VASA protein and gene expression analysis of human non-obstructive azoospermia and normal by immunohistochemistry, immunocytochemistry, and bioinformatics analysis

Abstract

VASA, also known as DDX4, is a member of the DEAD-box proteins and an RNA binding protein with an ATP-dependent RNA helicase. The VASA gene expression, which is required for human germ cell development, may lead to infertility. Immunocytochemistry and immunohistochemistry were used to examine the expression of VASA protein in the human testis sections of azoospermic patients, in-vitro and in-silico models. Some studies of fertile humans showed VASA expression in the basal and adluminal compartments of seminiferous tubules. Our Immunocytochemistry and immunohistochemistry in infertile humans showed expression of VASA in the luminal compartments of the seminiferous tubule. The immunohistochemical analysis of three human cases with different levels of non-obstructive azoospermia revealed a higher expression of VASA-positive cells. For this purpose, Enrichr and Shiny Gene Ontology databases were used for pathway enrichment analysis and gene ontology. STRING and Cytoscape online evaluation were applied to predict proteins' functional and molecular interactions and performed to recognize the master genes, respectively. According to the obtained results, the main molecular functions of the up-regulated and downregulated genes include the meiotic cell cycle, RNA binding, and differentiation. STRING and Cytoscape analyses presented seven genes, i.e., DDX5, TNP2, DDX3Y, TDRD6, SOHL2, DDX31, and SYCP3, as the hub genes involved in infertility with VASA co-function and protein-protein interaction. Our findings suggest that VASA and its interacting hub proteins could help determine the pathophysiology of germ cell abnormalities and infertility.

Figure 1

Immunohistochemy image of normal and non-obstructive azoospermia cells. (A) Immunohistochemy of non-obstructive azoospermia. (B) Immunohistochemy image of normal cells. VASA expression in normal is higher than non-obstructive azoospermia in-vivo; (1) VASA = red, (2) DAPI = blue nuclear staining, and (3) merge (Scale bar: 100 μm).

Figure 2

Immunohistochemical analysis of abnormal human testicular tissues from infertile men. (A) Low basal and high luminal VASA expression in this case: (A1) VASA = red, (A2) DAPI = blue nuclear staining, and (A3) merging; the white arrow shows undifferentiation spermatogonia, and the yellow arrow shows that differentiation cell (Scale bar: 50 μm). (B) Immunohistochemical analysis of abnormal human testicular tissues from infertile men (Sertoli cell). Vimentin expression in Sertoli cell: (B1) vimentin = green, (B2) Dapi = blue nuclear staining, and (B3) merging. The white arrow shows Sertoli cells (Scale bar: 50 μm). (C) Immunohistochemical study of infertile men's abnormal human testicular tissues. Low basal and high luminal VASA expression in cases. (C1) VASA = red , (C2) UTF1 = green, (C3) Dapi = blue nuclear staining, and (C4) merging. The white and yellow arrows show undifferentiated and differentiated spermatogonia, respectively (Scale bar: 50 μm).

Figure 3

Bright-field images of testicular cells from infertile humans in vitro. Testicular cells from human cases after expansion in culture: (A) Infertile human testicular cell expansion in culture, (B) Immunohistochemical analysis of VASA in human cells, (C) DAPI, (D) merge of DAPI and VASA, and (E) electron microscopy image representing cell features and the various diameters of the nucleus of infertility human SSCs in vitro. Scale bar (A–D) (25 μm) and (E) (2 μm).

Figure 4

Based on the Reactome and KEGG pathways and the STRING database, the STRING protein–protein interaction network is formed. (A) Spermatogenesis-regulated protein–protein interaction revealed significant coexpression. (B) Chosen regulation function interaction with VASA. Highlighting nodes by piRNA biosynthetic process, regulation of the reproductive process, piRNA binding, fertilization, male meiosis I, cell cycle, RNA binding, and other functions demonstrating hub genes.

Figure 5

The ratio of infertility genes expression: DDX5, TNP2, DDX3Y, TDRD6, SOHL2, DDX31, and SYCP3 in infertility cell (azoospermia) in comparison to a normal cell. There is a significant decrease in gene expression of DDX5, TNP2, and TDRD6 and an increase in DDX3Y, SOHL2, DDX31, and SYCP3 compared to a normal cell.