Skip to main content
SpringerLink
Log in

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

  • Original Paper
  • Published:
Histochemistry and Cell Biology Aims and scope Submit manuscript

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.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7
Fig. 8
Fig. 9
Fig. 10
Fig. 11
Fig. 12
Fig. 13
Fig. 14

Similar content being viewed by others

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

References

  • Albertini DF, Overstrom EW, Ebert KM (1987) Changes in the organization of the actin cytoskeleton during preimplantation development of the pig embryo. Biol Reprod 37:441–451

    Article  PubMed  CAS  Google Scholar 

  • Antczak M, Van Blerkom J (1997) Oocyte influences on early development: the regulatory proteins leptin and STAT3 are polarized in mouse and human oocytes and differentially distributed within the cells of the preimplantation stage embryo. Mol Hum Reprod 3:1067–1086

    Article  PubMed  CAS  Google Scholar 

  • Antczak M, Van Blerkom J (1999) Temporal and spatial aspects of fragmentation in early human embryos: possible effects on developmental competence and association with the differential elimination of regulatory proteins from polarized domains. Hum Reprod 14:429–447

    Article  PubMed  CAS  Google Scholar 

  • Beier HM (2002) Totipotenz und Pluripotenz. Von der klassischen Embryologie zu neuen Therapiestrategien. In: Oduncu FS, Schroth U, Vossenkuhl W (eds) Stammzellenforschung und therapeutisches Klonen. Vandenhoeck & Ruprecht, Göttingen, pp 36–54

    Google Scholar 

  • Bischoff A, Parfitt DE, Zernicka-Goetz M (2008) Formation of the embryonic-abembryonic axis of the mouse blastocyst: relationships between orientation of early cleavage divisions and pattern of symmetric/asymmetric divisions. Development 135(5):953–962

    Article  PubMed  CAS  Google Scholar 

  • Cervero A, Horcajadas JA, Dominguez F, Pellicer A, Simón C (2005) Leptin system in embryo development and implantation: a protein in search of a function. Reprod BioMed Online 10(2):217–223

    Article  PubMed  CAS  Google Scholar 

  • Dalcq AM (1954) Nouvelles données structurales et cytochimiques sur l’oeuf des Mammifères. Rev Gén Sci Pures Appl 61:19–41

    Google Scholar 

  • Denker H-W (1970a) Topochemie hochmolekularer Kohlenhydratsubstanzen in Frühentwicklung und Implantation des Kaninchens. I. Allgemeine Lokalisierung und Charakterisierung hochmolekularer Kohlenhydratsubstanzen in frühen Embryonalstadien. Zool Jahrb Physiol 75:141–245

    CAS  Google Scholar 

  • Denker H-W (1970b) Topochemie hochmolekularer Kohlenhydratsubstanzen in Frühentwicklung und Implantation des Kaninchens. II. Beiträge zu entwicklungsphysiologischen Fragestellungen. Zool Jahrb Physiol 75:246–308

    CAS  Google Scholar 

  • Denker H-W (1972) Furchung beim Säugetier: Differenzierung von Trophoblast- und Embryonalknotenzellen. Verh Anat Ges 66(Anat Anz Suppl 130):267–272

    Google Scholar 

  • Denker H-W (1976) Formation of the blastocyst: determination of trophoblast and embryonic knot. In: Gropp A, Benirschke K (eds) Developmental biology and pathology. (Current Topics in Pathology 62; Grundmann E and Kirsten WH, Series-eds). Springer, Berlin, pp 59–79

    Google Scholar 

  • Denker H-W (1983) Cell lineage, determination and differentiation in earliest developmental stages in mammals. In: Hilscher W (ed) Problems of the Keimbahn. New work on mammalian germ cell lineage. S. Karger AG, Basel (Bibliotheca Anatomica; Lierse W, Series-ed 24:22–58)

    Google Scholar 

  • Denker H-W (2000) Structural dynamics and function of early embryonic coats. Cells Tissues Organs 166:180–207

    Article  PubMed  CAS  Google Scholar 

  • Denker H-W (2004) Early human development: new data raise important embryological and ethical questions relevant for stem cell research. Naturwissenschaften 91:1–21

    Article  PubMed  CAS  Google Scholar 

  • Denker H-W (2006) Potentiality of embryonic stem cells: an ethical problem even with alternative stem cell sources. J Med Ethics 32:665–671

    Article  PubMed  Google Scholar 

  • Denker H-W (2009) Induced pluripotent stem cells: how to deal with the developmental potential. Reprod BioMed Online 19(Suppl 1):34–37

    Article  PubMed  Google Scholar 

  • Dietrich JE, Hiiragi T (2008) Stochastic processes during mouse blastocyst patterning. Cells Tissues Organs 188:46–51

    Article  PubMed  Google Scholar 

  • Fleming TP, Wilkins A, Mears A, Miller DJ, Thomas F, Ghassemifar MR, Fesenko I, Sheth B, Kwong WY, Eckert JJ (2004) The making of an embryo: short-term goals and long-term implications. Reprod Fertil Dev 16:325–337

    Article  PubMed  Google Scholar 

  • Fujimori T, Kurotaki Y, Komatsu K, Nabeshima Y (2009) Morphological organization of the mouse preimplantation embryo. Reprod Sci 16:171–177

    Article  PubMed  Google Scholar 

  • Gardner RL (1997) The early blastocyst is bilaterally symmetrical and its axis of symmetry is aligned with the animal–vegetal axis of the zygote in the mouse. Development 124:289–301

    PubMed  CAS  Google Scholar 

  • Gardner RL (2001) Specification of embryonic axes begins before cleavage in normal mouse development. Development 128(6):839–847

    PubMed  CAS  Google Scholar 

  • Gardner RL (2002) Experimental analysis of second cleavage in the mouse. Hum Reprod 17(12):3178–3189

    Article  PubMed  CAS  Google Scholar 

  • Gardner RL (2006a) The axis of polarity of the mouse blastocyst is specified before blastulation and independently of the zona pellucida. Hum Reprod 22:798–806

    Article  PubMed  Google Scholar 

  • Gardner RL (2006b) Weaknesses in the case against prepatterning in the mouse. Reprod BioMed Online 12:144–149

    Article  PubMed  CAS  Google Scholar 

  • Gardner RL, Davies TJ (2006) An investigation of the origin and significance of bilateral symmetry of the pronuclear zygote in the mouse. Hum Reprod 21:492–502

    Article  PubMed  CAS  Google Scholar 

  • Gilbert SF (2006) Developmental Biology, 8th edn. Sinauer Associates, Inc, Sunderland

    Google Scholar 

  • González RR, Caballero-Campo P, Jasper M, Mercader A, Devoto L, Pellicer A, Simón C (2000a) Leptin and leptin receptor are expressed in the human endometrium and endometrial leptin secretion is regulated by the human blastocyst. J Clin Endocrinol Metab 85(12):4883–4888

    Article  PubMed  Google Scholar 

  • González RR, Simón C, Caballero-Campo P, Norman R, Chardonnens D, Devoto L, Bischof P (2000b) Leptin and reproduction. Hum Reprod Update 6(3):290–300

    Article  PubMed  Google Scholar 

  • Gray D, Plusa B, Piotrowska K, Na J, Tom B, Glover DM, Zernicka-Goetz M (2004) First cleavage of the mouse embryo responds to change in egg shape at fertilization. Curr Biol 14:397–405

    Article  PubMed  CAS  Google Scholar 

  • Guo G, Huss M, Tong GQ, Wang C, Li Sun L, Clarke ND, Robson P (2010) Resolution of cell fate decisions revealed by single-cell gene expression analysis from zygote to blastocyst. Dev Cell 18:675–685

    Article  PubMed  CAS  Google Scholar 

  • Herrler A, von Wolff M, Beier HM (2002) Proteins in the extraembryonic matrix of preimplantation rabbit embryos. Anat Embryol (Berlin) 206:49–55

    Article  CAS  Google Scholar 

  • Hiiragi T, Solter D (2004) Forst cleavage plane of the mouse egg is not predetermined but defined by the topology of the two apposing pronuclei. Nature 430:360–364

    Article  PubMed  CAS  Google Scholar 

  • Hiiragi T, Alarcon VB, Fujimori T, Louvet-Vallee S, Maleszewski M, Marikawa Y, Maro B, Solter D (2006) Where do we stand now? Mouse early embryo patterning meeting in Freiburg, Germany 2005. Int J Dev Biol 50:581–588

    Article  PubMed  Google Scholar 

  • Honda H, Motosugi N, Nagai T, Tanemura M, Hiiragi T (2008) Computer simulation of emerging asymmetry in the mouse blastocyst. Development 135(8):1407–1414

    Article  PubMed  CAS  Google Scholar 

  • Jedrusik A, Parfitt DE, Guo G, Skamagki M, Grabarek JB, Johnson MH, Robson P, Zernicka-Goetz M (2008) Role of Cdx2 and cell polarity in cell allocation and specification of trophectoderm and inner cell mass in the mouse embryo. Genes Dev 22(19):2692–2706

    Article  PubMed  CAS  Google Scholar 

  • Johnson MH (2009) From mouse egg to mouse embryo: polarities, axes, and tissues. Ann Rev Cell Dev Biol 25:483–512

    Article  CAS  Google Scholar 

  • Johnson MH, Maro B (1984) The distribution of cytoplasmic actin in mouse 8-cell blastomeres. J Embryol Exp Morphol 82:97–117

    PubMed  CAS  Google Scholar 

  • Johnson MH, Pratt HPM, Handyside AH (1981) The generation and recognition of positional information in the preimplantation mouse embryo. In: Glasser SR, Bullock DW (eds) Cellular and molecular aspects of implantation. Plenum Press, New York, pp 55–74

    Google Scholar 

  • Kawamura K, Sato N, Fukuda J, Kodama H, Kumagai J, Tanikawa H, Nakamura A, Tanaka T (2002) Leptin promotes the development of mouse preimplantation embryos in vitro. Endocrinology 143(5):1922–1931

    Article  PubMed  CAS  Google Scholar 

  • Kawamura K, Sato N, Fukuda J, Kodama H, Kumagai J, Tanikawa H, Murata M, Tanaka T (2003) The role of leptin during the development of mouse preimplantation embryos. Mol Cell Endocrinol 202(1–2):185–189

    PubMed  CAS  Google Scholar 

  • Kunath T, Strumpf D, Rossant J (2004) Early trophoblast determination and stem cell maintenance in the mouse—A review. Placenta 25 Suppl. A:S32–S38

    Article  PubMed  Google Scholar 

  • Littwin T (2008) Zellpolaritäten und Achsenentwicklung beim präimplantativen Säugerembryo: Immunhistochemische Untersuchungen am Kaninchenembryo und entwicklungsbiologische Diskussion. Dissertation, Medizinische Fakultät der Universität Duisburg-Essen (Germany). URL:http://duepublico.uni-duisburgessen.de/servlets/DerivateServlet/Derivate-21311/Dissertation_Littwin.pdf

  • Maro B, Johnson MH, Pickering SJ, Flach G (1984) Changes in actin distribution during fertilization of the mouse egg. J Embryol Exp Morphol 81:211–237

    PubMed  CAS  Google Scholar 

  • Motosugi N, Dietrich JE, Polanski Z, Solter D, Hiiragi T (2006) Space asymmetry directs preferential sperm entry in the absence of polarity in the mouse oocyte. PLoS Biol 4(5):e135

    Article  PubMed  Google Scholar 

  • Nishioka N, Inoue K, Adachi K, Kiyonari H, Ota M, Ralston A, Yabuta N, Hirahara S, Stephenson RO, Ogonuki N, Makita R, Kurihara H, Morin-Kensicki EM, Nojima H, Rossant J, Nakao K, Niwa H, Sasaki H (2009) The hippo signalling pathway components lats and Yap pattern Tead4 activity to distinguish mouse trophectoderm from inner cell mass. Dev Cell 16(3):398–410

    Article  PubMed  CAS  Google Scholar 

  • Piotrowska K, Zernicka-Goetz M (2001) Role for sperm in spatial patterning of the early mouse embryo. Nature 409:517–521

    Article  PubMed  CAS  Google Scholar 

  • Ralston A, Rossant J (2008) Cdx2 acts downstream of cell polarization to cell-autonomously promote trophectoderm fate in the early mouse embryo. Dev Biol 313(2):614–629

    Article  PubMed  CAS  Google Scholar 

  • Rossant J, Tam PPL (2004) Emerging asymmetry and embryonic patterning in early mouse development. Developmental Cell 7:155–164

    Article  PubMed  CAS  Google Scholar 

  • Rossant J, Tam PP (2009) Blastocyst lineage formation, early embryonic asymmetries and axis patterning in the mouse. Development 136(5):701–713

    Article  PubMed  CAS  Google Scholar 

  • Seidel F (1960) Die Entwicklungsfähigkeiten isolierter Furchungszellen aus dem Ei des Kaninchens. Roux’ Arch Entwicklungsmech 152:43–130

    Article  Google Scholar 

  • Seidel F (1972) Entwicklungsphysiologie der Tiere. I. Ei und Furchung. (Sammlung Göschen, vol 7162), 2nd edn. Walter de Gruyter, Berlin

    Google Scholar 

  • Yamanaka Y, Ralston A, Stephenson RO, Rossant J (2006) Cell and molecular regulation of the mouse blastocyst. Dev Dyn 235(9):2301–2314

    Article  PubMed  CAS  Google Scholar 

  • Yoon SJ, Cha KY, Lee KA (2005) Leptin receptors are down-regulated in uterine implantation sites compared to interimplantation sites. Mol Cell Endocrinol 232(1–2):27–35

    Article  PubMed  CAS  Google Scholar 

  • Zernicka-Goetz M (2002) Patterning of the embryo: the first spatial decisions in the life of a mouse. Development 129(4):815–829

    PubMed  CAS  Google Scholar 

  • Zernicka-Goetz M (2006) The first cell-fate decisions in the mouse embryo: destiny is a matter of both chance and choice. Curr Opin Genet Dev 16:406–412

    Article  PubMed  CAS  Google Scholar 

Download references

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.

Author information

Authors and Affiliations

  1. Institut für Anatomie, Lehrstuhl für Anatomie und Entwicklungsbiologie, Universität Duisburg-Essen, Hufelandstr. 55, 45122, Essen, Germany

    T. Littwin & H. -W. Denker

Authors
  1. T. Littwin
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. H. -W. Denker
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to H. -W. Denker.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary material 1 (DOC 798 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

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

Download citation

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00418-011-0816-0

Keywords

Search

Navigation