FIGURE 7:

Effects of uroplakin knockout on mouse gamete fertilization. (A–C) Uroplakin-deficient eggs had a reduced fertilization rate. IVF assays using (A) uroplakin knockout eggs and sperms, (B) uroplakin knockout eggs and WT sperms, and (C) WT eggs and uroplakin knockout sperms. Note in A that uroplakin deficiency impaired the fertilization (2-Cell; blue bars) but not embryonic development (Blastocyst, BL; red bars), and in B and C that this impairment was caused by UP deficiency in eggs. Eggs and sperms were collected from 8 to 10 female and two to three male mice for each group, respectively. (D) Effects of anti-uroplakin antibodies on the in vitro fertilization of the mouse eggs; 5 WT female mice were used for each group. The antibodies used are affinity-purified rabbit antibodies to the extracellular domains of mouse UPIa (128) and UPIb (7727), and to a mouse UPIIIa synthetic peptide corresponding to a juxta-membrane epitope (position 179–191 QTLWSDPIWTNRP(C)). The ZP-intact WT eggs were preincubated with these antibodies (final concentration 200 µg/ml) for 30 min prior to incubation with WT sperms. Error bars are the SD of four independent groups. Asterisks mark values that are significantly different from the controls (p < 0.01, one-way analysis of variance [ANOVA]). (E) In vivo pup production: The litter sizes of various breeding combinations between the WT and double-UP-knockout mice. Note that pairs involving only female, but not male, KO mice had a reduced litter size. Five breeding pairs (8–12 wk old) were used for each group, and four to five litters were produced from each pair. Error bars are the SD of five independent groups. Asterisks denote statistical significance (p < 0.01, one-way ANOVA).