The luminal epithelium (LE) serves as the primary interface for embryo–maternal communication during implantation. In ruminants such as sheep, the LE orchestrates critical reproductive events, including noninvasive adhesion, conceptus elongation, and pregnancy recognition. However, existing models face significant limitations: conventional two-dimensional (2D) cultures fail to recapitulate endometrial complexity, while current organoid systems are predominantly derived from glandular epithelium. Herein, we report the establishment of a robust three-dimensional (3D) culture system for generating ovine endometrial luminal epithelial organoids using an optimized expansion medium (ExM) supplemented with CHIR99021, Y-27632, SB202190, and the EphrinA1 ligand. The resulting organoids exhibited characteristic luminal epithelium features, including polarized architecture, mucin secretion, and high expression of lineage markers (keratin 18 (KRT-18), trophoblast cell surface antigen 2 (TROP2), and epithelial cell adhesion molecule (EpCAM)). Moreover, we developed an apical-out polarity model that faithfully recapitulates the in vivo luminal orientation. Transcriptomic analysis confirmed the close resemblance between organoids and native luminal epithelium, as well as maintained hormonal responsiveness. Functional validation demonstrated the organoids’ capacity to promote blastocyst expansion and trophoblast proliferation in co-culture systems, as well as substrate-specific adhesion that was enhanced by β-estradiol (E2) + medroxyprogesterone acetate (MPA) treatment. Notably, we identified erythropoietin-producing hepatocellular receptor A (EphA) signaling as a novel regulator of stemness properties during organoid development. The successful generation of functional luminal epithelial organoids thus provides a physiologically relevant model that mirrors native endometrial structure, hormone responses, and embryo interactions. As such, it offers a valuable platform for investigating the mechanisms of endometrial receptivity and for screening potential therapeutics aimed at improving reproductive efficiency in ruminants.

In vitro embryo culture often leads to developmental arrest. The maternal-to-zygotic (MZT) transition, involving maternal mRNA clearance and zygotic genome activation (ZGA), is a critical step in early embryogenesis. Although major epigenetic regulators have been identified, the role of lactate-derived histone lactylation in early embryos remains unclear. Here, we used goat MII oocytes, in vivo-derived 8-cell embryos, and in vitro-cultured 8-cell embryos as models to systematically compare transcriptional, epigenetic, and chromatin accessibility differences through integrative multi-omics approaches, including RNA sequencing, cleavage under targets and tagmentation for H3K23 lactylation, and immunofluorescence staining. Our results revealed that in vitro 8-cell embryos exhibited pronounced abnormalities in global transcription, key splicing factor expression, and chromatin accessibility. Notably, H3K23 lactylation levels were significantly reduced, associated with downregulation of metabolic genes. Functional validation using mouse embryos treated with an LDH inhibitor confirmed that reduced H3K23la led to decreased blastocyst formation, accompanied by CDK9 downregulation, consistent with impaired transcriptional activation during MZT. Collectively, these findings indicate that aberrant histone lactylation contributes to developmental arrest in in vitro-cultured embryos and highlight the critical role of the metabolism-epigenetic axis in early embryonic development, providing theoretical and experimental insights for optimizing culture systems and improving assisted reproductive technology outcomes.

Zygotic genome activation (ZGA) is a pivotal process during early embryogenesis, marking the maternal-to-zygotic transition. ZGA is regulated by a variety of epigenetic and transcriptional factors. However, the transcriptional regulatory mechanisms underlying ZGA in livestock species remain largely unclear. By integrating ATAC-seq and RNA-seq, we characterized chromatin accessibility and transcriptional dynamics in goat embryos. Transcriptional inhibition with α-amanitin markedly reduced promoter accessibility and disrupted RNA polymerase II (Pol II)-mediated transcription. Motif enrichment analysis identified ZNF331 as a potential regulator with specific upregulation at the 8-cell stage. Functional knockdown of ZNF331 resulted in impaired embryonic development, reduced blastocyst formation, and widespread transcriptome alterations. Mechanistically, ZNF331 depletion caused abnormal elevation of Pol II Ser5 phosphorylation, excessive transcriptional activity, maternal mRNA retention, and excessive activation of zygotic genes. Our study identifies ZNF331 as a critical regulator of goat ZGA, functioning through fine-tuning Pol II Ser5 phosphorylation to balance maternal transcript clearance and zygotic gene activation. These findings highlight the essential role of the ZNF331-Pol II axis in goat embryogenesis and suggest a potentially conserved mechanism across mammals.

Timely and efficient degradation of maternal mRNA is essential for early embryonic development, which occurs from fertilization through the initiation of zygotic genome activation (ZGA). Yet, the regulatory mechanisms governing this process remain poorly characterized. In the present study, we investigated the function of CCR4-NOT transcription complex subunit 1 (CNOT1) during goat embryogenesis. We found that CNOT1 was upregulated during mammalian ZGA, and that its knockdown led to developmental arrest and a marked reduction in blastocyst formation. Moreover, CNOT1 knockdown impaired nascent RNA activity, resulting in 814 upregulated and 1014 downregulated genes, which were enriched for RNA splicing, regulation of chromosome organization, and RNA localization. RNA splicing analysis revealed differential splicing events in 2959 genes, of which 259 were downregulated following CNOT1 knockdown. Notably, CNOT1 was predicted to crosstalk with the m6A reader YTHDF2. Knockdown of YTHDF2 resulted in CNOT1 downregulation at the 8-cell stage in goats and increased transcription levels around polyadenylation sites during ZGA in mice. Together, these findings indicate that CNOT1 is a potential YTHDF2 target that orchestrates maternal mRNA decay and ZGA during goat embryogenesis. Our work provides new insight into the complex regulatory landscape underlying ZGA and may inform strategies to improve the efficiency of goat embryogenesis.

The pituitary gland plays a pivotal role in regulating puberty and reproductive physiology; however, the precise cellular and molecular mechanisms driving the pubertal transition in large animal, such as ewes, remain poorly understood. Here, we generated a comprehensive single-cell transcriptomic atlas of the ovine anterior pituitary, specifically comparing the pre-pubertal (3 month) and post-pubertal (6 month) stages. We identified 30 335 cells classified into ten distinct clusters. Comparative analysis revealed a global transcriptional reprogramming during puberty, characterized by a marked upregulation of genes associated with ribosome biogenesis, unfolded protein response, and hormone secretion across endocrine cells, reflecting an expanded biosynthetic capacity. Specifically, we identified SCG2 as a critical regulator of gonadotroph maturation. Functional validation demonstrated that SCG2 facilitates the biogenesis of secretory granules, thereby promoting FSH synthesis and secretion. Furthermore, intercellular communication analysis uncovered a distinct shift in the pituitary microenvironment: the 6 month pituitary exhibited enhanced regulatory networks, including IGF signaling mediated by non-endocrine cells and NT signaling (e.g., BDNF-NTRK2) driven by multiple cell types. These findings suggest that the onset of puberty relies on a coordinated "endocrine-to-endocrine" and "non-endocrine-to-endocrine" crosstalk. This study provides a high-resolution molecular blueprint of the pubertal transition, highlighting the key roles of biosynthetic machinery upgrades and microenvironmental remodeling in establishing the high reproductive performance of Hu sheep.

Myofiber type determines meat quality by governing postmortem glycolytic potential and lactate-driven pH decline. However, whether endogenous lactate actively regulates myofiber type through epigenetic signaling remains unknown. Here, we found that histone H3 lysine 27 lactylation (H3K27la), a modification driven by lactate, is a critical regulator of myoblast differentiation. Elevating lactate enhanced differentiation and H3K27la levels in myoblasts. Multi-omics analyses revealed that H3K27la enrichment is associated with transcriptional repression of the member of RAS oncogene family (RAP2C) during goat myoblast differentiation. This inactivation of the RAP2C-ERK-ELK1 signaling axis subsequently upregulates myosin light chain, phosphorylatable, fast skeletal muscle (MYLPF), thereby promoting glycolytic myofiber specification. Our study uncovers a lactate-H3K27la regulatory axis that translates the metabolic state of developing muscle into an epigenetic signal that orchestrates myofiber type, providing profound insights into the early-life programming of myofiber determination and meat quality.

The pituitary gland is a master regulator of endocrine and reproductive physiology; however, the lack of physiologically relevant in vitro models in livestock species has limited studies of pituitary development and endocrine function. Although pituitary organoids have been generated from human and mouse pluripotent stem cells, comparable models remain unavailable in large animal species. Here, we established functional pituitary-like organoids from ovine embryonic stem cells (oESCs) using a three-dimensional differentiation strategy. The resulting organoids self-organized into Rathke’s pouch-like structures and acquired pituitary progenitor characteristics, as demonstrated by the robust expression of LHX3, PITX1, ISLET1 and PROP1. Compared with conventional two-dimensional differentiation, the organoids displayed enhanced structural organization and improved lineage specification. Notably, the organoids exhibited functional gonadotroph maturation, with GnRH treatment significantly upregulating FSHB and LHB expression, as confirmed by immunohistochemistry, qPCR and Western blot. SMART-Seq transcriptome analysis revealed progressive loss of pluripotency-associated signatures and activation of pituitary developmental programs, including sustained expression of lineage-associated regulators such as PAX6. Collectively, this study establishes the first ovine pituitary-like organoid model and demonstrates its capacity to generate functional GnRH-responsive gonadotroph cells. This platform provides a valuable resource for investigating pituitary development, reproductive endocrinology, livestock fertility, and endocrine disease mechanisms in a large-animal context.

Elevated intramuscular fat (IMF) deposition is widely recognized as a hallmark of high-grade meat with improved sensory and nutritional properties. Thus, in the current study, the physicochemical and nutritional trajectories of Haimen goat meat slaughtered at different stages were systematically characterized. High-IMF (HF) and low-IMF (LF) were further dissected by lipidomics and amino-acid profiling to resolve fatty-acid and amino-acid signatures. IMF exhibited linear accretion with age. The HF group exhibited significantly higher proportions of SFA, MUFA and PUFA, concomitant with enhanced concentrations of essential and flavor-active amino acids. Proteomics analysis identified key regulatory genes related to intramuscular fat deposition including ECI1, HADH, and ACO2, highlighting the core roles of acetyl-CoA and succinyl-CoA in lipid metabolism. Overall, these results suggest that high-IMF goat meat may provide enhanced nutritional and flavor-related attributes.

This study investigated the effects of tributyrin supplementation on the immune and antioxidant status of lambs born to perinatal Hu ewes with negative energy balance (NEB) at 75%. Twenty healthy Hu ewes (average body weight 45.00 ± 5.00 kg) with similar body condition score, pregnant for 100 days with twin lambs, were randomly allocated into two groups: NEB group and 0.5% tributyrin supplementation group (TB). Lambs were artificially fed with milk from their respective maternal groups for 42 days and were classified as L-NEB (n = 20) and L-TB (n = 20). The experimental period lasted from 40 days before delivery to 42 days after delivery. At 42 days of age, five male lambs from each group were randomly selected and euthanized for analysis. Tributyrin supplementation significantly increased fat-corrected milk yield, milk fat content, and colostrum fat yield in perinatal ewes (p < 0.01). Lambs in the L-TB group exhibited improved growth performance compared with L-NEB lambs (p < 0.05). Serum triglyceride concentration at 7 days of age was 68.4% higher, and serum triglyceride and glucose concentrations at 42 days of age were 42.9% and 13.9% higher, respectively, in the L-TB group than in the L-NEB group (p < 0.05). In addition, the L-TB group showed a 39.2% lower serum IL-1β concentration at 7 days and significantly downregulated hepatic IL-8 mRNA expression at 42 days, together with enhanced antioxidant capacity (p < 0.05). These findings suggest that dietary tributyrin supplementation at 0.5% in perinatal undernourished ewes, applied under a controlled NEB feeding regimen, may improve milk composition and, consequently, enhance growth performance, immune status, and antioxidant capacity in lambs.

Reproduction is a fundamental biological process regulated by complex cellular and molecular networks across the neuroendocrine and reproductive systems. To explore conserved and species-specific mechanisms of fertility regulation, we constructed a high-resolution single-cell transcriptomic atlas of 15 reproductive and central nervous system (CNS) tissues from sheep and integrated it with human single-cell datasets from 13 matched tissues. This comparative atlas comprises over 1.09 million cells and identifies 76 major cell types across species. Cross-species integration based on 15 748 orthologous genes revealed that 54 cell types (71.1%) are shared between sheep and humans, showing strong conservation in transcriptional programs, cell lineage trajectories, and regulatory networks. Integrating genome-wide association studies (GWAS) for sheep lifetime average litter size with the single-cell atlas identified crucial fertility-associated genes and signaling pathways. Cell-cell communication analysis revealed UNC5-SLIT-BMP signaling cascades coordinating neuroendocrine regulation of fertility along the hypothalamus-pituitary-ovary (HPO) axis. Trait-cell type enrichment analyses for 41 human complex traits further demonstrated that conserved reproductive and CNS cell types in sheep recapitulate key human GWAS associations. Together, this cross-species single-cell atlas (https://csca.njau.edu.cn/) provides a valuable resource for understanding how conserved cellular programs and inter-organ signaling networks regulate fertility and other complex traits.

Background:Spermine has been shown to promote intestinal maturation and protect intestinal health. However, its potential as a dietary feed additive for improving intestinal health in Wuzhishan piglets remains unclear. In this study, eighteen weaned Wuzhishan piglets were randomly assigned to three groups (n = 6 per group) and provided with either a basal diet (control), a diet supplemented with 0.01% spermine, or a diet supplemented with 0.03% spermine for 28 days. Results:Dietary supplementation with 0.01% spermine significantly improved growth performance (p < 0.05) and increased small intestinal α-amylase and trypsin activities (p < 0.05). It also significantly upregulated anti-inflammatory cytokines and downregulated pro-inflammatory cytokines in small intestine (p < 0.05). Compared with the control group, the relative abundance of the beneficial gut bacterium Prevotella was significantly increased in the 0.01% spermine group, while that of the harmful bacterium Streptococcus was significantly decreased in both the 0.01 and 0.03% spermine groups (p < 0.05). Additionally, DNA synthesis-related metabolic capacity and immune-related metabolite levels were significantly elevated in both the 0.01 and 0.03% spermine groups (p < 0.05). Conclusion:These findings suggest that dietary supplementation with 0.01% spermine can effectively alleviate weaning-induced intestinal dysfunction in Wuzhishan piglets by improving intestinal morphology, digestive enzyme activity, and immune responses, modulating the relative abundance of specific gut bacteria, and enhancing metabolic capacity.

Although Mettl3-mediated N6-methyladenosine (m6A) modification has confirmed to regulate mammalian development, the precise mechanisms by which it controls myoblast differentiation and fusion remain unclear. Here, we found that Mettl3 expression and global m6A levels decreased during myogenic differentiation. Mettl3 knockdown inhibited myoblast proliferation, promoted myoblast differentiation and fusion, and reduced protein degradation in myotubes, whereas its overexpression had the opposite effects. High-throughput sequencing identified forkhead box O1 (FOXO1) as a potential target of Mettl3-mediated myogenesis. Mechanically, Mettl3-mediated methylation of FOXO1 3'untranslated region (3'UTR) promotes its translation in a Ythdf3-dependent manner, thereby suppressing the MyoD1-Myomaker/Myomixer axis and activating the ubiquitin-proteasome system (UPS). Furthermore, FOXO1 knockdown alleviates the impaired myoblast differentiation and fusion induced by Mettl3 overexpression by upregulating the MyoD1-Myomaker/Myomixer axis and suppressing the UPS. Collectively, these findings indicate that Mettl3 inhibits myoblast differentiation and fusion via the FOXO1-mediated MyoD1-Myomaker/Myomixer axis and UPS in an m6A-Ythdf3-dependent manner.

Sheep (Ovis aries) are valuable biomedical models for studying human reproductive biology, however, the transcriptional dynamics underlying their ovarian development remain insufficiently characterized. In this study, we profiled 61,649 single-cell transcriptomes from sheep ovarian tissues across key developmental stages: prenatal (E90), pre-puberty (M3), post-puberty (M6), and adulthood (Y2, Y4). We identified nine major cell types with distinct and dynamic gene expression patterns, highlighting significant developmental transitions. Notably, granulosa cells exhibited profound transcriptomic shifts, underscoring their critical role in oocyte maturation and follicular development. Key transcription factors, such as FOXO1 and ESR1, were identified as potential drivers of these transitions. Further cross-species comparisons revealed strong conservation in cell types, composition, and gene expression networks between sheep and human developing ovaries. Leveraging these conserved features, we uncovered context-dependent (e.g., cell-type-specific, developmental-stage-specific) associations with human reproductive traits and diseases. These findings advance our understanding of ovarian development in sheep and reinforce their utility as translational models in reproductive biology and disease research.

Histone lysine lactylation (Kla) is a novel epigenetic modification that plays a crucial role in cellular processes driven by glycolysis and lactate production. However, the mechanisms of histone lactylation and its interaction with m6A RNA methylation during early embryonic development remain underexplored. This study systematically investigated the effects of oxygen levels-atmospheric oxygen (atmosO2; 20% O2) and physiological oxygen (physO2; 5% O2)-on hallmark events during early embryonic development, revealing that lactylation modification regulates early goat embryonic development through the m6A methyltransferase-like 3 (METTL3). We observed that physO2 conditions significantly promote embryonic development, with higher expression levels of METTL3, global histone lactylation, and histone H3 lysine 18 lactylation (H3K18la) compared to atmosO2 exposure. Furthermore, the addition of lactate dehydrogenase inhibitors led to a decrease in global lactylation, which was accompanied by a significant reduction in METTL3 expression. Sequencing analysis of the METTL3 knockdown embryo revealed that the differentially expressed genes (DEGs) were primarily enriched in the ribosome, oxidative phosphorylation, thermogenesis, RNA degradation, and RNA polymerase pathways. These findings provide novel insights into the epigenetic regulatory mechanisms of histone lactylation during early embryonic development in livestock, highlighting potential molecular targets and strategies to enhance mammalian in vitro embryo production techniques.

Obesity has become a global epidemic with major implications for fertility. In particular, obesity can trigger follicular atresia by initiating the apoptosis of granulosa cells (GCs). Emerging evidence suggests that this process may be closely linked to the dysregulation of cellular autophagy. Metformin has been shown to restore autophagic flux and mitigate obesity-related cellular dysfunction in mice; however, the ability of metformin to alleviate lipid overload-induced damage in goat granulosa cells has yet to be investigated. Analyses showed that 400 μM palmitic acid (PA) significantly increased lipid accumulation and reduced cell viability (P < 0.05) in goat granulosa cells. Furthermore, PA impaired mitochondrial function, associated with a significant increase in the populations of both early and late apoptotic cells (P < 0.05). However, treatment with 5 μM metformin (MET) under PA exposure significantly enhanced the viability of GCs and reduced the expression levels of pro-apoptotic BAX (P < 0.05). Next, we evaluated the effect of MET on cellular autophagy and found that MET treatment significantly downregulated the expression levels of phosphorylated mTORC1 (Ser2448), LC3B, and P62 while upregulating the expression levels of ULK1 in PA-treated GCs (P < 0.05). Our findings indicate that metformin improved palmitate-induced granulosa cell dysfunction by activating ULK1-mediated autophagy. Our findings will advance our understanding of reproductive dysfunction in obese ruminants, and provide a theoretical foundation for improving fertility in obese mammals.

Placental development plays a pivotal role in ensuring successful pregnancy outcomes, yet its molecular regulatory mechanisms in sheep remain poorly characterized. This study aimed to systematically investigate stage-specific proteomic dynamics and functional adaptations in ovine placental tissues across gestation to elucidate molecular drivers of placental maturation. Using data-independent acquisition proteomics, we identified 7774 proteins in Hu sheep placental tissues at gestational days 50, 80, and 120. Comparative analysis revealed 1450, 1026, and 1964 differentially expressed proteins (DEPs) in the 50 d vs. 80 d, 80 d vs. 120 d, and 50 d vs. 120 d comparisons, respectively. DEPs were functionally enriched in biological processes including cell proliferation, apoptosis, angiogenesis, nutrient transport, and steroid synthesis, with prominent involvement of the PI3K-Akt, MAPK, and estrogen signaling pathways. Protein interaction networks identified SRC, MAP3K1, KRAS, and TJP1 as central regulators exhibiting dynamic expression patterns across gestation. Temporal expression trends showed progressive upregulation of tight junction, immune response, and glucose metabolism proteins, contrasting with downregulation of endoplasmic reticulum protein processing and proteasome components. Validation experiments confirmed elevated proliferation/transport gene expression at 80 d versus 50 d, followed by increased apoptosis/transport genes and decreased proliferation markers at 120 d. This comprehensive proteomic profiling reveals stage-specific regulatory networks governing placental development in sheep, highlighting coordinated shifts in proliferative, metabolic, and structural remodeling processes. These findings advance our understanding of placental adaptation mechanisms and provide valuable insights for improving reproductive management in livestock species.

BACKGROUND: Neuromedin B (NMB) has been implicated in the regulation of female reproductive functions, yet its precise role and underlying mechanisms in ovarian follicular development remain undefined. Granulosa cells (GCs), the principal functional cells within ovarian follicles, directly govern follicular growth and maturation through their proliferation and differentiation. In this study, we explored the regulatory effects and molecular mechanisms of NMB and its receptor neuromedin B receptor (NMBR) on goat GC proliferation. RESULTS: We documented dynamic expression patterns of NMB and NMBR throughout ovarian and follicular development. Exogenous NMB treatment significantly enhanced GC proliferation, as evidenced by an increased fraction of S-phase cells and upregulation of CCNE1 and CDK1/2/6. Mechanistically, NMB bound to NMBR to activate phospholipase C β1 (PLCβ1), triggering endoplasmic reticulum (ER) Ca²⁺ release and significantly raising cytosolic Ca²⁺ levels while alleviating ER stress. Further analyses revealed that NMB strengthened mitochondria-associated ER membranes (MAMs) formation via the IRE1α–IP3R–VDAC1 axis, facilitating Ca²⁺ transfer into mitochondria. This led to enhanced mitochondrial function, including increased mitochondrial membrane potential, elevated respiratory chain complex activities, augmented ATP production, and promotion of mitochondrial network fusion. Importantly, these effects were abolished by an NMBR antagonist. CONCLUSIONS: The molecular mechanism by which NMB-mediated activation of NMBR enhances mitochondrial metabolism through modulation of intracellular calcium homeostasis, thereby promoting proliferation of goat GCs. These findings shed new light on the regulation of follicular development and may inform strategies to improve reproductive efficiency in goats.

The VRTN (vertebrin) gene plays a crucial role in regulating thoracic vertebral number in mammals; however, its function in goats remains largely unexplored. This study aimed to investigate genetic variations in the VRTN gene in Hainan black goats (HNBGs) and evaluate their associations with growth and meat traits. Using whole-genome and Sanger sequencing, we identified four SNPs in the VRTN gene, including three missense mutations (p.Pro615Ser, p.Arg490Lys, p.Thr476Met) and one synonymous mutation (p.Asp688Asp). Tissue expression profiling revealed high VRTN mRNA expression in skeletal muscle and low expression in internal organs, suggesting a potential role in muscle development. Temporal expression analysis indicated dynamic regulation during growth, with higher expression levels observed in early developmental stages. Association analyses revealed significant correlations between specific SNPs and key traits, including body length, chest circumference, carcass weight, and meat quality parameters. Notably, the p.Pro615Ser mutation was associated with a 0.441-fold reduction in VRTN expression and showed strong associations with multiple traits, underscoring its functional importance. These findings demonstrate that VRTN polymorphisms influence growth and muscle development in HNBGs, providing valuable insights for marker-assisted selection in goat breeding.

Livestock growth performance is pivotal to meat production and skeletal muscle development. Identifying key genetic variants associated with growth traits can facilitate the selection of superior breeding stock. However, the role of Insulin-like Growth Factor Binding Protein 2 (IGFBP2) in livestock muscle development remains controversial, particularly regarding its genetic regulation in goats. In this study, the whole-genome resequencing of Hainan Black goats (HNBGs) revealed a growth-associated insertion/insertion variant (P1-Del-8-bp) in IGFBP2 3' UTR. Notably, individuals with the DD genotype exhibited significantly impaired growth performance, lower GH levels, and higher IGFBP2 levels compared to other genotypes. Mechanistically, IGFBP2 knockdown significantly enhanced the proliferation and differentiation of goat primary myoblasts (GPMs) by targeting Transforming Growth Factor Beta 2 (TGFβ2). Additionally, reduced IGFBP2 promoted myogenic differentiation by stimulating mitochondrial biogenesis. These findings provide novel insights into the molecular mechanisms governing skeletal muscle development in goats and offer potential genetic targets for livestock molecular breeding strategies aimed at improving meat production efficiency.