Insights into the role of hnRNPK in spermatogenesis via the piRNA pathway

Abstract

Deletion of hnRNPK in mouse spermatogonia leads to male sterility due to arrest permatogenesis, yet the underlying molecular mechanisms remain elusive. This study investigated the testicular proteome on postnatal day 28 (P28) to elucidate the infertility associated with Hnrnpk deficiency, identifying 791 proteins with altered expression: 256 were upregulated, and 535 were downregulated. Pathway enrichment analysis demonstrated that the downregulated proteins are primarily involved in spermatogenesis, fertilization, and piRNA metabolic processes. In Hnrnpk cKO mice, key proteins essential for piRNA metabolism, such as PIWIL1, TDRD7, DDX4, and MAEL, exhibited reduced expression, resulting in impaired piRNA production. Mechanistic studies employing RNA immunoprecipitation (RIP), dual-luciferase reporter assays, and fluorescence in situ hybridization/immunofluorescence (FISH/IF) assays demonstrated that hnRNPK directly interacts with the 3'UTR of piRNA pathway transcripts, enhancing their translational efficiency. These results establish that Hnrnpk deficiency disrupts the piRNA pathway by diminishing the expression of essential regulatory proteins, thereby impairing piRNA production and spermatogenesis. Our findings elucidate a novel molecular basis for infertility linked to hnRNPK dysfunction and advance understanding of post-transcriptional regulation in male germ cell development.

Keywords: Conditional knock-out mice; Proteomics; Spermatogenesis; Translational regulation; hnRNPK; piRNA pathway.

Fig. 1

Whole proteome analysis of Hnrnpk cKO mice testis. (a) PCA analysis of TMT-proteoome in each group. (b) Volcano plot of the differentially expressed proteins in Hnrnpk cKO testes compared with the WT control at P28. Green dots represent significantly downregulated proteins, red dots represent significantly upregulated proteins (p-adj value < 0.05, fold change > 2), and gray dots represent unchanged proteins. (c) The heatmap of differentially expressed proteins. (d) Validation of proteins expression by western blot analysis. (E) Statistical analysis of the results shown in D (n = 3). *p < 0.05; ** p < 0.01. Data are represented as mean ± SD.

Fig. 2

Proteins involved in spermatogenesis are influenced by hnRNPK. (a) Bubble graph of gene ontology (GO) analysis results from upregulated DEPs enriched in biological processes, cellular components, and molecular function. Gradation from pink to red indicates an increasing p-value (decreasing significance), the Bubble size represents the number of proteins in that term, and the ratio of proteins is in the category over total downregulated proteins. (b) For upregulated DEPs, three MCODE complexes automatically identified in Metascape, colored by their identities. Their functional labels are generated based on the top-three functional enriched terms, if available. (c) Bubble graph of gene ontology (GO) analysis results from downregulated DEPs enriched in biological processes, cellular components, and molecular function. (d) For downregulated DEPs, three MCODE complexes automatically identified in Metascape, colored by their identities.

Fig. 3

The level of piRNA-related proteins was reduced in the testes of Hnrnpk cKO mice. (a) Western blot analysis of hnRNPK, DDX4, ASZ1, PIWIL1, PIWIL2, and MAEL expression in WT and Hnrnpk cKO mouse testes. (b) Statistical analysis of the results shown in A (n = 3). *p < 0.05; ** p < 0.01. Data are represented as mean ± SD. (c) Immunofluorescence validation of PIWIL1 expression in WT and Hnrnpk cKO mouse testes. (d) Immunohistochemistry detection of PIWIL2 expression in WT and Hnrnpk cKO mouse testes. (e) Immunofluorescence detection of ASZ1 and γH2A.X expression in WT and Hnrnpk cKO mouse testes. (f) Immunofluorescence detection of MAEL and γH2A.X expression in WT and Hnrnpk cKO mouse testes. (g) Immunohistochemistry detection of DDX4 expression in WT and Hnrnpk cKO mouse testes. LS, leptotene spermatocyte; PS, pachytene spermatocyte; V, VI, X, XI, the stages of spermatogenic epithelial cycle. Red arrow points to the leptotene spermatocyte, wight arrow points to the pachytene spermatocyte. Scale bar, 50 μm.

Fig. 4

Hnrnpk depletion reduces the piRNA populations. (a) Length distribution of small RNAs in WT and Hnrnpk cKO testes. RPM, reads per million reads. (b) Distribution of the 1st and 10th nucleotides of piRNAs (24–35 nt in length) in WT and Hnrnpk cKO testes. (c) The expression of thirteen piRNAs was selected to verify by RT-qPCR analysis.

Fig. 5

Electron micrographs of WT and Hnrnpk cKO pachytene spermatocytes from 18 d testes. PS, pachytene spermatocyte; Mito, mitochondria; IMC, inter mitochondrial cement. Red arrow points to the typical mitochondria, peripherally located to the nuclear. Yellow arrow points to the IMC structure, clusters formed between mitochondria. The area within the white dashed box is the zoomed part of the image.

Fig. 6

hnRNPK interacts with the 3’UTR of piRNA metabolic process-related genes and regulates their translation. (a) RIP RT-qPCR assay was carried out to detect the interaction between piRNA metabolic process-related genes and hnRNPK in testis (n = 3). (b) FISH/IF images showing colocalization of Piwil1 (green) and hnRNP K (red) in pachytene spermatocyte (PS) and round sperm; scale bars, 100 µm. The results are representative of three biologically independent samples. (c) RT-qPCR was used to detect the expression of piRNA metabolic process-related DEPs (Piwil1, Tdrd7, Aszl, Mael, Piwil2, Tdrd1, Tdrd5, Tdrd6, Tdrd12, Ddx4 and Ddx25). (d-i) Luciferase activity of wild-type (WT) or mutant (MUT) Piwil1 (d), Ddx4 (e), Tdrd7 (f), Mael (g), Piwil2 (h) and Aszl (i) 3’ UTR in 293T cells after co-transfection with pcDNA3.1 or pcDNA3.1-Hnrnpk. *p < 0.05; ** p < 0.01; ns, not significant. Data are represented as mean ± SD, n = 3. (j) Luciferase assays were performed by co-transfection of Piwil1, Piwil2, Ddx4, Tdrd7, Aszl, and Mael 3’ UTR WT and MUT plasmids with pcDNA3.1-Hnrnpk in 293T cells. Data were expressed as mean ratio of relative luciferase activities (Renilla Luciferase / Firefly Luciferase) and normalized to that in cells transfected with pcDNA3.1 plasmid. *p < 0.05; ** p < 0.01; ns, not significant. Data are represented as mean ± SD, n = 3.

Fig. 7

Proposed action mechanism underlying hnRNPK-mediated piRNA metabolic process-related proteins regulation in spermatogenesis. Under the condition of hnRNPK deficient, the interaction between piRNA metabolic process-related proteins (such as Ddx4, Piwil1, Tdrd7 and Mael) mRNA 3′UTR and hnRNPK is disrupted, which results in decrease of these proteins expression. hnRNPK affects piRNA production in pachytene spermatocytes and plays an important role in the meiotic process of spermatogenesis meiosis.