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. 2024 Apr 26;24(1):55.
doi: 10.1186/s12862-024-02238-x.

Lack of detectable sex differences in the mitochondrial function of Caenorhabditis elegans

Dillon E King #  1   2 , A Clare Sparling #  1 , Abigail S Joyce  3 , Ian T Ryde  1 , Beverly DeSouza  4 , P Lee Ferguson  3 , Susan K Murphy  1   2 , Joel N Meyer  5
Affiliations

Lack of detectable sex differences in the mitochondrial function of Caenorhabditis elegans

Dillon E King et al. BMC Ecol Evol. .

Abstract

Background: Sex differences in mitochondrial function have been reported in multiple tissue and cell types. Additionally, sex-variable responses to stressors including environmental pollutants and drugs that cause mitochondrial toxicity have been observed. The mechanisms that establish these differences are thought to include hormonal modulation, epigenetic regulation, double dosing of X-linked genes, and the maternal inheritance of mtDNA. Understanding the drivers of sex differences in mitochondrial function and being able to model them in vitro is important for identifying toxic compounds with sex-variable effects. Additionally, understanding how sex differences in mitochondrial function compare across species may permit insight into the drivers of these differences, which is important for basic biology research. This study explored whether Caenorhabditis elegans, a model organism commonly used to study stress biology and toxicology, exhibits sex differences in mitochondrial function and toxicant susceptibility. To assess sex differences in mitochondrial function, we utilized four male enriched populations (N2 wild-type male enriched, fog-2(q71), him-5(e1490), and him-8(e1498)). We performed whole worm respirometry and determined whole worm ATP levels and mtDNA copy number. To probe whether sex differences manifest only after stress and inform the growing use of C. elegans as a mitochondrial health and toxicologic model, we also assessed susceptibility to a classic mitochondrial toxicant, rotenone.

Results: We detected few to no large differences in mitochondrial function between C. elegans sexes. Though we saw no sex differences in vulnerability to rotenone, we did observe sex differences in the uptake of this lipophilic compound, which may be of interest to those utilizing C. elegans as a model organism for toxicologic studies. Additionally, we observed altered non-mitochondrial respiration in two him strains, which may be of interest to other researchers utilizing these strains.

Conclusions: Basal mitochondrial parameters in male and hermaphrodite C. elegans are similar, at least at the whole-organism level, as is toxicity associated with a mitochondrial Complex I inhibitor, rotenone. Our data highlights the limitation of using C. elegans as a model to study sex-variable mitochondrial function and toxicological responses.

Keywords: C. elegans; Mitochondria; Mitochondrial toxicity; Sex differences.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1
Characteristics of C. elegans sexes and strains used in this study. (A) Representative image of hermaphrodite C. elegans at the L4 stage. (B) Representative image of male C. elegans at the L4 stage. Images were taken at 10X using a Keyence BZ-X710. The scale bar represents 100 μm. (C) Proportion of males in each strain used in this study. Statistical significance of p < 0.05, determined by a one-way ANOVA with a Tukey post hoc test for multiple comparisons, is represented as (a) denoting the comparison of N2 to other strains, (b) denoting the comparison of N2 male enriched (N2ME) to other strains, and (c) denoting the comparison between fog-2, him-5, and him-8 (N2ME vs. him-8: p = 0.0006, N2ME vs. him-5: p = 0.0032, N2ME vs. fog-2: p = 0.0032, N2ME vs. N2: p < 0.0001, him-8 vs. him-5: p = 0.9163, him-8 vs. fog-2: p = 0.9656, him-8 vs. N2: p < 0.0001, him-5 vs. fog-2: p = 0.9998, him-5 vs. N2: p < 0.0001, fog-2 vs. N2: p < 0.0001). Six biological replicates were assessed, each with > 75 worms assessed per replicate
Fig. 2
Fig. 2
Mitochondrial parameters of male enriched strains. For all copy number values, copy number is expressed per worm. (A) mtDNA copy number (CN) of each strain, separated by sex. For copy number analysis, three biological replicates were performed. Data was assessed by two-way ANOVA for both sex and strain. Sex: p = 0.0002***, strain: p = 0.6079, interaction: p = 0.4746. (B) nuclear genome (nucDNA) copy number of each strain, separated by sex. Data was assessed by two-way ANOVA for both sex and strain. Sex: p = 0.7707, strain: p = 0.9277, interaction: p = 0.5256. (C) mtDNA: nucDNA copy number ratio of each strain, separated by sex. Data was assessed by two-way ANOVA for both sex and strain. Sex: p < 0.0001***, strain: p = 0.2810, interaction: p = 0.6026. (D) Mean ATP levels of each strain, in pmol of ATP per microgram of protein in each strain. For ATP level analysis, four biological replicates were performed. Data was assessed by a one-way ANOVA, p = 0.9625
Fig. 3
Fig. 3
Seahorse respirometry analysis across male enriched strains. (A) Representative image of the mitochondrial function parameters assessed using the inhibitors dicyclohexylcarbodiimide (DCCD), carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP), and sodium azide. The Seahorse XFe24 Analyzer determines the oxygen consumption rate (OCR) in pmol O2 per minute prior to and following injections of the compounds. (B) Mean basal OCR across strains. (C) Mean mitochondrial OCR across strains. (D) Mean maximal OCR across strains. (E) Mean spare capacity across strains. (F) Mean proton leak across strains. (G) Mean non-mitochondrial OCR across strains (N2 to him-5p = 0.0419, N2 to him-8p = 0.0481). For figures B-G, the x-axis displays the strain and the y-axis displays the OCR associated with each parameter in pmol/min, normalized to the volume of worm in each well in picoliters. Statistical significance of all pairwise comparisons were assessed using a one-way ANOVA with a Tukey post hoc test for multiple comparisons when normally distributed and a Kruskal-Wallis test with a Dunn’s post-hoc test for multiple comparisons when not normally distributed, as was the case for proton leak. Data represents five biological replicates per strain
Fig. 4
Fig. 4
Wild type hermaphrodites and males have similar sensitivity to developmental rotenone exposure. (A) Dose response curves for rotenone exposure in males and hermaphrodites expressed in terms of the raw value of the worm volume following a 72-hour exposure to rotenone. Significance was assessed by a two-way ANOVA for sex and dose, sex: ***, p < 0.0001, dose: ***, p < 0.0001, interaction: *, p = 0.0196. (B) Dose response curves for rotenone exposure in males and hermaphrodites with worm volume expressed as a percentage of the control. Significance was assessed by a two-way ANOVA for sex and dose, sex: p = 0.0685, dose: ***, p < 0.0001, interaction: p = 0.5749. For both graphs, the x-axis represents the log transform of the rotenone concentration, where the doses were 0, 0.125, 0.25, and 0.5 µM. For the 0 µM dose, a value of 0.01 was used to log transform the data, so -2 represents the vehicle control. Three biological replicates were assessed, each with > 50 worms assessed per replicate
Fig. 5
Fig. 5
Analysis of rotenone uptake in N2 and him-5 strains. (A) Dose response curves for rotenone exposure in N2 and him-5 strains expressed in terms of the raw value of the worm volume following a 72-hour exposure to rotenone. Significance was assessed by a two-way ANOVA for strain and dose, strain: p = 0.1342, dose: ***, p < 0.0001, interaction: p = 0.6392. (B) Dose response curves for rotenone exposure in N2 and him-5 strains with worm volume expressed as a percentage of the control. Significance was assessed by a two-way ANOVA for strain and dose, strain: p = 0.4153, dose: ***, p < 0.0001, interaction: p = 0.6118. For graphs in panels A and B, the x-axis represents the log transform of the rotenone concentration, where the doses were 0, 0.125, 0.25, and 0.5 µM. For the 0 µM dose, a value of 0.01 was used to log transform the data, so -2 represents the vehicle control. (C) Detected concentrations of rotenone in the dosing media following the 72-hour exposure. On the x-axis is the intended dose and on the y-axis is the detected amount remaining at the time of harvesting worms for analysis. Significance was assessed by a two-way ANOVA assessing strain and dose, strain: p = 0.7838, dose: ***, p < 0.0001, interaction: p = 0.2697. (D) Detected internal concentrations of rotenone in the worms following the 72-hour exposure. On the x-axis is the intended dose and on the y-axis is the internal dose calculated using the worm volume determined using Wormsizer. Significance was assessed by a two-way ANOVA assessing strain and dose with a Šidák post hoc test for multiple comparisons, strain: p = 0.0513, dose: ***, p < 0.0001, interaction: *, p = 0.0501. Three biological replicates were assessed
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