Abstract
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.
Highlights
Spermatogenesis is characterized by a complex and highly specialized differentiation program, in which male germ cells show a cohesive expression of an array of testicular genes with a remarkable degree of cellular restructuring involved in a complex events of genomic and epigenetic reorganization to produce mature spermatozoa (Steger, 2001; Sassone-Corsi, 2002; Kimmins and Sassone-Corsi, 2005)
EM studies showed smaller and condensed chromatoid bodies (CBs) in KI mice, which is evident by a significant reduction in their size/diameter when compared to CBs of WT mice which display an amorphous “nuage” texture
Among proteins extracted from CBs of WT and KI mice, the levels of MVH/DDX4, mouse Argonaute/PIWI family RNA binding protein (MIWI) (CB control), and non-phospho GRTH were unaltered, while phospho-GRTH at T239 (pGRTH) protein was completely absent in the CBs of KI mice compared to WT (Figure 1F; Table 1A; Kavarthapu et al, 2019)
Summary
Spermatogenesis is characterized by a complex and highly specialized differentiation program, in which male germ cells show a cohesive expression of an array of testicular genes with a remarkable degree of cellular restructuring involved in a complex events of genomic and epigenetic reorganization to produce mature spermatozoa (Steger, 2001; Sassone-Corsi, 2002; Kimmins and Sassone-Corsi, 2005) During these events, several mRNAs transported from the nucleus to the cytoplasm undergo near-to-complete translational silencing/repression, extensive post-transcriptional processing, and storage at specific cytoplasmic sites, such as the ribonucleoprotein (RNP) granules called chromatoid bodies (CBs), for translation at later stages of spermiogenesis (Steger, 2001; Maclean and Wilkinson, 2005; Kotaja and Sassone-Corsi, 2007). CB contains several components of the RNA-induced silencing complex (Kotaja et al, 2006), mouse Vasa homolog (MVH/DDX4), a germ cell marker (Fujiwara et al, 1994; Noce et al, 2001), piRNA binding protein, and GRTH/DDX25 in high abundance (Sato et al, 2010; Meikar et al, 2014). piRNA and piRNA binding protein constitute a major part of the CB composition, and piRNAs derived from pachytene piRNA clusters and transposable elements. piRNAs are present both in the embryonic and the postnatal male germ cells, and their expression is induced abundantly in late meiotic cells and haploid round spermatids (Meikar et al, 2011, 2014)
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