Dynamics of Bovine Sperm Interaction with Epithelium Differ Between Oviductal Isthmus and Ampulla

Abstract

In mammals, many sperm that reach the oviduct are held in a reservoir by binding to epithelium. To leave the reservoir, sperm detach from the epithelium; however, they may bind and detach again as they ascend into the ampulla toward oocytes. In order to elucidate the nature of binding interactions along the oviduct, we compared the effects of bursts of strong fluid flow (as would be caused by oviductal contractions), heparin, and hyperactivation on detachment of bovine sperm bound in vitro to epithelium on intact folds of isthmic and ampullar mucosa. Intact folds of oviductal mucosa were used to represent the strong attachments of epithelial cells to each other and to underlying connective tissue that exist in vivo. Effects of heparin on binding were tested because heparin binds to the Binder of SPerm (BSP) proteins that attach sperm to oviductal epithelium. Sperm bound by their heads to beating cilia on both isthmic and ampullar epithelia and could not be detached by strong bursts of fluid flow. Addition of heparin immediately detached sperm from isthmic epithelium but not ampullar epithelium. Addition of 4-aminopyridine immediately stimulated hyperactivation of sperm but did not detach them from isthmic or ampullar epithelium unless added with heparin. These observations indicate that the nature of binding of sperm to ampullar epithelium differs from that of binding to isthmic epithelium; specifically, sperm bound to isthmic epithelium can be detached by heparin alone, while sperm bound to ampullar epithelium requires both heparin and hyperactivation to detach from the epithelium.

Keywords: fallopian tube; fertilization; heparin; oviduct; spermatozoa.

FIG. 1

Swimming patterns of activated (progressive, nonhyperactivated (A) and hyperactivated (B) bovine sperm. Note the highly asymmetrical flagellar beating pattern of the hyperactivated sperm. Each panel shows a pair of merged differential contrast images taken of the maximum bend achieved on each side of the flagellum. Images are taken from Supplemental Movie S1. Bar = 10 μm.

FIG. 2

Preparation of folds of oviductal epithelium. A) Primary folds of mucosa (arrows) run longitudinally through the length of the oviduct. Bar = 1 mm. B) Laminar flow chamber containing mucosal folds (red arrows). The folds are anchored by a plastic mesh held in place with a metal spring. White arrows indicate the inflow (right) and outflow (left) tubing. Bar = 1 cm.

FIG. 3

Several sperm attached to cilia on epithelium (image from Supplemental Movie S2). The length of one of the sperm tails is indicated by black arrows. One of the sperm heads, with a dark acrosomal region and light postacrosomal region, is indicated by a white arrow. The cilia, which appear as a pale layer between the sperm heads and the black apical surface of the epithelium, are also indicated by white arrows. Bar = 20 μm.

FIG. 4

Bursts of strong fluid flow did not detach sperm from epithelium. Arrows indicate the flagella of some of the attached sperm. Image taken from Supplemental Movie S3. Bar = 20 μm.

FIG. 5

Heparin elicited sperm agglutination. Arrow indicates agglutinated heads of four sperm bound to isthmic epithelium 10 min after addition of heparin. Image taken from Supplemental Movie S4. Bar = 20 μm.

FIG. 6

Heparin and 4-AP affected sperm detachment differently in epithelia from the isthmus and ampulla. Whereas heparin alone produced sperm detachment from isthmic epithelium, stimulation of hyperactivation by 4-AP was additionally required to detach sperm from ampullar epithelium (means ± SEM for four replicates; *P < 0.05 when treatment is compared with its paired control).

FIG. 7

Treatment of bound sperm with heparin and 4-AP stimulated sperm detachment from ampullar epithelium. A) Sperm (white arrows) attached to the cilia before treatment. B) One min after treatment, sperm displayed hyperactivation and detached from epithelium (white arrows). C) Ten min after treatment, very few sperm (white arrow) remained bound to the epithelium (images taken from Supplemental Movie S5). Bar = 20 μm.

FIG. 8

Pretreatment of sperm with 4-AP to induce hyperactivation or with heparin did not prevent attachment to oviductal epithelium. A) Hyperactivated sperm attached to epithelium. Arrows indicate some of the bound sperm. B) Sperm pretreated with heparin were able to bind in high numbers. Arrows indicate groups of bound, agglutinated sperm. C) In contrast, when both sperm and oviductal epithelium were pretreated with heparin for 10 min, few sperm bound to epithelium. Arrow indicates two bound sperm. Images taken from Supplemental Movie S6. Bar = 20 μm.

FIG. 9

Effects of 4-AP and heparin on sperm swimming patterns in the absence of epithelium. Heparin alone did not hyperactive sperm, whereas 4-AP elicited hyperactivation in nearly all sperm regardless of the presence of heparin (means ± SEM for three replicate experiments; different letters denote family-wise P < 0.05).