Limits...
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 cyclooxygenase 2 (COX2, PTGS2), PGE2-synthase (PGES, PTGES), PGE2 receptors: EP4 (PTGER4) and EP2 (PTGER2), PGF2α-synthase (PGFS/AKR1C3), PGF2α receptor (PTGFR, FP) and PG-transporter (PGT, SLCO2A1), 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 was applied (24 h in COX2 P < 0.005, 48 h in COX2 P < 0.01, 72 h in COX2 P < 0.0001; 48 h in PGES P < 0.01, 72 h in PGES < 0.03; 24 h in EP2 P < 0.0001, 72 h in PGFS P < 0.03, 48 h in FP P < 0.03, 72 h in FP P < 0.01), followed by the Tukey-Kramer Multiple Comparison Test; 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)
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig7: Expression of cyclooxygenase 2 (COX2, PTGS2), PGE2-synthase (PGES, PTGES), PGE2 receptors: EP4 (PTGER4) and EP2 (PTGER2), PGF2α-synthase (PGFS/AKR1C3), PGF2α receptor (PTGFR, FP) and PG-transporter (PGT, SLCO2A1), 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 was applied (24 h in COX2 P < 0.005, 48 h in COX2 P < 0.01, 72 h in COX2 P < 0.0001; 48 h in PGES P < 0.01, 72 h in PGES < 0.03; 24 h in EP2 P < 0.0001, 72 h in PGFS P < 0.03, 48 h in FP P < 0.03, 72 h in FP P < 0.01), followed by the Tukey-Kramer Multiple Comparison Test; 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: Among the major members of the prostaglandin system, the expression of COX2 (PTGS2) displayed an expression pattern similar to that observed for IGF2 (Fig. 7). It was, however, already significantly upregulated 24 h after stimulation in response to 0.5 mM dbcAMP (P < 0.01). As for IGF2, its highest expression above controls was noted at 72 h (P < 0.001) in response to 0.5 mM dbcAMP. Similarly, the expression of PGFS/AKR1C3 resembled that of IGF2 and was significantly increased at 72 h in response to both 0.3 mM and 0.5 mM dbcAMP compared to the respective controls (P < 0.05). The PGF2α receptor PTGFR (FP) and PTGES presented similar expression levels (Fig. 7). Thus, while displaying greater variations, their expression levels were increased (P < 0.05) at 48 h after stimulation. Especially for PTGES, this effect was obvious at 72 h in response to the highest dbcAMP dosage (P < 0.05). Contrasting with this was the expression of the respective PGE2 receptors, which either did not change significantly (P > 0.05) for EP4 (PTGER4) or decreased significantly for EP2 (PTGER2), both in response to dbcAMP within the first 24 h of incubation (P > 0.001) when compared with the respective control, as well as in all controls over the entire time course (P < 0.001 for 48 h and P < 0.01 for 72 h, respectively).Fig. 7


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 cyclooxygenase 2 (COX2, PTGS2), PGE2-synthase (PGES, PTGES), PGE2 receptors: EP4 (PTGER4) and EP2 (PTGER2), PGF2α-synthase (PGFS/AKR1C3), PGF2α receptor (PTGFR, FP) and PG-transporter (PGT, SLCO2A1), 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 was applied (24 h in COX2 P < 0.005, 48 h in COX2 P < 0.01, 72 h in COX2 P < 0.0001; 48 h in PGES P < 0.01, 72 h in PGES < 0.03; 24 h in EP2 P < 0.0001, 72 h in PGFS P < 0.03, 48 h in FP P < 0.03, 72 h in FP P < 0.01), followed by the Tukey-Kramer Multiple Comparison Test; 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)
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig7: Expression of cyclooxygenase 2 (COX2, PTGS2), PGE2-synthase (PGES, PTGES), PGE2 receptors: EP4 (PTGER4) and EP2 (PTGER2), PGF2α-synthase (PGFS/AKR1C3), PGF2α receptor (PTGFR, FP) and PG-transporter (PGT, SLCO2A1), 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 was applied (24 h in COX2 P < 0.005, 48 h in COX2 P < 0.01, 72 h in COX2 P < 0.0001; 48 h in PGES P < 0.01, 72 h in PGES < 0.03; 24 h in EP2 P < 0.0001, 72 h in PGFS P < 0.03, 48 h in FP P < 0.03, 72 h in FP P < 0.01), followed by the Tukey-Kramer Multiple Comparison Test; 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: Among the major members of the prostaglandin system, the expression of COX2 (PTGS2) displayed an expression pattern similar to that observed for IGF2 (Fig. 7). It was, however, already significantly upregulated 24 h after stimulation in response to 0.5 mM dbcAMP (P < 0.01). As for IGF2, its highest expression above controls was noted at 72 h (P < 0.001) in response to 0.5 mM dbcAMP. Similarly, the expression of PGFS/AKR1C3 resembled that of IGF2 and was significantly increased at 72 h in response to both 0.3 mM and 0.5 mM dbcAMP compared to the respective controls (P < 0.05). The PGF2α receptor PTGFR (FP) and PTGES presented similar expression levels (Fig. 7). Thus, while displaying greater variations, their expression levels were increased (P < 0.05) at 48 h after stimulation. Especially for PTGES, this effect was obvious at 72 h in response to the highest dbcAMP dosage (P < 0.05). Contrasting with this was the expression of the respective PGE2 receptors, which either did not change significantly (P > 0.05) for EP4 (PTGER4) or decreased significantly for EP2 (PTGER2), both in response to dbcAMP within the first 24 h of incubation (P > 0.001) when compared with the respective control, as well as in all controls over the entire time course (P < 0.001 for 48 h and P < 0.01 for 72 h, respectively).Fig. 7

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