(P-411) The Zinc-finger Domains of Wilms’ Tumor 1 Protein are Critical for Uterine Receptivity During Pregnancy .

Abstract Authors: Sudikshya Paudel, Magdalina J Cummings, Steven L. Young, Xiaoqiu Wang

Abstract Text: Wilms’ Tumor 1 (WT1) is a zinc-finger transcription factor required for cell survival, growth and differentiation. We and others have shown that WT1 is abundantly expressed in the stromal cells of the secretory endometrium and the decidua in the first and second trimesters in humans. However, the functional roles of WT1 in the uterus during pregnancy is unknown. Mice that carry the Wt1^f/f allele flanking exons 8 and 9 of the Wt1 homolog gene were bred to Pgr^cre/+ mice to generate Pgr^cre/+Wt1^f/f (Wt1^D/D) mice. The Wt1^D/D mice produced a shortened WT1 protein lacking the second and third of the four DNA-binding zinc-finger domains, validated via genotyping and western blot analyses. After 6-month breeding trial, Wt1^D/D female mice were infertile (P< 0.001) as compared to the Wt1^f/f control mice (6.0±0.4 pups per litter). Also, haploinsufficiency was spotted in which significant reductions (P< 0.01) in pups born per litter (3.4±0.5 versus 6.0±0.4), as well as survival pups per litter at weaning (1.0±0.5 versus 5.6±0.4) were observed in Wt1^+/D mothers as compared to the Wt1^f/f controls. The rates of stillbirth and second-day neonatal death were also high (P< 0.05) in Wt1^+/Dfemales. Among the stillborn pups from Wt1^+/D females were the underdeveloped fetuses with congenital limb defects. Ovarian functions were not affected by Wt1 mutation in which no differences in the number of blastocysts at gestational day (GD) 3.5 and serum P4 levels at GD 4.5 were observed between Wt1^D/Dfemales and the Wt1^f/f controls. Blue dye injection analyses of implantation sites showed that uteri of Wt1^D/Dfemales exhibited no implantation responses at GD 4.5 and 5.5. Histological analyses further revealed that blastocysts can only attach to the luminal epithelium but failed to invade it and initiate decidualization at GD 4.5. However, at GD 6.5 and 9.5, smaller decidual bulb with reabsorbing blastocysts was observed in Wt1^D/Duteri as compared to the control, suggesting delayed and defective implantation, decidualization and placentation. Wt1^+/D females also exhibited no implantation responses at GD4.5 and 5.5 and embryos were found attached to the luminal epithelium whereas at GD6.5 and 9.5 decidual bulb were observed on Wt1^+/Dmice as compared to Wt1^f/+ control mice. Immunohistochemical analyses further showed that ESR1 decreased in glandular epithelium (GE) but increased in the stroma of Wt1^D/D uteri at GD 3.5 as compared to the Wt1^f/f control. PGR was downregulated in the stromal cells of Wt1^D/D uteri between pre-decidual (GD 3.5) and post-decidual stage (GD 9.5). In addition, PTGS2 was failed to express in stromal cells of implantation sites at GD 4.5 Wt1^D/D uteri as compared to the Wt1^f/f control. At decidual day -1 (DD-1; one day before artificial decidualization to mimick GD3.5), Areg mRNA was decreased (P< 0.01) but Wnt4, Ltf, and Bmp2 were increased in Wt1^D/D uteri as compared to Wt1^f/f. Moreover, the ability of the endometrial stromal cells to undergo an artificially induced decidualization was severely compromised (P< 0.01) in Wt1^D/D uteri, with failure of induction in Prl, Igfbp1, Pgr and Areg at DD2 and DD5. These results suggest that Wt1, via its second and third DNA-binding zinc-finger domains, plays critical roles in implantation and decidualization, thereby governing placentation and fetal development during pregnancy.