A Transcriptome-Wide Screen for mRNAs Enriched in Fetal Leydig Cells: CRHR1 Agonism Stimulates Rat and Mouse Fetal Testis Steroidogenesis

Fetal testis steroidogenesis plays an important role in the reproductive development of the male fetus. While regulators of certain aspects of steroidogenesis are known, the initial driver of steroidogenesis in the human and rodent fetal testis is unclear. Through comparative analysis of rodent fetal testis microarray datasets, 54 candidate fetal Leydig cell-specific genes were identified. Fetal mouse testis interstitial expression of a subset of these genes with unknown expression (Crhr1, Gramd1b, Itih5, Vgll3, and Vsnl1) was verified by whole-mount in situ hybridization. Among the candidate fetal Leydig cell-specific factors, three receptors (CRHR1, PRLR, and PROKR2) were tested for a steroidogenic function using ex vivo fetal testes treated with receptor agonists (CRH, PRL, and PROK2). While PRL and PROK2 had no effect, CRH, at low (approximately 1 to 10) nM concentration, increased expression of the steroidogenic genes Cyp11a1, Cyp17a1, Scarb1, and Star in GD15 mouse and GD17 rat testes, and in conjunction, testosterone production was increased. Exposure of GD15 fetal mouse testis to a specific CRHR1 antagonist blunted the CRH-induced steroidogenic gene expression and testosterone responses. Similar to ex vivo rodent fetal testes, ≥10 nM CRH exposure of MA-10 Leydig cells increased steroidogenic pathway mRNA and progesterone levels, showing CRH can enhance steroidogenesis by directly targeting Leydig cells. Crh mRNA expression was observed in rodent fetal hypothalamus, and CRH peptide was detected in rodent amniotic fluid. Together, these data provide a resource for discovering factors controlling fetal Leydig cell biology and suggest that CRHR1 activation by CRH stimulates rat and mouse fetal Leydig cell steroidogenesis in vivo.     

Insulin Directly Regulates Steroidogenesis via Induction of the Orphan Nuclear Receptor DAX-1 in Testicular Leydig Cells

Testosterone level is low in insulin-resistant type 2 diabetes. Whether this is due to negative effects of high level of insulin on the testes caused by insulin resistance has not been studied in detail. In this study, we found that insulin directly binds to insulin receptors in Leydig cell membranes and activates phospho-insulin receptor-β (phospho-IR-β), phospho-IRS1, and phospho-AKT, leading to up-regulation of DAX-1 (dosage-sensitive sex reversal, adrenal hypoplasia critical region, on chromosome X, gene 1) gene expression in the MA-10 mouse Leydig cell line. Insulin also inhibits cAMP-induced and liver receptor homolog-1 (LRH-1)-induced steroidogenic enzyme gene expression and steroidogenesis. In contrast, knockdown of DAX-1 reversed insulin-mediated inhibition of steroidogenesis. Whether insulin directly represses steroidogenesis through regulation of steroidogenic enzyme gene expression was assessed in insulin-injected mouse models and high fat diet-induced obesity. In insulin-injected mouse models, insulin receptor signal pathway was activated and subsequently inhibited steroidogenesis via induction of DAX-1 without significant change of luteinizing hormone or FSH levels. Likewise, the levels of steroidogenic enzyme gene expression and steroidogenesis were low, but interestingly, the level of DAX-1 was high in the testes of high fat diet-fed mice. These results represent a novel regulatory mechanism of steroidogenesis in Leydig cells. Insulin-mediated induction of DAX-1 in Leydig cells of testis may be a key regulatory step of serum sex hormone level in insulin-resistant states.     

Effects of Etomidate on GABAergic and Glutamatergic Transmission in Rat Thalamocortical Slices

Although accumulative evidence indicates that the thalamocortical system is an important target for general anesthetics, the underlying mechanisms of anesthetic action on thalamocortical neurotransmission are not fully understood. The aim of the study is to explore the action of etomidate on glutamatergic and GABAergic transmission in rat thalamocortical slices by using whole cell patch-clamp recording. We found that etomidate mainly prolonged the decay time of spontaneous GABAergic inhibitory postsynaptic currents (sIPSCs), without changing the frequency. Furthermore, etomidate not only prolonged the decay time of miniature inhibitory postsynaptic currents (mIPSCs) but also increased the amplitude. On the other hand, etomidate significantly decreased the frequency of spontaneous glutamatergic excitatory postsynaptic currents (sEPSCs), without altering the amplitude or decay time in the absence of bicuculline. When GABA_A receptors were blocked using bicuculline, the effects of etomidate on sEPSCs were mostly eliminated. These results suggest that etomidate enhances GABAergic transmission mainly through postsynaptic mechanism in thalamocortical neuronal network. Etomidate attenuates glutamatergic transmission predominantly through presynaptic action and requires presynaptic GABA_A receptors involvement.     

大鼠骨髓来源未成熟与成熟树突状细胞的对比研究

目的:对比培养大鼠骨髓来源的未成熟树突状细胞与成熟树突状细胞,并从形态学、表型及功能检测等多方面进行对比研究,为后续的实验做出基础研究。方法:大鼠脱臼法处死后取两侧胫骨、股骨,PBS冲洗骨髓腔收集骨髓细胞,经GM-CSF和IL-4刺激培养六天后,对比研究经LPS刺激组与未经LPS刺激培养组细胞状况。结果:①成熟树突状细胞悬浮生长,集落分散,扫描电镜下见其突起数目明显多于未成熟树突状细胞。②成熟树突状细胞高表达表面标记分子CD80、CD86、MHCⅡ,而未成熟树突状细胞均低表达。③成熟树突状细胞培养基上清中IL-12水平高,而未成熟树突状细胞培养基上清中IL-12水平低。④成熟树突状细胞具有强的刺激T细胞增殖能力,而未成熟树突状细胞基本不具有诱导T细胞增殖能力。结论:未成熟状态的树突状细胞具备致耐受原性,可抑制T细胞的应答,而成熟状态的树突状细胞由于获得了免疫刺激潜能从而会对炎性刺激做出反应。     

Effects of Nandrolone Stimulation on Testosterone Biosynthesis in Leydig Cells

Anabolic androgenic steroids (AAS) are among the drugs most used by athletes for improving physical performance, as well as for aesthetic purposes. A number of papers have showed the side effects of AAS in different organs and tissues. For example, AAS are known to suppress gonadotropin‐releasing hormone, luteinizing hormone, and follicle‐stimulating hormone. This study investigates the effects of nandrolone on testosterone biosynthesis in Leydig cells using various methods, including mass spectrometry, western blotting, confocal microscopy and quantitative real‐time PCR. The results obtained show that testosterone levels increase at a 3.9 μM concentration of nandrolone and return to the basal level a 15.6 μM dose of nandrolone. Nandrolone‐induced testosterone increment was associated with upregulation of the steroidogenic acute regulatory protein (StAR) and downregulation of 17a‐hydroxylase/17, 20 lyase (CYP17A1). Instead, a 15.6 µM dose of nandrolone induced a down‐regulation of CYP17A1. Further in vivo studies based on these data are needed to better understand the relationship between disturbed testosterone homeostasis and reproductive system impairment in male subjects. J. Cell. Physiol. 231: 1385–1391, 2016. © 2015 The Authors. Journal of Cellular Physiology Published by Wiley Periodicals, Inc.     

Icariside II Reduces Testosterone Production by Inducing Necrosis in Rat Leydig Cells

The present study demonstrates that Icariside II (10, 20, and 40 µM) reduced Leydig cell testosterone production and cell viability in a concentration‐ and time‐dependent manner. Hoechst 33342/propidium iodide staining indicated that no morphological changes in Leydig cell nuclear chromatin occurred, caspase‐3 expression also showed no significant change, but cell death was caused by the 10‐µM Icariside II treatment. Furthermore, a significant reduction in NAD⁺ levels was observed following Icariside II exposure (10, 20, and 40 µM). Cell death was avoided when Icariside II treated cells were incubated with extracellular NAD⁺ (5 and 10 mM). Moreover, the addition of NAD⁺ (5 and 10 mM) could restore ATP production and prevent cell death. The results suggest that Icariside II can reduce testosterone production by inducing necrosis, but not apoptosis, in rat Leydig cells. This mechanism may also account for the Icariside II induced depletion of NAD⁺ and ATP levels. © 2013 Wiley Periodicals, Inc. J BiochemMol Toxicol 27:243‐250, 2013; View this article online at wileyonlinelibrary.com . DOI 10.1002/jbt.21481     

阿片肽对骨髓未成熟B细胞的免疫学效应

 已经证明一些细胞象脾淋巴细胞、血T细胞、自然杀伤细胞和血小板存在有阿片受体。本文在骨髓细胞上的研究提示未成熟B细胞表面也存在有这类神经肽的受体,通过阿片肤类结合到细胞上可以观察到不同的效应。试验结果显示M-脑啡肽、L-脑啡肽和α-内啡肽对骨髓未成熟B细胞抗体生成反应的抑制分别为76.6％、68.6％和67.7％。相反,β-内啡肽增进抗体反应达78.0％。M-脑啡肽和两种内啡肽在未成熟B细胞上的效应能够被拮抗剂纳络酮所逆转,提示反应涉及阿片肽受体机制。     

负载供者抗原的第三方树突状细胞延长大鼠移植心脏存活

目的观察负载供者抗原第三方未成熟DC对大鼠同种异体移植心脏存活期的影响。方法培养骨髓来源第三方大鼠未成熟DC,负载供者抗原,CTLA-4 Ig致耐受处理。建立大鼠心脏腹部移植模型,A组：心脏移植;B组：心脏移植,术前输注供者源未成熟DC;C组：心脏移植,术前输注第三方未成熟DC;D组：心脏移植,术前输注负载供者抗原致耐受第三方未成熟DC。分别行移植心病理、IFNγmRNA表达和移植心存活时间观察。结果D组的移植心病理改变轻,IFNγmRNA表达下调,移植心脏存活时间显著延长。结论负载供者抗原第三方致耐受未成熟DC能够诱导供者特异性免疫耐受,明显延长大鼠移植心脏存活时间。     

Effects of Fluoride on Surface Structure of Primary Culture Leydig Cells in Mouse

Fluoride (F) is known to induce reproduction toxicity, and the elucidation of its underlying mechanisms is an ongoing research. These findings aim to provide deeper insights into roles of soduim fluoride (NaF) in testis damage, which could contribute to a better understanding of fluoride-induced male reproductive toxicity. The Leydig cells were administrated by 0, 5, 10, and 20 mg/L NaF for 24 h, respectively. Scanning electron microscope was used to identify the change of surface structure in the Leydig cells. The results showed that fluoride exposure of high-dose induced bulged balloon in the membrane of the Leydig cell. We speculated that high doses of NaF inhibited cell proliferation and induced cell apoptosis in Leydig cells. This is the first time that we have reported that fluoride impaired cytomembrane and cytoskeleton of the Leydig cells in vitro treated with different doses of fluoride for 24 h by using a scanning electron microscope. Damage to the cytoskeleton and cytomembrane may be one of the reasons of male reproductive toxicity induced by fluoride.     

Exposure to Perfluorooctane Sulfonate In Utero Reduces Testosterone Production in Rat Fetal Leydig Cells

Background

Perfluorooctane sulfonate (PFOS) is a synthetic material that has been widely used in industrial applications for decades. Exposure to PFOS has been associated with decreased adult testosterone level, and Leydig cell impairment during the time of adulthood. However, little is known about PFOS effects in utero on fetal Leydig cells (FLC).

Methods and Results

The present study investigated effects of PFOS on FLC function. Pregnant Sprague Dawley female rats received vehicle (0.05% Tween20) or PFOS (5, 20 mg/kg) by oral gavage from gestational day (GD) 11–19. At GD20, testosterone (T) production, FLC numbers and ultrastructure, testicular gene and protein expression levels were examined. The results indicate that exposures to PFOS have affected FLC function as evidenced by decreased T production, impaired FLC, reduced FLC number, and decreased steroidogenic capacity and cholesterol level in utero.

Conclusion

The present study shows that PFOS is an endocrine disruptor of male reproductive system as it causes reduction of T production and impairment of rat fetal Leydig cells.     

A Yeast-Based Chemical Screen Identifies a PDE Inhibitor That Elevates Steroidogenesis in Mouse Leydig Cells via PDE8 and PDE4 Inhibition

A cell-based high-throughput screen (HTS) was developed to detect phosphodiesterase 8 (PDE8) and PDE4/8 combination inhibitors. By replacing the Schizosaccharomyces pombe PDE gene with the murine PDE8A1 gene in strains lacking adenylyl cyclase, we generated strains whose protein kinase A (PKA)-stimulated growth in 5-fluoro orotic acid (5FOA) medium reflects PDE8 activity. From our previously-identified PDE4 and PDE7 inhibitors, we identified a PDE4/8 inhibitor that allowed us to optimize screening conditions. Of 222,711 compounds screened, ∼0.2% displayed composite Z scores of >20. Additional yeast-based assays using the most effective 367 compounds identified 30 candidates for further characterization. Among these, compound BC8-15 displayed the lowest IC₅₀ value for both PDE4 and PDE8 inhibition in in vitro enzyme assays. This compound also displays significant activity against PDE10A and PDE11A. BC8-15 elevates steroidogenesis in mouse Leydig cells as a single pharmacological agent. Assays using BC8-15 and two structural derivatives support a model in which PDE8 is a primary regulator of testosterone production by Leydig cells, with an additional role for PDE4 in this process. BC8-15, BC8-15A, and BC8-15C, which are commercially available compounds, display distinct patterns of activity against PDE4, PDE8, PDE10A, and PDE11A, representing a chemical toolkit that could be used to examine the biological roles of these enzymes in cell culture systems.     

不同移植部位对成年大鼠睾丸间质细胞雄激素分泌功能的影响

目的:探索不同移植部位对移植的成年SD大鼠睾丸中睾丸间质细胞存活及雄激素分泌功能的影响。方法:将健康成年雄性SD大鼠随机分为对照组、假手术组、皮下组和肾包膜组。对照组大鼠不去势,其余大鼠于睾丸移植前1周行去势手术。对照组和假手术组去势后仅行背部皮肤切开,不进行睾丸移植;皮下组背部两侧各移植1/3个成年SD大鼠睾丸组织;肾包膜组每侧肾包膜下移植1/3个成年SD大鼠睾丸组织。4周后取材行HE和免疫组化染色,分析移植睾丸组织中睾丸间质细胞存活情况,ELISA法检测受体大鼠血清睾酮水平。结果:皮下组和肾包膜组移植物中难于见到完整的睾丸间质组织,但免疫组化染色发现大量HSD-17β1阳性细胞,对照组、皮下组和肾包膜组的HSD-17β1阳性细胞数分别为(24.33±4.30)、(9.83±4.05)和(12.67±2.81)个,对照组与皮下组相比差异具有统计学意义(p0.05);ELISA分析发现对照组、假手术组、皮下组和肾包膜组的血清睾酮浓度分别为(3.81±1.32)、(0.28±0.08)、(0.44±0.13)和(0.90±0.31)ng/m L,肾包膜组血清睾酮浓度高于假手术组(p0.01)和皮下组(p0.05),而皮下组血清睾酮水平高于假手术组,但两者差异无统计学意义(p0.05)。结论:移植的成年大鼠睾丸组织中的睾丸间质细胞可在受体肾包膜下或皮下存活,但肾包膜下移植可能更加有利于睾丸间质细胞存活和雄激素分泌。     

Studies on mRNA Electroporation of Immature and Mature Dendritic Cells: Effects on their Immunogenic Potential

Previous studies have shown that mRNA-electroporated dendritic cells (DCs) are able to process and present tumor-associated antigens, leading to the activation of tumor-specific T cells in vitro and in vivo. However, the optimal maturation state of antigen loading and half-life of the mRNA-translated protein product and its immunogenic epitopes are significant parameters, which needs to be clarified in order to establish an effective electroporation protocol. In addition, despite extensive experimental investigations and their widespread application in research and clinical environments, little is known of the extent to which the immunological properties of DCs are influenced by electrical fields of critical strengths. We found that the mRNA transfection of DCs after maturation with short and low-voltage square-wave electrical pulses resulted in higher level of antigen expression and viability in addition to higher T-cell stimulatory ability compared to transfection of DCs prior to maturation. Mature mRNA-electroporated DCs showed long-lived expression of EGFP and were able to stimulate influenza matrix protein M1 (M1)-specific T cells up to 24 h after electroporation. However, when DCs were subjected to increasing electrical pulses the level of transgene expression was four-fold upregulated, equipping these DCs to be more potent in inducing M1-specific T cells. Also, the application of long electrical pulses induced further upregulation of HLA-DR, CD80, and CD86 expression in mature DCs, but did not promote phenotypic or functional maturation in immature DCs. These findings support the concept of mRNA transfection of DCs after maturation and also highlight the possibility to use long electrical pulses for further improvement of the immune responses by mRNA-transfected DCs.     

供体来源ImDex联合抗原特异性Treg保护大鼠肝脏移植物的研究

目的:探讨供体来源低剂量未成熟树突状细胞外来体(immature dendritic cells exosome,im Dex)联合供体抗原特异型调节性T细胞(regulatory t cells,Tregs)对于肝脏移植物的保护作用。方法:以BN-Lewis大鼠为供受体,建立大鼠原位肝移植模型。利用超高速梯度离心法获得im Dex,采用流式细胞术鉴定该im Dex的表型。不同剂量im Dex用于本移植模型,生存分析探究其对肝脏移植物的保护作用及该作用与剂量的相关关系。利用磁珠分选技术获得Treg,混合淋巴细胞培养获得供体抗原特异性Treg,将不同性质的Treg用于移植受体,验证抗原特异性Treg保护肝脏移植物。进一步探究两者联合使用的效果,应用免疫荧光、流式细胞术研究Treg在移植受体中的分布。结果:超高速梯度离心法分选出的im Dex具有im DC表型,其保护肝脏移植物的最佳作用剂量为20μg,Treg发挥保护移植物作用具有抗原特异性。两者联合应用组生存时间长于对照组(P0.05)。病理显示,输注的Treg分布于受体移植物。结论:Im Dex联合抗原特异性Treg可以有效保护大鼠肝脏移植物。