Fatty acid synthesis and oxidation in cumulus cells support oocyte maturation in bovine

Abstract

Oocyte meiotic maturation requires energy from various substrates including glucose, amino acids, and lipids. Mitochondrial fatty acid (FA) β-oxidation (FAO) in the oocyte is required for meiotic maturation, which is accompanied by differential expression of numerous genes involved in FAs metabolism in surrounding cumulus cells (CCs) in vivo. The objective was to elucidate components involved in FAs metabolism in CCs during oocyte maturation. Twenty-seven genes related to lipogenesis, lipolysis, FA transport, and FAO were chosen from comparative transcriptome analysis of bovine CCs before and after maturation in vivo. Using real-time PCR, 22 were significantly upregulated at different times of in vitro maturation (IVM) in relation to oocyte meiosis progression from germinal vesicle breakdown to metaphase-II. Proteins FA synthase, acetyl-coenzyme-A carboxylase, carnitine palmitoyltransferase, perilipin 2, and FA binding protein 3 were detected by Western blot and immunolocalized to CCs and oocyte cytoplasm, with FA binding protein 3 concentrated around oocyte chromatin. By mass spectrometry, CCs lipid profiling was shown to be different before and after IVM. FAO inhibitors etomoxir and mildronate dose-dependently decreased the oocyte maturation rate in vitro. In terms of viability, cumulus enclosed oocytes were more sensitive to etomoxir than denuded oocytes. In CCs, etomoxir (150 μM) led to downregulation of lipogenesis genes and upregulated lipolysis and FAO genes. Moreover, the number of lipid droplets decreased, whereas several lipid species were more abundant compared with nontreated CCs after IVM. In conclusion, FAs metabolism in CCs is important to maintain metabolic homeostasis and may influence meiosis progression and survival of enclosed oocytes.

Figure 1.

Expression kinetics of lipid metabolism-related genes in bovine CCs during IVM. Total RNA (500 ng) of CCs recovered before (0 hour) and after 6, 18, and 24 hours IVM were converted into cDNA, and real-time PCR was performed. Relative gene expression was calculated using gene expression values normalized by 3 reference genes and presented as a mean of 4 independent samples ± SEM. Different letters indicate significant differences (P < .05).

Figure 2.

Detection of FASN, CPTA1, PLIN2, FABP3, and TUBA proteins in CCs and oocytes (Oo) by Western blot. A, Proteins were detected using 10 μg total proteins from bovine CCs surrounding the oocytes after IVM with 10 μg total proteins from bovine sc adipose tissue (AT) loaded as a control. B, Proteins were detected in bovine DOs (pools of 50 mature oocytes loaded per line) and in corresponding CCs (10 μg proteins).

Figure 3.

Immunofluorescent detection of CPT1, FABP3, PLIN2, FASN, and ACACA proteins by confocal microscopy in bovine COCs treated with specific primary and secondary Alexa488-coupled antibodies. For control, no primary antibody but rabbit IgG was used. Pictures of immature COCs are shown. For CPT1 labeling, CCs after IVM are also shown (inset). For FABP3, Meta-II in a mature oocyte (Oo) is also shown (inset). Chromatin is marked by Hoechst33342. Scale bars, 50 μm.

Figure 4.

Effect of FAO inhibitors on oocyte maturation rate. A, Schematic representation of the main pathways of mitochondria β-oxidation and action of FAO inhibitors. Mildronate blocks carnitine production, and etomoxir inhibits CPT1 activity; thus, the entry of FAs into the mitochondria is blocked. B, Dose-dependent effect of mildronate on oocyte maturation rate (n = 411) and viability after 24 hours IVM. C and E, Dose effect of etomoxir on relative maturation rate and viability of the oocytes after 24 hours IVM (CEOs, n = 391 [C], or DOs, n = 417 [E]). D, Representative pictures of LIVE/DEAD staining of COCs after 24 hours IVM in the presence of 0μM, 25μM, 100μM, and 250μM etomoxir. F, Representative pictures of LIVE/DEAD staining of CEOs and DOs after 24 hours IVM in the presence or absence of etomoxir (150μM). Green indicates cells are living; red indicates dead cells.

Figure 5.

A, Analysis of cell viability by LIVE/DEAD staining of bovine COCs after 18 hours IVM in the presence or absence of etomoxir (150μM). Bar, 500 μm. The histogram represents the percentage of living CCs (6620 cells counted in 3 experiments). B, Analysis of reversibility of etomoxir (150μM) effect on oocyte maturation rate during IVM, performed in 2 steps: 1) entire COCs (thus containing CEOs, n = 593, and DOs, n = 589) were matured during the first 18 hours (IVM 0–18h) without (−) or with 150μM etomoxir (+), and 2) after 18 hours, CEOs were stripped from CCs and IVM was continued for 22 hours (IVM 18h–40h) for all oocyte groups either without (−) or with etomoxir (+). At the end of IVM (18h or 40h), oocyte viability was checked and maturation rate was determined by chromatin staining using Hoechst33342. Ana-I/Meta-II oocytes were considered mature. Different letters indicate significant differences (P < .05). Graph bars are mean values ± SEM of 3 experiments. C, Effect of etomoxir (150μM) on MAPK3/1 protein phosphorylation in CEOs and DOs after 6 hours IVM. Proteins from CEOs and DOs recovered after 6 hours IVM were analyzed by Western blot using phospho-MAPK 3/1 and total MAPK3/1 antibodies (30 oocytes loaded per line, 4 replicates). Graph represents the mean values of pMAPK1/MAPK1 protein ratio quantification ± SEM. Different letters indicate significant differences (P < .05).

Figure 6.

Microscopic analysis of bovine CC morphology on semi-thin sections before IVM (A) and after 18 hours of IVM performed either in control medium (B) or in the medium complemented with 150μM etomoxir (C and D). Bar, 50 μm. LDs are marked by arrowheads and magnified in insets. Necrotic nuclei are marked with asterisks. Abbreviations: PB, polar body; V, vacuole; ZP, zona pellucida.

Figure 7.

Lipid droplets (LD) distribution and quantification in CCs as detected on semithin sections of cumulus-oocyte complexes (COCs) before and after 18 hours of IVM with or without etomoxir (150μM). A, Left histogram: Percentage of CCs with visible LDs in immature COCs (n = 10; 1130 CC), in COC after 18 hours IVM (n = 11, 1184 CC) and in COC after IVM in the presence of 150μM etomoxir (n = 11; 1047 CC). Right histogram: Number of LDs per LD-containing CCs in immature COCs (n = 477 CC), in COCs after 18 hours IVM (n = 836 CC) and in COCs after 18 hours IVM with 150μM etomoxir (n = 471 CC). Different letters mean significant difference (P < .01) B, Distribution of CCs according to the number of LDs per cell counted in individual cells either before IVM, or after 18 hours IVM without or with 150μM of etomoxir.

Figure 8.

Analysis of lipid composition by MALDI-MS profiling in bovine CCs. A, Representative MALDI-MS spectra obtained on bovine CCs. B, Examples of lipid species m/z peaks detected by MALDI-MS profiling in either immature CCs (0h), CCs after 18 hours IVM with or without 150μM etomoxir (18hEto and 18h, respectively). C, Principal component analysis was performed on 93 m/z peaks differentially abundant between CCs at 0 or 18 hours and 18hEto as calculated from correspondent mass spectra. Each point represents an independent CC sample analyzed in triplicate with 5000 spectra per replicate. The numbers represent different m/z species (Supplemental Table 2).

Figure 9.

Differential analysis of lipid species abundances in bovine CCs. A, Graphic representation of normalized peak height values of 93 differential m/z species in CCs at 0 and 18 hours and 18hEto (ANOVA, P < .01). B, Comparison of 5 lipid species between the different groups of CCs. Bars are mean values of normalized peak height ± SEM (n = 3). Different letters indicate significant differences (P < .05).

Figure 10.

Effect of etomoxir on gene expression in CCs. A, Effect of etomoxir (150μM) on expression of genes related to lipid metabolism, oocyte quality, and apoptosis in CCs after 6 or 18 hours of IVM. Total RNA was converted to cDNA, and real-time PCR was performed in triplicate. Values were normalized relative to 3 reference genes. Data are presented as heat map clustering of relative gene expression mean values obtained on 4 independent samples. B, Western blot quantification of ACACA and FASN proteins in COCs after 18 hours IVM with or without etomoxir (150μM). Total proteins extracted from 10 COCs were loaded per line. Western blots of the same membrane for the protein of interest and then TUBA as a loading control were performed. Graphs are mean protein of interest to TUBA ratio values ± SEM (n = 4). Different letters indicate significant differences (P < .05); #, trend to significance (P < .1).

Figure 11.

Schema of hypothetical disposition of proteins involved in lipid metabolism in bovine CCs during oocyte maturation according to expression data and their known functions in different cells. Those proteins for which gene expression was affected by etomoxir-mediated FAO inhibition are marked by asterisk. Abbreviations: AS, ATP synthase; ER, endoplasmic reticulum; RC, respiratory chain; V, vacuole.