Anjorin Oluwakemi I, Yamanaka Takahiro, Shimada Masayuki
Graduate School of Integrated Sciences for Life, Hiroshima University, 1-4-4 Kagamiyama, Higashihiroshima, Hiroshima, 739-8528, Japan.
Graduate School of Innovation and Practice for Smart Society, Hiroshima University, 1-5-1 Kagamiyama, Higashihiroshima, Hiroshima, 739-8529, Japan.
In Vitro Cell Dev Biol Anim. 2025 Jul 21. doi: 10.1007/s11626-025-01084-5.
Sperm capacitation, a prerequisite for fertilization, is regulated not only by intrinsic signaling but also by paracrine factors within the female tract. Analysis of previously published RNA-seq datasets identified the ectodysplasin-A2 receptor (EDA2R), an X-linked member of the TNF-receptor superfamily, as a candidate regulator of this process. This study was conducted to test the hypothesis that the EDA-A2/EDA2R axis is a regulator that directly regulates sperm capacitation during fertilization process. Western blotting and immunofluorescence showed that EDA2R was localized in late spermatogenic cells and in the midpiece of epididymal sperm. Incubation of mouse sperm in HTF medium containing the corresponding ligand EDA-A2 (0-1 µg/mL) resulted in a dose-dependent improvement in the amplitude of lateral head displacement and curvilinear velocities. Ligand exposure promoted the appearance of capacitation hallmarks: tyrosine phosphorylation level was elevated within 30 min and the proportion of FITC-PNA positive, acrosome-reacted cells increased at 30 and 60 min (p < 0.05). The EDA-A2 treated sperm yielded a higher cleavage rate (78.5% vs. 48.3%) and a higher blastocyst formation rate (97.6% vs. 88.4%) after in vitro fertilization. qPCR in hormonally synchronized females revealed transient ovarian and prolonged oviductal Eda-a2 upregulation surrounding ovulation, suggesting that the ligand is present at the site of sperm-oocytes fertilization. These results clarify that EDA-A2/EDA2R is a rapid physiological driver of sperm capacitation. This provides a tractable cytokine axis for optimizing assisted reproduction.
精子获能是受精的前提条件，其不仅受内在信号调控，还受雌性生殖道内旁分泌因子的调节。对先前发表的RNA测序数据集进行分析后，确定了外胚层发育不良蛋白A2受体（EDA2R），它是TNF受体超家族的一个X连锁成员，是这一过程的候选调节因子。本研究旨在验证EDA-A2/EDA2R轴是受精过程中直接调节精子获能的调节因子这一假说。蛋白质免疫印迹和免疫荧光显示，EDA2R定位于晚期生精细胞和附睾精子的中段。将小鼠精子在含有相应配体EDA-A2（0-1µg/mL）的HTF培养基中孵育，导致侧向头部位移幅度和曲线速度呈剂量依赖性改善。配体暴露促进了获能标志的出现：酪氨酸磷酸化水平在30分钟内升高，FITC-PNA阳性、顶体反应细胞的比例在30和60分钟时增加（p<0.05）。体外受精后，经EDA-A2处理的精子产生了更高的卵裂率（78.5%对48.3%）和更高的囊胚形成率（97.6%对88.4%）。对激素同步的雌性动物进行qPCR分析发现，排卵前后卵巢中Eda-a2短暂上调，输卵管中Eda-a2长期上调，这表明该配体存在于精子与卵子受精的部位。这些结果表明，EDA-A2/EDA2R是精子获能的快速生理驱动因素。这为优化辅助生殖提供了一个易于处理的细胞因子轴。
