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Method for improving parthenogenetic development potential of vitrified mature oocytes

A technology of vitrification and oocytes, which is applied in the field of improving the parthenogenetic development potential of vitrified mature oocytes, which can solve the problems of the influence of oocyte development potential and the lack of development ability, so as to achieve the reduction of ROS content and the development of oocytes. Potential enhancement and gene expression stabilization effects

Inactive Publication Date: 2019-02-01
SICHUAN AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The results of our previous experiments showed that after ultra-low temperature freezing and thawing of mouse MII stage oocytes, different concentrations (10 -9 、10 -7 、10 -5 、10 -3 mol / L) melatonin for 1 hour, the developmental ability of oocytes after parthenogenesis was not improved
A number of studies have shown that adding appropriate concentrations of melatonin to the in vitro culture medium of frozen oocytes and embryos in animals such as mice, cattle, pigs, sheep, and zebrafish can promote the developmental ability of embryos. The effects of adding melatonin on the developmental potential of oocytes in post-in vitro culture medium, parthenogenetic activation medium and in vitro culture medium are rarely reported

Method used

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  • Method for improving parthenogenetic development potential of vitrified mature oocytes
  • Method for improving parthenogenetic development potential of vitrified mature oocytes
  • Method for improving parthenogenetic development potential of vitrified mature oocytes

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Example 1 Melatonin reduces the level of ROS in the G1 phase of the parthenogenetic mouse zygote

[0043] After the frozen oocytes were thawed, the oocytes of each group cultured in vitro for 0 hour and 1 hour were tested for ROS levels. Such as figure 1 As shown in A-1C, there was no significant difference in ROS levels between the groups at 0 hour and 1 hour (P>0.05). Three hours after the parthenogenetic activation of oocytes in each group, the zygote was in the late G1 stage, and the ROS level at this time was detected. Compared with the freezing + melatonin group, the ROS level in the freezing group was significantly increased (P0.05). Moreover, with the prolongation of the culture time, the ROS levels of each group had an upward trend.

Embodiment 2

[0044] Example 2 Melatonin reduces the level of GSH in the G1 phase of parthenogenetic mice

[0045] GSH staining was performed on the oocytes of each group cultured in vitro for 0 hour and 1 hour and the combinatorial cells 3 hours after parthenogenetic activation. It was found by GSH staining analysis ( figure 2 ), there was no significant difference between the groups in GSH levels of 0 hour and 1 hour cultured in vitro (P>0.05). Three hours after the parthenogenetic activation of oocytes, the zygote was in the late G1 phase, and the GSH level in the freezing group was significantly higher than that in the freezing + melatonin group and the fresh group (P0.05).

Embodiment 3

[0046] Example 3 Melatonin changes the mRNA expression of G1 cycle-related genes in parthenozygous mice

[0047] Three hours after parthenogenetic activation of oocytes in each group, the zygote was in the late G1 phase. At this time, the relative mRNA expression levels of key genes regulated in cell cycle checkpoints P53, P21 and E2F1 were detected. Such as image 3 As shown in A, the mRNA expression of P53 in the frozen group was significantly higher than that in the fresh group and the frozen + melatonin group (P0.05). exist image 3 In B, the result is similar to that in Figure A, the mRNA expression level of gene P21 in the frozen group was significantly higher than that in the frozen + melatonin group and the fresh group (P image 3 As shown in C, the expression level of the frozen group was significantly lower than that of the fresh group and the frozen + melatonin group (P0.05).

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Abstract

The invention discloses a method for improving the parthenogenetic development potential of vitrified mature oocytes, wherein oocytes are frozen in a melatonin-containing freezing liquid, and then arethawed with a melatonin-containing sucrose solution, the thawed oocytes are cultured, the cultured oocytes are subjected to parthenogenetic activation, and finally the parthenogenetically activated embryo is cultured in vitro. According to the present invention, the method has advantages of simple process and short time consumption, and can effectively improve the parthenogenetic activation development potential of vitrified mature oocytes.

Description

technical field [0001] The invention relates to the technical field of animal reproduction, in particular to a method for improving the parthenogenesis potential of vitrified frozen mature oocytes. 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. Oocytes from mice, cows, humans, and rabbits can continue to develop after in vitro fertilization and produce healthy offspring after vitrification cryopreservation. However, the blastocyst development rate or offspring birth rate after in vitro fertilization of frozen-thawed oocytes is not ideal. After vitrification of mouse oocytes at the germinal vesicle (GV) stage, the rate of blastocysts in in vitro fertilization (IVF) is 20-42.9% after thawing; and after vitrification of mouse mature oocytes The blastocyst rate of IVF is 33%, the blastocyst rate of...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/073
CPCC12N5/0604C12N2501/30C12N2501/86
Inventor 周光斌潘波张月李伟杨昊轩
Owner SICHUAN AGRI UNIV
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