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Low-input lipidomics reveals lipid metabolism remodelling during early mammalian embryo development

Nature Cell Biology volume 26, pages 278–293 (2024)Cite this article

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Abstract

Lipids are indispensable for energy storage, membrane structure and cell signalling. However, dynamic changes in various categories of endogenous lipids in mammalian early embryonic development have not been systematically characterized. Here we comprehensively investigated the dynamic lipid landscape during mouse and human early embryo development. Lipid signatures of different developmental stages are distinct, particularly for the phospholipid classes. We highlight that the high degree of phospholipid unsaturation is a conserved feature as embryos develop to the blastocyst stage. Moreover, we show that lipid desaturases such as SCD1 are required for in vitro blastocyst development and blastocyst implantation. One of the mechanisms is through the regulation of unsaturated fatty-acid-mediated fluidity of the plasma membrane and apical proteins and the establishment of apical–basal polarity during development of the eight-cell embryo to the blastocyst. Overall, our study provides an invaluable resource about the remodelling of the endogenous lipidome in mammalian preimplantation embryo development and mechanistic insights into the regulation of embryogenesis and implantation by lipid unsaturation.

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Fig. 1: Stage-specific lipidomics signatures in mouse preimplantation embryo development.
Fig. 2: Dynamic changes in the contents of individual lipid classes during mouse preimplantation embryo development support lipid-mediated functions.
Fig. 3: The degree of lipid unsaturation is increased during mouse preimplantation embryo development.
Fig. 4: The level of MUFAs is increased during mouse and human preimplantation embryo development.
Fig. 5: Lipid unsaturation contributes to the developmental process towards blastocyst embryos.
Fig. 6: The desaturase SCD1 contributes to membrane fluidity and cytoskeleton organization in eight-cell-to-blastocyst development.
Fig. 7: The desaturases for PUFA synthesis contribute to blastocyst formation by mediating cytoskeleton organization and apical domain formation.
Fig. 8: Desaturases contribute to the establishment of apical–basal polarity of early embryos for successful implantation of blastocysts in the uterus.

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Data availability

Sequencing data have been deposited into the Gene Expression Omnibus (GEO) under accession code GSE213782. Previously published RNA-seq data4 that were re-analysed here are available at the GEO under accession code GSE98150. All other data supporting the findings of this study are available from the corresponding authors on reasonable request. Source data are provided with this paper.

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Acknowledgements

We thank members of J. Zhang’s laboratory and Dr. X. Huang for discussions and help with paper preparation; Y. Ding and S. Hong from the animal core facilities of Zhejiang University School of Medicine for their technical support; and Z. Lin and L. Xuan from the Core Facilities, Zhejiang University School of Medicine for their technical support. This work was funded by the National Natural Science Foundation of China (91857116, 82373238 and 31871453 to J. Zhang), the National Key Research and Development Program of China (2018YFA0107103 and 2018YFC1005002 to J. Zhang), a Zhejiang Innovation Team grant (2019R01004 to J. Zhang), the Zhejiang Natural Science Foundation (LR19C120001 to J. Zhang), the Key Research and Development Program of Zhejiang Province (2023C03036 to J. Zhang), the National Natural Science Foundation of China (92057202 to G.S.) and the National Natural Science Foundation projects of China (32000809 to L.Z.).

Author information

Author notes

  1. These authors contributed equally: Ling Zhang, Jing Zhao, Sin Man Lam, Lang Chen, Yingzhuo Gao, Wenjie Wang.

Authors and Affiliations

  1. Center for Stem Cell and Regenerative Medicine, Department of Basic Medical Sciences, and Bone Marrow Transplantation Center of the First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, China

    Ling Zhang, Jing Zhao, Lang Chen, Wenjie Wang, Yuyan Xu, Tianyu Tan, Hua Yu, Min Zhang, Xufeng Liao, Mengchen Wu & Jin Zhang

  2. Liangzhu Laboratory, Zhejiang University, Hangzhou, China

    Ling Zhang, Jing Zhao, Tianyun Zhang, Ning Shen & Jin Zhang

  3. State Key Laboratory of Molecular Developmental Biology, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, China

    Sin Man Lam & Guanghou Shui

  4. LipidALL Technologies, Changzhou, China

    Sin Man Lam & Bowen Li

  5. Center of Reproductive Medicine, Shengjing Hospital of China Medical University, Shenyang, China

    Yingzhuo Gao & Da Li

  6. NHC Key Laboratory of Advanced Reproductive Medicine and Fertility (China Medical University), National Health Commission, Shenyang, China

    Yingzhuo Gao & Da Li

  7. College of Biomedical Engineering and Instrument Science, Key Laboratory for Biomedical Engineering of Ministry of Education, Zhejiang University, Hangzhou, China

    Jie Huang & Hyeon Jeong Lee

  8. Institute of Immunology, Department of Surgical Oncology of The First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, China

    Quan D. Zhou

  9. Zhejiang Provincial Key Laboratory for Cancer Molecular Cell Biology, Life Sciences Institute, Zhejiang University, Hangzhou, China

    Cunqi Ye

  10. University of Chinese Academy of Sciences, Beijing, China

    Guanghou Shui

  11. Center of Gene and Cell Therapy and Genome Medicine of Zhejiang Province, Hangzhou, China

    Jin Zhang

Authors
  1. Ling Zhang
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Contributions

L.Z. and J. Zhang conceived and designed the experiments. L.Z., L.C., J. Zhao and W.W. collected mouse oocytes and early-stage embryos. Y.G. collected human embryos. S.M.L. performed lipidomics and W.W. performed blastoid experiments. L.Z., J. Zhao, W.W., Y.X., T.T., M.Z., X.L., M.W. and J.H. performed other experiments. L.Z., H.Y., B.L. and T.Z. performed bioinformatics analyses. L.Z. and J. Zhang wrote the paper with discussion from G.S., D.L., C.Y., N.S., H.J.L. and Q.D.Z.

Corresponding authors

Correspondence to Da Li, Guanghou Shui or Jin Zhang.

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Competing interests

S.M.L. and B.L. are employees of LipidALL Technologies. The other authors declare no competing interests.

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Nature Cell Biology thanks Satoshi Tsukamoto and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available.

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Extended data

Extended Data Fig. 1 Dynamic changes of lipidome in mouse preimplantation embryo development.

(a) A total of 120 zygotes and 120 two-cell embryos were used for embryos number testing in a lipidomic experiment. The ratio of sample/blank represents the sample signal/noise value. The dots (blue or pink) represent metabolites detected. (b) Levels of all lipid classes quantified by LC–MS in six developmental stages. Data are from four biological replicates of oocytes, zygote and two-cell stage embryos each and three biological replicates of four-cell, eight-cell and blastocyst stage embryos each and are presented as the mean +/-s.e.m. (c) A pie chart showing the proportion of each lipid class in every developmental stage. (d) Heatmaps showing distinct temporal patterns of lipids along the six developmental stages. Data are from averaged lipid concentrations in each stage of the same biological replicates in (b). (e) Linear plots showing the trends of lipid clusters along the six developmental stages. Black dots represent the averaged Z-scores of the concentration of clustered lipids in the six developmental stages, yellow lines connect the median points of all the samples at each developmental stage, and blue curves represent the fitted levels by the maSigPro model.

Source data

Extended Data Fig. 2 Dynamic changes of lipidome in mouse preimplantation embryo development using the Bligh & Dyer method.

(a) A PCA plot of the lipidomics profiling data showing clustering of seven biological replicates of oocytes and zygote stage embryos each, four biological replicates of two-cell stage embryos each and five biological replicates of four-cell, eight-cell and blastocyst stage embryos each. (b) Levels of all lipid classes quantified by LC–MS in six developmental stages. Data are from the same biological replicates as in (a) and are presented as the mean +/-s.e.m. (c) A pie chart showing the proportion of each lipid class in every developmental stage. (d) Heatmaps showing the relative abundance of lipids in oocytes, zygotes, two-cell, four-cell, eight-cell and blastocyst embryos. Scaled value bar indicates the relative concentration. Data are from the same biological replicates as in (a).

Source data

Extended Data Fig. 3 Dynamic changes of different lipid classes during mouse preimplantation embryo development support lipid-mediated functions.

(a) Immunofluorescence of PEs staining showing its dynamic change in abundance in the six developmental stages. (b-g) A heatmap of individual species CLs (b), GluCers (c), PGs (d), SLs (e), LPCs (f) and acylcarnitine (g) quantified by LC–MS in six developmental stages. Scaled value bar indicates relative concentration. Data are from four biological replicates of oocytes, zygote and two-cell stage embryos each and three biological replicates of four-cell, eight-cell and blastocyst stage embryos each.

Extended Data Fig. 4 Dynamic changes of individual lipid classes during mouse preimplantation embryo development using the Bligh & Dyer method.

(a-h), Heatmaps showing the levels of each class of species of PCs (a), PIs (b), PC-Os (c), Gb3 (d), GluCers (e), SMs (f), TAGs (g) and CE and Cho (h) in six developmental stages. Scaled value bar indicates relative concentration. Data are from seven biological replicates of oocytes, zygote stage embryos each, four biological replicates of two-cell stage embryos and three biological replicates of four-cell, eight-cell and blastocyst stage embryos each.

Extended Data Fig. 5 The degree of lipid unsaturation is increased during mammalian preimplantation embryo development.

(a) Nightingale rose chart showing the distribution of polyunsaturated, mono+diunsaturated and saturated lipids in different lipid classes. Numbers of species measured in each class are shown. (b-d) Bar graphs showing the polyunsaturated lipids levels of LPCs (b), PC-Os (c) and PSs (d) in the mouse two-cell and blastocyst stage embryos. (e-g) Bar graphs showing the mono+diunsaturated lipid levels of GluCers (e), SLs (f) and SMs (g) in the mouse two-cell and blastocyst stage embryos. (h-l) Box plots showing the polyunsaturated lipid levels of PGs (h), BMPs (i), CLs (j), PC-Os (k) and TAGs (l) in the human eight-cell and blastocyst stage embryos. (m-n) FPKM values of Scd gene (m) and Fads gene (n) transcripts at different developmental stages. Each dot represents one biological replicate. Data are from two biological replicates of oocytes, four biological replicates of two-cell and four-cell stage embryos, three biological replicates of eight-cell stage embryos, two biological replicates of morula stage embryos, four biological replicates of ICM and TE of E3.5 blastocyst stage embryos, and two biological replicates of Epi of E6.5 of post-implantation embryos (ref. 4) and are presented as the mean +/-s.e.m. (o) Bar graphs illustrating the levels of palmitic acid (C16:0), palmitoleic acid(C16:1) and the ratio of C16:1/C16:0 in the mouse two-cell and the blastocyst stage embryos. (p) Box plot showing the ratio of palmitoleic acid (C16:1)/palmitic acid (C16:0) in the human eight-cell and blastocyst stage embryos. (b-g, o) Each dot represents one biological replicate, and data are from four biological replicates of two-cell stage embryos each and three biological replicates of blastocyst stage embryos each and are presented as the mean +/-s.e.m. Statistical significance was determined by two-tailed unpaired t test. (h-l, p) Each dot represents one biological replicate, data are from four biological replicates of eight-cell and blastocyst stage embryos each and are presented as the mean +/-s.e.m. Statistical significance was determined by two-tailed unpaired t test. Center line, median; box, 25th and 75th percentiles, whiskers, 1.5xIQR.

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Extended Data Fig. 6 Inhibition of SCD1 during mouse embryo development.

(a) A schematic of the experimental approach. Zygotes were isolated at 24 h after injection of hCG and cultured in KSOM medium with 100 nM CAY10566 or 100 nM CAY10566 plus 100 nM oleic acid, or Scd1 siRNA injection was performed at the zygote stage, and the embryos were collected at the indicated times for further analysis. (b) TUNEL assay was performed at 3.5 d.p.c embryos cultured in medium with DMSO, 100 nM CAY10566 or 100 nM CAY10566 plus 100 nM oleic acid. Representative images of embryos under the above three conditions are shown. (c) Scd1 mRNA expression at 3.5 d.p.c after Scd1 siRNA injection at the zygote stage. Data are from three independent experiments and are presented as the mean +/-s.e.m. Statistical significance was determined by two-tailed unpaired t test. (d) FPKM values of Elovl gene transcripts at different developmental stages. Each dot represents one biological replicate. Data are from two biological replicates of oocytes, four biological replicates of two-cell and four-cell stage embryos, three biological replicates of eight-cell stage embryos, two biological replicates of morula stage embryos, four biological replicates of ICM and TE of E3.5 blastocyst stage embryos, and two biological replicates of Epi of E6.5 of post-implantation embryos (ref. 4) and are presented as the mean +/-s.e.m. (e) Representative images of mouse embryos at 2.5, 3.5 or 4.5 d.p.c that were treated with siRNA of scramble (NC) or Elovl5 from the zygote stage. The images shown are representative of three independent experiments. (f) Elovl5 mRNA expression at 4.5 d.p.c after Elovl5 siRNA injection at the zygote stage. Data are from three independent experiments and are presented as the mean +/-s.e.m. Statistical significance was determined by two-tailed unpaired t test.

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Extended Data Fig. 7 The lipid desaturase SCD1 mediates cell plasma membrane fluidity in mouse preimplantation embryo development.

(a) FRAP analysis showing recovery after photobleaching to indicate cell membrane fluidity in embryos of all developmental stages. The images shown are representative of three independent experiments. (b) FRAP analysis showing recovery after photobleaching to indicate cell membrane fluidity in 3.5 d.p.c embryos treated with DMSO-, CAY10566- or CAY10566 plus oleic acid. The images shown are representative of three independent experiments.

Extended Data Fig. 8 The desaturase SCD1 contributes to cytoskeleton organization and apical domain formation in eight-cell-to-blastocyst development.

(a) Immunofluorescence of K18 and CDX2 showing their location in 3.5 d.p.c blastocyst embryos treated with DMSO-, CAY10566- or CAY10566 plus oleic acid; the images shown are representative of one (for K18) and two (for CDX2) independent experiments. (b-d) Quantification of fluorescence intensity of the cell-cell boundary (b), cell number per embryo (c) and CDX2 intensity (d) of 3.5 d.p.c blastocyst embryos treated with DMSO-, CAY10566- or CAY10566 plus oleic acid. Each dot represents one blastocyst, Data (b, c) are from n = 5 for DMSO, n = 10 for CAY10566 and n = 6 for CAY10566 + OA-treated blastocysts, the experiment was performed for one time; data (d) are from n = 13 for DMSO, n = 20 for CAY10566 and n = 15 for CAY10566 + OA-treated blastocysts, and the experiments were repeated for two times. Data are presented as the mean +/-s.e.m. Statistical significance was determined by two-tailed unpaired t test. Center line, median; box, 25th and 75th percentiles, whiskers, 1.5xIQR. (e) Immunofluorescence of K18 and CDX2 of 3.5 d.p.c. blastocyst embryos treated with NC, siScd1 or siScd1 plus oleic acid. The images shown are representative of two (for siScd1) and one (for siScd1 + OA) independent experiments. (f, g) Quantification of fluorescence intensity of the cell-cell boundary (f), cell number per embryo (g) of 3.5 d.p.c blastocyst embryos treated with NC, siScd1 or siScd1 plus oleic acid. Each dot represents one blastocyst. Data are from n = 17 for siNC, n = 19 for siScd1 and n = 5 for siScd1 + OA-treated blastocysts, the experiments were repeated for two (for siScd1) and one (for siScd1 + OA) times. Data are presented as the mean +/-s.e.m. Statistical significance was determined by two-tailed unpaired t test. Center line, median; box, 25th and 75th percentiles, whiskers, 1.5xIQR.

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Extended Data Fig. 9 The desaturase SCD1 contributes to blastocyst implantation, and the desaturases for the PUFA and MUFA synthesis mediate apical-basolateral protein expression in TSCs and ESCs.

(a) A table showing the number of transfered embryos and implanted embryos with and without knocking down Scd1. (b) Immunofluorescence of E-cadherin in ESCs treated with DMSO, CAY10566(1uM), SC-26196(1uM), sesamin(1uM). Arrows indicate E-cadherin localization. (c) Immunofluorescence of E-cadherin in TSCs treated with DMSO, CAY10566(1uM), SC-26196(1uM), sesamin(1uM). Arrows indicate E-cadherin localization. (d, e) Immunofluorescence of K8 and K18 in TSCs treated with DMSO, CAY10566(1uM), SC-26196(1uM), sesamin(1uM). Arrows indicate K8 and K18 localization. (f, g) Immunofluorescence of phosphorylated-ezrin(Thr567) in ESCs (f) and TSCs (g), respectively, treated with DMSO, CAY10566(1uM), SC-26196(1uM), sesamin(1uM). Arrows indicate Phospho-ezrin localization. (b-g) The images shown are representative of three independent experiments; scale bar, 30uM.

Extended Data Fig. 10 Desaturases for the PUFA and MUFA synthesis are required for the generation of EPS-blastoids.

(a) Representative phase-contrast images of EPSC aggregates at the indicated time to show the EPS-blastoid formation; the images shown are representative of three independent experiments. (b) Phase-contrast images of blastoid formation in the DMSO-, SC-26196 or sesamin-treated conditions; the images shown are representative of two independent experiments. (c) Quantification of blastoid formation efficiency in the DMSO-, SC-26196 and sesamin-treated conditions. Data are from two independent experiments. (d) Immunofluorescence of E-cadherin and K18 localization in the blastoids treated with DMSO-, SC-26196 and sesamin; the images shown are representative of two independent experiments. (e) A schematic to show apical-basal polarity establishment, lateral expansion of apical domain protein phospho-ezrin and cytoskeleton proteins F-actin and Keratin, and the zippering process to seal the blastocyst embryo along the cell-cell junction during the eight-cell-to-blastocyst embryo development. The desaturase SCD1-mediated lipid unsaturation, membrane fluidity and cytoskeleton dynamics are required for these processes to take place in order to support successful blastocyst implantation in the uterus.

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Supplementary Table 1

Tables 1–4: Lipidomics raw data. Table 5: siRNA and primer information.

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Zhang, L., Zhao, J., Lam, S.M. et al. Low-input lipidomics reveals lipid metabolism remodelling during early mammalian embryo development. Nat Cell Biol 26, 278–293 (2024). https://doi.org/10.1038/s41556-023-01341-3

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