Identification of a syncytin gene in a non-rodent laboratory mammal, Suncus murinus

Abstract

An endogenous retrovirus-derived membrane protein, syncytin (SYN), contributes to placental function via trophoblast fusion. Multinuclear trophoblasts (syncytiotrophoblasts) physically and functionally mediate the interaction between fetal and maternal vessels in various ways. Suncus murinus (suncus) is a small mammalian species with a pregnancy duration of approximately 30 days, 1.5 times longer than mice. However, the molecular basis for the longer pregnancy duration is unknown. In this study, we first isolated two genes that encoded putative SYN proteins expressed in the suncus placenta, which were named syncytin-1-like proteins 1 and 2 (SYN1L1 and SYN1L2). When their expression vectors were introduced into cultured cells, suncus SYN1L2 was found to be active in cell fusion. Moreover, the SYN1L2 protein was homologous to a SYN1-like protein identified in greater mouse-eared bats (bat SYN1L) and was structurally compared with bat SYN1L and other SYN proteins, implying the presence of structural features of the SYN1L2 protein.

Mammals are mainly divided into three groups: monotremes, marsupials, and placentals [24]. The monotremes lay and warm their eggs, whereas the marsupials and placentals are viviparous. Placentas are formed temporally inside the uterine cavity of marsupials and placentals. The placenta of placentals (chorioallantoic placenta; hereafter, placenta) is morphologically and functionally distinct from that of marsupials, leading to different reproductive strategies.

From feto-maternal interface, placentas are classified into distinct types such as labyrinthine in mice and villous types in humans [15]. Placentas are also divided into several types based on the fetal and maternal barriers [20]. In hemochorial barriers, formed in humans and mice, one single or two layers of multinuclear trophoblasts (syncytiotrophoblasts) separate the maternal blood from the fetal vessels. On the other hand, endotheliochorial barriers are formed in most carnivores such as dogs and cats, and the barrier between fetal vessels and maternal blood consists of syncytiotrophoblasts and the maternal endothelium.

The Asian house musk shrew, Suncus murinus (laboratory name: suncus), is a small mammalian species that was previously classified into the order Insectivore and now belongs to the order Eulipotyphla [18, 24]. Due to its unique features, the suncus has been employed as a mouse-sized, non-rodent laboratory animal. The laboratory Ous:KAT strain, established in Japan, is easily bred and has a fetal life stage that is 10 days longer than that of mice [1]. The controversial suncus placenta has been classified as hemochorial, endotheliochorial, or endothelio-endothelial type [13].

In the suncus placenta, syncytiotrophoblasts interpose between the hypertrophied maternal endothelium and the fetal endothelium by embryonic day 20 (E20) of the pregnancy [9, 13]. Syncytiotrophoblasts become discontinuous in the labyrinthine zone after E24, and the maternal endothelium contacts the fetal endothelium by the end of the pregnancy [9, 13]. As reported previously [9, 13], the gross appearance of the placenta at the stage of E19 is similar to that of a mouse placenta, but the color is dusky-red. When the suncus placenta is compared with the mouse placenta, the suncus placenta is an intense red color due to broadly formed vessels. The maternal vessels at the fetal-maternal boundaries are easily distinguished from fetal vessels by their comparatively thick endothelium and smaller red blood cells, as reported previously [9]. In contrast, layers of syncytiotrophoblasts separate the maternal blood from the fetal vessels in mice. Moreover, the suncus placenta is highly invasive with a complex labyrinthine pattern [9]. This finding indicates that two phases (endotheliochorial and endothelio-endothelial) control the placental function in the suncus.

Mammalian genomes consist of massive copies of ancestrally infected retroviruses [22]. Their non-replicable copies are termed endogenous retroviruses, which are distinguished from replicative and infective exogenous retroviruses [14]. A subset of endogenous retroviruses still encodes functional proteins. Remarkably, a retroviral surface envelope protein (ENV) has been identified as syncytin (SYN), which promotes cell fusion between mononucleate trophoblasts to form a layer of syncytiotrophoblasts at the maternal-fetal interface [17]. Furthermore, Syn genes have developed from distinct types of endogenous retroviruses, and their functional domains are diversified in placental mammals [3].

Paired retroviral genes (syncytin-1 and -2 [SYN1 and 2], and syncytin-A and -B [Syn-A and -B]) have been identified in humans and mice, respectively [3]. These sets of genes were independently acquired through ancient retroviral infections. They commonly exhibit trophoblast fusion and are predominantly expressed in the placenta [3]. We also expect that endogenous retroviruses largely contribute to the formation of the suncus placenta. However, genes encoding SYN remain unidentified in the suncus genome. In the present study, we identified endogenous retrovirus-derived genes, expressed in the suncus placenta.

MATERIALS AND METHODS

Animal care

The male and female adult suncuses (12–24 weeks old) studied here are an outbred Ous:KAT strain established from a wild population in Kathmandu, Nepal. They were maintained at 25°C under a 12-hr light and 12-hr darkness cycle, and provided commercial trout pellets (No. 5P; Nippon Formula Feed Manufacturing, Yokohama, Japan) following an established procedure [1]. Individual suncus was housed in specific pathogen-free controlled conditions. Food and water were available ad libitum.

All experiments were approved by the Institutional Animal Care and Use Committee of the National Research Institute for Child Health and Development (Experimental number, A2007-001).

Isolation of placenta

For mating, the female was housed with the male. The day after mating was denoted as embryonic day 0 (E0). The gestation period was approximately 30 days. Embryos were collected from pregnant females and euthanized using CO₂. The embryonic stage was identified using the morphological features of the embryos, as described previously [18]. At least, three placentas from three animals were examined for each stage.

Amplification of DNA fragments

The sequences of primers selected were based on sequences conserved between mouse and human genes encoding SYNs (Accession No. NP_001013773.1, NP_775596.1, NP_001124397.1, and NP_997465.1). The following 2 primer sets were used: 5′-ACCTCGAGCATAATGAGTGG-3′ (forward-1) and 5′-ACAAGCTTTTTGTGTTGTGAC-3′ (reverse-1); 5′-ACGTCGACCATAATGAGTG-3′ (forward-2) and 5′-CCGGATCCTAGGAGTCAATG-3′ (reverse-2). The suncus genome was extracted from tail fragments of the adult suncus and subjected to PCR analysis. The PCR products were amplified by the following procedure: Initial denaturation at 95°C for 3 min; 35 cycles of denaturation at 98°C for 10 sec, annealing at 60°C for 5 sec, and extension at 72°C for 1.5 min. The final cycle was followed by extension at 72°C for 5 min. The amplified products with a set of primers (forward-1 and reverse-1) were digested with restriction enzymes (XhoI and HindIII). The amplified products combined with a set of primers (forward-2 and reverse-2) were digested with restriction enzymes (SalI and BamHI). The digested products were subcloned into pBluescript SK (−) (Agilent Technologies, Ltd., Santa Clara, CA, USA) using a DNA ligation kit (Takara-Bio Inc., Kusatsu, Japan). Direct sequencing analysis was performed with a BigDye Terminator v3.1 cycle sequencing kit and ABI sequencer (Thermo Fisher Scientific, Waltham, MA, USA; FASMAC, Atsugi, Japan).

Reverse transcription (RT)-PCR

Previous works [9, 13] indicate the existence of two steps, endotheliochorial, and endothelio-endothelial phases, during placental formation in the suncus. Since placentas at stages E14, E19, E24 and E28 have structural features, we selected placentas at these four stages.

After collection, the placentas were immediately treated with RNAlater (Thermo Fisher Scientific) and stored at −80°C until required for RNA extraction. Total RNA was extracted from samples using ISOGEN (Nippon Gene Co., Ltd., Tokyo, Japan) according to the manufacturer’s protocol. First-strand cDNA was synthesized from 100 ng of total RNA with oligo-dT primers using SuperScript^TM III Reverse Transcriptase (Thermo Fisher Scientific) according to the manufacturer’s protocol. The sequences of primers for RT-PCR analysis were based on the sequences of the Serv-1 and Serv-2 genes. The following two primer sets were used: Serv-1 (SERV1-F, 5′-CGGATGACGGAGGACCAAAA-3′ and SERV1-R, 5′-CCATACTCGTCCTGGGGGTA-3′); Serv-2 (SERV2-F, 5′-CTCCCTTCCCTCTGAAAAGGT-3′ and SERV2-R, 5′-TCCCCATCCCCACCTTAGTAG-3′). RT-PCR analysis was performed to estimate the expression of Serv-1 and Serv-2. The RT-PCR products were amplified as follows: initial annealing and denaturation at 50°C for 2 min, then repeated at 95°C for 2 min, followed by 40 cycles of denaturation at 95°C for 15 sec, and annealing and extending at 60°C for 1 min. The amplified products were electrophoresed on 2% agarose gels.

Real-time RT-PCR

The placenta and six organs (heart, muscle, intestine, liver, kidney, and lung) were removed from pregnant females and immediately treated with RNAlater (Thermo Fisher Scientific) and stored at −80°C until required for RNA extraction. Total RNA was extracted from samples using ISOGEN (Nippon Gene Co., Ltd.) according to the manufacturer’s protocol. First-strand cDNA was synthesized from 100 ng of total RNA with oligo-dT primers using SuperScript^TM III Reverse Transcriptase (Thermo Fisher Scientific) according to the manufacturer’s protocol. Real-time PCR was performed with SYBR Green dye and QuantStudio 12K Flex real-time PCR system (Thermo Fisher Scientific). Ten microliters of 2 × Platinum^TM SYBR^TM Green qPCR SuperMix-UDG (Thermo Fisher Scientific) were mixed with the template and primers. The total reaction volume was 20 μL. The sequences of primers for real-time RT-PCR analysis were also designed based on the sequences of the Serv-1 and Serv-2 genes. The primer sets for Serv-1, Serv-2, and a gene encoding ribosomal protein L19 (Rpl19) were the same as those used for RT-PCR. In the present study, the expression of Rpl19 was used for normalization. The following primer sets were used: Rpl19 (RPL19-F, 5′-TGCTAACTCGCGTCAACAGAT-3′ and RPL19-R, 5′-GCATTCTGGCATTGGCGGTA-3′). The reaction was performed by the following process: Initial annealing and denaturation at 50°C for 2 min and at 95°C for 2 min; 40 cycles of denaturation at 95°C for 15 sec, and annealing and extension at 60°C for 1 min. Experiments were performed in triplicate for each sample. For all genes evaluated, mRNA was normalized to Rpl19 mRNA by subtracting the cycle threshold (Ct) value of Rpl19 mRNA from the Ct value of the gene of interest (ΔCt). The fold difference (2-ΔΔCt) was calculated by subtracting the ΔCt (treated sample) −ΔCt (normoxic sample) to generate a ΔΔCt.

Transfection of expression vectors

As described above, amplified products with two sets of primers (forward-1 and reverse-1; forward-2 and reverse-2) were subcloned into pBluescript SK (−). After sequencing, these plasmids were digested with XhoI and BamHI and electrophoresed. The cDNA fragments were extracted from agarose gels using QIAEX II Gel Extraction Kit (QIAGEN, Venlo, Netherlands) and were inserted into XhoI and BamHI sites in multiple cloning sites (MCS) of pIRES2-EGFP (BD Bioscience Clontech, Franklin Lakes, NJ, USA), which carries the internal ribosome entry site of the encephalomyocarditis virus between the MCS and a coding region of the enhanced green fluorescent protein (EGFP). This plasmid permits both the gene of interest and the EGFP gene to be translated from a single bicistronic mRNA under the control of the immediate early promoter of cytomegalovirus. The expression vectors were transiently transfected into a derivative of human embryonic kidney HEK293 cell line (HEK293T) using lipofectamine 3000 (Thermo Fisher Scientific), and the expression of EGFP proteins was observed after 48-hr incubation. Their transfection efficiency was calculated as percentage of EGFP-expressed HEK293T. To explore a physiological role of SERV-2 proteins, we measured the percentage of multinuclear cells. Nuclei were counterstained with Hoechst 33342.

Statistical analysis

Significant differences (P-value) were calculated using Student’s t-test and statistical significance was defined as P<0.05. Results were expressed as mean ± standard deviation of the mean (SD).

RESULTS

Identification of endogenous retrovirus-derived genes

Approximately 10% of mammalian genomes are copies stemmed from ancestrally infected retroviruses. These can be easily traceable because of the fragmented and mutated sequences of three viral genes, encoding structural proteins, viral enzymes, and retroviral surface envelope proteins (ENVs) [7]. As depicted in Fig. 1a, an ENV protein has been identified as SYN, which is cleaved into two subunits (surface [SU] and transmembrane [TM]) at a furin cleavage site (arginine-serine-arginine-arginine, RSRR). A cysteine-tryptophan-leucine-cysteine (CWLC) motif localized in the SU subunit is thought to be involved in the interaction between SU and TM subunits. The immunosuppressive domain (ISD) motif localizes at the N-terminal region of the TM subunit [7].

Fig. 1.

Isolation of two retroviral surface envelope proteins from the suncus genome. (a) Schematic structure of syncytin (SYN) protein. SU: surface; CWLC: a conserved motif in the SU region; RSRR: a putative furin cleavage site; ISD: immunosuppressive domain; TM: transmembrane domain. (b) Amplification of two retroviral surface envelope proteins (ENVs) from the suncus genome with two sets of primers. The expression of two ENVs was examined in transcripts isolated from embryos staged at E14, E19, E24, and E28 by RT-PCR. G: suncus genome. (c) Schematic structures of two ENVs, Serv-1 and Serv-2. Full nucleic acid sequences and putative amino acid sequences were attached as supplementary information. (d) Real-time RT-PCR. The expression levels of Serv-1 and -2 were quantified from mRNAs extracted from seven organs. For all genes evaluated, mRNA was normalized to ribosomal protein L19 (Rpl19) mRNA by subtracting the cycle threshold (Ct) value of Rpl19 mRNA from the Ct value of the gene of interest (ΔCt). Experiments were performed in triplicate for each sample. Values are expressed as mean ± standard deviation.

To examine the unique features in placental formation of the suncus, we next explored the endogenous retrovirus-derived genes expressed in the placenta. Two primer sets were designed based on sequences conserved between mouse and human SYN genes (see Materials and Methods for more details). When PCR analysis was performed using the suncus genome, a single major band was amplified from each of the two primer sets (Fig. 1b). These two fragments (1,851 and 1,843 base pairs [bp]) encoded open-reading frames (605 and 607 amino acids [aa]) with four SYN-specific motifs mentioned above (Fig. 1c, and Supplementary Figs. 1 and 2). These two putative proteins were tentatively named suncus endogenous retrovirus-1 and -2 (SERV-1 and -2). Serv-1 was 97.7% and 98.1% homologous to Serv-2 at nucleic acid and amino acid sequences, respectively (Supplementary Figs. 3 and 4), and three sequences (CWLC, RSRR, and ISD motifs) matched completely.

To examine the expression of Serv-1 and -2 genes, expression analysis was performed for transcripts extracted from the placenta and six other organs (heart, muscle, intestine, liver, kidney, and lung). The expression of a gene encoding ribosomal protein L19 (RPL19) was examined as an internal control. When we carried out the real-time RT-PCR, Serv-1 was expressed dominantly in both the placenta and muscle (Fig. 1d). In contrast, Serv-2 was expressed in all seven tissues and was highly expressed in the muscle and lung. From this result, we considered that both Serv-1 and -2 were expressed in the placenta (Fig. 1d).

Transfection of expression vectors into cell lines

To examine the function of SERV-1 and -2 proteins, their expression vectors (pCMV-SERV-1 and pCMV-SERV-2) were transfected into a derivative of human embryonic kidney cell line (HEK293T) [6]. SERV-1 and SERV-2-expressed cells were identified by bicistronic expression of enhanced green fluorescent proteins (EGFPs). We calculated the percentages of EGFP-positive cells and multinuclear cells, presumably fused cells, in transfected cells, compared with those of empty vector-transfected cells as a control.

When pCMV-SERV-2 and control vector were transfected, EGFP-positive cells were observed (Fig. 2a). In contrast, the EGFP fluorescence was highly fragmented in pCMV-SEV-1-transfected cells (arrowheads in Fig. 2a). Correspondingly, the percentage of EGFP-positive cells was 20.5 ± 6.6% and 16.1 ± 6.5% in empty vector- and pCMV-SERV-2-transfected cells but was significantly lower in pCMV-SERV-1-EGFP-transfected cells (3.4 ± 0.8%; P=0.0010) (Fig. 2b). From this result, we assume that SERV-1 may have a toxic effect on HEK293T cells.

Fig. 2.

Transfection of SERV-1 and SERV-2 expression vectors into HEK293T cells. (a) HEK293T cells transfected of pCMV-SERV-1 or pCMV-SERV-2. SERV-1- and SERV-2-expressed cells were identified by bicistronic enhanced green fluorescent protein (EGFP) expression. Control: empty plasmid; BF: bright field; arrowheads: fragmented EGFP-expressed cells; boxes: areas enlarged in below. Scale bars: 50 µm. (b) Percentage of EGFP-positive cells. Experiments were performed in sextuplicate for each sample. N.S.: not significant. Values are expressed as mean ± SD. (c) Multinuclear cells formed in pCMV-SERV-2-transfected cells. After transfection, their nuclei were stained with Hoechst 33342 (H33342). Arrowheads: multinuclear cells. Scale bars: 50 µm. (d) Percentage of multinuclear cells in EGFP-positive cells. The percentage was compared between pCMV-SERV-2-transfected and control cells. Experiments were performed in triplicate for each sample. Values are expressed as mean ± SD.

To explore a possible role of SERV-2 in the formation of multinuclear cells, pCMV-SERV-2-transfected cells were further examined. After transfection, their nuclei were stained with Hoechst 33342 (H33342). The multinuclear cells were observed in pCMV-SERV-2-transfected cells (Fig. 2c). Correspondingly, when the number of multinuclear cells in EGFP-positive cells was counted, its percentage was significantly high (21.6 ± 2.1) in pCMV-SERV-2-transfected cells, compared with that of control cells (8.7 ± 3.0; P=0.0035) (Fig. 2d). This result suggests that SERV-2 has the ability to induce the formation of multinuclear cells.

Structure prediction of SERV-1 and SERV-2

To explore the structural difference between SERV-1 and SERV-2, these structures were predicted using AlphaFold2 program (Fig. 3). The SU region mediates receptor recognition, while the TM region acts as a class I viral fusion protein [4]. Since their functions are distinct and separated finally [4], their structures were predicted independently. During membrane fusion, coiled-coil regions in the TM region are assumed to form a trimer-of-hairpin structure [4]. According to the AlphaFold2 program, the accuracy was expressed as five colored regions (red: very low; yellow: low; green: OK; light blue: confident; blue: very high). Consequently, trimmer-of-harpins-like structures were predicted for both proteins (lower images in Fig. 3). The structure of the TM region was rated as highly accurate except for carboxyl (C)-terminus in both proteins, implying that the structure of the TM region may be conserved between SERV-1 and SERV-2.

Fig. 3.

Predicted structures of SERV-1 and SERV-2. These structures were predicted using AlphaFold2 program. Each predicted model has an average estimated reliability (predicted local distance difference test, pLDDT). Red: very low (<50); yellow: low (60); green: OK (70); light blue: confident (80); blue: very high (>90). SU: surface; TM: transmembrane. Arrowheads: outer loops in the SU region; arrows: coiled-coil regions.

On the other hand, SU regions appeared to be structurally distinct (upper images in Fig. 3). Two outer loops (arrowheads in Fig. 3), predicted for both proteins, were rated as poorly accurate. Therefore, the comparison of these regions was excluded. In contrast, coiled-coil regions (arrows in Fig. 3), rated as highly accurate, were predicted for both proteins, but this region was hidden in SERV-1 and exposed in SERV-2. Correspondingly, five amino acid substitutions with different charges were found in the SU region, but not in the TM region (Supplementary Fig. 4). From this result, we assumed that the structural change of the SU region could lead to the functional difference between these two proteins.

Search for proteins homologous to SERV-2

We then focused on SERV-2, which had the ability to promote the formation of multinuclear cells (Fig. 2). To search proteins homologous with SERV-2, the NCBI protein BLAST search was performed, and proteins were listed (Supplementary Fig. 5). Except for the top four sequences we have registered, the most homologous sequence was a SYN-1-like protein identified in the greater mouse-eared bat (Myotis myotis) (bat SYN1L) (sequence ID: XP_036157207.1). In addition, proteins identified in the common brushtail possum (Trichosurus vulpecula), the Middle East blind mole-rat (Nannospalax galili), the hunting common shrew (Sorex araneus), the velvety free-tailed bat (Molossus molossus), the koala (Phascolarctos cinereus), the big brown bat (Eptesicus fusus), and the ring-tailed lemur (Lemur catta) were listed. As shown in Fig. 4a, the sequence of bat SYN1L was compared with SERV-2. Throughout their whole sequences, 47% of amino acids were identical, whereas 39% in the SU region and 58% in the TM region were identical. This result suggests that the sequence of the TM region is more homologous than that of the SU region between SERV-2 and bat SYN1L.

Fig. 4.

Comparison of SERV-2 amino acid sequence with bat syncytin-1-like protein (SYN1L). (a) Comparison of their amino acid sequences. Gray-colored background: surface (SU) region; yellow-colored background: transmembrane (TM) region. Boxes indicate cysteine-tryptophan-leucine-cysteine (CWLC), furin cleavage site (arginine-serine-arginine-arginine; RSRR), and immunosuppressive domain (ISD) motifs, and TM domain. (b) The structure of bat SYN1L. Red: very low (<50); yellow: low (60); green: OK (70); light blue: confident (80); blue: very high (>90). Arrowheads: outer loops in the SU region; arrow: coiled-coil regions.

In turn, the structure of bat SYN1L was predicted using AlphaFold2 program (Fig. 4b). The trimmer-of-harpins-like structure was predicted with high accuracy. The coiled-coil region (an arrow in Fig. 4b) was exposed in the bat SYN1L.

Structural comparison with SYNs from different types of placentas

From feto-maternal interdigitation, human and mouse placentas are classified into different types: villous and labyrinthine types, respectively [15]. Hence, the structures of human SYN1 and SYN2 (Fig. 5a), and mouse SYN-A and SYN-B (Supplementary Fig. 6) were predicted using AlphaFold2 program. Consequently, trimmer-of-harpins-like structures were predicted for these four proteins. TM regions were rated as highly accurate except for both C- and amino (N)-termini in these proteins, implying that the structure of the TM region could be conserved beyond species.

Fig. 5.

Predicted structures of human and opossum syncytins. (a) The structures of human syncytin-1 (SYN1) and syncytin-2 (SYN2). (b) The structure of opossum syncytin (SYN-OPO-1). Red: very low (<50); yellow: low (60); green: OK (70); light blue: confident (80); blue: very high (>90). Arrowheads: outer loops in the SU region; arrows: coiled-coil regions.

SU regions were also predicted in these four proteins, but the structures of mouse SYN-A and SYN-B were rated as poorly accurate (Supplementary Fig. 6). In human SYN1 and SYN2, the SU regions were rated as highly accurate except for the outer regions (arrowheads in Fig. 5a). The coiled-coil region with high accuracy (arrows in Fig. 5a) was predicted for both proteins, but this region was exposed in human SYN1 and hidden in human SYN2.

In marsupials, the gray short-tailed opossum (Monodelphis domestica) has a Syn gene, Syn-Opo1, which encodes a fusogenic protein [4]. Interestingly, this gene is dominantly expressed in the feto-maternal interface of a placenta-like structure that forms transiently [4]. We next predicted the structure of SYN-OPO1. Consequently, trimmer-of-harpins-like structures were predicted for this protein. The TM region was rated as highly accurate except for both C- and N-termini in this protein, implying that the structure of the TM region could be conserved beyond species. The SU region was rated as highly accurate except for the outer region (an arrowhead in Fig. 5b), but the coiled-coil region was hidden in SYN-OPO1 (an arrow in Fig. 5b). This result reinforces that the structural change of the SU region could lead to the functional difference among SYNs.

DISCUSSION

To explore the molecular basis underlying placental formation in the suncus, we here identified two genes that encoded ENV proteins expressed in the suncus placenta. Our results indicate that one of these two genes, SERV-2, encodes SYN with fusogenic ability, possibly contributing to the unique placental formation and longer pregnancy duration, despite the small body size of the suncus. The homology search revealed that SERV-2 was highly homologous to bat SYN1L. SERV-1 and SERV-2 shared the considerable homology with human SYN-1, compared with human SYN-2, and mouse SYN-A and SYN-B (Supplementary Figs. 8 and 9). Based on their similarity to human SYN-1, these two putative proteins were named SYN-1 like proteins 1 and 2 (SYN1L1 and SYN1L2) (Accession No. LC547502 and LC547503).

Although the amino acid homology between suncus SYN1L1 and SYN1L2 is very high (Supplementary Figs. 3 and 4), structural differences, especially in the SU region, are observed. On the other hand, the amino acid sequences and their structures were conserved among suncus, bat, human, mouse, and opossum. Further analyses are needed, but the SU region may be important for the physiological activity of each SYN and their functional differences.

The human placenta is classified as a hemochorial type, which is a unique interface between fetal and maternal tissues [20]. The hemochorial placenta, found in all monkeys, is commonly linked with a reduced number of offspring. No other mammal has a placental type identical to that of humans and monkeys. Although the gross shapes are morphologically similar, the placentas of some non-human primates exhibit low trophoblast invasiveness [2]. The mouse placenta shares hemochorial and disc-like characters with the human placenta, but the syncytiotrophoblasts are less invasive and less branched in the mouse placenta [2].

The suncus placenta is categorized as a labyrinthine placenta that is similar to the mouse placenta, but has highly invasive and highly branched features [13]. In addition, the number of offspring in the suncus is lower than in mice. The formation of highly invaded and vascularized labyrinthine placenta contributes to the lower number of offspring in the suncus. SYN1L1 and SYN1L2 are more homologous to bat SYN1L (sequence ID: XP_036157207.1) than to those of humans [17] or mice [8]. Bats have either an endotheliochorial or a hemochorial placenta, depending on the bat species [11]. Indeed, placentation of bats is extremely diverse and therefore even placentas with the hemochorial interface may differ considerably. The emergence of Syn genes is predicted to be a widespread process and repeatedly happen in widely separate lineages amid mammalian expansion [4]. Therefore, the SYN family may be frequently homologous beyond species.

Cytotrophoblasts are morphologically analogous to malignant tumor cells [5]. They both are highly invasive with a multinuclear formation, leading to the formation of a highly vascularized placenta or solid tumors. In particular, suncus cytotrophoblasts are more invasive than those of mice and rats, and are comparable with the highly invasive cytotrophoblasts in the hemochorial placentas of primates, including humans [5]. Similarly, the placenta in moles also has highly invasive morphological features [21]. There is a critical difference between tumor progression and placental formation [5]. There are tight regulation controls on placental formation, whereas tumor cells depart from normal cellular rules. In mammals, tumors are formed at high frequencies, suggesting the reactivation of a mechanism controlling trophoblast function, such as the expression of SYN proteins, in tumor cells [5].

In general, both marsupials (which have a yolk sac [choriovitelline] placenta) and placentals are thought to have evolved from a common ancestor more than 100 million years ago [9]. In marsupials, opossums have a Syn gene, Syn-Opo1, which encodes a fusogenic protein SYN-OPO1 [4]. Interestingly, this gene is dominantly expressed in the feto-maternal interface of a placenta that forms transiently [4]. In contrast, non-fusogenic ENVs are widely distributed in marsupials [4]. Our results show that suncus SYN1L2 has an amino acid sequence shared with bat, opossum, human, and mouse SYNs, more specifically in the TM region (Fig. 4, and Supplementary Figs. 7, 8, and 9). Marsupials are predicted to have evolved from one group of ancestral placentals with the disruption of Syn genes, for example, the insertion of additional sequences. Since SYN-OPO1 retains fusogenic activity [4], the immunosuppressive function, in addition to fusogenic activity, may contribute to placental formation [10, 16].

As mentioned above, the placental formation of the suncus consists of two phases, endotheliochorial and endothelio-endothelial [13]. In particular, the endothelio-endothelial phase is unseen in other placentals, even in the placenta-like structures of marsupials. Multiple steps frequently regulate organogenesis; therefore, the linkage between two and more steps may promote the formation of elaborate tissues and upgraded tissue functions.

Two-phase organogenesis is also useful for the in vitro formation of organoids from human iPSCs [19]. In contrast, adhesion between the endometrium and endometrium occurs in cancer metastasis, and endothelial cell displacement by tumor cells occurs at the contact site between the endometrium and endometrium [12]. However, the occurrence of spontaneous tumors is quite rare in the suncus [23]. This implies suncus possess a mechanism that utilizes the characteristics of tumor cells and suppresses their uncontrollable behavior.

CONFLICT OF INTEREST

The authors declare no competing financial interests.