Investigation of CD133 and CD24 as candidate azoospermia markers and their relationship with spermatogenesis defects

Abstract

Spermatogenesis is initiated when spermatogonial stem cells (SSCs) in the mature testes enter mitosis and trigger differentiation. Thus, spermatogenesis and the ability to maintain a continuous source of spermatogonia relies on the ability to differentiate SSCs. Many studies around the world have been performed to investigate the etiology of male infertility and recent studies have focused on the presence and identification of biomarkers. CD133 and CD24 are stem cell markers locating in the testis and spermatogonia. The aim of this study was to investigate the relationship of the CD133 and CD24 genes with spermatogenesis defects and examine them as a candidate a useful biomarker for azoospermia men. The association of CD133 and CD24 with spermatogenesis defects was investigated in patients with obstructive (O) and non-obstructive azoospermia (NOA). NOA cases were histopathologically classified into Hypospermatogenesis (HS), Maturation Arrest (MA), and Sertoli Cell Only Syndrome (SCO) groups. A qRT-PCR analysis of these genes was performed and protein expression levels were measured by Western blot analysis. CD133 expression in NOA group was found to be significantly different from OA and this was confirmed by immunohistochemistry and immunocytochemical assays.The qRT-PCR analysis revealed that gene expression of CD133 and CD24 had fold changes of 0.80 ± 0.34 and 1.59 ± 0.31 compared to controls, respectively in the HS group (p > 0.05) and 0.04 ± 0.01 and 0.54 ± 0.08 in the MA group (p < 0.05). In the SCO group, CD24 showed a 1.55 ± 0.35-fold increase (p > 0.05). CD133 gene expression was not detected at the transcriptional level in the SCO group. Western blot analysis of CD133 protein expression revealed 1.83, 4.11, and 11.4-fold decreases in the HS, MA and SCO groups, respectively, compared to controls (p < 0.05). CD24 showed fold changes of 1.18, 0.38, (p < 0.05), and 0.89 in the HS, MA, and SCO groups, respectively. Immunohistochemical analysis of CD133 revealed moderate, partial staining in the HS group, compared to substantial, wide-spread staining in the OA group. No staining was detected in either the MA or SCO groups. The localization of CD133 in healthy sperm was determined to be prominent in the tail and partly expressed in the head by confocal laser scanning microscopy analysis. It was also found that the expression of CD133 protein was high in healthy commercially-sourced Sertoli cells as well as in the Sertoli cells of OA individuals. Data from this study show that CD133 exhibits different profiles in infertile patient groups and thus may be considered as a candidate biomarker. CD24 can be associated with blockage of germ cell maturation in the MA group. Curative protocols for spermatogenesis defects may be possible with the use of these markers and thus their identification is extremely valuable in terms of human reproductive health.