Characterization of DDX4 Gene Expression in Human Cases with Non-Obstructive Azoospermia and in Sterile and Fertile Mice

Abstract

Background: In mammals, spermatogenesis is the main process for male fertility that is initiated by spermatogonial stem cells (SSCs) proliferation. SSCs are unipotent progenitor cells accountable for transferring the genetic information to the following generation by differentiating to haploid cells during spermato-and spermiogenesis. DEAD-box helicase 4 (DDX4) is a specific germ cell marker and its expression pattern is localized to, spermatocytes, and spermatids. The expression in the SSCs on the basement membrane of the seminiferous tubules is low.

Methods: Immunohistochemistry (IHC) and Fluidigm reverse transcriptase-polymerase chain reaction (RT-PCR) were used to analyze the expression of DDX4 in testis tissue of fertile and sterile mice and human cases with non-obstructive azoospermia.

Results: Our immunohistochemical findings of fertile and busulfan-treated mice showed expression of DDX4 in the basal and luminal compartment of seminiferous tubules of fertile mice whereas no expression was detected in busulfan-treated mice. The immunohistochemical analysis of two human cases with different levels of non-obstructive azoospermia revealed more luminal DDX4 positive cells.

Conclusion: Our findings indicate that DDX4 might be a valuable germ cell marker for analyzing the pathology of germ cell tumors and infertility as global urological problems.

Keywords: DDX4 protein; Seminiferous tubule; Spermatogonial Stem Cell; Testicles.

Figure 1.

Immunohistochemical analysis of two abnormal human testicular tissues from infertile men. Low basal and high luminal DDX4 expression in case 1 (Merge, A1) and moderate expression in case 2 in luminal cells (Merge, B1). Dapi=blue nuclear staining (A3, B3)

Figure 2.

Immunohistochemical analysis of sterile and fertile mice. Non-detectable expression of DDX4 in busulfan-treated mice (Merge, A1). Expression of DDX4 in the germinal epithelium of fertile mice (Merge, A1)

Figure 3.

Bright field images of testicular cells in sterile and fertile mice and infertile human in vitro. Testicular cells from sterile mouse after expansion in culture (A). Infertile human testicular cell expansion in culture (B). Immunohistochemical analysis of fertile mouse testicular cells. Bright field of testicular cells of fertile mice (C1), testicular cells of fertile mice stained with blue DAPI (C2), testicular cells of fertile mice stained with DDX4 (C3), and merge of DAPI and DDX4 (C4)

Figure 4.

Immunohistochemical analysis and RT-PCR of DDX in 1 and 4 week old mice. Luminal expression of DDX4 in seminiferous tubules of 1 week old mouse (A). The expression pattern of DDX4 in a 4 week old mouse showed that the DDX4 positive cells were redistributed from the lumen to the basal compartment of seminiferous tubules (B). Significantly increased expression of DDX4 in 4 week old mouse testis in comparison to 1 week old mice by Fluidigm real-time PCR analysis (C)