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Variable expressivity of the tumour suppressor protein TRP53 in cryopreserved human blastocysts.

Chandrakanthan V, Chami O, Stojanov T, O'Neill C - Reprod. Biol. Endocrinol. (2007)

Bottom Line: A blastocyst that had a collapsed blastocoel also showed high levels of TRP53 compared to morphologically normal blastocysts.Most TRP53 staining was in the region of the nucleus.However, some cells within these embryos had high levels of nuclear TRP53 expression.

View Article: PubMed Central - HTML - PubMed

Affiliation: Human Reproduction Unit, Royal North Shore Hospital, St Leonards, NSW, 2065, Australia. cvas@physiol.usyd.edu.au

ABSTRACT
In a mouse model, in vitro fertilization or extended embryo culture leads to the increased expression of TRP53 in susceptible embryos. Ablation of the TRP53 gene improved embryo viability indicating that increased expression of TRP53 is a cause of the reduction of embryo viability resulting from in vitro fertilization or embryo culture. This study investigates the status of TRP53 expression in human embryos produced by intracytoplasmic sperm injection. Following fertilization, embryos were cultured for 96 h and then cryopreserved. Immediately upon thawing they were fixed in formaldehyde and subjected to immunostaining for TRP53. Staining was visualized by confocal microscopy. Negative controls were incubated with isotype control immunoglobulin and showed negligible staining. All embryos showed TRP53 staining above negative controls. TRP53 staining was heterogenous within and between embryos. An embryo that showed retarded development showed high levels of TRP53 expression. A blastocyst that had a collapsed blastocoel also showed high levels of TRP53 compared to morphologically normal blastocysts. Most TRP53 staining was in the region of the nucleus. Morphologically normal blastocysts tended to show little nuclear accumulation of stain. However, some cells within these embryos had high levels of nuclear TRP53 expression. The results show that embryos have varying sensitivity to the stresses of production and culture in vitro, and this resulted in variable expressivity of TRP53.

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Compilation of sequential z-sections through the entire depth of two embryos shown in Fig 1. A. compilation of sections from embryo shown Fig 1 2G; B. compilation of embryos from Fig1 2C. Greyscale images were converted to pseudocolor representation of staining intensity (blue lowest intensity, red greatest intensity).
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Figure 2: Compilation of sequential z-sections through the entire depth of two embryos shown in Fig 1. A. compilation of sections from embryo shown Fig 1 2G; B. compilation of embryos from Fig1 2C. Greyscale images were converted to pseudocolor representation of staining intensity (blue lowest intensity, red greatest intensity).

Mentions: The expression of TRP53 in human embryos was assessed by immunofluorescence with confocal microscopy. Staining of 7 human embryos for immunodetectable TRP53 showed considerable within and between embryo heterogeneity of TRP53 expression. Figure 1 shows single equatorial optical sections through each embryo. Nonimmune (control) immunoglobulin samples showed no staining for a blastocyst with some signs of degeneration (embryo grade 3BB) (Fig 1A–B) or in a morphologically normal blastocyst (grade 3AA) (Fig 1, 2A–B). Five embryos were stained for TRP53. Two embryos were graded as 3BA (Fig 1C,D and 2C,D). These blastocyst showed a relatively high levels of staining in some nuclei of the trophectoderm, but this was less apparent in the cells of the inner cell mass (Fig 1C, 2C). One blastocyst showed extensive expansion (grade 4AA) and there was little obvious TRP53 staining in this blastocyst (Fig 1E–F). One blastocyst had either failed to expand or had collapsed (grade 1) (Fig 1, 2E), and it displayed obvious nuclear accumulation of TRP53 staining in all cells. In a blastocyst with obvious signs of degeneration (grade < 1) (Fig 1, 2G,H) there was extensive TRP53 staining throughout the embryo.


Variable expressivity of the tumour suppressor protein TRP53 in cryopreserved human blastocysts.

Chandrakanthan V, Chami O, Stojanov T, O'Neill C - Reprod. Biol. Endocrinol. (2007)

Compilation of sequential z-sections through the entire depth of two embryos shown in Fig 1. A. compilation of sections from embryo shown Fig 1 2G; B. compilation of embryos from Fig1 2C. Greyscale images were converted to pseudocolor representation of staining intensity (blue lowest intensity, red greatest intensity).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2099431&req=5

Figure 2: Compilation of sequential z-sections through the entire depth of two embryos shown in Fig 1. A. compilation of sections from embryo shown Fig 1 2G; B. compilation of embryos from Fig1 2C. Greyscale images were converted to pseudocolor representation of staining intensity (blue lowest intensity, red greatest intensity).
Mentions: The expression of TRP53 in human embryos was assessed by immunofluorescence with confocal microscopy. Staining of 7 human embryos for immunodetectable TRP53 showed considerable within and between embryo heterogeneity of TRP53 expression. Figure 1 shows single equatorial optical sections through each embryo. Nonimmune (control) immunoglobulin samples showed no staining for a blastocyst with some signs of degeneration (embryo grade 3BB) (Fig 1A–B) or in a morphologically normal blastocyst (grade 3AA) (Fig 1, 2A–B). Five embryos were stained for TRP53. Two embryos were graded as 3BA (Fig 1C,D and 2C,D). These blastocyst showed a relatively high levels of staining in some nuclei of the trophectoderm, but this was less apparent in the cells of the inner cell mass (Fig 1C, 2C). One blastocyst showed extensive expansion (grade 4AA) and there was little obvious TRP53 staining in this blastocyst (Fig 1E–F). One blastocyst had either failed to expand or had collapsed (grade 1) (Fig 1, 2E), and it displayed obvious nuclear accumulation of TRP53 staining in all cells. In a blastocyst with obvious signs of degeneration (grade < 1) (Fig 1, 2G,H) there was extensive TRP53 staining throughout the embryo.

Bottom Line: A blastocyst that had a collapsed blastocoel also showed high levels of TRP53 compared to morphologically normal blastocysts.Most TRP53 staining was in the region of the nucleus.However, some cells within these embryos had high levels of nuclear TRP53 expression.

View Article: PubMed Central - HTML - PubMed

Affiliation: Human Reproduction Unit, Royal North Shore Hospital, St Leonards, NSW, 2065, Australia. cvas@physiol.usyd.edu.au

ABSTRACT
In a mouse model, in vitro fertilization or extended embryo culture leads to the increased expression of TRP53 in susceptible embryos. Ablation of the TRP53 gene improved embryo viability indicating that increased expression of TRP53 is a cause of the reduction of embryo viability resulting from in vitro fertilization or embryo culture. This study investigates the status of TRP53 expression in human embryos produced by intracytoplasmic sperm injection. Following fertilization, embryos were cultured for 96 h and then cryopreserved. Immediately upon thawing they were fixed in formaldehyde and subjected to immunostaining for TRP53. Staining was visualized by confocal microscopy. Negative controls were incubated with isotype control immunoglobulin and showed negligible staining. All embryos showed TRP53 staining above negative controls. TRP53 staining was heterogenous within and between embryos. An embryo that showed retarded development showed high levels of TRP53 expression. A blastocyst that had a collapsed blastocoel also showed high levels of TRP53 compared to morphologically normal blastocysts. Most TRP53 staining was in the region of the nucleus. Morphologically normal blastocysts tended to show little nuclear accumulation of stain. However, some cells within these embryos had high levels of nuclear TRP53 expression. The results show that embryos have varying sensitivity to the stresses of production and culture in vitro, and this resulted in variable expressivity of TRP53.

Show MeSH