Transcriptome Analysis to Identify Human Spermatogonial Cells from Sertoli Cell-Only Testes.

Abstract

We sought to determine whether gene products from spermatogenic cells could be detected in Sertoli cell-only testes. Transcriptome analysis using next generation sequencing was performed using human testicular samples. This study enrolled 20 men with nonobstructive azoospermia with a histological diagnosis of Sertoli cell-only and no germ cells on in vitro fertilization analysis and 5 with obstructive azoospermia and histologically normal spermatogenesis. Individual differences in the transcriptome of obstructive azoospermia testicular tissues generated on the Illumina® platform were compared as the number of fragments per kilobase of exon per million mapped sequence reads. We confirmed mRNA expression and targeted gene product localization by quantitative reverse transcriptase-polymerase chain reaction and immunohistochemical analysis, respectively. Using next generation sequencing we analyzed 5,666 of the 23,003 screened genes. As representative markers of immature germ cells, <i>UTF1</i>, <i>CD9</i>, <i>DDX4</i>, <i>EPCAM</i>, <i>GFRA1</i>, <i>KIT</i>, <i>LIN28</i>, <i>DMRT</i>, <i>GPR125</i>, <i>UCHL1</i> and <i>NANOG</i> transcripts were detected in 13 Sertoli cell-only samples (65%). Reverse transcriptase-polymerase chain reaction showed significant mRNA expression of <i>UTF1</i>, <i>CD9</i>, <i>DDX4</i> and <i>KIT</i> in Sertoli cell-only samples. Immunostaining revealed the localization of DDX4-positive cells, presumably spermatogonia, in 9 Sertoli cell-only samples (45%). These cells lacked proliferative cell nuclear antigen expression. A number of Sertoli cell-only testes contain immature germ cells (spermatogonia and spermatogenic stem cells) with various gene expression profiles. Sertoli cell-only is a heterogeneous pathophysiology which may be corrected by medical treatment or regenerative technologies.