Fig. 3: Spermatogenesis was impaired in Hsf5 knockout male.

a The targeted Hsf5 allele with deletion of Exon1-6 is shown. 5’- and 3’- homology sequences in the ssODN are shown in green and red, respectively. Arrowheads: synthetic gRNAs. Arrows: PCR primers for genotyping. b Immunoblot analysis of testis extracts prepared from mice with the indicated genotypes (P17). The arrow indicates a band of HSF5. * indicates nonspecific bands. Two technically independent experiments from two pairs of WT and Hsf5 KO siblings were repeated and showed similar results. c Seminiferous tubule sections (8 weeks old) were stained for SYCP3, HSF5, and DAPI. pL preleptotene, Pa pachytene spermatocyte, rS round spermatid, eS elongated spermatid. Boundaries of the seminiferous tubules are indicated by white dashed lines. Roman numbers indicate the seminiferous tubule stages. Biologically independent mice (n = 3) for each genotype were examined. Scale bars: 25 μm. d Testes from WT, Hsf5 +/- and Hsf5 KO (upper left: 4 weeks old, upper right: 8 weeks old left). Testis/body-weight ratio (mg/g) of WT, Hsf5 +/-, and Hsf5 KO mice (lower left: 4 weeks old, lower right: 8 weeks old) are shown below (Mean with SD). n: the number of animals examined. Statistical significance is shown by p value (Two-tailed t-test). Scale bar: 5 mm. e Hematoxylin and eosin staining of the sections from WT, Hsf5 +/- and Hsf5 KO testes (upper: 4 weeks old, lower: 8 weeks old). Biologically independent mice (n = 3) for each genotype were examined. Scale bar: 100 μm. f Hematoxylin and eosin staining of the sections from WT, Hsf5 +/- and Hsf5 KO epididymis (8 weeks old). Biologically independent mice (n = 3) for each genotype were examined. Scale bar: 100 μm. g Seminiferous tubule sections (8 weeks old) were stained for PNA lectin and DAPI. Scale bar: 25 μm. A single experiment was performed. h Number of pups born by mating Hsf5 + /- and Hsf5 KO males with WT females to examine fertility. Hsf5 +/- males and Hsf5 KO males were initially mated with WT females (all 4 weeks old at the start point of mating). This cage was observed for 24 weeks from the start of mating.