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Hypogonadic rats of the HGN inbred strain show male sterility controlled by an autosomal single recessive allele (hgn). The testis weight of adult hgn/hgn rats is about 1% that of normal rats, and only a few thin seminiferous tubules surrounded by islet-like conformations of Leydig cells are present in the fibrous interstitial tissue of the testis (Suzuki et al. 1988). Primordial germ cells do exist in the neonatal hgn/hgn testis, but cannot differentiate into spermatogonia and degenerate before entering meiosis (Suzuki et al, 1998). hgn/hgn females have reduced fertility, including a small litter size, a reduced number of oocytes, and early reproductive senescence (Suzuki et al. 1992). hgn/hgn rats of both sexes also show bilateral hypoplastic kidney (HPK) with a reduced number (only one-quarter) of glomeruli (Suzuki et al. 1991; Suzuki and Suzuki 1995). Whether the phenotypes of hypogonadism and HPK are caused by a mutation in a single gene or defects in different genes located close to each other remains unclear. No other mutant animal or human disease showing a phenotype similar to that of the hgn/hgn rat has been reported. The hgn/hgn rat would, therefore, be a useful model for investigating the development of mammalian reproductive and urinary organs. In this study, linkage analysis was performed to determine the chromosome location of the hgn locus and to obtain information about candidate genes for hgn. Thirteen hgn/hgn females were selected from the population of the HGN inbred strain by laparotomy for detection of HPK at weaning (Suzuki et al. 1991). One hgn/hgn female was mated with a BN male rat (+/+), and the resulting F1 males were mated with the remaining 12 hgn/hgn females to produce backcross progeny. After the mating, the hgn/hgn females were autopsied to confirm the hgn/hgn phenotype (Suzuki et al. 1992). All male backcross progeny were autopsied at the age of 5 days to avoid postnatal loss, and their phenotypes were determined by macroscopic observation of testicular size. Relative testis weight of the affected rats (0.49 ± 0.06g, average ± S.D.) was about half that of normal rats (1.16 ± 0.09g). The histological appearance of the testes in the affected rats was similar to that of hgn/hgn rats of the HGN strain (Suzuki et al. 1993). Since it was difficult to clearly categorize females of the backcross progeny into affected and normal phenotypes by comparison of renal size, we did not use female rats for linkage analysis. Consequently, 29 affected and 26 normal males were obtained. The segregation ratio of the affected to normal rats did not deviate significantly from the expected 1 : 1 ratio (x 4 0.16, P > 0.20). Because of physical limitations of the PCR machine with a block of 48 samples, high-molecular-weight genomic DNA was isolated from livers of 24 affected and 24 normal rats by phenol extraction. The samples of remaining seven progeny were saved for the next mapping experiment. The total of 48 progeny were initially typed for 33 microsatellite markers on rat chromosomes. Primers for the microsatellite markers were purchased from Research Genetics Inc. (Huntsville, Ala.). The PCR was carried out in 10 m1 of a reaction mixture containing 25 ng of the genomic DNA, 1.0 pmol of each of the oligonucleotide primers, 2.0 nmol of each dNTP, and 0.26 U of Taq polymerase (TaKaRa) in the reaction buffer recommended by the manufacturer. After initial denaturation for 180 s at 92°C, 35 cycles of amplification, each consisting of denaturation for 15 s at 92°C, annealing 60 s at 55°C, and extension for 120 s at 72°C, were carried out with a Thermal Cycler TP2000 (TaKaRa). The PCR products were separated by nondenatured 10% polyacrylamide gel electrophoresis and visualized by silver staining (Silver Stain Plus Kit, Bio-Rad). The recombination fractions and the lod scores among the hgn and the other loci were calculated with Map Manager ver. 2.6.5 (distributed via the World Wide Web, URL: http://mcbio.med.buffalo.edu/mapmagr.html). The order of loci was determined so that the number of recombination events was minimized. By typing the 33 rat microsatellite marker loci in the backcross progeny, significant linkage was observed between the hgn locus and the D10Mgh10 locus on rat Chromosome (Chr) 10 with a lod score (z) of 5.1 and a recombination fraction (u) of 0.167. The D10Mgh3 locus also was linked to the hgn locus (z42.7, u40.25). No significant linkage was observed between the hgn locus and the other 31 loci. Therefore, the 48 progeny were further typed for the following seven loci located on rat Chr 10: D10Mit2, D10Mgh6, D10Mgh8 (Myh3), D10Wox14 (Asgr1), D10Wox6, D10Wox16 (Ngfr), and D10Wox12 (Abp). The highest lod score (z 4 14.4) was obtained for the D10Mit2 locus with a u value of 0. Segregation of alleles of the nine microsatellite loci as well as the hgn locus in the 48 progeny (Fig. 1) and a linkage map of rat Chr 10 with those loci (Fig. 2) are shown. The order and distances of the nine loci on the linkage map are basically identical with linkage maps reported previously (Jacob et al. 1995; Bihoreau et al. 1997). We therefore concluded that the hgn locus is located in the region close to the D10Mit2 locus on rat Chr 10. The Abp gene (Shbg in mouse) encodes androgen binding proteins expressed in Sertoli cells (Sallivan et al. 1991), which are apparently affected in the testis of hgn/hgn rats, suggesting that the gene is a candidate for hgh. As shown in Fig. 1, the presence of recombinations between the hgn and Abp loci, however, ruled out the Abp gene as a candidate. As shown in Fig. 2, the order of the functional genes (Myh3/Myhse, Abp/Shbg, Asgr1, and Ngfr) on rat Chr 10 is identical to that of the corresponding region of mouse Chr 11. Thus, the mouse gene corresponding to the hgn locus might be located in the region between map positions 37 (Asgr1) and 56 (Ngfr) of mouse Chr 11 (derived from the Mouse Genome Database; MGD). Possible candidate genes for the hgn mutation, including the Tex4, Fert2, Ube2b-rs2, Gsg2, and Lhx1 genes, have been mapped to this region. The Ube2b gene, which is implicated in postreplication repair, is located on either Chr 13 (Ube2b-rs1) or Chr 11 (Ube2brs2). Inactivation of the Ube2b gene of the mouse causes male sterility associated with chromatin modification, although homoCorrespondence to: H. Suzuki Mammalian Genome 10, 1106–1107 (1999).

In rats with genetically hypoplastic kidneys (hpk/hpk) and associated hypogonadism (hgn/hgn), their kidneys contain only one-quarter the number of nephrons that are found in those of normal rats [26]. Not surprisingly, therefore, renal excretive function has been shown to be depressed in hpk/hpk rats [26]. In the study presented here, we have examined the process of the progression of renal failure and the development of renal secondary disease in hpk/hpk rats. The plasma concentrations of urea-nitrogen and creatinine were significantly higher in adult hpk/hpk rats than in normal rats. These values elevated gradually and the degree of renal histological damage also progressed with advancing age in the hpk/hpk rats. In addition, renal anemia appeared at 140 days of age or later in these rats, and hyperplasia of the parathyroid glands was visible macroscopically at 280 days of age. In the hpk/hpk rats plasma levels of calcium and phosphorus were significantly lower and higher than in normal rats, respectively, at 280 days of age. Pathologically, the left femora of hpk/hpk rats exhibited fibrous osteodystrophy at 280 days of age and the calcium content of the right femora (as a percentage of the dry weight of bone) was significantly lower than in normal rats at both 210 and 280 days of age. These results indicate that the reduced nephrogenesis of the hpk/hpk rats causes progressive renal failure, secondarily inducing anemia, hyperparathyroidism, and osteodystrophy.

In male hypogonadic mutant rat (hgn/hgn), gonocytes degenerate and peritubular cells form multiple layers around seminiferous tubules during early postnatal testicular development. Alkaline phosphatase (AP) activity has been used as not only a tracer for the primordial germ cells (PGCs) but also a histochemical marker for the peritubular myoid cells. In the present study, we examined the localization of AP activity during the postnatal testicular development in the hgn/hgn and phenotypically normal (+/+ or +/hgn) rat. In the normal testis, high AP activity was located in the surface of the PGCs on 3 days of age. As the PGCs differentiated into spermatogonia, the AP activity drastically decreased in intensity on 7 days and was completely lost by 12 days. In the hgn/hgn, the PGCs showing high AP activity occupied the inside of dilated seminiferous tubules on 3 and 7 days of age. The luminal AP activity declined gradually by 18 days and disappeared on 21 days, when the germ cells completely degenerated in the hgn/hgn testis. In the normal, high AP activity emerged in a layer of the peritubular cells surrounding the tubules on 7 days and afterwards, indicating that the peritubular cells were differentiating into myoid cells. In the hgn/hgn, the peritubular cells formed multiple layers around the tubules and showed weak AP activity on 3 to 18 days of age. On 21 days of age, high AP activity emerged in a single layer of the peritubular cells directly attached to the basement membrane in the hgn/hgn. These results indicate that the PGCs showing AP activity kept remained at later stage in the hgn/ hgn and that the single layer of mature myoid cells showing high AP activity appeared much later in the hgn/hgn testis than in normal.

Background Koala (Koa) is a dominant mutation in mice causing bushy muzzle and pinna, and is associated with a chromosomal inversion on the distal half of chromosome 15. To identify the gene responsible for the Koa phenotypes, we investigated phenotypes of Koa homozygous mice and determined the breakpoints of the inversion with a genetic method using recombination between two different chromosomal inversions. Results Skeletal preparation of Koa homozygotes showed marked deformity of the ribs and a wider skull with extended zygomatic arches, in addition to a general reduction in the lengths of long bones. They also had open eyelids at birth caused by a defect in the extension of eyelid anlagen during the embryonic stages. The proximal and distal breakpoints of the Koa inversion were determined to be 0.8-Mb distal to the Trsps1 gene and to 0.1-Mb distal to the Hoxc4 gene, respectively, as previously reported. The phenotypes of mice with the recombinant inverted chromosomes revealed the localization of the gene responsible the Koa phenotype in the vicinity of the proximal recombinant breakpoint. Expression of the Trsps1 gene in this region was significantly reduced in the Koa homozygous and heterozygous embryos. Conclusion While no gene was disrupted by the chromosomal inversion, an association between the Koa phenotype and the proximal recombinant breakpoint, phenotypic similarities with Trps1-deficient mice or human patients with TRSP1 mutations, and the reduced expression of the Trsps1 gene in Koa mice, indicated that the phenotypes of the Koa mice are caused by the altered expression of the Trps1 gene.