Journal: Scientific Reports

Article Title: A new model of sperm nuclear architecture following assessment of the organization of centromeres and telomeres in three-dimensions

doi: 10.1038/srep41585

Figure Lengend Snippet: Panels a–d display the 3D localization of telomeres in four representative sperm cells. Nuclei are stained blue with DAPI, with telomere signals shown in green. Nuclei were captured with a Z step of 0.2 μm, with a minimum of 92 sections per nuclei using wide-field microscopy (Deltavision) and subjected to deconvolution (SoftWoRx). The first image on the left side of panels a–d, shows the raw image following deconvolution. 3D models were rendered with Imaris software (Bitplane, Switzerland, the second image in panels a–d, display the formation of 3D models with rendered telomeres (merged composite). The third image subsequently shows the same models with the DAPI partially removed (DAPI plane clipped) to better observe the 3D radial distribution of telomeres throughout all regions of the nucleus. The fourth and fifth images show the same DAPI clipped models rotated along the X-axis to further depict the 3D radial organization. Scale bar = 3 μm. Panel e displays a single sperm cell following 3D modeling that clearly depicts the sperm tail that is utilized to evaluate the longitudinal organization of telomeres with full DAPI, partially clipped DAPI and no DAPI. This clearly displays that sperm telomeres are longitudinally localized throughout the sperm nucleus from the sperm head to the tail). Scale bar = 4 μm.

Article Snippet: 3D reconstruction of the captured images (as 3D stacks) from SoftWoRx were rendered in 3D and analyzed using the Imaris software (V.7.6.3 Bitplane – Zurich, Switzerland) by converting images to 32-bit float images.

Techniques: Staining, Microscopy, Software

Journal: Scientific Reports

Article Title: A new model of sperm nuclear architecture following assessment of the organization of centromeres and telomeres in three-dimensions

doi: 10.1038/srep41585

Figure Lengend Snippet: Panels a–e display the 3D localization of centromeres in two representative sperm cells. Nuclei were captured with a Z step of 0.2 μm, with a minimum of 92 sections per nuclei using wide-field microscopy (Deltavision) and subjected to deconvolution (SoftWoRx). The upper panel shows the raw image following deconvolution. The middle panel shows the 3D rendered models using the Imaris software, for the same cells. Nuclei are stained with DAPI (blue in the raw image; pseudocolored gray in the 3D rendered models); the pancentromeric probe staining all centromeres are red ( a ); nuclear organizing regions (NOR) are green ( b ); and centromeres 1, 5, and 19 are aqua ( c ). Image d shows the merged image of all investigated loci, and image e is the same merged image rotated 180°, showing the backside of the cell. The bottom panel, shows the same two sperm cells with the DAPI plane partially removed (clipped) and rotated along the X-axis to better observe the radial 3D distribution of centromeres and NOR throughout the sperm nucleus. Scale bar = 5 μm.

Article Snippet: 3D reconstruction of the captured images (as 3D stacks) from SoftWoRx were rendered in 3D and analyzed using the Imaris software (V.7.6.3 Bitplane – Zurich, Switzerland) by converting images to 32-bit float images.

Techniques: Microscopy, Software, Staining

Journal: Genes

Article Title: Human Sperm Chromosomes: To Form Hairpin-Loops, Or Not to Form Hairpin-Loops, That Is the Question

doi: 10.3390/genes10070504

Figure Lengend Snippet: 3D rendered models and schematic diagrams illustrating two different models of chromosome organization and hairpin-loop configurations in human sperm. Schematic cross-section of a sperm nucleus, with magnified boxes that display 3D rendered models following sperm-FISH of the organization of centromeres (aqua), p-arms (green) and q-arms (red) in human sperm. Panel A: Displays the original chromocenter hairpin-loop model of sperm chromosome organization, whereby centromeres cluster in the nuclear interior of the sperm nucleus, forming one or more chromocenters, with the p- and q-arms stretching out toward the nuclear periphery. In the current study of 1240 cells, we rarely observed this type of chromosome configuration. This, alongside previously published data , led us to propose a refined hairpin-loop model of organization that is more segmentally arranged. Panel B: Segmental model of chromosome organization in human sperm. Here, we observe centromeres distributed throughout the nucleus and the chromosome arms forming narrow and wide hairpin-loops as well as displaying different orientations within the sperm nucleus. Additionally, in contrast to one study , we rarely observed chromosome arms to lie in parallel to one another leading to intermingling or coiling of the chromosome arms; with the majority of cells examined displaying discrete non-overlapping territories for the chromosome arms.

Article Snippet: 3D stacks from SoftWoRx were reconstructed, rendered in 3D and analyzed utilizing Imaris software (V.7.6.3 Bitplane–Zurich, Switzerland) by converting images to 32-bit float images.

Techniques:

Journal: Genes

Article Title: Human Sperm Chromosomes: To Form Hairpin-Loops, Or Not to Form Hairpin-Loops, That Is the Question

doi: 10.3390/genes10070504

Figure Lengend Snippet: 3D FISH imaging and model rendering demonstrating the hairpin-loop configuration for chromosome 2 in a human sperm nucleus. FISH probes utilized corresponded to the centromere (aqua), p-arm (green), q-arm (red) of chromosome 2. The nucleus is counterstained with DAPI (blue: A) and is pseudo-colored (gray: B, C, and D) in 3D rendered models. ( A ) Depicts the raw FISH image following deconvolution (note: aqua signal at position 8 o’clock is background fluorescence from the sperm tail and is removed from 3D rendered models [panels B, C, and D], note the sperm tail was only visible in a small proportion of cells precluding assessment of longitudinal positioning). ( B ) Provides the 3D model reconstruction from the raw deconvolved image (A). ( C ) Depicts the geometric center for each of the three FISH probe targets as determined by the Imaris software. ( D ) Illustrates how the angle of the hairpin-loop configuration is calculated by measuring the angle formed between the p-arm (point a), and the q-arm (point c) through the centromere (point b), which in this cell is 87.5°. It is important to note that the angle created between the chromosome arms is determined in 3D models. In this figure the 3D data is reduced to 2D, which can give the impression that the geometric centers or FISH probes may lie in the same focal plane or z-stack, which is not the case rather this image has compressed the data from 92 sections taken at 0.2 µm intervals, into a single plane. Thus, the angle cannot be accurately assessed in the 2D image and the 3D model has to be utilized to account for differences in FISH probe localizations in different 3D focal planes (X, Y, Z). Scale bar is 4 µm.

Article Snippet: 3D stacks from SoftWoRx were reconstructed, rendered in 3D and analyzed utilizing Imaris software (V.7.6.3 Bitplane–Zurich, Switzerland) by converting images to 32-bit float images.

Techniques: Imaging, Fluorescence, Software