Fertility of HE6 knockout and wild-type control males at various ages, as measured by the median number of embryos resulting from a successful mating (left axis and line graph; WT, wild type; KO, knockout) and the fertility (percentage of successful matings resulting in pregnancy) (right axis and bar chart).

(A to D) Representative photomicrographs (magnification, ×100) of hematoxylin- and eosin-stained sections through the rete testis of wild-type (A) and knockout (B) 12-week-old mice and the seminiferous tubules of wild-type (C) and knockout (D) 12-week-old mice showing dilation and expansion of these testicular structures in knockout mice. (E) Photomicrographs (magnification, ×25) showing expanded rete testis (*) and unoccluded efferent ducts in 2-week-old knockout mice.

Photomicrographs (magnification, ×100) of hematoxylin- and eosin-stained sections through the efferent ductules of wild-type (A) and knockout (B) mice, the epididymal initial segment of wild-type (C) and knockout (D) mice, and the caput epididymis of wild-type (E) and knockout (F) mice. An abnormal accumulation of spermatozoa in knockout efferent ductules (B) and subsequent reduction in spermatozoa in the lumen in the knockout initial segment and caput epididymis (D and F) were observed. The results shown are representative of histological analyses of 17 knockout and 15 wild-type 12-week-old mice.

Photomicrographs of caudal sperm preparations from equal volumes obtained from the cauda epididymis of wild-type (A) and knockout (B) mice at 12 weeks of age. The knockout animals reveal a drastically reduced number of spermatozoa, and those present show frequent morphological abnormalities. The results shown are representative of analyses of 17 knockout and 15 wild-type spermatozoon samples.

(A) Targeting strategy for the deletion of exons 21 to 25, encoding the 7TM domain. A selection cassette (neo) and a reporter cassette (IRES LacZ) were inserted. Two copies of a herpes simplex virus thymidine kinase (TK) gene were included in the vector for selection against nonhomologous integration. WT, wild type; KO, knockout. (B) Southern blot analysis of tail genomic DNA from wild-type male (+/Y), heterozygous female (+/−), and hemizygous knockout male (−/Y) animals demonstrating disruption of the HE6 gene. The probe corresponding to exon 26 hybridizes to an 11-kb EcoRV wild-type fragment and a 6-kb EcoRV fragment from the targeted allele. (C) Northern blot analysis of initial-segment total RNA from HE6 mutant animals (−/Y) and wild-type littermates (+/Y) hybridized with cDNA probes corresponding to the 7TM domain (exons 24 to 25) (top) and an extracellular domain (exons 16 to 17) (middle). The bottom blot shows an S26 ribosomal protein loading control. (D) Western blot analysis of epididymal membrane suspensions from HE6 mutant (−/Y) and wild-type (+/Y) littermates using the mN2 antibody recognizing an epitope within the ectodomain (Ecto) (bottom) and the mA23 antibody recognizing an epitope within the endodomain (Endo) (top). (E) Photomicrograph of a BLUO-GAL-stained section through the epididymis and testis of an HE6 mutant animal showing the activity of the β-galactosidase gene.