Unexpected flagellar movement patterns and epithelial binding behavior of mouse sperm in the oviduct

Abstract

In order to better understand how sperm movement is regulated in the oviduct, we mated wild-type female mice with Acr-EGFP males that produce sperm with fluorescent acrosomes. The fluorescence improved our ability to detect sperm within the oviduct. Oviducts were removed shortly before or after ovulation and placed in chambers on a warm microscope stage for video recording. Hyperactivated sperm in the isthmic reservoir detached frequently from the epithelium and then reattached. Unexpectedly, most sperm found in the ampulla remained bound to epithelium throughout the observation period of several minutes. In both regions, most sperm produced deep flagellar bends in the direction opposite the hook of the sperm head. This was unexpected, because mouse sperm incubated under capacitating conditions in vitro primarily hyperactivate by producing deep flagellar bends in the same direction as the hook of the head. In vitro, sperm that are treated with thimerosal to release Ca(2+) from internal stores produce deep anti-hook bends; however, physical factors such as viscous oviduct fluid could also have influenced bending in oviductal sperm. Some sperm detached from epithelium in both the ampulla and isthmus during strong contractions of the oviduct. Blockage of oviduct contractions with nicardipine, however, did not stop sperm from forming a storage reservoir in the isthmus or prevent sperm from reaching the ampulla. These observations indicate that sperm continue to bind to oviductal epithelium after they leave the isthmic reservoir and that sperm motility is crucial in the transport of sperm to the fertilization site.

FIG. 1

Flagellar beating patterns in mouse sperm. The upper row of images (A–C) shows the maximal pro-hook flagellar bends in each flagellar beat pattern, whereas the lower row of images (D–F) shows the maximal anti-hook flagellar bends. A, D) Symmetrical flagellar bends on non-hyperactivated sperm. B, E) Pro-hook flagellar beat pattern. C, F) Anti-hook flagellar beat pattern. Bars = 20 μm.

FIG. 2

A) Two free-swimming sperm in the isthmus exhibiting deep anti-hook bends of the anti-hook beat pattern. B) A sperm exhibiting a deep anti-hook bend at the moment of detaching from epithelium. Yellow arrows indicate the tip of the hook-shaped head; insets are the tracings of sperm. Images are taken from the Supplemental Movies S1 and S2. Bars = 20 μm.

FIG. 3

Non-hyperactivated sperm in pockets formed by mucosal folds in the isthmus, taken from the Supplemental Movie S3. The yellow arrows indicate the locations of the heads of the sperm that can be seen more clearly in the movie. Bar = 20 μm.

FIG. 4

An example of a bound sperm showing a deep anti-hook bend in the ampulla. Yellow arrow indicate the tip of hook in sperm head; red arrows indicate a ridge of ciliated epithelial cells. The image is taken from the Supplemental Movie S4. Bar = 20 μm.

FIG. 5

An example of sperm detaching from the epithelium in the ampulla during strong contractions of the oviduct wall, taken from the Supplemental Movie S5. The sperm can be seen more clearly in the movie. The sperm head is indicated by yellow arrows. A) A sperm bound to the ampulla wall before contraction. Red arrows indicate the sperm flagellum. Inset: tracing of the sperm. B) This sperm still bound to the ampulla wall at the beginning of contraction. C) The moment that this sperm detached during wall contraction. Red arrows indicate the direction in which the sperm moved after detachment. Bars = 20 μm.

FIG. 6

Pendular movement of COCs in the ampulla caused by oviductal contractions. Yellow arrows indicate oocytes surrounded by the cumulus. The image is taken from the Supplemental Movie S6. The time interval between each sequential image (from left to right) is 3 sec. Bars = 100 μm.

FIG. 7

Examples of fluorescent sperm in the isthmic reservoir at 3 h postcoitus. A) Oviduct incubated in control medium. B) Oviduct incubated with 4 μM nicardipine. Ut, uterus; Amp, ampulla. Yellow arrows indicate the reservoir in the first loop of the isthmus. Images are taken from the Supplemental Movies S7 and S8. Bars = 300 μm.