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Rev7 is a subunit of Polζ, one of the translesion DNA synthesis (TLS) polymerases involved in DNA damage repair. We recently found that Rev7 is also essential for germ cell development in mouse. In the present study, we found the development of ovarian tumors in Rev7 mutant mouse, suggesting the involvement of TLS deficiency in the etiology of ovarian tumor. The Rev7 mutant mice showed complete lack of oocytes and follicles in the ovary. The lack of follicles causes a significant increase of gonadotropin level and an increase in the proliferation of ovarian cells. As a result, the weight of the ovaries of Rev7 mutant mice increased with age and they developed tubulostromal adenomas. However, the remarkable overgrowth of ovaries occurred after gonadotropin level decreases at older ages, suggesting gonadotropin-independent progression of the ovarian tumors. In addition, the Rev7 mutant fibroblasts and ovarian cells showed significant accumulation of DNA damage. These findings suggest that not only increased gonadotropin levels but also lack of DNA damage repair function could be responsible for the development of ovarian tumors in the Rev7 mutant mouse.

Repro22 is a mutant mouse produced via N-ethyl-N-nitrosourea-induced mutagenesis that shows sterility with germ cell depletion caused by defective proliferation of primordial germ cells, decreased body weight, and partial lethality during embryonic development. Using a positional cloning strategy, we identified a missense mutation in Rev7/Mad2l2 (Rev7(C70R)) and confirmed that the mutation is the cause of the defects in repro22 mice through transgenic rescue with normal Rev7. Rev7/Mad2l2 encodes a subunit of DNA polymerase ζ (Polζ), 1 of 10 translesion DNA synthesis polymerases known in mammals. The mutant REV7 did not interact with REV3, the catalytic subunit of Polζ. Rev7(C70R/C70R) cells showed decreased proliferation, increased apoptosis, and arrest in S phase with extensive γH2AX foci in nuclei that indicated accumulation of DNA damage after treatment with the genotoxic agent mitomycin C. The Rev7(C70R) mutation does not affect the mitotic spindle assembly checkpoint. These results demonstrated that Rev7 is essential in resolving the replication stalls caused by DNA damage during S phase. We concluded that Rev7 is required for primordial germ cell proliferation and embryonic viability and development through the translesion DNA synthesis activity of Polζ preserving DNA integrity during cell proliferation, which is required in highly proliferating embryonic cells.

Multiple ocular defects (MOD) in cattle is an autosomal recessive hereditary disorder characterized by dysplasia of the lens, retinal detachment, persistence of the hyaloid artery, and microphthalmia. The locus responsible for MOD has been mapped to the proximal region of bovine chromosome 18. In the present study, we refined the localization of the MOD locus to a 1.0-Mb interval by haplotype analysis using a pedigree of affected animals. Comparison of nucleotide sequence of genes in this region revealed a one-nucleotide insertion in the WFDC1 gene, which resulted in a frame shift mutation and premature termination codon at the middle of the protein. WFDC1 is a small secretory protein containing a WAP-type four disulfide core domain. Specific expression of Wfdc1 was observed in the lens, retina, and optic nerves of embryonic and adult mouse eyes by immunohistochemical staining and in situ hybridization. The present finding demonstrated the essential role of WFDC1 in mammalian eye development.

A coagulopathy with subcutaneous bleeding and muscular or peritracheal/periesophageal bleeding occurred in two male Japanese Brown calves of the same dam. One of the affected calves died three days after the onset of bleeding and the other survived normally until being slaughtered despite once suffering from subcutaneous hematoma. Hemostatic tests of the latter case showed prolonged activated partial thromboplastin time (APTT), and severely reduced factor VIII activity. In addition, von Willebrand factor activity, determined by the human platelet aggregation test, was within the normal range; therefore, the calf was diagnosed with hemophilia A. These are the first bovine cases of hemophilia A definitely diagnosed clinicopathologically.

Repro34 is an N-ethyl-N-nitrosourea (ENU)-induced mutation in mice showing male-specific infertility caused by defective spermatogenesis. In the present study, we investigated pathogenesis and molecular lesions in relation to spermatogenesis in the repro34/repro34 homozygous mouse. Histological examination of the testis showed that the seminiferous epithelium of the repro34/repro34 mouse contained spermatogonia and spermatocytes but no round and elongating spermatids. Instead of these haploid cells, multinucleated giant cells occupied the niche of the seminiferous tubules. Immunohistochemical staining for Hsc70t, an elongating spermatid specific protein, confirmed the absence of elongating spermatids. Furthermore, RT-PCR showed that there were significantly reduced expressions of the marker genes specifically expressed in the spermatid and that there was no difference in the expressions of the spermatocyte specific marker genes. These findings indicated interruption of the spermatogenesis during transition from the spermatocyte to spermatid in the repro34/repro34 mouse. The repro34 locus has been mapped on a 7.0-Mb region of mouse chromosome 5 containing the Syntaxin 2/Epimorphin (Stx2/Epim) gene, and targeted disruption of this gene has been reported to cause defective spermatogenesis. We therefore sequenced the entire coding region of the Stx2/Epim gene and found a nucleotide substitution that results in a nonsense mutation of this gene. The expression pattern of the Stx2/Epim gene during the first wave of spermatogenesis, increased expression at later stages of spermatogenesis, was in agreement with the affected phase of spermatogenesis in the adult repro34/repro34 testis. We therefore concluded that the male infertility of the repro34/repro34 mouse is caused by the interruption of spermatogenesis during transition from the spermatocyte to spermatid and that the nonsense mutation of the Stx2/Epim gene is responsible for the interruption of spermatogenesis.

Congenital multiple ocular defects (MOD) of Japanese black cattle is a hereditary ocular disorder with an autosomal recessive mode of inheritance showing developmental defects of the lens, retina and iris, persistent embryonic eye vascularization and microphthalmia. The MOD locus has been mapped by linkage analysis to a 6.6-cM interval on the proximal end of bovine chromosome 18, which corresponds to human chromosome 16q and mouse chromosome 8. To refine the MOD region in cattle, we constructed an integrated radiation hybrid (RH) map of the proximal region of bovine chromosome 18, which consisted of 17 genes and 10 microsatellite markers, using the SUNbRH7000 panel. Strong conservation of gene order was found among the corresponding chromosomal regions in cattle, human and mouse. The MOD-critical region was fine mapped to a 59.5-cR region that corresponds to a 6.3-Mb segment of human chromosome 16 and a 4.8-Mb segment of mouse chromosome 8. Several positional candidate genes, including FOXC2 and USP10, were identified in this region.

Factor XI deficiency in Japanese black cattle is an hereditary mild bleeding disorder with an autosomal recessive mode of inheritance. To characterize the molecular lesion causing factor XI deficiency in cattle, we isolated an entire coding region of the bovine F11 gene, which comprises 15 exons and 14 introns, and determined its nucleotide sequences. Comparison of the nucleotide sequences of the F11 gene between affected and unaffected animals revealed an insertion of 15 nucleotides in exon 9 of the affected animals. The insertion results in a substitution of one amino acid with six amino acids in a highly conserved amino acid sequence in the fourth apple domain of factor XI protein. Genotyping of the F11 gene in 109 Japanese black cattle revealed that the insertion clearly corresponded to the factor XI activities of the animals. We therefore concluded that the insertion of 15 nucleotides in the F11 gene is the causative mutation for factor XI deficiency in Japanese black cattle. Genotyping of the F11gene by detecting the insertion will be an effective DNA-based diagnostic system to prevent incidence of the disease.

Two cases of hereditary bleeding disorder diagnosed as haemophilia A were recently observed in Japanese Brown cattle. We sequenced the entire coding region of the factor VIII gene of the affected animals to find a causative mutation. A nucleotide substitution of T to A resulting in an amino acid substitution of leucine to histidine (p.Leu2153His) was identified in a highly conserved residue in the C1 domain of factor VIII. Genotyping of 254 normal animals including the pedigree of the affected animals and randomly sampled animals of different breeds confirmed that the substitution is the causative mutation of cattle haemophilia A.

Repro22 is an N-ethyl-N-nitrosourea (ENU)-induced mutation in mice showing depletion of both male and female germ cells. In the present study, we investigated the male phenotypes of the mutant mouse at the adult stage. The repro22/repro22 homozygous mice showed reduced body weights as well as markedly reduced testis weights. Histological examination of the testes at 4 and 10 months of age showed no germ cells in the seminiferous tubules of the affected testis while a number of Sertoli cells were observed in the tubules. In addition to the germ cell depletion, the testes of the affected mouse contained expanded intertubular spaces that were filled by Leydig cell-like interstitial cells. These interstitial cells were confirmed to be Leydig cells by immunohistochmical staining using anti-3beta-HSD antibody. The estimated number of Leydig cells in the affected testes at 10 months of age increased approximately 2 fold compared with those of normal testes. Furthermore, the plasma testosterone levels of the affected mice at 10 months of age were significantly higher than those of the normal mice. These findings indicated that the repro22/repro22 mouse developed hyperplasia of Leydig cells that was presumably caused by the absence of germ cells in the seminiferous tubules.