The non-renewable primordial follicle (PF) pool underlies female reproductive longevity, yet PF numbers decline sharply before puberty for reasons that remain unclear. Here, we identify histone deacetylase 3 (HDAC3) in pregranulosa cells (pGCs) as a key suppressor of ferroptosis in PFs. Constitutively low HDAC3 expression in pGCs is essential for PF survival: conditional deletion of Hdac3 caused profound PF depletion and ultimately premature ovarian insufficiency. Loss of HDAC3 preferentially triggered ferroptosis, as supported by ferroptosis-associated transcriptional signatures and dysregulation of genes governing lipoxin biosynthesis and cellular iron homeostasis following Hdac3 depletion. Correspondingly, alterations in proteins central to ferroptotic pathways corroborated this mechanism. Together, these findings demonstrate that constitutive HDAC3 expression in pGCs safeguards the PF reserve by restraining ferroptosis and is therefore indispensable for female fertility.

Progesterone receptor (PR)-mediated progesterone (P4) signaling plays a crucial role in the establishment of uterine receptivity which is the prerequisite for successful embryo implantation in mammals. However, detailed molecular mechanisms underlying PR-chromatin binding and transcriptional activity in the uterus remain largely elusive. Here, combining the P4-administrated ovariectomized mouse model and PR-chromatin immunoprecipitation sequencing, we identified transcription factor WT1 as a potential cooperator of PR in the uterus. WT1 was specifically expressed in uterine stromal cells. Uterine deletion of Wt1 resulted in implantation failure due to attenuated P4 responsiveness in stromal cells and aberrant uterine receptivity. Mechanistically, WT1 physically interacted with PR in stromal cells, and exhibited genome-wide co-occupancy with PR. Furthermore, WT1 was indispensable for directing PR onto the chromatin via corecruitment and tethering manners, and facilitated PR to activate the transcription of target genes that were critical for uterine receptivity. Collectively, our study provided substantial evidence that WT1 served as a functional partner of PR in uterine stromal cells, thus guaranteeing P4 responsiveness conducive to uterine receptivity.

Female reproductive health is troubled by oocyte maturation disorder. In mammals, granulosa cells (GCs) mediate luteinizing hormone (LH) action on oocyte maturation and ovulation. However, the pathogenesis of disordered GCs in oocyte maturation arrest is rarely studied. Our previous study has showed that HDAC3 (histone deacetylase 3) in GCs was decreased by LH at physiological conditions. Here, we observed significantly elevated HDAC3 levels in GCs from patients with oocyte maturation disorder following LH treatment compared with those with normal oocyte maturation. To clarify whether abnormally high levels of HDAC3 in ovulatory GCs resulted in female infertility, a mice model of GC-conditional over- expression of Hdac3 was constructed. The results showed that abnormally high levels of HDAC3 in ovulatory GCs inhibited LH induction on oocyte maturation and ovulation, resulting in female infertility. Further, in GCs with abnormal high levels of HDAC3, the upregulation of oocyte maturation-related genes induced by LH was attenuated by HDAC3 through a reduction in H3K14ac levels in the promoter regions, implying that the action of LH in GCs was largely negatively controlled by HDAC3. Applying HDAC3 inhibitors enhanced the expression of multiple genes associated with oocyte maturation in GCs from clinical patients, ultimately improving both the oocyte maturation rate and developmental quality, as demonstrated by a higher blastocyst development rate. The findings contribute to both enrich understanding upon the pathological mechanisms and supply optimal treatment strategies for patients with oocyte maturation disorder.

Decidualization, a highly programmed differentiation process of the uterine stroma, is characterized by significant biochemical remodeling and is essential for pregnancy. However, the functions and molecular mechanisms of lipid metabolism during decidualization remain poorly understood. In this study, a dynamic process of lipid droplet synthesis and degradation is observed during decidual progression, and GSK3 is identified as a potential regulator for lipolysis. Specifically, lipolysis is inhibited in uterine Gsk3b knockout mice, leading to impaired terminal differentiation of decidual cells. Mechanistically, GSK3β promots phosphorylation‐dependent lysosomal degradation of RNF213, which permits the localization of adipose triglyceride lipase (ATGL) on lipid droplets, thereby facilitating lipolysis. Furthermore, fatty acids released from lipolysis enter the mitochondria to undergo β‐oxidation and produce acetyl‐CoA. The inhibition of lipolysis caused by GSK3β deficiency leads to a reduction in acetyl‐CoA levels, which in turn epigenetically affects gene transcription through histone acetylation. This study provided evidence for the regulation of dynamic lipid metabolism in vivo, and its influences on gene transcription for decidualization, which emphasized the critical role of metabolic modulation in uteri during early pregnancy.

The decidual endometrial stromal cells play a critical role in the establishment of uterine receptivity and pregnancy in human. Our previous studies demonstrate that protein tyrosine phosphatase 2 SHP2 is highly expressed in decidualized cells and governs the decidualization progress. However, the role and mechanism of SHP2 in the function of decidual cells remain unclear. Here, we screened proteins interacting with SHP2 in decidual hTERT-immortalized human endometrial stromal cells (T-HESCs) and identified Hypoxia-inducible factor-1 (HIF-1) signaling pathway as a potential SHP2-mediated signaling pathway through proximity-dependent biotinylation (BioID) analysis. Immunoprecipitation (Co-IP) revealed an interaction between SHP2 and HIF-1α, which colocalized to the nucleus in decidual cells. Furthermore, the SHP2 expression correlated with the transcriptional activation of HIF-1α and its downstream genes Beta-enolase (Eno3), Pyruvate kinase 2 (Pkm2), Aldolase C (Aldoc), and Facilitative glucose transporter 1 (Glut1). Knockdown or inhibition of SHP2 significantly reduced the mRNA and protein levels of HIF-1α and its downstream genes, as well as lactate production in decidual cells. We also established a hypoxia model of T-HESCs and 293 T cells and found that hypoxic treatment induced the expression of SHP2 and HIF-1α, which colocalized in the nucleus. SHP2 forced-expression rescued the inhibitory effects of SHP2 deficiency on HIF-1α expression and lactate production. Finally, SHP2 binds to the promoter regions of HIF-1α and its target genes (Eno3, Pkm2, Aldoc, and Glut1). Collectively, our results suggest that SHP2 influences the function of decidual cells by HIF-1α signaling and provide a novel function mechanism of decidual stromal cells.

Zika virus (ZIKV) vertical transmission results in devastating congenital malformations and pregnancy complications; however, the specific receptor and host factors facilitating ZIKV maternal-fetal transmission remain elusive. Here, we employ a genome-wide CRISPR screening and identify multiple placenta-intrinsic factors modulating ZIKV infection. Our study unveils that hepatitis A virus cellular receptor 1 (HAVCR1) serves as a primary receptor governing ZIKV entry in placental trophoblasts. The GATA3-HAVCR1 axis regulates heterogeneous cell tropism in the placenta. Notably, placenta-specific Havcr1 deletion in mice significantly impairs ZIKV transplacental transmission and associated adverse pregnancy outcomes. Mechanistically, the immunoglobulin variable-like domain of HAVCR1 binds to ZIKV via domain III of envelope protein and virion-associated phosphatidylserine. Proteomic profiling and function analyses reveal that AP2S1 cooperates with HAVCR1 for ZIKV internalization through clathrin-mediated endocytosis. Overall, our work underscores the pivotal role of HAVCR1 in mediating ZIKV vertical transmission and highlights a therapeutic target for alleviating congenital Zika syndrome.

The placenta plays an essential role during pregnancy in mammals, with the placental endocrine trophoblast subtypes secreting many growth factors and cytokines to promote fetal growth and maternal adaptation. These endocrine cells, including trophoblast giant cells (TGCs), glycogen trophoblast cells (GlyTs) and spongiotrophoblast cells (SpTs), are mainly derived from Tpbpα-positive (Tpbpα+) trophoblast cells primarily located in the ectoplacental cone (EPC) and later junctional zone (JZ) of the mouse placenta. However, the mechanism driving Tpbpα+ trophoblast cell differentiation and the functions of the factors secreted by these endocrine cells remain largely unknown. In the present study, we generated the Tpbpα-iCre-EGFP knock-in mice with codon-improved Cre recombinase (iCre) inserted into the endogenous locus of the Tpbpα gene. To examine the specificity and efficiency of iCre recombinase, we crossed the Tpbpα-iCre-EGFP mice with ROSA26Sortm9(CAG-tdTomato)Hze reporter mice. The co-expression of EGFP and Td-tomato was detected obviously in the EPC at E8.5 and E9.5, and in the JZ at E13.5. Meanwhile, employing lineage tracing and in situ hybridization, we demonstrated that Tpbpα+ trophoblast cells could differentiate into SpTs, GlyTs, maternal blood canal (C)-TGCs, parietal (P)-TGCs, and spiral artery-associated (Spa)-TGCs. In addition, no Tpbpα expression or iCre recombinase activity was detected in other organs examined, indicating the specificity of the iCre activity in placental trophoblast cells. In summary, we successfully generated the Tpbpα-iCre-EGFP knock-in mice with enhanced Cre recombinase for modulating specific genes and investigating their functions during pregnancy.

Uterine luminal epithelia (LE), the first layer contacting with the blastocyst, acquire receptivity for normal embryo implantation. Besides the well-accepted transcriptional regulation dominated by ovarian estrogen and progesterone for receptivity establishment, the involvement of epigenetic mechanisms remains elusive. This study systematically profiles the transcriptome and genome-wide H3K27me3 distribution in the LE throughout the preimplantation. Combining genetic and pharmacological approaches targeting the PRC2 core enzyme Ezh1/2, we demonstrate that the defective remodeling of H3K27me3 in the preimplantation stage disrupts the differentiation of LE, and derails uterine receptivity, resulting in implantation failure. Specifically, crucial epithelial genes, Pgr, Gata2, and Sgk1, are transcriptionally silenced through de novo deposition of H3K27me3 for LE transformation, and their sustained expression in the absence of H3K27me3 synergistically confines the nuclear translocation of FOXO1. Further functional studies identify several actin-associated genes, including Arpin, Tmod1, and Pdlim2, as novel direct targets of H3K27me3. Their aberrantly elevated expression impedes the morphological remodeling of LE, a hindrance alleviated by treatment with cytochalasin D which depolymerizes F-actin. Collectively, this study uncovers a previously unappreciated epigenetic regulatory mechanism for the transcriptional silencing of key LE genes via H3K27me3, essential for LE differentiation and thus embryo implantation.

The differentiation of the stroma is a hallmark event during postnatal uterine development. However, the spatiotemporal changes that occur during this process and the underlying regulatory mechanisms remain elusive. Here, we comprehensively delineated the dynamic development of the neonatal uterus at single-cell resolution and characterized two distinct stromal subpopulations, inner and outer stroma. Furthermore, single-cell RNA sequencing revealed that uterine ablation of Pr-set7, the sole methyltransferase catalyzing H4K20me1, led to a reduced proportion of the inner stroma due to massive cell death, thus impeding uterine development. By combining RNA sequencing and epigenetic profiling of H4K20me1, we demonstrated that PR-SET7-H4K20me1 either directly repressed the transcription of interferon stimulated genes or indirectly restricted the interferon response via silencing endogenous retroviruses. Declined H4K20me1 level caused viral mimicry responses and ZBP1-mediated apoptosis and necroptosis in stromal cells. Collectively, our study provides insight into the epigenetic machinery governing postnatal uterine stromal development mediated by PR-SET7.

Non-alcoholic fatty liver disease (NAFLD) is the most common form of chronic liver disease. Little is known about how gene expression and chromatin structure are regulated in NAFLD due to lack of suitable model. Ducks naturally develop fatty liver similar to serious human non-alcoholic fatty liver (NAFL) without adipose inflammation and liver fibrosis, thus serves as a good model for investigating molecular mechanisms of adipose metabolism and anti-inflammation. Here, we constructed a NAFLD model without adipose inflammation and liver fibrosis in ducks. By performing dynamic pathological and transcriptomic analyses, we identified critical genes involving in regulation of the NF-κB and MHCII signaling, which usually lead to adipose inflammation and liver fibrosis. We further generated dynamic three-dimensional chromatin maps during liver fatty formation and recovery. This showed that ducks enlarged hepatocyte cell nuclei to reduce inter-chromosomal interaction, decompress chromatin structure, and alter strength of intra-TAD and loop interactions during fatty liver formation. These changes partially contributed to the tight control the NF-κB and the MHCII signaling. Our analysis uncovers duck chromatin reorganization might be advantageous to maintain liver regenerative capacity and reduce adipose inflammation. These findings shed light on new strategies for NAFLD control.

Nutrient sensing and adaptation in the placenta are essential for pregnancy viability and proper fetal growth. Our recent study demonstrated that the placenta adapts to nutrient insufficiency through mechanistic target of rapamycin (mTOR) inhibition-mediated trophoblast differentiation toward syncytiotrophoblasts (STBs), a highly specialized multinucleated trophoblast subtype mediating extensive maternal-fetal interactions. However, the underlying mechanism remains elusive. Here, we unravel the indispensable role of the mTORC1 downstream transcriptional factor TFEB in STB formation both in vitro and in vivo. TFEB deficiency significantly impaired STB differentiation in human trophoblasts and placenta organoids. Consistently, systemic or trophoblast-specific deletion of Tfeb compromised STB formation and placental vascular construction, leading to severe embryonic lethality. Mechanistically, TFEB conferred direct transcriptional activation of the fusogen ERVFRD-1 in human trophoblasts and thereby promoted STB formation, independent of its canonical function as a master regulator of the autophagy-lysosomal pathway. Moreover, we demonstrated that TFEB directed the trophoblast syncytialization response driven by mTOR complex 1 (mTORC1) signaling. TFEB expression positively correlated with the reinforced trophoblast syncytialization in human fetal growth-restricted placentas exhibiting suppressed mTORC1 activity. Our findings substantiate that the TFEB-fusogen axis ensures proper STB formation during placenta development and under nutrient stress, shedding light on TFEB as a mechanistic link between nutrient-sensing machinery and trophoblast differentiation.

Background The duck (Anas platyrhynchos) is one of the principal natural hosts of influenza A virus (IAV), harbors almost all subtypes of IAVs and resists to many IAVs which cause extreme virulence in chicken and human. However, the response of duck’s adaptive immune system to IAV infection is poorly characterized due to lack of a detailed gene map of the major histocompatibility complex (MHC). Results We herein reported a chromosome-scale Beijing duck assembly by integrating Nanopore, Bionano, and Hi-C data. This new reference genome SKLA1.0 covers 40 chromosomes, improves the contig N50 of the previous duck assembly with highest contiguity (ZJU1.0) of more than a 5.79-fold, surpasses the chicken and zebra finch references in sequence contiguity and contains a complete genomic map of the MHC. Our 3D MHC genomic map demonstrated that gene family arrangement in this region was primordial; however, families such as AnplMHCI, AnplMHCIIβ, AnplDMB, NKRL (NK cell receptor-like genes) and BTN underwent gene expansion events making this area complex. These gene families are distributed in two TADs and genes sharing the same TAD may work in a co-regulated model. Conclusions These observations supported the hypothesis that duck’s adaptive immunity had been optimized with expanded and diversified key immune genes which might help duck to combat influenza virus. This work provided a high-quality Beijing duck genome for biological research and shed light on new strategies for AIV control.

Abstract Backgroud abnormalities or defects in oocyte meiosis can result in decreased oocyte quality, reduced ovarian reserve, and female diseases. However, the mechanisms of oocyte meiosis remain largely unknown, especially epigenetic regulation. Here, we explored the role of EZH1/2 (histone methyltransferase of H3K27) in mouse oocyte meiosis by inhibiting its activity and deleting its gene. Results with embryonic ovary cultured in vitro, EZH1/2 was demonstrated to be essential for oocyte development during meiosis prophase I in mice. Activity inhibition or gene knockout of EZH1/2 resulted in cell apoptosis and a reduction in oocyte numbers within embryonic ovaries. By observing the expression of some meiotic marker protein (γ-H2AX, diplotene stage marker MSY2 and synapsis complex protein SCP1), we found that function deficiency of EZH1/2 resulted in failure of DNA double-strand breaks (DSBs) repair and break of meiotic progression in fetal mouse ovaries. Moreover, Ezh1/2 deficiency led to the suppression of ATM (Ataxia Telangiectasia Mutated kinase) phosphorylation and a decrease in the expression of key DNA repair proteins Hormad1, Mre11, Rad50, and Nbs1 in fetal mouse ovaries, underscoring the enzyme’s pivotal role in initiating DNA repair. RNA-seq analysis revealed that Ezh1/2-deletion induced abnormal expression of multiple genes involved into several function of oocyte development in embryonic ovaries. Knockout of Ezh1/2 in ovaries also affected the levels of H3K9me3 and H4K20me2, as well as the expression of their target genes L3mbtl4 and Fbxo44. Conclusions our study demonstrated that EZH1/2 plays a role in the DSBs repair in oocyte meiosis prophase I via multiple mechanisms and offers new insights into the physiological regulatory role of histone modification in fetal oocyte guardianship and female fertility.

The decidua plays a crucial role in providing structural and trophic support to the developing conceptus before placentation. Following embryo attachment, embryonic components intimately interact with the decidual tissue. While evidence indicates the participation of embryo-derived factors in crosstalk with the uterus, the extent of their impact on post-implantation decidual development requires further investigation. Here, we utilize transgenic mouse models to selectively eliminate primary trophoblast giant cells (pTGCs), the embryonic cells that interface with maternal tissue at the forefront. pTGC ablation impairs decidualization and compromises decidual interferon response and lipid metabolism. Mechanistically, pTGCs release factors such as interferon kappa (IFNK) to strengthen the decidual interferon response and lipoprotein lipase (LPL) to enhance lipid accumulation within the decidua, thereby promoting decidualization. This study presents genetic and metabolomic evidence reinforcing the proactive role of pTGC-derived factors in mobilizing maternal resources to strengthen decidualization, facilitating the normal progression of early pregnancy.

Endogenous retroviruses (ERVs) have been proposed as a driving force for the evolution of the mammalian placenta, however, the contribution of ERVs to placental development and the underlying regulatory mechanism remain largely elusive. A key process of placental development is the formation of multinucleated syncytiotrophoblasts (STBs) in direct contact with maternal blood, through which constitutes the maternal-fetal interface critical for nutrient allocation, hormone production and immunological modulation during pregnancy. We delineate that ERVs profoundly rewire the transcriptional program of trophoblast syncytialization. Here, we first determined the dynamic landscape of bivalent ERV-derived enhancers with dual occupancy of H3K27ac and H3K9me3 in human trophoblast stem cells (hTSCs). We further demonstrated that enhancers overlapping several ERV families tend to exhibit increased H3K27ac and reduced H3K9me3 occupancy in STBs relative to hTSCs. Particularly, bivalent enhancers derived from the Simiiformes-specific MER50 transposons were linked to a cluster of genes important for STB formation. Importantly, deletions of MER50 elements adjacent to several STB genes, including MFSD2A and TNFAIP2, significantly attenuated their expression concomitant to compromised syncytium formation. Together, we propose that ERV-derived enhancers, MER50 specifically, fine-tune the transcriptional networks accounting for human trophoblast syncytialization, which sheds light on a novel ERV-mediated regulatory mechanism underlying placental development.

Traditional Chinese medicine has been used for a long time to treat a variety of gynecological diseases. Among various traditional Chinese medicine, Dingkun Pill (DK) has been used for the treatment of female gynecological diseases. However, DK therapeutic effect on PCOS and the target tissue for its potential effect need to be explored. This study aims to explore the therapeutic effect of DK for PCOS in mice from three aspects: metabolism, endocrine and fertility, and determine whether the brown adipose tissue is the target organ to alleviate the PCOS phenotype. PCOS mouse model was constructed by subcutaneous injection of DHEA. The estrous cycle, ovulation, and pregnancy outcome was examined in mice. The level of hormone including the LH, FSH, estrogen and testosterone in the serum were measured by ELISA. Both the glucose sensitivity and insulin sensitivity were determined in mice with different treatment. The histomorphology and lipid contents in the brown adipose tissue were analyzed. RNA-Seq was conducted for the brown adipose tissue and different expression of critical metabolism marker genes was confirmed by real-time PCR. The data showed that the fertility in PCOS mice with DK treatment was significantly increased, and the metabolic disorder was partially restored. Both the whiten of brown adipose tissue and reduced UCP1 expression induced by DHEA was rescued by the DK. The RNA-Seq data further demonstrated both the DHEA induced downregulation of lipolysis genes and oxidative phosphorylation genes were at least partially rescued by DK in the brown adipose tissue. DK has therapeutic effect on PCOS in DHEA treated mice and the brown adipose tissue is at least one critical target organ to alleviate the PCOS. This is achieved by not only regulating the lipid mobilization of brown adipose, but also restoring its thermogenic function.

BACKGROUND:Non-alcoholic fatty liver disease (NAFLD) is one of most common diseases in the world. Recently, alternative splicing (AS) has been reported to play a key role in NAFLD processes in mammals. Ducks can quickly form fatty liver similar to human NAFLD after overfeeding and restore to normal liver in a short time, suggesting that ducks are an excellent model to unravel molecular mechanisms of lipid metabolism for NAFLD. However, how alternative splicing events (ASEs) affect the fatty liver process in ducks is still unclear. RESULTS:Here we identify 126,277 unique transcripts in liver tissue from an overfed duck (77,237 total transcripts) and its sibling control (69,618 total transcripts). We combined these full-length transcripts with Illumina RNA-seq data from five pairs of overfed ducks and control individuals. Full-length transcript sequencing provided us with structural information of transcripts and Illumina RNA-seq data reveals the expressional profile of each transcript. We found, among these unique transcripts, 30,618 were lncRNAs and 1,744 transcripts including 155 lncRNAs and 1,589 coding transcripts showed significantly differential expression in liver tissues between overfed ducks and control individuals. We also detected 27,317 ASEs and 142 of them showed significant relative abundance changes in ducks under different feeding conditions. Full-length transcript profiles together with Illumina RNA-seq data demonstrated that 10 genes involving in lipid metabolism had ASEs with significantly differential abundance in normally fed (control) and overfed ducks. Among these genes, protein products of five genes (CYP4F22, BTN, GSTA2, ADH5, and DHRS2 genes) were changed by ASEs. CONCLUSIONS:This study presents an example of how to identify ASEs related to important biological processes, such as fatty liver formation, using full-length transcripts alongside Illumina RNA-seq data. Based on these data, we screened out ASEs of lipid-metabolism related genes which might respond to overfeeding. Our future ability to explore the function of genes showing AS differences between overfed ducks and their sibling controls, using genetic manipulations and co-evolutionary studies, will certainly extend our knowledge of genes related to the non-pathogenic fatty liver process.

Recurrent miscarriage (RM) is a distressing pregnancy complication. While the etiology of RM remains unclear, growing evidence has indicated the relevance of trophoblast impairment to the pathogenesis of RM. PR-SET7 is the sole enzyme catalyzing monomethylation of H4K20 (H4K20me1) and has been implicated in many pathophysiological processes. However, how PR-SET7 functions in trophoblasts and its relevance to RM remain unknown. Here, we found that trophoblast-specific loss of Pr-set7 in mice led to defective trophoblasts, resulting in early embryonic loss. Mechanistic analysis revealed that PR-SET7 deficiency in trophoblasts derepressed endogenous retroviruses (ERVs), leading to double-stranded RNA stress and subsequent viral mimicry, which drove overwhelming interferon response and necroptosis. Further examination discovered that H4K20me1 and H4K20me3 mediated the inhibition of cell-intrinsic expression of ERVs. Importantly, dysregulation of PR-SET7 expression and the corresponding aberrant epigenetic modifications were observed in the placentas of RM. Collectively, our results demonstrate that PR-SET7 acts as an epigenetic transcriptional modulator essential for repressing ERVs in trophoblasts, ensuring normal pregnancy and fetal survival, which sheds new light on potential epigenetic causes contributing to RM.

The underlying mechanisms governing parturition remain largely elusive due to limited knowledge of parturition preparation and initiation. Accumulated evidences indicate that maternal decidua plays a critical role in parturition initiation. To comprehensively decrypt the cell heterogeneity in decidua approaching parturition, we investigate the roles of various cell types in mouse decidua process and reveal previously unappreciated insights in parturition initiation utilizing single-cell RNA sequencing (scRNAseq). We enumerate the cell types in decidua and identity five different stromal cells populations and one decidualized stromal cells. Furthermore, our study unravels that stromal cells prepare for parturition by regulating local retinol acid (RA) synthesis. RA supplement decreases expression of extracellular matrix-related genes in vitro and accelerates the timing of parturition in vivo. Collectively, the discovery of contribution of stromal cells in parturition expands current knowledge about parturition and opens up avenues for the intervention of preterm birth (PTB).