SUPPRESSION OF THE ABERRANTLY ACTIVATED TNFa-NFkB PATHWAY IN THE TESTICULAR NICHE OF PATIENTS WITH NONOBSTRUCTIVE AZOOSPERMIA (NOA) RESTORES SPERMATOGONIAL STEM CELL SELF-RENEWAL AND EXPANSION
Pamela L. Yango BS(1), James F. Smith MD MS(1,2), Eran Altman MD(1), Philip Logan Ph.D, Jared Rosen BS(2), and Nam D. Tran MD, PhD(1)
(1)Department of Obstetrics, Gynecology, and Reproductive Sciences; (2)Department of Urology, University of California, San Francisco. USA
OBJECTIVES: Normal spermatogenesis is maintained by the intricate balance between self-renewal and differentiation within the spermatogonial stem cell (SSC) population primarily regulated by the testicular somatic niche. The precise mechanisms behind the pathophysiology of unexplained NOA and potential therapeutic treatments remain to be investigated. The aims of this study are to: 1) Develop an effective in vitro model of human NOA; and 2) to utilize this model to develop a novel treatment for these patients.
DESIGN: In vitro co-culture of primitive spermatogonia (SPG) and testicular somatic cells (TSCs) from subjects with normal (n=12) spermatogenesis and with unexplained NOA (n=12).
MATERIALS AND METHODS: SPG and TSCs were isolated by fluorescence-activated cell sorting (FACS) from testicular biopsies using SSEA4 and THY1 as markers, respectively. Normal and disease SPGs were co-cultured on either normal or disease specific TSCs in the presence of TNFa, TNFa inhibitor (TNFi), or conditioned media (CM).
RESULTS: When normal SPG were co-cultured with either normal or NOA TSCs, NOA TSCs caused a 31% decrease in the rate of SSC colony formation in comparison to normal TSCs (p<0.05). In contrast, a similar rate of SSC formation was seen when either normal or NOA SPG were culture on normal TSCs. Aberrant activation of the TNFa-NFkB pathway in the NOA TSCs was detected by RNAseq and confirmed with translocation of p50 and p65 from the cytoplasm to nucleus. When normal SPG were cultured in the presence of TNFa, SSC colony formation significantly decreased by 31% and 61% in the presence of NOA and normal TSCs, respectively. In contrast, TNFi normalized the negative impact of NOA TSCs on SSC self-renewal and expansion. Interestingly, when SPG were cultured on normal TSCs using CM collected from NOA TSCs, there was also significant decrease (35%) in SSC colony formation. However, CM collected from normal TSCs failed to rescue SSCs growing on NOA TSCs.
CONCLUSIONS: The intricate interactions between somatic testicular niche and SSCs are critical to the proper balance of SSC proliferation and differentiation. Disruption of the TNFa-NFkB pathway within the normal testicular niche disrupts this balance and pushes SSCs into a prosurvival and quiescent state. Correcting the defect in this pathway allows NOA SSCs to exit their nonproliferative state and regain normal self-renewal properties. These results suggest new potential diagnostic tests and therapeutic applications for men with idiopathic NOA.