Cysteine dioxygenase and taurine are essential for embryo implantation by involving in E2-ERα and P4-PR signaling in mouse

Abstract

Background: Taurine performs multiple physiological functions, and the maintenance of taurine level for most mammals relies on active uptake from diet and endogenous taurine synthesis through its synthesis enzymes, including cysteine dioxygenase (CDO). In addition, uterus tissue and uterus fluid are rich in taurine, and taurine synthesis is regulated by estrogen (E₂) and progesterone (P₄), the key hormones priming embryo-uterine crosstalk during embryo implantation, but the functional interactions and mechanisms among which are largely unknown. The present study was thus proposed to identify the effects of CDO and taurine on embryo implantation and related mechanisms by using Cdo knockout (KO) and ovariectomy (OVX) mouse models.

Results: The uterine CDO expression was assayed from the first day of plugging (d 1) to d 8 and the results showed that CDO expression level increased from d 1 to d 4, followed by a significant decline on d 5 and persisted to d 8, which was highly correlated with serum and uterine taurine levels, and serum P₄ concentration. Next, Cdo KO mouse was established by CRISPER/Cas9. It was showed that Cdo deletion sharply decreased the taurine levels both in serum and uterus tissue, causing implantation defects and severe subfertility. However, the implantation defects in Cdo KO mice were partly rescued by the taurine supplementation. In addition, Cdo deletion led to a sharp decrease in the expressions of P₄ receptor (PR) and its responsive genes Ihh, Hoxa10 and Hand2. Although the expression of uterine estrogen receptor (ERα) had no significant change, the levels of ERα induced genes (Muc1, Ltf) during the implantation window were upregulated after Cdo deletion. These accompanied by the suppression of stroma cell proliferation. Meanwhile, E₂ inhibited CDO expression through ERα and P₄ upregulated CDO expression through PR.

Conclusion: The present study firstly demonstrates that taurine and CDO play prominent roles in uterine receptivity and embryo implantation by involving in E₂-ERα and P₄-PR signaling. These are crucial for our understanding the mechanism of embryo implantation, and infer that taurine is a potential agent for improving reproductive efficiency of livestock industry and reproductive medicine.

Keywords: CDO; E2; Embryo implantation; P4; Taurine.

Fig. 1

Uterine CDO mRNA and protein expressions and their relations to taurine levels in uterus from d 1 to d 8 of pregnancy mice. A, Cdo mRNA expression in mouse uteri detected by RT-qPCR (n = 3). B, CDO protein expression in uteri of d 1 to d 8 pregnancy mice assayed by WB. C, Serum and uterine taurine concentrations assayed by HPLC (n = 3). D, Serum P₄ concentrations assayed by RIA (n = 5). E, CDO IHC staining of uterine cross-sections. Data shown as Mean ± SEM. Different letters represent significant differences (P < 0.05). d, days post coitum; E₂, estrogen; GE, glandular epithelium; LE, luminal epithelium; P₄, progesterone; St, stromal cells. Bars: 50 μm

Fig. 2

Generation of Cdo KO mouse. A, Cas9n target scheme in the first and the third exons of Cdo gene. The blue lines indicate sgRNAs’ spacer and the red lines indicate PAM (protospacer adjacent motif). B, Sanger sequencing of the target region in F0 mice. The Cdo KO mice get a miss of 7489 nucleotides. C, Genotype identification results using PCR. The 325 bp strand indicates wild type and the 245 bp strand indicates mutant type. D, IHC staining of CDO in WT and Cdo KO mouse uterus. Bars, 50 μm. E, RT-qPCR analysis of Cdo mRNA levels in WT and Cdo KO mouse uteri. F, Representative patterns of WB analysis of CDO expressions. G-I, Taurine levels in WT and Cdo KO mice liver, serum and uterus detected by HPLC. Data are the Mean ± SEM. (n ≥ 3). Different letters above columns indicate significant differences (P < 0.05). GE: glandular epithelium. LE: luminal epithelium. Sm: smooth muscle cells. St: stromal cells. Scale bars: 50 μm

Fig. 3

CDO deletion causes impairment of embryo implantation and severe subfertility. A and B, Serum (A) and uterus (B) taurine concentrations of pregnancy Cdo KO and WT females from d 1 to d 8, assayed by HPLC (n = 3). C, Pregnancy rates of WT and KO females. The number within brackets indicate females with pups over total number of plug-positive females. D, Average litter sizes of WT and Cdo KO females (n = 7). E, Representative images of d 5 pregnant uteri from WT and Cdo KO females. Bars, 1 cm. F, Blastocysts flushed from Cdo KO uterus. Bar, 50 μm. G, Average numbers of implanted embryos represented by implantation sites (n = 5). H, Representative patterns of d 5 WT receptive mice uteri transferred with WT and Cdo KO embryos. I, d 5 KO receptive mice uteri transferred with WT embryos. Arrowheads indicate the location of blastocysts. Bars, 1 cm. K, IS numbers of different treated mice uteri (n = 3). Data are presented as Mean ± SEM. Different letters above columns indicate significant differences (P < 0.05)

Fig. 4

Effects of Cdo deletion on PR and ER signaling transduction and the cell proliferation in the uteri of d 4 pregnancy mice. A, RT-qPCR analysis of relative Pgr mRNA expression levels. B and C, WB detection and analysis of PR protein expression. D, RT-qPCR analysis of relative Esr1 mRNA levels. E and F, WB detection and analysis of ERα protein expression levels. G, IHC staining of PR and ERα. Scale bars: 50 μm. H, Relative mRNA expressions of Ihh, Areg and Hand2. I, Relative mRNA levels of Ltf and Muc1. J, IF staining of Hand2 and Muc1. Scale bars: 50 μm. K, Ki67 (proliferative cell marker) IHC staining. Scale bars: 50 μm. GE: glandular epithelium. LE: luminal epithelium. St: stroma. Data are presented as Mean ± SEM, n ≥ 4. Different letters represent significant differences (P < 0.05)

Fig. 5

The relations of P₄-PR and E₂-ERα signaling to uterine CDO expression. A and B, Relative Cdo mRNA levels in OVX mouse uteri after 0 (control), 2, 6 and 12 h treatments with 100 ng E₂ (A) and 2 mg P₄ (B). C, CDO IHC staining in OVX mouse uteri after 0 (control) and 12 h 100 ng E₂ or 2 mg P₄ treatments. Scale bars: 50 μm. D, E and F, Relative Cdo mRNA (D) and protein levels (E and F) in OVX mouse uteri treated with E₂ and E₂ inhibitor ICI182780 (ICI). G, H and I, Relative Cdo mRNA (G) and protein levels (H and I) in OVX mouse uteri treated with 2 mg P₄ and PR inhibitor RU486. Data are presented as Mean ± SEM. Different letters represent significant differences (P < 0.05, n = 3)

Fig. 6

Taurine supplement increases the taurine levels in serum and uteri, and partly rescues the defects of embryo implantation in the CDO KO mice. A, Serum taurine concentration. B, Taurine concentration in uterus tissues. C, Representative images of implantation sites. Bars: 1 cm. WT: wide type female mice. KO: Cdo KO female mice. KO + taurine: Cdo KO female mice supplied with taurine. n = 3 for each genotype. D, Blastocysts flushed out from Cdo KO uterus. Bar, 50 μm. E, Numbers of implantation sites. Data are presented as Mean ± SEM. Different letters represent significant differences (P < 0.05, n ≥ 3)