SPAG17 mediates nuclear translocation of protamines during spermiogenesis

Abstract

Protamines (PRM1 and PRM2) are small, arginine-rich, nuclear proteins that replace histones in the final stages of spermiogenesis, ensuring chromatin compaction and nuclear remodeling. Defects in protamination lead to increased DNA fragmentation and reduced male fertility. Since efficient sperm production requires the translocation of protamines from the cytoplasm to the nucleus, we investigated whether SPAG17, a protein crucial for intracellular protein trafficking during spermiogenesis, participates in protamine transport. Initially, we assessed the protein-protein interaction between SPAG17 and protamines using proximity ligation assays, revealing a significant interaction originating in the cytoplasm and persisting within the nucleus. Subsequently, immunoprecipitation and mass spectrometry (IP/MS) assays validated this initial observation. Sperm and spermatids from Spag17 knockout mice exhibited abnormal protamination, as revealed by chromomycin A3 staining, suggesting defects in protamine content. However, no differences were observed in the expression of Prm1 and Prm2 mRNA or in protein levels between testes of wild-type and Spag17 knockout mice. Conversely, immunofluorescence studies conducted on isolated mouse spermatids unveiled reduced nuclear/cytoplasm ratios of protamines in Spag17 knockout spermatids compared to wild-type controls, implying transport defects of protamines into the spermatid nucleus. In alignment with these findings, in vitro experiments involving somatic cells, including mouse embryonic fibroblasts, exhibited compromised nuclear translocation of PRM1 and PRM2 in the absence of SPAG17. Collectively, our results present compelling evidence that SPAG17 facilitates the transport of protamines from the cytoplasm to the nucleus.

Keywords: SPAG17; nucleocytoplasmic transport; protamine; spermatogenesis; spermiogenensis.

FIGURE 1

Proximity ligation assay (PLA) showing interaction of SPAG17 and protamines in mouse elongating spermatids. (A) Representative image showing interaction of SPAG17 and PRM1 in wild-type elongating spermatid step 12–13. (B) Representative images showing interaction of SPAG17 and PRM2 in wild-type elongating spermatid step 12–13. (C) Representative images showing positive control using anti-α and anti-β tubulin primary antibodies in wild-type elongating spermatids step 11. (D) Representative images showing lack of interaction in Spag17 knockout elongating spermatid step 14, which lack SPAG17 protein, when anti-SPAG17 and anti-PRM2 antibodies were used. Images were collected from 3 independent PLAs experiments.

FIGURE 2

Abnormal protamination is observed in elongated spermatids and sperm from Spag17 knockout mice. Spermatids and sperm were collected from the testes and cauda epididymis of wild-type (WT, n = 3) and Spag17 knockout (KO, n = 4) mice, respectively, and stained with CMA3 to assess protamination. A total of 100–200 cells per sample were counted, and the percentage of CMA3-positive heads (green labeled) was calculated. (A) Representative images of spermatids at different steps. (B) Quantification of the percentage of CMA3-positive spermatids from step 8 to 16. (C) Quantification of the percentage of CMA3-positive spermatids from steps 15–16. (D) Representative images displaying CMA3-positive sperm and quantification of the percentage of CMA3-positive sperm. Results are means ± SEM. Significant differences were observed in comparison to WT, with * indicating p = 0.01 and ** indicating p = 0.0034.

FIGURE 3

Expression of Prm1 and Prm2 mRNA is not different between testes from wild-type and Spag17 knockout mice. Testes from adult wild-type (WT, n = 4) and Spag17 knockout (KO, n = 5) mice were collected. Total RNA was extracted and used to determine Prm1 and Prm2 gene expression by qPCR using 18S rRNA as a housekeeping gene. (A) Prm1 mRNA expression showed no differences between WT and KO testes; p = 0.17. (B) Prm2 mRNA expression showed no differences between WT and KO testes; p = 0.31. (C) Prm1/Prm2 mRNA ratio was not different between WT and KO testes. Results are means ± SEM, p = 0.96.

FIGURE 4

Comparison of protamine protein content between wild-type (WT) and Spag17 knockout (KO). (A) Representative lanes of the acid-urea polyacrylamide gel indicating the bands corresponding to protamines (PRM1 and PRM2; pre-PRM2 = PRM2 precursor), histones and other extracted proteins. (B) Quantification showing protamine content as measured by protamine band density on a Coomassie blue stained acid-urea polyacrylamide gel as percent of the whole lane (p = 0.64). (C) Quantification of PRM1/PRM2 ratio (p = 0.38). Results are means ± SEM, n = 3 per genotype.

FIGURE 5

SPAG17 is important for nuclear translocation of protamines in spermatids. Spermatids were isolated from mouse testes and immunolabeled using anti-PRM1 and PRM2 antibodies. (A) Representative immunolabeling in wild-type (WT) spermatids showing nuclear localization of PRM1 and PRM2. (B) Representative immunolabeling in Spag17 knockout (KO) spermatids revealed disrupted nuclear translocation of PRM1 and PRM2 (C) Quantification of nuclear/cytoplasmic (N/C) ratio of PRM1 and PRM2 localization in WT (n = 4) and KO (n = 4) spermatids at different steps during spermiogenesis. Results are means ± SEM. *Significant differences in comparison to WT; p < 0.05.

FIGURE 6

Transport of protamines into the nucleus is disrupted in the absence of SPAG17 in cultured fibroblasts. Mouse embryonic fibroblasts (MEFs) collected from wild-type (WT) and Spag17 knockout (KO) embryos were transfected with mouse pPrm1-mCherry-N1 and mouse pPrm2-EGFP-N3 expressing vectors. (A) Representative images showing protamine localization in WT and KO MEFs after 24 h transfection with the PRM1 vector. (B) Representative images showing protamine localization in WT and KO MEFs after 24 h transfection with the PRM2 vector. (C) Quantification of nuclear localization of protamines. At 24 h post-transfection, the percentage of nuclear localization of PRM1 or PRM2 is significantly lower in Spag17 knockout MEFs. Results are means ± SEM from four independent experiments. *Significant differences in comparison to WT; p = 0.001).