A cryoprotective additive for human sperm and its preparation method

Abstract

The present invention provides a cryoprotective additive for human sperm 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 the pure commercially available protectant, the cryoprotective additive of the present invention has an increased number of forward-moving sperm after thawing and recovery; there are more forward-moving sperm after recovery, with better forward motility and better forward motility endurance. At the same time, the mitochondrial structure and function of the sperm cells after recovery are less damaged, the mitochondrial function recovers quickly, less mitochondrial ROS is produced, apoptosis is reduced, the ability to protect the function of sperm cells is better, and it is more conducive to the long-term cryopreservation of sperm-like cells.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technologies, and particularly relates to a cryoprotective additive for human sperm and a preparation method thereof. Background Art

[0002] The cryopreservation of germ cells has become an essential auxiliary means for human and animal assisted reproduction. It is a valuable tool for preserving fertility, gamete donation, preimplantation genetic diagnosis, reducing multiple pregnancies, and increasing the cumulative pregnancy rate. Over the years, successful species- and cell-specific procedures for cryopreserving sperm, oocytes, embryos, and gonadal tissues have been perfected. However, the viability and competence of germ cells are related to the quality and type of gametes and embryos, as well as the extent of mitochondrial damage and oxidative stress (OS) induced by cryopreservation. Persistent forward motility is required to achieve IVF. Sperm cryopreservation has become a routine procedure in human ART and is also a common method for preserving and storing genetic material in most domestic species. Nevertheless, a large amount of evidence in humans and livestock shows that sperm cryopreservation reduces the number of live sperm and affects the function of surviving cells by impairing the motility, mitochondrial activity, chromatin integrity, and reproductive potential of the surviving cells. The slow freezing procedure was the first successful technique for cryopreserving sperm historically and is still the most commonly used technique, probably because the high sperm numbers typically present in semen allow for an acceptable survival rate. However, due to individual differences in tolerance to freezing, the post-thaw sperm recovery rate is low, and the poor sperm quality ultimately leads to a low fertilization rate.

[0003] Mitochondria provide energy for sperm motility, and impaired mitochondrial function affects sperm motility. The disruption of mitochondrial structure leads to abnormal membrane potential, and the generation of excessive oxidative stress is a key factor affecting sperm fertilization ability. Mitochondria are considered 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 the total ROS production in different cells is not fully understood, mitochondrial ROS are thought to regulate the production of ROS through non-mitochondrial sources. OS is considered one of the main factors leading to male infertility and the decline in sperm survival rate and competence after cryopreservation. This is particularly concerning because, at least in non-human mammals, sperm OS is associated with a significant reduction in fertilization rate and in vitro embryo development. Additionally, the activation of apoptosis enzymes such as caspase can also be induced during cell cryopreservation. Members of the Caspase family are important effector proteases in 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 remedial measures for sperm damage related to cryopreservation. Summary of the Invention

[0004] In view of the technical problems existing in the prior art, the present invention provides a cryoprotective additive for human sperm 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 the pure commercially available protectant, the cryoprotective additive of the present invention has an increased number of forward-moving sperm after thawing and recovery; there are more forward-moving sperm after recovery, the forward-movement ability is better, the forward-movement endurance is better. At the same time, the mitochondrial structure and function of the sperm cells after recovery are less damaged, the mitochondrial function recovers quickly, less mitochondrial ROS is produced, apoptosis is reduced, the ability to protect the function of sperm cells is better, and it is more conducive to the long-term cryopreservation of sperm-like cells.

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

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

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

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

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

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

[0013] Preferably, the vials and screw caps of the lyoprotectant have passed the MEA test.

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

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

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

[0017] 1) Prepare the cryoprotectant for human sperm 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) according to the volume ratio of semen: cryoprotectant = 3 - 5:1 - 2, and mix well;

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

[0021] The advantages of the present invention are as follows: When using this cryoprotectant for cryopreservation of human sperm, a better resuscitation effect can be obtained after thawing. In particular, there is a synergistic effect between the mTORC1 activator and the Caspase-9 inhibitor in the lyoprotectant. When combined, the forward motility of sperm after resuscitation is better. At the same time, compared with the commercially available cryoprotectant alone, the mitochondrial structure and function of sperm cells after resuscitation are less damaged, the mitochondrial function recovers quickly, less ROS is generated, the apoptosis rate of cells is less, and the ability to protect the function of sperm cells is better, which is more conducive to the long-term cryopreservation of sperm. Description of the Drawings

[0022] Figure 1 . Transmission electron microscopy was used to detect the mitochondrial microstructure of sperm cells at 0 hour and 2 hours after cryopresuscitation in Comparative Examples 1 - 3 and Example 2. Among them, a corresponds to the fresh semen group, b corresponds to the Example 2 group, and c - e respectively correspond to the Comparative Examples 1 - 3 groups;

[0023] Figure 2 . The ATP content of sperm cells at 2 hours after cryopresuscitation in Comparative Examples 1 - 3 and Example 2 was detected by an ATP detection kit;

[0024] Figure 3 . The ROS content in sperm mitochondria at 2 hours after cryopresuscitation in Comparative Examples 1 - 3 and Example 2 was detected by a sperm mitochondrial ROS detection kit. Detailed Embodiments

[0025] The present invention will be further described in detail below with reference to specific embodiments, so that those skilled in the art can understand the present invention more clearly.

[0026] The following examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. Based on the specific examples of the present invention, all other examples obtained by those of ordinary skill in the art without creative efforts fall within the protection scope of the present invention.

[0027] In the embodiments 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 embodiments of the present invention, unless specifically 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), its corresponding main active binding epitopes were analyzed, and highly active antigenic epitopes were comprehensively screened as immunogens to prepare monoclonal antibodies. The antigenic epitope sequence obtained by screening was vevkgdltak kmvlallela qqdhgaldcc v (SEQ ID NO.1).

[0030] The coding sequence of the artificial antigenic epitope was synthesized and used to construct a prokaryotic expression plasmid pET-N-GST-PreScission (Shanghai Beyotime Biotechnology Co., Ltd., product number D2916), and a Caspase-9 recombinant protein with 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 cleavage with PreScission, and the Caspase-9 short peptide was collected. After SDS-PAGE electrophoresis detection and Western blot identification, it was confirmed that the target protein Caspase-9 short peptide was correctly expressed, and the protein concentration was adjusted to 15 mg / mL for standby.

[0031] Six-week-old female BALB / c mice were immunized at a dose of 15 μg recombinant protein / mouse; 14 days after the first immunization, a booster immunization was performed in the same manner; on the 14th day after the second immunization, a third immunization injection was performed, with the same method and dose as the second immunization. On the 10th day after the third immunization, the mice were bled by tail amputation, and the serum antibody titer was monitored by the indirect ELISA method. When the antibody level reached the requirement, 14 days after the third immunization, the immunized mouse with the highest titer was selected, and the pure antigen solution was used for booster immunization via the tail vein. Three days later, the spleen cells of the mice 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 heavy and light chain types of the antibody 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 a specific binding antibody targeting Caspase-9 was finally obtained and named 9C-4. After amplifying the monoclonal cells, ascites was prepared to purify the antibody. Identified by a kit, 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 sequences of CDR-H1-3 are shown in SEQ ID NOs.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 NOs.7-9.

[0033] The non-competitive ELISA method was used to detect the affinity of the specific binding antibody targeting Caspase-9 for the polypeptide of SEQ ID NO.1. The polypeptide of SEQ ID NO.1 was diluted 10-fold to 0.001 μg / mL, and the OD 450 value was read using an enzyme-linked immunosorbent assay reader. The detection results showed that the EC 50 of the specific binding antibody targeting Caspase-9 for the polypeptide of SEQ ID NO.1 could reach 0.08469 μg / mL, proving that it has good binding activity. Using Caspase-3, Caspase-7, and Caspase-8 as specific antigens, it was found by western blot that the specific binding antibody targeting Caspase-9 only formed a specific band with Caspase-9, and no bands were produced with others, indicating that the Caspase-9 monoclonal antibody has good specificity.

[0034] Example 2

[0035] A cryoprotectant for human sperm, which includes: 5.0 mmol / L sodium chloride, 0.15 mmol / L magnesium sulfate, 0.3 mmol / L potassium dihydrogen phosphate, 0.4 mmol / L sodium bicarbonate, 4.0 mmol / L potassium chloride, 1.5 mmol / L calcium lactate, 100 ml / L glycerol, 2.5 mmol / L glucose, 1.1 mmol / L sodium pyruvate, 3.5 g / L EDTA, 2.5 mmol / L HEPES, 25 nM mTORC1 activator, 50 μg / mL Caspase-9 inhibitor, and the balance is pure water. Among them, 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 cryoprotectant for human sperm, comprising: 5.5 mmol / L sodium chloride, 0.20 mmol / L magnesium sulfate, 0.4 mmol / L potassium dihydrogen phosphate, 0.45 mmol / L sodium bicarbonate, 4.5 mmol / L potassium chloride, 2.0 mmol / L calcium lactate, 120 ml / L glycerol, 3.0 mmol / L glucose, 1.2 mmol / L sodium pyruvate, 4.0 g / L EDTA, 3.0 mmol / L HEPES, 50 nM mTORC1 activator, 100 μg / mL Caspase-9 inhibitor, and the balance being pure water. Among them, 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 cryoprotectant for human sperm, comprising: 6.0 mmol / L sodium chloride, 0.25 mmol / L magnesium sulfate, 0.45 mmol / L potassium dihydrogen phosphate, 0.5 mmol / L sodium bicarbonate, 4.8 mmol / L potassium chloride, 2.5 mmol / L calcium lactate, 160 ml / L glycerol, 3.5 mmol / L glucose, 1.4 mmol / L sodium pyruvate, 5.5 g / L EDTA, 4.5 mmol / L HEPES, 100 nM mTORC1 activator, 200 μg / mL Caspase-9 inhibitor, and the balance being pure water. Among them, 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 2, it does not contain the mTORC1 activator.

[0042] Comparative Example 2

[0043] Compared with Example 2, it does not contain the Caspase-9 inhibitor.

[0044] Comparative Example 3

[0045] Compared with Example 2, it does not contain the mTORC1 activator and the Caspase-9 inhibitor.

[0046] Test Example

[0047] A comparative example was selected and compared with the human sperm cryoprotectant provided in Example 2 of the present invention: According to the sixth edition of the World Health Organization's semen routine analysis standard, 10 cases of normal human semen were randomly selected; after the semen was fully liquefied, computer-aided sperm analysis (CASA) was used to record the semen routine parameters.

[0048] Each portion of semen was randomly divided into four parts, and the cryoprotectants described in Example 2 and Comparative Examples 1-3 were used respectively; the liquefied semen and the sperm cryoprotectant were fully mixed at a ratio of semen: cryoprotectant = 3:1 (volume ratio); the above sperm mixture was equilibrated at room temperature for 15 minutes, 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, it was thawed in a 37 °C water bath for 10 minutes, and the fully thawed semen and cryoprotectant mixture were respectively used for the following experiments: CASA was used to detect the semen routine parameters after thawing; transmission electron microscopy was used to detect the mitochondrial microstructure of sperm cells after cryopreservation and recovery; after 2 hours of recovery, an ATP detection kit was used to detect the ATP level in the above sperm cells; flow cytometry combined with MitoROS was used to detect the change level of reactive oxygen species (ROS) in the mitochondria of the above sperm cells. The results are shown in Table 1 below:

[0050] Table 1. Semen routine parameters after thawing

[0051]

[0052] As can be seen from the data in Table 1 above, when the cryoprotective additive provided by the present invention is used, compared with Comparative Examples 1-3, the number of forward-moving sperm after thawing and recovery increases; there are more forward-moving sperm after recovery, the forward-moving ability is better, and the forward-moving endurance is better. At the same time, through the observation of mitochondrial microstructure, it is found that the mitochondrial structure of sperm cells recovered after being treated with the lyophilized cryoprotectant of Example 2 remains relatively intact, while the mitochondrial structure after being treated with Comparative Examples 1-3 shows obvious damage and deformation ( Figure 1 ). And the ATP detection results also confirm that the lyophilized cryoprotectant of Example 2 can effectively reduce mitochondrial function damage, and there is less mitochondrial ROS production, and the ability to protect sperm cell function is better ( Figures 2 - 3 ).

[0053] It is necessary to point out here that the above embodiments are only limited to further elaborating and explaining the technical solutions of the present invention, rather than further restricting the technical solutions 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 modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A cryoprotectant for human sperm, characterized in that, Comprising: 5.0 - 6.0 mmol / L sodium chloride, 0.15 - 0.25 mmol / L magnesium sulfate, 0.3 - 0.45 mmol / L potassium dihydrogen phosphate, 0.4 - 0.5 mmol / L sodium bicarbonate, 4.0 - 4.8 mmol / L potassium chloride, 1.5 - 2.5 mmol / L calcium lactate, 100 - 160 ml / L glycerol, 2.5 - 3.5 mmol / L glucose, 1.1 - 1.4 mmol / L sodium pyruvate, 3.5 - 5.5 g / L EDTA, 2.5 - 4.5 mmol / L HEPES, 25 - 100 nM mTORC1 activator, 50 - 200 μg / mL Caspase-9 inhibitor, with the balance being pure water; Wherein, the Caspase-9 inhibitor is a specific binding antibody targeting Caspase-9, and the heavy chain variable region sequence of the specific binding antibody targeting Caspase-9 is as shown in SEQ ID NO.2, and the light chain variable region sequence is as shown in SEQ ID NO.3; The mTORC1 activator is selected from one or more of MHY1485 and L-Leucine.

2. The cryoprotectant for human sperm as described in claim 1, wherein Comprising: 5.0 mmol / L sodium chloride, 0.15 mmol / L magnesium sulfate, 0.3 mmol / L potassium dihydrogen phosphate, 0.4 mmol / L sodium bicarbonate, 4.0 mmol / L potassium chloride, 1.5 mmol / L calcium lactate, 100 ml / L glycerol, 2.5 mmol / L glucose, 1.1 mmol / L sodium pyruvate, 3.5 g / L EDTA, 2.5 mmol / L HEPES, 25 nM mTORC1 activator, 50 μg / mL Caspase-9 inhibitor, with the balance being pure water.

3. The cryoprotectant for human sperm as claimed in claim 1, wherein, Comprising: 5.5 mmol / L sodium chloride, 0.20 mmol / L magnesium sulfate, 0.4 mmol / L potassium dihydrogen phosphate, 0.45 mmol / L sodium bicarbonate, 4.5 mmol / L potassium chloride, 2.0 mmol / L calcium lactate, 120 ml / L glycerol, 3.0 mmol / L glucose, 1.2 mmol / L sodium pyruvate, 4.0 g / L EDTA, 3.0 mmol / L HEPES, 50 nM mTORC1 activator, 100 μg / mL Caspase-9 inhibitor, with the balance being pure water.

4. The cryoprotectant for human sperm as described in claim 1, wherein Comprising: 6.0 mmol / L sodium chloride, 0.25 mmol / L magnesium sulfate, 0.45 mmol / L potassium dihydrogen phosphate, 0.5 mmol / L sodium bicarbonate, 4.8 mmol / L potassium chloride, 2.5 mmol / L calcium lactate, 160 ml / L glycerol, 3.5 mmol / L glucose, 1.4 mmol / L sodium pyruvate, 5.5 g / L EDTA, 4.5 mmol / L HEPES, 100 nM mTORC1 activator, 200 μg / mL Caspase-9 inhibitor, with the balance being pure water.

5. The cryoprotectant for human sperm according to any one of claims 1-4, characterized in that, The heavy chain variable region of the specific binding antibody targeting Caspase-9 includes CDR-H1, CDR-H2, and CDR-H3. 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. The sequences of CDR-L1-3 are shown in SEQ ID NO.7-9.

6. The cryoprotectant for human sperm according to any one of claims 1-4, characterized in that, Both the bottle and the screw cap of the human sperm cryoprotectant have passed the MEA test.

7. The cryoprotectant for human sperm according to any one of claims 1-4, characterized in that, The endotoxin of the human sperm cryoprotectant is <1.0 EU / mL, and the pH value of the human sperm cryoprotectant is 7.2-7.5.

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