| Project/Area Number |
04454030
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| Research Category |
Grant-in-Aid for General Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Research Field |
動物発生・生理学
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| Research Institution | THE UNIVERSITY OF THE AIR |
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Principal Investigator |
MORI Hideo The University of the Air, Faculty of Liberal Arts, Vice-president, 教養学部, 副学長 (70012268)
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| Co-Investigator(Kenkyū-buntansha) |
HIRAMOTO Yukio The University of the Air, Faculty of Liberal Arts, Professor, 教養学部, 教授 (50011440)
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| Project Period (FY) |
1992 – 1994
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| Project Status |
Completed (Fiscal Year 1994)
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| Budget Amount *help |
¥6,500,000 (Direct Cost: ¥6,500,000)
Fiscal Year 1994: ¥1,100,000 (Direct Cost: ¥1,100,000)
Fiscal Year 1993: ¥1,700,000 (Direct Cost: ¥1,700,000)
Fiscal Year 1992: ¥3,700,000 (Direct Cost: ¥3,700,000)
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| Keywords | x-bearing sperm / y-bearing sperm / micromanipulation / FISH / 無担体電気泳動 / PCR / SRY |
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| Research Abstract |
In order to identify human x- and y-bearing sperm more efficiently and accurately, an application of fluorescence insitu hybridization (FISH) to the sperm cells was examined. Washed sperm were treated with dithiothreitol to decondense the heads, fixed with Carnoy solution on glass-slide, and stored at -80ﾟC until use. Hybridization was made after hardening at 65ﾟC,denaturation and dehydration. Probes used were DXZ1 for x chromosome and DYZ1 and DYZ3 for y chromosome. Specimens were observed under a flourescence microscope. Biotin-labeled probe was stained with avidin-FITC,while digoxigenin-labeled one was stained with anti-digoxigenin rhodamine. Nucleus was stained with propidium iodide (PI) and DAPI.When biotin-labeled DXZ1 was examined about 50% of sperm were recognized as x-bearing sperm, whereas double staining with digoxigenin-labeled DXZ1 and biotin-labeled DYZ3 resulted in a ratio of x- and y-bearing sperm, 1 : 1. The technique needs the time less than one tenth that for quinacrine mustard staining. Unfortunately, the application of FISH to the sperm subjected to free-flow electrophoresis has not yet been done because of the troble with the electrophoresis apparatus, but favorable results were obtained with the x-bearing rich fraction after Percoll density gradient. In addition, a new technique of microinjection into the egg, which consists of pressure and electric potential was tried with sea urchin eggs. This would be useful to inject isolated x- or y-bearing sperm into the egg, because the number of sperm isolated by the electrophoresis used to be small
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