Abstract
Inorganic pyrophosphate (PPi) is generated by ATP hydrolysis in the cells and also present in extracellular matrix, cartilage and bodily fluids. Fueling an alternative pathway for energy production in cells, PPi is hydrolyzed by inorganic pyrophosphatase (PPA1) in a highly exergonic reaction that can under certain conditions substitute for ATP-derived energy. Recombinant PPA1 is used for energy-regeneration in the cell-free systems used to study the zymology of ATP-dependent ubiquitin-proteasome system, including the role of sperm-borne proteasomes in mammalian fertilization. Inspired by an observation of reduced in vitro fertilization (IVF) rates in the presence of external, recombinant PPA1, this study reveals, for the first time, the presence of PPi, PPA1 and PPi transporter, progressive ankylosis protein ANKH in mammalian spermatozoa. Addition of PPi during porcine IVF increased fertilization rates significantly and in a dose-dependent manner. Fluorometric assay detected high levels of PPi in porcine seminal plasma, oviductal fluid and spermatozoa. Immunofluorescence detected PPA1 in the postacrosomal sheath (PAS) and connecting piece of boar spermatozoa; ANKH was present in the sperm head PAS and equatorial segment. Both ANKH and PPA1 were also detected in human and mouse spermatozoa, and in porcine spermatids. Higher proteasomal-proteolytic activity, indispensable for fertilization, was measured in spermatozoa preserved with PPi. The identification of an alternative, PPi dependent pathway for ATP production in spermatozoa elevates our understanding of sperm physiology and sets the stage for the improvement of semen extenders, storage media and IVF media for animal biotechnology and human assisted reproductive therapies.
Highlights
Adenosine 59-triphosphate (ATP) is a fundamental factor to maintain the life, by providing energy, and controlling the cell function and metabolism
To determine if sperm storage in PPisupplemented Beltsville thawing solution (BTS) extender has a beneficial effect on sperm fertilizing ability, freshly ejaculated boar spermatozoa were stored in BTS with or without 10 mM PPi for 3–4 days, and used for in vitro fertilization (IVF) in the presence or absence of 10 mM PPi
The total fertilization rates of treatments with PPi in BTS or TBM were higher than a treatment without PPi, and the polyspermy was highest of all treatments with addition of PPi in BTS or TBM (IVF medium) (Fig. 2D)
Summary
Adenosine 59-triphosphate (ATP) is a fundamental factor to maintain the life, by providing energy, and controlling the cell function and metabolism. Human spermatozoa treated with extracellular ATP (ATPe) have an increased motility and fertilization rates, suggesting that ATPe may be helpful to combat the male infertility [1,2]. Addition of ATPe increased mouse fertilization rates in vitro without affecting the typical alterations of ATP-dependent protein tyrosine phosphorylation and acrosomal exocytosis in capacitated spermatozoa; addition of ATPe during mouse sperm capacitation altered sperm motility, and led to the elevation of intracellular calcium, which was not accompanied by hyperactivation [3]. Sperm capacitation is essential for attaining fertilizing ability. Sodium bicarbonate (NaHCO3), CaCl2 and bovine serum albumin (BSA) are required for sperm capacitation and increased protein tyrosine phosphorylation during capacitation of mouse spermatozoa [4,5]. ATP participated in sperm capacitation, and enhanced sperm motility during fertilization
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