Murine Abortion is Associated with Enhanced Hyaluronan Expression and Abnormal Localization at the Fetomaternal Interface
Introduction
Successful implantation and pregnancy establishment are unique biological events characterized by delicate interactions between the conceptus and the maternal environment. Development of the embryo to the blastocyst stage, its adhesion to the endometrium and subsequent invasion of the underlying stroma leading to formation of a functional placenta are the result of complex regulatory mechanisms that involve endometrial modifications as well as maternal immunological tolerance [1]. Despite being critical to the survival of the species, mammalian reproduction is relatively inefficient. Indeed, the highest probability of conception during a menstrual cycle is as low as 30 percent. Furthermore, in humans, approximately 25 percent of all conceptions last beyond 20 weeks of gestation. Although many studies have been developed, the mechanisms that determine pregnancy maintenance are still largely elusive.
In rodents, pregnancy lasts between 19 and 20 days. Implantation of the blastocyst involves adhesion and invasion of endometrium and takes place between gestation days (Gd) 4–5. During pregnancy establishment, the endometrium is transformed into a highly specialized decidual tissue; a process referred to as decidualization that not only implicates proliferation of endometrial stromal cells into decidual cells but also formation of new vasculature [2]. Afterwards, the placentation process takes place. The placenta plays a crucial role since it constitutes the contact surface between mother and fetus (the fetomaternal interface) and allows the physiological exchange of gases, nutrients and waste products. Mice, as well as humans, present a haemochorial placentation characterized by trophoblast cells eroding the maternal vasculature resulting in direct contact between maternal blood and embryonic trophoblast cells. In spite of the differences between mice and human placentation [3], mice still constitute a useful model to understand the gestation process.
The remodelling of the endometrial architecture is fundamental to create a suitable environment for blastocyst implantation. This process includes phases of cellular proliferation, differentiation and tissue breakdown along with alterations in the composition of maternal extracellular matrix (ECM) [4]. The ECM remodelling plays an important role providing a prosperous medium for implantation and modulating trophoblast invasion leading to the establishment of a functional placental unit [5].
Hyaluronan (HA) is a conspicuous component of the ECM especially in remodelling tissues [6]. HA is a linear glycosaminoglycan (GAG), composed of repeated disaccharide units of glucuronic acid and N-acetylglucosamine, that exists as a high molecular weight polymer ranging from 105 to 107 Da. HA differs from other GAGs specially in its synthesis and degradation. HA biosynthesis is carried out at the inner face of the plasma membrane by hyaluronan synthases (HAS) and the growing polymer is extruded through the membrane into the extracellular space. In mammals, there are three HAS isozymes: HAS-1, HAS-2 and HAS-3 [7]. Though the synthases can be regulated, the major control for HA deposition occurs at catabolism. Degradation of HA occurs by the concerted action of three enzymes: a hyaluronidase (Hyal) and two exoglycosidases. In humans, six hyaluronidase genes have been identified. These are HYAL-1, HYAL-2, HYAL-3, HYAL-4, SPAM1 and PHYAL-1, which, respectively, encode HYAL-1, HYAL-2, HYAL-3, HYAL-4, PH-20 and a pseudogene transcribed but not translated [8]. Mouse genome presents a seventh hyaluronidase-like gene called Hyal-5. Among the Hyals, HYAL-1 and HYAL-2 are of great importance in degradation of high molecular weight HA (HMW-HA > 1000 kDa). Differential expression of the enzymes related with HA synthesis and degradation has been observed in tumors and during development [6], [9]. However, their role remains to be completely clarified.
Despite its simple chemical composition, HA possesses several functions both in physiological and pathological conditions such as morphogenesis, tissue injury and repair, inflammation, and tumorigenesis [6], [10], [11], [12]. HA is prominent during embryonic development and at sites of wound healing. In healthy tissues, HA regulates water homeostasis, influencing hydration and physical properties of tissues. Interaction of HA with other ECM components (proteoglycan, aggrecan, or versican) allows the assembly of many tissues. Moreover, upon interaction with CD44, RHAMM (receptor for hyaluronic acid mediated motility), TLR-4 (Toll like receptor 4) cell surface receptors or different HA binding-proteins (TSG-6, SHAP), HA is able to modulate fundamental cell behaviours such as adhesion, migration, proliferation and differentiation [13]. The molecular weight and concentration of HA are also important in determining their functions. For example, HMW-HA has been shown to be anti-angiogenic, while HA fragments (3–25 disaccharide units) induced angiogenesis in vitro [14]. Besides, HA fragments have been found to be potent activators of dendritic cells and to induce proinflammatory cytokine secretion that could not be exerted by HMW-HA [15]. As nearly all of the studies have been performed in vitro, the challenge is to characterize in vivo the size of HA in the local microenvironment and to clarify its role.
During gestation, changes in HA deposition and distribution indicate that HA may participate in preparation of the endometrial stroma for reception and implantation of the embryo [16], [17], [18], [19]. Little is known however about the role of HA at the fetomaternal interface, especially regarding its influence in pregnancy outcome. Consequently, the aim of this work was to analyze the levels, distribution, synthesis and degradation, and molecular size of HA during murine normal and pathologic pregnancies. The present study was performed using a normal pregnancy mouse model, BALB/c-mated CBA/J females, and an abortion prone mating, DBA/2J-mated CBA/J females. Findings in the latter model are of great importance to further understand the role of HA during normal pregnancy and spontaneous abortion.
Section snippets
Animals
Mice were purchased from Charles River and maintained in an animal facility with a 12 h light/dark cycle. All animal experiments were conducted according to institutional guidelines of the Medicine University of Berlin. CBA/J females were caged with DBA/2J or BALB/c males overnight and examined for a vaginal plug the next morning. The presence of the plug determined the day 0.5 of pregnancy. After that the pregnant females were segregated. The CBA/J females mated both with BALB/c (normal
Changes of HA peripheral and local levels during pregnancy
Considering that modification in ECM components, particularly in HA deposition, takes place during pregnancy [17], [19], we first evaluated HA levels in normal pregnancy (BALB/c-mated CBA/J females) and spontaneous abortion (DBA/2J-mated CBA/J females) models. An ELISA assay was used to determine HA concentration in serum and decidua extracts on Gd 7.5 and 13.5 as well as in placenta extracts on Gd 13.5.
Serum HA levels in non-pregnant mice (∼2 μg/ml) were similar to those observed in normal
Discussion
Establishment of pregnancy constitutes a unique situation in which the integrated action of delicate immunoregulatory mechanisms as well as endometrial modifications (including alterations in ECM components such as HA) occurs.
In the present work, we observed differential systemic and local HA levels and distribution during normal pregnancy and spontaneous abortion. Serum HA levels during pregnancy have been assessed by Uchiyama et al., observing that HA increases during pregnancy [29].
Acknowledgments
We are indebted with Dr. P.C. Arck for comments on the manuscript. We are grateful to Farmatrade Argentina for providing HMW-HA. R.C-R. and G.B. thank the German Academic Exchange Program (DAAD) for the fellowships granted. M.G.G is a guest scientist at the Charité, supported by the Alexander von Humboldt Foundation in Germany. S.M.B is a fellow of the Habilitation program at Charité, University Medicine Berlin. A.O. is supported by the Turkish Higher Education Council. M.G.G., L.A. and S.H are
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