Regulation of Miwi-mediated mRNA stabilization by Ck137956/Tssa is essential for male fertility

Abstract

Background: Sperm is formed through spermiogenesis, a highly complex process involving chromatin condensation that results in cessation of transcription. mRNAs required for spermiogenesis are transcribed at earlier stages and translated in a delayed fashion during spermatid formation. However, it remains unknown that how these repressed mRNAs are stabilized.

Results: Here we report a Miwi-interacting testis-specific and spermiogenic arrest protein, Ck137956, which we rename Tssa. Deletion of Tssa led to male sterility and absence of sperm formation. The spermiogenesis arrested at the round spermatid stage and numerous spermiogenic mRNAs were down-regulated in Tssa^-/- mice. Deletion of Tssa disrupted the localization of Miwi to chromatoid body, a specialized assembly of cytoplasmic messenger ribonucleoproteins (mRNPs) foci present in germ cells. We found that Tssa interacted with Miwi in repressed mRNPs and stabilized Miwi-interacting spermiogenesis-essential mRNAs.

Conclusions: Our findings indicate that Tssa is indispensable in male fertility and has critical roles in post-transcriptional regulations by interacting with Miwi during spermiogenesis.

Keywords: Ck137956/Tssa; Male infertility; Miwi; Spermiogenesis; mRNA stability.

Fig. 1

Expression of Ck137956 and fertility of Ck137956 knockout mice. A Ck137956 mRNA expression was detected in various tissues from adult mice by RT-PCR with 18S as a loading control. NC, negative control. B qRT-PCR analysis of Ck137956 expression in postnatal mouse testes. n = 3. Data were presented as the mean ± SD. C The presence of Ck137956 mRNA was evaluated in different types of spermatogenic cells with 18S as a loading control. Spg, spermatogonia; Spc, pachytene spermatocytes; RS, round spermatids; ES, elongated spermatids. D Structure of mouse Ck137956 protein predicted by AlphaFold. E Phylogenetic tree showing the evolutionary distance of Ck137956 among different animals with branch lengths in units representing the number of amino acids substituted per site. The numbers exhibit bootstrap values. F Schematic diagram of knockout strategy by CRISPR/Cas9. Yellow and green: the single guide RNA (sgRNA) targeting sites. G Sanger sequencing showing eight bases deletion of the Ck137956 gene. H Expression of Ck137956 protein in Ck137956^+/- and Ck137956^−/− testis with Actin as a loading control. I Litter size of Ck137956^+/- and Ck137956^−/− male mice mated with wildtype female mice. n = 6. Data were presented as the mean ± SEM. ***p < 0.001. J Morphology of adult Ck137956^+/- and Ck137956^−/− testes and statistics of testis/body weight ratio. Scale bar: 3 mm. n = 6. Data were presented as the mean ± SEM. ***p < 0.001

Fig. 2

Phenotype analysis of Ck137956 knockout male mice. A HE-stained sections of the caput and cauda epididymis from Ck137956^+/- and Ck137956^−/− mice. Scale bars: 50 μm. B HE-stained sections of the testes from Ck137956^+/- and Ck137956^−/− mice. PAC, pachytene spermatocytes; RS, round spermatids; ES, elongated spermatids. Scale bars: 50 μm. C Immunofluorescence analysis of seminiferous tubules from Ck137956^+/- and Ck137956^−/− testes using PNA (red) stained the acrosome and DAPI (blue) stained the nuclei. Magnification of round spermatids were shown in white. Scale bars: 50 μm. D Ultrastructural analysis of Ck137956^+/- and Ck137956^−/− round spermatids by transmission electron microscopy. The developmental stage of round spermatids was determined by acrosomal angles. Nu, nucleus; Acr, acrosome. Scale bar: 1 μm. E Immunofluorescence staining of SYCP3 (green) and γH2AX (red) on spread nuclei of spermatocytes from Ck137956^+/- and Ck137956^−/− testis. Scale bar: 5 μm. n = 3. Data were presented as the mean ± SEM. NS, not significant. F TUNEL staining (green) and quantitative analysis of Ck137956^+/- and Ck137956.^−/− seminiferous tubules. DAPI (blue) stained the nuclei. Asterisks indicated TUNEL-positive tubules. Scale bar: 50 μm. n = 3. Data were presented as the mean ± SEM. **p < 0.01

Fig. 3

Gene expression analysis of Ck137956 knockout pachytene spermatocytes and round spermatids. A Number of differentially expressed genes in Ck137956^−/− pachytene spermatocytes and round spermatids. PAC, pachytene spermatocytes; RS, round spermatids. B Venn diagram showing overlap between down-regulated genes in Ck137956^−/− pachytene spermatocytes and those in Ck137956^−/− round spermatids. C Heatmap of genes with differential expression (Log₂FoldChange > 2 and adjusted P < 0.01) between Ck137956^+/- and Ck137956^−/− mice in pachytene spermatocytes or round spermatids. D Heatmap of gene expression during spermatogenesis for genes down-regulated in Ck137956^−/− RS. 2C: spermatogonia and somatic cells; LZ: leptotene/zygotene spermatocytes; PAC, pachytene spermatocytes; RS, round spermatids. E–F Enriched cellular component terms (E) and biological process terms (F) in the down-regulated genes in Ck137956^−/− round spermatids. G Western blot analysis of Ck137956 expression in cytoplasm, nuclei, and chromatin. Gapdh, Hdac1 and histone H4 served as cytoplasm, nuclei and chromatin controls, respectively. H-I qRT-PCR analysis of the levels of mRNAs (H) and pre-mRNAs (I) of spermatogenesis-related genes in day-24 Ck137956^+/- and Ck137956^−/− testes. n = 3. Data were presented as the mean ± SEM. NS, not significant, **p < 0.01, ***p < 0.001. J Scatterplot of Log₂FoldChange between transcriptome and proteome after Ck137956 deletion in round spermatids

Fig. 4

Ck137956 regulates ubiquitination and localization of Miwi. A Co-IP assay of interaction between HA-Ck137956 and Flag-Miwi in HEK-293 T cells. B Co-IP assay of interaction between Ck137956 and Miwi in testis with or without RnaseA/T treatment. C Domain mapping of the Ck137956-Miwi interaction by co-transfection with HA-tagged Ck137956 truncations and Flag-tagged Miwi truncations. The scheme of Ck137956 and Miwi truncations is shown in the upper panel. The grey indicates disordered region. D The levels of Miwi protein expression in day-24 Ck137956^+/- and Ck137956^−/− testis. Tubulin was used as a loading control. n = 3. Data were presented as the mean ± SEM. **p < 0.01. E mRNA expression levels of Miwi quantified by qRT-PCR in day-24 Ck137956^+/- and Ck137956^−/− testis. n = 3. Data were presented as the mean ± SEM. NS, not significant. F Overexpression of HA-tagged ubiquitin in HEK-293 T cells and analysis of the ubiquitylation of Miwi with or without Ck137956 overexpression by immunoprecipitation using anti-HA antibody. The cells were harvested after a 6-h treatment with 20 μM of MG132. G Seminiferous tubule sections were stained with Miwi (red) and DAPI (blue). Pachytene spermatocytes and round spermatids were indicated by red dotted box and solid box, respectively. Scale bar: 20 μm. H Venn diagram showing the overlap among down-regulated genes in Ck137956^−/− and Miwi^−/− RS, and target genes of Miwi

Fig. 5

Ck137956 alters the mRNA-stabilizing ability of Miwi. A Immunofluorescence analysis of LINE1 (green) in Ck137956^+/-, Ck137956^−/− and Pnldc1^−/− testes. DAPI (white) stained the nuclei. Scale bars: 50 μm. B qRT-PCR analysis of the mRNA expression levels of Ppp1cb, Atr, Tox4, Gfpt1, Psmag, Mdc1 and BC026590 in Ck137956^−/− and control round spermatids. n = 3. C Distribution of Ck137956 and Miwi proteins in different fractions collected from sucrose gradient sedimentation of testis lysate. Rps3 and Msy2 were used as mono/polysomes and mRNPs controls, respectively. D qRT-PCR analysis of Tssk1, Tssk2, Tnp1, Tnp2, Prm2 mRNA expression levels in mRNPs of Ck137956^+/- and Ck137956^−/− testes. n = 9. E qRT-PCR analysis of mRNAs expression levels in GC-2spd(ts) cells treated with siCk137956 or/and siMiwi with scrambled sequence as a control. DK, doubleknock down group. The statistical significance between the groups was plotted. n = 3. F Quantitation of mRNA stability in siMiwi-treated and control GC-2spd(ts) cells after Actinomycin D treatment (10 μg/ml). The upper right corner shows the half-life time (t_1/2) of gene expression in siMiwi-treated and control cells. n = 3. G qRT-PCR analysis of mRNA expression in siCk137956-treated cells with or without Miwi overexpression. n = 3. H qRT-PCR analysis of mRNA expression levels in siMiwi-treated cells with or without Ck137956 overexpression. n = 3. All data were represented as mean ± SEM. NS, not significant, *p < 0.05, **p < 0.01, ***p < 0.001