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A proteomic atlas of ligand–receptor interactions at the ovine maternal–fetal interface reveals the role of histone lactylation in uterine remodeling

2021; Elsevier BV; Volume: 298; Issue: 1 Linguagem: Inglês

10.1016/j.jbc.2021.101456

1083-351X

Qianying Yang, Juan Liu, Yue Wang, Wei Zhao, Wenjing Wang, Jian Cui, Jiajun Yang, Yuan Yue, Shuai Zhang, Meiqiang Chu, Qingji Lyu, Lizhu Ma, Yawen Tang, Yupei Hu, Kai Miao, Haichao Zhao, Jianhui Tian, Lei An,

Preterm Birth and Chorioamnionitis

Well-orchestrated maternal–fetal cross talk occurs via secreted ligands, interacting receptors, and coupled intracellular pathways between the conceptus and endometrium and is essential for successful embryo implantation. However, previous studies mostly focus on either the conceptus or the endometrium in isolation. The lack of integrated analysis impedes our understanding of early maternal–fetal cross talk. Herein, focusing on ligand–receptor complexes and coupled pathways at the maternal–fetal interface in sheep, we provide the first comprehensive proteomic map of ligand–receptor pathway cascades essential for embryo implantation. We demonstrate that these cascades are associated with cell adhesion and invasion, redox homeostasis, and the immune response. Candidate interactions and their physiological roles were further validated by functional experiments. We reveal the physical interaction of albumin and claudin 4 and their roles in facilitating embryo attachment to endometrium. We also demonstrate a novel function of enhanced conceptus glycolysis in remodeling uterine receptivity by inducing endometrial histone lactylation, a newly identified histone modification. Results from in vitro and in vivo models supported the essential role of lactate in inducing endometrial H3K18 lactylation and in regulating redox homeostasis and apoptotic balance to ensure successful implantation. By reconstructing a map of potential ligand–receptor pathway cascades at the maternal–fetal interface, our study presents new concepts for understanding molecular and cellular mechanisms that fine-tune conceptus–endometrium cross talk during implantation. This provides more direct and accurate insights for developing potential clinical intervention strategies to improve pregnancy outcomes following both natural and assisted conception. Well-orchestrated maternal–fetal cross talk occurs via secreted ligands, interacting receptors, and coupled intracellular pathways between the conceptus and endometrium and is essential for successful embryo implantation. However, previous studies mostly focus on either the conceptus or the endometrium in isolation. The lack of integrated analysis impedes our understanding of early maternal–fetal cross talk. Herein, focusing on ligand–receptor complexes and coupled pathways at the maternal–fetal interface in sheep, we provide the first comprehensive proteomic map of ligand–receptor pathway cascades essential for embryo implantation. We demonstrate that these cascades are associated with cell adhesion and invasion, redox homeostasis, and the immune response. Candidate interactions and their physiological roles were further validated by functional experiments. We reveal the physical interaction of albumin and claudin 4 and their roles in facilitating embryo attachment to endometrium. We also demonstrate a novel function of enhanced conceptus glycolysis in remodeling uterine receptivity by inducing endometrial histone lactylation, a newly identified histone modification. Results from in vitro and in vivo models supported the essential role of lactate in inducing endometrial H3K18 lactylation and in regulating redox homeostasis and apoptotic balance to ensure successful implantation. By reconstructing a map of potential ligand–receptor pathway cascades at the maternal–fetal interface, our study presents new concepts for understanding molecular and cellular mechanisms that fine-tune conceptus–endometrium cross talk during implantation. This provides more direct and accurate insights for developing potential clinical intervention strategies to improve pregnancy outcomes following both natural and assisted conception. In mammals, successful implantation and healthy pregnancy depend on well-orchestrated cross talk between the developmentally competent conceptus and the receptive endometrium (1Wang H. Dey S.K. Roadmap to embryo implantation: Clues from mouse models.Nat. Rev. Genet. 2006; 7: 185-199Crossref PubMed Scopus (897) Google Scholar, 2Bazer F.W. Spencer T.E. Johnson G.A. Burghardt R.C. Uterine receptivity to implantation of blastocysts in mammals.Front. Biosci. (Schol. Ed.). 2011; 3: 745-767Crossref PubMed Google Scholar, 3Cha J. Sun X. Dey S.K. Mechanisms of implantation: Strategies for successful pregnancy.Nat. Med. 2012; 18: 1754-1767Crossref PubMed Scopus (723) Google Scholar). Early pregnancy loss occurring during the peri-implantation period is an important and pervasive problem in both human and agricultural animals, especially low-ovulating species. In natural conception, it has been estimated that approximately 75% of failed pregnancies are caused by implantation failure (1Wang H. Dey S.K. Roadmap to embryo implantation: Clues from mouse models.Nat. Rev. Genet. 2006; 7: 185-199Crossref PubMed Scopus (897) Google Scholar, 4Zinaman M.J. Clegg E.D. Brown C.C. O'Connor J. Selevan S.G. Estimates of human fertility and pregnancy loss.Fertil. Steril. 1996; 65: 503-509Abstract Full Text PDF PubMed Google Scholar, 5Norwitz E.R. Schust D.J. Fisher S.J. Implantation and the survival of early pregnancy.N. Engl. J. Med. 2001; 345: 1400-1408Crossref PubMed Scopus (834) Google Scholar). Failed implantation is also a major limiting factor in assisted reproduction (6Edwards R.G. Human implantation: The last barrier in assisted reproduction technologies?.Reprod. Biomed. Online. 2006; 13: 887-904Abstract Full Text PDF PubMed Google Scholar). 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Reprod. 2020; 102: 571-587Crossref PubMed Scopus (13) Google Scholar) in isolation, the lack of integrated analysis between the paired conceptus and endometrium has made it challenging to advance our understanding of the pathways and functions that govern conceptus–endometrium cross talk during implantation. Thus, despite the importance of ligand–receptor-based maternal–fetal cross talk, there are no reports of systematic studies trying to elucidate the repertoire of signaling routes essential for the cross talk. Recently, an emerging method for evaluation of ligand–receptor interactions based on high-throughput data provides a statistical tool to map the cell–cell communications of diverse physiological and pathological processes (30Ramilowski J.A. Goldberg T. Harshbarger J. Kloppmann E. Lizio M. Satagopam V.P. Itoh M. Kawaji H. Carninci P. Rost B. Forrest A.R.R. A draft network of ligand–receptor-mediated multicellular signalling in human.Nat. 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In the present study, via the screening method to identify potential ligand–receptor interactions, we used sheep as the model and present a comprehensive proteomic atlas of cross talk at maternal–fetal interface. This atlas reconstructed the first direct and accurate map of the ligand–receptor pathway cascades essential for implantation. In addition, by highlighting the enhanced glycolysis in the conceptus, we have firstly reported the novel role of lactate-induced histone lactylation, a newly identified important histone modification, in remodeling endometrial receptivity and achieving implantation. To profile the proteome in the conceptus and endometrium by implantation stage, we collected conceptuses, endometrial caruncular (C), and intercaruncular (IC) areas (Figs. 1A and S1A) from 36 pregnant sheep on day 17 of pregnancy, which is the time of filamentous conceptus attachment in the sheep uterus (33Spencer T. Burghardt R. Johnson G. Bazer F. Conceptus signals for establishment and maintenance of pregnancy.Anim. Reprod. Sci. 2004; 82–83: 537-550Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar, 34Wan P.-C. Bao Z.-J. Wu Y. Yang L. Hao Z.-D. Yang Y.-L. Shi G.-Q. Liu Y. Zeng S.-M. αv β3 integrin may participate in conceptus attachment by regulating morphologic changes in the endometrium during peri-implantation in ovine.Reprod. Domest. Anim. 2011; 46: 840-847Crossref PubMed Scopus (0) Google Scholar) that is frequently selected to explore the mechanisms of conceptus–endometrium cross talk (35Song G. Satterfield M.C. Kim J. Bazer F.W. Spencer T.E. Gastrin-releasing peptide (GRP) in the ovine uterus: Regulation by interferon tau and progesterone.Biol. Reprod. 2008; 79: 376-386Crossref PubMed Scopus (0) Google Scholar, 36Bazer F.W. Spencer T.E. Ott T.L. Interferon tau: A novel pregnancy recognition signal.Am. J. Reprod. Immunol. 1997; 37: 412-420Crossref PubMed Google Scholar, 37Yang Q. Fu W. Wang Y. Miao K. Zhao H. Wang R. Guo M. Wang Z. Tian J. An L. The proteome of IVF-induced aberrant embryo-maternal crosstalk by implantation stage in ewes.J. Anim. Sci. Biotechnol. 2020; 11: 7Crossref PubMed Scopus (0) Google Scholar). In ruminants, uterine secreting glands are specifically localized in large endometrial areas (i.e., intercaruncular zones, IC), thus glandular IC areas are mainly responsible for the synthesis and secretion of histotroph, including cytokines, growth factors, and adhesion molecules, etc. By contrast, small aglandular caruncular areas of stromal origin (i.e., caruncles, C) are scattered over the endometrium surface. Aglandular C areas serve as the sites of superficial attachment and placentation (12Spencer T.E. Johnson G.A. Bazer F.W. Burghardt R.C. Implantation mechanisms: Insights from the sheep.Reproduction. 2004; 128: 657-668Crossref PubMed Scopus (235) Google Scholar, 38Wimsatt W.A. New histological observations on the placenta of the sheep.Am. J. Anat. 1950; 87: 391-457Crossref PubMed Google Scholar, 39Bazer F.W. Uterine protein secretions: Relationship to development of the conceptus.J. Anim. Sci. 1975; 41: 1376-1382Crossref PubMed Google Scholar). Therefore, given the structural and functional differences associated to the C and IC areas, these two distinct endometrial zones have to be analyzed separately (21Mansouri-Attia N. Sandra O. Aubert J. Degrelle S. Everts R.E. Giraud-Delville C. Heyman Y. Galio L. Hue I. Yang X. Tian X.C. Lewin H.A. Renard J.-P. Endometrium as an early sensor of in vitro embryo manipulation technologies.Proc. Natl. Acad. Sci. U. S. A. 2009; 106: 5687-5692Crossref PubMed Scopus (0) Google Scholar, 22Biase F.H. Hue I. Dickinson S.E. Jaffrezic F. Laloe D. Lewin H.A. Sandra O. Fine-tuned adaptation of embryo–endometrium pairs at implantation revealed by transcriptome analyses in Bos taurus.PLoS Biol. 2019; 17: 1-20Crossref Scopus (6) Google Scholar, 37Yang Q. Fu W. Wang Y. Miao K. Zhao H. Wang R. Guo M. Wang Z. Tian J. An L. The proteome of IVF-induced aberrant embryo-maternal crosstalk by implantation stage in ewes.J. Anim. Sci. Biotechnol. 2020; 11: 7Crossref PubMed Scopus (0) Google Scholar, 40Zhao H. Sui L. Miao K. An L. Wang D. Hou Z. Wang R. Guo M. Wang Z. Xu J. Wu Z. Tian J. Comparative analysis between endometrial proteomes of pregnant and non-pregnant ewes during the peri-implantation period.J. Anim. Sci. Biotechnol. 2015; 6: 1-14Crossref PubMed Scopus (9) Google Scholar). We divided the 36 samples into equal three pools (12 samples/pool) as biological replicates and sequenced their proteomes, which showed high reproducibility (Fig. S1B). Finally, we identified 1468, 1494, and 1575 proteins in the conceptus, C area, IC area, respectively (Fig. 1A and Table S1). When hierarchical classification was applied, the endometrial C and IC areas clustered closely together, whereas the conceptus samples were categorized separately (Fig. S1C), which was also recapitulated by the results of principal component analysis (Fig. 1B). Comparative analysis of differentially abundant proteins (false discovery rate (FDR) < 0.05, fold change (FC) > 2) identified 196 and 232 proteins that were significantly more abundant in the conceptus compared with those in the C or IC areas; whereas 224 and 325 proteins were significantly more abundant in the C and IC areas compared with those in the conceptus, and a substantial proportion of differentially abundant proteins were specifically enriched in endometrial or conceptus tissues (Fig. S1, D and E). To explore the potential interactions between the conceptus and endometrium systematically, we screened out differentially abundant membrane and secreted proteins based on subcellular localization annotations from UniProt (Fig. 1C). Secreted proteins appeared to be more enriched in the C or IC areas than in the conceptus, which supported the previous notion that the endometrium is an active site of cytokine production and action (41Tabibzadeh S. Human endometrium: An active site of cytokine production and action.Endocr. Rev. 1991; 12: 272-290Crossref PubMed Google Scholar). Next, we used 304 common differentially abundant proteins between the conceptus and endometrial tissues to construct a protein–protein network and found nine high-scoring proteins (interaction edges > 40) (Fig. S1F), including the secreted protein fibronectin (FN1) and the membrane protein 40S ribosomal protein SA (RPSA), both of which are well-known adhesion molecules that have been reported to mediate intercellular interaction in various cell types (42Li F. Redick S.D. Erickson H.P. Moy V.T. Force measurements of the α5β1 integrin–fibronectin interaction.Biophys. J. 2003; 84: 1252-1262Abstract Full Text Full Text PDF PubMed Google Scholar, 43Groulx J.F. Gagné D. Benoit Y.D. Martel D. Basora N. Beaulieu J.F. Collagen VI is a basement membrane component that regulates epithelial cell-fibronectin interactions.Matrix Biol. 2011; 30: 195-206Crossref PubMed Scopus (75) Google Scholar, 44Lefebvre T. Rybarczyk P. Bretaudeau C. Vanlaeys A. Cousin R. Brassart-Pasco S. Chatelain D. Dhennin-Duthille I. Ouadid-Ahidouch H. Brassart B. Gautier M. TRPM7/RPSA complex regulates pancreatic cancer cell migration.Front. Cell Dev. Biol. 2020; 8: 549Crossref PubMed Scopus (8) Google Scholar). To investigate the conceptus–endometrium cross talk deeply, we next focused on the following three aspects: differentially abundant membrane proteins, differentially abundant secreted proteins, and differentially enriched pathways. Having identified differentially abundant membrane and secreted proteins of the maternal–fetal interface, we next attempted to screen potential interacting partners that might play roles in conceptus–endometrium cross talk during implantation. To this end, we first focused on the membrane proteins that changed commonly between the conceptus and endometrial tissues (FDR < 0.05, FC > 2). We screened out their interacting secreted partners based on the interaction score from Search Tool for Retrieval of Interacting Genes/Proteins (STRING), a well-established database of known and predicted protein–protein interactions (45Szklarczyk D. Gable A.L. Lyon D. Junge A. Wyder S. Huerta-Cepas J. Simonovic M. Doncheva N.T. Morris J.H. Bork P. Jensen L.J. von Mering C. STRING v11: Protein–protein association networks with increased coverage, supporting functional discovery in genome-wide experimental datasets.Nucleic Acids Res. 2019; 47: D607-D613Crossref PubMed Scopus (5658) Google Scholar), as well as the protein subcellular location from UniProt (Table S2). Many of the screened high-scoring interactions involve proteins that have been reported to participate in transmembrane transport (solute carrier family 2 member 1 (SLC2A1), calcineurin-like EF-hand protein 1 (CHP1), glutamic-oxaloacetic transaminase 2 (GOT2)), heat stress response (heat-shock protein 90 alpha family class B member 1 (HSP90AB1), heat shock protein family A member 8 (HSPA8)), and cell adhesion (claudin 4 (CLDN4), basal cell adhesion molecule (BCAM)) (Fig. 1D). Phenotype annotations based on Mouse Genome Informatics (MGI) database suggested that these membrane proteins may be associated with embryonic and fetal development and survival (Fig. 1D). In addition to differentially abundant membrane proteins common to endometrial tissues relative to the conceptus, we also examined differentially abundant membrane proteins (FDR < 0.05, FC > 2) specific to the C or IC areas and their interacting secreted partners (Fig. S2, A and B), which might provide further candidates to investigate the different roles of the C and IC areas in supporting conceptus implantation. To further validate the role of the predicted membrane partners in supporting conceptus–endometrium cross talk, we next characterized the expression patterns of these candidates in the endometria of successful and failed pregnancies using our previously published proteomic data (40Zhao H. Sui L. Miao K. An L. Wang D. Hou Z. Wang R. Guo M. Wang Z. Xu J. Wu Z. Tian J. Comparative analysis between endometrial proteomes of pregnant and non-pregnant ewes during the peri-implantation period.J. Anim. Sci. Biotechnol. 2015; 6: 1-14Crossref PubMed Scopus (9) Google Scholar). We found that the levels of a majority of these candidates changed significantly (p < 0.05) in the endometrium that underwent pregnancy failure, implying the essential functions of these membrane partners in supporting a successful pregnancy (Fig. S2C). Interestingly, we noticed that protein tyrosine kinase 7 (PTK7), an evolutionarily conserved transmembrane receptor, was significantly more abundant in the endometrium that underwent pregnancy failure compared with that in the successful pregnancy (Fig. S2C). In addition, using transcriptomic data of human endometrium from the prereceptive to receptive phase, we found that the mRNA expression levels of PTK7, CLDN4, and STAT3 were also significantly change during establishment of human endometrial receptivity (Fig. S2D), suggesting that those candidates are essential for the normal physiological functions of the endometrium. Meanwhile, we also found that the levels of many of our screened membrane partners changed significantly (p < 0.05) in endometrial tissues of patients with endometriosis compared with those in the healthy endometrium (Fig. S2E), suggesting that these interacting membrane partners might also participate in uterine pathology. Having screened potential interacting partners based on differentially abundant membrane proteins, we next focused on differentially abundant secreted proteins (FDR < 0.05, FC > 2) and their interacting membrane partners (Figs. 1E and S3, A and B). Many of the high-scoring interactions involved cell adhesion molecules (FN1, collagen type VI alpha 1 chain (COL6A1), collagen type VI alpha 2 chain (COL6A2)), glycoprotein family members (alpha 2-HS glycoprotein (AHSG), alpha-1-B glycoprotein (A1BG)), and complement components (complement C5 (C5), complement C9 (C9), plasminogen (PLG)). The MGI phenotype annotations suggested that these membrane partners might be essential for embryonic and fetal development and survival (Fig. 1E). Similarly, reanalysis of secreted partners using previously published proteomic or transcriptomic data also supported the physiological or pathological significance of our screened candidates (Fig. S3, C–E). Having characterized conceptus–endometrium cross talk using interacting secreted and membrane partners, we next attempted to further screen out the associated pathways and biological processes that may couple to ligand–receptor complexes to support the cross talk at the maternal–fetal interface. Therefore, functional profiling was performed using proteins that were more abundant in endometrial or conceptus tissues (Fig. S4, A–D). We found that proteins that were abundant in endometrial tissues were functionally associated with glutathione metabolism, complement and coagulation cascades, and focal adhesion (Fig. S4A), as well as biological processes of cell adhesion, response to virus, and actin cross-link formation (Fig. S4B). By contrast, using proteins that were abundant in the conceptus, we identified that pathways or biological processes of energy metabo