N-formyl-l-aspartate: A novel sperm chemoattractant identified in ovulatory phase oviductal fluid using a microfluidic chip.

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 behavior 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 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.

Chemotactic behavior of spermatozoa captured using a microfluidic chip.

Chemotaxis, as a mechanism for sperm guidance in vivo, is an enigma which has been difficult to demonstrate. To address this issue, various devices have been designed to study sperm chemotaxis in vitro. Limitations of traditional chemotaxis devices were related to the inability to maintain a stable concentration gradient as well as track single sperm over long times. Microfluidics technology, which provides superior control over fluid flow, has been recently used to generate stable concentration gradients for investigating the chemotactic behavior of several cell types including spermatozoa. However, the chemotactic behavior of sperm has not been unequivocally demonstrated even in these studies due to the inability to distinguish it from rheotaxis, thermotaxis, and chemokinesis. For instance, the presence of fluid flow in the microchannels not only destabilizes the concentration gradient but also elicits a rheotactic response from sperm. In this work, we have designed a microfluidic device which can be used to establish both, a uniform concentration and a uniform concentration gradient in a stationary fluid. By facilitating measurement of sperm response in ascending, descending ,and uniform chemoattractant concentration, the assay could isolate sperm chemotactic response from rheotaxis and chemokinesis. The device was validated using acetylcholine, a known chemoattractant and further tested with rat oviductal fluid from the estrus phase.