Using an Extracellular Flux Analyzer to Measure Changes in Glycolysis and Oxidative Phosphorylation during Mouse Sperm Capacitation

10.3791/60815 ◽  
2020 ◽  
Author(s):  
Melanie Balbach ◽  
Jochen Buck ◽  
Lonny R. Levin
Keyword(s):  
Oxidative Phosphorylation ◽  
Sperm Capacitation ◽  
Mouse Sperm ◽  
Extracellular Flux

scholarly journals Sialylation of Asparagine 612 Inhibits Aconitase Activity during Mouse Sperm Capacitation; a Possible Mechanism for the Switch from Oxidative Phosphorylation to Glycolysis

2020 ◽  
Vol 19 (11) ◽  
pp. 1860-1875
Author(s):  
Ana Izabel Silva Balbin Villaverde ◽  
Rachel A. Ogle ◽  
Peter Lewis ◽  
Vincenzo Carbone ◽  
Tony Velkov ◽  
...  
Keyword(s):  
Oxidative Phosphorylation ◽  
Serine Proteases ◽  
Alpha Helix ◽  
Complete Loss ◽  
Loss Of Function ◽  
Sperm Capacitation ◽  
Aconitate Hydratase ◽  
Mouse Sperm ◽  
Cycle Enzyme ◽  
Aconitase Activity

After ejaculation, mammalian spermatozoa must undergo a process known as capacitation in order to successfully fertilize the oocyte. Several post-translational modifications occur during capacitation, including sialylation, which despite being limited to a few proteins, seems to be essential for proper sperm-oocyte interaction. Regardless of its importance, to date, no single study has ever identified nor quantified which glycoproteins bearing terminal sialic acid (Sia) are altered during capacitation. Here we characterize sialylation during mouse sperm capacitation. Using tandem MS coupled with liquid chromatography (LC–MS/MS), we found 142 nonreductant peptides, with 9 of them showing potential modifications on their sialylated oligosaccharides during capacitation. As such, N-linked sialoglycopeptides from C4b-binding protein, endothelial lipase (EL), serine proteases 39 and 52, testis-expressed protein 101 and zonadhesin were reduced following capacitation. In contrast, mitochondrial aconitate hydratase (aconitase; ACO2), a TCA cycle enzyme, was the only protein to show an increase in Sia content during capacitation. Interestingly, although the loss of Sia within EL (N62) was accompanied by a reduction in its phospholipase A1 activity, a decrease in the activity of ACO2 (i.e. stereospecific isomerization of citrate to isocitrate) occurred when sialylation increased (N612). The latter was confirmed by N612D recombinant protein tagged with both His and GFP. The replacement of Sia for the negatively charged Aspartic acid in the N612D mutant caused complete loss of aconitase activity compared with the WT. Computer modeling show that N612 sits atop the catalytic site of ACO2. The introduction of Sia causes a large conformational change in the alpha helix, essentially, distorting the active site, leading to complete loss of function. These findings suggest that the switch from oxidative phosphorylation, over to glycolysis that occurs during capacitation may come about through sialylation of ACO2.

scholarly journals Sialylation of Asparagine 612 inhibits Aconitase activity during mouse sperm capacitation; A possible mechanism for the switch from oxidative phosphorylation to glycolysis

2020 ◽  
Author(s):  
Ana Izabel Silva Balbin Villaverde ◽  
Rachel Ogle ◽  
Peter Lewis ◽  
Louise Hetherington ◽  
Vince Carbone ◽  
...  
Keyword(s):  
Sialic Acid ◽  
Oxidative Phosphorylation ◽  
Serine Proteases ◽  
Computer Modelling ◽  
Alpha Helix ◽  
Loss Of Function ◽  
Sperm Capacitation ◽  
Aconitate Hydratase ◽  
Mouse Sperm ◽  
Cycle Enzyme

AbstractAfter ejaculation, mammalian spermatozoa must undergo a process known as capacitation in order to successfully fertilize the oocyte. Several post-translational modifications occur during capacitation, including sialylation, which despite being limited to a few proteins, seems to be essential for proper sperm-oocyte interaction. Regardless of its importance, to date, no single study has ever identified nor quantified which glycoproteins bearing terminal sialic acid (Sia) are altered during capacitation. Here we characterize sialylation during mouse sperm capacitation. Using tandem mass spectrometry coupled with liquid chromatography (LC-MS/MS), we found 142 non-reductant peptides, with 9 of them showing potential modifications on their sialylated oligosaccharides during capacitation. As such, N-linked sialoglycopeptides from C4b-binding protein, endothelial lipase (EL), serine proteases 39 and 52, testis-expressed protein 101 and zonadhesin were reduced following capacitation. In contrast, mitochondrial aconitate hydratase (aconitase; ACO2) was the only protein to show an increase in Sia content during capacitation. Interestingly, while the loss of Sia within EL (N62) was accompanied by a reduction in its phospholipase A1 activity, the increase of sialylation in the ACO2 (N612) also resulted in a decrease of the activity of this TCA cycle enzyme. The latter was confirmed by N612D recombinant protein with both His and GFP tag, in which the N612D mutant had no activity compared to WT when protein. Computer modelling show that N612 sits atop the catalytic site of ACO2. The introduction of sialic acid causes a large confirmation change in the alpha helix, essentially, distorting the active site, leading to complete loss of function. These findings suggest that the switch from oxidative phosphorylation, over to glycolysis that occurs during capacitation may come about through sialylation of ACO2.

scholarly journals Metabolic changes in mouse sperm during capacitation†

2020 ◽  
Vol 103 (4) ◽  
pp. 791-801 ◽  
Author(s):  
Melanie Balbach ◽  
Maria Gracia Gervasi ◽  
David Martin Hidalgo ◽  
Pablo E Visconti ◽  
Lonny R Levin ◽  
...  
Keyword(s):  
Oxidative Phosphorylation ◽  
Experimental Evidence ◽  
Glucose Uptake ◽  
Energy Demand ◽  
Metabolic Changes ◽  
Mouse Sperm ◽  
Dormant State ◽  
Extracellular Flux ◽  
Mammalian Sperm ◽  
Sufficient Energy

Abstract Mammalian sperm are stored in the epididymis in a dormant state. Upon ejaculation, they must immediately start producing sufficient energy to maintain motility and support capacitation. While this increased energy demand during capacitation is well established, it remains unclear how mouse sperm modify their metabolism to meet this need. We now show that capacitating mouse sperm enhance glucose uptake, identifying glucose uptake as a functional marker of capacitation. Using an extracellular flux analyzer, we show that glycolysis and oxidative phosphorylation increase during capacitation. Furthermore, this increase in oxidative phosphorylation is dependent on glycolysis, providing experimental evidence for a link between glycolysis and oxidative phosphorylation in mouse sperm.

scholarly journals Involvement of a Na+/HCO Cotransporter in Mouse Sperm Capacitation

2002 ◽  
Vol 278 (9) ◽  
pp. 7001-7009 ◽  
Author(s):  
Ignacio A. Demarco ◽  
Felipe Espinosa ◽  
Jennifer Edwards ◽  
Julian Sosnik ◽  
José Luis de la Vega-Beltrán ◽  
...  
Keyword(s):  
Sperm Capacitation ◽  
Mouse Sperm

scholarly journals Inhibition of Mouse Sperm Capacitation by Ethanol

1982 ◽  
Vol 27 (4) ◽  
pp. 833-840 ◽  
Author(s):  
Robert A. Anderson ◽  
Jakkidi M. Reddy ◽  
Cathy Joyce ◽  
Brian R. Willis ◽  
Hans Van der Ven ◽  
...  
Keyword(s):  
Sperm Capacitation ◽  
Mouse Sperm

Levels of sulfogalactosylglycerolipid in capacitated motile and immotile mouse spermatozoa

1990 ◽  
Vol 68 (2) ◽  
pp. 528-535 ◽  
Author(s):  
Nongnuj Tanphaichitr ◽  
Jacqueline Smith ◽  
Morris Kates
Keyword(s):  
Sperm Motility ◽  
Culture Medium ◽  
Mature Male ◽  
Percoll Gradient ◽  
Direct Relation ◽  
Sperm Capacitation ◽  
Epididymal Sperm ◽  
Pellet Fraction ◽  
Mouse Sperm ◽  
Time Point

The purpose of this study was to determine whether sulfogalactosylglycerolipid (SGG) was desulfated during mouse sperm capacitation. Levels of [35S]SGG were determined in freshly retrieved caudal epididymal sperm, motile capacitated sperm, and immotile sperm, after feeding mature male mice with [35S]sulfate-laced chow for 32 days. Caudal epididymal sperm and coisolated epididymal cells were separated into pellet and interphase fractions by centrifugation through a two-step Percoll gradient (45 and 90%). Upon resuspension in Krebs–Ringer bicarbonate medium supplemented with 0.4% bovine serum albumin, the Percoll-gradient pellet fraction consisted mainly of motile capacitated sperm, whereas the interphase fraction comprised largely immotile sperm and fragmented epididymal epithelial cells. The level of [35S]SGG in the Percoll-gradient-pelleted sperm appeared to be much higher than that in the Percoll-gradient interphase sperm. Percoll-gradient-pelleted sperm were further incubated in the culture medium for 2 h. The level of [35S]SGG showed little or no change after 1 h, but was reduced appreciably after 2 h. At this time point, sperm motility was also decreased. Reduction of sperm SGG is correlated with sperm immotility and (or) senescence and may have no direct relation to the capacitation process.Key words: sulfogalactosylglycerolipid, sperm motility, sperm capacitation.

Glycolytic Substrates Have Differential Effects on Mouse Sperm Capacitation and Hyperactivation.

2011 ◽  
Vol 85 (Suppl_1) ◽  
pp. 187-187
Author(s):  
Summer G. Goodson ◽  
Yunping Qiu ◽  
Guoxiang Xie ◽  
Wei Jia ◽  
Deborah A. O'Brien
Keyword(s):  
Sperm Capacitation ◽  
Mouse Sperm ◽  
Differential Effects
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