Absence of CD9 reduces endometrial VEGF secretion and impairs uterine repair after parturition

Abstract

In mammals, uterine epithelium is remodeled cyclically throughout adult life for pregnancy. Despite the expression of CD9 in the uterine epithelium, its role in maternal reproduction is unclear. Here, we addressed this issue by examining uterine secretions collected from patients undergoing fertility treatment and fertilization-competent Cd9(-/-) mice expressing CD9-GFP in their eggs (Cd9(-/-)TG). CD9 in uterine secretions was observed as extracellular matrix-like feature, and its amount of the secretions associated with repeated pregnancy failures. We also found that the litter size of Cd9(-/-)TG female mice was significantly reduced after their first birth. Severely delayed re-epithelialization of the endometrium was then occurred. Concomitantly, vascular endothelial growth factor (VEGF) was remarkably reduced in the uterine secretions of Cd9(-/-)TG female mice. These results provide the first evidence that CD9-mediated VEGF secretion plays a role in re-epithelialization of the uterus.

Figure 1. Detection of extracellular CD9 in uterine secretions.

(a), Experimental schematic for immunostaining the endometrial epithelium with an anti-CD9 mAb. After estrus-stage mice were identified by a vaginal smear test, the uterus was isolated and examined by immunohistochemical analysis. (b), As depicted from the left, the endometrium includes epithelial layers (EL), a basement membrane (BM), and stromal layers (SL). The endometrial epithelium was incubated with the anti-CD9 mAb and then an Alexa Fluor 488-labeled secondary antibody. UC, uterine cavity. Arrowheads, CD9-reduced apical regions. Scale bar, 20 μm. (c), Experimental schematic for immunoblotting of uterine secretions at each stage of the estrous cycle, and immuno-electron microscopic analysis of uterine secretions at the estrus stage. (d), Immunoblotting of uterine secretions collected from each stage of the estrous cycle. (e), Immuno-electron microscopic images of uterine secretions at the estrus stage. (f) and (g), Enlarged images of boxes in (e). Scale bars, 100 nm. (h), Schematic of the two types of CD9.

Figure 2. Immunoblotting of uterine secretions collected from patients undergoing fertility treatment.

(a), Uterine secretions were collected from the uterine cavity and examined by immunoblotting. Human samples were immunoblotted with anti-CD9 and anti-β-actin-mAbs, and stained with Coomassie brilliant blue for detection of albumin. The number of samples is shown. (b), The presence of CD9 in uterine flushing in recurrent implantation failure (RIF) patients (n = 115) and the control patients (n = 56). A significant difference among the mean values with asterisk was observed (P < 0.05). (c), The association of the CD9 presence with a history of dilation and curettage (D&C) in the RIF patients (D&C[+], n = 22; D&C[−], n = 49). A significant difference among the mean values with asterisk was observed (P < 0.05). (d), The association of the CD9 presence with endometrial thickness in the RIF patients. When endometrium width measured by vaginal ultrasound was less than 8.5 mm at the mid luteal phase, the endometrium was categorized as thin endometrium (n = 22). When the width was 8.5 mm or more than that, the endometrium was categorized normal-width endometrium (n = 69). A significant difference among the mean values with asterisk was observed (P < 0.05). (e), The association of the CD9 presence with prognosis (miscarriage rates) in the RIF patients. The RIF patients were classified into four groups: those with CD9 (+) and normal endometrium (n = 7), those with CD9 (+) and thin endometrium (n = 6), those with CD9 (−) and normal endometrium (n = 20), and those with CD9 (−) and thin endometrium (n = 9). A significant difference among the mean values that have different superscripts was observed (P < 0.05). The endometrial thickness was measured by ultrasound and magnetic resonance imaging at the site indicated with the line in (a), and double-headed arrows in (f). (f), Schematic explanation of a hypothetical relationship between the absence of CD9 and endometrial thinning in the RIF patients.

Figure 3. Reduction of endometrial repair in Cd9^−/−TG mice.

(a), Experimental schematic for examining the litter size of Cd9^−/−TG female mice. (b), Age-independent reduction of the litter size of Cd9^−/−TG mice. (c), Reduction of the litter size dependent on parturition in Cd9^−/−TG mice. Parenthesis indicates the number of examined mice. Values are the mean ± SEM. (d) and (e), Histochemical analysis of endometrial repair in Cd9^−/−TG and Cd9^+/+ mice after parturition (the day of parturition = day 0). (f), Experimental schematic for in vitro wound healing assays of the endometrial epithelium of Cd9^−/−TG mice. After the uterus was isolated from Cd9^−/−TG mice at the estrus stage, collagenase was injected to the intrauterine cavity to collect the epithelial cells. (g), Wounded epithelial cells after scratching the monolayer with a pipette tip. Scale bars, 150 μm. (h), Graph of the wound width of wounded epithelial cells. Values are the mean ± SEM.

Figure 4. Decrease of VEGF in uterine secretions of Cd9^−/−TG mice.

(a), Comparison of the amount of VEGF in uterine secretions of Cd9^−/−TG mice at the estrus stage using a multiplex suspension array. (b), Immunoblotting of uterine secretions at the estrus stage in Cd9^−/−TG and Cd9^+/+ mice. (c), Immunohistochemical observation of the endometrial epithelium in Cd9^−/−TG and Cd9^+/+ mice. White arrowheads indicate the uterine cavity. Scale bars, 20 μm. (d), Electron microscopic images of endometrial epithelial cells in Cd9^−/−TG and Cd9^+/+ mice. Left panels, endometrial epithelial cells of Cd9^−/−TG and Cd9^+/+ mice at the estrus stage. Middle panels, enlarged images of the boxes in the left panels. Right panels, enlarged images of the boxes in the middle images. Hollow arrowheads indicate the secreted materials in Cd9^−/−TG mice. Arrowheads indicate the outer membrane consisting of lipid bilayers in Cd9^+/+ mice. Scale bars, 500 nm. (e), Length of microvilli. Left graph, endometrial epithelial cells at the estrus stage. Right graph, endometrial epithelial cells at the metestrus stage. Values are the mean ± SEM.

Figure 5. Endometrial epithelium repair by VEGF treatment in Cd9^−/−TG mice.

(a), Experimental schematic for treatment of the endometrial epithelium of Cd9^−/−TG mice with VEGF. (b), Ventral view of the horns of a uterus isolated from a Cd9^−/−TG mouse at 1 week after VEGF-linked microparticles were injected into the uterine cavity. Scale bar, 5 mm. (c), Endometrial epithelium treated with VEGF or BSA as a control. Lower panels, enlarged images of boxes in the upper panels. Double-headed arrows indicate the endometrial thickness. Hollow arrowheads indicate the re-epithelialized layers. The dotted circle indicates the fused site of endometrial stromal layers without epithelium with a mixture of microparticles and immune cells. Scale bars, 300 μm in upper panels and 100 μm in lower panels. (d), The rate of re-epithelialized sites in the Cd9^−/−TG endometrium treated with VEGF or BSA. (e), Thickness of the endometrium treated with VEGF or BSA. Values are the mean ± SE. (f), Schematic model of re-epithelialization of the endometrium treated with VEGF. The endometrial thickness was measured at the site indicated with double-headed arrows. SL, stromal layers; Myo, myometrium. (g), Schematic model of CD9 and VEGF secretion from epithelial cells. CD9-mediated VEGF release contributes to endometrial re-epithelialization.