Segregation during cleavage in the mammalian embryo? A critical comparison of whole-mount/CLSM and section immunohistochemistry casts doubts on segregation of axis-relevant leptin domains in the rabbit

Abstract

Segregation of certain cytoplasmic molecules during cleavage and blastocyst formation that was previously reported to occur in the human and the mouse (Antczak and Van Blerkom Mol Hum Reprod 3:1067–1086, 1997; Antczak and Van Blerkom Hum Reprod 14:429–447, 1999) has been reinvestigated in the rabbit model. Additional methodology was used and two approaches were compared: (1) whole-mount immunohistochemistry followed by confocal laser scanning microscopy (WM-IHC/CLSM) versus (2) IHC performed on histological sections of resin-embedded material (S-IHC). This study concentrates on leptin and cytoskeletal proteins (actin and cytokeratins). With S-IHC, leptin was localized predominantly on the surface of blastomeres which is facing the perivitelline space, and in the extracellular embryonic coats, without any polar asymmetry being detectable along (presumptive) embryonic axes. A polar distribution of leptin with a pattern that could be interpreted as predictive of the prospective embryonic-abembryonic axis was seen only with WM-IHC/CLSM, not with S-IHC, although the latter gave excellent resolution. With both techniques, no differences between blastomeres were detected with respect to actin and cytokeratin patterns, an increased expression of cytokeratin in trophoblast cells occurring no earlier than at blastocyst formation. Artifacts that can occur with the two methodological approaches are critically discussed, as is the possible significance of the findings for theories on the differentiation of trophoblast versus embryoblast and on axis formation in early mammalian development. It is concluded that these data call for cautioning when studying distribution patterns of diffusible molecules with WM-IHC/CLSM technology, whereas patterns obtained with S-IHC are more reliable. Specifically these data cast doubts on previous claims that leptin IHC would allow to monitor cytoplasmic domain segregation occurring during cleavage as an element of early embryonic pattern/axis formation.

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Abbreviations

CLSM:

Confocal laser scanning microscopy

d:

Days

e–a axis:

Embryonic–abembryonic axis

h:

Hours

IHC:

Immunohistochemistry

p.c.:

Post coitum

PBS:

Phosphate-buffered salt solution

S-IHC:

Section immunohistochemistry

WM-IHC:

Whole-mount immunohistochemistry

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Acknowledgments

Our special thanks are due to PD Dr. Bernhard B. Singer and Birgit Maranca-Hüwel for performing the Western blots and ELISAs with the leptin antibody. Most of the data presented here are derived from an MD thesis by TL (Littwin 2008). We are grateful to Prof. Dr. Andreas Herrler (Aachen/Maastricht) for sharing with us his experience with methods of removing rabbit embryo coats, and to Dr. Axel Schleicher (Forschungszentrum Jülich) for his help with using the Amira software for computing 3D reconstructions from serial sections. We are likewise grateful to Prof. Dr. Gero Hilken (Zentrales Tierlaboratorium, Universitätsklinikum Essen) and his staff for their help with rabbit mating. We cordially thank Dorothea Schünke for her support in histotechnology.

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Correspondence to H. -W. Denker.

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Littwin, T., Denker, H.W. Segregation during cleavage in the mammalian embryo? A critical comparison of whole-mount/CLSM and section immunohistochemistry casts doubts on segregation of axis-relevant leptin domains in the rabbit. Histochem Cell Biol 135, 553–570 (2011). https://doi.org/10.1007/s00418-011-0816-0

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Keywords

  • Blastocyst
  • Cleavage
  • Confocal laser scanning microscopy
  • Whole-mount versus section IHC
  • Leptin
  • Segregation