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In vitro decidualisation of canine uterine stromal cells.

Kautz E, de Carvalho Papa P, Reichler IM, Gram A, Boos A, Kowalewski MP - Reprod. Biol. Endocrinol. (2015)

Bottom Line: A part of this process is decidualisation, comprising morphological and biochemical changes that result in formation of maternal stroma-derived decidual cells.Expression of the PGE2 receptors, PTGER2 and PTGER4, was clearly detectable.An in vitro decidualisation model with canine uterine stromal cells was successfully established, allowing future, more detailed studies to be undertaken on the underlying molecular and endocrine mechanisms of canine decidualisation.

View Article: PubMed Central - PubMed

Affiliation: Institute of Veterinary Anatomy, Vetsuisse Faculty, University of Zurich, Winterthurerstrasse 260, CH-8057, Zurich, Switzerland. ewa.kautz@yahoo.com.

ABSTRACT

Background: The uterine response to the presence of embryos is poorly understood in the domestic dog (Canis familiaris). The intimate embryo-maternal cross-talk, which begins following the hatching of blastocysts and embryo attachment leads to strong structural and functional remodelling of the uterus. A part of this process is decidualisation, comprising morphological and biochemical changes that result in formation of maternal stroma-derived decidual cells. These are an integral part of the canine placenta materna, which together with the maternal vascular endothelium are the only cells of the canine endotheliochorial placenta able to resist trophoblast invasion. These cells are also the only ones within the canine placenta expressing the progesterone receptor (PGR). Understanding the decidualisation process thus appears essential for understanding canine reproductive physiology.

Methods: Here, we investigated the capability of canine uterine stromal cells to decidualise in vitro, thereby serving as a canine model of decidualisation. A dbcAMP-mediated approach was chosen during a time course of 24 - 72 h. Tissue material from six (n = 6) healthy, dioestric bitches was used (approximately 2 weeks after ovulation). Cells were characterized by differential staining, nearly 100 % of which were vimentin-positive. Scanning and transmission electron microscope analyses were applied, and morphological changes were recorded with a live cell imaging microscope. Expression of several decidualisation markers was investigated.

Results: The in vitro cultured stromal cells acquired characteristics of decidual cells when incubated with 0.5 mM dbcAMP for 72 h. Their shape changed from elongated to rounded, while ultrastructural analysis revealed higher numbers of mitochondria and secretory follicles, and an increased proliferation rate. Elevated expression levels of IGF1, IGF2, PRLR and ERα were observed in decidualised cells; PRL and ERβ remained mostly below the detection limit, while PGR remained unaffected. The expression of smooth muscle α actin (αSMA), another decidualisation marker, was strongly induced. Among prostaglandin system members, levels of COX2 (PTGS2) and of PGE2-synthase (PTGES) were upregulated. Expression of the PGE2 receptors, PTGER2 and PTGER4, was clearly detectable.

Conclusion: An in vitro decidualisation model with canine uterine stromal cells was successfully established, allowing future, more detailed studies to be undertaken on the underlying molecular and endocrine mechanisms of canine decidualisation.

No MeSH data available.


Related in: MedlinePlus

Expression of insulin-like growth factor 1 (IGF1), IGF2, IGF-receptor 1 (IGF1R), prolactin receptor (PRLR), progesterone receptor (PGR) and oestrogen receptor alpha (ERα, ESR1), as determined by Real Time (TaqMan) PCR. Canine primary stromal cells were cultured for 24, 48 and 72 h in the presence of increasing dbcAMP concentrations. One-way ANOVA (24 h in IGF1 P < 0.01, 72 h in IGF1 P < 0.02; 72 h in IGF2 P < 0.03; 24 h in PRLR P < 0.0003, 48 h in PRLR P < 0.0001, 72 h in PRLR P < 0.001; 48 h in ERα P < 0.02, 72 h in ERα P < 0.004), followed by the Tukey-Kramer Multiple Comparison Test was applied; all samples were compared against the non-treated control in each group. Different letters indicate P < 0.05. Numerical data are presented as the mean ± standard deviation (SD)
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Fig6: Expression of insulin-like growth factor 1 (IGF1), IGF2, IGF-receptor 1 (IGF1R), prolactin receptor (PRLR), progesterone receptor (PGR) and oestrogen receptor alpha (ERα, ESR1), as determined by Real Time (TaqMan) PCR. Canine primary stromal cells were cultured for 24, 48 and 72 h in the presence of increasing dbcAMP concentrations. One-way ANOVA (24 h in IGF1 P < 0.01, 72 h in IGF1 P < 0.02; 72 h in IGF2 P < 0.03; 24 h in PRLR P < 0.0003, 48 h in PRLR P < 0.0001, 72 h in PRLR P < 0.001; 48 h in ERα P < 0.02, 72 h in ERα P < 0.004), followed by the Tukey-Kramer Multiple Comparison Test was applied; all samples were compared against the non-treated control in each group. Different letters indicate P < 0.05. Numerical data are presented as the mean ± standard deviation (SD)

Mentions: Table 1 presents a list of several selected genes, the expression of which was investigated at the mRNA level during dbcAMP-induced in vitro decidualisation of canine uterine stromal cells. Non-stimulated cells served as controls over the 72 h time course of the study. Whereas the expression of IGF1R did not change significantly during the period of in vitro decidualisation, IGF1 was already strongly upregulated (P < 0.05) after 24 h in response to 0.5 mM dbcAMP, while the expression of IGF2 was significantly elevated at 72 h (p < 0.05) (Fig. 6). The expression of PRLR increased significantly over controls within 24 h, responding to low and high dbcAMP concentrations (P < 0.05, P < 0.01 and P < 0.001 for 0.1 mM, 0.3 mM and 0.5 mM dbcAMP, respectively). This effect was also apparent in cells incubated for 48 h (P < 0.001 and P < 0.01 for 0.3 mM and 0.5 mM dbcAMP, respectively). The highest PRLR response was noted at 72 h in response to 0.3 mM and 0.5 mM dbcAMP, P < 0.001 and P < 0.01, respectively (Fig. 6). In contrast, the expression of PRL mRNA remained low and frequently below the detection limit both in control and stimulated cells throughout the experimental time-course (not shown).Fig. 6


In vitro decidualisation of canine uterine stromal cells.

Kautz E, de Carvalho Papa P, Reichler IM, Gram A, Boos A, Kowalewski MP - Reprod. Biol. Endocrinol. (2015)

Expression of insulin-like growth factor 1 (IGF1), IGF2, IGF-receptor 1 (IGF1R), prolactin receptor (PRLR), progesterone receptor (PGR) and oestrogen receptor alpha (ERα, ESR1), as determined by Real Time (TaqMan) PCR. Canine primary stromal cells were cultured for 24, 48 and 72 h in the presence of increasing dbcAMP concentrations. One-way ANOVA (24 h in IGF1 P < 0.01, 72 h in IGF1 P < 0.02; 72 h in IGF2 P < 0.03; 24 h in PRLR P < 0.0003, 48 h in PRLR P < 0.0001, 72 h in PRLR P < 0.001; 48 h in ERα P < 0.02, 72 h in ERα P < 0.004), followed by the Tukey-Kramer Multiple Comparison Test was applied; all samples were compared against the non-treated control in each group. Different letters indicate P < 0.05. Numerical data are presented as the mean ± standard deviation (SD)
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Fig6: Expression of insulin-like growth factor 1 (IGF1), IGF2, IGF-receptor 1 (IGF1R), prolactin receptor (PRLR), progesterone receptor (PGR) and oestrogen receptor alpha (ERα, ESR1), as determined by Real Time (TaqMan) PCR. Canine primary stromal cells were cultured for 24, 48 and 72 h in the presence of increasing dbcAMP concentrations. One-way ANOVA (24 h in IGF1 P < 0.01, 72 h in IGF1 P < 0.02; 72 h in IGF2 P < 0.03; 24 h in PRLR P < 0.0003, 48 h in PRLR P < 0.0001, 72 h in PRLR P < 0.001; 48 h in ERα P < 0.02, 72 h in ERα P < 0.004), followed by the Tukey-Kramer Multiple Comparison Test was applied; all samples were compared against the non-treated control in each group. Different letters indicate P < 0.05. Numerical data are presented as the mean ± standard deviation (SD)
Mentions: Table 1 presents a list of several selected genes, the expression of which was investigated at the mRNA level during dbcAMP-induced in vitro decidualisation of canine uterine stromal cells. Non-stimulated cells served as controls over the 72 h time course of the study. Whereas the expression of IGF1R did not change significantly during the period of in vitro decidualisation, IGF1 was already strongly upregulated (P < 0.05) after 24 h in response to 0.5 mM dbcAMP, while the expression of IGF2 was significantly elevated at 72 h (p < 0.05) (Fig. 6). The expression of PRLR increased significantly over controls within 24 h, responding to low and high dbcAMP concentrations (P < 0.05, P < 0.01 and P < 0.001 for 0.1 mM, 0.3 mM and 0.5 mM dbcAMP, respectively). This effect was also apparent in cells incubated for 48 h (P < 0.001 and P < 0.01 for 0.3 mM and 0.5 mM dbcAMP, respectively). The highest PRLR response was noted at 72 h in response to 0.3 mM and 0.5 mM dbcAMP, P < 0.001 and P < 0.01, respectively (Fig. 6). In contrast, the expression of PRL mRNA remained low and frequently below the detection limit both in control and stimulated cells throughout the experimental time-course (not shown).Fig. 6

Bottom Line: A part of this process is decidualisation, comprising morphological and biochemical changes that result in formation of maternal stroma-derived decidual cells.Expression of the PGE2 receptors, PTGER2 and PTGER4, was clearly detectable.An in vitro decidualisation model with canine uterine stromal cells was successfully established, allowing future, more detailed studies to be undertaken on the underlying molecular and endocrine mechanisms of canine decidualisation.

View Article: PubMed Central - PubMed

Affiliation: Institute of Veterinary Anatomy, Vetsuisse Faculty, University of Zurich, Winterthurerstrasse 260, CH-8057, Zurich, Switzerland. ewa.kautz@yahoo.com.

ABSTRACT

Background: The uterine response to the presence of embryos is poorly understood in the domestic dog (Canis familiaris). The intimate embryo-maternal cross-talk, which begins following the hatching of blastocysts and embryo attachment leads to strong structural and functional remodelling of the uterus. A part of this process is decidualisation, comprising morphological and biochemical changes that result in formation of maternal stroma-derived decidual cells. These are an integral part of the canine placenta materna, which together with the maternal vascular endothelium are the only cells of the canine endotheliochorial placenta able to resist trophoblast invasion. These cells are also the only ones within the canine placenta expressing the progesterone receptor (PGR). Understanding the decidualisation process thus appears essential for understanding canine reproductive physiology.

Methods: Here, we investigated the capability of canine uterine stromal cells to decidualise in vitro, thereby serving as a canine model of decidualisation. A dbcAMP-mediated approach was chosen during a time course of 24 - 72 h. Tissue material from six (n = 6) healthy, dioestric bitches was used (approximately 2 weeks after ovulation). Cells were characterized by differential staining, nearly 100 % of which were vimentin-positive. Scanning and transmission electron microscope analyses were applied, and morphological changes were recorded with a live cell imaging microscope. Expression of several decidualisation markers was investigated.

Results: The in vitro cultured stromal cells acquired characteristics of decidual cells when incubated with 0.5 mM dbcAMP for 72 h. Their shape changed from elongated to rounded, while ultrastructural analysis revealed higher numbers of mitochondria and secretory follicles, and an increased proliferation rate. Elevated expression levels of IGF1, IGF2, PRLR and ERα were observed in decidualised cells; PRL and ERβ remained mostly below the detection limit, while PGR remained unaffected. The expression of smooth muscle α actin (αSMA), another decidualisation marker, was strongly induced. Among prostaglandin system members, levels of COX2 (PTGS2) and of PGE2-synthase (PTGES) were upregulated. Expression of the PGE2 receptors, PTGER2 and PTGER4, was clearly detectable.

Conclusion: An in vitro decidualisation model with canine uterine stromal cells was successfully established, allowing future, more detailed studies to be undertaken on the underlying molecular and endocrine mechanisms of canine decidualisation.

No MeSH data available.


Related in: MedlinePlus