At the basis of the development of a viable pregnancy in mammals there is the adhesion and implantation of a blastocyst to the endometrial epithelium. While the inner cell mass of the blastocyst will form the embryo, the outer layer, called the trophoblast, further develops into the maternal decidua and gives rise to the placenta. This latter represents the pivotal organ linking the developing embryo to the mother, providing the necessary oxygen supply and nutrient exchange [1]. Proliferation, apoptosis, and angiogenesis are crucial mechanisms involved in the correct development and remodeling of all the complex structures that make up the placenta. Impairments of this processes can result in several complications, including a deficient growth of ongoing pregnancy [2].

Chicken Ovalbumin Upstream Promoter-Transcription Factor I (COUP-TFI), also known as nuclear receptor subfamily 2, group F, member 1 (NR2F1) is an orphan nuclear receptor factor and member of the steroid/thyroid hormone receptor superfamily, mainly expressed in the central and peripheral nervous systems. The mammalian COUP-TFI plays a key role during metabolic homeostasis as well as organogenesis through cell fate determination, differentiation, proliferation, and apoptosis [3, 4]. Overall, the high conservation of amino acid sequence between species suggests vital evolutionary conserved functions worth being explored, also when considering the process of placental development [5, 6, 7].

Earlier studies conducted on human placenta highlight the potential key role played by COUP-TFI. An in-silico investigation of transcriptional profile obtained using a microarray approach revealed that COUP-TFI was highly associated with self-renewal and differentiation of human chorionic trophoblast progenitor cells [8]. Furthermore, COUP-TFI was indicated in a meta-analysis among the genes that seems to play a role in the development of pre-eclampsia, a complication of placental function often related to fetal growth restriction [9]. However, whether and how COUP-TFI could influence the process of placental development has not been evaluated yet.

Compared to the monolayer in human placenta (hemomonochorial), placenta in mice is composed by three trophoblast layers (hemotrichorial) [10]. At embryological (E) day 10.5, when mid-gestation begins, all the layers of the placenta are formed, including the outermost maternal part, called the decidua, and the fetal part with the triple trophoblastic layer [11, 12]. Embryonic development ends with the mid-gestation phase at E13.5, thereafter the fetus matures until the time of birth, around day E19.5 [13].

Transgenic COUP-TFI KO mice have been developed to shed more light into COUP-TFI functions [14]. Upon COUP-TFI loss, mice litter show a high incidence of perinatal mortality due to several neuronal malformations, particularly in the glossopharyngeal ganglion, defects in axonal arborization, and loss of cortical patterning due to the absence of thalamocortical connections [7, 15].

The aim of this study was to explore whether and how COUP-TFI deletion in mice could interfere with placental development in terms of expression of some genes related to proliferation, apoptosis, and angiogenesis and in terms of neonatal weight at birth.

According to RT-qPCR data, the most highly expressed gene in both COUP-TFI KO and WT placental tissue was HIF1$\alpha$, followed by Bax and p21. In addition, p21 resulted more expressed than p53, in both COUP-TFI KO and WT placental tissues (not shown). Interestingly, we found an increase of VEGF-A and Bax mRNA expression in placental tissue of COUP-TFI KO mice compared to their control counterparts. On the contrary, no significant differences were observed in mRNA expression of other marker of placental development, such as Flt-1, PlGF, Bcl-2, p21, p53, VEGF-A, HIF1$\alpha$, ENG, and INHA (Fig. 1 and Table 2).

Fig. 1.

Key placental genes expression pattern. Box plot showing fold change values (2${}^{(-\mathrm{\Delta }\mathrm{\Delta }Ct)}$) of key placental genes in COUP-TFI KO (grey boxes) compared to WT (black box) levels. The values refer to the median and IQR and the p-values to the Wilcoxon test.

Correlations between all evaluated transcripts in WT and COUP-TFI KO mice are shown in Fig. 2. We found significant positive correlations in the placental tissue of WT mice between the following mRNA pairs: Bcl-2 and INHA (rho = 1 and p 0.05); p21 and ENG (rho = 1 and p 0.05); HIF1$\alpha$ and Flt-1 (rho = 1 and p 0.05). Notably, such positive correlation between Bcl-2 and INHA, p21 and ENG, and between HIF1$\alpha$ and Flt-1 mRNA expression was lost in mutant placentas. Additional positive correlations were found in WT placental tissue between the following pairs, even though they did not reach statistical significance (not shown): p21 and p53 (rho = 0.9 and p = 0.083); p21 and HIF1$\alpha$ (rho = 0.9 and p = 0.083); p21 and Flt-1 (rho = 0.9 and p = 0.083); p21 and PlGF (rho = 0.9 and p = 0.083); ENG and p53 (rho = 0.9 and p = 0.083); ENG and HIF1$\alpha$ (rho = 0.9 and p = 0.083); ENG and Flt-1 (rho = 0.9 and p = 0.083); and ENG and PlGF (rho = 0.9 and p = 0.083). All tested correlations were no longer significant in the placental tissue of COUP-TFI KO samples, where we only found a negative correlation between Bcl-2 and PlGF, even though not significant (rho = –0.9 and p = 0.083).

Fig. 2.

Plots showing correlations between placental genes in WT and COUP-TFI KO mice. Panel (A) Shows correlation between INHA and Bcl-2. Panel (B) Shows correlation between ENG and p21. And Panel (C) Shows correlation between HIF1$\alpha$ and Flt-1. p-values and rho refer to Spearman test.

Finally, we found a reduction of weight in COUP-TFI KO pups compared to littermates controls. The mean weight of WT mice was 1.6 grams ($\pm$ 0.14), compared to 1.3 grams ($\pm$ 0.13) of COUP-TFI KO mice (p 0.05).

In this study, we assessed the role of mouse COUP-TFI in regulating the expression of major genes involved in placentation. We found that VEGF-A and Bax mRNA expression were increased in COUP-TFI KO compared to wild-type mouse placentas, suggesting an impairment of apoptotic and angiogenetic pathways in mutant placental tissue. The positive correlations observed in normal placental tissue between Bcl-2 and INHA, p21 and ENG, and HIF1$\alpha$ and Flt-1 were lost in COUP-TFI mutants, suggesting that key molecular networks could be imbalanced upon COUP-TFI loss. Interestingly, mutant mice also showed a significantly lower weight at birth compared to wild-type mice, raising the possibility that placental impairments described above could ultimately converge in sub-optimal weight gain during gestation.

We focused our attention on COUP-TFI because this family of nuclear receptors carries out vital roles in physiological processes, including proliferation, apoptosis and cell signaling [15, 20]. Thanks to the level of evolutionary conservation of COUP-TFI, understanding the pathological pathway in relation to its expression in mouse models could help to better focus future studies on genes known to be relevant during the process of physiological placental development. In particular, we analyzed the expression of HIF1$\alpha$, ENG, Flt1, PlGF, VEGF-A, which are main players in angiogenesis and vascular pathfinding, p21 and p53 in cellular proliferation, as well as Bax, Bcl2 and INHA, which are involved in apoptosis and cell survival. All these genes regulate crucial aspects of placental development, and their variation was shown to be associated with impaired development of the ongoing pregnancy [21, 22].

When considering the influence of COUP-TFI on the expression of the most common angiogenetic factors, we found that VEGF-A mRNA was consistently up-regulated in COUP-TFI KO placentas compared to control mice. The isoform A of the vascular endothelial growth factor (VEGF-A), belonging to the VEGF family, is considered the most crucial factor promoting the differentiation of mesenchymal cells in villi into hemangioblastic stem cells. VEGF-A expression is induced by hypoxia, as a potent stimulus, and is mediated via HIF1$\alpha$ expression [23, 24, 25, 26]. Indeed, VEGF-A is strongly expressed by the cytotrophoblast cells during the first trimester of pregnancy and strong evidence indicates that high VEGF-A expression in fetal growth restriction reflects the hypoxic status of the placenta [27, 28].

Supporting this evidence, we found in COUP-TFI mutants a loss in the positive correlation between mRNA expression of HIF1$\alpha$ and Flt-1, this latter encoding the vascular endothelial growth factor receptor 1 (VEGFR1), one of the receptors for vascular endothelial growth factors (VEGF). In a hypoxic environment, Hif1$\alpha$ could regulate the expression of VEGF-A, Flt-1 and other angiogenic factors, by means of a compensatory mechanism aimed to restore normal placental blood flow and to rescue normoxia [29, 30]. This finding overlaps with studies showing that VEGF-AmRNA and protein levels are significantly reduced in patients with growth restriction and that an adenovirus-mediated overexpression of VEGF can improve fetal growth in a sheep model [31, 32]. Regarding the other angiogenic factors considered in our study, PlGF and ENG, no significant differences were found in COUP-TFI KO placentas compared to control mice.

Considering the most common genes involved in cell proliferation and survival control, we observed an increase of Bax mRNA in mutant placentas. An augmented Bax expression is in line with other studies conducted on human placenta [33, 34, 35]. Bax is a pro-apoptotic protein that exerts, in concert with the anti-apoptotic protein Bcl-2, a crucial role in apoptosis. Both are regulated by the p53 tumor suppressor gene [36]. Apoptosis contributes to the turnover of villous trophoblasts and plays a crucial function in the remodeling of spiral arteries in human placenta. Apoptosis in placental villi changes throughout normal pregnancy: it is low in the first trimester, increases in the second, and then reaches the highest levels beyond 40 weeks of gestation [37]. Furthermore, the amount of apoptosis is increased in villous trophoblast in placental pathologies, including preeclampsia [38].

In addition, we observed a significant positive correlation of Bcl-2 and INHA in normal mice. This could be due to a regulatory role of on trophoblast growth through inhibition of the activin receptor, known to have a fundamental role in trophoblast development and correct placentation [39]. This, in turn, could result in reduced placenta proliferation and increased apoptosis characterizing old placentas at the end of gestation [40]. According to our study, this correlation seems to be altered by the absence of COUP-TFI.

Interestingly, we also observed a lower level of p53 expression than its downstream target p21 in COUP-TFI KO samples, and a positive correlation of the expression rate of the two genes. These data confirmed previous results on human placentas [21]. We can further hypothesize a role of p21 independent of p53. Usually p53, through p21, promotes cell cycle arrest or apoptosis via the augmented expression of Bax [36, 41]. In the current study, we found a significant positive correlation between p21 and ENG transcripts. ENG is part of the transforming growth factor-beta receptor complex. Angiogenesis, apoptosis, and cell cycle arrest could be promoted by the transforming growth factor-beta pathway, reported to be implicated in fetal growth restriction [42]. p21 could also possibly interact in placental tissue with this cascade triggered by the transforming growth factor-beta receptor complex [43]. As the correlation between p53 and p21 is lost in mutant mice, this interaction could be disturbed by the absence of COUP-TFI.

Finally, we looked at the link of the mouse phenotype to placental function in terms of weight of pups recorded at birth. Interestingly, our results showed that COUP-TFI KO pups presented a significant lower weight than WT littermate controls. These data further support the pathological significance of COUP-TFI in placental development, potentially related to fetal growth restriction, a common complication associated with impaired placental function in humans [9, 44, 45]. Our preliminary results invite further studies on specific downstream cascades of molecular markers linked to COUP-TFI, both in mouse models and humans.

This explorative study aimed to identify potential markers involved in impaired placental function linked to COUP-TFI loss-of-function but lacks a detailed analysis of mechanisms underlying the downstream regulation of angiogenic, cell regulation and apoptotic factors included in this mouse model. Moreover, in our experiments we have not assessed the function COUP transcription factor 2 (COUP-TFII), a homolog to COUP-TFI sometimes compensating COUP-TFI functions. COUP-TFI and COUP-TFII expression patterns overlaps in many regions and organs, possibly resulting in redundant functions [6, 46, 47]. Thus, COUP-TFII may be able to compensate for the absence of COUP-TFI in COUP-TFI KO mice. Further experiments on placentas lacking both COUP-TF members derive from crossing between COUP-TFI and COUP-TFII mutant mice, could shed new light on the interplay between these nuclear receptors during placental development. Exploring in more detail placental morphology could also be a topic of future interest.

The present study provides evidence that the absence of COUP-TFI influences the expression levels of two key effectors of mouse placental angiogenesis and apoptosis, VEGF-A and Bax. Consistently, we showed that COUP-TFI KO mice presented a significant lower weight at delivery than WT littermate controls. Hence, we propose that COUP-TFI plays an important role for placental development and function, even though further studies will be necessary to dissect the molecular dynamics governing a COUP-TFI-dependent placenta development.

Bax, BCL2-associated X protein; Bcl-2, B-cell lymphoma 2; COUP-TFI, Chicken Ovalbumin Upstream Promoter-Transcription Factor I; COUP-TFII, Chicken Ovalbumin Upstream Promoter-Transcription Factor II; ENG, Endoglin; FLT-1, Fms-like tyrosine kinase 1 (also known as VEGFR1); HIF1$\alpha$, Hypoxia inducible factor 1 alpha subunit; INHA, Inhibin alpha; mRNA, Messenger ribonucleic acid; VEGF-A: Vascular Endothelial Growth Factor A.

LV, SM, APL, MS, LM, AF—substantial contributions to conception and design. LV, SM, MB, APL, MO, SB, MS, LM, AF—substantial contributions to acquisition of data or to analysis and interpretation of data. LV, SM, MB, APL, MO, SB, LD, CDL, MS, LM, AF—drafting the article or revising it critically for important intellectual content. All authors read and approved the final manuscript.

All mouse experiments were conducted in accordance with the relevant national and international guidelines and regulations (European Union rules; 2010/63/UE), and with approval by the local ethical committee in France (CIEPAL NCE/2019-548).

The authors would like to thank Christian Alfano from iBV, Nice, France, for initial interest in this study and the Pathology of the AOU Santa Maria della Misericordia, University of Udine, Italy, who collaborated in the study.

This research received no external funding.

The authors declare no conflict of interest. APL is the Editor of this journal, given his role as Editor, had no involvement in the peer-review of this article and has no access to information regarding its peer-review.

The data that support the findings of this study are available, but restrictions apply to the availability of these data, which were used under license for the current study, and so are not publicly available. Data are however available from the authors upon reasonable request and with permission of the Internal Review Board.