CN114885940A

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

Info

Publication number: CN114885940A
Application number: CN202210620964.0A
Authority: CN
Inventors: 孟江萍; 郝小玲; 刘西茹; 辛贵邦; 陈莹; 王虹; 黄润; 叶柳
Original assignee: First Affiliated Hospital of Chongqing Medical University
Current assignee: First Affiliated Hospital of Chongqing Medical University
Priority date: 2022-06-01
Filing date: 2022-06-01
Publication date: 2022-08-12
Anticipated expiration: 2042-06-01
Also published as: CN114885940B

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 preparation of semen cryoprotectant

Technical Field

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

Background

Semen freezing is not only a conventional technical means for assisted reproduction in clinic, but also a daily task of a human sperm bank, and has great significance for vast male patients with infertility, such as patients with azoospermia or oligospermia and cancer patients with fertility requirements, however, irreversible damage of sperm quality reduction can be caused in the semen freezing process, wherein the most important factor is active oxygen. Current research results show that cryopreservation can lead to excessive ROS production. In semen, ROS are derived from many sources, and are considered to be mainly derived from three types of substances such as leukocytes, immature sperm, and abnormal sperm. The generation of a large amount of active oxygen breaks an oxidation-antioxidation system, causes the oxidative stress damage of sperms, and causes the structural and functional damage of the sperms. The plasma membrane of sperm is rich in PUFAs, and during the maturation of sperm, loss of large amounts of cytoplasm and mitochondria results in a limited enzymatic system for scavenging ROS in the mature sperm cytoplasm, rendering the sperm vulnerable to ROS. Excessive ROS can also oxidize DNA to cause base pairing, the deoxyribonucleotide chain skeleton is changed, genetic materials from the father are affected, and embryos can be changed in different degrees in the development and pregnancy processes, such as haploid or broken chromosome formation, and finally abnormal cleavage or abortion can be caused. The abnormal morphological rate of the frozen and thawed sperms is also obviously increased, and researches show that the sperm acrosome enzyme activity is reduced while the sperm head and tail membrane integrity is changed after the freezing and thawing, and finally the sperm motility is reduced.

Sperm cryoprotectants are the most important substances in sperm freezing technology. At present, the most commonly used sperm cryoprotectant is a compound refrigerating fluid containing glycerol and yolk and consists of glycerol, yolk, glucose, sodium citrate and the like. The most important of these is glycerol. Glycerol can permeate into cells, concentrate or combine with intracellular water, and has dilution effect to reduce salt concentration in solution and osmotic pressure of refrigerating fluid, thereby protecting sperm at low temperature, but glycerol has toxic effect on sperm. Optimizing and improving the semen freezing protective solution is an effective way to solve the problem, wherein the addition of antioxidant is a hot spot of current research. In recent years, researchers have found that adding components such as enzymatic antioxidants (e.g., superoxide dismutase, catalase, glutathione peroxidase, etc.) or non-enzymatic antioxidants (e.g., taurine, L-carnitine, tea polyphenols, vitamin C, E, etc.) to semen cryoprotective solutions can inhibit oxidative damage of sperm and improve the quality of recovered sperm.

Disclosure of Invention

In view of the defects of the prior art, the invention aims to provide the application of Fer-1 in the preparation of a semen cryoprotectant, solve the problems of oxidative stress damage and the like in the process of freezing and thawing sperm and provide the semen cryoprotectant which can effectively prolong the survival efficiency of the sperm during ultralow-temperature cryopreservation and maintain the sperm function.

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

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

Further, the semen cryoprotectant is a yolk and glycerol compound cryoprotectant.

The invention provides a semen cryoprotectant in a second aspect, which comprises Fer-1.

Further, the semen cryoprotectant comprises 2-10 mu mol/L Fer-1.

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

Further, the semen cryoprotectant comprises the following components in concentration: 15-25% of yolk, 15-25% of glycerol, 15 +/-0.5 g/L of glucose, 15 +/-0.5 g/L of 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.

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

dissolving Fer-1 in dimethyl sulfoxide to obtain Fer-1 solution, and adding the Fer-1 solution into the yolk-glycerol composite cryoprotectant to obtain semen cryoprotectant.

Because of the property of Fer-1, DMSO is preferably used for dissolving, and DMSO is also a commonly used permeability protective agent, which can reduce the freezing point of cells, reduce the formation of ice crystals, relieve the damage of free radicals to cells, and change the permeability of biological membranes to electrolytes, drugs, poisons and metabolites.

Further, the preparation method of the yolk-glycerol compound cryoprotectant comprises the following steps: adding glucose, sodium citrate or sodium citrate hydrate into water, and mixing; then adding glycerol, mixing uniformly and filtering; adding yolk to prepare a suspension; then heating and stirring are carried out; finally, the pH value is detected to be between 6.8 and 7.2.

It should be noted that the experimental water of the present invention is sterile and ion interference-free water, such as ultrapure water, deionized water, RO water, distilled water, double distilled water, etc.

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

Further, the yolk is extracted from fresh eggs.

Further, heating in a water bath mode, wherein the heating temperature is 50-60 ℃, preferably 56 ℃, and the heating and stirring time is 20-40 min, preferably 30 min.

A fourth aspect of the invention provides the use of a semen cryoprotectant according to the second aspect and/or a semen cryoprotectant prepared according to the method of the third aspect for semen cryoprotection.

The fifth aspect of the invention provides a semen freezing protective method, which adopts a semen freezing protective agent containing Fer-1.

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

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 preparation method, the novel semen cryoprotectant with excellent performance is prepared, and the rapid freezing and rapid thawing method is matched, so that the oxidative damage of the sperms can be reduced, the mitochondrial damage of the sperms can be reduced, and the forward motion ability and the survival rate of the thawed sperms after recovery can be effectively improved while the osmotic pressure inside and outside the frozen and thawed sperms is maintained.

Drawings

FIG. 1 shows the comparison of ROS levels after freezing and thawing of the semen of each group after freezing and preserving the semen according to example 1 of the present invention (A: flow-measuring ROS levels of each group; B: ROS quantification; p: 0.05; p: 0.01; p: 0.001).

FIG. 2 shows the comparison of MDA levels after freezing and thawing of various groups of semen after cryopreservation in example 1 of the present invention (.: p < 0.01;: p < 0.001).

FIG. 3 shows the comparison of MMP levels after freezing and thawing of various groups of semen after cryopreservation in example 1 of the present invention (.: p < 0.01;: p < 0.001).

Detailed Description

The embodiments of the present invention are described below with reference to specific embodiments, and other advantages and effects of the present invention will be easily understood by those skilled in the art from the disclosure of the present specification. The invention is capable of other and different embodiments and of being practiced or of being carried out in various ways, and its several details are capable of modification in various respects, all without departing from the spirit and scope of the present invention.

Ferrostatin-1(Fer-1) is used as a specific iron death inhibitor and is also an artificially synthesized antioxidant, can effectively inhibit lipid peroxidation process, and plays a role in reducing lipid peroxidation level in cells and protecting the cells from iron death damage. The invention discovers that the addition of Fer-1 into the semen freezing protective agent can reduce oxidative stress damage in the process and improve the function of sperms after freeze thawing while protecting the survival rate of cells, and the invention can be a potential and high-efficiency semen freezing protective additive.

Based on the method, the Fer-1 with a certain concentration is added into the conventional semen cryoprotectant to prepare the novel semen cryoprotectant with excellent performance, and the novel semen cryoprotectant is matched with a rapid freezing and rapid thawing method, so that the oxidative damage of the sperms can be reduced, the damage of the sperms mitochondria can be reduced while the internal and external osmotic pressure of the freeze-thawed sperms is maintained, and the forward motion ability and the survival rate of the freeze-thawed sperms after recovery can be effectively improved.

The conventional semen cryoprotectant adopted in the invention is a yolk-glycerol composite cryoprotectant, which comprises the following components in concentration: 15-25% of yolk, 15-25% of glycerin, 15 +/-0.5 g/L of glucose, 15 +/-0.5 g/L of sodium citrate or sodium citrate hydrate. Conventional semen cryoprotectants employed in the following examples included the following components in the following concentrations: 20% of yolk, 20% of glycerol, 15g/L of glucose, 15g/L of sodium citrate or sodium citrate hydrate, and the pH value is 6.8-7.2.

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

The specific implementation process of the invention is as follows:

example 1

Firstly, preparing yolk-glycerol composite cryoprotectant

(1) 1.5g of glucose and 1.3g of trisodium citrate dihydrate are weighed, 60mL of double distilled water is added, and the mixture is uniformly mixed. Then, 20mL of glycerin was added, and the mixture was mixed well and filtered through a 0.45-. mu.m microporous filter. Adding 20mL of yolk (extracted from fresh egg) to obtain suspension; heating in 56 deg.C water bath for 30min while stirring; the pH was measured to be between 6.8 and 7.2. Sterilized and subpackaged, and stored at-80 deg.C for 3 months.

Adding Fer-1, wherein the Fer-1 is purchased from MCE company, and the purity is more than 99%;

(1) unfreezing egg yolk and glycerol compound cryoprotectant in water bath at 37 ℃;

(2) fer-1 is taken out from a refrigerator at the temperature of-80 ℃, and 26.235 x 10 is taken ^-3 g is dissolved in 10ml of dimethyl sulfoxide to prepare a solution with the Fer-1 content of 10 mmol/L;

(3) adding 0.1mL of the above solution into 99.9mL of yolk-glycerol composite cryoprotectant, wherein the concentration of Fer-1 is 10 μmol/L, and storing at-80 deg.C.

Thirdly, the semen freezing method adopting the sperm freezing protective agent containing Fer-1 comprises the following steps:

20 semen samples were selected from 8 months 2021 to 2022 months 1 to the clinic of the reproductive center of the first hospital affiliated to Chongqing medical university, with semen parameters meeting the normal semen standards as specified by the world health organization (WHO, 2010), aged 28 to 35 years, and with no recent history of teratogenic factors.

(1) Unfreezing the sperm cryoprotectant containing the Fer-1 in water bath at 37 ℃ to prepare the sperm cryoprotectant containing the Fer-1 with different concentrations;

control group (F0): yolk and glycerol compound cryoprotectant group;

experimental group (F1): 1 part of semen cryoprotectant with 10 mu mol/L of Fer-1 in volume is dissolved in 5 parts of yolk-glycerol composite cryoprotectant to obtain a semen cryoprotectant group with 2 mu mol/L of Fer-1;

experimental group (F2): 1 part of semen cryoprotectant with volume of 10 mu mol/L Fer-1 is dissolved in 1 part of yolk-glycerol composite cryoprotectant to obtain a semen cryoprotectant group with volume of 5 mu mol/L Fer-1;

experimental group (F3): semen cryoprotectant containing 10 mu mol/L Fer-1.

(2) Adding 1 part of semen cryoprotectant containing Fer-1 with different concentrations into 1 part of semen, dropwise adding, slowly shaking the sperm freezing tube, and finally spin down the cap;

(3) the freezing process is as follows: the mixture is firstly balanced for 5 minutes at 25 ℃, then is placed in a refrigerator at 4 ℃ for 30 minutes, is suspended at the position of 15cm on the surface of liquid nitrogen (-80 ℃ to-100 ℃) for 15 minutes, and then is put into the liquid nitrogen. And (5) freezing and storing for 2 weeks.

(4) After freezing recovery: analyzing the forward motility of each group of sperms after freeze thawing by adopting a Beijing Weili computer-assisted semen analysis system; analyzing the survival rate of the semen of each group after freeze thawing by adopting an eosin staining method; detecting and analyzing the ROS level of each group of semen after freezing and thawing by using DCFH-DA; detecting each group of MDA by using the Nanjing constructed MDA kit; mitochondrial membrane potential was detected using JC-1 probe.

Fourthly, experimental detection and results:

(1) forward locomotor ability and survival detection:

the forward motility of each group of sperms after freeze thawing is analyzed by adopting a Beijing Weili computer-assisted sperm analysis system, and the survival rate of each group of sperms after freeze thawing is analyzed by adopting an eosin staining method, and the results are shown in Table 1. As shown in Table 1, the sperm forward movement percentage before semen freezing (fresh group) is 52.11 + -8.42, the sperm survival rate is 68.82 + -5.51, and the sperm forward movement percentage and the sperm survival rate of each group after freezing recovery are lower than those of the fresh unfrozen group (p is less than 0.001); after the semen added with different concentrations of Fer-1 is frozen and thawed, the sperm forward movement and the sperm survival rate in the F1(2 mu mol/L) are not changed significantly compared with the F0 group, while the sperm forward movement and the sperm survival rate in the F2(5 mu mol/L) and the sperm survival rate in the F3(10 mu mol/L) are higher than those in the F0 group.

TABLE 1 comparison of forward motility and survival of various groups of semen before and after cryopreservation

Note: a: compared with a fresh group, the gene has statistical significance (p is less than 0.001); #: compared with the F0 group, the gene has statistical significance (p is less than 0.05).

(2) And (3) detecting the ROS of the sperm:

the fluorescence probe DCFH-DA is used for measuring the ROS level of the sperm in the experiment. DCFH-DA is a dye that can freely pass through cell membranes, and is hydrolyzed by intracellular esterase after entering cells to generate DCFH, while DCFH can not pass through cell membranes, and DCFH is oxidized by intracellular ROS to generate DCFH with fluorescence. Thus indirectly reflecting the intracellular reactive oxygen species levels by flow cytometry. The specific operation is as follows:

200 mu L of frozen and recovered semen is taken and centrifuged at 3000rpm/min for 5 minutes to remove the supernatant. The sperm were resuspended by adding 1mL of diluted DCFH-DA (10. mu. mol/L). Then, the treated sperm cells are placed at 37 ℃, incubated for 30min in the dark, and inverted and mixed uniformly every 5 min. After incubation was complete, centrifugation was carried out at 3000rpm/min for 5min, the supernatant removed, and the sperm cell pellet washed with PBS three times to remove excess, unbound probe. Finally, 300 mu LPBS is added to resuspend the cell pellet and send to a flow cytometer to detect the active oxygen level of the sperm.

As shown in FIG. 1, the ROS level in freeze-thawed sperm was significantly reduced (p <0.05) and concentration-dependently reduced after addition of different concentrations of Fer-1 compared to the group without Fer-1 (F0).

(3) And (3) detecting sperm MDA:

taking 400 mu L of frozen and recovered semen, centrifuging at 3000rpm/min for 5 minutes to remove supernatant, adding 200 mu L of RIPA lysate into cells according to the steps of an MDA kit, ultrasonically breaking the cells on ice for 5s each time, repeating for 6 times, centrifuging at 4 ℃, 3000rpm/min for 10 minutes, and taking supernatant for MDA detection. The principle is that Malondialdehyde (MDA) in the lipid peroxide degradation product can be condensed with thiobarbituric acid (TBA) to form a red product, and the maximum absorption peak is at 532 nm.

As shown in FIG. 2, there was no significant change in MDA levels in freeze-thawed sperm when the concentration was 2. mu. mol/L, and a significant decrease in MDA levels in freeze-thawed sperm when the concentrations were 5. mu. mol/L and 10. mu. mol/L, as compared to the group without the addition of Fer-1 (F0).

(4) And (3) detecting the potential of the sperm mitochondrial membrane:

JC-1 is an ideal fluorescent probe for detecting mitochondrial membrane potential delta Ψ m. When the mitochondrial membrane potential is higher, JC-1 exists in a mitochondrial matrix in a polymer form and generates red fluorescence; when the mitochondrial membrane potential is low, JC-1 exists in a monomer form and emits green fluorescence. Therefore, the change of the mitochondrial membrane potential of the cell can be reflected by the shift of the fluorescence color. The specific operation is as follows:

collecting 200 μ L of frozen and recovered semen, centrifuging at 3000rpm/min for 5min to remove supernatant, adding 500 μ L JC-1 buffer solution, mixing, and adding 5% CO at 37 deg.C ₂ Incubating in an incubator for 20min, centrifuging at 600g and 4 ℃ for 3min, and removing supernatant; adding J1000 mul of C-1 staining buffer, gently blowing and resuspending the cells, centrifuging at 600g and 4 ℃ for 2-5min, repeating for 3 times to wash off the excess unbound probes; finally, 400 mu L of JC-1 staining buffer solution is added for resuspending the cells, and the cells are sent to a flow cytometer for detecting mitochondrial membrane potential change.

The results are shown in FIG. 3, where addition of Fer-1 improved mitochondrial membrane potential levels in freeze-thawed sperm. Compared to the group without added Fer-1(F0), there was no significant change in MMP levels in the freeze-thawed sperm when the concentration was 2 μmol/L, whereas there was a significant improvement (p <0.05) and some degree of concentration-dependent change in MMP levels in the freeze-thawed sperm when the concentration was increased to 5 μmol/L to 10 μmol/L. In conclusion, the addition of a certain concentration of Fer-1 can obviously improve the sperm freezing effect, and the recovered sperm functional parameters are as follows: the forward movement ability and the survival rate are obviously higher than those of a control group (Fer-1 is not added), and the potentials of ROS, MDA and mitochondrial membranes are also obviously higher than those of the control group, so that the method has good safety on sperm quality protection.

The foregoing embodiments are merely illustrative of the principles and utilities of the present invention and are not intended to limit the invention. Any person skilled in the art can modify or change the above-mentioned embodiments without departing from the spirit and scope of the present invention. Accordingly, it is intended that all equivalent modifications or changes which can be made by those skilled in the art without departing from the spirit and technical spirit of the present invention be covered by the claims of the present invention.

Claims

The use of Fer-1 in the protection of semen freezing and/or as additive for the protection of semen freezing and/or in the preparation of semen freezing protectants.
2. Use according to claim 1, characterized in that: the concentration of the Fer-1 in the semen cryoprotectant is 2-10 mu mol/L;

and/or the semen cryoprotectant is a yolk and glycerol compound cryoprotectant.
3. A semen cryoprotectant, characterized in that: including Fer-1.
4. A semen cryoprotectant according to claim 3, wherein: the semen cryoprotectant comprises 2-10 mu mol/L Fer-1;

and/or the semen cryoprotectant is a yolk and glycerol compound cryoprotectant.
5. The semen cryoprotectant of claim 4, wherein: the semen cryoprotectant also comprises yolk, glycerol, glucose, sodium citrate or sodium citrate hydrate;

and/or the pH value of the semen cryoprotectant is 6.8-7.2.
6. A semen cryoprotectant according to claim 5, characterized in that: the semen cryoprotectant comprises the following components in concentration: 15-25% of yolk, 15-25% of glycerin, 15 +/-0.5 g/L of glucose, 15 +/-0.5 g/L of sodium citrate or sodium citrate hydrate.
7. The method for preparing a semen cryoprotectant according to any one of claims 3 to 6, comprising the steps of: dissolving Fer-1 in dimethyl sulfoxide to obtain Fer-1 solution, and adding the Fer-1 solution into the yolk-glycerol composite cryoprotectant to obtain semen cryoprotectant.
8. The method of claim 1, wherein: the preparation method of the yolk-glycerol composite cryoprotectant comprises the following steps: adding glucose, sodium citrate or sodium citrate hydrate into water, and mixing; then adding glycerol, mixing uniformly and filtering; adding yolk to prepare a suspension; then heating and stirring are carried out; finally, the pH value is detected to be between 6.8 and 7.2.
9. Use of a semen cryoprotectant according to any one of claims 3 to 6 and/or a semen cryoprotectant prepared by a method according to any one of claims 7 to 8 for semen cryoprotection.
10. A semen freezing protection method is characterized in that: adopts a sperm cryoprotectant containing Fer-1.