The morphogenic effect of gonadotropic hormones on the Leydig cells of the boar testis. II. Effects of administration of chorionic gonadotropin after hypophysectomy. Effects in vivo and in organ culture

The longer ago the hypophysectomy has been performed, the more marked is Leydig cell atrophy in the testis. The effects of HCG on cellular morphology have been observed in vivo and in organ culture; qualitative quantitative and ultrastructural aspects were studied. In vivo, the effects of a daily injection of gonadotropin on the testis of 2 boars hypophysectomized 3 1/2 months ago are shown. Markedly atrophied cells are strongly stimulated by HCG during the 15 first days (the cell and nucleus recover nearly to standard size, with the typical histological and ultrastructural appearance with all the cell organelles which characterize a functional steroid cell). Then after 1 1/2 month injection it decreases again to the initial state (very small size cytoplasm strongly reduced with very low organelle content). The number of the Leydig cells is maintained during the first 15 days, then it progressively decreases. The effects of HCG on the testicular tissue of 4 boars were studied in organ culture. Interstitial tissue with a greater or lesser degree of atrophy was examined experimentally (1 month, 3 months and 4 months after hypophysectomy) in order to prove a possible irreversibility of the effects of hypophysectomy. In each case, cell changes were studied according to the duration of the culture. Control cultures without HCG in the medium were set up simultaneously. 1 month and/or 3 months after hypophysectomy, the Leydig cells in culture progressively recover the size and the histological and ultrastructural appearances of a typical Leydig cell. After 16 days of culture, the stimulation is highest, as in vivo. The number of Leydig cells is maintained. From the 17th day stimulation decreases and the cell enters a new atrophy phase. In the anhormonal control medium the atrophy continues as long as the culture is maintained, and the number of Leydig cells decreases. 4 months after hypophysectomy, stimulation in culture is still possible during the first 10 days (proved by the same tests); however the size of the cell remains small compared to the normal; then it atrophies again quickly. In this case the hormone does not maintain the number of the Leydig cells. In the control cultures, slight response of the cell is observed, but this effect is limited and disappears a few days later; the number of the cells rapidly decreases. It has been shown that markedly atrophied Leydig cells can highly be stimulated during the first 2 weeks under the influence of HCG as well in vivo as in organ culture. The lability of the effect is not yet explained. 4 months after hypophysectomy, stimulation is not so effective.     

Basement membrane and epithelial features of fetal-type Leydig cells in rat and human testis

The basement membranes of developing Leydig cells in fetal and newborn testis of rat were studied by ultrastructural and immunocytochemical methods. Fetal-type Leydig cells in prenatal rats were organized in irregularly outlined groups in the interstitium and were extensively surrounded by ultrastructurally identifiable basement membranes and immunocytochemically localized laminin and collagen type IV. Prenatal Leydig cell precursors had small patches of laminin and collagen type IV on their surfaces, which indicated that changes in extracellular matrix took place during their differentiation to mature fetal-type Leydig cells. Additionally, ultrastructural evidence was obtained for a basement membrane surrounding the fetal human Leydig cells similar to that in fetal rats. Soon after birth the rat fetal-type cells gathered into distinct clusters surrounded by delicate envelope cells and a discontinuous basement membrane. Basement-membrane structures, laminin, and collagen type IV were observed between the clustered cells as well. The basement membranes covering large cell surface areas of the fetal-type Leydig cells in fetal and newborn rats differed from those of the adult-type cells, which, according to our earlier study, are covered only by small patches of basement membrane. The difference between the basement membranes of the fetal- and adult-type rat Leydig cells further supports the concept of two different Leydig cell populations. The earlier findings of the epithelial nature of the Leydig cells agree with the observation of basement membranes in the Leydig cells.     

Gonadotropin-induced changes in the luteinizing hormone receptors of cultured Leydig cells. Evidence of up-regulation in vitro

Previous in vivo studies have demonstrated that gonadotropic desensitization of luteinizing hormone/human chorionic gonadotropin (hCG) receptors and steroid responses is preceded by an early phase of receptor up-regulation. Hormonal desensitization has been recently reproduced in an in vitro Leydig cell culture, which has now been applied to studies on the early up-regulation of receptors. We performed comparative studies on the binding of 125I-hCG in isolated Leydig cells in plated culture and in suspension. In plated cells the total binding was up to 200% higher than that measured in suspension. This difference was not due to differential internalization. Preincubation with hCG in plated culture for 2 to 6 h increased the number of binding sites measured in suspension. The kinetics of the binding of labeled hCG to plated cells showed a secondary increase which reached its maximum after 3 h of incubation. This increase in hCG binding was not prevented by preincubation with inhibitors of protein synthesis and steroidogenesis or of microtubule or microfilament function. The sensitivities of the testosterone and cAMP responses to hCG in the plated cells were lower than those observed in suspension. These differences were maintained in the presence of a phosphodiesterase inhibitor. These results demonstrated that the cell interaction with a solid substratum is required for the acute up-regulation of the luteinizing hormone receptor and can induce changes in the Leydig cell responsiveness to gonadotropin stimulation.     

Type I IGF receptors of Leydig cells are upregulated by human chorionic gonadotropin

The effects of human chorionic gonadotropin (hCG) on type I insulin-like growth factor (IGF) receptors of purified Leydig cells were investigated. Sprague-Dawley rats (50 day-old) were treated with a single injection of hCG 10 units intraperitoneally, type I IGF receptors were then determined daily for 4 days. HCG caused a rapid increase in type I IGF receptors within 24 h, which returned to basal by 72 h. There was no significant change in binding affinity. Our present study indicates that type I IGF receptors of Leydig cells are up regulated by hCG, and this may be one mechanism by which hCG and IGF-I interact to enhance Leydig cell steroidogenesis.     

Interactions between immature porcine Leydig and Sertoli cells in vitro

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

In vitro effects of Celiptium and MR 14504 on mature rat Leydig cell testosterone production

Percoll-purified mature rat Leydig cells have been used to evaluate the testicular toxicity of two highly potent intercalating agents (Celiptium and MR 14505). Testosterone secretion in the absence and in the presence of human chorionic gonadotropin (hCG) was measured to assess Leydig cell function. Celiptium and MR 14504 induce time- and dose-related inhibitory effects on the production of testosterone by Leydig cells, both in the presence and in the absence of hCG, whatever the concentration of hCG used. We have observed that MR 14504 is about 5 times more potent as an inhibitor of rat Leydig cell steroidogenesis than Celiptium without inducing any cell toxicity. The present study indicates that the Leydig cell is an additional potential site for the primary toxic effects of these drugs in the adult rat testis.     

Effect of sinusoidal 50 Hz magnetic field on the testosterone production of mouse primary Leydig cell culture

This study evaluated the effect of sinusoidal 50 Hz magnetic field on the basal and human chorionic gonadotropin (hCG)-stimulated testosterone (T) production of 48-h mouse Leydig cell culture. The luteinizing hormone (LH) analog hCG was used to check the T response of the controls and to evaluate the possible effect of the applied magnetic field on the steroidogenic capacity of the exposed cells. Leydig cells were obtained from the testes of 35- to 45-g CFLP mice and isolated by mechanical dissociation without enzyme treatment. The cell cultures were exposed to sinusoidal 50 Hz 100 μT (root mean square) AC magnetic field during the entire time of a 48-h incubation. Testosterone content of the culture media was measured by radioimmunoassay. In cultures exposed to the magnetic field, a marked increase of basal T production was found (P < .05), compared with the unexposed controls, whereas no significant difference was seen between the exposed or unexposed cultures in the presence of maximally stimulating concentration of hCG. These findings demonstrate that sinusoidal 50 Hz 100 μT magnetic fields are able to stimulate the basal T production of primary mouse Leydig cell culture, leaving the steroidogenic responsiveness to hCG unaltered. Bioelectromagnetics 19:429–431, 1998. © 1998 Wiley-Liss, Inc.     

Primary cultures of Leydig cells from rat, mouse and pig: advantages of porcine cells for the study of gonadotropin regulation of Leydig cell function

Primary cultures of interstitial cells were prepared from the testis of mice, rats, and pigs. The cells were grown in a defined medium supplemented with low (0.1%) serum and insulin, transferrin and epidermal growth factor. Comparisons of the interstitial cell cultures from the three species were made for plating efficiency, cell survival, maintenance of hCG receptors and maintenance of steroidogenic responsiveness to hCG. The porcine cultures had a higher plating efficiency and higher hCG receptor levels per cell than Leydig cells from either rodent. Additionally, the porcine cells showed an increase in testosterone (T) production with hCG stimulation throughout their lifespan in culture while the rodent cultures showed a decrease in T stimulation with time with no stimulation by day 6 in culture. These data indicate that species differences exist in hCG receptor concentrations per cell, the maintenance of hCG receptors and steroidogenic response in culture. The initial high survival, purity and continued functional response of porcine interstitial cell cultures make them a superior system for the study of gonadotropin regulation of Leydig cell function.     

Age-related morphological and functional changes in the Leydig cells of the horse

Two ultrastructurally distinct types of Leydig cells were observed in the equine testis. Whereas the adult testis exhibited both postpubertal and adult Leydig cells, the testis of the pubertal horse contained only the postpubertal type, and that of the aged horse contained only the adult type. However, Percoll-purified testicular preparations from pubertal, adult, and aged horses all exhibited two distinct Leydig cell populations. The quantitative distribution and the functional characteristics of these Leydig cell populations (ability to bind human chorionic gonadotropin [hCG] and increase of testosterone production after hCG stimulation) evolved with the age of the horse. It is concluded that equine Leydig cells derive from two redundant successive postnatal generations and that there is no strict correlation between the functional properties and the morphological characteristics of these cells.     

Studies with purified immature porcine Leydig cells in primary culture

The steroidogenic capacity of purified immature porcine Leydig cells in culture was studied over several days. The cells were obtained by fractionating crude testicular interstitial cell suspensions on a discontinuous Percoll gradient (d = 1.037, 1.042, 1.052, 1.098 g/ml), and characterized by specific binding of 125I-human chorionic gonadotropin (hCG), testosterone (T) and cyclic adenosine 3':5'-monophosphate (cAMP) production in response to hCG, and the enzymatic determination of delta 5-3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) activity. The Leydig cells were recovered in a density band between 1.052-1.068 g/ml and grown in a chemically defined medium (Mather et al., 1981). In the absence of hCG, T production was low throughout the 6 days of culture. However, in response to hCG (10 mIU/ml), the cultured Leydig cells showed a progressive increase in T synthesis, which reached a maximum at Days 3-4. 8-Br-cAMP (1 mM) induced a comparable rise in T production to that obtained with hCG throughout the culture period. In contrast, 8-Br-cAMP induced a near maximal increase in dehydroepiandrosterone (DHEA) production from Day 1. This paper demonstrates that purified immature porcine Leydig cells in primary culture are a valuable model to study the ontogeny of Leydig cell function.     

Reduction of testicular human chorionic gonadotropin receptors by human chorionic gonadotropin in vivo and in vitro

Changes in rat and human testicular human chorionic gonadotropin (hCG) binding sites induced by hCG were estimated in vivo and in vitro. After a single administration of hCG, the specific 125I-hCG bindings were significantly reduced for 7 and 5 days in rat and human testes, respectively. Thereafter, 125I-hCG bindings had recovered to pretreatment values by the 14th day after the administration. Occupied hCG bindings accounted for about half of the reduced bindings on the day after administration of hCG. After this time, however, the occupancy did not contribute so much to the reduction of the bindings. In experiments in vitro using the organ culture technique, an exposure to hCG for 24 h induced a dose-related significant loss of the specific 125I-hCG bindings for 7 and 5 days in rat and human testes, respectively. Thereafter, the loss was gradually recovered. These patterns of changes in 125I-hCG bindings in vitro were similar to those in vivo. These findings suggest that the reduction in hCG binding sites by hCG is due to not only occupancy but also downregulation of the binding sites and that the testicular organ culture method used in the present study is useful to study hormonal regulation of testicular function, especially in human testes.     

Transient activation of c-myc protooncogene expression in Leydig cells by human chorionic gonadotropin

This study evaluated the effects of in vivo administration of human chorionic gonadotropin (hCG) on c-myc oncogene expression in Leydig cells. Sprague-Dawley rats (46-50 days old) were treated with hCG (10 units, i.p.), and purified Leydig cells were isolated 1-24 h later. HCG caused a transient elevation of c-myc mRNA in 4 h and returned to normal or lower than normal levels at 24 h. There was no change in c-fos or beta-actin mRNA levels. Our results suggest that the growth promoting effects of hCG on Leydig cells may be mediated by the transient expression of c-myc protooncogene.     

Development of fetal rat intestine in organ and monolayer culture

Maturation and differentiation of intestinal epithelial cells was demonstrated in segments of fetal rat small intestine, maintained for more than a month in suspension organ culture, by ultrastructural, biochemical, and immunological criteria. Over a 5-7 d period, fragments of fetal intestine evolved into globular structures covered with a single columnar epithelium ultrastructurally similar to suckling villus cells. Loose mesenchymal cells, cellular debris, and collagen were present inside the structures. After 6 d in culture, goblet cells, not present in the fetal intestine at day 18, were numerous and well developed. Intestinal endocrine cells were also observed. Immunofluorescence studies employing monoclonal antibodies specific for villus and crypt cells in vivo, and various enzyme assays, have demonstrated a level of differentiation and maturation of the cultured epithelial cells similar but not identical to that of suckling intestinal mucosa in vivo. Crypts and crypt cell markers were not observed in the the cultures. Addition of glucocorticoids to the culture medium resulted in the induction of sucrase-isomaltase but failed to promote most of the functional changes characteristic of the intestinal epithelium at weaning in vivo. Epithelial cells were identified in explants derived from the organ cultures by their specific expression of intestinal cytokeratin. Differentiation-specific markers, present in the epithelial cells in primary cultures, were lost upon selection and subculturing of pure epithelial cell populations. These results suggest a requirement for mesenchymal and/or extracellular matrix components in the maintenance of the differentiated state of the epithelial cells. The fetal intestinal organ cultures described here present significant advantages over traditional organ and monolayer culture techniques for the study of the cellular and molecular interactions involved in the development and differentiation of the intestinal epithelium.     

Regulation of renin angiotensins by gonadotropic hormones in cultured murine Leydig tumor cells. Release of angiotensin but not renin

Renin and angiotensins coexist in various tissues. The mode of control of the extrarenal renin-angiotensin system is not clear. Whether it is renin or angiotensin that is secreted has not been identified. We have investigated gonadotropin-dependent synthesis and subsequent release of the components of the intracellular renin-angiotensin system in a cloned and cultured mouse Leydig tumor cell line (MA-10). Treatment of cultured Leydig cells with bovine luteinizing hormone (bLH, 100 ng/ml) or human chorionic gonadotropin (hCG, 25 ng/ml) resulted in greater than 150- and 40- fold increased formation of angiotensin I and angiotensin II. In cells incubated with bLH or hCG, the majority of AII (up to 90%) was found in the culture medium while most of angiotensin I (greater than 85%) was in the cell lysate. Treatment with gonadotropic hormones (bLH/ hCG) increased renin 35- to 40-fold. Renin activity was confined mainly in the cell lysate even after the stimulation by gonadotropins, and only 1-2% of the total renin activity was detectable in culture medium. These results were interpreted that, in these transformed cells, hormonally-induced renin functions to generate angiotensin I within the Leydig cell and it is the angiotensins which are secreted.     

Leydig cell number and function in the adult cynomolgus monkey (Macaca fascicularis) is increased by daily hCG treatment but not by daily FSH treatment

Daily treatment of adult cynomolgus monkeys with 450 i.u. hCG for 16 days resulted in a significant 163% increase in the number of Leydig cells, and a 9-fold rise in plasma testosterone concentrations. The number of proliferating Leydig cells was very low, even after 16 days of treatment with hCG. Daily FSH administration (2 injections of 15 i.u. per day) did not have any effect on the number of Leydig cells or plasma testosterone values. It can be concluded, therefore, that in adult cynomolgus monkeys daily hCG treatment results in an increase in the number of Leydig cells, which is mainly caused by the differentiation of precursor cells. Since plasma testosterone concentrations were increased to an even higher extent, the steroid production per Leydig cell was also stimulated.     

Effect of human chorionic gonadotropin derivatives on leydig cell function.

BACKGROUND: Several human chorionic gonadotropin (hCG) derivatives have been detected in healthy human subjects, indicating that they may play a role in cell function. These hCG derivatives include deglycosylated hCG, proteolytic digestion products of hCG and free alpha and beta subunits of the hormone. It is well documented that testicular Leydig cells are responsive to luteinising hormone (LH) or its analogue hCG. These hormones have high affinity for LH/hCG receptors on the plasma membrane. METHODS: We designed functional and binding studies to compare the effects of native hCG and several hCG derivatives on a rat Leydig cell system. The molecular weight of the hCG derivatives was determined by SDS-PAGE and the binding affinity to LH/hCG receptors was measured by a radioligand assay. In addition, their ability to produce testosterone, cyclic AMP and arachidonic acid release was also studied. RESULTS: These hCG derivatives, with the exception of the free beta subunit, were able to bind to LH/hCG plasma membrane receptors with different affinities than that of native hCG. In addition, hCG derivatives did not increase intracellular cAMP levels or arachidonic acid release. However, they did increase testosterone production. CONCLUSION: Taken together, the results of this study lead us to suggest that these hCG derivatives may regulate the action of the native hormone in Leydig cells and are, thus, molecules of physiological relevance.     

Identification of a stem cell candidate in the normal human prostate gland

Stem cells of the human prostate gland have not yet been identified utilizing a structural biomarker. We have discovered a new prostatic epithelial cell phenotype-expressing cytokeratin 6a (Ck6a+ cells). The Ck6a+ cells are present within a specialized niche in the basal cell compartment in fetal, juvenile and adult prostate tissue, and within the stem cell-enriched urogenital sinus. In adult normal prostate tissue, the average abundance of Ck6a+ cells was 4.9%. With proliferative stimuli in the prostate organ culture model, in which the epithelial-stromal interaction was maintained, a remarkable increase of Ck6a expression was noticed to up to 64.9%. The difference in cytokeratin 6a expression between the normal adult prostate and the prostate organ culture model was statistically significant (p<0.0001). Within the prostate organ culture model the increase of cytokeratin 6a-expressing cells significantly correlated with increased proliferation index (r = 0.7616, p = 0.0467). The Ck6a+ cells were capable of differentiation as indicated by their expression of luminal cell markers such as ZO-1 and prostate specific antigen (PSA). Our data indicate that Ck6a+ cells represent a prostatic epithelial stem cell candidate possessing high potential for proliferation and differentiation. Since the development of benign prostatic hyperplasia and prostate carcinogenesis are disorders of proliferation and differentiation, the Ck6a+ cells may represent a major element in the development of these diseases.     

Temporal changes in rat Leydig cell function after the induction of bilateral cryptorchidism

Adult rats were made bilaterally cryptorchid and studied at intervals of 3, 7, 14 or 21 days to study temporal changes in Leydig cell function. Serum FSH and LH levels were measured and the cross-sectional area of the Leydig cells assessed by morphometry. The function of the Leydig cells was judged by the binding of 125I-labelled hCG to testicular tissue in vitro and the testosterone response of the testis to hCG stimulation in vitro. By 3 days after cryptorchidism, the binding of labelled hCG to testicular tissue was significantly decreased compared to that of controls, but the testes were able to respond to hCG stimulation in vitro. At 7, 14 and 21 days after cryptorchidism, an enhanced testosterone response was observed and the size of the Leydig cells was significantly greater than that of the controls, which indicated increased secretory activity by the cryptorchid testis. Although serum FSH levels were significantly elevated after 3 days of cryptorchidism, serum LH levels did not rise until 7 days, thereby suggesting that the loss of receptors is unlikely to result from down-regulation by LH. The reduced testosterone response of the cryptorchid testis in vivo to low doses of hCG and the enhanced response at high doses are probably related to the reduced blood flow to the cryptorchid testis and the decreased sensitivity of the Leydig cells induced by LH/hCG receptor loss.     

Regulation of steroidogenesis in rat Leydig cells in culture: effect of human chorionic gonadotropin and dibutyryl cyclic AMP on the synthesis of cholesterol side chain cleavage cytochrome P-450 and adrenodoxin

Rat Leydig cells in primary culture were used as a model system to investigate the effects of human chorionic gonadotropin (hCG) and dibutyryl cyclic AMP (Bt2cAMP) on the synthesis of cholesterol side chain cleavage cytochrome P-450 (cytochrome P-450scc) and the iron-sulfur protein, adrenodoxin. Leydig cells isolated from the testes of mature rats were placed in monolayer culture in the absence of stimulatory factors for 8 days. HCG (10 mIU/ml) or Bt2cAMP (1 mM) were then added to some of the cultures and the incubations were continued for up to 48 h. Testosterone production was increased markedly in cells incubated with hCG or Bt2cAMP. A significant accumulation of pregnenolone in the medium of cells treated with Bt2cAMP was also observed. Both hCG and Bt2cAMP increased the rates of synthesis of cytochrome P-450scc and adrenodoxin. In hCG-treated cells the apparent rate of synthesis of cytochrome P-450scc was increased 13-fold over that of controls after 48 h of incubation; the rate of adrenodoxin synthesis was increased 4-fold by hCG treatment. In Bt2cAMP-treated cells the rate of synthesis of cytochrome P-450scc was 37-fold greater than that of control cells after 48 h of incubation; adrenodoxin synthesis was increased 36-fold over controls. In hCG- and Bt2cAMP-treated cells, the concentration of immunoreactive cytochrome P-450scc and adrenodoxin increased with increasing time of incubation, and were correlated with the stimulatory effects of these agents on cytochrome P-450scc activity and on total steroid production. The results of this study are indicative that the maintenance by LH/hCG of elevated levels of testosterone synthesis by the Leydig cell is mediated, in part, by induction of the synthesis of cytochrome P-450scc and its associated protein, adrenodoxin. Since Bt2cAMP had effects similar to those observed with hCG, it is suggested that the stimulatory effects of hCG on the synthesis of cytochrome P-450scc and adrenodoxin are mediated by increased cyclic AMP formation.