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A method for quantitative detection of Izumo protein 1 on the surface of sperm

A quantitative detection method, sperm technology, applied in the field of cell biology, can solve problems such as lack of fertilization ability, and achieve the effect of fast speed, easy operation and high precision

Active Publication Date: 2020-06-05
ZHEJIANG UNIV
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AI Technical Summary

Problems solved by technology

Izumo gene knockout studies have shown that the sperm morphology and motility of male Izumo- / - mice are the same as those of wild-type mice, but during fertilization, due to the lack of Izumo1 sperm can penetrate the zona pellucida but cannot penetrate the egg membrane, therefore incapable of fertilization

Method used

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Embodiment 1

[0031] The invention uses flow cytometry to quantitatively detect the Izumo1 protein on the surface of human sperm to evaluate the fertilization ability of male sperm.

[0032] 1. Collect semen samples from three subjects in sterile semen collection tanks, and place them in a water bath at 37 degrees Celsius for 30 minutes to fully liquefy them.

[0033] 2. Sperm Separation Pretreatment

[0034] (1) In a plastic test tube, add 6 ml of Hepes buffered human tubal fluid (HTF) containing 10% serum substitute (synthetic serum substitute) to 2 ml of semen specimen and mix well.

[0035] (2) Centrifuge at 200×g for 5 minutes to wash the sperm, carefully suck off the supernatant, and keep the precipitate. Add HTF solution to resuspend and repeat washing once.

[0036] (3) Carefully suck off the supernatant, keep the precipitated sperm at the bottom of the test tube, and pipette 1 ml of G-IVF solution containing 10% serum substitute and carefully add it on top of the washed sperm.

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Abstract

The invention provides a quantitative detection method of sperm surface Izumo1 protein. The method comprises the following steps: performing separation and pretreatment of a sperm, performing preliminary treatment of the sperm to expose the Izumo1 protein epitope of the sperm, marking the Izumo1 protein of the sperm by using fluorescein, and performing detection by using a flow cytometer. According to Tthe method provided by the invention, utilizes the flow cytometry is utilized to perform quantitative detection on the Izumo1 protein in the human sperm for the first time, directly realizes precise quantification of the Izumo1 protein is directly realized, is easy to realize standardization in operation is easy to realize, is quick and accurate effects are realized, and is high inthe precision is high. Compared with traditional methods for qualitative or semi-quantitative detection of Izumo1 protein, such as cell immunofluorescence and fluorescence in situ hybridization, the method provided by the invention has the advantages of convenient detection, high precision, high speed and the like; the method can be applied to quantitative detection of expression of sperm surface Izumo1 protein and is easy to popularize; and the detection result can be used for performing auxiliary evaluation on the fertilization ability of the sperm, so that the method is suitable for practical application.

Description

technical field [0001] The invention belongs to the field of cell biology, and relates to a method for quantitatively detecting Izumo1 (Izumo1) on the surface of human sperm by using flow cytometry. Background technique [0002] In in vitro fertilization-embryo transfer (IVF-ET), low fertilization rates (<30%) and cases of infertility occur frequently. It is comprehensively reported that the number of cases of fertilization failure (low fertilization rate and infertility) accounts for about 10%-25% of the total number of cycles, which greatly affects the therapeutic effect of assisted reproductive technology and also brings psychological benefits to infertile couples. severe trauma. [0003] The reasons for the failure of fertilization are sperm factors and egg factors. Sperm factors mainly include abnormal zona pellucida binding or penetration, acrosome reaction defects, etc. Satisfactory fertilization rate and clinical pregnancy rate can be obtained if ICSI is used f...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N15/14
CPCG01N15/14
Inventor 叶英辉崔龙曲凡
Owner ZHEJIANG UNIV
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