Abnormal expression of fission and fusion genes and the morphology of mitochondria in eutopic and ectopic endometrium

Abstract

Mitochondria play a pivotal role in physiological and metabolic function of the cell. Mitochondrial dynamics orchestrate mitochondrial function and morphology, involving fission and fusion as well as ultrastructural remodeling. Mounting evidence unravels the close link between mitochondria and endometriosis. However, how mitochondrial architecture changes through fission and fusion in eutopic and ectopic tissues of women with ovarian endometriosis remains unknown. We detected the expression of fission and fusion genes and the morphology of mitochondria in eutopic and ectopic endometrium in ovarian endometriosis. The results showed that the expression of DRP1 and LCLAT1 was upregulated in eutopic endometrial stromal cells (ESCs), and the expression of DRP1, OPA1, MFN1, MFN2, and LCLAT1 was significantly downregulated in ectopic ESCs, and reduced number of mitochondria, wider cristae width and narrower cristae junction width was observed, but there was no difference in cell survival rate. The altered mitochondrial dynamics and morphology might, respectively, provide an advantage for migration and adhesion in eutopic ESCs and be the adaptive response in ectopic endometrial cells to survive under hypoxic and oxidative stress environment.

Keywords: Cristae; Endometriosis; Fission; Fusion; Mitochondria; Mitochondrial morphology.

Fig. 1

Culture and identification of ESCs in vitro. a ESCs were isolated from endometrial tissues and the morphology of the cultured ESCs was observed under a microscope (scale bars, 100 μm). b Identification of ESCs by immunofluorescent staining. ESCs were labeled with vimentin (red), and nuclear were labeled with Hoechst 33342 (blue)

Fig. 2

The mRNA expression of genes related to mitochondrial dynamics. Analysis of mRNA expression of DRP1, OPA1, MFN1, MFN2, and LCLAT1 (a–e) in CE, EU, and EC by qRT-PCR (*P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001)

Fig. 3

The protein expression of proteins related to mitochondrial dynamics. Analysis of protein expression of DRP1, OPA1, MFN1, MFN2, and LCLAT1 (A–E) in CE, EU, and EC by western blotting (*P < 0.05; **P < 0.01; ***P < 0.001)

Fig. 4

Representative images and analysis of immunohistochemical staining. Representative images and analysis of immunohistochemical staining of DRP1, OPA1, MFN1, MFN2, and LCLAT1 in controlled, eutopic, and ectopic endometrial tissues (magnification, ×400; scale bar, 200 μm)

Fig. 5

Transmission electron microscopy images of mitochondria in CE, EU, and EC. a Representative images of cells in CE, EU, and EC tissues (magnification, ×6800; scale bar, 5 μm). b Representative images of mitochondria in CE, EU, and EC tissue (23,000×; scale bar, 2 μm) and their locally magnified images. Red arrows: mitochondrial cristae width; blue arrows: width of mitochondria cristae junction. c–f Quantification of mitochondrial number per 30 μm² (c), mitochondrial length (d), cristae width (e), and cristae junction width (f) between the three groups (n = 21–42 cells per group and 65–92 mitochondria per group and 45–69 cristae per group. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001)

Fig. 6

Analysis of mitochondrial mass between CE, EU, and EC using flow cytometry. The fluorescent signal was quantified by flow cytometry and is displayed as histograms (a–d) and a bar graph (e) (*P < 0.05; **P < 0.01)

Fig. 7

Immunofluorescence images of mitochondria in CE, EU, and EC. a Immunofluorescence images. b The processed images using MiNA plugin of Fiji software. c–f Quantitative results of individuals (c), networks (d), individuals/networks (e), and mean branch length (f) of mitochondria (n = 50–70 cells per group. Scale bar, 10 μm. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001)

Fig. 8

Mitochondrial membrane potential. Mitochondrial membrane potential of CE (a), EU (b), and EC (c) and statistic graph (d)