Purpose Our previous studies showed that nanotechnology improves derived adipose-derived stem cells (ADSCs) therapy for erectile dysfunction (ED). In this study, the Neuregulin-1(NRG1) gene was transfected into ADSCs with superparamagnetic iron oxide nanoparticles (SPION) further to improve the therapeutic effect of ADSCs on ED. Materials and Methods ADSCs were isolated from epididymal adipose tissue of Sprague–Dawley rats. The optimal concentration of PEI-SPION (SPION modified with polyethyleneimine) was selected to construct the gene complex. After electrostatic binding of PEI-SPION and DNA, a PEI layer was wrapped to make the PEI-SPION-NRG1-PEI gene transfection complex. Different groups were set up for transfection tests. Lipo2000 transfection reagent was used as the control. PEI-SPION-NRG1-PEI in the experimental group was transfected under an external magnetic field. Results When the concentration of PEI-SPION was 10 µg/mL, it had little cytotoxicity, and cell activity was not significantly affected. PEI-SPION-NRG1-PEI forms positively charged nanocomposites with a particle size of 72.6±14.9 nm when N/P ≥8. The PEI-SPION-NRG1-PEI gene complex can significantly improve the transfection efficiency of ADSCs, reaching 26.74%±4.62%, under the action of the external magnetic field. PCR and Western blot showed that the expression level of the NRG1 gene increased significantly, which proved that the transfection was effective. Conclusions PEI-SPION can be used as a vector for NRG1 gene transfection into ADSCs. PEI-SPION-NRG1-PEI packaging has the highest transfection efficiency under the external magnetic field than the other groups. These findings may provide a new strategy for ADSCs therapy for ED.

timepoints. A subcohort of animals (n[3) also underwent a 140 day period of THC discontinuation with testicular, serum and semen evaluations at 70 and 140 days. Random intercept mixed effects models with a linear spline at start of THC abstinence were used to determine the average change in testes, serum, and semen measurements with increasing THC dose and after discontinuation.RESULTS: For every 1 mg/7 kg/day increase in THC dosing, there was a significant decrease in total testicular volume bilaterally by 13 cm 3 (95% CI -15.40--10.9, p <0.001 (Fig. 1A). A similar doseresponse significant decrease in liquid semen ejaculate volume (p[0.043), mean total testosterone (p <0.001) and estradiol (p <0.001), but significant increase in FSH (p[0.017), LH (p[0.013), and prolactin (p[0.01) was observed (Fig 1B). No other significant changes in semen parameters were present (Fig 1C).Every 70 days of THC abstinence (n[3), total testicular volume increased significantly by 5 cm 3 (95% CI 3.2-6.9, p<0.001) to 82% of its original volume by 140 days after THC discontinuation (Fig. 1D). Serum T (p[0.001) and LH (p[0.019) both rose significantly, and FSH (p[0.008) decreased significantly (Fig. 1E). There were no significant changes to semen parameters after THC discontinuation (Fig. 1F).CONCLUSIONS: Chronic THC use in rhesus macaques resulted in significant dose-response testicular atrophy, increased gonadotropins, decreased serum sex steroids, and semen volume. Abstinence from THC resulted in incomplete recovery of testicular volume.

BackgroundThe ubiquitin ligase HECT, UBA, and WWE domain‐containing E3 ubiquitin protein ligase 1 is essential for the establishment and maintenance of spermatogonia. However, the role of HECT, UBA, and WWE domain‐containing E3 ubiquitin protein ligase 1 in regulating germ cell differentiation remains unclear, and clinical evidence linking HECT, UBA, and WWE domain‐containing E3 ubiquitin protein ligase 1 to male infertility pathogenesis is lacking.ObjectiveThis study aims to investigate the role of HUWE1 in germ cell differentiation and the mechanism by which a HUWE1 single nucleotide polymorphism increases male infertility risk.Materials and methodsWe analyzed HUWE1 single nucleotide polymorphisms in 190 non‐obstructive azoospermia patients of Han Chinese descent. We evaluated HECT, UBA, and WWE domain‐containing E3 ubiquitin protein ligase 1 regulation by retinoic acid receptor alpha using chromatin immunoprecipitation assays, electrophoretic mobility shift assays, and siRNA‐mediated RARα knockdown. Using C18‐4 spermatogonial cells, we determined whether HECT, UBA, and WWE domain‐containing E3 ubiquitin protein ligase 1 participated in retinoic acid‐mediated retinoic acid receptor alpha signaling. We performed luciferase assays, cell counting kit‐8 assays, immunofluorescence, quantitative real‐time polymerase chain reaction, and western blotting. We quantified HUWE1 and retinoic acid receptor alpha in testicular biopsies from non‐obstructive azoospermia and obstructive azoospermia patients using quantitative real‐time polymerase chain reaction and immunofluorescence.ResultsThree HUWE1 single nucleotide polymorphisms were significantly associated with spermatogenic failure in 190 non‐obstructive azoospermia patients; one (rs34492591) was in the HUWE1 promoter. Retinoic acid receptor alpha regulates HUWE1 gene expression by binding to its promoter. HECT, UBA, and WWE domain‐containing E3 ubiquitin protein ligase 1 participates in retinoic acid/retinoic acid receptor alpha signaling pathway and regulates the expression of germ cell differentiation genes STRA8 and SCP3 to inhibit cell proliferation and reduce γH2AX accumulation. Notably, significantly lower levels of HUWE1 and RARα were detected in testicular biopsy samples from non‐obstructive azoospermia patients.ConclusionsAn HUWE1 promoter single nucleotide polymorphism significantly downregulates its expression in non‐obstructive azoospermia patients. Mechanistically, HECT, UBA, and WWE domain‐containing E3 ubiquitin protein ligase 1 regulates germ cell differentiation during meiotic prophase through its participation in retinoic acid/retinoic acid receptor alpha signaling and subsequent modulation of γH2AX. Taken together, these results strongly suggest that the genetic polymorphisms of HUWE1 are closely related to spermatogenesis and non‐obstructive azoospermia pathogenesis.

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