The role of the microtubular system in LH/hCG receptor downregulation in rat Leydig cells

The mechanism by which hCG-induced LH/hCG receptor 'downregulation' occurs in rat Leydig cells is unknown. To study the role of microtubules in this process we used the microtubule inhibitors vinblastine and colchicine. 200 IU hCG causes a 92-95% decrease in [125I]hCG binding to testis homogenates within 6 h after injection. Both vinblastine and colchicine prevent this loss of hCG binding. Vinblastine or colchicine did not depress the raised serum testosterone levels following hCG injection, suggesting that these agents were not having a toxic effect on the Leydig cells or on the circulation to the testis such that access of the injected hCG was impeded. The morphological observation of vinblastine-induced bundles of filamentous material in Leydig cells is evidence of a direct action on the microtubular-microfilamentous system. This data strongly implicates a role for microtubules in the process of LH/hCG receptor downregulation that occurs in the Leydig cell following large injections of hCG.     

Modulation of prolactin receptors in cultured rat granulosa cells by FSH,LH and GnRH

The hormonal modulation of prolactin (PRL)-binding capacity of rat granulosa cells was studied. Granulosa cells obtained from immature, hypophysectomized, estrogen-treated rats were cultured for 2 days in a serum-free medium in the presence of various hormones. FSH treatment in vitro stimulated granulosa cell PRL-binding capacity by ~ 4–6-fold in a dose-dependent manner. Concomitant treatment with 10^?8 M GnRH inhibited the FSH-induced increase in PRL-binding capacity by 64%. In contrast, the inhibitory effect of GnRH was blocked by concomitant treatment with 10^?6 M of a GnRH antagonist, [D-pGlu¹, D-Phe², D-Trp^3,6]GnRH. PRL-binding capacity was also increased (~2-fold) by in vitro treatment with cholera toxin (10 μg/ml). In granulosa cells pre-treated with FSH in vitro for 2 days, hCG treatment for 2 additional days stimulated PRL-binding capacity in a dose-dependent manner (~ 2-fold). Likewise, treatment with LH (100 ng/ml) also stimulated PRL-binding capacity by ~ 2-fold. These in vitro studies demonstrated that gonadotropins (FSH, LH and hCG) directly enhanced PRL binding by granulosa cells, whereas GnRH inhibited FSH action.     

Regulation of seminiferous tubule-associated stem Leydig cells in adult rat testes

Testicular Leydig cells are the primary source of testosterone in males. Adult Leydig cells have been shown to arise from stem cells present in the neonatal testis. Once established, adult Leydig cells turn over only slowly during adult life, but when these cells are eliminated experimentally from the adult testis, new Leydig cells rapidly reappear. As in the neonatal testis, stem cells in the adult testis are presumed to be the source of the new Leydig cells. As yet, the mechanisms involved in regulating the proliferation and differentiation of these stem cells remain unknown. We developed a unique in vitro system of cultured seminiferous tubules to assess the ability of factors from the seminiferous tubules to regulate the proliferation of the tubule-associated stem cells, and their subsequent entry into the Leydig cell lineage. The proliferation of the stem Leydig cells was stimulated by paracrine factors including Desert hedgehog (DHH), basic fibroblast growth factor (FGF2), platelet-derived growth factor (PDGF), and activin. Suppression of proliferation occurred with transforming growth factor β (TGF-β). The differentiation of the stem cells was regulated positively by DHH, lithium- induced signaling, and activin, and negatively by TGF-β, PDGFBB, and FGF2. DHH functioned as a commitment factor, inducing the transition of stem cells to the progenitor stage and thus into the Leydig cell lineage. Additionally, CD90 (Thy1) was found to be a unique stem cell surface marker that was used to obtain purified stem cells by flow cytometry.Testicular Leydig cells are the primary source of testosterone (T) in males. T is essential for the development of the male reproductive system and the maintenance of male reproductive functions (1, 2). In addition to defects in reproductive system, its deficiency in the adult contributes to other symptoms that include increased body fat, decreased muscle mass, increased fatigue, depressed mood, decreased cognitive function (3, 4), and reduced immune response (5, 6). In aged men, low T has been reported to contribute to mortality (7). Thus, the formation and maintenance of a functional Leydig cell population throughout adult life is of fundamental importance.In rodents and humans, T production gradually increases from the peripubertal period through the adult, coincident with the development of adult Leydig cells. There now is compelling evidence that most, if not all, of the adult Leydig cells arise from stem cells, not from the transdifferentiation of fetal Leydig cells (8, 9). In previous studies, we and others isolated cells from neonatal testes that expressed platelet-derived growth factor receptor-α (PDGFα) or nestin (9–11). Depending on culture conditions, these cells were shown to be capable of proliferating indefinitely or of differentiating into T-producing Leydig cells and, thus, were identified as stem Leydig cells (9–11). During their differentiation, these cells proceeded through two intermediate stages, progenitor Leydig cells and immature Leydig cells, before becoming adult Leydig cells (12). The gene expression pattern by the stem Leydig cells was similar to that of bone marrow stem cells and quite different from the patterns of the cells in the Leydig cell lineage (13).The adult Leydig cells, once established, turn over slowly during adult life. However, when these cells are eliminated from adult testes by treating the rats with ethane dimethanesolfonate (EDS), new, fully functional adult Leydig cells reappear (14, 15). In initial efforts to localize the precursor cells, seminiferous tubules were isolated from EDS-treated testes and cultured with luteinizing hormone (LH) (16). Cells on the tubule surfaces first underwent division, and then differentiated and produced T (17). These results suggested that in addition to reported perivascular locations in the interstitial compartment (18), stem cells also were located on the surfaces of the seminiferous tubules (16, 19, 20).Studies of a number of tissues have shown that stem cell self-renewal and differentiation are regulated by the interactions between cues that are intrinsic to the cells and extracellular signaling from the local environment, the latter referred to as the niche. In many tissues, including the testis, anatomic complexity combined with the inability to specifically mark stem cells make it difficult to identify these cells, characterize the niche, or determine the extrinsic factors involved in stem cell functions. Thus, as yet the extent to which the testicular environment influences the ability of the stem cells to proliferate and/or differentiate remains unknown. In the present study, we used a unique in vitro system of cultured seminiferous tubule to identify stem Leydig cells on the surface of seminiferous tubules and to assess the ability of factors associated with the tubules to regulate their proliferation and entry into the Leydig cell lineage (i.e., their differentiation). We provide evidence that the proliferation and subsequent differentiation of the stem Leydig cells are regulated by multiple niche factors from the seminiferous tubules, including PDGF, basic fibroblast growth factor (FGF2), transforming growth factor β (TGF-β), activin, Notch, Wnt, and most importantly, Desert hedgehog (DHH). Additionally, we report on the isolation of the stem cells by flow cytometric sorting through a specific cell surface marker protein, CD90.     

Stimulation and inhibition by LHRH analogues of cultured rat Leydig cell function and lack of effect on mouse Leydig cells

The effect of 2 luteinizing hormone-releasing hormone (LHRH) analogues (10^?8–10^?6 M) on the functional activity (testosterone and cyclic AMP production and [¹²⁵I]hCG binding) of purified mouse Leydig cells in culture was examined. The analogues were found to have no significant effect on the cells over a period of 3 days. No specific binding of a labelled analogue to impure or pure mouse Leydig cells could be detected. In contrast high levels of specific binding to impure rat interstitial cells occurred. Centrifugation of the rat interstitial cells on 0–90% Percoll gradients showed that the LHRH analogue bound specifically to the active lutropin-responsive Leydig cells.The purified rat Leydig cells were cultured in the presence of LHRH analogue (ICI 118630) (10^?7 M) and after an initial lag period (2 h) a marked stimulation of testosterone production occurred over a 32-h period (up to 400 ng/10⁶ cells). The response to LH alone increased with time in culture up to 10 h, and the LHRH analogue enhanced this LH-stimulated testosterone production. When the cells were cultured for longer time periods (24 h) the LHRH analogue was found to inhibit LH-stimulated testosterone production at all concentrations of LH used (P < 0.01). The LHRH analogue had no consistent effect on LH-stimulated cyclic AMP production, although when added alone, cyclic AMP production was increased.These results show that LHRH analogues do not bind to or have any detectable effect on mouse Leydig cells in vitro. However, LHRH analogue does bind specifically to purified rat Leydig cells. After a short lag period the analogue stimulates testosterone production which turns to inhibition after 20 h in culture.     

Modulation of Leydig cell functions by culture with Sertoli cells or with Sertoli cell-conditioned medium: effect of insulin, somatomedin-C and FSH

The potential regulatory action of Sertoli cells on Leydig cell functions has been investigated by using a coculture system of Leydig and Sertoli cells obtained from immature pig or by culturing Leydig cells with Sertoli cell-conditioned medium (SCCM). Coculture of Leydig and Sertoli cells for 48 h in the absence of insulin or somatomedin-C (Sm-C), produced a small but significant increase in both hCG receptors and hCG-induced testosterone production. Addition to the medium of pFSH (100 ng/ml), insulin (5 μg/ml) or somatomedin-C (50 ng/ml) produced a marked increase in these two parameters of Leydig cell function. A further significant increase was observed when pFSH was associated with insulin or Sm-C. In contrast, coculture of Leydig cells with aortic endothelial cells decreased both the hCG receptor number and the hCG responsiveness. SCCM stimulated Leydig cell testosterone production following a 4 h incubation. The stimulation depended upon the amount of SCCM used and the conditions in which Sertoli cells were cultured. The most active was the medium from cells cultured in the presence of pFSH and insulin at high concentrations. Since pig Sertoli cells have specific somatomedin-C receptors, but not insulin receptors, it is likely that the effect of micromolar concentrations of insulin are exerted through Sm-C receptors. In addition, SCCM produced a long-term effect after 48 h incubation. SCCM from cells cultured in the absence of insulin and pFSH inhibited both the hCG receptor number and hCG responsiveness. A similar inhibition was observed with SCCM medium from cells cultured without insulin but with pFSH. However, in this case, the inhibition was overcome by Sm-C. SCCM from cells treated with insulin and pFSH had a slight chronic effect but much less so than in the coculture system. Conditioned medium from aortic endothelial cells was always inhibitory. These results suggest that Sertoli cell secretory products have two effects on Leydig cells: acute stimulation of steroidogenesis and long-term trophic effects the nature of which depends upon the in vitro model used and the hormonal supplementation of the culture medium. Whether the factors responsible for the acute and the chronic effect are identical remains to be investigated.     

HCG-induced changes in the number of rat testis LH/HCG receptors.

Adult male rats were injected subcutaneously with a mixture of hCG and [125I]hCG (3, 5, 10, 100 or 1000 IU), and killed 3, 6, 16, 24 or 48 h after injection. Specific uptake of [125I]hCG by the testes as well as the remaining in vitro binding capacity were measured. Cycloheximide (1.5 mg/kg) was given together with hCG to another group of animals. The effect of hCG on testicular LH/hCG receptors was found to consist of two phases: first, 6--16 h after injection, a significant increase in the binding capacity occurred, which was found with the lower 3--10 IU doses of hCG. Thereafter there was a pronounced decrease in receptor concentration, which was directly correlated to the dose of hCG. Experiments with cycloheximide suggest that the decrease in binding, but not the increase, is at least partly dependent on protein synthesis.     

The role of luteinizing hormone and prolactin in the regulation of insulin receptors in Leydig cells of the adult rat

The role of luteinizing hormone (LH), prolactin and their combination in the regulation of insulin receptors in Leydig cells was studied. Leydig cells were isolated from adult male Wistar rats and measurement of insulin binding and internalization was done by incubating the cells with a saturating concentration of 125I-insulin in the presence or absence of different doses of unlabeled LH/insulin. LH exposure (100 and 200 ng dose) caused a significant increase in Leydig cell surface and internalized insulin receptor concentrations. Prolactin at all doses was ineffective in inducing a significant change in insulin receptor concentration. Under basal condition, Leydig cell surface binding of 125I-insulin was greater than the internalization at 34 degrees C but at 4 degrees C, surface binding remained lower than that at 34 degrees C with negligible internalization. Internalization of insulin receptors was measured by incubating the cells at 4 degrees C for 16h and then rapidly incubating at 34 degrees C for various time intervals (60, 120, and 180 min). LH/PRL or LH + PRL did not induce any significant change in the internalization of 125I-insulin at 60 and 120 min. The rate of internalization was greater at 120 min in basal as well as LH/PRL exposed Leydig cells, compared to 60 min of incubation. Prolactin alone did not evoke any appreciable change in internalization of 125I-insulin compared to basal at all three time points tested. Total and acid soluble release of 125I-insulin recorded a significant increase in Leydig cells exposed to LH, which was marginally potentiated when prolactin was added along with LH. Monensin treatment of Leydig cells prevented the recycling of insulin receptors to the cell surface and thereby suppressed the surface binding and enhanced the internalized 125I-insulin. Under cycloheximide treatment, neither surface bound nor internalized 125I-insulin recorded a significant change compared to their respective basal values. It is concluded from the present study that LH has dose-dependent biphasic effects on insulin receptors in Leydig cells by modulating the internalization and intracellular processing of hormone-receptor complexes but prolactin has no such effects.     

催乳素及其受体与Jurkat细胞CD154表达之间的关联

目的探讨催乳素(PRL)/催乳素受体(PRLr)与免疫细胞活化有关的协同刺激信号间的关系,以期发现PRL/PRLr在免疫调节方面的作用机制。方法①将JurkatD1．1细胞随机分为4组,用重组人催乳素(rhPRL)及植物血凝素(PHA)共同刺激JurkatD1．1细胞,预先加或不加鼠抗人催乳素受体抗体,流式细胞仪检测JurkatD1．1细胞表面抗原CD154的表达。②免疫组织化学法再次证实以上结果。结果在rhPRL和PHA共同作用下,Jurkat细胞CD154表达量[(20．8±7．0)％]较空白对照组[(2．6±0．5)％]明显增加(t=8．84,P＜0．01)。预先加入鼠抗人催乳素受体抗体后再加入rhPRL和PHA,CD154的表达量[(2．6±0．6)％]无明显升高(t=0．04,P＞0．05)。结论PRL可通过PRL/PRLr介导的途径促进T细胞CD154的表达,参与免疫反应。     

Nitric oxide production of rat Leydig and Sertoli cells is stimulated by round spermatid factor(s)

In this study, we provide evidence of cell-to-cell interaction between rat germ cells and Leydig or Sertoli cells in relation to nitric oxide (NO) production and inducible nitric oxide synthase (iNOS) messenger RNA (mRNA) expression. As a result of being cultured in a round spermatid-conditioned medium (RSd-CM), NO production in both Leydig and Sertoli cells increased in proportion to the length of the culture period. iNOS mRNA expression in both types of cells also increased in a dose-dependent manner as a result of being cultured with RSd-CM. This increase was detected as early as 3 h and was maintained up to 24 h. In contrast, neither NO production nor iNOS mRNA increased in either type of cell following culture in a pachytene spermatocyte-conditioned medium (PS-CM). Our findings suggest that RSd may control NO production of Leydig and Sertoli cells. This cell-to-cell interaction may be an important mechanism of regulation of testicular function.     

In vitro heteroregulation of LH receptors by prolactin and FSH in rat granulosa cells

The purpose of the present study was to further characterize the regulation of LH/hCG receptors by FSH in granulosa cells and test the hypothesis that the LH/hCG receptor levels are heteroregulated by PRL. Granulosa cells from immature hypophysectomized, DES-treated rats were cultured for 2-4 days in defined medium containing androstenedione (10(-7) M) and/or FSH and PRL, after which [125I]iodo-hCG binding to the granulosa cells was measured. When granulosa cells were cultured for 2 days (days 0-2) with increasing concentrations of FSH (0.1-100 ng/ml), there was a dose related increase in [125I]iode-hCG binding from a control value of 1.05 +/- 0.2 fmoles/10(6) cells to a maximum of 20 +/- 1.8 fmoles/10(6) cells. The miminum, half-maximum (ED50) and maximum doses of FSH were 0.3, 0.5 and 3 ng/ml, respectively. At concentrations of FSH greater than 3 ng/ml there was a progressive decrease in [125I]-iodo-hCG binding to a low value of 6.1 +/- 1 fmoles/10(6) cells at 100 ng/ml of FSH. No changes in [125I]iodo-hCG binding were observed in response to PRL (1 microgram/ml) during the day 0-2 incubation. When granulosa cells were stimulated for 2 days with 20 ng/ml of FSH, washed, and then recultured for another 2 days (days 2-4) with FSH, the LH/hCG receptor content remained high (F leads to F = 17.4 +/- 2.8 fmoles/10(6) cells). In contrast, when FSH-primed cells were recultured for 2 days without FSH, the [125I]iodo-hCG binding decreased sharply to near control levels (F leads to C = 2.5 +/- 0.2 fmoles/10(6) cells). This marked loss of LH/hCG receptors was largely prevented when FSH primed cells were recultured with PRL (F leads to P = 10.3 +/- 1.5 fmoles/10(6) cells). This stimulatory effect of PRL on [125I]iodo-hCG binding was dose-dependent: minimum, ED50, and maximum doses of PRL were 0.2, 0.5 and 1 microgram/ml, respectively. Scatchard-plot analysis revealed that although the dissociation constant (Kd) of the LH/hCG receptors stimulated by FSH and PRL were of similar high affinity (approximately 8 x 10(-11) M), the maximum binding (Bmax) values in the PRL-treated cells were less. Addition of 10(-7) estradiol together with the PRL did not cause a further increase in Bmax values above that observed with PRL alone.     

Morphological and functional characteristics of rat leydig cells isolated on Percoll gradients: is Leydig cell heterogeneity in vitro an artifact?

Rat testicular intertubular cells have been isolated on Percoll density gradients. Detailed light and electron microscopic studies have determined the sedimentation positions for Leydig cells, macrophages, fibroblasts, endothelial cells, germ cells and residual bodies. Stereological techniques have been utilized to determine the number of cells in the region of the gradient where Leydig cells sediment. Morphologically intact Leydig cells were present in the more dense region of the gradient (1.0590-1.0900 g/ml), and they responded to hCG stimulation with an 11-fold increase in testosterone production and contained LH/hCG receptors. Leydig cells in the less dense region of the gradient (1.0440-1.0589 g/ml) secreted less testosterone and contained less hCG receptors than those obtained from denser regions. However, the morphological studies described herein provide evidence for the first time that the majority of these less functional 'Leydig cells' from the lighter region of the gradients do not contain a nucleus and represent pieces of Leydig cell cytoplasm with variable size, shape and complement of organelles.     

The expression of the RLF/INSL3 gene is reduced in Leydig cells of the aging rat testis

The relaxin-like factor (RLF), which is the product of the INSL3 gene, is highly expressed in the fetal and adult-type Leydig cells of all species so far examined. In adult testes it is upregulated at puberty but appears subsequently to be expressed in a constitutive manner, independently of acute changes in the hypothalamic-pituitary-gonadal (HPG) axis. Functional hypogonadism with decreased testosterone is prevalent in the aging male. In order to test whether this is a property of the HPG axis, or of the Leydig cells themselves, RLF/INSL3 was used as an independent marker to assess rat Leydig cell differentiation status. Hybridization analysis showed that in the testes of old (2 years) rats, RLF/INSL3 mRNA expression was dramatically reduced, compared to young (3 months) animals. This was also evident at the protein level using immunohistochemistry. The results suggest that increasing functional hypogonadism in older male mammals is likely caused by a dedifferentiation of the Leydig cells themselves.     

RNA synthesis in Leydig cells during sexual maturation in the rat: effect of LH

The trophic action of LH on Leydig cells involves the triggering of a number of cellular events including changes in protein synthesis. This latter change has led a number of workers to postulate an effect of LH on RNA synthesis. A direct action of LH on RNA synthesis, however, has been difficult to assess. The aim of the present work was to analyse the effect of LH on RNA synthesis in vitro during sexual development. Studies were performed using purified Leydig cells from rats of 20, 30, 40, 50, 60 and 90 days of age. The results obtained show that basal uridine incorporation into RNA increases in an age-dependent manner in rats from 20 to 60 days of age and then remains unchanged until 90 days of age. A stimulatory effect of LH on RNA synthesis was clearly demonstrated only in the youngest rats (20 and 30 days old). In order to differentiate the effect of LH on different RNA populations, the RNA synthesized by immature and mature rats was analysed using a poly(U)-Sepharose column. In 20-day-old rats, LH stimulated both unbound and poly(A) RNA, although a more marked effect was clearly demonstrated on the latter. On the other hand, LH had an identical effect on both unbound and poly(A) RNA obtained from Leydig cells of 60-day-old rats. This stimulatory effect of LH on RNA synthesis in Leydig cells from immature rats seemed specific, since effectors which act on interstitial cells, such as LH-releasing hormone, [Arg8]-vasopressin and FSH (which may act on macrophages) did not modify RNA synthesis.(ABSTRACT TRUNCATED AT 250 WORDS)     

Differential modulation of apoptotic gene expression by N-acetyl-L-cysteine in Leydig cells stimulated persistently with hCG in vivo

The present study was designed to investigate the molecular mechanisms of NAC (150 mg/kg bw twice/week) action in vivo under repeated hCG (100 IU/rat/day) stimulation to adult rats. Leydig cell refractoriness led to a significant decline in serum testosterone and intracellular cAMP by day 30 of chronic hCG intervention which improved significantly following NAC co-administration. It inhibited the rise in lipid peroxidation, improved the activity of antioxidant enzymes along with intracellular glutathione and total antioxidant capacity in the target cells. Leydig cell apoptosis declined significantly (P < 0.001) with down-regulation of upstream, Fas, FasL, caspase-8, Bax and caspase-9, JNK/pJNK and downstream caspase-3 and PARP. On the other hand, anti-apoptotic Bcl2, NF-kβ, and Akt were up-regulated. Taken together, the above findings indicate that the specificity of NAC action was not restricted to regulating marker proteins in the extrinsic and JNK pathways as seen in vitro but extended to include intrinsic pathway of metazoan apoptosis as well.     

Effects of lutropin (LH) receptor internalization and recycling on the apparent numbers of LH receptors in rat testis Leydig cells at different temperatures

The effect of different temperatures on the Scatchard analysis of binding studies with 125I-hCG and intact rat testis Leydig cells has been investigated. The results show that at temperatures greater than 4 degrees C an overestimation of the number of receptors/cell is likely to occur. A one-site analysis of the data gives values of 13,573, 61,924 and 3802 LH receptors/cell at 34 degrees C, 21 degrees C and 4 degrees C respectively. At 34 degrees C and 21 degrees C (but not at 4 degrees C) a two-site analysis of the data is possible, giving similar high affinity binding components at both temperatures (KD 1 X 10(-10) M) but with dissimilar low affinity binding components (34 degrees C, 2.17 X 10(-9) M and 21 degrees C, 6.3 X 10(-8) M). The calculated total number of LH receptors using a two-site analysis at 21 degrees C is equal to 114,766 receptors/cell, and at 34 degrees C it is 37,987 receptors/cell. It is proposed that the differences in the level of binding at different temperatures and the associated changes in the value of KD, reflect the temperature sensitivity of the endocytic pathway of the 125I-hCG/LH receptor complex within the rat testis Leydig cell. From previous studies it is known that at 34 degrees C and 21 degrees C, a large part of the cell-associated hormone is not receptor bound but rather it represents 'processed' hormone before it is released from the cell. At 4 degrees C the cell-associated 125I-hCG remains bound only to cell surface LH receptors and thus gives a more accurate measure of total receptor numbers/cell.     

IGF-Ⅰ,胰岛素和雌二醇对雌性大鼠垂体细胞分泌LH的影响

LH分泌受到性激素正、负反馈调节的控制.胰岛素样生长因子-Ⅰ(IGF-Ⅰ)可能也参与LH分泌的调节[1].本实验观察在有或无血清的培养条件下,IGF-Ⅰ和E2对雌性大鼠垂体细胞LH分泌的影响.     

The effect of hGH deficiency on the insulin response to glucagon after oral glucose loading

Summary Six children and adolescents (aged from 2 6/12 to 16 years) with isolated hGH deficiency were subjected to a standard oral glucose tolerance test (OGTT) followed by the administration of IV glucagon at 180 mins. Three of them underwent a second test after several months of hGH therapy. Nine patients underwent a separate IV glucagon test and two of these patients had both tests. As controls served 14 endocrinologically normal children and adolescents, who underwent both tests. It was found that the patients with isolated hGH deficiency had lower basal plasma insulin and blood glucose levels and that their insulin response to IV glucagon even after oral glucose preloading was significantly lower than in the control group. This response was partially restored by several months of hGH treatment in the three patients tested. These findings are interpreted as further evidence for an insulinotrophic effect of hGH.Established Investigator of the Chief Scientist's Bureau, Ministry of Health     

The functional activity of adult mouse Leydig cells in monolayer culture: effect of lipoproteins, pregnenolone and cholera-toxin

Further studies were carried out on purified mouse Leydig cells to determine why they lose their hormone responsiveness after several days in monolayer culture. The effects of cholera-toxin on cyclic AMP and testosterone production were examined. It was found that cyclic AMP production could still be maximally stimulated by cholera-toxin after 4 days in culture when response to luteinizing hormone (LH) has declined. Testosterone production was, however, not maintained. Because this decline in testosterone production may have been due to the lack of a suitable substrate after several days in culture, cells were cultured initially in the presence of exogenous pregnenolone and low-density lipoproteins (LDL). Both substances were found to enhance basal and LH-stimulated testosterone production and to extend responsiveness of the cells until at least day 4, but by day 7 response was lost. Cells were then cultured in the presence of rat and human LDL and HDL and in both cases LDL was found to enhance consistently testosterone production, but HDL was much less effective. Scanning and transmission electron micrographs showed that changes in cell shape occurred during culture, but indicated that the cells were not depleted of lipid droplets by the end of culture or after LH stimulation. It is concluded that the eventual decline in testosterone synthesis is not due to lack of substrate, although the addition of exogenous substrate does extend the period of responsiveness. Nor is it due to a decrease in adenylate cyclase activity. At least part of the lesion is caused by a decrease in the enzymes required for the conversion of pregnenolone to testosterone.