Sperm gatekeeping: 3D imaging reveals a constricted entrance to zebra finch sperm storage tubules

Females across many internally fertilising taxa store sperm, often in specialised storage organs in their reproductive tracts. In birds, several hundred sperm storage tubules exist in the utero-vaginal junction of the oviduct and there is growing evidence that sperm storage in these tubules is selective. The mechanisms underlying female sperm storage in birds remain unknown due to our limited ability to make three dimensional, live observations inside the large, muscular avian oviduct. Here, we describe a new application of fluorescence selective plane illumination microscopy to optically section oviduct tissue from zebra finch Taeniopygia guttata females label-free, by harnessing tissue autofluorescence. Our data provide the first description of the 3D structure of sperm storage organs in any vertebrate and reveal the presence of gate-like constricted openings that may play a role in sperm selection. Statement of Significance Female birds can store sperm in microscopic tubular structures in their reproductive tract for up to several months, depending on species. Studying these sperm storage tubules has been a major challenge due to the muscular and opaque nature of reproductive tracts in birds. We have developed a new method for imaging live reproductive tract tissue using selective plane illumination microscopy, a fluorescence microscopy technique. From these images, we could extract three-dimensional measurements of sperm storage tubules and found these structures to have a gate-like constriction, providing evidence that females can actively select sperm at storage and ultimately influence the paternity of her offspring. Understanding these reproductive adaptations can help improve captive breeding programs and similar conservation strategies.