Fig 1: Inhibition of ERp57 and other PDIs reduces mouse fertilization in vitro. A cumulus-intact and zona-free mouse IVF assay was performed in the presence and absence of A bacitracin, a broad-spectrum inhibitor of PDIs, and B punicalagin, a selective inhibitor of PDIA3 (ERp57) and PDIA1 family. The fertilization rate and fertilization index (mean ± SEM) were measured for the cumulus-intact or zona-free IVF assays, respectively. C Mouse ERp57 (expected band at ~ 60 kDa) was detected using the mouse anti-hERp57 antibody (Santa Cruz Biotechnology, clone 4E69). Western blot analysis demonstrates cross-reactivity with recombinant mouse ERp57 protein, as well as with mouse sperm. D Immunofluorescence staining of ERp57 on acrosome intact (AI) and acrosome reacted (AR) mouse spermatozoa (green). Acrosome staining was confirmed using rhodamine-conjugated Pisum Sativum Agglutinin (PSA) (red), and nuclei were stained with DAPI (blue). E A cumulus-intact and zona-free mouse IVF assay was performed with a mouse anti-hERp57 antibody or IgG1 isotype control antibody. ****p < 0.0001 by an ordinary one-way ANOVA test for cumulus-intact IVF in Fig. 1D and by Brown-Forsythe and Welch one-way ANOVA tests for all other experiments presented in Fig. 1. All experiments were performed with a minimum of three biological replicates. The number of oocytes in each experiment is indicated in parentheses.
Fig 2: ERp57 spermatozoa conditional knockout male mice show hypofertility in vivo and in vitro. A WT females were mated with control (ctrl) or scKO males, and litter size (mean ± SEM) was counted 3 weeks post-mating, confirmed by the presence of a vaginal plug. **p < 0.01 by Mann–Whitney test. B Superovulated WT females were mated with ctrl or scKO males to evaluate in vivo fertilization. ****p < 0.0001 by unpaired t-test. C The number of oocytes with spermatozoa in their PVS has been compared in both conditions and showed no significant difference (ns). p > 0.05 by unpaired t-test. D Cumulus-intact mouse IVF assays were performed with control or scKO sperm. ****p < 0.0001 by Welch’s t-test. E Zona-free mouse IVF assays were performed with control or scKO sperm. ****p < 0.0001 by Welch’s t-test. All experiments were performed with a minimum of three biological replicates. Numbers between parentheses indicate oocyte counts.
Supplier Page from Abcam for Recombinant mouse ERp57 protein (Active) (His tag N-Terminus)