GRTH: A Key to Understanding Androgen-Mediated Germ Cell Signaling

Testosterone produced by Leydig cells within the interstitium surrounding the seminiferous tubules is essential for the onset and maintenance of spermatogenesis. Mice with a global naturally occurring loss-of-function mutation (Tfm) or with targeted ablation of the androgen receptor (AR/NR3C4) gene (ARKO) are infertile due to the failure of germ cells to progress beyond meiosis. Although ARs are clearly localized to Leydig cells, Sertoli cells, and peritubular myoid cells, ARs are lacking in germ cells from adult testes (1, 2). Thus, germ cells are not believed to be direct targets of androgen action. Moreover, mice with conditional disruption of the AR gene in Sertoli cells (1, 3, 4) or peritubular myoid cells (5) are infertile. Because the Sertoli cells directly interact with and provide a supportive microenvironment for germ cells, it is most likely that androgens act through Sertoli cells to stimulate the production of a factor to promote germ cell maturation. However, despite years of intensive research, such a factor has remained elusive. Notably, a number of studies using microarray analysis of Sertoli cells from AR gene-targeted vs wild-type mice to search for androgen-regulated genes related to spermatogenesis, show little overlap. Furthermore, more genes were found to be downregulated by testosterone acting through AR than upregulated (see Ref. 6 for review). Moreover, the relatively high testosterone concentrations required for AR activation within Sertoli cells and the predominant absence of classical AR response elements (AREs) in testosterone-regulated genes suggest the existence of nonclassical pathways for androgen action. Such pathways have been suggested to involve possible interaction of testosterone with a G protein-coupled receptor, resulting in calcium influx and signaling, as well as interaction of AR with Src tyrosine kinase within the plasma membrane, resulting in an epidermal growth factor receptor-mediated signaling cascade (see Ref. 7 for review). Although a number of androgen-responsive genes have been identified in germ cells, the mechanisms that underlie their regulation remain unclear. Work from the laboratory of Maria Dufau has shown that one of these androgen-responsive genes, gonadotropin-regulated testicular helicase (GRTH/Ddx25), is testis-specific and essential for completion of spermatogenesis. GRTH, a member of the DEAD-box family of RNA helicases, is expressed specifically in pachytene and metaphase spermatocytes and in round and elongated spermatids (8). They have observed that within the germ cell compartment, GRTH serves as an integral component of ribonuclear protein particles and participates in the transport of mRNAs from the nucleus to cytoplasmic chromatoid bodies where they are stored before translation during spermatogenesis. Phosphorylated GRTH associates with polyribosomes and facilitates the translation of androgen-regulated transcripts that play critical roles in sperm maturation, including protamines Prm1/2, histone H4, high-mobility group protein HMG2, transition proteins TP1 and TP2, and angiotensin-converting enzyme (ACE) (9, 10). Importantly, they found that targeted ablation of the GRTH gene resulted in sterility in male mice, due to the failure of round spermatids to elongate and a complete block of spermatogenesis at stage 8 (11). Notably, chromatoid bodies were found to be markedly reduced in size in the GRTH gene-targeted mice. Thus, GRTH may serve as a master androgen-regulated gene in germ cells that controls translation of a number of mRNAs to proteins critical for sperm maturation. GRTH also is expressed in Leydig cells where it is directly regulated by the androgen/AR. Within the Leydig cell, testosterone acts in an autocrine manner to enhance GRTH transcription by facilitating AR binding to an ARE half-site at −827 bp upstream of the GRTH translation start site (12) (Figure 1). By contrast, androgen regulation of GRTH expression in germ cells likely occurs via a paracrine mechanism involving testosterone/AR action in Sertoli cells. However, to date, the transacting factors and response elements that mediate androgen induction of GRTH in germ cells remain unknown. Figure 1. GRTH mediates effects of androgen to promote germ cell maturation. Androgen produced by Leydig cells within the interstitial compartment of the testis acts in a paracrine manner via AR in Sertoli cells to stimulate the production of presently unidentified ... In their study published in this issue of Endocrinology, Kavarthapu et al (13) used a transgenic approach to provide novel insight into the mechanisms for androgen regulation and cell-specific expression of the GRTH gene in male germ cells. In previous investigations using transgenic mice, these investigators observed that a −1085-bp region flanking the 5′-end of the GRTH gene containing an ARE half-site was sufficient for androgen/AR-regulated Leydig cell-specific expression of GRTH promoter activity (Figure 1). Expression of this transgene construct was undetectable in male germ cells, even when the GRTH 5′-flanking sequence was extended to −3600 bp (14). In the present study, it was observed that a transgene containing −6400 bp of GRTH 5′-flanking region was expressed both in testicular Leydig cells and in germ cells. Remarkably, they found that a modified transgene containing upstream sequences from −3600 to −6400 bp fused to the basal GRTH promoter was exclusively expressed in germ cells; germ-cell–specific expression was blocked by the AR antagonist flutamide. Thus, it is likely that this upstream region contains binding sites for critical androgen-regulated transcription factors (Figure 1). By sequence comparison of the genomic region between −3600 and −6400 bp with those of other premeiotic, meiotic, and postmeiotic male germ-cell–specific androgen-regulated genes, similarities were noted in a number of potential clustered transcription factor binding sites. These included putative response elements for germ cell nuclear factor (GCNF), a member of the nuclear receptor family known to be selectively expressed in developing germ cells, as well as those for the testis-specific transcription factors mammalian doublesex homologs Dmrt1 and -7. Notably, Dmrt1- and Dmrt7-knockout mice are infertile (15, 16). Although considerable work to ascertain and functionally characterize these regulatory factors lies ahead, their identification may provide key insight into the signaling mechanisms whereby androgen action in the Sertoli cell mediates male germ cell maturation. Moreover, the identification of an androgen-regulated factor that controls expression of a critical gene involved in spermatogenesis could lead to the development of contraceptive strategies that selectively block sperm formation without impacting other aspects of androgen action.