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Epithelial-mesenchymal transition and bi- and multi-nucleated trophoblast cell formation in ovine conceptuses during the peri-implantation period

Epithelial-mesenchymal transition (EMT), which is common in cancer metastasis, is also observed during developmental processes such as embryo implantation into the maternal endometrium in humans and rodents. However, this process has not been well characterized in the non-invasive type of implantation that occurs in ruminants. To understand whether EMT occurs in ruminant ungulates, ovine conceptuses (embryo plus extraembryonic membranes) from days 15 (P15: pre-attachment), 17 (P17: during attachment), and 21 (P21: post-attachment, day 0 = day of estrus) were evaluated. RNA-seq analysis revealed that the expression of EMT-related transcripts increased on P21. Real-time PCR and western blotting analyses indicated that levels of transcripts and proteins indicative of mesenchyme-related molecules increased on P21, but a minor expression of epithelium-related molecules remained. Immunohistochemical analysis revealed that E-cadherin (CDH1) was localized in the elongated trophectoderm on P15 and P17. On P21, CDH1 was localized to the trophectoderm and on the conceptus cells undergoing differentiation. Vimentin (VIM) was localized in the uterine stroma on P15 and P17, and its expression was observed at the edge of elongating trophoblast on P21. Further, it was found that some bi-nucleated trophoblast cells were present on P17; however, numerous bi- and multi-nucleated trophoblast cells on the uterine epithelium or next to the uterine stroma were found on P21. A minor expression of pregnancy-associated glycoprotein (PAG) transcripts was found on P15 and P17, but a definitive expression of PAGs, transcripts, and proteins was found on P21. Although further investigation is required, these observations indicate that bi-nucleated trophoblast cell formation begins on the day conceptus implantation to the maternal endometrium is initiated, followed by EMT in trophoblast cells. These results suggest that these sequential events are required if pregnancy is to be established in ruminants.

in cancer metastasis, organ development, wound healing, and invasive modes of conceptus implantation [1]. EMT is a cellular process in which epithelial cells acquire a mesenchymal nature, characterized by a loss of apical-basal cell polarity, cytoskeleton remodeling, cell-cell adhesion weakening, and acquisition of cell motility [1]. We previously observed that bovine conceptuses (embryo plus extraembryonic membranes) exhibited features of EMT on day 22 (day 0 = day of estrus), 2-3 days after the initiation of conceptus attachment to the uterine epithelium. During this period, trophectodermal cells expressed mesenchymal markers such as vimentin (VIM) and N-cadherin (CDH2), while expression of the epithelial cell marker cytokeratin was maintained. However, it is still not fully understood why non-invasive, ruminant trophoblasts undergo EMT at the time of conceptus adhesion to the uterine endometrium.
These observations were supported by results from in vitro cell culture experiments in which bovine trophoblast CT-1 cells [3] were co-cultured with bovine endometrial epithelial cells [4,5].
Endometrial follistatin was shown to decrease the effect of the potent EMT inducer activin A. These results were further supported by the observation that activin A expression increased when follistatin expression was downregulated on day 22 [4]. Furthermore, the expression of an EMT-related transcription factor, ovo-like zinc finger 2 (OVOL2), was elevated on day 20. As OVOL2 expression declined on day 22, the expression of other EMT-related transcription factors such as zinc finger E-box binding homeobox1 (ZEB1) and snail family transcriptional repressor 1 (SNAI2) increased, as did VIM, a phenotypic marker for mesenchymal cells [5]. Collectively, these observations suggest that EMT occurs in bovine species during conceptus adhesion to the uterine endometrium. It has been well documented that bi-and multi-nucleated trophectodermal cells begin to form during the peri-implantation period in ruminants [6]. These cells are responsible for establishing syncytial plaques at the placenta-uterine interface. They can also express large amounts of secreted proteins, such as placental lactogen, prolactinrelated proteins, and pregnancy-associated glycoproteins (PAGs) [7,8]. Placental PAG expression is restricted to species within the order Cetartiodactyla (even-toed ungulates) [8]. Most PAG genes are present in the family Bovidae [9]. Cattle and sheep have more than two dozen intact PAG genes each, most of which are expressed locally in bi-and multi-nucleated trophectoderms [8]. Consequently, PAGs can serve as useful markers of ruminant trophoblasts, especially in the identification of bi-and multi-nucleated cells in the placenta [10].
Based on these observations, we hypothesized that EMT and trophoblast cell fusion are integral parts of the conceptus implantation processes required for subsequent placental formation in ruminants. In this study, we examined whether EMT-related transcripts and proteins were expressed in utero on days 15 (P15: pre-attachment), 17 (P17: during attachment), and 21 (P21: post-attachment) of gestation (day 0 = day of estrus) in the ovine species. We then determined the presence of bi-and multi-nucleated cells and the expression of PAGs in the maternal-fetal compartments.

Collection of ovine conceptuses/endometrial tissues and whole uterus fixation
The protocol for sheep experimentation with whiteface crossbred ewes, maintained at the farm of the University of Tennessee, Knoxville, TN, USA, was reviewed and approved by the Animal Care Committee of the University of Tennessee, the University of Tokyo, and Tokai University (approval number: 20-017-23). Animal husbandry, estrous synchronization, and tissue collection were performed as described previously [11]. For uterine fixation, hysterectomy was performed on P15, P17, and P21, and the uteri (n = 3 each) were subjected to whole uterus fixation within 15 min after slaughter [12]. Fixed whole uteri that were paraffin-embedded, and frozen conceptuses and endometria were initially transferred to the Laboratory of Animal Breeding, The University of Tokyo, and then transferred to Tokai University, Kumamoto, Japan. The fixed and paraffin-embedded uterine tissue blocks were sectioned (6 μm), stained with hematoxylin and eosin, and evaluated for intra-uterine structures, including the presence of conceptuses [13].

Quantitative reverse-transcription (RT)-PCR analysis
Using ISOGEN II (311-07361; NIPPON GENE Co., Ltd., Tokyo, Japan), total RNA was extracted from P15, P17, and P21 frozen sheep conceptuses (n = 3/day) according to the manufacturer's protocol. For real-time PCR, isolated RNA (1 µg) was reverse-transcribed to cDNA using a ReverTra Ace qPCR RT Master Mix (FSQ-301; Toyobo, Osaka, Japan) according to the manufacturer's instructions. Three microliters of each prepared cDNA, diluted 1:10 in DNase-and RNase-free water, was subjected to real-time PCR amplification using a Thunderbird SYBR qPCR Mix Kit (QPS-201; Toyobo) on a StepOnePlus real-time PCR system (Thermo Fisher Scientific K.K., Tokyo, Japan). The primers used are listed in Table 1. The amplification efficiencies of each target and the reference gene, ovine beta-actin (ACTB), were examined through their calibration curves and were found to be comparable [4,5]. The thermal profile consisted of 40 cycles at 95°C for 15 sec and annealing and extension at 60°C for 60 sec. The average threshold (Ct) values for each target were determined as described previously [4]. Melting curve analysis was performed for each run to confirm the specificity of amplification and the absence of primer dimers [4]. The expression levels of the transcripts were normalized to the levels of the reference gene, ACTB, using the 2 -ΔΔCt method [18].

Statistical analysis
All experiments were repeated independently at least three times. Statistical analyses were conducted using RStudio (Version 3.5.3), and the significance of each pairwise comparison was assessed using the Tukey-Kramer test. Statistical significance was set at P < 0.05.

Expression of EMT-related transcripts in conceptuses during the peri-implantation period
From our previous RNA-sequencing data [14], EMT-related transcripts were extracted, their expression was evaluated, and heatmap analysis was performed. The heatmap analysis revealed that transcripts encoding EMT-related genes were distinctively upregulated in P21 ovine conceptuses (Fig. 1). These analyses indicated that the increase in EMT-related transcripts and conceptus adhesion appeared to occur simultaneously in ovine conceptuses on P21 [14].

RT-qPCR, western blot, and immunohistochemical analyses of EMT-related factors in ovine conceptuses
Next, we examined the expression of EMT-related transcripts and proteins in P15, P17, and P21 ovine conceptuses. Quantitative PCR demonstrated the downregulation of cytokeratin (as KRT8), CDH1, and OVOL2 on P21, while there was an upregulation of VIM, CDH2, SNAI2, and ZEB1 ( Fig. 2A). These results are in agreement with previous reports [2,4,5]. Western blotting analysis revealed that cytokeratin and CDH1 levels decreased but were still present on P21, while CDH2 and VIM expression was only seen on P21 (Fig. 2B). The expression of CDH1 and VIM on these days was also confirmed through immunohistochemical analyses (Fig. 3). Although CDH1 expression was confined to the trophectoderm on P15 and P17, its expression on P21 appeared at the apical side of the trophectoderm, opposite to the attachment side. On P15 and P17, VIM expression was found in the stromal region, excluding the glandular epithelium or blood vessels. On P21, however, VIM expression appeared at the apical side of the trophectoderm as well as at the edge of the trophoblast undergoing possible cellular differentiation.

Determining the presence of bi-or multi-nucleated trophectoderms and expression of PAG transcripts, followed by immunohistochemical and immunofluorescence detection of PAG expression in ovine conceptuses
Immunohistochemical analysis revealed that some bi-nucleated trophoblast cells were detected on P17, but definitive bi-and multinucleated trophoblast cells, located on the uterine epithelial cells or next to the uterine stroma, were found on P21 (Figs. 3 and 5A).
From our previous RNA-sequencing data [14], PAG transcripts were extracted, and their expression patterns were analyzed (Fig. 4). Minute expression of PAG transcripts was found on P15 and P17, but an increase in PAG transcripts was observed on P21.
Immunohistochemical detection also revealed that while PAG expression was rarely found on P15, there were two patterns of PAG expression, presence or absence, in P17 conceptuses. Similar to PAG transcripts, distinct PAG expression was observed on P21 (Fig. 5A). These findings were confirmed via immunofluorescence analysis, in which PAG expression was associated with bi-and multi-nucleated trophoblast cells (Figs. 5B and 5C). It should be noted that bi-or multi-nucleated cells were not detected in the endometrial-stromal compartments on P15, P17, or P21 ( Fig. 3 and Fig. 5).

Discussion
We demonstrated that, similar to bovine conceptuses [2,4,5], sheep conceptuses underwent epithelial-mesenchymal transition (EMT) on P21, 3-4 days after the initial attachment of the trophectoderm to the uterine epithelium. Although sheep conceptuses expressed morphological and functional mesenchymal markers, VIM and CDH2, respectively, on P21, they also expressed epithelial markers such as cytokeratin and CDH1 on the same day. In particular, VIM expression in P21 conceptuses was restricted to the apical side, opposite the conceptus attachment to the uterine epithelium or the edge of the developing trophoblast. In contrast, CDH1 expression was found in conceptuses undergoing differentiation; thus, the trophectodermal cells that express VIM or CDH1 appear to differ. Yang et al. [1] provided a consensus statement in their EMT plasticity model that   In this study, some bi-nucleated cells appeared in P17 trophoblasts; however, distinctive bi-and multi-nucleated cells were found on P21. Although the molecular and cellular mechanisms in which EMT is required for multi-nucleated cell fusion or for when the two events occur simultaneously in P21 trophoblasts have not yet been elucidated, the observations in which P21 sheep trophoblasts undergo intermediate EMT suggest that they could have reduced cell-cell adhesion, increased cell individualization, or the acquisition of cell motility, possibly allowing active cell fusions between neighboring trophectodermal cells.
The cellular mechanisms associated with the formation of bi-and multi-nucleated trophoblast cells from mononucleated cells are not well understood; however, two theories on trophectoderm cell fusions currently exist: consecutive nuclear divisions without cytokinesis (mitotic polyploidy) [20] or the fusion of mononucleated trophoblast cells [21]. Among the numerous bi-and multi-nucleated trophoblast cells detected in our tissue sample sets, we did not find any fused cells containing both conceptus and endometrial origins or those in the endometrial-stromal compartments. This does not agree with those found in bovine species, wherein tri-or multi-nucleate cells of the trophectoderm and uterine epithelial origins often appear in the uterine endometrium [22]. However, our results agree with those rabbit antibody used in this study was raised against purified bovine PAG4 and PAG6, which detects bi-and multi-nucleated ovine trophoblast cells [18]. A: Immunohistochemical analysis and localization of PAG in P15, P17, and P21 uteri. As seen in Fig. 3, a few bi-nucleated trophectoderm cells are present in P17 uteri, and distinctive bi-and multi-nucleated trophectoderm cells exist in P21 uteri. There are two patterns of PAG expression on P17, one with PAG expression and the other without PAG expression; however, definitive PAG expression was seen on P21. Arrowheads indicate trophoblasts and small arrows indicate bi-nucleated cells. B: Immunofluorescence study of PAG expression in the P21 ovine uterus. A) and B) are bright-field images (BF) of PAG expression in C) and the control with normal rabbit IgG in D), respectively. The dotted white line indicates the separation between the trophoblast layer from the maternal tissues. Note that trophoblast cells are located next to the uterine epithelial layer in C). C: Various bi-and multi-nucleated trophoblasts are found in P21 uteri, all of which are associated with PAG expression. These cells were found next to the stroma as if they invaded the epithelial cells and replaced them, but trophoblast cells were not found within the stroma. LE: luminal epithelium of the endometrium, Str: uterine stroma, Tro: trophoblast. Scale bars = 50 μm or 100 μm.
from a recent publication [23], wherein fused cells comprised all trophectodermal cells in sheep. Although a definitive conclusion on the cell fusion between trophectodermal and maternal cells could not be made, the results from two studies ( [23] and this study) could challenge those believed for the last 40 years in which tri-or multi-nucleated cells result from the fusion between one endometrial epithelial and one bi-nucleate trophectodermal cell. We found that PAG transcripts were minutely expressed in P15 conceptuses and their expression slightly increased on P17. However, major PAG expression was found when bi-and multi-nucleated trophoblast cells were present on P21. Our previous proteome studies demonstrated that a predicted PAG2-like protein was detected in P15 ovine uterine flushing media, the expression of which increased on P17 [14]. In addition, the PAG proteins, PAG4 and PAG8, were detected in the uterine flushing media from P17 and P20 bovine uteri [4]. It should be noted that PAG expression is routinely found in the maternal circulation; thus, PAG expression is likely confined in the uterus until the conceptus adheres to the maternal endometrium. Based on our previous [4] and present experiments, PAG expression appeared prior to EMT; however, the elucidation of the functional relationship between these two events is beyond the scope of the current investigation.
In this study, bi-and multi-nucleated cells with PAG expression were often located in the epithelial layer next to the uterine stroma, as if multi-nucleated trophectodermal cells had replaced the uterine epithelial cells. To determine whether the disappearance of uterine epithelial cells was due to cell apoptosis, we attempted to identify the in utero expression of the apoptosis inducer caspase 3, followed by detecting DNA degradation through the TUNEL assay. Although minute expression of caspase 3 in one of the four sheep uteri examined was found in the epithelial cells, we did not find any TUNEL-positive staining (unpublished observations). Unlike the TUNEL-positive cells found previously by others [23], who concluded that the trophoblast could have triggered apoptosis in uterine epithelial cells, our data do not support this notion. Furthermore, the disappearance of uterine epithelial cells was observed during the implantation processes in mice; however, the mechanisms of epithelial cell removal, apoptosis, or necrosis have not been established [24]. Results from previous and present studies suggest that further investigation is required to determine the molecular and cellular mechanisms associated with conceptus implantation processes such as uterine epithelial cell removal, trophoblast EMT, and cell fusion beyond conceptus attachment and adhesion to the uterine endometrium.

Conclusion
We found that sheep conceptuses undergo epithelial-mesenchymal transition (EMT) during the period in which conceptus adhesion to the uterine epithelium takes place several days after the initiation of conceptus implantation to the maternal endometrium. Bi-nucleated cell formation and PAG expression begin before EMT, but the increase in the frequency of these events was found on day 21, coincident with EMT events in the trophectoderm in ruminants.