卵泡抑制蛋白对大鼠睾丸间质细胞分泌睾酮的影响

目的 :探讨卵泡抑制蛋白 ( follistatin,FS)对离体大鼠睾丸间质细胞 ( L eydigcell)分泌睾酮的影响。方法 :用 Percoll梯度分离法分离和培养 Wistar雄鼠 ( 2 2 0～ 2 50 g)睾丸的 L eydig细胞。分别观察 FS( rh FS-2 88)、Ca2 +以及 FS加 Ca2 +在基础状态 (不加h CG)和刺激状态 ( h CG 1 .0 IU/ L )对 Leydig细胞分泌睾酮的影响。结果 :FS抑制基础和刺激状态的睾酮分泌 ,并与剂量相关。 Ca2 +亦有抑制作用 ,但在 1 0 0 mmol/ L时出现逸脱现象。FS加 Ca2 +表现为与剂量相关的抑制作用。结果 :FS呈剂量依赖性抑制睾酮分泌 ,Ca2 +可能是 FS的第二信使 ,单独高浓度 Ca2 +出现抑制逸脱的机制未明。     

大鼠睾丸Leydig细胞的培养和鉴定

目的:研究体外培养大鼠睾丸Leyd ig细胞的有效方法。方法:原代培养大鼠睾丸Leyd ig细胞,用4 U/m l人绒毛膜促性腺激素(hCG)作用细胞,对照组未用hCG,放射免疫法测定培养液中睾酮浓度,3β羟类固醇脱氢酶(3β-HSD)免疫组化染色观察睾丸Leyd ig细胞形态和生物学特性。结果:培养细胞成分均一、增殖旺盛、分化率高。接种72 h后大鼠睾丸Leyd ig细胞纯度达95%。接种后24 h内,hCG刺激组较对照组睾酮分泌量明显提高(P<0.05)。结论:体外培养的睾丸Leyd ig细胞可分泌高浓度的睾酮;睾丸Leyd ig细胞的纯化和培养方法的建立,可为中老年男性雄激素部分缺乏综合征睾酮替代治疗的基础和临床研究提供一条可行的思路。     

环境污染物三丁基氯化锡和氯化三苯锡对大鼠睾丸Leydig细胞的影响

目的:探讨环境污染物三丁基氯化锡(TBT)和氯化三苯锡(TPT)对大鼠睾丸Leyd ig细胞的影响。方法:①用0~80 nmol/L浓度的TBT和TPT处理大鼠睾丸Leyd ig(LC-540)细胞24~96 h,用四唑蓝(MTT)法确定细胞的存活率;②用DNA片段法确定是否存在细胞凋亡;③观察细胞内Ca2+螯合剂氨基苯乙烷四乙酸(BAPTA)和蛋白激酶A(PKA)抑制剂H-89、蛋白激酶C(PKC)抑制剂GF109203X、酪氨酸蛋白激酶(TPK)抑制剂Gen iste in是否可以阻断TBT所致的细胞凋亡;④检测TBT处理的原代大鼠睾丸Leyd ig细胞分泌睾酮的变化。结果:①TBT和TPT影响Leyd ig细胞存活率的强度基本相同,在20~80 nmol/L浓度时细胞存活率呈剂量和时间依赖性降低;②DNA片段法研究证实TBT和TPT可引起细胞凋亡;③BAPTA可阻断20 nmol/L的TBT所致的细胞凋亡,而PKA、PKC和TPK抑制剂对细胞存活率没有影响;④TBT可减少Leyd ig细胞分泌睾酮,并使其对人绒毛膜促性腺激素(hCG)刺激的反应性降低。结论:环境污染物TBT和TPT能直接导致睾丸Leyd ig细胞凋亡,并抑制睾酮分泌,这种致凋亡作用可能与细胞内Ca2+浓度增加有关。     

Regulation of steroidogenesis by Cordyceps sinensis mycelium extracted fractions with (hCG) treatment in mouse Leydig cells

The in vitro effect of extracted fractions of Cordyceps sinensis (CS) mycelium on hCG-treated testosterone production from purified normal mouse Leydig cells was examined. Different fractions extracted from CS (F1-water soluble polysaccharide, F2- water soluble protein and F3- poorly water soluble polysaccharide, and protein) were added to Leydig cells with hCG, and the production of testosterone was determined by radioimmunoassay (RIA). Testosterone productions stimulated by hCG in mouse Leydig cells were suppressed by F2 at 10 mg/ml and F3 at doses from 3 to 10 mg/ml, respectively. F2 and F3 at 10 mg/ml did inhibit dbcAMP-stimulated testosterone productions which indicated that F2 and F3 might affect steroidogenesis at the site after the formation of cyclic AMP. Finally, cycloheximide inhibited F2- and F3-treated mouse Leydig cell testosterone production.     

Serum testosterone response to single injection of hCG ovine-LH and LHRH in male rats

A biphasic pattern of testosterone secretion in response to a single injection of 100 IU hCG has been observed in the rat. Serum testosterone increased from basal levels of 8.7 pL 3.1 ng/ml (mean pL SEM) to 23.0 pL 1.4 ng/ml within 2 h of hCG-stimulation and returned to control levels by 2 days. A second, delayed, but significant increase in serum testosterone occurred, reaching a peak of 24.6 pL 4.0 ng/ml at 3 days and declining to basal values at 5 days. To study this response further, lower doses of hCG were tried. Administration of 10 IU hCG produced a single peak of testosterone, which did not occur until 24 h. Differences in the serum testosterone response were related to the concentration of hCG measured in the serum after injection, as injection of 1 IU, which failed to increase serum hCG levels above detection, was also inadequate to increase serum testosterone. The response after stimulatin with 500 μg ovine-LH or 0.1–10.0 μg LHRH was also evaluated. Injection of 500 μg ovine-LH produced a significant rise in serum testosterone reaching a peak at 2 h of 25.2 pL 2.6 ng/ml and subsequently declining over the next 48 h to control levels where it remained for 5 days. Stimulation with doses of 0.1–10.0 μg LHRH produced rapid and short increases in serum LH concentration which induced peaks of testosterone up to 48.8 pL 14.1 ng/ml 1 h post injection. No secondary peak of testosterone followed. Failure of ovine-LH and LHRH to produce a second testosterone peak suggests that this response may be due to a re-stimulation of the Leydig cell by elevated levels of hCG which persist until the fourth day after injection.     

Characterization of insulin binding and comparative action of insulin and insulin-like growth factor I on purified Leydig cells from the adult rat

Insulin binding and insulin action were characterized in adult rat Leydig cells, purified on discontinuous Percoll gradients. Binding of [125I]-porcine insulin was found to be dependent on time, temperature, cell concentration and Leydig cell specific gravity. Competition relative to porcine insulin (100) was as follows: insulin-like growth factor I (IGF-I) : less than 1; proinsulin : 5; guinea-pig insulin : 2; hCG, ovine prolactin and bovine GH : 0. High and low affinity binding sites for insulin were identified on purified Leydig cells with Ka values of 1.2 X 10(9) and 0.3 X 10(8) M-1, with 10,300 and 34,000 binding sites per cells, respectively. Using primary cultures of Leydig cells in serum-free medium, the action of insulin on steroidogenesis was studied and compared with IGF-I action. Insulin and IGF-I used at 1-35 nM enhanced basal testosterone production in a dose-dependent manner; the effect was significant 4 h after administration. Insulin or IGF-I also potentiated the effect of hCG on steroidogenesis during short-term incubation (4 h). Insulin was shown to improve hCG responsiveness without modifying sensitivity to hCG. Moreover, neither cell number nor hCG-binding was altered by insulin, IGF-I or a combination of the two. Concomitant treatment with insulin and IGF-I at half-maximal and maximally effective doses, in the presence or absence of hCG, indicated that the two factors synergized in the stimulation of testosterone production via a common saturable mechanism.     

Heterogeneity of adult mouse Leydig cells with different buoyant densities.

The authors investigated the morphologic characteristics and human chorionic gonadotrophin (hCG)-stimulated testosterone production of adult mouse Leydig cells in vitro, which have different buoyant densities. Leydig cells from five testes of Swiss outbred male mice (15 weeks old) were isolated and purified by mechanical dispersion followed by density gradient centrifugation using Percoll. Two groups of Leydig cells were obtained with different buoyant densities: group 1 had densities of 1.0667 to 1.0515 g/cm3 and group 2 had densities of 1.0514 to 1.0366 g/cm3. In vitro testosterone production of these Leydig cells, in response to different doses of hCG (0, 5, 25, 125, 625, and 3125 pg/mL), was determined by radioimmunoassay. Leydig cells were fixed and processed for electron microscopic stereology to quantify the organelles by volumes and surface area. In Leydig cells of Group 1, testosterone production per cell in vitro in response to 0 and 5 pg/mL hCG was not significantly different (P greater than 0.05). Increases in the dose up to 25 pg/mL produced a significant increase (P less than 0.05) in testosterone production, although hCG doses of 125 and 625 pg/mL did not produce further increases in testosterone levels. However, 3125 pg/mL hCG further elevated the testosterone production by those Leydig cells with high buoyant density. In Leydig cells in group 2, the patterns of testosterone production in response to hCG doses of 0, 5, and 25 pg/mL were similar to those of Leydig cells in group 1. Those Leydig cells with low buoyant density, however, were unable to stimulate further testosterone production by an hCG dose of 3125 pg/mL.(ABSTRACT TRUNCATED AT 250 WORDS)     

c-jun在人绒毛膜促性腺激素诱导睾丸间质细胞睾酮分泌中的作用

目的 :本研究拟通过c jun反义寡脱氧核苷酸 (ASODNs)观察c jun在调节hCG诱导的睾丸间质细胞 (LC)睾酮分泌中的作用。　方法 :用c junASODNs拮抗c jun ,用hCG诱导体外培养大鼠LC的睾酮分泌 ,用放射免疫法检测睾酮水平。　结果 :hCG可刺激LC分泌睾酮 ,是LC功能研究的有用模型。c junASODNs呈剂量依赖性地抑制hCG诱导下的离体LC的睾酮分泌 (P <0 .0 5 )。　结论 :c jun促进hCG诱导的大鼠LC的睾酮分泌。     

维拉帕米对c-jun调节睾丸间质细胞睾酮分泌的影响

目的通过c-jun反义寡脱氧核苷酸(ASODNs)观察c-jun在调节人绒毛膜促性腺激素(hCG)诱导的睾丸间质细胞(LC)睾酮(T)分泌的作用。方法采用c-jun ASODNs拮抗c-jun,维拉帕米阻断钙通道,hCG诱导体外培养大鼠LC的T分泌,放射免疫方法检测T水平。结果c-jun ASODNs(0~2μmol/L)呈剂量依赖性抑制hCG诱导的离体LC的T分泌(P<0.01)。维拉帕米(10-5mol/L)可加强c-jun ASODNs抑制T分泌。结论c-jun促进hCG诱导大鼠LC的T分泌,c-jun表达可能与钙离子相关。     

Testosterone and androstanediol production by regenerating Leydig cells in the ethylene dimethane sulphonate-treated mature rat

Mature (60-65 day old) male Sprague-Dawley rats received a single intraperitoneal injection of ethylene dimethane sulphonate (EDS; 100 mg/kg) and were subsequently killed at various times from day 2 to day 40 post-treatment. Testes were removed from these animals and age-matched controls and utilized either for light and electron microscopical analyses or for in-vitro assessment of Leydig cell function. Interstitial cells were prepared by collagenase digestion and used to measure 125I-labelled human chorionic gonadotrophin (hCG) binding capacity and androgen production in the presence or absence of hCG or dibutyryl cyclic AMP (dbcAMP). At day 2 after EDS treatment, 125I-labelled hCG binding capacity was reduced to 10% of control values, while the production of testosterone and 5 alpha-androstane-3 alpha, 17 beta-diol (adiol) were non-detectable. Histological observations confirmed the lack of identifiable Leydig cells at day 2-16 after EDS treatment. Between days 24 and 40 post-treatment, Leydig cell regeneration occurred, as indicated by a rise in 125I-labelled hCG binding capacity, increased androgen production and the presence of histologically identifiable Leydig cells. A pattern of adiol production similar to that seen in the immature rat during Leydig cell development was observed with peak synthesis occurring at day 30 post-treatment. Adiol production fell to barely detectable levels by day 36 and remained low at day 40. It is concluded that the steroidogenic pattern of regenerating Leydig cells in the EDS-treated animal is similar to that of developing Leydig cells in the immature animal.     

Bromocriptine, a Dopamine Agonist, Directly Inhibits Testosterone Production by Rat Leydig Cells

We investigated the direct effects of bromocriptine (BR) on both basal and hCG-stimulated testosterone production by rat collagenase-dispersed Leydig cells. In a final volume of 2.2 ml, 2.10(6) Leydig cells were incubated at 33 degrees C for 3 h either alone or with various amounts of hCG (1. 10. 10(2). 10(3). 10(4) mUI/vial) and BR (1.5 10(-9), 1.5 10(-7), 1.5 10(-5) M); BR was dissolved in 20 microliters of ethanol. BR (1.5 10(-5) M) decreased significantly both basal and hCG-stimulated testosterone production whereas at lower doses, BR had no effect. These results suggest that the dopamine itself may regulate rat Leydig cell function and that there is room for criticism of BR-induced hypoprolactinemia as an experimental model to study the effect of prolactin on the androgenic function.     

Response to acute hCG stimulation and steroidogenic potential of Leydig cell fibroblastic precursors in humans

The process of early testosterone (T) secretion and Leydig cell differentiation in humans was studied to explore the steroidogenic capacity of Leydig cell fibroblastic precursors. Seven cryptorchid boys received hCG prior to orchidopexy. Patients CP, PB, and MR received one injection of 1000 IU; patients JR and GG, three daily injections of 1000 IU, and patients MP and MM, five daily injections of 1000 IU. A testicular biopsy was obtained at the time of operation, 24 hours after the last injection. Serum T (ng/dl) before and after hCG stimulation and testicular T (ng/g) were determined by RIA. A control prepubertal testis (tumoral orchidectomy) was incubated in vitro and showed a time-dependent accumulation of T both in the medium and the testicular tissue. Testosterone released into the medium at 1, 2, and 4 hours was 0.76, 1.43, and 4.03 ng/ml, respectively. Tissue T at 0, 1, 2, and 4 hours was 9, 11, 16, and 24 ng/g, respectively. This indicates synthesis and secretion of T into the medium. Control testes showed abundant fibroblastic precursors with scanty cytoplasm, few organelles, heterochromatic nuclei, and minute nucleoli. No Leydig cells were present. After 1 day of hCG stimulation, numerous fibroblasts were activated, displaying enlarged cytoplasms with increased numbers of organelles, nuclei rich in euchromatin, and bigger nucleoli. No Leydig cells were present. Basal serum testosterone was 58.2 +/- 45.3 ng/dl and 87.3 +/- 42.0 after hCG administration, while testicular T was 974.0 +/- 686.0 ng/g (control prepubertal testicular T is 10-50 ng/g). After 3 days of hCG, activated fibroblasts increased and immature Leydig cells appeared. Basal serum T was 35.5 +/- 7.8 ng/dl and 394.0 +/- 24.0 after hCG stimulation, while testicular T rose to 2797.5 +/- 1222.6 ng/g. After 5 days, mature Leydig cells appeared for the first time. Serum T was 58 +/- 59.3 ng/dl (basal) and 641.5 +/- 390 ng/dl (after hCG); testicular T was 789 ng/g (patient MM did not have a value for testicular T). HCG induced numerous coated pits and endocytic vesicles in activated fibroblasts and young Leydig cells, suggesting receptor aggregation and internalization of hormone-receptor complexes. Peroxidase-antiperoxidase (PAP) localization of T was positive in peritubular fibroblasts and Leydig cells.(ABSTRACT TRUNCATED AT 400 WORDS)     

Sites of Action of Cyproterone and Cyproterone Acetate in the Immature Male Rat

Plasma testosterone (Leydig cell function), LH and FSH (pituitary function), the epididymal content of androgen binding protein (ABP) (Sertoli cell function) and plasma corticosterone (adrenal cortical function) were determined after 10 daily injections of varying doses of cyproterone and cyproterone acetate, beginning at 21 days of age. Daily doses of 0.5 mg or greater of either antiandrogen resulted in a marked depression in the levels of plasma testosterone and intra-testicular testosterone (measured only in the cyproterone group) with a dose-dependent decrease in testis and epididymal weights; both effects occurring with only minor changes in the levels of circulating gonadotrophins. In addition, these compounds caused a marked decrease in Sertoli cell secretion as reflected in a significant fall in the epididymal content of ABP.
A more detailed examination of the apparently direct effects of cyproterone on Leydig cell function revealed: 1) no major effects of in vivo treatment for 6 days (5 mg/day) on [I¹²⁵]hLH binding to testis membrane particles or on the in vitro response of enriched Leydig cell suspensions to hCG, 2) a dose-dependent inhibition of the hCG responsiveness of normal Leydig cells in the presence of the drug in vitro . The inhibitory effects on steroidogenesis in vivo can well be explained by a direct inhibition of the 3β-steroid-dehydrogenase-isomerase exerted by both antiandrogens.     

Tumor necrosis factor and interleukin-1 stimulate testosterone secretion in adult male rat Leydig cells in vitro

The actions of two cytokines, tumor necrosis factor (TNF) and interleukin-1 (IL-1), on testosterone production by dispersed adult testis cells and purified Leydig cells in culture were studied. In one set of experiments, testis cells from adult (90-day-old) rats were enzymatically dispersed. In another set of experiments, the dispersed testis cells were placed on a Percoll density gradient and were centrifuged to yield purified (greater than 85%) Leydig cells. Both whole testis cells and purified Leydig cells were cultured in the presence of varying doses of TNF or IL-1 with or without maximally stimulating doses of human chorionic gonadotropin (hCG). Both TNF and IL-1 stimulated basal secretion of testosterone in whole testis cells, as well as purified Leydig cells. Additionally, both TNF and IL-1 augmented maximally hCG stimulated testosterone secretion. Both cytokines stimulated testosterone secretion by dispersed testis cells as early as 4 hours, and the effect continued for up to 72 hours. The cytokines slightly, but significantly, stimulated testosterone production in purified Leydig cells after 24 hours, and continued for up to 72 hours. We have concluded from this data that TNF and IL-1 stimulate the testosterone secretion by adult rat Leydig cells. While this effect might be mediated through the action of the cytokines on testicular macrophages, there might also be a direct effect on the Leydig cell since augmentation of secretion occurred in purified Leydig cells, as well as whole testis cells. Therefore, TNF and IL-1 may serve as local regulators of Leydig cell function.     

Does gonadotropin receptor complex have an amplifying role in cAMP/testosterone production in Leydig cells?

The dose-response relationship between luteinizing hormone/human chorionic gonadotropin (LH/hCG)-stimulated biological response and 125I-labeled hCG binding was studied in purified Leydig cells from adult rat testes. The concentration of hCG needed for one-half maximal stimulation of cyclic adenosine monophosphate (cAMP) and testosterone production (ED50) was 2.16 x 10(-11)mol/L and 5.6 x 10(-13)mol/L, respectively. This suggests that extremely low levels of hormone in the range of 10(-13)mol/L hCG are sufficient to generate enough cAMP (5.66 pmol; 2.83 x 10(-9)mol/L) for steroidogenesis, thereby preserving the catalytic potential of the receptor-cyclase system. Most of the cAMP formed at 10(-13)mol/L hCG was released into the medium, and the intracellular cAMP was much less and barely detectable (0.98 x 10(-9)mol/L; 1.96 pmol/2 x 10(6) cells). The specific binding of 125I-labeled hCG to purified Leydig cells at a correspondingly higher hCG concentration (3 x 10(-10)mol/L) was extremely low and did not display a dose-dependent increase in binding. Assuming the specific binding to represent 100% occupancy of high affinity receptors (14.2 fmol/2 x 10(6) cells per 2 ml), each mole of bound hCG generated 15,423 mol cAMP and 12,817 mol testosterone. The results show that the hormone interacts with cellular receptors as a catalyst to generate the biological response. Moreover, the true affinity of hormone-receptor interaction responsible for the physiologic action is possibly much greater than previously reported for this system. This information should prove useful for reconstitution studies using the hormone receptor/G-protein/adenylate cyclase system in vitro in soluble form.     

Development of transmembrane signaling in the fetal rat Leydig cell

Gonadotropin binding to the adult Leydig cell activates a GTP binding protein that interacts with adenylate cyclase to increase cAMP production within the cell. The increased production of cAMP stimulates steroidogenesis and leads to an increase in testosterone production and secretion. The fetal Leydig cell responds to LH with an increase in cAMP and testosterone production as early as 15.5 days of gestation, although the specific mechanism of transmembrane signaling has not been characterized. Fetal rat testis cells from 13.5-20.5 days of gestation were treated with dibutyryl cAMP (dbcAMP), cholera toxin, and hCG to determine the onset of steroidogenesis stimulation by activation of each moiety in the transmembrane signaling system of the fetal Leydig cell. Maximal stimulation at each age from 14.5 through 20.5 days of gestation was achieved with 1 mM dbcAMP, 500 ng/ml cholera toxin, or 10 ng/ml hCG. At 13.5 days of gestation, fetal testes did not produce any testosterone. These findings indicate that a cholera toxin-sensitive, stimulatory guanine-nucleotide regulatory protein is functional in the fetal Leydig cell as early as 14.5 days of gestation. The LH receptor becomes functional in the transmembrane signaling system of the fetal Leydig cell at 14.5 days of gestation.     

Studies on LH modulated 8kDa peptide involved regulation of testosterone production in rat Leydig cells

Aim: To demonstrate the role of the 8 kDa peptide in regulation of testosterone production by mt Leydig cells. Meth-ods: A peptide similar to 8 kDa peptide purified from immature rat Leydig cells was isolated and purified from rat lungcytosol. Immunological and structural similarity between the peptides purified from lung and Leydig cells was estab-lished by Western blot and tryptic map comparison respectively. Results: Addition of the 8 kDa peptide 10, 50, 100;and 150 ug decreased the production of testosterone in Leydig cells dose-dependently. But the addition of the peptide150 ug along with hCG had no effect on hCG-stimulated increase in testosterone production. Conclusion: In vitro ad-dition of the peptide purified from lung cytosol to adult rat Leydig cells resulted in a concentration-dependent decrease inbasal testosterone production although it had no effect on hCG-stimulated testosterone production. (Asian J Androl1999 Dec; 1: 191-194)     

Study of the effects of neurotransmitters on the hypothalamus-pituitary-testis function in in vitro cell suspension system.

The effects of different neurotransmitters were tested in vitro on a hypothalamic tissue, collagenase-digested isolated anterior pituitary and Leydig cell suspension system by measuring the testosterone production of the Leydig cells. Neurotransmitters were used in concentrations of 0.25, 1.0, 2.5, 5.0, and 10.0 micrograms/ml incubation medium. Dopamine in doses of 1.0, 2.5, and 5.0 micrograms/ml increased the hypothalamic tissue-induced pituitary-testis activation, while it had no direct effect on pituitary and Leydig cells. Noradrenaline in the concentration range 2.5--10.0 micrograms/ml decreased the luteinizing-hormone-releasing-hormone (LHRH) sensitivity of the pituitary cells. 5.0 and 10.0 micrograms/ml 5-hydroxytryptamine decreased the testosterone production and the hCG sensitivity of the isolated Leydig cells. Carbamylcholine and pilocarpine had no action on the in vitro system at the different levels studied.     

Effects of hCG stimulation after withdrawal of long-term oestrogen treatment in patients with prostate carcinoma

Testosterone depletion is the keystone for therapy of patients metastic prostatic carcinoma. Our objective was to investigate Leydig cell function and testosterone levels after withdrawal of long-term endocrine treatment in patients with prostatic carcinoma. Thirteen patients with prostatic carcinoma, previously treated with oestrogens for at least 4 y, were stimulated with 5000 IU human chorionic gonadotrophin (hCG). The stimulation was performed 3-6 y after cessation of the oestrogen therapy. Serum concentrations of testosterone, luteinizing hormone (LH) and follicle stimulating hormone (FSH) were measured before and 24 and 48 h after hCG stimulation. Before hCG stimulation all patients had low serum testosterone concentrations (mean 2.0+/-0.2 nmol/l) and 24 and 48 h after hCG stimulation the values had not significantly increased (mean 2.4+/-0.2 and 2.5+/-1.1 nmol/l, respectively). LH and FSH were within or above the normal range before but after hCG stimulation the values significantly increased. In conclusion, the study shows that the Leydig cells were unable to respond to hCG stimulation more than 3 y after cessation of oestrogen therapy. The Leydig cell function seems to be irreversibly impaired by long-term oestrogen treatment.