Panchal Durva, Bhagwat Shweta, Parte Priyanka
Department of Gamete Immunobiology, ICMR-National Institute for Research in Reproductive and Child Health, Mumbai, Maharashtra, India.
Department of Obstetrics and Gynecology, Washington University School of Medicine, St. Louis, MO, United States.
Front Cell Dev Biol. 2022 Sep 23;10:959094. doi: 10.3389/fcell.2022.959094. eCollection 2022.
Chemotaxis is a highly conserved physiological event required for directed sperm movement during fertilization. Recently, studies from our laboratory have identified N-formyl-L-aspartate (NFA) as a sperm chemoattractant. NFA is a known agonist for the beta-2-adrenergic receptor (β-2-AR) that regulates cAMP production and Ca mobilization in somatic cells. As these downstream signaling molecules are also reported to be involved in sperm chemotaxis, in the present study we investigated the putative mechanism/s by which NFA may mediate chemotaxis. Toward this, the expression and localization of β-2-AR in sperm were studied by Western blot and indirect immunofluorescence, respectively. The responses of sperm to various concentration gradients of NFA and ICI-118,551, a β-2-AR specific antagonist, were evaluated using the microfluidics device-based chemotaxis assay. The intracellular concentration of Ca, on exposure to NFA, was analyzed using FURA-2 AM-based fluorimetric assay. Furthermore, the effect of NFA on sperm capacitation and acrosome reaction was evaluated using Western blot and immunofluorescence. NFA exhibited a bell-shaped dose-response curve typical of chemotaxis, with maximum response observed at 0.01M NFA, beyond which it was inhibitory; β-2-AR localization was seen on the sperm head and the mid-piece region of the flagella. Inhibition of sperm chemotaxis by ICI-118,551 confirms that sperm respond chemotactically to NFA β-2-AR. Interestingly, at the concentration used for chemotaxis, NFA induced an increase in the intracellular Ca but decreased cAMP in capacitating sperm. However, NFA did not induce capacitation as seen from the lack of effect on tyrosine phosphorylation and membrane potential of uncapacitated sperm. Acrosome evaluation of NFA-treated sperm using PSA-FITC staining showed no effect on the acrosome structure. Our data thus provide evidence indicating that NFA induces sperm chemotaxis and the chemotactic response of sperm to NFA from the ovulatory phase of oviductal fluid is mediated through the β-2-AR on sperm possibly via non-canonical signaling.
趋化性是受精过程中精子定向运动所需的一种高度保守的生理事件。最近，我们实验室的研究已确定N-甲酰-L-天冬氨酸（NFA）为精子趋化因子。NFA是β-2-肾上腺素能受体（β-2-AR）的已知激动剂，该受体调节体细胞中的环磷酸腺苷（cAMP）生成和钙动员。由于据报道这些下游信号分子也参与精子趋化性，因此在本研究中，我们研究了NFA可能介导趋化性的假定机制。为此，分别通过蛋白质免疫印迹法和间接免疫荧光法研究了β-2-AR在精子中的表达和定位。使用基于微流控装置的趋化性测定法评估精子对不同浓度梯度的NFA和β-2-AR特异性拮抗剂ICI-118,551的反应。使用基于FURA-2 AM的荧光测定法分析暴露于NFA时细胞内的钙浓度。此外，使用蛋白质免疫印迹法和免疫荧光法评估NFA对精子获能和顶体反应的影响。NFA呈现出典型的趋化性钟形剂量反应曲线，在0.01M NFA时观察到最大反应，超过此浓度则具有抑制作用；在精子头部和鞭毛的中段区域可见β-2-AR定位。ICI-118,551对精子趋化性的抑制证实精子对NFA通过β-2-AR产生趋化反应。有趣的是，在用于趋化性测定的浓度下，NFA可诱导获能精子的细胞内钙增加，但cAMP减少。然而，从未获能精子的酪氨酸磷酸化和膜电位缺乏影响来看，NFA并未诱导获能。使用PSA-FITC染色对经NFA处理的精子进行顶体评估，结果显示对顶体结构无影响。因此，我们的数据提供了证据，表明NFA可诱导精子趋化性，并且输卵管液排卵期的精子对NFA的趋化反应可能通过精子上的β-2-AR经由非经典信号传导介导。
