Data Sheet 1_Potential role of 4-hydroxyisoleucine in enhancing fertility in male mice with diet-induced obesity.docx

BackgroundObesity is associated with hormonal imbalance, increased oxidative stress, and inflammation in the testis. These conditions adversely affect sperm quality, leading to impaired male fertility. Therefore, therapeutic interventions to counteract the adverse effects of obesity are crucial. This study explored the therapeutic effects of 4-hydroxyisoleucine (4-HIL) on fertility in male mice with diet-induced obesity.
MethodsC57BL6 male mice (n=45) were randomly divided into normal diet (ND, n=15) and high-fat diet (HFD, n=30) groups for 10 weeks. The HFD group was then randomized into untreated (HFD, n=15) and 4-HIL-treated (200 mg/kg/day, intraperitoneal injection, HFD + 4-HIL group, n=15) for 6 weeks. ND and HFD controls received saline (0.3 mL/30 g body weight) throughout the intervention period. Comprehensive evaluations included (1) metabolic assessments (body weight, glucose, insulin and pyruvate tolerance tests, and blood serum lipids), (2) sperm analysis (count, concentration, and morphology), (3) fertility testing (mating trials and in vitro fertilization), (4) testicular histopathology (fat deposition and apoptosis), (5) biochemical assays (reproductive hormones, oxidative stress markers, and inflammatory cytokines), and (6) molecular analyses (mRNA sequencing and qPCR validation of differentially expressed genes).
Results4-HIL treatment improved metabolic parameters, including reduced weight gain, enhanced glucose tolerance, and optimized blood serum lipids, compared to HFD controls. Treated mice exhibited superior sperm quality with increased count and concentration, reduced histomorphological abnormalities in the testis, and attenuated oxidative stress and inflammation. Furthermore, the key spermatogenic gene expressions, including spem1 and spata24, were significantly optimized in the testes of mice treated with 4-HIL compared to those of untreated mice (HFD group).
ConclusionThis study demonstrates that 4-HIL therapy ameliorates obesity-induced testicular dysfunction and improves fertility markers in mice. The beneficial effects of this compound on metabolic parameters, sperm quality, and spermatogenic gene expression suggest its potential as a therapeutic agent for obesity-related male infertility. Further research is warranted to elucidate the underlying mechanisms and assess the clinical translatability of these findings.

背景：肥胖与激素失衡、睾丸氧化应激增强及炎症反应密切相关。上述病理状态会对精子质量产生不利影响，进而导致男性生育能力受损。因此，采取治疗手段抵消肥胖带来的不良影响具有重要意义。本研究探讨了4-羟基异亮氨酸（4-hydroxyisoleucine, 4-HIL）对饮食诱导肥胖雄性小鼠生育能力的治疗作用。
方法：将45只C57BL6雄性小鼠随机分为正常饮食组（normal diet, ND，n=15）与高脂饮食组（high-fat diet, HFD，n=30），造模周期为10周。随后将高脂饮食组小鼠进一步随机分为未处理组（HFD组，n=15）与4-HIL处理组（200 mg/kg/天，腹腔注射，HFD+4-HIL组，n=15），处理周期为6周。正常饮食组与高脂饮食对照组在整个干预期间均给予生理盐水（0.3 mL/30 g体重）。本研究开展了全方位的综合评估，具体包括：(1) 代谢指标检测：体重、葡萄糖耐受试验、胰岛素耐受试验、丙酮酸耐受试验及血清脂质水平检测；(2) 精子分析：精子计数、浓度与形态学检测；(3) 生育能力检测：交配试验与体外受精（in vitro fertilization）；(4) 睾丸组织病理学检测：脂肪沉积与细胞凋亡情况；(5) 生化指标检测：生殖激素、氧化应激标志物与炎症细胞因子水平；(6) 分子生物学分析：mRNA测序及差异表达基因的定量聚合酶链反应（quantitative PCR, qPCR）验证。
结果：与高脂饮食对照组相比，4-HIL治疗可改善代谢指标，包括减轻体重增长、增强葡萄糖耐受能力及优化血清脂质水平。经4-HIL处理的小鼠精子质量更优：精子计数与浓度均升高，睾丸组织形态学异常情况减少，氧化应激与炎症反应得到缓解。此外，与未处理的高脂饮食组小鼠相比，4-HIL处理组小鼠睾丸内关键生精基因（包括spem1与spata24）的表达水平显著优化。
结论：本研究证实，4-HIL治疗可改善肥胖诱导的睾丸功能障碍，并提升小鼠的生育相关指标。该化合物对代谢参数、精子质量及生精基因表达的有益作用，表明其有望成为治疗肥胖相关性男性不育的候选药物。未来仍需开展进一步研究，以阐明其潜在作用机制，并评估该研究结果的临床转化潜力。