CN114885940A - Application of Fer-1 in preparation of semen cryoprotectant - Google Patents

Abstract

The invention belongs to the technical field of assisted reproduction, and particularly discloses application of Fer-1 in preparation of a semen cryoprotectant. The invention discovers that the addition of Fer-1 in the conventional semen cryoprotectant can reduce oxidative stress damage in the process and improve the function of the frozen and thawed sperms while protecting the survival rate of cells; based on the above, the invention provides a novel semen cryoprotectant with excellent performance, and the cryoprotectant is matched with a rapid freezing and rapid thawing method, so that the oxidative damage of the sperm can be reduced, the mitochondrial damage of the sperm can be reduced, and the forward motion capability and the survival rate of the thawed sperm after recovery can be effectively improved while the osmotic pressure inside and outside the frozen sperm is maintained.

Description

Application of Fer-1 in the preparation of semen cryoprotectant

technical field

The invention relates to the technical field of assisted reproduction, in particular to the application of Fer-1 in the preparation of a semen cryoprotectant.

Background technique

Semen cryopreservation is not only a conventional technical means for assisted reproduction in clinical practice, but also a daily task for human sperm banks. Significant, however, in the process of semen cryopreservation can cause irreversible damage to sperm quality reduction, the most important factor is reactive oxygen species. The results of the current study show that cryopreservation can lead to excessive production of ROS. In semen, there are many sources of ROS, which are generally believed to be mainly derived from three types of substances: leukocytes, immature sperm and abnormal sperm. The production of a large amount of reactive oxygen species breaks the oxidation-antioxidant system, causing oxidative stress damage to sperm, resulting in structural and functional damage to spermatogenesis. The sperm plasma membrane is rich in PUFAs, and during sperm maturation, the loss of a large amount of cytoplasm and mitochondria results in a limited enzyme system for scavenging ROS in the cytoplasm of mature sperm, making sperm vulnerable to ROS attack. Excessive ROS can also oxidize DNA, causing changes in the base pair and deoxyribonucleotide chain skeleton, affecting the genetic material from the father, so that embryos can undergo varying degrees of changes during development and pregnancy, such as the formation of single Ploidy or broken chromosomes, etc., eventually leading to abnormal cleavage or miscarriage. In the frozen-thawed sperm, the abnormal morphology rate also increased significantly. The study found that the change of the integrity of the sperm head and tail membrane after freezing and thawing was accompanied by the decrease of sperm acrosome enzyme activity, which eventually led to the reduction of sperm motility.

Sperm cryoprotectants are the most important substances in sperm freezing technology. At present, the most commonly used sperm cryoprotectants are composite freezing solutions containing glycerol and egg yolk, which are composed of glycerol, egg yolk, glucose, and sodium citrate. One of the most important cryoprotectants is glycerol. Glycerol can penetrate into cells, concentrate or bind intracellular water, and glycerol also has a diluting effect, which can reduce the concentration of salt in the solution and the osmotic pressure of the freezing liquid, thereby exerting its protective effect on sperm at low temperature, but glycerol is toxic to sperm. effect. Optimizing and improving semen cryoprotective solution is an effective way to solve this problem, among which the addition of antioxidants is the current research focus. In recent years, scholars have found that enzymatic antioxidants (such as superoxide dismutase, catalase, glutathione peroxidase, etc.) or non-enzymatic antioxidants (such as taurine) are added to semen cryoprotective fluid. Acid, L-carnitine, tea polyphenols, vitamins C, E, etc.) can inhibit sperm oxidative damage and improve sperm quality after recovery.

SUMMARY OF THE INVENTION

In view of the shortcomings of the above-mentioned prior art, the object of the present invention is to provide the application of Fer-1 in the preparation of semen cryoprotectant, to solve the problems such as oxidative stress damage in the process of freezing and thawing sperm, and to provide an effective A semen cryoprotectant that prolongs the survival effect of cryopreservation of sperm and maintains sperm function.

To achieve the above object and other related objects, the first aspect of the present invention provides the application of Fer-1 in semen cryoprotection and/or as a semen cryopreservation additive and/or in the preparation of a semen cryoprotectant.

Further, the concentration of Fer-1 in the semen cryoprotectant is 2-10 μmol/L.

Further, described semen cryoprotectant is egg yolk, glycerol composite cryoprotectant.

A second aspect of the present invention provides a semen cryoprotectant, comprising Fer-1.

Further, the semen cryoprotectant includes 2-10 μmol/L Fer-1.

Further, described semen cryoprotectant is egg yolk, glycerol composite cryoprotectant.

Further, the semen cryoprotectant also includes egg yolk, glycerol, glucose, sodium citrate or sodium citrate hydrate.

Further, the semen cryoprotectant includes components of the following concentrations: 15-25% egg yolk, 15-25% glycerol, 15±0.5g/L glucose, 15±0.5g/L sodium citrate or sodium citrate hydrate; Preferably, the semen cryoprotectant comprises the following concentrations of ingredients: 20% egg yolk, 20% glycerol, 15g/L glucose, 15g/L sodium citrate or sodium citrate hydrate.

Further, the pH value of the semen cryoprotectant is 6.8-7.2.

A third aspect of the present invention provides a method for preparing a semen cryoprotectant according to the second aspect, comprising the steps of:

Fer-1 was dissolved in dimethyl sulfoxide to prepare a Fer-1 solution, and the Fer-1 solution was added to a compound cryoprotectant of egg yolk and glycerol to obtain a semen cryoprotectant.

Due to the properties of Fer-1, it is best to dissolve it with DMSO. At the same time, DMSO is also a commonly used permeability protectant, which can reduce the freezing point of cells, reduce the formation of ice crystals, reduce the damage of free radicals to cells, and change the biological membrane's effect on electrolytes and drugs. , the permeability of toxicants and metabolites.

Further, the preparation method of the egg yolk and glycerol composite cryoprotectant comprises the following steps: adding glucose, sodium citrate or sodium citrate hydrate into water, and mixing; then adding glycerol, mixing and filtering; adding egg yolk, Suspension was made; followed by heating and stirring; and pH value was finally detected to be between 6.8-7.2.

It should be noted that the experimental water of the present invention adopts sterile water without ion interference, such as ultrapure water, deionized water, RO water, distilled water, double distilled water and the like.

Further, a 0.45 μm microporous filter or filter membrane is used for filtration.

Further, the yolk is extracted from fresh eggs.

Further, the heating is performed by means of a water bath, the heating temperature is 50-60° C., preferably 56° C., and the heating and stirring time is 20-40 min, preferably 30 min.

The fourth aspect of the present invention provides the application of the semen cryoprotectant according to the second aspect and/or the semen cryoprotectant prepared by the method according to the third aspect in semen cryoprotection.

A fifth aspect of the present invention provides a method for cryoprotection of semen, using a semen cryoprotectant containing Fer-1.

As mentioned above, the application of Fer-1 of the present invention in the preparation of semen cryoprotectant has the following beneficial effects:

The invention finds that adding Fer-1 to the conventional semen cryoprotectant can protect the cell survival rate, reduce the oxidative stress damage in the process, and improve the sperm function after freezing and thawing; based on this, the invention has prepared a A new type of semen cryoprotectant with excellent performance, combined with the methods of rapid freezing and rapid thawing, can reduce sperm oxidative damage and sperm mitochondrial damage while maintaining the internal and external osmotic pressure of frozen-thawed sperm, effectively improving the recovery of frozen-thawed sperm. To exercise capacity and survival.

Description of drawings

Figure 1 shows the comparison of ROS levels in each group of semen after freezing and thawing of semen in Example 1 of the present invention (A: Flow cytometry detection of ROS levels in each group; B: ROS quantitative analysis, *: p<0.05; ** :p<0.01;***:p<0.001).

Figure 2 shows the comparison of MDA levels in each group of semen after freezing and thawing of semen in Example 1 of the present invention (**: p<0.01; ***: p<0.001).

Figure 3 shows the comparison of the MMP levels of each group of semen after freezing and thawing of semen in Example 1 of the present invention (**: p<0.01; ***: p<0.001).

Detailed ways

The embodiments of the present invention are described below through specific specific examples, and those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and various details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention.

Ferrostatin-1 (Fer-1), as a specific ferroptosis inhibitor, is also a synthetic antioxidant, which can effectively inhibit the process of lipid peroxidation, reduce the level of lipid peroxidation in cells, and protect cells from immune Injured by ferroptosis. The present invention finds that adding Fer-1 to the semen cryoprotectant can protect the cell survival rate, reduce the oxidative stress damage in the process, and improve the sperm function after freezing and thawing, which may be a potential and efficient Semen cryoprotective additives.

Based on this, the present invention adds a certain concentration of Fer-1 to the conventional semen cryoprotectant, and prepares a new type of semen cryoprotectant with excellent performance. With the methods of rapid freezing and rapid thawing, it can maintain the inside and outside of the frozen and thawed sperm. At the same time of osmotic pressure, it can reduce sperm oxidative damage, reduce sperm mitochondrial damage, and effectively improve the forward motility and survival rate of frozen-thawed sperm after recovery.

The conventional semen cryoprotectant used in the present invention is egg yolk and glycerol composite cryoprotectant, which comprises the following concentrations: 15-25% egg yolk, 15-25% glycerol, 15±0.5g/L glucose, 15±0.5 g/L sodium citrate or sodium citrate hydrate. The conventional semen cryoprotectant used in the following examples includes the following concentrations of ingredients: 20% egg yolk, 20% glycerol, 15g/L glucose, 15g/L sodium citrate or sodium citrate hydrate, pH 6.8-7.2.

In the following examples, human semen is used for cryopreservation experiments, but the semen cryoprotectant of the present invention is also suitable for semen cryopreservation of other animals, such as pigs, horses and chickens.

The specific implementation process of the present invention is as follows:

Example 1

1. Preparation of egg yolk and glycerol composite cryoprotectant

(1) Weigh 1.5 g of glucose and 1.3 g of trisodium citrate dihydrate, add 60 mL of double distilled water, and mix well. Then 20 mL of glycerol was added, and after mixing, it was filtered through a 0.45 μm microporous filter. Then add 20 mL of egg yolk (obtained from fresh eggs) to make a suspension; place it in a water bath at 56°C for 30 minutes, stirring while heating; check the pH value to make it between 6.8-7.2. After aseptically distributed, store at -80°C and use within 3 months.

2. Add Fer-1, Fer-1 was purchased from MCE company, and the purity is more than 99%;

(1) Thaw the egg yolk and glycerol composite cryoprotectant in a 37°C water bath;

(2) Take out Fer-1 from the -80°C refrigerator, dissolve 26.235×10 ^-3 g in 10 ml of dimethyl sulfoxide, and prepare a solution with a Fer-1 content of 10 mmol/L;

(3) Take 0.1 mL of the above solution and add it to 99.9 mL of egg yolk and glycerol composite cryoprotectant, wherein the concentration of Fer-1 is 10 μmol/L, and it is stored at -80°C.

3. The method for freezing semen using the semen cryoprotectant containing Fer-1, comprising the following steps:

Select 20 semen samples from August 2021 to January 2022 to the outpatient department of the Reproductive Center of the First Affiliated Hospital of Chongqing Medical University. 35 years old with no recent history of exposure to teratogenic factors.

(1) Thaw the semen cryoprotectant containing Fer-1 in a 37°C water bath, and prepare semen cryoprotectants containing different concentrations of Fer-1;

Control group (F0): egg yolk and glycerol compound cryoprotectant group;

Experimental group (F1): 1 volume of semen cryoprotectant containing 10 μmol/L Fer-1 was dissolved in 5 parts of egg yolk and glycerol composite cryoprotectant to obtain a semen cryoprotectant group with 2 μmol/L Fer-1;

Experimental group (F2): 1 part of semen cryoprotectant containing 10 μmol/L Fer-1 was dissolved in 1 part of egg yolk and glycerol composite cryoprotectant to obtain a semen cryoprotectant group with 5 μmol/L Fer-1;

Experimental group (F3): semen cryoprotectant containing 10 μmol/L Fer-1.

(2) Take 1 volume of semen cryoprotectant containing different concentrations of Fer-1 and add it to 1 volume of semen, add dropwise, and slowly shake the sperm freezing tube, and finally tighten the cap;

(3) The freezing process is as follows: first equilibrate at 25°C for 5 minutes, then place it in a 4°C refrigerator for 30 minutes, and then suspend it at 15cm (-80°C ~ -100°C) on the surface of liquid nitrogen for 15 minutes, and then put it into liquid nitrogen. Freeze for 2 weeks.

(4) After freezing and resuscitation: Beijing Weili computer-aided semen analysis system was used to analyze the forward motility of sperm in each group after freezing and thawing; eosin staining was used to analyze the survival rate of semen in each group after freezing and thawing; DCFH-DA detection was used to analyze freezing and thawing ROS levels in semen of each group; MDA in each group was detected by Nanjing Jiancheng MDA kit; mitochondrial membrane potential was detected by JC-1 probe.

4. Experimental testing and results:

(1) Detection of forward movement ability and survival rate:

The Beijing Weili computer-assisted semen analysis system was used to analyze the forward motility of sperm in each group after freezing and thawing, and eosin staining was used to analyze the survival rate of semen in each group after freezing and thawing. The results are shown in Table 1. As shown in Table 1, the percentage of sperm forward movement before semen freezing (fresh group) was 52.11±8.42, and the sperm survival rate was 68.82±5.51. The survival group was low (p<0.001); after the addition of different concentrations of Fer-1 to the semen cryopreservation, compared with the F0 group, the sperm forward motility and sperm survival rate in F1 (2 μmol/L) had no significant changes, while in F2 ( 5μmol/L) and F3 (10μmol/L) groups were higher than the F0 group.

Table 1. Comparison of forward motility and survival rate of semen in each group before and after semen cryopreservation

Note: a: Compared with the fresh group, there is a statistical significance (p<0.001); #: Compared with the F0 group, there is a statistical significance (p<0.05).

(2) Sperm ROS detection:

In this experiment, the fluorescent probe DCFH-DA was used to measure the level of sperm ROS. DCFH-DA is a dye that can freely pass through the cell membrane. After entering the cell, it is hydrolyzed by intracellular esterase to generate DCFH, while DCFH cannot pass through the cell membrane, and DCFH is oxidized by intracellular ROS to generate fluorescent DCF. Therefore, the level of intracellular reactive oxygen species is indirectly reflected by flow cytometry. The specific operations are as follows:

Take 200 μL of frozen-thawed semen and centrifuge at 3000 rpm/min for 5 minutes to remove the supernatant. Add 1 mL of diluted DCFH-DA (10 μmol/L) solution to resuspend the sperm. The treated sperm cells were then placed at 37°C and incubated in the dark for 30 min, inverting and mixing every 5 min. After incubation, centrifuge at 3000 rpm/min for 5 min, remove the supernatant, add PBS to wash the sperm cell pellet, and repeat three times to remove excess and unbound probes. Finally, 300 μL of PBS was added to resuspend the cell pellet and sent to a flow cytometer to detect the level of sperm reactive oxygen species.

The results are shown in Figure 1. Compared with the group without Fer-1 (F0), adding different concentrations of Fer-1 can significantly reduce the level of ROS in frozen-thawed sperm (p < 0.05), and the decrease is concentration-dependent. .

(3) Sperm MDA detection:

Take 400 μL of frozen and resuscitated semen, centrifuge at 3000 rpm/min for 5 minutes to remove the supernatant, add 200 μL of RIPA lysate to the cells according to the MDA kit procedure, and sonicate the cells on ice for 5 s each time, repeat 6 times at 4°C, After centrifugation at 3000rpm/min for 10min, the supernatant was taken for MDA detection. The principle is that malondialdehyde (MDA) in lipid peroxide degradation products can condense with thiobarbituric acid (TBA) to form a red product with a maximum absorption peak at 532 nm.

The results are shown in Figure 2. Compared with the group without Fer-1 (F0), when the concentration was 2 μmol/L, there was no significant change in the level of MDA in frozen-thawed sperm, while when the concentration was 5 μmol/L and 10 μmol/L MDA levels in frozen-thawed sperm decreased significantly.

(4) Detection of sperm mitochondrial membrane potential:

JC-1 is an ideal fluorescent probe for detecting mitochondrial membrane potential ΔΨm. When the mitochondrial membrane potential is high, JC-1 exists in the form of polymer in the mitochondrial matrix, and produces red fluorescence concurrently; when the mitochondrial membrane potential is low, JC-1 exists in the form of monomer and emits green fluorescence. Therefore, the change of mitochondrial membrane potential can be reflected by the change of fluorescence color. The specific operations are as follows:

Take 200μL of frozen and resuscitated semen, centrifuge at 3000rpm/min for 5 minutes to remove the supernatant, add 500μL of JC-1 buffer, mix well, incubate in a 37℃, 5% _CO2 incubator for 20min, centrifuge at 600g at 4℃ 3 min, discard the supernatant; add 1000 μL of JC-1 staining buffer, gently blow off and resuspend the cells, centrifuge at 600 g for 2-5 min at 4°C, repeat 3 times to wash off excess unbound probes; finally add JC-1 staining The cells were resuspended in 400 μL of buffer and sent to a flow cytometer to detect changes in mitochondrial membrane potential.

The results are shown in Figure 3, adding Fer-1 can improve the mitochondrial membrane potential level in frozen-thawed sperm. Compared with the group without Fer-1 (F0), when the concentration was 2 μmol/L, there was no significant change in the level of MMP in frozen-thawed spermatozoa, while when the concentration was increased to 5 μmol/L to 10 μmol/L, the level of MMP in frozen-thawed sperm was not significantly changed. The level of MMP in sperm was significantly improved (p<0.05), and changed in a concentration-dependent manner to a certain extent. In summary, adding a certain concentration of Fer-1 can significantly improve the sperm freezing effect, and the sperm function parameters after recovery: forward motility and survival rate are significantly higher than those in the control group (without adding Fer-1), and ROS , MDA and mitochondrial membrane potential were also significantly higher than the control group, suggesting a good safety for sperm quality protection.

The above-mentioned embodiments merely illustrate the principles and effects of the present invention, but are not intended to limit the present invention. Anyone skilled in the art can modify or change the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or changes made by those with ordinary knowledge in the technical field without departing from the spirit and technical idea disclosed in the present invention should still be covered by the claims of the present invention.

Claims (10)

1. The application of Fer-1 in semen cryoprotection and/or as an additive for semen cryopreservation and/or in the preparation of semen cryoprotectant. 2. application according to claim 1 is characterized in that: the concentration of Fer-1 in semen cryoprotectant is 2～10 μmol/L; And/or, described semen cryoprotectant is egg yolk, glycerol composite cryoprotectant. 3. a semen cryoprotectant is characterized in that: comprise Fer-1. 4. semen cryoprotectant according to claim 3, is characterized in that: described semen cryoprotectant comprises 2～10 μmol/L Fer-1; And/or, described semen cryoprotectant is egg yolk, glycerol composite cryoprotectant. 5. semen cryoprotectant according to claim 4, is characterized in that: described semen cryoprotectant also comprises egg yolk, glycerol, glucose, sodium citrate or sodium citrate hydrate; And/or, the pH value of the semen cryoprotectant is 6.8-7.2. 6. semen cryoprotectant according to claim 5, is characterized in that: described semen cryoprotectant comprises the composition of following concentration: 15～25% egg yolk, 15～25% glycerol, 15±0.5g/L glucose, 15±0.5g/L sodium citrate or sodium citrate hydrate. 7. the preparation method of the semen cryoprotectant according to any one of claims 3～6, is characterized in that, comprises the steps: Fer-1 is dissolved in dimethyl sulfoxide, makes Fer-1 solution, Fer-1 solution was added to egg yolk and glycerol composite cryoprotectant to prepare semen cryoprotectant. 8. preparation method according to claim 1 is characterized in that: the preparation method of described egg yolk, glycerol composite cryoprotectant comprises the steps: adding glucose, sodium citrate or sodium citrate hydrate into water, mixing Then add glycerin, mix well and filter; add egg yolk again to make suspension; then heat and stir; finally, check the pH value to make it between 6.8-7.2. 9. The application of the semen cryoprotectant according to any one of claims 3 to 6 and/or the semen cryoprotectant prepared by the method according to any one of claims 7 to 8 in semen cryoprotection. 10. A method for cryoprotection of semen, characterized in that: adopt the cryoprotectant of semen containing Fer-1.

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