Altered three-dimensional organization of sperm genome in DPY19L2-deficient globozoospermic patients

Abstract

Purpose: To explore the three-dimensional (3D) organization of sperm genome in DPY19L2-deficient globozoospermic patients speculating a link between DPY19L2 and genome organization of sperm nucleus.

Methods: This is a study of chromatin organization in DPY19L2-deficient globozoospermic patients and healthy donors using three-dimensional fluorescence in situ hybridization (3D-FISH) combined with confocal laser scanning microscopy followed by 3D image analysis. The 3D structures of sperm nuclei, chromocenter, telomeric regions and chromosome territories (CTs), were reconstructed using IMARIS software, and the relative radial position for each individual signal was calculated. Statistical analysis used a non-parametric Mann-Whitney test was appropriate with significance at p < 0.05.

Results: DPY19L2-deficient globozoospermic patients display impaired sperm chromocenter organization resulting in an increased number of chromocenters (5.4 vs 3.5; p < 0.0001). Moreover, radial positions of telomeres are modified with a more central position in globozoospermic nuclei. 3D-FISH analysis of five chromosome territories (CTs) (X, Y, 7, 17, 18) showed that DPY19L2-deficient globozoospermic sperm nuclei display altered spatial organization of CT X, CT 7 and CT 18.

Conclusions: Our findings strengthen the hypothesis that DPY19L2 might be considered as a LINC-like protein having a crucial role in the organization of nuclear chromatin in sperm nucleus through its interaction with nuclear lamina. Our results might also explain defective embryonic development after intracytoplasmic sperm injection (ICSI) performed with DPY19L2-deficient globozoospermic sperm.

Keywords: Chromatin organization; DPY19L2 gene; Globozoospermia; Nuclear lamina; Three-dimensional fluorescence in situ hybridization (3D-FISH).

Fig. 1

3D reconstruction of nuclei and chromocenters using IMARIS software (Bitplane). 3D-FISH was performed using Human Chromosome Pan-Centromeric probe (green) on control sperm nuclei (n = 97) and globozoospermic sperm nuclei (n = 81). DAPI-counterstained nuclei. Scale bar, 2 μm. a Raw image of a control sperm nucleus after 3D-FISH. b Reconstructed nuclear territory and chromocenters of a control sperm nucleus. c Raw image of a globozoospermic sperm nucleus after 3D-FISH. d Reconstructed nuclear territory and chromocenters of a globozoospermic sperm nucleus

Fig. 2

Comparison of relative radial distribution of telomeric regions (a) and chromosome territories (CTs) of chromosome X (b), chromosome 7 (c) and chromosome 18 (d) in control sperm nuclei and globozoospermic sperm nuclei showed a significant difference between the two types of nuclei (p < 0.05)

Fig. 3

3D reconstruction of nuclei and chromosome X territory (CTX) using IMARIS software (Bitplane). 3D-FISH was performed using WCPX probe (red) on control sperm nuclei (n = 102) and globozoospermic sperm nuclei (n = 90). DAPI-counterstained nuclei. Scale bar, 2 μm. a Raw image of a control sperm nucleus after 3D-FISH. b Reconstructed nuclear territory and CTX of a control sperm nucleus. c Raw image of a globozoospermic sperm nucleus after 3D-FISH. d Reconstructed nuclear territory and CTX of a globozoospermic sperm nucleus

Fig. 4

Raw images of nuclei, chromosome 7 territory (CT 7) and chromosome 18 territory (CT 18). 3D-FISH using WCP 7 probe (red) on control sperm nucleus (a) and globozoospermic sperm nucleus (b). 3D-FISH using WCP18 probe (green) on control sperm nucleus (c) and globozoospermic sperm nucleus (d). Relative radial positions of CT 7 and CT 18 are significantly different between control and globozoospermic sperm nuclei with a more central position in patients. Scale bar, 2 μm

Fig. 5

Raw images of nuclei, chromosome Y territory (CT Y) and chromosome 17 territory (CT 17). 3D-FISH using WCPY probe (red) on control sperm nucleus (a) and globozoospermic sperm nucleus (b). 3D-FISH using WCP17 probe (red) on control sperm nucleus (c) and globozoospermic sperm nucleus (d). Relative radial positions of CT Y and CT 17 are not significantly different between control and globozoospermic sperm nuclei. Scale bar, 2 μm