PMC

Fertilization potential of Prnd^−/− and Prnp^−/−/Prnd^−/− sperm. a: Frequency of in vivo and in vitro fertilization by wild-type, Prnd^−/−, and Prnp^−/−/Prnd^−/− sperm. b: Frequency of two-cell stage embryos after in vitro fertilization. Two separate determinations were performed for wild-type, Prnd^−/−, and Prnp^−/−/Prnd^−/− sperm. c: Frequency of two-cell stage embryos, produced by in vitro fertilization, that develop to four-cell and morula stages. Two separate determinations were performed for wild-type, Prnd^−/−, and Prnp^−/−/Prnd^−/−.

Defective acrosome reaction in Prnd^−/− and Prnp^−/−/Prnd^−/− sperm. a: Illustration of acrosome-reacted and nonreacted (intact) sperm heads stained with Coomassie blue G-250. b: Frequency of acrosome-reacted sperm in the presence and absence of calcium ionophore A23187 for wild-type (n = 3 separate determinations), Prnd^−/− (n = 2), and Prnp^−/−/Prnd^−/− (n = 3) sperm.

a: Levels of DNA damage measured by the SCSA. Prnd^−/− and Prnp^−/−/Prnd^−/− samples show a significant increase in COMP αt values greater than the wild-type control. Open points represent individual data, filled points represent the means for each genotype (n = 4 independent samples for each genotype). b: Levels of DNA damage measured by the TUNEL assay. Prnd^−/− and Prnp^−/−/Prnd^−/− samples show a significant increase in the frequency of TUNEL-stained sperm greater than the wild-type control. Open points represent individual data, filled points represent the means for each genotype (n = 4 independent samples for each genotype).

Ablation of Dpl in Prnd^−/− and Prnp^−/−/Prnd^−/− mouse lines. a: Immunoblotting of wild-type (wt), Prnd^−/− (Dpl ko) and Prnp^−/−/Prnd^−/− (PrP/Dpl ko) testis lysates using an anti-Dpl antibody only detected a diffuse band of Dpl immunoreactivity in the wild-type sample. The mobility of molecular weight markers on the same gel is shown. Immunoreactivity against GAPDH in all samples provided an internal control for protein loading. b: Ablation of PrP in the Prnp^−/−/Prnd^−/− mouse line. Immunoblotting of wild-type (wt), Prnd^−/− (Dpl ko) and Prnp^−/−/Prnd^−/− (PrP/Dpl ko) brain lysates with an anti-PrP antibody only detected PrP immunoreactivity in the wild-type and Prnd^−/− samples.

Spermatogenesis in Prnd^−/− and Prnp^−/−/Prnd^−/− mouse lines. a: H&E-stained testis sections from wild-type, Prnd^−/−, and Prnp^−/−/Prnd^−/− mice, showing normal histology with all spermatogenic cell types present. b: Epididymal sperm counts. Normal counts in wild-type (n = 4 animals examined), Prnd^−/− (n = 2), and Prnp^−/−/Prnd^−/− (n = 4). c: Sperm motility. Normal motility in wild-type (n = 6), Prnd^−/− (n = 4), and Prnp^−/−/Prnd^−/− (n = 4). d: Sperm morphology. Normal morphology with low levels of head and tail abnormalities in wild-type, Prnd^−/−, and Prnp^−/−/Prnd^−/− (all n = 4).

Generation of Prnd^−/− and Prnp^−/−/Prnd^−/− mouse lines by gene targeting. a, Prnd; b, Prnp/Prnd. The structure of the Prnp and Prnd loci, targeting constructs, and targeted loci are shown schematically. Exons for each gene are indicated: filled boxes, coding regions; shaded boxes, untranslated regions. In the targeting constructs the positive (HPRT) and negative (HSV-TK) selectable markers are indicated. The locations of PCR primers, probes, and restriction fragments used to identify targeted clones are shown. Restriction sites: B, BamHI; E, EcoRI; P, PstI; S, ScaI; X, XhoI.