YPEL3 Negatively Regulates Endometrial Function via the Wnt/β-Catenin Pathways during Early Pregnancy in Goats

Abstract

In ruminants, the establishment of pregnancy requires a series of structural and functional changes in the endometrium under the action of hormones, thereby providing an optimal environment for the implantation of the embryo. In this study, we explored the molecular mechanism by which YPEL3 regulates endometrial function during gestation in goats. We found YPEL3 expression was significantly downregulated during early gestation and that YPEL3 overexpression inhibited the expression of ISG15, but had no significant effects on the expression of RSAD2 and CXCL10 in goat endometrial epithelial cells (gEECs). In addition, YPEL3 silencing significantly inhibited PGF_2α secretion and the expression of the prostaglandin synthesis-related rate-limiting enzyme-encoding genes PGFS and PTGES, with no significant effect on the expression of PTGS1 and PTGS2. Moreover, YPEL3 inhibited the expression of vimentin and β-catenin and pretreatment of gEECs with the β-catenin activator CHIR99021 prevented a YPEL3-induced decrease in vimentin expression. Collectively, our findings confirm that, as a hormone-regulated factor, YPEL3 regulates endometrial function by inhibiting the Wnt/β-catenin signaling pathway and provide new insights for further clarification of the mechanism by which YPEL3 functions during early pregnancy in ruminants.

Keywords: Wnt/β-catenin; YPEL3; endometrial function; goat endometrial epithelial cells; hormone.

Figure 1

YPEL3 expression in the goat endometrium during early pregnancy and hormone treatment. (A) Immunohistochemical staining of YPEL3 protein in goat uterine tissues during early pregnancy (days 5, 15 and 18 of the pregnancy). (B) Real-time quantitative PCR analysis of YPEL3 mRNA levels after E₂, P₄ and IFN-τ treatment. (C) Western blot analysis of YPEL3 protein expression following E₂, P₄ and IFN-τ treatment. L, luminal epithelium; G, glandular epithelium; S, stromal cells. Scale bars = 200 μm or scale bars = 50 μm. ** p < 0.01, ns, not significant (p > 0.05).

Figure 2

Confirmation of silencing and overexpression of YPEL3 in gEECs. (A) Real-time quantitative PCR analysis of YPEL3 mRNA overexpression efficiency in gEECs transfected with pcDNA3.1 and pcDNA3.1-YPEL3 for 48 h. (B) Western blot analysis of YPEL3 protein overexpression efficiency in gEECs transfected with pcDNA3.1 and pcDNA3.1-YPEL3 for 48 h. (C) Real-time quantitative PCR analysis of YPEL3 mRNA levels in gEECs transfected with shN and shYPEL3 for 48 h. (D) Western blot analysis of YPEL3 protein overexpression efficiency in gEECs transfected with shN and shYPEL3 for 48 h. (E) Confocal microscope images of YPEL3 expression in gEECs transfected with shN and shYPEL3 for 48 h. Scale bars = 20 μm. ** p < 0.01, ns, not significant (p > 0.05).

Figure 3

YPEL3 altered endometrial receptivity and the secretion of PGF_2α. (A–C) Real-time quantitative PCR analysis of the expression of genes (ISG15, RSAD2, CXCL10) associated with promoting conceptus elongation in gEECs overexpressing YPEL3 and treated with E₂, P₄ and IFN-τ. (D) ELISA analysis of PGF_2α secretion by gEECs with YEPL3 silencing and treated with E₂, P₄ and IFN-τ. (E) Real-time quantitative PCR analysis of the expression of genes encoding rate-limiting PG synthesis enzymes (PGFS, PTGES, PTGS1, PTGS2) in gEECs with YEPL3 silencing and treated with E₂, P₄ and IFN-τ. ** p < 0.01, ns, not significant (p > 0.05).

Figure 4

The effect of YPEL3 on EMT marker expression in gEECs. (A) Western blot analysis of the EMT marker proteins N-cadherin and vimentin after treatment with or without E₂, P₄ and IFN-τ. Western blot analysis of the EMT marker protein expression in gEECs with silencing (B) and overexpression (C) of YPEL3 and treated with E₂, P₄ and IFN-τ. * p < 0.05, ** p < 0.01, ns, not significant (p > 0.05).

Figure 5

YPEL3 regulated the Wnt/β-catenin signaling pathway. Western blot analysis of the expression of β-catenin in gEECs with YEPL3 silencing (A) and YEPL3 overexpression (B) and treated with E₂, P₄ and IFN-τ. Confocal microscope images of immunohistochemical staining of β-catenin expression and nuclear translocation in gEECs with YEPL3 overexpression (C) or YEPL3 silencing (D) and treated with E₂, P₄ and IFN-τ. ** p < 0.01.

Figure 6

The effect of β-catenin activation on gEEC EMT. Western blot analysis of the expression of EMT markers (N-cadherin, vimentin) and β-catenin proteins in gEECs pre-treated with 5 μM CHIR99021 before E₂, P₄ and IFN-τ treatment. * p < 0.05, ** p < 0.01, ns, not significant (p > 0.05).