Spermatozoa released from Sertoli cells must be transported to the epididymis. However, the mechanism of the luminal flow in seminiferous tubules has remained unclear to date. Therefore, in this study, we investigated luminal flow and movements in the seminiferous tubules by three-dimensional analysis and in vivo imaging. Serial 5-μm-thick mouse testicular sections at 50-µm-intervals were prepared and stained by Periodic Acid Schiff-hematoxylin. After three-dimensional reconstruction of the seminiferous tubules, the localization of the released spermatozoa and the stages observed in the sections were recorded in each reconstructed tubule. Luminal movements in the seminiferous tubules were observed by in vivo imaging using a fluorescent-reporter mouse and two-photon excitation microscopy system. Spermatozoa without contact to the seminiferous epithelium were not accumulated toward the rete testis. Additionally, such spermatozoa were found on their way not only to the most proximal rete testis but also a more distant rete testis from any stage VIII seminiferous epithelia. In vivo imaging demonstrated that the direction of the flagella of spermatozoa attached to the seminiferous epithelium was repeatedly reversed. The epithelium at the inner curve of the seminiferous tubule was shaken more actively and had fewer spermatozoa attached compared with the epithelium at the outer curve. Our results hence suggest that the luminal flow in the seminiferous tubules is repeatedly reversed and that this physical force helps spermatozoa to be released from Sertoli cells.

Introduction: Spermatozoa released from Sertoli cells must be transported to the epididymis. However, the contribution of the peristaltic motion in the seminiferous tubule to sperm release and transport remains unclear. We, therefore, investigated luminal flow and movements in the seminiferous tubules by three-dimensional analysis and in vivo imaging. Materials and Methods: Serial testicular sections were cut in 5-μm-thick and 50-μm-interval and stained by PAS-hematoxylin. After the three-dimensional reconstruction of the seminiferous tubules, the localization of the flowing spermatozoa and stages observed in the sections were recorded in each reconstructed tubule. The luminal movements in the seminiferous tubule were observed by in vivo imaging using a fluorescent-reporter mouse and two-photon excitation microscopy system. Results: Flowing spermatozoa were mainly scattered in the lumina at stage VII/VIII, and clustered spermatozoa were also found in some regions. The clustered spermatozoa were observed at zero to two regions in each seminiferous tubule. Flowing spermatozoa were also found in the opposite direction to the rete testis. The flagellum direction of the spermatozoa attached to the seminiferous epithelium was reversed within a few seconds to a few tens of seconds when observed by in vivo imaging. The epithelium at the inner curve of the seminiferous tubule moved more actively and attached fewer spermatozoa compared to that at the outer curve. Discussion: This study revealed the presence of repeatedly reversed luminal flow in the seminiferous tubule. Such movements are suggested to help the sperm release from the Sertoli cells and the following aggregation of the released spermatozoa.

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