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The sex determining gene is divergent among different animal species. However, sox9 is up-regulated in the male gonads in a number of species in which it is the essential regulator of testis determination. It is therefore often discussed that the sex determining gene-sox9 axis functions in several vertebrates. In our current study, we show that sox9b in the medaka (Oryzias latipes) is one of the orthologues of mammalian Sox9 at syntenic and expression levels. Medaka sox9b affects the organization of extracellular matrices, which represents a conserved role of sox9, but does not directly regulate testis determination. We made this determination via gene expression and phenotype analyses of medaka with different copy numbers of sox9b. Sox9b is involved in promoting cellular associations and is indispensible for the proper proliferation and survival of germ cells in both female and male medaka gonads. Medaka mutants that lack sox9b function exhibit a seemingly paradoxical phenotype of sex reversal to male. This is explained by a reduction in the germ cell number associated with aberrant extracellular matrices. Together with its identified roles in other vertebrate gonads, a testis-determining role for Sox9 in mammals is likely to have been neofunctionalized and appended to its conserved role in germ cell maintenance.

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“…1A, 1B) [24]. These variants have nonsense mutations in the 5′ coding region and HMG box domain, respectively.…”

Section: Resultsmentioning

confidence: 99%

Analysis of Medaka sox9 Orthologue Reveals a Conserved Role in Germ Cell Maintenance

et al. 2012

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“…1) and compared the results with those obtained previously by screening the same library with direct sequencing [9]. The p53 HRM assay yielded 5 HRM-positive samples that displayed aberrant melting curves.…”

Section: Resultsmentioning

confidence: 97%

“…We identified 55 independent mutations (Table 1) and found that the average mutation frequency of the library was 1 mutation per 314 kb. The value is slightly higher than that obtained by direct sequencing the same library; 1 mutation per 345 kb [9]. The higher rate of mutation detection by the HRM assay relative to that by the direct sequencing method is unexpected because direct sequencing is the gold standard for identifying mutations and thus would be expected to detect mutations more efficiently than any other method.…”

Section: Discussionmentioning

confidence: 99%

High-resolution melting curve analysis for rapid detection of mutations in a Medaka TILLING library

et al. 2010

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BackgroundDuring the last two decades, DNA sequencing has led to the identification of numerous genes in key species; however, in most cases, their functions are still unknown. In this situation, reverse genetics is the most suitable method to assign function to a gene. TILLING (Targeting Induced Local Lesions IN Genomes) is a reverse-genetic strategy that combines random chemical mutagenesis with high-throughput discovery of the induced mutations in target genes. The method has been applied to a variety of plant and animal species. Screening of the induced mutations is the most important step in TILLING. Currently, direct sequencing or nuclease-mediated screening of heteroduplexes is widely used for detection of mutations in TILLING. Both methods are useful, but the costs are substantial and turnaround times are relatively long. Thus, there is a need for an alternative method that is of higher throughput and more cost effective.ResultsIn this study, we developed a high resolution melting (HRM) assay and evaluated its effectiveness for screening ENU-induced mutations in a medaka TILLING library. We had previously screened mutations in the p53 gene by direct sequencing. Therefore, we first tested the efficiency of the HRM assay by screening mutations in p53, which indicated that the HRM assay is as useful as direct sequencing. Next, we screened mutations in the atr and atm genes with the HRM assay. Nonsense mutations were identified in each gene, and the phenotypes of these nonsense mutants confirmed their loss-of-function nature.ConclusionsThese results demonstrate that the HRM assay is useful for screening mutations in TILLING. Furthermore, the phenotype of the obtained mutants indicates that medaka is an excellent animal model for investigating genome stability and gene function, especially when combined with TILLING.

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“…Advances in genetic modification procedures, from the first transgenic medaka [20] to the most recent disruption (or knock-out) of specific gene function [21] have further increased the attractiveness of this species. In particular, procedures such as tilling (Targeted Induced Lesions In Genomes), in which random mutations are chemically introduced in the male genome followed by breeding with wild-type females to establish a library of genomic DNA and sperm from the F 1 generation male offspring hold significant promise in the use of medaka as an animal model [21].…”

Section: Introductionmentioning

confidence: 99%

“…In particular, procedures such as tilling (Targeted Induced Lesions In Genomes), in which random mutations are chemically introduced in the male genome followed by breeding with wild-type females to establish a library of genomic DNA and sperm from the F 1 generation male offspring hold significant promise in the use of medaka as an animal model [21]. These gene-targeted mutants are retrieved using in vitro fertilization with cryopreserved sperm, and the lineages are established by subsequent out crossing.…”

Section: Introductionmentioning

confidence: 99%

Evaluation of cryoprotectant and cooling rate for sperm cryopreservation in the euryhaline fish medaka Oryzias latipes

et al. 2010

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Medaka Oryzias latipes is a well-recognized biomedical fish model because of advantageous features such as small body size, transparency of embryos, and established techniques for gene knockout and modification. The goal of this study was to evaluate two critical factors, cryoprotectant and cooling rate, for sperm cryopreservation in 0.25-ml French straws. The objectives were to: 1) evaluate the acute toxicity of methanol, 2-methoxyethanol (ME), dimethyl sulfoxide (Me2SO), N, N- dimethylacetamide (DMA), N, N,-dimethyl formamide (DMF), and glycerol with concentrations of 5, 10, and 15% for 60 min of incubation at 4 °C; 2) evaluate cooling rates from 5 to 25 °C/min for freezing and their interaction with cryoprotectants, and 3) test fertility of thawed sperm cryopreserved with selected cryoprotectants and associated cooling rates. Evaluation of cryoprotectant toxicity showed that methanol and ME (5 and 10%) did not change the sperm motility after 30 min; Me2SO, DMA, and DMF (10 and 15%) and glycerol (5, 10 and 15%) significantly decreased the motility of sperm within 1 min after mixing. Based on these results, methanol and ME were selected as cryoprotectants (10%) to evaluate with different cooling rates (from 5 °C/min to 25 °C/min) and were compared to Me2SO and DMF (10%) (based on their use as cryoprotectants in previous publications). Post-thaw motility was affected by cryoprotectant, cooling rate, and their interaction (P ≤ 0.000). The highest post-thaw motility (50 ± 10%) was observed at a cooling rate of 10 °C/min with methanol as cryoprotectant. Comparable post-thaw motility (37 ± 12%) was obtained at a cooling rate of 15 °C/min with ME as cryoprotectant. With DMF, post-thaw motility at all cooling rates was ≤ 10% which was significantly lower than that of methanol and ME. With Me2SO, post-thaw motilities were less than 1% at all cooling rates, and significantly lower compared to the other three cryoprotectants (P ≤ 0.000). When sperm from individual males were cryopreserved with 10% methanol at a cooling rate of 10 °C/min and 10% ME with a rate of 15 °C/min, no difference was found in post-thaw motility. Fertility testing of thawed sperm cryopreserved with 10% methanol at a rate of 10 °C/min showed average hatching of 70 ± 30% which was comparable to that of fresh sperm (86 ± 15%). Overall, this study established a baseline for high-throughput sperm cryopreservation of medaka provides an outline for protocol standardization and use of automated processing equipment in the future.

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Cited by 136 publications

References 37 publications

Analysis of Medaka sox9 Orthologue Reveals a Conserved Role in Germ Cell Maintenance

Analysis of Medaka sox9 Orthologue Reveals a Conserved Role in Germ Cell Maintenance

High-resolution melting curve analysis for rapid detection of mutations in a Medaka TILLING library

Evaluation of cryoprotectant and cooling rate for sperm cryopreservation in the euryhaline fish medaka Oryzias latipes

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