Chemotaxis of capacitated ram spermatozoa to ewe follicular fluid

Abstract

Sperm orientation mechanisms, as chemotaxis, are essential for the sperm to reach the oocyte and then fertilize it. The follicular fluid is the biochemical environment where the oocyte develops until its ovulation, and its composition changes throughout the estrous cycle. The follicular fluid is released together with the oocyte at the moment of ovulation, and it has been proposed as a chemoattractant for spermatozoa. This chemotactic ability has been demonstrated in some mammals, but to date, no studies have been done in the ovine species. Also, it has been reported that only capacitated and hyperactivated spermatozoa can respond to chemotactic stimuli. Therefore, the aim of this work was to study the chemotactic response of ovine spermatozoa to ewe follicular fluid, obtained at various stages of the estrous cycle, and to evaluate the influence of the capacitation status in the chemotaxis mechanism. In order to perform the experiments, ovine follicular fluids were obtained in the early and late follicular and luteal phases, and two concentrations, 2 and 10%, were tested. Semen samples were collected from nine Rasa Aragonesa rams, and spermatozoa were selected by the swim-up/dextran method. In vitro capacitation was induced by incubation without (cap-TALP) or with cAMP-elevating agents (cap-Cocktail). After sperm quality evaluation (motility, viability, and capacitation state), the chemotactic assays were carried out with the aim of the commercial device (IBIDI® µ-Slide Chemotaxis chamber, Ibidi GmbH). Swim- up, cap-TALP or cap-Cocktail samples were loaded in the chambers in the presence of follicular fluid. Individual trajectories were analyzed by the OpenCASA software (Alquezar-Baeta et al., PLoS Comput Biol, 15(1), 2019), which calculates a chemotaxis index. Differences were analysed by χ² test, except for the chemotaxis index (ANOVA) using GraphPad PRISM® (Version 8.0.1). Follicular fluid at 2 and 10% concentrations, obtained in luteal and early follicular phases, induced sperm chemoattraction. This chemotactic response was only observed in capacitated spermatozoa, which supports previous evidence in other species. These results reveal, for the first time, the chemotactic effect of ewe follicular fluid in ram spermatozoa. Grants: AGL-2017- 83799-R and DGA A07-17R. S.M.-J. has a predoctoral contract from the Diputación General de Aragón (DGA).