Dynamic histone modifications mark sex chromosome inactivation and reactivation during mammalian spermatogenesis

Abstract

Based on the formation of the XY body at pachytene and expression studies of a few X-linked genes, the X and Y chromosomes seem to undergo transcriptional inactivation during mammalian spermatogenesis. However, the extent and the mechanism of X and Y inactivation are not known. Here, we show that both the X and Y chromosomes undergo sequential changes in their histone modifications beginning at the pachytene stage of meiosis. These changes usually are associated with transcriptional inactivation in somatic cells, and they coincide with the exclusion of the phosphorylated (active) form of RNA polymerase II from the XY body. Both sex chromosomes undergo extensive deacetylation at histones H3 and H4 and (di)methylation of lysine (K)9 on histone H3; however, there are no changes in H3-K4 methylation. These changes persist even when the XY body disappears in late pachytene, and the X and Y chromosomes segregate from one another after the first meiotic division. By the spermatid stage, histone modifications of the X and Y chromosomes revert to those of active chromatin and RNA polymerase II reengages with both chromosomes. Our observations indicate that X and Y inactivation is extensive and persists even when the X and Y chromosomes are separated in secondary spermatocytes. These findings provide insights into epigenetic programming and chromatin dynamics in the male germ line.

Fig. 1.

Stages of spermatogenesis showing the X and Y chromosomes. Chromosomal DNA (blue) is stained with 4′,6-diamidino-2-phenylindole (DAPI). Sex chromosomes are shown with DNA whole-chromosome paints for X (pink) and Y (green). The X and Y chromosomes pair at their pseudoautosomal regions to form the sex body at pachytene. After the first meiotic division (metaphase I), the X and Y chromosomes are in separate secondary spermatocytes (metaphase II). Four spermatids are formed from each spermatogonium. The duration of spermatogenesis, from spermatogonia to mature sperm, is ≈14–26 days in mice and 64–74 days in humans (1).

Fig. 2.

Changes in histone H4 acetylation of X and Y at pachytene and metaphase I. (A–D) At pachytene, the XY body is hyperacetylated at K5 and K8 (A and B) and underacetylated at K12 and K16 (C and D). (E–H) By metaphase I (MI), the X and Y chromosomes have become underacetylated at K5 and K8 (E and F) and are still underacetylated at K12 (G) and reacetylated at K16 (H). (E Lower) Note that the pericentromeric regions (arrows) of X and Y remain acetylated at K5.

Fig. 3.

Transition from H3-K9 hyperacetylation to H3-K9 hypermethylation. (A and B) In early pachytene, the XY body is hyperacetylated at H3-K9 (A) and undermethylated at H3-dimethyl-K9 (B). (C) At diplotene, the X and Y chromosomes are extensively underacetylated at H3-K9; however, there is a discrete region on the X chromosome that remains acetylated (Lower). (D) X and Y are extensively hypermethylated at H3-dimethyl-K9; however, there is a discrete region on the X chromosome that remains undermethylated (Lower) and appears to correspond to the same location that remains acetylated, as shown in C Lower.

Fig. 4.

RNA polymerase II distribution at pachytene and the round spermatid stage. (A) The phosphorylated (active) form of RNA polymerase II (H5) is excluded from XY body (arrow) at pachytene. (B) The phosphorylated form of RNA polymerase II is engaged with the X chromosome in the round spermatid (arrow) but excluded from the chromocenter. PI, propidium iodide.