Abstract
The precursor of heme, protoporphyrin IX (PPIX), accumulates abundantly in the uteri of birds, such as Japanese quail, Coturnix japonica, which has brown-speckled eggshells; however, the molecular basis of PPIX production in the uterus remains largely unknown. Here, we investigated the cause of low PPIX production in a classical Japanese quail mutant exhibiting white eggshells by comparing its gene expression in the uterus with that of the wild type using transcriptome analysis. We also performed genetic linkage analysis to identify the causative genomic region of the white eggshell phenotype. We found that 11 genes, including 5'-aminolevulinate synthase 1 (ALAS1) and hephaestin-like 1 (HEPHL1), were specifically upregulated in the wild-type uterus and downregulated in the mutant. We mapped the 172 kb candidate genomic region on chromosome 6, which contains several genes, including a part of the paired-like homeodomain 3 (PITX3), which encodes a transcription factor. ALAS1, HEPHL1, and PITX3 were expressed in the apical cells of the luminal epithelium and lamina propria cells of the uterine mucosa of the wild-type quail, while their expression levels were downregulated in the cells of the mutant quail. Biochemical analysis using uterine homogenates indicated that the restricted availability of 5'-aminolevulinic acid is the main cause of low PPIX production. These results suggest that uterus-specific transcriptional regulation of heme-biosynthesis-related genes is an evolutionarily acquired mechanism of eggshell pigment production in Japanese quail. Based on these findings, we discussed the molecular basis of PPIX production in the uteri of Japanese quails.
Highlights
protoporphyrin IX (PPIX) levels are low in most tissues because heme biosynthesis is tightly regulated to avoid the toxic effect of PPIX accumulation [11], and the synthesized PPIX is efficiently converted to heme in the presence of ferrous iron
Histological examination revealed that brown pigment granules were accumulated in the apical cells of the mucosal epithelium of the uterus in the wild-type quail before anticipated secretion (19–20 h after oviposition), whereas few brown pigment granules were observed in the mutant quail (Fig 1C and 1D)
This study demonstrated that aminolevulinate synthase 1 (ALAS1) is highly expressed in the apical cells of the epithelium and lamina propria (LP) cells of the uterine mucosa, indicating the possible involvement of these cells in PPIX synthesis through aminolevulinic acid (ALA) synthesis
Summary
Avian eggshells display diverse colors and patterns, which may be a result of adaptation to different habitats by birds. PPIX accumulates in the uterus, especially in the caudal part of the oviduct, of bird species that produce brown eggs, indicating that the biosynthesis pathway of heme is regulated in their uteri. The Japanese quail, Coturnix japonica, usually lays eggs with brown-speckled color patterns generated by PPIX. To elucidate the molecular basis of PPIX production in the uterus of Japanese quail, we focused on a classical quail mutant exhibiting white eggshells owing to the low PPIX production in its uterus [14, 15]. The cause of low PPIX production in this mutant quail remains unknown. We postulated that the genes involved in PPIX production should be upregulated in the uterus of wild-type quail and downregulated in the uterus of the mutant quail exhibiting white eggshells.
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