Autophagy in the corpus luteum correlates with tissue growth in pregnant rats.

The developmental activation of the corpus luteum (CL) structurally and functionally is critical for the temporally regulated establishment, maintenance, and termination of pregnancy in rats. In this study, we have investigated the possible involvement of autophagy in the regulation of the CL during pregnancy in rats. The expression ratio of microtubule-associated protein light chain 3 (LC3)-II/-I, a widely used indicator of autophagic activity, in the CL remained relatively stable until day 15 of pregnancy. Subsequently, it progressively increased until day 21, and then declined until day 3 postpartum. This fluctuation was closely associated with the tissue weight of the CL rather than progesterone (P4) production activity. Light and electron microscopy revealed the presence of immunoreactive LC3 aggregates and irregularly shaped autolysosome-like microstructures in the cytoplasm of luteal cells during late pregnancy. Notably, a bolus intrabursal injection of the autophagy inhibitor bafilomycin A1 on day 15 of pregnancy resulted in a significant reduction in luteal cell size and disrupted the normal alteration of circulating P4 levels. Consequently, treatment with this inhibitor increased the likelihood of the varied timing (both advanced and delayed) of delivery and led to reduced body weight in neonates when compared with the vehicle-treated control group. Our findings suggest that autophagy in the rat CL contributes to luteal tissue growth, influences P4 production, and thereby fine-tunes the regulation of gestation length in rats.

Histological species differences among chickens, rats, and mice in experimental cholestasis by bile duct ligation.

Cholestasis is a hepatic disease reported in humans, dogs, and chickens and is characterized by various signs. Bile duct ligation (BDL) is a standard model for research in cholestasis in male rats and mice. However, the timing and degree of structural changes in BDL-subjected liver differ in the two animal species. This study focused on chickens as a choice model for cholestasis. Specifically, we aimed to evaluate the features of BDL in hens and compare them with those in rats and mice. Eighteen hens, 19 female ICR mice, and 18 female SD rats were randomly divided into the sham-operated and BDL groups. At 2, 4, and 6 weeks after BDL, and 4 weeks after the sham operation, liver and blood samples were collected and analyzed histologically and biochemically. Histologically, bile duct proliferation in BDL-subjected livers was first observed in the chickens and then the rats and mice, whereas CD44-positive small hepatocytes were observed only in chickens in the BDL group. Biochemically, the mRNA expression of the hepatocyte growth factor was higher in BDL-subjected chickens, while Interleukin 6 expression was higher in the BDL-subjected rats and mice than in animals in the sham group. In addition, farnesoid X receptor mRNA expression was lower in the BDL-subjected chickens than in the sham chickens. The BDL group had significantly higher total bile acid blood concentration than the sham group. In conclusion, the signs of hepatopathy caused by BDL differ among animal species. Furthermore, we propose that compared to BDL-subjected mice and rats, BDL-subjected chickens are a novel cholestasis animal model that demonstrates severe hepatopathy and liver restructuring.

Critical Involvement of the Thioredoxin Reductase Gene (trxB) in Salmonella Gallinarum-Induced Systemic Infection in Chickens.

Salmonella enterica serovar Gallinarum biovar Gallinarum (SG) causes fowl typhoid, a notifiable infectious disease in poultry. However, the pathogenic mechanism of SG-induced systemic infection in chickens remains unclear. Thioredoxin reductase (TrxB) is a redox protein crucial for regulating various enzyme activities in Salmonella serovar, but the role in SG-induced chicken systemic infection has yet to be determined. Here, we constructed a mutant SG strain lacking the trxB gene (trxB::Cm) and used chicken embryo inoculation and chicken oral infection to investigate the role of trxB gene in the pathogenicity of SG. Our results showed that trxB::Cm exhibited no apparent differences in colony morphology and growth conditions but exhibited reduced tolerance to H2O2 and increased resistance to bile acids. In the chicken embryo inoculation model, there was no significant difference in the pathogenicity of trxB::Cm and wild-type (WT) strains. In the chicken oral infection, the WT-infected group exhibited typical clinical symptoms of fowl typhoid, with complete mortality between days 6 and 9 post infection. In contrast, the trxB::Cm group showed a 100% survival rate, with no apparent clinical symptoms or pathological changes observed. The viable bacterial counts in the liver and spleen of the trxB::Cm-infected group were significantly reduced, accompanied by decreased expression of cytokines and chemokines (IL-1ß, IL-6, IL-12, CXCLi1, TNF-α, and IFN-γ), which were significantly lower than those in the WT group. These results show that the pathogenicity of the trxB-deficient strain was significantly attenuated, indicating that the trxB gene is a crucial virulence factor in SG-induced systemic infection in chickens, suggesting that trxB may become a potentially effective target for controlling and preventing SG infection in chickens.

Salmonella pathogenicity island-14 is a critical virulence factor responsible for systemic infection in chickens caused by Salmonella gallinarum.

Salmonella enterica serovar Gallinarum (S. gallinarum) is an important host-specific pathogen that causes fowl typhoid, a severe systemic, septicemic, and fatal infection, in chickens. S. gallinarum causes high morbidity and mortality in chickens and poses a significant burden and economic losses to the poultry industry in many developing countries. However, the virulence factors and mechanisms of S. gallinarum-induced systemic infection in chickens remain poorly understood. In this study, we constructed a Salmonella pathogenicity island-14 (SPI-14) mutant strain (mSPI-14) of S. gallinarum and evaluated the pathogenicity of mSPI-14 in the chicken systemic infection model. The mSPI-14 exhibited the same level of bacterial growth and morphological characteristics but significantly reduced resistance to bile acids compared with the wild-type (WT) strain in vitro. The virulence of mSPI-14 was significantly attenuated in the chicken oral infection model in vivo. Chickens infected with WT showed typical clinical symptoms of fowl typhoid, with all birds succumbing to the infection within 6 to 9 days post-inoculation, and substantial increases in bacterial counts and significant pathological changes in the liver and spleen were observed. In contrast, all mSPI-14-infected chickens survived, the bacterial counts in the organs were significantly lower, and no significant pathological changes were observed in the liver and spleen. The expression of interleukin (IL)-1ß, IL-12, CXCLi1, tumor necrosis factor (TNF)-α, and interferon (IFN)-γ in the liver of mSPI-14-infected chickens were significantly lower than those in the WT-infected chickens. These results indicate that SPI-14 is a crucial virulence factor in systemic infection of chickens, and avirulent mSPI-14 could be used to develop a new attenuated live vaccine to prevent S. gallinarum infection in chickens.

Experimental infection of goats with pVAPN-harboring Rhodococcus equi causes latent infection in the lymph nodes.

Rhodococcus equi has recently been identified in various animals, including ruminants. Several studies have highlighted the emergence of pVAPN-harboring strains, isolated from multiple abscesses, in the liver and lungs of ruminants. Epidemiological evidence strongly suggests that pVAPN-harboring strains are pathogenic in ruminants. This study aims to replicate the disease in goats through experimental infection. Intravenous administration of the pVAPN-harboring strain (Yokkaichi), pVAPA-harboring strain (ATCC33701), and pVAPN-cured strain (Yokkaichi_P-), each at 1.0 × 107 CFU/head, was conducted in 24-month-old goats (n = 1 per group). During the observation period, goats treated with Yokkaichi or ATCC33701 exhibited transient increases in body temperature and white blood cell count, alongside a decrease in body weight from the administration day. Conversely, goats treated with Yokkaichi_P- displayed no significant changes in these values. The Yokkaichi-treated goat demonstrated a >10-fold increase in anti-VapN antibody titers from 11 to 14 days postadministration, whereas the other two goats exhibited no variation in anti-VapA and VapN antibody titers. Pathological autopsy analysis of organs harvested 28 days postadministration revealed no characteristic lesions on gross examination. However, the inoculated strain (vapN-positive R. equi) was exclusively recovered from the tracheobronchial lymph node in the Yokkaichi-treated goat. Immunohistochemistry detected a VapN-positive reaction in the tracheobronchial lymph node, confirming latent infection despite the absence of dramatic suppurative lesions seen in ruminants. Overall, this study highlights the latent infection in lymph nodes induced by the pVAPN-harboring strain, despite the absence of overt pathological manifestations.

Molecular characterization of an avian rotavirus a strain detected from a large-billed crow (Corvus macrorhynchos) in Japan.

Avian rotaviruses A (RVAs) are occasionally transmitted to animals other than the original hosts across species barriers. Information on RVAs carried by various bird species is important for identifying the origin of such interspecies transmission. In this study, to facilitate an understanding of the ecology of RVAs from wild birds, we characterized all of the genes of an RVA strain, JC-105, that was detected in a fecal sample of a large-billed crow (Corvus macrorhynchos) in Japan. All of the genes of this strain except for the VP4 and VP7 genes, which were classified as novel genotypes (P[56] and G40, respectively), were closely related to those of the avian-like RVA strain detected from a raccoon, indicating the possibility that crows had been involved in the transmission of avian RVAs to raccoons. Our findings highlight the need for further viral investigations in wild birds and mammals to understand the mechanisms of avian-to-mammal RVA transmission.

Toxicity Assessment of Mixed Exposure of Nine Perfluoroalkyl Substances at Concentrations Relevant to Daily Intake.

Per- and poly-fluoroalkyl substances (PFAS) exhibit high persistence in the environment and accumulate within the human body, warranting a thorough assessment of their toxicity. In this study, we exposed mice (male C57BL/6J mice aged 8 weeks) to a composite of nine PFAS, encompassing both long-chain PFAS (e.g., perfluorooctanoic acid and perfluorooctanesulfonic acid) and short-chain PFAS (e.g., perfluorobutanoic acid and perfluorobutanesulfonic acid). The exposure concentrations of PFAS were equivalent to the estimated daily human intake in the composition reported (1 µg/L (sum of the nine compounds), representing the maximum reported exposure concentration). Histological examination revealed hepatocyte vacuolization and irregular hepatocyte cord arrangement, indicating that exposure to low levels of the PFAS mixture causes morphological changes in liver tissues. Transcriptome analysis revealed that PFAS exposure mainly altered a group of genes related to metabolism and chemical carcinogenesis. Machine learning analysis of the liver metabolome showed a typical concentration-independent alteration upon PFAS exposure, with the annotation of substances such as glutathione and 5-aminovaleric acid. This study demonstrates that daily exposure to PFAS leads to morphological changes in liver tissues and alters the expression of metabolism- and cancer-related genes as well as phospholipid metabolism.

Expression of lysophosphatidic acid receptors in the rat uterus: cellular distribution of protein and gestation-associated changes in gene expression.

Though lysophosphatidic acid (LPA) shows a variety of regulatory roles in reproduction, its action mechanisms in the gestational organs are still largely unknown. We here characterized cellular distribution of its six kinds of specific receptors (LPA1-6) in rat uteri by immunohistochemistry and quantitatively analyzed changes in Lpar1-6 mRNAs expression throughout pregnancy. Among LPA1-6, evident expression of LPA3, LPA4, and LPA6 was immunologically detected and less expression of immunoreactive LPA1 and LPA2 was also found. Luminal and glandular epithelial cells, stromal cells, and myometrial cells are sites of positive immunoreactions, and they are all likely to express three or more subtypes. All of Lpar1-6 mRNAs were expressed, and their alterations were variable depending on subtypes and gestational age. The present information suggests that diverse actions of LPA in the uterus involve varied expression of LPA receptors dependent on tissue/cell types, receptor subtype(s), and organ reproductive states and helps to understand uterine biology of LPA.

Development of monoclonal antibodies against Rhodococcus equi virulence-associated protein N and their application to pathological diagnosis.

IMPORTANCE: Rhodococcus equi can cause infection in ruminants, and its pathogenicity is suggested to be associated with VapN. Despite its wide distribution, no immunological diagnostic method has been developed for VapN-producing R. equi. Against this background, we attempted to develop monoclonal antibodies targeting VapN and assess their application in immunostaining. In the study, mice were immunized with recombinant VapN, and cell fusion and cloning by limiting dilution permitted the generation of three antibody-producing hybridomas. The utility of the antibodies produced from the hybridomas in immunostaining was demonstrated using an infected mouse model, and the antibodies were further applied to previously reported cases of R. equi infection in goats and cattle. Although the 4H4 antibody induced the strongest reactions, the reactivity of two other antibodies was improved by antigen retrieval. Our monoclonal antibodies will be utilized to support the definitive diagnosis of suspected R. equi infection, including cases that were previously missed.

Aberrant activation of estrogen receptor-α signaling in Mettl14-deficient uteri impairs embryo implantation.

The precise control of endometrial receptivity is crucial for successful embryo implantation, which is strictly regulated by the ovarian steroid hormones estrogen and progesterone. Despite our improved understanding of the genetic regulation of implantation downstream of the action of hormones, we do not know much about the epigenetic regulation that occurs during early pregnancy. To investigate the role of the N6-methyladenosine (m6A) RNA modification in embryo implantation, we generated mice with conditional deletion of Mettl14, a core component of the m6A writer complex, in the uterus. These mice were infertile due to implantation failure. We showed that Mettl14-deficient uteri had aberrant upregulation of estrogen receptor α (ERα) signaling and ERα phosphorylation, but progesterone receptor (PGR) signaling was largely unaffected. Additionally, Mettl14 deletion led to abnormal activation of the innate immune pathway in Mettl14-deficient uteri. This effect was accompanied by the infiltration of immune cells, such as macrophages and dendritic cells, into the basal region of the endometrial epithelium. Methylated RNA immunoprecipitation sequencing (MeRIP-seq) showed that genes involved in the innate immune response had decreased m6A peaks in Mettl14-deficient mice. These results suggest that Mettl14 plays a crucial role in successful implantation by precisely regulating both ERα signaling and innate immunity in the uterus.

Male pseudohermaphroditism in a complex malformed calf born with an acardius amorphus cotwin-a case report.

BACKGROUND: Male pseudohermaphroditism is a developmental anomaly wherein animals are genetically and gonadally male, but their internal and/or external genitalia resemble those of females. In cattle, pseudohermaphroditism is often accompanied by multiple severe malformations. To the best of our knowledge, this is the first report of male pseudohermaphroditism in a complex malformed calf born with an acardius amorphous cotwin. CASE PRESENTATION: This report describes the case of a three-day-old, male anurous Japanese Black calf born with an acardius amorphous cotwin, complete absence of the tail, agenesis of the anus, separate scrota, and umbilical hernia. Transthoracic echocardiography and computed tomography revealed serious malformations in the skeletal system and the circulatory, digestive, urinary, and genital organs. Necropsy revealed rectal atresia, immature testes, epididymis, and penis, but no male accessory gonads. Histological analyses revealed vaginal- and uterine-like tissues adjacent to or fused to the rectum. Fluorescence in situ hybridization detected X and Y chromosomes, and some cells presented two X-probe signals in the same nucleus. CONCLUSIONS: In contrast to the male genitalia, the female genitalia derived from the Müllerian ducts were difficult to detect by necropsy in the presented case. Many similar cases may be overlooked in clinical practice.

Generation and characterization of a genetically modified live rabies vaccine strain with attenuating mutations in multiple viral proteins and evaluation of its potency in dogs.

Live rabies vaccines have advantageous features that can facilitate mass vaccination for dogs, the most important reservoirs/transmitters of rabies. However, some live vaccine strains have problems in their safety, namely, risks from the residual pathogenicity and the pathogenic reversion of live vaccine strains. The reverse genetics system of rabies virus provides a feasible option to improve the safety of a live vaccine strain by, for example, artificially introducing attenuating mutations into multiple viral proteins. It was previously demonstrated in separate studies that introduction of amino acid residues Leu at position 333 in the viral glycoprotein (G333), Ser at G194, and Leu/His at positions 273/394 in the nucleoprotein (N273/394) enhance the safety of a live vaccine strain. In this study, to test our hypothesis that combinational introduction of these residues would significantly increase the safety level of a vaccine strain, we generated a novel live vaccine candidate, ERA-NG2, that is attenuated by mutations at N273/394 and G194/333, and we examined its safety and immunogenicity in mice and dogs. ERA-NG2 did not cause any clinical signs in mice after intracerebral inoculation. After 10 passages in suckling mouse brains, ERA-NG2 retained all of the introduced mutations except the mutation at N394 and the highly attenuated phenotype. These findings indicate that the ERA-NG2 is highly and stably attenuated. After confirming that ERA-NG2 induced a virus-neutralizing antibody (VNA) response and protective immunity in mice, we immunized dogs intramuscularly with a single dose (105-7 focus-forming units) of ERA-NG2 and found that, at all of the tested doses, the strain induced a VNA response in dogs without inducing any clinical signs. These findings demonstrate that ERA-NG2 has a high level of safety and a substantial level of immunogenicity in dogs and thus is a promising live vaccine candidate that can facilitate vaccination in dogs.

Lysophosphatidic acid stimulates rat uterine contraction in vitro.

Lysophosphatidic acid (LPA) has been implicated in the uterine endometrial functions of implantation and decidualization; however, not much is known about its myometrial contractile function. Herein we characterized the uterotonic effects of LPA in non-pregnant (estrus) and peri-parturient rats in vitro. LPA dose-dependently (0.01-10 µM) stimulated the amplitude and integral, but not the frequency, of the uterine strip contraction of estrous rats. The stimulatory effect of LPA was enhanced 1 day before parturition but was lost 1 day postpartum. LPA did not cause the de novo synthesis of prostaglandin (PG) F2α but stimulated contractions cooperatively with the PG. LPA-induced contractions were significantly inhibited by an LPA1/2/3 antagonist in the uteri of estrous rats but not in term rats. This study characterized the uterotonic effect of a natural LPA that occurs at physiological concentrations, changes with reproductive states, and is independent of mediation by the newly synthesized PG.

Dietary olive oil intake induces female-specific hepatic lipid accumulation without metabolic impairment in mice.

Olive oil is one of the most widely researched Mediterranean diet components in both experimental models and clinical studies. However, the relationship between dietary olive oil intake and liver function in a healthy state of the body remains unclear. Because men are at a greater risk of developing hepatic diseases than women, and because hepatic metabolism is regulated by sex hormones, we hypothesized that olive oil-induced changes in hepatic metabolism would differ by sex. To test our hypothesis, 12-week-old C57BL/6JJcl male and female mice were fed an olive oil diet for 4 weeks. Blood was collected and serum biochemical components were analyzed. Hepatic lipid accumulation was determined via histological analysis using Sudan III staining. Finally, transcript expression levels of hepatic metabolism-related genes were analyzed using quantitative polymerase chain reaction. We observed significant increased hepatic lipid droplet accumulation in olive oil-fed female mice. Serum biochemical and liver messenger RNA expression analyses revealed that the hepatic lipid accumulation was nonpathological and did not involve inflammation. Moreover, the expression of genes related to triacylglycerol and fatty acid synthesis (Dgat1, Dgat2, Agpat3, and Fasn) was significantly upregulated in the liver of olive oil-fed female mice compared with control female mice. Our study demonstrates female-specific hepatic lipid accumulation without liver impairment in a dietary olive oil-fed mouse model. These findings provide a deeper mechanistic understanding of sex-dependent hepatic lipid metabolism of dietary oils.

Multiplexed genome editing by in vivo electroporation of Cas9 ribonucleoproteins effectively induces endometrial carcinoma in mice.

Synergistic effects among multiple gene mutations are involved in cancer development and progression. However, developing genetically modified mouse models to analyze various combinations of mutations is extremely labor-intensive and time-consuming. To address these problems, we developed a novel method for in vivo multiplexed genome editing of the murine uterus to model human endometrial carcinoma (EMC). To do this, we injected a CRISPR-Cas9 ribonucleoprotein complex into the uterine cavity of adult female mice, followed by electroporation. Evaluation of reporter mice demonstrated that genome editing occurred specifically in uterine epithelial cells, which are the origin of EMCs. Simultaneous targeting of Pten/Trp53/Lkb1, or targeting of Pten/Lkb1 along with the Ctnnb1ΔEx3 mutation, resulted in efficient generation of invasive tumors in wild-type females within 3 months. This novel method will enable rapid and easy validation of many combinations of gene mutations that lead to endometrial carcinogenesis.

Molecular characterisation of a novel avian rotavirus A strain detected from a gull species (Larus sp.).

A recent study demonstrated the possibility that migratory birds are responsible for the global spread of avian rotavirus A (RVA). However, little is known about what types of RVAs are retained in migratory birds. In this study, to obtain information on RVA strains in migratory birds, we characterised an RVA strain, Ho374, that was detected in a faecal sample from a gull species (Larus sp.). Genetic analysis revealed that all 11 genes of this strain were classified as new genotypes (G28-P[39]-I21-R14-C14-M13-A24-N14-T16-E21-H16). This clearly indicates that the genetic diversity of avian RVAs is greater than previously recognised. Our findings highlight the need for investigations of RVA strains retained in migratory birds, including gulls.

Establishment of a reverse genetics system for rabies virus strain Komatsugawa.

The rabies virus strain Komatsugawa isolated from a dog in Tokyo in the 1940s retains biological properties as a field strain, providing an effective model for studying rabies pathogenesis. To facilitate molecular studies on the pathogenesis, this study aimed to establish a reverse genetics system for the Komatsugawa strain. By transfecting the full-length genome plasmid of this strain, infectious virus with artificially introduced genetic markers in its genome was rescued. The recombinant strain had biological properties similar to those of the original strain. These findings indicate that a reverse genetics system for the Komatsugawa strain has successfully been established.

The Amino Acid at Position 95 in the Matrix Protein of Rabies Virus Is Involved in Antiviral Stress Granule Formation in Infected Cells.

Stress granules (SGs) are dynamic structures that store cytosolic messenger ribonucleoproteins. SGs have recently been shown to serve as a platform for activating antiviral innate immunity; however, several pathogenic viruses suppress SG formation to evade innate immunity. In this study, we investigated the relationship between rabies virus (RABV) virulence and SG formation, using viral strains with different levels of virulence. We found that the virulent Nishigahara strain did not induce SG formation, but its avirulent offshoot, the Ni-CE strain, strongly induced SG formation. Furthermore, we demonstrated that the amino acid at position 95 in the RABV matrix protein (M95), a pathogenic determinant for the Nishigahara strain, plays a key role in inhibiting SG formation, followed by protein kinase R (PKR)-dependent phosphorylation of the α subunit of eukaryotic initiation factor 2α (eIF2α). M95 was also implicated in the accumulation of RIG-I, a viral RNA sensor protein, in SGs and in the subsequent acceleration of interferon induction. Taken together, our findings strongly suggest that M95-related inhibition of SG formation contributes to the pathogenesis of RABV by allowing the virus to evade the innate immune responses of the host. IMPORTANCE Rabies virus (RABV) is a neglected zoonotic pathogen that causes lethal infections in almost all mammalian hosts, including humans. Recently, RABV has been reported to induce intracellular formation of stress granules (SGs), also known as platforms that activate innate immune responses. However, the relationship between SG formation capacity and pathogenicity of RABV has remained unclear. In this study, by comparing two RABV strains with completely different levels of virulence, we found that the amino acid mutation from valine to alanine at position 95 of matrix protein (M95), which is known to be one of the amino acid mutations that determine the difference in virulence between the strains, plays a major role in SG formation. Importantly, M95 was involved in the accumulation of RIG-I in SGs and in promoting interferon induction. These findings are the first report of the effect of a single amino acid substitution associated with SGs on viral virulence.

Dermocystid infection in Japanese fire-bellied newt, Cynops pyrrhogaster.

Here, we report details of a new infectious disease in wild-caught Japanese fire-bellied newts (Cynops pyrrhogaster), a Near Threatened species. Skin lesions consisting of numerous masses were found in the animals near Lake Biwa, Shiga Prefecture, Japan. The gross appearance of the skin lesions showed blister-, cyst-, and/or tumor-like morphology. Various sizes of skin lesions were observed on their entire body surface. Histologically, spherical basophilic cysts, including numerous spores, were observed in the dermis layer. Ultrastructural analysis indicated the presence of main bodies of flagellated zoospores within the spores. While 18s rRNA gene sequencing indicated that the skin lesions were due to dermocystid infection. To our knowledge, this is the first report of dermocystid infection in this amphibian in Japan. Further studies are needed to prevent epidemics and to establish diagnostic and treatment methods.

QTL Mapping for Age-Related Eye Pigmentation in the Pink-Eyed Dilution Castaneus Mutant Mouse.

Pink-eyed dilution castaneus (Oca2p-cas) is a mutant gene on mouse chromosome 7 that arose spontaneously in wild Mus musculus castaneus. Homozygotes for Oca2p-cas exhibit pink eyes and a light gray coat throughout life. In an ordinary mutant strain carrying Oca2p-cas, we previously discovered a novel spontaneous mutation that gradually increases melanin pigmentation in the eyes and coat with aging, and we developed a novel mutant strain that was fixed for the novel phenotype. The purpose of this study was to map major quantitative trait loci (QTLs) for the novel pigmentation phenotype and for expression levels of four important melanogenesis genes, microphthalmia-associated transcription factor (Mitf), tyrosinase (Tyr), tyrosinase-related protein-1 (Tyrp1) and dopachrome tautomerase (Dct). We developed 69 DNA markers and created 303 F2 mice from two reciprocal crosses between novel and ordinary mutant strains. The QTL analysis using a selective genotyping strategy revealed a significant QTL for eye pigmentation between 34 and 64 Mb on chromosome 13. This QTL explained approximately 20% of the phenotypic variance. The QTL allele derived from the novel strain increased pigmentation. Although eye pigmentation was positively correlated with Dct expression, no expression QTLs were found, suggesting that the pigmentation QTL on chromosome 13 may not be directly in the pathway of any of the four melanogenesis genes. This study is the first step toward identifying a causal gene for the novel spontaneous phenotype in mice and is expected to discover a new regulatory mechanism for complex melanin biosynthesis during aging.