Differential expression of microRNAs in TM3 Leydig cells of mice treated with brain‐derived neurotrophic factor

Brain‐derived neurotrophic factor (BDNF) is a neurotrophin that can promote the development and proliferation of neurons. BDNF has been found to be involved in male reproduction. Leydig cells in testicular interstitial tissues can secrete testosterone in a luteinizing hormone‐dependent manner. We showed that BDNF and its receptor TrkB were expressed in mice TM3 Leydig cells in the present study. Furthermore, BDNF can promote proliferation of mouse TM3 Leydig cells in vitro. Results of microRNA (miRNA) deep sequencing showed that BDNF can alter the expression profile of miRNAs in TM3 Leydig cells. Eighty‐three miRNAs were significantly different in the BDNF‐treated and control groups (fold change of >2.0 or <0.5, P < 0.05) wherein 40 were upregulated and 43 were downregulated. The expression levels of miR‐125a‐5p, miR‐22‐5p, miR‐342‐59, miR‐451a, miR‐148a‐5p, miR‐29b‐3p, miR‐199b‐5p, and miR‐145a‐5p were further confirmed by quantitative real‐time polymerase chain reaction. Bioinformatic analysis revealed that miRNAs regulated a large number of genes with different functions. Pathway analysis indicated that miRNAs participate in the pathways involved in signal transduction, cancer, metabolism, endocrine system, immune system, and nerve system. This study indicated that miRNAs might be involved in the BDNF‐regulated cellular functions of Leydig cells.     

Brain-derived neurotrophic factor promotes proliferation and progesterone synthesis in bovine granulosa cells

Brain-derived neurotrophic factor (BDNF) is involved in regulating the growth of ovarian follicles, maturation of the oocyte, and development of the early embryo through its receptor, tyrosine kinase receptor B (TrkB). However, it is still unclear as to how BDNF influences proliferation and steroidogenesis of bovine granulosa cells (GCs). In this paper, we confirmed that BDNF and TrkB were expressed in bovine GCs, and that proliferation and steroidogenesis by bovine GCs were reduced by knockdown of BDNF or inhibition of TrkB. With respect to GC proliferation, BDNF enhanced cellular viability and the percentage of cells in the S phase. BDNF also activated both protein kinase B (PKB, also known as AKT) and the extracellular signal-regulated protein kinase 1/2 (ERK1/2)-signaling pathway. Through the AKT-signaling pathway, BDNF increased the expression of proliferation-related genes, including cyclin A1 (CCNA1), cyclin E2 (CCNE2), cyclin D1 (CCND1), and cyclin-dependent kinase 1 (CDK1). However, through the ERK1/2 signaling pathway, BDNF only increased the expression of CCNA1 and CCNE2. Regarding steroidogenesis by bovine GCs, BDNF promoted progesterone (P ₄) synthesis, but had no effect on estradiol; it also activated the AKT-signaling pathway and increased the expression of steroidogenesis-related genes, including steroidogenic acute regulatory protein (STAR) and hydroxy-δ-5-steroid dehydrogenase, 3β- and steroid δ-isomerase 1 (HSD3B1). In summary, our data are the first to show that BDNF promotes the proliferation of bovine GCs through TrkB–AKT and ERK1/2 signaling pathways and increases P₄ synthesis by bovine GCs through the TrkB–AKT signaling pathway.     

Epidermal growth factor regulates the development of stem and progenitor Leydig cells in rats

Epidermal growth factor (EGF) has many physiological roles. However, its effects on stem and progenitor Leydig cell development remain unclear. Rat stem and progenitor Leydig cells were cultured with different concentrations of EGF alone or in combination with EGF antagonist, erlotinib or cetuximab. EGF (1 and 10 ng/mL) stimulated the proliferation of stem Leydig cells on the surface of seminiferous tubules and isolated CD90⁺ stem Leydig cells and progenitor Leydig cells but it blocked their differentiation. EGF also exerted anti‐apoptotic effects of progenitor Leydig cells. Erlotinib and cetuximab are able to reverse EGF‐mediated action. Gene microarray and qPCR of EGF‐treated progenitor Leydig cells revealed that the down‐regulation of steroidogenesis‐related proteins (Star and Hsd3b1) and antioxidative genes. It was found that EGF acted as a proliferative agent via increasing phosphorylation of AKT1. In conclusion, EGF stimulates the proliferation of rat stem and progenitor Leydig cells but blocks their differentiation.     

Galectin-1: biphasic growth regulation of Leydig tumor cells

Galectin-1 (Gal-1) is a widely expressed beta-galactoside-binding protein that exerts pleiotropic biological functions. To gain insight into the potential role of Gal-1 as a novel modulator of Leydig cells, we investigated its effect on the growth and death of MA-10 tumor Leydig cells. In this study, we identified cytoplasmic Gal-1 expression in these tumor cells by cytofluorometry. DNA fragmentation, caspase-3, -8, and -9 activation, loss of mitochondrial membrane potential (DeltaPsim), cytochrome c (Cyt c) release, and FasL expression suggested that relatively high concentrations of exogenously added recombinant Gal-1 (rGal-1) induced apoptosis by the mitochondrial and death receptor pathways. These pathways were independently activated, as the presence of the inhibitor of caspase-8 or -9 only partially prevented Gal-1-effect. On the contrary, low concentrations of Gal-1 significantly promoted cell proliferation, without inducing cell death. Importantly, the presence of the disaccharide lactose prevented Gal-1 effects, suggesting the involvement of the carbohydrate recognition domain (CRD). This study provides strong evidence that Gal-1 is a novel biphasic regulator of Leydig tumor cell number, suggesting a novel role for Gal-1 in the reproductive physiopathology.     

Analysis of the developmental expression of small VCP-interacting protein and its interaction with steroidogenic acute regulatory protein in Leydig cells

Small VCP-interacting protein (SVIP) is a 9-kDa protein that is composed of 76 amino acids, and it plays a role in the endoplasmic reticulum-associated protein degradation (ERAD) pathway. Recent studies have shown that SVIP is an androgen-responsive protein and its expression is regulated by androgens. Because no data are available regarding the cellular localization and expression of SVIP in the mouse testis, where androgens are highly expressed, immunohistochemistry and western blotting were performed. In the fetal testis, we found that moderate but consistent staining of SVIP is present in the cytoplasm of Leydig cells. In prepubertal and adult life, SVIP remains present in Leydig cells as well as in the cytoplasm of some peritubular and Sertoli cells. From postnatal day 15 onward, SVIP is strongly expressed in the cytoplasm of Leydig cells.Furthermore, TM3, MA-10 Leydig and Sertoli cell lines were also used to evaluate the expression of SVIP. To identify the interacting partners, such as steroidogenic acute regulatory (STAR) protein, colocalization studies were performed by fluorescence microscopy, showing that STAR colocalized with SVIP in the adult mouse testis. The expression changes of STAR were studied by using SVIP siRNAs in Leydig cell line cultures. Depletion of SVIP resulted in decreased expression of STAR. Additionally, the number and size of lipid droplets were significantly increased in SVIP-depleted Leydig cells. Taken together, our data identify SVIP as a marker of Leydig cell lineage and as a regulator of STAR protein expression and lipid droplet status in Leydig cells.     

Brain-derived neurotrophic factor transiently stabilizes silent synapses on developing neuromuscular junctions

A general feature of the developing nervous system is the activity-dependent rearrangement of genetically defined, synaptic connections. A parallel process occurs at the developing neuromuscular junction as activity-dependent synapse withdrawal reduces the initial polyneuronal innervation of individual muscle fibers to a mononeuronal innervation within the first few weeks after birth. Because members of the neurotrophin gene family influence motor neuron differentiation and survival, we examined whether or not they also influence synaptic rearrangements in neonatal muscles. We found that treatment with brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), or neurotrophin-4/5 (NT-4/5) causes the transient retention of multiple synaptic contacts on neonatal myofibers. However, the combination of both electrophysiological and histological assays revealed that the majority of such supernumerary synaptic contacts are functionally inactive or “silent.” There also occurs an increase in the number of retracting axons. Because BDNF mRNA is expressed in developing muscle and the trkB tyrosine kinase receptor for BDNF is expressed by neonatal motor neurons, our results suggest that BDNF may play an endogenous role in the refinement of synaptic connectivity that occurs in skeletal muscles after birth. © 1996 John Wiley & Sons, Inc.     

Effects of boric acid on cell death and oxidative stress of mouse TM3 Leydig cells in vitro

BackgroundBoron (B) is an abundant element on earth and presents at physiological pH in the form of boric acid (BA). It has both positive and negative effects on biological systems. BA and sodium borates have been considered as being toxic to the reproduction system in animal experiments. Unfortunately, the molecular mechanism underlying the toxic effects of BA is not fully understood.MethodsHere, we demonstrate the influence of BA on mouse TM3 Leydig cells which are male reproductive system cells targeted by BA exposure. The cytotoxicity was evaluated by MTT and NRU assays. Annexin V-FITC/PI double staining kit, mitochondria membrane potential (ΔΨm) assay kit with JC-1 and caspase-3 colorimetric assay kit were used to indicate the cell death pathway. To estimate the role of oxidative stress in BA induced toxicity, glutathione (GSH) level, catalase (CAT) and superoxide dismutase (SOD) activities were measured manually.ResultsThe cell viability assays showed that BA was not cytotoxic within the tested concentrations up to 1000 μM. Sub-toxic concentrations were used for detecting oxidative stress status. BA exposure was significantly reduced GSH level at 1000 μM and CAT activity in a concentration-dependent manner. However, SOD activity was increased at the tested concentrations (100–1000 μM). Moreover, ΔΨm was significantly decreased at 500 and 1000 μM of BA, while caspase-3 activity was not changed apparently.ConclusionThese findings demonstrated that BA is not cytotoxic and apoptotic but may slightly induces oxidative stress in TM3 Leydig cells at higher concentrations.     

Activation of NPFF2 receptor stimulates neurite outgrowth in Neuro 2A cells through activation of ERK signaling pathway

Neurite outgrowth is an important process in neural regeneration and plasticity, especially after neural injury, and recent evidence indicates that several G_αi/o protein-coupled receptors play an important role in neurite outgrowth. The neuropeptide (NP)FF system contains two G_αi/o protein-coupled receptors, NPFF₁ and NPFF₂ receptors, which are mainly distributed in the central nervous system. The aim of the present study was to determine whether the NPFF system is involved in neurite outgrowth in Neuro 2A cells. We showed that Neuro 2A cells endogenously expressed NPFF₂ receptor, and the NPFF₂ receptor agonist dNPA inhibited cyclic adenosine monophosphate (cAMP) production stimulated by forskolin in Neuro 2A cells. We also demonstrated that NPFF and dNPA dose-dependently induced neurite outgrowth in Neuro 2A cells, which was completely abolished by the NPFF receptor antagonist RF9. Pretreatment with mitogen-activated protein kinase inhibitors PD98059 and U0126 decreased dNPA-induced neurite outgrowth. In addition, dNPA increased phosphorylation of extracellular signal-regulated kinase (ERK) in Neuro 2A cells, which was completely antagonized by pretreatment with U0126. Our results suggest that activation of NPFF₂ receptor stimulates neurite outgrowth in Neuro 2A cells through activation of the ERK signaling pathway. Moreover, NPFF₂ receptor may be a potential therapeutic target for neural injury and degeneration in the future.     

Galectin-1, a cell adhesion modulator, induces apoptosis of rat Leydig cells in vitro

Galectin-1 (Gal-1), a beta galactoside-binding lectin, is involved in multiple biological functions, such as cell adhesion, apoptosis, and metastasis. On the basis of its ability to interact with extracellular matrix (ECM) glycoproteins, we investigated the Gal-1 effect on Leydig cells, which express and are influenced by ECM proteins. In this study, Gal-1 was identified in Leydig cell cultures by immunofluorescence. To gain insight into its biological role, Gal-1 was added to purified rat Leydig cells, under both basal and human chorionic gonadotrophin-stimulated conditions. Substantial morphological changes were observed, and cell viability showed an 80% decrease after 24 h culture. As a functional consequence of Gal-1 addition, testosterone production was reduced in a dose-dependent fashion, reaching a minimum of 26% after 24 h compared with basal values. cAMP showed a similar variation after 3 h. Assessment of DNA hypodiploidy and caspase activity determinations indicated that the reduction in viability and in steroidogenesis was caused by apoptosis induced by Gal-1. Besides, addition of Gal-1 caused Leydig cell detachment. Presence of laminin-1 or lactose prevented the effect of Gal-1, suggesting that the carbohydrate recognition domain is involved in inducing apoptosis. These findings demonstrate a novel mechanism, based on Gal-1 and laminin-1 interaction, which could help us better understand the molecular basis of Leydig cell function and survival control.     

Interaction of thyroid hormone and steroidogenic acute regulatory (StAR) protein in the regulation of murine Leydig cell steroidogenesis

The steroidogenic acute regulatory (StAR) protein, a novel phosphoprotein, is a crucial factor involved in intramitochondrial cholesterol transportation, the rate-limiting step in steroidogenesis. The present investigations were undertaken to elucidate involvement of thyroid hormone and StAR protein in the regulation of steroidogenesis in mouse Leydig cells. Treatment of cells with triiodothyronine (T₃) coordinately augmented the levels of StAR protein, StAR mRNA, and steroid production, and these responses were progressively dependent on expression of steroidogenic factor 1 (SF-1). With regard to steroidogenesis and StAR expression, the T₃ response requires both on-going mRNA and protein synthesis. In addition, the effects of T₃ were acutely modulated at the steroidogenic machinery and luteinizing hormone receptor (LHR) function, while these levels were suppressed following longer periods of exposure to T₃. Furthermore, the inhibition of SF-1 expression by DAX-1 markedly abolished T₃-mediated StAR expression in a time frame, which was consistent with decreased steroid biosynthesis. Specific involvement of SF-1 was further confirmed by assessing the 5′-flanking region of the mouse StAR gene, which identified a region between −254 and −110 bp that was essential for T₃ function. Importantly, it was found that the SF-1 binding site at position −135 bp of the 5′-flanking region was greatly involved in T₃-mediated reporter activity. Electrophoretic mobility shift assays (EMSA) also demonstrated involvement of SF-1 in T₃ function. The relevance of T₃-mediated LHR function was investigated in mice rendered hypo-and hyperthyroid, which accounted for up-regulation in the former and down-regulation in the latter group, respectively. These findings demonstrate a key role of thyroid hormone in maintaining mouse Leydig cell function, where thyroid hormone and StAR protein coordinately regulate steroid hormone biosynthesis.     

Fate determination of fetal Leydig cells

Leydig cell (LC) is one of the most important somatic cell types in testis, which localized in the interstitium between seminiferous tubules. The major function of Leydig cells is to produce steroid hormone, androgens. LC differentiation exhibits a biphasic pattern in rodent testes, which are divided into two different temporal mature populations, fetal Leydig cells (FLCs) and adult Leydig cells (ALCs). FLCs are transiently present in fetal testes and undergo involution or degeneration after birth. FLCs are completely devoid and replaced by ALCs in adult testes. Comparing to ALCs, FLCs display unique morphology, ultrastructure and functions. The origin of FLCs has been debated for many years, but it is still a mystery. Many factors have been reported regulating the specification, proliferation and differentiation of FLCs. FLCs degenerate in a few weeks postnatally, however, the underlying mechanism is still unknown. In this review, we will focus on the fate determination of FLCs, and summarize the resent progress on the morphology, ultrastructure, function, origin and involution of FLCs.     

Brain-derived neurotrophic factor: Its role in energy balance and cancer cachexia

Brain-derived neurotrophic factor (BDNF) plays an important role in the development of the central and peripheral nervous system during embryogenesis. In the mature central nervous system, BDNF is required for the maintenance and enhancement of synaptic transmissions and the survival of neurons. Particularly, it is involved in the modulation of neurocircuits that control energy balance through food intake, energy expenditure, and locomotion. Regulation of BDNF in the central nervous system is complex and environmental factors affect its expression in murine models which may reflect to phenotype dramatically. Furthermore, BDNF and its high-affinity receptor tropomyosin receptor kinase B (TrkB), as well as pan-neurotrophin receptor (p75^NTR) is expressed in peripheral tissues in adulthood and their signaling is associated with regulation of energy balance. BDNF/TrkB signaling is exploited by cancer cells as well and BDNF expression is increased in tumors. Intriguingly, previously demonstrated roles of BDNF in regulation of food intake, adipose tissue and muscle overlap with derangements observed in cancer cachexia. However, data about the involvement of BDNF in cachectic cancer patients and murine models are scarce and inconclusive. In the future, knock-in and/or knock-out experiments with murine cancer models could be helpful to explore potential new roles for BDNF in the development of cancer cachexia.     

Fibroblast growth factor homologous factor 1 stimulates Leydig cell regeneration from stem cells in male rats

Fibroblast growth factor homologous factor 1 (FHF1) is an intracellular protein that does not bind to cell surface fibroblast growth factor receptor. Here, we report that FHF1 is abundantly present in Leydig cells with up‐regulation during its development. Adult male Sprague Dawley rats were intraperitoneally injected with 75 mg/kg ethane dimethane sulphonate (EDS) to ablate Leydig cells to initiate their regeneration. Then, rats daily received intratesticular injection of FHF1 (0, 10 and 100 ng/testis) from post‐EDS day 14 for 14 days. FHF1 increased serum testosterone levels without affecting the levels of luteinizing hormone and follicle‐stimulating hormone. FHF1 increased the cell number staining with HSD11B1, a biomarker for Leydig cells at the advanced stage, without affecting the cell number staining with CYP11A1, a biomarker for all Leydig cells. FHF1 did not affect PCNA‐labelling index in Leydig cells. FHF1 increased Leydig cell mRNA (Lhcgr, Scarb1, Star, Cyp11a1, Hsd3b1, Cyp17a1, Hsd17b3, Insl3, Nr5a1 and Hsd11b1) and their protein levels in vivo. FHF1 increased preadipocyte biomarker Dlk1 mRNA level and decreased fully differentiated adipocyte biomarker (Fabp4 and Lpl) mRNA and their protein levels. In conclusion, FHF1 promotes Leydig cell regeneration from stem cells while inhibiting the differentiation of preadipocyte/stem cells into adipocytes in EDS‐treated testis.     

Brain-derived neurotrophic factor signal enhances and maintains the expression of AMPA receptor-associated PDZ proteins in developing cortical neurons

Postsynaptic molecules with PDZ domains (PDZ proteins) interact with various glutamate receptors and regulate their subcellular trafficking and stability. In rat neocortical development, the protein expression of AMPA-type glutamate receptor GluR1 lagged behind its mRNA expression and rather paralleled an increase in PDZ protein levels. One of the neurotrophins, brain-derived neurotrophic factor (BDNF), appeared to contribute to this process, regulating the PDZ protein expression. In neocortical cultures, BDNF treatment upregulated SAP97, GRIP1, and Pick1 PDZ proteins. Conversely, BDNF gene targeting downregulated these same PDZ molecules. The BDNF-triggered increases in PDZ proteins resulted in the elevation of their total association with the AMPA receptors GluR1 and GluR2/3, which led to the increase in AMPA receptor proteins. When Sindbis viruses carrying GluR1 or GluR2 C-terminal decoys disrupted their interactions, GluR2 C-terminal decoys inhibited both BDNF-triggered GluR1 and GluR2/3 increases, whereas GluR1 C-terminal decoys blocked only the BDNF-triggered GluR1 increase. In agreement, coexpression of SAP97 and GluR1 in nonneuronal HEK293 cells increased both proteins compared with their single transfection, implying mutual stabilization. This work reveals a novel function of BDNF in postsynaptic development by regulating the PDZ protein expression.     

Brain-derived neurotrophic factor promotes gephyrin protein expression and GABAA receptor clustering in immature cultured hippocampal cells

Fast synaptic inhibition in the adult brain is largely mediated by GABA_A receptors (GABA_AR). GABA_AR are anchored to synaptic sites by gephyrin, a scaffolding protein that appears to be assembled as a hexagonal lattice beneath the plasma membrane. Brain derived neurotrophic factor (BDNF) alters the clustering and synaptic distribution of GABA_AR but mechanisms behind this regulation are just starting to emerge. The current study was aimed to examine if BDNF alters the protein levels and/or clustering of gephyrin and to investigate whether the modulation of gephyrin is accompanied by changes in the distribution and/or clustering of GABA_AR. Exogenous application of BDNF to immature neuronal cultures from rat hippocampus increased the protein levels and clustering of gephyrin. BDNF also augmented the association of gephyrin with GABA_AR and promoted the formation of GABA_AR clusters. Together, these observations indicate that BDNF might regulate the assembly of GABAergic synapses by promoting the association of GABA_AR with gephyrin.     

Postnatal differentiation of Leydig cells in the African green monkey

At two years of age the interstitial tissue of Cercopithecus aethiops is composed principally of undifferentiated, fibroblast-like cells. Also present during this time are scattered differentiating Leydig cells, which are characterized by a large nucleus, numerous mitochondria, elements of smooth reticulum, and small cisternae of rough reticulum. A mean level of 1.69 ± 0.66 ng/ml of testosterone was found. At three years Leydig cells are much more numerous and developed; since all the elements of steroid secreting cells are present, even their morphology differs from that observed in mature cells. Lipid accumulation is characteristic during this period. A mean testosterone level of 2.28 ± 0.47 ng/ml was found. Mature Leydig cells are basically similar to that of other mammals, while they differ significantly from that of human Leydig cells.     

Brain-derived neurotrophic factor facilitates maturation of mesenchymal stem cell-derived dopamine progenitors to functional neurons

The generation of dopamine (DA) neurons from stem cells holds great promise in the treatment of Parkinson's disease and other neural disease associated with dysfunction of DA neurons. Mesenchymal stem cells (MSCs) derived from the adult bone marrow show plasticity with regards to generating cells of other germ layers. In addition to reduced ethical concerns, MSCs could be transplanted across allogeneic barriers, making them desirable stem cells for clinical applications. We have reported on the generation of DA cells from human MSCs using sonic hedgehog (SHH), fibroblast growth factor 8 and basic fibroblast growth factor. Despite the secretion of DA, the cells did not show evidence of functional neurons, and were therefore designated DA progenitors. Here, we report on the role of brain-derived neurotrophic factor (BDNF) in the maturation of the MSC-derived DA progenitors. 9-day induced MSCs show significant tropomyosin-receptor-kinase B expression, which correlate with its ligand, BDNF, being able to induce functional maturation. The latter was based on Ca²⁺ imaging analyses and electrophysiology. BDNF-treated cells showed the following: increases in intracellular Ca²⁺ upon depolarization and after stimulation with the neurotransmitters acetylcholine and GABA and, post-synaptic currents by electrophysiological analyses. In addition, BDNF induced increased DA release upon depolarization. Taken together, these results demonstrate the crucial role for BDNF in the functional maturation of MSC-derived DA progenitors.