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Mitofusin 2 plays a role in oocyte and follicle development, and is required to maintain ovarian follicular reserve during reproductive aging - PubMed

  • ️Tue Jan 01 2019

Mitofusin 2 plays a role in oocyte and follicle development, and is required to maintain ovarian follicular reserve during reproductive aging

Man Zhang et al. Aging (Albany NY). 2019.

Abstract

Mitochondria change their shape through fusion and fission in order to adapt to their metabolic milieu. Mitofusin-2 (MFN2) is a key regulatory protein in this process, mediating mitochondrial fusion and interaction with endoplasmic reticulum. Targeted deletion of Mfn2 in oocytes resulted in mitochondrial dysfunction and female subfertility associated with impaired oocyte maturation and follicle development. Oocytes lacking MFN2 showed shortened telomeres and increased apoptosis, resulting in compromised oocyte quality and accelerated follicular depletion, consistent with a reproductive aging phenotype.

Keywords: female fertility; follicle maturation; mitochondrial fusion; ovarian aging; ovarian function.

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Conflict of interest statement

CONFLICTS OF INTEREST: E.S. is a consultant for and receives research funding from the Foundation for Embryonic Competence.

Figures

Figure 1
Figure 1

Subfertility, and impaired follicle, oocyte and embryo development in Mfn2-/- mice. (A) Fertility of female Mfn2-/- (oocyte-specific Mfn2 knockout, Mfn2fl/fl/Zp3-Cre, referred to as Mfn2-/-) and WT mice (8-week-old, n = 7 for each genotype) was assessed by mating with WT males of proven fertility (male/female; 1:2) for 12 weeks. Mfn2-/- mice had smaller litter size (pups per litter) and litters per female compared with WT females. (B) Number of GV oocytes, MII oocytes, 2-cell embryos and blastocysts in Mfn2-/- and WT mice. (C, D) Oocytes at GV stage were collected from PMSG-primed Mfn2-/- and WT mice and analyzed after 18 h of culture under in vitro maturation conditions. Percentages of GVBD and of first polar body extrusion in Mfn2-/- and WT oocytes are shown. (E, G) After 18 h of IVM, Mfn2-/- and WT MII oocytes were stained with α-tubulin and DAPI. Left column, DAPI (blue); middle column, anti-α-tubulin antibody (green); right column, merged images of DAPI and anti-α-tubulin staining. Percentages of normal spindle morphology in Mfn2-/- and WT MII oocytes after IVM are shown. (F, H) Mature (MII) oocytes were collected from the oviducts of superovulated 8-week-old Mfn2-/- and WT mice and stained with α-tubulin and DAPI. Percentages of normal spindle morphology in Mfn2-/- and WT MII oocytes are shown. Data presented as mean ± SEM. *p < 0.05, ***p < 0.0001, vs. WT using t-test.

Figure 2
Figure 2

Mitochondrial function is impaired in Mfn2-/- oocytes. (A) ATP measurement in Mfn2-/- and WT mice GV stage oocytes. (B) mRNA expression of respiratory chain genes was assessed using qRT-PCR in GV stage oocytes collected from Mfn2-/- and WT mice. (C, F) Representative fluorescent micrographs of GV stage oocytes stained by mitochondria JC-1. Red fluorescence represents J-aggregate while green fluorescence represents JC-1 monomer. Mitochondrial membrane potential is indicated by the red/green fluorescence intensity ratio. (D, G) Fluorescence intensity of Carboxy-H2DCFDA was used to measure ROS levels after treatment with H2O2. (E) mtDNA copy number was determined by qRT-PCR in GV stage oocytes collected from Mfn2-/- and WT mice. (H) Mitochondria and ER were labeled by immunostaining with MitoTracker (red) and ER-Tracker (blue). (I) The percentages of oocytes with normal distribution of mitochondria in the Mfn2-/- and WT mice. (J) Representative electron microscopic graphs of oocytes from 8-week-old Mfn2-/- and WT mice (n=3 ovary from different mice assessed in each group). Arrows show mitochondria. (K, L) Mitochondrial size and aspect ratio in Mfn2-/- and WT oocytes. Data presented as mean ± SEM. *p < 0.05, **p < 0.01 vs. WT from t-test. ATP: Adenosine triphosphate. Ndufv1: NADH dehydrogenase (ubiquinone) flavoprotein 1; Sdhb: succinate dehydrogenase complex iron sulfur subunit B; Uqcrc2: ubiquinol cytochrome c reductase core protein 2; Cox1: cytochrome c oxidase subunit I; Atp5a1: ATP synthase, H+ transporting, mitochondrial F1 complex, alpha subunit 1.

Figure 3
Figure 3

Gene expression is altered in Mfn2-/- GV stage and secondary follicle enclosed oocytes. (A, E) Heatmaps showing differentially expressed genes in Mfn2-/- and WT GV oocytes and secondary follicle enclosed oocytes (SFO) from 8-week-old mice. The color spectrum ranging from red color to blue color indicates normalized levels of gene expression from high to low. (B, F) Volcano plots for RNA-seq comparing Mfn2-/- and WT GV oocytes and SFOs. Red spot represents –log10 (p-value) ≥ 2; blue spot represents the – log10 (p-value) < 2. (C, G) Gene ontology (GO) cluster analysis of the significant over-representation of elements in Mfn2-/- and WT GV oocytes and SFOs from 8-week-old mice. (D, H) Pathway enrichment analysis in Mfn2-/- oocytes compared to WT GV oocytes and SFOs.

Figure 4
Figure 4

Accelerated follicle depletion in Mfn2-/- mice. (A, C, E, G) Representative micrographs of 3-weeks and 2-, 6- and 12-months old Mfn2-/- and WT mice ovary sections stained with hematoxylin and eosin. (B, D, F, H) Follicle counts in 3-weeks and 2-, 6- and 12-months old Mfn2-/- and WT mice ovaries. Data presented as mean ± SEM. *p < 0.05 vs. WT using t-test.

Figure 5
Figure 5

Increased apoptosis in Mfn2-/- secondary follicle-enclosed oocytes is associated with increased ceramide and decreased junction protein expression. (A, C) Immunofluorescence staining of caspase-6 (green) and ceramide (green) in secondary follicles of Mfn2-/- and WT mice ovaries. DAPI was used to stain nuclei (blue). (B, D) Quantitative analysis of caspase-6 and ceramide immunofluorescence in secondary follicles of Mfn2-/- and WT mice ovaries. (E, G) Immunofluorescence staining of E-cadherin (red) and Connexin37 (red) in secondary follicles of Mfn2-/- and WT mice ovaries. (F, H) Quantitative analysis of E-cadherin and Connexin37 immunofluorescence in secondary follicles of Mfn2-/- and WT mice ovaries. Data presented as mean ± SEM. *p < 0.05 vs. WT using t-test.

Figure 6
Figure 6

The telomere is shorter in Mfn2-/- oocyte. (A) Ct values from quantitative real-time PCR (qRT-PCR) of GV oocytes, cumulus cells and white blood cells form Mfn2-/- and WT mice. (B) Standard curves were generated by serial dilution of known amounts of DNA to calculate relative DNA concentrations (log DNA) from Ct values of the qRT-PCR products. Orange squares, telomeres; blue diamonds, 36B4 single copy gene control. The correlation regression equation and coefficients (R2) of Ct versus log DNA are shown. (C) The relative telomere length of GV oocytes, cumulus cells and white blood are represented as ratio of T/S. (D) Immunofluorescence double staining of 53BP1 (green) and TRF1 (red) in cumulus oophorus complexes of Mfn2-/- and WT mice. DAPI was used to stain nuclei (blue). (E) Quantitative analysis of TRF1 immunofluorescence in Mfn2-/- and WT GV oocytes. The arrow showed the co-localization of 53BP1 and TRF1. Data presented as mean ± SEM. **p < 0.01, *p < 0.05 vs. WT using t-test.

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