Cold-shock eliminates female nucleus in fertilized eggs to induce androgenesis in the loach (Misgurnus anguillicaudatus), a teleost fish

<p>Abstract</p> <p>Background</p> <p>Androgenesis (all-male inheritance) is generally induced by means of irradiating the eggs to inactivate the maternal genome, followed by fertilization with normal sperm. In fish, the conventional technique for induced androgenesis has been applied for rapid fixation to traits, recovery of cryopreserved genotypes, sex-control, etc. A new method of androgenesis that eliminates the need to irradiate the egg was proposed using the loach, <it>Misgurnus anguillicaudatus </it>(a teleost fish).</p> <p>Results</p> <p>When the eggs of wild-type females were fertilized with sperm of albino or orange phenotype males and cold-shocked at 0 to 3°C for 60 min duration just after fertilization, generally more than 30% (with a peak of 100%) of the hatched progeny were androgenotes. While a few of them were the normal diploid, most of them turned out to be abnormal haploid. All-male inheritance was verified by the expression of the recessive color trait (albino or orange) and microsatellite genotypes comprising only paternally derived alleles. Nuclear behavior after the cold-shock treatment was traced by microscopic observation of DAPI (4'6-diamidino-2-phenylindole)-stained samples and hematoxylin-eosin stained histological sections, and the extrusion of egg (maternal) nucleus was observed in eggs treated in the optimum timing.</p> <p>Conclusion</p> <p>In this paper, we demonstrate that cold-shock treatment (at 0 and 3°C) of loach eggs for 60 min just after fertilization successfully induces androgenetic haploid development. The most likely mechanism of cold-shock induced androgenesis is an elimination of the egg nucleus together along with the second polar body and subsequent development of a decondensed sperm nucleus or male pronucleus.</p

Validation of the Burden Index of Caregivers (BIC), a multidimensional short care burden scale from Japan

BACKGROUND: We constructed a concise multidimensional care burden scale that reflects circumstances unique to Japan, with a focus on intractable neurological diseases. We surveyed 646 family caregivers of patients with intractable neurological diseases or stroke using 28 preliminary care burden scale items obtained from qualitative research. The results were used to finalize the feeling of care burden scale (BIC: burden index of caregivers), and verify its reliability and validity. METHODS: The survey was conducted among caregivers providing home health care to patients with intractable neurological diseases (PD [Parkinson's disease], SCD [spinocerebellar degeneration], MSA [multiple system atrophy], and ALS [amyotrophic lateral sclerosis]) or CVA (cerebrovascular accident) using a mailed, self-administered questionnaire between November, 2003 and May, 2004. RESULTS: Response rates for neurological and CVA caregivers were 50% and 67%, respectively, or 646 in total (PD, 279; SCD, 78; MSA, 39; ALS, 30; and CVA, 220). Item and exploratory factor analyses led to a reduction to 11 items, comprising 10 items from the 5 domains of time-dependent burden, emotional burden, existential burden, physical burden, and service-related burden; and 1 item on total burden. Examination of validity showed a moderate correlation between each domain of the BIC and the SF-8 (Health related quality of life scale, Short Form-8), while the correlation coefficient of the overall BIC and CES-D was 0.62. Correlation between the BIC and ZBI, a preexisting care burden scale, was high (r = 0.84), while that with the time spent on providing care was 0.47. The ICC (Intraclass correlation coefficient) by test-retest reliability was 0.83, and 0.68 to 0.80 by individual domain. CONCLUSION: These results show that the BIC, a new care burden scale comprising 11 items, is highly reliable and valid

Sensitivity of CT perfusion for the diagnosis of cerebral infarction

We aimed to determine the sensitivity of CT perfusion (CTP) for the diagnosis of cerebral infarction in the acute stage. We retrospectively reviewed patients with ischemic stroke who underwent brain CTP on arrival and MRI-diffusion weighted image (DWI) after hospitalization between October 2008 and October 2011. Final diagnosis was made from MRI-DWI findings and 87 patients were identified. Fifty-five out of 87 patients (63%) could be diagnosed with cerebral infarction by initial CTP. The sensitivity depends on the area size (s) : 29% for S<3 cm2, 83% for S≥3 cm2-<6 cm2, 88% for S≥6 cm2-<9 cm2, 80% for S≥9 cm2-<12 cm2, and 96% for S≥12 cm2 (p<0.001). Sensitivity depends on the type of infarction : 0% for lacunar, 74% for atherothrombotic, and 92% for cardioembolism (p<0.001). Sensitivity is not correlated with hours after onset. CT perfusion is an effective imaging modality for the diagnosis and treatment decisions for acute stroke, particularly more serious strokes

Generation of Germline-Competent Rat Induced Pluripotent Stem Cells

Recent progress in rat pluripotent stem cell technology has been remarkable. Particularly salient is the demonstration that embryonic stem cells (ESCs) in the rat (rESCs) can contribute to germline transmission, permitting generation of gene-modified rats as is now done using mouse ESCs (mESCs) or mouse induced pluripotent stem cells (iPSCs; miPSCs). However, determinations of whether rat iPSCs (riPSCs) can contribute to germ cells are not published. Here we report the germline competency of riPSCs.We generated riPSCs by transducing three mouse reprogramming factors (Oct3/4, Klf4, and Sox2) into rat somatic cells, followed by culture in the presence of exogenous rat leukemia inhibitory factor (rLIF) and small molecules that specifically inhibit GSK3, MEK, and FGF receptor tyrosine kinases. We found that, like rESCs, our riPSCs can contribute to germline transmission. Furthermore we found, by immunostaining of testis from mouse-rat interspecific chimeras with antibody against mouse vasa homolog, that riPSCs can contribute to embryonic development with chimera formation in mice (rat-mouse interspecific chimeras) and to interspecific germlines.Our data clearly demonstrate that using only three reprogramming factors (Oct3/4, Klf4, and Sox2) rat somatic cells can be reprogrammed into a ground state. Our generated riPSCs exhibited germline transmission in either rat-rat intraspecific or mouse-rat interspecific chimeras

The occurrence of hypertetraploid and other unusual polyploid loaches Misgurnus anguillicaudatus among market specimens in Japan

Exotic animals may cause a genetic contamination of indigenous species if they escape and reproduce in wild populations. Loach Misgurnus anguillicaudatus and its related species have been imported to Japan for commercial uses. We collected live loach specimens from central wholesale market in Tokyo. Among 451 specimens, ploidy status was examined by DNA content flow cytometry and polyploid loaches with triploid, tetraploid and other higher DNA content ranges were detected. Hyper-triploid and hyper-tetraploid individuals could be easily detected by flow cytometry using a standard eudiploid, eutriploid and eutetraploid controls and then reproductive capacity of these hyper-polyploid males was examined. Sperm of hyper-triploid males did not exhibit active progressive motility and major populations of spermatozoa or spermatozoon-like cells were detected in triploid and hexaploid ranges. Motile haploid spermatozoa were very few in sperm from hyper-triploid males. Therefore, hyper-triploid males were sterile, whereas hyper-tetraploid males produced fertile hyper-diploid spermatozoa with active progressive motility after contact with ambient water. Viable progeny occurred in the cross between normal wild-type diploid female and hyper-tetraploid males, but androgenotes induced by fertilization of UV-irradiated eggs with sperm of hyper-tetraploid males were inviable hyper-diploid. Cytogenetic analyses in such androgenotes indicated that hyper-tetraploid males should produce hyper-diploid spermatozoa with 2n=54, i.e. presence of four supernumerary micro-chromosomes