Mouse denuded oocyte in vitro maturation technology

An in vitro maturation, mouse technology, applied in the field of in vitro maturation of mouse nude eggs

Inactive Publication Date: 2009-10-21
SHANDONG AGRICULTURAL UNIVERSITY
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  • Abstract
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  • Claims
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Problems solved by technology

[0003] In recent years, many researchers have attempted to improve the in vitro maturation quality of DO by co-culturing with cumulus cells, but the results have proved that co-culture with cumulus cells or cumulus-oocyte complex (COC) is not effective unles

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  • Mouse denuded oocyte in vitro maturation technology

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Embodiment Construction

[0014] 1. Preparation of mature medium

[0015] TCM-199 culture medium (Gibco, Grand Island, NY, USA) add 1μg / ml 17β-E 2 , 24.2mg / l sodium pyruvate, 0.05IU / ml FSH, 0.05IU / ml LH and 10ng / ml EGF are made into mature culture medium. Prepare cysteamine and cystine into concentrated stock solutions of 20mmol / l and 100mmol / l respectively, store them at -20°C, dilute to the required concentration with mature culture broth before use (cysteamine: 100μmol / l; cysteamine) Acid: 200μmol / l). Since the commercial TCM-199 culture medium itself contains 83 μmol / l cystine, the final concentration of cystine used is 283 μmol / l.

[0016] 2. Preparation of mouse DO

[0017] Obtain ovaries from superovulated mice and puncture large follicles to obtain COC. Use a thin glass tube with a diameter slightly larger than that of the oocytes by pipetting repeatedly to remove the cumulus cells to obtain DO.

[0018] 3. Preparation of mouse or goat granule cell monolayer

[0019] Mouse or goat COC shed cumulus ...

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Abstract

The invention relates to a mouse denuded oocyte (DO) in vitro maturation technology, belonging to the technical field of biology. The invention is characterized in that a cumulus cell uses cystine and cysteamine to improve GSH synthesis and developmental capacities of the DO and, through cumulus cell co-culture and addition of cysteamine and cystine, an in vitro maturation system is established, which completely recovers the developmental capacity of the DO. The DO is co-cultured with mouse or goat cumulus cells in TCM-199 mature culture fluid added with 100-200 mumol/l of cysteamine and 250-300 mumol/l of cystine, the blastocyst rate reaches the level of mature COC cultured by adding the same mercapto-compound. After transplanting 2-cell embryos from the mature DO through co-culture and from COC respectively, the differences in pregnancy rate and number born are not obvious. The invention, for the first time in the world, establishes an in vitro mature system for completely recovering the developmental capacity of mouse denuded oocyte, which is of great importance in the research and application of both animal embryo engineering and human-assisted reproductive technology.

Description

(1) Technical field [0001] The invention relates to a technique for in vitro culturing and maturation of naked mouse eggs, which belongs to the field of biotechnology. (2) Background technology [0002] Many studies have proved that the removal of cumulus cells before in vitro maturation (IVM) will seriously affect the embryonic development ability of oocytes (Schroeder AC, Eppig JJ., The developmental capacity of mouse oocytes that matured spontaneously in vitro is normal. Dev Biol 1984; 102 : 493-497) and glutathione (GSH) synthesis ability (de Matos DG, Furnus CC, Moses DF., Glutathione synthesis during in vitro maturation of bovineoocytes: role of cumulus cells. Biol Reprod 1997; 57: 1420-1425 ). Although the naked eggs (DO) of some animals can complete nuclear maturation in vitro, their developmental ability is greatly reduced after in vitro fertilization (Ge L, Han D, Lan GC et al. Factors affecting the in vitro action of cumulus cells on the maturing mouse oocytes. Mol Rep...

Claims

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Application Information

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IPC IPC(8): C12N5/06
Inventor 谭景和周萍吴延光罗明久李青王刚高达魏德莉
Owner SHANDONG AGRICULTURAL UNIVERSITY
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