Journal: Journal of Advanced Research

Article Title: Integrated ultrasensitive metabolomics and single-cell transcriptomics identify crucial regulators of sheep oocyte maturation and early embryo development in vitro

doi: 10.1016/j.jare.2024.08.040

Figure Lengend Snippet: Metabolic profile analysis for oocyte maturation and early embryo development. (A) Schematic overview of the workflow for metabolome profiling in oocytes and early embryos (created by biorender.com ). (B-D) Volcano plot shows the number of upregulated metabolites (orange dots) or downregulated metabolites (blue dots). The black dotted line indicates that the P value is equal to 0.05. (E-F) Upregulated metabolic KEGG pathways in oocytes or early embryos based on the upregulated metabolites. (G-H) Downregulated metabolic KEGG pathways in oocytes or early embryos based on the downregulated metabolites. IVM, in vitro maturation; IVF, in vitro fertilization; IVC, in vitro culture; OO, oocyte; EM, embryo.

Article Snippet: Subsequently, cumulus-oocyte complexes (COCs) displaying evenly granulated cytoplasm and at least three layers of compacted cumulus cells (CCs) were transferred to in vitro maturation medium composed of bicarbonate-buffered medium 199 (11150059, Gibco), 0.33 mmol/L sodium pyruvate (P5280, Sigma), 10 % FBS (Gibco, 10099141), 0.5 IU/mL FSH (F8470, Solarbio), 0.5 IU/mL LH (L8040, Solarbio), 1 μg/mL E 2 (E2758, Sigma), 50 μmol/L L-cystine (C7602, Sigma) and 1 % penicillin–streptomycin medium (516106, Millipore) under mineral oil at 38.5 °C with 5 % CO 2 in a humidified atmosphere.

Techniques: In Vitro

Journal: Journal of Advanced Research

Article Title: Integrated ultrasensitive metabolomics and single-cell transcriptomics identify crucial regulators of sheep oocyte maturation and early embryo development in vitro

doi: 10.1016/j.jare.2024.08.040

Figure Lengend Snippet: Functional analysis of betaine and L-carnitine. (A) Developmental competence analysis of oocytes when betaine, L-carnitine or both were added to in vitro maturation and in vitro culture systems. a, b, c Values with different superscripts are significantly different ( P <0.05). (B) Effect of a glycine transporter 1 inhibitor (GlyT1 inhibitor) and GlyT1 inhibitor combined with betaine on blastocyst formation. “F”, cultured in normal IVC medium; “F+G”, cultured in IVC medium supplemented with GlyT1 inhibitor. “F+G+B”, cultured in IVC medium supplemented with GlyT1 inhibitor and betaine. Red arrows represent blastocyst formation. (C) Evaluation of spindle morphology of MⅡ oocyte.The spindle and DNA were labeled with α-Tubulin antibody and DAPI, respectively. (D) Effect of L-carnitine transport and synthesis inhibitors on lipid content in matured sheep oocytes. The lipid content was determined by BODIPY 493/503 labeling. (E) Effect of L-carnitine transport and synthesis inhibitors on lipid peroxidation in matured sheep oocytes. Lipid peroxidation was evaluated by BODIPY 581/591 C11. Lipids were oxidized and exhibited a green color. The lipid was in a reductive state with a red color. (F) Blastocyst rate corresponding to B. (G) Oocyte maturation rate after different treatments. (H) Effect of L-carnitine transport and synthesis inhibitors on the average grade of spindles of matured oocyte. (I) and (J) Lipid content and lipid peroxidation levels corresponding to D and E, respectively. COCs, cumulus-oocyte complexes; PA, parthenogenetic activation; F, fresh, without any treatment; B, betaine treatment; LC, L-carnitine treatment; FBL, fresh group with betaine and L-carnitine treatment; G, GlyT1 inhibitor; M, Mildronate, inhibitor of BBOX1 and OCTN2; E, Etomoxir, inhibitor of CPT-1A.

Article Snippet: Subsequently, cumulus-oocyte complexes (COCs) displaying evenly granulated cytoplasm and at least three layers of compacted cumulus cells (CCs) were transferred to in vitro maturation medium composed of bicarbonate-buffered medium 199 (11150059, Gibco), 0.33 mmol/L sodium pyruvate (P5280, Sigma), 10 % FBS (Gibco, 10099141), 0.5 IU/mL FSH (F8470, Solarbio), 0.5 IU/mL LH (L8040, Solarbio), 1 μg/mL E 2 (E2758, Sigma), 50 μmol/L L-cystine (C7602, Sigma) and 1 % penicillin–streptomycin medium (516106, Millipore) under mineral oil at 38.5 °C with 5 % CO 2 in a humidified atmosphere.

Techniques: Functional Assay, In Vitro, Cell Culture, Labeling, Activation Assay

Journal: Journal of Advanced Research

Article Title: Integrated ultrasensitive metabolomics and single-cell transcriptomics identify crucial regulators of sheep oocyte maturation and early embryo development in vitro

doi: 10.1016/j.jare.2024.08.040

Figure Lengend Snippet: Metabolic characteristic analysis of oocytes and embryos based on transcriptomics and metabolomics. (A) Heatmap of KEGG pathway analysis related to metabolism regulation from oocyte maturation to embryonic development. C1-C5 represented 5 different gene clusters. (B-D) KEGG enrichment analysis by combining transcriptomics and metabolomics.

Article Snippet: Subsequently, cumulus-oocyte complexes (COCs) displaying evenly granulated cytoplasm and at least three layers of compacted cumulus cells (CCs) were transferred to in vitro maturation medium composed of bicarbonate-buffered medium 199 (11150059, Gibco), 0.33 mmol/L sodium pyruvate (P5280, Sigma), 10 % FBS (Gibco, 10099141), 0.5 IU/mL FSH (F8470, Solarbio), 0.5 IU/mL LH (L8040, Solarbio), 1 μg/mL E 2 (E2758, Sigma), 50 μmol/L L-cystine (C7602, Sigma) and 1 % penicillin–streptomycin medium (516106, Millipore) under mineral oil at 38.5 °C with 5 % CO 2 in a humidified atmosphere.

Techniques: