Abstract Oocyte development requires coordinated metabolic and signaling support from granulosa and theca cells. By performing integrated single-cell RNA sequencing and spatial transcriptomic analyses of murine and human ovaries, we discovered a functionally specialized stromal subtype essential for folliculogenesis. These stromal cells (SCs) with glutamyl aminopeptidase (ENPEP) function, designated perifollicular SCs based on their circumferential follicle localization, exhibit two hallmark features: (1) dynamic proliferation synchronized with follicular maturation from primary to secondary to antral stages, and (2) secretion of midkine (MDK), which activates nucleolin (NCL) receptor signaling to drive granulosa cell (GC) expansion. Furthermore, analyses of ovarian aging revealed the concurrent depletion of perifollicular SCs and the attenuation of MDK–NCL signaling between perifollicular SCs and GCs. The unique spatial confinement and regulatory capacity of perifollicular SCs endow them with the potential to become important components of the follicular functional unit, providing new theoretical support for understanding the molecular regulatory mechanisms of ovarian aging from the perspective of the follicular microenvironment.

With the increasing trend of delayed childbearing, the decline in oocyte quality associated with advanced maternal age has emerged as a pressing concern. However, the mechanism remains unclear, and effective strategies for improvement are currently lacking. Previously, we reported that the downregulation of the mevalonate pathway in aged granulosa cells (GCs) contributed to meiotic defects in oocytes, which may implicate farnesyl pyrophosphate-mediated protein farnesylation. Nevertheless, the role of farnesylation in ovarian aging and its impact on oocytes requires further investigation. In this study, using cumulus-oocyte complexes (COCs) from young and aged female mice, we observed impaired cumulus expansion and concurrent meiotic defects during aged oocyte maturation, accompanied by significantly reduced protein farnesylation in aged GCs. Furthermore, inhibiting farnesylation with FTI-277 in young COCs recapitulated the aging phenotype, disrupting cumulus expansion and inducing meiotic defects similar to those in aged COCs. Conversely, restoring farnesylation via farnesol supplementation effectively ameliorated these deficits in both aged COCs (in vitro) and aged mice (in vivo). Proteomic analysis and experimental validation identified prostaglandin E2 synthase 2 (PTGES2) as a farnesylated protein. Mechanistically, age-related decline in PTGES2 farnesylation in GCs reduces its endoplasmic reticulum localization and impairs prostaglandin E2 (PGE2) production, thereby compromising PGE2-dependent cumulus expansion and oocyte maturation. Collectively, our findings highlight the detrimental effects of decreased farnesylation in aged GCs on oocyte quality and propose a potential therapeutic strategy for improving the developmental competence of aged oocytes.

The human endometrial immune landscape is critical for homeostasis and pregnancy success, yet its dynamic remodeling across the menstrual cycle and in pathology remains poorly characterized. Here, we construct a comprehensive immune cell atlas of the human endometrium, profiling 53 healthy women and 34 chronic endometritis (CE) patients across precisely staged menstrual cycle phases. Our analysis reveals NK and T cells as dominant constituents, with NK cells increasing while T cells decreasing progressively from proliferative to secretory phase. B cells, mast cells, and innate lymphoid cells remain consistently low. Of the two tissue-resident NK subsets, CD11c⁺ NK cells accumulate during the secretory phase and early pregnancy. CE disrupts this landscape, marked by CD11c⁺ NK cell reduction, local B cell proliferation and differentiation, and formation of ectopic lymphoid aggregates comprising germinal-center-B-like cells and T follicular helper-like cells. Using a murine CE model and a subsequent multi-center cohort study we demonstrate that these aggregates compromise pregnancy outcomes in a Tfh-like-cell-dependent manner. Collectively, we establish a detailed immune cell atlas of the cycling human endometrium and define a pathogenic mechanism for adverse pregnancy outcomes. The immune landscape of human endometrium is remodeled across the menstrual cycle. The authors here provide a comprehensive immune atlas of these changes, and show that ectopic lymphoid aggregates, composed of germinal-center-B-like cells and T follicular helper-like cells, form in mouse and human chronic endometritis, which might lead to adverse pregnancy outcomes.

Early uterine development in humans is a critical determinant of reproductive health and disease susceptibility during adolescence. While single-cell analyses have extensively characterized the adult uterus, the developmental origins of this organ remain poorly understood. In this study, single-cell RNA sequencing and spatial transcriptomics of the human uterus from gestational week 12 (GW12) to GW24 and from adolescents revealed a population of hyaluronan-mediated motility receptor-expressing (HMMR+) stromal cells essential for uterine endometrial and myometrial development. These HMMR+ stromal cells were highly proliferative and differentiated into stromal- and muscle-like lineages upon in vitro induction. Following xenotransplantation into immunodeficient mice, they engrafted and formed mesenchymal-like tissue expressing both stromal and smooth muscle markers. We determined that HMMR+ stromal cells promote epithelial cell differentiation and development through pleiotrophin signaling. Notably, these HMMR+ stromal cells were significantly reduced in the thin endometrium. Collectively, our findings define the cellular ontogeny of the human uterus and identify a pivotal stromal progenitor-like population with implications for developmental disorders and endometrial pathologies.

Precise control of cell-surface glycosylation remains challenging due to the dynamic and spatially complex nature of glycans. Here, we present GLOBE, a platform for optical regulation of glycoenzyme activity via site-specific incorporation of photocaged unnatural amino acids. Using galactose oxidase (GAO) as a model, o-nitrobenzyl-tyrosine (ONBY) installed at a proximal tyrosine suppresses activity until photoactivation, enabling rapid cell-surface glycan oxidation with micrometer-scale spatial and temporal resolution. Coupling GAO with MUC1-targeting aptamer produces binding-gated, protein-selective glycan oxidation, achieving precise control in mixed cellular populations. GLOBE is further extended to in situ suppression of a sialidase catalytic domain, and integrated into an orthogonal photoresponsive cascade, where sialic acid removal exposes substrates for subsequent GAO oxidation, enhancing labeling efficiency. Finally, upconversion nanoparticle-assisted photoactivation enables in vivo glycan editing in a murine tumor model, overcoming UV tissue penetration limits. Together, GLOBE provides a modular, spatiotemporally programmable framework for probing and manipulating dynamic glycosylation at single-cell and tissue levels, offering a versatile strategy to interrogate and engineer the complex sugar code underlying biological and pathological processes.

Ovarian aging-induced decline in oocyte quality has been a main issue in women of advanced maternal age. However, the potential mechanism remains elusive, and there are no effective strategies to ameliorate aged oocyte quality. The lipid metabolism of oocytes has drawn great attention, but the intrinsic regulation of oocyte quality by metabolites, metabolic enzymes, and intracellular mediators is less well-characterized. Targeted lipidomics was employed to detect the neutral lipids in oocytes during maturation. We used 4,4-difluoro-1,3,5,7,8-pentamethyl-4-bora-3a,4a-diaza-s-indacene (BODIPY 493/503) and Filipin to stain cholesteryl ester and free cholesterol, respectively. The Cholesterol/Cholesteryl Ester Quantification Assay kit was used further to quantify cholesterol-related metabolites. Western blotting was performed to evaluate acyl-coenzyme A: cholesterol acyltransferase 1/2 (ACAT1/2) expression. Immunofluorescence and quantitative real-time polymerase chain reaction (qRT-PCR) were conducted to validate the knockdown efficiency of ACAT1. Avasimibe treatment and ACAT1 small interfering RNA (siRNA) microinjection were performed to investigate the effect of impaired cholesterol–cholesteryl ester metabolism on oocyte quality. Single-oocyte RNA sequencing was conducted to explore the mechanism. Mitochondrial membrane potential (MMP), adenosine triphosphate (ATP) production, reactive oxygen species (ROS), and mitochondrial autophagosomes were detected to evaluate mitochondrial function and mitophagy. There is a profound increase in the conversion of cholesterol to cholesteryl ester in oocytes during maturation, which depends on ACAT1. Conversely, disturbing the homeostasis of cholesterol–cholesteryl ester metabolism by manipulating ACAT1 impairs oocyte quality, primarily manifested as decreased polar body extrusion (PBE), increased meiotic defects, and abnormal early embryonic development. Mechanistically, the impaired conversion of cholesterol to cholesteryl ester reduces oocyte mitophagy, leading to mitochondrial dysfunction, including reduced MMP and ATP production, and excessive accumulation of ROS. Notably, we also reveal that this metabolic homeostasis is impaired in aged oocytes, accompanied by decreased ACAT1 levels. Moreover, cholesteryl ester supplementation via cholesterol conjugated to methyl-β-cyclodextrin (CCM) can effectively ameliorate aged oocyte quality by enhancing mitophagy. This study reveals the mechanism by which cholesterol–cholesteryl ester metabolism regulates oocyte quality and thus participates in the process of oocyte aging by influencing mitophagy and mitochondrial function.

Perfluorooctanoic acid (PFOA) is an environmentally persistent chemical that poses significant risks to human health. Studies have shown that PFOA affects female reproduction, but the specific impact on endometrial receptivity and the underlying mechanisms remain poorly understood. In this study, we investigated the effects of low-dose PFOA exposure through drinking water on endometrial receptivity in a murine model. Our results demonstrate that PFOA exposure significantly impaired endometrial receptivity, which led to a marked decrease in embryo implantation rates. Utilizing single-cell RNA sequencing technology, we conducted a comprehensive analysis that revealed specific mechanisms by which PFOA disrupts the function and development of endometrial epithelial cells. Notably, we identified dysregulation of the ANGPTL (angiopoietin-like) signaling pathway, which is critical for communication between endometrial stromal and epithelial cells, ultimately contributing to embryo implantation failure. These findings provide novel insights into the reproductive toxicity of PFOA and highlight potential targets for therapeutic interventions aimed at addressing infertility associated with environmental contaminants.

With the development of modern society and prolonged education, more women choose to delay their childbearing age, which greatly increases the number of women aged older than 35 years with childbearing needs. However, with increasing age, the quantity and quality of oocytes continue to fall, especially with increasing aneuploidy, which leads to a low in vitro fertilization (IVF) success rate, high abortion rate and high teratogenesis rate in assisted reproduction in women with advanced maternal age. In addition to genetics and epigenetics, follicular metabolism homeostasis is closely related to ovarian aging and oocyte aneuploidy. Glucose, lipid, and amino acid metabolism not only provide energy for follicle genesis but also regulate oocyte development and maturation. This review focuses on the relationships among follicular metabolism, oocyte aneuploidy, and ovarian aging and discusses potential therapeutic metabolites for ovarian aging.

Ovarian aging is characterized by a progressive decline in oocyte quality and quantity with age. Icariin (ICA), a flavonoid compound derived from Epimedium species, has demonstrated potential as an agent for ovarian restoration. In this study, a subcutaneous implantation system using gelatin methacryloyl (GelMA) hydrogel embedded with ICA was developed to restore ovarian function in aged female mice. Mice were assigned to receive subcutaneous implantation of GelMA alone (GelMA group), GelMA containing ICA (GelMA/ICA group), or a sham operation. Ovarian morphology, serum hormone levels, follicle counts across developmental stages, and reproductive outcomes were evaluated. In vitro fertilization (IVF) and embryo culture assays were performed to assess oocyte developmental potential, while a 10 day natural mating trial was conducted to determine fertility restoration. RNA sequencing (RNA-seq) and RT-qPCR were performed to elucidate the underlying molecular mechanisms. Results showed that GelMA/ICA treatment significantly increased ovarian index (0.19±0.01 vs. 0.13±0.01, P<0.0001) and follicle numbers at all developmental stages, including primordial (383.33±151.65 vs. 107.14±32.26, P<0.0001), primary (203.33±83.22 vs. 91.43±27.04, P=0.003), and secondary follicles (154.17±52.00 vs. 59.28±20.50, P=0.029) compared to the sham controls. Hormonal analyses revealed a significant reduction in serum follicle-stimulating hormone (FSH, 11.97±3.53 vs. 53.10±17.89 ng/mL, P=0.0008), accompanied by elevated anti-Müllerian hormone (AMH, 22.97±2.26 vs. 5.54±1.56 ng/mL, P<0.0001) and estradiol (E 2, 315.30±37.62 vs. 168.5±14.78 pg/mL, P<0.0001). Oocyte yield and developmental potential improved significantly, as reflected by the increased number of superovulated MII oocytes (17.83±5.15 vs. 4.83±4.79, P=0.0002), and higher proportions of two-cell (85.90%±6.16% vs. 50.00%±10.00%, P=0.0009), four-cell (81.67%±9.76% vs. 50.00%±10.00%, P=0.0061), and blastocyst stage embryos (64.25%±10.55% vs. 23.33%±15.28%, P=0.0067). Live birth numbers were significantly increased following GelMA/ICA treatment (6.90±3.21 vs. 1.72±2.05, P=0.0001). Transcriptomic analysis revealed up-regulation of genes associated with cytoskeletal organization ( Vil1, Tubb3), lipid storage ( Soat2, Plin4), oocyte maturation ( Oosp2), and cytokine secretion ( Cxcl12). Collectively, these findings suggest that GelMA/ICA hydrogels effectively reverse key hallmarks of ovarian aging and restore reproductive function in aged mice, offering a promising platform for fertility preservation and a novel therapeutic for future investigations into ovarian aging.

BACKGROUND:The immunosuppressive tumor microenvironment (TME) is a key characteristic of human cancer. Immunotherapy has emerged as a promising treatment strategy to overcome immune escape and has gained widespread use in recent years. In particular, the blockade of PD-1/PD-L1 interaction holds significant importance in oncotherapy. Combining anti-PD-1/PD-L1 with small molecule inhibitors targeting key pathways represents an emerging trend in therapeutic development. METHODS:To validate our findings biologically, we employed qRT-PCR or Western blotting and immunofluorescence staining techniques to assess the expression levels of DIAPH1 and PD-L1 in cells. Additionally, CCK8 and clone formation assays were utilized to evaluate cell proliferation ability, while flow assays were conducted to detect apoptosis in T cells. RESULTS:Knockdown of DIAPH1 restored the tumor-killing capacity of T cells, effectively suppressing tumor immune escape. We observed a highly positive correlation between the expression levels of DIAPH1 and 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR), which can be competitively inhibited by lovastatin. Through Sybyl analysis followed by confirmation via micro scale thermophoresis, we identified lovastatin as a potential inhibitor targeting DIAPH1. Lovastatin downregulated DIAPH1 expression both in tumor cell lines and xenograft lung cancer tissues within a mouse lung cancer model. Furthermore, we found that lovastatin degraded DIAPH1 through lysosomal degradation pathway. Treatment with lovastatin was strongly associated with improved response rates and prolonged overall survival among patients with lung adenocarcinoma. Finally, overexpression of DIAPH1 reversed the inhibitory effects mediated by lovastatin on tumor development. CONCLUSIONS:Lovastatin downregulates PD-L1 expression by targeting DIAPH1 and restores the tumor-killing ability of T cells to block tumor immune escape. Lovastatin may become a potential drug for cancer patients to enhance immunotherapy response in the clinic.

Declining oocyte quality is the major contributor to female subfertility in aged mammals. Currently, there are no effective interventions to ameliorate aged oocyte quality. Here we found that oocytes at metaphase I from the cumulus–oocyte complexes of aged mice showed reduced cortical F-actin and lower levels of mevalonate (MVA) pathway metabolites, including MVA, farnesyl pyrophosphate (FPP) and geranylgeranyl pyrophosphate. We further showed that MVA supplementation improved FPP levels, cortical F-actin and the quality of aged oocytes. Mechanistically, we found that MVA supplementation induced granulosa cells to synthesize FPP, which was subsequently transferred to aged oocytes. Transported FPP increased the prenylation of small GTPases, including CDC42 and RAC1, and promoted membrane localization of CDC42–N-WASP–Arp2/3 and RAC1–WAVE2–Arp2/3 complexes, promoting cortical F-actin reassembly and reducing aneuploidy of aged oocytes. We also identified a natural chemical compound, 8-isopentenyl flavone, with an isopentenyl side chain from Epimedium brevicornu Maxim, which could increase CDC42 and RAC1 prenylation, improving the cortical F-actin and the competence of aged oocytes, and ameliorating reproductive outcomes in aged female mice. Collectively, increasing the prenylation of small GTPases via MVA metabolites or 8-isopentenyl flavone provides a therapeutic approach for boosting female fertility during reproductive aging. Declining oocyte quality contributes to age-related reduction in fertility; however, the underlying mechanisms are incompletely understood. Here Liu et al. reveal that replenishing mevalonate pathway metabolites and supplementation with a natural compound, 8-isopentenyl flavone, improve aged oocyte quality by restoring cortical F-actin through CDC42 and RAC1 prenylation.

Thin endometrium (TE) has been widely recognized as a critical cause of various obstetric and gynecological conditions. Although the role of stem cells in endometrial functions and their pathologies has been suggested, the identity and molecular mechanisms of such stem cells remain unclear. Data analysis of a publicly available single-cell RNA sequencing (scRNA-seq) was performed to unravel the cellular and molecular characteristics of endometrial CD9+ SUSD2+ cells in normal endometria (n = 3 ) across the menstrual cycle and proliferative phase of thin endometrium (n = 3 ). Then, CD9+ SUSD2+ cells were isolated for analysis. Flow cytometry and colony-forming assays were utilized to assess changes in CD9+ SUSD2+ cell proliferation and differentiation. Additionally, CellChat, Western blotting, and multiplex immunofluorescent analysis were performed to elucidate the tissue distribution of the CD9+ SUSD2+ cells and their molecular regulatory effects on the pathogenesis of TE. A total of 59,770 cells were grouped into 13 distinct clusters in normal proliferative, secretory endometrium, and thin endometrium. Our findings revealed that perivascular CD9+ SUSD2+ cells as putative progenitor stem cells based on pseudotime trajectory and enriched functions in ossification, stem cell development, and wound healing. Histological analysis unveiled a significant perivascular expression pattern of CD9+ SUSD2+ cells in different menstrual cycle phases. The scRNA-seq of endometrial samples during the proliferative phase from patients with TE and controls revealed TE-associated shifts in cell function, manifesting as increased fibrosis and attenuated cell cycle and adipogenic differentiation. Cell-cell communication network mapping underscored aberrant crosstalk among specific cell types, implicating crucial pathways such as collagen over-deposition around perivascular CD9+ SUSD2+ cells, indicating a disrupted response to endometrial repair in TE, particularly remodeling of the extracellular matrix. Our study provides potential molecular mechanisms underpinning perivascular CD9+ SUSD2+ cells in the context of thin endometrium. The mechanistic insights could establish new therapeutic strategies for endometrial regeneration and repair.

This study aimed to evaluate the clinical outcomes of umbilical cord mesenchymal stem cell (UC-MSC) transplantation on ovarian function in women with premature ovarian insufficiency (POI), and its potential to improve in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) outcomes. A clinical cohort study was conducted at the Reproductive Medicine Center of Nanjing Drum Tower Hospital from 2019 to 2023. The study included POI patients who underwent either UC-MSC ovarian tissue injection or traditional IVF/ICSI-FET treatment. UC-MSCs were isolated from Wharton’s jelly of the umbilical cord, cultured, and transplanted into the ovaries of patients via transvaginal ultrasound-guided injection. The embryo freezing rate, number of oocytes retrieved, and ovarian function indicators (e.g., serum FSH levels, antral follicle count (AFC)) were compared between the UC-MSC-treated group and the traditional IVF/ICSI-FET group. A total of 71 POI patients were included, with 43 in the UC-MSC group and 28 in the IVF/ICSI-FET control group. After treatment, the AFC in the UC-MSC group was significantly higher compared to the control group (3.86 ± 2.93 vs. 2.39 ± 1.66, p = 0.019). Furthermore, the number of oocytes retrieved in the initial controlled ovarian hyperstimulation (COS) cycle following treatment was significantly greater in the UC-MSC group (1.88 ± 1.00 vs. 1.39 ± 1.13, p = 0.034). Patients undergoing UC-MSC treatment exhibited a substantial increase in the number of oocytes retrieved in their first COS cycle compared to their pre-treatment cycle (1.88 ± 1.00 vs. 1.28 ± 1.01, p = 0.007). Additionally, the number of frozen embryos significantly increased (0.74 ± 0.82 vs. 0.14 ± 0.41, p < 0.001), and the proportion of cycles resulting in frozen embryos was notably higher (55.81

STUDY QUESTION:Does the downregulation of cell division cycle 42 (CDC42) protein in endometrial stroma lead to endometrial senescence in patients with recurrent implantation failure (RIF), and what is the potential mechanism? SUMMARY ANSWER:CDC42 deficiency causes endometrial stromal senescence and decidualization defects, impairing uterine receptivity of RIF patients, via activation of Wnt signaling pathway. WHAT IS KNOWN ALREADY:Uterine aging is unique due to the cyclic remodeling and decidualization of endometrial tissue. Several transcriptomic studies have reported increased senescence in the endometrium in young patients with RIF. Our previous transcriptomic sequencing study discovered that endometrium from women with RIF showed downregulation of CDC42, which is an essential molecule affected by various senescence-related diseases. STUDY DESIGN, SIZE, DURATION:The endometrial samples of a total of 71 fertile control patients and 37 RIF patients were collected to verify the association between CDC42 expression and endometrial senescence of RIF patients. Primary endometrial stromal cells (EnSCs) were isolated from endometrial biopsies taken from patients without any endometrial complications and planning to undergo IVF, then subjected to adenovirus-mediated CDC42 knockdown and decidualization induction to explore the detailed mechanism by which CDC42 governs stromal senescence and decidualization. Wnt inhibitor XAV-939 was used to correct the endometrial senescence and decidualization defect. PARTICIPANTS/MATERIALS, SETTING, METHODS:Senescence was determined by cell cycle arrest markers (e.g. P16, P21, and P53), SASP molecules (e.g. IL6 and CXCL8), and SA-β-gal staining. Masson's staining and Sirius Red staining were used to detect the endometrial fibrosis. Decidualization was evaluated by the mRNA expression and protein secretion of PRL and IGFBP1, F-actin immunostaining, and the BeWo spheroids 'in vitro implantation' model. Methods used to assess cell function included adenovirus transduction, RNA-sequencing, bioinformatic analysis, western blotting, RT-qPCR, ELISA, and immunofluorescence. MAIN RESULTS AND THE ROLE OF CHANCE:Here, we observed remarkably increased levels of stromal senescence and fibrosis, along with stromal CDC42 deficiency, in the endometrium of patients with RIF (P < 0.001). Knockdown of CDC42 effectively induced premature senescence in EnSCs, leading to aberrant accumulation of senescent EnSCs and collagen deposition during decidualization. CDC42 deficiency in EnSCs restrained the decidualization differentiation and receptivity to trophoblast cells. Transcriptomic analysis revealed Wnt signaling activation as a critical downstream alteration in CDC42-deficient EnSCs. Mechanistically, CDC42 interacted with AKT competitively to impede the binding of GSK3β to AKT. Knockdown of CDC42 increased AKT-mediated phosphorylation of GSK3β to inactivate the Axin-GSK3β destruction complex, leading to accumulation and nuclear translocation of β-catenin. Importantly, Wnt signaling inhibitors partially corrected the endometrial senescence caused by CDC42 deficiency, and improved both decidualization and trophoblast invasion. LARGE SCALE DATA:RNA-seq data sets generated in this study have been deposited at the NCBI database with BioProject accession number PRJNA1102745. LIMITATIONS, REASONS FOR CAUTION:The present study was based on in vitro cell cultures. Further studies involving CDC42-regulated endometrial senescence are needed in knockout mice model and human endometrial assembloids. WIDER IMPLICATIONS OF THE FINDINGS:In addition to uncovering endometrial senescence in RIF, our findings underscore the significance of CDC42 in modulating EnSC senescence to maintain the decidualization function, and suggest Wnt signaling inhibitors as potential therapeutic agents for alleviating endometrial senescence. STUDY FUNDING/COMPETING INTEREST(S):This work was supported by the National Natural Science Foundation of China [82271698 (R.J.), 82030040 (H.S.), 82288102 (H.W.), and 82371680 (G.Y.)]; the Natural Science Foundation of Jiangsu Province [BK20231117 (R.J.)]; and the Medical Science and Technology Development Foundation of Nanjing Department of Health [YKK23097 (Y.Z.)]. The authors declare no potential conflicts of interest.

Obtaining information about cellular interactions is fundamental to the elucidation of physiological and pathological processes. Proximity labeling technologies have been widely used to report cellular interactions in situ; however, the reliance on addition of tag molecules typically restricts their application to regions where tags can readily diffuse, while the application in, for example, solid tissues, is susceptible. Here, we propose an "in‐situ‐tag‐generation mechanism" and develop the GalTag technology based on galactose oxidase (GAO) for recording cellular interactions within three‐dimensional biological solid regions. GAO mounted on bait cells can in situ generate bio‐orthogonal aldehyde tags as interaction reporters on prey cells. Using GalTag, we monitored the dynamics of cellular interactions and assessed the targeting ability of engineered cells. In particular, we recorded, for the first time, the footprints of Bacillus Calmette‐Guérin (BCG) invasion into the bladder tissue of living mice, providing a valuable perspective to elucidate the anti‐tumor mechanism of BCG.

Various posttranslational modifications (PTMs) have been implicated in endometrial stromal cell (EnSC) differentiation, but the potential role of PTM crosstalk has not been identified. Here, we report that protein arginine methyltransferase 5 (PRMT5) is indispensable for human endometrial decidualization, functioning as a key regulator of decidualization defect in recurrent implantation failure (RIF) patients. Uterine-selective deletion of Prmt5 led to defective embryo implantation in mice due to impaired EnSC decidualization. Mechanistically, we find that PRMT5 catalyzes symmetric dimethylation of orphan nuclear receptor Nur77 at arginine 346, which in turn promotes Nur77 nuclear localization and increases its transcriptional activity in EnSC. Moreover, we demonstrate that PRMT5-mediated Nur77 methylation antagonizes AKT-induced phosphorylation of Nur77 at serine 351 in the transition from proliferation to differentiation of EnSC and disruption of the balance between methylation and phosphorylation of Nur77 is essentially involved in the endometrium of RIF patients. Furthermore, by modulating the methylation-phosphorylation of Nur77 and its transcriptional activity, we rescued impaired decidualization in RIF, further highlighting the critical role of the PRMT5/AKT/Nur77 complex in uterine receptivity to embryo implantation.### Competing Interest StatementThe authors have declared no competing interest.

SUMMARYThe removal of the sperm nuclear membrane is the first step in the formation of the male pronucleus and the initiation of parental genome reprogramming during fertilization. However, the initiation of this process remains poorly understood. In this study, we found that the maternal storage of cholesterol in oocytes transferred from granulosa cells during oocyte maturation was essential for removing the sperm nuclear membrane. Oocytes with low levels of cholesterol storage fail to form male pronuclei and develop into four-cell embryos. Mechanistically, cholesterol regulates the activity of protein kinase C (PKC) to disassemble the sperm nuclear envelope. These findings establish an important function of the maternal storage of cholesterol in initiating sperm decondensation and catalysing early embryonic development in mammals.

Abstract Background Intrauterine adhesions (IUAs) jeopardise uterine function in women, which is a great challenge in the clinic. Previous studies have shown that endometrial perivascular cells (En-PSCs) can improve the healing of scarred uteri and that hydroxysafflor yellow A (HSYA) promotes angiogenesis. The purpose of this study was to observe whether the combination of En-PSCs with HSYA could improve the blood supply and fertility in the rat uterus after full-thickness injury. Methods En-PSCs were sorted by flow cytometry, and the effect of HSYA on the proliferation and angiogenesis of the En-PSCs was detected using CCK-8 and tube formation assays. Based on a previously reported rat IUA model, the rat uteri were sham-operated, spontaneously regenerated, or treated with collagen-loaded PBS, collagen-loaded HSYA, collagen-loaded En-PSCs, or collagen-loaded En-PSCs with HSYA, and then collected at both 30 and 90 days postsurgery. HE staining and Masson staining were used to evaluate uterine structure and collagen fibre deposition, and immunohistochemical staining for α-SMA and vWF was used to evaluate myometrial regeneration and neovascularization in each group. A fertility assay was performed to detect the recovery of pregnancy function in each group. RNA-seq was performed to determine the potential mechanism underlying En-PSCs/HSYA treatment. Immunofluorescence, tube formation assays, and Western blot were used to validate the molecular mechanism involved. Results The transplantation of Collagen/En-PSCs/HSYA markedly promoted uterine repair in rats with full-thickness injury by reducing fibrosis, increasing endometrial thickness, regenerating myometrium, promoting angiogenesis, and facilitated live births. RNA sequencing results suggested that En-PSCs/HSYA activated the NRG1/ErbB4 signaling pathway. In vitro tube formation experiments revealed that the addition of an ErbB inhibitor diminished the tube formation ability of cocultured En-PSCs and HUVECs. Western blot results further showed that elevated levels of NRG1 and ErbB4 proteins were detected in the Collagen/En-PSCs/HSYA group compared to the Collagen/En-PSCs group. These collective results suggested that the beneficial effects of the transplantation of Collagen/En-PSCs/HSYA might be attributed to the modulation of the NRG1/ErbB4 signaling pathway. Conclusions The combination of En-PSCs/HSYA facilitated morphological and functional repair in rats with full-thickness uterine injury and may promote endometrial angiogenesis by regulating the NRG1/ErbB4 signaling pathway.