Background
Ruminants have an epitheliochorial-cotyledonary placenta, which is formed at the interface between endometrial caruncles (CAR) and fetal cotyledon (COT) and especially characterizes the migration of trophoblast binucleate cells (BNCs) [
1]. Both fetal and maternal components of bovine placenta develop vasculature at the first trimester of gestation, which become more elaborate structures during the course of pregnancy [
2]. A variety of angiogenic and vasoactive substances released from vascular endothelial cells and placental cells is thought to regulate local vascular functions in the bovine placenta [
3‐
6].
Adrenomedullin (AM) is a potent vasodilator peptide that was originally isolated from human phaeochromocytoma [
7]. Bovine AM consists of 52 amino acids and its sequence is highly conserved with human, porcine, canine, rat and mouse AM [
8]. Adrenomedullin acts through a complex of calcitonin receptor-like receptor (CRLR) and its associated receptor activity modifying proteins (RAMPs) [
9]. CRLR combines with RAMP2 and RAMP3 to form AM1 receptor and AM2 receptor, respectively. In addition to potent vasodilatory effects, AM is involved in multiple physiological activities such as angiogenesis, apoptosis, inflammation, cell proliferation and endocrine secretion [
10‐
12]. Previous studies have demonstrated that AM function is required for normal fetal growth and placental development in humans and rodents. In the human placenta, AM and CRLR are mainly expressed in syncytiotrophoblast cells and fetal membranes including amnion and extravillous cytotrophoblast cells [
13‐
17]. In the mouse placenta, trophoblast giant cells abundantly express
AM mRNA [
18]. Both
AM and
RAMP3 mRNA levels in rat placenta were higher in mid than in late gestation [
19]. In rats, AM antagonist treatment during early or late gestation decreased placental size, restricted fetal growth and induced deficient placental vascular formation [
20,
21]. AM heterozygous knockout mice had reduced fertility caused by restricted fetal growth due to a high incidence of abnormal trophoblast cell invasion followed by morphological placental defects [
22].
The ruminant placenta has different structures from humans and rodents, which is characterized by non-invasive trophoblast cells that never migrate into the basement membrane of the uterine endometrial unlike the placenta of humans and rodents. Since AM plays a role in various physiological functions such as angiogenesis, tissue remodeling, hormone secretion and placental development, we expect that AM is locally produced and its receptors are also distributed in the bovine placenta. However, there is no detailed information available about the involvement of AM in feto-placental development in ruminants. The objective of this study was 1) to determine mRNA expression patterns of AM, CRLR, RAMP2 and RAMP3 in the bovine uteroplacental unit during pregnancy; and 2) to investigate mRNA and protein localization of AM, CRLR and RAMPs in the bovine placentome and interplacentomal tissues.
Discussion
This is the first study to show that AM and its receptors are expressed in the bovine uteroplacental unit and that their mRNA expression pattern changes considerably during the course of pregnancy. We also demonstrated for the first time that AM mRNA and protein in the bovine placentome were detected only in BNCs, whereas those of CRLR, RAMP2 and RAMP3 were localized in trophoblast cells, including the BNCs, and caruncular epithelial cells. In interplacentomal tissues, mRNA and protein of AM were mainly detected in BNCs of fetal membrane and CRLR, RAMP2 and RAMP3 were found in fetal membrane, luminal epithelium, stroma under the epithelium, endothelial lineage of blood vessels and glandular epithelium. The results of
in situ hybridization and immunohistochemistry were almost match, suggesting transcripted and translated AM, CRLR and RAMP2 proteins continue to localize in the same cells in bovine utero-placenta throughout gestation period. It seems that locally produced AM is an important factor in the regulation of bovine placental function, as has been previously suggested in human and mouse studies [
10].
In this study, the mRNA expression profiles of
AM,
CRLR,
RAMP2 and
RAMP3 in the uteroplacental unit were summarized as follows:
AM mRNA was expressed in all tissues from the beginning of placentation to mid gestation transition (Days 60–200).
CRLR mRNA was lowest expression but most abundant in maternal tissue at the beginning of placentation.
RAMP2 mRNA was also most abundant in maternal tissue at the beginning of placentation whereas
RAMP3 mRNA was present in the fetal and maternal tissues more abundantly mid to late gestation.
AM mRNA in ICOT and
CRLR and
RAMP2 mRNA in all regions were highly expressed on Day 60 of gestation. AM promotes the migration and invasion of endothelial cells through CRLR/RAMP2 and CRLR/RAMP3 receptors [
27]. Homozygous knockout of
RAMP2 causes embryonic death in the mouse due to severe vascular abnormality, suggesting that the CRLR/RAMP2 receptor is required for angiogenesis [
28]. Both maternal and fetal vasculature of the bovine placentome at the third and fourth month of gestation are still immature and consist only of simple capillary loops [
2]. Although changes in mRNA expression of two of the primary angiogenic factors, fibroblast growth factor (FGF) and vascular endothelial growth factor (VEGF), have been reported in the bovine placentome during pregnancy, VEGF and its receptors on both the fetal and maternal side of the placentome increase after Day 80 of gestation and FGF1 and FGF type 2 receptor in the total placentome increase from around Day 80 until Day 200 of gestation [
5]. Therefore, increased
CRLR and
RAMP2 mRNA expression in the bovine uteroplacental unit at Day 60 of gestation may be involved in the stimulation of angiogenesis at an early stage of placentation before the increase in VEGF and FGF.
AM and
RAMP3 mRNA were highly expressed in EEM-COT, CAR and ICAR from Day 150 to Day 200 as compared with early or mid-gestation. The AM signal during this period is likely to act via the CRLR/RAMP3 receptor. The weight and length of bovine placentomes increase exponentially and their growth rates peak at Day 200 of gestation [
29]. In addition, bovine uterine artery blood flow increases linearly throughout gestation, suggesting an increase in blood supply to the placenta [
30]. In the bovine uteroplacental unit, gene expression of several major vasoactive substances such as endothelin-1 system, angiotensin II system and endothelial nitric oxide (NO) synthase increase during the mid-gestation period [
3,
4,
6]. The AM-induced vasodilatation is partially mediated through NO release [
31]. Locally produced AM in bovine placenta may regulate the vascular tonus in response to increasing blood supply by coordinating with other vasoactive substances to support placental development and feto-maternal exchange. Treatment with AM antagonist during late gestation in the rat causes necrosis in the decidua and labyrinthine trophoblast resulting in decreased placental and fetal weight and an increased incidence of fetal reabsorption [
20]. One possible reason for these abnormalities is deficient vascular development in the placenta [
20]. Therefore, it is plausible that AM plays certain roles in not only functional, but also morphological regulation of uteroplacental vasculature during mid to late gestation in bovine.
We revealed by
in situ hybridization and immunohistochemistry that the BNC is the primary source of AM production in the bovine placenta. In addition, CRLR and RAMPs are localized in same cell types within bovine uteroplacental tissues. This suggests that CRLR and RAMPs form receptor complex and exist as functional AM receptors in these cells. The secreted AM may act on fetal and maternal placental cells through an autocrine/paracrine mechanism. This finding is in agreement with previous report that
AM mRNA is most highly expressed in trophoblast giant cells in the mouse placenta [
18]. The bovine BNC secretes various placental-specific molecules such as placental lactogen (PL), pregnancy-associated glycoproteins (PAGs) and prolactin-related proteins (PRPs) in COT and ICOT, which play a crucial role in the regulation of placentation, maintenance of pregnancy and stimulation of fetal growth [
32]. Some of these molecules show a similar temporal expression profile with AM during pregnancy, that is the mRNA expression of
PRP1 in ICOT peaks at Day 60 of gestation and
PL,
PAG1,
PAG9 and
PRP-VII in COT begins to increase at mid gestation [
24,
33]. Thus, we speculate that AM may be involved in the regulation of secretory function of the BNC to interact with these placental-specific molecules in both placentome and interplacentomal regions. In addition, it has been reported that AM affects the secretory activities of endocrine organs including the pituitary gland, adrenal cortex and ovary [
34‐
38]. In rats, AM inhibited FSH-induced estradiol secretion in follicles and also suppressed eCG-stimulated progesterone release in corpus luteum [
37].
In vitro treatment of preantral follicular culture with AM increased estradiol production [
38]. The regulation of progesterone production by AM in corpus luteum in culture was pregnancy-stage dependent, inhibitory at early and late pregnancy but stimulatory at mid pregnancy [
38]. Since the utero-placenta is a major source of estrogen secretion during bovine pregnancy [
39,
40], AM may act as an important regulator in steroids production to maintain pregnancy.
Adrenomedullin enhances both cell invasion and proliferation via CRLR and RAMP2 in human choriocarcinoma cells [
41]. In mice, invasive trophoblast giant cells show dramatic upregulation of AM genes compared with undifferentiated trophectoderm cells, indicating the involvement of AM in trophoblast invasion and guidance of developing placental tissue [
22]. The bovine BNC is known to appear around Day 20 of gestation and migrate and fuse with endometrial epithelium from the trophoblast epithelium throughout gestation [
1].
AM,
CRLR and
RAMP2 mRNA were expressed in BNCs throughout gestation, suggesting that AM may also have a regulatory effect on migration, proliferation and/or turnover of the BNC itself during bovine placental development.
Treatment with AM antagonist during early rat pregnancy causes severely retarded placental development and restricted fetal growth through apoptosis of the placenta and uterus [
21]. This activation of apoptosis is due to decreased antiapoptotic protein Bcl-2 levels and increased mitochondrial proapoptotic Bcl2-associated X protein (Bax) levels with a decrease in cytochrome C levels in both the placenta and uterus [
21]. In the bovine placenta, mRNA expression of Bcl-2 related antiapoptotic protein
Bcl2A1 in bovine COT is higher on Day 60 and 150 of gestation than in EEM on Day 28 of gestation and the expression ratio between Bcl2A1 and BAX is highest on Day 60 of gestation [
25]. In addition, the mean number of apoptotic cells in bovine fetal and maternal placenta increases significantly from the first to the third trimester [
42]. Both
CRLR and
RAMP2 mRNA in the bovine placenta may also be involved in regulation of local apoptosis for adequate placentation.
In the present study, AM, CRLR, RAMP2 and RAMP3 mRNA and protein were also found in interplacentomal endometrium (ICAR) by both QPCR and histological studies. It has been reported that uterine secretions are essential for survival and development of the embryo and associated extraembryonic membranes [
43] and uterine artery blood flow increase throughout bovine pregnancy [
30]. The AM system may affect the regulation of cellular remodeling of luminal epithelium, angiogenesis, vascular permeability and uterine gland function via AM receptors within endometrium. Further studies are required to determine the functional role of AM system in bovine placental development and maintenance of pregnancy.
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
KGH participated in the design of the study, collected the materials, carried out all experiments and drafted the manuscript. MH was responsible for all animal care, collected the materials and helped to carry out all experiments. RS collected the materials and helped to carry out QPCR and in situ hybridization. TT supervised the study, collected the materials and helped to draft the manuscript. All authors read and approved the final manuscript.