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Abstract
BackgroundOestrogens 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).ResultsMorphology, 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.ConclusionsThe 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.
Highlights
Oestrogens and progesterone have a significant impact on the endometrium during the canine oestrous cycle
Histological and immunohistochemical evaluation of the native endometrial tissue The canine uterine tissues used in this study for the Three dimensional (3D) cell culture systems were classified as late metestrous (n = 3) and anestrous (n = 6) by means of histological and immunohistochemical assessment, including oestrogen and progesterone receptor expression (Figure 1) and proliferative activity (Figure 2)
Vimentin was used as a mesenchymal marker to identify stromal cells in the native tissue and the cultured endometrial structures, and to distinguish them from epithelial cells, which were used as an internal negative control (Figure 3B)
Summary
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. 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). Oestrogens stimulate growth, vascularity and edema of the endometrium as well as proliferation of the glandular epithelia, whereas progesterone promotes proliferation of SCs and secretory activity of the endometrial glands [3,11,12,16] These results underline the distinct responsiveness of the different endometrial cell populations to the respective steroid hormones. In veterinary medicine only a few 3D cell cultures have been established for experimental approaches [23,24,25,26], and a cell culture system of complete endometrial glands with their specific environment has not existed until now
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