Method for improving in vitro maturation of oocytes in foaming stage after vitrification freezing

A technology of vitrification and oocytes, which is applied in the field of vitrification to improve the in vitro maturation of oocytes in the germinal and vesicular stage, to achieve the stable expression of genes related to the spindle assembly checkpoint, the increase of mitochondrial membrane potential and ATP levels, and the rate of abnormal morphology Falling effect

Pending Publication Date: 2019-09-20
SICHUAN AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the effect of adding melatonin on the in vitro maturation of germinal vesicular oocytes in the vitrified thawing solution and in vitro culture medium after thawing is seldom reported.

Method used

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  • Method for improving in vitro maturation of oocytes in foaming stage after vitrification freezing
  • Method for improving in vitro maturation of oocytes in foaming stage after vitrification freezing
  • Method for improving in vitro maturation of oocytes in foaming stage after vitrification freezing

Examples

Experimental program
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Effect test

Embodiment 1

[0028] This example provides a method for improving vitrification in vitro maturation of oocytes in germinal and vesicular stages, specifically through the following steps:

[0029] 1) Oocyte collection

[0030] 5-6 week-old female mice were raised at 18-22°C, with free access to food and water, and controlled light (14 hours / day). After two weeks of adaptive feeding, each mouse was injected with 10IU PMSG, and the ovary was collected 44-48 hours later, and the oocytes in the germinal vesicle stage with 2-3 layers of granulosa cells were collected and passed through M 2 Washed 3 times before use.

[0031] 2) Oocytes were vitrified, thawed and cultured in vitro for 1 hour

[0032] The plastic thin tube (250mL; IMV, L’Aigle, France) was heated and softened, and manually drawn into a thin tube with an inner diameter of 0.10mm and an outer diameter of 0.15mm, and a razor blade was used to cut OPS about 3cm long from the thin end for later use.

[0033] Freezing of oocytes: adop...

Embodiment 2

[0037] Example 2 Melatonin reduces the ROS level of mouse oocytes during in vitro maturation

[0038] After the frozen oocytes were thawed, the oocytes in different stages (GV, MI, and MII stages) of each group cultured in vitro for 0, 8, and 12 hours were tested for ROS levels. Oocytes in different stages (GV, MI and MII stages) of each group were washed three times in 1 mmol / LDCFH-DA (ROS staining solution) staining solution (DPBS as solvent), and then placed in 37.5 ° C, 5% CO 2 Stain in saturated humidity incubator for 30 minutes, after M 2 After washing three times with DAPI staining, the slides were stained with DAPI and observed under a fluorescent microscope (IX53 Olympus, Tokyo, Japan) after standing at 4°C in the dark for 2 hours. ROS detection was excited with blue light. The acquired image was saved in TIFF format, and the image was analyzed with ImageJ 1.48 to obtain the fluorescence intensity of each oocyte, and the background value was subtracted.

[0039] li...

Embodiment 3

[0040] Example 3 Melatonin improves the GSH level of mouse oocytes during in vitro maturation

[0041] After the frozen oocytes were thawed, the oocytes in different stages (GV, MI and MII stages) of each group cultured in vitro for 0, 8, and 12 hours were subjected to GSH staining. Oocytes in different stages (GV, MI and MII stages) of each group were washed three times in 1 mmol / LDCFH-DA (ROS staining solution) staining solution (DPBS as solvent), and then placed in 37.5 ° C, 5% CO 2 Stain in saturated humidity incubator for 30 minutes, after M 2 After washing three times with DAPI staining, the slides were stained with DAPI and observed under a fluorescent microscope (IX53 Olympus, Tokyo, Japan) after standing at 4°C in the dark for 2 hours. GSH was stained with 10 μmol / L Cell Tracker Blue, the specific operation was as above, and excited with purple light.

[0042] It was found by GSH staining analysis ( figure 2 ), compared with the fresh group, the GSH levels of oocy...

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Abstract

The invention discloses a method for improving in vitro maturation of oocytes in a foaming stage after vitrification freezing. The method includes freezing the oocytes in a freezing solution containing melatonin, thawing the oocytes in a sucrose solution containing melatonin, and finally performing in vitro maturation culture on the oocytes in a culture solution containing melatonin. The method has simple process and short time consumption, and can effectively improve the in vitro maturation of oocytes in the foaming stage after vitrification freezing.

Description

technical field [0001] The invention belongs to the technical field of animal reproduction, and in particular relates to a method for improving in vitro maturation of oocytes in the germinal and vesicular stages of vitrification. Background technique [0002] Oocyte ultra-low temperature cryopreservation is a very important assisted reproductive technology, which is of great application value in the fields of biological science, agriculture and medicine. In earlier studies, it has been reported that oocytes in the germinal vesicle (GV) stage were successfully cryopreserved, and their survival rate, fertilization rate and developmental ability were all low. At present, the in vitro maturation rate of mouse germinal vesicular oocytes after freezing and thawing is 53-92.6%, and the rate of blastocysts after in vitro fertilization is 20-42.9%. The blastocyst rate was 9.6-35.6%. After freezing and thawing oocytes, the level of reactive oxygen species (reactive oxygen species, R...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/075A01N1/02
CPCC12N5/0609A01N1/0221A61K31/4045
Inventor 周光斌吴祯峥潘波杨昊轩
Owner SICHUAN AGRI UNIV
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