To investigate the mechanism by which peripubertal exposure to di-2-ethylhexyl phthalate (DEHP) induces reproductive injury in adult male mice through impairment of Sertoli cell function and the blood-testis barrier.

A total of 180 male C57BL/6J mice (3 weeks old) were randomly divided into corn oil group (control group), and 5, 25, 125, 250 and 500 mg/kg DEHP exposure groups. All groups received daily oral gavage for 5 consecutive weeks. Ten mice from each group were sacrificed for first material collection when they were 9 weeks old. The remaining F0 generation mice were mated with unexposed adult female mice at a male-to-female ratio of 1∶2 at 14 weeks of age for a period of 2 weeks. The second material collection was completed at 16 weeks of age, with 20 mice taken from each group. A computer-assisted analysis system was used to detect sperm density and motility in 9-week-old and 16-week-old male mice. The average birth body weight of F1 generation pups, survival rate of pups at 4 d after birth per litter, number of pups per litter, and male-to-female sex ratio within the litter were recorded. ELISA was used to detect hormone levels in the serum. Sperm abnormalities were observed using eosin staining. HE staining was used to observe pathological changes in testicular tissue. Immunofluorescence assay was utilized to detect the protein expression of tight junction protein 1(zonula occludens-1, ZO-1) and gap junction protein 43 (connexin43, Cx43) related to the blood-testis barrier in testicular tissue, as well as the protein expression of apoptosis-related molecule Cleaved-Caspase3. Immunohistochemistry was used to detect the expression of Sertoli cell marker SOX9 protein in testicular tissue. TUNEL was used to detect apoptosis in testicular tissue. Comparative toxicogenomics database (CTD) was used to screen for mono-2-ethylhexyl phthalate (MEHP) induced reproductive damage-associated genes and combined with GO/KEGG and Reactome enrichment analysis to predict possible signaling pathways. An in vitro exposure model was established by treating TM4 cells with MEHP at concentrations of 0, 50, 100, 200 and 400 μmol/L, respectively. CCK-8 assay, flow cytometry and Western blotting were applied to detect the cell viability, apoptosis, and protein expression of apoptosis-related proteins B-cell lymphoma 2 (Bcl-2) and Bcl-2-associated X protein (Bax), and blood-testis barrier-related proteins ZO-1 and Cx43.

Compared with the control group, the pregnancy rate was decreased from 80. 0% to 52. 5%, and the total number of pups was decreased from 212 to 125 in the female mice mating with the male from the 500 mg/kg group. Compared with the control group, the sperm density and motility were significantly reduced, and the total rate of sperm abnormalities was obviously increased in the 9-week-old DEHP-exposed mice (P<0. 05), and even in the 16-week-old mice, the sperm motility was still in a dose-dependent downward trend (P<0. 05). In the 25, 125, 250 and 500 mg/kg DEHP exposure groups, the thickness of the seminiferous epithelium was obviously decreased, with more cellular vacuolization and notable epithelial atrophy and degeneration. Cell apoptosis in the testicular tissue was enhanced, while the expression of ZO-1 and Cx43 were decreased in the 250 and 500 mg/kg exposure groups. The levels of androgen-binding protein, gonadotropin-releasing hormone, and testosterone in the serum were decreased in a dose-dependent manner (P<0. 05), while those of luteinizing hormone and follicle-stimulating hormone showed a dose-dependent increase (P<0. 05). Bioinformatics analysis showed that 153 genes related to male system diseases were screened out by CTD for MEHP. GO enrichment, KEGG pathway, and Reactome enrichment analyses displayed that the apoptosis signaling pathway was significantly enriched. CCK-8 assay showed that TM4 cell viability was decreased in a dose-dependent manner after MEHP treatment when compared with the control group (P<0. 05). Western blotting indicated that MEHP treatment reduced the expression levels of Bcl-2, ZO-1 and Cx43 (P<0. 05), and enhanced that of Bax in comparison with the control group (P<0. 05).

Peripubertal DEHP exposure may lead to reduced sperm quality and reproductive damage in adult male mice by disrupting the blood-testis barrier and activating Sertoli cell apoptosis.

To explore the role and mechanism of RNA demethylase fat mass and obesity-associated protein (FTO) in the ferroptosis in testicular interstitial cells induced by di (2-ethylhexyl) phthalate (DEHP).

Forty 3-week-old C57BL/6 male mice were randomly divided into a control group (corn oil) and 3 dosed DEHP treatment groups (5, 250 and 500 mg/kg), and received an intragastric infusion of corresponding agents for 35 d, respectively. After mouse testicular interstitial TM3 cells was treated with 0, 100, 200 and 400 μmol/L mono-2-ethylhexyl phthalate (MEHP) for 24 h, corresponding plasmids were transfected to construct Fto overexpressing TM3 cells. Serum testosterone level was detected by ELISA, expression of testicular proteins was detected with immunohistochemical assay, and contents of Fe²⁺, malondialdehyde (MDA) and lipid peroxides in the testicle were detected by colorimetry. Methylated RNA immunoprecipitation, RT-PCR, and Western blotting were used to detect the level of N6-methyladenosine (m6A) modification.

In the mice exposed to 250 and 500 mg/kg DEHP, the serum testosterone level was significantly reduced (P<0.01), contents of Fe²⁺, MAD and lipid peroxides in testicular tissue were obviously increased (P<0.01), and protein levels of RNA demethylase FTO, and ferroptosis related molecules ferritin heavy chain 1 (FTH1) and glutathione peroxidase 4 (GPX4) were significantly down-regulated (P<0.05), while those of transferrin receptor (TFRC), ferroportin (FPN), cyclooxygenase-2 (COX-2), and acyl-CoA synthetase long-chain family member 4 (ACSL4) were notably up-regulated (P<0.05). MEHP treatment for 24 h resulted in remarkably decreased cell viability in the TM3 cells, increased production of intracellular reactive oxygen species (ROS), reduced mitochondrial membrane potential (MMP) (P<0.01), down-regulated mRNA and protein levels of Fto (P<0.01), and the changes in other ferroptosis related proteins were consistent with the trend in testicular tissue, indicating ferroptosis in testicular interstitial cells. Intervention with ferroptosis inhibitor Fer-1 or overexpression of Fto significantly inhibited MEHP-induced toxicity and ferroptosis in TM3 cells (P<0.05), and overexpression of Fto reduced the m6A modification of Gpx4 and Fth1 mRNA (P<0.05).

Abnormal m6A modification of Gpx4 and Fth1 caused by inhibiting FTO expression may be the mechanism of ferroptosis in testicular interstitial cells induced by DEHP.

To explore the effect of physical exercise on semen quality in order to provide basic data and theoretical basis for the improvement of male reproductive health.

A cross-sectional study was conducted on 1 059 males who visited the Reproductive Medicine Center of Shandong Maternal and Child Health Hospital for medical treatment and physical examination during July 2022 and April 2023. Their demographic data and physical exercise data were surveyed with questionnaires. Total sperm count, sperm concentration, total sperm motility, forward movement and normal sperm morphology were analyzed with computer aided analysis. Logistic regression model and multiple linear regression model were applied to analyze the effects of physical exercise on semen quality.

After adjustment for confounding factors such as age, body mass index, alcohol consumption and smoking, logistic regression analysis showed that the risk of abnormal semen quality was increased in patients with moderate and heavy exercise intensity (OR=2.103, OR=2.229). Compared with the participants with physical exercise ≤10 min per session, those with >20 min per session had a lower risk of abnormal semen quality (OR=0.357, 0.256, 0.289 for exercise time for >20~30, >30~60, >60 min, respectively). There was no statistical significance between physical exercise frequency and semen quality (P > 0.05). The participants having exercise well were at a lower risk for abnormal semen quality (OR=0.711). Multiple linear regression analysis revealed that the frequency of physical exercise was an influencing factor of sperm concentration (β=7.474, 95%CI: 4.800~10.149, P < 0.05); the time of physical exercise per session was an influencing factor for total sperm count (β=20.632, 95%CI: 7.634~33.629); the intensity of physical exercise (β=-1.461, 95%CI: -2.392~-0.530) and time of physical exercise per session (β=2.608, 95%CI: 1.404~3.812, P < 0.05) were influencing factors for percentage of forward motility sperm (P < 0.05); and physical exercise intensity (β=-1.934, 95%CI: -3.238~-0.630), time of physical exercise per session (β=4.211, 95%CI: 2.525~5.897) and frequency of physical exercise (β=-2.008, 95%CI: -3.480~-0.536) were influencing factors of total sperm motility (P < 0.05).

Physical exercise may affect semen quality, greater intensity of physical exercise may be a risk factor for abnormal semen quality, and longer physical exercise time may be related to improving semen quality. Therefore, proper physical exercise can help improve semen quality.

To explore the role and underlying mechanisms of Acyl-CoA synthetase short chain family member 2（ACSS2）in ethanol-promoted migration of colorectal cancer（CRC）cells.

The ACSS2 expression level in cancer tissues and adjacent normal tissues was analyzed by online tools Gene Expression Profiling Interactive Analysis（GEPIA）and Human Protein Atlas（HPA）. The transcription factor enrichment analysis tool ChIP-X Enrichment Analysis 3（ChEA3）was used to identify ACSS2 upstream transcription factors. The ACSS2 expression level in cancer tissue and related clinical characteristic parameters of 513 CRC patients were obtained from The Cancer Genome Atlas（TCGA）database. After HCT116 and SW480 cells were treated with ethanol at a dose of 0, 22, 44 and 88 mmol/L for 24, 48 and 72 h, respectively, cell viability, migration and expression of related molecules at mRNA and protein levels were detected with CCK-8 assay, cell scratch test and transwell assay, and RT-qPCR and Western blotting respectively.

① After treated by ethanol for 48 and 72 h, the 3 doses of 22, 44 and 88 mmol/L showed a significant inhibition of proliferation for both HCT116 and SW480 cells（P<0.05）. ②After 48 h of ethanol treatment, RT-qPCR and Western blotting revealed that the expression levels of ACSS2 were increased significantly in HCT116 and SW480 cells, cell scratch test and Transwell assay indicated that the cell healing rate and number of perforated cells were also increased obviously（P<0.05）, and epithelial-mesenchymal transformation（EMT）related proteins, E-cadherin was down-regulated, while N-cadherin and Vimentin were up-regulated（all P<0.05）. ③ Inhibition of ACSS2 expression resulted in obviously decreased cell healing rate and number of perforated cells（P<0.05）, enhanced expression level of E-cadherin and decrased levels of N-cadherin and Vimentin（all P<0.05）. ④ HPA database analysis showed that ACSS2 has a significantly higher expression level in CRC cancer tissues than in normal tissues, and upstream regulators enrichment analysis suggested 8 transcription factors which probably regulate ACSS2 expression. Western blotting showed CEBPB, SMC1A and CTCF had notably increased expression in both SW480 and HCT116 cells after ethanol treatment, which was consistent with the increase trend of ACSS2 expression. ⑤ Of the 513 CRC patients, those with higher ACSS2 expression had a significantly higher incidence of peripheral nerve or vascular infiltration, and higher rate of existed distant metastases than those with lower expression（Chi-square=6.411, P=0.011）.

Ethanol treatment may probably induce EMT by upregulating ACSS2 expression and its upstream transcription factors to promote the migration of CRC cells.