Figure 1.
Generation of inorganic pyrophosphate (PPi).
PPi is produced by the hydrolysis of ATP into AMP in cells. Inorganic pyrophosphatase (PPA1) catalyzes the hydrolysis of PPi to form 2 orthophosphates (2Pi), resulting in energy release.
Figure 2.
Effect of PPi on total and polyspermic fertilization during porcine IVF.
Diagram indicates % monospermic (□) and % polyspermic (▪) fertilization. Values are expressed as the mean percentages of total fertilization ± SEM. Different superscripts a, b & c in each histogram denote a significant difference at p<0.05, meaning that column a is significantly different from columns b and c, column b is significantly different from columns a and c, column ab is not significantly different from either a or b, and column bc is not significantly different from columns b and c. Numbers of inseminated ova are indicated in parentheses. (A) Porcine oocytes matured in vitro were inseminated with a standard concentration of 1×106 spermatozoa/ml, in the presence of ascending concentrations of PPi. Experiments were repeated five times. (B) The polyspermy rates, reflective of sperm fertilizing ability in vitro (same as panel A) dramatically increased in the presence of PPi. (C) Fertilization rates of porcine oocytes inseminated with different concentrations of spermatozoa in the presence/absence of 10 µM PPi. Experiments were repeated three times. (D) Porcine oocytes were inseminated (sperm conc. 5×105 spermatozoa/ml) with (+) or without (−) 10 µM PPi in TBM, using spermatozoa stored with (+) or without (−) PPi in BTS. Experiments were repeated three times. (E) Effect of extrinsic PPA1 enzyme on porcine IVF. Oocytes were inseminated with different concentrations of purified PPA1 protein. Experiments were repeated twice. (F) Porcine oocytes were inseminated in the presence of rabbit polyclonal anti-PPA1 antibody or non-immune rabbit serum (a control of PPA1 antibody). Experiments were repeated twice.
Figure 3.
Measurement of pyrophosphate (PPi) content by fluorometric assay.
PPi assay with boar seminal plasma (SP), porcine oviductal fluids (pOVF), rabbit sera, mouse sera (final conc. 10 µg/ml), boar spermatozoa (1×106 spermatozoa/ml) and 10 mM H2O2 working solution (a negative control). The emitted fluorescence (no units) was measured at multiple time points to follow the kinetics of the reaction (excitation 530 nm; emission 590 nm). Experiments were repeated three times. Values are expressed as the mean of fluorescence intensity ± SEM.
Figure 4.
Detection of inorganic pyrophosphatase (PPA1) by Western blotting.
Boar seminal plasma (SP; 20 µg/ml), porcine oviductal fluid (pOVF; 100 µg/ml), and boar, bull, mouse and human spermatozoa (all at 1×106 spermatozoa/ml) were extracted to perform the protein analysis. Equal protein loads were used. Distinct band at ∼32 kDa was detected by rabbit polyclonal anti-PPA1 antibody. The purified PPA1 (extreme right lane; 1 µg/ml; Sigma I1643) from S. cerevisiae was used as a control protein.
Figure 5.
Localization of inorganic pyrophosphatase (PPA1; red) in spermatozoa by immunofluorescence.
(A, B) Whole mount immunofluorescence of boar spermatozoa. Most prominent labeling is observed in the sperm tail connecting piece and in the postacrosomal sheath of the sperm head. (C) Identical labeling (arrows) is observed in spermatozoa attached to oocyte zona pellucida at 30 min after gamete mixing during IVF. (D) Negative control with anti-PPA1 antibody immunosaturated with full length PPA1 protein. DNA was counterstained with DAPI (blue). Epifluorescence micrographs were overlapped with parfocal transmitted light photographs acquired with DIC optics.
Figure 6.
Sperm viability during sperm storage with/without PPi.
Percentages of viable spermatozoa are based on dual SYBR14 (live sperm) and PI (dead sperm) labeling. Experiments were repeated three times. Values are expressed as the mean percentages ± SEM. Different superscripts a & b in histogram denote significant differences at p<0.05, meaning that column a is significantly different from column b.
Figure 7.
Effect of PPi on proteasomal enzymatic activities of stored boar spermatozoa.
Fresh boar spermatozoa were stored in BTS with/without 10 µM PPi for 3 or 10 days (No treat/PPi+BTS). Proteasomal proteolytic and deubiquitinating activities were measured using specific fluorometric substrates Z-LLE-AMC (A), Z-LLVY-AMC (B), Z-LLL-AMC (C) and ubiquitin-AMC (D). In a separate treatment group, PPi (10 µM) was added before measurement to spermatozoa preserved without PPi in BTS (PPi). The emitted fluorescence (no units) was measured at multiple time points to follow the kinetics of the reaction (Excitation 380 nm; emission 460 nm). Experiments were repeated three times. Values are expressed as the mean of fluorescence intensity ± SEM.
Figure 8.
Western blotting of PPi transporter protein ANKH in boar spermatozoa (anticipated mass: 76 kD).
Figure 9.
Immunofluorescence of ANKH (red) in boar spermatozoa (A) and spermatids (B).
(A) Prominent labeling is present in the postacrosomal sheath, and in the equatorial segment (arrowheads in panel b) of ejaculated spermatozoa. (B) During spermiogenesis, ANKH colocalizes with tubulin (TUBB, green) in the microtubules of the spermatid caudal manchette (a–c) at all steps of spermatid elongation during which the manchette is present (late round/early elongating spermatid-a; late elongating spermatid-b & c; top and side view-d, respectively). This pattern is typical of the deposition of proteins into postacrosomal sheath that serve a structural or metabolic/signaling purpose in fully differentiated spermatozoa. Microtubules were labeled with monoclonal antibody against tubulin beta (TUBB). DNA was counterstained with DAPI (blue). Epifluorescence micrographs were overlaid with parfocal transmitted light photos acquired with DIC optics.