Immunohistochemical Analysis of Histone H3 Modifications in Germ Cells during Mouse Spermatogenesis

Abstract

Histone modification has been implicated in the regulation of mammalian spermatogenesis. However, the association of differently modified histone H3 with a specific stage of germ cells during spermatogenesis is not fully understood. In this study, we examined the localization of variously modified histone H3 in paraffin-embedded sections of adult mouse testis immunohistochemically, focusing on acetylation at lysine 9 (H3K9ac), lysine 18 (H3K18ac), and lysine 23 (H3K23ac); tri-methylation at lysine 4 (H3K4me3) and lysine 27 (H3K27me3); and phosphorylation at serine 10 (H3S10phos). As a result, we found that there was a significant fluctuation in the modifications; in spermatogonia, the stainings for H3K9ac, H3K18ac, and H3K23ac were strong while that for H3K4me3 was weak. In spermatocytes, the stainings for H3K9ac, H3K18ac, H3K23ac, and H3K4me3 were reduced in the preleptotene to pachytene stage, but in diplotene stage the stainings for H3K18ac, H3K23ac, and H3K4me3 seemed to become intense again. The staining for H3K27me3 was nearly constant throughout these stages. In the ensuing spermiogenesis, a dramatic acetylation and methylation of histone H3 was found in the early elongated spermatids and then almost all signals disappeared in the late elongated spermatids, in parallel with the replacement from histones to protamines. In addition, we confirmed that the staining of histone H3S10phos was exclusively associated with mitotic and meiotic cell division. Based upon the above results, we indicated that the modification pattern of histone H3 is subject to dynamic change and specific to a certain stage of germ cell differentiation during mouse spermatogenesis.

Keywords: epigenetics; histone H3 modification; immunohistochemistry; mouse; spermatogenesis.

Fig. 1

Hematoxylin and eosin (H-E) staining (A) and immunohistochemical detection of histone H3 (B) in mirror sections of mouse testis. A stage IV seminiferous tubule is shown. (C) As a negative control, an adjacent section was reacted with normal rabbit IgG instead of histone H3 antibody. Bar=50 µm.

Fig. 2

Immunohistochemical detection of acetylated histone H3 at lysine 9, 18 and 23 in paraffin-embedded mouse testis. (A, C, F, I) Serial sections were used for H-E staining (A), immunohistochemical staining for H3K9ac (C), H3K18ac (F), and H3K23ac (I). A stage X seminiferous tubule was shown. (B, D, G, J) Another set of serial sections with a stage VIII seminiferous tubule is shown. (E, H, K) As a negative control, sections adjacent to B were reacted with normal rabbit IgG instead of anti-H3K9ac, anti-H3K18ac, and anti-H3K23ac, respectively. Bar=50 µm.

Fig. 3

Immunohistochemical detection of tri-methylated of histone H3 at lysine 4 and 27 in paraffin-embedded mouse testis. (A–C) Serial sections were used for H-E staining (A), immunohistochemical staining for H3K4me3 (B) and H3K27me3 (C). A stage VI seminiferous tubule is shown. (D and E) As a negative control, adjacent sections were reacted with normal rabbit IgG instead of anti-H3K4me3 and anti-H3K27me3, respectively. (F–M) A set of mirror sections was used for the detection of H3K4me3 and H3K27me3. Identical spermatogonia (F and G), leptotene spermatocytes (H and I), pachytene spermatocytes (J and K) and elongating spermatids (L and M) are shown. Bar=50 µm (A–E) or 10 µm (F–M).

Fig. 4

H-E staining and immunohistochemical detection of phosphorylated histone H3 at serine 10 in mirror sections of mouse testis (A and B, E and F). (A and B) A stage V seminiferous tubule. (E and F) A stage XII seminiferous tubule. Arrows indicate H3S10phos positive spermatogonia in B. C and D are enlarged from B. (G) The inset of F is enlarged. Bar=50 µm (A, B, E, F) or 10 µm (C, D, G).