Differential Signals From TNFα-Treated and Untreated Embryos in Uterine Tissues and Splenic CD4+ T Lymphocytes During Preimplantation Pregnancy in Mice

Abstract

The main aim of this study was to examine if a female mouse body in preimplantation pregnancy can distinguish between embryos of normal and impaired biological quality in the local and peripheral compartments. Normal (control group) and TNFα (tumor necrosis factor-α)-treated embryos (experimental group) at the morula stage were non-surgically transferred into the uteri of CD-1 strain [Crl:CD1(Icr)] female murine recipients. Twenty-four hours after the embryo transfer, females were euthanised, and uteri and spleens were dissected. In uterine tissues (local compartment), we assessed the expression of 84 genes comprising nine signal transduction pathways, using a modified RT² Profiler PCR Array. In the spleen (peripheral compartment), we determined the proteome of splenic CD4⁺ lymphocytes using 2D protein electrophoresis with subsequent protein identification by mass spectrometry. Sample clustering and differential gene expression analyses within individual signal transduction pathways revealed differential expression of genes in the uteri of females after transplantation of normal vs. TNFα-treated embryos. The most affected signal transduction cascade was the NFKB (Nuclear factor NF-kappa-B) pathway, where 87.5% of the examined genes were significantly differentially expressed. Proteomic analysis of splenic CD4⁺ T lymphocytes revealed significant differential expression of 8 out of 132 protein spots. Identified proteins were classified as proteins influenced by cell stress, proteins engaged in the regulation of cytoskeleton stabilization and cell motility, and proteins having immunomodulatory function. These results support the hypothesis that even before embryo implantation, the body of pregnant female mice can sense the biological quality of an embryo both at the local and peripheral level.

Keywords: CD4 lymphocytes; T cells; TNF; embryo; pregnancy; preimplantation; proteome; uterus.

Figure 1

The layout of the entire experiment. Female mouse donors were superovulated by PMSG (Pregnant Mare Serum Gonadotropin) and hCG (human Choriogonadotropin) injections and mated with fertile males. Embryos were collected at the 1st day post-coitum (dpc) and cultured for the next 24 h in the presence or without TNFα. Embryos were transferred into progesterone-treated and mated with vasectomized males, female recipients. Uteri and spleens from recipient females were dissected. Gene expression and proteome of CD4⁺ lymphocytes were examined in obtained tissues. A separate group of embryos was examined in vitro for checking the developmental progress and phosphatidylserine expression.

Figure 2

The uniform pattern of phosphatidylserine expression in blastomeres of TNFα-treated embryos (A,B) and normal embryos (C,D).

Figure 3

Hierarchical clustering heatmap of mRNA transcripts of nine signal transduction pathways differentially expressed in recipients' uteri of normal (NE) and TNFα- (TNE) treated embryos. Gene expression level is color coded.

Figure 4

A representative proteomic 2D map of splenic CD4⁺ T cells. The 2-DE gel illustrates a Coomassie-stained protein pattern. Spot numbers correspond to the numbers in Table 4.

Figure 5

The graphic trend for spots with a variable expression of (A) HSP70, (B) LSP1, (C) TPM3, (D) IL-24, (E) GDIR2, (F) PRDX2, (G) ATP5H and (H) SEMA7A proteins between TNE (dark gray bars) and NE group of mice (light gray bars). The data concerning the protein expression are given as arithmetic mean with SD (group n = 6). Student's t-test *p < 0.05, **p < 0.01.