CN113041261B - Method for improving oligospermia sperm function by using exosomes - Google Patents

Method for improving oligospermia sperm function by using exosomes Download PDF

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CN113041261B
CN113041261B CN202110280768.9A CN202110280768A CN113041261B CN 113041261 B CN113041261 B CN 113041261B CN 202110280768 A CN202110280768 A CN 202110280768A CN 113041261 B CN113041261 B CN 113041261B
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exosomes
sperm
semen
asthenospermia
asthenozoospermia
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张小宁
曾旭辉
周阳
康爱文
宋建
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Nantong University
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Abstract

The invention discloses a method for improving the sperm function of asthenospermia by using exosomes, belonging to the technical field of medicine. The method comprises the following steps: separating normal human seminal plasma exosomes, screening and preprocessing asthenospermia sperms, and intervening the exosomes on the asthenospermia spermia. The invention improves the sperm motility and other functions by utilizing the seminal plasma exosome from the human body to interfere with sperm in vitro, so as to interfere with and treat oligospermia by simple non-invasive means such as artificial fertilization and the like, and improve the fertility outcome of patients.

Description

Method for improving oligospermia sperm function by using exosomes
Technical Field
The invention belongs to the technical field of medicine, and particularly relates to a method for improving the functions of asthenospermia sperms by using exosomes.
Background
Male reproductive health has become a focus of close attention in today's human society. Based on WHO statistics, the sterility rate of the couples of the worldwide childbearing age is 10-15%, wherein the sterility caused by factors of men accounts for about half. Sperm quality problems are a major cause of male infertility. Epidemic investigation has found that asthenozoospermia has become a major factor in causing male infertility and has an increasing trend year by year. Asthenozoospermia refers to a type of infertility caused by a percentage of sperm forward movement in semen below 32% (WHO fifth edition). Meanwhile, weak sperm not only has reduced forward movement ability, but also is accompanied by abnormal functions of other sperm, such as capacitation, super-activation, acrosome reaction and the like, and the comprehensive factors lead to male sterility. However, the pathogenesis is not clear due to the complex etiology of the asthenozoospermia, and research models and means are relatively deficient, so that effective treatment means are not available at present. Although offspring can be produced by abnormal conception means (e.g. egg cytoplasmatic single sperm microinjection technique, genetic diagnosis/screening prior to embryo implantation), there is still a major risk to the health of the test tube infant. Therefore, the conventional treatment of asthenozoospermia patients makes the birth of the asthenozoospermia patients by natural means or by less invasive artificial fertilization means of great significance for human health.
Semen is composed of cells (sperm cells) and non-cellular components (seminal plasma). The seminal plasma is rich in various substances required by sperm vital activities such as lipid, sugar, growth factors, transcription factors, proteins and the like, can provide stable and comfortable environment for sperm survival and movement, can also be used as a carrier for transporting sperm to the fertilization part of female genital tract, and has several important functions: providing nutrition for sperm transport; the sperm can be protected and prevented from being infected by harmful substances; so that the sperm can overcome the chemical and immunological environment of the hostile vagina. Studies have shown that seminal plasma also contains a large number of exosomes which are excreted with the prostatic secretions during ejaculation and are involved in the maturation and functional regulation of sperm cells, and therefore exosomes have great potential in the treatment of asthenozoospermia.
At present, development of a method for treating oligospermia with small toxic and side effects and convenient clinical application is urgently needed to improve sperm function, so that a patient can naturally give birth after intervention or can give birth by using an artificial fertilization method with small wound.
Disclosure of Invention
Aiming at the technical problems that the conventional treatment means of the current asthenozoospermia are limited in clinic and most of the asthenospermia with unknown etiology do not have very effective treatment means, the invention provides a method for improving the sperm function of the asthenospermia by using exosomes.
In order to achieve the above object, the present invention adopts the following technical means:
a method for improving oligospermia sperm function using exosomes, comprising the steps of:
step 1, separation of normal human seminal plasma exosomes: collecting normal fertility semen, centrifuging 12000 g for 10 min, removing cells and impurities, sequentially filtering the seminal plasma with microporous filter membranes of 0.45 μm and 0.22 μm, removing cell debris and macromolecular aggregate impurities, adding 2.5 mL supernatant into 300 μl of sucrose pad solution containing 20 mM Tris, 30 wt% sucrose and deuterium oxide, ultracentrifugating at 100000 and 4 ℃ for 90 min, collecting supernatant, ultracentrifugating at 100000 g and 4 ℃ in sucrose pad solution containing 20 mM Tris, 25 wt% sucrose and deuterium oxide, mixing exosomes obtained by the two centrifugation, washing with HS solution once, dissolving the obtained exosomes with HS solution, adjusting 10 times of physiological concentration, and preserving at-80 ℃ for standby;
step 2, screening and pretreatment of asthenozoospermia spermia: liquefying a patient semen sample collected on the same day in a constant-temperature water bath at 37 ℃ for 60 min, blowing and uniformly mixing, taking 10 mu L of liquefied semen, analyzing various parameters of human semen by using a computer-aided semen analyzer, screening samples meeting the requirements on diagnostic parameters of asthenozoospermia, and staining samples for eliminating obvious abnormal sperm morphology;
step 3, intervention of exosomes on asthenospermia sperms: washing asthenospermia with HS twice, 1000 g min each time, 5 min each time, adding 90% of HS solution or HTF solution, re-suspending sperm, adding 10% of exosomes, and incubating at 37deg.C for 10 min.
Further, the sample satisfying the diagnostic parameter requirements for asthenozoospermia in step 2 is a sample with sperm motility < 32%.
The invention improves the sperm motility and other functions by utilizing the seminal plasma exosome from the human body to interfere with sperm in vitro, so as to interfere with and treat oligospermia by simple non-invasive means such as artificial fertilization and the like, and improve the fertility outcome of patients.
Drawings
Figure 1 shows the result of the enhancement of the forward motor capacity of the asthenospermia sperm by the exosomes. Wherein: HS is solvent control group, N-exosome is normal exosome, A-exosome is exosome from oligospermia, HSA is human serum albumin positive control.
FIG. 2 shows the effect of exosomes on sperm hyperactivation ability. Wherein: HTF is the solvent control group and exosomes are normal exosomes.
FIG. 3 shows the effect of exosomes on sperm climbing capacity. Wherein: HTF is solvent control group, N-exosome is normal exosome, A-exosome is exosome from oligospermia, and P4 is progesterone.
Detailed Description
The invention will now be described in further detail with reference to the drawings and specific examples, which should not be construed as limiting the invention. Modifications and substitutions to methods, procedures, or conditions of the present invention without departing from the spirit and nature of the invention are intended to be within the scope of the present invention. The experimental procedures and reagents not shown in the formulation of the examples were all in accordance with the conventional conditions in the art.
The formula of the HS solution adopted in the invention is as follows: 135 mM NaCl,5 mM KCl,1 mM MgSO 4 ,2 mM CaCl 2 20 mM HEPES,5 mM sucrose, 10 mM lactic acid, 1 mM sodium pyruvate, pH was adjusted to 7.4 using NaOH.
The HTF solution formulation was: 93.8 mM NaCl,4.69 mM KCl,0.2 mM MgSO 4 ,0.37 mM KH 2 PO 4 ,2.04 mM CaCl 2 ,21.4 mM lactic acid,2.78 mM glucose,21 mM HEPES,4 mM NaHCO 3 The pH was adjusted to 7.4 and 330 mM Na-Pyruvate (1000:1), HSA (25:1), 21 mM NaHCO were added prior to use 3
Example 1
Step 1, separation of normal human seminal plasma exosomes
The specific separation method is as follows: collecting semen of normal fertile volunteers, centrifuging 12000 g for 10 min, and removing cells such as sperms and other impurities; sequentially filtering the refined pulp with microporous filter membranes of 0.45 μm and 0.22 μm, and removing cell fragments, macromolecular aggregate impurities, etc.; 2.5. 2.5 mL supernatant was added to 100000 g of a 300. Mu.L sucrose cushion solution containing 20 mM Tris/30% sucrose/deuterium oxide and ultracentrifuged at 4℃for 90 min; the supernatant was collected and then centrifuged at 100000 g in sucrose pad solution containing 20 mM Tris/25% sucrose/deuterium oxide at 4℃for 5 h; mixing exosomes in 30% and 25% sucrose pads, washing with HS solution once (100000 g,4 ℃ C., ultracentrifugation for 90 min); dissolving the obtained exosomes with HS; finally, BCA protein is quantified, and the concentration is adjusted to 10 times of physiological concentration and then stored at-80 ℃ for standby.
Step 2, screening and pretreatment of asthenospermia sperm
The patient semen samples collected on the same day were placed in a constant temperature water bath at 37℃for liquefaction for 60 min. Blowing and mixing evenly, taking 10 mu L of liquefied semen, analyzing various parameters of human sperms by using a computer-aided semen analyzer (CASA), screening samples meeting the requirements on the diagnostic parameters (sperm motility < 32%) of asthenozoospermia in the standard parameters of 5 th edition (WHO 5) of semen in the handbook of human semen inspection and treatment laboratory of world health organization, and staining samples for eliminating obvious abnormal sperm morphology.
Step 3, intervention of exosome on asthenospermia sperm
Washing asthenospermia sperm twice with HS (high speed) for 1000 g and 5 min each time; sperm were re-selected with HS at 90% of the initial semen (e.g., semen of 2 mL was re-suspended with 1.8 mL HS or HTF solution) and 10% exosomes were added and incubated for 10 min at 37 ℃.
And then, analyzing sperm motility parameters and super-activation motility levels treated by exosomes by using CASA, so as to evaluate the improvement level of exosomes on sperm functions. When sperm motility parameters VCL > =150 um, lin < =50%, ALH > =7 μm, we consider that super-activation occurs, the percentage of super-activation being the number of sperm/total number of sperm that have super-activated.
The ability of sperm to penetrate viscous media was tested using a climbing experiment, the specific method was as follows:
the mucolytic ability of sperm mimics the ability of sperm to traverse the female reproductive tract and uses 1% methyl cellulose to mimic the environment of the female reproductive tract, since sperm can only combine with an egg to complete fertilization after traversing the female reproductive tract. After exosomes and sperm were treated in HTF solution for 30 min, aspirated in capillary glass tubes, capillaries containing 20 μl of 1% methylcellulose were placed in the well mixed sperm samples, and after 1h the number of sperm 1, 2, 3cm in the capillaries was recorded under a microscope.
As can be seen from fig. 1, normal exosomes at physiological concentrations can significantly increase the forward motor capacity of sperm, about 60% above the group without exosomes, but exosomes derived from severe asthenozoospermia do not. As can be seen from fig. 2, exosomes can significantly increase the level of hyperactivated motility of sperm (about 60% increase). In addition, another parameter that reflects sperm fertility, the ability to cross mucus was also significantly improved by the addition of exosomes (fig. 3, about 70% increase), with an effect similar to that produced by 1 μm progesterone. These data indicate that the addition of normal exosomes has the effect of significantly improving sperm function, and is expected to be used for treating asthenozoospermia in future.

Claims (1)

1. A method for improving the functions of asthenospermia sperms by utilizing exosomes for non-diagnostic treatment purposes, which is characterized by comprising the following steps of: the method comprises the following steps:
step 1, separation of normal human seminal plasma exosomes: collecting normal fertility semen, centrifuging 12000 g for 10 min, removing cells and impurities, sequentially filtering the seminal plasma with microporous filter membranes of 0.45 μm and 0.22 μm, removing cell debris and macromolecular aggregate impurities, adding 2.5 mL supernatant into 300 μl of sucrose pad solution containing 20 mM Tris, 30 wt% sucrose and deuterium oxide, ultracentrifugating at 100000 and 4 ℃ for 90 min, collecting supernatant, ultracentrifugating at 100000 g and 4 ℃ in sucrose pad solution containing 20 mM Tris, 25 wt% sucrose and deuterium oxide, mixing exosomes obtained by the two centrifugation, washing with HS solution once, dissolving the obtained exosomes with HS solution, adjusting 10 times of physiological concentration, and preserving at-80 ℃ for standby;
step 2, screening and pretreatment of asthenozoospermia spermia: liquefying a patient semen sample collected on the same day in a constant-temperature water bath at 37 ℃ for 60 min, blowing and uniformly mixing, taking 10 mu L of liquefied semen, analyzing various parameters of human semen by using a computer-aided semen analyzer, screening samples meeting the requirements on diagnostic parameters of asthenozoospermia, and staining samples for eliminating obvious abnormal sperm morphology;
the sample meeting the requirements of the diagnostic parameters for asthenozoospermia is a sample with sperm forward movement sperm motility < 32%;
step 3, intervention of exosomes on asthenospermia sperms: washing asthenospermia with HS twice, 1000 g min each time, 5 min each time, adding 90% of HS solution or HTF solution, re-suspending sperm, adding 10% of exosomes, and incubating at 37deg.C for 10 min.
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