Sertoli cells from non-obstructive azoospermia and obstructive azoospermia patients show distinct morphology, Raman spectrum and biochemical phenotype

Abstract

Are there differences in the morphology, spectrum and biochemical phenotype between Sertoli cells from non-obstructive azoospermia (NOA) patients and those from obstructive azoospermia (OA) patients with normal spermatogenesis? Sertoli cells from NOA patients are distinct from those from OA patients in terms of morphological features, Raman spectrum and phenotype including the expression of genes and proteins (e.g. SCF, BMP4 and GDNF). NOA affects 10% of infertile men and has been diagnosed in 60% of azoospermic men. In contrast with OA patients who have normal spermatogenesis, NOA patients have an impaired spermatogenesis. This case-control study included 100 NOA patients (as cases) and 100 OA patients with normal spermatogenesis (as controls). The study was performed between January 2012 and January 2013. Karyotype analysis was performed to check the chromosome content and multiplex PCR was carried out to determine the expression of numerous Y chromosome genes in NOA patients. Human Sertoli cells were then isolated from the testes of NOA and OA patients by two-step enzymatic digestion and differential plating. Transmission electron microscopy was used to determine the ultrastructure of the Sertoli cells and real-time Raman microspectroscopy was used to assess their spectrum. We further compared the two groups of patients for expression of SCF, GDNF and BMP4 in Sertoli cells, using RT-PCR, microarray analysis, immunofluorescence, immunohistochemistry and Western blots. NOA patients had normal chromosome karyotypes and Y chromosome microdeletions were excluded. In morphology, Sertoli cells isolated from NOA patients had a series of abnormal ultrastructural features compared with the control Sertoli cells: (i) existence of small and spindle-shaped nuclei, (ii) smaller diameter, (iii) deficient nucleolus or endoplasmic reticulum and (iv) more vacuoles. Spectral intensities in Sertoli cells of NOA patients were distinct at four typical Raman peaks compared with the control Sertoli cells. In phenotype, SCF, BMP4 and GDNF transcripts and proteins were significantly lower in Sertoli cells of NOA patients than in the control Sertoli cells. The Sertoli cells of NOA patients were not compared with Sertoli cells of normal fertile men due to the fact that it is hard to obtain adult testes from normal donors. This study provides novel insights into understanding the underlying causes for NOA and might offer a basis for developing new therapeutic strategies for patients with NOA.