Effects of steroid hormones on differentiated glandular epithelial and stromal cells in a three dimensional cell culture model of the canine endometrium

Abstract

Background: Oestrogens and progesterone have a significant impact on the endometrium during the canine oestrous cycle. Their receptors mediate plasma steroid hormone levels and are expressed in several endometrial cell types. Altered steroid receptor expression patterns are involved in serious uterine diseases; however the mechanisms of hormone action during pathogenesis in these tissues remain unclear. The development of 3D culture systems of canine endometrial cells provides an opportunity for the effects of steroid hormones to be quantitatively assessed in a more in vivo-like setting. The present study aimed to determine the effects of the steroid hormones 17β-estradiol (E) and progesterone (P) on the expression of the oestrogen and progesterone receptors (ER and PR), and on proliferative activity, in a 3D co-culture system of canine uterine origin, comprising differentiated endometrial glands, and stromal cells (SCs).

Results: Morphology, differentiation, and apical-basolateral polarity of cultured glandular epithelial cells (GECs) were comparable to those in native uterine tissue as assessed by immunohistochemistry using differentiation markers (β-catenin, laminin), lectin histochemistry, and transmission electron microscopy. Supplementation of our 3D-culture system with E (at 15, 30 and 100 pg/mL) resulted in constant levels of ER expression in GECs, but reduced expression levels in SCs. PR expression was reduced in both GECs and SCs following treatment with E. 3 ng/mL P resulted in increased ER expression in GECs, but a decrease in SCs. PR expression in GECs increased in all P-treated groups, whereas PRs in SCs decreased with the lowest and highest doses, but increased with the middle dose of treatment. Proliferative activity, assessed by Ki67 staining, remained below 1% in all assays and cell types.

Conclusions: The present study demonstrates the applicability of our 3D organotypic canine endometrium-derived culture system for cellular-level studies. 3D cultures represent near-physiological systems allowing reproducible quantitative experimentation, thus reducing the need to experiment on living animals. The results of the present investigation emphasize the importance of co-culture of the uterine glands with SCs, as it was shown that the responsiveness of the different cell types to steroid hormones were divergent in the 3D cell culture model.

Figure 1

Estrogen and progesterone receptor expression in 3D cultured and native canine endometrial cells. Percentage of oestrogen receptor ER (A) and progesterone receptor PR (B) positive glandular epithelial cells (GECs) and stromal cells (SCs) in the canine uterine tissue of origin (native) as well as after 24 and 48 hr of cell culture in standard medium and hormone free medium. Statistical significances (p < 0.05) are indicated as letters (A-E for GECs, a-e for SCs) above the related columns.

Figure 2

Proliferative activity in 3D cultured and native canine endometrial cells. Percentage of anti-Ki67 positive glandular epithelial cells (GECs) and stromal cells (SCs) indicating proliferative activity in the canine uterine tissue of origin (native) as well as after 24 and 48 hr of cell culture in standard medium and hormone free medium. Statistical significances (p < 0.05) are indicated as letters (A for GECs, a-d for SCs) above the related columns.

Figure 3

A-H: 3D cell culture of canine endometrial glands with surrounding stromal cells cultured for 48 hr in standard medium (M199 + 10% FCS). (A) H&E-stained histological section of 3D-cultured canine endometrial glands (asterisk) and surrounding stromal cells (arrows) featuring physiological morphology with luminal formation and secretory activity(s). (B) Immunohistochemical characterization of stromal cells by anti-vimentin staining (green); glandular epithelial cells lack any staining reaction,counterstained with DAPI (blue). (C) Glandular epithelial cells were identified by immunohistochemical anti-cytokeratin staining (green), counterstained with DAPI (blue). (D) Demonstration of the intact basement membrane (green) surrounding the glandular structures with anti-laminin immunohistochemical staining, counterstained with DAPI (blue). (E) Transmission electron micrograph of 3D-cultured canine endometrial epithelial cells to demonstrate apical polarity (mv, microvilli)and intact junctional complex (arrows) featuring tight junctions, zonulaadherens and desmosomes, characteristic for differentiated glandular epithelial cells. (F) Basolateral polarization of the 3D-cultured glandular epithelial cells, ensuring correct luminal formation and function,demonstrated with anti-b-catenin immunohistochemical staining (green),counterstained with DAPI (blue nuclei). (G) Combined demonstration of apical polarization, with histochemical WGA lectin binding to the glycocalyx (red), and basolateral polarisation demonstrated with immunohistochemical anti-b-catenin staining (green) on 3D-cultured canine endometrial glands(nuclei demonstrated by DAPI counterstaining, blue). (H)The same combined double-staining method (compare with panel G)of the canine endometrium in vivo, featuring apical and basolateral polarization of the endometrial glands demonstrated histochemically byapical WGA lectin binding to the glycocalyx (red), and immunohistochemically bybasolateralanti-b-catenin staining (green),comparable to the 3D in vitro cultured glandular structures (panel G); DAPI counterstaining (blue). Scale bars A-D, F-H25 µm; E 1 µm.

Figure 4

Immunohistochemical demonstration of oestrogen and progesterone receptorexpression and proliferative activityin 3D cell cultured canine endometrial cells compared to the native tissue. Oestrogen (A) and progesterone (B) receptor expression of glandular epithelial and stromal cells cultured for 48 hr in standard mediumcompared to the native tissue (Coestrogen receptor, Dprogesterone receptor) indicated by immunohistochemical nuclear staining. Proliferative activity in 3D cell-cultured canine glandular epithelial cells (E) (arrows) compared to the in vivo situation (F) of uterine glands and surrounding stromal cells by means of anti-Ki67 immunohistochemical staining. Scale bars A-F, 25µm.

Figure 5

Alterations of oestrogen receptor expression in 3D cultured canine endometrial cells due to 17β-estradiol and progesterone supplementation. Percentage of oestrogen receptor ER positive glandular epithelial cells (GECs) and stromal cells (SCs) after 24 and 48 hr of 3D cell culture with (A) 17β-estradiol supplementation (E) and (B) progesterone (P) in the respective three different concentrations. Due to the high number of statistical significant results concerning the comparison with the control group (hormone free) statistical non-significant (p > 0.05) results are indicated as italic letters (A-B for GECs, a-e for SCs) above the related columns.

Figure 6

Alterations of progesterone receptor expression in 3D cultured canine endometrial cells due to 17β-estradiol and progesterone supplementation. Percentage of progesterone receptor PR positive glandular epithelial cells (GECs) and stromal cells (SCs) after 24 and 48 hr of 3D cell culture with (A) 17β-estradiol (E) and (B) progesterone (P) supplementation in the respective three different concentrations. Statistical significances (p < 0.05) are indicated as letters (A-E for GECs, a-e for SCs) above the related columns for comparison with the control group (hormone free medium).

Figure 7

Alterations of proliferative activity in 3D cultured canine endometrial cells due to 17β-estradiol and progesterone supplementation. Percentage of anti-Ki67 positive glandular epithelial cells (GECs) and stromal cells (SCs) indicating proliferative activity after 24 and 48 hr of 3D cell culture with (A) 17β-estradiol (E) and (B) progesterone (P) supplementation in the respective three different concentrations. Statistical significances (p < 0.05) are indicated as letters (A-E for GECs, a-e for SCs) above the related columns.