CN103472044A - Lectin composition for detecting male fertility and kit of lectin composition - Google Patents

Abstract

The invention discloses a lectin composition for detecting male fertility. The lectin composition includes two or more of the following lectins: Jacalin, GHA, MPL, ACL, VVL and ABA. The present invention also provides a kit for detecting male fertility, comprising the lectin composition and PBS washing solution. The present invention utilizes the combination of the above six lectins and the difference in the binding strength of different human sperm samples to evaluate the quality of sperm by detecting the intensity of fluorescence through flow cytometer, fluorescence microscope, liquid chip and other methods, and realizes the evaluation of male fertility , to provide a theoretical basis for the study of the molecular mechanism of male infertility and the diagnosis of the etiology of male infertility.

Description

Lectin composition and kit for detecting male fertility

technical field

The present invention relates to a lectin composition and a kit thereof, in particular to a lectin composition for detecting male fertility and a kit thereof.

Background technique

In mammals, when spermatozoa are produced from the testes and pass through the epididymis, their surface is covered with high-density sugar molecules, thus forming a glycocalyx with a thickness of 20-60nm. Most of the sugar residues are attached to proteins. These sugars Some proteins are integrated on the sperm membrane, and some are loosely connected to the surface of the sperm. At present, the structural map of the glycocalyx on the surface of human sperm is unknown, and only the structure of the sperm membrane glycocalyx is known to be closely related to male fertility. In cynomolgus monkeys, highly glycosylated β-defensin126 (DEFB126), which is secreted from the body of the epididymis and covers the entire sperm surface, is the main glycoprotein component of the sperm glycocalyx, and is the main glycoprotein component of the sperm glycocalyx. It plays an important role in the immune surveillance and immune response, mediating the combination of sperm and fallopian tube epithelial cells. In humans, about 20% of males have frameshift mutations with two nucleotide deletions in the coding region of the DEFB126 gene, and males with homozygous mutations (del/del) have significantly reduced natural fertility.

At present, there are not many technical means to study the sugar structure on the cell surface, mainly flow cytometry (FCM) and fluorescence microscopy using the specific combination of lectins and oligosaccharides. Among them, peanut agglutinin (PNA) and pea agglutinin (PSA) can specifically recognize the acrosome glycoprotein of sperm, and are used to detect the acrosome reaction ability of sperm. However, at present, there is no comprehensive analysis of the sugar structure map on the surface of human sperm with the lectin chip method, and the screening of differentially bound lectins on the surface of male sperm with different fertility through the lectin chip method, so as to obtain a method that can be used for male fertility screening. As for the lectin composition and its kit, there is no similar product in the prior art.

Contents of the invention

The problem to be solved by the present invention is to provide a lectin composition that specifically binds to the sugar structure on the surface of human sperm, and use the different binding strengths of sperm cells of men with different fertility to this lectin combination to evaluate the fertility of men, and provide a basis for male fertility. It provides a theoretical basis for the study of the molecular mechanism of infertility and the diagnosis of the etiology of male infertility. The present invention also provides a kit mainly composed of the above-mentioned lectin combination for screening male fertility.

The present invention is realized through the following technical solutions:

A lectin composition for detecting male fertility, comprising two or more of the following lectins: Jacalin, GHA, MPL, ACL, VVL and ABA. Preferably, the Jacalin, GHA, MPL, ACL, VVL and ABA are labeled with fluorescent materials. More preferably, the fluorescent material is FITC, AIexaFluor488 or fluorescent nanospheres.

A kit for detecting male fertility, comprising the above-mentioned lectin composition and PBS washing solution.

In the present invention, Jacalin refers to Artocarpus integrifolia lectin (Artocarpus integrifolia, AIA), GHA refers to cotton lectin (Gossypium hirsutum agglutinin), ACL refers to Amaranthuscaudatus lectin, and MPL refers to orange mulberry agglutinin Maclura pomifera lectin, VVL refers to Viciavillosa lectin and ABA refers to Agaricus bisporus.

Compared with the prior art, the present invention has the following beneficial effects: the DEFB126 gene defect will lead to male fertility decline, but in the present invention, 6 lectins are screened out with a lectin chip, and the combination with DEFB126 mutant sperm is reduced. These 6 lectins labeled with FITC (or other fluorescently labeled probes, such as AIexaFluor488) are incubated with the sperm samples to be tested and combined, and the fluorescence intensity of the samples is detected by flow cytometry, liquid chips or fluorescence microscopes to determine male fertility force. In the present invention, the combination of these lectins is applied clinically as a screening method for male infertility, which can evaluate the changes in the sugar structure fingerprints on the surface of sperm in semen, and provide a theory for the study of the molecular mechanism of male infertility and the diagnosis of the etiology of male infertility in accordance with.

Description of drawings

Other characteristics, objects and advantages of the present invention will become more apparent by reading the detailed description of non-limiting embodiments made with reference to the following drawings:

Figure 1 is a graph showing the results of lectins that specifically bind to human sperm;

Figure 2 is a diagram showing differences in the binding of male sperm and lectins of different genotypes according to the DEFB126 double-base deletion mutation;

Figure 3 is a graph showing the results of lectin fluorescence signals used to evaluate male fertility.

Detailed ways

The present invention will be described in detail below in conjunction with specific embodiments. The following examples will help those skilled in the art to further understand the present invention, but do not limit the present invention in any form. It should be noted that those skilled in the art can make several modifications and improvements without departing from the concept of the present invention. These all belong to the protection scope of the present invention.

In order to facilitate the study of the sugar structure changes on the sperm surface of infertile men caused by changes in the glycocalyx, the present invention uses one of the main components of sperm surface glycoproteins - β-defensin 126 protein (DEFB126), and DEFB126 expression-deficient as the case group, Analyze the changes in the sugar chain map caused by the change of the glycoprotein, and find the change rule of the sugar structure map that can be used as an indicator of male infertility. The present invention will be further described in detail below in conjunction with the detection of specific lectin chips.

Example 1. Collection and fluorescent labeling of human sperm

Step (1) Grouping of semen samples

Genomic DNA was extracted from male whole blood or sperm, and the rs140685149 site of the DEFB126 gene coding region was genotyped according to the following primer sequences and fluorescent quantitative PCR conditions, and the semen samples were divided into wild-type group and heterozygous group and pure mutation group, amplified with 2W/W and 2D/D primers respectively in two qPCR tubes, and analyzed the data after amplification; the specific formula of qPCR reaction system is shown in Table 1, and the qPCR amplification procedure is shown in Table 2 shows:

The forward primer of 2W/W_169bp: the nucleotide sequence shown in SEQ ID NO:1;

The reverse primer of 2W/W_169bp: the nucleotide sequence shown in SEQ ID NO:2;

The forward primer of 2D/D_295bp: the nucleotide sequence shown in SEQ ID NO:3;

The reverse primer of 2D/D_295bp: the nucleotide sequence shown in SEQ ID NO:4;

Table 1

volume 2x Master Mix 7.5μL Template (about 5ng/μl) 2.0 μL F/R primers (2.5 μmol/L each) 1.5μL ddH ₂ O 4.0 μL

Table 2

Step (2) Collection of semen samples

The sample subjects were abstinent for 2 to 7 days, and the whole semen was obtained by masturbation, and placed in a 37°C water bath for 30 minutes for liquefaction.

Step (3) Fixation of semen samples

After the liquefied semen was centrifuged at 500g for 10 minutes, the seminal plasma was discarded, then resuspended and washed with PBS (pH7.4), and centrifuged at 500g for 5 minutes; the sperm pellet was fixed with freshly prepared 2% paraformaldehyde and 0.2% glutaraldehyde After 30 minutes, wash twice again with PBS, and finally resuspend in PBS (pH 7.4) containing 0.02% sodium azide. After counting on a sperm counting board, adjust the sperm density to 40×10 ⁶ sperm/mL, and set Store in a refrigerator at 4°C for later use;

The purpose of counting and quantifying each sperm sample is to ensure the consistency of the loading amount of the lectin chip and improve the accuracy of gellin chip detection;

Step (4) Fluorescence labeling of semen samples

Take 0.5 mL of refrigerated sperm, add propidium iodide (20 μg/mL) and incubate in the dark for 30 minutes for fluorescent staining, wash once with PBS, resuspend with 800 μL binding buffer, and set aside.

Example 2. Using a lectin chip to detect sperm samples of different genotypes of DEFB126

Step (1) Chip reheating: Take the chip out of the 4°C refrigerator and place it at room temperature for 20-30 minutes to evaporate water vapor and restore the temperature;

Step (2) chip sealing: configure 50mL TBST, slowly add to the tank, place on a shaker at room temperature and shake gently for 1 hour;

Step (3) Chip washing: wash with 50mL PBST for 10 minutes, then wash twice with PBS for 10 minutes each time, and dry for later use;

Step (4) Preparation of the chip incubation tank: put the glass slide with the marked array position as a template on the bottom of the chip, then stick the chip incubation tank on the chip according to the position of the array, and press firmly;

Step (5) Sperm and chip binding: add 200 μL of fluorescently labeled sperm samples to each incubation tank of the lectin chip, and incubate at room temperature for 1 hour in the dark;

Step (6) Wash non-specific binding: prepare 500mL PBST for each chip; slowly pour it into the washing box, hold the chip in the right hand, gently lift off the incubation tank, slowly flip the chip about 10-20 times for washing;

Step (7) Chip scanning: The dried lectin chip was scanned with a GenePix4100B (Axon, Sunnyvale, CA) chip scanner, with the resolution set to 5 μm and the photomultiplier tube (PMT) set to 100%, and a pre-scan was performed first , and then select the spotting area, perform precise scanning, adjust the brightness and contrast to achieve the best visual effect; obtain the fluorescent image of the combination of lectin and sugar chains on the surface of the labeled sperm;

Step (8) Perform average analysis on the fluorescence images by GenePix3.0 software.

Embodiment 3, fluorescence microscopy and flow cytometry detection

Resuspend 5×10 ⁶ sperm cells in 100 μl PBS, add 100 μg/ml FITC-MPL and FITC-ABA to resuspend and mix, place at 37°C, and incubate in the dark for 30 minutes. Then wash once with 0.5ml PBS, take 20μl smear, observe the fluorescence signal with fluorescence microscope, then analyze the fluorescence intensity with Facs Calibur flow cytometer, and analyze and identify the results with WinMID2.9 software.

Analysis and evaluation of implementation results

Through statistical analysis and comparison of the fluorescent signals of the lectin chips of the DEFB126 gene wild-type, heterozygous and homozygous mutant sperm samples, it can be seen that: (1) Among the 91 lectins on the chip, the DEFB126 wild-type sperm The sample can bind to 54 lectins (as shown in Figure 1); (2) Comparing the lectin binding signals of the three genotype samples, it was found that 6 lectins (as shown in Figure 2) had significant differences, and Sperm samples with DEFB126 homozygous mutations showed significantly reduced binding signals to these six lectins; (3) The binding strength of sperm carrying different DEFB126 genotypes to FITC-MPL and FITC-ABA was detected by fluorescence microscopy and flow cytometry , as shown in Figure 3, it was found that consistent with the results of lectin microarray screening, the sperm samples with homozygous mutation of DEFB126 had a significantly reduced binding signal to FITC-MPL and FITC-ABA, which can be used to screen sperm fertility .

Specific embodiments of the present invention have been described above. It should be understood that the present invention is not limited to the specific embodiments described above, and those skilled in the art may make various changes or modifications within the scope of the claims, which do not affect the essence of the present invention.

Claims (4)

1. A lectin composition for detecting male fertility, characterized in that it comprises two or more of the following lectins: Jacalin, GHA, MPL, ACL, VVL and ABA. 2. The lectin composition according to claim 1, wherein the Jacalin, GHA, MPL, ACL, VVL and ABA are labeled with fluorescent materials. 3. The lectin composition according to claim 2, wherein the fluorescent material is FITC, AIexaFluor488 or fluorescent nanospheres. 4. A kit for detecting male fertility, characterized in that it comprises the lectin composition according to claim 1, 2 or 3, and PBS washing solution.

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