In vitro decidualisation of canine uterine stromal cells.

Ewa Kautz,Aykut Gram,Alois Boos,Paula De Carvalho Papa,Iris M Reichler,Mariusz P Kowalewski

doi:10.1186/s12958-015-0066-4

Ewa Kautz, Aykut Gram + Show 4 more

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https://doi.org/10.1186/s12958-015-0066-4

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Abstract

BackgroundThe 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.MethodsHere, 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.ResultsThe 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.ConclusionAn 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s12958-015-0066-4) contains supplementary material, which is available to authorized users.

Highlights

Successful establishment of pregnancy requires highly orchestrated interactions between embryonic and maternal uterine tissues, which undergo specific morphological and biochemical changes to establish the uterine milieu required for proper embryo development
Characterisation and in vitro decidualisation of isolated canine uterine stromal cells Primary stromal cells were isolated from uteri of dogs collected during the early dioestrus phase using enzymatic dissociation and using the differential adhesion time
Devoid of an anti-luteolytic mechanism that serves in other domestic animal species to prevent luteolysis, the canine uterus was recently shown to respond to the presence of pre-implantation, free-floating embryos by activating the expression of markers of ongoing, Fig. 4 Transmission electron microscopy (TEM) analysis of canine uterine primary stromal cells during in vitro decidualisation. a-d control cells at 72 h of culture. e-i in vitro decidualised cells, after 72 h treatment with 0.5 mM dbcAMP early decidualisation [15]

Summary

Introduction

Successful establishment of pregnancy requires highly orchestrated interactions between embryonic and maternal uterine tissues, which undergo specific morphological and biochemical changes to establish the uterine milieu required for proper embryo development. Reprogramming of the endometrial stromal compartment during decidualisation involves transformation of stromal cells into decidual cells, which are involved in coordinating the processes of embryo implantation, placenta formation and development of the conceptus [1, 2] This specific tissue differentiation remains under the control of progesterone (P4) and oestrogens [3, 4]. 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. Understanding the decidualisation process appears essential for understanding canine reproductive physiology

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