CN106290120A - The test kit of detection human spermatogoa surface sugar structure change - Google Patents

Abstract

The present invention proposes a kit for detecting the change of sugar structure on the surface of human sperm. The kit includes: Agaricus bisporus lectin, datura lectin, jackfruit lectin, black bean lectin, and bindweed lectin , gorse agglutinin, pinto bean agglutinin, snail agglutinin, Huaihuai agglutinin, Huaihuai agglutinin, pea agglutinin, Polygonum multiflorum agglutinin, gore agglutinin, villous vetch agglutinin one or a combination of several. The kit is used clinically, can evaluate the quality of sperm in semen, and provides a theoretical basis for the study of the molecular mechanism of male infertility and the diagnosis of the etiology of male infertility.

Description

Kit for detection of sugar structure changes on the surface of human sperm

technical field

The invention relates to the field of sperm detection kits, in particular to a kit for detecting sugar structure changes on the surface of human sperm.

Background technique

Cryopreservation of spermoids has a history of more than 70 years. Nowadays, sperm cryopreservation technology has become an important part of assisted reproductive technology and sperm bank. The improvement of this technology level and the stability of recovery rate will directly affect the final conception rate. On the one hand, by freezing and resuscitating the donor’s semen, normal sperm can be provided to patients with azoospermia and severe genetic diseases that are not suitable for fertility, so that many infertile couples can realize their dream of having children; Patients with malignant diseases undergoing radiotherapy, staff who need to be exposed to radiation, radiation, poisons, etc. and lose sperm, and patients who are about to undergo orchiectomy or vasectomy, should carry out sperm freezing storage in advance to provide insurance for reproductive health. Although freezing methods have been continuously improved, cryopreservation still causes irreversible damage to sperm structure and function. The surface of the sperm is covered with a layer of glycocalyx, and when the sperm is produced from the testis and passes through the epididymis, various sugar residues are covalently bound to the proteins or lipids on the surface of the sperm membrane to form a 20-60nm thick glycocalyx. Compared with the egg zona pellucida, which is composed of only three glycoproteins, the composition of the sperm glycocalyx is much more complex. It is estimated that there are more than 300 different glycoproteins and glycolipids. The sperm glycocalyx is the main interface for the interaction between male gametes and the external environment. During the process of sperm formation, maturation, capacitation and acrosome reaction, the sperm surface glycoproteins undergo a huge rearrangement, and the subtle changes in the sperm glycocalyx have great impact on sperm fertility. With a major impact, the maturation of the sperm glycocalyx is closely related to sperm fertility.

At present, there are not many technical means to study the sugar structure on the cell surface, mainly using flow cytometry (FCM) and fluorescence microscopy techniques that use the specific combination of lectin and oligosaccharides, and lectin chip technology.

Contents of the invention

The invention proposes a kit for detecting the change of sugar structure on the surface of human sperm, which enriches the detection methods in the prior art, can evaluate the quality of sperm in semen, and provides a theoretical basis for the research on the molecular mechanism of male infertility and the diagnosis of the etiology of male infertility .

Technical scheme of the present invention is realized like this:

A test kit for detecting changes in the sugar structure on the surface of human sperm, the test kit comprising:

Agaricus bisporus agglutinin (ABA), datura agglutinin (DSL), jackfruit agglutinin (AIA), black bean agglutinin (Black bean crude), field bindweed agglutinin (CALSEPA), gorse agglutinin (CSA), pinto bean agglutinin (GSL I-B4), snail agglutinin (HPA), Huaihuai agglutinin (MAA), Huaihuai agglutinin (MAL II), pea agglutinin (PEA), Polygonum multiflorum agglutinin ( PMA), gore agglutinin (UEA I), villous vetch lectin (VVL) or a combination of several.

As a preferred technical solution, the above-mentioned lectins are all fluorescently labeled.

As a preferred technical solution, the kit further includes: propidium iodide, 50-100 ug/ml fluorescein isothiocyanate, TBST washing solution, PBST washing solution and PBS washing solution.

As a preferred technical solution, the concentration of the propidium iodide is 5-20ug/ml.

Principle: Because the sperm quality of different people is different, we screened out 14 lectins with different degrees of binding to the sperm of men with different freezing resistance by using the lectin chip. The 14 lectins labeled with FITC and PI are incubated with the sperm sample to be detected, and the fluorescence intensity of the sample is detected by flow cytometry, so as to determine the freezing resistance of the sperm. The present invention uses 14 kinds of lectin combinations as a screening method for poor freezing resistance. It can be used clinically to evaluate the quality of sperm in semen, and provide a theoretical basis for the study of the molecular mechanism of male infertility and the diagnosis of the etiology of male infertility.

Compared with the prior art, the present invention has the following beneficial effects:

(1) The kit of the present invention provides a new method for detecting sperm quality, and broadens the way of studying the sugar structure on the cell surface.

(2) With the kit of the present invention, the whole detection process is quick and easy.

(3) The present invention utilizes the difference between the binding of lectin and the binding strength of different human sperm samples, and evaluates the freezing resistance of sperm by detecting the fluorescence intensity with flow cytometer, so as to realize the evaluation of whether it can be frozen, and improve the health of male reproductive health. and insurance, and provide a theoretical basis for the research on the molecular mechanism of male infertility and the diagnosis of the etiology of male infertility.

Description of drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that need to be used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the accompanying drawings in the following description are only These are some implementations of the present invention. For those skilled in the art, other drawings can also be obtained according to these drawings on the premise of not paying creative efforts.

Fig. 1 Differences in the binding of sperm and lectins in males with different freezing tolerance; where a-freeze-resistant, b-freeze-intolerant.

Figure 2 Differences in binding to lectin in male sperm with different freezing performances before and after freezing and thawing Figure 1 (freezing tolerance group); where c-before freezing and thawing, d-after freezing and thawing.

Figure 3 Differences in binding to lectin in male sperm with different freezing tolerance before and after freezing and thawing Figure 2 (freeze-intolerant group); where c-before freezing and thawing, d-after freezing and thawing.

Fig. 4 is a graph showing the results of lectin fluorescence signals used to evaluate the freezing tolerance of male sperm; where e-freeze-resistant, f-freeze-intolerant.

detailed description

The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some of the embodiments of the present invention, but not all of them. Based on the embodiments of the present invention, all other embodiments obtained by persons of ordinary skill in the art without creative efforts fall within the protection scope of the present invention.

Example 1

Collection and fluorescent labeling of human spermatozoa

Step (1) collection of semen samples

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

Step (2) grouping of semen samples

The liquefied semen samples were divided into two parts, one part was the fresh group for subsequent processing. The other is the cryopreservation group, which adopts a one-step fumigation method for ultra-low temperature freezing treatment. Firstly, mix the liquefied semen sample with an equal amount of cryoprotectant immediately and slowly, and after mixing gently and fully, quickly transfer the balanced sample to a centrifuge tube, put it in a one-step fumigation apparatus for fumigation for more than 2 hours, and transfer quickly Store in liquid nitrogen until use. After 7 days, thaw and recover in a constant temperature water bath at 37°C for 5 minutes.

For the recovery rate (i.e., forward motility rate after freezing/thawed group/forward motility rate of fresh group) less than 35%, it was defined as poor freeze-tolerant sperm samples, and the recovery rate greater than 70% was defined as good freeze-tolerant spermatozoa sample.

Step (3) fixation of semen sample

After the above 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 for 30 minutes Afterwards, wash twice with PBS again, and finally resuspend in PBS (pH7.4) containing 0.02% sodium azide. After counting on a sperm counting board, adjust the sperm density to 40×10 ⁶ sperm/mL, place in 4 ℃ refrigerator for future 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 the detection of the lectin chip;

Step (4) fluorescent 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.

Detection of sperm samples with different freezing resistance by using lectin chip

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, and slowly flip the chip about 10-20 times for washing;

Step (7) chip scanning: the dried lectin chip is scanned with a GenePix4100B (Axon, Sunnyvale, CA) chip scanner, the resolution is set to 5 μm, the photomultiplier tube (PMT) is set to 100%, and a pre-scan is 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.

Fluorescence microscopy and flow cytometry detection

Resuspend 5×106 sperm cells in 100 μl PBS, add 100 μg/ml FITC-ABA to resuspend and mix, place at 37°C, incubate in the dark for 30 minutes, then add 20 μg/ml PI to resuspend and mix, place at 37°C, avoid Incubate with light for 10min, then wash once with 0.5ml PBS, take 20μl smear, observe fluorescence signal with fluorescence microscope, then analyze fluorescence intensity with Facs Calibur flow cytometer, and analyze and identify the results with FlowJo7.6.1 software.

Analysis and evaluation of implementation results

(1) For the results of the lectin chip, we performed an independent T test on the data of the fresh sample (not frozen and thawed) for the data of the freeze-resistant group and the freeze-resistant group. Comparing the binding signals between sperm and lectins with different freezing resistance properties, it was found that there were significant differences in the lectins Agaricus bisporus agglutinin (ABA) and datura lectin (DSL) (as shown in Figure 1). Compared with sperm samples with good freeze tolerance, the signals of sperm samples with poor freeze tolerance and ABA lectin were significantly increased, and the signals of sperm samples with poor freeze tolerance were significantly decreased with DSL lectin;

(2) For the results of the lectin chip, we performed a paired T-test on the data of the samples before and after freezing and thawing. There was a difference in the data of the freeze-resistant group before and after freezing, but there was no difference in the data of the intolerant group before and after freezing. Performance 12 kinds of lectins: jackfruit agglutinin (AIA), black bean agglutinin (Black bean crude), field bindweed agglutinin (CALSEPA), gorse agglutinin (CSA), pinto bean agglutinin (GSL I-B4 ), snail agglutinin (HPA), Huaihuai agglutinin (MAA), Huaihuai agglutinin (MAL II), pea agglutinin (PEA), Polygonum multiflorum agglutinin (PMA), gorse agglutinin (UEAI), long soft Vegetable vetch lectin (VVL) (Figure 2 and Figure 3).

(3) Using fluorescence microscopy and flow cytometry to detect the binding strength of sperm with different freeze-resistant properties to FITC-ABA and PI, it was found that the sperm samples with poor freeze-resistant performance were consistent with the results of lectin chip screening, and their binding strength with FITC-ABA The binding signal increased significantly (as shown in Figure 4).

name Freeze-resistant group-freeze-resistant group Lectin Specificity ABA * Gal(β1-3)GalNAc

Datura agglutinin (DSL), jackfruit agglutinin (AIA), black bean agglutinin (Black beancrude), bindweed agglutinin (CALSEPA), gorse agglutinin (CSA), pinto bean agglutinin (GSL I-B4), snail agglutinin (HPA), Huaihuai agglutinin (MAA), Huaihuai agglutinin (MAL II), pea agglutinin (PEA), Polygonum multiflorum agglutinin (PMA), gore agglutinin (UEAI) The detection results of lectin (VVL) in villous vetch were also consistent with the screening results.

The above descriptions are only preferred embodiments of the present invention, and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention shall be included in the scope of the present invention. within the scope of protection.

Claims (4)

1. a test kit for detecting changes in human sperm surface sugar structure, characterized in that, the test kit comprises: Agaricus bisporus lectin, Datura phytolectin, jackfruit lectin, black bean lectin, bindweed lectin, gorse lectin, pinto bean lectin, snail lectin, Huaihuai lectin, Huaihuai lectin One or a combination of lectins, pea lectins, Polygonum multiflorum lectins, gore lectins, and vetch villous lectins. 2. a kind of test kit for detecting the change of sugar structure on the surface of human sperm according to claim 1, characterized in that, said Agaricus bisporus lectin, datura lectin, jackfruit lectin, black kidney bean lectin, Convolvulus agglutinin, gorse agglutinin, pinto bean agglutinin, snail agglutinin, locust agglutinin, locust agglutinin, pea agglutinin, Polygonum multiflorum agglutinin, gore agglutinin or villous vetch The source lectins are all fluorescently labeled. 3. A test kit for detecting changes in human sperm surface sugar structure according to claim 1, wherein said test kit also includes: propidium iodide, fluorescein isothiocyanate of 50-100ug/ml , TBST washing solution, PBST washing solution and PBS washing solution. 4. A test kit for detecting changes in the surface sugar structure of human sperm according to claim 4, wherein the concentration of the propidium iodide is 5-20ug/ml.