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Method for maintaining meiotic arrest of oocytes by storing/culturing oocytes in vitro

A technology of oocyte and meiosis, which is applied in the field of reproductive biology, can solve the problems of lack of oocyte meiosis arrest, etc., and achieve the effect of improving the quality of in vitro maturation, improving utilization rate, and easy source

Inactive Publication Date: 2013-07-17
INST OF ZOOLOGY CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Therefore, there is still a lack of an effective method that can maintain the meiotic arrest of oocytes for a long period of time

Method used

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  • Method for maintaining meiotic arrest of oocytes by storing/culturing oocytes in vitro
  • Method for maintaining meiotic arrest of oocytes by storing/culturing oocytes in vitro
  • Method for maintaining meiotic arrest of oocytes by storing/culturing oocytes in vitro

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Example 1: Acquisition and preservation of pig oocytes in GV stage

[0030] Young sows aged 3-5 months who had just been slaughtered in Beijing No. 5 Meat Co., Ltd. were selected, and the ovaries were obtained immediately after slaughter, and it was confirmed that there was no corpus luteum on the ovaries. Put the ovaries in 25-30°C physiological saline containing 75 μg / ml penicillin and 50 μg / ml streptomycin, and transport them to the laboratory within 2 hours.

[0031] On the JJ-CJ-1FD type clean workbench (purification level is 100, produced by Wujiang Purification Equipment General Factory), use a 20ml disposable syringe with a fixed 18-gauge needle (produced by Shanghai Mishawai Yike Industrial Co., Ltd.) Extract follicular fluid from follicles with a medium diameter (2-6mm), and suck off the supernatant after settling, and wash the settling precipitate twice with TCM-199, which contains the cumulus-oocyte complex. Under a C-DS microscope (manufactured by Nikon),...

Embodiment 2

[0046] Embodiment 2: the inspection of oocyte maturation in vitro

[0047] The porcine oocyte complex preserved for 3 days in Example 1 was taken out, washed three times in TCM-199 operating fluid together with fresh oocytes, then washed three times in maturation culture medium, and put into preheated ( In mature culture drops preheated at 38.5° C., they were cultured in a Hera Cell 150 cell culture incubator (manufactured by Thermo) at 38.5° C., 5% CO 2 and 100% humidity. Among them, the mature culture medium is improved TCM-199 (Gibco, Grand Island, NY) with 75 μg / ml penicillin, 50 μg / ml streptomycin, 0.57 mM cysteine, 0.5 μg / ml FSH, 0.5 μg / ml LH and 10ng / ml EGF; the mature culture drop is a 100μl micro-drop made of the mature culture solution, and each drop cultures 25 oocytes.

[0048] Observe under a solid microscope whether the oocyte excretes the first polar body. Since the polar body of the porcine oocyte is extremely small, use a pipette to move the oocyte under a...

Embodiment 3

[0052] Example 3: Detection of Oocyte Microfilament Integrity Rate and GSH Content

[0053] 1. Detection of microfilament integrity in oocytes

[0054] The microfilament integrity rate of fresh oocytes and oocytes preserved for 3 days in Example 1 was detected. The oocytes were removed with acidic Tyrode's solution (pH2.5) to remove the zona pellucida, and fixed with 4% paraformaldehyde (pH7.4 diluted in PBS) at room temperature for at least 30min. 1% Triton X-100 was permeabilized overnight at 37°C, the blocking solution was PBS containing 1% BSA, blocked for 1 hour, and then transferred to 1:100 diluted FITC-coupled phalloidin (1 μg / ml diluted in blocking solution) for 2 hours at room temperature. The cells were washed three times with PBS containing 0.1% Tween 20 and 0.01% Triton X-100, 5 min each time, and then stained with PI (10 μg / ml in PBS) for 10 min. Finally, the fully washed oocytes were mounted on glass slides and observed with a laser confocal scanning micros...

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Abstract

The invention provides a method for maintaining the meiotic arrest of oocytes by storing / culturing the oocytes in vitro. In the method, the oocytes are stored / cultured in vitro by whole serum serving as preserving fluid or culture solution for storing / culturing the oocytes in vitro at the temperature of between 20 and 30 DEG C to maintain the meiotic arrest of the oocytes. When the method is used, the utilization rate of ovarian oocytes in scientific research and the production of animal husbandry can be improved greatly, the time limit is relieved for the utilization of ovaries of slaughterhouses in the scientific research, and the arrangement of in-vitro operating time is facilitated; by storing / culturing the oocytes during the germinal vesicle period at normal temperature, the in-vitromature quality of the oocytes is improved and the synchronization of nucleus mature and cytoplasm mature is promoted; therefore, the method is an optimal method for controlling the process of the nucleus mature physiologically without depending on inhibitors, and has the important significance on the study and application of animal embryo engineering and the assisted reproductive technology of human.

Description

technical field [0001] The invention belongs to the field of reproductive biology and relates to a method for maintaining meiotic arrest of oocytes in the Germinal Vesicle (GV) stage and inhibiting Germinal Vesicle Breakdown (GVBD) of the oocytes by utilizing physiological components. Background technique [0002] The developmental ability of in vitro matured oocytes was significantly lower than that of in vivo matured oocytes. Oocytes undergo cytoplasmic maturation concurrently with nuclear maturation, thereby supporting subsequent successful developmental maturation. A series of events occur in oocytes in vivo, including transcriptional and translational cytoplasmic maturation in prophase of meiosis. However, oocytes obtained from follicles in vitro undergo premature attenuation before they have acquired cytoplasmic maturation after being transferred into the culture medium. Data split recovery. [0003] In order to improve the quality of in vitro matured oocytes, pharma...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/075
Inventor 杨彩荣苗德强郭磊张清华欧阳迎春王震波侯毅孙青原
Owner INST OF ZOOLOGY CHINESE ACAD OF SCI
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