The Differential Metabolomes in Cumulus and Mural Granulosa Cells from Human Preovulatory Follicles

Abstract

This study evaluated the differences in metabolites between cumulus cells (CCs) and mural granulosa cells (MGCs) from human preovulatory follicles to understand the mechanism of oocyte maturation involving CCs and MGCs. CCs and MGCs were collected from women who were undergoing in vitro fertilization (IVF)/intracytoplasmic sperm injection (ICSI) treatment. The differences in morphology were determined by immunofluorescence. The metabolomics of CCs and MGCs was measured by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) followed by quantitative polymerase chain reaction (qPCR) and western blot analysis to further confirm the genes and proteins involved in oocyte maturation. CCs and MGCs were cultured for 48 h in vitro, and the medium was collected for detection of hormone levels. There were minor morphological differences between CCs and MGCs. LC-MS/MS analysis showed that there were differences in 101 metabolites between CCs and MGCs: 7 metabolites were upregulated in CCs, and 94 metabolites were upregulated in MGCs. The metabolites related to cholesterol transport and estradiol production were enriched in CCs, while metabolites related to antiapoptosis were enriched in MGCs. The expression of genes and proteins involved in cholesterol transport (ABCA1, LDLR, and SCARB1) and estradiol production (SULT2B1 and CYP19A1) was significantly higher in CCs, and the expression of genes and proteins involved in antiapoptosis (CRLS1, LPCAT3, and PLA2G4A) was significantly higher in MGCs. The level of estrogen in CCs was significantly higher than that in MGCs, while the progesterone level showed no significant differences. There are differences between the metabolomes of CCs and MGCs. These differences may be involved in the regulation of oocyte maturation.

Keywords: Cumulus cells; Follicle; Human; Metabolomes; Mural granulosa cells.

Fig. 1

Cultured CCs and MGCs were observed under a laser confocal microscope. A MGCs; B CCs. Magnification: 1000×

Fig. 2

The picture shows the metabolite correlations between CCs and MGCs. A is in anion mode, and B is in positive mode

Fig. 3

Volcano plots depict differentially detected metabolites and the differences between MGCs and CCs. A is in anion mode, and B is in positive mode. The x-coordinate in the figure is the logarithm (log 2) of the fold change (MGC/CC), and the y-coordinate is the logarithm (−log 10) of the P value significance. The red dots in the figure are metabolites with FC > 1.5 and P value < 0.05, that is, the different metabolites screened by univariate statistical analysis

Fig. 4

The metabolite contents of MGCs and CCs were analyzed by hierarchical clustering, and the left colored bar indicates the range of fold change values. A is in anion mode, and B is in positive mode

Fig. 5

PCA (A), PLS-DA (B), and OPLS-DA (C) plots obtained for metabolite extracts from MGCs and CCs. The x-coordinate represents the first principal component with t [1], and the y-coordinate represents the second principal component with t [2]

Fig. 6

Metabolic pathways in which significant metabolites are involved. The P value in the KEGG pathway enrichment results was small (P < 0.05), and KEGG pathway enrichment was more statistically significant

Fig. 7

Gene expression of MGCs and CCs was evaluated by qPCR. A shows the gene expression of ABCA1, ABCG1, LDLR, and SCARB1; B shows the gene expression of SULT2B1, STS, and CYP19A1. In A and B, the gene expression of MGCs is considered to be 1; C shows the gene expression of LPCAT3 and PLA2G4A; D shows the gene expression of PTPMT1 and CRLS1. In C and D, the gene expression of MGCs is considered to be 1. *P < 0.05, **P < 0.01, Student’s t-test

Fig. 8

Protein expression in CCs and MGCs. Primary antibodies: mouse anti–human ABCA1 polyclonal antibody (Abcam; diluted 1:3000); rabbit anti–human LDLR polyclonal antibody (PTG; diluted 1:1000); rabbit anti–human SCARB1 polyclonal antibody (Abcam; diluted 1:1000); rabbit anti–human SULT2B1 polyclonal antibody (Abclonal; diluted 1:1000); rabbit anti–human CYP19A1 polyclonal antibody (Abclonal; diluted 1:1000); rabbit anti–human CRLS1 polyclonal antibody (PTG; diluted 1:1000); mouse anti–human LPCAT3 polyclonal antibody (Abcam; diluted 1:1000); rabbit anti–human PLA2G4A polyclonal antibody (Abclonal; diluted 1:1000). Secondary antibodies: goat anti-mouse IgG conjugated with horseradish peroxidase (HRP) diluted 1:3000 in TTBS for ABCA1 and LPCAT3 or goat anti–rabbit IgG conjugated with HRP diluted 1:3000 in TTBS for LDLR, SCARB1, SULT2B1, CYP19A1, CRLS1, and PLA2G4A. The expression of mouse anti–human ACTIN polyclonal antibody (Servicebio; diluted 1:3000) was used as an internal reference. A shows the protein expression of ABCA1, LDLR, SCARB1, SULT2B1, and CYP19A1; B shows the protein expression of CRLS1, LPCAT3, and PLA2G4A

Fig. 9

Steroid production in the culture medium from MGCs and CCs. The black color represents MGCs, and the gray color represents CCs. *P < 0.05, Student’s t-test