Polyploidy in Xenopus lowers metabolic rate by decreasing total cell surface area

Abstract

Although polyploidization is frequent in development, cancer, and evolution, impacts on animal metabolism are poorly understood. In Xenopus frogs, the number of genome copies (ploidy) varies across species and can be manipulated within a species. Here, we show that triploid tadpoles contain fewer, larger cells than diploids and consume oxygen at a lower rate. Drug treatments revealed that the major processes accounting for tadpole energy expenditure include cell proliferation, biosynthesis, and maintenance of plasma membrane potential. While inhibiting cell proliferation did not abolish the oxygen consumption difference between diploids and triploids, treatments that altered cellular biosynthesis or electrical potential did. Combining these results with a simple mathematical framework, we propose that the decrease in total cell surface area lowered production and activity of plasma membrane components including the Na⁺/K⁺ ATPase, reducing energy consumption in triploids. Comparison of Xenopus species that evolved through polyploidization revealed that metabolic differences emerged during development when cell size scaled with genome size. Thus, ploidy affects metabolism by altering the cell surface area to volume ratio in a multicellular organism.

Keywords: Kleiber’s law; Xenopus; cell size; energy budget; metabolism; oxygen consumption rate; polyploidy; scaling.

Figure 1.. Triploid X. laevis develop normally.

(A) Triploid X. laevis were obtained by blocking polar body extrusion post fertilization (p.f.) using a cold shock. (B) The 1.5-fold increase in chromosome number in triploid (3N) compared to diploid (2N) embryos was verified by metaphase chromosome spreads. Scale bar = 10 μm. (C) Representative images of diploid and triploid embryos obtained from the same clutch of eggs. Scale bar = 1 mm. (D) Compared to diploids, triploids showed a comparable mass increase except for a transient ~ 5% reduction during day 3 (19 clutches, n=10 to 82 embryos per ploidy and stage, details in Table S1). (E) TMR-phalloidin staining of actin in ventral epithelial cells of stage 41 diploid and triploid X. laevis embryos showing cell outlines and strong actin enrichment at the surface of multiciliated cells. (F) Area of diploid and triploid multiciliated cells in embryos at stages 33–34, 37–38 and 41, normalized to diploids of each clutch. (3 clutches, n>150 for each condition). (D), (F), Welch two sample t-test comparing the means, *: p<0.5, **: p<0.01,***: p<0.001. See also Figure S1.

Figure 2.. Metabolism assessed by single-tadpole oxygen consumption rates vary between diploids and triploids of both X. laevis and X. borealis.

(A) Single tadpoles were placed in individual glass vials sealed from air. The decrease in O₂ levels over time was used to calculate the oxygen consumption rate (OCR) for each tadpole. (B) OCR as a function of body mass in diploid X. laevis embryos from day 2 to 5 p.f. The curve shows a fit B = B₀m^α with values B₀ = 0.0089 ± 0.0002 and α = 0.58 ± 0.02 (n=271) (see Methods and Table S2). (C) OCR for bins of body mass and stages in X. laevis (left panel) and X. borealis (right panel) of diploid and triploid embryos (t-test comparing the means *= p<0.5, **= p<0.01; 3 to 5 clutches per condition, details in Table S3). (D) OCR as a function of body mass in diploid (n=189) and triploid (n=165) tadpoles from day 3 (stage 41) to day 5 (stage 46). The lines represent linear fits of the logarithmic values, which yielded the same allometric scaling component exponent α (α = 0.46 ± 0.03), but different Y-intercepts that indicate a lower basal metabolic rate (B₀) in triploids (see Methods and Table S2). See also Figure S2.

Figure 3.. An embryo energy budget establishes the costs of proliferation, biosynthesis, and maintenance.

(A) OCR per mass as a function of palbociclib doses for stage 41 diploid embryos and two bins of mass (6 clutches, n = 2 to 57 per bin of mass and palbociclib concentration). (B) Representative image of phospho-histone H3 immunostaining and Hoescht DNA dye staining of tadpole tails (scale bar = 200 μm). (C) Mitotic cell density in the tails of stage 41 diploid embryos assessed by quantifying phospho-histone H3-positive cells (n=2, 5–6 embryos per clutch, Welch two samples t-test comparing the means, ***: p<0.001). (D) OCR per mass for stage 41 diploid embryos in water, 6 μg/mL palbociclib, 12.5 nmol/mL torkinib, 1 μg/mL torin-1, 0.1% DMSO, or 200 μg/mL ouabain. (E) The percentage decrease of OCR in diploids upon treatment with drugs in (D) provides an estimate of the contribution of proliferation (palbociclib), growth (torkinib, torin-1), and maintenance (ouabain) to the overall embryo energy expenditure. See also Figure S3 and S4.

Figure 4.. The energetic cost of cell membrane maintenance, not proliferation, accounts for the difference in metabolism between diploids and triploids.

(A) Mitotic cell density in the tails of diploid and triploid embryos assessed by quantifying phosphor-histone H3-positive cells (n=3, 5–6 embryos per clutch) (B) OCR per mass normalized to the median diploid value for each condition in stage 41 tadpoles during a 6 hr incubation in water, 6 μg/mL palbociclib, 12.5 nmol/mL torkinib, 0.1% DMSO, or 200 μg/mL ouabain. Results are shown for 3–4 mg embryos except 5–6 mg for ouabain (n=3 to 5 clutches per condition, details in Table S4). Dashed line indicates the average in untreated triploids. (A), (B), Welch two samples t-test comparing the means, *: p<0.5, **: p<0.01,***: p<0.001. See also Figure S4.

Figure 5:. Metabolic rates of Xenopus species scale with cell size, not ploidy.

(A) X. longipes, X. laevis and X. tropicalis are 12N, 4N and 2N, respectively, with corresponding differences in chromosome (ch.) number; tree not to scale with phylogenetic distances. (B) Cell size measurements show scaling with genome size at stage 48, but not stage 41 (2–5 clutches, n>40 per condition, X. tropicalis and X. longipes stage 48 data from). (C) OCR across species: (top) before the onset of cell size scaling with genome size at day 3–4 (stage 41–46) and (bottom) after scaling onset (stage 48–50) at day 6–8 (X. laevis), 7–13 (X. longipes), and 26–46 (X. tropicalis) (see Table S5 for n of each bin). (D) OCR data at stages 48 or later (from Figure 5C, bottom) normalized to the mean X. laevis diploid value for each bin of mass (each dot is the value for a different bin of mass and species) and plotted as a function of mean cell area at stage 48 (Figure 5B) normalized to the X. laevis diploid value. (B) and (D): ρ is the Pearson’s correlation coefficient of the means weighted by the SD (*= p value <0.05, **= p value <0.01). See also Figure S2 and S5.