Kulikova T et al. (2016), Low-voltage scanning electron microscopy study ...

XB-ART-52795

Sci Rep 2016 Jan 22;6:36878. doi: 10.1038/srep36878.

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Low-voltage scanning electron microscopy study of lampbrush chromosomes and nuclear bodies in avian and amphibian oocytes.

Kulikova T , Khodyuchenko T , Petrov Y , Krasikova A .

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Nucleus is a highly compartmentalized part of the cell where the key processes of genome functionality are realized through the formation of non-membranous nuclear domains. Physically nuclear domains appear as liquid droplets with different viscosity stably maintained throughout the interphase or during the long diplotene stage of meiosis. Since nuclear body surface represents boundary between two liquid phases, the ultrastructural surface topography of nuclear domains is of an outstanding interest. The aim of this study was to examine ultrathin surface topography of the amphibian and avian oocyte nuclear structures such as lampbrush chromosomes, nucleoli, histone-locus bodies, Cajal body-like bodies, and the interchromatin granule clusters via low-voltage scanning electron microscopy. Our results demonstrate that nuclear bodies with similar molecular composition may differ dramatically in the surface topography and vice versa, nuclear bodies that do not share common molecular components may possess similar topographical characteristics. We also have analyzed surface distribution of particular nuclear antigens (double stranded DNA, coilin and splicing snRNA) using indirect immunogold labeling with subsequent secondary electron detection of gold nanoparticles. We suggest that ultrastructural surface morphology reflects functional status of a nuclear body.

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Species referenced: Xenopus
Genes referenced: coil

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	Figure 1. The surface morphology of avian lampbrush chromosomes: chromomeres, lateral loops, and associated structures.Lampbrush chromosomes of Coturnix coturnix japonica (a–c,f), Gallus gallus domesticus (d,e) and Columba livia (g,h). (a–c) Surface morphology of chromomeres in the ZW-lampbrush bivalent. (c) Enlarged fragment of W-lampbrush chromosome demonstrates compact neighboring chromomeres (chr) and interchromomeric regions (ich). (d,e) Fragment of macrobivalent and whole microbivalent demonstrate simple lateral loop morphology (arrows); gr–large 100–200 nm RNP-granules. (f) Enlarged fragment of simple lateral loop; RNP-matrix of a transcription unit (arrowhead), short regions of “nacked” loop axis (arrows). (g,h) The morphology of ZW-bivalent with pericentromeric dense loops, centromere protein bodies (arrowheads) and giant terminal RNP-aggregates (GITERA) (asterisks). (h) Enlarged fragment of panel (g), W-chromosome with centromere protein body (arrowhead) and pericentromere dense loops (arrows); uf–untwisted RNP-fibrils. Low-voltage scanning electron microscopy. Scale bars: (a) 5 μm, (b,f) 1 μm, (c) 200 nm, (d,e) 2 μm, (g) 10 μm, (h) 2 μm.
	Figure 2. The surface morphology of nuclear bodies from growing oocytes of Xenopus laevis.(a) Numerous nuclear bodies of Xenopus laevis on nuclear content preparations: lampbrush chromosomes, amplified nucleoli, HLB and IGC; (b–d) sequential enlarged images demonstrate nucleolus (Nu) with patches (arrowheads) connected to the surface of nucleolus by fibrillar material (arrows); (e) – nucleolus (Nu) and HLB (H) with attached IGC (I); (f) surface of the enlarged fragment of HLB bearing patches (arrowheads). (g) Two HLB (H) with attached IGC (I). (h) Enlarged fragment of IGC with the area of contact between HLB and IGC demonstrating fibrillar material (arrows); arrowheads indicates patches. Low-voltage scanning electron microscopy. Scale bars: (a) 10 μm, (b) 2 μm, (c) 1 μm, (d) 200 nm, (e) 1 μm, (f) 200 nm, (g) 1 μm, (h) 200 nm.
	Figure 3. The surface morphology of nuclear bodies from growing oocytes of Columba livia.(a,b) centromere protein body; (c) CB-like bodies (C) and centromere protein body (PB); (d,g)–CB-like body; (e,f,h) enlarged fragments of CB-like bodies shown on panels (c,g); single or grouped pores–arrowheads. Low-voltage scanning electron microscopy. Scale bars: (a) 2 μm, (b) 200 nm, (c) 2 μm, (d) 1 μm, (e–h) 200 nm.
	Figure 4. Surface distribution of dsDNA along transcriptionally active regions of lateral loop axes.(a–c) Fragment of a lampbrush chromosome of Coturnix coturnix japonica, immunogold labelling with antibodies against dsDNA. (b) DsDNA revealed by 18 nm gold nanoparticles (electron dense granules) along the DNP-axis (arrows) of individual lateral loop with growing nascent RNP fibrils (arrowheads); (c) gold nanoparicles pseudocolured with yellow; (d) the distribution pattern of gold nanoparticles.
	Figure 5. The relative distribution of coilin and snRNA on the surface of HLB and attached IGC of Xenopus laevis growing oocyte.Double immunogold labeling with antibodies against coilin and snRNAs (a,b). Distribution of coilin and snRNA on the surface of HLB (H) and IGC (I) revealed by 10 nm and 18 nm gold nanoparticles (electron dense granules) correspondingly. (c,d) Distribution pattern of gold nanoparticles reflects surface distribution of snRNA. (e,f) Distribution patterns of gold nanoparticles reflect surface distribution of coilin; (g,h) Distribution pattern gold nanoparticles reflects surface distribution of both coilin (green) and snRNA(magenta). Low-voltage scanning electron microscopy. Scale bars: 200 nm.
	Figure 6. The distribution of coilin and snRNA on the surface of CB-like bodies of Columba livia growing oocytes.Immunogold labeling with antibodies against coilin (a,c,e) and snRNAs (b,d,f); centromere protein body (PB). (a,b) Distribution of coilin or snRNA on the surface of CB-like bodies revealed by 10 nm and 18 nm gold nanoparticles (electron dense granules) correspondingly; (c,d) gold nanoparicles pseudocolured with yellow. (e,f) Distribution patterns of gold nanoparticles. Low-voltage scanning electron microscopy. Scale bars: (a,b) 1 µm.
	Figure 7. The relative distribution of coilin and snRNA on the surface of CB-like bodies of Columba livia growing oocyte.Double immunogold labeling with antibodies against coilin and snRNAs (a,b). Distribution of coilin and snRNA on the surface of CB-like body revealed by 10 nm and 18 nm gold nanoparticles (electron dense granules) correspondingly. (b) Gold nanoparicles corresponding to coilin pseudocolured with yellow. (c,d) Distribution patterns of gold nanoparticles reflect snRNA and coilin surface distribution. (e,f) Enlarged images of CB-like body shown on panel (b); gold nanoparicles corresponding to coilin and snRNAs pseudocolured with yellow. Low-voltage scanning electron microscopy. Scale bar: 1 μm.

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Allen, Scanning electron microscopy of human metaphase chromosomes. 1986, Pubmed