Specific dose-dependent effects of ethane 1,2-dimethanesulfonate in rat and mouse Leydig cells and non-steroidogenic cells on programmed cell death.

The mechanism by which ethane 1,2-dimethanesulfonate (EDS) selectively kills Leydig cells is poorly understood. To characterize further the cell-specific actions of EDS, we studied biochemical and morphological changes during apoptosis in different Leydig cell and non-steroidogenic cell models. Rat testicular and H540 tumor Leydig cells were killed by 1-2 mM EDS, whereas 20 mM EDS were required for MA-10 cells. This higher concentration of EDS was also necessary for activation of apoptosis in non-steroidogenic Chinese hamster ovary cells, whereas COS-1 monkey kidney cells were resistant. These variable effects of EDS on apoptosis were independent of new protein synthesis and, interestingly, could be delayed by co-incubation with dibutyrl cyclic AMP. Along with cell death, we also observed chromosomal fragmentation and other hallmarks indicative of apoptosis as evidenced by DNA laddering and fluorescent microscopy. Time-lapse photography with a confocal microscope showed that the time of onset, duration and even the sequence of apoptotic events between individual H540 cells was heterogeneous. When the dose of EDS was gradually increased from 2 to 10 mM, the proportion of cells showing normal apoptotic features gradually decreased. Intriguingly, treatment with 10 mM EDS did not result in death for most cells and was marked by an absence of DNA laddering and ultrastructural features of apoptosis and necrosis. However, incubation with 20 mM EDS resulted in necrosis.These results demonstrated that the effects of EDS on cell survival are not specific to Leydig cells, that different cell types have different sensitivities to EDS and that stimulation of the cAMP pathway may mitigate EDS action. The data obtained with H540 cells further revealed that EDS can induce two types of programmed cell death.

Implications of progesterone metabolism in MA-10 cells for accurate measurement of the rate of steroidogenesis.

In virtually all studies with MA-10 cells, progesterone RIAs have been used to measure steroid synthesis. To test whether progesterone is a stable end product, we investigated the metabolism of added tritiated progesterone and pregnenolone in MA-10 cells over a period of 3 h. Steroids were then extracted, separated by HPLC, and identified by GC/MS. We found that more than 70% of radiolabeled steroids were converted to at least five different metabolites. A major metabolite (40%) was 5 alpha-pregnan-3 alpha or 3 beta-ol-20one. Similar studies, using radiolabeled T, demonstrated conversion to dihydrotestosterone and two forms of 5 alpha-androstane-diols. These data indicate the presence of active 5 alpha-reductase and 3 alpha- and/or 3 beta-hydroxysteroid dehydrogenase activities in MA-10 cells. Because these results suggest that progesterone is an unstable end product, to gauge the level of active metabolism, we incubated cells in the presence of inhibitors of pregnenolone metabolism and assessed pregnenolone levels by RIA. We discovered that basal levels of steroidogenesis in MA-10 cells were considerably higher than previously estimated. Moreover, dibutyryl cAMP-stimulated steroid production was linear over more than 13 h, in contrast to previous findings that measured progesterone levels. Other consequences of inaccurate assessment of steroidogenic activity in MA-10 cells because of the application of the progesterone assay are discussed.

Apoptosis related gene products in differentiated and tumorigenic rat Leydig cells and following regression induced by the cytotoxin ethane dimethanesulphonate.

Androgen secreting Leydig cells in the adult are differentiated with a very low turnover, however, Leydig cell tumours can arise spontaneously or after treatment with toxins. This study in the rat investigated whether changes in components of programmed cell death could be involved. In contrast to their absence in differentiated Leydig cells, antiapoptotic Bcl-2 and proapoptotic Bax were expressed in tumours. Bak and Bcl-xl were found in both tumour and normal Leydig cells. Apoptosis was induced in subcutaneous implants of Leydig cell tumour by ethane dimethanesulphonate (EDS) which is known to kill differentiated Leydig cells. The marked regression of the tumour following EDS treatment was transient and re-growth occurred between 6 and 14 days later. Tumour regression and growth was associated with a similar weight pattern in the seminal vesicles caused by changes in serum testosterone. During tumour regression, clusterin and Bax proteins were elevated but Bak, Bcl-xl and Bcl-2 were unchanged. Fas-R, Fas-L and Bax were upregulated after tumour regression had taken place. These data show that Leydig cell tumours possess many of the apoptosis related gene products and can die by apoptosis, however, regulation is clearly different in differentiated and mitotic Leydig cells.

Calcium confusion--is the variability in calcium response by Sertoli cells to specific hormones meaningful or simply redundant?

When results of more than ten different studies on hormone-induced calcium signals in Sertoli cells are taken together, a wide variety of responses emerges. The reported changes range from increased concentrations, via no response at all, to decreased calcium concentrations. Minor variations in cell isolation techniques, culture conditions, or techniques for measuring the intracellular calcium could explain some of these differences. However, erratic variations in response are also observed within research groups under very similar experimental conditions. Such 'negative' findings are mainly reported orally and do not further penetrate the scientific community. As hormone-dependent calcium responses evidently may depend very much on the context of the cells, calcium transients would appear to be unreliable bioassay principles with which to detect the primary actions of FSH and effectors such as androgens on Sertoli cells. A more important biological question is whether these sometimes opposed calcium transients are connected with a particular cellular response. To date there is no evidence for such a tight coupling in Sertoli cells, implying that, at least under in vitro conditions, calcium signals might even be redundant altogether. Such calcium variability is probably not unique to Sertoli cells, and the aim of this commentary is to promote an open debate that may help to transform the current state of 'calcium confusion' into a better understanding of the intracellular calcium language.

Rapid androgen actions on calcium signaling in rat sertoli cells and two human prostatic cell lines: similar biphasic responses between 1 picomolar and 100 nanomolar concentrations.

Androgen-induced calcium fluxes and gap junctional intercellular communication (GJIC) were studied in three different cell types. A transient (2-3 min duration) increase in intracellular calcium levels was observed within 20-30 sec of androgen addition, which was followed by a plateau phase with steroid concentrations higher than 1 nM. The kinetics of the calcium responses were similar in immature rat Sertoli cells, which contain normal nuclear receptors; the human prostatic tumor cell line, LNCaP, which contains a mutated nuclear receptor; and the human prostatic cell line, PC3, which does not contain a nuclear receptor. The human A431 tumor cell line did not respond to androgens. Concentrations of testosterone and the synthetic androgen, R1881, between 1-1000 pM induced transient calcium increases with ED(50) values near 1 pM and 1 nM, whereas dihydrotestosterone (DHT) was not active at these concentrations. At concentrations higher than 1 nM, testosterone, R1881, and DHT were equipotent in stimulating an increase in calcium that lasted for more than 10 min, with ED(50) values between 5 and 20 nM. Testosterone covalently bound to albumin was also active, whereas 11 related androstane compounds as well as progesterone and estradiol-17beta were inactive at 1000 nM. The calcium response induced by the three androgens (10 nM) was abolished in all cell types by hydroxyflutamide (1000 nM) and finasteride (1000 nM), but not by cyproterone acetate (1000 nM). The calcium response was also abolished in the absence of extracellular calcium and strongly inhibited by the presence of verapamil. Exposure of the responsive cells to brief (150-sec) pulses of androgens generated calcium responses that were similar to those after continuous exposure. After exposure of Sertoli cells for only 30 sec to 100 nM testosterone, the calcium response lasted for at least 50 min. Although nuclear binding of androgens could be demonstrated, there was no evidence for tight binding to the plasma membrane under similar conditions. When protein synthesis was inhibited, an enhancement of GJIC between rat Sertoli cells, but not between LNCaP cells or PC3 cells, was observed within 15 min of the addition of 10 nM testosterone. Because nuclear androgens are not present in PC3 cells and many functional properties of the responsive system are different from the nuclear receptor in all three cell types, we postulate the existence of an alternative cell surface receptor system with biphasic response characteristics (high and low affinity). The calcium signals are probably coupled to the regulation of gap junctional efficiency between Sertoli cells. The low-affinity receptors may convey complementary androgen signals at elevated local levels such as in the testis, when nuclear receptors are (over)saturated.

Ethane dimethylsulphonate selectively destroys Leydig cells in the adult bonnet monkeys (Macaca radiata).

AIM: To study the effect of intratesticular administration of ethane-1,2-dimethylsulphonate (EDS) which has been extensively used to selectively destroy Leydig cells in rats and study the role of gonadotropin in regulation of differentiation of Leydig cells (LC) in the adult male bonnet monkey. METHODS AND RESULTS: In vitro studies with cultured interstitial cells isolated from monkey testis revealed an inhibitory effect of EDS on LC as assessed by decrease in testosterone production. Intratesticular administration of EDS (5, 10, 20, 50 mg/testis) resulted in a dose-dependent rapid decrease in serum testosterone levels, with a 65% decrease with 5 mg of EDS by the 3rd day, which returned to control levels by the 45th day. EDS treatment resulted in a significant decrease in testicular testosterone. In addition a significant decrease in [125I]hCG binding and phenylesterase activity in the interstitial cells was noticed. Histological analysis of the testes on the 5th day after administration of EDS revealed an interstitium devoid of LC indicating the destructive action of EDS. CONCLUSION: The monkey LC are sensitive to destructive action of EDS.

Ethane dimethane sulfonate and NNN'N'-tetrakis-(2-pyridylmethyl)ethylenediamine inhibit steroidogenic acute regulatory (StAR) protein expression in MA-10 Leydig cells and rat Sertoli cells.

Apoptosis inhibits steroid biosynthesis, but it is not clear how the Steroidogenic Acute Regulatory (StAR) protein, is affected. To characterize StAR expression during apoptosis, mouse MA-10 Leydig tumor cells were treated with ethane dimethane sulfonate (EDS), an inducer of apoptosis, and the metal ion chelator NNN'N'-tetrakis-(2-pyridylmethyl)ethylenediamine (TPEN), an inducer of cell death. Both chemicals induced cell death and similarly inhibited dbcAMP-stimulated steroidogenesis and accumulation of the 30 kDa form of StAR. Utilizing the dye JC-1, it was found that TPEN and EDS also impaired the mitochondrial electrochemical potential (delta psi). In Sertoli cells, which also express StAR, EDS induced cell death and attenuated StAR expression. We conclude 1) steroidogenesis and accumulation of mature StAR protein are inhibited as a consequence of the induction of apoptosis; 2) reduced levels of StAR may be partially attributed to inhibition of import because of the loss of delta psi; 3) loss of steroidogenesis is probably due to loss of StAR synthesis and disruption of delta psi.

Low levels of follicle-stimulating hormone receptor-activation inhibitors in serum and follicular fluid from normal controls and anovulatory patients with or without polycystic ovary syndrome.

In patients with normogonadotropic anovulation, either with or without polycystic ovary syndrome (PCOS), factors interfering with FSH action may be involved in arrested follicle development. The aim of this study is to assess whether factors inhibiting FSH receptor activation are elevated in serum or follicular fluid from anovulatory patients, as compared with regularly cycling women. For this purpose, a Chinese hamster ovary cell line, stably transfected with the human FSH receptor, has been applied. FSH-stimulated cAMP secretion in culture medium was measured in the presence of serum or follicular fluid. Chinese hamster ovary cells were stimulated with a fixed concentration of FSH (3 or 6 mIU/mL) to mimic FSH levels in serum or follicular fluid. Samples were added in concentrations ranging from 3-90% vol/vol to approach protein concentrations occurring in serum or follicular fluid. In the presence of 10% vol/vol serum from regularly cycling women (n = 8), FSH-stimulated cAMP production was inhibited to 42 +/- 2% (mean +/- SEM of 2 experiments, each performed in duplicate) of cAMP production in the absence of serum, whereas a similar cAMP level (up to 38 +/- 4% of the serum-free level) was observed at higher concentrations of serum (30-90% vol/vol). The inhibition of FSH-stimulated cAMP production in the presence of serum samples from normogonadotropic anovulatory patients, without (n = 13) or with (n = 16) PCOS, was similar to controls. Follicular fluid samples (n = 57) obtained during the follicular phase in 25 regularly cycling women and follicular fluid samples (n = 25) from 5 PCOS patients were tested in a slightly modified assay system. In the presence of 10 or 30% (vol/vol) follicular fluid, FSH-stimulated cAMP levels were decreased to 68 +/- 2% and 55 +/- 2% (mean +/- SEM of a single experiment in triplicate) of the cAMP levels in the absence of follicular fluid, respectively. There was no correlation between the degree of cAMP inhibition and follicle size, steroid content (androstenedione or estradiol concentrations), or menstrual cycle phase. Furthermore, no differences in inhibition were found, comparing PCOS follicles with size- and steroid content-matched follicles obtained during the normal follicular phase. It is concluded that inhibition of FSH receptor activation by proteins present in serum or follicular fluid is constant (60 and 40%, respectively) and independent from the developmental stage of the follicle, either during the normal follicular phase or in patients with normogonadotropic anovulation. Inhibition of FSH receptor activation may be of limited significance for normal and arrested follicle development.

Application of a CHO cell line transfected with the human FSH receptor for the measurement of specific FSH receptor activation inhibitors in human serum.

Effects of FSH on ovarian follicular development can be modulated by factors present in serum or by locally produced factors in follicular fluid. Some of these factors may act directly on the FSH receptor. A Chinese hamster ovary cell line (CHO-F3B4) stably transfected with the human FSH receptor has been used to measure the effects of these modulators on FSH-stimulated adenylate cyclase activity. After incubation of CHO-F3B4 cells with human recombinant FSH (recFSH) for 4 h, cAMP levels were elevated 100-230 times above basal levels (ED50 24.9 mU/ml recFSH). cAMP production was inhibited after the addition of increasing amounts (up to 90% of the incubation volume) of hypogonadotrophic human serum (HS) at a fixed stimulatory dose of 30 mU/ml recFSH. At 10% HS the cAMP response was diminished to approximately 40-60% of the original value, whereas at a concentration of 90% HS the cAMP values were diminished to 30%. Effects of serum components on cell viability could be excluded, since forskolin- and cholera-toxin-stimulated cAMP production were not affected by preincubation of the cells in the presence of HS. The FSH-stimulated oestradiol production in rat Sertoli cells, which has been used frequently for in vitro bioassays of FSH, was almost completely inhibited by the addition of human serum, suggesting that serum has more pronounced effects on events downstream of receptor activation. Various specific FSH binding inhibitors have been demonstrated by radioreceptor assays to be present in serum. In order to assess whether such FSH receptor binding inhibitors would also inhibit receptor activation, the specific conditions used in the radioreceptor assays (buffers of low ionic strength) were also used to measure the effects of serum on FSH receptor activation. Under these conditions (a low-salt buffer, corrected for low osmolarity with 200 mM sucrose), CHO-F3B4 cells responded to FSH stimulation in a similar way to that observed in normal buffers. When CHO-F3B4 cells were incubated in this low-salt buffer with a fixed low dose of FSH (3 mU/ml), the addition of 3-90% (v/v) dialysed HS inhibited the FSH-stimulated cAMP accumulation to a similar extent to that in standard conditions. The observed inhibition of adenylate cyclase activation by the low-molecular-mass fraction (< 10 kDa) of HS could be attributed to the presence of salts in this fraction, since the addition of PBS in similar concentrations displayed an equal degree of inhibition. It is concluded that the inhibitory effects of serum on FSH-stimulated cAMP production in CHO-F3B4 cells are small, compared with the inhibition of aromatase induction in rat Sertoli cells. The strong inhibition of aromatase in rat Sertoli cells may result from the effects of serum acting on the FSH receptors as well as on other pathways not related to the FSH receptor. Therefore, measurement of aromatase in Sertoli cells is not suitable for the detection of inhibitors of FSH receptor activation. The CHO-F3B4 cells are useful for the measurement of whether inhibition of FSH receptor activation occurs in serum or follicular fluid from patients with disturbed follicle development.

The relative importance of the oligosaccharide units in human chorionic gonadotropin (CG) for LH/CG receptor activation in rat Leydig cells and mouse Leydig tumor cells.

The biological properties of deglycosylated human chorionic gonadotropin (DhCG), obtained by hydrogen fluoride treatment (HF-DhCG) of intact hCG or by oligonucleotide-directed mutagenesis (CHO-DhCG), and that of their fully glycosylated counterparts, were tested in terms of cAMP and steroid production in rat Leydig cells and in mouse Leydig tumor cells (MA-10 cells). In both cell types, HF-DhCG and CHO-DhCG possessed comparable biological activities. The maximum for DhCG-induced cAMP production was approximately 12% of that of intact hCG when tested in rat Leydig cells, and only 2% when tested in MA-10 cells. DhCG possessed significant steroidogenic activity in both cell types. In MA-10 cells the maximum for DhCG-induced steroidogenesis was 30-50% of that of intact hCG, while in rat Leydig cells DhCG and hCG induced similar steroidogenic maxima. Based on its ED50, DhCG possessed 10-17% of the steroidogenic potency of intact hCG in rat Leydig cells, while in MA-10 cells DhCG was only 2-fold less potent than hCG. When accurate hormone-receptor binding data are absent, the intrinsic receptor-stimulating activity of a ligand can still be estimated at full receptor occupancy, provided that over the whole dose range the biological response is proportional to receptor stimulation. The present data show that in transfected MA-10(P+29) cells which over-express rat phosphodiesterase, the hormone-induced stimulation of cAMP and steroid production is directly coupled to receptor activation up to maximal occupation of the LH/CG receptor.(ABSTRACT TRUNCATED AT 250 WORDS)

Inhibition of the luteinizing hormone-dependent induction of cholesterol side chain cleavage enzyme in immature rat Leydig cells by Sertoli cell products.

The modulation of the luteinizing hormone (LH) induction of cholesterol side chain cleavage (CSCC) enzyme in immature rat Leydig cells was studied using rat Sertoli cell-conditioned medium (SCCM), which stimulates short-term endogenous steroid production. Luteinizing hormone increased the CSCC enzyme activity 10-fold in cells cultured for 7 days in the absence of hormones. This enzyme induction was abolished almost completely in the presence of SCCM. The inhibition was dose dependent (half-maximal effect at 5 mg protein/l) and paralleled by a decrease in the amount of cytochrome P-450scc (P-450scc) enzyme. There were no indications for loss of cell viability. The inhibitory action of SCCM could be localized at the level of adenylate cyclase activation and at steps beyond cyclic adenosine monophosphate production. The inhibition was not specific for Sertoli cell products because conditioned media from different cell lines and media from isolated rat hepatocytes displayed similar effects. Trypsin treatment of SCCM destroyed the activity whereas the bioactivity could resist heating for 5 min at 100 degrees C. Generally occurring (growth) factors, such as epidermal growth factor or tumor necrosis factor alpha, may have contributed to the observed inhibitory effects of SCCM. These inhibitory effects of Sertoli cell products in vitro are in contrast to stimulatory effects of Sertoli cells on Leydig cell steroidogenesis in vivo after FSH administration.

Temporal changes in sulphated glycoprotein-2 (clusterin) and ornithine decarboxylase mRNA levels in the rat testis after ethane-dimethane sulphonate-induced degeneration of Leydig cells.

Short- (3-24 h) and long-term (4-50 days) changes in sulphated glycoprotein-2 (SGP-2) and ornithine decarboxylase (ODC) mRNA levels in the adult rat testis were studied following a single dose of ethane-dimethane sulphonate (EDS), to destroy the Leydig cells. Distribution patterns of SGP-2 and ODC labelling were consistent with prevailing expression of the two transcripts in Sertoli cells and germ cells, respectively. This pattern did not show appreciable changes following EDS administration. No labelling of SGP-2 mRNA was noted in the interstitium of control and EDS-treated rats. This finding indicates that Leydig cell death induced by EDS is not associated with increased SGP-2 mRNA levels, a phenomenon related to apoptotic cell death in many tissues. Semi-quantitative densitometric analysis of the preparations demonstrated differential changes in SGP-2 and ODC mRNA levels in the tubular compartment following EDS treatment. At 6, but not at 3 and 12, h following EDS administration, SGP-2 mRNA levels showed a significant increase, possibly secondary to a direct effect of the alkylating agent on Sertoli cells. A significant decrease in ODC mRNA levels was observed from day 7 to day 28, matching degenerative changes in the seminiferous epithelium. In contrast, a decrease in SGP-2 transcript levels was observed from days 21-35 after treatment. In conclusion, our findings demonstrate that SGP-2 mRNA, a putative marker of apoptosis, is not altered in the testicular interstitium during EDS-induced degeneration of Leydig cells.(ABSTRACT TRUNCATED AT 250 WORDS)

[Application of confocal laser microscopy in studying the effect of epidermal growth factor on the seminiferous epithelium]. / Application de la microscopie laser confocale à l'étude de l'effet de l'EGF sur l'épithélium séminifère.

Effects of epidermal growth factor (EGF) on pH transients in aggregates of Sertoli cells and germinal cells have been investigated with confocal microscopy using a fluorescent pH sensitive indicator. In some Sertoli cells EGF caused a rapid rise in the pH whereas other Sertoli cells did not respond to EGF. Some Sertoli cells showed a delayed response which coincidated with a similar pH change in neighbouring germinal cells. Since isolated germinal cells never showed a pH response after exposure to EGF, we have concluded that some Sertoli cells may communicate with germinal cells via gap junctions.

The stimulation of rat Leydig cell steroidogenesis by human ovarian steroidogenesis-inducing protein (SIP) may not require endogenous cAMP.

Recently a protein from ovarian follicular fluid was isolated which stimulates steroid production in different cells (Khan et al., 1990). The present study was performed to further characterize the short term effects of this steroidogensis-inducing protein (SIP) on steroid production in isolated rat Leydig cells and to compare the effects with LH/hCG and LHRH. SIP stimulated testosterone production in a dose-dependent manner. In Leydig cells isolated from adult rats the degree of stimulation was much higher than that obtained with hCG, dibutyryl cAMP (db cAMP) or LHRH. Moreover, the stimulated steroid production in the presence of hCG or db cAMP was further enhanced by SIP. The time courses of hCG and SIP action on testosterone production were comparable and maximal stimulation of steroid production was obtained within one hour. In contrast to hCG, SIP did not stimulate cAMP production. An antagonist of LHRH action was unable to block the effects of SIP on Leydig cells indicating that SIP does not act via LHRH receptors. Neither SIP nor LH could further stimulate the steroid production in the presence of 22-R-OH-cholesterol, illustrating that both stimulators control the availability of cholesterol as substrate. An inhibitor of mitochondrial cholesterol side chain cleavage (CSCC), aminoglutethemide, completely blocked the stimulatory effects of SIP and LH/hCG. Thus the effects of SIP on steroid production are not the result of conversion of contaminating steroids in the SIP-preparation. SIP and LH/hCG actions were also blocked when the cells were incubated in the presence of cycloheximide.(ABSTRACT TRUNCATED AT 250 WORDS)

Luteinizing hormone induction of the cholesterol side-chain cleavage enzyme in cultured immature rat Leydig cells: no role of insulin-like growth factor-I?

Long-term inductive effects of luteinizing hormone (LH) on cholesterol side-chain cleavage (CSCC) enzyme activity were studied, using cultured Leydig cells isolated from 21-day-old rats. Particular reference was given to the role of insulin-like growth factor-I (IGF-I) as an autocrine or paracrine modulator or as an essential extracellular mediator of LH action. The CSCC enzyme activity was measured using an excess of 22(R)-hydroxycholesterol as substrate to saturate the enzyme, and inhibitors of pregnenolone metabolism to concentrate all the products of the enzyme reaction in pregnenolone. The rate of sterol conversion into pregnenolone (CSCC enzyme activity) reflected the amount of cytochrome P-450scc (P-450scc), as was shown by Western blotting. In cells cultured without LH, the CSCC enzyme activity decreased to 10% on day 7 of the culture period. In the presence of various doses of LH ranging from 0.01 to 100 ng/ml, the CSCC enzyme activity also diminished during the first 3 days of culture, but during the following days, the amount of CSCC enzyme was stimulated by LH. In contrast to the absence of any LH effect on the activity of the CSCC enzyme during the first days of the culture, the endogenous steroid production (no added 22(R)-hydroxycholesterol) could be stimulated at least 10-fold by high doses of LH. When LH (1 ng/ml) was added to cells which had been cultured for 7 days without hormones, CSCC enzyme activity was elevated 8-fold after 4 days of exposure of LH. These effects of LH could be mimicked by dbcAMP (0.5 mM). No evidence could be provided that IGF-I plays any role in the LH induction of the CSCC enzyme; neither the addition of exogenous IGF-I or analogs that do not bind to IGF-I binding proteins (IGFBPs) nor the inactivation of endogenous IGF-I action (through binding to IGFBP and antibodies to IGF-I or via masking of IGF-I receptor by antibodies) could influence the LH induced CSCC enzyme activity. The present data raise the question under which conditions IGF-I is capable of modulating Leydig cell steroidogenesis.

Hormone-induced resistance of rat Leydig cells to the cytotoxic effects of ethane-1,2-dimethane sulphonate.

Several studies have shown that the cytotoxic agent ethane-1,2-dimethane sulphonate (EDS) specifically destroys Leydig cells in the adult rat testis. It has also been reported that when rats are pretreated with human chorionic gonadotrophin (hCG), administration of EDS does not result in the complete destruction of the Leydig cell population. It has been suggested that hCG pretreatment 'protects' Leydig cells against the cytotoxic action of EDS. In the present study the underlying principles for this resistance to the cytotoxic effects of EDS have been investigated. Within 48 h of the start of daily hCG treatment the number of nuclear profiles of Leydig cells (henceforth called relative number of Leydig cells) had increased from 1014 +/- 40 to 1368 +/- 30 cells per 1000 Sertoli cell nuclei. Previous experiments have indicated that these newly formed Leydig cells probably develop from differentiating Leydig cell precursors. When EDS is administered concomitantly with the third injection of hCG (2 days after the start of hCG treatment), the relative number of Leydig cells surviving EDS treatment was 388 +/- 52 per 1000 Sertoli cells. Hence, there is a similarity between the increase in the relative number of Leydig cells after 2 days of hCG treatment and the relative number of EDS-resistant Leydig cells. The Leydig cells that survived EDS administration showed characteristics which also occur in developing Leydig cells in the immature testis. It is concluded that, in rats pretreated with hCG for 2 days before EDS administration, new Leydig cells with some immature characteristics are formed. One of these characteristics is that these cells are insensitive to EDS.

Sterol carrier protein 2 (non-specific lipid transfer protein) is localized in membranous fractions of Leydig cells and Sertoli cells but not in germ cells.

The cellular and subcellular distribution of sterol carrier protein 2 (SCP2; nsL-TP) was reinvestigated in rat testicular cells by Western blotting and immunocytochemistry, using the affinity purified antibody against rat liver SCP2. Western blot analysis revealed high levels of the protein in the somatic cells of the testis, e.g., Leydig and Sertoli cells whereas it could not be detected in germ cells. This cellular localization of SCP2 was confirmed by Northern blotting. Immunocytochemical techniques revealed that in Leydig cells, immunoreactive proteins were concentrated in peroxisomes. Although SCP2 was also detected in Sertoli cells, a specific subcellular localization could not be shown. SCP2 was absent from germ cells. Analysis of subcellular fractions of Leydig cells showed that SCP2 is membrane bound without detectable amounts in the cytosolic fraction. These results are at variance with data published previously which suggested that in Leydig cells a substantial amount of SCP2 was present in the cytosol and that the distribution between membranes and cytosol was regulated by luteinizing hormone. The present data raise the question in what way SCP2 is involved in cholesterol transport between membranes in steroidogenic cells but also in non-steroidogenic cells.

Testicular Leydig cells in vitro secrete only inhibin alpha-subunits, whereas Leydig cell tumors can secrete bioactive inhibin.

The secretion of inhibin and inhibin-related proteins by testicular Leydig cells was studied by estimation of inhibin immunoreactivity and bioactivity in spent media of preparations of immature and mature rat Leydig cells and of tumor Leydig cells. Immature and mature rat Leydig cells expressed inhibin alpha-subunit mRNA and secreted immunoreactive inhibin. The immunoreactive material did not contain inhibin bioactivity as measured by an in vitro rat pituitary bioassay system. Results of pulse labeling with [35S]methionine followed by immunoprecipitation indicated that the inhibin-related proteins secreted by the immature Leydig cell preparations are 26 kDa and 44 kDa molecules. Mature rat Leydig cells only secreted the 44 kDa inhibin-related protein. Tumor Leydig cells (rat H540 and mouse MA10) secreted immunoreactive and bioactive inhibin, which could be immunoneutralized by an antibody against inhibin. In the culture medium of some H540 tumor Leydig cells 26 kDa and 42 kDa inhibin-related proteins and 30 kDa inhibin were detected. In culture medium of other H540 tumor Leydig cells, not secreting bioactive inhibin, only 26 kDa and 42 kDa inhibin-related proteins were found. No activin bioactivity was detected in culture media of immature rat Leydig cells, H540 and MA10 tumor Leydig cells. It is concluded that normal Leydig cells secrete inhibin alpha-subunits, while Leydig cell tumors can also secrete bioactive inhibin. Neither normal Leydig cells nor Leydig cell tumors produce activin.

The stimulatory effect of albumin on luteinizing hormone-stimulated Leydig cell steroid production depends on its fatty acid content and correlates with conformational changes.

The effects of purified albumin species and albumin fragments (0.2-1% w/v) on short-term (4 h) steroid secretion by immature rat Leydig cells, in the presence of a maximally stimulating dose of luteinizing hormone (LH), were investigated. Human albumin and the peptic fragment (comprising residues 1-387) enhanced pregnenolone production in isolated rat Leydig cells, whereas chicken albumin and the tryptic fragment (comprising residues 198-585) were not active. This stimulatory effect of human albumin and the peptic fragment correlated with the potential of these proteins to undergo a pH-dependent neutral-to-base transition as measured by circular dichroism. The tryptic fragment and chicken albumin did not have the potential to undergo such a transition. The pH-dependent conformational changes of albumin and fragments thereof occurred in parallel with a change in the binding affinity for testosterone and pregnenolone. The fatty acid oleic acid and the drug suramin, only when present in a molar ligand-to-albumin ratio equal to or higher than 2, inhibited the albumin-mediated stimulation of steroid production. These data show that the stimulatory effects of albumin species on LH-induced Leydig cell pregnenolone production depend on their fatty acid content and correlate with the potential of these molecules to undergo conformational changes. It is unknown via which mechanisms albumin exerts its stimulatory effect, but the LH action through the cyclic AMP pathway seems not to be affected.