Human Testicular Endothelial Cells

Characterizations and Proteomic Analysis of TECs-Exos

TECs play an integral role in maintaining spermatogonial stem cells by producing specific factors vital for spermatogenesis. Exosomes facilitate cell communication, thus influencing physiological processes including sperm formation. However, the involvement of TECs in spermatogenesis has not been extensively studied.

Song et al. leveraged ultracentrifugation to isolate HTEC-Exos, followed by characterization through nanoparticle tracking analysis, TEM, and western blotting. The exosomes were round or elliptical, most of them between 70-200 nm in diameter (Fig. 1a and 1b). Exosomal markers TSG101, CD63 and CD9 were found by Western blotting but not the endoplasmic reticulum marker calnexin (Fig. 1c). HTEC-Exos had 945 proteins and 5470 peptides in LC-MS. The proteins were identified with more than half (562 of 945, 59.5%) containing 2 different peptides (Fig. 2a), which made the identification confidence high. KEGG ranking indicated the top 20 signaling pathways, where the number of "Ribosome", "Focal adhesion" and "ECM-receptor interaction" pathways are very rich (Fig. 2b). GO analysis oriented around molecular activity, cellular component, and biology. Proteins were high in "cell-cell adhesion", "signal transduction", and "translational initiation" (Fig. 2c), but also in "extracellular exosome", "cytosol" and "cytoplasm" (Fig. 2d). Molecular activities were "binding proteins", "binding poly(A) RNA" and "binding ATP" (Fig. 2e).

Fig. 1. Proteomic analyses of the exosomes from testicular endothelial cells (Song WP, Gu SJ, et al., 2022).

Fig. 2. miRNA profiling of the exosomes from testicular endothelial cells (Song WP, Gu SJ, et al., 2022).

Human-Mouse Comparisons in Intra-Niche and Niche-Germline Interaction

Human spermatogenesis involves SSC differentiation into mature sperm, regulated by the testis niche. While progress in understanding these processes has been made in mice, less is known about human SSC regulation. Using single-cell RNA sequencing, Guo et al. analyzed ~6500 unselected testicular cells from young adults via the 10× Genomics Chromium platform, providing a transcriptional atlas.

In human testicular endothelial cells (Cluster 10, marked by VWF and PECAM1), NOTCH signaling components (NOTCH4, JAG1, HES1, and MAML1) were up-regulated (Fig. 2b). Hedgehog pathway receptors (PTCH1, PTCH2) and downstream elements (GLI and IGFBP6) were highly expressed in human adult myoid (Cluster 11, marked by MYH11 and ACTA2) and Leydig cells, indicating ongoing Hedgehog and NOTCH signaling in adult human testes (Fig. 3c). Sertoli cells (Cluster 12, marked by SOX9 and AMH) express ITGA6, present in seminiferous tubule membranes (Fig. 3d). Sertoli cells also express WFDC2 and PRND, related to sperm maturation and interaction (Fig. 3d). Leydig cells (Cluster 13, marked by DLK1 and IGF1) expressed IGF binding proteins (IGFBP5, IGFBP3), INHBA, VIT, and testosterone biosynthesis genes STAR and HSD17B3. Responsive genes SHBG and SRD5A2 appeared in maturing sperm (Fig. 3f). Leydig and myoid cells expressed RA synthesis enzymes ALDH1A1 and ALDH1A3; STRA8 was present during spermatogonia to spermatocytes transition (Fig. 3f). WNT2B was in myoid cells, its receptors in primary spermatocytes, suggesting a meiosis role (Fig. 3f). PDGFB in endothelial cells, with receptors in Leydig and myoid cells, suggests endothelial influence via niche cell interaction. These data reveal similarities and differences in germline-niche interactions between humans and mice, paving the way for detailed studies.

Fig. 3. Expression patterns of representative genes marking niche cells, and Niche-Germline interactions (Guo J, Groe E J, et al., 2018).