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

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.

Figure 1

Staining of several embryos from 2 couples (1 & 2) with either nonimmune IgG (1A and 2A) or TRP53 antibody (all other embryos). Two images of each embryo (except 2C) are provided: an equatorial confocal section (confocal) or phase contrast (phase). All confocal images were taken with identical microscope, laser and magnification settings. The position of the inner cell mass is identified by a white arrow where applicable. The bar is 10 μm, N.A. is not available.

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).