[Supplemental Data] -
"Supplemental Figure 1: Control experiments for IVF with anti-UCHL1/L3 antiserum. A) Rabbit polyclonal anti-UCHL1/L3 antiserum caused 100% polyspermy in this experiment, while control non-immune sera at comparable concentration showed low rates of fertilization and polyspermy. B) Antibodies against unrelated sperm head/acrosome antigens ZPBP/IAM38, IAM32 [47], and PT32/PAWP [49] showed low fertilization and polyspermy rates, not comparable to high polyspermy induced in the same trial by anti-UCHL1/L3 antibody. The ZPBP/IAM38 antigen synonymous with porcine ZPBP protein, also called SP38 (accession #Q29108) becomes exposed on the inner acrosomal membrane during acrosome reaction and participates in secondary sperm-zona binding, explaining why this antiserum specifically inhibits porcine IVF [47]. C) Sodium azide at concentrations up to 1 mM (equals 65 µg/ml) did not impair porcine IVF. Highest content of sodium azide in our antibody experiments was 0.25 µg/ml. D) Anti-UCHL1/L3 antisera did not reduce sperm-ZP binding during IVF, even if the ova were parthenogenetically-activated prior to insemination. E) Anti-UCH-antiserum
treatment increased polyspermy regardless of whether fresh or frozen-thawed
spermatozoa were used. Higher overall rate of fertilization seen with fresh semen was anticipated, as freeze-thawing reduced sperm viability, and may cause acrosome-damage and sperm premature capacitation. F) An unrelated anti-UCHL1/L3 antiserum from Chemicon (cat # AB1761) increased polyspermy in a fashion similar to Biomol sourced antiserum (cat. # PG 9500) used for most of our IVF studies. As expected, control antiproteasome antibodies LMP2 and 20S (both from Biomol, described in [26]) caused fertilization failure due to inhibition of sperm proteasomal core proteolytic activity.
Supplemental Figure 2: UCHL3 is present on the surface of boar sperm acrosome. Immunolocalization of UCHL3 (green) was performed both in spermatozoa permeabilized with 0.1% Triton X-100 (A) and in spermatozoa processed without permeabilization (B). Negative control with non-immune rabbit serum followed by goatanti-rabbit-IgG-FITC is shown in panel C.
Supplemental Figure 3: Antibodies used to detect UCHL1 in porcine ova also recognized UCHL1 in mouse oocyte cortex, as reported by others [41]. Besides of detecting UCHL1 in the cortex of porcine and bovine ova, we detected it in mouse ova before (A-A") and after meiotic maturation (B-C"). Intriguingly, the process of oocyte maturation in mouse resulted in the extrusion of UCHL1-immunoreactive, granular material in the perivitelline space (C-C" and insert). This is consistent with the suggestion that a major anti-polyspermy mechanism in the mouse operates at the oolemma level [37].
Supplemental Figure 4: Supplemental data illustrating lack of the effect of oocytederived UCHs on anti-polyspermy defense in the pig. (A, B) Detection of the anti-UCHL1/L3 antiserum-binding to the oocyte-surface. Some immunoreactive material was observed in the perivitelline space of fertilized porcine ova (A-A'''), but not in the metaphase II ova (B-B'''). (C) Despite this observation, parthenogenetic activation and subsequent oocyte preincubation with anti-UCHL1/L3 antiserum did not alter fertilization rates. (D) Numbers of accessory spermatozoa (arrowheads) present in perivitelline space suggested that the addition of anti-UCHL1/L3 during fertilization
(columns B & D), but not the preincubation of parthenogenetically activated ova with said antiserum (column C) increased the rate of sperm-ZP penetration. Columns in diagram correspond to columns in oocyte-figure plate.
Supplemental Figure 5: Immunolocalization of UCHL1 (green) and UCHL3 (red) in bovine ova. Similar to porcine ova, UCHL1 is predominantly detected in the cortex of unfertilized metaphase II bovine ova (A), while UCHL3 is sequestered in the oocyte’s meiotic spindle (A, B), in the midbody formed during extrusion of the second polar body (C), and in the sperm aster of pronuclear zygotes (D). Both UCH-isozymes are also present in bovine cumulus cells (E).
Supplemental Figure 6: Besides testis, UCHL3 could be acquired by the boar spermatozoa during sperm maturation in the epididymis (A-E') or from oviductal secretions (F-H), known to have an anti-polyspermic action."
