Human sperm cryoprotection additive and preparation method thereof

Abstract

The invention provides a human sperm cryoprotection additive and a preparation method thereof. According to the method, an mTORC1 activator and a Caspase-9 inhibitor are added into a commercially available sperm cryoprotectant, and compared with a pure commercially available sperm cryoprotectant, the number of forward movement sperms after unfreezing and resuscitation of the cryoprotectant is increased; the resuscitated forward movement sperms are more, the forward movement ability is better, the forward movement persistence is better, meanwhile, the mitochondrial structure and function damage of the resuscitated sperm cells is small, the mitochondrial function recovery is fast, the mitochondrial ROS generation is less, the cell apoptosis is reduced, the sperm cell function protection ability is better, and the long-term cryopreservation of the sperm-like is more facilitated.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and in particular to a human sperm cryoprotectant additive and a preparation method thereof. Background Art

[0002] Cryopreservation of germ cells has become an essential adjunct to assisted reproduction in humans and animals. It is a valuable tool for preserving fertility, gamete donation, preimplantation genetic diagnosis, reducing multiple pregnancies, and improving cumulative pregnancy rates. Over the years, successful species- and cell-specific procedures for cryopreservation of sperm, oocytes, embryos, and gonadal tissue have been refined. However, the viability and capacity of germ cells are related to the quality and species of gametes and embryos and the extent of mitochondrial damage and oxidative stress (OS) induced by cryopreservation. Only with persistent forward motility can IVF be achieved. Sperm cryopreservation has become a routine procedure for human ART and a common method for preserving and storing genetic material in most domestic species. Despite this, a large body of evidence from humans and domestic animals shows that sperm cryopreservation reduces the number of viable sperm and affects the function of surviving cells by impairing their motility, mitochondrial activity, chromatin integrity, and reproductive potential. The slow freezing procedure was the first successful technique for cryopreserving sperm in history and is still the most commonly used technique, probably because the high sperm count normally present in semen makes the survival rate acceptable. However, due to individual differences, there is heterogeneity in tolerance to freezing, low sperm recovery rate after freezing, and poor sperm quality ultimately lead to low fertilization rate.

[0003] Mitochondria provide energy for sperm motility, and impaired mitochondrial function affects sperm motility. Disruption of mitochondrial structure leads to abnormal membrane potential, and excessive oxidative stress is a key factor affecting sperm fertilization ability. Mitochondria are considered to be the main producers of ROS in cells, although other organelles and enzymes also contribute to this function. The two main sources of intracellular ROS are plasma membrane NADPH oxidase and mitochondrial respiratory chain enzyme complexes. In addition, other enzymes in the cytosol, endoplasmic reticulum, and peroxisomes contribute to the production of ROS. Although the exact contribution of mitochondria to total ROS production in different cells is not fully understood, mitochondrial ROS are thought to regulate ROS production through non-mitochondrial sources. OS is considered to be one of the main factors leading to male infertility and decreased sperm survival and potency after cryopreservation. This is particularly worrying because, at least in non-human mammals, sperm OS is associated with a significant decrease in fertilization rate and in vitro embryo development. In addition, the activation of apoptotic enzymes such as caspases can also be caused during cell cryopreservation. Caspase family members are important effector proteases of the apoptotic cascade, and inhibiting Caspase expression can improve the survival rate of various cells after cryopreservation. Therefore, it is extremely important to find the causes and remedies for sperm damage associated with cryopreservation. Summary of the invention

[0004] In view of the technical problems existing in the prior art, the present invention provides a human sperm cryoprotectant additive and a preparation method thereof. The method is to add an mTORC1 activator and a Caspase-9 inhibitor to a commercially available sperm cryoprotectant. Compared with a simple commercially available protectant, the cryoprotectant additive of the present invention increases the number of forward-moving sperm after thawing and recovery; the forward-moving sperm after recovery are more, the forward-moving ability is better, and the forward-moving endurance is better. At the same time, the mitochondrial structure and function of the sperm cells after recovery are less damaged and the mitochondrial function recovers quickly, the mitochondrial ROS production is less, the cell apoptosis is reduced, the protection ability of the sperm cell function is better, and it is more conducive to the long-term cryopreservation of sperm.

[0005] Specifically, the present invention first provides a human sperm cryoprotectant, comprising: 5.0-6.0mmol / L sodium chloride, 0.15-0.25mmol / L magnesium sulfate, 0.3-0.45mmol / L potassium dihydrogen phosphate, 0.4-0.5mmol / L sodium bicarbonate, 4.0-4.8mmol / L potassium chloride, 1.5-2.5mmol / L calcium lactate, 100-160ml / L glycerol, 2.5-3.5mmol / L glucose, 1.1-1.4mmol / L sodium pyruvate, 3.5-5.5g / L EDTA, 2.5-4.5mmol / L HEPES, 25-100nM mTORC1 activator, 50-200μg / mL Caspase-9 inhibitor, and the balance is purified water.

[0006] Preferably, the human sperm cryoprotectant comprises: 5.0mmol / L sodium chloride, 0.15mmol / L magnesium sulfate, 0.3mmol / L potassium dihydrogen phosphate, 0.4mmol / L sodium bicarbonate, 4.0mmol / L potassium chloride, 1.5mmol / L calcium lactate, 100ml / L glycerol, 2.5mmol / L glucose, 1.1mmol / L sodium pyruvate, 3.5g / L EDTA, 2.5mmol / L HEPES, 25nM mTORC1 activator, 50μg / mL Caspase-9 inhibitor, and the balance is purified water.

[0007] Preferably, the human sperm cryoprotectant comprises: 5.5mmol / L sodium chloride, 0.20mmol / L magnesium sulfate, 0.4mmol / L potassium dihydrogen phosphate, 0.45mmol / L sodium bicarbonate, 4.5mmol / L potassium chloride, 2.0mmol / L calcium lactate, 120ml / L glycerol, 3.0mmol / L glucose, 1.2mmol / L sodium pyruvate, 4.0g / L EDTA, 3.0mmol / L HEPES, 50nM mTORC1 activator, 100μg / mL Caspase-9 inhibitor, and the balance is purified water.

[0008] Preferably, the human sperm cryoprotectant comprises: 6.0mmol / L sodium chloride, 0.25mmol / L magnesium sulfate, 0.45mmol / L potassium dihydrogen phosphate, 0.5mmol / L sodium bicarbonate, 4.8mmol / L potassium chloride, 2.5mmol / L calcium lactate, 160ml / L glycerol, 3.5mmol / L glucose, 1.4mmol / L sodium pyruvate, 5.5g / L EDTA, 4.5mmol / L HEPES, 100nM mTORC1 activator, 200μg / mL Caspase-9 inhibitor, and the balance is purified water.

[0009] Preferably, the mTORC1 activator is selected from one or more of MHY1485 and L-Leucine.

[0010] Preferably, the Caspase-9 inhibitor is one or more of a specific binding antibody, siRNA, sgRNA, miRNA, and ASO targeting Caspase-9.

[0011] More preferably, the heavy chain variable region sequence of the specific binding antibody targeting Caspase-9 is shown as SEQ ID NO.2, and the light chain variable region sequence is shown as SEQ ID NO.3.

[0012] Further preferably, the heavy chain variable region of the specific binding antibody targeting Caspase-9 includes CDR-H1, CDR-H2, and CDR-H3, and the sequences of CDR-H1-3 are shown in SEQ ID NO.4-6, and the light chain variable region includes CDR-L1, CDR-L2, and CDR-L3, and the sequences of CDR-L1-3 are shown in SEQ ID NO.7-9.

[0013] Preferably, the bottle and screw cap of the lyoprotectant are both MEA tested.

[0014] Preferably, the endotoxin of the lyoprotectant is less than 1.0 EU / mL.

[0015] Preferably, the pH value of the lyoprotectant is 7.2-7.5.

[0016] Furthermore, the present invention also provides an application of the human sperm cryoprotectant, which comprises the following steps:

[0017] 1) preparing the human sperm cryoprotectant according to the formula;

[0018] 2) Take the fully liquefied semen and put it into a labeled cryopreservation tube;

[0019] 3) Slowly add the cryoprotectant to the semen in step 2) at a volume ratio of semen: cryoprotectant = 3-5:1-2, and mix thoroughly;

[0020] 4) Freeze the sperm according to the programmed freezing method. After completing the freezing procedure, freeze the cryopreservation tube in a liquid nitrogen tank for storage.

[0021] The advantages of the present invention are as follows: using the cryoprotectant for cryopreservation of human sperm can achieve a good recovery effect after thawing. In particular, the mTORC1 activator and the Caspase-9 inhibitor in the lyoprotectant have a synergistic effect, and the forward motility of the sperm after recovery is better in the case of combination. At the same time, compared with the commercially available cryoprotectant alone, the mitochondrial structure and function of the sperm cells after recovery are less damaged, the mitochondrial function recovers quickly, the ROS generation is less, the cell apoptosis rate is less, the protection ability of the sperm cell function is better, and it is more conducive to the long-term cryopreservation of sperm. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 Transmission electron microscopy was used to detect the mitochondrial microstructure of sperm cells in Comparative Examples 1-3 and Example 1 at 0 hours and 2 hours after freezing and thawing, wherein a corresponds to the fresh semen group, b corresponds to the Example 1 group, and ce corresponds to the Comparative Examples 1-3 groups respectively;

[0023] Figure 2 . The ATP detection kit was used to detect the ATP content of sperm cells in Comparative Examples 1-3 and Example 1 2 hours after freezing and thawing;

[0024] Figure 3 . The sperm mitochondrial ROS detection kit was used to detect the ROS content in the sperm mitochondria of Comparative Examples 1-3 and Example 1 2 hours after freezing and thawing. DETAILED DESCRIPTION

[0025] The present invention is further described in detail below in conjunction with specific embodiments so that those skilled in the art can understand the present invention more clearly.

[0026] The following embodiments are only used to illustrate the present invention and are not intended to limit the scope of the present invention. Based on the specific embodiments of the present invention, all other embodiments obtained by ordinary technicians in the field without creative work belong to the protection scope of the present invention.

[0027] In the examples of the present invention, unless otherwise specified, all raw material components are commercially available products well known to those skilled in the art; in the examples of the present invention, unless otherwise specified, the technical means used are conventional means well known to those skilled in the art.

[0028] Example 1

[0029] According to the amino acid sequence of Caspase-9 (GenPept: BAA82697.1), the corresponding main active binding epitopes were analyzed, and highly active antigenic epitopes were screened comprehensively to prepare monoclonal antibodies as immunogens. The antigenic epitope sequence obtained by screening was vevkgdltak kmvlallela qqdhgaldcc v (SEQ ID NO.1).

[0030] The coding sequence of the artificially synthesized antigen epitope was used to construct the prokaryotic expression plasmid pET-N-GST-PreScission (Shanghai Biyuntian Biotechnology Co., Ltd., product number D2916), and the Caspase-9 recombinant protein containing a GST tag at the N-terminus was expressed in Escherichia coli DH5α. The recombinant protein was purified by glutathione agarose gel affinity chromatography, and then the GST tag was removed by enzymatic excision by PreScission, and the Caspase-9 short peptide was collected. SDS-PAGE electrophoresis detection and Western blot identification confirmed that the target protein Caspase-9 short peptide was correctly expressed, and the protein concentration was adjusted to 15 mg / mL for standby use.

[0031] Six-week-old female BALB / c mice were immunized at a dose of 15 μg recombinant protein / mouse; the same method was used to boost immunization once on the 14th day after the first immunization; the third immunization injection was performed on the 14th day after the second immunization, and the method and dose were the same as the second immunization. On the 10th day after the third immunization, the mice were cut off from their tails and blood was collected, and the serum antibody titer was monitored by indirect ELISA. When the antibody level reached the requirement, on the 14th day after the third immunization, the immunized mice with the highest titer were selected, and the tail vein was boosted with pure antigen liquid. After 3 days, the mouse spleen cells were aseptically taken and fused with SP2 / 0 cells to prepare hybridoma cells. The culture supernatant of the hybridoma cell line was taken, and the antibody heavy chain and light chain types were determined using the IsoStrip Mouse Monoclonal Antibody Isotyping Kit (Roche).

[0032] After three rounds of immunization, a monoclonal cell line with high expression of specific binding antibodies targeting Caspase-9 was finally obtained, named 9C-4. After the monoclonal cells were amplified, ascites was used to prepare purified antibodies. According to the kit identification, the heavy chain variable region sequence of the specific binding antibody targeting Caspase-9 is shown in SEQ ID NO.2, and the light chain variable region sequence is shown in SEQ ID NO.3. The heavy chain variable region includes CDR-H1, CDR-H2, and CDR-H3, and the sequence of CDR-H1-3 is shown in SEQ ID NO.4-6. The light chain variable region includes CDR-L1, CDR-L2, and CDR-L3, and the sequence of CDR-L1-3 is shown in SEQ ID NO.7-9.

[0033] The affinity of the specific binding antibody targeting Caspase-9 to the SEQ ID NO.1 polypeptide was detected by non-competitive ELISA method. The SEQ ID NO.1 polypeptide was diluted 10 times to 0.001 μg / mL and the OD was read using an ELISA reader. 450 The test results show that the EC value of the specific binding antibody targeting Caspase-9 to the polypeptide of SEQ ID NO.1 50 It can reach 0.08469μg / mL, proving that it has good binding activity. Caspase-3, Caspase-7, and Caspase-8 were used as specific antigens, and western blot detection showed that the specific binding antibody targeting Caspase-9 only formed specific bands with Caspase-9, and no bands were produced with other antibodies, indicating that the Caspase-9 monoclonal antibody has good specificity.

[0034] Example 2

[0035] A human sperm cryoprotectant, comprising: 5.0mmol / L sodium chloride, 0.15mmol / L magnesium sulfate, 0.3mmol / L potassium dihydrogen phosphate, 0.4mmol / L sodium bicarbonate, 4.0mmol / L potassium chloride, 1.5mmol / L calcium lactate, 100ml / L glycerol, 2.5mmol / L glucose, 1.1mmol / L sodium pyruvate, 3.5g / L EDTA, 2.5mmol / L HEPES, 25nM mTORC1 activator, 50μg / mL Caspase-9 inhibitor, and the remainder is purified water. The mTORC1 activator is MHY1485, and the Caspase-9 inhibitor is the specific binding antibody targeting Caspase-9 in Example 1.

[0036] Example 3

[0037] A human sperm cryoprotectant, comprising: 5.5mmol / L sodium chloride, 0.20mmol / L magnesium sulfate, 0.4mmol / L potassium dihydrogen phosphate, 0.45mmol / L sodium bicarbonate, 4.5mmol / L potassium chloride, 2.0mmol / L calcium lactate, 120ml / L glycerol, 3.0mmol / L glucose, 1.2mmol / L sodium pyruvate, 4.0g / L EDTA, 3.0mmol / L HEPES, 50nM mTORC1 activator, 100μg / mL Caspase-9 inhibitor, and the remainder is purified water. The mTORC1 activator is MHY1485, and the Caspase-9 inhibitor is the specific binding antibody targeting Caspase-9 in Example 1.

[0038] Example 4

[0039] A human sperm cryoprotectant, comprising: 6.0mmol / L sodium chloride, 0.25mmol / L magnesium sulfate, 0.45mmol / L potassium dihydrogen phosphate, 0.5mmol / L sodium bicarbonate, 4.8mmol / L potassium chloride, 2.5mmol / L calcium lactate, 160ml / L glycerol, 3.5mmol / L glucose, 1.4mmol / L sodium pyruvate, 5.5g / L EDTA, 4.5mmol / L HEPES, 100nM mTORC1 activator, 200μg / mL Caspase-9 inhibitor, and the remainder is purified water. The mTORC1 activator is MHY1485, and the Caspase-9 inhibitor is the specific binding antibody targeting Caspase-9 in Example 1.

[0040] Comparative Example 1

[0041] Compared with Example 1, it does not contain an mTORC1 activator.

[0042] Comparative Example 2

[0043] Compared with Example 1, it does not contain Caspase-9 inhibitor.

[0044] Comparative Example 3

[0045] Compared with Example 1, it does not contain mTORC1 activator and Caspase-9 inhibitor.

[0046] Test example

[0047] A comparative example was selected for comparison with the human sperm cryoprotectant provided in Example 1 of the present invention: according to the sixth edition of the World Health Organization's semen routine analysis standards, 10 normal human semens were randomly selected; after the semens were fully liquefied, computer-aided sperm analysis (CASA) was used and the semen routine parameters were recorded.

[0048] Each semen sample was randomly divided into four parts, and the protective agents described in Example 1 and Comparative Examples 1-3 were used respectively; the liquefied semen and the sperm cryoprotectant were fully mixed at a ratio of semen: protective agent = 3:1 (volume ratio); the sperm mixture was balanced at room temperature for 15 minutes, and then the sample was suspended 10 cm above liquid nitrogen for 1 hour, and finally transferred to liquid nitrogen for storage for three weeks.

[0049] Subsequently, the sperm was thawed in a 37°C water bath for 10 minutes, and the fully thawed semen and cryoprotectant mixture were used in the following experiments: CASA was used to detect the routine parameters of the thawed semen; the mitochondrial microstructure of the sperm cells after freezing and thawing was detected by a perspective electron microscope; after 2 hours of thawing, the ATP level in the sperm cells was detected by an ATP detection kit; MitoROS combined with flow cytometry was used to detect the change level of reactive oxygen species (ROS) in the mitochondria of the sperm cells. The results are shown in Table 1 below:

[0050] Table 1. General parameters of thawed semen

[0051]

[0052] From the data in Table 1 above, it can be seen that the use of the cryoprotectant provided by the present invention increases the number of forward-moving sperm after thawing and recovery compared with Comparative Examples 1-3; the forward-moving sperm after recovery are more, the forward-moving ability is better, and the forward-moving endurance is better. At the same time, the mitochondrial microstructure observation found that the mitochondrial structure of the sperm cells recovered by the lyophilized protective agent of Example 1 remained relatively intact, while the mitochondrial structure after the treatment of Comparative Examples 1-3 was obviously damaged and deformed ( Figure 1 ). And the ATP test results also confirmed that the freeze-dried protective agent of Example 1 can effectively reduce mitochondrial function damage, and the mitochondrial ROS production is small, and the protective ability of sperm cell function is better ( Figure 2-3 ).

[0053] It is necessary to point out that the above embodiments are limited to further elaboration and explanation of the technical solution of the present invention, and are not further limitations of the technical solution of the present invention. The method of the present invention is only a preferred implementation scheme, and is not used to limit the protection scope of the present invention. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A human sperm cryoprotectant, characterized in that: include: 5.0-6.0mmol / L sodium chloride, 0.15-0.25mmol / L magnesium sulfate, 0.3-0.45mmol / L potassium dihydrogen phosphate, 0.4-0.5mmol / L sodium bicarbonate, 4.0-4.8mmol / L potassium chloride, 1.5-2.5mmol / L calcium lactate, 100-160ml / L glycerol, 2.5-3.5mmol / L glucose, 1.1-1.4mmol / L sodium pyruvate, 3.5-5.5g / L EDTA, 2.5-4.5mmol / L HEPES, 25-100nM mTORC1 activator, 50-200μg / mL Caspase-9 inhibitor, and the balance is purified water.

2. The human sperm cryoprotectant according to claim 1, characterized in that include: 5.0mmol / L sodium chloride, 0.15mmol / L magnesium sulfate, 0.3mmol / L potassium dihydrogen phosphate, 0.4mmol / L sodium bicarbonate, 4.0mmol / L potassium chloride, 1.5mmol / L calcium lactate, 100ml / L glycerol, 2.5mmol / L glucose, 1.1mmol / L sodium pyruvate, 3.5g / L EDTA, 2.5mmol / L HEPES, 25nM mTORC1 activator, 50μg / mL Caspase-9 inhibitor, and the remainder is purified water.

3. The human sperm cryoprotectant according to claim 1, characterized in that include: 5.5mmol / L sodium chloride, 0.20mmol / L magnesium sulfate, 0.4mmol / L potassium dihydrogen phosphate, 0.45mmol / L sodium bicarbonate, 4.5mmol / L potassium chloride, 2.0mmol / L calcium lactate, 120ml / L glycerol, 3.0mmol / L glucose, 1.2mmol / L sodium pyruvate, 4.0g / L EDTA, 3.0mmol / L HEPES, 50nM mTORC1 activator, 100μg / mL Caspase-9 inhibitor, and the remainder is purified water.

4. The human sperm cryoprotectant according to claim 1, characterized in that include: 6.0mmol / L sodium chloride, 0.25mmol / L magnesium sulfate, 0.45mmol / L potassium dihydrogen phosphate, 0.5mmol / L sodium bicarbonate, 4.8mmol / L potassium chloride, 2.5mmol / L calcium lactate, 160ml / L glycerol, 3.5mmol / L glucose, 1.4mmol / L sodium pyruvate, 5.5g / L EDTA, 4.5mmol / L HEPES, 100nM mTORC1 activator, 200μg / mL Caspase-9 inhibitor, and the balance is purified water.

5. The human sperm cryoprotectant according to any one of claims 1 to 4, characterized in that The mTORC1 activator is selected from one or more of MHY1485 and L-Leucine.

6. The human sperm cryoprotectant according to any one of claims 1 to 4, characterized in that The Caspase-9 inhibitor is one or more of a specific binding antibody, siRNA, sgRNA, miRNA, and ASO targeting Caspase-9.

7. The human sperm cryoprotectant according to claim 6, characterized in that The heavy chain variable region sequence of the specific binding antibody targeting Caspase-9 is shown in SEQ ID NO.2, and the light chain variable region sequence is shown in SEQ ID NO.

3.

8. The human sperm cryoprotectant according to claim 6, characterized in that The heavy chain variable region of the specific binding antibody targeting Caspase-9 includes CDR-H1, CDR-H2, and CDR-H3, and the sequences of CDR-H1-3 are shown in SEQ ID NO.4-6; the light chain variable region includes CDR-L1, CDR-L2, and CDR-L3, and the sequences of CDR-L1-3 are shown in SEQ ID NO.7-9.

9. The human sperm cryoprotectant according to any one of claims 1 to 8, characterized in that The bottles and screw caps of the lyoprotectant were MEA tested.

10. The human sperm cryoprotectant according to any one of claims 1 to 9, characterized in that The endotoxin of the lyophilization protectant is less than 1.0 EU / mL, and the pH value of the lyophilization protectant is 7.2-7.5.

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