Tetraspanin-interacting protein IGSF8 is dispensable for mouse fertility  Naokazu Inoue, Ph.D., Takao Nishikawa, M.S., Masahito Ikawa, Ph.D., Masaru Okabe, Ph.D.  Fertility and Sterility  Volume 98, Issue 2, Pages 465-470 (August 2012) DOI: 10.1016/j.fertnstert.2012.04.029 Copyright © 2012 American Society for Reproductive Medicine Terms and Conditions

Figure 1 Targeted disruption of the Igsf8 gene. (A) Complete structures of the wild-type mouse Igsf8 allele. Exons and introns are represented by boxes and horizontal lines, respectively. For the targeted disruption of mouse Igsf8, the neomycin-resistance gene (Neor) was inserted between exons 2 and 3. A herpes simplex virus thymidine kinase gene (tk) was introduced into the targeting construct for negative selection. The floxed gene within the targeting vector was used to introduce a mutated Igsf8 locus by homologous recombination and spans the region from upstream of exon 2 through exon 7. LoxP sites were inserted within introns 2 and 5. A Neor cassette driven by the phosphoglycerate kinase (PGK) promoter and flanked by Flip recombinant target (FRT) sites was inserted into intron 2 downstream of the 5′-loxP site. Cre recombinase excised the region between the loxP sites to generate an Igsf8-null allele after removing the Neor cassette with Flpe recombinase. (B) Genotyping of tail tip DNA by PCR amplification with primers indicated in the figure (arrows). The PCR for floxed and knockout alleles yielded 1.6- and 2.0-kb bands, respectively. (C) Multitissue blot analysis of IGSF8. All solubilized proteins were loaded at 30 μg on each lane and detected by 1 μg/mL anti-IGSF8 antibody. (D) Western blotting analysis of 10 egg lysates from Igsf8F/− and Igsf8−/− female mice. Anti-ZP3 antibody was used as a loading control. “F” stands for floxed allele. Fertility and Sterility 2012 98, 465-470DOI: (10.1016/j.fertnstert.2012.04.029) Copyright © 2012 American Society for Reproductive Medicine Terms and Conditions

Figure 2 Fertility analysis of Igsf8−/− mice. (A) Fecundity of Igsf8F/− and Igsf8−/− males and females. The numbers in parentheses indicate the numbers of mating pairs. Values are presented as the mean ± SEM. (B) Comparison of the fusing ability of Igsf8F/− and Igsf8−/− eggs. Average numbers of fused spermatozoa observed 30 minutes after insemination (n = 5). Values are presented as the mean ± SEM. (C) Representative photo of sperm-egg binding and sperm-egg fusion assay. Arrowhead and asterisks indicate fused spermatozoa and egg chromosomes, respectively. Scale bar = 100 μm. Fertility and Sterility 2012 98, 465-470DOI: (10.1016/j.fertnstert.2012.04.029) Copyright © 2012 American Society for Reproductive Medicine Terms and Conditions

Figure 3 Immunolocalization of CD9 and IGSF8 proteins in the wild-type, Cd9−/−, and Igsf8−/− eggs. All eggs were immunostained with 5 μg/mL anti-IGSF8 (red) and 1 μg/mL anti-CD9 (green) antibodies. The staining area of IGSF8 in the Cd9−/− egg was reduced compared with the wild-type. Nuclei were stained by Hoechst 33342 (blue). On the other hand, despite the absence of the IGSF8 protein, Igsf8−/− eggs did not display differential CD9 expression or localization. Scale bar = 20 μm. Fertility and Sterility 2012 98, 465-470DOI: (10.1016/j.fertnstert.2012.04.029) Copyright © 2012 American Society for Reproductive Medicine Terms and Conditions