Optical Clearing and 3D‐Histology of Murine Testis

Abstract

Optical clearing techniques (e.g., CLARITY, Scale, SeeDB, and 3DISCO) provide unprecedented opportunities to study large tissue samples at histological resolution, eliminating the need for physical sectioning while preserving the three‐dimensional structure of intact biological systems. Although these methods have been used primarily to investigate nervous system tissues, there is significant potential for applying optical clearing to reproductive tissues. In testicular biology, for example, the study of spermatogenesis and the use of spermatogonial stem cells offer high‐impact applications in fertility medicine and reproductive biotechnology. Therefore, the objective of our study is to apply optical clearing and immunofluorescence to testicular tissue in order to reconstruct its three‐dimensional microstructure in intact large samples. We used Triton‐X detergent‐based clearing in combination with Refractive Index Matching Solution to achieve optical transparency of fixed mouse testes. An antibody against smooth muscle actin (SMA) was used to label peritubular myoid cells of seminiferous tubules while an antibody against ubiquitin C‐terminal hydrolase (UCHL1) was used to label Sertoli cells and spermatogonia in the seminiferous epithelium. Specimens were then imaged using confocal fluorescence microscopy. Our results demonstrate that we were able to successfully clear testicular tissue and utilize immunofluorescent probes. Even with a basic confocal microscope and conventional objective lenses, we were able to image through as much as 500 microns of cleared tissue (the mechanical limit of our microscope). Moreover, we successfully visualized the histological compartments of testicular tissue in three‐dimensional reconstructions. The anti‐SMA antibody labeled the peritubular myoid cells in striking detail and exhibited their characteristic polygonal morphology. The antibody against UCHL1 provided clear visualization of mouse Sertoli cells and spermatogonia. When complemented with DAPI nuclear staining, the progressive stages of spermatogenesis could be observed proceeding from the basal compartment to the adluminal compartment of the seminiferous epithelium. In summary, optical clearing combined with immunofluorescence and confocal imaging offers a powerful new method to analyze the cytoarchitecture of testicular tissue at histological resolution while maintaining the large‐scale perspective of the intact system.Support or Funding InformationThis research was generously funded by the Arizona Alzheimer's Consortium and Midwestern University.