Researchers led by Irina V. Larina from Baylor College of Medicine have developed a new imaging approach that incorporates Imaris for three-dimensional (3-D) tracking of sperm in the fallopian tube– or oviduct–of a mouse. The new technique could make it possible to integrate the complex biochemical and biomechanical properties of the oviduct into the analysis of sperm activities to understand natural sperm-oviduct interactions.
How the oviduct guides and regulates sperm towards fertilization has long been a major question in mammalian reproduction. An in-depth understanding of the underlying mechanisms involved in this process would improve management of infertility and aid in the development of novel contraceptive methods.
Imaging the fertilization site
Studying sperm at their fertilization site in the mammalian oviduct is challenging because it is hard to gain access to the area for imaging. Thus, most studies examining sperm dynamics have relied on in vitro and ex vivo experiments where the sperm or the oviduct containing the sperm is imaged in a petri dish, away from the natural fertilization environment. For this reason, sperm behaviors prior to fertilization remain largely unknown and in vivo evidence is greatly needed to confirm in vitro and ex vivo findings.
Imaris was used to visualize the structure of the oviduct and produce the 3D trajectory of sperm. The trajectory shows the sperm being collected by the epithelium of the mouse oviduct ampulla in vivo. In this process, the sperm swimming in the isthmus-to-ovary direction moves from the central lumen towards the ampulla epithelium. The sperm trajectory is color-coded according to time. The arrow shows the overall direction of sperm movement. Courtesy of Shang Wang and Irina V Larina, Baylor College of Medicine.
To address this technical hurdle and enable novel in vivo studies of ejaculated sperm, the researchers developed an optical imaging approach based on optical coherence tomography (OCT), a 3-D depth-resolved micro-scale-resolution imaging modality. The new method relies on the movement of motile sperm for identification, and thus doesn’t require transgenic mouse lines or exogenous labeling. Using this approach, the researchers captured and quantitatively analyzed sperm behaviors in the oviduct ampulla, where fertilization occurs.
The new imaging approach made use of Imaris TrackLineage for 3-D tracking of sperm inside the oviduct lumen and for determining the 3-D location, velocity, and distance to the epithelium for the sperm. “After a trajectory is built, Imaris provides a number of very useful measurements of the tracks, such as the 3D position and velocity over time, which has allowed us to generate quantitative measures of the sperm behaviors,” Larina said. “The Imaris Spots function with a scalable labeling ball and the Imaris Ssurfaces rendering function were also important for measuring the 3-D distance between the sperm and the oviduct epithelium.”
The researchers used Imaris 3-D rendering the 4-D rendering feature of Imaris to reconstruct time-lapse volumetric OCT data that contained the oviduct tissue and images of tissue scatters inside the oviduct lumen. With the Imaris tracking function, they located and track the scatters to form a 3-D trajectory. For each time point within the trajectory, the researchers exported the spatial position and velocity amplitude from Imaris and calculated a newly defined parameter, known as the standard deviation of direction variation, for the trajectory. This new parameter was used to identify the motile sperm.
To quantitatively test the relationship between a sperm’s velocity and its distance to the oviduct epithelium, they measured the 3-D distance in Imaris. After using the Imaris Surfaces rRendering feature to create a surface of the oviduct epithelium, they scaled the ball used to label the sperm location so that it just touched the surface of the epithelium. This allowed them to use the radius of the ball to determine the 3D distance between the sperm and the oviduct epithelium.
New sperm behaviors
“Our new method revealed a number of interesting sperm behaviors in the oviduct that were previously unknown,” said Larina. “Some of the behaviors we observed suggest the sperm are possibly guided by rheotaxis, or swimming against the flow, in response to cilia-generated fluid flows in the ampulla. We also saw cooperation-related behaviors and movements that indicated the possible existence of multiple regulatory cues that guide sperm towards fertilization or to avoid polyspermy.”
Next, the researchers plan to use genetically manipulated mouse models and their new in vivo sperm imaging approach to test whether oviduct ciliary activity regulates the sperm behaviors in the ampulla. They also want to image the fertilization process in vivo as it happens in the mouse oviduct ampulla. For both studies, they plan to use Imaris for rendering, visualization, tracking, and quantification.