Presentation Authors: Russell Hayden*, Anna Mielnik, New York, NY, Ryan Flannigan, Vancouver, Canada, Peter Schlegel, Darius Paduch, New York, NY
Introduction: The SOX family of transcription factors play critical roles in development, stem cell maintenance, and cellular differentiation. SOX2 is one of the 4 Yamanaka factors, which also include OCT4, KLF4, and MYC, that are used to induce pluripotent stem cells. SOX2 is normally not expressed in the testis, in contrast to SOX3 which localizes to spermatogonial stem cells. However, preliminary RNA-sequencing results in our lab demonstrated expression of SOX2 in men with Sertoli Cell Only Syndrome (SCO). In this study we sought to substantiate these results with real time PCR (RT-PCR).
Methods: Testis biopsies were obtained during testicular sperm extraction for infertility. RNA-seq was performed on testis biopsies from 11 men with SCO and 10 with normal spermatogenesis using the Illumina HiSeq2000 platform. Reads were mapped using the STAR Aligner v2.5 against human genome hg38. Raw counts were normalized with Limma v3.6 using the R statistical package v3.4. RT-PCR was conducted on 3 men with SCO and 3 men with normal spermatogenesis on a LightCycler 480 (Roche). The RT2 qPCR platform was used in all experiments (Qiagen), and all assays were conducted in triplicate. Quality control was assured using melting point analysis, interplate calibrators, and negative controls.
Results: RNA-seq data demonstrated a 6-fold (p < 0.01) increase of SOX2 expression in men with SCO as compared to normal controls. The mean counts per million (CPM) for men with SCO was 46.5 (CI 35.2 - 57.8), whereas men with normal spermatogenesis had a CPM value of 7.8 (CI 5.9 - 9.8). RT-PCR results demonstrated a 2.7-fold (CI 1.7 - 4.1) increase in SOX2 expression of SCO compared to normal controls.
Conclusions: SOX2 is over expressed in men with SCO as compared to normal controls. Although RT-PCR produced an attenuated result, the fold change in expression levels between SCO and normal men remained significant. Further study is needed to delineate how SOX2 overexpression may contribute to the pathophysiology of SCO, especially regarding its ability to suppress stem cell differentiation.
Source of Funding: P50 HD076210, U1 1U01HD074542-01; Frederick J. and Theresa Dow Wallace Fund of the New York Community Trust, the Mr. Robert S. Dow Foundation; Irena and Howard Laks Foundation.