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. 2014 Jul 18;22(4):452–461. doi: 10.1177/1933719114542009

Figure 1.

Figure 1.

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.