Relevance of gonadotropin-regulated testicular RNA helicase (GRTH/DDX25) in the structural integrity of the chromatoid body during spermatogenesis

Abstract

Gonadotropin-regulated testicular RNA helicase (GRTH/DDX25), a multifunctional protein and a component of ribonucleoprotein complexes, is essential for the completion of spermatogenesis. We investigated the nuclear/cytoplasmic shuttling of GRTH in germ cells and its impact on the chromatoid body (CB)-a perinuclear organelle viewed as a storage/processing site of mRNAs. GRTH resides in the nucleus, cytoplasm and CB of round spermatids. Treatment of these cells with inhibitors of nuclear export or RNA synthesis caused nuclear retention of GRTH and its absence in the cytoplasm and CB. The nuclear levels of GRTH bound RNA messages were significantly enhanced and major reduction was observed in the cytoplasm. This indicated GRTH main transport function of mRNAs to the cytoplasm and CB. MVH, a germ cell helicase, and MIWI, a component of the RNA-induced-silencing complex (RISC), confined to the CB/cytoplasm, were absent in the CB and accumulated in the cytoplasm upon treatment. This also occurred in spermatids of GRTH-KO mice. The CB changed from lobular-filamentous to a small condensed structure after treatment resembling the CB of GRTH-KO. No interaction of GRTH with MVH or RISC members in both protein and RNA were observed. Besides of participating in the transport of messages of relevant spermatogenic genes, GRTH was found to transport its own message to cytoplasmic sites. Our studies suggest that GRTH through its export/transport function as a component of mRNP is essential to govern the CB structure in spermatids and to maintain systems that may participate in mRNA storage and their processing during spermatogenesis.

Fig. 1. A–C. Subcellular localization of RNA helicase (GRTH/DDX25 and MVH/DDX4) and MIWI in spermatids during spermatogenesis

Top: Specific stages of spermatogenic cells in seminiferous tubules of wild type mice compared to those of GRTH^−/− mice were characterized by their trans-illumination properties under phase-contrast microscopy. Dried-down slides of male germ cells from different stages were immuno-stained with antibodies (2^nd panel) to GRTH (A), MVH (B), MIWI (C), nuclear staining by DAPI (1^st panel), merged image (3^rd panel) and phase contrast of the cells (4th panel). Alexa Fluor 568 anti-rabbit IgG was used as a secondary antibody. Arrow-heads indicate chromatoid bodies (CB).

Fig. 2. Disappearance of GRTH and MVH and MIWI signal from CBs via a different mechanism after LMB and DRB treatment

Spermatids isolated from stages VII–VIII were incubated with vehicle (control), nuclear protein export inhibitor (LMB) or RNA polymerase II inhibitor (DRB) for 3 h. Dried-down slides were immuno-stained with GRTH (A), or MVH (B) or MIWI antibodies (C) (2^nd panel), nuclear staining by DAPI (1^st panel), and merged image (3^rd panel) and phase contrast (4th panel). Alexa Fluor 568 anti-IgG was used as a secondary antibody. Arrow-heads indicate the CBs. A′–C′: Single cell magnification of merged images. C: cytoplasm., N: Nucleus., CB: chromatoid body.

Fig. 3. Real time-PCR analysis of RNA message associated with GRTH

A. Evaluation of messages: GRTH, mouse homolog of drosophila VASA (MVH), mouse PIWI Like homolog 1 (MIWI), protamine 2 (Prm2) and cAMP responsive element modulator (CREM) in immuno-precipitated testicular GRTH complexes (GRTH-IP) from wild type (WT) and GRTH knockout (KO) adult mice. Data was expressed as a relative ratio of messages from GRTH-IP to IgG immuno-precipitated negative control group (IgG-IP). Inset: Gel image of messages with the expected fragment size (bp). B. Total message was determined and normalized by β-actin in isolated cytoplasm (C) and nuclear (N) fractions of round spermatids treated with nuclear export inhibitor LMB (200 nM) for 3 hours. C. GRTH and Prm2 messages were determined in immuno-precipitated GRTH complexes from isolated cytoplasm and nuclear fractions of round spermatids treated with LMB. Specific sets of primers for genes of interest were designed accordingly. The values are the means ± S.E. of three independent experiments in triplicate. *, p< 0.05. The ratios of nuclear to cytoplasmic (N/C), nuclear to total (N/T) or cytoplasm to total (C/T) RNA of specific genes were used to represent the effect of LMB on the export of messages from nucleus to cytoplasm.

Fig. 4. Panel I. EM analysis of the CB after LMB treatment

A. Spermatids isolated from stages VII–VIII tubular fragments were incubated with vehicle (control), LMB or DRB for 3 and 6 h. Typical CB structure (red arrow). Abnormal compact CB (pink arrows). N, nuclear. Panel II. CB of spermatids from WT and GRTH^−/− previously reported (7) are presented for comparative purposes. Green arrow: CB-wild type, Pink arrow: CB - null mice. Panel III. Diagram illustrates the relevance GRTH for the structural integrity of the CB in the spermatids of mouse testis. Distribution of GRTH/Ddx25 (red circle), MVH/Ddx4 (solid blue circle) and MIWI (blue-stripped circle) in cellular compartments of round spermatids; Nucleus (N), cytoplasm (C) and CB (green, hexagon). Abnormal compact CB noted in GRTH^−/− mice and after treatment with inhibitor (LMB or DRB) compared with the CB in spermatids of wild type (stages VII–VIII).

Fig. 5. Assessment of expression of selected protein members of the si/mi/pi RNA pathway and their interaction with GRTH

Protein extracts from whole testis (A) or purified round spermatids (RS) (B) were utilized for Western analysis of wild type (WT) and GRTH knockout samples (KO) for assessment of protein expression with antibodies to the indicated relevant proteins (CRM1, MVH, MIWI, Dicer and Argonaute protein 2 [Ago2]) and β-actin. These preparations were also used for studies of GRTH association with relevant proteins in immuno-precipitation (IP) studies with GRTH antibody followed by Western analysis utilizing specific antibodies for the specified proteins indicated above. CRM1 was used as the control for IP procedure. IgG was used as a negative control.

Fig. 6. Postulated model of GRTH action in the CB of male germ cells

During spermatogenesis, specific RNA transcripts associated to GRTH in mRNP complexes are found in the nucleus of round spermatids. These GRTH-RNP complexes are transported from nucleus to cytoplasm (a) and the CB (b) via a CRM1-dependent export pathway. GRTH-RNP complexes are required to maintain CB filamentous-lobulated perinuclear structure (CB-EM image). GRTH does not directly interact with protein factors of si/mi/pi RNA pathway that might occur in the CB. We propose that specific messages associated with GRTH are presented to members of this system (RISC: RNA induced silencing complex- [decapping enzyme Dcp1a, MIWI, MVH, Dicer]) for either storage (noted as 1) or degradation (noted as 2). GRTH most probably dissociate from mRNA at the CB site during this process. Messages are transported by GRTH from CB to polyribosomes for translation.