IκBα controls dormancy in hematopoietic stem cells via retinoic acid during embryonic development.

Recent findings suggest that Hematopoietic Stem Cells (HSC) and progenitors arise simultaneously and independently of each other already in the embryonic aorta-gonad mesonephros region, but it is still unknown how their different features are established. Here, we uncover IκBα (Nfkbia, the inhibitor of NF-κB) as a critical regulator of HSC proliferation throughout development. IκBα balances retinoic acid signaling levels together with the epigenetic silencer, PRC2, specifically in HSCs. Loss of IκBα decreases proliferation of HSC and induces a dormancy related gene expression signature instead. Also, IκBα deficient HSCs respond with superior activation to in vitro culture and in serial transplantation. At the molecular level, chromatin regions harboring binding motifs for retinoic acid signaling are hypo-methylated for the PRC2 dependent H3K27me3 mark in IκBα deficient HSCs. Overall, we show that the proliferation index in the developing HSCs is regulated by a IκBα-PRC2 axis, which controls retinoic acid signaling.

All-trans retinoic acid downregulates HBx levels via E6-associated protein-mediated proteasomal degradation to suppress hepatitis B virus replication.

All-trans retinoic acid (ATRA), recognized as the principal and most biologically potent metabolite of vitamin A, has been identified for its inhibitory effects on hepatitis B virus (HBV) replication. Nevertheless, the underlying mechanism remains elusive. The present study reveals that ATRA induces E6-associated protein (E6AP)-mediated proteasomal degradation of HBx to suppress HBV replication in human hepatoma cells in a p53-dependent pathway. For this effect, ATRA induced promoter hypomethylation of E6AP in the presence of HBx, which resulted in the upregulation of E6AP levels in HepG2 but not in Hep3B cells, emphasizing the p53-dependent nature of this effect. As a consequence, ATRA augmented the interaction between E6AP and HBx, resulting in substantial ubiquitination of HBx and consequent reduction in HBx protein levels in both the HBx overexpression system and the in vitro HBV replication model. Additionally, the knockdown of E6AP under ATRA treatment reduced the interaction between HBx and E6AP and decreased the ubiquitin-dependent proteasomal degradation of HBx, which prompted a recovery of HBV replication in the presence of ATRA, as confirmed by increased levels of intracellular HBV proteins and secreted HBV levels. This study not only contributes to the understanding of the complex interactions between ATRA, p53, E6AP, and HBx but also provides an academic basis for the clinical employment of ATRA in the treatment of HBV infection.

ADH1B, the adipocyte-enriched alcohol dehydrogenase, plays an essential, cell-autonomous role in human adipogenesis.

Alcohol dehydrogenase 1B (ADH1B) is a primate-specific enzyme which, uniquely among the ADH class 1 family, is highly expressed both in adipose tissue and liver. Its expression in adipose tissue is reduced in obesity and increased by insulin stimulation. Interference with ADH1B expression has also been reported to impair adipocyte function. To better understand the role of ADH1B in adipocytes, we used CRISPR/Cas9 to delete ADH1B in human adipose stem cells (ASC). Cells lacking ADH1B failed to differentiate into mature adipocytes manifested by minimal triglyceride accumulation and a marked reduction in expression of established adipocyte markers. As ADH1B is capable of converting retinol to retinoic acid (RA), we conducted rescue experiments. Incubation of ADH1B-deficient preadipocytes with 9-cis-RA, but not with all-transretinol, significantly rescued their ability to accumulate lipids and express markers of adipocyte differentiation. A homozygous missense variant in ADH1B (p.Arg313Cys) was found in a patient with congenital lipodystrophy of unknown cause. This variant significantly impaired the protein's dimerization, enzymatic activity, and its ability to rescue differentiation in ADH1B-deficient ASC. The allele frequency of this variant in the Middle Eastern population suggests that it is unlikely to be a fully penetrant cause of severe lipodystrophy. In conclusion, ADH1B appears to play an unexpected, crucial and cell-autonomous role in human adipocyte differentiation by serving as a necessary source of endogenous retinoic acid.

Development and validation of CYP26A1 inhibition assay for high-throughput screening.

All-trans retinoic acid (atRA) is an endogenous ligand of the retinoic acid receptors, which heterodimerize with retinoid X receptors. AtRA is generated in tissues from vitamin A (retinol) metabolism to form a paracrine signal and is locally degraded by cytochrome P450 family 26 (CYP26) enzymes. The CYP26 family consists of three subtypes: A1, B1, and C1, which are differentially expressed during development. This study aims to develop and validate a high throughput screening assay to identify CYP26A1 inhibitors in a cell-free system using a luminescent P450-Glo assay technology. The assay performed well with a signal to background ratio of 25.7, a coefficient of variation of 8.9%, and a Z-factor of 0.7. To validate the assay, we tested a subset of 39 compounds that included known CYP26 inhibitors and retinoids, as well as positive and negative control compounds selected from the literature and/or the ToxCast/Tox21 portfolio. Known CYP26A1 inhibitors were confirmed, and predicted CYP26A1 inhibitors, such as chlorothalonil, prochloraz, and SSR126768, were identified, demonstrating the reliability and robustness of the assay. Given the general importance of atRA as a morphogenetic signal and the localized expression of Cyp26a1 in embryonic tissues, a validated CYP26A1 assay has important implications for evaluating the potential developmental toxicity of chemicals.

Molecular Pathways and Animal Models of d-Transposition of the Great Arteries.

During normal cardiovascular development, the outflow tract becomes septated and rotates so that the separate aorta and pulmonary trunk are correctly aligned with the left and right ventricles, respectively. However, when this process goes wrong, the aorta and pulmonary trunk are incorrectly positioned, resulting in oxygenated blood being directly returned to the lungs, with deoxygenated blood being delivered to the systemic circulation. This is termed transposition of the great arteries (TGA). The precise etiology of TGA is not known, but the use of animal models has elucidated that genes involved in determination of the left- embryonic body axis play key roles. Other factors such as retinoic acid levels are also crucial. This chapter reviews the animal models presenting with TGA that have been generated by genetic manipulation or with exogenous agents.

Airway hillocks are injury-resistant reservoirs of unique plastic stem cells.

Airway hillocks are stratified epithelial structures of unknown function1. Hillocks persist for months and have a unique population of basal stem cells that express genes associated with barrier function and cell adhesion. Hillock basal stem cells continually replenish overlying squamous barrier cells. They exhibit dramatically higher turnover than the abundant, largely quiescent classic pseudostratified airway epithelium. Hillocks resist a remarkably broad spectrum of injuries, including toxins, infection, acid and physical injury because hillock squamous cells shield underlying hillock basal stem cells from injury. Hillock basal stem cells are capable of massive clonal expansion that is sufficient to resurface denuded airway, and eventually regenerate normal airway epithelium with each of its six component cell types. Hillock basal stem cells preferentially stratify and keratinize in the setting of retinoic acid signalling inhibition, a known cause of squamous metaplasia2,3. Here we show that mouse hillock expansion is the cause of vitamin A deficiency-induced squamous metaplasia. Finally, we identify human hillocks whose basal stem cells generate functional squamous barrier structures in culture. The existence of hillocks reframes our understanding of airway epithelial regeneration. Furthermore, we show that hillocks are one origin of 'squamous metaplasia', which is long thought to be a precursor of lung cancer.

Oct4 redox sensitivity potentiates reprogramming and differentiation.

The transcription factor Oct4/Pou5f1 is a component of the regulatory circuitry governing pluripotency and is widely used to induce pluripotency from somatic cells. Here we used domain swapping and mutagenesis to study Oct4's reprogramming ability, identifying a redox-sensitive DNA binding domain, cysteine residue (Cys48), as a key determinant of reprogramming and differentiation. Oct4 Cys48 sensitizes the protein to oxidative inhibition of DNA binding activity and promotes oxidation-mediated protein ubiquitylation. Pou5f1 C48S point mutation has little effect on undifferentiated embryonic stem cells (ESCs) but upon retinoic acid (RA) treatment causes retention of Oct4 expression, deregulated gene expression, and aberrant differentiation. Pou5f1 C48S ESCs also form less differentiated teratomas and contribute poorly to adult somatic tissues. Finally, we describe Pou5f1 C48S (Janky) mice, which in the homozygous condition are severely developmentally restricted after E4.5. Rare animals bypassing this restriction appear normal at birth but are sterile. Collectively, these findings uncover a novel Oct4 redox mechanism involved in both entry into and exit from pluripotency.

Retinoic acid tiers mitochondrial metabolism to Sertoli Cell-Mediated efferocytosis via a non-RAR-dependent mechanism.

Efferocytosis of massive non-viable germ cells by Sertoli cells (SCs), the specialized phagocytes, is essential for maintaining testis homeostasis. What elusive is the contribution of mitochondrial metabolism to this energy-consuming process, as SC has a preference of aerobic glycolysis. All-trans retinoic acid (ATRA, hereafter referred to as RA) is a well-known morphogen that primarily acts through the nuclear RA receptor (RAR). It sustains SC blood-testisbarrier integrity, and it's SC-derived RA sets the timing of meiotic commitment. In this study, we revisited RA in SC biology, from the perspective of SC-mediated efferocytosis. We provide evidence that RA induces transcriptional programming of multiple regulators involved in efferocytosis, which thereby represses SC-mediated efferocytosis, via a RAR-independent mechanism, as blocking pan-RAR activity fails to rescue RA-induced defective efferocytosis. RA-treated SCs exhibit alternations in mitochondrial dynamics and metabolism, and the hindered efferocytosis can be rescued by stimulating mitochondrial OXPHOS via pharmacological targeting of AMPK and PDK. We thus prefer to propose a signaling axis of RA-mitochondrial metabolism-efferocytosis. Our study uncovers a hitherto unappreciated role of RA in SC biology and tiers mitochondria metabolism to SC-mediated efferocytosis, contributing a deeper understanding of SC in male reproduction.

Spectral and mass characterization of kinetic conversion from retinoids to retinoic acid in an in vitro 3-D human skin equivalent model.

To investigate the effect of retinoids, such as retinol (ROL), retinal (RAL), and retinyl palmitate (RP), on epidermal integrity, skin deposition, and bioconversion to retinoic acid (RA). 3-D human skin equivalent model (EpiDermFT™) was used. Epidermal cellular integrity measured by TEER values was significantly higher for a topical treatment of ROL and RAL than RP (p < 0.05). The skin deposition (µM) of ROL and RAL was approximately 269.54 ± 73.94 and 211.35 ± 20.96, respectively, greater than that of RP (63.70 ± 37.97) over 2 h incubation. Spectral changes were revealed that the CO maximum absorbance occurred between 1600∼1800 cm-1 and was greater from ROL than that from RAL and RP, indicating conjugation of R-OH to R-CHO or R-COOH could strongly occur after ROL treatment. Subsequently, a metabolite from the bioconversion of ROL and RAL was identified as RA, which has a product ion of m/z 283.06, by using liquid a chromatography-mass spectrometry (LC-MS) - total ion chromatogram (TIC). The amount of bioconversion from ROL and RAL to RA in artificial skin was 0.68 ± 0.13 and 0.70 ± 0.10 µM at 2 h and 0.60 ± 0.04 and 0.57 ± 0.06 µM at 24 h, respectively. RA was not detected in the skin and the receiver compartment after RP treatment. ROL could be a useful dermatological ingredient to maintain epidermal integrity more effectively, more stably deposit on the skin, and more steadily metabolize to RA than other retinoids such as RAL and RP.

Risk and Resilience Variants in the Retinoic Acid Metabolic and Developmental Pathways Associated with Risk of FASD Outcomes.

Fetal Alcohol Spectrum Disorder (FASD) is a common neurodevelopmental disorder that affects an estimated 2-5% of North Americans. FASD is induced by prenatal alcohol exposure (PAE) during pregnancy and while there is a clear genetic contribution, few genetic factors are currently identified or understood. In this study, using a candidate gene approach, we performed a genetic variant analysis of retinoic acid (RA) metabolic and developmental signaling pathway genes on whole exome sequencing data of 23 FASD-diagnosed individuals. We found risk and resilience alleles in ADH and ALDH genes known to normally be involved in alcohol detoxification at the expense of RA production, causing RA deficiency, following PAE. Risk and resilience variants were also identified in RA-regulated developmental pathway genes, especially in SHH and WNT pathways. Notably, we also identified significant variants in the causative genes of rare neurodevelopmental disorders sharing comorbidities with FASD, including STRA6 (Matthew-Wood), SOX9 (Campomelic Dysplasia), FDG1 (Aarskog), and 22q11.2 deletion syndrome (TBX1). Although this is a small exploratory study, the findings support PAE-induced RA deficiency as a major etiology underlying FASD and suggest risk and resilience variants may be suitable biomarkers to determine the risk of FASD outcomes following PAE.

Retinoic Acid-Mediated Control of Energy Metabolism Is Essential for Lung Branching Morphogenesis.

Lung branching morphogenesis relies on intricate epithelial-mesenchymal interactions and signaling networks. Still, the interplay between signaling and energy metabolism in shaping embryonic lung development remains unexplored. Retinoic acid (RA) signaling influences lung proximal-distal patterning and branching morphogenesis, but its role as a metabolic modulator is unknown. Hence, this study investigates how RA signaling affects the metabolic profile of lung branching. We performed ex vivo lung explant culture of embryonic chicken lungs treated with DMSO, 1 µM RA, or 10 µM BMS493. Extracellular metabolite consumption/production was evaluated by using 1H-NMR spectroscopy. Mitochondrial respiration and biogenesis were also analyzed. Proliferation was assessed using an EdU-based assay. The expression of crucial metabolic/signaling components was examined through Western blot, qPCR, and in situ hybridization. RA signaling stimulation redirects glucose towards pyruvate and succinate production rather than to alanine or lactate. Inhibition of RA signaling reduces lung branching, resulting in a cystic-like phenotype while promoting mitochondrial function. Here, RA signaling emerges as a regulator of tissue proliferation and lactate dehydrogenase expression. Furthermore, RA governs fatty acid metabolism through an AMPK-dependent mechanism. These findings underscore RA's pivotal role in shaping lung metabolism during branching morphogenesis, contributing to our understanding of lung development and cystic-related lung disorders.

Defining a TFAP2C-centered transcription factor network during murine peri-implantation.

Transcription factors (TFs) play important roles in early embryonic development, but factors regulating TF action, relationships in signaling cascade, genome-wide localizations, and impacts on cell fate transitions during this process have not been clearly elucidated. In this study, we used uliCUT&RUN-seq to delineate a TFAP2C-centered regulatory network, showing that it involves promoter-enhancer interactions and regulates TEAD4 and KLF5 function to mediate cell polarization. Notably, we found that maternal retinoic acid metabolism regulates TFAP2C expression and function by inducing the active demethylation of SINEs, indicating that the RARG-TFAP2C-TEAD4/KLF5 axis connects the maternal-to-zygotic transition to polarization. Moreover, we found that both genomic imprinting and SNP-transferred genetic information can influence TF positioning to regulate parental gene expressions in a sophisticated manner. In summary, we propose a ternary model of TF regulation in murine embryonic development with TFAP2C as the core element and metabolic, epigenetic, and genetic information as nodes connecting the pathways.

The retinoid X receptor has a critical role in synthetic rexinoid-induced increase in cellular all-trans-retinoic acid.

Rexinoids are agonists of nuclear rexinoid X receptors (RXR) that heterodimerize with other nuclear receptors to regulate gene transcription. A number of selective RXR agonists have been developed for clinical use but their application has been hampered by the unwanted side effects associated with the use of rexinoids and a limited understanding of their mechanisms of action across different cell types. Our previous studies showed that treatment of organotypic human epidermis with the low toxicity UAB30 and UAB110 rexinoids resulted in increased steady-state levels of all-trans-retinoic acid (ATRA), the obligatory ligand of the RXR-RAR heterodimers. Here, we investigated the molecular mechanism underlying the increase in ATRA levels using a dominant negative RXRα that lacks the activation function 2 (AF-2) domain. The results demonstrated that overexpression of dnRXRα in human organotypic epidermis markedly reduced signaling by resident ATRA, suggesting the existence of endogenous RXR ligand, diminished the biological effects of UAB30 and UAB110 on epidermis morphology and gene expression, and nearly abolished the rexinoid-induced increase in ATRA levels. Global transcriptome analysis of dnRXRα-rafts in comparison to empty vector-transduced rafts showed that over 95% of the differentially expressed genes in rexinoid-treated rafts constitute direct or indirect ATRA-regulated genes. Thus, the biological effects of UAB30 and UAB110 are mediated through the AF-2 domain of RXRα with minimal side effects in human epidermis. As ATRA levels are known to be reduced in certain epithelial pathologies, treatment with UAB30 and UAB110 may represent a promising therapy for normalizing the endogenous ATRA concentration and signaling in epithelial tissues.

Fucosylation inhibitor 6-alkynylfucose enhances the ATRA-induced differentiation effect on acute promyelocytic leukemia cells.

For acute promyelocytic leukemia (APL), differentiation therapy with all-trans retinoic acid (ATRA) is well established. However, the narrow application and tolerance development of ATRA remain to be improved. In this study, we investigated the effects of combinations of glycosylation inhibitors with ATRA to achieve better efficiency than ATRA alone. We found that the combination of fucosylation inhibitor 6-alkynylfucose (6AF) and ATRA had an additional effect on cell differentiation, as revealed by expression changes in two differentiation markers, CD11b and CD11c, and significant morphological changes in NB4 APL and HL-60 acute myeloid leukemia (AML) cells. In AAL lectin blot analyses, ATRA or 6AF alone could decrease fucosylation, while their combination decreased fucosylation more efficiently. To clarify the molecular mechanism for the 6AF effect on ATRA-induced differentiation, we performed microarray analyses using NB4 cells. In a pathway analysis using DAVID software, we found that the C-type lectin receptor (CLR) signaling pathway was enriched with high significance. In real-time PCR analyses using NB4 and HL-60 cells, FcεRIγ, CLEC6A, CLEC7A, CASP1, IL-1ß, and EGR3, as components of the CLR pathway, as well as CD45 and AKT3 were upregulated by 6AF in ATRA-induced differentiation. Taken together, the present findings suggest that the CLR signaling pathway is involved in the 6AF effect on ATRA-induced differentiation.

Lineage specific transcription factor waves reprogram neuroblastoma from self-renewal to differentiation.

Temporal regulation of super-enhancer (SE) driven transcription factors (TFs) underlies normal developmental programs. Neuroblastoma (NB) arises from an inability of sympathoadrenal progenitors to exit a self-renewal program and terminally differentiate. To identify SEs driving TF regulators, we use all-trans retinoic acid (ATRA) to induce NB growth arrest and differentiation. Time-course H3K27ac ChIP-seq and RNA-seq reveal ATRA coordinated SE waves. SEs that decrease with ATRA link to stem cell development (MYCN, GATA3, SOX11). CRISPR-Cas9 and siRNA verify SOX11 dependency, in vitro and in vivo. Silencing the SOX11 SE using dCAS9-KRAB decreases SOX11 mRNA and inhibits cell growth. Other TFs activate in sequential waves at 2, 4 and 8 days of ATRA treatment that regulate neural development (GATA2 and SOX4). Silencing the gained SOX4 SE using dCAS9-KRAB decreases SOX4 expression and attenuates ATRA-induced differentiation genes. Our study identifies oncogenic lineage drivers of NB self-renewal and TFs critical for implementing a differentiation program.

Vitamin A resolves lineage plasticity to orchestrate stem cell lineage choices.

Lineage plasticity-a state of dual fate expression-is required to release stem cells from their niche constraints and redirect them to tissue compartments where they are most needed. In this work, we found that without resolving lineage plasticity, skin stem cells cannot effectively generate each lineage in vitro nor regrow hair and repair wounded epidermis in vivo. A small-molecule screen unearthed retinoic acid as a critical regulator. Combining high-throughput approaches, cell culture, and in vivo mouse genetics, we dissected its roles in tissue regeneration. We found that retinoic acid is made locally in hair follicle stem cell niches, where its levels determine identity and usage. Our findings have therapeutic implications for hair growth as well as chronic wounds and cancers, where lineage plasticity is unresolved.

Differentiating SH-SY5Y Cells into Polarized Human Neurons for Studying Endogenous and Exogenous Tau Trafficking: Four Protocols to Obtain Neurons with Noradrenergic, Dopaminergic, and Cholinergic Properties.

Pathological alterations of the neuronal Tau protein are characteristic for many neurodegenerative diseases, called tauopathies. To investigate the underlying mechanisms of tauopathies, human neuronal cell models are required to study Tau physiology and pathology in vitro. Primary rodent neurons are an often used model for studying Tau, but rodent Tau differs in sequence, splicing, and aggregation propensity, and rodent neuronal physiology cannot be compared to humans. Human-induced pluripotent stem cell (hiPSC)-derived neurons are expensive and time-consuming. Therefore, the human neuroblastoma SH-SY5Y cell line is a commonly used cell model in neuroscience as it combines convenient handling and low costs with the advantages of human-derived cells. Since naïve SH-SY5Y cells show little similarity to human neurons and almost no Tau expression, differentiation is necessary to obtain human-like neurons for studying Tau protein-related aspects of health and disease. As they express in principle all six Tau isoforms seen in the human brain, differentiated SH-SY5Y-derived neurons are suitable for investigating the human microtubule-associated protein Tau and, for example, its sorting and trafficking. Here, we describe and discuss a general cultivation procedure as well as four differentiation methods to obtain SH-SY5Y-derived neurons resembling noradrenergic, dopaminergic, and cholinergic properties, based on the treatment with retinoic acid (RA), brain-derived neurotrophic factor (BDNF), and 12-O-tetrade canoylphorbol-13-acetate (TPA). TPA and RA-/TPA-based protocols achieve differentiation efficiencies of 40-50% after 9 days of treatment. The highest differentiation efficiency (~75%) is accomplished by a combination of RA and BDNF; treatment only with RA is the most time-efficient method as ~50% differentiated cells can be obtained already after 7 days.

High OXPHOS efficiency in RA-FUdr-differentiated SH-SY5Y cells: involvement of cAMP signalling and respiratory supercomplexes.

Neurons are highly dependent on mitochondria to meet their bioenergetic needs and understanding the metabolic changes during the differentiation process is crucial in the neurodegeneration context. Several in vitro approaches have been developed to study neuronal differentiation and bioenergetic changes. The human SH-SY5Y cell line is a widely used cellular model and several differentiation protocols have been developed to induce a neuron-like phenotype including retinoic acid (RA) treatment. In this work we obtained a homogeneous functional population of neuron-like cells by a two-step differentiation protocol in which SH-SY5Y cells were treated with RA plus the mitotic inhibitor 2-deoxy-5-fluorouridine (FUdr). RA-FUdr treatment induced a neuronal phenotype characterized by increased expression of neuronal markers and electrical properties specific to excitable cells. In addition, the RA-FUdr differentiated cells showed an enrichment of long chain and unsaturated fatty acids (FA) in the acyl chain composition of cardiolipin (CL) and the bioenergetic analysis evidences a high coupled and maximal respiration associated with high mitochondrial ATP levels. Our results suggest that the observed high oxidative phosphorylation (OXPHOS) capacity may be related to the activation of the cyclic adenosine monophosphate (cAMP) pathway and the assembly of respiratory supercomplexes (SCs), highlighting the change in mitochondrial phenotype during neuronal differentiation.

Retinoic acid induced specific changes in the phosphoproteome of C17.2 neural stem cells.

Retinoic acid (RA), a vitamin A derivative, is an effective cell differentiating factor which plays critical roles in neuronal differentiation induction and the production of neurotransmitters in neurons. However, the specific changes in phosphorylation levels and downstream signalling pathways associated with RA remain unclear. This study employed qualitative and quantitative phosphoproteomics approaches based on mass spectrometry to investigate the phosphorylation changes induced by RA in C17.2 neural stem cells (NSCs). Dimethyl labelling, in conjunction with TiO2 phosphopeptide enrichment, was utilized to profile the phosphoproteome of self-renewing and RA-induced differentiated cells in C17.2 NSCs. The results of our study revealed that, qualitatively, 230 and 14 phosphoproteins were exclusively identified in the self-renewal and RA-induced groups respectively. Quantitatively, we successfully identified and quantified 177 unique phosphoproteins, among which 70 exhibited differential phosphorylation levels. Analysis of conserved phosphorylation motifs demonstrated enrichment of motifs corresponding to cyclin-dependent kinase and MAPK in the RA-induced group. Additionally, through a comprehensive literature and database survey, we found that the differentially expressed proteins were associated with the Wnt/ß-catenin and Hippo signalling pathways. This work sheds light on the changes in phosphorylation levels induced by RA in C17.2 NSCs, thereby expanding our understanding of the molecular mechanisms underlying RA-induced neuronal differentiation.

The role of Sertoli cells-secreted factors in different stages of germ cells development in mice exposed to BDE-209.

Decabromodiphenyl ether (BDE-209), a frequently used brominated flame retardant, readily enters the environment and is difficult to degrade with bioaccumulation. BDE-209 could cause male reproductive toxicity, but the regulatory functions of Sertoli cells-secreted factors remain uncertain. In present study, male mice were treated with 75 mg/kg BDE-209 and then stopped exposure for 50 days. Exogenous Glial cell line-derived neurotrophic factor (GDNF), a Sertoli cell-secreted factor, was injected into testes of mice treated with BDE-209 for 50 days to explore the role of GDNF in BDE-209-induced reproductive toxicity. The mouse spermatogonia cell line GC-1 spg was used in vitro to further verify regulatory effects of Sertoli cells-secreted factors on meiotic initiation. The results showed that BDE-209 inhibited expressions of the self-renewal pathway GFRα-1/RAS/ERK1/2 in spermatogonial stem cells (SSCs), and reduced expressions of spermatogonia proliferation-related pathway NRG3/ERBB4 and meiosis initiation factor Stra8. Furthermore, BDE-209 decreased the levels of both GDNF and retinoic acid (RA) secreted by Sertoli cells in testes. Importantly, the alterations of above indicators induced by BDE-209 did not recover after 50-day recovery period. After exogenous GDNF injection, the decreased expression of GFRα-1/RAS/ERK in SSCs was reversed. However, the level of RA and expressions of NRG3/ERBB4/Stra8 were not restored. The in vitro experimental results showed that exogenous RA reversed the reductions in NRG3/ERBB4/Stra8 and ameliorated inhibition of GC-1 spg cells proliferation induced by BDE-209. These results suggested that Sertoli cells-secreted factors play roles in regulating various stages of germ cell development. Specifically, BDE-209 affected the self-renewal of SSCs by decreasing GDNF secretion resulting in the inhibition of GFRα-1/RAS/ERK pathway; BDE-209 hindered the proliferation of spermatogonia and initiation of meiosis by inhibiting the secretion of RA and preventing RA from binding to RARα, resulting in the suppression of NRG3/ERBB4/Stra8 pathway. As a consequence, spermatogenesis was compromised, leading to persistent male reproductive toxicity.