Cellular Characteristics of Cultured Canine Trophoblasts

Abstract

Contents Shallow trophoblast invasion is detrimental in human pregnancies, but represents normal endotheliochorial placentation in dogs. Factors regulating shallow trophoblast invasion into the canine decidua are not well described, but it is known that matrix metalloproteinases (MMPs) play a crucial role in trophoblast invasion in many species. Following the methods previously described for isolating human trophoblasts, canine trophoblasts were isolated using collagenase and trypsin digestions with Percoll density gradient centrifugation. In addition, placental pieces were cryopreserved prior to primary culture following methods previously described for human tissue. Expression of cytokeratin‐7, MMP2 and MMP9 was confirmed using fluorescent immunocytochemistry. Cellular morphology was similar to that reported for trophoblasts. More than 97% of the cells cultured expressed cytokeratin‐7. More cultured canine trophoblasts expressed MMP9 (54.7 ± 3.4%) compared with MMP2 (40.3 ± 1.8%) (p = 0.02). Although both MMPs were immunolocalized to the cytoplasm, MMP2 was found in large, coalescing granules, whereas MMP9 was more diffusely expressed throughout the cell. Cryogenic freezing of placental tissue prior to primary cell culture had no effect on cell proliferation (p = 0.37). This research has established a baseline for future studies investigating the canine placenta as a model for disorders of shallow trophoblast invasion in humans.

Placental Evolution within the Supraordinal Clades of Eutheria with the Perspective of Alternative Animal Models for Human Placentation

“…Data show that the shallow trophoblast invasion in the endotheliochorial placentation of dogs is mainly regulated by matrix metalloproteinases, similar to what is known from humans [165]. Thus, the dog may be worth to consider as a model for disorders of shallow trophoblast invasion in humans [165].…”

“…Since the dog has shallow trophoblast invasion throughout gestation, it may improve the understanding of the underlying reasons and exact mechanisms for pre-eclampsia [164]. Data show that the shallow trophoblast invasion in the endotheliochorial placentation of dogs is mainly regulated by matrix metalloproteinases, similar to what is known from humans [165]. Thus, the dog may be worth to consider as a model for disorders of shallow trophoblast invasion in humans [165].…”

“…Finally, there are recent studies on preeclampsia in the dog model [164,165]. In humans, this disease results from shallow trophoblast invasion into the endometrium and myometrium, followed life-threatening conditions for both the mother and the fetus.…”

Trophoblast of domestic and companion nimals: basic and applied clinical perspectives

“…Reportedly (Staun-Ram and Shalev, 2005), the MMP-TIMP interaction controls trophoblast invasion by restricting over or underinvasion into the uterus. In support of this concept, immunohistochemical expression of MMP-2, MMP-9 and TIMP-2 have been detected in isolated canine trophoblast of term placenta (Sahlfeld et al, 2012). Temporally, from pre-parturition to parturition, there appears to be an increase in TIMP-2 mRNA expression as pregnancy advances.…”

“…Bone morphogenetic protein 4 is believed to regulate the terminal differentiation of chorionic girdle acting paracrinally from chorion, allantochorion and yolk sac through the Sma-and Mad-related protein, a mechanism of signaling by TGFβ superfamily proteins (CabreraSharp et al, 2014). Similarly in dogs, many factors have been shown or suspected to be involved in the regulation of trophoblast migration, invasion, and proliferation in carnivores including endopeptidases and their inhibitors, cytokines, growth factors, hormones, and other factors (Kovacs and Ojeda, 2012 (Sahlfeld et al, 2012) and endometrium (Beceriklisoy et al, 2007). The expression of MMP-2 and MMP-9 peaked at the time of implantation with more MMP-2 than MMP-9 (Beceriklisoy et al, 2007).…”