Extracellular adenosine 5'-triphosphate (ATP) previously has been shown to increase the fertilization percentage in human in vitro fertilization (IVF) performed for male factor infertility. The objective of this study is to determine the effects of extracellular adenosine 5'-triphosphate (ATPe) on human sperm function by examining its effects on end points of sperm capacitation. Sperm obtained from healthy volunteers with normal semen parameters, asthenozoospermic men, and cryopreserved samples were incubated in medium with or without 2.5 mM ATPe. The effects of ATPe on acrosomal exocytosis, protein tyrosine phosphorylation, and sperm motility parameters were quantified. Although ATPe did not affect acrosomal exocytosis or protein tyrosine phosphorylation in sperm from healthy donors, it significantly altered several motility parameters, with the largest effects manifested in increased curvilinear velocity and percentage hyperactivation. ATPe similarly affected sperm selected for poor motility and thawed cryopreserved sperm but to a lesser extent than its effects on sperm with normal motility. ATPe increased straight-line velocity and linearity of sperm obtained from asthenozoospermic men. Human sperm motility characteristics are altered by ATPe; this finding may explain its previously reported beneficial effect on human IVF. These results suggest that ATPe could constitute a new therapeutic modality in the treatment of male infertility.
"These fundamentally important events serve to increase the concentration of intracellular second messenger . It has also been suggested that ATP increases in vitro fertilization in mouse spermatozoa without affecting the typical alterations of ATP-dependent protein tyrosine phosphorylation and acrosomal exocytosis in capacitated spermatozoa . Simultaneously, [Ca2+]i play an important role in the regulation of capacitation and the acrosome reaction, an exocytotic event that allows sperm to penetrate the zona pellucida and fuse with the oocyte plasma membrane towards fertilization . "
[Show abstract][Hide abstract] ABSTRACT: Ubiquinol-cytochrome-c reductase core protein 2 (UQCRC2) is a component of ubiquinol-cytochrome c reductase complex that is known to correlate with male fertility via spermatogenesis. Simultaneously, nutlin-3a is a small molecule antagonist of mouse double minute 2 repressor (MDM2), activate p53 and induce apoptosis responsible for spermatogenesis. To date, however there are no known effects of nutlin-3a on reproduction. Therefore, present study was designed to investigate the effect of nutlin-3a on male fertility via UQCRC2. In this in vitro trial with mice spermatozoa, we utilized CASA, CTC staining, ATP assay, western blotting, and IVF to measure the main study outcome. The short-term exposure of spermatozoa in nutlin-3a decreases sperm motion kinematics, intracellular ATP production, capacitation, the acrosome reaction, UQCRC2, and tyrosine phosphorylation (TYP) of sperm proteins in a dose-dependent manner. Notably, the decreased UQCRC2 and TYP were associated with reduced sperm kinematics, ATP production, and capacitation, which ultimately led to adverse effects on male fertility such as poor fertilization rates and embryo development. Thus, nutlin-3a may be considered as a potential male contraceptive agent due to its ability to decrease fertility secondary to changes in overall sperm physiology and embryonic development. However, the results of this preliminary study have to be confirmed by additional independent trial.
PLoS ONE 10/2013; 8(10):e76959. DOI:10.1371/journal.pone.0076959 · 3.23 Impact Factor
"In spermatozoa, ATP plays important roles for the movement to female reproductive tract, viability and penetration to fertilize with oocyte. 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 , . 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 . "
[Show abstract][Hide abstract] 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.
PLoS ONE 04/2012; 7(4):e34524. DOI:10.1371/journal.pone.0034524 · 3.23 Impact Factor
"Assuming that ATP depletion may occur during the re-warming phase (Holt et al., 1992) and that incorporation of exogenous ATP occurs coincident with membrane structural rearrangements, the potential of supplemental ATP improving the viability of thawed sperm seems plausible. In fact, extracellular ATP increases the motility and fertility of frozen/thawed murine (Rodríguez-Miranda et al., 2008) and human (Edwards et al., 2007) sperm. "
[Show abstract][Hide abstract] ABSTRACT: A comparative approach was used to evaluate the cryosurvival of turkey and crane sperm frozen in a dimethylacetamide (DMA) cryodiluent supplemented with osmoprotectants and ATP. A range (6-26%) of DMA concentrations was used alone or in combination with ATP (30, 60 or 118mM) or one of the following osmoprotectants: (1) sucrose (turkey, 8.0%; crane, 5.0%); (2) 5.0% sucrose and 5.0% trehalose; or (3) betaine hydrochloride (0.1, 0.2 or 0.4mM). The viability of thawed sperm was assessed using the nigrosin-eosin stain and sperm motility was determined using the hanging-drop technique. For semen frozen only with DMA, post-thaw sperm motility was greatest (P<0.05) for the 6.0%, 10.0% and 18% concentrations, regardless of species. Turkey sperm frozen with the sucrose/trehalose combination had greater (P<0.05) post-thaw motility for all DMA treatments compared to DMA alone. The lowest concentration of the osmoprotectant betaine hydrochloride substantially improved turkey sperm viability post-thaw in all treatments compared to DMA alone (P<0.05). The post-thaw motility of crane sperm was improved (P<0.05) with a combination of 18.0%, 24.0% or 26.0% DMA and 30mM ATP. Moreover, in the presence of osmoprotectants, crane sperm motility decreased as the osmoprotectant concentration increased. The lowest concentration of ATP also improved crane sperm viability post-thaw, especially for DMA concentrations 18% or greater. The combination of sucrose and trehalose improved (P<0.05) crane sperm viability only with 6% and 10% DMA. These data affirm that there are avian-specific differences in sperm survival after cryopreservation and suggest that post-thaw survival can be enhanced by including species-based osmoprotectant/ATP combinations in a cryodiluent where DMA is the cryoprotectant.
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