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. 2020 Nov 19;5(22):e135775. doi: 10.1172/jci.insight.135775

Figure 9. Trophectoderm overexpression of HAS-2 resulted in early embryonic lethality and attenuated decidual angiogenesis.

Figure 9

(A) H&E staining of implantation sites revealed embryo resorption upon HAS-2 OEx (n = 4 dams). (B) Remnants of embryo overexpressing HAS-2 by trophoblast cells (CK-7) as opposed to decidual cells expressing HAS-2 in the embryonic niche in the control (n = 3 dams). (C) Profound embryonic cell death indicated by TUNEL staining was detected upon HAS-2 OEx, (n = 3 dams). (D) Similar pattern of MAC-2+ macrophages, confined to the mesometrial pole, away from the embryonic niche, observed in both groups (n = 3 dams). (E) Quantification MAC-2 staining by fluorescent microscopy (1.066-fold change ± 0.18 [control]; 0.41 [overexpression]; n = 5 dams, 7 implantation sites, P = 0.879). (F) Number of embryo implantation site per dam was assessed, at E6.5 by gross morphology inspection, as well as by examination of histological sections (3 ± 0.63 [control]; 2.88 ± 0.61 [overexpression], P = 0.88) (n = 6 dams in control; 8 dams in HAS-2 OEx). (G) Representative image of DBA+ NK cells in E6.5 decidua (n = 3 dams). (H) Representative image of MMP-9 in E6.5 decidua (n = 3 dams). (I) Impaired development of CD34+ newly formed blood vessels was observed in pregnant mice carrying HAS-2 OEx embryos, reflected by confinement of vessels away from the embryonic niche (n = 3 dams). (J) T1 weighted GE-MRI of embryo implantation sites, acquired from pregnant mice at E6.5 30 minutes after administration of biotin-BSA-GdDTPA. Little accumulation of biotin-BSA-GdDTPA was observed in the in dams carrying HAS-2 OEx embryos in comparison with control (white arrows indicate implantation sites; n = 6 dams). (K) Visualization of hyperpermeable blood vessels in the embryonic niche was achieved by staining of biotin-BSA-GdDTPA, 40 minutes after i.v. injection. Hyperpermeable vessels were not detected at HAS-2 OEx decidua in contrast to those detected in the control group (n = 3 dams). (L–N) These observations were consistent with DCE-MRI of biotin-BSA-GdDTPA (L), fBV (M) (0.041 ± 0.005; 0.023 ± 0.002; P = 0.004) and PS (N) (0.0017 ± 0.0003; 0.0009 ± 0.00031; P = 0.2) (control: 5 dams 9 implantation sites; HAS-2 OEx: 6 dams 19 implantation sites). The statistical analysis applied was Student’s t test (E, F, M, and N).