Trophoblastic Signals Facilitate Endometrial Interferon Response and Lipid Metabolism PDF

Article Trophoblastic signals facilitate endometrial interferon response and lipid metabolism, ensuring normal decidualization Graphical abstract...

Article Trophoblastic signals facilitate endometrial interferon response and lipid metabolism, ensuring normal decidualization Graphical abstract Authors Ningjie Yang, Yang Sun, Bing Han,..., Decidua Shuangbo Kong, Jinhua Lu, Haibin Wang Endothelium Correspondence Vessels [email protected] (H.B.), Lipid [email protected] (S.K.), LPL metabolism [email protected] (J.L.), [email protected] (H.W.) In brief Trophoblast Embryonic factors are essential for IFNAR endometrial decidualization during early IFNK pregnancy. Yang et al. report that primary trophoblast giant cells, the embryonic cells directly contacting the endometrium, can secrete type I interferon IFNK and lipoprotein lipase to strengthen the decidual interferon response and lipid accumulation, Decidual Conceptus stromal cell respectively, thus ensuring early pregnancy. Highlights d Embryonic pTGC deficiency impairs maternal decidualization in early pregnancy d Type I interferon IFNK from pTGCs elevates decidual interferon responses d pTGCs affect decidual lipid metabolism and distribution in the decidua d pTGCs secrete LPL to facilitate decidual lipid accumulation and decidualization Yang et al., 2024, Cell Reports 43, 114246 June 25, 2024 ª 2024 The Author(s). Published by Elsevier Inc. https://doi.org/10.1016/j.celrep.2024.114246 ll  ll OPEN ACCESS Article Trophoblastic signals facilitate endometrial interferon response and lipid metabolism, ensuring normal decidualization Ningjie Yang,1,3 Yang Sun,1,3 Bing Han,1 Na Deng,1 Gaizhen Li,1 Qian Han,1 Yinan Wang,1 Han Cai,1 Fan Liu,1,2 Bin Cao,1 Wenbo Deng,1 Haili Bao,1,* Shuangbo Kong,1,* Jinhua Lu,1,* and Haibin Wang1,2,4,* 1Fujian Provincial Key Laboratory of Reproductive Health Research, Department of Obstetrics and Gynecology, The First Affiliated Hospital of Xiamen University, School of Medicine, Xiamen University, Xiamen, Fujian 361102, China 2State Key Laboratory of Vaccines for Infectious Diseases, Xiang An Biomedicine Laboratory, School of Medicine, Xiamen University, Xiamen, Fujian 361102, China 3These authors contributed equally 4Lead contact *Correspondence: [email protected] (H.B.), [email protected] (S.K.), [email protected] (J.L.), [email protected]. com (H.W.) https://doi.org/10.1016/j.celrep.2024.114246 SUMMARY 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 pre- sents 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. INTRODUCTION lipid metabolism, and energy supply capacity. These changes are consistent with the histotrophic role of the decidua in sup- The process of decidua development, known as decidualization, porting embryo development before placentation. The decidua entails the proliferation and differentiation of endometrial stromal provides lipids as vital components for constructing plasma cells, accompanied by structural and functional changes membranes, facilitating steroid synthesis, and participating in throughout the entire uterus. The in vivo decidualization process the regulation of signal transduction processes.5 Lipid meta- exhibits features of spatiotemporal and regional progression. In bolism assumes a pivotal role in both embryonic development mice, embryo attachment to the hormone-primed uterus initiates and decidualization.6,7 decidualization on the uterine anti-mesometrial (AM) side on day Following embryo attachment, primary trophoblast giant cells 5 of pregnancy (day 5).1,2 Initially, stromal cells enclosing the em- (pTGCs), derived from the trophectoderm (TE), invade the decidua, bryo decidualize into an avascular, impermeable, tight structure and participate in maternal tissue remodeling, forming the initial known as the primary decidual zone (PDZ), forming a protective maternal-embryonic interface.8 Trophoblast invasion determines barrier surrounding the conceptus.3 Subsequently, peripheral the depth of decidualization in a given species,9 and certain stromal cells undergo decidualization, forming the secondary trophoblast-derived molecules mediate the maternal-embryonic decidual zone (SDZ), which gradually expands toward the myo- crosstalk during early pregnancy. For instance, interferon-tau metrium until day 8 of pregnancy (day 8), leaving a layer of undif- from ruminant trophoblasts prevents luteolysis and reduced uter- ferentiated stromal cells adjacent to the myometrium. Stromal- ine prostaglandin production, ensuring normal progression of early decidual transformation is marked by notable changes in cellular pregnancy.10,11 Additionally, the induction of uterine fibroblast ultrastructure, including the dilation of the endoplasmic reticu- growth factor occurred exclusively in the presence of embryos in lum (ER) and Golgi complexes, an increase in mitochondrial den- rats.12 It has been proposed that trophoblast signaling influences sity, and the accumulation of glycogen and lipid droplets.4 These decidualization; however, early experimental data from various ar- alterations indicate that the decidual transformation is concur- tificially induced decidualization models have yielded conflicting rent with a substantial increase in macromolecular synthesis, perspectives. In mice, decidualization can be effectively induced Cell Reports 43, 114246, June 25, 2024 ª 2024 The Author(s). Published by Elsevier Inc. 1 This is an open access article under the CC BY-NC license (http://creativecommons.org/licenses/by-nc/4.0/).  ll OPEN ACCESS Article Figure 1. Ablation of Prl3d1-positive TGCs impairs decidualization in early pregnancy (A) In situ hybridization of pTGC marker Prl3d1 mRNA in uteri of WT mice. (B) Schematic of the pTGC ablation strategy. A Prl3d1Cre/Cre male was mated with an R26DTA/DTA female. The endogenic promoter of Prl3d1 drives the expression of Cre to remove the stop cassette before the DTA transgene, inducing the expression of DTA, which ablated the pTGCs in Prl3d1Cre/+, R26DTA/+ concepti. (C) The efficiency of Prl3d1-Cre was indicated by Prl3d1Cre/+, R26mTmG/+ concepti. (D) Co-immunofluorescence staining of PL-I (encoded by Prl3d1) and CK8 revealed that pTGCs were efficiently ablated in the uterus. (E) Representative images of day 6.5 and day 8 uteri in mice of the WT and DTA groups. Arrowheads indicate the ISs in each group. (F) The weight of ISs in WT and DTA mice on day 6.5 and day 8. Data represent the mean ± SEM. Two-tailed unpaired Student’s t test. (legend continued on next page) 2 Cell Reports 43, 114246, June 25, 2024  ll Article OPEN ACCESS in a receptive uterus by artificial stimuli such as endometrial decidua with DTA conceptus is referred to as DTA decidua) (Fig- scratching, intrauterine injection of sesame oil, or intrauterine ure 1B). The efficiency of Prl3d1 driving Cre recombinase was transfer of lectin concanavalin a (ConA)-coated beads. The ses- verified in advance by crossing Prl3d1Cre/Cre mice with ame oil-induced deciduoma exhibits significant differential gene R26mTmG/mTmG reporter mice, which expressed Td-tomato ubiq- expression compared to the normal pregnant decidua.13,14 More- uitously and EGFP exclusively in cells expressing the Cre recom- over, sesame oil or isolated inner cell mass of the embryo fails to binase (Figure 1C). Then the pTGCs were efficiently ablated in induce a functional PDZ in the deciduoma, while trophoblast ves- the Prl3d1Cre/+; R26DTA/+ conceptus at the maternal-embryonic icles can do so.15 On the contrary, the artificial deciduoma ignited interface, indicated by the absence of CK8 and PL-I (encoded by ConA-coated beads can induce a functional PDZ and exhibits by Prl3d1) double-positive TGCs on day 6.5 and day 8 (Fig- a gene expression profile similar to that of the pregnant ure 1D). The absent expression of the Prl3d1-positive TGC decidua.16–18 Nevertheless, several recent studies have supported markers Pfpl, Pla2g5, and Prl4a1 (based on recently published the unique trophoblast contribution to decidualization. Tropho- placental single-cell transcriptome data26) further confirmed blast-derived tumor necrosis factor alpha is involved in implanta- the ablation in both day 6.5 and day8 DTA deciduae (Figure S1). tion and decidualization in mice.19,20 Furthermore, blastoids Both wild type (WT) and DTA embryos implanted normally into formed with trophoblast stem cells (TSCs) exhibiting elevated the uteri (Figure 1E), while the average weight of DTA implanta- Cdx2 expression have been shown to regulate the secretion of tion sites (ISs) was significantly lower than that of WT ISs on Wnt6/7b, enhancing decidualization capacity compared to TSC day 6.5 and day 8 (Figure 1F). Accordingly, embryonic growth blastoids.21,22 Single-cell multiomics analyses provide insights was significantly retarded in the DTA group on day 8 (Figure 1G). into potential trophoblast-decidua interactions at the early The observed abnormalities in DTA conceptus and decidua on maternal-embryonic interface.23,24 While these findings under- day 6.5 were not caused by apoptosis, as evidenced by the score the significance of trophoblast signaling in decidualization absence of cleaved caspase-3 immunostaining signals (Fig- and early pregnancy, the extent and mechanisms of trophoblast ure 1H). Furthermore, both mRNA expression and protein abun- influence on post-implantation decidualization remain insuffi- dance of the decidua marker Prl8a2 were reduced in DTA ciently understood. decidua compared to WT decidua (Figures 1I–1K). These results To further explore this issue, we employed a mouse model demonstrate that the specific ablation of pTGCs impaired de- with selective ablation of pTGCs, using the Rosa26-DTA system cidualization during early pregnancy. driven by pTGC-specific Prl3d1-Cre. In this study, we observed impaired decidualization as well as a reduction in interferon Ablation of pTGCs derails the patterns of gene response and downregulation of numerous lipid-related path- expression in the decidua ways in the decidua with pTGC ablation during early pregnancy. To further investigate the molecular mechanisms underlying the Spatial metabolomics data depicted changes in lipid distribution impaired decidualization upon the ablation of pTGCs, we per- and a decrease in triglyceride accumulation within the decidua formed bulk RNA sequencing (RNA-seq) on uterine tissues upon pTGC ablation. Furthermore, we demonstrated the from WT and DTA groups after removal of the conceptus and involvement of pTGC-derived interferon kappa ( IFNK) and lipo- myometrium on day 6.5 and day 8. According to the prin- protein lipase (LPL) in these processes. These findings substan- cipal-component analysis (PCA) of transcriptome data, the tiate the contribution of trophoblast signaling to post-implanta- variance between the WT and DTA groups was marked on tion decidual development, providing valuable insights into the day 6.5 and reduced on day 8 (Figure 2A). The day 6.5 DTA maternal-embryonic interactions during early pregnancy. group exhibited the most significant trend of difference among all groups when combining all differentially expressed genes RESULTS (false discovery rate [FDR] < 0.05 and fold change > 1.5) iden- tified in the comparisons of day 6.5 DTA vs. WT and day 8 DTA Ablation of Prl3d1-positive TGCs impairs vs. WT (Figure 2B). Critical genes for decidualization, such as decidualization in early pregnancy Wnt4, Bmp2, and Alpl,27,28 or genes related to decidual func- pTGCs specifically express Prl3d1 from day 5.5 of pregnancy tion, such as Hsd11b2,29 were expressed abnormally in day (day 5.5) and form the outermost layer of the conceptus (Fig- 6.5 DTA decidua (Figure 2C). The changes of these genes ure 1A). To explore the physiological importance of pTGCs dur- were validated by quantitative real-time PCR (real-time qPCR) ing pregnancy, pTGCs were eliminated by crossing Prl3d1Cre/Cre analysis (Figure 2D). mice25 with diphtheria toxin A (R26DTA/DTA) mice to obtain In addition to the expression levels, the spatiotemporal Prl3d1Cre/+; R26DTA/+ concepti (hereafter referred to as DTA; expression patterns of these genes differed between the WT (G) Hematoxylin and eosin (H&E) staining of WT and DTA embryos in early pregnancy, indicating retarded embryonic development in DTA uteri on day 8. (H) Immunohistochemical staining of cleaved caspase-3, indicating comparable outcomes between day 6.5 WT and DTA uteri, with a day 2 WT uterus serving as a positive control (luminal epithelium). (I) In situ hybridization of decidual marker Prl8a2 mRNA (top) and immunofluorescence staining of PRL8A2 protein (bottom) in day 6.5 WT and DTA uteri. (J) Real-time qPCR analysis of Prl8a2 in WT and DTA ISs on day 6.5. Values were normalized to Gapdh and represent the mean ± SEM. Two-tailed unpaired Student’s t test. (K) Immunoblot analysis of PRL8A2 in day 6.5 WT and DTA uteri. E, embryo; M, mesometrial; AM, anti-mesometrial. Scale bars: 100 mm. Cell Reports 43, 114246, June 25, 2024 3  ll OPEN ACCESS Article Figure 2. Ablation of pTGCs derails the patterns of gene expression in the decidua (A) PCA plot of RNA-seq profiles characterizing the differences between WT and DTA decidual tissues on day 6.5 and day 8. PCA was unsupervised and based on normalized RNA expression values by variance stabilizing transformation in the DESeq2 package of R. (B) Heatmap of DEGs (FDR < 0.05 and fold change > 1.5) in day 6.5 DTA vs. WT and day 8 DTA vs. WT. (C) Volcano plot of DEGs (FDR < 0.05 and fold change > 1.5) in day 6.5 DTA vs. WT decidual tissues as determined by negative binomial distribution in the DESeq2 package of R. Genes that were not significantly changed are shown in gray. (D) Real-time qPCR analysis of Wnt4, Bmp2, Alpl, and Hsd11b2 relative expression levels in WT and DTA uteri on day 6.5. Values were normalized to Gapdh and represent the mean ± SEM. Two-tailed unpaired Student’s t test. (E) In situ hybridization of Wnt4, Bmp2, Alpl, and Hsd11b2 in WT and DTA uteri on day 6.5 and day 8. Dotted rings sketch out the embryo locations. Arrowheads highlight the abnormal expression regions in DTA uteri. Scale bars: 100 mm. and DTA groups. Consistent with a previous study,30 the regionalized expression in the DTA decidua (Figure S2). These expression of Wnt4 and Bmp2 was localized in the SDZ of data indicate that the ablation of pTGCs lead to maldevelop- WT decidua on day 6.5 and day 8. However, in the DTA group, ment of presumptive PDZ and disturbs spatiotemporal Wnt4 and Bmp2 were expressed adjacent to the conceptus in decidualization. the presumptive PDZ (Figure 2E). Similarly, the expression of Alpl showed impaired localization in the presumptive PDZ adja- pTGC-derived IFNK reinforces decidual interferon cent to the conceptus in day 6.5 DTA decidua (Figure 2E). response during early pregnancy Furthermore, the expression of Hsd11b2 appeared abnormally Through an unbiased ranking of EnrichR analysis, it was in the presumptive PDZ of DTA decidua on D6.5 (Figure 2E). In observed that one of the most significantly altered biological pro- addition, other genes, such as Bmp7, also exhibited impaired cesses with downregulated genes in D6.5 DTA decidua was 4 Cell Reports 43, 114246, June 25, 2024  ll Article OPEN ACCESS (legend on next page) Cell Reports 43, 114246, June 25, 2024 5  ll OPEN ACCESS Article interferon (IFN) signaling pathway (Figure 3A), which was also (Figure 3J). These data suggest that pTGC-derived IFNK may be evidenced by gene set enrichment analysis (GSEA) (Figure 3B). involved in triggering uterine IFN response at peri-implantation. Furthermore, concomitant with the downregulation of the Subsequently, we sought to validate whether IFNK could janus kinase signal transducer and activator of transcription induce an IFN response in the decidua. The attachment reaction (JAK-STAT) signaling pathway (Figure 3B), which plays a during implantation is indispensable for the following deciduali- pivotal role in both IFN signaling and inflammation, there was zation and is essential for investigating the decidual IFN a notable reduction in the inflammatory response and response post implantation. Our previous findings have demon- the expression of Ptges (Figures S3A–S3C). Ptges is a key strated that the TE-derived proinflammatory signal S100A9 trig- enzyme in the cyclooxygenase (Cox)-2-mediated pros- gers an attachment-like reaction in vivo,19 indicating potential taglandin E2 biosynthetic pathway crucially involved in the availability of other early trophoblast-derived proinflammatory inflammatory response.31 Accordingly, the prostaglandin E2 factors. In line with this, upon reanalyzing the published RNA- (PGE2) level was also decreased in day 6.5 DTA decidua seq data mentioned above,32 we observed high expression of (Figure S3D). proinflammatory factors, including S100a8, S100a9, and Il6, in Day 6.5 DTA decidua exhibited significant downregulation the day 5 TE (Figure S4A). Furthermore, Il6 was expressed in of IFN-stimulated genes, including Stat1, Stat2, Irf7, Irf9, and TE and TGCs, while its receptor Il6ra was detected in the decidua Isg15 (Figure 3C). The expression changes of Stat1, Stat2, as well as TE and TGCs (Figure S4B). Single-cell-resolution Isg15, and Irf7 were confirmed by real-time qPCR analysis (Fig- in situ hybridization on tissues further confirmed the expression ure 3D). Additionally, Stat1 mRNA exhibited a significant reduc- of Il6 in the PL-I-positive pTGCs in vivo (Figure S4C). Accord- tion in the upper region of the day 6.5 DTA SDZ flanking the ingly, we tested the possibility of TE- and pTGC-derived inflam- conceptus (Figure 3E). Furthermore, both total STAT1 and phos- matory factor interleukin-6 (IL-6) to induce the attachment-like phorylated STAT1 (p-STAT1) proteins were markedly decreased reaction. As expected, the transfer of Affi-Gel beads coated in day 6.5 DTA decidua (Figure 3F). These data indicate a signif- with eukaryotic recombinant IL-6 protein into the uteri of icant impairment of decidual IFN response following pTGC abla- pseudo-pregnant mice on day 4 (Figure 3K) effectively initiated tion. Since our RNA-seq data showed no significant expression bead attachment to the receptive uterus (Figure S4D). Subse- of any IFN ligand in day 6.5 decidual tissue, our focus shifted to quently, we coated Affi-Gel beads with IL-6 along with IFNK re- investigating the embryonic factors contributing to the decidual combinant protein or an equal amount of bovine serum albumin IFN response. By reanalyzing the published data from RNA- (BSA), which were used to determine the IFNK-induced seq performed on day 5 and day 5.25 mural TE, as well as response in the decidua. We observed that the expression of TGCs differentiated from day 5 mural TE for 3 days (equivalent Stat1 was higher in the decidua surrounding the IFNK-coated to the pTGCs),32 we found that the type I IFN Ifnk was expressed beads (IFNK+) compared to that around the BSA-coated beads in both the TE and pTGCs, while its receptors, Ifnar1 and Ifnar2, (IFNK
) (Figure 3L). Furthermore, the expression levels of Isg15 existed in both trophoblastic and decidual cells (Figures 3G and and Irf7 were upregulated in the decidua surrounding the IFNK- 3H). Double in situ hybridization revealed that Ifnk was mainly ex- coated beads (Figure 3M). This evidence suggests that pTGC- pressed in the pTGCs on day 6.5 and day 8 (Figure 3I), while If- derived IFNK strengthens IFN response in the early pregnant nar1 and Ifnar2 were expressed in the decidua as well as pTGCs uterus. Figure 3. pTGC-derived IFNK reinforces the decidual IFN response during early pregnancy (A) Bar plot of the top downregulated terms from EnrichR analysis of day 6.5 DTA vs. WT using Reactome 2022 database, ranked by significance. (B) GSEA plots of genes related to the receptor signaling pathway via JAK-STAT (Gene Ontology:0007259, Biological Process) and the type I IFN -mediated signaling pathway (Gene Ontology:0060337, Biological Process) in DTA vs. WT uteri on day 6.5. Permutation was based on p-value calculation. NES, normalized enrichment score. (C) Heatmap of downregulated IFN-responsive genes (FDR < 0.05 and fold change > 1.5) in day 6.5 DTA vs. WT uteri. (D) Validation of Stat1, Stat2, Isg15, and Irf7 relative expression levels in WT and DTA uteri on day 6.5 by real-time qPCR analysis. Values were normalized to Gapdh and represent the mean ± SEM. Two-tailed unpaired Student’s t test. (E) In situ hybridization of Stat1 mRNA in day 6.5 WT and DTA deciduae. Dotted rings sketch out the conceptus locations. Arrowheads indicate the regions with higher gene expression levels. (F) Immunoblotting analysis of p-STAT1 and STAT1 in WT and DTA uteri on day 6.5. (G) Fragments per kilo base of transcript per million mapped fragments (FPKM) values of Ifnk in day 5 mural TE, day 5.25 mural TE, and TGCs differentiated from day 5 mural TE for 3 days (TGC). (H) Comparison of average FPKM values of Ifnk, Ifnar1, and Ifnar2 in TGC and day 6.5 decidual tissues (day 6.5 decidua). Data represent the mean ± SEM. Two- tailed unpaired Student’s t test. (I) Double in situ hybridization of Ifnk and Prl3d1 mRNAs in day 6.5 and day 8 WT pregnant uteri. Arrowheads indicated the typical dual-colored TGCs. (J) Double in situ hybridization images of Ifnar1 and Prl3d1 mRNAs (with magnified image) and Ifnar2 and Prl3d1 mRNAs (with magnified image) in day 6.5 WT pregnant uteri. Arrowheads indicate the typical dual-colored TGCs. (K) Schematic of the protein-carrying bead transfer test. Affi-Gel beads were soaked with diluted recombinant proteins and transferred into the uterine horn of pseudo-pregnant mice on the morning of day 4. Implantation sites (ISs) with blue band reactions were checked on day 6.5. (L) Stat1 mRNA expression was examined via in situ hybridization in day 6.5 deciduomas from mice that received beads soaked with 5 ng/mL IL-6 and 50 ng/mL BSA (IFNK
) or 5 ng/mL IL-6 and 50 ng/mL IFNK (IFNK+). (M) Real-time qPCR analysis of Isg15 and Irf7 relative expression levels in IFNK
and IFNK+ deciduae and WT decidua on day 6.5. Values were normalized to Gapdh and represent the mean ± SEM. Two-tailed unpaired Student’s t test. Scale bars: 100 mm. 6 Cell Reports 43, 114246, June 25, 2024  ll Article OPEN ACCESS Figure 4. Ablation of pTGCs impairs lipid metabolism pathways in the decidua (A) Bar plot of the top downregulated terms from EnrichR analysis of day 6.5 DTA vs. WT using the GO Cellular Component 2023 and Metabolomics Workbench Metabolites 2022 databases, ranked by significance. (B) Bubble chart of GSEA showing the enrichment of genes related to biological effects of lipid metabolism in day 6.5 DTA vs. WT. Permutation was based on p value calculation. (C) Heatmap of representative downregulated genes related to sphingolipid metabolism, the PPAR signaling pathway, GP metabolism and GL metabolism in day 6.5 DTA vs. WT. Genes in black font indicate FDR < 0.05 and fold change > 1.5. (D) In situ hybridization of Dgat2, Fads3, and Ppard mRNA in day 6.5 WT and DTA deciduae, indicating the regionalized expression of these genes. Dotted rings sketch out the conceptus locations. Arrowheads indicate the high expression regions. Scale bars: 100 mm. Ablation of pTGCs impairs lipid metabolism pathways in pathways in day 6.5 GSEA, encompassing sphingolipid meta- the decidua bolism, glycerophospholipid metabolism, glycerolipid meta- Previous studies have demonstrated the importance of lipid- bolism, lipid storage, and FAO (Figure 4B). In addition, processes related pathways in decidualization. For instance, disruptions in related to lipid metabolism pathways were also significantly sphingolipid metabolism compromise decidualization and result downregulated in D8 DTA group (Figure S5). In D6.5 DTA decidua, in early pregnancy loss.33 Additionally, the inhibition of fatty acid the expression of several genes encoding enzymes related to b-oxidation (FAO) hampered early decidualization in both humans biosynthesis and metabolism of ceramide (Cer), a membrane and mice.34 Following the pTGC ablation on day 6.5, notable signaling metabolite produced by the sphingolipid metabolism downregulation observed through unbiased ranking of EnrichR pathway, were decreased, including Kdsr, Acer2, Fads3, and analysis included metabolism of lipids (Figure 3A), lipid droplets, Enpp7 (Figure 4C). Furthermore, Dgat2, which controls the rate- and triacylglycerol (Figure 4A). It was further indicated by the limiting step to synthesize the bulk of triglycerides (TGs) and incor- consistent downregulation of several lipid metabolism-related porate them into lipid droplets,35 markedly decreased in day 6.5 Cell Reports 43, 114246, June 25, 2024 7  ll OPEN ACCESS Article (legend on next page) 8 Cell Reports 43, 114246, June 25, 2024  ll Article OPEN ACCESS DTA decidua (Figures 4C and 4D). The peroxisome-proliferator- energy supply essential for energy homeostasis.37 Lipid droplets associated receptor (PPAR) signaling pathway, which is activated were extensively accumulated in the day 6.5 decidua, primarily by unsaturated fatty acids and derivatives from TG lipolysis, was flanking the conceptus (Figure 5G), and the lipid droplets were also downregulated in DTA decidua (Figure 4B). Consistently, significantly reduced upon pTGC deficiency (Figure 5G). Addi- the expression of Ppard was significantly decreased (Figures 4C tionally, phosphatidylcholines (PCs) are constituents of plasma and 4D). Moreover, the biological process related to ER function membranes, including the lipid droplet membrane. Two PCs and calcium homeostasis, which is crucial for regulating the activ- with the highest variable importance in projection (VIP) scores, ities of numerous enzymes in lipid metabolism, was also downre- PC (m/z 922.7353) and PC (m/z 898.7343), were reduced simul- gulated in DTA decidua (Figures 4A and 4B). These data indicate taneously with TGs in the DTA decidua (Figure S7A). that pTGC ablation impairs lipid metabolism in the decidua. The ablation of pTGCs also caused alterations in other decidual Notably, the expression of Dgat2 and Ppard was significantly lipid distributions. The enrichment of PC (m/z 746.6079) shifted enriched in the mesometrial (M) side of the uteri, whereas from the M to the AM side of the uterus following pTGC ablation Fads3 showed predominant expression in the AM side (Fig- (Figure S7A). The only cholesteryl ester detected in the day 6.5 ure 4D). Moreover, Fads3 exhibited localization in the presump- uterus, cholesteryl docosahexaenoic acid (m/z 719.5728), was tive PDZ, whereas Dgat2 was located in the SDZ (Figure 4D). decreased in DTA decidua and myometrium and increased in re- These findings forebode the spatiotemporal features of lipid dis- gions covering the conceptus (Figure S7B). C24:1-OH sphingo- tribution in the decidua. myelin (m/z 867.6297) showed alterations similar to cholesteryl docosahexaenoic acid (Figure S7B). Additionally, consistent pTGC ablation disturbs decidual accumulation of TGs with the upregulated oxidative phosphorylation pathway genes and lipid droplets in DTA groups, coenzyme Q10 was increased at the AM side of To confirm that pTGC ablation affected lipid distribution in the the DTA uterus, suggesting a possible compensatory energy sup- uteri, MALDI-mass spectrometry (MS) imaging was performed ply in DTA decidua (Figures S8A and S8B). These findings demon- on day 6.5 WT and DTA deciduae (three biological replicates strate that pTGC ablation reduces TGs and lipid droplets in the each group). Orthogonal projections to latent structures discrim- decidua and disturbs the proper distribution of decidual lipids. inant analysis was performed to identify differential metabolites following unsupervised PCA and permutation tests. The analysis pTGC-derived lipoprotein lipase facilitates lipid droplet revealed that 68% of the differential glycerolipids (GLs) and glyc- accumulation to ensure normal decidualization erophospholipids (GPs) exhibited a decrease in the DTA groups, To investigate the pTGC-derived factors in regulating accumula- with TGs contributing significantly to the decrease (Figures 5A tion of lipid droplets, we reanalyzed published RNA-seq data32 and 5B). The decrease in TGs exhibited a higher number and and identified trophoblast-specific molecules that participate in greater amplitude of changes compared to the increase in TGs lipid metabolism (Figure 6A). Pla2g10 and Pla2g5, which encode (Figure 5C), indicating that the metabolic variation is dominated enzymes of the secretory phospholipase A2 family, known to by the decreased TGs in the decidua with pTGC deficiency. generate inflammatory mediators like lysophospholipids and The ion images demonstrated that all seven decreased TGs arachidonic acid,38 exhibited high expression in day 5.25 TE predominantly accumulated in the decidualizing regions, espe- and TGCs, respectively (Figure 6A). LPL serves as the rate-limiting cially flanking the conceptus, and significantly decreased enzyme that regulates the tissue import of fatty acids derived from following pTGC ablation (Figure 5D). In contrast, the two circulating lipoproteins by endothelial cells, providing energy sup- increased TGs showed less pronounced increases in the DTA ply and facilitating TG storage.39 Apoc1, which encodes an inhib- decidua (Figure 5E). The remaining unchanged TGs were local- itor of LPL known to impede TG metabolism,40 was expressed ized either universally in the uterus or intensively in non-decidu- before TGC formation in day 5 TE, while TGCs expressed alizing regions of the uterus (Figure S6). The relative quantifica- Apoc2, an activator of LPL41 (Figure 6A). These findings suggest tion of total TG levels further confirmed a significant decrease a stage-specific regulation of lipolysis at the maternal-embryonic in TGs in the DTA decidua (Figure 5F). Excessive TGs are stored interface. Notably, the expression of Lpl was significantly higher as lipid droplets,36 which act as hubs for lipid metabolism and at the TGC stage compared to the TE stage (Figure 6A). Figure 5. pTGC ablation disturbs decidual accumulation of TGs and lipid droplets (A) Bubble chart indicating changes in the relative content of differential metabolites in day 6.5 DTA vs. WT decidua, categorized by the classification of metabolites represented in different colors. (B) Bubble chart displaying the top 20 metabolites with the highest variable importance in projection (VIP) scores in day6.5 DTA vs. WT decidua. Lipid metabolites are marked in black font and shown by values of m/z. (C) Volcano plot of differential metabolites in day 6.5 DTA vs. WT decidua. Metabolites without significant changes are shown in gray. The plots of TGs are circled with black rings and labeled with values of m/z. (D) Ion images of significantly decreased TGs in a day 6.5 DTA uterus. Scale bar: 300 mm (n = 3 biologically independent samples). (E) Ion images of increased TGs in a day 6.5 DTA uterus. Scale bars: 300 mm (n = 3 biologically independent samples). (F) Relative quantification of total TG levels (using MS) in both day 6.5 WT and DTA deciduae. Values represent the mean ± SEM. Two-tailed unpaired Student’s t test. (G) Bodipy fluorescent staining of lipid droplets and immunofluorescence of PL-I in day 6.5 WT and DTA uteri (left). Scale bars: 100 mm. The values of integrated density/area of Bodipy staining were measured by ImageJ software to quantify fluorescence intensity between day 6.5 WT and DTA uteri (right). Values represent the mean ± SEM. Two-tailed unpaired Student’s t test. Cell Reports 43, 114246, June 25, 2024 9  ll OPEN ACCESS Article (legend on next page) 10 Cell Reports 43, 114246, June 25, 2024  ll Article OPEN ACCESS Furthermore, we observed the localization of Lpl expression in the pregnancy outcomes through early pregnancy interventions, a pTGCs among day 6.5 uteri (Figure 6B and S9). Through co-im- more profound understanding of the maternal-embryonic inter- munostaining of LPL and Claudin 5, which marks the tight junc- actions during post-implantation decidual development is tions of endothelial cells,42 we demonstrated that the LPL protein, imperative.44 This study provides evidence regarding the influ- upon being secreted from pTGCs, was recruited to the peri-em- ence of pTGC-derived signals on decidualization. It also enumer- bryonic decidual zone and localized adjacent to the tight junctions ates two pTGC-derived factors involved in this process: IFNK, of decidual endothelial cells (Figure 6C). Moreover, the ablation of which reinforces the decidual IFN response, and LPL, which pro- pTGCs eliminated LPL signals in the day 6.5 DTA decidua (Fig- motes lipid accumulation and, thus, facilitates decidualization. ure 6D). We speculated that LPL is involved in the accumulation The induction of the IFN response and the classic IFN-stimu- of lipid droplets in the decidua, as overexpression of Lpl has lated gene Isg15 in the endometrium is a conserved phenome- been shown to increase the number of lipid droplets in muscle non in mammalian pregnancy, allowing for a rapid immune and liver.43 To test this hypothesis, we administered either the response to protect the developing embryo.45 Nevertheless, in LPL inhibitor orlistat or DMSO to WT mice and found that LPL ac- both human and mouse endometria, the specific type of embry- tivity and TG content were significantly reduced in the day 6.5 onic IFN responsible for inducing Isg15 has not been clearly deciduae of pregnant mice with orlistat injection (Figures 6E and identified.8,46 Previous studies have suggested that the IFN de- 6F). Consistently, orlistat injection resulted in a reduction of lipid tected on day 7 of pregnancy in mice is mainly produced by droplets in day 6.5 deciduae (Figure 6G). Importantly, the expres- the conceptus.47 Furthermore, Isg15 is induced to a significantly sion of Prl8a2 was downregulated in the orlistat-treated deciduae, lower extent in sesame oil-induced artificial decidua compared indicating that the LPL deficiency could contribute to the impair- to the pregnant uterus, pointing to an embryonic contribution ment of decidualization (Figures 6H and 6I). to IFN response induction.8,13 However, subsequent research Subsequently, we employed the Affi-Gel bead assay (Fig- employing lectin (ConA)-coated beads induced a similar level ure 6J) to further confirm the functions of pTGC-derived LPL in of decidual Isg15 expression compared to the pregnant uterus.17 lipid accumulation and decidualization. The Affi-Gel beads These findings indicate that the process of decidualization oc- were coated with recombinant LPL (LPL+) or an equal amount curs along with the induction of Isg15 and IFN response, but of BSA (LPL
), along with the priming level of IL-6 (to induce the initiating trigger for this response may not be limited to an attachment-like reaction), and then transferred into the uteri IFNs. Moreover, the existence and contribution of embryonic of pseudo-pregnant mice on day 4. Although both LPL+ and IFNs to the decidual IFN response cannot be clearly excluded LPL
beads showed similar efficiencies in triggering an attach- because the artificial decidua models may not be the ideal ment-like reaction (Figure 6K), LPL+ beads induced superior de- means to evaluate the contribution of embryonic signals in the cidualization, indicated by larger and heavier ISs (Figures 6J and decidual IFN response, as any factor that enhances the decidu- 6L). Importantly, the LPL-treated group displayed significantly alization response also induces Isg15 expression. For instance, greater accumulation of lipid droplets (Figure 6M) and improved the proinflammatory factor IL-1b, which promotes the decidual- decidualization, as indicated by increased Prl8a2 expression ization response in primates, can also upregulate ISG15 in hu- (Figure 6N). These data further support the essentiality of em- man uterine fibroblast cells.45 Treatment with human chorionic bryo-derived LPL in ensuring normal decidualization. gonadotropin elevates Isg15 in heifers.48 Different types of ‘‘de- ciduogenic stimuli’’ that induce artificial deciduomas are likely to DISCUSSION up-regulate Isg15 in various ways and with different intensities. To expand the understanding that the decidua serves as a pro- The decidua plays a pivotal role in ensuring the survival and vider of energy to support the developing embryo before placen- development of the embryo during early pregnancy. To improve tation, this study presents evidence that the energy supplier role Figure 6. pTGC-derived LPL facilitates lipid droplet accumulation to ensure normal decidualization (A) Heatmap of genes encoding secretory proteins involved in lipid metabolism in day 5 mural TE, day 5.25 mural TE, and TGCs differentiated from day 5 mural TE for 3 days (TGC). (B) Double in situ hybridization of Lpl and Prl3d1 mRNAs in a day 6.5 WT uterus. Scale bar: 50 mm. (C) Co-immunofluorescence staining of LPL and CLDN5 in a day 6.5 WT uterus. Scale bar: 50 mm. (D) Co-immunofluorescence staining of LPL and PL-I in day 6.5 WT and DTA uteri. Scale bar: 50 mm. (E and F) LPL activity assay (E) and TG content assay (F) by spectrophotometer. (G and H) Bodipy fluorescent staining of lipid droplets (G) and in situ hybridization of Prl8a2 mRNAs (H) in day 6.5 WT uteri of mice injected with DMSO and orlistat. Scale bar: 100 mm. (I) Real-time qPCR analysis of Prl8a2 relative expression levels in day 6.5 WT deciduae of mice injected with DMSO and orlistat. (J) Representative uteri images showing responses to transfer of beads coated with 100 ng/mL BSA only (BSA), 5 ng/mL IL-6 plus 100 ng/mL BSA (LPL
), and 5 ng/mL IL-6 plus 100 ng/mL LPL (LPL+) on day 6.5. Blue bumps indicate the deciduomas of ISs induced by the beads. Scale bar: 10 mm. (K) Quantitative results of the bead transfer test with BSA beads (mice transferred = 10), LPL
beads (mice transferred = 18), and LPL+ beads (mice transferred = 20). (L) The weight comparisons of ISs induced by LPL
and LPL+ beads on day 6.5. (M) Oil red O staining of lipid droplets in uteri receiving with LPL
and LPL+ beads (left). Scale bar: 100 mm. The values of integrated density/area of oil red O staining were measured by ImageJ software to quantify gray value (right). (N) Adjacent images of (M). In situ hybridization of Prl8a2 mRNAs in LPL
and LPL+ uteri. Scale bar: 100 mm. Data represent the mean ± SEM. Two-tailed unpaired Student’s t test. Cell Reports 43, 114246, June 25, 2024 11  ll OPEN ACCESS Article of the decidua at least partially relies on pTGCs. The pivotal role 3D-cultured human pre-gastrulation embryos, according to sin- of LPL in supplying lipids for storage and fuel consumption is gle-cell transcriptome data.62 As a fundamental process in evident.49 Given that pTGCs and its surrounding tissues have mammalian development, trophoblast in the fetal-maternal inter- direct access to maternal blood,18 we demonstrate that pTGC- face during the peri-implantation stage would be expected to derived LPL enhances the decidua’s capacity to accumulate exhibit some remnants of conservation despite evolutionary lipid droplets from the maternal circulation, thereby fulfilling its changes. Our study presents novel evidence supporting the crucial role in energy supply. Lipid droplets in contact with the role of post-implantation maternal-fetal interactions in decidual- ER and numerous organelles are highly competent in regulating ization. It illustrates two effective mechanisms through which the energy homeostasis37 and fueling FAO, which is essential for de- embryo actively engages in the decidualization process. Howev- cidualization and embryo development.6,34,50 Consequently, the er, these mechanisms represent only a partial insight into the sufficient accumulation of lipid droplets promotes decidual broader implications of trophoblast signaling in post-implanta- development and benefits the conceptus. However, the histotro- tion decidual development, holding the potential for advancing phic role of the decidua also involves the uterine glands, which clinical science by introducing novel ideas and perspectives. secrete nutrients encompassing a wide spectrum of growth fac- tors, cytokines, glycogen, glycoproteins, and lipids.51,52 While Limitation of the study significant numbers of lipid droplets accumulate in the decidua, Since pTGC deficiency led to inefficient decidual development, the relationship between these lipid droplets and gland-derived some of the decidual defects resulting from pTGC ablation might histotrophs needs to be explored further. be attributable to the underdevelopment of decidua, rather than Previous studies have provided evidence for the correlation directly due to pTGC ablation. However, limitations may arise between lipid metabolism and the inflammatory-like process due to the constraints of our current knowledge and experi- during the initiation of decidualization in mice. At the onset of im- mental approaches, which affect the scope of our analysis. For plantation, uterine luminal epithelial cells accumulate lipid drop- instance, we observed tissue remodeling defects (Figure 3A), lets and lysosomes.4 This coincides with an increase of enzymes including expression alteration in laminins and integrins, in the such as phospholipase A2 and Cox which are implicated in the DTA decidua. Due to a lack of sufficient and direct evidence, production of inflammation-related lipids like arachidonic acid, we tentatively attributed these defects to inefficient decidualiza- lysophosphatidic acid, and prostaglandins. These lipid com- tion rather than directly to pTGC ablation. Furthermore, we pounds have been shown to be critical for implantation and de- recognized that certain decidual biological processes can be cidualization induction.2,20,53–55 In our study, we also suggest a influenced by both pTGCs and the decidua itself. For example, correlation between inflammatory responses and lipid-related IFN response genes such as Isg15, Isg12, and Stat1 are induced effects in the overlapping decidual regions where these biolog- by a deciduogenic stimulus rather than by an embryo13 or IFNK- ical processes occur. The reduction of TGs and PCs in the coated beads (Figure 3L). The presence of an embryo enhances DTA group was primarily concentrated in the decidual region this initial IFN -like response, suggesting that other maternal fac- flanking the conceptus, where lipid droplets and pTGC-derived tors63 or the JAK-STAT pathway may have primed this response. LPL accumulated. Genes associated with inflammatory re- This observation suggests the presence of mutual promotion sponses, such as Ptges and Stat1, exhibited higher expression and compensation mechanisms within decidual biological pro- in this region and were downregulated in DTA decidua. Addition- cesses, where both the embryo and decidua contribute collabo- ally, Ppard, which plays multiple roles in both inflammatory ratively, highlighting their joint efforts in responding to the chal- response and lipid metabolism56,57 and is crucial for implantation lenges presented by the pregnancy environment. Future and initial decidualization through the Cox2-Ppard signaling models will facilitate a more nuanced understanding of the mutu- pathway,2 also displayed a higher expression in this decidual re- ally reinforcing and distinct contributions from both the embryo gion and downregulation in DTA decidua. It is worth noting that and mother. artificial ‘‘deciduogenic stimuli,’’ such as intrauterine injection of sesame oil, endometrial scratching, or the use of lectin STAR+METHODS (ConA)-coated beads, may give rise to metabolic stress and/or inflammatory responses.58,59 In some cases, these models Detailed methods are provided in the online version of this paper and include might even amplify certain effects, leading to increased expres- the following: sion of specific decidualization markers.13 d KEY RESOURCES TABLE The initiation of human decidualization occurs in response to d RESOURCE AVAILABILITY the postovulatory progesterone.60 However, it is contended B Lead contact that the degree of decidualization in humans is intensified B Materials availability following embryo attachment.2 While investigating human B Data and code availability maternal-embryonic interactions in vivo presents ethical and d EXPERIMENTAL MODEL AND STUDY PARTICIPANT DETAILS technical challenges, coculture experiments involving endome- B Mice trial stromal cells and first-trimester trophoblast explants have d METHOD DETAILS B Implantation sites Collection revealed the upregulation of genes associated with inflammatory B Preparation of protein carrying beads response, immune response, glucose metabolism, and lipid B Intraperitoneal injection of orlistat metabolism in the presence of trophoblast.61 Furthermore, the B Measurement of LPL activity and TG contents in uterine tissues expression of Ifnk and LPL was detected in trophoblast cells of B Measurement of PGE2 level in uterine tissues 12 Cell Reports 43, 114246, June 25, 2024  ll Article OPEN ACCESS B In situ hybridization thesis and maintenance of lipid droplets are essential for mouse preim- B Single-cell resolution In situ hybridization on tissues (SCRINSHOT) plantation embryonic development. Development 146, dev181925. B RNA extraction and quantitative real-time PCR https://doi.org/10.1242/dev.181925. B RNA-seq and data analysis 8. Austin, K.J., Bany, B.M., Belden, E.L., Rempel, L.A., Cross, J.C., and Han- B Mass spectrometry imaging and data analysis sen, T.R. (2003). Interferon-stimulated gene-15 (Isg15) expression is up- B Relative quantification of TG in decidua regulated in the mouse uterus in response to the implanting conceptus. B Immunofluorescence (IF) Endocrinology 144, 3107–3113. https://doi.org/10.1210/en.2002-0031. B Detection of lipid droplets with lipophilic dyes 9. Ramsey, E.M., Houston, M.L., and Harris, J.W. (1976). Interactions of the B Western blot trophoblast and maternal tissues in three closely related primate species. d QUANTIFICATION AND STATISTICAL ANALYSIS Am. J. Obstet. Gynecol. 124, 647–652. https://doi.org/10.1016/0002- 9378(76)90068-5. SUPPLEMENTAL INFORMATION 10. 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Reprod. 49, 653–665. https:// and 2021YFC2700302 to H.W.) and the National Natural Science Foundation doi.org/10.1095/biolreprod49.4.653. of China (82288102 to H.W., 82222026 to S.K., 82171660 to J.L., and 13. Kashiwagi, A., DiGirolamo, C.M., Kanda, Y., Niikura, Y., Esmon, C.T., Han- 82301886 to H.B.). sen, T.R., Shioda, T., and Pru, J.K. (2007). The postimplantation embryo differentially regulates endometrial gene expression and decidualization. AUTHOR CONTRIBUTIONS Endocrinology 148, 4173–4184. https://doi.org/10.1210/en.2007-0268. 14. Bany, B.M., and Cross, J.C. (2006). Post-implantation mouse concep- Y.S., N.Y., and H.W. designed the research. N.Y., Y.S., B.H., N.D., G.L., Q.H., tuses produce paracrine signals that regulate the uterine endometrium un- Y.W., and H.C. performed the research. Y.S. and W.D. analyzed the RNA-seq dergoing decidualization. Dev. Biol. 294, 445–456. https://doi.org/10. data. J.L., S.K., H.B., B.C., and F.L., supervised the study. 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Cell Reports 43, 114246, June 25, 2024 15  ll OPEN ACCESS Article STAR+METHODS KEY RESOURCES TABLE REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Anti-Cytokeratin 8 (CK8) Millipore Cat# MABT329; RRID:AB_2891089 Anti-Placental lactogen I (PL-1) Santa Cruz Biotechnology Cat# sc-34713; RRID:AB_654194 Anti-LPL (LPL) GeneTex Cat# GTX101125; RRID:AB_1950830 Anti-Claudin 5 (CLDN5) Thermo Fisher Scientific Cat# 35–2500; RRID:AB_2533200 Anti-Stat1 (STAT1) Cell Signaling Technology Cat# 9172; RRID:AB_2198300 Anti-Phospho-Stat1 (p-STAT1) Cell Signaling Technology Cat# 9167; RRID:AB_561284 Anti-b-Tubulin ABclonal Cat# AC008; RRID:AB_2773006 Anti-Dtprp (PRL8A2) Homemade64 N/A Chemicals, peptides, and recombinant proteins Affi-Gel Blue Gel Beads Bio-rad Cat# 1537302 Recombinant mouse IL-6 MedChemExpress Cat# HY-P7378 Recombinant mouse IFN-Kappa R&D Systems Cat# 8437-MK Recombinant mouse Lipoprotein lipase CUSABIO Cat# CSB-MP013065MO BSA Sigma-Aldrich Cat# A9418 Orlistat (Tetrahydrolipstatin) MedchemExpress Cat# HY-B0218 NBT/BCIP Sangon Biotech Cat# C520019 INT/BCIP Sangon Biotech Cat# C500033 SplintR ligase NEB Cat# M0375L phi29 DNA Polymerase NEB Cat# M0269L CY3-Tyramide Servicebio Cat# G1223 TRIzol Invitrogen Cat# 15596018 BODIPY 493/503 GlpBio Cat# GC42959 4, 6-diamidino 2-phenyindiol Beyotime Cat# C1002 Critical commercial assays PrimeScript RT reagent Kit TAKARA Cat# RR047A TB Green Permix Ex Taq II TAKARA Cat# RR820A Prostaglandin (PG) E2 ELISA kit (Mouse) Solarbio Cat# SEKM-0173 Triglyceride content determination kit Solarbio Cat# BC0620 lipoprotein lipase assay kit Solarbio Cat# BC2445 Deposited data Raw and analyzed bulk RNA-seq data This paper GEO: GSE240086 TE and pTGC bulk RNA-seq data Niraimathi et al.34 GEO: GSE151329 Experimental models: Organisms/strains Pl1cre (Prl3d1-Cre) mouse line Ouseph et al.27 N/A Rosa26-DTA (R26-DTA) mouse line The Jackson Laboratory RRID:IMSR_JAX:032087 Rosa26-mTmG (R26-mTmG) mouse line The Jackson Laboratory RRID:IMSR_JAX:007576 Oligonucleotides Primers for in situ hybridization probes (Table S1) This paper N/A Primers for SCRINSHOT probes (Table S1) This paper N/A Primers for qRT-PCR analysis (Table S1) This paper N/A Software and algorithms R (v4.3.2) The R Development Core Team https://www.r-project.org Rstudio (v2023.09.1 + 494) RStudio Team http://www.rstudio.com (Continued on next page) 16 Cell Reports 43, 114246, June 25, 2024  ll Article OPEN ACCESS Continued REAGENT or RESOURCE SOURCE IDENTIFIER Trim Galore The Babraham Institute https://www.bioinformatics.babraham. ac.uk/projects/trim_galore STAR Dobin et al.65 https://github.com/alexdobin/STAR EdgeR Bioconductor Chen Y et al.66 http://bioconductor.org DESeq2 Bioconductor (v1.42.0) Love et al.67 http://bioconductor.org Clusterprofiler Bioconductor (v4.10.0) Yu G et al.68 http://bioconductor.org ComplexHeatmap Bioconductor (v2.18.0) Gu Z et al.69 http://bioconductor.org ggplot2 (v3.4.4) H. Wickham et al. https://ggplot2.tidyverse.org MetaboAnalystR Pang Z et al.70 https://www.metaboanalyst.ca Prism 9.0 GraphPad software https://www.graphpad.com ImageJ Schindelin et al.71 https://imagej.net RESOURCE AVAILABILITY Lead contact Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Haibin Wang ([email protected]). Materials availability Unique reagents generated for this work are available upon request. Data and code availability d RNA-seq data have been deposited at GEO with the accession code GSE240086 and are publicly available. This paper also analyzed existing public data which are listed in the key resources table. d This paper does not report original code. All software packages and their accessibility are described in the key resources table. d Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request. EXPERIMENTAL MODEL AND STUDY PARTICIPANT DETAILS Mice The Prl3d1-Cre knockin mouse line which targets cre-mediated recombination in pTGCs was generated as previously described25 and kindly provided by Dr. Gustavo Leone (Ohio State University, Columbus, USA). The Rosa26-DTA (R26-DTA) transgenic mice line72,73 and the Rosa26-mTmG (R26-mTmG) mice line74 were purchased from the Jackson Laboratory (Bar Harbor, ME, USA). All mice were bred under a 12-h light/12-h dark cycle at temperatures of 22 C–24 C with 50–60% humidity and free access of water and food. 8-week-old R26DTA/DTA female mice were mated with fertile Prl3d1Cre/Cre male mice (8–12-week-old) to generate Prl3d1Cre/+; R26DTA/+ conceptus which expresses DTA to ablate Prl3d1-positive pTGCs. The decidua of these conceptuses served as the DTA group. The decidua of R26+/+ or R26DTA/+ conceptus served as the wild-type (WT) group. All animal experiments were conducted following the guidelines for the care and use of laboratory animals, which were approved by the Animal Welfare Commit- tee of the Research Organization (X200811) at Xiamen University. METHOD DETAILS Implantation sites Collection Plug-positive females were checked out for experiments (vaginal plug = day 1 of pregnancy). Implantation sites (ISs) of D6.5 pregnant mouse were visualized by mouse tail intravenous injection of 100mL of 1% Chicago blue in saline. The number and average weight of ISs demarcated by distinct blue bands was recorded (ISs of D8 were demarcated by clear boundary between ISs). Preparation of protein carrying beads Blastocyst-sized Affi-Gel Blue Gel Beads (Bio-rad; 100–200 mesh, 1537302) were selected and washed three times with sterile PBS, then incubated with different combinations of recombinant mouse IL-6 (5 ng/mL, MedChemExpress, HY-P7378), recombinant mouse IFN-Kappa (IFNK) (50 ng/mL, R&D, 8437-MK), recombinant mouse Lipoprotein lipase (LPL) (100 ng/mL, CUSABIO, CSB- MP013065MO), or BSA (100 ng/mL, Sigma-Aldrich, A9418) at 4 C overnight. Following incubation, the beads were washed in PBS three times and promptly loaded into siliconized pipette tips (soaked with 1%BSA/PBS in advance) before being transferred Cell Reports 43, 114246, June 25, 2024 17  ll OPEN ACCESS Article into the uteri of D4 pseudo-pregnant mice with 6–8 beads per horn. The mice were euthanized on D6.5 after injection of the blue dye. Uteri without blue bands were flushed to recover beads for verification of successful transfer. Intraperitoneal injection of orlistat Female mice were administered orlistat (Tetrahydrolipstatin, MedchemExpress, HY-B0218) twice, at 10:00 a.m. on D5 and D6, via intraperitoneal injection (50 mg/kg/day) using a solution of 10% DMSO, 40% PEG300, 5% Tween 80, 45% saline, with a final volume 100 mL/mouse/day. The mice were euthanized at 10:00 p.m. on day 6 to collect ISs. Measurement of LPL activity and TG contents in uterine tissues Triglyceride content determination kit (Solarbio, BC0620) and lipoprotein lipase assay kit (Solarbio, BC2445) were utilized to assay the total triglyceride content and the lipoprotein lipase activity of D6.5 uterine tissues. After removal of myometrium and embryo tis- sues from the whole uteri, the decidua was homogenized at 4 C. Supernatant after refrigerated centrifugation was collected and pro- cessed according to the manufacturer’s protocol. The OD values were measured spectrophotometrically by SpectraMax 190 Micro- plate Reader (Molecular Devices). Measurement of PGE2 level in uterine tissues Prostaglandin (PG) E2 ELISA kit (Solarbio, SEKM-0173) was utilized to assay the total PGE2 level in D6.5 uterine tissues. After dis- secting myometrium and embryo tissues from whole uteri, the D6.5 WT and DTA deciduae were individually harvested. Each exper- imental group consisted of five to six deciduae, pooled together to achieve a total weight of approximately 15mg, with precise mea- surements. Subsequently, the pooled samples were homogenized on ice with PBS (100mL/10mg) and protease inhibitors. The homogenized samples was centrifuged at 4 C, 10000g for 10min, and the supernatant was taken for ELISA according to the man- ufacturer’s protocol. The OD values were measured spectrophotometrically by SpectraMax 190 Microplate Reader (Molecular Devices). In situ hybridization In situ hybridization using digoxigenin (DIG) or fluorescein was performed following the previously described methods.64 Briefly, cryo- stat sections (10 mm) of WT and DTA ISs were processed on the same slide. The slides were fixed at room temperature in 4% para- formaldehyde for 1 h, followed by three washes with PBS. After prehybridization, uterus tissue sections were hybridized with specific cRNA probes and incubated overnight at 65 C. Following hybridization, the sections were incubated with RNase A (10 mg/mL) at 37 C for 30 min. RNase A-resistant hybrids were then detected using chromogenic agents, specifically NBT/BCIP (Sangon Biotech, C520019) or INT/BCIP (Sangon Biotech, C500033). The primers to make specific mouse cRNA probes labeled with digoxin or fluo- rescein can be found in Table S1. Single-cell resolution In situ hybridization on tissues (SCRINSHOT) The padlock probes were designed based on the published method.75 Briefly, the 50 - and 30 -arm-sequences of the padlock probes, which are complementary to the corresponding mRNA sequence, were acquired online using the PrimerQuest Tool. The ‘‘Padlock Design Assistant’’ (https://github.com/AlexSount/SCRINSHOT) was then utilized to design the padlock probes. Frozen tissue sec- tions (10 mm) were fixed in 4% paraformaldehyde in PBS for 1 h at room temperature. Foll