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Method and culture medium for carrying out oocyte in vitro maturation culture

A technology for in vitro culture of oocytes, applied in the field of assisted reproduction, to improve the quality, increase the rate of oocyte division and blastocyst development, and improve the expression

Inactive Publication Date: 2018-08-10
SDIVF R&D CENT LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the role of resveratrol in reproductive development, including in oocyte development, remains to be further studied, especially whether resveratrol plays a role in the maturation of GV stage oocytes and the development of aged animal embryos and cysts. developmental potential for embryogenesis

Method used

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  • Method and culture medium for carrying out oocyte in vitro maturation culture
  • Method and culture medium for carrying out oocyte in vitro maturation culture
  • Method and culture medium for carrying out oocyte in vitro maturation culture

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Embodiment 1 Materials and methods

[0027] Solutions and Chemicals

[0028] All chemicals and reagents in the studies were purchased from Sigma-Aldrich Chemical Company (USA) unless otherwise stated. Resveratrol was dissolved in dimethylsulfoxide (DMSO) and stored at -20°C until use. The M199 medium used for IVM culture was supplemented with 10% human serum albumin (HSA), 50 IU / mL penicillin, 5 μg streptomycin, 0.29 mmol / L sodium pyruvate, 0.15 IU / ml human chorionic gonadotropin (HCG ), 0.075 U / mL recombinant follicle stimulating hormone, 10 ng / mL recombinant human epidermal growth factor (EGF) and 10 μg / ml estradiol-17β (E2).

[0029] Statistical Analysis

[0030] At least three replicates were assessed for each treatment. Data (mean ± SEM) were analyzed by ANOVA using SPSS software (IBM Corp, USA), followed by Pearson chi-square test. P<0.05 was considered statistically significant.

Embodiment 2

[0031] Example 2 Resveratrol promotes maturation and fertilization of oocytes and blastocyst formation in aged mice

[0032] Oocytes at the GV stage were obtained by puncturing the ovary of aged mice (C57BL / 6J, purchased from Victoria Lee, 48-52W) with a sterile needle, and washed thoroughly in M199 medium. By gently pipetting cumulus cells in hyaluronidase medium. 1138 oocytes from aged mice (48-52W) were randomly divided into 4 groups. In the presence of three different concentrations of resveratrol (0.1, 1.0 and 10 μM / L) or DMSO blank control IVM medium, in the presence of 5% CO 2 The oocytes from which the cumulus had been removed were cultured at 37°C in an environment. MI and MII stage oocytes were assessed after 16 hours. Obtain sperm from the vas deferens of male mice over 8 weeks old, wash with in vitro fertilization medium, and press 2 to 5×10 in a 6% carbon dioxide incubator at 37°C. 6 / mL density for 2 hours. Transfer the resulting MII stage oocytes into sperm...

Embodiment 3

[0039] Example 3 Resveratrol improves the oocyte quality of aged mice

[0040] Oocyte quality is reflected by spindle morphology, chromosome alignment and mitochondrial function.

[0041] Detection by immunofluorescence and confocal analysis methods: MII stage oocytes were fixed in 2% paraformaldehyde for 10 min at 37°C and permeabilized with 0.2% Triton X-100 for 45 min. Next, the oocytes were incubated with monoclonal anti-mouse α-tubulin antibody (1 / 100 in PBS; Sigma) for 90 minutes and incubated with FITC-labeled goat antibody diluted 1 / 300 in PBS. Mouse IgG (Sigma) was incubated for 60 minutes. To specifically assess the distribution of mitochondria, MII-oocytes were incubated with 400 nmol / L Mito Tracker Green FM diluted in PBS for 30 min and fixed in 2% paraformaldehyde for 10 min (all steps at 37 °C). Chromatin in MII-oocytes was stained with Hoechst (B-2261, Sigma) for 10 min. Finally, oocytes were examined under a LSM780 confocal scanning microscope (Zeiss, German...

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Abstract

The invention provides a method and a culture medium for carrying out oocyte in vitro maturation culture during assisted reproduction. The invention in particular provides a method for carrying out invitro culture on oocytes of mammals which are of high child-bearing age and are subjected to an assisted reproductive technology for promoting the development of the oocytes; the method comprises thestep of adding resveratrol into a nutrient solution in which the oocytes are cultured in vitro. The invention also provides an oocyte in vitro culture medium composition for the mammals of high child-bearing age; the composition contains the resveratrol.

Description

technical field [0001] The invention relates to the technical field of assisted reproduction. Specifically, the present invention relates to a method for in vitro maturation of oocytes in assisted reproduction and a medium for in vitro maturation of oocytes. Background technique [0002] In mammals, oocytes grow and develop in follicles within the ovary. Maturation of the oocyte (cytoplasmic and nuclear) is critical for successful fertilization, embryo development, and for the embryo's ability to implant, ultimately affecting pregnancy outcome. Mitochondria play an important role in the cellular energy metabolism of oocytes. Adenosine triphosphate (ATP) is produced in oocytes by oxidative phosphorylation and is required for several functions such as maintenance of cellular homeostasis, regulation of apoptosis, meiosis, and calcium signaling. [0003] Age-related female fertility declines with increasing maternal age. This reproductive aging appears to be primarily due to...

Claims

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Application Information

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IPC IPC(8): C12N5/075
CPCC12N5/0609C12N2501/01
Inventor 赵涵刘洪彬马金龙赵世刚熊志强路钢
Owner SDIVF R&D CENT LTD
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