MicroRNA-34c Enhances Murine Male Germ Cell Apoptosis through Targeting ATF1

Abstract

BackgroundMicroRNAs (miRNAs) play vital regulatory roles in many cellular processes. The expression of miRNA (miR)-34c is highly enriched in adult mouse testis, but its roles and underlying mechanisms of action are not well understood.Methodology/Principal FindingsIn the present study, we show that miR-34c is detected in mouse pachytene spermatocytes and continues to be highly expressed in spermatids. To explore the specific functions of miR-34c, we have established an in vivo model by transfecting miR-34c inhibitors into primary spermatocytes to study the loss-of-function of miR-34c. The results show that silencing of miR-34c significantly increases the Bcl-2/Bax ratio and prevents germ cell from apoptosis induced by deprivation of testosterone. Moreover, ectopic expression of the miR-34c in GC-2 cell trigger the cell apoptosis with a decreased Bcl-2/Bax ratio and miR-34c inhibition lead to a low spontaneous apoptotic ratio and an increased Bcl-2/Bax ratio. Furthermore, ectopic expression of miR-34c reduces ATF1 protein expression without affecting ATF1 mRNA level via directly binding to ATF1's 3′UTR, indicating that ATF1 is one of miR-34c's target genes. Meanwhile, the knockdown of ATF1 significantly decreases the Bcl-2/Bax ratio and triggers GC-2 cell apoptosis. Inhibition of miR-34c does not decrease the GC-2 cell apoptosis ratio in ATF1 knockdown cells.Conclusions/SignificanceOur study shows for the first time that miR-34c functions, at least partially, by targeting the ATF1 gene in germ cell apoptosis, providing a novel mechanism with involvement of miRNA in the regulation of germ cell apoptosis.

The role of epigenetics in idiopathic male infertility

“…Although how the inactivation of this family in a cancer setting reconciles with its downregulation in infertile males is yet not clear, miR-34c seems to relate through a novel pathway by promoting germinal lineage differentiation [121]. Interestingly, miR-34c has also been shown to promote murine male germ cell apoptosis by targeting the transcription factor ATF1 in a p53-independent manner [122]. In mouse models, miR-34c and miR-34b were found to be highly expressed in post-mitotic male germ cells from primary spermatocytes up to round spermatids [121,123], and deletion of this locus along with miR-449 led to OAT and infertility [124].…”

MicroRNAs and DNA methylation as epigenetic regulators of mitosis, meiosis and spermiogenesis

“…miR-34a, -34b and -34c) are found in bovine sperm (Tscherner et al 2014). Additionally, miR-34c seems to be important for the later steps of spermatogenesis because it is highly expressed in mouse pachytene spermatocytes and round spermatids in murine testis by targeting the activating transcription factor 1 (ATF1; Liang et al 2012). Moreover, the induction of ATF1 in spermatocytes of the pachytene stage is essential for maintaining cell viability and mouse early development (Bleckmann et al 2002, Persengiev & Green 2003.…”

Role of microRNAs in mammalian spermatogenesis and testicular germ cell tumors

“…Tgfb signaling inhibits the second meiotic division in spermatogenesis (Damestoy et al 2005), whereas notch signaling promotes spermatogonia differentiation (Kostereva & Hofmann 2008). miR449 and miR34b/ miR34c regulate germ cell differentiation and survival by targeting NOTCH1 and DAZL, as well as germ cell apoptosis by targeting BCL2 and ATF1 (Yan et al 2009, Luo et al 2010, Liang et al 2012. miR214 is preferentially expressed in pachytene spermatocytes and plays a key role in meiosis by targeting heat shock proteins (Marcon et al 2008, Dai et al 2011.…”