Steroid hormones and human choriogonadotropin influence the distribution of alpha6-integrin and desmoplakin 1 in gland-like endometrial epithelial spheroids

Abstract

In human glandular endometrial epithelial cells, desmosomal and adherens junction proteins have been shown to extend from a subapically restricted lateral position to the entire lateral membrane during the implantation window of the menstrual cycle. Similarly, a menstrual cycle stage-dependent redistribution of the extracellular matrix adhesion protein α6-integrin has been reported. These changes are believed to be important for endometrial receptiveness and successful embryo implantation. To prove the hypothesis that steroid hormones and human choriogonadotropin can induce the redistribution of these adhesion molecules, we used the human endometrial cell line Ishikawa in a 3D culture system. Gland-like spheroids were grown in reconstituted basement membrane (Matrigel™). The lumen-bearing spheroids were treated for 2 or 4 days with ovarian steroids or human choriogonadotropin and then assessed by immunofluorescence microscopy. In addition, human endometrial biopsies were obtained from patients, who were in therapy for assisted reproductive technology, and were examined in parallel. Lateral redistribution of the desmosomal plaque protein desmoplakin 1 was observed in the spheroids treated either with progesterone, medroxyprogesterone acetate or human choriogonadotropin. Furthermore, the extracellular matrix adhesion protein α6-integrin showed an increased lateral membrane localization upon gestagen stimulation in the 3D culture system. The results of this study demonstrate that the 3D endometrial Ishikawa cell culture might be suited as an experimental model system to prove the effect of hormonal changes like those occurring during the window of implantation.

Keywords: 3D cell culture system; Cell adhesion; Endometrial receptivity; Epithelial polarity; Human endometrium; Ishikawa cell line.

Fig. 1

Ishikawa spheroids in cell culture and morphological confirmation of cell polarity. a–c show the development of Ishikawa spheroids in cell culture from day 0 to day 6. Microscopic phase contrast images were inverted. Scale bars: 150 µm. d shows a representative polarized Ishikawa spheroid on day 6. Nuclei are stained with Hoechst 33,342 (blue), tight junctions with anti-ZO-1 antibody (green). Scale bar: 30 μm

Fig. 2

Influence of ovarian steroid hormones and hCG on Ishikawa spheroid proliferation. Images show Ishikawa spheroids stained for the proliferation marker Ki-67 after 4 days of stimulation with estradiol/E2 (b/b’), progesterone/P4 (c/c’), medroxyprogesterone acetate/MPA (d/d’) or human choriogonadotropin/hCG (e/e’). a/a’ show the control without hormones. Arrows highlight the Ki-67 positive nuclei. Grey-scale pictures for Ki-67 in (a–e) and combination with nuclear staining (Hoechst; blue) in (a’–e’). Scale bar: 20 µm

Fig. 3

Influence of ovarian steroid hormones and hCG on localization of desmoplakin 1. Images show Ishikawa spheroids after 4 days of stimulation with estradiol/E2 (b/b’), progesterone/P4 (c/c’), medroxyprogesterone acetate/MPA (d/d’) or human choriogonadotropin/hCG (e/e’). a/a’ show the control without hormones. Arrowhead highlights subapical accumulation of Dsp-1 expression (b). Arrows highlight Dsp-1 redistribution to the basolateral membrane (c and d). Grey-scale pictures for Dsp-1 (a–e) or in green combined with tight junctional staining (ZO-1, magenta) and Hoechst (blue) in (a’–e’). Scale bar: 20 µm

Fig. 4

Influence of ovarian steroid hormones and hCG on localization of α6-integrin. Images show Ishikawa spheroids after 2 days of stimulation with E2/estradiol (b/b’), P4/progesterone (c/c’), medroxyprogesterone acetate/MPA (d/d’) or human choriogonadotropin/hCG (e/e’). a/a’ depict the control without hormones. Arrowheads highlight the basal localization of α6-integrin in (a, b). Arrows highlight the lateralization of the α6-integrin signal in (c–e). Grey-scale pictures for α6-integrin (a–e) or in green combined with tight junctional staining (ZO-1, magenta) and Hoechst (blue) in (a’–e’). Scale bar: 20 µm

Fig. 5

Influence of ovarian steroid hormones and hCG on localization of β4-integrin. Images show Ishikawa spheroids after 2 days of stimulation with E2/estradiol (b/b’), P4/progesterone (c/c’), medroxyprogesterone acetate/MPA (d/d’) or human choriogonadotropin/hCG (e/e’). a/a’ represent the control without any hormones. Grey-scale pictures for β4-integrin (a–e) or in green combined with tight junctional staining (ZO-1, magenta) and Hoechst (blue) in (a’–e’). Scale bar: 20 µm

Fig. 6

α6-Integrin staining during days 17–21 of the menstrual cycle of ART patients. Images show the expression pattern of α6-integrin during the menstrual cycle between days 17 and 21 in biopsies of women who underwent an ART cycle. a (menstrual cycle days 17–19), b/b’ (days 18–19) and c (day 19) represent non-receptive biopsies before the WOI. d/d’ (days 20–21) represent a potential receptive state during the WOI. Arrowheads depict a basal localization of α6-integrin (b’), while arrows highlight more laterally localized α6-integrin positive membrane staining (d’). Chromogen: AEC. Nuclear staining: Hematoxylin. Scale bar: 50 µm