GRTH Gene Research Articles

LBMON272 Chromatoid Bodies In The Regulation Of Spermatogenesis: Novel Role Of Grth

Abstract More than 5 million couples in US are affected by infertility. Male infertility is a major cause (>30%), and understanding spermatogenesis is important to address the problem. During early spermatogenesis, mRNAs are actively transcribed, transported and stored transiently in chromatoid bodies (CBs). CBs are membrane-less perinuclear organelles which serve as storehouses of mRNAs until later stages of spermatogenesis. Gonadotropin-regulated testicular RNA helicase (GRTH/DDX25) plays critical role in spermatogenesis. In addition to its inherent helicase activity, GRTH transports mRNAs from nucleus to cytoplasm and from there to CBs for storage until translation. In Germ cells, there are two species of GRTH, the 56 kDa non-phospho and 61 kDa phospho (pGRTH) forms. Our early studies revealed a missense mutation (R242H) of GRTH in Japanese azoospermic men which resulted in the lack of pGRTH in in vitro studies. GRTH knock-in (KI) mice with the human mutant GRTH gene (R242H), show loss of the phospho-species from cytoplasm and CBs with preservation of the non-phospho form in the cytoplasm, Nucleus and CBs. GRTH-KI mice are sterile, lack elongated spermatids and spermatozoa with spermatogenic arrest at step 8 of round spermatids (RS). CBs isolated from GRTH KI mice are smaller, highly condensed, lack pGRTH and RNA-Seq analysis of isolated mRNA (from CBs) revealed differential abundance of mRNAs related to spermatid differentiation. Integrative analysis of small RNA and mRNA expression will provide cohesive outlook of molecular mechanisms of gene regulation during spermatogenesis. Hence, we proceeded with small RNAseq analysis which reveal differential expression (Padj<0. 05) of piRNAs and miRNAs. In total 226706 piRNAs upregulated and 166567 piRNAs downregulated in the round spermatids. Several miRNAs like miR32, miR7, miR184, miR335, miR140, miR141, miR1981, miR202, miR880, miR669c were downregulated. These are involved in the positive regulation of spermatid differentiation, sperm motility, mRNA processing and decay. The miRNAs, miR150, miR196a-2, miR7b, miR652, miR146, miR10b, miR379, miR122a, miR27a, miR127 and miR328 were upregulated, which negatively regulate the spermatid differentiation. Gene ontology analysis revealed that the majority of downregulated miRNAs belong to regulation of gene expression. Majority of upregulated miRNAs belong to miRNA mediated suppression of translation mostly associated with spermiogenesis and CBs. The identified miRNAs regulate essential genes like Tnp2, TSSK6, Pabpc1, Ppp1cc and UPF2 etc. These indicates that the CBs mediate highly specialized mRNA metabolism process and serve as a processing center of essential mRNAs. These results demonstrate the importance of GRTH and phospho-GRTH in the regulation of CB and their role in mRNA storage and stability of germ cell specific mRNAs during spermiogenesis and healthy sperm formation. Presentation: Monday, June 13, 2022 12:30 p.m. - 2:30 p.m.

Role of Phosphorylated Gonadotropin-Regulated Testicular RNA Helicase (GRTH/DDX25) in the Regulation of Germ Cell Specific mRNAs in Chromatoid Bodies During Spermatogenesis.

GRTH/DDX25 is a member of the DEAD-box family of RNA helicases that play an essential role in spermatogenesis. GRTH knock-in (KI) mice with the human mutant GRTH gene (R242H) show loss of the phospho-species from cytoplasm with preservation of the non-phospho form in the cytoplasm and nucleus. GRTH KI mice are sterile and lack elongated spermatids and spermatozoa, with spermatogenic arrest at step 8 of round spermatids which contain chromatoid body (CB) markedly reduced in size. We observed an absence of phospho-GRTH in CB of GRTH KI mice. RNA-Seq analysis of mRNA isolated from CB revealed that 1,421 genes show differential abundance, of which 947 genes showed a decrease in abundance and 474 genes showed an increase in abundance in GRTH KI mice. The transcripts related to spermatid development, differentiation, and chromatin remodeling (Tnp1/2, Prm1/2/3, Spem1/2, Tssk 2/3/6, Grth, tAce, and Upf2) were reduced, and the transcripts encoding for factors involved in RNA transport, regulation, and surveillance and transcriptional and translational regulation (Eef1a1, Ppp1cc, Pabpc1, Ybx3, Tent5b, H2al1m, Dctn2, and Dync1h1) were increased in the CB of KI mice and were further validated by qPCR. In the round spermatids of wild-type mice, mRNAs of Tnp2, Prm2, and Grth were abundantly co-localized with MVH protein in the CB, while in GRTH KI mice these were minimally present. In addition, GRTH binding to Tnp1/2, Prm1/2, Grth, and Tssk6 mRNAs was found to be markedly decreased in KI. These results demonstrate the importance of phospho-GRTH in the maintenance of the structure of CB and its role in the storage and stability of germ cell-specific mRNAs during spermiogenesis.

Linking Phospho-Gonadotropin Regulated Testicular RNA Helicase (GRTH/DDX25) to Histone Ubiquitination and Acetylation Essential for Spermatid Development During Spermiogenesis.

GRTH/DDX25 is a testicular RNA helicase expressed in germ cells that plays a crucial role in completion of spermatogenesis. Previously, we demonstrated a missense mutation (R242H) of GRTH gene in Japanese infertile patients (5.8%) with non-obstructive azoospermia. This mutation upon expression in COS-1 cells revealed absence of the 61 kDa phosphorylated GRTH in cytoplasm and the presence of the 56 kDa non-phosphorylated GRTH in the nucleus. GRTH knock-in (KI) mice carrying the human GRTH (R242H) mutation, lack phosphorylated GRTH, and sperm due to failure of round spermatid elongation during spermiogenesis. To determine the impact of phosphorylated GRTH on molecular events/pathways participating in spermatid development during spermiogenesis, we analyzed transcriptome profiles obtained from RNA-Seq of germ cells from KI and WT mice. RNA-Seq analysis of 2624 differentially expressed genes revealed 1404 down-regulated and 1220 up-regulated genes in KI mice. Genes relevant to spermatogenesis, spermatid development and spermatid differentiation were significantly down-regulated. KEGG enrichment analysis showed genes related to ubiquitin-mediated proteolysis and protein processing in endoplasmic reticulum pathway genes were significantly down-regulated while the up-regulated genes were found to be involved in Focal adhesion and ECM-receptor interaction pathways. Real-Time PCR analysis confirmed considerable reduction in transcripts of ubiquitination related genes Ube2j1, Ube2k, Ube2w, Rnf8, Rnf133, Rnf138, Cul3 and increased expression of Ccnd2, Col1a, Lamb1, Cav1, Igf1, Itga9 mRNA’s in KI mice compared to WT. Also, marked reduction in protein expression of UBE2J1, RNF8, RNF138 (ubiquitination network), MOF (histone acetyltransferase), their modified Histone substrates (H2AUb, H2BUb) and H4Ac, H4K16Ac were observed in KI mice. GRTH-IP mRNA binding studies revealed that Rnf8 and Ube2J1 mRNAs from WT mice associated with GRTH protein and the binding is greatly impaired in the KI mice. Immunohistochemistry analysis showed significantly reduced expression of RNF8, MOF, H4Ac and H4K16Ac in round spermatids of KI mice. Absence of phosphorylated GRTH impairs UBE2J1, RNF8 and MOF-dependent histone ubiquitination and acetylation essential for histone replacement, chromatin condensation and spermatid elongation during spermiogenesis.

OR02-03 Role of Phosphorylated Gonadotropin-Regulated Testicular RNA Helicase (GRTH/DDX25) in the Regulation of Germ Cell Specific MRNAs in Chromatoid Bodies During Spermiogenesis

Gonadotropin-regulated testicular RNA helicase (GRTH/DDX 25) is a member of the DEAD-box family of RNA helicases which play an essential role in spermatogenesis. There are two species of GRTH, the 56 kDa non-phospho and 61 kDa phospho forms. Our early studies revealed a missense mutation (R242H) of GRTH in the Japanese azoospermic men which resulted in the lack of phospho-GRTH (pGRTH) in in vitro studies. GRTH knock-in (KI) mice with insertion of the human mutant GRTH gene show loss of the cytoplasmic 61 KDa phospho-species with preservation of the non-phospho nuclear form. KI mice are sterile, lack elongated spermatids and spermatozoa with arrest at step 8 of round spermatids (RS) which contain chromatoid bodies (CB) markedly reduced in size. CB is a non-membranous, cytoplasmic organelle present adjacent to the nucleus of RS, where mRNAs bound to GRTH transported from nucleus to cytoplasmic sites are temporarily stored, translationally repressed for later transport to polyribosomes for translation at specific stages of spermiogenesis. Owing to the specific function of CBs and importance of pGRTH in spermatid elongation, CBs isolated from germ cells of WT and GRTH KI mice were used for subsequent experiments. CBs isolated from GRTH KI mice are smaller, highly condensed and lack the nuage texture of CBs in WT mice. We observed the absence of pGRTH in CB of round spermatids of GRTH KI mice. Also, MVH protein (recognized CB marker protein) was decreased in the CB of GRTH KI mice. Expression of genes related to spermatid regulation, chromatin compaction, remodeling (TP1 and 2, PRM1 and 2, GRTH, TSSK6, HMG2, GCNF, RNF8, TDRD 1, 6, 7 and 9) analyzed by qPCR were markedly reduced in the CB of GRTH KI mice compared to WT. No change was observed in the expression of bromodomain mRNAs and protein, indicating that pGRTH does not participate in the translational regulation of this protein class at the level of this organelle. Notably, mRNAs of TP2, PRM2 and GRTH which associated with GRTH protein were co-localized with MVH protein in the CB. This indicated the relevance of GRTH as a binder/transport protein of key chromatin remodelers for ensuring their mRNA repression/stability within the CB. In addition, GRTH binding to genes essential for spermatid development and regulation (TP1 and 2, PRM1 and 2, GRTH, TSSK6, RNF8 and GCNF) were also found to be markedly decreased in the CB KI mice. These results demonstrate the importance of pGRTH in the maintenance of biochemical composition/structure of the CB and role in spermatid regulation, chromatin compaction, spermatid development and completion of spermatogenesis.

SAT-031 Linking Gonadotropin-Regulated Testicular RNA Helicase (GRTH/DDX25) to Histone Ubiquitination Network and Acetylation During Spermiogenesis

Gonadotropin Regulated Testicular Helicase (GRTH/DDX25), a testis specific RNA helicase essential for the completion of spermatogenesis. Our early studies discovered a missense mutation (R242H) of GRTH gene in 5.8 % of a Japanese population with azoospermia. This mutation in COS-1 cells showed loss of the 61 kDa cytoplasmic phospho-species with preservation of the 56Kda nuclear non-phospho form of GRTH. Mice with knock-in (KI) of the human GRTH mutation, lack the phospho-form of GRTH, are sterile and lack sperm, with spermatogenic arrest at stage 8 of round spermatids. To determine the impact of phospho-GRTH on gene expression comparative studies of germ cells transcriptome profiles from WT and KI mice were conducted using Illumina RNA sequencing. RNA-Seq analysis revealed 1614 differentially expressed genes of which 886 down-regulated and 728 genes up-regulated genes. Gene Ontology analysis revealed several genes relevant to spermatogenesis, spermatid development and differentiation significantly downregulated. KEGG analysis showed genes related to Ubiquitin mediated proteolysis, protein processing in ER, RNA transport, Glycolysis pathways are down-regulated, and genes related to Focal adhesion and ECM interaction are up-regulated. RealTime-PCR analysis confirmed dramatic reduction in mRNA expression of ubiquitination related genes Ube2j1, Ube2k, Ube2w, Rnf8, Rnf133, Rnf138 and increased expression of Ccnd2, Col1a, Lamb1, Cav1, Igf1, Itga9 mRNA’s in KI mice compared to WT. Western blot analysis revealed marked reduction in protein expression of UBE2J1, RNF8, RNF138 (ubiquitination network), MOF (histone acetyltransferase) and their modified Histone substrates (H2AUb, H2BUb), as well as H4Ac, H4K16Ac in KI mice. Immunohistochemistry analysis showed significantly reduced expression of RNF8, MOF, H4Ac and H4K16Ac in round spermatids of KI mice compared to WT. In KI mice absence of phospho-GRTH impairs RNF8 and MOF dependent ubiquitination and acetylation of histones required for histone replacement, chromatin condensation and spermatid elongation during spermiogenesis which finally results in germ cell arrest in step 8 of round spermatids. Thus, we conclude that phospho-GRTH affects the network which is critical for the replacement of histones during spermiogenesis.

Gonadotropin Regulation Testicular RNA Helicase, Two Decades of Studies on Its Structure Function and Regulation From Its Discovery Opens a Window for Development of a Non-hormonal Oral Male Contraceptive.

Gonadotropin Regulated Testicular Helicase (GRTH/DDX25) is member of the DEAD-box family of RNA helicases present in Leydig and germ cells. GRTH is the only family member regulated by hormones, luteinizing hormone, through androgen action. Male mice with knock-out of the GRTH gene are sterile, lack sperm with arrest at round spermatids. GRTH participates on the nuclear export and transport of specific mRNAs, the structural integrity of Chromatoid Bodies of round spermatids, where mRNAs are processed and stored, and in their transit to polyribosomes, where it may regulate translation of relevant genes. GRTH has a central role in the control of germ cell apoptosis and acts as negative regulator of miRNAs which regulate expression of genes involved in the progress of spermatogenesis. In Leydig cells, GRTH gene transcription is regulated by LH via autocrine actions of androgen/androgen receptor and has regulatory effects in steroidogenesis. In germ cells, androgen actions are indirect via receptors in Sertoli cells. Transgenic mice carrying GRTH 5′ flanking region-GFP permitted to discern regions in the gene which directs its expression upstream, in germ cells, and downstream in Leydig cells, and the androgen-regulated transcription at interstitial (autocrine), and germ cell (paracrine) compartments. Further evidence for paracrine actions of androgen/androgen receptor is their transcriptional induction of Germ Cell Nuclear Factor as requisite up-regulator of GRTH gene transcription in round spermatids, linking androgen action to two relevant germ cell genes essential for the progress of spermatogenesis. A missense mutation of R to H at amino acid 242 of GRTH found in 5.8% of a patient population with azoospermia causes loss of the cytoplasmic phospho-GRTH species with preservation of the non-phospho form in transfected cells. Mice with knock-in of the human mutation, lack sperm due to arrest at round spermatids. This model permits to discern the function of phospho-GRTH. The GRTH phospho-site resides at a Threonine structurally adjacent to the mutant site found in patients. Molecular modeling of this site elucidated the amino acids that form the GRTH/PKA interphase and provide the basis for drug design for use as male contraceptive.

Investigation of GRTH gene single nucleotide polymorphism in association with male infertility in Iranian population

IntroductionGRTH gene is located on chromosome 11q24 and consists of 14 exons. GRTH codes for a protein product which is involved in testicular function and spermatogenesis. The absence of this gene can lead to gametogenesis arrest. The purpose of this study was to investigate the association of SNP IVS6+55G→T (rs551373) in DDX25/GRTH gene with male infertility. MethodsIn this case-control study, DNA samples were extracted from 180 men, including 90 fertile men and 90 infertile cases with azoospermia by Salting out method. PCR-RFLP was conducted to identify the SNP in DDX25/GRTH gene. ResultsAccording to statistical analyses carried out in SPSS and SNPstats online software, the SNP rs551373 is significantly associated with azoospermic cases studied. ConclusionThe results of our study suggest that positive association of rs551373 SNP probably reveals an important role in azoospermia male infertility. Further studies with larger sample size are required to support our findings. Investigation of association of this SNP with other types of non-obstructive male infertility is also suggested.

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

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

A 5′-Flanking Region of Gonadotropin-Regulated Testicular RNA Helicase (GRTH/DDX25) Gene Directs Its Cell-Specific Androgen-Regulated Gene Expression in Testicular Germ Cells

Gonadotropin-regulated testicular RNA helicase (GRTH/Ddx25) is a posttranscriptional regulator of genes that are essential for spermatid elongation and completion of spermatogenesis. It also prevents Leydig cells (LCs) from gonadotropin overstimulation of androgen production. In transgenic (Tg) mice carrying deletions of the GRTH 5'-flanking regions, we previously demonstrated that the -1085 bp to ATG contains the elements for basal and androgen-induced LC-specific expression. No expression in germ cells (GCs) was found with sequences extended up to -3.6 kb. To define regulatory regions of GRTH required for expression in GC, Tg mice were generated with 5'-flanking sequence 6.4 kb (6.4 Kb-Tg) and/or deletion using green fluorescent protein (GFP) as reporter gene in the present study. GFP was expressed in all lines. Immunohistochemistry analysis showed that 6.4 Kb-Tg directed GFP expression in both GCs and LCs. Deletion of the sequence -205 bp to -3.6 kb (6.4 Kb/del-Tg) directs GFP expression only in meiotic and haploid GCs. This indicated that the distal region -6.4 kb/-3.6 kb is required for GRTH cell-specific expression in GC. Also, it inhibits the expression of GRTH in LC directed by the 205-bp promoter, an effect that is neutralized by the -3.6-kb/-205-bp sequence. Androgen receptor antagonist, flutamide treatment prevents GFP/GRTH expression in Tg lines, demonstrating in vivo direct and indirect effects of endogenous androgen on LCs and GCs, respectively. Our studies have generated and characterized Tg lines that can be used to define requirements for cell-specific expression of the GRTH gene and to further advance our knowledge on the regulation of GRTH by androgen in GCs.

Gonadotropin-regulated Testicular RNA Helicase (GRTH/DDX25), a Negative Regulator of Luteinizing/Chorionic Gonadotropin Hormone-induced Steroidogenesis in Leydig Cells: CENTRAL ROLE OF STEROIDOGENIC ACUTE REGULATORY PROTEIN (StAR)

Gonadotropin-regulated testicular RNA helicase (GRTH/DDX25) is a testis-specific gonadotropin-regulated RNA helicase that is present in Leydig cells (LCs) and germ cells and is essential for spermatid development and completion of spermatogenesis. Normal basal levels of testosterone in serum and LCs were observed in GRTH null (GRTH(-/-)) mice. However, testosterone production was enhanced in LCs of GRTH(-/-) mice compared with WT mice by both in vivo and in vitro human chorionic gonadotropin stimulation. LCs of GRTH(-/-) mice had swollen mitochondria with a significantly increased cholesterol content in the inner mitochondrial membrane. Basal protein levels of SREBP2, HMG-CoA reductase, and steroidogenic acute regulatory protein (StAR; a protein that transports cholesterol to the inner mitochondrial membrane) were markedly increased in LCs of GRTH(-/-) mice compared with WT mice. Gonadotropin stimulation caused an increase in StAR mRNA levels and protein expression in GRTH(-/-) mice versus WT mice, with no further increase in SREBP2 and down-regulation of HMG-CoA reductase protein. The half-life of StAR mRNA was significantly increased in GRTH(-/-) mice. Moreover, association of StAR mRNA with GRTH protein was observed in WT mice. Human chorionic gonadotropin increased GRTH gene expression and its associated StAR protein at cytoplasmic sites. Taken together, these findings indicate that, through its negative role in StAR message stability, GRTH regulates cholesterol availability at the mitochondrial level. The finding of an inhibitory action of GRTH associated with gonadotropin-mediated steroidogenesis has provided insights into a novel negative autocrine molecular control mechanism of this helicase in the regulation of steroid production in the male.

An Androgen Response‐Like Element Half‐Site Is Essential For Androgen Regulation Of Gonadotropin Regulated Testicular RNA Helicase (GRTH/DDX25) Transcription

GRTH is a testis‐specific member of the DEAD‐box family of RNA helicases which is present in Leydig and germ cells and is essential for spermatogenesis. It is the only known RNA helicase to be hormonally regulated. GRTH is transcriptionally up‐regulated by hCG via cAMP‐induced androgen (A) in Leydig cells. To gain insights into the mechanism of androgen regulation, immortalized rat hypothalamic GnRH neurons that express GRTH mRNA were used to generate a stable cell line expressing the A receptor (GT1‐7AR). The significantly A‐induced GRTH expression (mRNA/protein) in GT1‐7AR was prevented by the AR antagonist Nilutamide. Two putative ARE atypical half sites were present at −800 (ARE2) and −200 bp (ARE1) of GRTH gene. Functional analysis of sequential deleted GRTH 5' flanking sequence in GT1‐7AR treated with dihydrotestosterone (DHT) and/or Nilutamide revealed an active AR response region at −1085/−500 bp. Point mutation of ARE2 prevented A‐induced up‐regulation of GRTH transcription. ChIP using AR antibody in GT1‐7AR cells treated with DHT revealed AR recruitment to GRTH (−980/−702 bp) containing ARE2 compared to control. No binding activity was associated with ARE1. We conclude that ARE2 plays a key role in the androgen regulation of the GRTH gene transcription. Our findings provide valuable insights for future elucidation of androgen regulated GRTH function during germ cell development. Supported‐IRP, NICHD

Gonadotropin‐regulated testicular RNA helicase (GRTH/DDX25) gene: Cell‐specific expression and transcriptional regulation by androgen in transgenic mouse testis

Gonadotropin-regulated testicular RNA helicase is a multifunctional enzyme present in both Leydig and germ cells that is essential for the progress of spermatogenesis. GRTH gene expression is transcriptionally upregulated by human chorionic gonadotropin (hCG) via second messenger (cAMP) and androgen in Leydig cells. The regulatory region(s) in the GRTH gene that is/are required for its cell-specific expression in the testis and hCG/androgen dependent expression were investigated in transgenic mice carrying sequential deletions of 5' flanking sequences of the GRTH gene. GFP-reporter gene expression directed by the GRTH 5' flanking sequences extending to -3.6 kb was specifically located in Leydig cells and the 205 bp minimal promoter domain was sufficient for this cell-specific expression. The 1 kb (5' to the ATG codon) transgene-directed expression was markedly increased by in vivo hCG treatment. Administration of the androgen receptor inhibitor Flutamide blocked the basal and hCG stimulated GFP expression in Leydig cells. We conclude that the expression of GRTH in testicular cells is differentially regulated by its 5' flanking sequence and that the 1 kb fragment of GRTH gene contains sequences for androgen regulation of its expression in Leydig cells.

Polymorphism of the GRTH/DDX25 gene in normal and infertile Japanese men: a missense mutation associated with loss of GRTH phosphorylation

The gonadotropin-regulated testicular RNA helicase (GRTH/Ddx25) is present in Leydig and germ cells of rodents, and is essential for fertility in mice. This study evaluated the incidence of GRTH/DDX25 gene mutations in a group of infertile patients with non-obstructive azoospermia (NOA), 85% with a preponderance of Sertoli cells in the seminiferous tubule and 15% with spermatogenic arrest, and compared them to a group of fertile subjects. Exonic sequences in the GRTH gene were screened using denaturing high-performance liquid chromatography of the genomic DNA from 143 NOA and 100 fertile Japanese men. A unique heterozygous missense mutation Arg(242)His in exon 8 was identified in 5.8% of Sertoli cell-only patients and in 1% of normal subjects. Although the mutant protein was efficiently expressed in COS-1 cells, only the 56 kDa nuclear/cytoplasmic non-phosphorylated species was present, whereas the 61 kDa cytosolic phosporylated species was absent. In addition, a silent mutation was identified in exon 11 in NOA subjects. The Arg(242)His missense mutation of the GRTH/DDX25 gene associated with expression of a protein with reduced basicity, and the absence of the phospho-GRTH species, could be of relevance to some of the functional aspects of the protein that impact on germ cell development and/or function.

Gonadotropin-regulated testicular RNA helicase (GRTH/Ddx25) is essential for spermatid development and completion of spermatogenesis.

Gonadotropin-regulated testicular RNA helicase (GRTH/Ddx25), a member of the DEAD-box protein family, is a testis-specific gonadotropin-regulated RNA helicase that is present in Leydig cells and germ cells (meiotic spermatocytes and spermatids). In this study, we observed that GRTH is present in the nucleus, cytoplasm and chromatoid body of germ cells, and is an integral component of messenger ribonuclear protein particles. Male mice with a null mutation in the GRTH gene displayed normal gonadotropin and androgen profiles. However, they were sterile, with azoospermia caused by a complete arrest of spermiogenesis at step 8 of round spermatids and failure to elongate. Round spermatids of the null mice showed marked diminution in the size of chromatoid bodies. The transcription of relevant messages was not altered, but their translation was abrogated in a selective manner. Protein expression of transition proteins 1 and 2 and angiotensin-converting enzyme was completely absent, whereas that of the transcriptional activator cAMP responsive element modulator was intact. These findings indicate that GRTH participates in translational-associated events during germ cell development. Although significant apoptosis was present at the metaphase of meiosis in the GRTH-null mice, spermatogenesis proceeded to step 8 of spermiogenesis when complete arrest occurred. This progression may relate to compensatory gene function(s) and/or the observed up-regulation of DNA repair proteins Rad51 and Dmc1. This study (i) demonstrates that GRTH is essential for completion of spermatogenesis, (ii) provides insights into intrinsic requirements for spermiogenesis, and (iii) establishes a model for studies of male infertility and contraception.