Maternal undernutrition inhibits fetal rumen development： Novel miRNA-736-mediated dual targeting of E2F2 and MYBL2 in sheep

Maternal undernutrition inhibits fetal rumen development： Novel miRNA-736-mediated dual targeting of E2F2 and MYBL2 in sheep | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Maternal undernutrition inhibits fetal rumen development： Novel miRNA-736-mediated dual targeting of E2F2 and MYBL2 in sheep Peng Jiao, Yun Xu, Yamei Gu, Baoyuan Li, Huizhen Lu, Caiyun Fan, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6598539/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background Undernutrition disrupts pregnant ewe’s metabolic homeostasis and severely inhibits fetal growth and development. In this study, undernourished and nutrition-recovery pregnant sheep models and rumen epithelial cells were utilized to investigate the mechanisms behind undernutrition-induced disruptions in fetal rumen metabolism and development. Results Maternal undernutrition significantly reduced fetal rumen weight and papilla length, width and surface area. Maternal undernutrition extremely suppressed nutrient metabolism and energy production in fetal rumen via JAK3 / STAT3 signaling to inhibit cell cycle progression and fetal rumen development, while maternal nutritional recovery partially restored metabolic inhibition but failed to alleviate fetal rumen development. Meanwhile, 64 differentially expressed miRNAs (DEMs) were identified in fetal rumen between undernourished ewes and controls. Novel miR-736 was overexpressed both in fetal rumen of undernourished and nutrition-recovery models. E2F transcription factor 2 ( E2F2 ) and MYB proto-oncogene like 2 ( MYBL2 ) were the intersection of fetal rumen differentially expressed genes (DEGs) and DEMs target genes integrated analysis and were predicted as miR-736 target genes. Further, we confirmed that miR-736 targeted and downregulated E2F2 and MYBL2 expressional levels. Silencing E2F2 and MYBL2 promoted apoptosis and inhibited S-phase entry in rumen epithelial cells. Conclusions In summary, maternal undernutrition disrupted fetal rumen metabolism and elevated miR-736, which targeted and downregulated E2F2 and MYBL2 to inhibit cell cycle progression and promote apoptosis, finally inhibited fetal rumen development. This study provides new insights into the epigenetic mechanisms underlying maternal undernutrition-induced fetal rumen developmental deficits. Maternal undernutrition Fetal rumen development Novel miRNA-736 E2F2 MYBL2 Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction In ruminant production system, many animals are subjected to undernutrition, a condition that may be exacerbated by seasonal fluctuations of feed availability or by economic factors-driven artificial control [ 1 ]. Due to the multi-fetus characteristic of Hu-sheep and the rapid fetal growth and development during late gestation, pregnant ewes are prone to undernutrition which ultimately inhibits fetal development [ 2 ]. Rumen, as a critical digestive organ in ruminant, requires early and optimal development to ensure both animal health and productivity [ 3 ]. The transitional period (pre-rumination) in young ruminants represents a sensitive window for influencing the development of rumen wall [ 4 ]. A large body of researches have shown that varying kinds of nutritional regulation promote rumen development of lambs [ 5 – 7 ]. The enhancement of rumen wall morphology and physiological function through early-stage nutritional strategies holds significant potential for supporting the lifelong health and productivity of ruminants. However, the effect of maternal nutritional status or nutritional regulation on the morphology and development of fetal rumen before fetal birth remains underexplored. The loss of physical function caused by undernutrition in ruminants may be compensated by subsequent nutritional recovery [ 8 ]. Metabolic disorders and pregnancy toxemia resulting from malnutrition during late pregnancy can be effectively ameliorated through nutritional recovery [9; 10]. However, whether the effects of maternal undernutrition on fetal rumen development can be alleviated by maternal nutritional recovery has not been studied. Ruminal papilla are basic structures of ruminal epithelium and associated with great contact with chyme, enhancing the digestion and absorption of nutrients [ 11 ]. Our previous study has shown undernutrition significantly decreases maternal rumen weight and the length, width, and surface area of rumen papilla [ 12 ]. Meanwhile, maternal undernutrition seriously affects fetal weight and fetal liver development [ 2 ]. However, the effects of maternal undernutrition on fetal rumen development and the potential regulatory mechanisms remain poorly understood. Undernutrition of pregnant ewes has been found to inhibit the metabolism of carbohydrates, amino acids, and energy in the rumen and to suppress DNA replication and cell cycle in rumen epithelium through JAK3/STAT2 signaling pathway, ultimately leading to the narrower, shorter, and small rumen papilla [ 12 ]. Therefore, it is supposed that maternal undernutrition may affect nutrient metabolism and cell cycle in fetal remen to inhibit its development via some key signaling pathways. As known, miRNAs are key regulators of various biological processes, and fetal plasticity during pregnancy was regulated by miRNAs [13; 14]. miR-21-3p has been identified in sheep rumen where it regulates amino acid metabolism by targeting ATP1A2 [ 15 ], while miR-148a-3p inhibits the proliferation of rumen epithelial cells by targeting QKI5 [ 16 ]. However, whether maternal undernutrition can induce DEMs in fetal rumen and further regulate the growth and development of rumen epithelial cells remains unclear. This study postulated that maternal undernutrition during late gestation might inhibit fetal rumen development through regulating miRNA expression to disrupt cell cycle and rumen nutrient metabolism, and maternal nutritional recovery after undernutrition could only partially restore the nutrient metabolism homeostasis and fetal rumen development. Undernourished and nutrition-recovery pregnant sheep model were established using dietary nutrition control. Both in vivo animal models and in vitro sheep primary rumen epithelial cells were integrated to investigate the molecular mechanism of how maternal nutrition affects fetal rumen development. This study would provide theoretical foundations for the development of molecular therapeutic strategies to promote fetal rumen development. Methods Animal experimental design and sample collection The animal experiments were approved by the Institutional Animal Care and Use Committee of Anhui Agricultural University (AHAUXMSQ2024073). This study was part of a larger project aimed at exploring how undernutrition and nutritional recovery during late gestation affects maternal and fetal metabolic homeostasis. In undernourished and nutrition-recovery ewe models, the diet formula and nutrient composition are detailed in Table S1 . The metabolic energy and crude protein content of the diet were 11.25 MJ/kg and 12.60%, respectively. Undernourished ewe model: Twenty ewes (body weight 54.4 ± 2.48 kg, 2–3 fetuses, pregnancy for 108 days) were fed ad libitum for a 7-day adaptation period to detect feed intake and make sure the dietary nutritional level could meet the requirements of pregnant ewes carrying multiple fetuses. Subsequently, these ewes were randomly assigned to the CON group (n = 10, fed the baseline feed intake) or the UN group (n = 10, restricted to 30% of the baseline feed intake) for 15 days. Ewes were slaughtered at 4 hours after morning feeding, and fetal rumen epithelium samples were collected from the male fetuses (CON: n = 10, UN: n = 10) and stored in liquid nitrogen. Nutrition-recovery ewe model: Twenty four ewes (body weight 54.6 ± 1.69 kg, 2–3 fetuses, pregnancy for 108 days) were similarly fed ad libitum for a 7-day adaptation period to detect the baseline feed intake. Ewes were then randomly assigned to the CON group (n = 12, fed the baseline feed intake for 15 days followed by ad libitum feeding for another 15 days) or the REC group (n = 12, feed intake restricted to 30% of baseline for 15 days followed by ad libitum feeding for another 15 days). Due the predelivery of 3 pregnant ewes in the nutritional recovery period, they were removed from the research subjects. Subsequently, fetal rumen epithelium samples were collected from male fetuses (CON: n = 9, REC: n = 9) and stored in liquid nitrogen. Fetal rumen weight and morphological analysis Chyme was immediately removed from the fetal rumen after slaughter, and the weight of the empty fetal rumen was measured. Fetal rumen specimens were collected, fixed in 4% paraformaldehyde for 24 hours, embedded in paraffin, and sectioned for hematoxylin and eosin staining for morphological observation [12]. Transcriptome assay of rumen epithelium Total RNA was extracted from cryopreserved fetal rumen tissues using TRIzol reagent (Takara Bio, Otsu, Japan). RNA quality was verified by spectrophotometric analysis (NanoDrop ND-1000) with A260/230 and A260/280 ratios between 1.80–2.10, and RNA integrity was confirmed using an Agilent 2100 Bioanalyzer. A total of twenty RNA samples were randomly selected for transcriptome analysis, with ten samples from undernourished model (five CON and five UN) and another ten samples from nutritional recover model (five CON and five REC). The mRNA samples were purified using the magnetic bead method, fragmented, and reversely transcribed into cDNA, which were then ligated to sequencing adapters and amplified. Fragments with appropriate length were selected to construct cDNA library using NEBNext® Ultra™ RNA Library Preparation Kit and sequenced on an Illumina NovaSeq 6000 (Biomarker, Beijing, China). Clean reads were aligned to the Sheep Reference Genome 3.0 using HiSAT2, and gene expression was quantified using FPKM method. DEGs were identified with DESeq2 (v.1.6.3) based on P 1.5 or < 0.667. Principal component analysis (PCA) and partial least squares discriminant analysis (PLS-DA) were performed using SIMCA v.14.1 (Umetrics, Umea, Sweden). Data analyses including clustering heatmaps, KOG functional classification, and GSEA analysis were conducted using the Biomarker platform (www.biocloud.net). miRNA-Seq library construction, sequencing, and data analysis Small RNA library was constructed using 1.5 µg of total RNA with a NextFlex Small RNA Sequencing Kit (Illumina, San Diego, CA, USA). Briefly, small RNAs (18–30 nucleotides) were size-selected via gel electrophoresis, followed by sequential ligation of 3’ and 5’ adapters. After adapter ligation, reverse transcription and PCR amplification were performed with 12 cycles to enrich the library. Library quality was assessed using an Agilent Bioanalyzer 2100, and the quantification was performed by Qubit 3.0 Fluorometer (Thermo Fisher Scientific). The library was subsequently sequenced on the Illumina HiSeq 6000 platform using single-end sequencing. Raw sequencing reads underwent quality control using FastQC to remove low-quality reads, adapter sequences, and potential contaminants. Clean reads were aligned to the Sheep Reference Genome 3.0 using Bowtie2, allowing for the identification of known miRNAs and the discovery of novel miRNAs. The analysis of miRNA expressional levels and the identification of DEMs were conducted using DESeq2 (v.1.6.3) with the thresholds of P 1.5 or < 0.667. Additionally, potential target genes of DEMs were predicted using miRanda software. KEGG pathway enrichment analysis, network map of miRNA target genes, and Venn diagram analysis were conducted to explore the biological functions and regulatory pathways of these miRNAs in fetal rumen development. Subsequently, STRING software was used to analyze the protein-protein interactions (PPIs) of the target genes ( E2F2 and MYBL2 ) and to perform Gene Ontology (GO) pathway enrichment analysis. Cell culture assay Primary rumen epithelial cells were cultured in DMEM/F12 medium (GE Healthcare Life Sciences, Hyclone Laboratories, South Logan, Utah) supplemented with 10% fetal bovine serum (FBS; BI, Israel Beit Haemek Ltd.). Human embryonic kidney (HEK) 293T cells were cultured in high-glucose DMEM (GE Healthcare Life Sciences, Hyclone Laboratories, South Logan, Utah) containing 5% FBS (BI, Israel Beit Haemek Ltd.). All cell cultures were maintained under standard incubation conditions of 37°C and 5% CO 2 . miRNA mimic, inhibitors, and siRNA The mimic of miR-736 (double-strand oligonucleotides), mismatched negative control mimic (mimic NC), miR-736 inhibitor (single-strand oligonucleotides), and mismatched negative control inhibitor (inhibitor NC) were synthesized by RIBOBIO Co., Ltd. (Guangzhou, China). These mimics and inhibitors were used to assess the effects of miR-736 overexpression and inhibition on its activity in HEK-293 cells and rumen epithelial cells. The small interference RNA (siRNA) for E2F2 and MYBL2 were synthesized by SANGON Biotech (Shanghai, China). The sequences for the si-NC and siRNA primers were provided in Table S2 . 3′ UTR luciferase reporter assays Primers targeting the 3' UTR regions of E2F2 and MYBL2 were designed using Primer Premier 5.0 (Premier Biosoft, Palo Alto, CA). Detailed primer sequences are provided in Table S3 . Then, RNA from rumen epithelial cells were extracted and reversely transcribed into cDNA using PrimescriptTM RT reagent Kit (TaKaRa, Dalian, China). The 3′ UTR fragments of E2F2 and MYBL2 were amplified using the following 20 µL reaction system: 0.8 µL of forward and reverse primers, 1 µL cDNA, 10 µL Taq PCR Master Mix, and 7.4 µL ddH₂O. The resulting 3′ UTR double-stranded cDNA fragments of E2F2 and MYBL2 were enzymatically cleaved and cloned into the XhoI - NotI sites of the psiCHECK™-2 vector (Promega Corp.), which contained both Renilla and Firefly luciferase reporter genes, forming the wild-type dual luciferase reporter gene recombinant vector. The psiCHECK2-E2F2 and psiCHECK2-MYBL2 recombinant plasmids were extracted using the EndoFree Plasmid Mini Kit (Omega Bio-Tek, Norcross, GA), and the reliability of these recombinant vectors was verified by DNA sequencing. HEK-293T cells were seeded into 24-well plates at a density of 1.0 × 10 5 cells/well. After 24 hours, cells were co-transfected with 0.5 µg of either psiCHECK2-E2F2 or psiCHECK2-MYBL2 recombinant plasmid and 0.5 µg of miR-736 mimic (or control NC_mimic) using 2 µL of X-tremeGENE HP DNA transfection reagent (Roche, Penzberg, Germany) following the manufacturer’s protocol (06365752001a.fm). After 48 hours, Renilla luciferase activity, normalized to Firefly luciferase activity, was measured using the Dual-Luciferase® Reporter Assay System (Promega, Madison, WI) according to the manufacturer’s instructions. A decrease of more than 30% in the Renilla to Firefly luciferase ratio in the psiCHECK2 3′ UTR + mimic-transfected group compared to the control group indicated that miRNA interacted with the target gene 3′ UTR. Rumen epithelial cell transfection assays Rumen epithelial cells were plated into 6-well plates and allowed to reach 60–70% confluence. Transfections were then performed following the previously described protocol [17]. Briefly, rumen epithelial cells were transfected with 5 µL of miR-736 mimic or mimic NC, 10 µL of miR-736 inhibitor or inhibitor NC, and 5 µL of siRNA or si-NC, respectively, when the cells reached 60–70% confluence. There were three replicates for each group. After 48 hours, cells were harvested for RT-qPCR and flow cytometry analysis. Real-time quantitative polymerase chain reaction The mRNA expression levels of key genes involved in nutrient metabolism, cell cycle, and apoptosis in both rumen samples and cultured cells were quantitatively measured by RT-qPCR. The primer sequences for the target genes, which were designed using Primer3 software and synthesized by General Biology Co., LTD (Chuzhou, China), were provided in Table S4 . RT-qPCR was performed in a 20 µL reaction mixture, consisting of 10 µL SYBR Green I Master Mix, 0.8 µL of each primer, 0.4 µL of ROX Reference Dye II (50x), 2 µL of cDNA template, and 6 µL of enzyme-free deionized water. Fluorescence detection was carried out using the ABI 7500 real-time PCR system (Thermo Fisher Scientific), and gene expression was normalized to β-actin using the 2 −ΔΔCt method. Apoptosis and cell cycle detected by flow cytometry The Annexin V-FITC/PI Apoptosis and Cell Cycle Detection Kit (No. C1052) from Biyun Tian Biotechnology was used to assess cell apoptosis and cell cycle progression. This kit utilizes Annexin V-FITC in combination with propidium iodide (PI) staining to distinguish viable cells, early apoptotic cells, and late apoptotic or necrotic cells. In the experiment, cells were first washed with pre-cooled PBS and digested with EDTA-free trypsin to prevent interference with Annexin V binding to phosphatidylserine (PS). The cells were then resuspended in 1× Binding Buffer, stained with Annexin V-FITC and PI, and incubated at room temperature for 15–20 minutes in the dark. Apoptotic cells were analyzed by flow cytometry (Beckman Coulter, USA), and data were processed using FlowJo software. For cell cycle analysis, rumen epithelial cells were fixed with 70% ethanol, washed with PBS, and stained with RNase A and PI solution. DNA content was analyzed by flow cytometry (Beckman Coulter, USA), and the cell cycle phases were determined using ModFit software. Statistical analysis The RT-qPCR data were analyzed using a two-tailed t-test for comparisons between two groups or one-way ANOVA for comparisons among three groups. Pearson’s correlation test was applied to analyze the relationships among rumen weight, length, width, surface area, nutrient metabolism, energy production, and cell cycle related gene expression in fetal rumen. GraphPad Prism v.9.0 software was used for graphical representations, and all data are presented as the mean ± SEM. Statistical significance was set at P < 0.05. Results Maternal undernutrition inhibited fetal rumen development via impeding cell cycle and nutrient metabolism To investigate the effect of maternal undernutrition on fetal rumen development, an undernourished pregnant sheep model was established using 15-day 70% nutritional restriction. At the experimental outset, no significant difference in initial body weight was observed between the CON and UN groups ( P = 0.259) (Table 1 ). After 15 days of treatment, the body weight of ewes in the UN group was significantly lower than that in the CON group ( P < 0.001), and the average daily gain was also significantly lower than that in the CON group ( P < 0.001) (Table 1 ). Furthermore, compared to the CON group, the fetal rumen tissue weight was significantly reduced in the UN group ( P = 0.039) (Fig. 1 A). Meanwhile, maternal undernutrition caused a significant reduction in the length ( P < 0.001), width ( P = 0.019), and surface area ( P < 0.001) of fetal rumen papilla (Fig. 1 B-D). Transcriptome analysis revealed the clear distinctions of the general transcriptional profiles of fetal rumen between the CON and UN groups, as shown in PCA and PLS-DA plots of total genes (Fig. 1 E-F). The volcano plot analysis identified 38 upregulated genes, 111 downregulated genes, and 15,771 non-significant genes ( Fig. S1 A ). Further GSEA identified the enrichment of genes involved in cytokine-cytokine receptor interaction ( P = 0.153, NES = -1.152) and the cell cycle ( P = 0.645, NES = -0.921) (Fig. 1 G, 1 H). RT-qPCR results also demonstrated the downregulation of cytokine-cytokine receptor-related genes such as JAK3 ( P = 0.032) and STAT3 ( P = 0.060) and cell cycle-related genes including BUB1 ( P = 0.037), CCNB1 ( P = 0.022), CCNE1 ( P = 0.037), CDC25C ( P = 0.014), E2F2 ( P = 0.018), E2F8 ( P = 0.011), MYBL2 ( P = 0.037), and TTK ( P = 0.018) (Fig. 1 I). Additionally, fetal rumen weight and the length, width, and surface area of rumen papilla were positively correlated with the expressional levels of genes related to JAK3 / STAT3 signaling pathway and cell cycle (Fig. 1 J). KOG classification revealed that lipid, carbohydrate, and amino acid transport and metabolism, as well as energy production and conversion, were notably enriched by DEGs between the CON and UN groups (Fig. 1 K). DEGs associated with fatty acid and cholesterol synthesis as well as energy, carbohydrate, and amino acid metabolism were all downregulated (Fig. 1 L). RT-qPCR results confirmed the transcriptome findings, showing downregulation of genes related to fatty acid synthesis ( ACACA , ACACB , ELVOL6 , FASN , SCD , and FADS1 ), cholesterol synthesis ( HMGCR and HMGCS1 ), ketogenesis ( HMGCS2 ), and carbohydrate metabolism ( ARG1 , DPP4 , FGGY , GLUD1 , and PDK4 ) (Fig. 1 M). Meanwhile, the gene expressional levels of JAK3 and STAT3 were positively correlated with the expressional levels of genes related to nutrient metabolism including fatty acid and cholesterol synthesis as well as carbohydrate metabolism ( Fig. S1 C ). Thus, maternal undernutrition inhibited nutrient metabolism and cell cycle via JAK3 / STAT3 signaling pathway in fetal rumen, which suppressed fetal rumen development (Fig. 1 N). Table 1 Growth performance and daily feed intake of ewes in the CON and UN groups ( n = 10). 1 Item 3 Groups 2 P -value CON UN Initial body weight, kg 55.07 ± 0.847 53.74 ± 0.693 0.240 Final body weight, kg 56.83 ± 0.677 a 44.65 ± 0.798 b < 0.001 Daily weight gain, g/d 117.33 ± 28.025 a -606.00 ± 50.776 b < 0.001 Daily feed intake, kg/d 1.64 ± 0.013 a 0.52 ± 0.001 b < 0.001 1 The data were expressed as the mean ± SEM. 2 CON, control group; UN, undernutrition group. 3 Initial body weight, body weight measured on day 0; Final body weight, body weight measured on day 15. a−b Means in a row not sharing a common letter were significantly different ( P < 0.05). Maternal nutritional recovery partially restored undernutrition-disrupted metabolism but failed to alleviate fetal rumen maldevelopment To find out whether maternal nutritional recovery can restore fetal rumen development inhibited by previous maternal undernutrition, a maternal nutritional recovery model was established using 15-day 70% nutritional restriction followed by 15-day ad libitum feeding. At trial initiation, ewe body weights did not differ between CON and REC groups ( P = 0.229) (Table 2 ). At both 15-day and 30-day time points, the body weight and average daily gain of REC group ewes were significantly lower than those in the CON group ( P 0.050) (Table 2 ). Rumen tissue weight in the REC group was significantly lighter ( P = 0.005) than the CON group (Fig. 2 A). Further, fetal rumen papillary width ( P < 0.001) and surface area ( P = 0.002) in the REC group were significantly lower than the CON group while fetal rumen papillary length was almost recovered ( P = 0.207) (Fig. 2 B-D). Transcriptome analysis of the fetal rumen still revealed clear distinctions between the CON and REC groups, as shown in PCA and PLS-DA plots of total genes (Fig. 2 E-F). The volcano plot analysis identified 159 upregulated genes, 82 downregulated genes, and 14,971 non-significant genes ( Fig. S1 B ). Further, GSEA identified significant enrichment of genes involved in cytokine-cytokine receptor interaction ( P = 0.008, NES = 1.448) and the cell cycle ( P = 0.002, NES = -1.713) (Fig. 2 G-H). RT-qPCR results demonstrated the downregulation of cell cycle-related genes including CDK2AP2 ( P = 0.002), E2F2 ( P = 0.023), MYBL2 ( P = 0.011), SFN ( P = 0.004), TGM1 ( P < 0.001), and TUBA4A ( P < 0.001) in fetal rumen of REC group, moreover, the expression of JAK3 ( P = 0.062) and STAT3 ( P = 0.068) also showed downregulated trend (Fig. 2 I). Additionally, fetal rumen weight and the length, width, and surface area of rumen papilla were positively correlated with the expressional levels of key genes related to JAK3/STAT3 signaling pathway and cell cycle (Fig. 2 J). KOG classification revealed that lipid, carbohydrate, and amino acid transport and metabolism and energy production and conversion, were also notably enriched by DEGs between the CON and REC groups (Fig. 2 K). DEGs associated with cholesterol synthesis, energy metabolism, carbohydrate metabolism, and fatty acid synthesis were partially upregulated in fetal rumen of REC group, while DEGs related to amino acid metabolism were still downregulated (Fig. 2 L). RT-qPCR analysis revealed that maternal nutritional recovery after nutritional restriction upregulated genes associated with fatty acid synthesis ( DEGS1, ACACA , and ELVOL6 ) and downregulated genes related to cholesterol synthesis ( HMGCR and HMGCS1 ) in fetal rumen (Fig. 2 M). Meanwhile, the gene expressional levels of JAK3 and STAT3 were negatively correlated with the expressional levels of genes related to fatty acid synthesis and positively correlated with the expressional levels of genes related to cholesterol synthesis ( Fig. S1 D ). Thus, maternal nutritional recovery could partially alleviate the inhibited nutrient metabolism in fetal rumen caused by maternal undernutrition, while the suppressed cell cycle and the retarded fetal rumen development still existed (Fig. 2 N). Table 2 Growth performance and daily feed intake of ewes in the CON and REC groups ( n = 12). 1 Item Groups 2 P -value CON REC d0 body weight, kg 55.06 ± 0.413 54.22 ± 0.541 0.229 d15 body weight, kg 56.70 ± 0.498 a 45.45 ± 0.630 b < 0.001 d30 body weight, kg 58.26 ± 0.633 a 47.20 ± 0.353 b < 0.001 d0-d15 daily weight gain, g/d 109.44 ± 23.733 a -584.44 ± 37.947 b < 0.001 d15-d30 daily weight gain, g/d 103.89 ± 14.940 116.67 ± 32.240 0.723 d0-d30 daily weight gain, g/d 106.67 ± 18.142 a -233.89 ± 11.115 b < 0.001 d0-d15 daily feed intake, kg/d 1.63 ± 0.013 a 0.48 ± 0.009 b < 0.001 d15-d30 daily feed intake, kg/d 1.66 ± 0.013 1.61 ± 0.026 0.140 d0-d30 daily feed intake, kg/d 1.65 ± 0.020 a 0.84 ± 0.023 b < 0.001 1 The data were expressed as the mean ± SEM. 2 CON, control group; REC, nutritional recovery group. a−b Means in a row not sharing a common letter were significantly different ( P < 0.05). Maternal undernutrition altered fetal rumen miRNA profile and upregulated miRNA-736 expression It was hypothesized that miRNA might play a role in the epigenetic regulation of fetal rumen plasticity during pregnancy. In the analysis of miRNAs expressional profile, 1,253 miRNAs were detected including 152 known miRNAs and 1,101 novel miRNAs ( Table S5 ). To investigate the clustering of samples, PCA of total miRNA read counts in fetal rumen was performed. PCA plot showed clear distinction between the CON and UN groups, which indicated the changed fetal rumen miRNA profile upon maternal undernutrition ( Fig. S2 A ). Compared to the CON group, 34 DEMs were upregulated (Fig. 3 A), while 29 DEMs were downregulated in fetal rumen of UN group (Fig. 3 B). RT-qPCR results confirmed the findings from miRNA sequencing (Fig. 3 C). Excitingly, the significant upregulation of miR-736 ( P = 0.039) was also found in fetal rumen of ewes from the REC group compared to the gestational-age-matched controls (Fig. 3 D). To further explore the functions of these putative miRNA gene targets, KEGG enrichment analysis was conducted in which necroptosis and cytokine-cytokine receptor interaction were significantly enriched (Fig. 3 E). Interestingly, E2F2 and MYBL2 were the intersection of DEGs dataset in fetal rumen induced by maternal undernutrition, DEGs dataset in fetal rumen induced by maternal nutritional recovery, and DEMs target genes dataset ( Fig. S2 B ). Notably, miR-736 was found to target E2F2 and MYBL2 (Fig. 3 F). STRING analysis indicated that E2F2 and MYBL2 , the potential target genes of miR-736, were closely associated with cell cycle-related genes, including CCNA2 , CCNE1 , CCNE2 , and CCNA2 (Fig. 3 G-H). GO enrichment analysis further revealed that proteins interacting with E2F2 and MYBL2 were significantly enriched in cell cycle-related processes ( Fig. S2 C-D ). Thus, maternal undernutrition might inhibit fetal rumen development by upregulating miR-736, which then downregulated the expression of its target genes, E2F2 and MYBL2 , ultimately leading to the suppression of cell cycle-related gene expression. miRNA-736 targeted E2F2 and MYBL2 and regulated rumen epithelial cells development via cell cycle and apoptosis The binding sites of miRNA-736 to the 3' UTR regions of E2F2 and MYBL2 were predicted using miRanda software (Fig. 4 A). To further validate E2F2 and MYBL2 were target genes of miR-736, the 3' UTRs of E2F2 and MYBL2 were cloned into the psiCHECK2 vector, respectively. The inserted fragments lengths of these two recombinant vectors were verified using XhoI - ApaI double enzyme digestion and gel electrophoresis and the inserted fragments sequences were confirmed by DNA sequencing ( Fig. S3 A-B ). The psiCHECK2 + 3'UTR constructs and miR-736 mimics (or negative control mimic NC) were co-transfected into HEK-293 cells, and luciferase activity was measured using a dual luciferase reporter assay. The results showed that the luciferase activity in the co-transfected psiCHECK2 + 3' UTR ( E2F2 or MYBL2 ) and miR-736 mimic group was significantly downregulated compared to the negative control groups, confirming that miRNA-736 targeted both E2F2 and MYBL2 (Fig. 4 B-C). To explore the regulatory function of miR-736 in rumen epithelial cells, overexpression and silencing experiments were performed using miR-736 mimics and inhibitors, respectively. RT-qPCR results demonstrated that miR-736 mimics significantly increased the expression of mature miR-736 in rumen epithelial cells ( P = 0.002) (Fig. 4 D), while miR-736 inhibitors significantly decreased its expressional level ( P = 0.018) (Fig. 4 E). Overexpression of miR-736 resulted in a significant downregulation of E2F2 ( P = 0.027) and MYBL2 ( P = 0.017) gene expression in rumen epithelial cells, while inhibition of miR-736 caused a significant upregulation of E2F2 ( P = 0.008) and MYBL2 ( P < 0.001) gene expression (Fig. 4 F-G). Additionally, overexpression of miR-736 significantly reduced the expression of cell cycle-related genes ( CCNA2 , CCNE1 , CCNE2 , and CDC20 ) and the anti-apoptosis-related gene ( BCL2 ), while upregulated apoptosis-related genes ( BAX and CASP3 ) in rumen epithelial cells (Fig. 4 H). Conversely, inhibition of miR-736 produced the completely opposite effects on the expression of genes associated with cell cycle and apoptosis (Fig. 4 I). Flow cytometry analysis revealed that overexpression of miR-736 significantly increased the apoptosis level of rumen epithelial cells ( P < 0.001) compared to the mimic NC group, while inhibition of miR-736 significantly decreased the apoptosis level ( P < 0.001) (Fig. 4 J-K). Thus, miRNA-736 targeted E2F2 and MYBL2 and acted as a negative regulator of fetal rumen development by inhibiting cell cycle progression and promoting apoptosis. Silencing of E2F2 and MYBL2 inhibited cell cycle and promoted apoptosis of rumen epithelial cells To further confirm that miR-736 regulated the development of rumen epithelial cells by targeting the inhibition of E2F2 and MYBL2 , E2F2 and MYBL2 were silenced in rumen epithelial cells to examine their effects on the cell cycle and apoptosis. The results showed that the transfection of siRNA2 resulted in an 80.85% reduction in E2F2 gene expression in rumen epithelial cells (Fig. 5 A), so siRNA2 was used for the following assays. Silencing of E2F2 significantly reduced the mRNA expression of cell cycle-related genes ( CCNA2 , CCNE1 , CCNE2 , and CDC20 ) and the anti-apoptosis-related gene BCL2 , while upregulated the apoptosis-related gene BAX in rumen epithelial cells (Fig. 5 B). Further analysis revealed that silencing E2F2 expression significantly reduced the cell viability of rumen epithelial cells (Fig. 5 C). Flow cytometry analysis showed that silencing E2F2 significantly increased the apoptosis level ( P < 0.001) of rumen epithelial cells (Fig. 5 D-E), which was consistent with the results observed following miR-736 overexpression. Silencing E2F2 significantly inhibited cell proliferation in rumen epithelial cells and negatively regulated the entry into the S-phase of the cell cycle (Fig. 5 F-G). Additionally, silencing MYBL2 by transfecting siRNA4 resulted in a 53.97% reduction in MYBL2 gene expression in rumen epithelial cells (Fig. 5 H). Silencing MYBL2 also significantly inhibited the mRNA expression of cell cycle-related genes ( CCNA2 , CCNE1 , CCNE2 , and CDC20 ) and the anti-apoptosis-related gene BCL2 , while upregulated the apoptosis-related gene BAX in rumen epithelial cells (Fig. 5 I). Moreover, silencing MYBL2 significantly reduced the cell viability of rumen epithelial cells (Fig. 5 J). Flow cytometry analysis showed that silencing MYBL2 also significantly increased the apoptosis level ( P < 0.001) (Fig. 5 K-L) and negatively regulated entry into the S-phase of the cell cycle (Fig. 5 M-N). Thus, maternal undernutrition induced the overexpression of miRNA-736 in fetal rumen, which targeted E2F2 and MYBL2 , leading to enhanced apoptosis and the negative regulation of S-phase entry in rumen epithelial cells, ultimately inhibiting fetal rumen development. Discussion Rumen development during early life critically determines lifelong nutrient utilization in ruminants [ 18 ]. Maternal nutritional status profoundly influences offspring organogenesis, particularly in Hu-sheep during late gestation due to multi-fetal pregnancies induced high nutritional demand and low nutritional intake [19; 20]. However, the effect of maternal undernutrition on the metabolic homeostasis and development of fetal rumen is poorly understood. Dissecting the phenotypes and molecular regulatory mechanisms of maternal undernutrition-affected fetal rumen will enrich the mother-child nutrition theory and contribute to finding novel strategies to promote fetal development in utero and improve productivity after birth. Previous studies showed that fetal small intestinal and total gastrointestinal tract mass [ 21 ] and liver mass [ 22 ] were decreased by mid-gestation feed restriction in ewes. In the current study, maternal undernutrition significantly reduced fetal rumen weight which was consistent with a previous report about the effects of maternal nutritional restriction on fetal rumen development in cows during pregnancy [ 23 ]. Furthermore, maternal undernutrition reduced the length, width, and surface area of ruminal papilla in fetal rumen. Interestingly, nutritional recovery partially restored fetal rumen development reduced by previous maternal undernutrition; however, fetal rumen weight and the width and surface area of rumen papilla were still lower than the normal levels. Ewe undernutrition during late gestation affected lipid, carbohydrate, amino acid, and energy metabolism in the rumen [ 12 ], jejunum and ileum [ 24 ], cecum [ 25 ], pituitary [ 26 ], fat [ 27 ], and liver [ 2 ]. In this study, maternal undernutrition extremely reduced nutrient metabolism including fatty acid synthesis, cholesterol synthesis, carbohydrate metabolism, amino acid metabolism, and energy production in fetal rumen. It was worthy to note that lipid metabolism and transport were closely linked to cell survival and growth, the inhibition of lipid metabolism induced a suppression of cell proliferation [28; 29]. In addition, carbohydrate, amino acid, and energy metabolism played important roles in rumen development [30; 31]. Systemic inhibition of JAK / STAT signaling pathway induced multi-organ metabolic dysregulation [ 32 ]. Hepatic STAT5 deficiency exacerbated hyperglycemia and dyslipidemia [ 33 ] and liver-specific STAT6 deletion directly mediated hepatic steatosis and insulin resistance [ 34 ]. Adipose STAT3 / STAT5 ablation promoted obesity and suppresses lipolytic capacity [35; 36]. Skeletal muscle STAT5 depletion reduced lean mass and impaired glucose tolerance [ 37 ]. In this study, JAK3 and STAT3 expressional levels were significantly correlated with the expressional levels of genes related to nutrient metabolism and energy production. Therefore, maternal undernutrition suppressed nutrient metabolism and energy production via JAK3 / STAT3 signaling pathway and further inhibited fetal rumen development, while maternal nutritional recovery only partially restored metabolic homeostasis to alleviate undernutrition-induced fetal rumen maldevelopment. Metabolic disorders and energy production barriers could affect cell cycle and proliferation. Notably, CCNB1 played a crucial role in cell division and had been shown to accelerate the cell cycle and then promote rumen growth in goats [ 38 ]. The enhancement of rumen cell proliferation was associated with the increased expression of CCNB1 and CCNE1 , which contributed to the development of rumen epithelium [ 38 ]. In addition, BUB1 played an active role in promoting the proliferation of Human endometrial epithelial cells and endometrial cancer Ishikawa cells [ 39 ]. Interestingly, E2F family proteins were transcriptional activators of the genes encoding cell cycle-regulatory proteins including CCNA, CCNE, CDK2, and CDC25 [ 40 ]. In this study, it was found maternal undernutrition downregulated these genes related to cell cycle and cell proliferation in fetal rumen and inhibited fetal rumen development. As a critical post-transcriptional regulatory mechanism, miRNAs play a much larger role in gene expression regulation than previously understood [41; 42]. Although miRNAs had been widely identified in various ruminant tissues, their roles in regulating gastrointestinal development and function remain underexplored [ 43 ]. In calves, miR-143 was the most highly expressed miRNA in rumen, and its expression pattern changed over the first 6 weeks after birth [ 44 ]. The targeted genes of miR-143 were associated with immune response and developmental processes [ 44 ]. In this study, we identified a total of 1,253 miRNAs in sheep fetal rumen, including 1,101 newly predicted miRNAs and 152 known miRNAs. Moreover, we found the newly identified miR-736 targeted E2F2 and MYBL2 . In mouse lung cancer cells, miR-637 targeted E2F2 and reduced cell proliferation [ 45 ]. However, to date, no E2F2 -targeting miRNAs had been reported in the rumen. In addition, MYBL2 expression was downregulated during the maturation of colon epithelial cells, in part through miR-365 [ 46 ]. In this study, it was confirmed for the first time that the newly identified miRNA-736 inhibited E2F2 and MYBL2 , thereby promoted apoptosis and negatively regulated entry into the S-phase of the cell cycle for rumen epithelial cells. Therefore, maternal undernutrition induced the overexpression of miRNA-736 in fetal rumen, which targeted E2F2 and MYBL2 , promoted rumen epithelial cell apoptosis, and inhibited cell cycle and cell proliferation. The oncogenic effects of E2F family genes ( E2F1 and E2F7 ) in gastric cancer were at least partially mediated through transcriptional activation of MYBL2 [ 47 ]. E2F1 , E2F2 , and E2F3 were transcriptional activators while E2F7 and E2F8 acted as atypical transcriptional suppressors [ 48 ]. E2F2 was closely involved in several critical cellular processes including cell cycle, apoptosis, differentiation, and stress response [ 49 ]. Silencing E2F2 had been shown to impair cell growth, invasion, and migration in gastric cancer cells [ 50 ]. In this study, silencing E2F2 significantly inhibited the proliferation of rumen epithelial cells and promoted apoptosis. In addition, knockdown of MYBL2 significantly inhibited the proliferation and migration of ovarian cancer cells [ 51 ]. In our study, silencing MYBL2 also significantly inhibited cell proliferation and promoted apoptosis of rumen epithelial cells. Therefore, maternal undernutrition downregulated E2F2 and MYBL2 in fetal rumen, which further suppressed cell cycle and cell proliferation, promoted cell apoptosis, and inhibited fetal rumen development. Conclusions In conclusion, maternal undernutrition suppressed nutrient metabolism and energy production via JAK3 / STAT3 signaling pathway and further inhibited fetal rumen development, while maternal nutritional recovery partially restored metabolic homeostasis and energy production to alleviate undernutrition-induced fetal rumen maldevelopment (Fig. 6 ). Interestingly, maternal undernutrition induced the overexpression of miRNA-736 in fetal rumen, which inhibited the expression of its target genes E2F2 and MYBL2 , thereby disrupted cell cycle, promoted apoptosis, and ultimately inhibited fetal rumen development (Fig. 6 ). These findings provided valuable insights into the molecular mechanisms underlying the impact of maternal nutrition on fetal growth and development and contributed to developing potential nutritional strategies to improve fetal health outcomes. Abbreviations DEMs : Differentially expressed miRNAs DEGs : Differentially expressed genes E2F2 : E2F transcription factor 2 MYBL2 : MYB proto-oncogene like 2 FC : Fold change PCA : Principal component analysis PLS-DA : Partial least squares discriminant analysis NC : Negative control PI : Propidium iodide PS : Phosphatidylserine Declarations Authors' contributions The author’s contributions are as follows: Y. X., P. J., S. M., and J. C. conceived and designed the study; P. J., Y. L., and Y. X. conducted the research; P. J. and Y. X. analyzed and interpreted the data; P. J. and Y. X. wrote the manuscript; and Y. X., H. L., Y. G., C. F., W. Z., S. M., and J. C. revised the manuscript. All authors read and approved the final version of the manuscript. Funding This work was supported by the National Natural Science Foundation of China (32402767), National Key Research and Development Program of China (2022YFD1301102), Anhui Province Natural Science Foundation Youth Project (2308085QC104), and AAU Introduction of High-level Talent Funds (RC392107), Key Laboratory of Utilization of Livestock and Forage Resources in Circum-Tarim Region (Co-construction by Ministry and Province), Ministry of Agriculture and Rural Affairs (BSGJSYS202502). Acknowledgments We thank Biomarker Biotechnology Co., Ltd. (Beijing, China) for technical assistance on transcriptome sequencing and miRNA sequencing. Rumen epithelial cells were generously provided by Zhu Wen's team at Anhui Agricultural University. Data availability The raw sequencing data supporting this study have been deposited in the Gene Expression Omnibus (GEO) under controlled access, with rumen transcriptome data available through accession number GSE284288 (security token: qdqbggywvpehjif; direct link: https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE284288&token=qdqbggywvpehjif) and rumen miRNA sequencing data accessible via accession number GSE285445 (security token: wlqxqkmwbtoxtml; direct link: https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE285445&token=wlqxqkmwbtoxtml). References Skurková, L., L. Matulníková, B. Peťková, M. Florian, M. Slivková, L. Lešková, L. Mesarčová, and J. Kottferová. Seasonal pattern of cortisol fluctuation in horsehair samples from three different body areas: A year long study. Journal of Equine Veterinary Science. 2025; 147:105387. https://doi.org/10.1016/j.jevs.2025.105387. Xue, Y., C. Guo, F. Hu, W. Zhu, and S. Mao. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6598539","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":454119950,"identity":"3942b295-cfef-444a-87ad-b1dc1c33a5da","order_by":0,"name":"Peng Jiao","email":"","orcid":"","institution":"Anhui Agricultural University College of Animal Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"Peng","middleName":"","lastName":"Jiao","suffix":""},{"id":454119951,"identity":"4d057cf6-1962-4cf1-ac8a-d4dda8c336aa","order_by":1,"name":"Yun Xu","email":"","orcid":"","institution":"Anhui Agricultural University 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University","correspondingAuthor":false,"prefix":"","firstName":"Huizhen","middleName":"","lastName":"Lu","suffix":""},{"id":454119955,"identity":"8736999c-5c8b-4376-8ca4-63ae71dc8bee","order_by":5,"name":"Caiyun Fan","email":"","orcid":"","institution":"Anhui Agricultural University College of Animal Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"Caiyun","middleName":"","lastName":"Fan","suffix":""},{"id":454119956,"identity":"f089e1a3-6ce9-48f6-b54e-96748d5f6790","order_by":6,"name":"Wen Zhu","email":"","orcid":"","institution":"Anhui Agricultural University College of Animal Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"Wen","middleName":"","lastName":"Zhu","suffix":""},{"id":454119957,"identity":"797968e3-46fc-4280-8b3a-2bca782634d3","order_by":7,"name":"Jianbo Cheng","email":"","orcid":"","institution":"Anhui Agricultural University College of Animal Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"Jianbo","middleName":"","lastName":"Cheng","suffix":""},{"id":454119958,"identity":"154e11b0-c54f-4096-ade4-74155e16328c","order_by":8,"name":"Mianqun Zhang","email":"","orcid":"","institution":"Anhui Agricultural University College of Animal Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"Mianqun","middleName":"","lastName":"Zhang","suffix":""},{"id":454119959,"identity":"6d08145a-9c01-47d7-86f0-eb27d7dbe32a","order_by":9,"name":"Shengyong Mao","email":"","orcid":"","institution":"Nanjing Agricultural University College of Animal Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"Shengyong","middleName":"","lastName":"Mao","suffix":""},{"id":454119960,"identity":"5eb5be87-4fc4-4ed5-8c1a-6e7a237a342b","order_by":10,"name":"Yanfeng Xue","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABBklEQVRIiWNgGAWjYPACOSBmbHzwwcCGh5+/gSgtxkDM3Gw4oyJNRnLGAaK1sLdJ85w5bGPQkIBfrcGN5GMSP3cYyJnzL2yT5m07z2PAcIDxw8cc3FokZ6SlSfaeMTC2nPGw2XJu220ec+YGZsmZ23Br4ZfIMZPgbfuTuOHGwcYbb4FaLBsOsDHz4tHCJpH/TfJvm0E9UEsDUO85HoMDCfi1AG1hA3rBIMHgfGOTJM+ZA4S1SPY8M7aWbTMw3HCDERTIyTySMw424/WLwfHkhzffthnIG5w//hAYlXb2/PzNBz98xKMFCFgkwJREAkyAsQGveiBg/gDx1QFCCkfBKBgFo2CkAgCICFd9RYeOZgAAAABJRU5ErkJggg==","orcid":"https://orcid.org/0000-0003-0257-3825","institution":"Anhui Agricultural University College of Animal Science and Technology","correspondingAuthor":true,"prefix":"","firstName":"Yanfeng","middleName":"","lastName":"Xue","suffix":""}],"badges":[],"createdAt":"2025-05-06 03:28:27","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6598539/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6598539/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":82642728,"identity":"08149993-ec13-46e3-a97d-91e51b5f7540","added_by":"auto","created_at":"2025-05-13 15:33:37","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":614812,"visible":true,"origin":"","legend":"\u003cp\u003eMaternal undernutrition suppressed nutrient metabolism and cell cycle in fetal rumen to inhibit fetal rumen development. (\u003cstrong\u003eA\u003c/strong\u003e) Fetal rumen empty weight. (\u003cstrong\u003eB\u003c/strong\u003e) Fetal rumen hematoxylin-eosin staining sections. (\u003cstrong\u003eC\u003c/strong\u003e) Fetal rumen papilla size. (\u003cstrong\u003eD\u003c/strong\u003e) Fetal rumen papilla surface area. (\u003cstrong\u003eE-F\u003c/strong\u003e) PCA and PLS-DA of total gene expression in rumen of the CON and UN groups. PCA score scatter plot (predictive ability parameter (Q2) (cum) = 0.200, goodness-of-fit parameter (R2) (Y) = 0.146). PLS-DA score scatter plot (predictive ability parameter (Q2) (cum) = 0.131, goodness-of-fit parameter (R2) (Y) = 0.141). (\u003cstrong\u003eG-H\u003c/strong\u003e) GSEA maps of enriched pathways associated with cytokine-cytokine receptor and cell cycle. (\u003cstrong\u003eI\u003c/strong\u003e) The expression of genes related to cytokine-cytokine receptor and cell cycle by RT-qPCR. (\u003cstrong\u003eJ\u003c/strong\u003e) Correlation analysis of rumen phenotypes and gene expression of cytokine-cytokine receptor and cell cycle. (\u003cstrong\u003eK\u003c/strong\u003e) KOG functional classification of DEGs in fetal rumen between the CON and UN groups. (\u003cstrong\u003eL\u003c/strong\u003e) The expression of DEGs related to nutrient metabolism by RNA-Seq. (\u003cstrong\u003eM\u003c/strong\u003e) The expression of genes related to nutrient metabolism by RT-qPCR. (\u003cstrong\u003eN\u003c/strong\u003e) Maternal undernutrition inhibited fetal rumen growth and development by reducing nutrient metabolism, cytokines and cytokine receptors, and cell cycle. Rumen weight, size, and surface area and RT-qPCR date were analyzed using two-tailed t-test (n = 10, * \u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"Onlinefloatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-6598539/v1/48b6bbf87f18750996ed64a3.png"},{"id":82642270,"identity":"c7c7914d-20e4-49c6-9ea5-0832bc49ddaa","added_by":"auto","created_at":"2025-05-13 15:25:37","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":635927,"visible":true,"origin":"","legend":"\u003cp\u003eMaternal nutritional recovery partially restored metabolic inhibition but failed to alleviate fetal rumen development. (\u003cstrong\u003eA\u003c/strong\u003e) Fetal rumen hematoxylin-eosin staining sections. (\u003cstrong\u003eB\u003c/strong\u003e) Fetal rumen empty weight. (\u003cstrong\u003eC\u003c/strong\u003e) Fetal rumen papilla size. (\u003cstrong\u003eD\u003c/strong\u003e) Fetal rumen papilla surface area. (\u003cstrong\u003eE-F\u003c/strong\u003e) PCA and PLS-DA of total gene expression in rumen of the CON and REC groups. PCA score scatter plot (predictive ability parameter (Q2) (cum) = 0.269, goodness-of-fit parameter (R2) (Y) = 0.165). PLS-DA score scatter plot (predictive ability parameter (Q2) (cum) = 0.148, goodness-of-fit parameter (R2) (Y) = 0.153). (\u003cstrong\u003eG-H\u003c/strong\u003e) GSEA maps of enriched pathways associated with cytokine-cytokine receptor and cell cycle. (\u003cstrong\u003eI\u003c/strong\u003e) The expression of genes related to cytokine-cytokine receptor and cell cycle by RT-qPCR. (\u003cstrong\u003eJ\u003c/strong\u003e) Correlation analysis of rumen phenotypes and gene expression of cytokine-cytokine receptor and cell cycle. (\u003cstrong\u003eK\u003c/strong\u003e) KOG functional classification of DEGs in fetal rumen between the CON and REC groups. (\u003cstrong\u003eL\u003c/strong\u003e) The expression of DEGs related to nutrient metabolism by RNA-Seq. (\u003cstrong\u003eM\u003c/strong\u003e) The expression of genes related to nutrient metabolism by RT-qPCR. (\u003cstrong\u003eN\u003c/strong\u003e) Maternal nutritional recovery partially restored metabolic inhibition to alleviate undernutrition-induced fetal rumen maldevelopment. Rumen weight, size, and surface area and RT-qPCR date were analyzed using two-tailed t-test (n = 7, * \u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"Onlinefloatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-6598539/v1/18f5669cb1c8d0c3345f1b8b.png"},{"id":82642279,"identity":"d0974312-62e7-48bb-9fcc-dabc89903af3","added_by":"auto","created_at":"2025-05-13 15:25:37","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":332452,"visible":true,"origin":"","legend":"\u003cp\u003eDifferentially expressed miRNA analysis in fetal rumen induced by maternal undernutrition. (\u003cstrong\u003eA-B\u003c/strong\u003e) DEMs in fetal rumen of maternal undernutrition model (n=3). (\u003cstrong\u003eC\u003c/strong\u003e) DEMs verification by RT-qPCR. (\u003cstrong\u003eD\u003c/strong\u003e) miRNA-736 expressional level in fetal rumen of maternal nutrition-recovery model via RT-qPCR. (\u003cstrong\u003eE\u003c/strong\u003e) KEGG analysis of DEMs between the CON and UN groups. (\u003cstrong\u003eF\u003c/strong\u003e) Network map of miRNA-736 target genes. (\u003cstrong\u003eG-H\u003c/strong\u003e) Protein-protein interaction networks of E2F2 and MYBL2 constructed using STRING database. RT-qPCR data were analyzed using two-tailed t-test (n = 10, * \u003cem\u003eP\u003c/em\u003e\u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"Onlinefloatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-6598539/v1/0dbeff7e3082c84bacea2396.png"},{"id":82642274,"identity":"623c2d0c-9c92-43ef-9896-808a0c2d0a7a","added_by":"auto","created_at":"2025-05-13 15:25:37","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":315468,"visible":true,"origin":"","legend":"\u003cp\u003emiRNA-736 regulated rumen epithelial cell cycle and apoptosis by targeting \u003cem\u003eE2F2\u003c/em\u003e and \u003cem\u003eMYBL2\u003c/em\u003e. (\u003cstrong\u003eA\u003c/strong\u003e) miRNA-736 target sites prediction for \u003cem\u003eE2F2\u003c/em\u003e and \u003cem\u003eMYBL2\u003c/em\u003e. (\u003cstrong\u003eB-C\u003c/strong\u003e) Verification of the presence of miRNA-736 binding site in the \u003cem\u003eE2F2\u003c/em\u003e and \u003cem\u003eMYBL2\u003c/em\u003e3'UTR by a dual-luciferase reporter assay in 293T cells. (\u003cstrong\u003eD-E\u003c/strong\u003e) Transfection efficiency of miRNA-736 mimic and inhibitor in Hu-sheep rumen epithelial cells via RT-qPCR. (\u003cstrong\u003eF-G\u003c/strong\u003e) The mRNA expressional levels of \u003cem\u003eE2F2\u003c/em\u003eand \u003cem\u003eMYBL2\u003c/em\u003e transfected with miRNA-736 mimic and inhibitor. (\u003cstrong\u003eH-I\u003c/strong\u003e) The expression of genes related to cell cycle and apoptosis transfected with miRNA-736 mimic and inhibitor. (\u003cstrong\u003eJ-K\u003c/strong\u003e) Effects of miRNA-736 mimic and inhibitor transfection on rumen epithelial cell apoptosis. mimic NC or inhibitor NC, negative control; mimic, overexpression of miRNA-736; inhibitor, inhibition of miRNA-736. pisCHECK2+3' UTR= dual-luciferase reporter recombinant vectors \u003cem\u003eE2F2\u003c/em\u003e or \u003cem\u003eMYBL2\u003c/em\u003e -wt-3' UTR. Rluc/Fluc = Renilla luciferase/firefly luciferase. The data were analyzed using two-tailed t-test for comparisons between two groups (n = 3) or one-way ANOVA for comparisons among three groups (n = 3), *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05.\u003c/p\u003e","description":"","filename":"Onlinefloatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-6598539/v1/2f4d7246cd6b3066c90a84c1.png"},{"id":82642729,"identity":"95bb1be2-61be-4da5-be38-2a5e82bec225","added_by":"auto","created_at":"2025-05-13 15:33:37","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":367457,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eE2F2\u003c/em\u003e and \u003cem\u003eMYBL2\u003c/em\u003e regulated rumen epithelial cell cycle and apoptosis. (\u003cstrong\u003eA\u003c/strong\u003e) \u003cem\u003eE2F2\u003c/em\u003e silencing efficiency assay. (\u003cstrong\u003eB\u003c/strong\u003e) The expression of genes related to cell cycle and apoptosis transfected with siRNA-\u003cem\u003eE2F2\u003c/em\u003e. (\u003cstrong\u003eC\u003c/strong\u003e) Detection of rumen epithelial cell viability transfected with siRNA-\u003cem\u003eE2F2\u003c/em\u003e. (\u003cstrong\u003eD-E\u003c/strong\u003e) Detection of rumen epithelial cell apoptosis rate transfected with siRNA-\u003cem\u003eE2F2\u003c/em\u003e. (\u003cstrong\u003eF-G\u003c/strong\u003e) Cell cycle detection of rumen epithelial cells transfected with siRNA-\u003cem\u003eE2F2\u003c/em\u003e. (\u003cstrong\u003eH\u003c/strong\u003e) MYBL2 silencing efficiency assay. (\u003cstrong\u003eI\u003c/strong\u003e) The expression of genes related to cell cycle and apoptosis transfected with siRNA-\u003cem\u003eMYBL2\u003c/em\u003e. (\u003cstrong\u003eJ\u003c/strong\u003e) Detection of rumen epithelial cell viability transfected with siRNA-\u003cem\u003eMYBL2\u003c/em\u003e. (\u003cstrong\u003eK-L\u003c/strong\u003e) Detection of rumen epithelial cell apoptosis rate transfected with siRNA-\u003cem\u003eMYBL2\u003c/em\u003e. (\u003cstrong\u003eM-N\u003c/strong\u003e) Cell cycle detection of rumen epithelial cells transfected with siRNA-\u003cem\u003eMYBL2. \u003c/em\u003eThe data were analyzed using two-tailed t-test for comparisons between two groups (n = 3) or one-way ANOVA for comparisons among three groups (n = 3), *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05.\u003c/p\u003e","description":"","filename":"Onlinefloatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-6598539/v1/7326018628c9e03aa9b700e5.png"},{"id":82642276,"identity":"a4fda8be-794f-44e4-a200-ad005c25d1c3","added_by":"auto","created_at":"2025-05-13 15:25:37","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":2212376,"visible":true,"origin":"","legend":"\u003cp\u003eMechanism summary diagram of maternal undernutrition induced fetal rumen nutrient metabolism disorders and development retardation. Red font and background indicated enhancement, green font and background indicated decrease.\u003c/p\u003e","description":"","filename":"Onlinefloatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-6598539/v1/a468be31c850eff5a0c64d47.png"},{"id":90157676,"identity":"80cae3be-b918-4df5-abb7-55452820462f","added_by":"auto","created_at":"2025-08-29 08:32:34","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3651795,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6598539/v1/f9634255-ddf5-4327-ab27-4057a955a23d.pdf"},{"id":82642731,"identity":"066b50e9-1fd5-487e-98c2-638b60049d0e","added_by":"auto","created_at":"2025-05-13 15:33:37","extension":"docx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":1591048,"visible":true,"origin":"","legend":"","description":"","filename":"Fig.S1.docx","url":"https://assets-eu.researchsquare.com/files/rs-6598539/v1/ce62222a8d8db975064723d4.docx"},{"id":82642298,"identity":"622a71cb-a573-489e-b82a-56e9004f5381","added_by":"auto","created_at":"2025-05-13 15:25:38","extension":"docx","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":2699350,"visible":true,"origin":"","legend":"","description":"","filename":"Fig.S2.docx","url":"https://assets-eu.researchsquare.com/files/rs-6598539/v1/133483c9c6256daa0c6c5a9d.docx"},{"id":82642293,"identity":"3972a801-dddc-4b83-bcdc-b962223b20fa","added_by":"auto","created_at":"2025-05-13 15:25:38","extension":"docx","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":3091699,"visible":true,"origin":"","legend":"","description":"","filename":"Fig.S3.docx","url":"https://assets-eu.researchsquare.com/files/rs-6598539/v1/43b4366e732c33669b6e5e5a.docx"},{"id":82642730,"identity":"c7f726e0-9826-47b0-8036-cca610e64bf0","added_by":"auto","created_at":"2025-05-13 15:33:37","extension":"docx","order_by":7,"title":"","display":"","copyAsset":false,"role":"supplement","size":17682,"visible":true,"origin":"","legend":"","description":"","filename":"TableS1.docx","url":"https://assets-eu.researchsquare.com/files/rs-6598539/v1/248b1e7b91b09362a6312078.docx"},{"id":82642285,"identity":"2ea6d249-f905-45ef-a4bb-be66265feb5a","added_by":"auto","created_at":"2025-05-13 15:25:37","extension":"docx","order_by":8,"title":"","display":"","copyAsset":false,"role":"supplement","size":17029,"visible":true,"origin":"","legend":"","description":"","filename":"TableS2.docx","url":"https://assets-eu.researchsquare.com/files/rs-6598539/v1/3d703a3937c5c0d79c26e026.docx"},{"id":82642297,"identity":"b4ba3c15-272e-48e4-b439-48092984d4cc","added_by":"auto","created_at":"2025-05-13 15:25:38","extension":"docx","order_by":9,"title":"","display":"","copyAsset":false,"role":"supplement","size":16693,"visible":true,"origin":"","legend":"","description":"","filename":"TableS3.docx","url":"https://assets-eu.researchsquare.com/files/rs-6598539/v1/6fb9b937ba1c782cf905fdb9.docx"},{"id":82642280,"identity":"8e4ef1ae-6a4d-4dfa-b04d-4e287313f1ba","added_by":"auto","created_at":"2025-05-13 15:25:37","extension":"docx","order_by":10,"title":"","display":"","copyAsset":false,"role":"supplement","size":22035,"visible":true,"origin":"","legend":"","description":"","filename":"TableS4.docx","url":"https://assets-eu.researchsquare.com/files/rs-6598539/v1/81daf8c616bf90c138190bf5.docx"},{"id":82642737,"identity":"d8e092b0-5c4e-4917-9fb9-06ddb06655ac","added_by":"auto","created_at":"2025-05-13 15:33:38","extension":"xlsx","order_by":11,"title":"","display":"","copyAsset":false,"role":"supplement","size":177497,"visible":true,"origin":"","legend":"","description":"","filename":"TableS5.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-6598539/v1/197990c5f994815d52194993.xlsx"}],"financialInterests":"","formattedTitle":"Maternal undernutrition inhibits fetal rumen development： Novel miRNA-736-mediated dual targeting of E2F2 and MYBL2 in sheep","fulltext":[{"header":"Introduction","content":"\u003cp\u003eIn ruminant production system, many animals are subjected to undernutrition, a condition that may be exacerbated by seasonal fluctuations of feed availability or by economic factors-driven artificial control [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Due to the multi-fetus characteristic of Hu-sheep and the rapid fetal growth and development during late gestation, pregnant ewes are prone to undernutrition which ultimately inhibits fetal development [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Rumen, as a critical digestive organ in ruminant, requires early and optimal development to ensure both animal health and productivity [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. The transitional period (pre-rumination) in young ruminants represents a sensitive window for influencing the development of rumen wall [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. A large body of researches have shown that varying kinds of nutritional regulation promote rumen development of lambs [\u003cspan additionalcitationids=\"CR6\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. The enhancement of rumen wall morphology and physiological function through early-stage nutritional strategies holds significant potential for supporting the lifelong health and productivity of ruminants. However, the effect of maternal nutritional status or nutritional regulation on the morphology and development of fetal rumen before fetal birth remains underexplored. The loss of physical function caused by undernutrition in ruminants may be compensated by subsequent nutritional recovery [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Metabolic disorders and pregnancy toxemia resulting from malnutrition during late pregnancy can be effectively ameliorated through nutritional recovery [9; 10]. However, whether the effects of maternal undernutrition on fetal rumen development can be alleviated by maternal nutritional recovery has not been studied.\u003c/p\u003e \u003cp\u003eRuminal papilla are basic structures of ruminal epithelium and associated with great contact with chyme, enhancing the digestion and absorption of nutrients [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Our previous study has shown undernutrition significantly decreases maternal rumen weight and the length, width, and surface area of rumen papilla [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Meanwhile, maternal undernutrition seriously affects fetal weight and fetal liver development [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. However, the effects of maternal undernutrition on fetal rumen development and the potential regulatory mechanisms remain poorly understood. Undernutrition of pregnant ewes has been found to inhibit the metabolism of carbohydrates, amino acids, and energy in the rumen and to suppress DNA replication and cell cycle in rumen epithelium through JAK3/STAT2 signaling pathway, ultimately leading to the narrower, shorter, and small rumen papilla [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Therefore, it is supposed that maternal undernutrition may affect nutrient metabolism and cell cycle in fetal remen to inhibit its development via some key signaling pathways. As known, miRNAs are key regulators of various biological processes, and fetal plasticity during pregnancy was regulated by miRNAs [13; 14]. miR-21-3p has been identified in sheep rumen where it regulates amino acid metabolism by targeting \u003cem\u003eATP1A2\u003c/em\u003e [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e], while miR-148a-3p inhibits the proliferation of rumen epithelial cells by targeting \u003cem\u003eQKI5\u003c/em\u003e [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. However, whether maternal undernutrition can induce DEMs in fetal rumen and further regulate the growth and development of rumen epithelial cells remains unclear.\u003c/p\u003e \u003cp\u003eThis study postulated that maternal undernutrition during late gestation might inhibit fetal rumen development through regulating miRNA expression to disrupt cell cycle and rumen nutrient metabolism, and maternal nutritional recovery after undernutrition could only partially restore the nutrient metabolism homeostasis and fetal rumen development. Undernourished and nutrition-recovery pregnant sheep model were established using dietary nutrition control. Both \u003cem\u003ein vivo\u003c/em\u003e animal models and \u003cem\u003ein vitro\u003c/em\u003e sheep primary rumen epithelial cells were integrated to investigate the molecular mechanism of how maternal nutrition affects fetal rumen development. This study would provide theoretical foundations for the development of molecular therapeutic strategies to promote fetal rumen development.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\"\u003e\n \u003ch2\u003eAnimal experimental design and sample collection\u003c/h2\u003e\n \u003cp\u003eThe animal experiments were approved by the Institutional Animal Care and Use Committee of Anhui Agricultural University (AHAUXMSQ2024073). This study was part of a larger project aimed at exploring how undernutrition and nutritional recovery during late gestation affects maternal and fetal metabolic homeostasis. In undernourished and nutrition-recovery ewe models, the diet formula and nutrient composition are detailed in \u003cstrong\u003eTable S1\u003c/strong\u003e. The metabolic energy and crude protein content of the diet were 11.25 MJ/kg and 12.60%, respectively.\u003c/p\u003e\n \u003cp\u003eUndernourished ewe model: Twenty ewes (body weight 54.4\u0026thinsp;\u0026plusmn;\u0026thinsp;2.48 kg, 2\u0026ndash;3 fetuses, pregnancy for 108 days) were fed \u003cem\u003ead libitum\u003c/em\u003e for a 7-day adaptation period to detect feed intake and make sure the dietary nutritional level could meet the requirements of pregnant ewes carrying multiple fetuses. Subsequently, these ewes were randomly assigned to the CON group (n\u0026thinsp;=\u0026thinsp;10, fed the baseline feed intake) or the UN group (n\u0026thinsp;=\u0026thinsp;10, restricted to 30% of the baseline feed intake) for 15 days. Ewes were slaughtered at 4 hours after morning feeding, and fetal rumen epithelium samples were collected from the male fetuses (CON: n\u0026thinsp;=\u0026thinsp;10, UN: n\u0026thinsp;=\u0026thinsp;10) and stored in liquid nitrogen.\u003c/p\u003e\n \u003cp\u003eNutrition-recovery ewe model: Twenty four ewes (body weight 54.6\u0026thinsp;\u0026plusmn;\u0026thinsp;1.69 kg, 2\u0026ndash;3 fetuses, pregnancy for 108 days) were similarly fed \u003cem\u003ead libitum\u003c/em\u003e for a 7-day adaptation period to detect the baseline feed intake. Ewes were then randomly assigned to the CON group (n\u0026thinsp;=\u0026thinsp;12, fed the baseline feed intake for 15 days followed by \u003cem\u003ead libitum\u003c/em\u003e feeding for another 15 days) or the REC group (n\u0026thinsp;=\u0026thinsp;12, feed intake restricted to 30% of baseline for 15 days followed by \u003cem\u003ead libitum\u003c/em\u003e feeding for another 15 days). Due the predelivery of 3 pregnant ewes in the nutritional recovery period, they were removed from the research subjects. Subsequently, fetal rumen epithelium samples were collected from male fetuses (CON: n\u0026thinsp;=\u0026thinsp;9, REC: n\u0026thinsp;=\u0026thinsp;9) and stored in liquid nitrogen.\u003c/p\u003e\n\u003c/div\u003e\n\u003ch3\u003eFetal rumen weight and morphological analysis\u003c/h3\u003e\n\u003cp\u003eChyme was immediately removed from the fetal rumen after slaughter, and the weight of the empty fetal rumen was measured. Fetal rumen specimens were collected, fixed in 4% paraformaldehyde for 24 hours, embedded in paraffin, and sectioned for hematoxylin and eosin staining for morphological observation [12].\u003c/p\u003e\n\u003ch3\u003eTranscriptome assay of rumen epithelium\u003c/h3\u003e\n\u003cp\u003eTotal RNA was extracted from cryopreserved fetal rumen tissues using TRIzol reagent (Takara Bio, Otsu, Japan). RNA quality was verified by spectrophotometric analysis (NanoDrop ND-1000) with A260/230 and A260/280 ratios between 1.80\u0026ndash;2.10, and RNA integrity was confirmed using an Agilent 2100 Bioanalyzer. A total of twenty RNA samples were randomly selected for transcriptome analysis, with ten samples from undernourished model (five CON and five UN) and another ten samples from nutritional recover model (five CON and five REC). The mRNA samples were purified using the magnetic bead method, fragmented, and reversely transcribed into cDNA, which were then ligated to sequencing adapters and amplified. Fragments with appropriate length were selected to construct cDNA library using NEBNext\u0026reg; Ultra\u0026trade; RNA Library Preparation Kit and sequenced on an Illumina NovaSeq 6000 (Biomarker, Beijing, China). Clean reads were aligned to the Sheep Reference Genome 3.0 using HiSAT2, and gene expression was quantified using FPKM method. DEGs were identified with DESeq2 (v.1.6.3) based on \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01 and fold change (FC)\u0026thinsp;\u0026gt;\u0026thinsp;1.5 or \u0026lt;\u0026thinsp;0.667. Principal component analysis (PCA) and partial least squares discriminant analysis (PLS-DA) were performed using SIMCA v.14.1 (Umetrics, Umea, Sweden). Data analyses including clustering heatmaps, KOG functional classification, and GSEA analysis were conducted using the Biomarker platform (www.biocloud.net).\u003c/p\u003e\n\u003ch3\u003emiRNA-Seq library construction, sequencing, and data analysis\u003c/h3\u003e\n\u003cp\u003eSmall RNA library was constructed using 1.5 \u0026micro;g of total RNA with a NextFlex Small RNA Sequencing Kit (Illumina, San Diego, CA, USA). Briefly, small RNAs (18\u0026ndash;30 nucleotides) were size-selected via gel electrophoresis, followed by sequential ligation of 3\u0026rsquo; and 5\u0026rsquo; adapters. After adapter ligation, reverse transcription and PCR amplification were performed with 12 cycles to enrich the library. Library quality was assessed using an Agilent Bioanalyzer 2100, and the quantification was performed by Qubit 3.0 Fluorometer (Thermo Fisher Scientific). The library was subsequently sequenced on the Illumina HiSeq 6000 platform using single-end sequencing. Raw sequencing reads underwent quality control using FastQC to remove low-quality reads, adapter sequences, and potential contaminants. Clean reads were aligned to the Sheep Reference Genome 3.0 using Bowtie2, allowing for the identification of known miRNAs and the discovery of novel miRNAs. The analysis of miRNA expressional levels and the identification of DEMs were conducted using DESeq2 (v.1.6.3) with the thresholds of \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 and FC\u0026thinsp;\u0026gt;\u0026thinsp;1.5 or \u0026lt;\u0026thinsp;0.667. Additionally, potential target genes of DEMs were predicted using miRanda software. KEGG pathway enrichment analysis, network map of miRNA target genes, and Venn diagram analysis were conducted to explore the biological functions and regulatory pathways of these miRNAs in fetal rumen development. Subsequently, STRING software was used to analyze the protein-protein interactions (PPIs) of the target genes (\u003cem\u003eE2F2\u003c/em\u003e and \u003cem\u003eMYBL2\u003c/em\u003e) and to perform Gene Ontology (GO) pathway enrichment analysis.\u003c/p\u003e\n\u003ch3\u003eCell culture assay\u003c/h3\u003e\n\u003cp\u003ePrimary rumen epithelial cells were cultured in DMEM/F12 medium (GE Healthcare Life Sciences, Hyclone Laboratories, South Logan, Utah) supplemented with 10% fetal bovine serum (FBS; BI, Israel Beit Haemek Ltd.). Human embryonic kidney (HEK) 293T cells were cultured in high-glucose DMEM (GE Healthcare Life Sciences, Hyclone Laboratories, South Logan, Utah) containing 5% FBS (BI, Israel Beit Haemek Ltd.). All cell cultures were maintained under standard incubation conditions of 37\u0026deg;C and 5% CO\u003csub\u003e2\u003c/sub\u003e.\u003c/p\u003e\n\u003cdiv id=\"Sec8\"\u003e\n \u003ch2\u003emiRNA mimic, inhibitors, and siRNA\u003c/h2\u003e\n \u003cp\u003eThe mimic of miR-736 (double-strand oligonucleotides), mismatched negative control mimic (mimic NC), miR-736 inhibitor (single-strand oligonucleotides), and mismatched negative control inhibitor (inhibitor NC) were synthesized by RIBOBIO Co., Ltd. (Guangzhou, China). These mimics and inhibitors were used to assess the effects of miR-736 overexpression and inhibition on its activity in HEK-293 cells and rumen epithelial cells. The small interference RNA (siRNA) for \u003cem\u003eE2F2\u003c/em\u003e and \u003cem\u003eMYBL2\u003c/em\u003e were synthesized by SANGON Biotech (Shanghai, China). The sequences for the si-NC and siRNA primers were provided in \u003cstrong\u003eTable S2\u003c/strong\u003e.\u003c/p\u003e\n\u003c/div\u003e\n\u003ch3\u003e3\u0026prime; UTR luciferase reporter assays\u003c/h3\u003e\n\u003cp\u003ePrimers targeting the 3\u0026apos; UTR regions of \u003cem\u003eE2F2\u003c/em\u003e and \u003cem\u003eMYBL2\u003c/em\u003e were designed using Primer Premier 5.0 (Premier Biosoft, Palo Alto, CA). Detailed primer sequences are provided in \u003cstrong\u003eTable S3\u003c/strong\u003e. Then, RNA from rumen epithelial cells were extracted and reversely transcribed into cDNA using PrimescriptTM RT reagent Kit (TaKaRa, Dalian, China). The 3\u0026prime; UTR fragments of \u003cem\u003eE2F2\u003c/em\u003e and \u003cem\u003eMYBL2\u003c/em\u003e were amplified using the following 20 \u0026micro;L reaction system: 0.8 \u0026micro;L of forward and reverse primers, 1 \u0026micro;L cDNA, 10 \u0026micro;L Taq PCR Master Mix, and 7.4 \u0026micro;L ddH₂O. The resulting 3\u0026prime; UTR double-stranded cDNA fragments of \u003cem\u003eE2F2\u003c/em\u003e and \u003cem\u003eMYBL2\u003c/em\u003e were enzymatically cleaved and cloned into the \u003cem\u003eXhoI\u003c/em\u003e-\u003cem\u003eNotI\u003c/em\u003e sites of the psiCHECK\u0026trade;-2 vector (Promega Corp.), which contained both Renilla and Firefly luciferase reporter genes, forming the wild-type dual luciferase reporter gene recombinant vector. The psiCHECK2-E2F2 and psiCHECK2-MYBL2 recombinant plasmids were extracted using the EndoFree Plasmid Mini Kit (Omega Bio-Tek, Norcross, GA), and the reliability of these recombinant vectors was verified by DNA sequencing.\u003c/p\u003e\n\u003cp\u003eHEK-293T cells were seeded into 24-well plates at a density of 1.0 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e cells/well. After 24 hours, cells were co-transfected with 0.5 \u0026micro;g of either psiCHECK2-E2F2 or psiCHECK2-MYBL2 recombinant plasmid and 0.5 \u0026micro;g of miR-736 mimic (or control NC_mimic) using 2 \u0026micro;L of X-tremeGENE HP DNA transfection reagent (Roche, Penzberg, Germany) following the manufacturer\u0026rsquo;s protocol (06365752001a.fm). After 48 hours, Renilla luciferase activity, normalized to Firefly luciferase activity, was measured using the Dual-Luciferase\u0026reg; Reporter Assay System (Promega, Madison, WI) according to the manufacturer\u0026rsquo;s instructions. A decrease of more than 30% in the Renilla to Firefly luciferase ratio in the psiCHECK2 3\u0026prime; UTR\u0026thinsp;+\u0026thinsp;mimic-transfected group compared to the control group indicated that miRNA interacted with the target gene 3\u0026prime; UTR.\u003c/p\u003e\n\u003ch3\u003eRumen epithelial cell transfection assays\u003c/h3\u003e\n\u003cp\u003eRumen epithelial cells were plated into 6-well plates and allowed to reach 60\u0026ndash;70% confluence. Transfections were then performed following the previously described protocol [17]. Briefly, rumen epithelial cells were transfected with 5 \u0026micro;L of miR-736 mimic or mimic NC, 10 \u0026micro;L of miR-736 inhibitor or inhibitor NC, and 5 \u0026micro;L of siRNA or si-NC, respectively, when the cells reached 60\u0026ndash;70% confluence. There were three replicates for each group. After 48 hours, cells were harvested for RT-qPCR and flow cytometry analysis.\u003c/p\u003e\n\u003cdiv id=\"Sec11\"\u003e\n \u003ch2\u003eReal-time quantitative polymerase chain reaction\u003c/h2\u003e\n \u003cp\u003eThe mRNA expression levels of key genes involved in nutrient metabolism, cell cycle, and apoptosis in both rumen samples and cultured cells were quantitatively measured by RT-qPCR. The primer sequences for the target genes, which were designed using Primer3 software and synthesized by General Biology Co., LTD (Chuzhou, China), were provided in \u003cstrong\u003eTable S4\u003c/strong\u003e. RT-qPCR was performed in a 20 \u0026micro;L reaction mixture, consisting of 10 \u0026micro;L SYBR Green I Master Mix, 0.8 \u0026micro;L of each primer, 0.4 \u0026micro;L of ROX Reference Dye II (50x), 2 \u0026micro;L of cDNA template, and 6 \u0026micro;L of enzyme-free deionized water. Fluorescence detection was carried out using the ABI 7500 real-time PCR system (Thermo Fisher Scientific), and gene expression was normalized to \u0026beta;-actin using the 2\u003csup\u003e\u0026minus;\u0026Delta;\u0026Delta;Ct\u003c/sup\u003e method.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec12\"\u003e\n \u003ch2\u003eApoptosis and cell cycle detected by flow cytometry\u003c/h2\u003e\n \u003cp\u003eThe Annexin V-FITC/PI Apoptosis and Cell Cycle Detection Kit (No. C1052) from Biyun Tian Biotechnology was used to assess cell apoptosis and cell cycle progression. This kit utilizes Annexin V-FITC in combination with propidium iodide (PI) staining to distinguish viable cells, early apoptotic cells, and late apoptotic or necrotic cells. In the experiment, cells were first washed with pre-cooled PBS and digested with EDTA-free trypsin to prevent interference with Annexin V binding to phosphatidylserine (PS). The cells were then resuspended in 1\u0026times; Binding Buffer, stained with Annexin V-FITC and PI, and incubated at room temperature for 15\u0026ndash;20 minutes in the dark. Apoptotic cells were analyzed by flow cytometry (Beckman Coulter, USA), and data were processed using FlowJo software. For cell cycle analysis, rumen epithelial cells were fixed with 70% ethanol, washed with PBS, and stained with RNase A and PI solution. DNA content was analyzed by flow cytometry (Beckman Coulter, USA), and the cell cycle phases were determined using ModFit software.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec13\"\u003e\n \u003ch2\u003eStatistical analysis\u003c/h2\u003e\n \u003cp\u003eThe RT-qPCR data were analyzed using a two-tailed t-test for comparisons between two groups or one-way ANOVA for comparisons among three groups. Pearson\u0026rsquo;s correlation test was applied to analyze the relationships among rumen weight, length, width, surface area, nutrient metabolism, energy production, and cell cycle related gene expression in fetal rumen. GraphPad Prism v.9.0 software was used for graphical representations, and all data are presented as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM. Statistical significance was set at \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eMaternal undernutrition inhibited fetal rumen development via impeding cell cycle and nutrient metabolism\u003c/h2\u003e \u003cp\u003eTo investigate the effect of maternal undernutrition on fetal rumen development, an undernourished pregnant sheep model was established using 15-day 70% nutritional restriction. At the experimental outset, no significant difference in initial body weight was observed between the CON and UN groups (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.259) (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). After 15 days of treatment, the body weight of ewes in the UN group was significantly lower than that in the CON group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001), and the average daily gain was also significantly lower than that in the CON group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001) (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Furthermore, compared to the CON group, the fetal rumen tissue weight was significantly reduced in the UN group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.039) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Meanwhile, maternal undernutrition caused a significant reduction in the length (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001), width (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.019), and surface area (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001) of fetal rumen papilla (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB-D). Transcriptome analysis revealed the clear distinctions of the general transcriptional profiles of fetal rumen between the CON and UN groups, as shown in PCA and PLS-DA plots of total genes (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE-F). The volcano plot analysis identified 38 upregulated genes, 111 downregulated genes, and 15,771 non-significant genes (\u003cb\u003eFig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eA\u003c/b\u003e). Further GSEA identified the enrichment of genes involved in cytokine-cytokine receptor interaction (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.153, NES = -1.152) and the cell cycle (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.645, NES = -0.921) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG, \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eH). RT-qPCR results also demonstrated the downregulation of cytokine-cytokine receptor-related genes such as \u003cem\u003eJAK3\u003c/em\u003e (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.032) and \u003cem\u003eSTAT3\u003c/em\u003e (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.060) and cell cycle-related genes including \u003cem\u003eBUB1\u003c/em\u003e (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.037), \u003cem\u003eCCNB1\u003c/em\u003e (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.022), \u003cem\u003eCCNE1\u003c/em\u003e (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.037), \u003cem\u003eCDC25C\u003c/em\u003e (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.014), \u003cem\u003eE2F2\u003c/em\u003e (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.018), \u003cem\u003eE2F8\u003c/em\u003e (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.011), \u003cem\u003eMYBL2\u003c/em\u003e (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.037), and \u003cem\u003eTTK\u003c/em\u003e (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.018) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eI). Additionally, fetal rumen weight and the length, width, and surface area of rumen papilla were positively correlated with the expressional levels of genes related to \u003cem\u003eJAK3\u003c/em\u003e/\u003cem\u003eSTAT3\u003c/em\u003e signaling pathway and cell cycle (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eJ). KOG classification revealed that lipid, carbohydrate, and amino acid transport and metabolism, as well as energy production and conversion, were notably enriched by DEGs between the CON and UN groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eK). DEGs associated with fatty acid and cholesterol synthesis as well as energy, carbohydrate, and amino acid metabolism were all downregulated (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eL). RT-qPCR results confirmed the transcriptome findings, showing downregulation of genes related to fatty acid synthesis (\u003cem\u003eACACA\u003c/em\u003e, \u003cem\u003eACACB\u003c/em\u003e, \u003cem\u003eELVOL6\u003c/em\u003e, \u003cem\u003eFASN\u003c/em\u003e, \u003cem\u003eSCD\u003c/em\u003e, and \u003cem\u003eFADS1\u003c/em\u003e), cholesterol synthesis (\u003cem\u003eHMGCR\u003c/em\u003e and \u003cem\u003eHMGCS1\u003c/em\u003e), ketogenesis (\u003cem\u003eHMGCS2\u003c/em\u003e), and carbohydrate metabolism (\u003cem\u003eARG1\u003c/em\u003e, \u003cem\u003eDPP4\u003c/em\u003e, \u003cem\u003eFGGY\u003c/em\u003e, \u003cem\u003eGLUD1\u003c/em\u003e, and \u003cem\u003ePDK4\u003c/em\u003e) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eM). Meanwhile, the gene expressional levels of \u003cem\u003eJAK3\u003c/em\u003e and \u003cem\u003eSTAT3\u003c/em\u003e were positively correlated with the expressional levels of genes related to nutrient metabolism including fatty acid and cholesterol synthesis as well as carbohydrate metabolism (\u003cb\u003eFig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eC\u003c/b\u003e). Thus, maternal undernutrition inhibited nutrient metabolism and cell cycle via \u003cem\u003eJAK3\u003c/em\u003e/\u003cem\u003eSTAT3\u003c/em\u003e signaling pathway in fetal rumen, which suppressed fetal rumen development (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eN).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eGrowth performance and daily feed intake of ewes in the CON and UN groups (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;10).\u003csup\u003e1\u003c/sup\u003e\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eItem\u003csup\u003e3\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003eGroups\u003csup\u003e2\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eP\u003c/em\u003e-value\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCON\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eUN\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eInitial body weight, kg\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e55.07\u0026thinsp;\u0026plusmn;\u0026thinsp;0.847\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e53.74\u0026thinsp;\u0026plusmn;\u0026thinsp;0.693\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.240\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFinal body weight, kg\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e56.83\u0026thinsp;\u0026plusmn;\u0026thinsp;0.677\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e44.65\u0026thinsp;\u0026plusmn;\u0026thinsp;0.798\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDaily weight gain, g/d\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e117.33\u0026thinsp;\u0026plusmn;\u0026thinsp;28.025\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-606.00\u0026thinsp;\u0026plusmn;\u0026thinsp;50.776\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDaily feed intake, kg/d\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.64\u0026thinsp;\u0026plusmn;\u0026thinsp;0.013\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.52\u0026thinsp;\u0026plusmn;\u0026thinsp;0.001\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"4\"\u003e\u003csup\u003e1\u003c/sup\u003e The data were expressed as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM.\u003c/td\u003e\u003c/tr\u003e \u003ctr\u003e\u003ctd colspan=\"4\"\u003e\u003csup\u003e2\u003c/sup\u003e CON, control group; UN, undernutrition group.\u003c/td\u003e\u003c/tr\u003e \u003ctr\u003e\u003ctd colspan=\"4\"\u003e\u003csup\u003e3\u003c/sup\u003e Initial body weight, body weight measured on day 0; Final body weight, body weight measured on day 15.\u003c/td\u003e\u003c/tr\u003e \u003ctr\u003e\u003ctd colspan=\"4\"\u003e\u003csup\u003ea\u0026minus;b\u003c/sup\u003e Means in a row not sharing a common letter were significantly different (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eMaternal nutritional recovery partially restored undernutrition-disrupted metabolism but failed to alleviate fetal rumen maldevelopment\u003c/h2\u003e \u003cp\u003eTo find out whether maternal nutritional recovery can restore fetal rumen development inhibited by previous maternal undernutrition, a maternal nutritional recovery model was established using 15-day 70% nutritional restriction followed by 15-day \u003cem\u003ead libitum\u003c/em\u003e feeding. At trial initiation, ewe body weights did not differ between CON and REC groups (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.229) (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). At both 15-day and 30-day time points, the body weight and average daily gain of REC group ewes were significantly lower than those in the CON group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001) (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). From day 15 to 30, daily weight gain and daily feed intake did not differ between CON and REC groups (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.050) (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Rumen tissue weight in the REC group was significantly lighter (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.005) than the CON group (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Further, fetal rumen papillary width (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001) and surface area (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.002) in the REC group were significantly lower than the CON group while fetal rumen papillary length was almost recovered (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.207) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB-D). Transcriptome analysis of the fetal rumen still revealed clear distinctions between the CON and REC groups, as shown in PCA and PLS-DA plots of total genes (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE-F). The volcano plot analysis identified 159 upregulated genes, 82 downregulated genes, and 14,971 non-significant genes (\u003cb\u003eFig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eB\u003c/b\u003e). Further, GSEA identified significant enrichment of genes involved in cytokine-cytokine receptor interaction (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.008, NES\u0026thinsp;=\u0026thinsp;1.448) and the cell cycle (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.002, NES = -1.713) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eG-H). RT-qPCR results demonstrated the downregulation of cell cycle-related genes including \u003cem\u003eCDK2AP2\u003c/em\u003e (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.002), \u003cem\u003eE2F2\u003c/em\u003e (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.023), \u003cem\u003eMYBL2\u003c/em\u003e (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.011), \u003cem\u003eSFN\u003c/em\u003e (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.004), \u003cem\u003eTGM1\u003c/em\u003e (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001), and \u003cem\u003eTUBA4A\u003c/em\u003e (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001) in fetal rumen of REC group, moreover, the expression of \u003cem\u003eJAK3\u003c/em\u003e (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.062) and \u003cem\u003eSTAT3\u003c/em\u003e (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.068) also showed downregulated trend (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eI). Additionally, fetal rumen weight and the length, width, and surface area of rumen papilla were positively correlated with the expressional levels of key genes related to JAK3/STAT3 signaling pathway and cell cycle (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eJ). KOG classification revealed that lipid, carbohydrate, and amino acid transport and metabolism and energy production and conversion, were also notably enriched by DEGs between the CON and REC groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eK). DEGs associated with cholesterol synthesis, energy metabolism, carbohydrate metabolism, and fatty acid synthesis were partially upregulated in fetal rumen of REC group, while DEGs related to amino acid metabolism were still downregulated (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eL). RT-qPCR analysis revealed that maternal nutritional recovery after nutritional restriction upregulated genes associated with fatty acid synthesis (\u003cem\u003eDEGS1, ACACA\u003c/em\u003e, and \u003cem\u003eELVOL6\u003c/em\u003e) and downregulated genes related to cholesterol synthesis (\u003cem\u003eHMGCR\u003c/em\u003e and \u003cem\u003eHMGCS1\u003c/em\u003e) in fetal rumen (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eM). Meanwhile, the gene expressional levels of \u003cem\u003eJAK3\u003c/em\u003e and \u003cem\u003eSTAT3\u003c/em\u003e were negatively correlated with the expressional levels of genes related to fatty acid synthesis and positively correlated with the expressional levels of genes related to cholesterol synthesis (\u003cb\u003eFig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eD\u003c/b\u003e). Thus, maternal nutritional recovery could partially alleviate the inhibited nutrient metabolism in fetal rumen caused by maternal undernutrition, while the suppressed cell cycle and the retarded fetal rumen development still existed (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eN).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eGrowth performance and daily feed intake of ewes in the CON and REC groups (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;12).\u003csup\u003e1\u003c/sup\u003e\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eItem\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003eGroups\u003csup\u003e2\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eP\u003c/em\u003e-value\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCON\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eREC\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ed0 body weight, kg\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e55.06\u0026thinsp;\u0026plusmn;\u0026thinsp;0.413\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e54.22\u0026thinsp;\u0026plusmn;\u0026thinsp;0.541\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.229\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ed15 body weight, kg\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e56.70\u0026thinsp;\u0026plusmn;\u0026thinsp;0.498\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e45.45\u0026thinsp;\u0026plusmn;\u0026thinsp;0.630\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ed30 body weight, kg\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e58.26\u0026thinsp;\u0026plusmn;\u0026thinsp;0.633\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e47.20\u0026thinsp;\u0026plusmn;\u0026thinsp;0.353\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ed0-d15 daily weight gain, g/d\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e109.44\u0026thinsp;\u0026plusmn;\u0026thinsp;23.733\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-584.44\u0026thinsp;\u0026plusmn;\u0026thinsp;37.947\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ed15-d30 daily weight gain, g/d\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e103.89\u0026thinsp;\u0026plusmn;\u0026thinsp;14.940\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e116.67\u0026thinsp;\u0026plusmn;\u0026thinsp;32.240\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.723\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ed0-d30 daily weight gain, g/d\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e106.67\u0026thinsp;\u0026plusmn;\u0026thinsp;18.142\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-233.89\u0026thinsp;\u0026plusmn;\u0026thinsp;11.115\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ed0-d15 daily feed intake, kg/d\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.63\u0026thinsp;\u0026plusmn;\u0026thinsp;0.013\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.48\u0026thinsp;\u0026plusmn;\u0026thinsp;0.009\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ed15-d30 daily feed intake, kg/d\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.66\u0026thinsp;\u0026plusmn;\u0026thinsp;0.013\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.61\u0026thinsp;\u0026plusmn;\u0026thinsp;0.026\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.140\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ed0-d30 daily feed intake, kg/d\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.65\u0026thinsp;\u0026plusmn;\u0026thinsp;0.020\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.84\u0026thinsp;\u0026plusmn;\u0026thinsp;0.023\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"4\"\u003e\u003csup\u003e1\u003c/sup\u003e The data were expressed as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM.\u003c/td\u003e\u003c/tr\u003e \u003ctr\u003e\u003ctd colspan=\"4\"\u003e\u003csup\u003e2\u003c/sup\u003e CON, control group; REC, nutritional recovery group.\u003c/td\u003e\u003c/tr\u003e \u003ctr\u003e\u003ctd colspan=\"4\"\u003e\u003csup\u003ea\u0026minus;b\u003c/sup\u003e Means in a row not sharing a common letter were significantly different (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eMaternal undernutrition altered fetal rumen miRNA profile and upregulated miRNA-736 expression\u003c/h2\u003e \u003cp\u003eIt was hypothesized that miRNA might play a role in the epigenetic regulation of fetal rumen plasticity during pregnancy. In the analysis of miRNAs expressional profile, 1,253 miRNAs were detected including 152 known miRNAs and 1,101 novel miRNAs (\u003cb\u003eTable \u003cspan refid=\"MOESM5\" class=\"InternalRef\"\u003eS5\u003c/span\u003e\u003c/b\u003e). To investigate the clustering of samples, PCA of total miRNA read counts in fetal rumen was performed. PCA plot showed clear distinction between the CON and UN groups, which indicated the changed fetal rumen miRNA profile upon maternal undernutrition (\u003cb\u003eFig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003eA\u003c/b\u003e). Compared to the CON group, 34 DEMs were upregulated (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA), while 29 DEMs were downregulated in fetal rumen of UN group (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). RT-qPCR results confirmed the findings from miRNA sequencing (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). Excitingly, the significant upregulation of miR-736 (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.039) was also found in fetal rumen of ewes from the REC group compared to the gestational-age-matched controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). To further explore the functions of these putative miRNA gene targets, KEGG enrichment analysis was conducted in which necroptosis and cytokine-cytokine receptor interaction were significantly enriched (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE). Interestingly, \u003cem\u003eE2F2\u003c/em\u003e and \u003cem\u003eMYBL2\u003c/em\u003e were the intersection of DEGs dataset in fetal rumen induced by maternal undernutrition, DEGs dataset in fetal rumen induced by maternal nutritional recovery, and DEMs target genes dataset (\u003cb\u003eFig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003eB\u003c/b\u003e). Notably, miR-736 was found to target \u003cem\u003eE2F2\u003c/em\u003e and \u003cem\u003eMYBL2\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF). STRING analysis indicated that \u003cem\u003eE2F2\u003c/em\u003e and \u003cem\u003eMYBL2\u003c/em\u003e, the potential target genes of miR-736, were closely associated with cell cycle-related genes, including \u003cem\u003eCCNA2\u003c/em\u003e, \u003cem\u003eCCNE1\u003c/em\u003e, \u003cem\u003eCCNE2\u003c/em\u003e, and \u003cem\u003eCCNA2\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eG-H). GO enrichment analysis further revealed that proteins interacting with \u003cem\u003eE2F2\u003c/em\u003e and \u003cem\u003eMYBL2\u003c/em\u003e were significantly enriched in cell cycle-related processes (\u003cb\u003eFig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003eC-D\u003c/b\u003e). Thus, maternal undernutrition might inhibit fetal rumen development by upregulating miR-736, which then downregulated the expression of its target genes, \u003cem\u003eE2F2\u003c/em\u003e and \u003cem\u003eMYBL2\u003c/em\u003e, ultimately leading to the suppression of cell cycle-related gene expression.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003emiRNA-736 targeted\u003c/b\u003e \u003cb\u003eE2F2\u003c/b\u003e \u003cb\u003eand\u003c/b\u003e \u003cb\u003eMYBL2\u003c/b\u003e \u003cb\u003eand regulated rumen epithelial cells development via cell cycle and apoptosis\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe binding sites of miRNA-736 to the 3' UTR regions of \u003cem\u003eE2F2\u003c/em\u003e and \u003cem\u003eMYBL2\u003c/em\u003e were predicted using miRanda software (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). To further validate \u003cem\u003eE2F2\u003c/em\u003e and \u003cem\u003eMYBL2\u003c/em\u003e were target genes of miR-736, the 3' UTRs of \u003cem\u003eE2F2\u003c/em\u003e and \u003cem\u003eMYBL2\u003c/em\u003e were cloned into the psiCHECK2 vector, respectively. The inserted fragments lengths of these two recombinant vectors were verified using \u003cem\u003eXhoI\u003c/em\u003e-\u003cem\u003eApaI\u003c/em\u003e double enzyme digestion and gel electrophoresis and the inserted fragments sequences were confirmed by DNA sequencing (\u003cb\u003eFig. \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003eA-B\u003c/b\u003e). The psiCHECK2\u0026thinsp;+\u0026thinsp;3'UTR constructs and miR-736 mimics (or negative control mimic NC) were co-transfected into HEK-293 cells, and luciferase activity was measured using a dual luciferase reporter assay. The results showed that the luciferase activity in the co-transfected psiCHECK2\u0026thinsp;+\u0026thinsp;3' UTR (\u003cem\u003eE2F2\u003c/em\u003e or \u003cem\u003eMYBL2\u003c/em\u003e) and miR-736 mimic group was significantly downregulated compared to the negative control groups, confirming that miRNA-736 targeted both \u003cem\u003eE2F2\u003c/em\u003e and \u003cem\u003eMYBL2\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB-C). To explore the regulatory function of miR-736 in rumen epithelial cells, overexpression and silencing experiments were performed using miR-736 mimics and inhibitors, respectively. RT-qPCR results demonstrated that miR-736 mimics significantly increased the expression of mature miR-736 in rumen epithelial cells (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.002) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD), while miR-736 inhibitors significantly decreased its expressional level (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.018) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE). Overexpression of miR-736 resulted in a significant downregulation of \u003cem\u003eE2F2\u003c/em\u003e (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.027) and \u003cem\u003eMYBL2\u003c/em\u003e (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.017) gene expression in rumen epithelial cells, while inhibition of miR-736 caused a significant upregulation of \u003cem\u003eE2F2\u003c/em\u003e (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.008) and \u003cem\u003eMYBL2\u003c/em\u003e (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001) gene expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF-G). Additionally, overexpression of miR-736 significantly reduced the expression of cell cycle-related genes (\u003cem\u003eCCNA2\u003c/em\u003e, \u003cem\u003eCCNE1\u003c/em\u003e, \u003cem\u003eCCNE2\u003c/em\u003e, and \u003cem\u003eCDC20\u003c/em\u003e) and the anti-apoptosis-related gene (\u003cem\u003eBCL2\u003c/em\u003e), while upregulated apoptosis-related genes (\u003cem\u003eBAX\u003c/em\u003e and \u003cem\u003eCASP3\u003c/em\u003e) in rumen epithelial cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eH). Conversely, inhibition of miR-736 produced the completely opposite effects on the expression of genes associated with cell cycle and apoptosis (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eI). Flow cytometry analysis revealed that overexpression of miR-736 significantly increased the apoptosis level of rumen epithelial cells (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001) compared to the mimic NC group, while inhibition of miR-736 significantly decreased the apoptosis level (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eJ-K). Thus, miRNA-736 targeted \u003cem\u003eE2F2\u003c/em\u003e and \u003cem\u003eMYBL2\u003c/em\u003e and acted as a negative regulator of fetal rumen development by inhibiting cell cycle progression and promoting apoptosis.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eSilencing of\u003c/b\u003e \u003cb\u003eE2F2\u003c/b\u003e \u003cb\u003eand\u003c/b\u003e \u003cb\u003eMYBL2\u003c/b\u003e \u003cb\u003einhibited cell cycle and promoted apoptosis of rumen epithelial cells\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo further confirm that miR-736 regulated the development of rumen epithelial cells by targeting the inhibition of \u003cem\u003eE2F2\u003c/em\u003e and \u003cem\u003eMYBL2\u003c/em\u003e, \u003cem\u003eE2F2\u003c/em\u003e and \u003cem\u003eMYBL2\u003c/em\u003e were silenced in rumen epithelial cells to examine their effects on the cell cycle and apoptosis. The results showed that the transfection of siRNA2 resulted in an 80.85% reduction in \u003cem\u003eE2F2\u003c/em\u003e gene expression in rumen epithelial cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA), so siRNA2 was used for the following assays. Silencing of E2F2 significantly reduced the mRNA expression of cell cycle-related genes (\u003cem\u003eCCNA2\u003c/em\u003e, \u003cem\u003eCCNE1\u003c/em\u003e, \u003cem\u003eCCNE2\u003c/em\u003e, and \u003cem\u003eCDC20\u003c/em\u003e) and the anti-apoptosis-related gene \u003cem\u003eBCL2\u003c/em\u003e, while upregulated the apoptosis-related gene \u003cem\u003eBAX\u003c/em\u003e in rumen epithelial cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). Further analysis revealed that silencing \u003cem\u003eE2F2\u003c/em\u003e expression significantly reduced the cell viability of rumen epithelial cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). Flow cytometry analysis showed that silencing E2F2 significantly increased the apoptosis level (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001) of rumen epithelial cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD-E), which was consistent with the results observed following miR-736 overexpression. Silencing E2F2 significantly inhibited cell proliferation in rumen epithelial cells and negatively regulated the entry into the S-phase of the cell cycle (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF-G). Additionally, silencing \u003cem\u003eMYBL2\u003c/em\u003e by transfecting siRNA4 resulted in a 53.97% reduction in \u003cem\u003eMYBL2\u003c/em\u003e gene expression in rumen epithelial cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eH). Silencing \u003cem\u003eMYBL2\u003c/em\u003e also significantly inhibited the mRNA expression of cell cycle-related genes (\u003cem\u003eCCNA2\u003c/em\u003e, \u003cem\u003eCCNE1\u003c/em\u003e, \u003cem\u003eCCNE2\u003c/em\u003e, and \u003cem\u003eCDC20\u003c/em\u003e) and the anti-apoptosis-related gene \u003cem\u003eBCL2\u003c/em\u003e, while upregulated the apoptosis-related gene \u003cem\u003eBAX\u003c/em\u003e in rumen epithelial cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eI). Moreover, silencing \u003cem\u003eMYBL2\u003c/em\u003e significantly reduced the cell viability of rumen epithelial cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eJ). Flow cytometry analysis showed that silencing \u003cem\u003eMYBL2\u003c/em\u003e also significantly increased the apoptosis level (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eK-L) and negatively regulated entry into the S-phase of the cell cycle (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eM-N). Thus, maternal undernutrition induced the overexpression of miRNA-736 in fetal rumen, which targeted \u003cem\u003eE2F2\u003c/em\u003e and \u003cem\u003eMYBL2\u003c/em\u003e, leading to enhanced apoptosis and the negative regulation of S-phase entry in rumen epithelial cells, ultimately inhibiting fetal rumen development.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eRumen development during early life critically determines lifelong nutrient utilization in ruminants [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Maternal nutritional status profoundly influences offspring organogenesis, particularly in Hu-sheep during late gestation due to multi-fetal pregnancies induced high nutritional demand and low nutritional intake [19; 20]. However, the effect of maternal undernutrition on the metabolic homeostasis and development of fetal rumen is poorly understood. Dissecting the phenotypes and molecular regulatory mechanisms of maternal undernutrition-affected fetal rumen will enrich the mother-child nutrition theory and contribute to finding novel strategies to promote fetal development in utero and improve productivity after birth.\u003c/p\u003e \u003cp\u003ePrevious studies showed that fetal small intestinal and total gastrointestinal tract mass [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e] and liver mass [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e] were decreased by mid-gestation feed restriction in ewes. In the current study, maternal undernutrition significantly reduced fetal rumen weight which was consistent with a previous report about the effects of maternal nutritional restriction on fetal rumen development in cows during pregnancy [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Furthermore, maternal undernutrition reduced the length, width, and surface area of ruminal papilla in fetal rumen. Interestingly, nutritional recovery partially restored fetal rumen development reduced by previous maternal undernutrition; however, fetal rumen weight and the width and surface area of rumen papilla were still lower than the normal levels.\u003c/p\u003e \u003cp\u003eEwe undernutrition during late gestation affected lipid, carbohydrate, amino acid, and energy metabolism in the rumen [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e], jejunum and ileum [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e], cecum [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e], pituitary [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e], fat [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e], and liver [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. In this study, maternal undernutrition extremely reduced nutrient metabolism including fatty acid synthesis, cholesterol synthesis, carbohydrate metabolism, amino acid metabolism, and energy production in fetal rumen. It was worthy to note that lipid metabolism and transport were closely linked to cell survival and growth, the inhibition of lipid metabolism induced a suppression of cell proliferation [28; 29]. In addition, carbohydrate, amino acid, and energy metabolism played important roles in rumen development [30; 31]. Systemic inhibition of \u003cem\u003eJAK\u003c/em\u003e/\u003cem\u003eSTAT\u003c/em\u003e signaling pathway induced multi-organ metabolic dysregulation [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Hepatic \u003cem\u003eSTAT5\u003c/em\u003e deficiency exacerbated hyperglycemia and dyslipidemia [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e] and liver-specific \u003cem\u003eSTAT6\u003c/em\u003e deletion directly mediated hepatic steatosis and insulin resistance [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Adipose \u003cem\u003eSTAT3\u003c/em\u003e/\u003cem\u003eSTAT5\u003c/em\u003e ablation promoted obesity and suppresses lipolytic capacity [35; 36]. Skeletal muscle \u003cem\u003eSTAT5\u003c/em\u003e depletion reduced lean mass and impaired glucose tolerance [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. In this study, \u003cem\u003eJAK3\u003c/em\u003e and \u003cem\u003eSTAT3\u003c/em\u003e expressional levels were significantly correlated with the expressional levels of genes related to nutrient metabolism and energy production. Therefore, maternal undernutrition suppressed nutrient metabolism and energy production via \u003cem\u003eJAK3\u003c/em\u003e/\u003cem\u003eSTAT3\u003c/em\u003e signaling pathway and further inhibited fetal rumen development, while maternal nutritional recovery only partially restored metabolic homeostasis to alleviate undernutrition-induced fetal rumen maldevelopment.\u003c/p\u003e \u003cp\u003eMetabolic disorders and energy production barriers could affect cell cycle and proliferation. Notably, \u003cem\u003eCCNB1\u003c/em\u003e played a crucial role in cell division and had been shown to accelerate the cell cycle and then promote rumen growth in goats [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. The enhancement of rumen cell proliferation was associated with the increased expression of \u003cem\u003eCCNB1\u003c/em\u003e and \u003cem\u003eCCNE1\u003c/em\u003e, which contributed to the development of rumen epithelium [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. In addition, BUB1 played an active role in promoting the proliferation of Human endometrial epithelial cells and endometrial cancer Ishikawa cells [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. Interestingly, E2F family proteins were transcriptional activators of the genes encoding cell cycle-regulatory proteins including CCNA, CCNE, CDK2, and CDC25 [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. In this study, it was found maternal undernutrition downregulated these genes related to cell cycle and cell proliferation in fetal rumen and inhibited fetal rumen development.\u003c/p\u003e \u003cp\u003eAs a critical post-transcriptional regulatory mechanism, miRNAs play a much larger role in gene expression regulation than previously understood [41; 42]. Although miRNAs had been widely identified in various ruminant tissues, their roles in regulating gastrointestinal development and function remain underexplored [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. In calves, miR-143 was the most highly expressed miRNA in rumen, and its expression pattern changed over the first 6 weeks after birth [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. The targeted genes of miR-143 were associated with immune response and developmental processes [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. In this study, we identified a total of 1,253 miRNAs in sheep fetal rumen, including 1,101 newly predicted miRNAs and 152 known miRNAs. Moreover, we found the newly identified miR-736 targeted \u003cem\u003eE2F2\u003c/em\u003e and \u003cem\u003eMYBL2\u003c/em\u003e. In mouse lung cancer cells, miR-637 targeted \u003cem\u003eE2F2\u003c/em\u003e and reduced cell proliferation [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. However, to date, no \u003cem\u003eE2F2\u003c/em\u003e-targeting miRNAs had been reported in the rumen. In addition, \u003cem\u003eMYBL2\u003c/em\u003e expression was downregulated during the maturation of colon epithelial cells, in part through miR-365 [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. In this study, it was confirmed for the first time that the newly identified miRNA-736 inhibited \u003cem\u003eE2F2\u003c/em\u003e and \u003cem\u003eMYBL2\u003c/em\u003e, thereby promoted apoptosis and negatively regulated entry into the S-phase of the cell cycle for rumen epithelial cells. Therefore, maternal undernutrition induced the overexpression of miRNA-736 in fetal rumen, which targeted \u003cem\u003eE2F2\u003c/em\u003e and \u003cem\u003eMYBL2\u003c/em\u003e, promoted rumen epithelial cell apoptosis, and inhibited cell cycle and cell proliferation.\u003c/p\u003e \u003cp\u003eThe oncogenic effects of E2F family genes (\u003cem\u003eE2F1\u003c/em\u003e and \u003cem\u003eE2F7\u003c/em\u003e) in gastric cancer were at least partially mediated through transcriptional activation of \u003cem\u003eMYBL2\u003c/em\u003e [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. \u003cem\u003eE2F1\u003c/em\u003e, \u003cem\u003eE2F2\u003c/em\u003e, and \u003cem\u003eE2F3\u003c/em\u003e were transcriptional activators while \u003cem\u003eE2F7\u003c/em\u003e and \u003cem\u003eE2F8\u003c/em\u003e acted as atypical transcriptional suppressors [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. \u003cem\u003eE2F2\u003c/em\u003e was closely involved in several critical cellular processes including cell cycle, apoptosis, differentiation, and stress response [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. Silencing \u003cem\u003eE2F2\u003c/em\u003e had been shown to impair cell growth, invasion, and migration in gastric cancer cells [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. In this study, silencing \u003cem\u003eE2F2\u003c/em\u003e significantly inhibited the proliferation of rumen epithelial cells and promoted apoptosis. In addition, knockdown of \u003cem\u003eMYBL2\u003c/em\u003e significantly inhibited the proliferation and migration of ovarian cancer cells [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. In our study, silencing \u003cem\u003eMYBL2\u003c/em\u003e also significantly inhibited cell proliferation and promoted apoptosis of rumen epithelial cells. Therefore, maternal undernutrition downregulated \u003cem\u003eE2F2\u003c/em\u003e and \u003cem\u003eMYBL2\u003c/em\u003e in fetal rumen, which further suppressed cell cycle and cell proliferation, promoted cell apoptosis, and inhibited fetal rumen development.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eIn conclusion, maternal undernutrition suppressed nutrient metabolism and energy production via \u003cem\u003eJAK3\u003c/em\u003e/\u003cem\u003eSTAT3\u003c/em\u003e signaling pathway and further inhibited fetal rumen development, while maternal nutritional recovery partially restored metabolic homeostasis and energy production to alleviate undernutrition-induced fetal rumen maldevelopment (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). Interestingly, maternal undernutrition induced the overexpression of miRNA-736 in fetal rumen, which inhibited the expression of its target genes \u003cem\u003eE2F2\u003c/em\u003e and \u003cem\u003eMYBL2\u003c/em\u003e, thereby disrupted cell cycle, promoted apoptosis, and ultimately inhibited fetal rumen development (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). These findings provided valuable insights into the molecular mechanisms underlying the impact of maternal nutrition on fetal growth and development and contributed to developing potential nutritional strategies to improve fetal health outcomes.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003e\u003cstrong\u003e\u003cem\u003eDEMs\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e:\u003c/strong\u003e Differentially expressed miRNAs\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eDEGs\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e:\u003c/strong\u003e Differentially expressed genes\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eE2F2\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e:\u0026nbsp;\u003c/strong\u003eE2F transcription factor 2\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eMYBL2\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e:\u0026nbsp;\u003c/strong\u003eMYB proto-oncogene like 2\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eFC\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e:\u0026nbsp;\u003c/strong\u003eFold change\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003ePCA\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e:\u003c/strong\u003e Principal component analysis\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003ePLS-DA\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e:\u003c/strong\u003e Partial least squares discriminant analysis\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eNC\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e:\u003c/strong\u003e Negative control\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003ePI\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e:\u003c/strong\u003e Propidium iodide\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003ePS\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e:\u003c/strong\u003e Phosphatidylserine\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthors' contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe author’s contributions are as follows: Y. X., P. J., S. M., and J. C. conceived and designed the study; P. J., Y. L., and Y. X. conducted the research; P. J. and Y. X. analyzed and interpreted the data; P. J. and Y. X. wrote the manuscript; and Y. X., H. L., Y. G., C. F., W. Z., S. M., and J. C. revised the manuscript. All authors read and approved the final version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the National Natural Science Foundation of China (32402767), National Key Research and Development Program of China (2022YFD1301102), Anhui Province Natural Science Foundation Youth Project (2308085QC104), and AAU Introduction of High-level Talent Funds (RC392107), Key Laboratory of Utilization of Livestock and Forage Resources in Circum-Tarim Region (Co-construction by Ministry and Province), Ministry of Agriculture and Rural Affairs (BSGJSYS202502).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank Biomarker Biotechnology Co., Ltd. (Beijing, China) for technical assistance on transcriptome sequencing and miRNA sequencing. Rumen epithelial cells were generously provided by Zhu Wen's team at Anhui Agricultural University.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe raw sequencing data supporting this study have been deposited in the Gene Expression Omnibus (GEO) under controlled access, with rumen transcriptome data available through accession number GSE284288 (security token: qdqbggywvpehjif; direct link: https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE284288\u0026amp;token=qdqbggywvpehjif) and rumen miRNA sequencing data accessible via accession number GSE285445 (security token: wlqxqkmwbtoxtml; direct link: https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE285445\u0026amp;token=wlqxqkmwbtoxtml).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eSkurkov\u0026aacute;, L., L. Matuln\u0026iacute;kov\u0026aacute;, B. Peťkov\u0026aacute;, M. Florian, M. Slivkov\u0026aacute;, L. 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Frontiers in Immunology. 2024; 15:1438198. https://doi.org/10.3389/fimmu.2024.1438198.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Maternal undernutrition, Fetal rumen development, Novel miRNA-736, E2F2, MYBL2","lastPublishedDoi":"10.21203/rs.3.rs-6598539/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6598539/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eUndernutrition disrupts pregnant ewe\u0026rsquo;s metabolic homeostasis and severely inhibits fetal growth and development. In this study, undernourished and nutrition-recovery pregnant sheep models and rumen epithelial cells were utilized to investigate the mechanisms behind undernutrition-induced disruptions in fetal rumen metabolism and development.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eMaternal undernutrition significantly reduced fetal rumen weight and papilla length, width and surface area. Maternal undernutrition extremely suppressed nutrient metabolism and energy production in fetal rumen via \u003cem\u003eJAK3\u003c/em\u003e/\u003cem\u003eSTAT3\u003c/em\u003e signaling to inhibit cell cycle progression and fetal rumen development, while maternal nutritional recovery partially restored metabolic inhibition but failed to alleviate fetal rumen development. Meanwhile, 64 differentially expressed miRNAs (DEMs) were identified in fetal rumen between undernourished ewes and controls. Novel miR-736 was overexpressed both in fetal rumen of undernourished and nutrition-recovery models. E2F transcription factor 2 (\u003cem\u003eE2F2\u003c/em\u003e) and MYB proto-oncogene like 2 (\u003cem\u003eMYBL2\u003c/em\u003e) were the intersection of fetal rumen differentially expressed genes (DEGs) and DEMs target genes integrated analysis and were predicted as miR-736 target genes. Further, we confirmed that miR-736 targeted and downregulated \u003cem\u003eE2F2\u003c/em\u003e and \u003cem\u003eMYBL2\u003c/em\u003e expressional levels. Silencing \u003cem\u003eE2F2\u003c/em\u003e and \u003cem\u003eMYBL2\u003c/em\u003e promoted apoptosis and inhibited S-phase entry in rumen epithelial cells.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eIn summary, maternal undernutrition disrupted fetal rumen metabolism and elevated miR-736, which targeted and downregulated \u003cem\u003eE2F2\u003c/em\u003e and \u003cem\u003eMYBL2\u003c/em\u003e to inhibit cell cycle progression and promote apoptosis, finally inhibited fetal rumen development. This study provides new insights into the epigenetic mechanisms underlying maternal undernutrition-induced fetal rumen developmental deficits.\u003c/p\u003e","manuscriptTitle":"Maternal undernutrition inhibits fetal rumen development： Novel miRNA-736-mediated dual targeting of E2F2 and MYBL2 in sheep","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-05-13 15:25:32","doi":"10.21203/rs.3.rs-6598539/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"b4e3fc22-a990-4cd5-aed6-5452c907710c","owner":[],"postedDate":"May 13th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-08-29T08:24:23+00:00","versionOfRecord":[],"versionCreatedAt":"2025-05-13 15:25:32","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6598539","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6598539","identity":"rs-6598539","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}