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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

Transmission electron microscopy (TEM) analysis of canine uterine primary stromal cells during in vitro decidualisation. a-d control cells at 72 h of culture (solid arrowheads = nucleus, open arrows = mitochondria, solid arrows = microfilaments, open arrowheads = cisternae of rough endoplasmic reticulum). e-iin vitro decidualised cells, after 72 h treatment with 0.5 mM dbcAMP (solid arrowheads = large secretory vesicles, open arrows = mitochondria, solid arrows = Golgi apparatus, open arrowheads = cisternae of rough endoplasmic reticulum)
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Fig4: Transmission electron microscopy (TEM) analysis of canine uterine primary stromal cells during in vitro decidualisation. a-d control cells at 72 h of culture (solid arrowheads = nucleus, open arrows = mitochondria, solid arrows = microfilaments, open arrowheads = cisternae of rough endoplasmic reticulum). e-iin vitro decidualised cells, after 72 h treatment with 0.5 mM dbcAMP (solid arrowheads = large secretory vesicles, open arrows = mitochondria, solid arrows = Golgi apparatus, open arrowheads = cisternae of rough endoplasmic reticulum)

Mentions: Transmission electron microscopic analysis (TEM) of control stromal cells revealed the presence of long, often branched mitochondria, frequently visible microfilaments and abundant cisternae of rough endoplasmic reticulum (Fig. 4a-d). Cells undergoing in vitro decidualisation were characterized by higher abundance of Golgi apparatus which is responsible for protein sorting, modification and packaging for secretion, and consequently, greater amounts of secretory vesicles (Fig. 4e-i). These cells also displayed higher numbers of small, round-shaped mitochondria. As revealed by scanning electron microscopic analysis (SEM), the increased secretory activity of decidualised cells was also clearly visible on cell surfaces, displaying higher amounts of secreted deposits than in non-treated controls (Fig. 5a-f).Fig. 4


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)

Transmission electron microscopy (TEM) analysis of canine uterine primary stromal cells during in vitro decidualisation. a-d control cells at 72 h of culture (solid arrowheads = nucleus, open arrows = mitochondria, solid arrows = microfilaments, open arrowheads = cisternae of rough endoplasmic reticulum). e-iin vitro decidualised cells, after 72 h treatment with 0.5 mM dbcAMP (solid arrowheads = large secretory vesicles, open arrows = mitochondria, solid arrows = Golgi apparatus, open arrowheads = cisternae of rough endoplasmic reticulum)
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig4: Transmission electron microscopy (TEM) analysis of canine uterine primary stromal cells during in vitro decidualisation. a-d control cells at 72 h of culture (solid arrowheads = nucleus, open arrows = mitochondria, solid arrows = microfilaments, open arrowheads = cisternae of rough endoplasmic reticulum). e-iin vitro decidualised cells, after 72 h treatment with 0.5 mM dbcAMP (solid arrowheads = large secretory vesicles, open arrows = mitochondria, solid arrows = Golgi apparatus, open arrowheads = cisternae of rough endoplasmic reticulum)
Mentions: Transmission electron microscopic analysis (TEM) of control stromal cells revealed the presence of long, often branched mitochondria, frequently visible microfilaments and abundant cisternae of rough endoplasmic reticulum (Fig. 4a-d). Cells undergoing in vitro decidualisation were characterized by higher abundance of Golgi apparatus which is responsible for protein sorting, modification and packaging for secretion, and consequently, greater amounts of secretory vesicles (Fig. 4e-i). These cells also displayed higher numbers of small, round-shaped mitochondria. As revealed by scanning electron microscopic analysis (SEM), the increased secretory activity of decidualised cells was also clearly visible on cell surfaces, displaying higher amounts of secreted deposits than in non-treated controls (Fig. 5a-f).Fig. 4

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