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Method for in-vitro fertilization by taking testicular sertoli cells as trophoblasts

A technology of Sertoli cells and in vitro fertilization, applied in the field of in vitro fertilization using Sertoli cells as trophoblasts, can solve the problems of low insemination rate in in vitro fertilization, improve microenvironment, increase utilization value, and increase insemination rate and efficiency Effect

Inactive Publication Date: 2016-06-08
QINGDAO AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] In the existing research reports, the improvement of in vitro fertilization technology mainly focuses on the formula of sperm capacitation fluid and insemination fluid, and there is no related report on the improvement of the whole system, and the insemination rate of existing in vitro fertilization technology is low

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] This example is an experimental group, and the treatment of the experimental group is as follows:

[0027] A method for in vitro insemination using Sertoli cells as a trophoblast includes the following steps:

[0028] (1) Separation and culture of Sertoli testicular cells for 2 days, cell monolayer formation, discard the old medium 12h before in vitro fertilization, wash once with PBS, and add a-MEM medium containing 1% FBS and 1% PS , Put the petri dish at 37℃, 5% CO 2 Incubate the balance overnight in the incubator, and put the HTF sperm capacitation solution into 37℃, 5% CO 2 Balance overnight in the incubator;

[0029] (2) Put the mature male mouse to death by the method of vertebral dislocation, immediately remove the testes and epididymis, remove the blood and fat tissue on sterilized filter paper, cut the epididymal tail with ophthalmic scissors, take out the sperm mass, and introduce the sperm mass into the balance In a good HTF capacitation solution, at 37℃, 5% CO 2 ...

Embodiment 2

[0039] This example is a specific implementation of the control group, and the treatment of the control group is as follows:

[0040] (1) Add a-MEM medium containing 1% FBS and 1% PS to a 35mm petri dish without supporting cell monolayer, and place the petri dish at 37°C, 5% CO 2 Incubate the balance overnight in the incubator, and put the HTF sperm capacitation solution into 37℃, 5% CO 2 Balance overnight in the incubator;

[0041] (2) Put the mature male mouse to death by the method of vertebral dislocation, immediately remove the testes and epididymis, remove the blood and fat tissue on sterilized filter paper, cut the epididymal tail with ophthalmic scissors, take out the sperm mass, and introduce the sperm mass into the balance In a good HTF capacitation solution, at 37℃, 5% CO 2 Incubate for 1h in the incubator;

[0042] (3) Collection of eggs:

[0043] a. Inject 5IU of PMSG into mature female mice, and then inject 5IU of hCG 48h later to induce ovulation;

[0044] b. 14 hours af...

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Abstract

The invention relates to a method for in-vitro fertilization by taking testicular sertoli cells as trophoblasts. The method includes the steps of (1), subjecting the testicular sertoli cells to isolated culture to prepare the sertoli cell trophoblasts; (2), guiding sperms into capacitation liquid containing well balanced HTF for incubation for 1 hour; (3), collecting ova; (4), transferring washed ova into a balanced culture medium with a testicular sertoli cell monolayer; (5), adding 20 microliters of incubated sperm suspension liquid into a culture solution containing the ova; (6), placing a culture dish back to an incubator with 5% CO2 at the temperature of 37 DEG C for incubation, and holding for 5-6 hours; (7), picking out the ova with second polar bodies and pronuclei and transferring into the culture medium for culturing. The method for in-vitro fertilization by taking the testicular sertoli cells as the trophoblasts has the advantages of high insemination rate and developmental rate.

Description

Technical field [0001] The invention belongs to the technical field of in vitro insemination, and in particular relates to a method for in vitro insemination using testicular supporting cells as a trophoblast. Background technique [0002] In vitro insemination refers to the technology in which the sperm and eggs of mammals complete the insemination process in an artificially controlled environment in vitro, which is referred to as "Working VF" in English. Because it is inseparable from embryo transfer technology (ET), it is also referred to as IVF-ET. In biology, the animals obtained after the in vitro fertilization embryos are transferred to the mother are called test-tube animals. Artificial insemination technology is a high technology applied to the breeding of mammals. The use of artificial insemination, combined with embryo transfer technology, can develop the reproductive potential of female animals with excellent genetic characteristics and quickly expand the herd of im...

Claims

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Application Information

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IPC IPC(8): C12N5/073C12N5/075C12N5/076
CPCC12N5/0603C12N5/0609C12N5/061
Inventor 董焕声王明明潘庆杰
Owner QINGDAO AGRI UNIV
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