Method for preserving/culturing oocytes in vitro to inhibit oocytes from germinal vesicle breakdown

A technology of cell germinal vesicles and oocytes, applied in germ cells, animal cells, vertebrate cells, etc., can solve the problem of short maintenance time, and achieve the effects of easy access, good clinical effect, 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

It has been reported that adding part of the follicle fluid to the culture medium (Leibfried and First, Bio reprod, 23:699-704 (1980)) can be used to maintain meiotic arrest, but the maintenance time is very short

Method used

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  • Method for preserving/culturing oocytes in vitro to inhibit oocytes from germinal vesicle breakdown
  • Method for preserving/culturing oocytes in vitro to inhibit oocytes from germinal vesicle breakdown
  • Method for preserving/culturing oocytes in vitro to inhibit oocytes from germinal vesicle breakdown

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Example 1: Preparation of Preservation Medium for Culturing / Preserving Pig Oocytes

[0037] 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.

[0038] 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 porcine follicular fluid (porcine follicular fluid, pFF) from porcine follicles with a diameter of 2-6 mm; this step can also be carried out under low vacuum using a suction pump with a fixed 18-gauge needle, and the porcine follicular flu...

Embodiment 2

[0039] Example 2: Acquisition and preservation of pig oocytes in GV stage

[0040] On the JJ-CJ-1FD type clean workbench, the precipitate obtained by the natural sedimentation of the porcine follicular fluid in Example 1 was washed twice with TCM-199, and the eggs were selected with a mouth suction pipe under a C-DS type microscope (produced by Nikon Corporation). The cumulus-oocyte complex (COCs) with tightly wrapped cumulus cells and uniform distribution of oocyte cytoplasmic granules was rinsed three times with TCM-199, and then washed three times in porcine follicular fluid.

[0041] A. Comparison experiment of using pFF culture drops and TCM-199 culture drops to store porcine oocytes in GV phase at different temperatures

[0042] Prepare pFF culture drops with 200 μl of preserved culture solution / drop; prepare TCM-199, prepare TCM-199 control culture drops with 200 μl of prepared TCM-199 / drop, and then place the two culture drops in SPX- Preheat for 2 hours in advance ...

Embodiment 3

[0053] Embodiment 3: the inspection of oocyte maturation in vitro

[0054] The porcine oocyte complex preserved for 3 days in Example 2 was taken out, washed three times in the TCM-199 operating fluid together with fresh oocytes, then washed three times in the mature culture medium, and put into the preheated ( In a mature culture drop preheated to a temperature of 38.5° C., culture in a cell culture incubator 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-droplet made of the mature culture medium, and each droplet cultures 25 oocytes.

[0055] 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 40× microscope to r...

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Abstract

The invention provides a method for preserving / culturing oocytes in vitro to inhibit oocytes from germinal vesicle breakdown. The method provided by the invention is characterized by adopting follicular fluid as preserving fluid or culture fluid for preserving / culturing oocytes in vitro and preserving / culturing oocytes in vitro at 20-30 DEG C so as to inhibit oocytes from germinal vesicle breakdown. The method has the following advantages: the use ratios of ovary oocytes in scientific research and production in animal husbandry can be greatly improved, thus removing the time restriction on utilization of ovaries from slaughter houses on scientific research and being convenient for arrangement of in vitro operation time; preservation / culture of oocytes in the germinal vesicle period under normal temperature is conductive to improvement of in vitro oocyte maturation quality and promotion of synchronization of nucleus maturation and cytoplasmic maturation; therefore, the method is an optimal method which is independent of inhibitors and is used for controlling the nucleus maturation process in physiology; the porcine follicular fluid is easy to prepare and can be prepared into commercialized preparations; and the method has great significance for research and application of animal embryo engineering and human assisted reproductive technology.

Description

technical field [0001] The invention belongs to the field of reproductive biology, and is a method for maintaining meiotic arrest of oocytes in the germinal vesicle (GV) stage and inhibiting the germinal vesicle breakdown (Germinal Vesicle Breakdown, GVBD) of oocytes by utilizing physiological components. It is the development and utilization of a new application of porcine follicular fluid. 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 cultu...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/075
Inventor 苗德强杨彩荣郭磊梁兴伟戚树涛欧湘红侯毅孙青原
Owner INST OF ZOOLOGY CHINESE ACAD OF SCI
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