In Vivo and In Vitro Transcriptional Regulation of Gonadotropin-Regulated Testicular RNA Helicase (GRTH/DDX25) By Germ Cell Specific Nuclear Factor (GCNF/RTR) in Transgenic Mice Model

Presentation Number: THR-115
Date of Presentation: March 5th, 2015

Raghuveer Kavarthapu*1, Chon-Hwa Tsai-Morris2 and Maria L Dufau3
1National Institutes of Health, Bethesda, MD, 2National Institutes of Health, 3NIH-NICHD, Bethesda, MD

Abstract

Gonadotropin-Regulated Testicular RNA Helicase (GRTH/Ddx25) is a testis specific member of the DEAD-box protein family, which is essential for round spermatid elongation and completion of spermatogenesis, is expressed in germ (spermatocytes, round spermatids) and Leydig cells. The transgenic (Tg) mice model carrying 5’ flanking regions of the GRTH-GFP reporter provides a unique in vivo system that permits differential elucidation of 5’ UTR regulatory regions of GRTH gene that directs its expression in germ cells and Leydig cells. The 5’ UTR distal region directs its expression in germ cells (GC) and the downstream region in Leydig cells. We showed an indirect action of androgen on GRTH expression in GC of Tg mice carrying this specific -6.4 Kb/-3.6 Kb 5’ flanking sequence which lacks ARE (Androgen Response Element) (1), and autocrine regulation via androgen/androgen receptor/ARE in Leydig cells.  A putative cis-binding element for a germ cell specific nuclear factor (GCNF/RTR), predominantly expressing in the developing GCs and required to direct gene expression in the postmeiotic phase of GC of the testis, was identified on -5270/-5252 of GRTH gene. In subsequent studies we explore the potential androgen action through GCNF on GRTH gene transcription in GC.  Western blot analysis showed GCNF protein expression significantly decreased in the testis of Tg mice carrying -6.4 kb/-3.6 kb sequence upon flutamide (androgen receptor antagonist) treatment, indicating the presence of a regulatory network for androgen/ GCNF-upstream 5’ GRTH sequence to direct GRTH expression in GC. A marked reduction of GCNF level was also noted in purified round spermatids from wild type mice treated in vivo with flutamide. ChIP analysis and gel shift further demonstrated the association of GCNF with GRTH sequence spanning the GCNF binding site on -5270/-5252 in both round spermatids and spermatocytes. This interaction was abolished by flutamide treatment. In vitro studies with seminiferous tubules in culture (dark zone stages VII-VIII), from -6.4 kb/-3.6 kb-GFP Tg mice using GCNF siRNA revealed marked decrease in the expression of GFP expression. Also tubules exposed to flutamide showed mark reduction of GFP expression. Our studies provide evidence for indirect action of androgen on GCNF regulation of GRTH specific expression in GC. This model permits to elucidate the mechanism of indirect actions of androgen via Sertoli cells in GC and thus facilitate the identification of androgen regulated factors that control expression of critical gene(s) required for GRTH expression in GC involved in the progression of spermatogenesis. This could lead to the development of effective male contraceptive strategies in Sertoli cells to block sperm formation without impacting other aspects of androgen action.

 

Nothing to Disclose: RK, CHT, MLD