N-Formyl L-Aspartate: a novel sperm chemoattractant identified in Ovulatory Phase Oviductal Fluid using a microfluidic chip.

Bhagwat, Shweta; Sontakke, Shraddha; Desai, Sneha; Panchal, Durva; Jadhav, Sameer; Parte, Priyanka

BACKGROUND 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. OBJECTIVES To investigate sperm motility behaviour in response to ovulatory (OV) and preovulatory (preOV) oviductal fluid (OF) and identify potential chemotactic metabolites. MATERIALS AND METHODS Intracellular calcium ([Ca2+ ]I ) 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. RESULTS Spectrofluorimetric analysis depicts a significant increase in sperm [Ca2+ ]I 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 5 and 4 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 tested for its ability to influence sperm movement, shows chemotaxis potential. DISCUSSION AND CONCLUSION(S) This is the first study that has systematically demonstrated sperm chemotaxis with ovulatory phase rat oviductal fluid, 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 of infertile women, and whether it may likely be a reason for the failure of natural conception in idiopathic infertile women.