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The effect of malonate, succinate, oxaloacetate and 2-deoxy-D-glucose on boar sperm metabolism using ¹³C MRS

Nurul Fadhlina Ismail¹, Steven Reynolds¹, Sarah Calvert², Allan Pacey², and Martyn Paley¹

¹Academic Unit of Radiology, University of Sheffield, Sheffield, United Kingdom, ²Academic Unit of Reproductive & Developmental Medicine, University of Sheffield, Sheffield, United Kingdom

Synopsis

One in five young men has poor semen quality, including low motility. Studying energy metabolism may provide better understanding of sperm motility. We acquired ¹³C Magnetic Resonance spectra of sperm incubated with ¹³C-glucose with different concentrations of inhibitors: malonate, oxaloacetate, succinate, and 2-deoxy-D-glucose. This study examined the effect of these inhibitors on sperm lactate production and vitality, with a secondary aim to observe Krebs cycle intermediates in the MR spectrum. Glucose signal significantly decreased with increasing oxaloacetate concentration. Malonate and oxaloacetate and 2DG significantly decreased in lactate production. These inhibitors did not lead to observable ¹³C labelled Krebs cycle intermediates.

Introduction

One in five young men has poor semen quality¹, including low motility. An understanding of energy metabolism may play an important role in understanding male infertility. In order to understand sperm metabolism, we acquired ¹³C Magnetic Resonance (MR) spectra of sperm incubated with ¹³C labelled glucose with different concentrations of metabolic inhibitors. The inhibitors chosen were: malonate and oxaloacetate inhibitors for succinate dehydrogenase²; succinate, an inhibitor of α-ketoglutarate (αKG)³ and 2-deoxy-D-glucose(2DG) a competitive inhibitor with glucose⁴. This study examined the effect of these inhibitors on sperm lactate production and vitality, with a secondary aim to observe Krebs cycle intermediates in the MR spectrum.

Methods

Boar sperm (n=3 ejaculates) were washed using a 30/70% Percoll density centrifugation method⁵. 60µl of 100mM ¹³C_u-glucose, 12µl penicillin/streptomycin was added to 100µl of sperm. Inhibitors were added with final concentrations of 3-500mM, with PBS added to maintain a constant volume (570µl). Control samples, without inhibitor, were also prepared. Samples were incubated in a water bath at 37^oC for 18 hours and subsequently frozen at -80^oC (without preservative). For MRS experiments, 570µl of thawed sample was added to a 5mm MR tube with 10µl of 200mM ¹³C urea (frequency/concentration reference), 10µl D₂O and scanned using a 9.4T MR spectrometer with a 5mm BBO probe at 21°C. ¹³C spectra were acquired using a ¹³C{1H} inverse-gated pulse sequence (SW=239ppm, NS=4096, AQ=0.5s, TR=2s, flip angle=90°). Spectra were phase and baseline corrected using Bruker Topspin v2.1 software. Integral ranges for lactate, urea and glucose peaks were defined based on a control spectrum and applied to all spectra (Figure 1). Absolute lactate integrals only were normalized by sperm concentration. Sperm count, vitality and motility assays were carried out according to WHO guidelines⁶. Data analysis was performed using MATLAB and GraphPad PRISM 7. All glucose and lactate integrals were relative to their respective integrals in the control spectrum for each sample. Linear regression with Pearson correlation were applied to integrals versus log₁₀(inhibitor concentration). One-way ANOVA was used for comparison of mean values of the counts and motilities. The significance value for all tests were p <0.05. Values quoted are mean±SD.

Results

Sperm concentrations used for MRS experiments were 55.6-84.8x10⁶/ml with progressive motility between 3.5% and 8.9%(Table 1). Though the motility was low, there was no significant difference between sample counts and motilities (ANOVA) for the MRS experiments. There was no significant correlation for glucose integral versus inhibitor concentration for succinate, malonate and 2-deoxy-D-glucose (Figure 2). However, glucose significantly decreased with increasing oxaloacetate concentration. Increasing concentration of malonate caused the lactate integral and percentage vitality to decreased at a similar rate (lactate=15.3±0.6; vitality=15.4±4.3), suggesting that malonate had a toxic effect on sperm (Figure 3). Oxaloacetate had a similar effect on sperm vitality (slope=-17.6±0.9) as malonate (and succinate). However, oxaloacetate had the greatest rate of decrease in lactate signal (slope=-34.9±4.5), suggesting that, in addition to toxicity, there was an inhibitory effect. Sperm incubated with increasing 2DG concentration caused a decreased rate in lactate production that was in between that of malonate and oxaloacetate (2DG=-23±2). 2DG had the least toxicity effect on sperm vitality (slope=-7.1±0.7). Increasing concentration of succinate reduced sperm vitality comparable to malonate and oxaloacetate (slope=-18.4±1.6) (Figure 4). However, lactate integrals remained almost constant with increasing succinate concentration (slope=-3.6±5.5).

Discussion

At the concentrations used, both malonate and oxaloacetate showed similar levels of toxicity to sperm that would result in decreased lactate production. However, oxaloacetate, caused the greatest rate of decline in lactate production with increasing inhibitor concentration. This suggests that oxaloacetate, a known inhibitor of succinate dehydrogenase in the Krebs cycle, interfered with the availability of cofactors which were responsible for the observed change in lactate rather than oxaloacetate acting directly on the glycolytic pathways, as 2DG does. The observed result for succinate was surprising as it would be expected that the lactate integral would decrease at a similar rate to the vitality. Whether succinate feeds directly into the Krebs cycle to promote lactate production requires further investigation⁷. The usage of these inhibitors (malonate, succinate and oxaloacetate) to block steps within the Krebs cycle did not lead to an accumulation of ¹³C labelled intermediates at the blocked enzymes. Glucose signal significantly decreased with increasing oxaloacetate concentration but did not lead to increased lactate. It would be expected the total ¹³C signal for remain constant at all concentration. It is possible that ¹³C-glucose was converted into labelled substrate below the level of detection. Further study, including DNP and mass spectrometry, would help to confirm these findings.

Acknowledgements

1-Medical Research Council(MRC)

2-Ministry of Higher Education Malaysia

References

1. Kamel RM. Management of the infertile couple?: an evidence- based protocol. Reprod Biol Endocrinol. 2010;8(1):21.

2. Pardee A, Potter V. Inhibition of succinic dehydrogenase by oxalacetate. J Biol Chem. 1948.

3. Selak MA, Armour SM, MacKenzie ED, et al. Succinate links TCA cycle dysfunction to oncogenesis by inhibiting HIF-alpha prolyl hydroxylase. Cancer Cell. 2005;7(1):77-85.

4. Lahnsteiner F, Berger B, Weismann T. Sperm metabolism of the teleost fishes Chalcalburnus chalcoides and Oncorhynchus mykiss and its relation to motility and viability. J Exp Zool. 1999;284(4):454-465.

5. Lessley BA, Garner DL. Isolation of motile spermatozoa by density gradient centrifugation in Percoll®. Gamete Res. 1983;7(1):49-61.

6. World Health Organization (WHO). WHO laboratory manual for the Examination and processing of human semen. 2010;Fifth Edit:286.

Figures

Figure 1: ¹³C MR spectrum after incubation of sperm with ¹³Cu glucose for 18 hours and storage at -18°C. 10µl of 200mM ¹³C urea was added as a concentration and frequency reference. The spectrum from control sample shown here was used to define integral range for lactate, urea, and glucose (glucose = 63.80-62.99 ppm, 98.98-98.29 ppm; lactate = 23.01-22.56 ppm; and urea = 165.58-165.42 ppm). Red boxes showed the peaks integrated used in the analysis.

Table 1: Counts and motilities analyzed from samples (n= 5). Colored rows represent the animal sample that was used in each experiment respectively.

Figure 2: Percentage normalized mean of glucose integral after adding malonate, succinate, oxaloacetate, 2-Deoxy-D-glucose, sample number, n = 3, sig represents significant (p ≤ 0.05), ns = not significant. Concentration used: 3, 7, 15, 31, 62, 125, 250 and 500 mM.

Figure 3: Percentage normalized mean of lactate integral after adding malonate, succinate, oxaloacetate, 2-Deoxy-D-glucose, sample number, n = 3, sig represents significant (p ≤ 0.05), ns = not significant. Concentration used: 3, 7, 15, 31, 62, 125, 250 and 500 mM.

Figure 4: Regression plot for percentage of live sperm with increasing concentration of inhibitors after incubation for 1 hour, n = 3. Concentration used: 3, 7, 15, 31, 62, 125, 250 and 500 mM.

Proc. Intl. Soc. Mag. Reson. Med. 25 (2017)

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