325 PROLONGING THE LIFE OF RAM SPERMATOZOA IN VITRO USING OVIDUCTAL EPITHELIAL CELLS

Abstract

Although binding to oviductal epithelial cells (OEC) enhances both bull (Ellington et al. 1991 Theriogenology 35, 970–977) and boar (Fazeli et al. 1997 J. Reprod. Fertil. 20, 49 abst) sperm survival in vitro, the ram sperm-OEC interaction is largely uninvestigated. Consequently, this study aimed to determine (1) whether ram spermatozoa bind to OEC, and (2) whether this binding prolongs ram sperm survival in vitro. Four types of OEC (follicular isthmus, follicular ampulla, luteal isthmus, and luteal ampulla), isolated from the oviducts of ewes slaughtered locally, and sheep kidney epithelial (MDOK)cells, purchased commercially, were used in this study. Each cell type was grown in separate wells of chamber slides in a humidified incubator (39°C and 5 % CO₂ in air) overnight. The next day, the growth medium was removed and the cells were washed once with PBS. Ficoll-washed ram spermatozoa, resuspended in Tyrode's medium, were added to the cells; as controls, spermatozoa were added to medium only (no OEC) in some wells. The viability of spermatozoa, unbound and bound to the cells, and in medium alone, was determined at 0, 1, 6, 24, and 48 h by incubating with the live and dead stains, Sybr-14 (100 nm) and ethidium homodimer (1 µm), respectively, for 15 min. In each case, 200 spermatozoa were counted and classified as live or dead using a fluorescence microscope. The experiment was replicated 4 times using an ejaculate from a different ram each time. The mean percentage (%) of viable spermatozoa was determined in each case by normalizing to the viability determined for each ram ejaculate at 0 h. Data were log transformed and analyzed using factorial ANOVA. Statistical significancewas defined as P < 0.05. Following 1, 6, 24, and 48 h of co-culture, the % of viable spermatozoa bound to the OEC for each of the culture periods (mean ± SEM: 129.87 ± 7.34, 101.21 ± 7.34, 126.72 ± 7.34, and 135.97 ± 7.34, respectively) was significantly greater (P < 0.001) than the % unbound to the OEC (20.94 ± 7.34, 9.55 ± 7.34, 14.47 ± 7.34, and 31.38 ± 7.34, respectively), and in medium alone (35.44 ± 14.67, 31.38 ± 14.67, 24.45 ± 14.67, and 28.49 ± 14.67, respectively). This effect was irrespective of the oviductal region and the reproductive cycle of the ewe used to derive the OEC. Interestingly, the MDOK cells, like the OEC, selectively bound viable spermatozoa following 1 and 6 h of co-culture (136.75 ± 39.41 and 153.31 ± 39.41, respectively). However, the % of viable spermatozoa bound to the MDOK was significantly less than to the OEC following 24 h (P = 0.05) and 48 h (P < 0.01) of co-culture (93.52 ± 39.41 and 72.85 ± 39.41, respectively). These data show that epithelial cells originating from reproductive and non-reproductive tissues both are capable of binding and prolonging sperm viability over the first 6 h of co-culture, but only OEC are capable of prolonging sperm viability for longer periods of time.

This work was funded by DEFRA (UK), Innovis (UK), and IMV (France).