Sperm binding to oviductal epithelium is thought to be an important mechanism regulating sperm reservoir formation in the oviduct. On the basis of evidence in the hamster, we hypothesized that capacitation affects release of bovine sperm, allowing them to fertilize. Oviducts were obtained from the ovulatory side of estrous Holstein heifers. The isthmic and ampullar epithelia were milked out and reduced to fragments, which formed everted vesicles (explants). Explants were placed in tissue culture wells in TALP medium and incubated at 39 degrees C in 5% CO2. Frozen-thawed sperm were prepared by swim-up in TALP and capacitated by incubation for 4 h in TALP with 20 micrograms/ml heparin (without glucose). Uncapacitated sperm were used immediately after dilution into capacitation medium. Within 2 h of surgery, sperm were added to the explants and incubated with them for 15 min. Sperm and explants were videotaped, and the tapes were analyzed to determine the numbers of sperm bound per surface area. ANOVA did not show a difference between the number of sperm bound/0.1 mm2 in the isthmus and ampulla (p > 0.05); however, an effect of capacitation was detected (p = 0.0015). Also, the percentage of capacitated sperm, determined by chlortetracycline labeling, was greater in sperm that remained free-swimming in the presence of explants than in the absence of explants (p = 0.001). In conclusion, capacitation appears to be involved in the release of bovine sperm from oviductal epithelium and therefore could enable sperm to leave the reservoir and fertilize oocytes.

A reservoir for sperm has been found in the oviductal isthmus in several species. Sperm are apparently trapped in the reservoir by binding to the oviductal epithelium, although other factors may be involved. We hypothesized that binding sites for bovine sperm are limited to the isthmus and are regulated by the hormonal state of the cow. Ipsilateral oviducts were obtained from heifers that were preovulatory (in estrus), had ovulated recently (within 12 h), or were in diestrus (Day 10). The isthmic and the ampullar epithelium were milked out and incubated separately in serum-free (SFRE-199-2) medium, at 39 degrees C in 5% CO2. Frozen-thawed sperm from bulls were added to the epithelium and coincubated for 15 min. The number of spermatozoa that bound to explants was not affected by stage of cycle or by anatomic origin of the explants (p > 0.05). In an additional experiment, oviducts were infused with sperm in vivo and then prepared for scanning electron microscopy, which revealed that sperm were associated with ciliated epithelium in both the isthmus and ampulla. Thus, bovine sperm may form a reservoir in the isthmic end of the oviduct because it is the first oviductal region that they encounter.

Sperm binding to oviductal epithelium probably serves to form the isthmic sperm reservoir. This interaction of sperm and oviductal epithelium may involve species-specific carbohydrate recognition. We tested a series of carbohydrates and glycoproteins for inhibition of bovine sperm binding to oviductal epithelium in vitro. Explants of isthmic and ampullar epithelium were obtained from oviducts that had been surgically removed from preovulatory heifers. The explants were incubated (39 degrees C, 5% CO2) with fetuin, asialofetuin, ovalbumin, fucoidan, fucose, N-acetyl glucosamine, or N-acetyl glucosamine sulfate dissolved in a modified Tyrode's balanced salt solution, termed sperm-TALP (pH 7.4, 295 mOsm) for 10 min before frozen-thawed motile sperm obtained by swim-up were added. After 15 min, the explants were rinsed, and sperm binding density was evaluated. Oviductal explants treated with fucoidan (3 mg/ml; p < 0.001, n = 5) or fucose (31 mM, p < 0.01, n = 6) had reduced densities of bound sperm compared to the controls. Incubation of explants in increasing concentrations of fucose (4-62 mM) resulted in increased inhibition of sperm binding. Pretreating explants with fucosidase also reduced sperm binding (p < 0.001, n = 3) compared to that in controls containing the fucosidase inhibitor deoxyfuconojirimycin. The presence of fucosylated molecules on the surface of the oviductal epithelium was confirmed by labeling with fucose lectins from Ulex europeus and Lotus tetragonolobus. We conclude that fucose is involved in a specific interaction between bovine sperm and oviductal epithelium.

The lower isthmus of the mammalian oviduct appears to serve as a reservoir for sperm that are retained by adherence to the epithelium. By inhibiting sperm binding within excised hamster oviducts and making use of carbohydrate probes, we have characterized the adherence of sperm in the reservoir and established a potential biochemical mechanism for the adherence and release of sperm. Fetuin and its terminal sugar, N-acetylneuraminic acid, interfered with the adherence of sperm to the oviductal epithelium. Labeled fetuin bound to the acrosomal region of fresh epididymal sperm, but not hyperactivated sperm, which have previously been reported to release from epithelial binding. Western blots labeled with fetuin and sialic acid-recognizing lectins identified proteins at several molecular masses that are candidates for the sperm surface component involved in adherence. Labeling of some of these candidates was reduced in samples from hyperactivated sperm. These results indicate that a sialylated oligosaccharide similar to that found on fetuin may be recognized by a sperm surface component and mediate binding to the oviductal epithelium. Release may be accomplished by loss or modification of the component during capacitation or hyperactivation.