Chromosome Research

, Volume 15, Issue 2, pp 163–174 | Cite as

Lamins A and C are present in the nuclei of early porcine embryos, with lamin A being distributed in large intranuclear foci

  • Helen A. Foster
  • Paula Stokes
  • Katherine Forsey
  • Henry J. Leese
  • Joanna M. Bridger
Article

Abstract

Gametogenesis and embryogenesis are dynamic developmental stages marked by extensive modifications in the organization of the genome and nuclear architecture. In the literature it is conveyed that only B-type lamins are required in these early stages of development and that A-type lamins are not present or required until differentiation of specific cell types associated with specialized tissue is initiated. To assess the presence of nuclear structures that are putatively involved in genome regulation, we investigated the distribution of lamin proteins throughout the early stages of porcine embryonic development, using testes tissue sections, oocytes and in-vitro fertilized (IVF) porcine embryos and employing anti-lamin antibodies. We have shown that anti-lamin A staining is present at the one-cell, two-cell, four-cell, and six- to eight-cell stages of early porcine embryo development, but diminishes at the morulae and blastocyst stages. Large intranuclear anti-lamin A foci are prominent in the early preimplantation stages. Both anti-lamin A/C and anti-lamin B staining were clearly present in all embryonic stages. Immature porcine oocytes revealed lamin rings using the monoclonal anti-lamin A/C antibody and many immature oocytes exhibited a pale rim staining pattern with anti-lamin A antibody. A-type lamins were not observed in sperm precursor cells. Thus, we have shown that A-type lamins and B-type lamins are present at the nuclear envelope in very early porcine embryos and that lamin A is also found in large intranuclear aggregates in two-cell to eight-cell embryos but is lacking from later embryonic stages.

Key words

embryogenesis lamin nuclear organization oogenesis spermatogenesis 

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Supplementary material

cro01088ESM.jpg (738 kb)
Supplementary Figure 1

Indirect immunofluorescence on porcine testes tissue. Monoclonal anti-lamin A staining 133A2 (A) polyclonal anti-lamin A C-20 (B) monoclonal anti-lamin A/C 636 (C) and polyclonal anti-lamin B C-20 (D). Heterogeneous positive anti-lamin A/C 636 staining was determined in certain nuclei (C- C-). Nuclei were counterstained with propidium iodide. The solid arrow depicts a spermatocyte nucleus, whereas the hatched arrow portrays a spermatid nucleus. Bar=10 μm. Porcine testes were incubated with sterile 40% sucrose for 3 days in order to replace the water and to allow easier sectioning when frozen. The testes tissue was cut into small pieces, frozen in hexane bath, and stored at -80°C. Sections of frozen tissue (60 μm ) were cut using a cryomicrotome (Bright 5030 microtome), and adhered to slides coated with 3% 3-aminopropyltriethoxysilane (APES). The tissue sections were stored at -80°C until required, they were then air-dried for at least 1 h prior to fixation for 10 min with ice-cold acetone. They were then rinsed three times with PBS and incubated with 10% (NCS) for 10 min, before application of the primary antibody. The tissue sections were incubated in primary antibodies mouse anti-lamin A 133A2 antibody diluted 1:400, goat anti-lamin A C-20 antibody diluted 1:100, mouse anti-lamin A/C 636 antibody and goat anti-lamin B C-20 antibody diluted 1:100, all diluted with 1% (v/v) NCS in PBS and incubated with the slide for 24 h at 4°C. The tissue sections were washed three times with PBS and incubated with the appropriate secondary antibody (donkey anti-mouse FITC diluted 1:60; Jackson Laboratory or donkey anti-goat FITC diluted 1:60; Jackson Laboratory) for 30 min at 37°C. The sections were rinsed three times in PBS prior to mounting in Vectashield anti-fade mountant (Vectorlabs) containing propidium iodide (PI) as a counterstain. (JPG 755 993)

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Copyright information

© Springer 2007

Authors and Affiliations

  • Helen A. Foster
    • 1
  • Paula Stokes
    • 2
  • Katherine Forsey
    • 2
  • Henry J. Leese
    • 2
  • Joanna M. Bridger
    • 1
  1. 1.Laboratory of Nuclear and Genomic Health, Centre for Cell and Chromosome Biology, Division of Biosciences, School of Health Sciences and Social CareBrunel UniversityWest LondonUK
  2. 2.Department of Biology (Area 3)University of YorkYorkUK

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