Tryptophan in the mouse diet is essential for embryo implantation and decidualization

Abstract

Introduction: Nutritional deficiency occurs frequently during pregnancy and breastfeeding. Tryptophan (Trp), an essential amino acid which is critical for protein synthesis, serves as the precursor for serotonin, melatonin, and kynurenine (Kyn). The imbalance between serotonin and kynurenine pathways in Trp metabolism is closely related to inflammation and depression. This study assessed the effects of Trp deficiency on mouse early pregnancy.

Methods: Embryo implantation and decidualization were analyzed after female mice had been fed diets containing 0.2% Trp (for the control group), 0.062% Trp (for the low Trp group) and 0% Trp (for the Trp-free group) for two months. The uteri of the mice were collected on days 4, 5, and 8 of pregnancy for further analysis.

Results: On day 8 of pregnancy, the number of implantation sites were found to be similar between the control and the low Trp groups. However, no implantation sites were detected in the Trp-free group. On day 5 of pregnancy, plane polarity- and decidualization-related molecules showed abnormal expression pattern in the Trp-free group. On day 4 of pregnancy, there was no significant difference in uterine receptivity molecules between the low-Trp group and the control group, but uterine receptivity was abnormal in the Trp-free group. At implantation sites of the Trp-free group, IDO and AHR levels were markedly elevated. This potentially increased levels of Kyn, 2-hydroxy estradiol, and 4-hydroxy estradiol to affect decidualization.

Conclusions: Trp-free diet may impair decidualization via the IDO-KYN-AHR pathway.

Keywords: aryl hydrocarbon receptor; decidualization; tryptophan deficiency; uterine receptivity; uterus.

Figure 1

Complete Trp deficiency results in abnormal decidualization. (A) The body weight of female mice after feeding with different diet for two months. (B) A representative photograph showing the number of implantation sites on day 8 of pregnancy in each group (N=5 mice). (C) A representative photograph showing the number of implantation sites on day 5 of pregnancy in each group (N=5 mice). (D) Statistical analysis of the number of implantation sites on day 5 of pregnancy in each group. (E) qPCR analysis of Prl8a2 mRNA level in mouse uteri at implantation sites on day 5 in each group. (F) Alkaline phosphatase staining in mouse uteri on day 5 of implantation sites in each group. * Embryo. Scale bar, 50 μm. **, p < 0.01; ***, p < 0.001, ns, not significant.

Figure 2

The morphology of implantation chambers and planar cell polarity signaling on day 5 of pregnancy. (A) The morphology of implantation chambers in each group. There were abnormal luminal closure and increased epithelial branching in Trp-free group. (B) A diagram showing normal and abnormal implantation chamber at implantation sites. (C) The number of abnormal implantation chambers and statistical analysis by the chi-square test (N=15 mice). IS: implantation sites. (D) Immunofluorescence of RBPJ and VANGL2 at implantation sites. * Embryo. Scale bar, 50 μm. **, p < 0.01, ns, not significant.

Figure 3

Uterine receptivity on day 4 of pregnancy. (A) Ki67 immunostaining and HAND2 immunofluorescence. * Embryo. Scale bar, 50 μm. (B) qPCR analysis of Ltf mRNA level. (C) qPCR analysis of C3 mRNA level. *, p < 0.05; ***, p < 0.001; ns, not significant.

Figure 4

IDO-kynurenine pathway at implantation site on day 5 of pregnancy. (A) Immunofluorescence of IDO1 and TPH1. * Embryo. Scale bar, 50 μm. (B) Uterine Kyn concentration at implantation site. (C) Uterine 5-HT concentration at implantation site. (D) qPCR analysis of Prl8a2 mRNA level after mouse stromal cells were treated with Kyn for 48 h under in vitro decidualization. (E) qPCR analysis of Prl8a2 mRNA level after mouse stromal cells were treated with 5-HT for 48 h under in vitro decidualization. *, p < 0.05; **, p < 0.01; ***, p < 0.001, ns, not significant.

Figure 5

AHR signaling and its effects on mouse decidualization. (A) AhR immunofluorescence at implantation site on day 5 of pregnancy. * Embryo. Scale bar, 50 μm. (B) qPCR analysis of Cyp1a1 mRNA level in mouse uterus at implantation sites on day 5 of pregnancy. (C) qPCR analysis of Cyp1b1 mRNA level in mouse uterus at implantation site on day 5 of pregnancy. (D) qPCR analysis of Prl8a2 mRNA level after mouse stromal cells were treated with 2OE-E2 for 48 h under in vitro decidualization. (E) qPCR analysis of Prl8a2 mRNA level after mouse stromal cells were treated with 4OE-E2 for 48 h under in vitro decidualization. (F) qPCR analysis of Cypla1 mRNA level after mouse stromal cells were treated with CH223191 (AHR inhibitor) for 48 h in the presence or absence of Kyn under in vitro decidualization. (G) qPCR analysis of Cyplb1 mRNA level after mouse stromal cells were treated with CH223191 for 48 h in the presence or absence of Kyn under in vitro decidualization. (H) qPCR analysis of Prl8a2 mRNA level after mouse stromal cells were treated with CH223191 for 48 h in the presence or absence of Kyn under in vitro decidualization. *, p < 0.05; **, p < 0.01; ***, p < 0.001, ns, not significant.