Glycosaminoglycans isolated from follicular fluid reduce PKA activity during capacitation in porcine spermatozoa.

AGUILERA AC; VIERA, LA; MATAS, C

Bath

Congreso; 33rd Annual Meeting A.E.T.E.; 2017

A.E.T.E.

Glycosaminoglycans isolated from follicular fluid reduce PKA activity during capacitation in porcine spermatozoa.Aguilera AC1, Vieira LA2, and Matás C2,3**cmatas@um.es1Faculty of Natural Sciences (FCEN), National University of Cuyo, Mendoza, Argentina2Dept Physiology. Veterinary School, University of Murcia. Spain 3Institute for Biomedical Research of Murcia (IMIB), Murcia, SpainIntroduction: Glycosaminoglycans are linear polysaccharides comprised of repeating hexosamine-containing disaccharides that are found in the female genital tract and follicular fluid. Different studies showed that they may be involved in capacitation and acrosome reaction in vivo and their presence in sperm reservoir could maintain membrane stability due they interaction with sperm membrane (Tienthai, Journal of Reproduction and Development, 61, 245-250, 2015). The aim of this study was to investigate the effect of hyaluronic acid (HA) and glycosaminoglycans (GAGs) isolated from folicullar fluid (G-FF) and cumulus oophorus secretion obtained after oocyte porcine in vitro maturation (G-COS) on the activity of PKA and acrosome status after in vitro capacitation. Material and Methods: Glycosaminoglycans were isolated from COS and FF (from ovaries in the periovulatory phase) by protease digestion, lipid extraction and by different precipitation conditions according to Bellin and Ax (J Dairy Sci 70:1913?1919. 1987). Spermatozoa from fertile porcine was incubated for 3 h in TALP (at 38.5ºC and 5% CO2) or PBS (38.5ºC in air) supplemented or not with G-FF, G-COS or HA (100 ug/mL or 500 ug/mL). The activity of PKA was assessed by Western Blot (9624, Cell Signaling Technology, Massachusetts, USA). Viability was evaluated by propidium iodide 500 mg/mL (Sigma P 4170, Madrid, Spain) and carboxifluorescein 0.46 mg/mL (Sigma D6883, Madrid, Spain), while the acrosome status was analysed by fluorescein isothiocyanate-conjugated peanut agglutinin (PNA-FICT). Data were analysed by one-way ANOVA followed by Dunnett´s multiple comparisons test (p< 0.05).Results: The activity of PKA was significantly reduced after the incubation with G-FF in TALP compared to the other conditions. No effect was observed after the incubation with AH (100 ug/mL or 500 ug/mL) and G-COS. Spermatozoa incubated in TALP showed the lowest percentage of viability and a highest percentage of acrosomal damage (p