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

(A) Both VCL (top) and VSL (bottom) showed a decrease 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). The y-axis of each figure is displayed in log-scale except for LIN.

The sperm hook plays an important role in sperm migration through the female reproductive tract by facilitating interactions between sperm and epithelia.

(A) Sperm alter their travel direction based on their head orientation upon reaching the uterine wall (sperm hook functions as a pivot, Movie S2A). The trajectory of a sperm cell is depicted in colours representing different time points. Scale bar: 50 µm. (B) The illustration shows how sperm travel direction changes. Numbers in the illustration indicate the sequences of sperm movement. (C) The direction of sperm travel may be influenced by the orientation of sperm hook to the wall (pro-wall or anti-wall) while they migrate along the uterine epithelium. (D) When sperm reach the uterine epithelium, their trajectories predominantly follow the pro-wall-hook direction, where the sperm hook is directed towards the uterine epithelium. (E) The sperm hook may assist a spermatozoon in anchoring to the epithelia (hook as an anchor). This anchoring may facilitate sperm attachment to the uterine and UTJ epithelium and help them resist changes in internal flow direction due to peristaltic movement. (F) The sperm hook and thin sperm head may aid sperm in squeezing through the sperm-crowded UTJ entrance (CT) and attaching to the epithelium by acting as an anchor (Movie S3 and Fig. S3). Note that the principal and terminal pieces of sperm in all illustrated diagrams do not represent the entire sperm shape and beating motion of the sperm tail due to a lack of fluorescence.

The apical sperm hook may facilitate sperm entry into the UTJ through the CT by aiding in sperm attachment and sliding.

(A) The structure of the CT (entrance to the UTJ) of the intramural UTJ of an unmated female is shown. There are only a few small gaps indicated by arrows between mucosal folds, which may limit sperm migration into the UTJ from the uterus (Movie S4). Scale bar: 100 µm. (B) Asynchronised movement of mucosal folds at the CT due to uterine and UTJ contractions may enable sperm to penetrate or 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. (C) The apical shape of the sperm head, due to the sperm hook, results in head asymmetry. This asymmetrical falciform head shape may also facilitate sperm re-arrangement and clustering at crypts in the uterus (Movie S6). The circle highlights sperm undergoing unidirectional re-arrangement over time. The elapsed time after the first frame is shown in the upper left of the images. The right upper zoom-in inset shows an instant of synchronised motion and unidirectional re-arrangement. Scale bar: 50 µm. (D) Unidirectional sperm clustering in the uterus at the entrance of the intramural UTJ (indicated by a dashed line) is marked with arrows. Such large sperm clustering sometimes results in synchronised sperm beating at the entrance of the UTJ (Movie S7). Scale bar: 200 µm. Note that due to a lack of fluorescence, the principal and terminal pieces of sperm in all illustrated diagrams do not represent the actual sperm shape and beating motion of the entire sperm tail.

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

(A) The projected images, comprising 60 frames, depict the trajectories of sperm trains and unlinked single spermatozoa. 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. In the few observed cases, the sperm trains 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.

Illustration of sperm migration through narrow inter-luminal gaps in UTJ and various size of accumulated sperm in the female reproductive tract.

(A and B) The pointed sperm hook and thin head shape may assist sperm in passing through narrow gaps between mucosal folds during their migration through the UTJ (Movie S9A). Scale bar: 50 µm. (C) Sperm can only migrate through the UTJ when the luminal space is extended due to oviductal contraction and relaxation in the UTJ. (D) Various sizes of sperm accumulations (sperm trains) in the uterus. Sperm trains (or sperm assemblage) were not observed to swim faster than individual sperm in the uterus. Scale bar: 100 µm. (E) Accumulations of sperm in the oviduct, including the UTJ and isthmus, were primarily composed of dead and inactive sperm. These accumulated sperm may obstruct the migration of other active sperm or could cause damage to live sperm. Scale bar: 50 µm. Note that due to a lack of fluorescence, the principal and terminal pieces of sperm in all illustrated diagrams do not represent the actual shape and beating motion of the sperm tail.

Schematic diagram of the custom-built 2PSLM.

DsRed and eGFP were excited nonlinearly using a tunable high peak power femtosecond laser (Chameleon discovery/Coherent) and a water-immersion objective lens (CFI75 LWD 16X W/Nikon). The fluorescence emitted was collected by the same objective lens and detected by a pair of GaAsP photomultiplier tubes (PMT, H10770PA-40/Hamamatsu). Dichroic mirrors and filters; DM1(T735lpxrxt-UF3/Chroma), DM2(T565lxr/Chroma), BF(ET720SP-2P8/Chroma), F1(ET605/70m/Chroma), and F2(ET525/70m/Chroma) were used to split the excitation beam and emission light. A resonant-galvo scanner (RESCAN-GEN/Sutter instrument) enabled real-time fluorescence imaging of sperm behaviour at a speed of 30 frames per second for 512 pixels per line acquisition. A Pockels cell (M350-80-LA-02 KDP/Conoptics) allowed for rapid control of the laser beam intensity, homogenising the illumination across the field of view, and applying varying laser power per tissue depth. The sample position in all three dimensions was controlled by a Piezo-driven objective scanner (P-725.4CA/PI) and a motorised 3-axes stage (3DMS/Sutter instrument). A small animal heating plate (HP-4M/Physitemp) maintained the warmth of the mouse female reproductive tract at body temperatures.

Uterus wall and parameters that were used to measure sperm migration speed and linearity.

(A) Areas along the uterine wall that were relatively straight were selected. The boundary of these areas was identified using the object selection tool in Adobe Photoshop CC (23.1.0 version). (B) The uterine wall was approximated as a linear line using linear regression (①). The distance between a spermatozoon and the uterine wall was defined as the minimum distance between the midpoint of the track displacement and a sperm trajectory (②). The angle between sperm trajectories and the uterine wall was calculated as the angle (in radians) between the uterine wall (approximated line) and the straight line that connected the first and last points of a sperm trajectory – track displacement line (③). The maximum sideward movement was determined as the greatest distance between the parallel lines that aligned with the track displacement line at the positions of the sperm trajectory (④). SWR was then computed by dividing the track displacement by the maximum sideward movement.

A hypothetical model for sperm migration from the uterus to UTJ.

The upper left inset represents a uterine horn that moves to the left or right due to muscle contraction (exaggerated for visualization). The 5 subfigures with numbering represent zoom-in of the two red square frames in the inset. When the uterine horn moves from the centre to the right (① to ②), two facing surfaces between the two mucosal folds slide against each other. This sliding results in opening space where sperm can ascend – note that the sperm moves from cell 1 to cell 2 of the right mucosal fold (②). When the uterine horn moves from right to left (③), the two surfaces between the mucosal folds slide in opposite directions where the sperm can now reach cell 4 of the left mucosal fold. If sperm can turn over, its head can be attached to cell 4 of the left mucosal fold (④ to ⑤). Repetition of these procedures will make the sperm finally pass the CT and migration through narrow gaps between mucosal folds in intramural UTJ. This process appears to be as if sperm may slide through the space between mucosal folds when the space is too small for normal beating (Movie S5C).

Basic information of 4 males that were used for the mating experiment.

Mating records and the information of the females for sperm tracking.

Summary results of the generalized linear mixed models (GLMM).

Each model represents sperm trajectory parameters that were log-transformed. In all models, we examined the effect of sperm to uterine wall distance (Distance from wall), angle between a sperm trajectory and uterine wall (respective angle with wall) and cropping of the acquired image (O: cropped vs X: uncropped). The GLMM for SWR showed a boundary (singular) fit warning message. However, two models that omitted one of random variables (Male or Date) did not result in any significant changes in the predictor variables (p > 0.05).