The regulation of trophoblast invasion and decidual reaction by matrix metalloproteinase‐2, metalloproteinase‐7, and metalloproteinase‐9 expressions in the rat endometrium

Abstract Purpose We aimed to evaluate how matrix metalloproteinases (MMPs) regulate the trophoblast invasion and placentation. Methods Female rats were divided into the estrous cycle and early pregnancy day groups. Obtained uterine tissues and implantation sites were processed for immunofluorescence and real‐time PCR examinations. Results The mRNA expression of MMP‐7 was higher than MMP‐2 and MMP‐9. Immunofluorescence findings confirmed that MMP‐2, MMP‐7, and MMP‐9 were localized in the endometrial stroma, while MMP‐7 was high in glandular and lining epithelial cells throughout the entire estrous cycle. However, their immunolocalizations and mRNA expressions were dramatically changed with the early pregnancy days. The MMP‐7 reached very strong immunostaining in the giant trophoblast cells (GTCs), and the cytoplasm of mature and differentiating decidual cells, whereas MMP‐2 and MMP‐9 were mostly seen in the primary decidual zone (PDZ), GTCs, and the endothelium of blood vessels. Conclusions All three MMPs seemed likely to be a key mediator of trophoblast invasion into the decidual region as well as angiogenesis during the placentation process. Due to the strong and wide expression of MMP‐7 in the mature decidua, it could be suggested that MMP‐7 is important for decidual ECM remodeling and it might be used as a new marker of decidual reaction.

thrombospondin motifs (ADAMTS), and BMP/tolloid proteases found in mammals that function in the ECM metabolism, and contain a large number of endopeptidases, each containing an active site Zn 2+ ion. Some BMP/tolloid and ADAMTS proteases are required not only for the ECM turnover but also for its assembly through molecular activation or maturation of matrix precursor proteins as well. 3 MMPs are responsible for the ECM degradation 4 and are highly regulated to maintain tissue-specific activity and a variety of physiological processes. 5,6 These proteases are related to the decomposition of the ECM components to produce a variety of cellular environments for the execution of fully coordinated mechanisms. 7 during the implantation process, 9,10 at different stages ranging from the transcriptional level to activation inhibition of other ECM components. 11,12 Uncontrolled activity of proteases may lead to various defects such as arthritis, cancer, chronic tissue ulcer, fibrosis, aneurysms, nephritis, encephalomyelitis, and atherosclerosis. [13][14][15][16][17] In addition to the re-arrangement of the extracellular structure is critical for the uterine physiology, ECM remodeling plays also a vital role in the successful pregnancy by regulating decidualization 18 trophoblast invasion, placental development, 19,20 and spiral artery regeneration (especially MMP-2 and MMP-9). 21,22 It has been reported that the proliferation and differentiation of uterine stromal cells during decidualization are partially regulated by MMPs and tissue inhibitors of metalloproteinases (TIMPs). 18 The TIMPs differ in their selectivity for different MMPs. 23,24 Although TIMP-1 and TIMP-2 inhibit the active forms of all MMPs, they are linked to both active and latent forms of MMP-9 and MMP-2, which are involved in the breakdown of collagen IV that is the main component of the maternal basal membrane, and are considered key enzymes during implantation. The end of tissue breakdown and bleeding are initiated by the increase in expression of TIMPs, mainly TIMP-1 and TIMP-2. 25 TIMPs produced by trophoblastic and decidual tissues during pregnancy have also an inhibitory effect on activated MMPs, thus limits excessive trophoblast invasion. Uterine trophoblasts and vascular cells are the major sources of MMPs. 26,27 MMP-2 and MMP-9 play a role in the endometrial tissue remodeling during the estrous and menstrual cycles and during pregnancy. 28,29 Most MMPs are expressed in the human endometrium 30 and are present in large amounts during the menstruation and blastocyst implantation. 31 While the menstrual cycle in humans lasts 28 days, the estrous cycle takes only five days in rats. Therefore to work with rats in such studies is easy to conduct and it was shown that trophoblast invasion, placentation, and decidualization mechanisms are highly similar both in rats and in humans. Moreover, in addition to limited studies and the specific roles of MMP-2, MMP-7, and MMP-9 proteins are not fully highlighted, we think that most of the data obtained from in vitro models are insufficient to understand the mechanism of implantation biology since many critical factors may not be supplied in in vitro cultured systems. In this study, we employed immunofluorescence (IF) staining and real-time PCR methods to give a more comprehensive tissue function of these proteins during the estrous cycle and early days of pregnancy in the rat endometrium.
We showed here that all three MMPs might be crucial for controlling trophoblast invasion and placental development, while only MMP-7 has a role of decidual cell differentiation and maintain proper/permanent mature decidual matrix. According to our knowledge, this study could be the first to demonstrate the presence of MMP-7 expression in mature and differentiating decidual cells during implantation, and thus, it might be predicted as a new marker for decidual reaction.

| Animals
For the present study, 21 adult female Wistar albino rats (6-8 weeks old) weighing between 220 and 250 grams were obtained from the Animal Laboratory of Cumhuriyet University (Sivas, Turkey). The rats were maintained in a temperature-controlled room (23 ± 2°C) at 60-70% relative humidity with a 12-L:12-D photoperiod cycle. They were fed with standard pellet feed and tap water ad libitum throughout the experiment. Of the total, twelve animals were used for the estrous cycle days (three for each stage), whereas the remaining nine animals were divided into three groups of three for pregnancy days (7.5, 8.5, and 9.5). The status of the estrous cycle, that is, the proestrus, estrus, metaestrus, and diestrus stages, was determined in the non-pregnant rats by the vaginal smear method. 32 The rats were mated with fertile males of the same strain overnight in standard plastic cages in the Animal Laboratory of Cumhuriyet University, Sivas, Turkey, to establish a pregnancy. The next morning, mating was confirmed through the presence of a vaginal plug, and the males were prevented from further contact with such females. The sighting of a vaginal plug was declared as Day 1 of pregnancy (1.0 day post-coium, dpc). All females were sacrificed by rapid decapitation in the adult period. The pregnant females were laparotomized on days 7.5, 8.5, and 9.5 to obtain implantation sites. Uterine horns were dissected. All efforts were made to minimize the number of animals used and their suffering.

| Tissue processing
The tissues were fixed in 4% PBS-buffered paraformaldehyde overnight at 4°C. After dehydration, the tissues were embedded in paraffin, sectioned (thickness, 2 μm), dewaxed in xylene, and stained using hematoxylin & eosin and immunofluorescence staining methods. However, we did not include hematoxylin-eosin photographs in this article. Serial sections were made.

| Immunofluorescence labeling
Following deparaffinization in xylene and rehydration in decreasing concentrations of ethanol, the sections were washed in distilled water.
Antigen retrieval was applied to sections at 95°C, 550W in 10 mmol/L citrate buffer at pH:6 for 5 minutes in a microwave oven. The sections were washed once in phosphate-buffered saline (PBS)-Triton-X 100. In order to prevent non-specific staining, the sections were de- in PBS-Triton-X 100, the sections were evaluated using a fluorescence microscope (Olympus BX51). All steps in the immunostaining process were applied to the negative control sections without the primary antibody incubation. Photographs from the convenient fields of view were taken using an Olympus BX51 (Tokyo, Japan), and a semi-quantitative scoring method was applied.

| Quantification of mRNA expression
The uterine tissues were removed from the non-pregnant and pregnant rats, followed by euthanasia, and tissue was obtained by scraping the uterine epithelium through curettage. The scraped samples were placed in RNA later, which is an aqueous, non-toxic solution that rapidly permeates tissue to stabilize and protect the cellular RNA content, 33 and the samples were stored at − 20°C. The quantity and quality of total RNA were determined by spectrometry and denaturing agarose gel electrophoresis, respectively. Total RNA was obtained using the RNA isolation kit (RNeasy Mini Kit, Invitrogen) to determine the expression of MMP-2, MMP-7, and MMP-9 mRNAs. cDNAs were synthesized from the RNAs obtained from these mRNAs using the cDNA kit (Superscript II-RT, Thermo Fisher Scientific), and the expression of these mRNAs was determined by real-time PCR using relative quantitation. PCR amplification was performed using Sso Advanced Universal SYBR Green Supermix compatible with Bio-Rad CFX Connect. Primers were designed to amplify cDNAs of around 100 bp to maximize efficiency and are summarized in Table 1. Own designed primers were used in the present study. All cDNA concentrations were set to 100 ng/μl. Reference GAPDH and MMP genes expression levels in the estrous cycle and early pregnancy days were given in Figure 1. A value of 2 ΔΔCt (ΔΔCt=(Ct target gene1 -Ct reference gene(GAPDH) ) estrous cycles/early pregnancy days -(Ct target gene2 -Ct reference gene(GAPDH) ) estrous cycles/early pregnancy days ) was calculated over Ct values and normalized to the selected reference genes. The aim is to determine the increase or decrease in the relative quantification of expression levels of genes. Relative expression levels which calculated from the formula of 2 −ΔΔCt of the estrous cycle and the early pregnancy days were given in Figure 2 and Figure 3, respectively.

| RE SULTS
The immunolocalizations and expressions of MMP-2, MMP-7, and MMP-9 were determined using immunofluorescence and real-time PCR methods, and the results were presented in tables (Tables 2 and 3) and figures (Figures 2 and 3). All sections were examined by two independent observers, and evaluations were made in the uterine tissue based on the degree of staining of these proteins.

| Immunofluorescence of MMP-2, MMP-7, and MMP-9 proteins in the estrous cycle
Semi-quantitative results of the MMPs immunolocalizations during the estrous cycle in rat endometrium are shown in Table 2.

| Proestrus
The immunolocalization of MMP-7 in the luminal epithelium was strong; however, there was found that no MMP-2 and MMP-9 immunostainings in the glandular epithelium. (

| Estrus
The immunolocalization of MMP-7 in the luminal and glandular epithelia was very strong ( Figure 4E). MMP-2 and MMP-9 immunolocalizations, except MMP-7, increased gradually from subepithelial stroma to basal stroma as in the proestrus stage, and there was also found that strong MMP-7 immunostaining in the cytoplasm of stromal cells rather than the stromal region ( Figure 4E). MMP-2 and MMP-7 showed high levels of immunolocalization in the endothelium of the blood vessels, but MMP-9 was stronger ( Figure 4F-h) ( Table 2).

F I G U R E 1
Ct values of MMP-2, MMP-7, and MMP-9 mRNAs for estrous cycle and pregnancy days F I G U R E 2 Relative expression ratios of MMP genes showing relative changes in the estrous cycle stages (n = 3) after normalization to GAPDH. It has been determined that MMP-2 gene was expressed at decreasing levels in transition from the proestrus to estrus and the diestrus to proestrus, and it was expressed in increasing levels in the transition from the estrus to metaestrus and metaestrus to diestrus. The MMP-7 gene was expressed as upregulated in the transition from the proestrus to estrus, while the MMP-9 gene was downregulated. Therefore, it can be said that MMP-7 and MMP-9 genes display inverse-related expression patterns F I G U R E 3 Relative expression ratios of MMP genes showing relative changes in the early pregnancy days (n = 3) after normalization to GAPDH. It was observed that MMP-2 had an increased level of expression in the transition from pregnancy days of 7.5-8.5 and 8.5-9.5. MMP-7 and MMP-9 genes were downregulated in the transition from the pregnancy days of 7.5-8.5, while they were upregulated in the transition from the days of 8.5-9.5

| Metaestrus
Immunolocalization levels of all MMPs in luminal and glandular epithelia were quite low ( Figure 5A-C). Increasing and strong immunolocalization levels of all MMPs from the subepithelial stroma to the basal stroma were observed, but this was slightly weaker in MMP-9 ( Figure 5C) ( Table 2).

| Diestrus
MMP-7 immunolocalization was detected as strong and very strong in the luminal and glandular epithelia, respectively ( Figure 5E). MMP-7 expression levels were also detected very strong in stromal cells located in the endometrial stromal region as in the proestrus and estrus stages ( Figure 5E). MMP-2 and MMP-9 immunolocalization levels were found to be strong in the endothelial part of the blood vessels, whereas MMP-7 was relatively weak ( Figure 5D and F) ( Table 2).  TA B L E 2 MMP-2, MMP-7, and MMP-9 immunolocalizations during the estrous cycle

| Day 7.5 of pregnancy
Immunolocalization levels of all three proteins were observed to be less strong in the PDZ surrounding the embryo site ( Figure 6A,D,g and h). Immunolocalization levels of MMP-2 and MMP-9 increased in SDZ than PDZ ( Figure 6A, g and h); however, the level of MMP-7 was the opposite in these decidual regions ( Figure 6D and E). The level of MMP-7 immunolocalization in the cytoplasm of stromal/undifferentiated decidual cells in the mesometrial basal zone was quite strong ( Figure 6F). The immunostaining level of MMP-7 was found to be less strong than other proteins in the endothelial part surrounding the lumen of blood vessels ( Figure 6C,i) ( Table 3).

| Day 8.5 of pregnancy
Immunolocalization of MMP-7 was stronger in the embryonal region compared to other proteins ( Figure 7B,F and j). Immunostaining of MMP-2 and MMP-9 was found to be strong in PDZ ( Figure 7C,i,j).
The immunolocalization of all three proteins in GTC regions was determined as strong ( Figure 7B,C,g and j). Immunostaining of MMP-2 and MMP-9 proteins was determined to be strong in the endothelial part of the blood vessels ( Figure 7D and l). The expression levels of MMP-7 protein in the cytoplasm of decidual cells in the anti-mesometrial region (especially basal zone), cells in the embryo site ( Figure 7A and B), and undifferentiated stromal cells in the mesometrial region ( Figure 7H) were quite strong during this period of pregnancy (Table 3).

| Day 9.5 of pregnancy
It could be easily said that MMP-7 exhibited a tremendously strong immunolocalization, mainly in the cytoplasm of decidual cells located in the anti-mesometrial and mesometrial regions, GTCs, and EPC, as well as the embryo region, compared to other MMPs ( Figure 8E-h).
The immunolocalization level of MMP-2 and MMP-9 was particularly strong in GTCs ( Figure 8A,B and j). Immunolocalization of all three proteins was found to be strong in the endothelial part of the blood vessels in this region ( Figure 8D,h and l) ( Table 3).

| Expression levels of MMP genes in early pregnancy
MMPs genes were highly induced by day 9.5 of pregnancy, but especially the level of MMP-2 and MMP-7 mRNAs was higher than those of MMP-9 during days 7.5-9.5 of pregnancy. It was determined that MMP-7 reached the highest level when three genes were compared to each other (Figure 1).

| D ISCUSS I ON
MMPs are responsible for the tissue degradation and turnover of ECM components in several physiological processes. 34  Although there are many factors secreted from decidual tissue that may regulate the trophoblast invasion, it seems that MMP-TIMP interaction is also a very critical event at the embryo-uterine interface. 43  invasion. 43 At weeks 6-8 of a human pregnancy, high expression of MMP-2 (at later decreasing concentrations) dominated the condition on MMP-9, whereas MMP-9 expression increased between 8th and 11th weeks, predominating until the end of pregnancy, 49 which led to the conclusion that MMP-2 plays a vital role during implantation and MMP-9 plays an essential role during invasion. 50 The expression of MMP-9 by trophoblast cells, as we also showed here, which possesses basement membrane-degrading proteolytic activity, is required to facilitate successful implantation and endometrial invasion. 49 Moreover, in recent reports, through a reduced hatching rate and MMP-9 expression in in vitro culture condition has been linked to low implantation rate. [51][52][53] In rodents, the primary decidual zone consisted of the closely packed decidual cells with tight junctions, which has been considered as a barrier zone against the trophoblasts. 54 Moreover, since this area has collapsed blood vessels, this barrier should be removed gradually for the trophoblasts to reach the blood vessels and to form yolk sac placenta and chorioallantoic placenta at the anti-mesometrial and mesometrial pole, respectively. 55 As observed in the present disorders. [69][70][71][72] In conclusion, considering the importance of MMPs for invasion and remodeling processes, our results may provide that the temporal regulation of all three MMPs may be crucial for controlling trophoblast invasion and placental development, while MMP-7 has a role of decidual cell differentiation and may be predicted a new marker for decidual reaction because it is wide distribution through the mature decidua during the peri-implantation period. Further studies are needed to identify the molecular signals involved in the regulation of expression and activity of studied MMPs.

ACK N OWLED G M ENTS
We acknowledge Sivas-Cumhuriyet University, Scientific Research Project for supporting our work. This work was supported by Sivas-Cumhuriyet University Scientific Research Project (No. T-725).

Conflicts of Interest:
The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.
Human and animal rights: This article does not describe any experiments involving human participants. All of the institutional and national guidelines for the care and use of laboratory animals were followed. The protocol for the research project was approved by a suitably constituted ethics committee.