Binding Regulation of Porcine Spermatozoa to Oviductal Vesicles In Vitro

Abstract

ABSTRACT: In vivo, en route to the site of fertilization, mammalian spermatozoa bind to oviduct epithelial cells (OECs). This binding may favor sperm survival and capacitation, but little is known about the regulation of detachment of sperm from the oviduct in vivo. Therefore, we studied sperm‐oviduct interaction in vitro using vesicles formed from OECs in primary culture. Porcine oviducts were collected from gilts at the slaughterhouse. OECs were obtained by compressing the oviduct and culturing them in TCM‐199 medium with 10% fetal calf serum for 48 hours. For the first experiment, to test the hypothesis that progesterone (P4) and estradiol (E2) affect sperm‐OEC binding, OECs were pretreated for 48 hours with 100 ng/mL of P4 or E2. In the second experiment, porcine follicular fluid (pFF, 5%), caffeine (1 μM), calcium ionophore A23187 (1 μM), and DMSO (0.01%) were added to the incubation medium to provide insights on the mechanisms of sperm release from the oviduct. For both experiments, 50 × 106 sperm were coincubated with 50 μL of OEC vesicles in 1 mL of incubation medium for up to 24 hours at 37°C and 5% CO2. After an initial 30 minutes of coincubation, the vesicles settled and a sample of the supernatant was removed to evaluate sperm release and acrosomal status; subsequent samples were removed after 2, 4, and 24 hours of coincubation. To evaluate the effect of the different treatments on sperm integrity, the acrosome status of the spermatozoa was determined using fluorescein‐labeled Pisum sativum agglutinin staining. Experiment 1 showed that P4 pretreatment of OECs interferes with sperm binding compared with pretreatment with E2 or controls (P < .05). In experiment 2, coincubation in the presence of A23187 increased sperm detachment compared with pretreatment with DMSO, pFF, caffeine, or controls (P < .05). For each experiment, the treatments did not affect the percentage of acrosome‐reacted sperm compared with controls (P < .05).