Histone modifications in the male germ line of Drosophila

Abstract

Background: In the male germ line of Drosophila chromatin remains decondensed and highly transcribed during meiotic prophase until it is rapidly compacted. A large proportion of the cell cycle-regulated histone H3.1 is replaced by H3.3, a histone variant encoded outside the histone repeat cluster and not subject to cell cycle controlled expression.

Results: We investigated histone modification patterns in testes of D. melanogaster and D. hydei. In somatic cells of the testis envelope and in germ cells these modification patterns differ from those typically seen in eu- and heterochromatin of other somatic cells. During the meiotic prophase some modifications expected in active chromatin are not found or are found at low level. The absence of H4K16ac suggests that dosage compensation does not take place. Certain histone modifications correspond to either the cell cycle-regulated histone H3.1 or to the testis-specific variant H3.3. In spermatogonia we found H3K9 methylation in cytoplasmic histones, most likely corresponding to the H3.3 histone variant. Most histone modifications persist throughout the meiotic divisions. The majority of modifications persist until the early spermatid nuclei, and only a minority further persist until the final chromatin compaction stages before individualization of the spermatozoa.

Conclusion: Histone modification patterns in the male germ line differ from expected patterns. They are consistent with an absence of dosage compensation of the X chromosome during the male meiotic prophase. The cell cycle-regulated histone variant H3.1 and H3.3, expressed throughout the cell cycle, also vary in their modification patterns. Postmeiotically, we observed a highly complex pattern of the histone modifications until late spermatid nuclear elongation stages. This may be in part due to postmeiotic transcription and in part to differential histone replacement during chromatin condensation.

Figure 1

Spermatogonia and somatic testis cells. (a-c) H3K9me2; D. melanogaster, spermatogonia (Stg). Label in cytoplasm (Cy) and in heterochromatin (N: nucleus). (d-f) H4K20me1; D. melanogaster, small diploid cells of the testis envelope (dc) and one cell of low polyteny (pc). The label appears in a dotted pattern in the euchromatin (EU) while heterochromatin (HET) remains unlabeled. (g-i) H3K9me2; D. hydei, diploid cells of the testis envelope. Heterochromatin (chromocenter) and a few spots in the nucleus react with this antibody. (j-l) H3K9me3; D. melanogaster, polytene cell with labeled heterochromatin (HET). Also here, few spots appear in euchromatin. Cytoplasm and nucleus display background only. (a, d, g, j) DAPI; (b, e, h, k) antibody reaction; (c, f, i, l) merged pictures of DAPI and antibody.

Figure 2

Meiotic and postmeiotic cells. (a, b) D. melanogaster, H4K12ac; late spermatid elongation stages (PM VI) and unlabeled sperm (stage PM VII). In the upper part elongating spermatids (PM VI/VII) with decreasing acetylated H4K12 are seen. (c, d) D. hydei, H4K16ac; two labeled daughter nuclei (N) after the first meiotic division. (e, f) D. melanogaster, H3S10p; elongating spermatids (PM V). (g, h) D. hydei, H4K16ac; young spermatids (PM III), very early elongation of the nucleus with chromatin in a restricted region. NK: Nebenkern, N: nucleus. (i, l) D. hydei, H4K20me2; early elongating spermatid nuclei (early PM V) with asymmetric label concentrated in the posterior part of the nucleus (p). (a, c, e, g, i, l) antibody reaction; (b, f, j, k) merged picture of DAPI and antibody; (d, h) DAPI. Staging of postmeiotic cells according to [2].

Figure 3

Primary spermatocytes of D. melanogaster. (a, b) H4K20me2; (c, d) H4K20me1; (e) H3K9me1. (a, c) Antibody reactions; (b, d, e) merged pictures of DAPI and antibody. (c-e) Show the characteristic patch-like patterns of prominent reactions with X chromosome and autosomes. In the smaller young spermatocytes (Stc(y)) the chromatin is more compact while in the larger nuclei of older stags (Stc(o)) is becomes more diffuse. In (e) two spermatogonia (Stg) are seen which still display some low cytoplasmic label (Cy) while the spermatocytes are not labeled around their nuclei (N). (a, b) display a more uniform label with some prominent patches in older spermatocytes. One of these patches represents the nucleolus.

Figure 4

Spermatocytes of D. hydei (a-f) and D. melanogaster (g-i). (a-c) H4K12ac. Autosomes (A) and X chromosome (X) and nucleolus (NO) are stained. (d-i) H3K9ac. The Y chromosomal chromatin (Y) is stained. (a, d, g) DAPI; (b, e, h) antibody; (c, f, i) combination of DAPI and antibody.

Figure 5

Overviews of label in the testis tube of D. melanogaster. (a-c) H4K12ac; different stages of primary spermatocytes show the gradual decondensation of the chromatin. In older spermatocytes (Stc(o)) mainly X chromosome and autosomes as well as the nucleolus are labeled. Younger spermatocytes (Stc(y)) and spermatogonia (Stg) have a more compact chromatin. The signal strength increases from spermatogonia to young spermatocyte stages (compare DAPI with antibody fluorescence!). (d-f) H3K9me3; apical tip of the testis tube. Spermatogonia are only slightly labeled and the cytoplasm seems slightly labeled (compare (e) with spermatogonia in (b)!). The hub cells (hub) display a strong reaction. Spermatocytes (Stc) show a more diffuse label compared with (b), as is characteristic for Y chromosomal chromatin label (cf. Figures 4d-i). Cytoplasm is unlabeled. (a, d) DAPI; (b, e) antibody; (c, f) merged pictures of DAPI and antibody.