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.     

A partial characterization of a Sertoli cell-secreted protein stimulating Leydig cell testosterone production.

To examine whether immature rat Sertoli cells in culture secrete a factor(s) which stimulates testosterone production by mature mouse Leydig cells, Sertoli cell-enriched cultures were prepared from 3-week-old male rats with trypsin and collagenase. Sertoli cells were plated at an initial density of 3-5 x 10(6) cells/35 mm well and cultured in 3 ml serum free media supplemented with insulin (10 micrograms/ml). Sertoli cell culture medium (SCCM) collected every 3rd day was added to Leydig cells (10(6) cells in 1 ml of MEM with 2% steroid-free FCS) prepared from 10-week-old mice by mechanical separation and incubated for 3 h at 34 degrees C. Secreted testosterone was determined by RIA. SCCM 15 times concentrated by Amicon YM10 membrane demonstrated a dose-dependent stimulation of testosterone production, whereas there was no effect on testosterone secretion when Leydig cells were maximally stimulated by LH. Leydig cell stimulating activity was retained by both a dialysis membrane with a pore size of 24 A and an ultrafiltration membrane with a molecular weight cut-off of 10 kDa. However, activity was reduced by heating at 60 degrees C for 30 min and almost lost after incubation with 0.1% trypsin for 1 h at 37 degrees C. This activity was not retained by means of a Con A-Agarose column and was demonstrated only in break-through fractions. HPLC gel filtration of a 15 times concentrated SCCM preparation on a TSK gel G3000SW revealed Leydig cell-stimulating activity at approximately 13 kDa.(ABSTRACT TRUNCATED AT 250 WORDS)     

Optimizing testosterone production by purified adult rat Leydig cells in vitro

Summary We sought to establish conditions that increased the duration of testosterone production by fully differentiated adult rat Leydig cells in primary culture. A freshly isolated suspension of highly purified adult rat Leydig cells produced 83 ng testosterone/10⁶ Leydig cells·h⁻¹ when incubated in Medium 199 in a 1.5 ml microfuge tube with shaking for 3 h with a maximally stimulating concentration of ovine luteinizing hormone (LH). Unfortunately, adult rat Leydig cells that were allowed to attach only to a plastic culture dish flattened out, and testosterone production diminished rapidly. Leydig cells in Dulbecco's modified Eagles' medium-Ham's F12 (1∶1; vol/vol) containing Cytodex 3 beads pre-equilibrated in culture medium containing fetal bovine serum attached to the beads and remained viable, but produced only 30 ng testosterone/10⁶ Leydig cells·h⁻¹ when incubated for 24 h with similar stimulation. Leydig cells similarly cultured and maximally stimulated with LH, responded to bovine lipoproteins (<1.222 g/ml) producing 105 ng of testosterone/10⁶ Leydig cells·h⁻¹ when incubated with 1 mg/ml bovine lipoprotein. Therefore, lipoproteins maintain the steroidogenic capacity of purified adult rat Leydig cells in primary culture for 24 h. Paper presented at the 38th Annual Meeting of the Tissue Culture Association in Arlington, Virginia, in May 1987. The session was chaired by Dr. Carlton H. Nadolney, member of the TCA Committee on Toxicity, Carcinogenesis and Mutagenesis Evaluation. This research was supported in part by the National Institutes of Health (grant HD-07204), The Population Center (grant HD-06268), and EPA cooperative agreement (CR81-2765), an NSF equipment grant, and a Mellon Foundation Postdoctoral Fellowship for Gary Klinefelter. Although the research described herein has been funded in part by the U.S. Environmental Protection Agency through cooperative agreement (CR81-2765) to the Division of Reproductive Biology at Johns Hopkins University, it has not been subjected to the agency's peer and policy review, and therefore, does not necessarily reflect the views of the agency and no official endorsement should be inferred.     

Interaction between Leydig and Sertoli cells in vitro

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

Delta-9-tetrahydrocannabinol stimulates ABP secretion from rat Sertoli cells in vitro

The effect of delta-9-tetrahydrocannabinol (THC) on rat Sertoli cell function was investigated. THC significantly increased ABP secretion by 1.5- to 2.1-fold but did not consistently enhance the stimulation of ABP induced by FSH, testosterone or dibutyryl cyclic AMP. ABP was measured by steady-state polyacrylamide gel electrophoresis, DEAE Bio-Gel and immunoassay; all three methods gave similar results. The minimal concentration of THC that stimulated ABP was 10 ng/ml; maximal stimulation was observed with 100-200 ng/ml. This effect was specific since THC did not affect gamma glutamyl transpeptidase activity or the secretion of plasminogen activator, lactate and transferrin. This observation that THC affects ABP secretion specifically is the first report of any differential effect of a drug on Sertoli cell secretion.     

Microsomal ethanol-oxidizing system in purified rat Leydig cells

These studies provide evidence for the presence of a microsomal ethanol oxidizing system in rat Leydig cells. Activity of the microsomal ethanol oxidizing system in Leydig cells was 47.4 +/- 4.1 nmol acetaldehyde per 20 min per mg protein, while activity in crude interstitial cells was 26.0 +/- 5.4 nmol. This suggests that among cells comprising interstitial cells, activity is concentrated in Leydig cells. Activity was linear with respect to protein concentration and incubation time. The highest specific activity was observed in the microsomal fraction. The most effective cofactor was NADPH. The apparent Km for ethanol was 4 mM, suggesting that this system could effectively metabolize ethanol at concentrations found in the blood of males who drink. The apparent Km for NADPH was 11 microM. The activity in Leydig cells was unaffected by 4-methylpyrazole or potassium cyanide, which inhibit alcohol dehydrogenase and catalase activities, respectively. These data provide strong evidence for an enzyme system in Leydig cell microsomes which is capable of metabolizing ethanol.     

Calmodulin stimulates DNA synthesis by rat liver cells

Incubation in low (0.02 mM)-calcium medium prevented T51B rat liver cells from initiating DNA synthesis. Raising the calcium concentration in the medium from 0.02 to 1.25 mM caused these arrested cells to initiate DNA synthesis 1–2 hours later. The possibility of this rapid DNA-synthetic response to calcium addition being mediated through Ca-calmodulin complexes was suggested by the following observations: It was blocked by the putative Ca-calmodulin blockers chlorpromazine and trifluoperazine; the trifluoperazine-inhibited cells were stimulated by purified rat calmodulin; and purified rat calmodulin itself (10^?7 to 10^?6 moles/l) mimicked calcium action, unless the already low ionic calcium concentration in the calcium-deficient medium was reduced further by adding the specific calcium chelator EGTA.     

Interactions between immature porcine Leydig and Sertoli cells in vitro

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

In vitro effects of growth factors on rat germ cell RNA synthesis and their modulation by Sertoli cell-secreted proteins

In vitro rat germ cell RNA synthesis is influenced by growth factors. Basic fibroblast growth factor (0.1 to 100 ng/ml) increases [3H]uridine incorporation in round spermatids (RS) but not in pachytene spermatocytes (PS); this effect is potentiated by insulin (10 micrograms/ml) and blocked in the presence of Sertoli cell-secreted proteins (SCSP). Somatomedin C (0.1 to 100 ng/ml) exhibits a similar effect when used alone without an influence by SCSP. Transforming growth factor beta (0.1 to 10 ng/ml) acts on both cell types, but SCSP amplify this effect only in PS. These data suggest that growth factors synthesized in situ may play a role in the germ cell development and that their effects are modulated by SCSP.     

Cisatracurium stimulates testosterone synthesis in rat and mouse Leydig cells via nicotinic acetylcholine receptor

As a cis‐acting non‐depolarizing neuromuscular blocker through a nicotinic acetylcholine receptor (nAChR), cisatracurium (CAC) is widely used in anaesthesia and intensive care units. nAChR may be present on Leydig cells to mediate the action of CAC. Here, by Western blotting, immunohistochemistry and immunofluorescence, we identified that CHRNA4 (a subunit of nAChR) exists only on rat adult Leydig cells. We studied the effect of CAC on the synthesis of testosterone in rat adult Leydig cells and mouse MLTC‐1 tumour cells. Rat Leydig cells and MLTC‐1 cells were treated with CAC (5, 10 and 50 μmol/L) or nAChR agonists (50 μmol/L nicotine or 50 μmol/L lobeline) for 12 hours, respectively. We found that CAC significantly increased testosterone output in rat Leydig cells and mouse MLTC‐1 cells at 5 μmol/L and higher concentrations. However, nicotine and lobeline inhibited testosterone synthesis. CAC increased intracellular cAMP levels, and nicotine and lobeline reversed this change in rat Leydig cells. CAC may increase testosterone synthesis in rat Leydig cells and mouse MLTC‐1 cells by up‐regulating the expression of Lhcgr and Star. Up‐regulation of Scarb1 and Hsd3b1 expression by CAC was also observed in rat Leydig cells. In addition to cAMP signal transduction, CAC can induce ERK1/2 phosphorylation in rat Leydig cells. In conclusion, CAC binds to nAChR on Leydig cells, and activates cAMP and ERK1/2 phosphorylation, thereby up‐regulating the expression of key genes and proteins in the steroidogenic cascade, resulting in increased testosterone synthesis in Leydig cells.     

Growth hormone (GH) stimulates protein synthesis in cells transfected with GH receptor complementary DNA

An expression vector containing a rat GH receptor cDNA was transfected into Chinese hamster ovary (CHO) cells, and stable cell lines expressing GH receptors were established. In contrast to nontransfected CHO cells, expression of GH receptors in transfected cells resulted in the appearance of high affinity (Kd = 1.53 nM) specific binding of GH. Cross-linking of [125I]hGH to the receptors and subsequent sodium dodecyl sulfate (SDS)-electrophoresis gave an estimated receptor mol wt of 84,000. GH treatment stimulated protein synthesis 60% over basal levels in GH receptor-expressing CHO cells, but not in the receptor-negative parental cells. The effect was observed only under serum-free conditions and was time and dose dependent. These results show that heterologous expression of the rat GH receptor results in the appearance of specific binding of GH and the acquisition of a functional GH response.     

Regulation of the synthesis of lutropin-induced protein in rat testis Leydig cells

The mechanism of action of lutropin on the stimulation of the synthesis of a specific lutropin-induced protein in rat testis Leydig cells was investigated. Lutropin-induced protein has a mol.wt. of approx. 21000 and is detected by labelling the Leydig-cell proteins with [³⁵S]methionine, followed by separation by polyacrylamide-gel electrophoresis and radioautography of the dried gel. The incorporation of ³⁵S into lutropin-induced protein was used as an estimate for the synthesis of the protein. Incubation of Leydig cells with dibutyryl cyclic AMP or cholera toxin also resulted in the stimulation of synthesis of the protein. Synthesis of lutropin-induced protein, when maximally stimulated with 100ng of lutropin/ml, could not be stimulated further by addition of dibutyryl cyclic AMP. Addition of 3-isobutyl-1-methylxanthine, a phosphodiesterase inhibitor, further increased synthesis of the protein in the presence of a submaximal dose of lutropin (10ng/ml) but not in the absence of lutropin or with maximal amounts of lutropin (100 and 1000ng/ml). Actinomycin D prevented the effect of lutropin on the stimulation of lutropin-induced protein synthesis when added immediately or 1h after the start of the incubation, but not when added after 5–6h. This is interpreted as reflecting that, after induction of mRNA coding for lutropin-induced protein, lutropin had no influence on the synthesis of the protein in the presence of actinomycin D. Synthesis of the protein was also stimulated in vivo by injection of choriogonadotropin into rats 1 day after hypophysectomy, and the time course of this stimulation of lutropin-induced protein synthesis in vivo was similar to that obtained by incubating Leydig cells in vitro with lutropin. From these results it is concluded that stimulation of lutropin-induced protein synthesis by lutropin is most probably mediated by cyclic AMP and involves synthesis of mRNA.     

Nicotine stimulates DNA synthesis and proliferation in vascular endothelial cells in vitro

Nicotine is a major component of cigarette smokeand has been postulated to play an important role in atherogenesis andmalignancy. Endothelial cell growth may be regulated by nicotine, yetoperative mechanisms at the endothelial level are poorly understood. We studied the effects of nicotine(10¹⁴-10⁴M) on endothelial DNA synthesis, DNA repair, proliferation, and cytotoxicity by using cultures of bovine pulmonary artery endothelial cells. Assays were performed on cells incubated with nicotine in thepresence and absence of hydroxyurea (an inhibitor of scheduled DNAsynthesis), serum, human platelet-poor plasma, and platelet-derived growth factor and endothelial cell growth factor (PDGF andPDECGF, respectively). Nicotine significantly stimulatedendothelial cell DNA synthesis and proliferation at concentrationslower than those obtained in blood after smoking(<10⁸ M). The stimulatoryeffects of nicotine were enhanced by serum (0.5%) and PDECGF and wereblocked by the nicotinic-receptor antagonist hexamethonium. Theresponse to nicotine was bimodal because cytotoxicity was observed athigher concentrations(>10⁶ M). This study hasimplications for understanding cellular mechanisms of nicotine action.The results may be important in tumor angiogenesis, atherogenesis, andvascular dysfunction in smokers.

     

Activation of protein kinase C increases proteoglycan synthesis in immature rat Sertoli cells

In order to determine the signal transduction pathways involved in the regulation of proteoglycan (PG) synthesis in immature rat Sertoli cells (SC), we have examined the effect of the tumor promoter phorbol ester PMA (phorbol myristate acetate) on [35S]sulfate and [3H]glucosamine incorporation into PG molecules neosynthesized by cultured rat SC. PMA induced a dose- and time-dependent stimulation of labeled cell-associated PG as determined by quantitative solid phase assay. The overall effect of PMA resulted from enhancement of both glycosylation and catabolism of cell PG, this latter effect leading to a drastic decrease of their residence time in the membrane. Besides these quantitative effects, activation of protein kinase C by PMA induced qualitative changes as reflected by increase in relative proportion of heparan sulfate PG (HSPG) in cell membrane PG. In light of our previous results suggesting an inverse relationship between PG synthesis and FSH responsiveness in immature rat Sertoli cells, the PMA-induced upregulation of cell membrane PG, and particularly HSPG, could constitute one mechanism involved in the repression of FSH-stimulated steroidogenesis induced by PKC activation.     

Arginine-vasopressin stimulates the formation of phosphatidic acid in rat Leydig cells

Arginine-vasopressin (AVP) stimulated the formation of labelled phosphatidic acid (PA) in [14C]arachidonic acid-prelabelled rat Leydig cells. After addition of 10(-6)M AVP [14C]arachidonoylphosphatidic acid reached a maximum within 2 min. The increase was dose-dependent (10(-11)-10(-6)M). No change in labelling of other phospholipids and diacylglycerol could be detected. The V1 antagonist dPTyr(Me)AVP inhibited in a dose-dependent manner the AVP-stimulated accumulation of PA. The V2 agonist dPVDAVP was without effect. The present results suggest that AVP binds to V1 receptors in rat Leydig cells resulting in stimulation of PA turnover. We suggest that the AVP-stimulated PA formation is an indication of phosphoinositide turnover.     

Prolactin administration stimulates rat hepatic DNA synthesis

Prolactin is an important growth modulatory hormone in fetal and adult tissues. Its administration stimulates enzymatic markers of the G1 phase of cell cycle in rat liver and other tissues. To determine the effects of prolactin administration on hepatic DNA synthesis (S phase), rats received prolactin at 12 hour intervals for 48 hours and DNA synthesis was assessed by [3H]-thymidine incorporation. Prolactin administration stimulated DNA synthesis 2-4 fold above controls in the livers of adult and weanling animals. Increased incorporation of radiolabel was associated with the nucleus of hepatoparenchymal cells. These data support the hypothesis that prolactin may be a physiological regulator of hepatic DNA synthesis. Further, since stress stimulates prolactin secretion, we suggest that prolactin may participate in the hepatic compensatory hyperplasia elicited by the stress associated with partial hepatectomy.     

D-Aspartate stimulation of testosterone synthesis in rat Leydig cells

D-Aspartate increases human chorionic gonadotropin-induced testosterone production in purified rat Leydig cells. L-Aspartate, D-,L-glutamate or D-,L-asparagine could not substitute for D-aspartate and this effect was independent of glutamate receptor activation. Testosterone production was enhanced only in cells cultured with D-aspartate for more than 3 h. The increased production of testosterone was well correlated with the amounts of D-aspartate incorporated into the Leydig cells, and L-cysteine sulfinic acid, an inhibitor of D-aspartate uptake, suppressed both testosterone production and intracellular D-aspartate levels. D-Aspartate therefore is presumably taken up into cells to increase steroidogenesis. Intracellular D-aspartate probably acts on cholesterol translocation into the inner mitochondrial membrane, the rate-limiting process in steroidogenesis.     

Germ cell binding to rat Sertoli cells in vitro

The interaction between male germ cells and Sertoli cells was studied in vitro by co-incubation experiments using isolated rat germ cells and primary cultures of Sertoli cells made germ cell-free by the differential sensitivity of germ cells to hypotonic shock. The germ cell/Sertoli cell interaction was examined morphologically with phase-contrast and scanning electron microscopy and then quantified by measuring radioactivity bound to Sertoli cell cultures after co-incubation with added [3H]leucine-labeled germ cells. Germ cell binding to Sertoli cell cultures was the result of specific adhesion between these two cell types, and several features of this specific adhesion were observed. First, germ cells adhered to Sertoli cell cultures under conditions during which spleen cells and red blood cells did not. Second, germ cells had a greater affinity for Sertoli cell cultures than they had for cultures of testicular peritubular cells or cerebellar astrocytes. Third, germ cells fixed with paraformaldehyde adhered to live Sertoli cultures while similarly fixed spleen cells adhered less tightly. Neither live nor paraformaldehyde-fixed germ cells adhered to fixed Sertoli cell cultures. Fourth, germ cell binding to Sertoli cell cultures was not immediate but increased steadily and approached a maximum at 4 h of co-incubation. Saturation of germ cell binding to Sertoli cell cultures occurred when more than 4200 germ cells were added per mm2 of Sertoli cell culture surface. Finally, germ cell binding to Sertoli cell cultures was eliminated when co-incubation was performed on ice. Based on these observations, we concluded that germ cell adhesion to Sertoli cells was specific, temperature-dependent, and required a viable Sertoli cell but not necessarily a viable germ cell. These results have important implications for understanding the complex interaction between Sertoli cells and germ cells within the seminiferous tubule and in the design of future experiments probing details of this interaction.     

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.