Abstract
Histone acetylation is involved in the regulation of chromatin structure and gene function. We reported previously that histone H3 acetylation pattern is subject to dynamic changes and limited to certain stages of germ cell differentiation during murine spermatogenesis, suggesting a crucial role for acetylation in the process. In the present study, we investigated the effects of hyper- and hypo-acetylation on spermatogenesis. Changes in acetylation level were induced by either in vivo administration of sodium phenylbutyrate, a histone deacetylase inhibitor, or by knockdown of histone acetyltransferases using short hairpin RNA plasmids transfection. Administration of sodium phenylbutyrate induced accumulation of acetylated histone H3 at lysine 9 and lysine 18 in round spermatids, together with spermatid morphological abnormalities and induction of apoptosis through a Bax-related pathway. Knockdown of steroid receptor coactivator 1, a member of histone acetyltransferases, but not general control of amino acid synthesis 5 nor elongator protein 3 by in vivo electroporation of shRNA plasmids, reduced acetylated histone H3 at lysine 9 in round spermatids, and induced morphological abnormalities. We concluded that the proper regulation of histone H3 acetylation levels is important for spermatid differentiation and complex chromatin remodeling during spermiogenesis.
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Acknowledgments
This study was supported in part by a Grant-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (No. 18390060 to Koji, T.).
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Fig. S1 TUNEL staining and immunohistochemical detection of 8-OHdG in adult NaPB (a-d)-treated mouse testes at 6 h after treatment and SAHA (e-h)-treated mouse testes at 12 h after treatment. Serial sections were used for H&E staining (a, e), TUNEL staining (b, f), immunohistochemical staining for 8-OHdG (c, g). Arrowheads TUNEL-positive cells without 8-OHdG signals; arrows TUNEL-positive cells with 8-OHdG signals. The insets (b′) in b and (c′) in c are enlarged. As a negative control, sections of NaPB (d)- and SAHA (h)-treated testes were reacted with normal mouse IgG instead of the specific antibody. Scale bars 50 μm. (JPEG 1463 kb)
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Fig. S2 Immunohistochemical detection of histone H3K9ac, H3K18ac and H3K23ac in paraffin-embedded sections of adult DMSO (a-e)- and SAHA (f-j)-treated mouse testes at 12 h after treatment. Serial sections were used for H&E staining (a, f), immunohistochemical staining for H3K9ac (b, g), H3K18ac (c, h), and H3K23ac (d, i). As a negative control, DMSO (e)- and SAHA (j)-treated sections of testes were reacted with normal rabbit IgG instead of specific antibodies. Seminiferous tubules at stage VI-VII are shown. Scale bars 50 μm. (JPEG 2003 kb)
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Fig. S3 Immunofluorescence detection of GFP in LacZ (a-c)- , SRC-1 (d-f)-shRNA transfected and non-transfected (g-i) 28-day-old neonatal mouse testes. (a, d, g) immunofluorescence staining of GFP. (b, e, h) As a negative control, sections were reacted with normal mouse IgG instead of the specific antibody. (c, f, i) DAPI staining. Arrowheads GFP-positive cells; asterisks nonspecific fluorescence signals from the secondary antibody in interstitial Leydig cells. Scale bars 50 μm. (JPEG 2039 kb)
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Fig. S4 Double immunofluorescence staining of GFP and GCN5 in LacZ- and GCN5-shRNA plasmid transfected 28-day-old neonatal mouse testes at day 10 post-transfection. (a-f) Double immunofluorescence staining of GFP (a, d) and GCN5 (b, e) in LacZ (a-c)- and GCN5 (d-f)-shRNA transfected testes. Arrows GFP-positive round spermatids in LacZ-shRNA transfected testes; arrowheads GFP-positive round spermatids in GCN5-shRNA transfected testes. Scale bars 10 μm (JPEG 2089 kb)
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Fig. S5 Double immunofluorescence staining of GFP and ELP3 in LacZ- and ELP3-shRNA plasmid transfected 28-day-old neonatal mouse testes at day 10 post-transfection. (a-f) Double immunofluorescence staining of GFP (a, d) and ELP3 (b, e) in LacZ (a-c)- and ELP3 (d-f)-shRNA transfected testes. Arrows GFP-positive round spermatids in LacZ-shRNA transfected testes; arrowheads GFP-positive round spermatids in ELP3-shRNA transfected testes. Scale bars 10 μm. (JPEG 1935 kb)
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Fig. S6 Double immunofluorescence staining of GFP and H3K9ac; GFP and H3K18ac; GFP and H3K23ac in shRNA plasmid transfected 28-day-old neonatal mouse testes at day 10 post-transfection. (a-d) Double immunofluorescence staining of GFP and H3K9ac in LacZ (a)- , SRC-1 (b)-, GCN5 (c)- and ELP3 (d)-shRNA transfected testes. (e-h) Double immunofluorescence staining of GFP and H3K18ac in LacZ (e)- , SRC-1 (f)- , GCN5 (g)- and ELP3 (h)-shRNA transfected testes. (i-l) Double immunofluorescence staining of GFP and H3K23ac in LacZ (i)- , SRC-1 (j)- , GCN5 (k)- and ELP3 (l)-shRNA transfected testes. Arrowheads GFP-positive round spermatid in SRC-1-shRNA transfected testes with reduced H3K9ac staining. Scale bar 20 μm. (JPEG 2750 kb)
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Fig. S7 Methylation level of CCGG sites in adjacent section of NaPB-treated testis at 6 h after treatment by HELMET method using DAB staining. Adjacent sections of paraffin-embedded mouse testes were used for H&E staining, TUNEL, non-methylated CCGG sites and methylated CCGG sites. (a) H&E staining. (b) TUNEL staining. (c) Blockade of 3′-OH ends with dideoxynucleotides by TdT. After blockade, the section was labeled with biotin-16-dUTP by TdT and the incorporated biotin was detected with HRP-anti-biotin. No signals were observed. (d) Staining for non-methylated CCGG sites. After the blockade procedure described in (c), the section was digested with HpaII, labeled with biotin-16-dUTP and visualized by enzyme immunohistochemistry with HRP-anti-biotin. (e) Blockade of HpaII cutting sites with dideoxynucleotides by TdT. The section was digested with HpaII, and the cutting sites were blocked with a dideoxynucleotide mixture. Then, the section was processed in a manner similar to that described in (c). (f) Staining for methylated CCGG sites. After blockade of HpaII cutting sites with dideoxynucleotides, the section was digested with MspI and the cutting sites were labeled with biotin-16-dUTP and visualized with HRP-anti biotin. Arrows TUNEL-positive round spermatids with elevated non-methylated CCGG sites; arrowheads TUNEL-negative round spermatids with elevated non-methylated CCGG sites. Scale bars 50 μm. (JPEG 1996 kb)
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Dai, L., Endo, D., Akiyama, N. et al. Aberrant levels of histone H3 acetylation induce spermatid anomaly in mouse testis. Histochem Cell Biol 143, 209–224 (2015). https://doi.org/10.1007/s00418-014-1283-1
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DOI: https://doi.org/10.1007/s00418-014-1283-1