In the human sperm nucleus, nucleosomes form spatially restricted domains consistent with programmed nucleosome positioning

Abstract

In human sperm, a fraction of its chromatin retains nucleosomes that are positioned on specific sequences containing genes and regulatory units essential for embryonic development. This nucleosome positioning (NP) feature provides an inherited epigenetic mark for sperm. However, it is not known whether there is a structural constraint for these nucleosomes and, if so, how they are localized in a three-dimensional (3D) context of the sperm nucleus. In this study, we examine the 3D organization of sperm chromatin and specifically determine its 3D localization of nucleosomes using structured illumination microscopy. A fraction of the sperm chromatin form nucleosome domains (NDs), visible as microscopic puncta ranging from 40 μm to 700 μm in diameter, and these NDs are precisely localized in the post acrosome region (PAR), outside the sperm's core chromatin. Further, NDs exist mainly in sperm from fertile men in a pilot survey with a small sample size. Together, this study uncovers a new spatially-restricted sub-nuclear structure containing NDs that are consistent with NPs of the sperm, which might represent a novel mark for healthy sperm in human.

Keywords: Chromatin; Domain; Human sperm; Nucleosome.

Fig. 1.

Nucleosomes in human sperm localize in PAR with sparse or aggregated dots to form NDs. (Top row) Type I NDs with sparse dots. The diameter of NDs ranges from 40–700 μm. (Middle row) Type II NDs with aggregated dots. Type I is major, while type II is minor. (Bottom row) Negative controls were performed in the absence of primary antibody. H4, histone H4; NC, negative control. Scale bars: (upper and lower rows) 1 µm, (middle row) 2 µm.

Fig. 2.

Using SIM the 3D positioning of NDs is shown. (A) After rotation around the Y-axis, NDs were shown to localize outside the core chromatin in PAR. The NDs were not shown to be inside of sperm chromatin. (B) A diagram of NDs positioning outside the major dense sperm chromatin. Scale bars: 1 µm.

Fig. 3.

After cutting through AR and PAR, localization of NDs is shown. (A) After sperm was cut on both sides, the inner side stuck to the slide without antibody staining, but the outer side was stained by both H4 and protamine 1 antibodies. H4 signals were confined in the PAR and outside of the inner chromatin, but not in the inner chromatin. However, protamine 1 signals ranged from the AR to the PAR in inner chromatin in the longitude section. (B) A diagram showing that NDs are not localized in the inner chromatin, but outside of the sperm chromatin in the PAR. Scale bars: 1 µm.

Fig. 4.

After cutting through AR, NDs are not detected. (A) Sperm was cut in AR. After staining by both H4 and protamine 1 antibodies, H4 signals were not detected, while protamine 1 signal was full in the inner chromatin in the longitude section. (B) A diagram showing that NDs are not detected in the inner and outer sides of sperm chromatin in AR. Scale bars: 1 µm.