Fig. 1.

Characterization of testicular extracellular vesicles and their involvement in cell communication in the testicular microenvironment. A Representative transmission electron microscopy image of testicular EVs isolated from one-step testis dissociation followed by affinity columns. Scale bar: 500 nm. B Size distribution of testicular EVs isolated as in A determined by dynamic light scattering. C Representative Western blot analysis of EV markers CD81, CD63, and β-tubulin, Golgi marker Golgin 97 and endoplasmic reticulum marker Calnexin in testicular EVs isolated by affinity columns (column) or in exosomes (exo) isolated by differential centrifugation (D.C.). Dissociation buffer and elusion buffer were used as negative control. D Representative fluorescent Z-stack images of spermatogonia cell lines (C18-4 and GC1-spg) and Sertoli cell line (TM4) incubated with PKH67 labeled (green) testicular EVs for 24 h. The cells were stained for F-actin (phalloidin, red) and nucleus (Hoechst 33342, blue). Scale bar: 20 μm. E Representative histograms showing the percentage of C18-4, GC1-spg, and TM4 cells taken up the testicular EVs as analyzed by flow cytometry. Data is presented as mean ± S.D. F Representative fluorescent images showing the uptake of PKH67 labeled testicular EVs in interstitial space (left panel) (n=3) and in seminiferous tubules (right panel) (n=3). Immunohistochemical staining of Sertoli cell marker SOX9 (red) was shown. Nucleus were counterstained with DAPI (blue). Scale bar: 25 μm. Key: arrow - spermatogonia, arrowhead - spermatocytes, number sign - round spermatids, asterisk - elongated spermatids, and yellow arrowhead - Sertoli cells. G Representative fluorescent Z-stack images of isolated round spermatid (RS), elongated spermatid (ES), and testicular sperm incubated with PKH67 (green) labeled testicular EVs for 3h. Nucleus were stained by DAPI (blue), and the acrosomes were stained by PNA (red). Scale bar: 20 μm. Supporting data values available in Additional file 6: Table S5. Uncropped blots available in Additional file 7: Fig S8