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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

Morphological appearance of canine uterine stromal cells during in vitro decidualisation (a). Quantitation of cell density: percentage (%) of surface determined under live cell imaging conditions (b; average from three independent experiments). One-way ANOVA followed by Dunnett’s Multiple Comparison Test was applied to test the effects of time on cell density in all control or treated samples ((*) indicates P < 0.0001). Student’s t-test was applied to test the effect of treatment on cell density: (**) indicates P < 0.0002, (***) indicates P < 0.0007
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Fig2: Morphological appearance of canine uterine stromal cells during in vitro decidualisation (a). Quantitation of cell density: percentage (%) of surface determined under live cell imaging conditions (b; average from three independent experiments). One-way ANOVA followed by Dunnett’s Multiple Comparison Test was applied to test the effects of time on cell density in all control or treated samples ((*) indicates P < 0.0001). Student’s t-test was applied to test the effect of treatment on cell density: (**) indicates P < 0.0002, (***) indicates P < 0.0007

Mentions: Primary stromal cells were isolated from uteri of dogs collected during the early dioestrus phase using enzymatic dissociation and using the differential adhesion time. Nearly 100 % of cells stained positively for vimentin, thus verifying their mesenchymal origin (Fig. 1). Only sporadically were epithelial cell-derived cytokeratin signals detected. The morphological changes observed during in vitro decidualisation were consistent with the acquired characteristics of decidual cells. The shape of cells changed from elongated and spindle-shaped to rounded (Fig. 2a) when cells were incubated for 72 h with 0.5 mM dbcAMP. Morphological changes observed during in vitro decidualisation of canine stromal cells were captured using time-lapse microscopy and are presented in the supplemental material (Additional file 1). Simultaneously with the morphological changes, the in vitro decidualised cells revealed an accelerated proliferation rate as determined by quantification of cell densities in time-lapse derived pictures at the selected time points of 0, 12, 24, 48 and 72 h (Fig. 2a). Whereas both dbcAMP-stimulated and control cell densities increased significantly over time (P < 0.0001), the density of decidualised cells significantly exceeded that of control cells by 48 h and 72 h (P < 0.0002 and p < 0.0007, respectively) (Fig. 2b). Nearly 100 % confluence was observed in decidualised cells by 72 h.Fig. 1


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)

Morphological appearance of canine uterine stromal cells during in vitro decidualisation (a). Quantitation of cell density: percentage (%) of surface determined under live cell imaging conditions (b; average from three independent experiments). One-way ANOVA followed by Dunnett’s Multiple Comparison Test was applied to test the effects of time on cell density in all control or treated samples ((*) indicates P < 0.0001). Student’s t-test was applied to test the effect of treatment on cell density: (**) indicates P < 0.0002, (***) indicates P < 0.0007
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4526293&req=5

Fig2: Morphological appearance of canine uterine stromal cells during in vitro decidualisation (a). Quantitation of cell density: percentage (%) of surface determined under live cell imaging conditions (b; average from three independent experiments). One-way ANOVA followed by Dunnett’s Multiple Comparison Test was applied to test the effects of time on cell density in all control or treated samples ((*) indicates P < 0.0001). Student’s t-test was applied to test the effect of treatment on cell density: (**) indicates P < 0.0002, (***) indicates P < 0.0007
Mentions: Primary stromal cells were isolated from uteri of dogs collected during the early dioestrus phase using enzymatic dissociation and using the differential adhesion time. Nearly 100 % of cells stained positively for vimentin, thus verifying their mesenchymal origin (Fig. 1). Only sporadically were epithelial cell-derived cytokeratin signals detected. The morphological changes observed during in vitro decidualisation were consistent with the acquired characteristics of decidual cells. The shape of cells changed from elongated and spindle-shaped to rounded (Fig. 2a) when cells were incubated for 72 h with 0.5 mM dbcAMP. Morphological changes observed during in vitro decidualisation of canine stromal cells were captured using time-lapse microscopy and are presented in the supplemental material (Additional file 1). Simultaneously with the morphological changes, the in vitro decidualised cells revealed an accelerated proliferation rate as determined by quantification of cell densities in time-lapse derived pictures at the selected time points of 0, 12, 24, 48 and 72 h (Fig. 2a). Whereas both dbcAMP-stimulated and control cell densities increased significantly over time (P < 0.0001), the density of decidualised cells significantly exceeded that of control cells by 48 h and 72 h (P < 0.0002 and p < 0.0007, respectively) (Fig. 2b). Nearly 100 % confluence was observed in decidualised cells by 72 h.Fig. 1

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