Ca2+ free medium and heparin can induce release of bull sperm attached to oviductal epithelial cell monolayers

BOSCH, P.; ELLINGTON, J. E.; DE AVILA, J. M.; WRIGHT, R. W.

Maastricht, Holanda

Congreso; 26 Annual Congress of the International Embryo Transfer Society; 2000

International Embryo Transfer Society

Sperm cells attach to the oviductal epithelial cells (OEC) both in vivo and in vitro. In addition, a motile and morphologically normal sperm subpopulation binds to OEC (Thomas et al. Biol Reprod 1994; 51:303-309). The objective of the present study was to investigate the ability of heparin and Ca2+ free medium to induce sperm release from bovine OEC attachment. Oviductal epithelial cells were obtained from cows in follicular phase. Cells from at least 4 animals were pooled and grown to confluence in 24-well plates in F-12 Coon´s modification medium with 10% fetal bovine serum, 5 mg/mL insulin, 5 mg/mL transferrin, 5 ng/mL selenium, 10 ng/mL EGF, and 0.01 mg/mL gentamicin. Pooled frozen bull semen was thawed, washed twice in Sperm-TALP and the concentration was adjusted to 2 x 107 sperm cells/mL in the same medium. Medium in each well was replaced by 0.5 mL of Sperm-TALP before adding the sperm suspension to a final concentration of 2 x 106 sperm/mL. After 2 h binding, the contents of each well were mixed by pipetting, followed by recovery of medium with unbound sperm. Medium in each well was replaced by either Sperm-TALP alone (control), or Ca2+ free Sperm-TALP with 2 mM EGTA, or Sperm-TALP containing heparin (5, 10, or 15 IU/mL). Treatments were left on sperm-OEC cocultures for 0.5, 1, 2, 3, or 5 h. At each time point, the media were recovered and sperm from each treatment were evaluated for motility and counted in a hemocytometer. All cultures were performed at 38.5°C in humidified 5% CO2 in air. Sperm release is expressed as percentage of the total number of sperm attached to OEC after 2 h of coculture, which is considered 100%. Data were analyzed by ANOVA and treatment means compared by protected LSD. Results are expressed as mean±SEM. At 2 h 40.4±2.0% of sperm were attached to OEC and the motility of unbound sperm was 41.5±2.9%. At 0.5 h more sperm were released from heparin treatments as compared to control (Table 1) (Ver la tabla en versión PDF). Ca2+ free medium also induced sperm release when compared to control, but the effect was significant at 3 h (38.2±1.9% vs 59.5±6.9%; P<0.05). Sperm motility at 5 h was lower (P<0.05) in heparin treatments (15.8±2.9, 23.3±0.6, and 18.4±1.4% for heparin 5, 10, and 15 IU/mL respectively) than control (32.6±0.5%). It is concluded that both Ca2+ free medium and heparin are equally able to promote sperm displacement from OEC attachment. However, heparin reduces unbound sperm motility at 5 h of coculture. P. Bosch supported by a Scholarship from Fulbright-CONICOR, Argentina.