164 POTENTIAL REGULATION OF PERI-IMPLANTATION OVINE CONCEPTUS GROWTH AND DIFFERENTIATION BY THE WNT SIGNALING SYSTEM

Abstract

In mice WNT signaling regulates cell fate, differentiation, and growth in the conceptus (embryo and associated extra-embryonic membranes), as well as implantation. We studied various components of the WNT signaling pathway in the ovine uterus during the estrous cycle (C) and pregnancy (P) and in the peri-implantation conceptus. Expression of WNT2, WNT2B, and WNT4 mRNAs was very low in endometria of C and P ewes from Days 10 to 16 and in conceptus trophectoderm (Tr). WNT5A/5B mRNAs were abundant in the stratum compactum stroma, whereas WNT11 mRNA was detected in endometrial epithelia of C and P ewes, but not in conceptus Tr. WNT7A mRNA was localized specifically to luminal (LE) and superficial glandular (sGE) epithelia of Day 10 C and P ewes, was undetectable by Day 12, and then increased up to Day 16 and was maximum on Day 20 only in P ewes. Frizzled receptor (FZD6/8) mRNAs were detected primarily in conceptus Tr and uterine LE and GE, whereas the co-receptor LRP5/6 (low density lipoprotein receptor-related protein) mRNAs were expressed in all uterine cell types and conceptus Tr. Dickkopf (DKK1), a negative regulator of WNT signaling, was detected in stratum compactum stroma after Day 14 in P ewes. CTNNB1 (beta-catenin), a key mediator of canonical WNT signaling, and GSK3B (glycogen synthase kinase-3 beta) and CHD1 (E-cadherin) mRNAs were abundant in endometrial epithelia and in conceptus Tr. Immunoreactive CTNNB1 protein was abundant in LE and GE, and present at lower levels in stroma and myometrium in uteri from C and P ewes. In the conceptus Tr, immunoreactive CTNNB1 protein was abundant in nuclei of the mononuclear and binuclear cells (BNC), as well as in cell adherens junctions. Nuclear CTNNB1 interacts with transcription factors, most notably LEF1/TCF7 (lymphoid enhancer-binding factor 1/transcription factor 7), to regulate gene transcription. LEF1 mRNA was detected in LE and sGE, whereas nuclear TCF7L2 protein was particularly abundant in trophoblast giant BNC. WNT/beta-catenin/TCF7 target genes were also studied. MSX2 mRNA was abundant in conceptus Tr, and MYC mRNA was abundant in BNC of conceptus Tr and endometrial epithelia. Next, ovine Tr (oTr) cells and endometrial stromal (oST) cells were used for mechanistic studies that revealed that transfection of mouse WNT7A stimulated a LEF/TCF-responsive reporter (TOPFLASH), and co-transfection of either dnTCF or SFRP2 (a secreted FZD inhibitor) inhibited WNT7A effects. WNT7A stimulated expression of MSX2 and MYC in oTr cells, and this effect was inhibited by SFRP2. These results implicate the canonical WNT system as a regulator of peri-implantation conceptus growth and differentiation in sheep.

This work was supported by NIH HD38274 and 5 P30 ES09106 funding.