Pyrophosphate Supplementation Prevents Chronic and Acute Calcification in ABCC6-Deficient Mice.

Soft tissue calcification occurs in several common acquired pathologies, such as diabetes and hypercholesterolemia, or can result from genetic disorders. ABCC6, a transmembrane transporter primarily expressed in liver and kidneys, initiates a molecular pathway inhibiting ectopic calcification. ABCC6 facilitates the cellular efflux of ATP, which is rapidly converted into pyrophosphate (PPi), a major calcification inhibitor. Heritable mutations in ABCC6 underlie the incurable calcification disorder pseudoxanthoma elasticum and some cases of generalized arterial calcification of infancy. Herein, we determined that the administration of PPi and the bisphosphonate etidronate to Abcc6-/- mice fully inhibited the acute dystrophic cardiac calcification phenotype, whereas alendronate had no significant effect. We also found that daily injection of PPi to Abcc6-/- mice over several months prevented the development of pseudoxanthoma elasticum-like spontaneous calcification, but failed to reverse already established lesions. Furthermore, we found that the expression of low amounts of the human ABCC6 in liver of transgenic Abcc6-/- mice, resulting in only a 27% increase in plasma PPi levels, led to a major reduction in acute and chronic calcification phenotypes. This proof-of-concept study shows that the development of both acute and chronic calcification associated with ABCC6 deficiency can be prevented by compensating PPi deficits, even partially. Our work indicates that PPi substitution represents a promising strategy to treat ABCC6-dependent calcification disorders.

Lactate and adenosine triphosphate in the extender enhance the cryosurvival of rat epididymal sperm.

We evaluated the cryosurvival of rat epididymal sperm preserved in raffinose-modified Krebs-Ringer bicarbonate-egg yolk extender supplemented with various energy-yielding substrates (glucose, pyruvate, lactate, and ATP) and assessed the effect on sperm oxygen consumption. The incubation of sperm at 37 degrees C for 10 min in lactate-free extender decreased sperm motility and oxygen consumption before and after thawing compared with those of sperm in glucose- and pyruvate-free mediums. We then focused on the effect of supplementing the extender with lactate (0, 10.79, 21.58, 32.37, and 43.16 mM) and found that sperm frozen and thawed in extender supplemented with 32.37 mM lactate exhibited the highest motility. When we supplemented extender containing 32.37 mM lactate with ATP (0, 0.92, 1.85, 3.70, and 5.55 mM), sperm frozen and thawed in the extender supplemented with 1.85 mM ATP exhibited considerably higher motility and viability than those of sperm frozen and thawed in ATP-free extender. These results provide the first evidence that supplementation of the raffinose-modified Krebs-Ringer bicarbonate-egg yolk extender with 32.37 mM lactate and 1.85 mM ATP increases of number of motile sperm before freezing and enhances the cryosurvival of rat sperm. These supplements to the extender may enhance sperm cryosurvival by improving the metabolic capacity of sperm before freezing.

Extracellular ATP and dibutyryl cAMP enhance the freezability of rat epididymal sperm.

We studied the effects of ATP, ionomycin, and dibutyryl cAMP (dbcAMP) on the motility, freezability, and oxygen consumption of rat epididymal sperm. In vitro fertilization and intrauterine insemination were performed by using frozen-thawed rat sperm. Frozen-thawed sperm diluted in raffinose-modified Krebs-Ringer bicarbonate solution-egg yolk extender containing 1.85 mM ATP and 100 microM dbcAMP exhibited considerably higher motility and viability than sperm diluted in dbcAMP-free extender. Addition of ionomycin and dbcAMP to ATP-containing extenders did not alter the oxygen consumption rate of sperm, suggesting that extracellular ionomycin and dbcAMP are not involved in the mobilization of mitochondrial energy substrates in sperm. Further, high rates of pronucleus formation and progression to the blastocyst stage were observed in embryos produced by the fertilization of oocytes with fresh sperm in an in vitro fertilization medium supplemented with ATP and dbcAMP. Oocytes were not penetrated by frozen-thawed sperm when cocultured with cumulus-oocyte complexes in a medium without ATP and dbcAMP. In contrast, cryopreserved sperm penetrated oocytes when the gametes were cultured in an ATP- and dbcAMP-containing medium, and the resultant embryos formed blastocysts. Our results show that the dilution of rat sperm in raffinose-modified Krebs-Ringer bicarbonate solution-egg yolk extender supplemented with ATP and dbcAMP prior to sperm cryopreservation enhances the freezability of the cryopreserved sperm. Furthermore, the in vitro fertilization medium we developed effectively supports the production of embryos from both fresh and cryopreserved rat sperm.