The standard protocols used in clinics to assess quality of embryos are still unable to reliably assess the embryo's ability to implant and develop full term. On the other hand, being able to predict the implantation potential of a given embryo appears to be crucial, as implantation abnormalities are one of the major causes of reproductive loss in mammals. Notwithstanding the differences in the implantation mechanism between different species, the functional trophectoderm is prerequisite in this process. In the present study we investigated whether the velocity of cytoplasmic movement in mural and polar TE cells of mouse blastocysts can be used to assess the embryo implantation potential. The cytoplasmic velocity was measured using time-lapse imaging in E3.5 and E4.5 mouse embryos and analyzed using Particle Image Velocimetry. We found that it decreases during late preimplantation embryo development and is higher in polar than in mural trophectoderm cells of mouse blastocysts. Our data indicate that cytoplasmic movement velocity was only minimally affected by alterations in the keratin cytoskeleton, despite its established role in determining the biomechanical properties of trophectoderm cells. Furthermore, cytoplasmic movement velocity was not affected by either maternal or postovulatory aging, both of which are known to impair the developmental potential of embryos. However, using an outgrowth assay, we showed that the velocity of cytoplasmic movement in polar, but not mural, trophectodermal cells in E4.5 blastocysts reflects the embryo's ability to implant in vitro. Therefore, analysis of cytoplasmic speed may support evaluation of the embryo quality.
Primordial germ cells (PGCs) are the embryonic precursors of gametes, essential for transmitting genetic and epigenetic information across generations. However, PGC specification occurs within a narrow developmental window and involves only a small number of cells, making it difficult to study in vivo. In vitro models using pluripotent stem cells have enabled the generation of primordial germ cell-like cells, but these systems often rely on exogenous signaling and exhibit variability in efficiency and epigenetic fidelity. In this review, we synthesize current understanding of PGC specification in mouse and human systems, emphasizing the integration of signaling pathways, transcriptional networks, epigenetic reprogramming, and metabolic regulation. Canonical regulators, including PRDM1, PRDM14, TFAP2C, and SOX17, function within a broader, interconnected network that establishes for PGC competence. Understanding these interactions will be crucial for advancing in vitro gametogenesis and improving mammalian reproduction.
Porcine conceptus elongation during the peri-implantation period is essential for establishment of pregnancy and involves rapid morphological and biochemical remodeling. However, metabolomic alterations within developing porcine conceptuses during this developmental interval are poorly understood. Therefore, the objective of this study was to characterize metabolomic remodeling in porcine conceptuses between gestational Days (GD) 10 and 16 using liquid chromatography-mass spectrometry (LC-MS)-based metabolomics and targeted amino acid analyses. A total of 230 metabolites were identified in conceptuses collected on GD10, 12, 13, 14, 15, and 16. Principal component analyses revealed progressive temporal separation of metabolomic profiles during conceptus elongation and implantation. Conceptus elongation was associated with progressive remodeling of metabolites related to nucleotide biosynthesis, phospholipid metabolism, amino acid metabolism, redox regulation, and energy metabolism. Random forest analyses identified cytidine diphosphate-ethanolamine (CDP-ethanolamine) and nucleotide-associated metabolites as key discriminators of progression in conceptus development. Targeted amino acid analyses of conceptuses demonstrated progressive increases in glutamine, glutamate, histidine, valine, arginine, and cystine, whereas tryptophan, glycine, and serine decreased during later stages of development. These coordinated changes support increased requirements for nucleotide synthesis, membrane biogenesis, redox homeostasis, and nutrient metabolism during rapid elongation and subsequent extraembryonic growth and differentiation. Collectively, these findings define metabolic transitions associated with porcine conceptus elongation and post-elongation development and provide candidate metabolic processes for future mechanistic investigation.
Phosphate is critical for pregnancy maintenance, placental, and fetal development. Although the mechanisms regulating postnatal phosphate homeostasis are well described, those regulating phosphate availability during gestation in cattle remain poorly understood. Tissue non-specific alkaline phosphatase (TNSALP, encoded by ALPL), is a postnatal regulator of phosphate availability, yet its role in the regulation of utero-placental phosphate availability in ruminants remains poorly understood. This study characterized phosphate abundance and TNSALP activity in bovine utero-placental tissues and fetal fluids, and the expression of ALPL mRNA, and TNSALP protein and activity localization in bovine utero-placental tissues during early-mid gestation. Fetal fluids and utero-placental tissues were collected (n=3-10 per group; range: 48-133 days). Phosphate concentrations and TNSALP activity were quantified spectrophotometrically. Expression of ALPL mRNA was quantified using qPCR. TNSALP protein and activity localization were determined using histological staining. Phosphate concentrations increased in allantoic fluid and decreased in amniotic fluid as gestation progressed (P ≤ 0.0001). Phosphate abundance did not change in utero-placental tissues throughout gestation. Gestational day affected TNSALP activity in allantoic fluids, amniotic fluids, and endometria (P ≤ 0.001), but not placentomes. Gestational day affected expression of ALPL mRNA in endometria and placentomes (P ≤ 0.01). TNSALP protein and enzymatic activity localized to the endometrial luminal and glandular epithelia, endometrial and placental vasculature, and the caruncular-cotyledonary epithelial interface. Collectively, these findings establish a spatiotemporal profile of phosphate abundance and TNSALP activity in bovine utero-placental tissues and fetal fluids and demonstrate TNSALP is localized to regions associated with maternal-fetal nutrient exchange.
THOR (testis-associated highly conserved oncogenic long non-coding RNA) is a highly conserved and testis-enriched lncRNA across vertebrates that plays diverse roles in various cancers. However, its physiological function and regulatory mechanism in testes remain largely unknown. Here, we investigated the genomic location and expression pattern of THOR in the model organism zebrafish, and generated a homozygous THOR knockout model using CRISPR-Cas9 technology. Loss of THOR in zebrafish impaired spermatogenesis, leading to oligospermia (38.7% reduction in sperm count), reduced sperm motility, sperm ultrastructural defects, and decreased fertilization rates. RNA-seq analysis of WT and THOR knockout testes revealed dysregulation of cell cycle-related genes, including cdkn1d, foxo1a, tsc1a, tsc2, atrx, and rad21b. RNA pulldown assays in zebrafish testes identified 486 potential THOR-interacting proteins primarily involved in ribosome biogenesis, RNA splicing, chromatin architecture, and meiotic progression. Notably, the core synaptonemal complex components Sycp1, Sycp2, and Sycp3 were all captured as THOR-binding partners. We further demonstrated that THOR directly interacts with Sycp3 and positively regulates its protein levels. Immunostaining assays on chromosome spreads revealed a significantly higher frequency of discontinuous Sycp3 signals in THOR-/- testes, suggesting the presence of meiosis defects caused by Sycp3 downregulation. Our findings expand the understanding of lncRNA-mediated control of spermatogenesis and male infertility by providing the first evidence that lncRNA THOR interacts with the synaptonemal complex to regulate meiosis progression.
Early embryonic development occurs in a low oxygen environment, and mitochondrial morphology and function are distinct in embryonic cells and pluripotent stem cells (PSC), which rely less on oxidative phosphorylation than differentiated cells. Oxidative phosphorylation increases with differentiation to trophoblast (TB) and this process is reversed by reprogramming of adult cells to pluripotency, but the influence of oxygen conditions on this process has not been characterized. When PSC were differentiated to trophoblast by treatment with BAP (BMP4, A83-01 and PD173074), cellular ATP concentrations increased equally in 5% and 20% oxygen conditions. Although oxygen conditions in culture altered transcripts encoding mitochondrial proteins, particularly by suppressing COX4l2 at 20% oxygen, there were no consistent differences in mitochondrial morphology in either cytotrophoblast (CTB) or syncytiotrophoblast (STB) cells with changing oxygen. These results suggest that TB adapt to maintain mitochondrial function at high and low oxygen during differentiation. In a previous study, iPSCs were derived from both control and early onset preeclampsia (EOPE) pregnancies and differentiated with BAP; high oxygen conditions impaired TB invasion only in cells from EOPE pregnancies. Here, the increase in ATP concentration with TB differentiation was less robust in EOPE cells, and cytochrome C and ATPase subunit transcripts differed between EOPE and control cells at high oxygen. However, investigation of mitochondrial morphology revealed no excess damage in EOPE-derived lines, and no difference in mitochondrial respiration was detected. Collectively, these data provide limited support for the hypothesis that intrinsic differences in mitochondria underlie poor TB invasion in EOPE.
Testicular tissue cryopreservation (TTC) is currently the only option to preserve fertility in pre-pubertal individuals with testes. However, reimplantation of tissue collected during TTC has yet to result in a live human birth, despite recent progress in rhesus macaque models. Additionally, cell or tissue implantations are not options for individuals with metastatic disease, making it critical to develop alternative fertility restoration strategies using TTC specimens. Our goal was to generate primary cell-derived testicular assembloids with native-similar internal cell organization, enclosed seminiferous tubule-like structures (TLS), and surviving germ cells. Organoids derived from primary testicular cells harvested from 5-day old mice developed native-similar organization and enclosed TLSs when seeded at ~9,300 cells/microwell. We assessed the fusion and internal organization of merged organoids ("assembloids") in multiple open channel designs with differing physical constraints and access to media and oxygen. These conditions impacted assembloid ellipticity, cellular organization, TLS formation, TLS diameter, and germ cell survival. Open channels with 400- and 600-micron widths that were submerged within culture media most consistently produced assembloids with native-similar architecture. These data support a robust framework for the generation of murine testicular assembloids with germ cell-containing tubule-like structures and interstitial compartments. Optimization of this in vitro platform is an important step towards expanding fertility restoration options for prepubertal patients at increased risk for infertility.
Fish oogonial stem cells (OSCs) are vital for fish reproduction research and germplasm conservation, yet stable long-term in vitro culture of fish OSCs remains a major technical bottleneck. Herein, we established an efficient in vitro culture system (termed L15SP) for OSCs derived from Paralichthys olivaceus, consisting of L-15 medium supplemented with15% fetal bovine serum (FBS), 2 μg/L basic fibroblast growth factor (bFGF), 2 μg/L leukemia inhibitory factor (LIF), 50 μmol/L β-mercaptoethanol (β-ME), 1% fish serum, and 18 g/L embryo extract protein, cultured at 23 °C. This system enabled stable long-term passage of P. olivaceus OSCs. Further investigations revealed that interferon-induced transmembrane protein 3 (Ifitm3) was highly expressed on the membrane of P. olivaceus OSCs. Functional assays demonstrated that Ifitm3 significantly enhanced proliferation, migration, and stemness maintenance of long-term cultured OSCs. Ifitm3 positively modulated the expression of key PI3K-AKT signaling components, including pik3cb, pik3r1, and akt2, to regulate downstream OSC functional genes. Upon pathway activation, Ifitm3 facilitated PIP3 enrichment at the OSC plasma membrane, thereby amplifying PI3K-AKT signaling. In conclusion, Ifitm3 governed OSC biological functions by potentiating PI3K-AKT signal transduction. This study establishes a reliable long-term culture system for fish OSCs and identifies a novel functional marker for OSC identification, providing valuable support for fish germplasm preservation and genetic breeding.
Stress adversely affects various aspects of the female reproductive process, including follicle development, maturation, ovulation, luteal activity, and embryo implantation. Restraint stress reduces the number of implantated embryos. The precise mechanisms by which stress affects embryo implantation are not fully understood. In this study, a mouse model of restraint stress was used to investigate the effects of stress on uterine receptivity and the underlying mechanisms. Mice were randomly divided into three groups. The groups were the Control group, Stress group, and Ab-IL-6 treatment group. Mice in the Stress and Ab-IL-6 treatment groups underwent restraint stress in perforated 50 ml centrifuge tubes for 4 hours daily over 7 consecutive days. Results indicated that the number of implanted blastocysts and total litter size in the stress group were significantly lower than those in the control group. These findings indicate that restraint stress leads to high IL-6 expression in the endometrium. This increased expression may affect the endometrium's receptivity, resulting in a decreased number of implanted blastocysts. Furthermore, we demonstrated that IL-6 regulated autophagy through the IL-6/JAK2/BECN1 pathway in Ishikawa cells and mouse endometrial epithelial cells, and treatment with an IL-6 antibody significantly mitigated the adverse effects of stress-induced blastocyst implantation. Our findings demonstrated that restraint stress had a negative influence on endometrial receptivity via the IL-6/JAK2/BECN1 pathway. This effect led to a reduction in the number of implanted embryos. Consequently, inhibiting IL-6 expression may represent a potential therapeutic strategy for preventing stress-induced implantation failure.
Preeclampsia (PE) is a severe pregnancy-specific disorder featured by insufficient extravillous trophoblast (EVT) invasion and impaired spiral artery remodeling. Pregnancy-specific beta-1-glycoprotein 2 (PSG2), a placenta-derived protein, is significantly elevated in the serum of PE patients, yet its exact role and underlying mechanism in trophoblast function remain largely unknown. We demonstrated that PSG2 was remarkably upregulated in PE placentas and its expression correlated positively with blood pressure and proteinuria, and negatively with gestational age and birth weight. In HTR-8/SVneo trophoblast cells, PSG2 overexpression suppressed proliferation, migration, invasion, and epithelial-mesenchymal transition (EMT), downregulated MMP2 and MMP9 expression, and induced S-phase cell cycle arrest and late apoptosis. Conversely, PSG2 knockdown exerted opposing effects without influencing cell cycle distribution or apoptosis. Immunofluorescence staining verified the co-localization of PSG2 and TGF-β1 in trophoblasts and placental tissues. Mechanistically, PSG2 activated the TGF-β/Smad3 signaling pathway, and the TGFBR1 inhibitor SB431542 effectively reversed PSG2-mediated inhibition of EMT, MMP2/MMP9 expression, and trophoblast functional impairment. In conclusion, highly expressed PSG2 in PE placentas contributes to PE pathogenesis by activating the TGF-β/Smad3 pathway, thereby inhibiting EMT and MMPs expression and attenuating trophoblast proliferation, migration, and invasion. These findings provide a novel molecular basis for understanding PE pathogenesis.
Flame retardants are a diverse class of environmental contaminants widely used to reduce flammability in consumer products, leading to pervasive human exposure. Among these, polybrominated flame retardants (PBFRs), including polybrominated diphenyl ethers (PBDEs), and organophosphate ester flame retardants (OPEs) have raised significant concern due to their potential to disrupt reproductive health. Increasing experimental, epidemiological, and mechanistic evidence demonstrates that both PBDEs and OPEs adversely affect both the male and female reproductive systems. In males, exposure to these compounds is associated with impaired spermatogenesis, altered sperm quality, disrupted steroidogenesis, and structural and functional damage to the testis. Mechanistic studies reveal direct toxicity to Leydig, Sertoli, and germ cells, driven by mitochondrial dysfunction, oxidative stress, endocrine disruption, and cytoskeletal disorganization that may compromise both fertility and offspring health. In females, PBDEs and OPEs disrupt ovarian folliculogenesis, steroid hormone synthesis, and oocyte quality, while also altering uterine receptivity. These effects are mediated through perturbations of the hypothalamic-pituitary-gonadal axis, interference with nuclear and membrane hormone receptors, mitochondrial injury, oxidative stress, and dysregulation of signaling pathways essential for follicle maturation and ovulation. Both classes of flame retardants can bioaccumulate and cross critical biological barriers, including the placenta, raising concerns about developmental and transgenerational effects. Collectively, the evidence underscores that PBFRs and OPEs pose significant risks to reproductive function in both sexes, acting through molecular mechanisms that disrupt gonadal development, endocrine signaling, and cellular homeostasis. Understanding these shared and sex-specific pathways is essential for improving human health risk assessment and guiding regulatory strategies.
Endocrine-disrupting chemicals (EDCs) constitute a broad class of toxicants capable of altering hormone synthesis, transport, and receptor activity. Phthalate esters, a major subgroup of EDCs, are pervasive in consumer, industrial, and medical products and readily leach from these materials, resulting in human exposure through ingestion, inhalation, dermal absorption, and medical treatments. After absorption, phthalates undergo extensive biotransformation and distribute systemically, including to ovarian tissue and follicular fluid. Epidemiological studies consistently associate phthalate burden with diminished ovarian reserve, altered reproductive hormones, early menopause, pregnancy loss, and reproductive disorders. Notably, phthalate metabolites detected in follicular fluid correlate with reduced antral follicle count, impaired oocyte maturation, decreased fertilization rates, and poor medically assisted reproduction outcomes. Experimental evidence from mouse and bovine models supports these associations, demonstrating that phthalate exposure reduces antral follicle counts, disrupts steroidogenesis, causes oxidative stress, and promotes apoptosis. In vitro and mechanistic studies show that phthalates impair follicular function and hormone biosynthesis by disrupting lipid metabolism, mitochondrial function, microRNA expression, and pathways governing redox balance, inflammation, cell cycle and apoptosis. These disruptions contribute to impaired folliculogenesis, increased follicular atresia, ovulation deficits, and compromised oocyte competence. Although substantial descriptive evidence exists, significant mechanistic gaps still remain, highlighting the need for research using biologically relevant phthalate doses and approaches capable of distinguishing effects of individual congeners from mixtures.
Maternal obesity compromises placental development and fetal growth, yet the mechanisms remain unknown. Here, we investigated the impact of high-fat diet (HFD) on placental energy homeostasis in mice. Female C57BL/6 mice were fed a control diet or an HFD, with a subset receiving FerroTerminator-1 (FOT1) to modulate metabolic dysfunction. At embryonic day 18.5, maternal metabolic status, fetal growth, placental morphology, energy metabolism, and oxidative stress were assessed. HFD-fed dams developed dyslipidemia and insulin resistance, and exhibited placental lipid accumulation and impaired labyrinth zone development, resulting in reduced fetal weight and altered fetal liver lipid and glycogen contents. The placenta showed mitochondrial damage, accompanied by reduced ATP content, mitochondrial membrane potential, mitochondrial DNA copy number, and suppressed respiratory chain complex activities. Placental metabolomic profiling showed that HFD was associated with altered levels of metabolites in glycolysis and oxidative phosphorylation pathways. These alterations coincided with placental iron-associated oxidative stress, evidenced by elevated non-heme iron, lipid peroxidation products, upregulation of transferrin receptor (TFRC) and lysophosphatidylcholine acyltransferase 3 (LPCAT3), downregulation of ferritin heavy chain 1 (FTH1) and solute carrier family 40 member 1 (SLC40A1), a reduced glutathione to oxidized glutathione ratio, and impaired glutathione peroxidase 4 (GPX4) expression and activity. Meanwhile, FOT1 administration attenuated placental oxidative stress, partially restored ATP production and mitochondrial function, and improved fetal growth in HFD-fed mice. Collectively, maternal obesity was associated with placental iron-associated oxidative stress and bioenergetic impairment, which may contribute to reduced placental efficiency and FGR.
Endometriosis (EMS) is characterised by a disrupted peritoneal immune microenvironment where M2 macrophage polarization and impaired phagocytosis promote lesion survival. Single-cell RNA-seq (ScRNAseq) of the peritoneal macrophages from EMS patient revealed elevated Triggering Receptor Expressed on Myeloid cells 2 (TREM2) and signal regulatory protein α (SIRPα). The expression of TREM2 and SIRPa is positively correlated, and each are positively correlated with estrogen response. Mouse EMS model was established by intraperitoneal injection of estrogen primed mouse endometrial fragments into recipient female mice. In vitro estrogen treatment of RAW 264.7 cells indicated an increasing TREM2/SIRPα expression and enhancing phagocytosis of lesion cells derived from WT EMS ectopic lesions. Peritoneal macrophage from mice with EMS were analyzed using flow cytometry for the expression of TREM2 and SIRPα. The assessment of F4/80, CD206, and SIRPα expressing cells within the lesions were performed through the implementation of multi-color immunohistochemistry (mIHC). Small peritoneal macrophages (SpMs, F4/80low CD11blow) were markedly increased in the EMS model. Trem2 knockout mice, as the recipient, showed smaller ectopic lesion size and less lesion formation. TREM2 deficiency inhibited SpM polarization into M2 and downregulated their SIRPα expression, while enhancing phagocytic activity. Mechanistically, estrogen pretreatment upregulated TREM2, which correlated with SIRPα upregulation and impaired phagocytosis. Under estrogen exposure, TREM2 knockdown RAW 264.7 cells potentiated phagocytosis. Thus, TREM2 is an important contributing pathogenic factor in promoting EMS by enhancing M2 polarization and suppressing phagocytosis via SIRPα upregulation.
The golden hamster, renowned for its representative PIWI-piRNA pathway profile among mammals, has emerged as a promising animal model for investigating female mammalian fertility. Prior research uncovered that the disruption of Piwil1 leads to complete infertility of female golden hamsters, with embryos derived from homozygous knockout (Piwil1-/-) females mated with wild-type males arresting at the two-cell stage. However, the underlying deep mechanisms remain unexplored. In the current study, we utilized highly sensitive single-cell mass spectrometry to generate proteome data of five developmental stages from oocytes to embryos in both wild-type (WT) and Piwil1-deficient golden hamsters, thus profiling the dynamic proteome landscape during oocyte-to-embryo transition (OET). Integrative analyses highlighted the temporal dynamics and complexity of hamster oocytes and embryos. Notably, we observed that classical maternal proteins in hamsters exhibit a more intricate pattern compared to mice, while PIWIL1 deficiency led to aberrant expression of proteins with diverse functions in oocytes and early embryos. Analysis of Multi-omics data indicated that PIWIL1 primarily regulates differentially expressed proteins at the post-translational level during OET. Specifically, the stability of TDRD1, essential for embryogenesis and gametogenesis, is modulated by PIWIL1, and could be its downstream target. Our study provides an extensive database that offers valuable insights into mammalian oocyte and early embryo development, and represents an invaluable resource for further mechanistic studies to deepen the understanding of developmental regulation.
Normal neurodevelopment relies on effective placental oxygen transfer and the specialized circulatory shunts that direct oxygen-rich blood to key fetal organs. The tone of these shunts, particularly the ductus venosus, is critical for routing oxygen and nutrient-rich blood towards the fetal brain. However, these shunts are sensitive to both oxygenation status and sympathetic innervation. Notably, fetal hypoxia increases shunting through the ductus venosus via enhanced hepatic sympathetic activation, a mechanism that redistributes oxygen rich blood toward vital organs. Thus, we hypothesized that hyperoxygenation may decrease shunting through the ductus venosus via hepatic sympathetic regulation but may be reversed if combined with an α-adrenergic agonist. Pregnant ewes (n = 8) underwent fetal catheterization surgery at 116-117 days gestation. At 120-124 days gestation, ewes were anaesthetized, and MRI scans were performed during normoxia, hyperoxia, and hyperoxia + phenylephrine infusion to measure blood flow and oxygenation within the major fetal vessels using phase-contrast and T2 oximetry MRI techniques, respectively. Hyperoxia had minimal effects on fetal haemodynamics. However, with the addition of phenylephrine, there was increased flow through the ductus venosus and ascending aorta but decreased flow toward the brain via the carotid arteries. Hyperoxia + phenylephrine increased overall fetal oxygen delivery but decreased cerebral oxygen delivery, despite no change in ductus venosus shunting (DV/UV flow ratio). There was no change in fetal or cerebral oxygen consumption. In conclusion, hyperoxia + phenylephrine reduced cerebral oxygenation, indicating that increased ductus venosus flow does not translate to improved cerebral oxygen delivery when carotid vascular resistance is elevated.
Mammalian fertility is dependent upon meiosis, a specialized cell division by which diploid progenitors undergo one round of DNA replication followed by two rounds of chromosomal segregation to produce haploid gametes. The germ line in the testis and ovary undergoes several rounds of mitotic divisions before ultimately transitioning to the meiotic cell cycle. This transition is achieved by replacing the mitotic cell cycle program with the meiotic one. Here, we discuss the molecular players that regulate the transition from mitosis to meiosis. In spermatogenesis, MEIOC, YTHDC2, and RBM46 form an RNA-binding complex that post-transcriptionally represses the mitotic cell cycle program, while in oogenesis, MEIOC inhibits mitotic cycling prior to meiotic initiation. STRA8 and MEIOSIN act as a transcription factor complex to drive meiotic initiation by upregulating genes involved in cell cycle progression and the unique chromosomal events of meiosis in oogenesis and spermatogenesis. These complexes are activated by upstream molecular players, including transcription factors, epigenetic regulators of chromatin structure, and extrinsic signaling factors, that form an intricate and reinforced molecular network to precisely regulate the transition from the mitotic to meiotic cell cycle. Here, we integrate current knowledge of the regulation of meiotic initiation in mammals and highlight key gaps in this regulatory program that remain to be explored.
The placenta establishes the maternal-fetal interface during pregnancy, enabling the selective exchange of nutrients and gases between maternal and fetal circulations. In humans and mice, this exchange surface is lined by multinucleated epithelial layers called the syncytiotrophoblast (SynT), which in the mouse placenta is organized into two distinct layers, SynT-I and SynT-II. SynT formation from trophoblast precursors requires epithelial plasticity to permit cell differentiation, fusion, and morphogenesis, while preserving epithelial integrity to maintain the maternal-fetal exchange barrier. However, the mechanisms that safeguard epithelial features during SynT development remain unclear. OVO-like 2 (OVOL2), a transcriptional repressor of mesenchymal-associated programs and key regulator of epithelial identity, is highly expressed in the mouse placenta and essential for its development. We hypothesized that OVOL2 promotes SynT lineage formation by restraining mesenchymal-associated transcriptional programs during trophoblast differentiation. To test this, placental development was examined following Ovol2+/- matings, and wild-type and Ovol2-deficient trophoblast stem cells were analyzed under stem conditions or differentiated with CHIR99021 to enrich for SynT lineages. SynT-I lineage development was disrupted in both Ovol2-deficient placentas and differentiating trophoblast stem cells, whereas SynT-II-associated differentiation appeared less severely affected. Chromatin profiling identified OVOL2 binding near genes associated with epithelial-to-mesenchymal transition, including Id1, Zeb1, and Vim, which were upregulated in Ovol2-deficient trophoblasts. Consistent with these observations, Ovol2-deficient cells showed elevated levels of mesenchymal markers such as ZEB1 and Vimentin and reduced levels of epithelial markers including E-cadherin. These findings identify OVOL2 as a critical regulator of SynT-I lineage formation and epithelial identity in the mouse placenta.
