Entosis allows timely elimination of the luminal epithelial barrier for embryo implantation

Abstract

During implantation, uterine luminal epithelial (LE) cells first interact with the blastocyst trophectoderm. Within 30 hr after the initiation of attachment, LE cells surrounding the blastocyst in the implantation chamber (crypt) disappear, allowing trophoblast cells to make direct physical contact with the underneath stroma for successful implantation. The mechanism for the extraction of LE cells was thought to be mediated by apoptosis. Here, we show that LE cells in direct contact with the blastocyst are endocytosed by trophoblast cells by adopting the nonapoptotic cell-in-cell invasion process (entosis) in the absence of caspase 3 activation. Our in vivo observations were reinforced by the results of co-culture experiments with primary uterine epithelial cells with trophoblast stem cells or blastocysts showing internalization of epithelial cells by trophoblasts. We have identified entosis as a mechanism to remove LE cells by trophoblast cells in implantation, conferring a role for entosis in an important physiological process.

Figure 1. Uterine LE Surrounding the Blastocyst Is Breached on Day 5 Evening

(A) H&E-stained sections of implantation sites on days 5 (D5) and 6 (D6). Arrows point to the gaps on the LE. Em, embryo. (B) Immunofluorescence of E-cad and β-catenin in sections of implantation sites on days 5 and 6. Signals for E-cad show LE is removed in the evening of day 5. An area with breached epithelium delineated by a rectangle is presented at a larger magnification. Arrows point to the breaches in the LE. Em, embryo; M, mesometrial pole; AM, antimesometrial pole.

Figure 2. Extraction of LE Cells Surrounding the Embryo Is Independent of Caspase 3 Activation

(A) Immunofluorescence of cleaved caspase 3 and CK8 in sections of implantation sites on days 5 and 6. Signals for activated caspase 3 are absent from the LE in direct contact with the embryo. The area of LE breaching is indicated by arrows. Asterisks depict the position of the embryo. Le, luminal epithelium; S, stroma; M, mesometrial pole; AM, antimesometrial pole. (B) TUNEL and CK8 staining in sections of implantation sites on days 5 and 6. TUNEL-positive cells were absent in the LE prior to and at the time of its breaching (D5, 20:00 hr). Arrowheads point to the TUNEL-positive cells engulfed by trophoblast cells. Very few TUNEL-positive cells were observed in the anti-mesometrial extension of LE not in contact with the blastocyst as outlined by dotted lines on day 5 (24:00 hr); these cells became TUNEL positive on day 6 morning. Scale bars represent 100 μm.

Figure 3. Trophoblast Cells Engulf LE Cells in the Evening of Day 5

(A) Immunofluorescence of CK8 and ZO1 in sections of implantation sites on days 5 and 6. Arrowheads point to the protruding trophoblast cells. Areas containing protruding trophoblast cells outlined by dotted lines are presented at higher magnification. Red arrows show epithelial cells engulfed by the protruding trophoblast cells, which are outlined by dotted circles. Asterisks indicate the position of the embryo. Le, luminal epithelium; S, stroma; M, mesometrial pole; AM, antimesometrial pole. Scale bar represents 100 μm. (B) Immunohistochemistry of E-cad in a section of day 5 implantation site at 20:00 hr. The section is counterstained with hematoxylin. E-cad signals depict the borders of the trophectoderm and LE. At the LE (Le) gaps, trophoblast cells are in direct contact with the basal membrane of LE cells. Blue arrows point to the nuclei of trophoblast cells, while red arrows point to cells engulfed by trophoblast cells. Scale bar represents 50 μm. (C) Immunofluorescence of LAMP1 and CK8 in sections of a day 5 implantation site at 20:00h. The detached LE cell as indicated by an arrowhead is within a LAMP1-positive endosome, a marker of endosome. Trophoblast (Tr) nuclei are indicated by arrows. Scale bar represents 50 μm. (D) LE cells undergo apoptosis on day 6 of pseudopregnancy after an induction of decidual reaction by intraluminal oil infusion on day 4. Immunofluorescence of E-cad in oil-infused pseudopregnant uterine sections on days 5 and 6. LE cells start to disintegrate on day 6 of pseudopregnancy. O, oil drop; Le, luminal epithelia; S, stroma. (E) Immunofluorescence of cleaved-caspase3 and CK8 in sections of pseudopregnant uteri of day 6 following intraluminal oil infusion. caspase 3 is activated in LE (Le) on day 6 (16:00 hr). Two representative sections from two different mice are shown. Scale bars represent 100 μm.

Figure 4. TSCs and Blastocysts Endocytose Primary Uterine Epithelial Cells In Vitro

(A) TSCs engulf primary epithelial cells. Red arrowhead points toward the nucleus of one TSC (red), whose nucleus is squeezed to a crescent shape by an epithelial cell (green). The green arrowhead points toward the engulfed epithelial cell. The bottom panels show two other engulfed epithelial cells (green) by TSCs (red). Scale bar represents 10 μm. (B) Immunofluorescence of β-catenin in engulfed epithelial cells. Epithelial cells were stained with either red or green tracker as indicated. The boundary of the engulfed epithelial cells and the TSCs is highlighted by β-catenin staining. Scale bar represents 10 μm. (C) Trophoblast cells in a Rosa-tomato reporter blastocyst showing engulfment of primary epithelial cells (green). Green arrowheads point to the nuclei of engulfed epithelial cells. A white arrow points to an enlarged trophoblast cell which had engulfed an epithelial cell. BL, blastocyst; ICM, inner cell mass. Tr, trophectoderm. Scale bar represents 50 μm. (D) A ROCK inhibitor Fasudil impedes the removal of LE cells at 20:00 hr of day 5. The LE barrier begins to disappear in control (vehicle) females, as indicated by the white arrow, whereas the LE encasing the blastocyst remained intact in Fasudil-treated females at 20:00 hr of day 5. Le, luminal epithelium; S, stroma; Em, embryo; M, mesometrial pole; AM, antimesometrial pole. Scale bar represents 100 μm. (E) Percentage of mice with breached luminal epithelia versus total mice examined at 20:00 hr on day 5. All five mice treated with Fasudil had intact LE surrounding embryos, whereas all six control (vehicle-treated) mice had implantation sites (IS) with breached LE.