Fetal growth restriction exhibits various mTOR signaling in different regions of mouse placentas with altered lipid metabolism

doi:10.21203/rs.3.rs-3557723/v1

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Fetal growth restriction exhibits various mTOR signaling in different regions of mouse placentas with altered lipid metabolism

https://doi.org/10.21203/rs.3.rs-3557723/v1

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published 07 Mar, 2024

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Fetal growth restriction (FGR) is a common complication of pregnancy and can have significant impact on obstetric and neonatal outcomes. Increasing evidence has shown that the inhibited mechanistic target of rapamycin (mTOR) signaling in placenta is associated with FGR. However, interpretation of existing research is limited due to inconsistent methodologies and varying understanding of the mechanism by which mTOR activity contributes to FGR. Hereby, we have demonstrated that different anatomic zones of human and mouse placentas exhibited different levels of mTOR activity in normal compared to FGR pregnancies. When using the rapamycin-induced FGR mouse model, we found that placentas of FGR pregnancies exhibited abnormal morphological changes and reduced mTOR activity in the decidual-junctional zone. Using transcriptomics and lipidomics, we revealed that lipid and energy metabolism was significantly disrupted in the placentas of FGR mice. Finally, we demonstrated that maternal physical exercise during gestation in our FGR mouse model was associated with increased fetal and placental weight as well as increased placental mTOR activity and lipid metabolism. Collectively, our data indicate that the inhibited placental mTOR signaling contributes to FGR by disrupting lipid metabolism in different anatomical regions of mouse placentas, and maternal exercise could be an effective method to reduce the occurrence of FGR or alleviate the adverse outcomes associated with FGR.

fetal growth restriction

placenta

mTOR signaling

lipid metabolism

maternal exercise

Fetal growth restriction (FGR) is a commonly encountered complication during human pregnancy and is frequently associated with low birth weight or small for gestational age (SGA) neonates. The global prevalence of FGR ranges between 3–7% of all pregnancies (Chew and Verma 2022), yet developing countries experience a strikingly higher incidence of approximately 20% among live births (Lee et al. 2017). FGR can be associated with significant perinatal morbidity and mortality as well as adverse long-term developmental and neurologic outcomes (von Beckerath et al. 2013). The underlying etiology of FGR can be heterogenous and associated with maternal conditions (e.g., malnutrition, hypertension, preeclampsia, environmental toxins), fetal anomalies (e.g., genetic aberrations, multiple fetuses), or placental dysfunction (Malhotra et al. 2019). Inadequate placental perfusion is thought to be responsible in the majority of FGR cases (Burton and Jauniaux 2018).

The placenta is a vital organ of pregnancy and proper function is required to ensure adequate fetal growth and development. The placenta acts as a maternal-fetal immune barrier and is responsible for the transfer of nutrients and oxygen from the maternal circulation to the developing fetus while also secreting hormones and cytokines (Mess and Carter 2007). In response to suboptimal uterine environments (including gestational diabetes, malnutrition, inflammation, or infection) the placenta exhibits a remarkable adaptive ability to regulate fetal growth (Sferruzzi-Perri and Camm 2016). However, when placental function becomes severely compromised due to maternal or genetic conditions significant fetal affects can be observed, including the development of FGR. Nevertheless, the specific molecular mechanisms underlying the relationship between abnormal placental function and FGR remain inadequately understood.

The mTOR signaling pathway is critical in the regulation of cellular metabolism, growth, and survival, responding to cues from nutrient availability, energy levels, and growth factors (Saxton and Sabatini 2017). Two distinct forms of mTOR exist, mTORC1 and mTORC2. mTORC1 governs cellular growth and metabolism via the acceleration of protein, lipid, and nucleic acid synthesis, while mTORC2 is primarily implicated in cell proliferation and survival (Saxton and Sabatini 2017). A salient function of mTORC1 is its ability to promote protein translation via the phosphorylation of downstream effector molecules, including ribosomal S6 kinase (S6K) and eukaryotic translation initiation factor 4E binding protein 1 (4EBP1) (Burnett et al. 1998). Notably, the initiation and elongation of mTORC1-dependent protein translation hinges on the phosphorylation of ribosomal protein S6 (RPS6), which serves as a direct downstream effector of S6K (Holz et al. 2005). Consequently, the phosphorylation levels of mTOR, S6K, RPS6, and 4EBP1 often represent useful indices of mTOR signaling activity (Dong et al. 2020).

Increasing evidence indicates that the placental mTOR signaling pathway plays a crucial role in balancing maternal nutrient availability to support fetal growth (Dong et al. 2020; Roos et al. 2009). However, the underlying mechanism by which reduced mTOR activity in the placenta contributes to FGR remains fully unclear. In fact, despite the majority of studies showing inhibition of mTOR activity in FGR placentas, inconsistencies remain which hinder the ability to clearly understand the relationship between placental mTOR activity and fetal growth (Dong et al. 2020). The heterogeneity in research results among studies may due to not using identical methods to measure mTOR activity, or the potential impact of different cell types (or anatomic zones) on mTOR signaling in the placenta. Our previous research with mouse placentas demonstrated that different anatomical regions exhibit varying mTOR activity, as indicated by downstream effectors, when exposed to maternal inflammation (Dong et al. 2019). Based on these findings, we proposed that key molecules involved in mTOR signaling, such as mTOR, S6K, RPS6, and 4EBP1, may exhibit distinct activities across different placental zones.

In the present study, we conducted a comprehensive investigation of mTOR signaling activity in various anatomic layers of human and mouse placentas under normal conditions. We then examined differences in mTOR signaling in various placental zones in human placentas from SGA pregnancies and mouse placentas using the rapamycin-induced FGR model (rapamycin being a known inhibitor of mTOR activity). We utilized transcriptomic and lipidomic analyses to understand the molecular mechanisms underlying the role of mTOR signaling in FGR. Lastly, since we observed an increased high fat state in mouse placenta of rapamycin-induced FGR in mice, we investigated the potential of maternal exercise as a moderator to promote fetal growth. The objective of our study was to elucidate the precise mechanism by which mTOR signaling may contribute to FGR, therefore identifying interventional targets in the prevention of FGR.

Sample collection

Human placenta and umbilical cord blood collection

All placentas were collected immediately after delivery of full-term newborns by elective cesarean section without pregnancy-related complications (such as gestational hypertension, diabetes, placenta abruption etc.). Neonates were classified as either SGA, if the birth weight was below the 10th percentiles for gestational age, or appropriate for gestational age (AGA), if the birth weight was between the 10th to 90th percentiles for gestational age. Placental tissue (~ 0.5 cm thick) was harvested from both the fetal and maternal placental sides, near the umbilical cord insertion site. The biopsies were washed in cold PBS to remove blood, immediately frozen in liquid nitrogen, and stored at -80℃ for subsequent use.

Mouse sample preparation

All animal experiment procedures were conducted in Air Force Medical University according to the Animal Protection Guidelines of Air Force Medical University (SYXK2019-001). Six-week-old ICR female mice and adult ICR male mice were obtained from Beijing HFK Bioscience Company (Beijing, China). All mice were housed at 22–24 ℃ with constant light between 8 am and 8 pm and had unrestricted access to food and water. Female and male mice were caged together at ratio of 1:1 to facilitate mating. Embryonic day 0.5 (E0.5) was defined based on identification of a vaginal plug the day after mating. Pregnant mice were sacrificed by cervical dislocation. We collected mouse placentas at E14.5 (a time point for mouse placenta maturation) or E18.5 and performed tissue separation with the aid of a dissecting microscope. To obtain the different zones of mouse placentas, we transected the mouse placenta in half, then identified divided one half to decidual and junctional (DJ) layer and labyrinth (L) layer according to placental structure. The isolated samples were preserved in liquid nitrogen and stored at -80℃. A subset of intact samples was fixed in 4% paraformaldehyde to facilitate histological analysis.

Mouse model of FGR

The FGR mouse model was developed using pregnant 6-week-old ICR female mice, as illustrated by Shao et al (Shao et al. 2021). They were subjected to daily intraperitoneal injection of either 0.01 mg or 0.02 mg rapamycin (Selleck, #1039, USA) or Dimethyl sulfoxide (DMSO, Sigma, #D8418, USA) diluted in phosphate-buffered saline (PBS) from E10.5 to E13.5. Pregnant mice were sacrificed by cervical dislocation at E14.5 or E18.5. At that time maternal blood samples were collected and fetal and placental data were recorded. The same procedures used for placental dissection was performed as the above mentioned.

Maternal exercise

To explore the effects of exercise during pregnancy on fetal weight in the rapamycin-induced FGR model pregnant mice were subjected to flat treadmill exercise. The running parameters were set according to the method published by Son et al. (Son et al. 2019). In Brief, pregnant mice were separated into three stages, E1.5 – E7.5, E8.5 – E14.5, and E15.5 – E16.5. The exercise protocol consisted of three phases: warming up (5 m/min for 10 min), exercise (10–14 m/min for 40 min), and cooling down (5 m/min for 10 min), which were performed at the same time every morning. The treadmill speed during the exercise phase was set to 11, 14, and 12.5 m/min for E1.5 – E7.5, E8.5 – E14.5 and E15.5 – E16.5, respectively. Mice were not included in running exercise between E16.5 and E18.5 to avoid possible acute effects of this activity on sample collection at E18.5.

Histological analysis of mouse placenta

Mouse placentas were fixed in paraformaldehyde overnight before being processed and embedded into paraffin wax for histological staining. Each sample was sectioned at 6 µm thickness perpendicular to the chorionic plate (Leica RM2235, Germany). Slides were then stained with hematoxylin & eosin (HE). All stained slides were covered by dibutyl phthalate polystyrene xylene mounting medium (Sigma) and scanned (Olympus VS120, Japan) to assess placental thickness and distinguish DJ and L layers. For immunohistochemistry (IHC) staining, the slides were incubated in heated antigen unmasking solution (Vector Laboratories, USA) for antigen retrieval, and the remaining steps were similar to those previously described (Dong et al. 2019). The primary antibodies for IHC included Tpbpa (a marker for spongiotrophoblast cells, Abcam, #ab104401), Vimentin (a marker for interstitial cell, Abcam, #ab8978) and CD31 (a marker for endothelial cells, Abcam, #ab182981). The secondary antibodies included Goat anti-rabbit IgG Alexa Flour 568 or 488 and Goat anti-mouse IgG Alexa Flour 568 (Life Technologies). All IHC slides were finally covered using Fluromount-G (eBioscience). Images were photographed and scanned using the Olympus VS120. We analyzed the intensity and density of positive regions on IHC slides by Image J software (NIH, Bethesda, USA).

Western blot (WB)

For protein sample extraction, placental tissues were homogenized on ice in Protein Extraction Reagent (Thermo, #78510) with protease inhibitor (MCE, #HT-K0011) and phosphatase inhibitor (Roche, #04906837001). The homogenized specimens were then placed on ice for 20 minutes and centrifuged at 14,000 rpm for 20 minutes at 4°C, the resultant supernatant was isolated. We used Coomassie Staining solution (Beyotime, #P0017F, China) to determine the quality and loading volume of protein samples. Total protein was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis using 4–20% gels (Biorad, #4561096, USA) and then transferred onto PVDF membranes using Trans-Blot Turbo RTA Transfer Kit (Biorad, #1704272) via a semidry transfer device (Bio-Rad). Membranes were blocked with 5% bovine serum albumin (MP, #FC0077) in Tris-buffered saline containing 0.1% Tween-20 (TBST) for 30 minutes at room temperature, incubated with primary antibodies in 5% BSA at 4°C overnight, then washed with TBST three times (10 minutes each). The secondary antibody was HRP-labelled goat anti-rabbit IgG. Protein bands were visualized using an Immobilon Chemiluminescence Reagent (Millipore, #P90720) and recorded by a ChemiDox Gel imaging system (Biorad). The primary antibodies (diluted at 1:1000) used were Tpbpa (Abcam, #ab104401), phospho-mTOR Ser2448 (p-mTOR, CST, #5536), total mTOR (t-mTOR, CST, #2983), total S6K (t-S6K, CST, #2708), phospho-RPS6 Ser235/6 (p-RPS6, CST, #4858), total RPS6 (t-RPS6, CST, #2217), phospho-4EBP1 Thr37/46 (p-4EBP1, CST, #2855), and total 4EBP1 (t-4Ebp1, CST, #9644). Although the level of phosphor-S6K Thr389 (p-S6K, CST, #9234) was also detected, difficulty in identifying specific bands in both mouse and human placentas made it impossible to accurately analyze placental p-S6K and therefore was not analyzed. To determine the oxidative phosphorylation (OXPHOS) level, we utilized an antibody kit (Thermo, #45-8099) suitable for detecting the relative levels of the five OXPHOS complexes (CⅠ-Ⅴ) by WB. Immunological blots corresponding to CⅠ, CⅡ and CⅤ were observed. For analyzing the target protein levels, total protein shown by Coomassie staining was used as the reference and Image J software was applied to calculate protein band gray values.

mRNA sequencing

Total RNA was extracted from mouse placentas using RNeasy mini kits (Qiagen, Germany) according to the operation manual. The integrity of RNA was identified by Agilent Bioanalyzer 2100, and the concentration and purity were measured by Qubit®3.0 Fluorometer (Life Technologies, USA) and Nanodrop One spectrophotometer (Thermo). The mRNA sequencing libraries were constructed using NEBNext® UltraTM RNA Library Prep Kits for Illumina NovaSeq 6000 (USA) following the manufacturer’s protocol. Paired-end mRNA libraries were constructed by using the TruSeq™ RNA Sample Preparation Kits (Illumina, USA) following the guidebook. Briefly, the poly-A containing mRNA molecules were purified using poly-T oligo-attached magnetic beads. After purification, the mRNAs were fragmented into small pieces using divalent cations under 94℃ for 8 minutes. The cleaved RNA fragments were copied into first strand cDNA using reverse transcriptase and random primers. This was followed by second strand cDNA synthesis using DNA Polymerase I and RNase H. These cDNA fragments then went through an end repair process, the addition of a single A’ base, and ligation of the adapters. The products were then purified and enriched by PCR to create the final cDNA library. Purified libraries were quantified by Qubit® 2.0 Fluorometer (Life Technologies, USA) and validated by Agilent 2100 bioanalyzer (Agilent Technologies, USA) to confirm the insert size and calculate the mole concentration. Clusters were generated by cBot with the library diluted to 10 pM then sequenced on the Illumina NovaSeq 6000 (Illumina, USA). Differential expression analysis for mRNA was performed using R package. The thresholds of the differentially expressed (DE) mRNA were set as |log2(fold change)| ≥ 1.5 and p-value < 0.05, and retained for further analysis. Functional enriched signaling pathways were analyzed for the overlapped DE mRNAs. Gene Ontology (GO) biological process, Kyoto Encyclopedia of Genes and Genomes (KEGG) Pathway, Reactome Gene Sets and WikiPathway terms were searched and enriched by Metascape.

Real-time quantitative polymerase chain reaction (RT-qPCR)

Total RNA of mouse placentas was extracted using Trizol reagent (Invitrogen, #15596026) according to the instructions and added DNaseI to avoid DNA disruption. The purity and concentration of RNA were determined by a Nanodrop 2000 spectrophotometer (Thermo). A total of 0.5 µg of total RNA was converted to cDNA using a commercial reverse transcription kit (Takara, #6210A). Each cDNA sample was diluted 10-fold, followed with reaction system construction. The reaction mixture for RT-qPCR consisted of 2 µL of diluted cDNA, 0.8 µL of each primer (0.1 µM), 10 µL of SYBR premix Ex Taq™ Ⅱ (Takara, #RR42LR) and 6.4 µL of nuclease-free water. RT-qPCR amplification was performed under the following steps: 95°C for 3 minutes, 40 cycles at 95°C for 15 seconds, 58°C for 30 seconds and 72°C for 2 minutes. All samples were run in duplicate. The expression levels of target genes were normalized by housekeeping gene β-Actin. The primer sequences were obtained from Primer Bank, provided from Sangon biotech company (Shanghai, China) and shown in Supplementary Tables 4 and 5. The ΔΔCt method was used for analyzing the mRNA relative expressions.

Lipidomics analysis

Lipids were extracted from approximately 30 mg of frozen placental tissue using a modified version of the Bligh and Dyer's method as described by Lam et al. (Lam et al. 2022). Briefly, tissues were homogenized in 750 µL of chloroform, methanol, and MilliQ H₂O solution (3:6:1, v/v/v). The homogenate was then incubated at 1500 rpm for 1 hour at 4℃. Phase separation when then induced by adding 350 µL of deionized water and 250 µL of chloroform. The samples were then centrifuged and the lower organic phase containing lipids was extracted.

Lipidomic analyses were conducted at LipidALL Technologies using a Shimadzu Nexera 20-AD coupled with Sciex QTRAP 6500 PLUS. Separation of individual lipid classes of polar lipids by normal phase (NP)-HPLC was carried out using a TUP-HB silica column (i.d. 150x2.1 mm, 3 µm) with the following conditions: mobile phase A (chloroform, methanol, ammonium hydroxide, 89.5:10:0.5) and mobile phase B (chloroform, methanol, ammonium hydroxide, water, 55:39:0.5:5.5). Multiple reaction monitoring (MRM) transitions were set up for comparative analysis of various polar lipids. Individual lipid species were quantified by referencing to spiked internal standards. Free fatty acids were quantitated using d31-16:0 (Sigma) and d8-20:4 (Cayman Chemicals). Glycerol lipids including diacylglycerols (DAG) and triacylglycerols (TAG) were quantified using a modified version of reverse phase HPLC/MRM. Separation of neutral lipids were achieved on a Phenomenex Kinetex-C18 column (i.d. 4.6x100 mm, 2.6 µm) using an isocratic mobile phase containing chloroform, methanol, 0.1 M ammonium acetate 100:100:4 (v/v/v) at a flow rate of 300 µL for 10 minutes. Levels of short-, medium-, and long-chain TAGs were calculated by referencing to spiked internal standards of TAG(14:0)3-d5, TAG(16:0)3-d5 and TAG(18:0)3-d5 obtained from CDN isotopes, respectively. DAGs were quantified using d5-DAG17:0/17:0 and d5-DAG18:1/18:1 as the internal references (Avanti Polar Lipids). Free cholesterols (Cho) and cholesteryl esters (CE) were analyzed under atmospheric pressure chemical ionization mode on a Jasper HPLC coupled to Sciex 4500 MD as reported by Shui et al. (Shui et al. 2011), using d6-cholesterol and d6-C18:0 CE as internal standards.

Assay of (free fatty acid) FFA, TAG and Cho

For measuring FFA, TAG, and Cho concentrations in mouse placentas, we utilized the relevant commercial kits purchased from Solarbio (Beijing, China), including FFA detection kit (Cat: #BC0590), TAG detection kit (Cat: #BC0620), total Cho detection kit (Cat: #BC1980), and free Cho detection kit (Cat: #BC1890). The spectrophotometry testing procedures were in accordance with the instructions. The level of Cho Easter was calculated by subtraction of free Cho from total Cho.

Statistical analysis

Data processing and graphic production were achieved using Graphpad Prism software 8. Continuous variables were presented as mean ± standard deviation. For two-group comparison, the student’s t-test was used for parametric data while the Mann–Whitney test for nonparametric data. For multiple group analysis, one-way ANOVA was employed and Tukey’s test was for subsequent pairwise comparisons. A p-value < 0.05 was considered significant.

Differential expression of key molecules in mTOR pathway in different anatomic zones of human placentas

We firstly compared the levels of the total and phosphorylated mTOR-related proteins (including mTOR, S6K, RPS6 and 4EBP1) in the placentas of normal human pregnancies (clinical information shown in Supplementary Table 1). We found that p-RPS6 and p-4EBP1 had significantly higher levels in the fetal side of placenta compared to the maternal side, and the levels of p-mTOR, t-mTOR, t-S6K, t-RPS6 and t-4EBP1 were similar between the fetal and maternal sides (Fig. 1a and b). The results suggested that the activity of placental mTOR signaling in the fetal side was greater than that in the maternal side.

Subsequently, we analyzed the changes in mTOR activity in the fetal and maternal sides of placentas from SGA compared to control, or AGA, neonates. The baseline clinical data are reported in Supplementary Table 2. Our results showed that in the maternal side of SGA placentas the expression of t-mTOR was significantly decreased while the level of p-4EBP1 was increased, compared to controls (Fig. 1c). However, in the fetal side of SGA placentas, we only observed a robust reduction in the level of p-RPS6 (Fig. 1d). These findings suggest that mTOR activity exhibits inconsistent patterns in different anatomic locations of human placentas under both normal and SGA conditions.

Differential expression of mTOR-related proteins in different anatomic layers of mouse placentas

As depicted in Fig. 2a, we partitioned the mouse placenta into three segments: the decidua junction (DJ) layer, the labyrinth (L) layer, and the composite DJ and L (DJ + L) layer. By means of IHC staining of Tpbpa, a specific biomarker for spongiotrophoblast cells, we distinguished the DJ and L layers in the mouse placenta (Fig. 2b). The RT-qPCR and WB assays demonstrated that Tpbpa mRNA and protein were preferentially expressed in the DJ layer compared to the L layer, indicating the significance and purpose of our mouse placenta separation methodology for subsequent analyses (Fig. 2c and 2e). We assessed the protein levels and phosphorylation activities of four key mTOR-related genes (mTOR, S6k, Rps6, and 4Ebp1) in mouse placentas. Our results indicated that the levels of p-mTOR, t-mTOR, and t-S6k were significantly lower in the DJ layer than in the L layer, whereas the levels of p-4Ebp1 and t-4Ebp1 were markedly higher in the DJ layer than in the L layer. No differences were found in the levels of p-Rps6 and t-Rps6 between the DJ and L layers (Fig. 2d and 2e). Our findings demonstrate that the key molecules in mTOR signaling exhibit variable expression and phosphorylation activity across distinct anatomic regions in mouse placentas.

Differential expression of mTOR-related proteins in different anatomic layers of rapamycin-induced FGR mouse placentas

Given the differential expression and phosphorylation of key proteins in the mTOR signaling pathway across anatomical regions of normal mouse placenta, we sought to investigate whether there would be variation in these findings with FGR placentas. Utilizing the rapamycin-induced FGR mouse model (Fig. 3a), pregnant mice receiving low-dose (0.01 mg/kg) or high-dose (0.02 mg/kg) rapamycin exhibited significantly reduced fetal and placental weight at E14.5, with the survival rate of fetuses being lower in the high-dose group (Fig. 3b, 3c). We primarily compared control and low-dose rapamycin groups in the subsequent experiments.

To confirm the inhibitory effect of rapamycin on placental mTOR signaling in mice, we examined mTOR activity in the three parts of mouse placentas, as depicted in Fig. 2a. Our results showed that in the DJ layer the levels of p-mTOR, p-Rps6, and t-Rps6 were significantly reduced in rapamycin-treated compared to control placentas (Fig. 3d and Figure S1a-d). In contrast, no changes were observed in the levels and activities of mTOR-related targets in the L layer between the two groups (Fig. 3e and Figure S1e-h). Notably, in placentas containing both DJ and L components, there was a substantial decrease in the levels of p-Rps6 and t-Rps6 in the rapamycin group compared to control (Fig. 3f and Figure S1i-l). These data collectively suggest that FGR established by inhibiting placental mTOR signaling in our mouse model develops through variable levels of activity of key mTOR signaling molecules in different layers or zones of the placenta.

Histological findings in rapamycin-induced FGR mouse placentas

To investigate the potential mechanisms underlying FGR induced by rapamycin treatment, we conducted morphological analyses of control and FGR placentas. HE staining revealed that FGR placentas were smaller in size and thinner than control placentas, especially in the junctional zone (Fig. 4a). To study the change of the spongiotrophoblast distribution in mouse placentas, we analyzed Tpbpa protein, which labels spongiotrophoblast cells. We observed that the Tpbpa-positive area was relatively flat in FGR placentas exposed to rapamycin, compared to the irregular shape observed in control placentas (Fig. 4b). Moreover, the ratio of Tpbpa-positive area was significantly reduced in FGR placentas (Fig. 4c). To examine the overall distribution of mesenchymal cells and vascular endothelial cells in mouse placentas, we performed IHC staining using Vimentin and CD31, respectively. Our results revealed that Vimentin expression was similar, but CD31 intensity was significantly decreased in FGR placentas compared to controls (Fig. 4d-f). Collectively, our findings suggest that the mouse placenta displays abnormal morphological characteristics in the FGR model induced by rapamycin.

Analysis of mRNA sequencing in control and rapamycin-induced FGR mouse placentas

To explore the underlying molecular mechanisms involved in FGR induced by rapamycin, we conducted mRNA transcriptome analysis on mouse placentas from three groups: control, low-dose rapamycin, and high-dose rapamycin. Compared to control placentas, we identified 68 differentially expressed (DE) mRNA in the low-dose rapamycin group and 106 DE mRNA in the high-dose group (Fig. 5a). To further refine the DE transcripts, we focused on the 37 overlapped DE mRNA (12 upregulated and 25 downregulated) among the three groups and generated a heatmap (Fig. 5b). Subsequent enrichment analysis of the DE mRNAs revealed multiple biological processes including regulation of development growth, regulation of lipid localization, organ growth, intestinal lipid absorption, and receptor ligand activity. (Fig. 5c). Cnetplot of KEGG enrichment analysis also reflected the involvement of the lipid-related pathways and corresponding genes (Fig. 5d). Given the crucial role of lipid metabolism in placental and fetal growth, we further identified the expressions of five genes (Enpp7, Acacb, Cyp4f18, Msr1 and Clps) associated with lipid metabolism. The mRNA levels of Enpp7, Acacb, and Clps were significantly decreased in both the DJ and L layers of FGR placentas compared to control (consistent with the mRNA sequencing results), while the expressions of Cyp4f18 and Msr1 were not altered in FGR placentas (Fig. 5e and 5f). Collectively, these findings suggest that FGR induced by rapamycin may be attributed to the disruption of lipid metabolism within the mouse placenta.

Analysis of lipid molecules in different anatomic layers in rapamycin-induced FGR placentas

Lipids are essential for placental and fetal development and their activity may be altered in FGR placentas, as indicated by the enrichment analysis results of DE mRNAs mentioned previously. We thusly compared the lipid components between control and low-dose rapamycin groups using lipidomics analysis in both the DJ and L layers of mouse placentas. The original data are listed in Supplementary Excel 1 and 2, respectively. The principal component analysis revealed that the concentration of various lipid molecules in FGR placentas differed significantly from controls, regardless of the tissue origin (DJ layer or L layer) (Fig. 6a). Specifically, we analyzed 29 different types of lipids in the DJ and L layers of mouse placentas, comparing lipid content of rapamycin-induced FGR placentas to those of control placentas (Fig. 6b, c). We found that in the DJ layer four types of lipids (FFA, CE, Gb3 and CL) were substantially upregulated, while GM3 was downregulated (Fig. 6d), and in the L layer seven lipid types (DAG, PS, CE, TAG, FFA, CL and BMP) were significantly increased, whereas acylcarnitine was decreased (Fig. 6e). We quantified the comparative values of individual lipid types and found that 138 lipid molecules were changed in the DJ layer and 246 lipids changed in the L layer of FGR placentas compared to controls (Fig. 6f). A total of 61 different lipid components were simultaneously changed in the DJ and L layers of mouse placentas between the two groups (Fig. 6F, Supplementary Table 3). When further examining lipid types, we found that in general those lipids involved in energy metabolism, such as TAG and FFA, were up-regulated in FGR placentas while structural type lipids, such as CL, PE, and PS, were down-regulated (Supplementary Table 3).

Previous studies have reported that rapamycin administration can impact lipid concentrations in circulating blood (Morrisett et al. 2002; Framarino-dei-Malatesta et al. 2014). We measured the serum concentration of the three major lipids FFA, TAG, and Cho in pregnant mice and did not observe a difference between control and rapamycin groups (Figure S2a-c). Our data suggests that FGR affects the levels of multiple lipids in mouse placentas without impacting maternal circulating lipids.

To further understand the potential mechanism underlying the lipid changes observed in FGR placentas, we assessed the mRNA levels of several genes involved in lipid oxidation (Cpt1a, Cpt1b, Cpt1c, Acaca and Acacb), lipogenesis (Fasn), lipid transport (Slc27a2, Slc27a4, Slc27a6 and Cd36), and relevant regulation factors (Srebf1, Srebf2, Soat1, Soat2, Ppar-α, Ppar-β/δ and Ppar-γ). The results showed that in the DJ layer the most investigated genes (including Acaca, Fasn, Slc27a6, Srebf1, Srebf2, Soat1, Soat2, Ppar-β/δ and Ppar-γ) were lower mRNA levels in FGR placentas than in controls (Figure S3a). Similarly, in the L layer, several genes (including Cpt1a, Cpt1b, Fasn, Slc27a6, Cd36, Srebf2, Soat1 and Ppar-β/δ) were expressed at lower levels in FGR placentas than in controls (Figure S3b). The findings suggest that many of the genes associated with lipid metabolism are downregulated in FGR placentas, which may explain the abnormal changes observed in several lipid components. We found the concentration of multiple lipids to be increased in the DJ and L layers of FGR mouse placentas. We investigated whether energy supply was altered in FGR placentas by measuring the expression of ATP regulatory proteins. We recognized that the expression of CⅡ was unaltered in the DJ layer of FGR placentas; however, the abundance of CⅤ was significantly increased by 23.9% and 48.0% in the DJ or L layers of FGR placentas, respectively (Fig. 6g). These findings indicate that energy supply may be altered in the placental labyrinth region of our FGR model.

Effect of maternal exercise on rapamycin-induced FGR in mice

As we found that FGR placentas exhibited high lipid state despite down-regulated genes associated with lipid transport and metabolism, we speculated that the availability of placental lipids was limited, resulting in reduced energy utilization. Studies report that physical exercise during pregnancy is beneficial to placental perfusion and fetal growth (Genest et al. 2012; Brett et al. 2015). We suggest that maternal exercise may promote lipid utilization in FGR placentas and therefore mitigate FGR induced by rapamycin. The experimental design diagram was shown in Figure S4 and consisted of control groups with or without exercise (Ctrl and Ctrl + Ex), and Rapa group with or without exercise (Rapa and Rapa + Ex). The data demonstrated that maternal exercise contributed to a significant increase in fetal and placental weight at late gestation (E18.5) in FGR pregnancies, although it did not fully reverse the reduced fetal growth (Fig. 7a) or placental weight (Fig. 7b). Additionally, we observed increased invasion of spongiotrophoblast cells, as evidenced by Tpbpa staining, in the labyrinth of FGR placentas exposed to maternal exercise (Fig. 7c). IF staining revealed that CD31 intensity was decreased in FGR placentas compared to controls at E18.5, but restored with exposure to maternal exercise (Fig. 7c).

We investigated the effect of maternal exercise on the mTOR signaling pathway in rapamycin-induced FGR mouse placentas. As presented in Fig. 7d, the phosphorylation of Rps6 (p-Rps6) in the DJ layer was significantly decreased in the FGR group compared to controls, but was restored with maternal exercise exposure. In the L layer, we observed that placentas from pregnancies exposed to maternal exercise had an elevated p-Rps6 level, despite no significant difference seen between the unexercised FGR and control groups at E18.5 (Fig. 7e). Additionally, we found the phosphorylation of 4Ebp1 in the L layer of mouse placentas to be significantly reduced in the FGR group, regardless of maternal exercise, compared to the control group (Fig. 7e).

Due to the changes in mTOR signaling observed in different placental zones of FGR mice exposed to maternal exercise, we supposed that placental lipid metabolism may also be affected. We tested the three lipids FFA, TAG, and Cho in mouse placentas. We found that, similar to E14.5, at E18.5 FGR placentas had increased FAA levels in the L layer compared with controls, but maternal exercise exposure lowered this level in FGR placentas closer to that of controls (Fig. 8a). When examining TAG levels in the L layer, in contrast to E14.5 when similar levels were observed in FGR and control placentas, at E18.5 decreased TAG levels were identified in FGR placentas and maternal exercise was not found to alter these levels (Fig. 8a). Total and free Cho as well as CE were measured and showed no significant differences in the L layer of placentas among the four groups at E18.5 (Fig. 8a). Unexpectedly, we did not find the levels of FFA, TAG, or Cho to be different in the DJ layer of FGR and control placentas with or without maternal exercise (Fig. 8b).

Finally, we tested whether maternal exercise altered the expression of ATP regulatory proteins. In the DJ layer of FGR placentas at E18.5 we found that CⅡ expression was significantly decreased and not reversed by maternal exercise, whereas CⅤ expression was not found to be different between the four groups (Fig. 8c). In the L level of FGR placentas at E18.5 we found that CⅡ expression was not changed but CⅤ level was increased and maternal exercise seemed to further increase CⅤ expression (Fig. 8d).

These results suggest that FGR associated with inhibited mTOR signaling may be mitigated by maternal exercise through increased availability of FFA in the DJ layer of mouse placentas.

The placenta is a complex organ with different structural subregions consisting of various cell types including syncytiotrophoblasts, cytotrophoblasts, mesenchymal cells, and fetal vascular cells (Wang and Zhao 2010). Different anatomic placental areas could play varying roles in normal fetal growth and development. Our previous study revealed that intrauterine injection of lipopolysaccharide resulted in different mTORC1 activities in different anatomic locations of the mouse placenta (Dong et al. 2019). These findings suggest that it is necessary to distinguish placental zones when analyzing molecular expressions and functional changes. In this study, we identified increased mTOR activity in fetal-side compared to maternal-side of human placentas. Similarly, in mouse placentas the key molecules of mTOR signaling exhibited different expression levels and phosphorylation activity in different anatomical zones (DJ layer vs L layer). A study by Napso et al. (Napso et al. 2022) reported that the change in respiratory capacity differed in different placental zones under the condition of maternal diet-induced obesity, suggesting that the junctional and labyrinth zones have different functionality and response abilities.

FGR is a common complication of pregnancy and can have significant effects on long term neonatal outcomes Although a growing number of studies support mTOR signaling inhibition as an underlying mechanism of FGR (Gupta and Jansson 2019; Larqué et al. 2013; Roos et al. 2009), there remains inconsistencies in the literature, likely owing to differences in study methodology (Dong et al. 2020). Therefore, our study aimed to clarify the precise mechanism of placental mTOR activity in FGR.

We investigated the placental mTOR signaling pathway in full-term human SGA and AGA pregnancies. In contrast to previously published studies (Hung et al. 2017; Roos et al. 2007; Zhang et al. 2017), we found lower levels of p-RPS6 and higher levels of p-4EBP1 in the fetal-side of human SGA placentas, while t-mTOR level was decreased in the maternal-side of SGA placentas. The variation in these results compared to previously published findings is thought to be due to optimization of detection methods in our study. For example, we tested mTOR-related molecules in the maternal- and fetal-side of placentas. In addition, we utilized Coomassie staining as the total protein reference used for calculating the relative values of protein bands in WB, which may be superior to the housekeeping proteins in terms of quantitative analysis (Welinder and Ekblad 2011).

Many different FGR models exist, including folate deficiency, maternal diet restriction, and hypoxia. As summarized in our review (Dong et al. 2020), studies of these models have identified the commonality of placental mTOR signaling pathway inhibition in FGR, indicating that it may be a key regulatory hub in the development of growth restriction. Based on this, we assume that inhibiting placental mTOR activity with rapamycin to induce FGR is a valid model to study the mechanism underlying FGR, especially the type without specific pathogenic factors. In 2021, Shao et al. (Shao et al. 2021) developed the rapamycin-induced FGR mouse model by intraperitoneally injecting rapamycin at the dose of 1 mg/kg/day (approximately 0.04 mg/day per dam) during E10.5 to E13.5, and found the phosphorylation activities of mTOR and S6k (an upstream of Rps6) were significantly reduced in the bulk placentas from FGR group. In this study, we utilized the FGR model induced by a low-dose rapamycin (0.01 mg/day), we observed that the decreased mTOR activity occurred in the DJ layer of mouse FGR placentas. Our data demonstrate that low-dose rapamycin is associated with decreased intrauterine fetal growth and that mTOR signaling is affected to a greater extent in the DJ layer than the L layer of mouse placentas.

To understand the potential mechanism responsible for FGR due to the decreased placental mTOR signaling, we analyzed the morphology of mouse placentas and found that rapamycin-induced FGR placentas demonstrated reduced placental size and thickness, decreased ratio of junctional zone, and decreased vascular density. These morphological changes may be associated with abnormal placental structure and function. By the methods of whole placenta transcriptomics, we characterized that lipid metabolism may be affected in FGR placentas based on the enrichment analysis and expression identification of the DE mRNAs (Enpp7, Acacb and Clps). In addition, lipidomic examination of the DJ and L layers of mouse placentas, identified the increased levels of multiple lipid components linked with energy supply (such as FFA and TAG) in rapamycin-induced FGR placentas. However, other lipids (such as CL, PE, and PS) involved in cellular structure assembly were significantly reduced in FGR placentas, which may explain their reduced placental size. When analyzing the expression of genes related to lipid metabolism, despite the differential expression between the DJ and L layers, most of the relevant genes demonstrated reduced expression levels in FGR placentas compared to controls. As mTOR signaling plays a fundamental role in regulating lipid biosynthesis and metabolism, the observed changes in lipid molecules and corresponding regulatory genes could be attributed to mTOR inhibition in these FGR placentas (Caron et al. 2015). With the exclusion of the potential effect of maternal blood lipids on placental lipid levels, we suppose that the increased major lipids (FFA, CE and TAG) are due to suppressed lipid metabolism (including lipid oxidation, synthesis, and transport) in rapamycin-induced FGR mouse placentas. Importantly, we observed that mitochondrial respiratory chain (MRC) complexes V (CV, also called ATP synthase) were significantly increased in the L layer of FGR placentas compared to controls. These findings indicate that, as a consequence of altered lipid metabolism, there may be a compensatory increase in energy supply in FGR placentas to maintain fetal growth.

Strategies to prevent unexplained FGR are critical but limited in obstetric management. At present, effective interventions to reduce the occurrence of FGR mainly emphasize maintaining a healthy lifestyle (ex. avoiding tobacco and alcohol) and routine ultrasound monitoring (Berghella 2007). There remains a significant need for effective preventive and therapeutic interventions. Since rapamycin-induced FGR placentas had increased lipid accumulation, and may have decreased utilization, we explored the effect of maternal exercise on fetal weight in the FGR model. Our data showed that maternal exercise in rapamycin-treated pregnancies, conducted during E0.5 to E16.5, increased fetal and placental weight. Maternal exercise was also associated with improved placental morphology in the FGR model and increased phosphorylation activity of Rps6 in the DJ and L layers of FGR placentas. Additionally, we observed that the FFA level in the DJ zone of FGR placentas returned to normal level with maternal exercise. These results suggest that maternal exercise improves placental insufficiency and promotes FFA metabolism in rapamycin-induced FGR. According to our findings, maternal exercise may be an effective method to mitigate the development of FGR. In fact, some researchers have reported that pregnant women who participate in moderate physical activity have a less risk of low-birth weight babies (Xi et al. 2020; Walasik et al. 2020; Gollenberg et al. 2011; Takito and Benício 2010). However, other studies found no effect of maternal physical activity in reducing the risk for SGA neonates (Pastorino et al. 2019; Beetham et al. 2019; Davenport et al. 2018), with some actually reporting that vigorous physical activity may increase the risk (Legesse et al. 2020; Ehrlich et al. 2020). These discrepancies may be related to differences in exercise definition and study design. Currently, there is no strong evidence that moderate exercise during pregnancy is detrimental. Further research on maternal exercise is important and may assist in the development of recommendations for clinical practice to improve pregnancy outcomes, including in cases of FGR.

Although we found that maternal exercise mitigated the abnormal findings in fetuses and placentas in the rapamycin-induced FGR mouse model, we cannot be certain that maternal activity directly regulates placental mTOR signaling or promotes lipid utilization and energy supply to the fetuses. The placenta has the ability to adapt to intrinsic and extrinsic environmental abnormalities in order to support fetal growth (Sandovici et al. 2012; Myatt 2006). We found that, compared to controls, p-mTOR level was decreased in the DJ layer of FGR placentas at E14.5, but restored to normal levels at E18.5. In the L layer, the levels of FFA, TAG, and Cho were no different at E18.5 between the FGR and control groups, despite an increase identified at E14.5. Additionally, the increased CV expression in the L layer of FGR placentas at E14.5 was found to further increase at E18.5 under the treatment of maternal exercise. Placental junctional and labyrinth zones support intrauterine fetal growth. The junctional zone provides the main endocrine function of the placenta by generating hormones, growth factors, and cytokines while labyrinth zone transports nutrients and gases from mother to fetus (Woods et al. 2018). Previous studies have identified the junctional zone structure as being particularly affected in cases of FGR, much more so than the labyrinth zone (Tao et al. 2022; Gualdoni et al. 2021; Roberts et al. 2021). We speculate that the junctional zone of the placenta may be more sensitive to insults associated with FGR compared to the labyrinth zone, and the labyrinth zone may primarily act as an adaptive regulator to sustain fetal growth.

It is important to note that there are also some limitations in this study. First, evidence reported by Tshering S et al.(Tshering et al. 2021) showed that rapamycin can transfer to the fetal body through the placenta, and it may simultaneously restrict placental and fetal growth despite the controversial findings(Chu et al. 2008; Tshering et al. 2021; Ebrahimi-Fakhari et al. 2021). Additionally, the inhibition of placental mTOR activity is common in FGR caused by specific (like maternal malnutrition, pre-eclampsia, chemical exposures, etc.) or unknown factors. Therefore, the rapamycin-reduced FGR model may be appropriate to simulate clinical FGR appeared in pregnant women who receive organ transplantation and take sirolimus, or mimic FGR with unknown pathogenic factors. Second, although we found that mTOR signaling were changed in different anatomic zones of human and mouse placentas, from a detailed perspective, the changed molecular markers were not fully consistent between human and mouse placentas. We speculated there may exist different adaptive responsive mechanisms in FGR placentas between the two species. Moreover, despite the benefit of maternal exercise in mitigating mouse FGR, the clinical significance of maternal exercise in preventing FGR is required to be further proven.

In summary, we have demonstrated that human and mouse placentas have different mTOR signaling activities in different anatomic regions in normal and FGR pregnancies. By utilizing the rapamycin-induced FGR mouse model, we revealed that the inhibition of mTOR signaling in DJ zone of mouse placenta leads to abnormal lipid metabolism appeared in the whole placenta, which may be involved in the occurrence of FGR. In addition, we found that maternal exercise is associated with improved placental mTOR activity and lipid metabolism, as well as increased fetal and placental weight in our FGR model. Our data indicate that maternal exercise could be an effective method to reduce the risk of FGR. Future studies are needed to expand upon these findings in order to improve pregnancy outcomes.

AGA, Appropriate for gestational age; ATP, Adenosine triphosphate; BMP, Bis-monoacylglycerol phosphate; CE, Cholesteryl esters; Cho, cholesterols; CL, Cardiolipins; DAG, Diacylglycerols; DJ, Decidua junction; DMSO, Dimethyl sulfoxide; FFA, Free fatty acid; FGR, Fetal growth restriction; Gb3, Ceramide trihexoside; GO, Gene Ontology; HE, Hematoxylin & eosin; IHC, Immunohistochemistry; KEGG, Kyoto Encyclopedia of Genes and Genomes; L, Labyrinth; LPE, Lyso- phosphatidylethanolamines; LPS, Lysophosphatidylserines; mTOR, Mechanistic target of rapamycin; PBS, Phosphate-buffered saline; PC, Phosphatidylcholines; PE, Phosphatidylethanolamines; PS, Phosphatidylserines; RT-qPCR, Real-time quantitative polymerase chain reaction; SGA, Small for gestational age; TAG, Triacylglycerols; WB, Western blot.

Author contribution

Jie Dong and Qian Xu designed and performed experiments, analyzed data, and wrote the manuscript. Chenxi Qian collected clinical data collection and molecular experiments. Lu Wang analyzed the sequencing data. Alison DiSciullo and Jun Lei edited the manuscript. Hui Lei, Song Yan, Jingjing Wang, Ni Jin and Yujing Xiong contributed experiment guidance and data analysis. Jianhua Zhang collected human placentas. Irina Burd and Xiaohong Wang designed this project, provided financial support, and approved the manuscript. All authors read and approved the final manuscript.

Funding

This work was supported by grants from National Natural Science Foundation of China (Grant No. 82101794, 82071717), and the Platform Construction Fund of Tangdu Hospital (2020XKPT003).

Data Availability

According to our department regulations, the original data of lipidomics and RNA sequencing data can be uploaded publicly or will be shared via e-mail from corresponding author on reasonable request.

Ethics approval

In this study, the utilization of human placentas was approved by the Tangdu Hospital Ethics Committee (TDLL-202305-08) and the informed consent of delivering women during December 2019 to February 2020 was obtained. All animal experiment procedures were conducted in Air Force Medical University according to the Animal Protection Guidelines of Air Force Medical University (SYXK2019-001).

Competing interests

The authors declare that there are no competing interests associated with the manuscript.

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Fetal growth restriction exhibits various mTOR signaling in different regions of mouse placentas with altered lipid metabolism

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Abstract

Figures

Introduction

Materials and Methods

Sample collection

Human placenta and umbilical cord blood collection

Mouse sample preparation

Mouse model of FGR

Maternal exercise

Histological analysis of mouse placenta

Western blot (WB)

mRNA sequencing

Real-time quantitative polymerase chain reaction (RT-qPCR)

Lipidomics analysis

Assay of (free fatty acid) FFA, TAG and Cho

Statistical analysis

Results

Differential expression of key molecules in mTOR pathway in different anatomic zones of human placentas

Differential expression of mTOR-related proteins in different anatomic layers of mouse placentas

Differential expression of mTOR-related proteins in different anatomic layers of rapamycin-induced FGR mouse placentas

Histological findings in rapamycin-induced FGR mouse placentas

Analysis of mRNA sequencing in control and rapamycin-induced FGR mouse placentas

Analysis of lipid molecules in different anatomic layers in rapamycin-induced FGR placentas

Effect of maternal exercise on rapamycin-induced FGR in mice

Discussion

Conclusions

Abbreviations

Declarations

References

Additional Declarations

Supplementary Files

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Journal Publication

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