The structure of female reproductive tract and various sperm behaviours and interactions with epithelia in the uterus.

(A) An illustration of the female reproductive tract and sperm migration direction (arrows in orange colour) inside the tract. Ova (egg cells) are in the ampulla. (B) Spermatozoa alter their travel direction based on their head orientation upon reaching the uterine wall (sperm hook functions as a pivot, Movie S2A). Left image shows a sperm trajectory exhibiting pro-wall-hook direction, and the right image shows anti-wall-hook direction. The trajectory is depicted in colours representing different time points. Dashed lines are guides to the eye to visualize the uterus wall. Scale bar: 50 µm. (C) Sperm moving direction changes over time when they reach the uterine epithelia. Numbers indicate sequences of sperm movement. (1) During sperm migration, they reach the uterine wall. (2) After reaching the uterine wall, they beat several times while facing their head towards the uterine wall. (3) Sperm beating results in a change in sperm orientation (1 to 3′: pro-wall-hook, 1 to 3″: anti-wall-hook). The pro-wall-hook orientation coincides with sperm travelling along the wall and the anti-wall-hook orientation coincides with sperm moving away from the wall. (D) When spermatozoa reach the uterine wall, their trajectories predominantly follow the pro-wall-hook direction, where the sperm hook is directed towards the uterine wall. (E) The sperm hook assists a spermatozoon in anchoring to the epithelia (hook as an anchor). This anchoring facilitates sperm attachment to the uterine and UTJ epithelium and prevents spermatozoa from being swept away by internal flow caused by peristaltic movement. (F) The sperm hook and thin sperm head aid spermatozoa in squeezing through the sperm-crowded UTJ entrance (CT) and attaching to the epithelium by acting as an anchor (Movie S3). Note that in our experiments, we do not observe the principal and terminal pieces of spermatozoa due to a lack of fluorescence in our animal model. The observed acrosome and midpiece are shown coloured in the schematic figures.

Analysis of sperm kinetic parameters relative to the distance and angle between the sperm trajectory and uterine wall.

(A) Both VCL (top) and VSL (bottom) decreased with an increase in the distance between the sperm trajectory and the uterine wall. Similarly, VCL and VSL decreased as the angle between the sperm trajectory and uterine wall increased. (B) The distance between the sperm trajectory and uterine wall did not significantly affect LIN (top left) and SWR (bottom left). However, both LIN and SWR decreased when the angle between the sperm trajectory and uterine wall increased. Data from different males are represented in different colours and shapes. The dotted lines indicate regression lines from simple regressions to aid visual interpretation. Check model estimates for more details and precise interpretation of the models (Table S3; Fig. S3A, B). The y-axis of each figure is displayed in log-scale except for LIN. Images for sperm tracking were acquired around the area labelled as ‘Wall’ in the uterus in Figure 1A. Sperm trajectories used in this analysis are presented in Movie S1B. The effect of the two random variables (males and females) are visualised in Fig. S3.

The structure of the entrance of intramural UTJ and sperm passage.

(A) The upper three images are the vertical view of the CT (entrance to the UTJ) of the intramural UTJ from an unmated female. The bottom right image is a horizontal view of the intramural UTJ. There are only a few small gaps, indicated by arrows, between mucosal folds, which control sperm migration into the UTJ from the uterus (Movie S4). Illustrations at upper and lower right conners indicate imaging planes (blue rectangles) and the intramural UTJ (orange cylindrical structure) with the CT (black tilde). Scale bar: 100 µm, Arrow line thickness: 10 µm for top three images, 5 µm right bottom image. (B) Asynchronised movement of mucosal folds (sliding against each other) at the CT due to uterine and UTJ contractions enable spermatozoa to slide into the intramural UTJ from the uterus (Movie S5). Two dashed arrows (in blue) indicate the direction of sperm migration from the uterus to the UTJ, and the dashed arrow in the centre (in orange) indicates the direction of sperm sliding in the intramural UTJ. The two curved black arrows indicate asynchronised (opposite) movement of confronting mucosal folds in the intramural UTJ.

Self-organised sperm behaviour at CT inside the uterus.

(A) The apical shape of the sperm head, due to the sperm hook, results in head asymmetry. This asymmetrical falciform head shape may facilitate sperm re-arrangement and clustering at uterine wall (Movie S6). The circle highlights spermatozoa undergoing unidirectional re-arrangement over time. The elapsed time after the first frame is shown in the upper left of the images. The right lower zoom-in inset shows an instant of synchronised motion and unidirectional re-arrangement. Scale bar: 50 µm. (B) Unidirectional sperm clustering at the entrance of the intramural UTJ (CT, indicated by a dashed line) is marked with arrows. Such large sperm clustering resulted in synchronised sperm beating at the CT (Movie S7). Scale bar: 200 µm. Note that due to a lack of fluorescence, the principal and terminal pieces of sperm tails are not seen in the images.

Comparative trajectories and kinetics of linked spermatozoa (sperm trains) and unlinked single spermatozoa

(A) The projected images, comprising 60 frames, depict the trajectories of sperm trains and unlinked single spermatozoa. Each of the images in the lower left corner shows a sperm train that was traced. Movie S8 shows how the three traced sperm trains are moving. The colour bar located at the bottom centre represents the VCL of each sperm trajectory, with blue indicating slower speeds and red indicating faster speeds. (B) The boxplots, which include individual data points, represent the kinetic parameters of the sperm trains and unlinked single spermatozoa. The parameters, including curvilinear velocity (VCL), straight-line velocity (VSL), linearity of forward progression (LIN), and straight line-to-sideward movement ratio (SWR), were computed using images of 100x100 pixels that contained a sperm train. The three sperm trains that were traced did not exhibit a faster VCL or VSL, nor a higher LIN. However, it is still possible that the SWR is higher in the sperm train. The lines within the boxes represent the medians, and the whiskers represent 1.5 times the interquartile ranges, and the symbols show the individual data points.

Sperm migration through narrow luminal space in UTJ and various sizes of accumulated sperm in the oviduct.

(A and B) Spermatozoa can penetrate narrow space with their thin head and anchor their hook when they pass through the narrow gaps between mucosal folds during migration in the UTJ (Movie S9A). Scale bar: 50 µm. (C) Spermatozoa can only migrate through the UTJ when the luminal space is extended due to oviductal contraction and relaxation in the UTJ. (D) Entangled spermatozoa in the oviduct, including the UTJ and isthmus, were predominantly made up of inactive acrosome reacted spermatozoa. These entangled spermatozoa obstruct the migration of other active spermatozoa and can cause damage to live spermatozoa. Scale bar: 50 µm.