49 Expression and actions of the dickkopf-1 receptors KREMEN1 and KREMEN2 in the bovine pre-implantation embryo

Abstract

Dickkopf-1 (DKK1) is a secreted inhibitor of canonical WNT signaling expressed in the endometrium that can increase the competence of bovine blastocysts to establish pregnancy after transfer into recipients. The mechanism by which DKK1 regulates embryo function is not known. KREMEN1 and KREMEN2 are paralogs that function as DKK1 transmembrane receptors. Binding of DKK1 to KREMEN leads to internalization of the LRP5/6 WNT co-receptor and inhibition of β-catentin (CTNNB1)-dependent WNT signaling. Here we evaluated whether the bovine pre-implantation embryo expresses KREMEN1 and KREMEN2 and whether knockdown of KREMEN1 would alter embryonic development and accumulation of CTNNB1. In the first experiment, expression of KREMEN1 and KREMEN2 was determined in individual matured oocytes and embryos at the 2-cell, 3-4-cell, 5-8-cell, 9-16-cell, morula, and blastocyst stages of development (n=4) using RT-qPCR with GAPDH and YWHAZ as internal controls. Data were analysed by analysis of variance using GLM procedure of SAS (SAS Institute Inc.; stage, replicate in model), and Tukey test for multiple comparisons. Expression of both KREMEN1 (P<0.0001) and KREMEN2 (P=0.003) varied between stages of development. There was a large decline in transcript abundance for KREMEN1 after the 9-16-cell stage. Expression (fold-change) relative to housekeeping genes was 0.29, 0.47, 0.27, 0.47, 0.34, 0.05, and 0.01 (standard error=0.08) for oocyte, 2-cell, 4-cell, 5-8 cell, 9-16-cell, morula, and blastocyst stages, respectively. Expression of KREMEN2 was low at early stages of development, increased at the 8-16 cell and morula stages (i.e. after embryonic genome activation), and then declined. Expression (fold-change) relative to housekeeping genes was 0.00, 0.04, 0.03, 0.05, 0.12, 0.15, and 0.01 (standard error=0.04) for oocyte, 2-cell, 4-cell, 5-8-cell, 9-16-cell, morula, and blastocyst stages, respectively. In the second experiment, we determined effects of a GapmeR antisense oligonucleotide directed against KREMEN1 on development to the blastocyst stage and amounts of immunoreactive CTNNB1 in the blastocyst. The experiment was performed in 3 replicates using 217 putative zygotes per treatment. The GapmeR treatment did not affect cleavage rate (78.5±0.02% for GapmeR vs. 71.7%±0.02% for vehicle; P=0.21), percent putative zygotes becoming blastocysts (23.4±0.02% vs. 20.0±0.02%; P=0.57), or the percent of cleaved embryos becoming blastocysts (30.6±0.03% vs. 28.8±0.03%; P=0.88). Amounts of immunoreactive CTNNB1 were determined in blastocysts collected at 168h after insemination (n=7/treatment). Treatment with GapmeR tended (P=0.08) to increase the fluorescence intensity of CTNNB1 (1761±68 vs. 1583±72 units). In summary, KREMEN1 is expressed before genome embryonic activation, whereas KREMEN2 is expressed in 8-16 cells and morula. Knocking down KREMEN1 does not compromise embryonic development, but preliminary studies indicate KREMEN1 can regulate CTNNB1 abundance. Support was provided by USDA-NIFA 2017-67015-26452.