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Part 2 - Regulation of Testis Function

Gonadotropin-Regulated Testicular RNA Helicase (GRTH/DDX25): A Multifunctional Protein Essential for Spermatogenesis

From the Section on Molecular Endocrinology, Program in Developmental Endocrinology and Genetics, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland.

Correspondence to: Chon-Hwa Tsai-Morris, Bldg 49, Rm 6B-03, 49 Convent Dr, MSC 4510, NIH, Bethesda, MD 20892-4510 (e-mail: morrisch{at}mail.nih.gov).

Received for publication May 18, 2009; accepted for publication October 5, 2009.

Abstract

Male germ cell maturation is governed by the expression of specific protein(s) in a precise temporal sequence during development. Gonadotropin-regulated testicular RNA helicase (GRTH/DDX25), a member of the Glu-Asp-Ala-Glu (DEAD)-box protein family, is a testis-specific gonadotropin/androgen-regulated RNA helicase that is present in germ cells (meiotic spermatocytes and round spermatids) and Leydig cells. GRTH is essential for completion of spermatogenesis as a posttranscriptional regulator of relevant genes during germ cell development. Male mice lacking GRTH are sterile with spermatogenic arrest due to failure of round spermatids to elongate, where striking structural changes and reduction in size of chromatoid bodies are observed. GRTH also plays a central role in preventing germ cell apoptosis. In addition to its inherent helicase unwinding/adenosine triphosphatase activities, GRTH binds to specific mRNAs as an integral component of ribonuclear protein particles. As a shuttle protein, GRTH transports target mRNAs from nucleus to the cytoplasm for storage in chromatoid bodies of spermatids, where they await translation during spermatogenesis. GRTH is also associated with polyribosomes to regulate target gene translation. The finding of a missense mutation associated with male infertility, where its expression associates with loss of GRTH phosphorylation, supports the relevance of GRTH to human germ cell development. We conclude that the mammalian GRTH/DDX25 is a multifunctional RNA helicase that is an essential regulator of spermatogenesis and is highly relevant for studies of male infertility and contraception.

     Key words: Testis, spermiogenesis, messenger ribonuclear protein particle, shuttling protein, translation, phosphorylation, chromatoid body, apoptosis, infertility, single-nucleotide polymorphism (SNP), missense mutation

RNA helicase members of Glu-Asp-Ala-Glu (DEAD)-box protein family are known to modulate the RNA structure that is a crucial step in many fundamental biological processes. Although these proteins participate in various aspects of RNA metabolism and translational events (de la Cruz et al, 1999; Silverman et al, 2003), their precise role during testicular germ cell development is poorly understood. Current knowledge of mouse PL10/DDX3, a male germ cell–specific helicase, is limited to its functional replacement of Saccharomyces cerevisiae Ded 1 protein (DED1p)/homolog of mammalian translational initiation factor IF4A required for the initiation step of translation in the yeast (Chuang et al, 1997). Mouse Vasa (MVH)/DDX4, homolog of Drosophila VASA that is present in the CB and cytoplasm of germ cells and is commonly used as a CB marker of germ cells (Fujiwara et al, 1994; Noce et al, 2001), has been recently proposed to participate in the small interfering RNA pathway to regulate RNA processing in the CB of spermatids (Kotaja and Sassone-Corsi, 2007). However, because MVH function is confined to the premeiotic stage of male germ cells before the formation of CB as determined by knockout mouse model (Tanaka et al, 2000), it is difficult to determine its role in the translational delay of messages during spermiogenesis.

The discovery of a hormonally regulated gonadotropin-regulated testicular RNA helicase (GRTH/DDX25), which is a testis-specific member of the DEAD-box family of RNA helicases present in Leydig and germ cells (Tang et al, 1999; Dufau and Tsai-Morris, 2007), has greatly advanced understanding of essential requirements and mechanisms associated with germ cell development. It has also provided insights linking the actions of gonadotropin/androgen in the transcriptional and posttranscriptional regulation of GRTH during testicular cell maturation. The regulation of GRTH by androgen occurs through direct actions of androgen in Leydig cells, and presumably in germ cells indirectly by the participation of androgen-responsive genes from Sertoli cells. Here, we summarize the current status of our findings on the essential role of GRTH/DDX25 in germ cell development, with emphasis on its multifunctional control of spermatogenesis.

GRTH Structure, Biochemical Properties, Genomic Organization, and Tissue/Cell Distribution

GRTH contains 483 amino acids (aa) and shares all conserved signature motifs of members of the DEAD-box family of RNA helicases (Tang et al, 1999; Sheng et al, 2003) (Figure 1). In general, this family of proteins binds RNA through specific motifs, interacts with adenosine triphosphate (ATP), and possesses ATPase and helicase activities that unwind RNA (Cordin et al, 2006; Linder, 2006). It is also a translational regulator because it enhances translation of in vitro–transcribed messages in a dose-dependent manner (Tang et al, 1999). Aside from the conserved motifs related to inherent biochemical functions, GRTH displays low aa sequence similarity with other members of the DEAD-box protein family. Unique 5' and 3' extension sequences, as well as the overall structure of GRTH, determine its specific physiologic function in germ cells (Sheng et al, 2003, 2006). Phylogenic comparative analysis of aa sequences revealed that the GRTH gene is only distantly related to other germ cell stage–specific DEAD-box RNA helicases, including PL10/DDX3 (Leroy et al, 1989) and MVH/DDX4 (Fujiwara et al, 1994). GRTH is most closely related to human/mouse/yeast DBP5/DDX19, which is expressed ubiquitously and has a perinuclear localization (Gee and Conboy, 1994) that is required for its poly(A) RNA export function (Tseng et al, 1998). The GRTH/Ddx25 gene contains 12 coding exons, and all but one of its conserved helicase motifs are contained within single exons. GRTH is a TATA-less gene, and its basal promoter activity is driven by transcription factors Sp1/Sp3, which bind GC-rich sequences at the promoter (Tsai-Morris et al, 2004a). Cell-specific transcriptional activation in expressing (pituitary -T3 and hypothalamic GT1-7) and nonexpressing (mouse Leydig tumor) cells could be accomplished by the presence or absence of as yet unidentified transcription factor(s) binding to upstream sequences.

View larger version (45K): [in this window] [in a new window]   Figure 1. Domain structure and function of gonadotropin-regulated testicular RNA helicase (GRTH). (Top) Conserved motifs with specific intrinsic functions shared by GRTH with members of the Glu-Asp-Ala-Glu (DEAD)-box family of proteins are indicated as Q, I, Ia, Ib, and II-VI. Amino acid position of each motif is indicated. (Bottom) Summary of specific physiologic function of GRTH. (Tang et al, 1999; Sheng et al, 2003, 2006).

Regulation of GRTH Gene Expression

Gonadotropin-induced androgen increases cause autocrine stimulation of GRTH gene transcription in Leydig cells (see below), whereas in germ cells (SPs and RSs), the major induction of the GRTH gene observed at puberty presumably results from paracrine actions of androgen through cognate receptors in Sertoli cells. This latter aspect will require further investigation.

Time-course studies showed up-regulation of GRTH mRNA and 43/48-kDa protein species in Leydig cells from adult rats treated with a single dose of hCG (Tang et al, 1999; Sheng et al, 2003). Significant increases were observed at 12 hours and reached maximal levels at 24 hours, then returned to near-basal levels at 96 hours. The increases in mRNA expression were not related to changes in message stability. Nuclear runoff studies demonstrated that in vitro, newly transcribed GRTH mRNA from Leydig cell nuclear extracts was significantly increased in animals treated with hCG, indicating that the gonadotropin-induced up-regulation of GRTH gene expression occurs at the transcriptional level. The in vivo hCG effect was reproduced by in vitro exposure of Leydig cell cultures to gonadotropin (10 ng/mL) for 24 hours, and treatment with cAMP caused comparable increases in GRTH expression. The hCG-induced increases were prevented when Leydig cells were preincubated with a mixture of enzyme inhibitor(s) of the steroidogenic pathway (Sheng et al, 2003) that effectively abolished androgen production. Similar to hCG and cAMP, dihydrotestosterone but not estradiol significantly increased the expression of GRTH. This action of androgen was confirmed by in vivo studies on animals treated with the androgen receptor inhibitor flutamide, which prevented the GRTH increases induced by hCG (Sheng et al, 2003). Taken together, these findings indicate that GRTH expression is up-regulated in Leydig cells at the transcriptional level by gonadotropin via cAMP through the actions of androgen generated by the hormonal stimulus.

GRTH-Targeted Null Mouse

Considerable information on the functional role of GRTH has been derived from studies on GRTH-null mice (Tsai-Morris et al, 2004b). Adult male homozygous knockout (KO) mice are sterile but display normal sexual behavior. GRTH heterozygous male mice and null female mice are fertile. The endocrine profiles are normal in all groups. Although Leydig cells of KO mice have reduced lipid droplets and swollen mitochondria lacking normal central cristae, normal basal circulating levels of testosterone are maintained. This excludes reduced androgen production as the cause of the spermatogenic arrrest. No morphologic changes in germ cell development were observed in GRTH-null mice from birth to day 21 of age. In the adult testis, RSs of null mice were arrested at step 8 of spermiogenesis and failed to elongate. The ultrastructure of the germ cells was generally normal up to the point of arrest. However, the CB of GRTH-null mice was unusually condensed, reduced in size, and lacked the typical filamentous-lobular structure during all steps of spermiogenesis. Immunoelectron microscopy gold labeling analysis showed GRTH expression in the nucleus, cytoplasm, and the CB of wild-type spermatids. Because the CB shares components (argonaute/small RNA induced silencing complex [RISC]/dicer) of its functional somatic P-body homolog that participate in the RNA-mediated gene silencing/degradation pathway (Eulalio et al, 2007; Kotaja and Sassone-Corsi, 2007), this organelle might function as an RNA storage site to control RNA processing and translation of relevant spermatogenic messages in male germ cells. The tightly compact CB of reduced size found in the GRTH KO mice with spermatogenic arrest implicates an essential requirement of GRTH to maintain the structural integrity of the CB for posttranscriptional regulation of the expression of genes that presumably control the elongating process of the germ cells.

Comparative studies in wild-type and KO of genes that are normally expressed early in spermatocytes, including histone H4 and high-mobility group protein 2 (HMG2), showed unchanged levels of their mRNA in total cell extracts and the cytoplasmic fraction of KO mice (Sheng et al, 2006). However, their protein expression was completely abolished, suggesting a functional role of GRTH in translational regulation. Similarly, in total extracts of round spermatids of KO mice, no change was observed in the steady-state level of transcripts of a set of genes that are normally expressed at later stages of spermatogenesis, including phosphoglycerate kinase 2 (PGK2), testicular angiotensin-converting enzyme (tACE), and transition protein 2 (Tp2). In contrast, their cytosolic mRNA levels were significantly reduced, and their protein expression was completely abolished. This indicated that in addition to its function as a translational regulator, GRTH is required for the nuclear export of mRNAs of specific genes during later stages of germ cell development. The lack of GRTH does not affect the expression of other proteins, including acrosin, any of the cAMP response element modulator isoforms, transcription factor SP1, steroidogenic enzymes (P450 scc and P450c17), and RNA helicases p68 and PL10 (Tsai-Morris et al, 2004b; Sheng et al, 2006). Taken together, these data indicate that GRTH is crucial for the posttranscriptional control of the expression of subsets of stage-relevant genes that might have an impact on histone-associated chromatin remodeling processes (H4 and Tp2) and the nonhistone component of chromatin modifications (HMG2), including DNA bending and unwinding and on specific metabolic processes to generate ATP (PGK2) during spermatogenesis.

The prominent apoptosis observed in KO spermatocytes entering the metaphase of meiosis before the appearance of RSs indicates an additional role for GRTH in determining the survival and apoptotic fate of adult germ cells (Tsai-Morris et al, 2004b). The degree of apoptosis appears to be related to the reduction of GRTH protein expression in germ cells, because about 30% terminal deoxynucleotidyl transferase dUTP nickend labeling–positive cells per tubule were observed in null mice, whereas only 8% positive cells were found in heterozygous mice and less than 2% in wild type. Comparative studies on the global expression of proapoptotic and antiapoptotic protein profiles of individual apoptotic pathways in spermatocytes of wild-type and GRTH-null mice indicated that GRTH acts as a negative regulator of mitochondrial, death receptor, and nuclear factor -B (NF-B) pathways to prevent apoptosis in the adult testis (Gutti et al, 2008). Lack of GRTH causes increases in the protein levels of proapoptotic factors and decreases of antiapoptotic factors and reduced phospho-BAD, leading to disruption of mitochondrial integrity, which promotes release of cytochrome c (Donovan and Cotter, 2004) and induction of the apoptotic cascade. This, together with the enhanced caspase 3 transcript stability, the higher level of caspase 3–binding factor, Smac, and the diminished level of binding competitor (IAP) observed in the absence of GRTH facilitates the generation of caspase 3/9 active products and PARP (poly[ADP ribose] polymerase), which induce DNA fragmentation in the apoptotic process. GRTH has an impact on NF-B signaling at both cytoplasmic and nuclear levels. The nonphosphorylate IB/β, which sequesters NF-B dimer and prevents its translocation to the nucleus to stimulate the transcription of antiapoptotic genes, was highly elevated in the KO mice. Lack of GRTH also could alter the acetylation status of NF-B in the nucleus through the observed increases in histone deacetylase (HDAC) and decreases in acetylase p300 expression in KO, which could lead to the decrease in transcription of antiapoptotic genes and apoptosis. Regarding the tumor necrosis factor receptor-1 (TNF-R1) signaling pathway, the increase in TNFR-associated adaptor protein TNF-R1–associated DEATH domain protein (TRADD) in GRTH KO could cause the observed activation of the downstream caspase 8 cascade that leads to apoptosis. GRTH-mediated apoptotic regulation is also indicated by its selective binding to proapoptotic and antiapoptotic mRNAs. Thus, as a component of mRNP particles, GRTH acts as a negative regulator of the TNF-R1 and caspase pathways, and it promotes NF-B function to control apoptosis in spermatocytes of adult mice.

GRTH Function

In addition to the intrinsic biochemical function as an RNA helicase (Tang et al, 1999), GRTH has multiple functions (Figure 1): it 1) associates with mRNA as an integral component of mRNPs (Tsai-Morris et al, 2004b); 2) is a shuttling protein that exports mRNAs from the nucleus to cytoplasmic sites (Sheng et al, 2006); 3) is a translational regulator through its association with active elongating polyribosomes, where it participates in the translation of specific RNA transcripts at certain stages in germ cell development (Sheng et al, 2006). The GRTH protein was present in oligo(dT) pull-down RNA-protein complexes from rodent testicular lysates. Also, reverse transcriptase–polymerase chain reaction analysis of RNAs extracted from immunoprecipitated testicular GRTH-mRNA complexes revealed their association with a specific set of testicular gene transcripts, including those of chromatin-remodeling proteins (transition protein 1/2 [Tp1 and Tp2] and protamine 1/2 [Prm1 and Prm2]), cytoskeletal structural proteins sperm fibrous sheath component 1/outer dense fiber 1 (Fsc1/Odf1), and tACE. GRTH also selectively binds mRNAs of proapoptotic and antiapoptotic genes and mRNAs from death receptor and NF-B pathways to mediate apoptotic regulation (Gutti et al, 2008). Confocal studies on living cells showed that GRTH–green fluorescent protein was retained in the nucleus of COS1 cells after treatment of RNA polymerase II inhibitor (DRB), which inhibits RNA synthesis, or a nuclear protein export inhibitor (LMB), which inhibits nuclear protein export through a chromosome region maintenance-1 protein (CRM1)–dependent pathway. These findings indicate that GRTH functions as a transport protein to export mRNAs through the CRM1-dependent pathway, and they support our studies in germ cells that demonstrated that GRTH is required for the export of long-lived transcripts (PGK2, tACE, and Tp2) from the nucleus to the cytoplasm.

Individual GRTH protein species were preferentially associated with cellular compartments (Sheng et al, 2006). The 61-kDa species is the phosphorylated form of GRTH primarily present in the cytoplasm, whereas the 56-kDa protein is the unphosphorylated form found predominantly in the nucleus of germ cells. GRTH protein is phosphorylated at threonine residues by cAMP-dependent protein kinase. This posttranslational species does not participate in nuclear export but is required for cytoplasmic events, including its association with polyribosomes to initiate translation of target genes. The 56-kDa protein species associates with CRM1. Nuclear export and localization signals are present at the N terminus of GRTH, which contains two stretches of leucine-rich sequences. The first leucine-rich region (aa 61–74) functions as nuclear export signal and as the binding site to CRM1, and the second leucine-rich region (aa 100–114) functions as the nuclear localization signal. GRTH shuttles between cellular compartments and exports RNAs.

View larger version (42K): [in this window] [in a new window]   Figure 2. Model of gonadotropin-regulated testicular RNA helicase (GRTH) action in male germ cells during development. Routes 1 to 6 (in red) represent functions and transit of GRTH protein entry to the nucleous (1), where it binds messages and associates with CRM1 (2), and as an integral component of ribonuclear protein particles (RNPs) exports messages through nuclear pores via the CRM1 pathway to the cytoplasm (3a) and to the chromatoid body (CB) (3b and 4b), either directly (via nuclear pores adjacent to or associated with the CB) (3b) or indirectly (via the cytoplasmic route) (4b). It is phosphorylated at cytoplasmic sites to deliver messages, associates with polyribosomes (4a), and participates in translation. In the CB, messages are potentially regulated via si/mi/pi RNA pathway (RNA storage and degradation) (5a and b). Stored messages are subsequently translated in polyribosomes (4a and 6).

Conclusion

GRTH is a testis-specific protein and is the only RNA helicase known to be hormonally regulated. Gonadotropin action via cAMP stimulates androgen and regulates GRTH in Leydig cells at the translational and transcriptional levels, and in germ cells indirectly through androgen actions on Sertoli cells. GRTH has multifunctional roles in the processing of germ cell–specific RNAs and is essential for spermatid development and completion of spermatogenesis. The phosphorylation status at designated cellular sites of GRTH determines its regulatory action. GRTH is required for the integrity of CB, where it could participate in the regulation of messages awaiting translation during germ cell development.

Based on current knowledge, a model of GRTH action in male germ cells during development is presented in Figure 2. After translation in germ cells, the 56-kDa GRTH species is transported into the nucleus (route 1), where it binds selectively to a subset of nuclear RNAs, and as a component of mRNP complexes associates with CRM1 (route 2) and exports messages to cytoplasmic sites of germ cells through the CRM1-dependent exporting pathway via nuclear pores (route 3a). GRTH undergoes phosphorylation in the cytoplasm (61-kDa species). Phospho-GRTH associates with polyribosomes, where it regulates translation of associated mRNAs (route 4a) or transports mRNAs to the CB (route 4b) to silence/store/degrade transcripts through the RISC and the putative si/mi/pi RNA complex (route 5a and 5b). Messages are released at specific times from the CB to proceed with translation (route 6). Alternatively, GRTH exports mRNAs through the CRM1 pathway directly to the CB via adjacent/associated nuclear pores (route 3b). GRTH is a master posttranslational regulator of spermatogenesis, and understanding its basic regulatory mechanisms in germ cell development provides avenues to the study of male infertility and contraception.

Acknowledgments

This work was supported by the Intramural Research Program of the National Institute of Child Health and Human Development, National Institutes of Health.

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