Expression pattern implicates a potential role for luman recruitment factor in the process of implantation in uteri and development of preimplantation embryos in mice

Abstract

Luman/CREB3 recruitment factor (LRF or CREBRF) was identified as a regulator of Luman (or CREB3) that is involved in the unfolded protein response during endoplasmic reticulum stress. Luman is implicated in a multitude of functions ranging from viral infection and immunity to cancer. The biological function of LRF, however, is unknown. In this paper, we report that uteri of pregnant mice and embryos displayed enhanced LRF expression at all stages, and the expressed LRF was found to be localized specifically at implantation sites. On the other hand, uteri of mice induced for delayed implantation or pseudopregnant mice showed low levels of LRF expression, suggesting that LRF mediates uterine receptivity during implantation. Further, expression of LRF was found to be modulated by steroid hormones such as progesterone and estradiol. This study thereby identifies a potential role for LRF in the process of implantation in uteri and development of preimplantation embryos in mice.

Fig. 1.

a: Uteri isolated from pregnant and pseudopregnant mice at different stages were analyzed for LRF transcripts using qRT-PCR as described in Materials and Methods. The figure represents the quantification of LRF transcripts, with the black and white bars corresponding to uteri isolated from pregnant and pseudopregnant mice, respectively. Different letters indicate significant differences in P value with P<0.05. b: Comparative analysis of LRF transcripts by qRT-PCR, in uteri isolated from mice treated to induce delayed implantation and implantation activated mice. The figure represents the quantification of LRF transcripts. *Indicates significant difference in P value with P<0.01. c: Comparative analysis of LRF transcripts by qRT-PCR in uteri isolated from mice treated to induce decidualization and non-decidual mice. The figure represents the quantification of LRF transcripts. *Indicates a significant difference in P value with P<0.01. d: Analysis of LRF transcripts by qRT-PCR, in the uteri of mice treated with either estradiol (E₂), progesterone (P₄) or both E2 and P₄. Mice administered with sesame oil were used as controls. The figure represents the quantification of LRF transcripts. Asterisks (** and *) indicate significant differences in P value with P<0.01 and P<0.05, respectively.

Fig. 2.

The figure represents the immunohistochemistry of LRF protein in uteri of pregnant mice during embryonic implantation at days 1 through 5 (panels a–e, respectively), at day 5 without embryonic implantation (panel f), at days 6 and 7 showing embryonic implantation (panels g and h, respectively), during delayed implantation (panel i), during activated implantation (panel j) and during induced decidualization (panel k); in the control uterus (panel l), in the untreated control uterus (panel m); after estradiol treatment (panel n); after progesterone treatment (panel o); and after treatment with both estradiol and progesterone (panel p). Each panel shows the images taken at both low and high magnifications. H and H' in the panels indicate images taken at higher magnifications, while L indicates the images taken at lower magnifications. The scale bars for H, H' and L reperesent 40 μm, 100 μm and 20 μm, respectively. DC, decidual cell; EM, embryo; GE, glandular epithelium; IS, implantation site; LE, luminal epithelium; NIS, non-implantation site; PDZ, primary decidual zone; SDZ, secondary decidual zone.

Fig. 3.

a–g: Immunofluorescence analyses of LRF expression in embryos at different stages of preimplantation, i.e., a– zygote, b– 2-cell embryo, c– 4-cell embryo, d– 8-cell embryo, e– morula; f– early blastocyst and g–hatching blastocyst. Scale bar: 100 µm. h: Quantification of the immunofluorescence data obtained in panels a–g. **P<0.05; *P<0.01.