Seasonal variation in the activity of the Leydig cells in Egyptian Nubian goat (Zaraibi) bucks

Ten Egyptian Nubian goat bucks were used to evaluate the effect of season on testicular hormonal activity and ultrastructure. Parameters were recorded for 7 consecutive weeks in the middle of the four seasons, with blood samples being collected weekly. At the end of each of these seasons, testicular biopsies were obtained surgically for histological and cytological studies. Season had a significant effect on plasma testosterone concentration, being at its lowest level (P < 0.01) during winter and spring (1.2 and 2.6 ng/ml, respectively), while at its highest during summer (10 ng/ml). The effect of season on plasma LH concentration was higher (P < 0.01) in autumn (2.9 mIU/ml) and less in spring and summer (0.4 mIU/ml). Season of the year influenced the percentage of sectional tissue area occupied by the seminiferous tubules and interstitial tissue. Seminiferous tubules occupied the majority of the testicular tissue during winter (76.6%), with the least being occupied during spring (49.8%). The thickness of the seminiferous tubules was maximal during autumn, followed by summer (53 and 36 μm, respectively). In summer the Leydig cells contained abundant smooth endoplasmic reticulum (sER), while some areas of the cytoplasm were occupied exclusively by tubular sER, arranged in parallel—indicating the highest activity of these cells. A characteristic multivesicular structure with numerous large lipid droplets and vacuoles was recorded in the Leydig cells during spring and winter, denoting low or even arrested activity of the cells. It could be concluded that season influences the activity of the Leydig cells of Egyptian Nubian bucks, and this is reflected by their ultrastructure and secretive activity.     

细菌脂多糖(LPS)在糖尿病视网膜微血管病变中的作用

本研究旨在通过Akita小鼠糖尿病模型及糖尿病人群血浆样本,探讨病原体相关性分子细菌脂多糖(lipopolysaccharide,LPS)在糖尿病视网膜病变中的重要作用。本研究选择6个月糖尿病病程的Akita小鼠(Ins2+/Akita)及其同年龄组野生型(wild type,WT)小鼠(C57BL/6J)尾静脉内注射脂多糖(LPS)或生理盐水对照共7 d,从影像学、电生理及病理学水平评估糖尿病视网膜眼病进展。最后收集糖尿病视网膜眼病患者及对照人群血标本,通过ELISA测定血浆LPS表达水平。通过光学相干断层扫描技术分析,发现Akita小鼠的视网膜层间厚度较WT小鼠组相比明显变薄(p=0.000 2),LPS处理进一步加重糖尿病小鼠视网膜结构损害(p=0.000 7)。视网膜电图检测发现LPS处理Akita小鼠组的视网膜细胞幅值较生理盐水处理Akita小鼠显著减慢,有统计学意义(p<0.05)。胰酶消化法分离及PAS染色小鼠眼球视网膜微血管网后,计数测得LPS处理显著增加了Akita小鼠视网膜中无细胞毛细血管数量(p=0.002 6),提示LPS在糖尿病微血管损伤中的重要作用。为保证该研究的临床转化性,我们进一步检测了糖尿病视网膜病变患者(n=19)、糖尿病患者(无微血管并发症)(n=23)及健康对照组(n=20)的血浆LPS水平,发现糖尿病患者血浆LPS水平较健康对照组显著升高(p=0.002 3),其中糖尿病视网膜病变患者LPS升高最为显著(p<0.000 1)。本研究表明,循环中细菌脂多糖增加在糖尿病视网膜病变进展中起到重要作用。     

The ultrastructure of the cells of Leydig in the white-crowned sparrow (Zonotrichia leucophrys gambelii) in relation to plasma levels of luteinizing hormone and testosterone

In male White-crowned Sparrows subjected to 20 h daily photoperiods there is an approximately 3-fold increase in the plasma concentration of luteinizing hormone (LH) on the first long day after which a quasi-stable level is maintained for at least 42 days. This increase is followed by an increase in numbers of cells of Leydig and an enhancement of their steroidogenic features, a decrease in transitional interstitial cells, and an increase in plasma level of testosterone. With the decline in plasma LH, as photorefractoriness develops, the steroidogenic features of the cells of Leydig undergo disorganization. For as yet unexplainable reasons the plasma levels of testosterone decline before the decrease in plasma LH and before the degeneration of the steroidogenic features of the cells of Leydig.     

Ultrastructure and morphometry of testicular Leydig cells and the interstitial components correlated with testosterone in aging rats

The ultrastructure of testicular interstitium in young and aged adult rats was analysed using morphometric methods, and the plasma testosterone concentration was measured. With increasing age there was an augumentation in the volume of collagen fibrils in the intercellular matrix and in blood vessels. During the aging process (approximately two years) the average volume of the Leydig cell decreased from 1364 m³ to 637 m³, but the number of Leydig cells in paired testes increased from 53x10⁶ to 113x10⁶. The absolute volume of smooth surfaced endoplasmic reticulum (SER) per Leydig cell amounted in aged rats to 78% of that in young adult rats. The total amount of SER in paired testes increased by 62% with aging. The present analysis suggests that the ability of SER to maintain peripheral testosterone concentration decreases with age. In young adult rats the absolute volume of peroxisomes per Leydig cell correlated significantly with the concentration of testosterone in blood and also with the absolute volume of SER per Leydig cell. These results combined with ultrastructural observations of close apposition of peroxisomes and SER suggest that peroxisomes have a role in testosterone secretion by Leydig cells.Visiting scientist to Laboratory of Electron Microscopy (Director: Prof. L.J. Pelliniemi)     

Diphosphoryl lipid A derived from the lipopolysaccharide (LPS) of Rhodobacter sphaeroides ATCC 17023 is a potent competitive LPS inhibitor in murine macrophage-like J774.1 cells

Abstract Pentaacyl diphosphoryllipid A derived from the nontoxic lipopolysaccharide (LPS) of Rhodobacter sphaeroides ATCC 17023 (RsDPLA) did not induce tumour necrosis factor-α nor interleukin-6 release in the murine macrophage-like cell line J774.1. However, it effectively inhibited the induction of these two cytokines by LPS of Salmonella minnesota Re mutant R595 (ReLPA) in a concentration-dependent manner. Maximal inhibition and half-maximal inhibition occured when the ReLPS to RsDPLA mass ratio was 1:30 and 1:1, respectively. A binding study was performed in the presence of serum to determine whether RsDPLA is competing with ReLPS for LPS binding sites on J774.1 cells. This assay allows the determination of LPS binding to J774.1 cells via a mechanism involving CD14, a receptor for complexes of LPS with LPS binding protein (LBP), and its possible inhibition. The results show that RsDPLA strongly inhibits the binding of ¹²⁵I-labelled ReLPS to J774.1 cells. Maximal and one-half maximal inhibition of binding occured when the ReLPS to RsDPLA mass ratios were 1:2.5 and 1:0.5, respectively. It was found that the inhibition of binding by RsDPLA was much stronger than that by unlabelled ReLPS. These results suggest that RsDPLA is competing with ReLPS for CD14-dependent recognition of LPS on J774.1 cells.     

Studies on the protective role of vitamin C and E against polychlorinated biphenyl (Aroclor 1254)--induced oxidative damage in Leydig cells

Free radical production and lipid peroxidation are potentially important mediators in testicular physiology and toxicology. Polychlorinated biphenyls (PCBs) are global environmental contaminants that cause disruption of the endocrine system in human and animals. The present study was conducted to elucidate the protective role of vitamin C and E against Aroclor 1254-induced changes in Leydig cell steroidogenesis and antioxidant system. Adult male rats were dosed for 30 days with daily intraperitoneal (ip) injection of 2 mg/kg Aroclor or vehicle (corn oil). One group of rats was treated with vitamin C (100 mg/kg bw/day) while the other group was treated with vitamin E (50 mg/kg bw/day) orally, simultaneously with Aroclor 1254 for 30 days. One day after the last treatment, animals were euthanized and blood was collected for the assay of serum hormones such as luteinizing hormone (LH), thyroid stimulating hormone (TSH), prolactin (PRL), triiodothyronine (T₃), thyroxine (T₄), testosterone and estradiol. Testes were quickly removed and Leydig cells were isolated in aseptic condition. Purity of Leydig cells was determined by 3β-hydroxysteroid dehydrogenase (3β-HSD) staining method. Purified Leydig cells were used for quantification of cell surface LH receptors and steroidogenic enzymes such as cytochrome P₄₅₀ side chain cleavage enzyme (P₄₅₀scc), 3β-hydroxysteroid dehydrogenase (3β-HSD) and 17β-hydroxysteroid dehydrogenase (17β- HSD). Leydig cellular enzymatic antioxidants superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), glutathione reductase (GR), γ-glutamyl transpeptidase (γ-GT), glutathione-S-transferase (GST) and non-enzymatic antioxidants such as vitamin C and E were assayed. Lipid peroxidation (LPO) and reactive oxygen species (ROS) were also estimated in Leydig cells. Aroclor 1254 treatment significantly reduced the serum LH, TSH, PRL, T₃, T₄, testosterone and estradiol. In addition to this, Leydig cell surface LH receptors, activities of the steroidogenic enzymes such as cytochrome P₄₅₀scc, 3β-HSD, 17β-HSD, antioxidant enzymes SOD, CAT, GPX, GR, γ-GT, GST and non-enzymatic antioxidants such as vitamin C and E were significantly diminished whereas, LPO and ROS were markedly elevated. However, the simultaneous administration of vitamin C and E in Aroclor 1254 exposed rats resulted a significant restoration of all the above-mentioned parameters to the control level. These observations suggest that vitamin C and E have ameliorative role against adverse effects of PCB on Leydig cell steroidogenesis.     

Selective destruction and regeneration of rat Leydig cells in vivo

Summary The effect of a single i.p. administration of ethane dimethanesulphonate (EDS) upon rat testicular histology was studied by light microscopy and morphometry up to 4 weeks after treatment. One day after injection the interstitial tissue exhibited degenerating Leydig cells, abundant pyknotic interstitial cells, deposition of cellular debris and extensive networks of fibrillar material. Macrophages contained greatly increased numbers of cytoplasmic inclusion bodies. From 3 to 7 days morphometric analysis showed that Leydig cells and cellular debris had disappeared from the interstitial tissue, leaving only macrophages, fibroblasts and lymphatic endothelial tissue. A very small number of new Leydig cells were seen on day 14, often located in peritubular or perivascular positions. Regeneration of foetal-like Leydig cells occurred by 4 weeks, their cytoplasm containing large lipid inclusions and, numerous Leydig cells were often observed closely applied to the walls of the seminiferous tubules. The observations suggest that, after experimental destruction and depletion of Leydig cells, an interstitial precursor cell, as yet unidentified, gives rise to a new Leydig cell population. EDS thus offers a valuable opportunity to study further the interactions between the seminiferous tubules and the interstitial tissue following the destruction and subsequent regeneration of the Leydig cells.     

Ultrastructure of germ cells, the Leydig cells, and Sertoli cells during spermatogenesis in Boleophthalmus pectinirostris (Teleostei, Perciformes, Gobiidae)

The ultrastructures of germ cells, Leydig cells, and Sertoli cells during spermatogenesis in male Boleophthalmus pectinirostris were investigated by electron microscopic observations. During the period of maturation divisions, well-developed Leydig cells have three major morphological characteristics: a vesicular nucleus, mitochondria with tubular cristae, and a number of smooth endoplasmic reticulum. Based on cytoplasmic features, it appears that Leydig cells are responsible for the synthesis of male sex steroids. Although no clear evidence of steroidogenesis was found in the Sertoli cells, they were found to perform a phagocytic function in the seminiferous lobules. Most Sertoli cells contain granules thought to represent deposited glycogen or lipid but there is no indication of a transfer of nutrients to the spermatids. During the period of germ cell degeneration, several characteristics of phagocytosis appear in the cytoplasm of the Sertoli cells. In particular, it is assumed that the Sertoli cells are involved in the degeneration and resorption of undischarged spermatids after spermiation. No acrosome of the sperm is formed. The structure of the spermatozoon in B. pectinirostris is very similar and closely resembles to those of suborder Gobioidei (perciform type teleosts). The flagellum or sperm tail shows the typical 9+2 array of microtubules.     

乌梢蛇精巢间质细胞微细结构与血清 睾酮浓度的相关性

用光镜及透射电镜观察了乌梢蛇(Zaocys dhumnades)精巢间质细胞的显微与超微结构,并利用放射免疫测定法测定了血清中睾酮浓度.结果表明,在一个年生殖周期中,乌梢蛇间质组织所占区域相对大小、间质细胞数量和显微结构均存在较明显的变化;5月份的间质细胞具有发达的管状嵴线粒体、丰富的滑面内质网、大量的脂滴等合成和分泌...     

Stem cell therapy for the treatment of Leydig cell dysfunction in primary hypogonadism

The production of testosterone occurs within the Leydig cells of the testes. When production fails at this level from either congenital, acquired, or systemic disorders,the result is primary hypogonadism. While numerous testosterone formulations have been developed, none are yet fully capable of replicating the physiological patterns of testosterone secretion. Multiple stem cell therapies to restore androgenic function of the testes are under investigation. Leydig cells derived from bone marrow, adipose tissue, umbilical cord, and the testes have shown promise for future therapy for primary hypogonadism. In particular, the discovery and utilization of a group of progenitor stem cells within the testes, known as stem Leydig cells(SLCs), has led not only to a better understanding of testicular development, but of treatment as well. When combining this with an understanding of the mechanisms that lead to Leydig cell dysfunction, researchers and physicians will be able to develop stem cell therapies that target the specific step in the steroidogenic process that is deficient. The current preclinical studies highlight the complex nature of regenerating this steroidogenic process and the problems remain unresolved. In summary, there appears to be two current directions for stem cell therapy in male primary hypogonadism. The first method involves differentiating adult Leydig cells from stem cells of various origins from bone marrow, adipose, or embryonic sources. The second method involves isolating, identifying, and transplanting stem Leydig cells into testicular tissue. Theoretically, in-vivo re-activation of SLCs in men with primary hypogonadism due to age would be another alternative method to treat hypogonadism while eliminating the need for transplantation.     

C1q/TNF-related protein-3 (CTRP-3) attenuates lipopolysaccharide (LPS)-induced systemic inflammation and adipose tissue Erk-1/-2 phosphorylation in mice in vivo

Background

The C1q/TNF-related proteins comprise a growing family of adiponectin paralogous proteins. CTRP-3 represents a novel adipokine with strong expression in adipose tissue and was shown to inhibit chemokine and cytokine release in adipocytes and monocytes in vitro. The aim of the study was to gain the proof of principle that CTRP-3 is a potent anti-inflammatory adipokine in vivo.

Methods

C57BL/6N mice were treated intraperitoneally (i.p.) with bacterial lipopolysaccharide (LPS) for 2 h. The effects of a 30 min pre-treatment with CTRP-3 i.p. or intravenously (i.v.) on systemic and on epididymal, perirenal and subcutaneous adipose tissue inflammation was analyzed via real-time RT-PCR, ELISA and Western blot analysis.

Results

LPS (1 μg i.p.) significantly increased serum IL-6 and MIP-2 levels as well as epididymal adipose tissue expression of IL-6 and MIP-2 in mice, whereas CTRP-3 (10 μg i.p.) alone or PBS (i.p.) had no effect. Pre-treatment of mice by CTRP-3 i.p. prior to LPS application significantly attenuated LPS-induced cytokine levels but had no effect on adipose tissue cytokine mRNA expression. In contrast to i.p. application of CTRP-3, systemic i.v. application was not sufficient to inhibit LPS-induced cytokine levels or mRNA tissue expression. CTRP-3 given i.p. significantly attenuated LPS-induced phosphorylation of Erk-1/-2 in inguinal adipose tissue.

Conclusion

The present study shows the proof of principle that the novel adipokine CTRP-3 is a potent inhibitor of LPS-induced systemic inflammation and LPS-induced signaling in adipose tissue in vivo.     

Effects of prolactin on the morphology and function of rat Leydig cells: short-term versus long-term administration

Summary The bolus administration of prolactin (PRL) to adult rats did not cause any apparent change in the basal and luteinizing hormone (LH)-stimulated blood levels of testosterone (as estimated by radioimmune assay). Prolonged PRL infusion did not affect either basal testosterone plasma concentration or the morphology of Leydig cells (as evaluated by electron microscopy and stereology). Conversely, prolonged PRL treatment notably increased the gonadotrophic effects of chronic LH administration; this mainly consisted of a rise in the blood concentration of testosterone and a conspicuous hypertrophy of Leydig cells. The LH-induced increase in the volume of Leydig cells was the result of an increase in the volumes of all the organelles involved in steroid synthesis (i.e., smooth endoplasmic reticulum, peroxisomes and mitochondria). However, the trophic effects of PRL infusion exclusively concerned smooth endoplasmic reticulum and peroxisomes. In the light of these findings, the hypothesis is advanced that the mechanism underlying the gonadotrophic action of PRL involves an enhancement of the endogenous cholesterol synthesis, which could provide an abundance of precursors for testosterone synthesis, the post-cholesterol steps of which, in turn, would be exclusively controlled by LH.     

Leydig cell loss and spermatogenic arrest in platelet-derived growth factor (PDGF)-A-deficient mice

Platelet-derived growth factor (PDGF)- A-deficient male mice were found to develop progressive reduction of testicular size, Leydig cells loss, and spermatogenic arrest. In normal mice, the PDGF-A and PDGF-Ralpha expression pattern showed positive cells in the seminiferous epithelium and in interstitial mesenchymal cells, respectively. The testicular defects seen in PDGF-A-/- mice, combined with the normal developmental expression of PDGF-A and PDGF-Ralpha, indicate that through an epithelial-mesenchymal signaling, the PDGF-A gene is essential for the development of the Leydig cell lineage. These findings suggest that PDGF-A may play a role in the cascade of genes involved in male gonad differentiation. The Leydig cell loss and the spermatogenic impairment in the mutant mice are reminiscent of cases of testicular failure in man.     

Characterization and age dependence of the stimulatory effect of VIP on cyclic AMP accumulation in rat Leydig cells

The neuropeptide vasoactive intestinal peptide (VIP) has been shown to stimulate cyclic AMP accumulation in Leydig cells isolated from rat testis. The effect was dependent on time, temperature and cell concentration. At 15° half-maximal and maximal stimulation were observed at about 1 and 100 nM VIP, respectively. The interaction was specific since an order of potencies chicken VIP> rat VIP> secretin>glucagon and no effect of neurotensin and substance P were obtained. The efficiency of VIP was lower in pubertal rats and then increased in young-adult and adult animals. These results together with the known presence of VIP in the testis support the idea that VIP may be involved in the regulation and function of Leydig cells during development.     

Immunobiology of Lipopolysaccharide (LPS) and LPS-Derived Immunoconjugates Vaccinate Mice against Salmonella typhimurium

The immunobiology of lipopolysaccharide (LPS) of Salmonella typhimurium LT2–71 was studied in its native, modified and conjugated states using mice as the experimental model. An alkali-treated detoxified fraction of LPS (D-LPS) was found to be not only non-toxic but also equally immunogenic, like LPS. In addition D-LPS alone or conjugated with enterotoxin or hemolysin was also non-pyrogenic and non-indurogenic. The immunoprophylactic activity of D-LPS conjugates to a 100 ID₅₀ challenge dose of S. typhimurium was also higher than that of detoxified LPS or native LPS.     

Role of tumor necrosis factor-α and glucocorticoid on lipopolysaccharide (LPS)-induced apoptosis of thymocytes

Abstract Administration of bacterial lipopolysaccharide (LPS) into mice markedly induced the apoptosis of CD4⁺8⁺ thymocytes. The injection of anti-tumor necrosis factor (TNF)-α antibody or RU38486, a glucocorticoid receptor antagonist, into mice definitely inhibited LPS-induced apoptosis of thymocytes. Addition of the sera 1 h after injection of LPS into in vitro cultures of thymocytes caused thymocyte apoptosis. It was also prevented by either anti-TNF-α antibody or RU38486. Further, recombinant TNF-α and hydrocortisone collaborated in induction of the thymocyte apoptosis in vitro. The in vivo phenomenon of LPS-induced apoptosis of thymocytes was reproducible by the in vitro experimental system. It was therefore suggested that both TNF-α and glucocorticoid participate and collaborate as effector molecules in LPS-induced apoptosis of thymocytes.