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.

Differences in blastomere totipotency in 2-cell mouse embryos are a maternal trait mediated by asymmetric mRNA distribution

“…, 2003) and is the most differently expressed among the EPI-related mRNAs that are detected at the 2-cell stage, directly followed by Pou5f1 ( Oct4 ). As we queried the expression of Cops3 in situ using Stellaris® probes in confocal imaging, we knew we had to be very cautious, because: (i) non-homogeneous signal distribution may be caused by the fixative (Littwin and Denker, 2011); (ii) extended focus in a spherical object is bound to create projection images with a gradient of fluorescence between the centre and periphery and (iii) differences in fluorescence intensity in the Z dimension can be due to bleaching. Therefore, we examined the sister blastomeres next to each other in the sole equatorial region (10 confocal slices corresponding to a total of 2.22 μm) to minimize these problems.…”

Recent progress in histochemistry and cell biology

“…Sadly, abovementioned findings have emerged to be not reproducible, possibly due to the methodology used [immunohistochemistry, IHC, performed on whole-mount (WM) in conjunction with confocal laser scanning microscopy, WMIHC/CLSM]. This prompted Littwin and Denker (2011) to resume work on this open issue using a rabbit model. The authors performed, as previously, WMIHC/ CLSM, but this time compared acquired results with section immunohistochemistry (S-ICH).…”

Adipokines Expression and Effects in Oocyte Maturation, Fertilization and Early Embryo Development: Lessons from Mammals and Birds

“…In embryos of mouse, human, bovine, chicken, and rabbit species, leptin is present and has specific spatial localization [121,122,128,[156][157][158] (Table 5). In these studies, authors demonstrated that after fertilization, leptin becomes differentially distributed among the inner and outer blastomeres of the morula stage embryo in a pattern that persists with respect to the ICM and trophoblast through the hatched blastocyst stage, suggesting an important role of leptin in early embryo development.…”