Department of Chemical Engineering, Indian Institute of Technology Bombay, Powai, Mumbai, India.
Department of Gamete Immunobiology, ICMR-National Institute for Research in Reproductive Health, Parel, Mumbai, India.
Andrology. 2021 Jul;9(4):1214-1226. doi: 10.1111/andr.12988. Epub 2021 Mar 2.
Chemotaxis, as a mechanism for sperm guidance although known, has been difficult to demonstrate in vitro. Consequently, very few chemoattractants have been identified till date.
To investigate sperm motility behavior in response to ovulatory (OV) and preovulatory (preOV) oviductal fluid (OF) and identify potential chemotactic metabolites.
Intracellular calcium ([Ca ] ) influx in capacitating sperm was determined by spectrofluorimetry. The chemotactic response of rat caudal sperm to OF from the preOV- and OV- phases of normally cycling female rats was assessed in a microfluidic device developed by us. Hydrophilic metabolites extracted from the OF of both the phases were resolved and identified by LC-MS/MS, followed by data analysis using XCMS and MetaboAnalyst software, and chemotactic potential of the most promising compound was validated using the microfluidic device.
Spectrofluorimetric analysis depicts a significant increase in sperm [Ca ] in response to OV-OF. With the microfluidic chemotaxis assay, sperm population shows a significantly increased directionality and velocity to an ascending gradient of 0.06 µg/µl OV-OF compared to preOV-OF. LC-MS/MS of the OFs demonstrates five and four metabolites to be exclusive to the OV-OF and preOV-OF, respectively, and 25 metabolites common to both, of which 14 metabolites, including N-formyl-l-aspartate (NFA), are increased in OV-OF; NFA was tested for its ability to influence sperm movement, and shows chemotaxis potential.
DISCUSSION AND CONCLUSION(S): This is the first study that has systematically demonstrated sperm chemotaxis with OV phase rat OF, identified NFA present in this fluid as a novel chemoattractant to sperm, and proven the utility of the device to test putative chemoattractants. It remains to be seen whether NFA is present in the follicular fluid (FF) of infertile women, and whether it may likely be a reason for the failure of natural conception in idiopathic infertile women.
虽然已知趋化作用是精子导向的一种机制，但在体外很难证明。因此，迄今为止，很少有趋化剂被鉴定出来。
研究精子对排卵（OV）和排卵前（preOV）输卵管液（OF）的运动行为，并鉴定潜在的趋化代谢产物。
通过荧光光谱法测定获能精子的细胞内钙（[Ca 2+ ]）内流。使用我们开发的微流控装置评估来自正常周期雌性大鼠 preOV 和 OV 阶段的大鼠尾部精子对 OF 的趋化反应。使用 LC-MS/MS 分离和鉴定来自这两个阶段 OF 的亲水性代谢物，然后使用 XCMS 和 MetaboAnalyst 软件进行数据分析，并使用微流控装置验证最有前途的化合物的趋化潜力。
荧光光谱分析显示，精子对 OV-OF 的反应显著增加[Ca 2+ ]。使用微流控趋化分析，与 preOV-OF 相比，精子群体对 0.06μg/μl OV-OF 的上升梯度表现出显著增加的方向性和速度。OF 的 LC-MS/MS 显示，OV-OF 中分别有 5 种和 4 种代谢物是独特的，preOV-OF 中分别有 25 种代谢物是共同的，其中包括 N-甲酰-L-天冬氨酸（NFA）在内的 14 种代谢物在 OV-OF 中增加；测试了 NFA 影响精子运动的能力，显示出趋化潜力。
这是第一项系统地证明 OV 期大鼠 OF 中精子趋化作用的研究，鉴定出这种液体中的 NFA 作为一种新的精子趋化剂，并证明了该装置用于测试潜在趋化剂的效用。目前尚不清楚 NFA 是否存在于不孕妇女的卵泡液（FF）中，以及它是否可能是特发性不孕妇女自然受孕失败的原因。
