Targeted depletion of uterine glandular Foxa2 induces embryonic diapause in mice

Abstract

Embryonic diapause is a reproductive strategy in which embryo development and growth is temporarily arrested within the uterus to ensure the survival of neonates and mothers during unfavorable conditions. Pregnancy is reinitiated when conditions become favorable for neonatal survival. The mechanism of how the uterus enters diapause in various species remains unclear. Mice with uterine depletion of Foxa2, a transcription factor, are infertile. In this study, we show that dormant blastocysts are recovered from these mice on day 8 of pregnancy with persistent expression of uterine Msx1, a gene critical to maintaining the uterine quiescent state, suggesting that these mice enter embryonic diapause. Leukemia inhibitory factor (LIF) can resume implantation in these mice. Although estrogen is critical for implantation in progesterone-primed uterus, our current model reveals that FOXA2-independent estrogenic effects are detrimental to sustaining uterine quiescence. Interestingly, progesterone and anti-estrogen can prolong uterine quiescence in the absence of FOXA2. Although we find that Msx1 expression persists in the uterus deficient in Foxa2, the complex relationship of FOXA2 with Msx genes and estrogen receptors remains to be explored.

Keywords: Foxa2; delayed pregnancy; developmental biology; diapause; dormant; estrogen; mouse; uterus.

Figure 1.. Uterine conditional depletion of Foxa2 causes loss of leukemia inhibitory factor (LIF) secretion and female infertility.

(a) Percentage of mice with pups per total mated mice.Numbers on bars indicate mice with pups over total number of mated mice. *p < 0.05 by Student's t-test. (b) Cre recombinase activity starts differently in Foxa2^f/fLtf^Cre+ and Foxa2^f/fPgr^Cre+ females. (c) Immunostaining of FOXA2 in the uteri on postnatal day 30 and day 4 pregnancy of Foxa2^f/f, Foxa2^f/fLtf^Cre+, and Foxa2^f/fPgr^Cre+females. Epithelial cells are outlined by cytokeratin 8 (CK8) staining. Scale bars: 100 μm. (d) In situ hybridization of Lif in day 4 of pregnant uteri from Foxa2^f/f, Foxa2^f/fLtf^Cre+, and Foxa2^f/fPgr^Cre+ females. White arrows point to uterine glands. Scale bar: 200 μm. Le, luminal epithelia; Ge, glandular epithelia; S, stroma.

Figure 1—figure supplement 1.. Quantitative PCR of Foxa2 in Foxa2^f/f, Foxa2^f/fLtf^Cre+, and Foxa2^f/fPgr^Cre+uteri tissues on day 4 of pregnancy.

RNA levels of Foxa2 are normalized against Rpl7. Values are mean ± SEM; *p < 0.05 by Student's t-test.

Figure 2.. Dormant blastocysts are present in Foxa2^f/fLtf^Cre+ and Foxa2^f/fPgr^Cre+uteri on day 8 pregnancy.

(a) Representative photographs of day 8 pregnant uteri from Foxa2^f/f, Foxa2^f/fLtf^Cre+, and Foxa2^f/fPgr^Cre+ females. An ovariectomy-induced delayed model of Foxa2^f/f mice served as a prototypical control in maintaining dormant blastocysts. Scale bar: 10 mm. Ov, ovary. (b) Blastocysts recovered from Foxa2^f/fLtf^Cre+ and Foxa2^f/fPgr^Cre+uteri on day 8. Blastocysts retrieved from ovariectomized Foxa2^f/f mice in delay served as controls. ICM, inner cell mass. Scale bar: 100 µm. Quantification of blastocyst numbers were shown in panel c. Numbers on bars indicate numbers of animals examined. Values are expressed as mean + SEM. (d) Representative photographs of nuclear staining of dormant blastocysts recovered from mice without implantation sites. Scale bar: 50 µm. (e) Average cell numbers per blastocyst. Numbers of embryos examined are shown on bars. Values are expressed as mean + SEM.

Figure 2—figure supplement 1.. A schematic outline of sample collection from ovariectomy-induced delayed model.

P₄, progesterone (2 mg) administration.

Figure 3.. Foxa2^f/fLtf^Cre+ and Foxa2^f/fPgr^Cre+ females maintain uterine quiescence when examined on day 8 of pregnancy.

(a) Fluorescence in situ hybridization of Msx1 in days 4 and 8 pregnant uteri from Foxa2^f/f, Foxa2^f/fLtf^Cre+, and Foxa2^f/fPgr^Cre+ females. Scale bar: 200 μm. (b) Epidermal growth factor receptor (EGFR) immunostaining on dormant blastocysts. Positive signals were observed in activated blastocysts recovered from Foxa2^f/f uteri on day 4 of pregnancy. Scale bar: 25 μm. (c) Ki67 immunostaining on dormant blastocysts collected from day 8 Foxa2^f/fLtf^Cre+ and Foxa2^f/fPgr^Cre+ females. Scale bar: 25 μm. ICM, inner cell mass.

Figure 4.. Pregnancy in Foxa2^f/fLtf^Cre+ and Foxa2^f/fPgr^Cre+ females with leukemia inhibitory factor (LIF) treatment.

(a) Schematic outline of sample collection. LIF, LIF administration (20 μg). (b) Representative photograph of uteri from Foxa2^f/fLtf^Cre+ and Foxa2^f/fPgr^Cre+ females (days 6 and 10) with LIF treatment. Foxa2^f/f uteri on day 6 serve as control. Scale bar: 10 mm. Histological pictures of implantation sites in panel b were presented in panel c. Arrowheads point to embryos. Em, embryo. Scale bar: 500 μm. (d) Average number of implantation sites in Foxa2^f/fLtf^Cre+ and Foxa2^f/fPgr^Cre+ mice treated with LIF (20 μg) on day 4 or 8 of pregnancy. Numbers and percentage on bars indicate mice with implantation sites over total number of mated mice. (e) Litter sizes of Foxa2^f/fLtf^Cre+ and Foxa2^f/fPgr^Cre+mice treated with LIF (20 μg) or vehicle on days 4 or 8 of pregnancy. Numbers and percentage on bars indicate mice with pups over total number of mated mice. *p<0.05. (f) 3D visualization of day 6 implantation sites in Foxa2^f/f, Foxa2^f/fLtf^Cre+, and Foxa2^f/fPgr^Cre+females. Images of E-cadherin immunostaining, segmented, and 3D rendered images of day 6 implantation sites in each genotype show defects in Foxa2^f/fPgr^Cre+ females with a LIF injection on day 4 of pregnancy. Scale bar: 200 μm.

Figure 5.. Counterbalance of estrogenic effects by P₄ improves maintenance of diapause in Foxa2^f/fLtf^Cre+ and Foxa2^f/fPgr^Cre+ mice.

(a) Scheme of P₄ treatment. Foxa2^f/fLtf^Cre+ and Foxa2^f/fPgr^Cre+ mice were treated with leukemia inhibitory factor (LIF) (20 μg) on day 8. Pregnancy was evaluated on day 10, 2 days after LIF administration. (b) Representative photographs of uteri in Foxa2^f/fLtf^Cre+ and Foxa2^f/fPgr^Cre+ mice with P₄ supplement 2 days after LIF administration. Scale bar: 10 mm. Histological pictures of implantation sites in panel b were presented in panel c. Scale bar: 500 μm. (d) Average number of implantation sites in Foxa2^f/fLtf^Cre+ and Foxa2^f/fPgr^Cre+ mice with P₄ supplement. Numbers and percentage on bars indicate mice with implantation sites over total number of mated mice. (e) Litter sizes of Foxa2^f/fLtf^Cre+ and Foxa2^f/fPgr^Cre+mice with P₄ supplement. Numbers and percentage on bars indicate mice with pups over total number of mated mice.

Figure 5—figure supplement 1.. Serums levels of P₄ and E₂ in Foxa2^f/f, Foxa2^f/fLtf^Cre+, and Foxa2^f/fPgr^Cre+females on days 4 and 8 of pregnancy.

Numbers of animals examined are presented on bars.

Figure 5—figure supplement 2.. Fluorescence in situ hybridization of Bmp2.

Implantation sites of Foxa2^f/f females were collected on day 6 of pregnancy. Implantation sites of Foxa2^f/fLtf^Cre+ females with different treatments were collected 2 days after leukemia inhibitory factor (LIF) injections. Scale bar: 500 μm. Le, luminal epithelia; Ge, glandular epithelia; S, stroma. Asterisks represent the locations of embryos.

Figure 6.. Neutralization of estrogenic effects by ICI improves diapause in Foxa2^f/fLtf^Cre+ and Foxa2^f/fPgr^Cre+ mice.

(a) Scheme of ICI treatment. Foxa2^f/fLtf^Cre+ and Foxa2^f/fPgr^Cre+ mice were treated with leukemia inhibitory factor (LIF) (20 μg) on day 8. Pregnancy was evaluated on day 10, 2 days after LIF administration. (b) Representative photographs of uteri in Foxa2^f/fLtf^Cre+ and Foxa2^f/fPgr^Cre+ mice with ICI treatment 2 days after LIF administration. Foxa2^f/f mice have normal day 10 implantation sites, suggesting implantation occurs under 25 μg ICI treatment in Foxa2^f/f mice. Scale bar: 10 mm. Histological pictures of implantation sites in panel b were presented in panel c. Scale bar: 500 μm. (d) Average number of implantation sites in Foxa2^f/fLtf^Cre+ and Foxa2^f/fPgr^Cre+ mice with ICI treatment. Numbers and percentage on bars indicate mice with implantation sites over total number of mated mice. (e) Litter sizes of Foxa2^f/fLtf^Cre+ and Foxa2^f/fPgr^Cre+mice with ICI treatment. Numbers and percentage on bars indicate mice with pups over total number of mated mice.

Figure 7.. A representative scheme depicting roles of E₂, FOXA2 (forkhead box protein A2), and leukemia inhibitory factor (LIF) in mouse diapause.

In natural pregnancy, a uterus enters a prereceptive phase before E₂ secretion in the morning of day 4. In the presence of FOXA2, LIF is induced by E₂ which renders the uterus into a receptive phase. In the event of implantation failure, the uterus becomes refractory on day 5 of pregnancy. The transition to the receptive phase is stopped if the E₂ secretion on day 4 is prevented by ovariectomy. The uterus remains quiescent as long as P₄ is supplemented; the embryonic development is arrested. A similar extension of prereceptive phase can be achieved by deleting FOXA2 or LIF. But the uterine quiescence is gradually compromised indicating that a LIF-independent estrogenic effect is detrimental to uterine quiescence.