PMC

Titration of monkey serum samples diluted from 1:25 in a twofold series against constant antigen concentration of 10 mg/L.
Anti-peptide antiserum is indicated by ▴ with the corresponding preimmune serum, indicated by ▵, as negative control. Anti KLH-conjugated peptide antiserum is indicated by ▪, and the corresponding preimmune serum is indicated by □ as negative control. (A), M_S1 antiserum (▴) and M_S1_KLH antiserum (▪); (B), M_S2 antiserum (▴) and M_S2_KLH antiserum (▪); (C), M_S3 antiserum (▴) and M_S3_KLH antiserum (▪); (D), M_S4 antiserum (▴) and M_S4_KLH antiserum (▪); (E), M_S5 antiserum (▴) and M_S5_KLH antiserum (▪); (F), M_S6 antiserum (▴) and M_S6_KLH antiserum (▪); (G), M_MIX antiserum (▴) and M_MIX_KLH antiserum (▪).

Titration of rabbit serum samples diluted from 1:25 in a twofold series against constant antigen concentration of 10 mg/L.
Anti-peptide antiserum is indicated by ▴ with the corresponding preimmune serum, indicated by ▵, as negative control. Anti KLH-conjugated peptide antiserum is indicated by ▪, and the corresponding preimmune serum is indicated by □ as negative control. (A), R_S1 antiserum (▴) and R_S1_KLH antiserum (▪); (B), R_S2 antiserum (▴) and R_S2_KLH antiserum (▪); (C), R_S3 antiserum (▴) and R_S3_KLH antiserum (▪); (D), R_S4 antiserum (▴) and R_S4_KLH antiserum (▪); (E), R_S5 antiserum (▴) and R_S5_KLH antiserum (▪); (F), R_S6 antiserum (▴) and R_S6_KLH antiserum (▪); (G), R_MIX antiserum (▴) and R_MIX_KLH antiserum (▪).

(A) Anti-GST IgG depletion from the original antiserum analyzed by SDS-PAGE and Coomassie Blue staining. Lane 1: protein ladder; Lane 2: original antiserum; Lane 3: antiserum depleted of anti-GST IgG; Lane 4: GST-bound GSH Sepharose beads incubating with original antiserum. (B) Depletion of anti-GST IgG from the antiserum was analyzed by immunoblots. Original antiserum and purified antiserum were respectively used to probe with purified GST protein. The purified GST-ERK8(28aa) fusion protein was used as a positive control.

The SPRASA antiserum significantly reduced the numbers of sperm binding to the oolemma, the fertilization rate, and the development to morula stage; SPRASA was localized to the zona pellucida and oolemma of bovine oocytes. Bovine oocytes and sperm were used to access the effect of SPRASA antiserum on sperm binding to oocytes, sperm motility, fertilization, and embryonic development; as well as to localize SPRASA expression. For binding assays, sperm and oocytes were coincubated with an SPRASA or control antiserum for 2 hours. Sperm–oocyte complexes were stained and the number of sperm nuclei attached per oocyte was counted. For assessment of sperm motility, viable sperm was incubated with SPRASA or control antiserum for 3 hours. The number of motile sperm were counted after 0, 1/2, 1, 2, and 3 hours. For IVF, oocytes and sperm were incubated for 18 to 22 hours. Three treatment groups were investigated: (1; sperm treatment) sperm incubated with antiserum for 30 minutes, washed, then incubated with oocytes; (2; oocyte treatment) oocytes incubated with antiserum for 30 minutes, washed, and incubated with sperm; and (3; coincubation) sperm and oocytes coincubated with antiserum for IVF culture. Presumptive embryos were selected and allowed to develop for 8 days. To determine whether oocytes express SPRASA, oocytes were fixed and incubated with antiserum, then detected visualized using fluorescent microscopy. A, The SPRASA antiserum did not affect the number of sperm bound to zona-intact oocytes but (B) significantly reduced the numbers of acrosome-reacted sperm binding to the oolemma of zona-free oocytes. C, The percentage of motile sperm was not affected by incubation with SPRASA antiserum. D, The fertilization rate was significantly reduced when oocytes, or sperm and oocytes, were incubated with SPRASA antiserum prior to fertilization with untreated bovine sperm. Development to morula stage was significantly inhibited by SPRASA antiserum when the antiserum was incubated with either oocytes alone, or with oocytes and sperm, but not when sperm was treated with the antiserum. Data are presented as percentage ± standard deviation (SD); *P = .05; **P = .01; ***P = .001. E, Immunofluorescent confocal photomicrographs demonstrating the localization of SPRASA on oocytes. Zona-intact or zona-free oocytes were incubated with (E1 and E3) SPRASA antiserum or (E2 and E4) control antiserum. SPRASA was localized to the zona pellucida (grey arrow) and oolemma (white arrow) of zona-intact oocytes and to the oolemma of zona-free oocytes. Scale bar = 50 µm and applies to all images. SPRASA indicates sperm protein reactive with antisperm antibody. IVF indicates in vitro fertilization; SPRASA, sperm protein reactive with antisperm antibody.