Targeted deletion of liver-expressed Choriogenin L results in the production of soft eggs and infertility in medaka, Oryzias latipes

Egg envelopes (chorions) in medaka, Oryzias latipes, are composed of three major glycoproteins: ZI-1, − 2, and − 3. These gene-encoded chorion glycoproteins are expressed in the liver and/or ovarian oocytes of sexually mature female fish. In medaka, the glycoproteins produced in the female liver are induced by estrogen as Choriogenin (Chg.) H and Chg. H minor (m), which correspond to the zona pellucida (ZP) B (ZPB) protein in mammals, and Chg. L, which corresponds to ZPC in mammals. Chg. H, Chg. Hm, and Chg. L, are then converted to ZI-1, − 2, and − 3, respectively, during oogenesis in medaka ovaries.In the present study, we established a medaka line in which the chg.l gene was inactivated using the transcription activator-like effector nuclease (TALEN) technique. Neither intact chg.l transcripts nor Chg. L proteins were detected in livers of sexually mature female homozygotes for the mutation (homozygous chg.l knockout: chg.l−/−). The chg.l−/− females spawned string-like materials containing “smashed eggs.” Closer examination revealed the oocytes in the ovaries of chg.l−/− females had thin chorions, particularly at the inner layer, despite a normal growth rate. In comparing chorions from normal (chg.l+/+) and chg.l−/− oocytes, the latter exhibited abnormal architecture in the chorion pore canals through which the oocyte microvilli pass. These microvilli mediate the nutritional exchange between the oocyte and surrounding spaces and promote sperm-egg interactions during fertilization. Thus, following in vitro fertilization, no embryos developed in the artificially inseminated oocytes isolated from chg.l−/− ovaries. These results demonstrated that medaka ZI-3 (Chg.L) is the major component of the inner layer of the chorion, as it supports and maintains the oocyte’s structural shape, enabling it to withstand the pressures exerted against the chorion during spawning, and is essential for successful fertilization. Therefore, gene products of oocyte-specific ZP genes that may be expressed in medaka oocytes cannot compensate for the loss Chg. L function to produce offspring for this species.

Myosin phosphatase target subunit 1 governs integrity of the embryonic gut epithelium to circumvent atresia development in medaka, Oryzias latipes

BackgroundIntestinal atresia (IA) is a congenital gut obstruction caused by the absence of gut opening. Genetic factors are assumed to be critical for the development of IA, in addition to accidental vascular insufficiency or mechanical strangulation. However, the molecular mechanism underlying IA remains poorly understood.ResultsIn this study, to better understand such a mechanism, we isolated a mutant of Oryzias latipes (the Japanese rice fish known as medaka) generated by N-ethyl-N-nitrosourea mutagenesis, in which IA develops during embryogenesis. Positional cloning identified a nonsense mutation in the myosin phosphatase target subunit 1 (mypt1) gene. Consistent with known Mypt1 function, the active form of myosin regulatory light chain (MRLC), which is essential for actomyosin contraction, and F-actin were ectopically accumulated in the intestinal epithelium of mutant embryos, whereas cell motility, proliferation and cell death were not substantially affected. Corresponding to the accumulation site of F-actin/active MRLC, the intestinal epithelium architecture was disordered. Importantly, blebbistatin, a non-muscle myosin inhibitor, attenuated the development of IA in the mutant.ConclusionsCytoskeletal contraction governed by mypt1 regulates the integrity of the embryonic intestinal epithelium. This study provides new insight into our understanding of the mechanism of IA development in humans.Bullet PointsMedaka mypt1 mutants display intestinal atresia.The level of phosphorylated myosin regulatory light chain was higher in mypt1 mutant embryos than in wild-type embryos.The levels of F-actin appeared elevated in the intestinal epithelium of mypt1 mutants.Blebbistatin, an inhibitor of non-muscle myosin II, rescued intestinal atresia in mypt1 mutant embryos.

Low Dose-Rate Irradiation of Gamma-Rays-Induced Cytotoxic and Genotoxic Alterations in Peripheral Erythrocytes of p53-Deficient Medaka (Oryzias latipes)

Morphological alterations and nuclear abnormalities in fish erythrocytes have been used in many studies as bioindicators of environmental mutagens including ionizing radiation. In this study, adult Japanese medaka (Oryzias latipes) were irradiated with gamma rays at a low dose rate (9.92 μGy/min) for 7 days, giving a total dose of 100 mGy; and morphological alterations, nuclear abnormalities, and apoptotic cell death induced in peripheral erythrocytes were investigated 8 h and 7 days after the end of the irradiation. A variety of abnormalities, such as tear-drop cell, crenated cell, acanthocyte, sickled cell, micronucleated cell, eccentric nucleus, notched nucleus, and schistocyte, were induced in the peripheral erythrocytes of the wild-type fish, and a less number of abnormalities and apoptotic cell death were induced in the p53-deficient fish. These results indicate that low dose-rate chronic irradiation of gamma rays can induce cytotoxic and genotoxic effects in the peripheral erythrocytes of medaka, and p53-deficient medaka are tolerant to the gamma-ray irradiation than the wild type on the surface.