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Acrosome reaction kit, method for inducing acrosome reaction and , and method for evaluating acrosome reaction condition

A technology of acrosome reaction and kit, which is applied in the field of sperm acrosome function screening, can solve the problems of high experimental intensity, poor methodological stability and repeatability, and affect the accuracy of result interpretation, so as to ensure accuracy and repeatability , ease the work intensity, compact test process effect

Active Publication Date: 2012-06-13
BRED LIFE SCI TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, sperm acrosome function tests include sperm morphology and integrity test, zona pellucida-induced sperm acrosome reaction test, etc. Intra-injection) has a positive effect on the success rate, but because most of these detection methods are in the research stage, the methodology recommended by the World Health Organization is too cumbersome and complicated, and the stability and repeatability of the methodology are poor, so it is difficult to be used as a routine clinical practice. The method is popularized, but there is a lack of industrialized products
Existing experimental methods require the entire test process to be completed in one go, from the pretreatment of semen samples, in vitro capacitation, calcium ion induction to fluorescent staining of smears and result interpretation, the entire test takes 6 to 7 hours, and the test is intensive and difficult to apply clinically and popularity
Moreover, the intensity of the fluorescent dyeing system of the existing test method is not high, and it usually decays rapidly within half an hour, which affects the accuracy of result interpretation and will cause instability of test results

Method used

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  • Acrosome reaction kit, method for inducing acrosome reaction and , and method for evaluating acrosome reaction condition
  • Acrosome reaction kit, method for inducing acrosome reaction and , and method for evaluating acrosome reaction condition
  • Acrosome reaction kit, method for inducing acrosome reaction and , and method for evaluating acrosome reaction condition

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0089] 1. Kit preparation

[0090] 1. Select the general product in the prior art for use, prepare 80% density gradient solution, 40% density gradient solution, sperm washing solution A (pH is 7.2~7.6), sperm culture fluid, 5 × sperm washing solution according to the above specifications and dosage Solution B, calcium ionophore, control solution, fixative solution, tissue slide, long-shaped cover slip. The calcium ionophore is the A23187 calcium ionophore provided by Sigma. The materials of these existing products may not be included in the kit, and the users prepare these materials by themselves before the experiment.

[0091] 2. Prepare tissue slice preservation solution. Mix 0.5ml of glutaraldehyde, 5ml of methanol, 2ml of ethanol, 0.2g of PVP (polyvinylpyrrolidone) and 1ml of formalin with purified water to obtain 100ml of tissue slice preservation solution.

[0092] 3. Preparation of fluorescent conjugates. First prepare the fluorescent conjugate preservation solution...

Embodiment 2

[0151] 1. Kit preparation

[0152] The preparation of the kit in Example 2 is roughly the same as in Example 1, except that when preparing the fluorescent conjugate, during the preparation of the fluorescent conjugate preservation solution, fluorescein markers: 5 ng, methacrylic acid, methyl methacrylate Ester copolymer (1:2): 1.5g, Tris buffer: 2.5g, NaCl: 0.9g, casein: 0.3g, sodium salicylate: 1.4mg, preservative Proclin300: 60ul, the preparation method is the same as in Example 1 . When preparing fluorescent mounting solution, take N-(1,3-dimethylbutyl)-N'-phenyl-p-phenylenediamine: 0.2g, polyvinyl alcohol PVA: 0.9g, polyethylene glycol 8000: 0.8 g, Tris buffer solution: 2.5 g, NaCl: 1.0 g, bovine serum albumin BSA provided by Sigma Company: 3 g, thimerosal provided by Sigma Company 0.03 g, glycerol: 30%, and the preparation method is the same as in Example 1.

[0153] 2. Detection of induced sperm acrosome reaction

[0154] (1) Reagent preparation

[0155] With embodim...

Embodiment 3

[0178] 1. Kit preparation

[0179] The preparation of the kit in Example 2 is roughly the same as in Example 1, except that when preparing the fluorescent conjugate, during the preparation of the fluorescent conjugate preservation solution, fluorescein markers: 10 ng, methacrylic acid, methyl methacrylate Ester copolymer (1:2): 2.4g, Tris buffer: 3.5g, NaCl: 1.5g, casein: 0.5g, sodium salicylate: 2.0mg, preservative Proclin300: 100ul, the preparation method is the same as in Example 1 . When preparing fluorescent mounting solution, take N-(1,3-dimethylbutyl) N'-phenyl-p-phenylenediamine: 0.5g, polyvinyl alcohol PVA: 1.6g, polyethylene glycol 8000: 1.5g , Tris buffer: 5.0g, NaCl: 1.6g, bovine serum albumin BSA: 5g, NaN 3 0.05g, glycerol: 60%, and the preparation method is the same as in Example 1.

[0180] 2. Detection of induced sperm acrosome reaction

[0181] (1) Reagent preparation

[0182] With embodiment 1.

[0183] (2) Carry out testing procedures

[0184] Steps 1...

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Abstract

The invention discloses an acrosome reaction kit, which comprises tissue slide preserving fluid, a fluorescent conjugate and a fluorescent sealing solid liquid, wherein each 100 ml of the tissue slide preserving fluid contains 0.5ml of glutaraldehyde, 5ml of methanol, 2ml of ethanol, 0.2g of polyvinylpyrrolidone (PVP), 1ml of formalin and the balance of purified water. The kit can carry out a sperm-induced acrosome reaction and detection; and if calcium ion induction is not carried out, the kit can carry out the acrosome reaction condition evaluation and the acrosome integrity determination. By the tissue slide preserving fluid, the test process for the sperm-induced acrosome reaction and detection is carried out by two parts, samples and test results which are obtained in the first part can be preserved and the final test result cannot be influenced if the operations of the second part are carried out in the subsequent 72 hours, so that the work intensity can be relieved. The fluorescence of a fluorescent staining system is strong and cannot be attenuated after being stabilized for 15 days, so that the detection result is more stable and is easy to interprete and the repeatability of the result can be ensured. The kit and a determination method can be clinically applied and popularized.

Description

technical field [0001] The present invention relates to sperm acrosome function screening, especially simulating the fertilization environment in the human body in vitro, initiating sperm capacitation and inducing acrosome reaction in vitro, and evaluating the ability of capacitated sperm to undergo acrosome reaction by fluorescent staining, thereby evaluating The ability of sperm to penetrate oocytes and fertilize them provides a theoretical basis for the analysis of the etiology of infertility and the selection of assisted reproductive treatment options. technical background [0002] Male sex and its associated factors account for at least 50% of the causes of infertile couples. In clinical practice, male fertility is mainly evaluated by routine semen analysis that detects indicators such as sperm density, motility, and morphology. However, the indicators provided by routine semen analysis only reflect the quality of sperm, and only the function of sperm can reflect the in...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N21/64C09K11/06
Inventor 邓少君程锦军胡勇华
Owner BRED LIFE SCI TECH
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