Extended proliferation of chicken- and Okinawa rail-derived fibroblasts by expression of cell cycle regulators.

Although immortalized cultured cells are useful for various functional assays or transcriptome analysis, highly efficient and reproducible immortalization methods have not been developed in avian-derived cells. We introduced the simian virus 40 T antigen (SV40T) and human papillomavirus (HPV)-E6E7 to chick and Okinawa rail (endangered species)derived fibroblast. As a result, neither the SV40T nor E6E7 genes could induce avian cell immortality. Accordingly, we attempted to use a recently developed immortalization method, which involved the coexpression of mutant cyclin-dependent kinase 4 (CDK4), Cyclin D, and TERT (K4DT method) in these avian cells. Although the K4DT method could not efficiently induce the efficient immortalization in mass cell population, cellular division until the senescence was significantly extended by K4DT, we succeeded to obtain the immortalized avian cells (chick K4DT: one clone, Okinawa rail K4DT: three clones, Okinawa rail K4DT + telomerase RNA component: one clone) with K4DT expression. We conclude that K4DT expression is used to extend the cell division and immortalization of avian-derived cells.

Characterization of feline cytochrome P450 2B6.

1. Little is known about drug metabolism in carnivores. Although the domestic cat (Felis catus) is an obligate carnivore and is the most common companion animal, usage and dosage of many drugs are determined according to information obtained from humans and dogs. We determined the complete cDNA sequence of CYP2B6 from the feline lung. 2. Feline CYP2B6 consists of 494 deduced amino acids, showing highest identity with the dog CYP2B ortholog, followed by those of horse, pig, primate and human. 3. Feline CYP2B6 transcripts were expressed predominantly in the lung and slightly in the small intestine but not in the liver without significant sex-dependent differences. Western blot analysis with an anti-human CYP2B6 antibody confirmed the presence of CYP2B protein in the lung but not in the liver. 4. Feline CYP2B6 proteins heterologously expressed in Escherichia coli metabolized several substrates specific to human CYP2B6, including 7-ethoxy-4-(trifluoromethyl) coumarin (EFC). The metabolic activity was strongly inhibited by medetomidine and atipamezole, potent inhibitors of canine CYP2B11 (now officially CYP2B6) as well as by ticlopidine and sertraline, inhibitors selective to human CYP2B6. 5. The results suggest that feline CYP2B6 is a functional CYP2B ortholog that plays a role in the local defense mechanism in the cat respiratory system and intestine.

Parrot bornavirus-2 and -4 RNA detected in wild bird samples in Japan are phylogenetically adjacent to those found in pet birds in Japan.

Bornaviruses (family Bornaviridae) are non-segmented negative-strand RNA viruses. Avian bornaviruses (ABVs), which are causative agents of proventricular dilatation disease, are a genetically diverse group with at least 15 genotypes, including parrot bornaviruses (PaBVs) and aquatic bird bornavirus 1(ABBV-1). Borna disease virus 1(BoDV-1), which infects mammals and causes neurological diseases, has also been reported to infect avian species, although the numbers of the cases have been markedly fewer than those of ABVs. In this study, we conducted genetic surveillance to detect ABVs (PaBV-1 to -5 and ABBV-1) and BoDV-1 in wild birds in Japan. A total of 2078 fecal or cloacal swab samples were collected from wild birds in 2006, 2007, 2008, and 2011, in two regions of Japan. The results demonstrated the presence of PaBV-2 and -4 RNA, while no positive results for other PaBVs, ABBV-1, and BoDV-1 were obtained. PaBV-2 and -4 RNA were detected in 18 samples (0.9 %) of the genera Anas, Grus, Larus, Calidris, Haliaeetus, and Emberiza, in which either PaBV-2 RNA or PaBV-4 RNA, or both PaBV-2 and -4 RNA were detected in 15 (0.7 %), 5 (0.2 %), and 2 (0.1 %) samples, respectively. The nucleotide sequences of PaBV-2 and -4 detected in these samples from wild birds are phylogenetically close to those found in samples from pet birds in Japan, with identities ranging from 99.8 to 100 % and from 98.2 to 99.4 %, respectively. To the best of our knowledge, this is the first report on the detection of PaBV-2 and -4 RNA detected in samples from wild birds.

Degenerate polymerase chain reaction strategy with DNA microarray for detection of multiple and various subtypes of virus during blood screening.

BACKGROUND: The risk of transferring blood-borne infections during transfusion is continually increasing because of newly emerging and reemerging viruses. Development of a rapid screening method for emerging viruses that might be transmitted by transfusion is required to eliminate such pathogens during blood donor screening. Owing to increased use of human materials in organ transplants and cell therapy, the risk of donor-transmitted viral infections is also increasing. Although nucleic acid amplification technology (NAT) is dedicated to blood screening, a small, convenient detection system is needed at the laboratory and hospital level. STUDY DESIGN AND METHODS: We developed a new pathogen detection system that can detect multiple viruses simultaneously, using originally designed degenerate polymerase chain reaction primers to amplify a wide range of viral genotypes. Amplified samples were identified using a DNA microarray of pathogen-specific probes. RESULTS: We detected very low copy numbers of multiple subtypes of viruses, such as human hepatitis C virus (HCV), human hepatitis B virus (HBV), human parvovirus B19 (PVB19), and West Nile virus (WNV), using a single plate. We also detected all genotypes of human immunodeficiency virus (HIV) but sensitivity was less than for the other viruses. CONCLUSION: We developed a microarray assay using novel primers for detection of a wide range of multiple pathogens and subtypes. Our NAT system was accurate and reliable for detection of HIV, HBV, HCV, PVB19, and WNV, with respect to specificity, sensitivity, and genotype inclusivity. Our system could be customized and extended for emerging pathogens and is suitable as a future NAT system.

Phylogeny analysis of whole protein-coding genes in metagenomic data detected an environmental gradient for the microbiota.

Environmental factors affect the growth of microorganisms and therefore alter the composition of microbiota. Correlative analysis of the relationship between metagenomic composition and the environmental gradient can help elucidate key environmental factors and establishment principles for microbial communities. However, a reasonable method to quantitatively compare whole metagenomic data and identify the primary environmental factors for the establishment of microbiota has not been reported so far. In this study, we developed a method to compare whole proteomes deduced from metagenomic shotgun sequencing data, and quantitatively display their phylogenetic relationships as metagenomic trees. We called this method Metagenomic Phylogeny by Average Sequence Similarity (MPASS). We also compared one of the metagenomic trees with dendrograms of environmental factors using a comparison tool for phylogenetic trees. The MPASS method correctly constructed metagenomic trees of simulated metagenomes and soil and water samples. The topology of the metagenomic tree of samples from the Kirishima hot springs area in Japan was highly similarity to that of the dendrograms based on previously reported environmental factors for this area. The topology of the metagenomic tree also reflected the dynamics of microbiota at the taxonomic and functional levels. Our results strongly suggest that MPASS can successfully classify metagenomic shotgun sequencing data based on the similarity of whole protein-coding sequences, and will be useful for the identification of principal environmental factors for the establishment of microbial communities. Custom Perl script for the MPASS pipeline is available at https://github.com/s0sat/MPASS.

PCR-based gene synthesis with overlapping unisense-oligomers asymmetric extension supported by a simulator for oligonucleotide extension achieved 1 kbp dsDNA.

We formulated a method to synthesize 1 kbp DNA fragments using 'oligomer unidirectional joining method' via asymmetric extension supported by a simulator for oligonucleotide extension (AESOE). In this study, trials were conducted on 41 sets of different genomic pieces of ten flaviviral genomes, and 31 bacterial 16s rRNA fragments with sizes ranging from 500 bases to 1.0 kbp. Synthetic gene production was found to be successful in all those sets. The synthesis method has three steps: the first step is a seven-linked AESOE, the second step is the linking of the 400-base fragments from the first step, and the third step is the final amplification. Our present approach is highly reproducible and may no longer require optimization of oligomer design.

Analysis of Crop Consumption Using Scatological Samples from the Red-Crowned Crane Grus japonensis in Eastern Hokkaido, Japan.

Total DNA extracts from the intestinal contents of 60 flying red-crowned cranes (juveniles, subadults and adults) found dead in 2006-2021, and the feces of 25 chicks collected in June and July of 2016-2018, were used for PCR reactions with primers specific for 16 crops, followed by high-throughput sequencing. The most predominant crop detected was corn in adult and subadult cranes (61.7%). Other grains (barley, wheat, soybean) (5.0-8.3%) and vegetables (tomatoes, Chinese cabbage, etc.) (1.7-6.7%) were also detected in flying cranes. Surprisingly, some of the detected crops were not grown in the Kushiro and Nemuro regions. There was no significant difference in crop intake status in winter and that in other seasons for most of the crops. Corn (28.0%), soybeans (8.0%), wheat and beet (4.0%) were detected in crane chicks in summer, though the detection rates were generally lower than those in flying cranes. Alfalfa, which is not grown in eastern Hokkaido but is used in some cattle feed, was detected in some cranes. Rice, buckwheat, adzuki beans, common beans, potatoes and carrots were not detected at any life stage, indicating the preferences of red-crowned cranes. The results suggest that red-crowned cranes in Hokkaido are dependent on dairy farmers for their feed supply.

Detection of bat coronaviruses from Miniopterus fuliginosus in Japan.

Bats have great potential as reservoirs for emerging viruses such as severe acute respiratory syndrome-coronavirus. In this study, bat coronaviruses (BtCoVs) were detected by RT-PCR from intestinal and fecal specimens of Miniopterus fuliginosus breeding colonies in Wakayama Prefecture caves, where we previously identified bat betaherpesvirus 2. Two primer sets were used for the detection of BtCoV: one was for the RNA-dependent RNA polymerase (RdRp) region and the other was for the spike (S) protein region. Eleven and 73% of intestinal and fecal specimens, respectively, were positive for RdRp region, and 2 and 40% of those were positive for S protein region. Sequencing and phylogenetic analysis showed that the detected BtCoV belonged to the group 1 (alpha) coronaviruses. These data suggest that BtCoV is endemic in M. fuliginosus in Japan.

Differential display system with vertebrate-common degenerate oligonucleotide primers: uncovering genes responsive to dioxin in avian embryonic liver.

To assess possible impacts of environmental pollutants on gene expression profiles in a variety of organisms, we developed a novel differential display system with primer sets that are common in seven vertebrate species, based on degenerate oligonucleotide-primed PCR (DOP-PCR). An 8-mer inverse repeat motif was found in most transcripts from the seven vertebrates including fish to primates with detailed transcriptome information; more than 10,000 motifs were recognized in common in the transcripts of the seven species. Among them, we selected 275 common motifs that cover about 40-70% of transcripts throughout these species, and designed 275 DOP-PCR primers that were common to seven vertebrate species (common DOP-PCR primers). To detect genes responsive to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in developing embryos, differential display with common DOP-PCR primers was applied to embryonic liver of two avian species, the chicken (Gallus gallus) and the common cormorant (Phalacrocorax carbo), which were exposed in ovo to TCDD. The cDNA bands that showed differences between the control and TCDD-treated groups were sequenced and the mRNA expression levels were confirmed by real-time RT-PCR. This approach succeeded in isolating novel dioxin-responsive genes that include 10 coding genes in the chicken, and 1 coding gene and 1 unknown transcript in the cormorant, together with cytochrome P450 1As that have already been well established as dioxin markers. These results highlighted the usefulness of systematically designed novel differential display systems to search genes responsive to chemicals in vertebrates, including wild species, for which transcriptome information is not available.

Radioprotective effect of alk(en)yl thiosulfates derived from allium vegetables against DNA damage caused by X-ray irradiation in cultured cells: antiradiation potential of onions and garlic.

To evaluate a radioprotective effect of sodium n-propyl thiosulfate (NPTS) and sodium 2-propenyl thiosulfate (2PTS) derived from onions and garlic, respectively, rat hepatoma H4IIE cells and mouse lymphoma L5178Y cells were preincubated with each of these compounds for 48 hours at 37°C before receiving 10 Gy of X-ray irradiation. Cell damage caused by the irradiation was quantified as comet tail moment, which represents the degree of DNA damage. X-ray-induced DNA damage was significantly decreased in both H4IIE and L5178Y cells by micromolar concentrations of NPTS and 2PTS compared with the control without the compounds. The protective effect was more potent with 2PTS than NPTS. Onions and garlic have antiradiation potential.

Evaluation of Renal Blood Flow in Dogs during Short-Term Human-Dose Epoprostenol Administration Using Pulsed Doppler and Contrast-Enhanced Ultrasonography.

Prostacyclin is an in vivo bioactive substance that regulates renal blood flow (RBF). Information regarding how epoprostenol, a prostacyclin preparation, affects RBF in dogs is lacking. We investigated the effects of short-term epoprostenol administration on RBF in six healthy dogs under anesthesia by administering it intravenously at human doses-2, 5, and 10 ng/kg/min for 20 min. RBF was evaluated before and during epoprostenol administration using pulsed Doppler ultrasonography, and renal perfusion was evaluated using contrast-enhanced ultrasonography. Effects on renal and systemic circulation were evaluated by measuring systolic arterial, mean arterial, diastolic arterial, pulmonary arterial, mean right atrial, and pulmonary capillary wedge pressures; heart rate; and cardiac output. Kruskal-Wallis and Bonferroni multiple comparison tests and Spearman's rank correlation coefficient were used for statistical analyses. As epoprostenol dosage increased, the peak systolic and end diastolic velocity of the renal artery, maximum and minimum venous flow velocities of the interlobular and renal veins, and heart rate all tended to increase, although not significantly. Our results indicate that human-dose epoprostenol administration in dogs does not cause significant changes in renal or systemic circulation. However, the human doses used may have been too low to produce a clinical effect in dogs.

Conventional Gel Electrophoresis-Resolvable Insertion/Deletion Markers for Individual Identification and Analysis of Population Genetics in Red-Crowned Cranes in Eastern Hokkaido, Japan.

Red-crowned crane Grus japonensis is an endangered species in two separate populations: the mainland population in the Eurasian continent and the island population in eastern Hokkaido, Japan. We found 11 insertion/deletion (InDel) markers in the genome of the red-crowned crane and designed primer sets across these InDels that can be analyzed with conventional agarose gel electrophoresis. Sixty-six samples of whole blood and skeletal muscle obtained from red-crowned cranes, including 12 families in eastern Hokkaido from 1994 to 2021, showed different patterns in gel images of 11 InDel PCR reactions except for two pairs. The combined non-exclusion probability of the 11 markers indicates that individuals can be determined with a probability of 99.9%. In 39 non-relative chicks, the expected heterozygosity (He) was 0.316, suggesting low genetic diversity. This might not be caused by high levels of inbreeding since the average FIS was not significantly different from zero (0.095, p = 0.075). The results suggest that the 11 InDel primer sets can be used for fairly accurate individual identification as well as genetic population analyses in red-crowned cranes in the island population.

Evaluation of caudal vena cava size using computed tomography in dogs under general anesthesia.

This study investigated the association between caudal vena cava (CVC) size and circulatory dynamics in dogs using computed tomography (CT) under general anesthesia. The subjects were 104 dogs who had undergone CT under general anesthesia in the past. The ratio of short diameter of the CVC to aortic diameter (CVCS/Ao) and the ratio of long to short diameter of the CVC (CVCL/CVCS) in the thorax and abdomen, respectively, were calculated using factors such as mean blood pressure (MBP), shock index (SI), anemia, hypoproteinemia, presence of intra-abdominal mass, and cardiac disease. There was a significant but negligible negative correlation between CVCS/Ao and MBP. In contrast, no significant correlation was found between CVC size and SI. The low MBP group had significantly higher CVCS/Ao of the thorax than the normal MBP group. The group with intra-abdominal mass had significantly lower CVCS/Ao of the abdomen than the group without intra-abdominal mass. The group with cardiac disease had significantly lower CVCL/CVCS of the thorax than the group without cardiac disease. In multiple regression analysis, low MBP, cardiac disease, intra-abdominal mass, and anemia were significant factors for CVCS/Ao of the thorax, CVCL/CVCS of the thorax, CVCS/Ao of the abdomen, and CVCL/CVCS of the abdomen, respectively. In conclusion, CVC size assessment using CT in dogs under general anesthesia is influenced by various factors.

Metabarcoding of feces and intestinal contents to determine carnivorous diets in red-crowned cranes in eastern Hokkaido, Japan.

The red-crowned crane Grus japonensis in Hokkaido, Japan forms a closed population as a residence that is independent of the mainland population. Based on observations of a limited number of individuals as well as cranes in captivity, red-crowned cranes are omnivores and eat fish, worms, insects and plants in their own territories except in winter, when they are fed with dent corn that is supplied in eastern Hokkaido. DNA metabarcoding based on high throughput sequencing was carried out using universal primer sets for cytochrome oxidase subunit I gene. Feces from 27 chicks collected in June and July in the period from 2016 to 2018 and intestinal contents from 33 adult and subadult cranes that were found dead almost throughout year in 2006-2013 in the field in eastern Hokkaido were used. Although compositions varied considerably in the cranes, both insects and fish were found in adults and subadults to the same extents, while insects were predominant in chicks. Both insects and fish were detected in all seasons for adults and subadults. Horse flies, scarab beetles and weevils accounted for the most of the insects regardless of the life stage. Dace, stickleback, flatfish and sculpin were the major fish species in adults, while chicks ate almost only stickleback. The results provide the first comprehensive data on carnivorous diets in wild red-crowned cranes in eastern Hokkaido as basis for conservation of red-crowned cranes, for which the life style and area continue to change.

Radiofrequency radiation at 40 kHz induces hepatic injury in Long-Evans Cinnamon (LEC) rats, an animal model for human Wilson disease.

In the present study, we examined effects of radiofrequency (RF) radiation at 40 kHz on hepatic injury in Long-Evans Cinnamon (LEC) rats, an animal model for human Wilson disease, which is a heritable disease of copper metabolism in the liver. The activities of ALT and AST in serum of LEC rats exposed to RF radiation for 2 weeks were approximately 3.8-fold and 2-fold higher than those in serum of sham-exposed rats, respectively. Although there were no significant differences in hepatic copper contents between LEC rats exposed to RF radiation for 2 weeks and sham-exposed rats, copper contents in the kidney and serum of exposed LEC rats were approximately 4.2-fold and 12.9-fold higher than those in sham-exposed rats, respectively. Relative O2⁻-scavenging activities in the S-100 fraction of the liver of LEC rats exposed to RF radiation for 2 weeks were 1.6-fold higher than those in sham-exposed rats. No significant differences were observed in activities of AST and ALT in serum and relative O2⁻-scavenging activity in the S-100 fraction of the liver of normal control WKAH rats that were sham-exposed and exposed to RF radiation. No significant differences were observed in copper contents in the liver, kidney and serum of WKAH rats that were sham-exposed and exposed to RF radiation for 2 weeks. The results show that RF radiation at 40 kHz induced hepatic injury in LEC rats.

Prediction of PCR amplification from primer and template sequences using recurrent neural network.

We have developed a novel method to predict the success of PCR amplification for a specific primer set and DNA template based on the relationship between the primer sequence and the template. To perform the prediction using a recurrent neural network, the usual double-stranded formation between the primer and template nucleotide sequences was herein expressed as a five-lettered word. The set of words (pseudo-sentences) was placed to indicate the success or failure of PCR targeted to learn recurrent neural network (RNN). After learning pseudo-sentences, RNN predicted PCR results from pseudo-sentences which were created by primer and template sequences with 70% accuracy. These results suggest that PCR results could be predicted using learned RNN and the trained RNN could be used as a replacement for preliminary PCR experimentation. This is the first report which utilized the application of neural network for primer design and prediction of PCR results.

The effects of gel pad thickness on the evaluation of skin structures using ultrasonography in normal dogs.

Gel pads are commonly used in skin ultrasonography; however, the effects of their thickness are unknown. This study investigated the effects of pad thickness on measurements of skin thickness in 10 beagle dogs. Sonograms to measure neck skin thickness were captured without pads and using pads with thicknesses of 3, 5, 10, and 20 mm. Without pads, acoustic shading was observed due to air bubbles in the coupling gel. With 20-mm pads, echogenic artifacts were observed on the skin surface. Entry echo with 20-mm pads was significantly higher than with 3-mm pads. This suggests that visibility of the skin structure could be affected when a gel pad is not used or when a thick gel pad is selected.

Local overexpression of interleukin-1 family, member 6 relates to the development of tubulointerstitial lesions.

Identification of factors that exacerbate a disease is important for the development of biomarkers. In this study, we discovered ectopic overexpression of interleukin-1 family, member-6 (IL-1F6) in several murine renal diseases. IL-1F6 participates in cytokine/chemokine production in the epithelium. In PCR array analysis for inflammatory mediators, Il1f6 showed the highest expression in the kidney of the B6.MRLc1 glomerulonephritis model. IL-1F6 was localized in the epithelium from the DCTs to CCDs, which showed tubular dilations or epithelial deciduations. Ultrastructual examination of the epithelial cells revealed that IL-1F6 was localized on the cytoplasmic ribosome, vesicles, and nucleus. In and around these tubules, we found infiltrations of CD3-positive T-cells and nestin- or alpha-smooth-muscle actin-positive mesenchymal cells. Expression of the IL-1F6 protein and Il1f6 mRNA in the kidney was increased by the development of TILs in the B6.MRLc1 model and in lupus (BXSB, NZB/WF1, and MRL/lpr), nephrotic syndrome (ICGN), and streptozotocin-induced diabetic models. IL-1F6 was also detected in the epithelia having squamous or deciduous contours in other organs such as the skin, esophagus, thymus, or uterus. In vitro analysis using M-1 cells from the murine collecting duct revealed that Il1f6 mRNA induction was related to the upregulation of IL-6, TGF-beta receptor-1, and mesenchymal markers and to the downregulation of epithelial markers and changes in the squamous cells of the epithelium. Interestingly, urine Il1f6 mRNA expression was detected earlier than renal dysfunctions in these mouse models. Ectopic overexpression of IL-1F6 in kidneys is associated with TILs and especially with cell infiltrations and changes in epithelial morphology. We propose that local overexpression of IL-1F6 is related to the development of TILs.

BLV-CoCoMo-qPCR: Quantitation of bovine leukemia virus proviral load using the CoCoMo algorithm.

BACKGROUND: Bovine leukemia virus (BLV) is closely related to human T-cell leukemia virus (HTLV) and is the etiological agent of enzootic bovine leukosis, a disease characterized by a highly extended course that often involves persistent lymphocytosis and culminates in B-cell lymphomas. BLV provirus remains integrated in cellular genomes, even in the absence of detectable BLV antibodies. Therefore, to understand the mechanism of BLV-induced leukemogenesis and carry out the selection of BLV-infected animals, a detailed evaluation of changes in proviral load throughout the course of disease in BLV-infected cattle is required. The aim of this study was to develop a new quantitative real-time polymerase chain reaction (PCR) method using Coordination of Common Motifs (CoCoMo) primers to measure the proviral load of known and novel BLV variants in clinical animals. RESULTS: Degenerate primers were designed from 52 individual BLV long terminal repeat (LTR) sequences identified from 356 BLV sequences in GenBank using the CoCoMo algorithm, which has been developed specifically for the detection of multiple virus species. Among 72 primer sets from 49 candidate primers, the most specific primer set was selected for detection of BLV LTR by melting curve analysis after real-time PCR amplification. An internal BLV TaqMan probe was used to enhance the specificity and sensitivity of the assay, and a parallel amplification of a single-copy host gene (the bovine leukocyte antigen DRA gene) was used to normalize genomic DNA. The assay is highly specific, sensitive, quantitative and reproducible, and was able to detect BLV in a number of samples that were negative using the previously developed nested PCR assay. The assay was also highly effective in detecting BLV in cattle from a range of international locations. Finally, this assay enabled us to demonstrate that proviral load correlates not only with BLV infection capacity as assessed by syncytium formation, but also with BLV disease progression. CONCLUSIONS: Using our newly developed BLV-CoCoMo-qPCR assay, we were able to detect a wide range of mutated BLV viruses. CoCoMo algorithm may be a useful tool to design degenerate primers for quantification of proviral load for other retroviruses including HTLV and human immunodeficiency virus type 1.

Analysis of factors decreasing testis weight in MRL mice.

MRL/MpJ (MRL) mouse testes have several unique characteristics, including the appearance of oocytes, the occurrence of metaphase-specific apoptosis of meiotic spermatocytes, and the presence of heat-shock-resistant spermatocytes. In the present study we used chromosomal mapping to determine the genomic background associated with small testis size in MRL mice. We prepared and analyzed C57BL/6-based congenic mice carrying MRL mouse loci. Quantitative trait loci (QTL) analysis revealed susceptibility loci for small testis size at 100 cM on chromosome (Chr) 1 and at around 80 cM on Chr 2. Analysis with B6.MRLc1 and B6.MRLc2 congenic mice and double-congenic mice confirmed the QTL data and showed that low testis weight in MRL mice was caused by germ cell apoptosis. Through histological examinations we found that B6.MRLc1 and B6.MRLc2 mice showed stage-specific apoptosis in their testes, the former at metaphase stage XII and the later at pachytene stage IV. Metaphase-specific apoptosis of spermatocytes occurs due to mutation of the exonuclease 1 (Exo1) gene located at 100 cM on Chr 1. Thus, the mutation of the Exo1 gene is also responsible for low testis weight caused by metaphase-specific apoptosis. In conclusion, testis weight is reduced in MRL mice due to apoptosis of germ cells caused by mutations in loci on Chrs 1 and 2.