Desert Hedgehog调控Leydig细胞增殖分化的研究进展

Hedgehog信号通路是内分泌腺体发育的一个重要调控因子通路。近年来,Desert Hedgehog(Dhh)在睾丸性腺发育中的调控作用备受关注,尤其是对Leydig细胞的调节作用。在睾丸中,Dhh由Sertoli细胞旁分泌产生而作用于Leydig细胞,对其增殖、分化过程及其分泌睾酮的功能起调控作用。睾酮的分泌对睾丸发育、精子发生及雄性表型的维持至关重要,因而对Dhh因子的正确调控是生精功能的前提条件。对睾丸中Hedgehog信号通路的研究,也将对雄激素不足及生精障碍等疾病的病理生理机制研究和临床治疗具有一定的潜在意义。     

Autophagic deficiency is related to steroidogenic decline in aged rat Leydig cells

Late-onset hypogonadism (LOH) is closely related to secondary androgen deficiency in aged males, but the mechanism remains unclear. In this study, we found that reduced testosterone production in aged rat Leydig cells is associated with decreased autophagic activity. Primary rat Leydig cells and the TM3 mouse Leydig cell line were used to study the effect of autophagic deficiency on Leydig cell testosterone production. In Leydig cells from young and aged rats, treatment with wortmannin, an autophagy inhibitor, inhibited luteinising hormone (LH)-stimulated steroidogenic acute regulatory (StAR) protein expression and decreased testosterone production. In contrast, treatment with rapamycin, an autophagy activator, enhanced LH-stimulated steroidogenesis in Leydig cells from aged, but not young, rats. Intracellular reactive oxygen species (ROS) levels were increased in both young and aged Leydig cells treated with wortmannin but decreased only in aged Leydig cells treated with rapamycin. Furthermore, an increased level of ROS, induced by H(2)O(2), resulted in LH-stimulated steroidogenic inhibition. Finally, knockdown of Beclin 1 decreased LH-stimulated StAR expression and testosterone production in TM3 mouse Leydig cells, which were associated with increased intracellular ROS level. These results suggested that autophagic deficiency is related to steroidogenic decline in aged rat Leydig cells, which might be influenced by intracellular ROS levels.     

老年大鼠睾丸间质细胞结构和功能变化的实验研究

目的:研究老年SD大鼠(PADAM动物模型)睾丸间质细胞形态、分泌功能变化,探讨老年大鼠睾丸间质细胞的功能状态。方法:分别取青年SD大鼠和老年各20只,静脉血测定血清总睾酮和游离睾酮的浓度,并通过组织切片和透射电镜观察两个年龄组大鼠睾丸间质细胞形态学变化;此外,分别用hCG、Forskolin刺激体外培养的两个年龄组大鼠的睾丸间质细胞,比较培养基中睾酮和孕酮的浓度。结果:老年大鼠的血清总睾酮[(3.07±0.75)nmol/L]和游离睾酮[(0.71±0.65)nmol/L]均比青年大鼠[(10.89±6.11)nmol/L和(2.42±1.02)nmol/L]显著降低(P<0.05);细胞形态有较显著差异;体外培养的大鼠睾丸间质细胞分泌能力显著降低(P<0.05)。结论:老年SD大鼠血清睾酮和游离睾酮浓度显著低于青年SD大鼠,原因在于睾酮合成酶系统整体功能衰退。     

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.     

Evaluation of the effect of peritubular cell secretions and the testicular paracrine factor P-Mod-S on Leydig cell steroidogenesis and immunoactive inhibin production

Testicular peritubular cells have been shown to produce a paracrine factor, termed P-Mod-S, under androgen control that has dramatic effects on Sertoli cell function and may provide an important mode of androgen action in the testis. Therefore, the current study was designed to investigate the possibility that peritubular cell secretory products could feedback and regulate Leydig cell function. The Leydig cell functional parameters that were examined included testosterone production and inhibin secretion. Purified forms of P-Mod-S (P-Mod-S(A) and P-Mod-S(B) shown to be biologically active on Sertoli cells) had no effect on basal or gonadotrophin-stimulated production of testosterone or inhibin by Leydig cells. A preparation of peritubular cell-secreted proteins (PSP) with molecular weights greater than 3 kDa did not influence testosterone production by Leydig cells. PSP, however, did influence cultured Leydig cell morphology and improved cell viability. PSP also had no effect on the ability of LH to stimulate Leydig cell testosterone production. Whilst determining the effect of PSP on Leydig cell inhibin production, PSP was found to contain endogenous levels of inhibin apparently due to 2% contamination of the peritubular cell cultures with Sertoli cells. When this endogenous inhibin level was considered, PSP was found to have no influence on basal or hormone-stimulated production of inhibin by Leydig cells. Results of the current study indicate that peritubular cell secretory products, including the paracrine factor P-Mod-S, do not appear to play a major role in the regulation of Leydig cell function. Therefore, the regulation of Leydig cell function by the seminiferous tubule will primarily be due to Sertoli cell secretory products.     

Morphometric and Functional Investigation on the Leydig Cells in Experimental Unilateral Cryptorchism in the Rat

By distal gubernaculotomy on one side in newborn rats testicular descent was prevented on that side, and unilateral cryptorchid rats were obtained. Leydig cell structure and function was studied at 20, 30 and 100 days of age, using both morphometric and biochemical techniques. Up to 30 days of age no differences in structure of the interstitium and Leydig cells, or testicular testosterone concentration were observed between the scrotal and the abdominal testicles. In contrast the tubular cells in the abdominal testicle were highly damaged already at 30 days of age. The abdominal testicles of the 100-day-old rats contained the same total number of Leydig cells as the scrotal testicles, but the size of these cells, and the testicular testosterone concentration were highly reduced. Thus Leydig cell function in the cryptorchid testicle was maintained at 30 days but highly impaired at 100 days of age. Tubular damage occurred prior to the Leydig cell malfunction.     

Effects of varicocele on testosterone, apoptosis and expression of StAR mRNA in rat Leydig cells

The aim of this study was to explore the effects of varicocele on the morphology and function of Leydig cells in the rat testis. Forty male Sprague-Dawley rats were divided into two groups: the experimental group underwent surgery to create a left varicocele (VC), and the control group underwent a sham operation. Serum testosterone and intratesticular testosterone levels were measured using a radioimmunoassay after 4 and 8 weeks of operation. Leydig cells were studied for apoptosis and expression of steroidogenetic acute regulatory (StAR) protein mRNA levels. Serum testosterone levels declined after 4 and 8 weeks of operation but were not significant (P>0.05). However, the intratesticular testosterone levels after 8 weeks were significantly decreased compared with the control group (P<0.01). The mean apoptosis index of Leydig cells in the experimental group was significantly higher than that in the control group after 4 or 8 weeks (P<0.01). StAR mRNA levels in the Leydig cells of the experimental group were significantly lower compared to those of the control group (P<0.01). Our data show that varicocele did impair Leydig cell function by increasing apoptosis and suppressing the expression of the StAR protein.     

A morphological study of the testis in patients with idiopathic male infertility--quantification and ultrastructure of Leydig cells

Testicular biopsy was performed on 51 patients with idiopathic male infertility and 13 normal fertile adults. The biopsied specimens were examined by light and electron microscopy. A quantitative evaluation of Leydig cell number was made by determining the mean number of Leydig cells per one cross section of seminiferous tubule in the entire histologic section of each specimen, which was defined as "Leydig cell index". In both oligospermic and azoospermic groups, the Leydig cell index was significantly elevated as compared with in that the normal group, which indicated the presence of Leydig cell hyperplasia in the infertile testis. In addition, this index significantly correlated with serum luteinizing hormone and follicle stimulating hormone levels but not with serum testosterone level. Leydig cells were classified into three types based upon their morphological characteristics. Type I Leydig cells were functionally active and mature ones, with a smooth-surfaced round or oval nucleus, had abundant smooth endoplasmic reticulum (SER), characteristic mitochondria and relatively few lysosomes. Type II Leydig cells were functionally less active, and contained an irregularly shaped nucleus, less abundant SER, mitochondria with undeveloped cristae and relatively few lysosomes. Type III Leydig cells included immature and regressive cells, which were considered to have almost no hormone secreting function. The immature Leydig cells were spindle-shaped and had few cell organelles. The regressive cells had poorly developed SER and many secondary lysosomes in the cytoplasm. In the normal group, type I Leydig cells are the most numerous but in the infertile groups type II Leydig cells are the most numerous. However, in the latter, there is no significant difference in relative number of each Leydig cell type among the groups classified according to the number of sperm or Leydig cell. In summary, Leydig cell hyperplasia observed in the testis of the infertile patients was supposed to be induced by the elevation of the serum LH. Despite of the significant increase in Leydig cell number, the serum testosterone was not elevated, and this was considered to be due to the fact that the hyperplastic cells are mainly composed of functionally less active type II Leydig cells. In addition, especially in the severe hyperplastic group, not only type II but also type I Leydig cells increased in number. However, in this group, the serum testosterone level was not elevated. Therefore, the function of type I Leydig cells was suggested to be impaired.     

Sertoli cell — Leydig cell interaction in the regulation of testicular function

Considerable evidence exists to indicate that the Leydig cells influence the seminiferous tubule by maintaining a high concentration of testosterone in the peritubular compartment of the testes. Recent studies using various types of agents to disrupt spermatogenesis, have shown that significant changes occur in the structure and function of the Leydig cells. In these situations, Leydig cells enlarge, show hyperresponsiveness to hCG stimulation in vitro and contain a reduced number of receptors for LH/hCG. Evidence is presented to support the hypothesis that the changes in Leydig cell structure and function are the result of local factors. The data provide support for the concept that the functioning of Leydig cells is modulated by the seminiferous tubules.     

低剂量睾酮疗法降低TM3睾丸间质细胞的氧化损伤

睾酮替代疗法对老年男性及性腺机能减退症的治疗是有意义的。然而,外源性睾酮对睾丸间质细胞的作用尚不清楚,需要进一步阐明。本研究表明睾酮补充疗法能降低睾丸间质细胞的氧化损伤。本文用睾丸间质细胞TM3作为体外细胞模型。研究发现睾酮剂量为100nmol L-1治疗时可以产生细胞保护作用,但睾酮补充剂量≥500nmol L-1时就会产生细胞毒作用。在睾酮剂量为100nmol L-1治疗时能够显著降低ROS的产生,脂质过氧化物含量,缺氧诱导因子（HIF）-1α的稳定性和活性。睾酮剂量为500nmol L-1时产生的活性氧比100nmol L-1时增加了1．72倍。与对照组相比,睾酮剂量为50nmol L-1时类固醇激素合成急性调节蛋白（STAR）的表达增加了1．58倍（P〈0．01）．睾酬补充疗法降低了化学性诱导缺氧。低剂量睾酮补充疗法治疗睾丸间质细胞通过降低ROS和脂质过氧化物,增加StAR蛋白表达,减缓缺氧应激即降低HIF-1α测子的稳定性性产生细胞保护作用。研究中发现当睾酮剂量≥500nmol L-1时,氧化损伤增加。为阐明睾酮替代疗法的效果,需进一步阐明睾丸间质细胞中华酮量效关系受哪种机制调控。     

Leydig cell cooperation in vitro: evidence for communication between adult rat Leydig cells

In short-term incubations (32 C, 3 h) of purified adult rat Leydig cells, increasing the density from 5000 to 50,000 cells/16 mm diameter culture well caused a significant increase in human chorionic gonadotropin (hCG)-stimulated testosterone secretion/cell. Density-dependent stimulation was also observed under basal conditions and in the presence of dibutyryl cyclic adenosine monophosphate, luteinizing hormone-releasing hormone, or 22-hydroxy cholesterol. In contrast, increasing the incubation density of purified Leydig cells by addition of other testicular cells had no effect on basal or hCG-stimulated testosterone secretion. hCG-stimulated testosterone secretion by Leydig cells incubated at low density was also increased by addition of Leydig cell-conditioned medium. This stimulatory activity was removed by charcoal extraction and by ultrafiltration (approximately 30 kDa cut-off). The data indicate that Leydig cells cooperate by secretion of low molecular weight, cell-specific stimulatory factors that support Leydig cell steroidogenesis in vitro, and may also play a role in regulating Leydig cell function in vivo.     

Morphologic response of rat Leydig cells to hemicastration

Hemiorchidectomized rats were followed up to 15 days postsurgery for morphologic evaluation of compensatory testicular response and its correlation to serum testosterone levels. Although gross compensatory testicular hypertrophy (CTH) was not noted, an enlarged interstitium was observed with hypertrophy and hyperplasia of Leydig cells with morphologic changes suggestive of increased cellular activity. These histologic changes were accompanied by compensatory testicular hypersecretion (CTHS) illustrated by the return of the serum testosterone levels to near the intact-control value in the later groups. Ultrastructural studies of the Leydig cells indicated an increase in the amount of smooth endoplasmic reticulum as an underlying mechanism for this response. In view of the previously reported normal serum luteinizing hormone levels after hemicastration, the compensatory hyperactivity/hypersecretion should be considered primarily an intrinsic Leydig cell response, not related to changes in the hypothalamo-hypophyseal axis.     

衰老大鼠睾丸间质细胞形态学观察

目的通过对老年SD大鼠睾丸间质组织学和Leydig细胞超微结构的观察,探讨老年大鼠性腺功能减退的机制.方法测定3月龄和24月龄SD大鼠血清睾酮水平,并进一步对不同年龄大鼠睾丸间质和Leydig细胞进行光镜和电镜检查.结果老年大鼠血清T浓度显著低于青年大鼠;老年大鼠睾丸色泽灰暗、质地松弛;睾丸间质中成纤维细胞增生明显而Leydig细胞数量减少,单个Leydig细胞变小,并出现退行性改变;hCG刺激8d后,青年和老年大鼠两组睾丸形态学均无明显变化.结论老年大鼠的Leydig细胞出现明显的形态学异常,可能与睾酮合成功能减退有关.     

Effects of Basella alba and Hibiscus macranthus extracts on testosterone production of adult rat and bull Leydig cells

Aim： To determine the androgenic effects of Basella alba and Hibiscus macranthus extracts in the rat and the bull, and to develop a novel in vitro test system using Leydig cells from bull testes. Methods： The effect of methanol extracts from both plants on testosterone production in isolated Leydig cells from the rat and the bull was analyzed using ^125I-radioimmunoassay （^125I-RIA）. Rat Leydig cells were obtained by common methods, whereas a novel technique was used to purify Leydig cells from bull testes. Results： Bull testes from the slaughter house were a cheap source of pure Leydig cells. In culture, these cells produced testosterone for 5-6 days, which can be stimulated by human chorionic gonadotrophin （hCG）. Basella alba extracts significantly enhanced testosterone production in bull and rat Leydig cells in a concentration-dependent manner. Hibiscus macranthus showed no androgenic effect but was shown to inhibit testosterone production at higher concentrations. Conclusion： Leydig cells purified from bull testes can be used as an alternative tool in experimental animal research. Certain fractions of Basella alba extract demonstrated androgenic potential whereas Hibiscus macranthus extracts did not.     

Cholesterol transport,peripheral benzodiazepine receptor,and steroidogenesis in aging Leydig cells

The cellular mechanisms responsible for age-related decline in the ability of Leydig cells to produce testosterone are not yet fully understood. The decline in testosterone production could result from a reduction in the Leydig cell enzymatic activities mediating testosterone synthesis, the amount of substrate available for these enzymes, or both. In the present study, we examined the effect of age on a critical early step in the steroidogenic pathway, the transport of cholesterol into mitochondria. Leydig cells were isolated from the testes of young and old Brown Norway rats and incubated with human chorionic gonadotropin (hCG) and the side-chain cleavage cytochrome P450scc inhibitor aminoglutethimide (AMG). Mitochondria were isolated from these cells in the presence of AMG. Upon removal of AMG, the mitochondria from old cells produced 80% less steroid than those from young cells, only a fraction of which could be accounted for by a decrease in P450scc activity. These results suggest that the accumulation of hormonally recruited cholesterol into mitochondria is defective in old Leydig cells. With this in mind, we turned our attention to peripheral benzodiazepine receptor (PBR), a mitochondrial cholesterol-binding protein known to be involved in mediating cholesterol transport. PBR messenger RNA (mRNA) and protein expression were decreased in old cells. Moreover, both the dissociation constant (Kd) and the number of binding sites (Bmax) of the PBR were decreased in the old cells by 50% and 30%, respectively. Taken together, these results suggest that alterations in cholesterol transport and in PBR may play critical roles in age-related decreases in testosterone production in Brown Norway rat Leydig cells.     

壬基酚对体外培养大鼠睾丸间质细胞分泌雄激素影响的研究

目的:探讨壬基酚(NP)对大鼠睾丸间质细胞分泌雄激素的影响。方法:建立体外培养大鼠睾丸间质细胞原代培养体系,并采用3β-HSD染色对间质细胞进行鉴定;以不同浓度的NP(分别为较低浓度0.005、0.015、0.025、0.05、0.1μmol/L及稍高浓度0.5、5.0、10.0、15.0、25.0μmol/L)作用于间质细胞,采用3β-HSD染色观察间质细胞形态的变化,并检测间质细胞分泌睾酮含量的变化。结果:间质细胞经NP处理后,细胞形态发生变化,细胞密度降低,NP作用浓度达到25μmol/L时,细胞发生裂解。0.005及0.015μmol/L NP处理后,间质细胞睾酮分泌量增加,与对照组比较差异有显著性(P<0.05)。NP浓度达到0.5μmol/L后,睾酮分泌量与对照组相比显著下降(P<0.05),且当NP浓度为5,10,15,25μmol/L时睾酮分泌量下降极为明显(P<0.01)。结论:低浓度NP能促进间质细胞分泌睾酮,而高浓度NP抑制睾酮分泌,且高浓度NP能诱导间质细胞坏死。     

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)     

Association of selenoprotein P with testosterone production in cultured Leydig cells.

Selenoprotein P, a plasma selenoprotein, is thought to act as an antioxidant in the testis, similar to glutathione peroxidase. mRNA encoding selenoprotein P was selectively expressed by Leydig cells, suggesting participation in testosterone production. On the other hand, testosterone production has been linked to O2 toxicity in cultured Leydig cells. The authors, therefore, examined changes in selenoprotein P mRNA expression and testosterone production following stimulation by a stable analog cyclic adenosine 3',5'-monophosphate (cAMP) in cultured Leydig cells (MLTC-1 cells) under normal O2 concentrations. Selenoprotein P mRNA was analyzed by Northern blotting, while testosterone concentration in culture medium was measured by radioimmunoassay. When cAMP was added to cultures at 0, 0.01, 0.1, or 1 mM, selenoprotein P mRNA expression showed dose-dependent stimulation. cAMP was added at 0.1 mM to cultures, and the selenoprotein P mRNA expression and testosterone concentration were evaluated after incubation times of 2, 5, 9, 15, or 24 h. Selenoprotein P mRNA expression was maximal at 9 h. Testosterone concentration in the medium also increased, becoming maximal at 15 h. Selenoprotein P induced in Leydig cells following cAMP stimulation may counteract O2 toxicity from cAMP-mediated increases in testosterone production.     

睾丸间质干细胞分化和移植

睾丸间质细胞是男性体内合成雄激素的主要细胞,胚胎发育期中肾胚的间质细胞及生精小管周成纤维样细胞可能是睾丸间质细胞的干细胞。在胚胎期间质干细胞分化为胎儿型间质细胞;出生后间质干细胞经间质祖细胞、未成熟间质细胞分化为成熟间质细胞。老年期间质细胞数量可能不变,但雄激素合成下降。间充质干细胞及脂肪干细胞等干细胞经诱导可分化为分泌雄激素的睾丸间质细胞,因此,间质干细胞移植可望成为治疗男性性腺功能不全和中老年雄激素缺乏的创新方法,本文对睾丸间质干细胞的分化及移植方面研究进行综述。     

Local regulation of Leydig cells by the seminiferous tubules. Effect of short-term cryptorchidism

Unilateral cryptorchism was induced in adult rats for 24 h, and its effect on testicular morphology and intratesticular testosterone concentration after hCG-stimulation were studied. In seminiferous, tubules from abdominal testes an increased number of degenerating germ cells was noted in stages XIV-III of the spermatogenic cycle and Sertoli cells contained an increased amount of lipid droplets in stages XIV-VIII. However, germ cells and Sertoli cells from tubules at other stages of the cycle appeared unaffected. In scrotal testes the size of peritubular Leydig cells varied in phase with the spermatogenic cycle. The largest cells were found adjacent to stage VII-VIII and the smallest adjacent to stage XI-XII. In abdominal testes no stage-dependent variation in the size of peritubular Leydig cells was seen. Perivascular Leydig cells were of equal size in abdominal and scrotal testes. The testicular testosterone concentration following stimulation with a low dose of hCG was significantly lower in abdominal testes. It is suggested that the seminiferous tubules locally modulate Leydig cell function and that the stage specific stimulatory influence from stage VII-VIII is rapidly lost during experimental cryptorchidism.