Clearing, immunofluorescence, and confocal microscopy for the three-dimensional imaging of murine testes and study of testis biology.

Optical clearing techniques 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. 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. The objective of our study is to apply optical clearing, immunofluorescence, and confocal microscopy to testicular tissue in order to reconstruct its three-dimensional microstructure in intact samples. We used Triton-X/DMSO clearing in combination with refractive index matching to achieve optical transparency of fixed mouse testes. An antibody against smooth muscle actin was used to label peritubular myoid cells of seminiferous tubules while an antibody against ubiquitin C-terminal hydrolase was used to label Sertoli cells and spermatogonia in the seminiferous epithelium. Specimens were then imaged using confocal fluorescence microscopy. We were able to successfully clear testicular tissue and utilize immunofluorescent probes. Additionally, we successfully visualized the histological compartments of testicular tissue in three-dimensional reconstructions. 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 macro-scale perspective of the intact system. Considering the importance of the murine model, our developed method represents a significant contribution to the field of male reproductive biology, enabling the study of testicular function.

Altered Energy Metabolism Pathways in the Posterior Cingulate in Young Adult Apolipoprotein E ɛ4 Carriers.

The APOE gene, encoding apolipoprotein E, is the primary genetic risk factor for late-onset Alzheimer's disease (AD). Apolipoprotein E ɛ4 allele (APOE4) carriers have alterations in brain structure and function (as measured by brain imaging) even as young adults. Examination of this population is valuable in further identifying details of these functional changes and their association with vulnerability to AD decades later. Previous work demonstrates functional declines in mitochondrial activity in the posterior cingulate cortex, a key region in the default mode network, which appears to be strongly associated with functional changes relevant to AD risk. Here, we demonstrate alterations in the pathways underlying glucose, ketone, and mitochondrial energy metabolism. Young adult APOE4 carriers displayed upregulation of specific glucose (GLUT1 & GLUT3) and monocarboxylate (MCT2) transporters, the glucose metabolism enzyme hexokinase, the SCOT & AACS enzymes involved in ketone metabolism, and complexes I, II, and IV of the mitochondrial electron transport chain. The monocarboxylate transporter (MCT4) was found to be downregulated in APOE4 carriers. These data suggest that widespread dysregulation of energy metabolism in this at-risk population, even decades before possible disease onset. Therefore, these findings support the idea that alterations in brain energy metabolism may contribute significantly to the risk that APOE4 confers for AD.