In the present study, we investigated the distribution of histone H3 and its modified molecules with acetylation (H3K9ac, H3K18ac, H3K23ac), methylation (H3K4me3, H3K27me3) and phosphorylation (H3S10phos) immunohistochemically in adult mouse testis, and we determined the differentiation stage-specific modification patterns of histone H3 in spermatogenic cells. We believe these basic findings are quite useful to better understand the roles of epigenetic factors in the regulation of mammalian spermatogenesis.
Spermatogonia undergo self-renewal to ensure a constant supply of germ cells to form spermatozoa [24
]. During the process, many distinct genes have been identified with specific expression profiles and the expression was thought to be influenced by histone modifications [14
]. In the case of somatic cells, acetylation of histone H3 is generally linked with active transcription [18
], whereas methylated histone H3 at lysine 27 is associated with transcriptionally silent states of genes [20
]. Moreover, methylated histone H3 at lysine 4 is known to be closely linked with the transcriptionally active states of genes [25
]. In the present study, we detected significant acetylation of histone H3 at lysine 9, 18, and 23 in spermatogonia, which is consistent with active chromatin patterns in somatic cells. However, the staining for methylated histone H3 at lysine 4 and 27 in spermatogonia seems to indicate transcriptionally silent states of genes, raising an apparent controversy. In this context, it is of interest to note that similar conflicting histone patterns were also identified in mouse embryonic stem cells that displayed both active and repressive histone modifications [3
]. Considering that spermatogonia in adult mouse may retain the capacity to generate pluripotent cells which are able to differentiate into derivatives of three embryonic germ layers [7
], the unique pattern of histone H3 modifications found in the present study may extend our understanding of pluripotency of spermatogonia.
In spermatocytic differentiation, the spermatocytes undergo a long prophase of meiosis, consisting of leptotene, zygotene, pachytene, and diplotene stage, which leads to meiotic cell division. During the differentiation, chromatin becomes continuously condensed and the homologous chromosomes are paired and then exchange DNA segments through a process of homologous recombination. In this stage, most of the genes are transcriptionally inactive [13
]. For this inactivation, a few studies reported potential requirements for specific histone modification during meiosis in mouse and Drosophila
]. In the present study, the signals for H3K9ac, H3K18ac, H3K23ac, and H3K4me3 were significantly reduced from preleptotene to pachytene stage, indicating that genes may be generally transcriptionally inactivated. In addition, the staining intensity for H3K18ac, H3K23ac and H3K4me3 was increased from the stage of diplotene spermatocytes. Although the roles of the modification change are still unknown, these changes should be involved in the progression of meiotic cell division.
In spermiogenesis, a process of postmeiotic metamorphosis of male haploid germ cells, spermatids undergo the extreme condensation of chromatin into sperm head, in which histones are sequentially replaced by protamines [15
]. In mouse, histones are firstly replaced with transition proteins, and subsequently with protamine 1 and 2. However, our knowledge about the mechanisms controlling the process of histone-protamine exchange is still rather limited. In the present study, we showed that histone H3 became significantly acetylated at lysine 9, 18, and 23 in spermatids. Since a few in vitro
studies proposed that certain histone modifications could facilitate histone-protamine exchange [21
], the hyperacetylation of these residues, which is usually related to the relaxation of chromatin structure [6
], might be a possible mechanism.
The phosphorylation of histone H3 at serine 10 was already well-known to be correlated with the M-phase in mitosis [9
]. In this study, we also confirmed that phosphorylation of histone H3 at serine 10 was strictly associated with meiotic cell division of diplotene spermatocytes under a pattern similar to mitosis. Thus, we believe that the phosphorylation of histone H3 at serine 10 may be essential for the movement of condensed chromosomes during cell division to proceed.
In conclusion, we have shown here the unique pattern of acetylation, methylation, and phosphorylation of histone H3 in male germ cells, which was substantially different from that of somatic cells. Our results may extend our understanding of male germ cell specific epigenetic regulation in the mouse, which would therefore be an excellent source for the investigation of pluripotency, chromatin reorganization, and nucleosome disassembly machinery. However, a precise correlation of these modifications of histone H3 with the chromatin structure in spermatogenic cells remains to be clarified in the future.