Do the known capacitating agents HCO3− and serum albumin regulate the generation of ATP required for sperm motility and capacitation?
Serum albumin and HCO3− seem to regulate two separate pools of ATP by different mechanisms in human spermatozoa.
WHAT IS KNOWN ALREADY
Sperm capacitation is a maturation process that naturally occurs in the female reproductive tract preparing the sperm cell for fertilization. It is a highly energy-depending process as it involves hyperactive motility and substantial levels of protein phosphorylation.
STUDY DESIGN, SIZE, DURATION
Human sperm cells from four (motility experiments) and three (all other experiments) healthy donors were used. Untreated cells were compared with cells treated with HCO3− and serum albumin for up to 4 h.
PARTICIPANTS/MATERIALS, SETTING, METHODS
Changes in glycolysis and mitochondrial respiration rates upon treatment with serum albumin and HCO3− were analysed by metabolic tracing of 13C-labelled substrates and respirometry studies, respectively. Levels of hyperactive spermatozoa and ATP content were measured during 4 h of incubation under capacitating conditions.
MAIN RESULTS AND THE ROLE OF CHANCE
We found that HCO3− significantly (P < 0.05) increased glycolytic flux by >3-folds via a cAMP/PKA sensitive pathway. This was accompanied by an increase in hyperactive motility. In contrast, serum albumin significantly increased endogenous ATP levels by 50% without stimulating hyperactive motility or glycolysis, indicating that this pool of ATP is separately located from the HCO3−-induced ATP. The increase in ATP induced by albumin could be mimicked by treatment with the cholesterol acceptors 2-hydroxypropyl- and methyl-β-cyclodextrin and counteracted by co-incubation with cholesterol sulphate to the level of the non-treated control (P < 0.05), pointing to cholesterol extraction from the sperm cell membrane as the main mechanism. However, the concentration of cyclodextrins needed to directly detect cholesterol extraction from the sperm cells was not compatible with maintenance of sperm viability. The increase in ATP seemed not to be dependent on the sperm-specific Ca2+ channel CatSper. Finally, we demonstrated that neither HCO3− nor serum albumin stimulated mitochondrial respiration rates. However, serum albumin increased the respiratory capacity of mitochondria by >50%, an effect that was counteracted by HCO3−.
LIMITATIONS, REASONS FOR CAUTION
Great variation in motility and capacitation is observed between sperm cells from different species. Hence, caution should be taken when extrapolating the findings in this work on human spermatozoa to sperm from other species.
WIDER IMPLICATIONS OF THE FINDINGS
It is already established that an efficient energy-generation is required to support sperm motility and capacitation. However, the mechanisms explaining how ATP production is regulated in spermatozoa are not fully understood. Our findings indicate that HCO3− stimulates hyperactive motility by increasing glycolytic flux and ATP production in a cAMP/PKA sensitive fashion. On the other hand, serum albumin seems to increase ATP concentration at a different location and by a mechanism different from glycolysis that involves extraction of cholesterol from the sperm cell membrane. These new insights into sperm metabolism may pave the way for both the development of new and improved male contraceptives and optimized assisted reproduction techniques.
The work was funded by Spermatech AS, The University of Oslo and the Research Council of Norway.
T.H.H. and K.R.R. are employees at Spermatech. B.S.S is a shareholder in Spermatech.
Keywords: human serum albumin; HCO3−; sperm metabolism; protein kinase A; capacitation
Journal Article. 8477 words. Illustrated.
Subjects: Reproductive Medicine
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