Syncytin-A knockout induces placental developmental abnormalities partially through calpain1-apoptosis-inducing factor-mediated trophoblast apoptosis
Dan Sun1, Hua-Yang Long2, Xiao He1, Wei-Wei Kang1, Juan Zhou1, Jian-Lei Huang1
1 Department of Obstetrics and Gynaecology, Second Affiliated Hospital, Air Force Medical University, Xi'an 710038, China
2 Department of Assisted Reproductive Medical Center, Sichuan Academy of Medical Sciences and Sichuan Provincial People's Hospital, Chengdu 610000, China
Source of Support: None, Conflict of Interest: None
DOI: 10.4103/2096-2924.320885
Objective: Structural abnormalities and dysfunction of the placenta contribute to pregnancy-related complications, such as preeclampsia. Syncytin-A (synA) has been reported to be expressed in the placenta. The contribution of synA to developmental abnormalities and dysfunction of the placenta remains elusive. In this study, we aimed to explore the role of synA in placental development and functions.
Methods: SynA-knockout mice were generated using the CRISPR-Cas9 method, and the phenotypes of the placenta and fetus of synA-knockout mice were observed. Real-time quantitative polymerase chain reaction (PCR) and routine PCR were employed to detect the genotypes of the offspring. CD31 immunohistochemistry was used to evaluate the vessel density of the placenta, and the protein levels of key molecules were measured by western blotting.
Results: SynA knockout caused fetal death. Furthermore, synA-knockout mice showed placental developmental abnormalities, indicated by a thinner labyrinth layer, thicker spongiotrophoblast layer, lower blood vessel density, and significantly higher numbers of apoptotic trophoblasts, when compared with wild-type littermates. Mechanistically, synA ablation induced apoptosis-inducing factor (AIF) cleavage and nuclear localization and promoted placental trophoblast apoptosis. In addition, synA knockout increased the calpain1 protein levels. The calpain1 inhibitor calpeptin blocked synA knockout-induced AIF cleavage, partially restoring the placental structural abnormalities of synA-knockout mice.
Conclusions: SynA knockout leads to placental developmental abnormalities by inducing trophoblastic apoptosis via the calpain1-AIF pathway.
Keywords: Apoptosis; Apoptosis-Inducing Factor; Placental Abnormality; Preeclampsia; Syncytin-A
Preeclampsia (PE) is a hypertensive complication of pregnancy that affects approximately 4%–5% of pregnancies worldwide.[1] PE patients often present with hypertension, proteinuria, and subsequent systemic organ dysfunction, which may increase maternal and prenatal morbidity and mortality.[2] The pathogenesis of PE is complicated and requires further exploration.
The placenta sits at the interface between the maternal and fetal vascular beds, where it mediates normal nutrient and waste exchange. Placental dysfunction and structural abnormalities, such as defective placentation, placental ischemia, and abnormal spiral artery remodeling, are recognized as the main causes of PE.[3] In patients with PE, placental syncytiotrophoblasts are always abnormal, characterized by significantly thinner, weakened, and disordered microvilli and a thickened basement membrane.[4],[5] These lesions seriously disrupt fetal-maternal exchange, resulting in fetal ischemia or even fetal death.[4],[5] Dysfunction and loss of placental syncytiotrophoblast cells contribute to the development of PE. However, the molecular mechanism underlying syncytiotrophoblast cell injury in PE remains elusive and requires further research.
The syncytin-1 gene is located on human chromosome 7q21–7q22, belonging to the various endogenous retrovirus family. It is reported that syncytin-1 is involved in human placental morphogenesis.[6] Syncytin-A (SynA) in mice is highly homologous to the syncytin-1 gene in humans. SynA localizes in the syncytiotrophoblast-containing labyrinthine zone, and the transcription level of synA increases from day 9.5 to day postcoitum.[7]SynA-mediated cell-cell fusion may provide a crucial clue to placental cytotrophoblast morphogenesis.[8] In addition, clinical studies have shown that the expression of syncytin protein is significantly downregulated and that it abnormally localizes to the apical syncytiotrophoblast microvillous membrane in the placenta of patients with PE,[9] which suggests that synA may be involved in syncytiotrophoblast injury during the development of PE. However, the exact molecular mechanisms remain unknown.
Apoptosis plays a critical role in inducing placental injury during the development of PE. Apoptosis-inducing factor (AIF) is a type of flavoprotein involved in mitochondrial energy production and redox reactions.[9],[10],[11] Under prolonged or severe stress, AIF is cleaved into a short fragment (approximately 57 kDa), and the cleaved AIF fragments then translocate into the nucleus, causing chromosome condensation, DNA fragmentation, and finally apoptosis.[9],[12]
In this study, we aimed to explore whether synA deletion induced placental structural and functional abnormalities in synA transgene mice and investigate the molecular mechanism underlying synA ablation-induced placental developmental defects. We hope that this study will be helpful in identifying novel targets for preventing and treating PE.
MethodsAnimals
Mice were raised in a temperature-controlled specific-pathogen-free grade facility and maintained at 26°C ± 2°C and 55% ± 15% relative humidity with a constant 12 h light/dark cycle. All procedures were conducted with the approval of the Animal Care and Use Committee of the National Institute of Biological Sciences, Beijing, in accordance with the governmental regulations of China. Calpeptin (Selleck, Houston, TX, USA) was dissolved in 5% dimethyl sulfoxide and injected intraperitoneally 0.04 mg every other day for 14 days, as previously reported.[13] The mice were then euthanized using pentobarbital. The tissues were harvested after animal sacrifice, fixed in 4% paraformaldehyde, or frozen immediately in liquid nitrogen.
Gene targeting
We utilized the CRISPR-Cas9 method to construct synA-knockout mice, as previously reported.[14] Two sgRNAs were found in the noncoding region of synA, and double-stranded sgRNAs were obtained using polymerase chain reaction (PCR). After in vitro transcription, they were injected with Cas9 RNA into fertilized eggs of C57BL/6J mice via prokaryotic microinjection methods, and these eggs were transplanted into pseudo-pregnant ICR mice. The F0 synA hetero mice were confirmed by PCR, and synA-knockout homozygous mice were generated using the germ-line transmission method.
Transferase dUTP nick end labeling assay
Placental cellular apoptosis was detected using the in situ cell death detection kit (Roche, USA). Following deparaffinization and rehydration, the slides were examined according to the manufacturer's protocol. Apoptotic cells and total cells were counted in 10 randomly chosen microscopic fields from three different slides for each group, and the ratio of apoptotic cells to total cells was calculated and compared with that in the wild-type (WT) littermates.
Histology and immunohistochemistry
Mice were sacrificed and carotid artery blood was collected; the fresh placentas were harvested, fixed at 4°C in 4% paraformaldehyde, embedded in paraffin, and stained with hematoxylin and eosin (HE). For AIF immunohistochemistry, paraffin sections were treated for thermally induced antigen repair and incubated with rabbit anti-mouse AIF antibody (1:100; Abcam, USA) and goat anti-rabbit antibody (1:200; ZSGB-BIO, China). CD31 immunochemistry was performed using rabbit anti-mouse CD31 antibody (Abcam, USA, 1:50). Tpbpa immunochemistry was conducted using rabbit anti-mouse Tpbpa antibody (Abcam, USA, 1:500).
Western blot analysis
Western blot analysis was performed as described previously.[15] The following antibodies were used for western blot analysis: anti-AIF polyclonal antibody (Abcam, USA, 1:5,000), anti-calpain1 (Abcam, USA, 1:1,000), and anti-caspase3 (Abcam, USA, 1:3,000). β-actin (Abcam, 1:5,000) was used as a protein loading reference.
Statistical analysis
GraphPad Prism Software 8 (Graphpad, California, USA) was used to analyze the data. All data are presented as mean ± standard deviation. Datasets with more than two groups were analyzed using analysis of variance, and datasets with two groups were analyzed using an unpaired t-test. Statistical significance was set at P < 0.05.
ResultsGeneration of Syncytin A-knockout mice
To study the role of synA in the placenta, we deleted the synA exon in mice using the CRISPR-Cas9 system, as previously reported.[14] Two Cas9 target loci (target 1 in the non-coding region (−9,354) and target 2 in the intron (−12,021)) were found in the synA gene [Figure 1]a and [Figure 1]b. We employed T7-driven expression of sgRNA1 and sgRNA2 cassettes, targeting the two targets within the synA gene [Figure 1]b. After amplifying gRNA in vitro, a mixture of Cas9 mRNA and gRNA for synA was microinjected into the zygote and transplanted into pseudo-pregnant ICR mice. PCR primer pair1 (forward primer1 [F1] + reverse primer [R1]) and PCR primer pair2 (F1 + R2) were designed for the WT allele (product size: 398 bp) and synA-knockout allele (product size: 367 bp), respectively [Figure 1]c and 1d]. The genotypes of the offspring were identified by PCR. PCR-based genotyping of E13.5 embryos showed that 1# was a WT mouse, 3#, 5#, and7# were synA+/− mice, and 2#, 4#, and 6# were synA−/− mice [Figure 1]d. As shown in [Figure 1]e, real-time quantitative PCR revealed that synA mRNA levels in synA−/− mice were significantly decreased compared with those in WT littermates. Taken together, we successfully generated synA−/− mice using the CRISPR-Cas9 method.
Syncytin A knockout results in embryo growth restriction and placental dysfunction in mice
To illustrate the effect of synA on the development and function of the placenta and fetus, the morphology, size, and weight of the placentas and embryos were observed. We first observed the distribution of offspring genotypes during intercrossing of synA+/− mice. SynA+/− mice were used to establish germ-line chimeras. As shown in [Supplementary Table 1], synA+/− mice were normal without significant differences in pregnancy rate, body weight, and the proportion of male and female offspring when compared with the WT mice. PCR-based measurement of embryo genotypes on embryonic day 13.5 (E13.5) showed that the genotype distribution of synA+/+, synA+/−, and synA−/− was nearly 1:2:1, which is consistent with the expected Mendelian ratio [Supplementary Table 2]. Surprisingly, we could not find any live synA−/− fetuses after E14.5 during the intercrosses of synA+/− mice, which suggests that synA knockout leads to embryo death on E14.5. To better clarify the mechanism by which synA affects the development of embryos, the size and weight of E11.5, E12.5, and E 13.5 embryos were observed. As shown in [Figure 2]a, 1# was the WT embryo, 3#, 5#, and 7# were synA+/− embryos, and 2#, 4#, and 6# were synA−/− embryos. SynA knockout induced abnormal embryonic development, leading to fetal loss in the 8#synA−/− embryo [Figure 2]a. In addition, synA knockout significantly inhibited embryo growth, as indicated by significantly decreased embryonic weight on E12.5 and E13.5 [Figure 2]b and [Figure 2]c. SynA−/− embryos exhibited pale and organ hemolysis on E13.5 [Figure 2]b. These data suggest that synA−/− adversely influences embryo development. Placental abnormalities contribute to embryonic developmental defects. We further studied the general morphology of the placenta. As shown in [Figure 2]d and [Figure 2]e, the weight of synA−/− mouse placentas showed no significant difference compared to that of WT littermates, but the fetal sides of synA−/− mouse placentas were paler and thinner than those of WT littermates [Figure 2]d, suggesting that synA knockout induced placental structural abnormalities. Taken together, these results suggest that synA knockout severely impairs placental function and restricts embryo development, suggesting that synA plays an essential role in placental and embryonic development.
Syncytin A knockout leads to abnormal placental structure in labyrinth and spongiotrophoblast layers
To better elucidate the mechanism responsible for defects in embryo development and placental dysfunction, we further analyzed placental histological changes in vivo. As shown in [Figure 3]a, a visible layer structure between the labyrinth (La) and spongiotrophoblast was observed in the placental section. SynA knockout led to significantly decreased width of the La layer and significantly increased width of the spongiotrophoblast layer in the placenta on E12.5, E13.5, and E14.5 [Figure 3]a, [Figure 3]b, [Figure 3]c. The significantly thickened spongiotrophoblast layer was further confirmed by immunofluorescence with anti-Tpbpa antibody, a marker of spongiotrophoblast cells [Figure 3]b. As reduced blood supply adversely influences placental development and function, we further studied the placental vessel density on E12.5, E13.5 and E14.5. As shown in [Figure 3]d, [Figure 3]e, [Figure 3]f, synA knockout notably decreased the vascular density in the synA−/− trophoblastic layer, as revealed by immunohistochemistry with CD31 antibody (endothelial cell marker). The number of fetal blood vessels significantly decreased on E12.5, E13.5, and E14.5 compared with that in WT placentas. In addition, synA−/− placenta exhibited significantly decreased and disorganized villous vasculature in the placental La on E12.5, E13.5, and E14.5 [Figure 3]e. These results suggest that synA knockout results in placental structural abnormalities and pathological microvasculature.
Syncytin A knockout induces placental cell apoptosis in the labyrinth layer through activation of apoptosis-inducing factor
To further determine the mechanism contributing to synA knockout-induced developmental defects in the placenta, we detected placental autophagy and cellular apoptosis in WT and synA−/− mice. As shown in [Supplementary Figure 1]a, synA knockout did not influence the placental autophagy level, as indicated by comparable protein levels of P62 and LC3 between the WT and synA−/− groups. SynA knockout led to significantly increased numbers of transferase dUTP nick end labeling (TUNEL)-positive cells in the placental La layer, compared with those in WT littermates [Figure 4]a and [Figure 4]b. In addition, we performed HE staining and merged with the TUNEL-positive cells, and found that the nuclei of TUNEL-positive cells were relatively large [Figure 4]a, suggesting that these apoptotic cells may be placental giant cells. AIF, caspase3, and caspase9 are commonly regarded as effectors mediating cellular apoptosis. AIF-mediated apoptosis has been implicated in tissue remodeling, hypoxia-induced cell death, and embryo development.[10],[16] AIF translocates into the nucleus upon pro-apoptotic signals and induces apoptosis by inducing DNA fragmentation.[11] To explore the mechanism underlying synA-induced apoptosis, we examined the AIF, caspase3, and caspase9 pathways in synA−/− mice. As shown in [Supplementary Figure 1]b, synA knockout did not induce increased protein levels of caspase9, pro-caspase3, cleaved-caspase3, Bax, and Bcl2. However, synA knockout elevated the protein levels of pro-AIF and cleaved AIF fragments [Figure 4]c. In addition, immunostaining showed that synA knockout significantly enhanced the colocalization of AIF in the nucleus [Figure 4]d. The ratio of AIF-nucleus-localized cells to total cells in the placenta also significantly increased [Figure 4]e. These results suggest that synA knockout induces placental cell apoptosis through an AIF-mediated mechanism.
Syncytin A knockout induces placental cell apoptosis through the calpain1-apoptosis-inducing factor pathway
Calpain1 is reportedly involved in AIF cleavage and activation.[12] To determine the role of calpain1 in AIF-induced placental cell apoptosis, we examined the protein level of calpain1. As shown in [Figure 5]a, synA knockout significantly increased calpain1 protein levels. The calpain1 specific inhibitor calpeptin significantly decreased the protein level of cleaved AIF [Figure 5]b and inhibited the nuclear entry of AIF in vivo [Figure 5]c. As shown in [Figure 5]d and 5e, the TUNEL assay showed that calpeptin significantly decreased synA knockout-induced placental cell apoptosis and partially restored the structural abnormalities in synA−/− mice [Figure 5]f. Taken together, these results suggest that synA knockout induced placental cell apoptosis and impaired placental development, partially through the calpain1-AIF pathway.
In this study, we made several important observations. First, we generated synA-knockout mice using the CRISPR-Cas9 method and found that synA knockout resulted in embryonic growth retardation and embryonic lethality on E14.5. We found that synA−/− embryos displayed significantly decreased body weight during embryonic development. Some synA−/− fetuses exhibited organ hemolysis, which suggests that synA knockout severely impaired embryonic development. As synA is mainly expressed in placental tissues,[7] we assume that synA knockout severely restricts normal placental development.
Second, we demonstrated that synA knockout led to placental structural abnormalities, such as a thinner La and thicker spongiotrophoblast layer. The abnormal placental structure may limit the normal oxygen and nutrient exchange between the mother and embryo and hinder fetal development. In addition, we found that synA knockout induced a significant decrease in vessel density in the placenta, which may disrupt blood supply to the placenta and cause placental developmental defects. Placental vascular abnormalities contribute to placental dysfunction in patients with PE.[17] However, the effect of synA knockout that leads to disordered placental vasculature requires further exploration. SynA receptor Ly6e deletion impairs syncytiotrophoblast fusion and placental morphogenesis, causing embryonic lethality in mice, suggesting that synA plays a crucial role in placental development. The findings of a previous study are consistent with our findings.[18]
Third, we demonstrated that synA knockout induced syncytiotrophoblast apoptosis through the calpain1-AIF pathway. Autophagy and apoptosis contribute to placental abnormalities and dysfunction in patients and mice with PE.[19],[20] Caspase3, caspase9, and AIF-correlated apoptotic pathways are involved in placental developmental defects in PE.[21],[22] AIF-mediated apoptosis is also thought to play a critical role in autophagy during neurological maturation and remodeling.[23],[24] We found that synA knockout did not affect the autophagy level and caspase3- and caspase9-mediated apoptotic pathway in E14.5 placental tissues but significantly increased AIF cleavage and nucleus translocation. In addition, we found that synA knockout elevated placental calpain1 protein levels. Pharmacological inhibition of calpain1 partially reversed AIF activation-induced placental apoptosis and structural abnormalities in synA−/− mice. Our results suggest that synA knockout induces syncytiotrophoblast dysfunction through calpain1-AIF-mediated apoptosis. SynA has been reported to affect placentogenesis by inducing cell-cell fusion.[25] However, the non-fusion function of synA upon placental development has received little attention. Our research demonstrated that synA knockout resulted in placental apoptosis through the calpain1-AIF pathway, which provides novel evidence of the non-fusion role of synA in placental development.
Although there were no preeclamptic symptoms, such as hypertension and proteinuria, in synA−/− mice (data not shown), the histological changes in the placenta of synA−/− mice were similar to those in PE mice.[26] In addition, studies have reported that syncytin-1 expression is decreased in preeclamptic placentas[9] and that reduced expression of syncytin 1 is correlated with the severity of PE.[27] AIF protein levels are known to be increased in preeclamptic placental tissues.[22] Our observation is consistent with these studies reporting that synA and AIF participate in placental developmental abnormalities, thus contributing to the development of PE. Currently, the treatment of PE is limited to symptom control and early termination of pregnancy owing to insufficient knowledge of the development of PE. Abnormalities of the placenta in synA−/− mice were similar to those of the placenta from patients with PE, suggesting that synA-knockout mice may serve as a suitable animal model for PE research.
This study demonstrated that synA exerts anti-apoptotic effects on placental development and that synA may be involved in the occurrence and development of PE. However, the underlying molecular mechanism requires further exploration.
Supplementary information is linked to the online version of the paper on the Reproductive and Developmental Medicine website.
Acknowledgement
We thank Hui-Shou Zhao for technical and material supports.
Financial support and sponsorship
Nil.
Conflicts of interest
There are no conflicts of interest.
References
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