Hostname: page-component-74656f9884-rngvg Total loading time: 0 Render date: 2026-11-06T00:31:10.485Z Has data issue: false hasContentIssue false
  • English
  • Français

Effects of maternal restraint stress on offspring intestinal microbiota and adipogenesis: insights from in vivo and in vitro studies

Published online by Cambridge University Press:  28 February 2025

Takako Kondo Open the ORCID record for Takako Kondo [Opens in a new window] ,
Yuta Tsunematsu ,
Yu Aoki ,
Noriyuki Miyoshi ,
Mitsuo Iinuma  and
Kumiko Yamada
Takako Kondo*
Affiliation:
Department of Health and Nutrition, Faculty of Health and Sciences, Nagoya Women’s University, Nagoya-shi, Aichi, Japan Department of Anatomy and Neuroscience, Graduate School of Medical Sciences, Nagoya City University, Nagoya, Aichi, Japan
Yuta Tsunematsu
Affiliation:
Graduate School of Bioagricultural Sciences Department of Applied Biosciences, Nagoya University, Nagoya, Aichi, Japan
Yu Aoki
Affiliation:
Graduate School of Integrated Pharmaceutical and Nutritional Sciences, University of Shizuoka, Shizuoka, Japan
Noriyuki Miyoshi
Affiliation:
Graduate School of Integrated Pharmaceutical and Nutritional Sciences, University of Shizuoka, Shizuoka, Japan
Mitsuo Iinuma
Affiliation:
Department of Pediatric Dentistry Division of Oral Structure, Asahi University, Gifu, Japan
Kumiko Yamada
Affiliation:
Department of Health and Nutrition, Faculty of Health and Sciences, Nagoya Women’s University, Nagoya-shi, Aichi, Japan
*
Corresponding author: Takako Kondo; Email: tkondo@nagoya-wu.ac.jp
Rights & Permissions [Opens in a new window]

Abstract

The maternal restraint stress animal model is based on a long-term stress paradigm administered to pregnant maternal animals, and these offspring have been shown to exhibit a variety of biochemical defects including obesity. This study aimed to investigate whether maternal restraint stress affects obesity-associated changes in offspring intestinal microbiota and the adipogenic differentiation of mesenchymal stem cells (MSCs).

Pregnant mice were subjected to restraint stress three times daily from gestational Day12 to delivery. Changes in the composition of the intestinal microbiota of mothers (during pregnancy and lactation) and their lactating offspring exposed to maternal restraint stress were analyzed using next-generation sequencing. Maternal stress altered the maternal microbiota, with reduced Bacteroidetes and increased Firmicutes. While similar trends were observed in offspring, these changes were not statistically significant. However, maternal stress notably reduced microbial diversity in the offspring’s intestinal microbiota. Bone marrow-derived MSCs from offspring at weaning were analyzed for adipogenic transcription factors and hormone receptor expression using quantitative PCR. Maternal stress enhanced the adipogenic phenotype of offspring MSCs, as evidenced by increased expression of adipogenic markers (PPARγ, leptin receptor) and a reduced osteogenic phenotype. In vitro induction further confirmed the higher adipocyte differentiation potential in stressed offspring MSCs compared to controls.

Our results revealed that maternal restraint stress altered the maternal intestinal microbiota, leading to reduced microbial diversity in offspring, predisposing their MSCs toward an adipocyte phenotype. These finding suggest that modulating the intestinal microbiota of stressed pregnant women may improve the susceptibility to obesity in their children.

Keywords

Maternal restrain stressoffspringintestinal microbiotamesenchymal stem cellsadipogenesis

Information

Type
Original Article
Information
Journal of Developmental Origins of Health and Disease , Volume 16 , 2025 , e14
Check for updates
Copyright
© The Author(s), 2025. Published by Cambridge University Press in association with The International Society for Developmental Origins of Health and Disease (DOHaD)

Introduction

Stress during pregnancy correlates with an increased risk for physical and mental disability in children Reference Antonson, Evans and Galley1,Reference Eberle, Fasig and Bruseke2 . Expansion of research into fetal programing has revealed that the fetal environment influences the risk of obesity and chronic diseases in adulthood. Fetal programing can result in structural and functional changes in fetal body components, which may through interactions with subsequent developmental environments, including diets, inflict critical long-term consequences for future disease susceptibility. Maternal stress has been identified as the cause of adverse effects on the intrauterine environment. Stress during pregnancy is known to influence postnatal body composition, energy balance, and metabolic function through exposure to cortisol and proinflammatory cytokines Reference Entringer3 . Stress exposure early in pregnancy tends to result in pregnancy loss, whereas stress exposure later in pregnancy tends to result in low birth weight and increased risk factors for various adulthood diseases in offspring Reference Brunton4 .

There is a bidirectional relationship between stress and the intestinal flora, which influences obesity induction. The maternal microbiota has been shown to be involved in offspring inflammation, glucose metabolism and neural development in rodents and humans Reference Di Simone, Santamaria Ortiz and Specchia5,Reference Ferguson6 . Maternal restraint stress has been shown to activate the hypothalamic–pituitary–adrenal (HPA) axis with increased cortisol and disrupted maternal microbial homeostasis Reference Joseph and Whirledge7 . Since reduced intestinal microbiota diversity correlates with obesity, maternal restraint stress may induce obesity through changes in the offspring’s intestinal microbiota.

Previous studies have reported that metabolites produced by intestinal microbiota influence the proliferation and differentiation of mesenchymal stromal cells (MSCs) Reference Ren, Su and Liu8,Reference Xiao, He and Wu9 . MSCs derived from bone marrow are multipotent stromal cells that can differentiate into mesenchymal lineage cells, such as osteoblasts and adipocytes, as well as chondrocytes and myoblasts. MSCs are isolated by in vitro plastic surface adherence and colony-forming capacity, which consists of a heterogeneous population of cells that differ in their cell surface marker expression, proliferation and differentiation. The identified microenvironments with growth factors, hormones and cytokines control the proliferation and differentiation of MSCs in vivo and in vitro. Transcription factors, runt-related transcription factor 2 (Runx2) and peroxisome proliferator-activated receptor γ (PPARγ) have been identified as key molecular switches in the involvement of undifferentiated MSCs in osteoblast and adipocyte lineages. It has been reported that MSCs in a rat model of maternal food restriction in late pregnancy exhibit an adipogenic phenotype through regulation of Runx2 and PPARγ expression Reference Gong, Antony and Sakurai10 . There have been reports on the relationship between the intestinal microbiota and fat differentiation of MSCs. It has been revealed that activation of free fatty acid receptor 2 (FFAR2) by short-chain fatty acids produced by the intestinal microbiota suppresses insulin signaling in adipose tissue and inhibits fat accumulation. It has also been shown that activation of FFAR2 reduces the fat differentiation ability of human chorionic-derived MSCs, contributing to the inhibition of fat accumulation Reference Kimura, Ozawa and Inoue11,Reference Iván, Major and Sipos12 .

Maternal restraint stress has been shown in animal models to influence diet-induced obesity in offspring, ultimately leading to adult chronic diseases. In addition, it has been reported that restraint stress on over- or undernourished pregnant model animals results in significant visceral fat accumulation in offspring fed a high-fat diet Reference Juarez, Quiroga and Prochnik13 . In this study, we investigated whether maternal stress induces obesity-prone intestinal microbiota and alters the bone marrow MSC phenotype in offspring. Specifically, we examined the changes in the gene expression of markers associated with adipogenic and osteogenic differentiation, as well as hormone and short-chain fatty acid receptors, in offspring MSCs in response to maternal stress.

Materials and methods

Animal

Pregnant ddY mice (gestation Day 8) were purchased from Chubu Kagaku Shizai Co., Ltd (Nagoya, Japan). The room temperature was 23 ± 3°C, with a humidity of 50 ± 5% and a light /dark cycle of 12 h (light 7:00–19:00, dark 19:00–7:00). All animals had free access to tap water and standard rodent pellet chow (CE-2, CLEA Japan, Inc., Tokyo, Japan). The environment, welfare and general condition of the animals were continuously monitored during the experimental period. All animal studies were approved by the Nagoya Women’s University institutional animal experimentation committee (approval number 2021-3), and conducted according to the standards outlined by the Care and Management of Experimental Animals (Notification number 88, from the Prime Minister’s Office, Tokyo, Japan).

Pregnant mice were randomly assigned to either the control or stress condition group (n = 6 / group). In the stress condition group, mice (n = 6) were exposed to restraint stress for 45 min, three times a day, from Day 12 of pregnancy until delivery, by placing them in a ventilated plastic restraint tube, in which they could move back and forth but not turn around Reference Azuma, Furuzawa and Fujiwara14 . All dams delivered their offspring vaginally. The offspring were raised by their biological mothers (n = 6 / group), and to avoid variability due to postnatal nutrition, litter size (6 pups/ mother) was normalized within 1 week. At 4 weeks of age, male pups (2-5 pups / mother) were selected from the control and stress groups and assigned to the control and stress groups, respectively.

Next-generation 16S rRNA sequencing

At pregnant period (at the last of restraint stress) and the end of the experimental period (at 4 weeks after birth), the feces were collected fresh in tubes directly and stored at − 80°C until use (n = 6). Microbial DNA in feces was extracted, and the V3-V4 variable region of the 16S ribosomal RNA (rRNA) was amplified using specific primers (F5′-TCG TCG GCA GCG TCA GAT GTG TAT AAG AGA CAG CCT ACG GGN GGC WGC AG-3′; R5′-GTC TCG TGG GCT CGG AGA TGT GTA TAA GAG ACA GGA CTA CHV GGG TAT CTA ATC C-3′). Quantified amplicons were tagged with a barcode, which were then sequenced using MiSeq (Illumina, San Diego, CA, USA) Reference Kondo, Saigo and Ugawa15 . Data were analyzed by QIIME (Quantitative Insights Into Microbial Ecology), Nephele (National Institutes of Health, Bethesda, MD, USA) and Mass Profiler Professional software (Agilent Technology, Santa Clara, CA, USA).

MSC culture

Bone marrow was isolated from the femurs and tibias of offspring mice at 4 weeks of age. Epiphyses of femurs and tibias were removed, and the marrow was flushed out into a culture dish. The isolated bone marrow, composed of hematopoietic and stromal cells, was maintained in high-glucose DMEM (Invitrogen, Grand Island, NY, USA) supplemented with 10% FBS (Invitrogen)/1% Gluta-Max (Invitrogen)/100 units/ml penicillin (Sigma, St. Louis, MO, USA) at 37°C with 5% CO2. Hematopoietic and nonadherent cells were removed by changes of medium. Cell Proliferation Assay was performed as previously reported Reference Kondo, Johnson and Yoder16 . Three days after plating cells on chamber slides, BrdU (BD Bioscience, San Jose, CA, USA) at 10 μM was added to the culture medium and incubation continued for an additional 24 h. At the end of the incubation, the cultures were fixed with 4% paraformaldehyde for 30 min. The cells were denatured with 2 N HCl for 30 min. After blocking of nonspecific binding, the specimens were incubated with anti-BrdU antibody (BD Biosciences). BrdU immunoreactivity was visualized with an Alexa Fluor 488-conjugated anti-mouse IgG1 and cell nuclei were counterstained with DAPI.

RNA isolation and RT-PCR

The expression of genes was assayed by conventional or quantitative RT-PCR by a previously described procedure with modifications Reference Kondo, Matsuoka and Shimomura17 . Total RNA was isolated from MSCs by using the RNeasy Minikit (Qiagen, CA, US) and then treated with TURBO DNase (Ambion, TX, USA). Single-stranded cDNA was synthesized by using Omniscript reverse transcriptase (Qiagen). For nonquantitative RT-PCR, the resultant cDNA was amplified with Platinum taq DNA polymerase (Invitrogen) for 30 cycles. For quantitative RT-PCR, the cDNA samples were amplified on an ABI PRISM 7900HT Sequence Detection System (Applied Biosystems, Waltham, MA, USA) by using the SYBR Green PCR Master Mix (Applied Biosystems). To check whether amplification yields PCR products with a single molecular weight, the PCR products were electrophoresed on 2% agarose gels containing ethidium bromide. The mRNA level for each probe (x) relative to L27 mRNA (internal control) was calculated as follows: mRNA(x %) = 2Ct (L27)-Ct(x) × 100.

Flow cytometry

Cultured MSCs were dissociated by a brief exposure to trypsin-EDTA. After being blocked, cells were incubated with one of the following primary antibodies: anti-CD29-PE, anti-CD31-APC, anti-CD44-PerCP, anti-45-PerCP, and mouse immunoglobulinG (IgG) isotype as a control. Alternatively, cells were fixed and permeabilized using the Cytofix/Cytoperm Fixation/Permeabilization kit (BD Biosciences, San Jose, CA). Cell sorting and analysis were performed with a FACS Calibur flow cytometry system (BD Biosciences) Reference Kondo, Matsuoka and Shimomura17 .

Adipocyte induction

MSCs were subjected to adipogenic induction according to the protocol of the AdipoInducer Reagent (for animal cells) kit (Takara, Shiga, Japan) for 14 days. After induction, cultured MSCs were fixed with 4% paraformaldehyde and stained Oil red O solution (Cosmo Bio, Tokyo, Japan) for 15 min. After drying, the dye was eluted by adding an extraction reagent and measured with a plate reader (540 nm).

Cell based ELISA

Cultured MSCs and adipocyte induced MSCs were fixed with 4% paraformaldehyde and permeabilized with TritonX-100 according to the protocol of In-cell ELISA kit (abcam, Cambridge, UK). After being blocked, cells were incubated with one of the following primary antibodies: anti-FFAR2, anti-adipose differentiation-related protein (ADRP), anti-fatty acid binding protein 4 (FABP4), and anti-glyceraldehyde-3-phosphate dehydrogenase as an internal control. The immunoreactive signal was measured with Horseradish Peroxidase conjugated secondary antibodies and KPL SureBlue TMB Microwell Substrate (Sera care, Milford, MA, USA).

Results

Pregnant mice were subjected to restraint stress three times per day from embryonic Day12 until delivery. There was no significant difference in litter size by maternal restraint stress, and the size of the individual depended on litter size. On postnatal Day7, male pups were selected and decimated to a specific number to unify the breastfeeding burden of their mothers. Maternal feces were collected at the last stress loading and lactation (28 days), and offspring feces were collected 28 days after birth (Fig. 1a). The analysis of genomic DNA extracted from feces clarified the compositional variation of the intestinal microbiota in mice in response to maternal restraint stress. At the phylum level, maternal exposure to restraint stress resulted in an approximately 7.1% increase in Firmicutes (p < 0.050), while Bacteroidota (p = 0.068) and Actinobacteriota (p = 0.050) decreased by approximately 6.0% and 0.6%, respectively (Fig. 1b). Restraint stress increased Firmicutes by approximately 7.0% (p = 0.070) and decreased Bacteroidota by approximately 7.4% (p = 0.340) in lactating mothers. A similar trend was observed in the offspring, with Firmicutes increasing by approximately 7.3% and Bacteroidota decreasing by approximately 6.1%, but due to the difference in the number of bacteria among individuals was large, no significant difference could be confirmed in their offspring. At the class level, maternal restraint stress was associated with an increase in Bacilli and a decrease in Clostridium within the Firmicutes phylum. Similar trends were observed in mothers during pregnancy and lactation and in their offspring, but these differences were not statistically significant (Table 1). No significant differences were found in other bacterial taxa between the control and stress groups in pregnant and lactating mothers or their offspring. A comparison of the intestinal microbiota composition showed significant differences in Coriobacteriia in the stress group and in Bacteroidia across both the control and stress groups.

Figure 1.

Effects of maternal restraint stress on the composition of intestinal microbiota. (a) Experimental scheme (from embryonic day 12 to postnatal day 28) and (b) comparative analysis of intestinal microbiota at the phylum level in late pregnancy and lactation mothers and weaning offspring in the absence of stress (CNT) and in the presence of stress (STR). Arrows indicated stress loads. The day of fecal collection was marked with a white circle (mother) and a gray circle (offspring).

Table 1.

Effects of maternal restraint stress on the composition of intestinal microbiota

Comparative analysis of intestinal microbiota at the class level in late pregnancy and lactation mothers and weaning offspring in the absence of stress (CNT) and in the presence of stress (STR). a Significant difference (P < 0.050) between pregnant mother and lactating mother. b Significant difference (P < 0.050) between pregnant mother and offspring. c Significant difference (P < 0.050) between lactating mother and offspring. P values indicate significance between pregnant, lactating mother and offspring groups by ANOVA analysis. **P < 0.010, *P < 0.050.

Reduced intestinal microbiota diversity following exposure to restraint stress and a high-fat diet has been reported Reference Karl, Hatch and Arcidiacono18 . Our results showed that restraint stress did not significantly affect the evenness and richness of the intestinal microbiota species in mothers. However, maternal restraint stress reduced the evenness and richness of intestinal microbiota species in offspring. Shannon index in the control group was 4.40 ± 0.25, whereas in the stress group it was 4.05 ± 0.22 (Fig. 2a). Chao1 index in the control group was 113.70 ± 20.24, whereas in the stress group it was 79.10 ± 17.49 (Fig. 2b). Changes in the offspring’s intestinal microbiota due to maternal stress were correlated with the mother’s changes, but a decrease in intestinal microbiota diversity was observed only in the offspring.

Figure 2.

Effect of maternal restraint stress on intestinal microbiota diversity. (a and b) Beta diversity analysis reveals that maternal stress decreases the species richness and diversity of the intestinal microbiota in offspring. (c) Principal coordinates analysis (PCA) score plot of fecal 16S rRNA sequences. PCA was performed by Mass Profiler Professional software. CNTs are shown as circles and STRs are shown as squares for mothers in late pregnancy (gray), lactating mothers (black), and weaning offsprings (white). (d) Representation of the Firmicutes/Bacteroidetes ratios. Data are expressed as the mean ± SD (n = 6 control and 6 stressed pregnant mothers/group; 5 control and 6 stressed lactating mothers/group; 5 control and 6 stressed offspring/group). *P < 0.050.

Changes in the intestinal environment due to restraint stress were analyzed by principal coordinate analysis (PCoA) based on UniFrac distance. The PCoA results revealed variations in the relative abundance of taxonomic classifications at the family level (Fig. 2c). Because the difference in intestinal microbiota among individuals was largely reflected, significant changes in the intestinal environment by restraint stress could not be evaluated. However, the first principal component (PCo1) and the second principal component (PCo2) indicated similar patterns among pregnant mothers and their offspring within the stress group. The Firmicutes/Bacteroidetes (F/B) ratio has been frequently considered a possible hallmark of obesity in animal models and humans Reference Ley, Turnbaugh and Klein19,Reference Grigor’eva20 . Our results indicated that restraint stress caused an increase in the F/B ratio in pregnant and lactating mothers, while its effect on their offspring was relatively weak (Fig. 2d). Specifically, the F/B ratio increased from 10.80 ± 4.19 to 35.88 ± 18.75 in pregnant mothers, from 3.81 ± 1.65 to 18.85 ± 14.09 in lactating mothers, and from 2.33 ± 0.67 to 6.15 ± 3.68 in their offspring.

MSCs were isolated from the femurs and tibias of offspring mice at 4 weeks. We observed no differences in cell morphology between with MSCs derived from control mother (CNT-MSCs) and MSCs derived from mother with maternal stress (STR-MSCs) (Fig. 3b and c). Both types of MSCs were characterized using specific cell surface markers. CD31 and CD45 were negative, and CD29 and CD44 were positive in both of CNT-MSCs and STR-MSCs (Fig. 3g). The bone marrow-derived mesenchymal stromal cell markers Meflin and Endoglin (CD105), which are known to be spontaneously expressed by undifferentiated MSCs, were detected in CNT-MSCs and STR-MSCs by RT-PCR (Fig. 3a). To evaluate the rate of cell proliferation, BrdU incorporation assays were performed with MSCs. Approximately 34.3% of CNT-MSCs exhibited BrdU-positive cellular nuclei, whereas 63.4% of STR-MSCs that had experienced prenatal stress were positive for BrdU staining (Fig. 3d and e). STR-MSCs significantly increased the cell proliferation rate compared to CNT-MSCs.

Figure 3.

Effects of maternal restraint stress on mesenchymal stem cells (MSCs) proliferation in offspring. (a) Expression of mesenchymal stem cell markers (Meflin and CD105) by PT-PCR. 1: CNT-MSCs, 2: STR-MSCs, 3: negative control (no cDNA). (b and c) Phase contrast, (d and e) BrdU immunofluorescence (green) and DAPI (blue) are shown. Scale bar:100 µm. (f) Quantitative analysis of the percentage of BrdU-positive cells in MSCs isolated from offspring weaned from control (CNT) and stressed (STR) mothers. Data are expressed as the mean ± SD (n = 4 CNT and 4 STR-MSC cell culture chamber wells). Positive cells were counted in 25 random areas per well and analyzed on an Incell Analyzer 6000. *P < 0.050. (g) Cell surface marker expression profiles of CNT-MSCs and STR-MSCs. Expression of specific cluster of differentiation molecules (CD29, CD44, CD31 and CD45) by flow cytometry.

The expression levels of markers involved in adipogenesis and osteogenesis were compared between CNT-MSCs and STR-MSCs (Fig. 4). PPARγ and C/EBPα (CCAAT/enhancer-binding proteinα) are essential transcription factors for adipogenesis and are involved in determining fat accumulation and insulin sensitivity. FOXC1 (Forkhead Box C1) is a transcription factor expressed in CXCL12-abundant reticular cells that suppresses adipocyte differentiation Reference Omatsu, Seike and Sugiyama21 . Adipoq (Adiponectin) is known as an essential gene for adipocytes, but it is also expressed in adipogenic precursors of MSCs. On the other hand, RUNX2 and Osterix are transcription factors that promote the differentiation of MSCs into osteoblasts. Compared to CNT-MSCs, the expression of PPARγ and Adipoq was increased, and the expression of Foxc1, Runx2, and Osterix was decreased in STR-MSCs. No significant changes in the expression of C/EBPα were observed. The expression of glucocorticoid receptor (Nr3C, nuclear receptor subfamily 3 Group C) and adrenergic receptor (Adrb3, adrenoceptor beta 3), which mediate the effects of stress hormones, was lower in STR-MSCs than in CNT-MSCs. Perinatal stress reduces transcription through methylation of the NR3C1 gene in newborns Reference Chalfun, Reis and de Oliveira22 . Hypermethylation of the Adrb3 gene is associated with obesity Reference Lima, Ribeiro and de Farias Lima23 . A similar decrease in receptor expression was obtained in MSCs upon maternal stress.

Figure 4.

Effects of maternal restraint stress on the expression of adipogenic, osteogenic genes and hormone receptors in mesenchymal stem cells (MSCs) of offspring. Quantitative RT-PCR analysis for adipogenic differentiation-related transcription factors (PPARγ, C/EBPα, Foxc1 and Adipoq), osteoblast differentiation-related transcription factors (Runx2 and Osterix), and stress hormone receptors (Nr3c and Adrb3) in progeny MSCs. The relative expression of LepR gene and LepR protein. Values are means ± SDs. Significant differences between the control and STR groups (n = 3 per group). *P < 0.050.

Leptin receptors have been reported to promote adipogenesis and reduce bone formation by regulating mesenchymal stromal cells in adult bone marrow. In our results, leptin receptor was significantly highly expressed in STR-MSCs at both the gene and protein levels (Fig. 4). The expression trends of MSCs revealed in this study suggest that MSCs in offspring subjected to maternal stress are more likely to differentiate into adipocyte cells.

Next, we investigated the effect of maternal stress on the adipogenic differentiation potential of offspring MSCs. After 14 days of adipogenic induction in vitro, Oil red O staining revealed a significant increase in the number of positive cells in STR-MSCs (Fig. 5a and b). The expression of the short-chain fatty acid receptor FFAR2 and the adipogenic differentiation markers ADRP and FABP4 in CNT-MSCs and STR-MSCs were compared before and after adipogenic induction. Before adipogenic induction, the expression of these proteins was not significantly different between CNT-MSCs and STR-MSCs (Fig. 5c). After adipogenic induction, the expression of ADRP, which is involved in lipid droplet formation, was increased in STR-MSCs, while the expression of FABP4, which encodes a lipid transport protein, remained unchanged (Fig. 5d). As a result, maternal stress increased the expression of ADRP, a downstream target gene of PPARγ, in offspring MSCs without activating FFAR2.

Figure 5.

Effects of maternal restraint stress on the adipogenic differentiation of offspring mesenchymal stem cells (MSCs) in vitro. (a) Oil red O staining positive cells in CNT-MSCs and STR-MSCs after 14 days of adipogenic induction in vitro. Scale bar:100 µm. (b) The dye extractions were measured by spectrophotometer (540 nm). (c and d) Quantitative In Cell ELISA analysis for short-chain fatty acid receptor (FFAR2) and adipogenic differentiation markers (ADRP and FABP4) in CNT-MSCs and STR-MSCs before (c) and after (d) adipogenic induction. Data are expressed as the mean ± SD (n = 6 CNT and STR-MSC cell culture chamber wells). *P < 0.050.

Discussion

Recent studies have revealed the presence of bacteria in amniotic fluid, fetal membranes, and placenta, indicating possible exposure to the fetus in utero Reference Younge, McCann and Ballard24 . Since maternal stress causes a deterioration of the intrauterine environment and affects fetal development, the changes in the offspring’s intestinal microbiota seen in the present results may be due to differences in the intrauterine environment. Furthermore, it is known that components of breast milk are involved in the intestinal microbiota of newborns Reference Xu, Zhou and Liu25 . It is also possible that some changes in the composition of breast milk caused by restraint stress during pregnancy determined the intestinal microbiota of the offspring.

Compelling evidence for the association between intestinal microbiota and obesity has been reported. The report that transplanting feces from an obese person into germ-free mice made the mice obese had an enormous impact revealing a correlation between intestinal microbiota and obesity Reference Goodrich, Waters and Poole26 . Changes such as an increase in the ratio of the major phyla Firmicutes and Bacteroidetes have been shown to potentially promote the development of obesity in both dietary and genetic models of obesity in mice Reference Ley, Turnbaugh and Klein19,Reference Wu, Chen and Hoffmann27 . Our results also showed that the ratio of Firmicutes and Bacteroidetes was increased in mothers and offspring exposed to maternal stress. However, the association between obesity and changes in the Firmicutes/Bacteroidetes ratio may be inconsistent, as the relative abundances of Firmicutes and Bacteroidetes have been reported to vary widely between subjects in the same population Reference Walters, Xu and Knight28 . Numerous studies have focused on identifying intestinal bacteria associated with obesity. Verrucomicrobiota phylum, a mucin-degrading bacterium that contributes to stabilizing intestinal barrier function and controlling inflammation, has been reported to decrease with obesity Reference Liu, Yang and Yan29 . In others, an increase in the Proteobacteria phylum has been observed in obese humans and animals. Furthermore, maternal stress elevates cortisol levels, leading to offspring with a higher proportion of Proteobacteria, which is linked to allergy development Reference Ahmad, Yang and Chen30,Reference Aatsinki, Keskitalo and Laitinen31 . Additionally, the Lachnospiraceae family, which belongs to the Clostridia class, has been suggested to be related to obesity and hyperglycemia Reference Takeuchi, Kameyama and Miyauchi32 . In our study, Verrucomicrobiota was identified only in the intestinal microbiota of offspring at proportions exceeding 1.0%, with a tendency to decrease under maternal stress (control: 4.29 ± 7.28, stress: 2.20 ± 3.07). Proteobacteria were only detected in offspring exposed to maternal stress at a prevalence above 1.0%. Nevertheless, these changes were minor and not statistically significant when compared to the control group (Fig. 1b). Analysis of the Clostridium listed in Table 1 at the family level revealed no significant differences in the Lachnospiraceae family within the offspring’s intestinal microbiota. The prevalence in the control group was 11.29 ± 7.69, whereas in the stress group it was 13.87 ± 7.15. Although we did not identify significant changes in bacteria associated with obesity before weaning, the intestinal microbiota of offspring exposed to maternal restraint stress showed a decrease in diversity, which was consistent with the changes observed in obesity.

LepR-positive MSCs in adult bone marrow are confirmed to be the main source of new osteoblasts and adipocytes in vivo. Leptin/LepR signaling promotes adipogenesis and reduces osteogenesis by bone marrow-derived MSCs in response to diet and fat accumulation Reference Yue, Zhou and Shimada33 . It has been reported that maternal restraint stress causes increased corticosterone levels in offspring, leading to decreased bone mass Reference Azuma, Furuzawa and Fujiwara14 . In addition, M Gong et al. showed that bone marrow-derived MSCs isolated from the offspring of rats that were nutritionally restricted during pregnancy had significantly enhanced adipogenesis, along with increased cell proliferation Reference Gong, Antony and Sakurai10 . Similarly, progeny MSCs isolated from a restraint stress mouse model in our study showed increased adipogenic differentiation genes and decreased osteoblast differentiation genes. However, the expression of C/EBPα, which is involved in adipogenic differentiation, was not induced in STR-MSCs. PPARγ is an essential factor for the maturation of adipocytes, and C/EBPs and Foxc1 also contribute to the adipogenic pathway. Since C/EBPα has the function of suppressing cell proliferation, lower expression of C/EBPα may be associated with the high cell proliferation of STR-MSCs. Utilizing scRNAseq datasets, Adipoq was found to be highly expressed in cluster mesenchymal progenitor cell-adipo lineages Reference Baryawno, Przybylski and Kowalczyk34,Reference Baccin, Al-Sabah and Velten35 . Although the MSCs used in this study were heterogeneous and expression in individual cell clusters was not confirmed, the result of high expression of Adipoq in STR-MSCs was consistent with previous reports of high expression in LEPR-positive MSCs Reference Baryawno, Przybylski and Kowalczyk34 .

Recently, the relationship between short-chain fatty acids produced by intestinal bacteria and host obesity has been elucidated. Short-chain fatty acids such as acetic acid, propionic acid, and butyric acid play a role in suppressing obesity by controlling energy metabolism, including suppressing fat accumulation and increasing energy consumption through short-chain fatty acid receptors Reference Kimura, Ozawa and Inoue11 . Short-chain fatty acids produced by Bacteroidetes have been shown to interact with FFAR2 in adipocytes, inhibiting energy uptake and preventing adipocyte hypertrophy. It has also been shown that short-chain fatty acids produced by the intestinal bacteria of pregnant mothers are involved in the maturation of metabolic functions in offspring after birth, leading to the suppression of the onset of obesity Reference Kimura, Miyamoto and Kitano36 . In addition, it was reported that short-chain fatty acids inhibit adipogenesis of human chorionic membrane-derived mesenchymal stem cells via FFAR2 Reference Iván, Major and Sipos12 . In our study, we did not observe a decrease in FFAR2 expression in progeny MSCs due to maternal restraint stress. Similarly, a study on chronic restraint stress exposure reported a reduction in short-chain fatty acid levels in outbred CD-1 mice, while FFAR2 expression in the colon remained unaffected Reference Maltz, Keirsey and Kim37 . Therefore, future research should focus on metabolic products produced by intestinal bacteria, such as short-chain fatty acids, and their signaling pathways. Additionally, it is essential to investigate whether adipose-derived MSCs in offspring exhibit similar metabolic effects.

In conclusion, maternal restraint stress altered intestinal microbiota composition in mothers leading to reduced microbial diversity in the offspring. MSCs derived from offspring exposed maternal stress exhibited enhanced cell proliferation and an increased propensity for adipogenic differentiation, as evidenced by elevated adipogenic markers and leptin receptor expression, alongside reduced osteogenic markers. These findings suggest that maternal stress predisposes offspring MSCs to adipogenesis, potentially influencing their metabolic health.

This study focused on analyzing the effects of maternal restraint stress without dietary restrictions on lactating offspring, but in the future it will also be necessary to analyze these effects after weaning. It has been reported that when exposed to both stress and a high-fat diet during pregnancy, female offspring have higher body fat percentage and impaired glucose tolerance in adulthood than stress loading alone Reference Tamashiro, Terrillion and Hyun38 . Conversely, if mothers exposed to stress during pregnancy consume prebiotics that produce short-chain fatty acids, their offspring are less prone to obesity Reference Kimura, Miyamoto and Kitano36 . Further research is required to investigate how maternal dietary factors (e.g., high-fat diets, prebiotics, and energy restriction) influence the obesity-prone cellular characteristics of MSCs in offspring exposed to maternal stress.

Acknowledgements

None.

Financial support

This work was supported by JSPS KAKENHI Grant Number JP 19K11679.

Competing interests

The authors declare that there are no conflicts of interest.

Ethical standards

The authors assert that all procedures contributing to this work comply with the ethical standards of the relevant national guides on the Care and Management of Experimental Animals (Notification number 88, from the Prime Minister’s Office, Tokyo, Japan) and has been approved by the Nagoya Women’s University institutional animal experimentation committee (approval number 2021-3).”

References

Antonson, AM, Evans, MV, Galley, JD, et al. Unique maternal immune and functional microbial profiles during prenatal stress. Sci. Rep. 2020; 10(1), 20288. DOI: 10.1038/s41598-020-77265-x.CrossRefGoogle ScholarPubMed
Eberle, C, Fasig, T, Bruseke, F, et al. Impact of maternal prenatal stress by glucocorticoids on metabolic and cardiovascular outcomes in their offspring: a systematic scoping review. PLoS One. 2021; 16(1), e0245386. DOI: 10.1371/journal.pone.0245386.CrossRefGoogle ScholarPubMed
Entringer, S. Prenatal stress exposure and fetal programming of complex phenotypes: interactive effects with multiple risk factors, Neurosci. Biobehav. Rev. 2020; 117, 3–4. DOI: 10.1016/j.neubiorev.2020.04.002.CrossRefGoogle Scholar
Brunton, PJ. Effects of maternal exposure to social stress during pregnancy: consequences for mother and offspring. Reproduction. 2013; 146(5), 175–189. DOI: 10.1530/REP-13-0258.CrossRefGoogle ScholarPubMed
Di Simone, N, Santamaria Ortiz, A, Specchia, M et al. Recent insights on the maternal microbiota: impact on pregnancy outcomes. Front. Immunol. 2020; 11, 28202. DOI: 10.3389/fimmu.2020.528202.CrossRefGoogle ScholarPubMed
Ferguson, J. Maternal microbial molecules affect offspring health. Science. 2020; 367(6481), 978–979. DOI: 10.1126/science.aba7673.CrossRefGoogle ScholarPubMed
Joseph, DN, Whirledge, S. Stress and the HPA axis: balancing homeostasis and fertility. Int. J. Mol. Sci. 2017; 18(10), 18. DOI: 10.3390/ijms18102224.CrossRefGoogle ScholarPubMed
Ren, Y, Su, S, Liu, X et al. Microbiota-derived short-chain fatty acids promote BMP signaling by inhibiting histone deacetylation and contribute to dentinogenic differentiation in murine incisor regeneration. Stem Cells Dev. 2020; 29(18), 1201–1214. DOI: 10.1089/scd.2020.0057.CrossRefGoogle ScholarPubMed
Xiao, E, He, L, Wu, Q et al. Microbiota regulates bone marrow mesenchymal stem cell lineage differentiation and immunomodulation. Stem Cell Res Ther. 2017; 8(1), 213. DOI: 10.1186/s13287-017-0670-7.CrossRefGoogle ScholarPubMed
Gong, M, Antony, S, Sakurai, R, et al. Bone marrow mesenchymal stem cells of the intrauterine growth-restricted rat offspring exhibit enhanced adipogenic phenotype. Int. J. Obes. (Lond.). 2016; 40(11), 40 1768–1775. DOI: 10.1038/ijo.2016.157.CrossRefGoogle ScholarPubMed
Kimura, I, Ozawa, K, Inoue, D, et al. The gut microbiota suppresses insulin-mediated fat accumulation via the short-chain fatty acid receptor GPR43. Nat Commun. 2013; 4(1), 4,1829. DOI: 10.1038/ncomms2852.CrossRefGoogle ScholarPubMed
Iván, J, Major, E, Sipos, A, et al. The short-chain fatty acid propionate inhibits adipogenic differentiation of human chorion-derived mesenchymal stem cells through the free fatty acid receptor 2. Stem Cells Dev. 2017; 26(23), 1724–1733. DOI: 10.1089/scd.2017.0035.CrossRefGoogle ScholarPubMed
Juarez, YR, Quiroga, S, Prochnik, A, et al. Influence of prenatal stress on metabolic abnormalities induced by postnatal intake of a high-fat diet in BALB/c mice. J. Dev. Orig. Health Dis. 2021; 12(5), 721–730. DOI: 10.1017/S2040174420000987.CrossRefGoogle ScholarPubMed
Azuma, K, Furuzawa, M, Fujiwara, S, et al. Effects of active mastication on chronic stress-induced bone loss in mice. Int. J. Med. Sci. 2015; 12(12), 952–957. DOI: 10.7150/ijms.13298.CrossRefGoogle ScholarPubMed
Kondo, T, Saigo, S, Ugawa, S, et al. Prebiotic effect of fructo-oligosaccharides on the inner ear of DBA/2 J mice with early-onset progressive hearing loss. J. Nutr. Biochem. 2020; 75, 108247. DOI: 10.1016/j.jnutbio.2019.108247.CrossRefGoogle ScholarPubMed
Kondo, T, Johnson, SA, Yoder, MC, et al. Sonic hedgehog and retinoic acid synergistically promote sensory fate specification from bone marrow-derived pluripotent stem cells. Proc. Natl. Acad. Sci. U. S. A. 2005; 102(13), 4789–4794. DOI: 10.1073/pnas.0408239102.CrossRefGoogle ScholarPubMed
Kondo, T, Matsuoka, AJ, Shimomura, A, et al. Wnt signaling promotes neuronal differentiation from mesenchymal stem cells through activation of Tlx3. Stem Cells. 2011; 29(5), 836–846. DOI: 10.1002/stem.624.CrossRefGoogle ScholarPubMed
Karl, JP, Hatch, AM, Arcidiacono, SM et al. Effects of psychological, environmental and physical stressors on the gut microbiota. Front. Microbiol. 2018; 9, 2013. DOI: 10.3389/fmicb.2018.02013.CrossRefGoogle ScholarPubMed
Ley, RE, Turnbaugh, PJ, Klein, S, et al. Microbial ecology: human gut microbes associated with obesity. Nature. 2006; 444(7122), 1022–1023. DOI: 10.1038/4441022a.CrossRefGoogle ScholarPubMed
Grigor’eva, IN. Gallstone disease, obesity and the firmicutes/Bacteroidetes ratio as a possible biomarker of gut dysbiosis. J Pers Med.2020. 2021; 11(1), 13. DOI: 10.3390/jpm11010013.Google Scholar
Omatsu, Y, Seike, M, Sugiyama, T, et al. Foxc1 is a critical regulator of haematopoietic stem/progenitor cell niche formation. Nature. 2014; 508(7497), 536–540. DOI: 10.1038/nature13071.CrossRefGoogle ScholarPubMed
Chalfun, G, Reis, MM, de Oliveira, MBG et al. Perinatal stress and methylation of the NR3C1 gene in newborns: systematic review. Ciba F Symp. 2022; 17(9), 1003–1019. DOI: 10.1080/15592294.2021.1980691.Google ScholarPubMed
Lima, RPA, Ribeiro, MR, de Farias Lima, KQ et al. Methylation profile of the ADRB3 gene and its association with lipid profile and nutritional status in adults. Biol. Res. 2019; 52(1), 21. DOI: 10.1186/s40659-019-0226-7.CrossRefGoogle ScholarPubMed
Younge, N, McCann, JR, Ballard, J, et al. Fetal exposure to the maternal microbiota in humans and mice. JCI Insight. 2019; 44(19), e127806. DOI: 10.1172/jci.insight.127806.CrossRefGoogle Scholar
Xu, D, Zhou, S, Liu, Y, et al. Complement in breast milk modifies offspring gut microbiota to promote infant health. Cell. 2024; 187(3), 750–763. DOI: 10.1016/j.cell.2023.12.019.CrossRefGoogle ScholarPubMed
Goodrich, JK, Waters, JL, Poole, AC, et al. Human genetics shape the gut microbiome. Cell. 2014; 159(4), 789–799. DOI: 10.1016/j.cell.2014.09.053.CrossRefGoogle ScholarPubMed
Wu, GD, Chen, J, Hoffmann, C, et al. Linking long-term dietary patterns with gut microbial enterotypes. Science. 2011; 334(6052), 105–108. DOI: 10.1126/science.1208344.CrossRefGoogle ScholarPubMed
Walters, WA, Xu, Z, Knight, R. Meta-analyses of human gut microbes associated with obesity and IBD. FEBS Lett. 2014; 588(22), 4223–4233. DOI: 10.1016/j.febslet.2014.09.039.CrossRefGoogle ScholarPubMed
Liu, MJ, Yang, JY, Yan, ZH et al. Recent findings in Akkermansia muciniphila-regulated metabolism and its role in intestinal diseases. Clin Nutr. 2022; 41(10), 2333–2344. DOI: 10.1016/j.clnu.2022.08.029.CrossRefGoogle ScholarPubMed
Ahmad, A, Yang, W, Chen, G, et al. Analysis of gut microbiota of obese individuals with type 2 diabetes and healthy individuals. PLoS One. 2019; 14(12), e0226372. DOI: 10.1371/journal.pone.0226372.CrossRefGoogle ScholarPubMed
Aatsinki, AK, Keskitalo, A, Laitinen, V, et al. Maternal prenatal psychological distress and hair cortisol levels associate with infant fecal microbiota composition at 2.5 months of age. Psychoneuroendocrino. 2020; 119, 104754. DOI: 10.1016/j.psyneuen.2020.104754.CrossRefGoogle ScholarPubMed
Takeuchi, T, Kameyama, K, Miyauchi, E, et al. Fatty acid overproduction by gut commensal microbiota exacerbates obesity. Cell Metab. 2023; 35(2), 361–375, e369. DOI: 10.1016/j.cmet.2022.12.013 .CrossRefGoogle ScholarPubMed
Yue, R, Zhou, BO, Shimada, IS, et al. Leptin receptor promotes adipogenesis and reduces osteogenesis by regulating mesenchymal stromal cells in adult bone marrow. Cell Stem Cell. 2016; 18(6), 782–796. DOI: 10.1016/j.stem.2016.02.015.CrossRefGoogle ScholarPubMed
Baryawno, N, Przybylski, D, Kowalczyk, MS, et al. A cellular taxonomy of the bone marrow stroma in homeostasis and Leukemia. Cell. 2019; 177(7), 1915–1932, e1916. DOI: 10.1016/j.cell.2019.04.040 .CrossRefGoogle ScholarPubMed
Baccin, C, Al-Sabah, J, Velten, L, et al. Combined single-cell and spatial transcriptomics reveal the molecular, cellular and spatial bone marrow niche organization. Nat. Cell Biol. 2020; 22(1), 38–48. DOI: 10.1038/s41556-019-0439-6.CrossRefGoogle ScholarPubMed
Kimura, I, Miyamoto, J, Kitano, RO et al. Maternal gut microbiota in pregnancy influences offspring metabolic phenotype in mice. Science. 2020; 367(6481), 6481. DOI: 10.1126/science.aaw8429.CrossRefGoogle ScholarPubMed
Maltz, RM, Keirsey, J, Kim, SC, et al. Prolonged restraint stressor exposure in outbred CD-1 mice impacts microbiota, colonic inflammation, and short chain fatty acids. PLoS One. 2018; 13(5), e0196961. DOI: 10.1371/journal.pone.0196961.CrossRefGoogle ScholarPubMed
Tamashiro, KL, Terrillion, CE, Hyun, J, et al. Prenatal stress or high-fat diet increases susceptibility to diet-induced obesity in rat offspring. Diabetes. 2009; 58(5), 1116–1125. DOI: 10.2337/db08-1129.CrossRefGoogle ScholarPubMed
Figure 0

Figure 1. Effects of maternal restraint stress on the composition of intestinal microbiota. (a) Experimental scheme (from embryonic day 12 to postnatal day 28) and (b) comparative analysis of intestinal microbiota at the phylum level in late pregnancy and lactation mothers and weaning offspring in the absence of stress (CNT) and in the presence of stress (STR). Arrows indicated stress loads. The day of fecal collection was marked with a white circle (mother) and a gray circle (offspring).

Figure 1

Table 1. Effects of maternal restraint stress on the composition of intestinal microbiota

Figure 2

Figure 2. Effect of maternal restraint stress on intestinal microbiota diversity. (a and b) Beta diversity analysis reveals that maternal stress decreases the species richness and diversity of the intestinal microbiota in offspring. (c) Principal coordinates analysis (PCA) score plot of fecal 16S rRNA sequences. PCA was performed by Mass Profiler Professional software. CNTs are shown as circles and STRs are shown as squares for mothers in late pregnancy (gray), lactating mothers (black), and weaning offsprings (white). (d) Representation of the Firmicutes/Bacteroidetes ratios. Data are expressed as the mean ± SD (n = 6 control and 6 stressed pregnant mothers/group; 5 control and 6 stressed lactating mothers/group; 5 control and 6 stressed offspring/group). *P < 0.050.

Figure 3

Figure 3. Effects of maternal restraint stress on mesenchymal stem cells (MSCs) proliferation in offspring. (a) Expression of mesenchymal stem cell markers (Meflin and CD105) by PT-PCR. 1: CNT-MSCs, 2: STR-MSCs, 3: negative control (no cDNA). (b and c) Phase contrast, (d and e) BrdU immunofluorescence (green) and DAPI (blue) are shown. Scale bar:100 µm. (f) Quantitative analysis of the percentage of BrdU-positive cells in MSCs isolated from offspring weaned from control (CNT) and stressed (STR) mothers. Data are expressed as the mean ± SD (n = 4 CNT and 4 STR-MSC cell culture chamber wells). Positive cells were counted in 25 random areas per well and analyzed on an Incell Analyzer 6000. *P < 0.050. (g) Cell surface marker expression profiles of CNT-MSCs and STR-MSCs. Expression of specific cluster of differentiation molecules (CD29, CD44, CD31 and CD45) by flow cytometry.

Figure 4

Figure 4. Effects of maternal restraint stress on the expression of adipogenic, osteogenic genes and hormone receptors in mesenchymal stem cells (MSCs) of offspring. Quantitative RT-PCR analysis for adipogenic differentiation-related transcription factors (PPARγ, C/EBPα, Foxc1 and Adipoq), osteoblast differentiation-related transcription factors (Runx2 and Osterix), and stress hormone receptors (Nr3c and Adrb3) in progeny MSCs. The relative expression of LepR gene and LepR protein. Values are means ± SDs. Significant differences between the control and STR groups (n = 3 per group). *P < 0.050.

Figure 5

Figure 5. Effects of maternal restraint stress on the adipogenic differentiation of offspring mesenchymal stem cells (MSCs) in vitro. (a) Oil red O staining positive cells in CNT-MSCs and STR-MSCs after 14 days of adipogenic induction in vitro. Scale bar:100 µm. (b) The dye extractions were measured by spectrophotometer (540 nm). (c and d) Quantitative In Cell ELISA analysis for short-chain fatty acid receptor (FFAR2) and adipogenic differentiation markers (ADRP and FABP4) in CNT-MSCs and STR-MSCs before (c) and after (d) adipogenic induction. Data are expressed as the mean ± SD (n = 6 CNT and STR-MSC cell culture chamber wells). *P < 0.050.