Analysis of the chemotactic response of ram spermatozoa to steroid hormones

Abstract

Some substances in follicular fluid may be acting as sperm attractants, and steroid hormones progesterone (P4) and estradiol (E2), both secreted by the cumulus cells, are possible candidates. We have previously observed a chemotactic effect of the ewe follicular fluid in ram spermatozoa (data not published). The presence of progesterone and estrogen receptors in ram spermatozoa suggests the involvement of these hormones in the sperm functionality, including guided movement towards the oocyte. P4 acts as a chemoattractant in human and rabbit sperm, but there is no information on its effect, or E2 effect, on ram spermatozoa. The objective of this work was to study the putative chemotactic action of P4 and E2 on ram spermatozoa, analyzing the influence of the sperm capacitation state. Swim-up selected spermatozoa from nine Rasa Aragonesa rams were incubated without (cap-TALP) or with cAMP-elevating agents (cap-Cocktail) to induce in vitro capacitation. After evaluating the sperm quality parameters in non- capacitated and incubated samples (motility, viability, capacitation state), IBIDI® µ-Slide Chemotaxis chamber (Ibidi GmbH) was loaded with swim-up, cap-TALP or cap-Cocktail sperm samples according to the manufacturer’s instructions. P4 or E2 were added to the chambers at 100 pM or 1 µM concentration each. Medium with 0.01% dimethyl sulfoxide (DMSO) was used as control treatment. After 5 min of equilibration, 200 fps videos were recorded for 3 sec with a video camera mounted on a microscope. Sperm trajectories and chemotaxis were analyzed with 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® (versión 8.0.1). A chemotactic response was only observed in capacitated spermatozoa, as we previously described in presence of follicular fluid. P4 at both concentrations had a chemotactic effect on capacitated ram spermatozoa. However, E2 had no effect on any of the studied samples. These results reveal, for the first time, the chemotactic effect of P4 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).