Three-dimensional analysis and in vivo imaging for sperm release and transport in the murine seminiferous tubule.

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. In brief: Spermatozoa are released from Sertoli cells and flow in the seminiferous tubule to the rete testis. Our results suggest that the luminal flow in the tubules is repeatedly reversed and that this physical force helps spermatozoa release from the Sertoli cells.

Three-dimensional analysis of seminiferous tubules and spermatogenic waves in mice.

The aim of the present study was to reconstruct seminiferous tubules and analyze spermatogenic waves in seminiferous epithelia in developing and adult mice using serial paraffin sections and high-performance three-dimensional (3D) reconstruction software. By labeling the basement membrane of seminiferous tubules with fluorescent immunohistochemistry or periodic acid-Schiff-hematoxylin staining, all seminiferous tubules were reconstructed in 9 testes from 9 different mice, 3 each at 0, 21 and 90 days (adult) postpartum. The 3D structure of seminiferous tubules, including the number and length of tubules as well as the number of connections with the rete testis, branching points and blind ends, was assessed accurately. Although tubules showed marked variations among individual mice, their overall structure was regular and retained from newborn to adult mice. Some seminiferous tubules contained inner portions running distant from the testis surface. In a representative testis at 21 days, the sites at which spermatids initially occurred were examined by labeling acrosomes and were found to be preferentially distributed in the upper and medial portions of the testis close to the rete testis. In a representative adult testis, 76 complete waves with an average length of 16.9 mm were found and their directions were analyzed. The methods used in the present study will be useful for investigating the structure and function of seminiferous tubules in mice and humans under normal and pathological conditions, such as infertility.