Mitochondrial Dysfunction in Advanced Maternal Aged Cumulus Cells: A Possible Link to ATP Synthase Impairment?

Abstract

Age-related changes in the mitochondrial status of human cumulus cells (hCCs) impact oocyte quality; however, the relationship between hCC mitochondrial (dys)function and reproductive aging remains poorly understood. This study aimed to establish the interplay between hCC mitochondrial dysfunction and women's reproductive potential. In this investigation, 266 women were enrolled and categorized into two groups based on their age: a young group (<35 years old) and an advanced maternal age (AMA) group (≥35 years old). Comprehensive analysis of reproductive outcomes was conducted in our population. Various mitochondrial-related parameters were analyzed across distinct subsets. Specifically, mitochondrial membrane potential (∆Ψm) and mitochondrial mass were examined in 53 samples, mtDNA content in 25 samples, protein levels in 23 samples, bioenergetic profiles using an XF24 Extracellular Flux Analyzer in 6 samples, and levels of reactive oxygen species (ROS) and adenosine triphosphate (ATP) in 39 and 43 samples, respectively. In our study, the reproductive potential of AMA women sharply decreased, as expected. Additionally, an impairment in the mitochondrial function of hCCs in older women was observed; however, no differences were found in terms of mitochondrial content. Regarding oxidative phosphorylation, metabolic profiling of hCCs from AMA women indicated a decrease in respiratory capacity, which was correlated with an age-dependent decrease in the ATP synthase (ATP5A1) protein level. However, intracellular ROS and ATP levels did not differ between groups. In conclusion, our study indicates that age-related dysfunction in hCCs is associated with impaired mitochondrial function, and, although further studies are required, ATP synthase could be relevant in this impairment.

Keywords: ATP synthase; cumulus cells; maternal aging; mitochondria; oxidative phosphorylation.

Figure 1

Schematic representation of the experimental design and samples analyzed. A total of 266 women were enrolled in the study and divided into two groups according to the age cut-off (35 years old). To ensure that age was the main relevant factor, a subgroup of fertile young women was created (n = 40) and compared with the young group. To analyse mitochondrial parameters, the samples were allocated into different groups: ∆Ψm (n = 53), mitochondrial mass (n = 53), ROS levels (n = 39), OXPHOS protein levels (n = 23), bioenergetic prolife (n = 6), mtDNA content (n = 25), and ATP levels (n = 43).

Figure 2

Mitochondrial function was impaired in hCCs from older women. TMRM (50 nM) and Mitotracker^® Green (100 nM) fluorescence area were monitored under fluorescence microscopy at 525 nm and 488 nm to indicate Δψm and mitochondrial content, respectively. (A) Representative fluorescence images of TMRM and Mitotracker^® Green among groups. Scale bar 20 μm (B) Median fluorescence area of cells positive TMRM for each group. Results are presented as mean ± SD (<35 n = 27 and ≥35 n = 26). A decrease in Δψm (p = 0.0017) in hCCs from the AMA group was observed. (C) Regression analysis of Δψm in the hCCs with age of patients (n = 53). The linear correlation between TMRM fluorescence in hCCs and age was analyzed, and Δψm tended to negatively correlate with maternal age. The linear equation was Y = −10.59×x + 37.98, with n = 53, r = −0.26, R² = 0.07, and p = 0.05. (D) Mitochondrial mass was detected by the fluorescence area of Mitotracker^® Green, and no difference was observed between groups (p = 0.81) (<35 n = 27 and ≥35 n = 26). (E) mtDNA content in hCCs from the young group and old group was analyzed by RT-qPCR. The box plot shows that the mtDNA content in hCCs from patients over 35 years old (n = 13) was equal (p > 0.99) to that of younger patients (n = 12). Statistical significance was considered when ** p < 0.01. Specific statistical tests: a t-test was performed for the ∆ψm and mitochondrial mass; a Mann–Whitney test was performed for mtDNA content.

Figure 3

Aerobic respiratory capacity is impaired in aged hCCs. (A) The OCR was measured using the Seahorse XFe24 Analyzer in hCCs from young (n = 3) and AMA (n = 3) women to detect several parameters related to aerobic respiratory capacity, including non-mitochondrial respiration (B), basal respiration (C), maximum respiration (D), ATP turnover (E), proton leak (F), and spare capacity (G). The aerobic respiratory capacity of the aged group was decreased in terms of basal respiration (p = 0.037), maximum respiration (p = 0.0037), and spare capacity (p = 0.002); no differences were observed in non-mitochondrial respiration (p = 0.25) or in proton leak (p = 0.105). In terms of ATP turnover, data suggest a decrease in ATP production (p = 0.054). In each assay, 5 × 10⁴ cells were plated overnight in experimental conditions and three measurements of OCR were performed before and after the sequential injection of each of the four compounds: Oligomycin (1 µM); FCCP (2 µM), and Antimycin A + Rotenone (1 µM each), respectively. Parameters of aerobic respiration were obtained as follows: non-mitochondrial respiration = OCR (AA/Rot); basal respiration = OCR (basal) − OCR (AA/Rot); ATP turnover = OCR (basal) − OCR (oligo); maximum respiration = OCR (FCCP) − OCR (AA/Rot). Spare capacity = OCR (FCCP) − OCR (basal) and Proton leak = OCR (Oligo) − OCR (AA/Rot). The arrow indicates the moment of compound injection. Specific statistical tests: a t-test was performed. Results are presented as the mean ± SD of three replicates. Statistical significance was considered when * p < 0.05 and ** p < 0.01.

Figure 4

OXPHOS complex levels in aged hCCs. OXPHOS components in hCCs from the young group and the AMA group were monitored by Western Blot (WB). In each assay, 20 μg of protein lysate was separated using a 12% SDS-PAGE, and protein loads were evaluated using calnexin, suggesting that the samples were loaded with a similar amount of protein in each lane. (A) Representative analysis of the WB of OXPHOS subunits (Complex I—NDUFB8, Complex II—SDHB, Complex III—UQCRC2, Complex IV—COX2, and ATP synthase—ATP5A) of hCCs in the young and AMA groups. (B) Densitometry of the OXPHOS subunit protein detection for young (n = 12) and AMA (n = 11) groups, relative to calnexin. The expression levels of ATP5A-related proteins were significantly decreased (p = 0.046) in the aged group compared with the young group. On the other hand, NDUFB8 (p = 0.89), SDHB (p = 0.75), UQCRC2 (p = 0.98), and COX2 (p > 0.99) subunits showed no differences between groups. (C) Representative analysis of the WB of OXPHOS subunits of hCCs in three young women and five AMA women. A one-way ANOVA test was performed for WB. Statistical significance was considered when * p < 0.05. Original western blotting figures can be found in Figure S2.

Figure 5

ATP and reactive oxygen species (ROS) levels in hCCs were not affected by aging. (A) Intracellular ATP content was measured by a luciferin-luciferase assay in hCCs from the young group (n = 24) and AMA group (n = 19). The box plot showed that the median intracellular ATP level for hCCs from the young and AMA groups was equal (p = 0.48). (B,C) Intracellular ROS in hCCs of the young group (n = 21) and AMA group (n = 18) were detected by fluorescence microscopy using H2DCFDA (10 μM), and nuclei were counterstained with Hoechst 33342 (5 μg/mL). (B) Representative fluorescence images of H2DCFDA and Hoechst 33342 for each group. (C) The boxplot represents the median fluorescence intensity of hCCs in women under 35 years old (n = 16) and above 35 years old (n = 13). The analysis indicates that there was no statistically significant difference in terms of intracellular ROS levels between the two groups (p = 0.79). A Mann–Whitney test was performed for both intracellular ATP and ROS levels. Scale bar: 20 μm.

Figure 5

ATP and reactive oxygen species (ROS) levels in hCCs were not affected by aging. (A) Intracellular ATP content was measured by a luciferin-luciferase assay in hCCs from the young group (n = 24) and AMA group (n = 19). The box plot showed that the median intracellular ATP level for hCCs from the young and AMA groups was equal (p = 0.48). (B,C) Intracellular ROS in hCCs of the young group (n = 21) and AMA group (n = 18) were detected by fluorescence microscopy using H2DCFDA (10 μM), and nuclei were counterstained with Hoechst 33342 (5 μg/mL). (B) Representative fluorescence images of H2DCFDA and Hoechst 33342 for each group. (C) The boxplot represents the median fluorescence intensity of hCCs in women under 35 years old (n = 16) and above 35 years old (n = 13). The analysis indicates that there was no statistically significant difference in terms of intracellular ROS levels between the two groups (p = 0.79). A Mann–Whitney test was performed for both intracellular ATP and ROS levels. Scale bar: 20 μm.