Title Page
Contents
ABSTRACT 9
Ⅰ. Introduction 11
Ⅱ. Materials and Methods 13
2.1. Animals and diet 13
2.2. Collection of blood serum 14
2.3. Measurement of body weights and fasting glucose levels 14
2.4. Isolation of ovarian granulosa cells 15
2.5. H&E staining of liver and visceral adipose tissues 16
2.6. RNA extraction and quantitative RT-PCR 16
2.7. RNA-sequencing 18
2.8. Immunofluorescence staining 19
2.9. Isolation of human granulosa cells 22
2.10. Statistical analysis 24
Ⅲ. Results 27
3.1. Evaluations of physiological and histological alterations in ND and HFD mice 27
3.2. RNA-sequencing analysis in granulosa cells (GCs) of ND and HFD mice 33
3.3. Downregulation of several immediate-early transcription factors (IE-TFs) in GCs of HFD mice 38
3.4. Localization of FOS and FOXL2 in isolated GCs and whole ovaries 43
3.5. Upregulation of inflammation-associated genes in GCs of HFD mice 48
3.6. Increased infiltration of F4/80- and CD68-positive cells in HFD ovaries 60
3.7. Downregulation of several IE-TFs in GCs of short-term HFD mice 63
3.8. Expression analysis of the selected genes in human GCs based on BMI 66
Ⅳ. Discussion 69
References 72
Abstract (in Korean) 79
Table 1. Primers for qRT-PCR in mouse GCs 17
Table 2. List of primary antibodies 20
Table 3. List of secondary antibodies 21
Table 4. Primers for RT-PCR in human GCs 23
Figure 1. A diagram depicting diet-induced obesity mouse model in this study. 26
Figure 2. Body weights and metabolic parameters of mice. 30
Figure 3. Histological analysis of liver and visceral adipose tissues. 32
Figure 4. A diagram depicting procedures for the isolation of mouse GCs. 35
Figure 5. Volcano plot of DEGs identified in RNA-seq analysis. 37
Figure 6. Heatmaps of downregulated DEGs in RNA-seq data. 40
Figure 7. Analysis of IE-TFs expression in GCs by qRT-PCR. 42
Figure 8. Immunofluorescence staining of FOS and FOXL2 in isolated GCs. 45
Figure 9. Immunofluorescence staining of FOS in ovaries. 47
Figure 10. Heatmap analysis of upregulated DEGs in RNA-seq data. 51
Figure 11. Analysis of Hpgds expression in isolated GCs by qRT-PCR. 53
Figure 12. Immunofluorescence staining of HPGDS and FOXL2 in isolated GCs. 55
Figure 13. Immunofluorescence staining of HPGDS in ovaries. 57
Figure 14. Immunofluorescence staining of CD45 in ovaries. 59
Figure 15. Immunofluorescence staining of F4/80 and CD68 in ovaries. 62
Figure 16. Expression of Egr1 and Fos in GCs of short-term groups by qRT-PCR. 65
Figure 17. Analysis of target genes in isolated human GCs by RT-PCR. 68