321 CO-CULTURE WITH OVIDUCT EPITHELIAL CELLS PROMOTES CAPACITATION OF BUFFALO (BUBALUS BUBALIS) SPERM

Abstract

The process of capacitation, necessary for spermatozoa to acquire fertilizing ability, normally takes place in the oviduct. It has been demonstrated that oviduct fluid improves capacitation in bovine spermatozoa (McNutt TL and Killian GJ 1991 J. Androl. 12, 244-252). The aim of this study was to evaluate whether co-culturing buffalo sperm with bovine oviduct epithelial cells (BOEC) promotes in vitro capacitation of buffalo spermatozoa. Bovine oviducts were obtained at a local abattoir from cows that were in the preovulatory phase of a normal estrous cycle. BOEC recovered as previously described (Gualtieri R and Talevi R 2000 Biol. Reprod. 62, 1754-1762) from 5 oviducts were pooled and plated in dishes of TCM-199 + 10% FCS, 100 U mL^-1 penicillin, 100 μg mL^-1 streptomycin, and 0.25 μg mL^-1 amphotericin B. Medium was changed every 48 h up to Day 6, when cell confluence and cilia activity were optimal. On day of trial, the medium was removed from the BOEC dishes and replaced with modified TALP containing 30 μM penicillamine, 15 μM hypotaurin, 0.15 μM epinephrine, and 1% bovine serum (mTALP). Buffalo frozen- thawed sperm from 4 bulls, pooled and treated by Percoll gradients, were incubated for 3 h (1 million sperm/mL): in mTALP with 30 μg mL^-1 of heparin (Group A), on the BOEC monolayer (Group B), on the BOEC monolayer with 30 μg mL^-1 of heparin (Group C), and in medium lacking capacitating agents, as negative control (Group D). After 3 h, sperm were recovered by centrifugation and capacitation was indirectly evaluated by assessing their ability to undergo acrosome reaction (AR) after 10 min of exposure to 10 μM calcium ionophore A23187. The AR was determined by the double-staining technique with trypan blue-Giemsa (Kovács A andFoote RH 1992 Biotec. Histochem. 67, 119-124). Spermatozoa (n = 294, 204, 225, and 222 for Groups A, B, C, and D, respectively) were then examined by microscopic evaluation. Differences in the percentages of AR among groups were analyzed by chi-square test. After Percoll separation, sperm viability was 95.8% and acrosomal loss was observed only in 5% of the sperm population. After 3 h of incubation, sperm viability decreased (P < 0.01) in all groups compared to the post-Percoll time, whereas no differences were found among groups (73, 80.4, 80.0, and 76.2%, respectively, for Groups A, B, C, and D). Sperm co-incubation with both BOEC and heparin for 3 h improved capacitation, as indicated by the significant increase (P < 0.01) of AR compared to the negative control (29.9, 30.5, 30.6, and 14%, respectively, for Groups A, B, C, and D). Furthermore, both BOEC groups gave percentages of AR similar to those observed after incubation with heparin, that is, the most widely used capacitating agent. In conclusion, it was demonstrated that co-culturing sperm with BOEC, both in the presence or absence of heparin, enhances buffalo sperm capacitation in vitro.