding of luteinizing hormone increased to 1.7-fold ofthat in the control group. From these results it is concluded that prolactin acts to up-regulate the binding of luteinizing hormone to rat testicular Leydig cells in serum-free culture     

Isolation of human Leydig cell mesenchymal precursors from patients with the androgen insensitivity syndrome: testosterone production and response to human chorionic gonadotropin stimulation in culture.

Mature Leydig cells, the main source of testicular testosterone in mammals, arise from immature mesenchymal precursors through an LH-dependent differentiation process. In order to study the steroidogenic potential of these precursors, undifferentiated mesenchymal cells were obtained from the testicular interstitium of two patients with androgen insensitivity syndrome. After double digestion with collagenase and separation of the suspensions in a Percoll density gradient, the cells were cultured in Ham's F12 medium: Dulbecco's Modified Eagle Medium (1:1) supplemented with antibiotics, transferrin, insulin, hydrocortisone, and vitamin E with or without 1 IU of hCG/ml. At 11 days in culture, samples were removed for morphological characterization and determination of 3 beta-hydroxysteroid dehydrogenase activity (3 beta-HSD). Testosterone concentration was determined by RIA in the culture medium at different intervals. Cultured cells were mesenchymal in appearance, elongated in shape, with numerous processes running in different directions. No mature Leydig cells were present. In basal conditions, the percentages of 3 beta-HSD-positive cells at 11 days on patients 1 and 2 were 33% and 28%, respectively, and the testosterone concentrations in the culture media were 4.8 and 8.4 ng.10(6) cells.24 h, respectively. In cultures stimulated with hCG, there was an increase of histochemical reactivity (47% and 42% in patients 1 and 2, respectively) and in the amount of testosterone secreted (10.2 and 12.0 ng.10(6) cells, respectively). Electron microscopic studies of cultures grown in the absence of hCG demonstrated a homogenous population of poorly differentiated, fibroblastic-type mesenchymal cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Steroid hormone responsive cells in serum-free culture--analyses of hormone-mediated gene expression and cell growth

Steroid hormone-responsive cell lines were clones from mouse mammary cancer (Shionogi Carcinoma 115) and Leydig cell tumor. SC-3 and SC-4 cells from Shionogi Carcinoma were androgen-responsive and -unresponsive in a serum-free medium, respectively. SC-3 cells secreted FGF-like growth factor as well as 24 K glycoprotein in response to androgen stimuli. B-1 and B-1F cells from mouse Leydig cell tumor were growth-stimulated in a serum-free medium by estrogen, androgen or retinoic acid. Transfection of ERE-TK-CAT gene into B-1F cells revealed that both estrogen and retinoic acid activated the CAT activity. In addition, the presence of corresponding receptors for steroid hormones or retinoic acid was demonstrated by hormone binding assays and/or Northern blot analysis. Thus, these serum-free culture systems seem to be very useful for analysing hormone action mechanisms in vitro.     

Effect of testosterone on testicular steroidogenesis in the hypogonadal (hpg) mouse

Previous studies have shown that androgens have direct inhibitory effects on steroidogenesis in active Leydig cells. It is not clear what effect androgens have on inactive Leydig cell either through direct action on the cell itself or indirectly through stimulation of Sertoli cell activity. The hpg mouse has undetectable levels of circulating gonadotrophins and the gonads fail to develop post-natally. The effect of androgen treatment on testicular steroidogenesis and morphology was examined in these animals. Treatment with testosterone propionate for two weeks significantly increased testicular and seminal vesicle weight. Seminiferous tubules showed marked development in androgen-treated animals, indicating increased Sertoli cell activity, but the abnormal Leydig cell morphology of the hpg testis was unchanged. Androgen production per testis in vitro was low in control hpg animals and remained unaffected by treatment with androgen. Similarly, the pattern of [3H]pregnenolone metabolism was not significantly affected by androgen treatment. The androgen content of the testis was higher in androgen-treated animals but this could be accounted for by uptake of administered steroid from the circulation. It is concluded that androgens have no direct trophic effect on Leydig cells and that stimulation of Sertoli cell activity is not, in itself, sufficient to affect Leydig cell function.     

The effect of prenatal treatment with busulfan on in vitro androgen production by testes from rats of various ages

Treatment of rats with busulfan in utero severely depletes the germ cell population of the seminiferous tubules. These studies have examined the in vitro capacity of testicular tissue and Leydig cells from such testes to secrete androgens. Leydig cells were identified by staining for 3 beta-hydroxy steroid dehydrogenase. Rats were studied at several ages to identify any developmental changes in the androgen-secreting capacity of control and treated gonads. At 30 days of age, no effect of treatment on serum androgen was found. At 60 and 90 days of age, treatment caused decreased androgen and increased LH content of the serum. At 12, 30, 60, and 90 days of age, the amount of androgen secreted per milligram of testicular tissue in response to LH was higher in busulfan-treated rats. Leydig cells from 60- and 90-day-old rats which had received busulfan were also hyperresponsive to LH. It was concluded that Leydig cells from testes essentially devoid of germ cells were hyperresponsive to LH. Serum androgen levels were decreased yet androgen production per Leydig cell was increased. A possible explanation of this apparent paradox is that busulfan treatment resulted in decreased numbers of Leydig cells in the gonads.     

Histochemical changes in the Leydig cells of rats drinking the aqueous hollyhock extract

The effect of aqueous hollyhock flower (Althaea rosea Cav. var. nigra) extract on the rat Leydig cell metabolism and morphology was studied using histochemical, morphometric and radioimmunological methods. The rats were drinking the extract for 30 days (group A1) and for 180 days (group A2). Leydig cells of group A1 manifested marked increase in the 3beta-HSD, G6PD and NADPD activities and in the Khanolkar reaction intensity. These findings were accompanied by the increase in the volume of Leydig cells and their nuclei. In group A2 Leydig cells, statistically insignificant changes in the G6PD and NADPD activities were observed, however, the significant increase in the 3beta-HSD activity and the Khanolkar reaction intensity indicated compensatory changes. The statistically significant elevation of the androgen level accompanied by a decrease in estrogen content in homogenates of group A2 testes pointed to weak antiestrogenic effect of the extract. The obtained results indicate an influence of the hollyhock extract on steroid metabolism.     

Hormonal regulation of oestrogen formation by Leydig cells in vitro: immunocytochemical approach

The ability of the testis to convert androgens into oestrogens is related to the presence of a microsomal enzyme, aromatase, in testicular cells. The aim of this study was to show whether the supplementation of culture media with LH or an aromatase inhibitor could affect the process of aromatisation in Leydig cells of the bank vole in vitro. This was investigated by means of immunocytochemistry and radioimmunological assays. In control cultures of Leydig cells, both steroid hormones secretion as well as immunoreactivities for aromatase and oestrogen receptor were weaker than in those treated with LH. On the contrary, the addition of aromatase inhibitor into the culture medium resulted in a decreased intensity of immunocytochemical stainings in comparison with the control. Concomitantly, the androgen level was slightly higher, whereas that of oestrogen significantly lower than in the control cultures. Additionally, to check whether steroid hormones are able to regulate aromatase or oestrogen receptor immunoexpressions, some of the Leydig cell cultures were enriched with testosterone or oestradiol, respectively. Strong immunoreactivities for both aromatase and oestrogen receptor were observed. This suggests that Leydig cells in vitro are able to regulate directly the secretion of oestrogens by active aromatase. Finally, it is concluded that oestrogen formation in bank vole Leydig cells in vitro can be influenced by various factors. It should be stressed, however, that the effect of hormone stimulation or aromatase inhibitor action appeared to be dependent on the length of light cycles that bank voles were exposed prior to the isolation of Leydig cells.     

A comparison of Leydig cell function after unilateral and bilateral cryptorchidism and efferent-duct-ligation

Surgical induction of cryptorchidism or ligation of the efferent ducts disrupts spermatogenesis. The response of Leydig cells to disrupted gametogenesis was studied in vitro in tissue and collagenase dispersed Leydig cells obtained from the testes of rats that were made unilaterally or bilaterally cryptorchid or had been efferent-duct-ligated. Four wks after surgery, androgen secretion per mg of tissue or per Leydig cell in response to maximal luteinizing hormone (LH) stimulation was greater in tissue from damaged than from sham-operated testes. It was concluded that disruption of spermatogenesis resulted in Leydig cells that were hyperresponsive to LH stimulation in vitro. Unilateral lesions produced different responsiveness of Leydig cells from the testes ipsilateral and contralateral to the lesion, supporting the hypothesis that intragonadal modulation of Leydig cells function occurs when the function of seminiferous tubules is impaired. Stimulated androgen production of Leydig cells from the contralateral nonligated testis did not differ from that of the sham-operated controls. With unilateral cryptorchidism, which is accompanied by an increase in the temperature of the operated testis, Leydig cells from the scrotal testis were also hyperresponsive compared to those from sham-operated controls. This suggests a possible intergonadal influence of aspermatogenesis caused by cryptorchidism.     

Visualization of the cytoskeleton in Leydig cells in vitro. Effect of luteinizing hormone and cytoskeletal disrupting drugs

Effect of LH, vinblastine and cytochalasin B on the cytoskeleton of cultured Leydig cells was investigated using monoclonal antibodies and fluorescence microscopy. After LH addition and treatment with cytoskeletal disrupting drugs, three main effects were observed: 1) increase of androgen level secreted by cultured mouse Leydig cells, 2) changes of cell-shape towards regular and rounded, 3) increase of delta 5,3 beta-HSD activity. The results are discussed in respect to possible involvement of cytoskeleton in the regulation of steroidogenesis.     

Enzyme histochemistry of cultured ovarian cells. II. Histochemistry of porcine theca interna cells in tissue culture.

By means of histochemical technique the activities of delta53beta-, 17beta-, 20alpha-hydroxysteroid dehydrogenases and glucose-6-phosphate dehydrogenase, as well as alkaline and acid phosphatase were investigated in monolayer cultures of theca interna cells, isolated from preovulatory porcine ovarian follicles. It was found that theca interna cells exhibited high and constant activity of delta53beta-hydroxysteroid dehydrogenase and G6P-DH, whereas activities of both 17beta- and 20alpha-hydroxysteroid dehydrogenase were lower and showed some fluctuations during in vitro culture. Addition of LH to the medium brought about the increase of all studied dehydrogenases. FSH was less effective. Estradiol showed quite and inhibiting effect. All the hormones mentioned above caused the increase of alkaline and acid phosphatase activity in cultured porcine theca interna cells.     

The expression of aromatase, estrogen receptor alpha and estrogen receptor beta in mouse Leydig cells in vitro that derived from cryptorchid males

A broad expression of aromatase and estrogen receptors (ERs) in the testis suggests an important role for estrogens in regulating testicular cell function and reproductive events. The aim of the present study was to show whether Leydig cells in vitro isolated from cryptorchid testes of two inbred strains of mice, KE and CBA, are a site of estrogen synthesis. Using immunocytochemistry, aromatase, estrogen receptor alpha(ERalpha), and estrogen receptor beta(ERbeta) were localized in cultured Leydig cells. Immunoreactive aromatase was found in the cytoplasm of control Leydig cells and those isolated from cryptorchid males, however the intensity of immunostaining was different, being stronger in Leydig cells deriving from cryptorchid mice. The strongest aromatase immunostaining was found in cryptorchid-KE Leydig cells. Strong immunoexpression of ERalpha was detected in the nuclei of both KE-and CBA-Leydig cells. The intensity of ERalpha immunostaining was stronger in cultured cells deriving from cryptorchid testes. ERbeta immunoexpression was detected predominantly in KE-Leydig cells. Control CBA-Leydig cells were negative for ERbeta or the result was inconclusive, whereas in cryptorchid CBA-Leydig cells a weak immunostaining was present in their nuclei. Western blot analysis confirmed the results obtained by immunocytochemistry. In KE- and CBA-Leydig cells aromatase as a band of 55 kDa protein was present, whereas ERalpha molecular weight was 67 kDa on Western blots. No band was detected for ERbeta. Radioimmunological analysis revealed that androgen and estrogen levels secreted by Leydig cells in vitro were strain-dependent. Additionally, in KE-Leydig cells that derived from cryptorchid mice estrogen level was distinctly higher in comparison with that of the respective control.