The effects of voluntary exercise on oocyte quality in a diet-induced obese murine model

Abstract

Obesity negatively affects many aspects of the human body including reproductive function. In females, the root of the decline in fertility is linked to problems in the oocyte. Problems seen in oocytes that positively correlate with increasing BMI include changes to the metabolism, lipid accumulation, meiosis, and metaphase II (MII) spindle structure. Studies in mice indicate that dietary interventions fail to reverse these problems. How exercise affects the oocytes has not been addressed. Therefore, we hypothesized an exercise intervention would improve oocyte quality. Here we show that in a mouse model of an exercise, intervention can improve lipid metabolism in germinal vesicle (GV) stage oocytes. Oocytes significantly increased activity and transcription of the β-oxidation enzyme hydroxyacyl-coenzyme A dehydrogenase in response to exercise training only if the mice had been fed a high-fat diet (HFD). An exercise intervention also reversed the lipid accumulation seen in GV stage oocytes of HFD females. However, delays in meiosis and disorganized MII spindles remained present. Therefore, exercise is able to improve, but not reverse, damage imparted on oocytes as a result of an HFD and obesity. By utilizing an exercise intervention on an HFD, we determined only lipid content, and lipid metabolism is changed in GV oocytes. Moving forward, interventions to improve oocyte quality may need to be more targeted to the oocyte specifically. Because of the HFD-induced deficiency in β-oxidation, dietary supplementation with substrates to improve lipid utilization may be more beneficial.

Figure 1. Body weights and body composition and metabolic parameters of mice

A) Average body weights; B) Fat Mass and C) Lean Mass as measured by magnetic resonance imaging. D) Homeostatic Model Assessment Indexes (HOMA1-IR) calculated as described in Materials and Methods. E) Glucose Tolerance Test, F) Fasting serum triglycerides different letters designate statistical significance, same letter indicates no significance between groups, p < 0.05.. In A–D and F) different letters designate statistical significance between groups whereas same letter indicates no significance between groups, p < 0.05. In E) letters above each time point represent statistical significance between groups at that time as follows; a) CD Exercise vs HFD exercise, b) CD Exercise vs HFD Sedentary, c) CD Sedentary vs. HFD Exercise, d) CD Sedentary vs. HFD Sedentary, e) HFD Exercise vs. HFD Sedentary, P<0.05. All Data was analyzed by two-way ANOVA with Tukey-Kramer correction for multiple comparisons. CD: Control Diet, HFD: High Fat Diet.

Figure 2. Lipid droplet accumulation in germinal vesicle stage oocytes in response to diet and exercise training

A) Representative images of oocytes from BODIPY 493/503 staining. All images were taken at the same keeping confocal settings identical, scale bars: 25 μm B) Mean relative gray value of fluorescence of individual oocyte area measured using ImageJ. C) Representative images of sections of oocytes taken by transmission electron microscopy at 2,000x magnification, scale bars: 2μm. Black arrowheads indicate lipid droplets. D) Total number of lipid droplets counted in images from each group. Different letters designate statistical significance, same letter indicates no significance between groups, p < 0.05; two-way ANOVA with Tukey-Kramer correction for multiple comparisons.

Figure 3. Metabolic enzyme activity, metabolite levels, and transcript levels are altered by diet and exercise

A) Hadha enzymatic activity was measured in individual GV-stage oocytes, (n=15 oocytes/group). Two-way ANOVA with multiple comparisons; different letters represent statistical significance between groups, p<0.001 B) ATP and C) citrate content of GV-stage oocytes as measured by enzymatic cycling assay (n=45 oocytes/group). Two way ANOVA with multiple comparisons; * P<0.05, **P<0.01 D)Relative abundance of Cpt2, Hadh, Idh3b, and Aco2 transcripts. 3 oocytes per mouse were collected from 6 individual mice per experimental condition and relative abundance of transcripts was calculated using the ΔΔC_T method, with Actb as the reference gene. Two-way ANOVA with multiple-comparisons; For Idh3b, a – no change between groups; b – P<0.001for Hadh Sedentary vs. Exercise. For Aco2, the difference was not statistically significant but is trending towards significance (p=0.015).

Figure 4. Transmission electron microscopy of GV stage oocytes

A) Representative image of round (normal), elliptical and dumbbell shaped oocyte mitochondria. B) Percentages of shape of mitochondria from each cohort of mice (n=7–9 oocytes/cohort). C) Representative image of normal oocyte mitochondria and rose petal mitochondria. D) Comparison of the number of rose petal mitochondria counted in oocytes from each cohort (n=7–9 oocytes/cohort); two-way ANOVA with multiple comparisons; different letters designate statistical significance, same letter indicates no significance between groups, p < 0.05. All images are at 15,000x magnification, scale bars: 500 nm.

Figure 5. Meiotic progression and spindle structure of meiosis-II stage (MII) oocytes

A) Representative images of each classification category of meiotic anomalies. Arrow indicates polar body. Apoptotic indicates and apoptotic oocyte contained within the zona pellucida, scale bars: 25 μm. B) Graph depicting percentage of oocyte in each category from each of the four experimental conditions. CD: Control Diet, HFD: High Fat Diet