Kinetic and autoradiographic studies on binding of hCG to the testicular LH receptors of neonatal rats

The properties of hCG binding to LH receptors of the neonatal (5-day-old) rat testis were analysed and compared with those of the adult testis. The equilibrium association constants (Ka) of hCG-binding were similar at both ages, 2-4 X 10(10) M-1. In contrast, kinetic binding studies revealed that the association and dissociation rate constants of hCG binding were more rapid in the neonatal testis. Likewise, it was observed that the progression from loose (easily dissociable) to tight (non-dissociable) binding was less complete in the young than in the adult testis. Autoradiography of 125I-labelled hCG binding to interstitial cell suspensions at the two ages showed that the gonadotrophin binding per Leydig cell was about 50% lower in the neonatal testis. Conversely, since the surface area of adult Leydig cells was about 4-fold larger, the receptor density appeared to be higher in the neonatal Leydig cells. The rapid recovery of LH receptors after hCG stimulation, typical of the neonatal cells, was due to rapid replenishment of binding in the cells initially occupied by the injected hormone, rather than to an hCG-induced increase of Leydig cell number. Finally, in-vivo experiments with cycloheximide revealed that the rapid recovery of LH receptors was dependent on protein synthesis. These differences in the kinetics of neonatal testicular LH receptor turnover may be involved in the unique functional features of the fetal-neonatal growth phase of rat testicular Leydig cells.     

Gonadotropin stimulation of cyclic adenosine monophosphate and testosterone production without detectable high-affinity binding sites in purified Leydig cells from rat testis.

Rat testicular interstitial cells were separated by three different gradient-density procedures and, with each, two biochemically and morphologically distinct cell fractions were isolated. The lighter density cells in fraction-I bound iodine 125-labeled human chorionic gonadotropin (hCG) with high-affinity (apparent equilibrium dissociation constant, Kd, approximately 10(-10) M) without producing either cyclic adenosine monophosphate or testosterone in response to hormone action. The heavier-density cells displayed morphologic features typical of Leydig cells and produced cyclic adenosine monophosphate and testosterone in the presence of hCG without detectable 125I-labeled hCG high-affinity binding. These cell fractions were further characterized by studies using deglycosylated hCG, a known antagonist to hCG action. Cell concentration-dependent studies with purified Leydig cells revealed that maximal testosterone production was achieved when lower cell concentrations (0.5 x 10(6) cells/250 microliters) were used for in vitro hCG stimulation assays. Under these conditions, the 125I-labeled hCG binding was barely detectable (2.24 fmol; 2,698 sites/cell). Furthermore, these studies revealed that the hCG-specific binding in Leydig cells is overestimated by the classic method for nonspecific binding correction using excess unlabeled hormone. An alternate method is presented.     

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.     

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.     

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.     

AVP receptors of mouse Leydig cells are regulated by LH and E2 and influenced by experimental cryptorchidism

Exposure of pubertal mouse Leydig cells for 24 h to increasing concentrations (1-100 ng/ml) of LH elicited a dose-dependent decrease in AVP receptor content. Maximal reduction (50%) was obtained at a dose of 10 ng/ml LH. A similar treatment applied to adult Leydig cells did not influence AVP receptor density. Treatment of adult Leydig cells for 24 h by E2 (5-500 ng/ml) resulted in a dose-dependent increase in AVP receptor content. About 50% increase was achieved with 500 ng/ml E2. AVP receptor content in pubertal Leydig cells was not modified irrespective of the concentration of E2 tested. These changes in AVP receptor number were well correlated with the response of Leydig cells to AVP (10(-6) M) in terms of testosterone production. 2 weeks bilateral cryptorchidism resulted in reduction of testicular weight, circulating testosterone levels associated with a marked rise in Leydig cell AVP receptor density with no change of affinity. Testosterone production by Leydig cells from cryptorchid testes in response to AVP (10(-6) M) or hCG (100 ng/ml) stimulation was reduced compared to that of control Leydig cells. This study provides new arguments supporting the concept that AVP could be involved in local regulation of testicular steroidogenesis.     

Luteinizing hormone receptor-mediated effects on initiation of spermatogenesis in gonadotropin-deficient (hpg) mice are replicated by testosterone

Testosterone (T) is an absolute requirement for spermatogenesis and is supplied by mature Leydig cells stimulated by LH. We previously showed in gonadotropin-deficient hpg mice that T alone initiates qualitatively complete spermatogenesis bypassing LH-dependent Leydig cell maturation and steroidogenesis. However, because maximal T effects do not restore testis weight or germ cell number to wild-type control levels, additional Leydig cell factors may be involved. We therefore examined 1). whether chronic hCG administration to restore Leydig cell maturation and steroidogenesis can restore quantitatively normal spermatogenesis and testis development and 2). whether nonandrogenic Leydig cell products are required to initiate spermatogenesis. Weanling hpg mice were administered hCG (0.1-100 IU i.p. injection three times weekly) or T (1-cm subdermal Silastic implant) for 6 weeks, after which stereological estimates of germinal cell populations, serum and testicular T content, and testis weight were evaluated. Human CG stimulated Leydig cell maturation and normalized testicular T content compared with T treatment where Leydig cells remained immature and inactive. The maximal hCG-induced increases in testis weight and serum T concentrations were similar to those for T treatment and produced complete spermatogenesis characterized by mature, basally located Sertoli cells (SCs) with tripartite nucleoli, condensed haploid sperm, and lumen development. Compared with T treatment, hCG increased spermatogonial numbers, but both hCG and T had similar effects on numbers of spermatocytes and round and elongated spermatids per testis as well as per SC. Nevertheless, testis weight and germ cell numbers per testis and per SC remained well below phenotypically normal controls, confirming the involvement of non-Leydig cell factors such as FSH for quantitative normalization of spermatogenesis. We conclude that hCG stimulation of Leydig cell maturation and steroidogenesis is not required, and that T alone mostly replicates the effects of hCG, to initiate spermatogenesis. Because T is both necessary and sufficient for initiation of spermatogenesis, it is likely that T is the main Leydig cell secretory product involved and that additional LH-dependent Leydig cell factors are not essential for induction of murine spermatogenesis.     

An enzymatic method for the isolation of mouse Leydig cells

Mouse Leydig cells were obtained by dispersion of testes of adult animals (aged 6-15 months) with a neutral protease from B. polymxa (dispase; EC 3.4.24.4). The crude Leydig cell suspension was purified by centrifugation on a discontinuous Percoll gradient using a special centrifugation procedure similar to elutriation. The crude cell suspension obtained from 50 testes could be processed in one run. The combination of these two methods yielded 320000 +/- 53000 Leydig cells/testis (n = 554 testes). The purity of the Leydig cell fraction was greater than or equal to 95% (nucleated cells) based on morphological and histochemical (staining for naphthyl esterase) identification. The purified Leydig cells showed an excellent ultrastructural appearance. More than 98% excluded trypan blue. In the presence of NADPH, testosterone biosynthesis was increased only 1.15 +/- 0.1-fold yielding a "quality factor" of 34.8. Maximal hCG doses induced 10(6) purified Leydig cells to produce 5 nmol testosterone/hr. (40-fold stimulation in comparison to basal values). The Leydig cells showed 43100 +/- 2500 LH/hCG receptors and an association constant of Ka = 1.95 x 10(9) M-1. Due to the reproducibility of the method, to the yield as well as to the morphological and functional state of the purified Leydig cells at least 25% of laboratory animals could be saved.     

Percoll-gradient separation of Leydig cells from postnatal rat testes

The characteristics of the fetal and adult populations of Leydig cells from postnatal rat testes were compared by Percoll gradient centrifugation. A single peak of hCG binding, due to the presence of fetal Leydig cells, was obtained after purification of intertubular cells from 8-day-old animals. Two peaks of specific hCG binding were obtained after purification of intertubular cells from 15-day-old rats: it was confirmed by autoradiographic techniques that the hCG was bound by adult-like Leydig cells in one peak and fetal Leydig cells in the other. Similarly, intertubular cell preparations from 21- and 25-day-old rats resolved into two peaks of hCG binding; adult-like Leydig cells were observed in the first peak, but fetal Leydig cells were rarely observed in the second of these peaks. These results demonstrate the separation of two Leydig cell populations from intertubular cells obtained from animals aged up to 15 days. Thereafter the pattern of the hCG binding profile is similar but is not due to the presence of the same cell types. Therefore these results emphasize the necessity for morphological identification of cell types to permit the correct interpretation of the corresponding biochemical data.     

Photoperiodic modulation of testicular LH receptors in the bank vole (Clethrionomys glareolus)

The temporal changes in testicular binding of 125I-labelled hCG in juvenile bank voles (18 days of age, born and reared in a 18L:6D photoperiod) exposed to a long (18L:6D, Group L) or short (6L:18D, Group S) photoperiod for 0, 3, 7, 14 and 42-56 days were investigated. During testicular maturation, in Group L, there was a slight initial decrease in LH receptor numbers per testis followed by a marked prepubertal rise during the initial phase of rapid testicular growth after which a decrease took place. In Group S, during testicular regression, the temporal changes in LH receptor numbers per testis resembled those of Group L except that the corresponding increase in hCG binding during the initial week was considerably less marked and the receptor numbers remained thereafter at a significantly lower level than in Group L. Leydig cell count indicated that the observed changes in LH receptors per testis were due to changes in the number of Leydig cells as well as in LH receptors per Leydig cell. The present results indicate, that (1) photoperiod is an important modulator of testicular LH receptor numbers in this species, (2) photoperiod or age has no significant effect on the binding affinity of LH receptors, (3) short photoperiods arrest the induction of LH receptors as well as the increase in Leydig cell numbers associated with normal testicular maturation, and (4) changes in LH receptor numbers per testis correlate well with the photoperiod-induced changes in androgen biosynthesis, spermatogenesis and Leydig cell morphology observed in our previous studies.     

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.     

Effects of hyper- and hypoprolactinemia on gonadotropin secretion, rat testicular luteinizing hormone/human chorionic gonadotropin receptors and testosterone production by isolated Leydig cells

The effect of prolactin (Prl) on gonadotropin secretion, testicular luteinizing hormone (LH)/human chorionic gonadotropin (hCG) receptors, and testosterone (T) production by isolated Leydig cells has been studied in 60-day-old rats treated for 4 days, 4 and 8 weeks with sulpiride (SLP), a dopaminergic antagonist, or for 4 days and 4 weeks with bromocriptine (CB), a dopaminergic agonist. Plasma Prl concentrations were significantly greater in the SLP groups (204 +/- 6 ng/ml) and lower in the CB groups (3.0 +/- 0.2 ng/ml) than those measured in the control groups (54 +/- 6 ng/ml). The plasma concentrations of gonadotropin were not affected by a 4-day treatment with SLP or CB, nor were they after a 4-week treatment with CB. However, the hyperprolactinemia induced by an 8-week treatment with SLP was associated with a reduced secretion of gonadotropin (LH, 16 +/- 4 vs. 35 +/- 6 ng/ml; FSH, 166 +/- 12 vs. 307 +/- 14 ng/ml). In SLP-induced hyperprolactinemia, a 30% increase in the density of the LH/hCG testicular binding sites was observed (178 +/- 12 fmol/mg protein), whereas a 60% decrease was measured in hypoprolactinemia (55 +/- 5 vs. control 133 +/- 5 fmol/mg protein). Plasma T levels were increased in 4-day and 4-week hyperprolactinemic animals (4.3 +/- 0.4 and 3.9 +/- 0.4 ng/ml, respectively), but returned to normal levels in the 8-week group (3.0 +/- 0.5 vs. C: 2.3 +/- 0.2 ng/ml). No T modifications were observed in hypoprolactinemic animals. Two distinct populations of Leydig cells (I and II) were obtained by centrifugation of dispersed testicular cells on a 0-45% continuous Metrizamide gradient. Both possess LH/hCG binding sites. However, the T production from Leydig cells of population II increased in the presence of hCG, whereas that of cell population I which also contain immature germinal cells did not respond. The basal and stimulated T secretions from cell populations I and II obtained from CB-treated animals were similar to controls, whereas from 4 days to 8 weeks of hyperprolactinemia, basal and hCG induced T productions from cell population II decreased progressively. These data show that hyperprolactinemia causes, in a time-dependent manner, a trophic effect on the density of LH/hCG testicular receptors; reduces basal and hCG-stimulated T production from isolated Leydig cells type II; and results in an elevated plasma T concentration which decreases with time. The latter suggests a slower T catabolism and/or an impaired peripheral conversion of T into 5 alpha-dihydrotestosterone (DHT). Although hypoprolactinemia is associated with a marked reduction in testicular LH receptors, it does not affect T production.     

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

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

Adrenomedullin in the rat testis. I: Its production, actions on testosterone secretion, regulation by human chorionic gonadotropin, and its interaction with endothelin 1 in the leydig cell

Based on the finding of gene expression of adrenomedullin (Adm) and its receptor components in the rat testis, a paracrine effect of ADM on testicular steroidogenesis has been suggested by our group. The present study demonstrates the gene expression of Adm and the effect of ADM on testosterone production in the Leydig cell. The regulation of ADM by hCG and its interaction with endothelin 1 (EDN1) in the rat Leydig cells are also observed. Primary culture of Leydig cells produced Adm mRNA and secreted 275+/-19 pg immunoreactive ADM per 10(6) cells in 24 h. In addition, the Leydig cell was shown to coexpress mRNAs encoding for the calcitonin receptor-like receptor (CALCRL) and receptor activity-modifying protein (RAMP1, RAMP2, and RAMP3). These may account for the specific binding of ADM to the Leydig cells. Administration of ADM to Leydig cells resulted in an inhibition of hCG- and EDN1-stimulated testosterone production. Correlated with this, ADM reduced EDN1 production, whereas its production was increased by EDN1. Furthermore, the production of ADM and the mRNA levels of Calcrl and Ramp2 were suppressed by hCG. Our results suggest that ADM has an autocrine effect on Leydig cell steroidogenesis, possibly by interacting with EDN1 and under the control of gonadotropin. We propose that there is an ADM/EDN1 local regulatory mechanism that may be important in modulating the control of testicular functions by gonadotropins.     

Paracrine regulation of testicular function

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

Effects of prolonged cyclosporine-A treatment on the Leydig cells of the rat testis

The effects of a prolonged (30-day) treatment with daily therapeutical doses of cyclosporin A (CAS) (20 mg/kg) on testicular Leydig cells were studied in adult rats. CSA administration provoked a significant decrease in both basal and human chorionic gonadotropin (hCG)-stimulated testosterone concentration in the peripheral blood without affecting the volume of the testes or the interstitial space. However, there was conspicuous atrophy of the Leydig cells, due mainly to a decrease in mitochondria and smooth endoplasmic reticulum, the organelles containing the enzymes of testosterone synthesis. Lipid droplets, in which cholesterol is stored, were notably increased. The nuclear volume and the surface area per cell of rough endoplasmic reticulum fell significantly in Leydig cells of CAS-treated animals. In light of these findings, it is concluded that CSA inhibits the growth and steroidogenic capacity of rat Leydig cells, probably by depressing their protein synthesis. Whether the mechanism underlying the action of CSA on Leydig cells is only indirect, by blockade of hypophyseal gonadotropin release, or also direct is unsettled and requires further investigation.     

Effects of restraint stress on plasma LH and testosterone concentrations, Leydig cell LH/hCG receptors, and in vitro testicular steroidogenesis in adult rats

We examined the effect of restraint stress (3 hr) on plasma LH and testosterone levels, on the Leydig cell LH/hCG receptor, and on the activity of enzymes in the testicular steroidogenic pathway of the adult rat. Restraint stress caused a 47% reduction in plasma testosterone concentrations, but had no effect on plasma LH levels. The binding capacity and affinity of Leydig cell LH/hCG receptors were not affected by restraint. Stress did not affect the testicular activity of 20,22 desmolase or 3 beta-hydroxysteroid dehydrogenase, but testicular interstitial cells of stressed rats incubated in vitro with progesterone as a substrate produced more 17 alpha-hydroxyprogesterone but less testosterone than control cells, and when incubated with 17 alpha-hydroxypregnenolone, produced 39% less androstenedione and 40% less testosterone than control cells. These results suggest that restraint stress inhibited 17,20 desmolase but not 17 alpha-hydroxylase activity. When the delta 4 pathway was blocked with cyanoketone (3 beta-HSD inhibitor), stress did not alter the production of pregnenolone or 17 alpha-hydroxypregnenolone, but the production of dehydroepiandrosterone by cells from stressed rats was subnormal, suggesting again a reduction of 17,20 desmolase activity. The data suggest that a major site of the inhibitory action of restraint stress on testicular steroidogenesis is the 17,20 desmolase step. The disruption of androgen production by restraint appears to be LH independent since stress did not affect plasma LH levels, the binding capacity or affinity of LH/hCG receptors, or the activity of 20,22 desmolase.     

Separation of germ cells from somatic cells in mouse testis by affinity for a lectin, peanut agglutinin

A new method for the separation of germ cells from somatic cells in the mouse testis was accomplished by making use of the differences in cell surface affinity for a lectin, peanut agglutinin (PNA). The separation procedure was based on the specific presence of PNA receptor on testicular germ cells and its absence on somatic cells, such as Leydig, Sertoli and peritubular cells. As a result, more than 99% of cells in PNA receptor-positive (PNA+) fractions were identified as germ cells by immunoperoxidase reaction with specific antiserum to mouse testicular germ cells. In contrast, Leydig cells were enriched in PNA receptor-negative (PNA-) fractions, i.e., 65% of cells in these fractions were cytochemically stained for 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) activity.