227 PHOSPHOINOSITIDE-3-KINASE SIGNALING AND THE CONTROL OF SPERM MOTILITY IN THE PORCINE OVIDUCT

Abstract

Prior to ovulation, interactions of spermatozoa with the oviductal epithelium lead to the formation of a sperm reservoir; here we investigate the hypothesis that the physiological state of the stored spermatozoa is modulated by the epithelial cells. In the pig, where modulation of sperm motility is sensitive to environmental components, the high bicarbonate content of the oviductal fluid would be expected to stimulate motility to its maximal extent; however, as this is incompatible with reservoir formation, we postulate that a signaling pathway counteracts bicarbonate stimulation and depresses motility (Satake et al. 2006 J. Exp. Biol. 209, 1560–1572). We chose to investigate a signaling mechanism which may suppress sperm motility through phosphoinositide-3-kinase (PI3K). Boar spermatozoa were exposed to solubilized proteins derived from the oviduct epithelial apical plasma membrane (sAPM). We exploited the motility activation effects of bicarbonate on Percoll-washed boar spermatozoa to see whether it was countered by sAPM and reversed by PI3K inhibitors. Washed spermatozoa (n = 9 boars) were incubated (10 min at 38°C) in Tyrode's medium in the presence of sAPM (25 and 50 µg mL^–1) or PI3K inhibitors (LY294002 or wortmannin) prior to activation with 15 mm bicarbonate/CO₂, and then incubated for a further 35 min. Sperm motility was monitored at 5-min intervals using a Hobson Sperm Tracker (Hobson Tracker, Ltd., Sheffield, UK). Samples were also analyzed with a flow cytometer for mitochondrial activity using JC-1. PATN analysis (Belbin 1993 PATN Pattern Analysis Package, Div. Wildlife & Ecol., CSIRO, Canberra, Australia) was used to distinguish four motility-based sperm subgroups within the data. The proportions of fast and progressive spermatozoa showed significant and rapid (<5 min) bicarbonate-induced increases when there was no sAPM treatment, or when PI3K inhibitors were present. However, there was no significant bicarbonate-induced increase in the presence of sAPM alone, or when spermatozoa were exposed to a mixture of wortmannin and sAPM. PI3K inhibition by LY294002 reversed the sAPM-induced motility suppression (P < 0.001). Mitochondrial membrane potential was also suppressed by sAPM and restored by the presence of LY294002 (P < 0.01). These results support the hypothesis that the sperm–oviduct interaction is modulated by an intracellular signaling pathway involving PI3K and appropriate receptors.