RESUMO
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 (ß-2-AR) that regulates cAMP production and Ca2+ 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 ß-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 ß-2-AR specific antagonist, were evaluated using the microfluidics device-based chemotaxis assay. The intracellular concentration of Ca2+, 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; ß-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 via ß-2-AR. Interestingly, at the concentration used for chemotaxis, NFA induced an increase in the intracellular Ca2+ but decreased cAMP in capacitating sperm. However, NFA per se 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 ß-2-AR on sperm possibly via non-canonical signaling.
RESUMO
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.
