Humans are inevitably exposed to ubiquitous phthalate esters (PAEs). In utero exposure to di-n-butyl phthalate (DBP) induces abnormal development of the testis and reproductive tract in male offspring, which correspond closely with the human condition of testicular dysgenesis syndrome (TDS)-like syndrome. However, the underlying mechanisms have not been elucidated in detail. In this study, pregnant rats were orally exposed to either corn oil (controls) or DBP at three different doses by gavage during Gestational Days 12.5–21.5. Pathological examinations were performed for toxicity evaluation. Proliferation and apoptosis related proteins (ras related dexamethasone induced 1 (Rasd1), mitogen-activated protein kinase kinases1/2 (MEK1/2), Bcl-2, and Bax) were measured for mechanisms exploration. The results showed that different doses of DBP caused male developmental and reproductive toxicity in rats, including the decrease of anogenital distance (AGD), the histological damage of testis, and apoptosis of seminiferous tubule cells. Our data suggested that DBP played chronic and continuous toxic roles on male reproductive system by disrupting expression of Rasd1 and MEK1/2 as well as Bcl-2/Bax ratio. Further research is warranted.

Xp11.2 translocation renal cell carcinoma (tRCC) is mainly caused by translocation of the TFE3 gene located on chromosome Xp11.2 and is characterized by overexpression of the TFE3 fusion gene. Patients are diagnosed with tRCC usually before 45 years of age with poor prognosis. We investigated this disease using two tRCC cell lines, UOK109 and UOK120, in this study. The purpose of this study was to investigate the pathogenic mechanism of TFE3 fusions in tRCC based on its subcellular localization, nuclear translocation and transcriptional activity. The expression of TFE3 fusions and other related genes were analyzed by quantitative reverse transcription PCR (qRT-PCR) and Western blot. The subcellular localization of TFE3 was determined using immunofluorescence. The transcriptional activity of TFE3 fusions was measured using a luciferase reporter assay and ChIP analysis. In some experiments, TFE3 fusions were depleted by RNAi or gene knockdown. The TFE3 fusion segments were cloned into a plasmid expression system for expression in cells. Our results demonstrated that TFE3 fusions were overexpressed in tRCC with a strong nuclear retention irrespective of treatment with an mTORC1 inhibitor or not. TFE3 fusions lost its co-localization with lysosomal proteins and decreased its interaction with the chaperone 14–3-3 proteins in UOK109 and UOK120 cells. However, the fusion segments of TFE3 could not translocate to the nucleus and inhibition of Gsk3β could increase the cytoplasmic retention of TFE3 fusions. Both the luciferase reporter assay and ChIP analysis demonstrated that TFE3 fusions could bind to the promoters of the target genes as a wild-type TFE3 protein. Knockdown of TFE3 results in decreased expression of those genes responsible for lysosomal biogenesis and other target genes. The ChIP-seq data further verified that, in addition to lysosomal genes, TFE3 fusions could regulate genes involved in cellular responses to hypoxic stress and transcription. Our results indicated that the overexpressed TFE3 fusions were capable of escaping from the control by the mTOR signaling pathway and were accumulated in the nucleus in UOK109 and UOK120 cells. The nuclear retention of TFE3 fusions promoted the expression of lysosomal genes and other target genes, facilitating cancer cell resistance against an extreme environment.

N⁶-methyladenosine (m⁶A) plays a crucial role in the development and functional homeostasis of the central nervous system. The fat mass and obesity-associated (FTO) gene, which is highly expressed in the hypothalamus, is closely related to female pubertal development. In this study, we found that m⁶A methylation decreased in the hypothalamus gradually with puberty and decreased in female rats with precocious puberty. FTO expression was increased at the same time. Methylated RNA immunoprecipitation sequencing (MeRIP-seq) showed that the m⁶A methylation of PLCβ₃, a key enzyme of the Ca²⁺ signalling pathway, was decreased significantly in the hypothalamus in precocious rats. Upregulating FTO increased PLCβ3 expression and activated the Ca²⁺ signalling pathway, which promoted GnRH expression. Dual-luciferase reporter and MeRIP-qPCR assays confirmed that FTO regulated m⁶A demethylation of PLCβ₃ and promoted PLCβ₃ expression. Upon overexpressing FTO in the hypothalamic arcuate nucleus (ARC) in female rats, we observed advanced puberty onset. Meanwhile, PLCβ₃ and GnRH expression in the hypothalamus increased significantly, and the Ca²⁺ signalling pathway was activated. Our study demonstrates that FTO enhances GnRH expression, which promotes puberty onset, by regulating m⁶A demethylation of PLCβ3 and activating the Ca²⁺ signalling pathway.

Precocious puberty (PP) is a common pediatric endocrine disorder that affects children's growth and sexual development. Environmental pollutants, especially microplastics (MPs), may be an important triggering factors in PP. MPs were widely found in children's living environment, while the impact of MPs on pubertal development remains unknown. In this study, 14 serum samples were collected from central precocious puberty (CPP) girls and healthy controls. The concentration of total MPs in CPP girls (68.19 ± 26.49 mg/kg) showed significant increase than healthy controls (36.94 ± 13.22 mg/kg). 5 of 11 types of MPs were detected, among which PE (CPP, 28.22 %; Control, 31.62 %) and PVC (CPP, 62.67 %; Control, 58.14 %) being the dominant ones. Correlation analysis between MPs and sex hormone revealed that total MPs were negatively correlated with progesterone and positively correlated with dehydroepiandrosterone (DHEAS). In order to investigate the toxic effects of MPs on pubertal onset, young female mice at 21 days were orally administered various concentrations of MPs for two weeks. Female mice exposed to MPs (PE, 3 mg/day; PVC, 6 mg/day) had an advanced vaginal opening, while none significant changes in body weight. Immunohistochemical staining demonstrated that MPs exposure during puberty could activate microglia, astrocytes and promote GnRH expression in the hypothalamus, which leading to an earlier pubertal onset. These data suggested that MPs can be absorbed and accumulated in brain and gonad after gavage, and accelerate female pubertal initiation. This study provided valuable evidence that MPs threatened the normal progression of female pubertal development.

Background: NONO-TFE3 translocation renal cell carcinoma (tRCC), one of the RCCs that are associated with Xp11.2 translocation/TFE3 gene fusion (Xp11.2 tRCCs), involves an X chromosome inversion between NONO and TFE3 with the characteristics of endonuclear aggregation of NONO-TFE3 fusion protein. The oncogenic mechanisms of NONO-TFE3 fusion have not yet been fully elucidated. Objective: This study aimed at investigating the mechanism of NONO-TFE3 fusion regulating HIF1A as well as the role of HIF-1α in the progression of NONO-TFE3 tRCC under hypoxia. Methods: Immunohistochemistry and Western Blotting assays were performed to profile HIF-1α expression in renal clear cell carcinoma (ccRCC) or in Xp11.2 tRCC. Chromatin immunoprecipitation (ChIP), a luciferase reporter assay, and real-time quantitative PCR (RT-qPCR) were used to evaluate the regulation of HIF1A expression by NONO-TFE3 fusion. Then, the flow cytometry analysis, tube formation assays, and cell migration assays were used as well as glucose or lactic acid levels were measured to establish the impact of HIF-1α on the progression of NONO-TFE3 tRCC. Besides, the effect of HIF-1α inhibitor (PX-478) on UOK109 cells was analyzed. Results: We found that HIF1A was the target gene of NONO-TFE3 fusion. In UOK109 cells, which were isolated from NONO-TFE3 tRCC samples, NONO-TFE3 fusion promoted aerobic glycolysis and angiogenesis by up-regulating the expression of HIF-1α under hypoxia. Furthermore, the inhibition of HIF-1α mediated by PX-478 suppressed the development of NONO-TFE3 tRCC under hypoxia. Conclusion: HIF-1α is a potential target for therapy of NONO-TFE3 tRCC under hypoxia.

Background: Puberty is among the most important stages in human development. Timely activation of hypothalamic gonadotropin-releasing hormone (GnRH) neurons underlies the initiation of pubertal development. The fat mass and obesity-associated (FTO), which is expressed in the hypothalamus, may regulate the m6A methylation of its target genes, influencing GnRH expression in the hypothalamus during puberty onset. This study aimed to explore the mechanism by which FTO regulates the function of its target neurotrophins within the hypothalamus and to clarify the molecular pathway underlying precocious puberty. Methods: Methylated RNA immunoprecipitation sequencing (MeRIP-seq) was used to assess m6A methylation in the hypothalamus of female rats. Serum brain-derived neurotrophic factor (BDNF) levels were measured in girls with central precocious puberty (CPP) and matched controls. BDNF was applied to GnRH neurons in vitro . The functions of FTO were assessed through overexpression and knockdown in animal models, with downstream signaling evaluated via BDNF/PI3K/Akt pathway analysis. Intracerebroventricular (ICV) injections of a BDNF-overexpressing lentiviral vector or a negative control (NC) were used to investigate the role of central BDNF in regulating puberty onset and reproductive function. Results: Hypothalamic GnRH and FTO expression progressively increased whereas m6A methylation decreased during puberty. MeRIP-seq revealed significantly reduced m6A methylation of Bdnf mRNA in the arcuate nucleus (ARC) of female rats during early puberty. In the ARC, BDNF was expressed adjacent to GnRH-positive fibers and terminals. In addition, serum BDNF levels were higher in girls with CPP girls than in the control group. In vitro , BDNF treatment stimulated GnRH expression in GT1–7 cells. FTO positively regulated BDNF expression in an m6A methylation-dependent manner. FTO overexpression activated BDNF/PI3K/Akt signaling in the ARC and accelerated puberty onset. Conversely, FTO knockdown delayed vaginal opening and suppressed BDNF/PI3K/Akt signaling. ICV delivery of a BDNF-overexpressing lentivirus increased hypothalamic BDNF and GnRH expression, leading to a distinct endocrine profile. Conclusion: These findings suggest that FTO regulates the m6A demethylation of BDNF and promotes GnRH expression through the BDNF/PI3K/Akt signaling pathway. This study improves our understanding of the function of BDNF, which is critical for the development of therapeutic strategies for preventing and treating of precocious puberty.