Application of melatonin nuclear receptor RORα in improving the quality of frozen semen

Abstract

The present invention provides the use of melatonin nuclear receptor RORα in improving semen quality. By increasing the expression level of melatonin nuclear receptor RORα, the quality of frozen semen after thawing can be further improved after adding melatonin. The present invention also provides a method for improving the quality of frozen bovine semen, which method adds melatonin to the frozen semen after thawing and adds a melatonin nuclear receptor RORα agonist for incubation. The sperm motility and acrosome integrity are detected. The results show that melatonin improves semen quality through the nuclear receptor gene RORα. The method of the present invention has good application prospects.

Description

[0001] This application is a divisional application of the patent application with application number 202210924812X, application date 20220803, and invention name “Use of melatonin nuclear receptor RORα in improving the quality of frozen semen”. Technical Field

[0002] The present invention relates to the field of reproduction, and in particular to the improvement of the quality of frozen semen. Background Art

[0003] With the rapid development of biotechnology, gamete and embryo cryopreservation is gradually becoming the most effective way to replace live animal preservation. Semen cryopreservation is to store semen at ultra-low temperatures (usually liquid nitrogen -196℃) after special treatment and adding protective agents to achieve long-term preservation. Its biggest advantage is that it can be stored for a long time and its use is not limited by time, region and the life span of male animals. At the same time, it can greatly improve the utilization efficiency of breeding bulls.

[0004] Ice crystal damage, cryoprotectant toxicity, and oxidative stress in the freezing process of semen are the main reasons for the low quality of semen after thawing and the low conception rate after insemination. This is largely related to the large amount of ROS produced during the freezing process of sperm. Many studies have shown that adding melatonin to frozen semen can help protect sperm from freezing. In frozen semen after adding melatonin, the number of live sperm, motility, and kinetic-related parameters were significantly improved, and the integrity of sperm membrane systems such as acrosome membranes and plasma membranes was significantly improved. However, when melatonin is added to a certain amount, the quality of sperm cannot be further improved. Summary of the invention

[0005] The first object of the present invention is to provide the use of melatonin nuclear receptor RORα.

[0006] The second object of the present invention is to provide a method for improving the quality of frozen semen.

[0007] The present invention has found that it is very difficult to significantly improve the quality of frozen semen due to the bottleneck when using melatonin to improve the quality of frozen semen. When the expression of melatonin nuclear receptor RORα is increased, the quality of semen can be further significantly improved. Accordingly, the present invention first provides the use of melatonin nuclear receptor RORα in improving semen quality.

[0008] In the above use, improving semen quality includes improving sperm motility and improving sperm acrosome integrity.

[0009] The present invention further provides a method for improving semen quality, which improves semen quality by increasing the expression level of melatonin nuclear receptor protein RORα and adding melatonin to semen.

[0010] Further, the semen is frozen semen, including but not limited to semen from cattle, horses or other livestock or humans. In practice, sex-controlled frozen semen of dairy cows is the most widely used frozen semen in the breeding field.

[0011] Furthermore, the method for improving semen quality comprises the steps of: thawing frozen semen to recover sperm, adding PBS and semen diluent to resuspend, and then adding melatonin nuclear receptor protein RORα agonist and melatonin and incubating.

[0012] Preferably, the melatonin nuclear receptor protein RORα agonist is added to a final concentration of 0.001 to 0.1 mM. More preferably, the melatonin nuclear receptor protein RORα agonist is added to a final concentration of 0.01 mM. The added concentration of the melatonin nuclear receptor protein RORα agonist is an effective amount to achieve an increase in the expression of the melatonin nuclear receptor protein RORα. In practice, the effective amount of the agonist added can be determined according to the method provided in the embodiment of the present invention.

[0013] Preferably, the melatonin is added to a final concentration of 0.001 to 0.1 mM. More preferably, the melatonin is added to a final concentration of 0.01 mM. The concentration of melatonin added is an effective amount to improve the quality of semen. In practice, the effective amount of melatonin added can be determined according to the method provided in the embodiments of the present invention.

[0014] Thawing and recovery of frozen semen can be carried out by conventional means. Preferably, the method in the embodiment of the present invention can be used. The sex-controlled frozen semen is quickly taken out from liquid nitrogen with tweezers, placed in a 37°C constant temperature water bath and shaken for 30 seconds. After thawing, the capillary is wiped with absorbent paper, and the semen is collected in a 1.5 mL centrifuge tube, centrifuged at room temperature at 378×g for 5 minutes, the supernatant is discarded, and 1 mL of 37°C PBS is added again to resuspend the sperm, centrifuged at room temperature at 378×g for 5 minutes, the supernatant is discarded, and this is repeated twice.

[0015] The recovered sperm can be resuspended by conventional methods. For example, for sex-controlled frozen semen, 170 μL PBS and 170 μL semen diluent can be added to resuspend it after centrifugation in a 1.5 mL centrifuge tube.

[0016] The increase in the expression of melatonin nuclear receptor protein RORα in the present invention can be achieved by, for example, using a melatonin nuclear receptor protein RORα agonist, or by overexpression, or by other regulatory pathways. However, for the treatment of frozen semen, adding an agonist is a simple and effective way.

[0017] The present invention also provides the use of a melatonin nuclear receptor protein RORα agonist, that is, the expression of melatonin nuclear receptor protein RORα is increased by the melatonin nuclear receptor protein RORα agonist, thereby improving the quality of semen. In other words, the quality of semen can be improved by the melatonin nuclear receptor protein RORα agonist. That is, the use of a melatonin nuclear receptor protein RORα agonist in improving the quality of semen is provided.

[0018] Furthermore, the present invention also provides the use of melatonin and melatonin nuclear receptor protein RORα agonist in the preparation of a preparation for improving the quality of frozen semen.

[0019] The present invention further provides a preparation comprising melatonin and a melatonin nuclear receptor protein RORα agonist. It is used to improve the quality of frozen semen. This preparation contains an effective amount of melatonin and an effective amount of a melatonin nuclear receptor protein RORα agonist. Preferably, the molar ratio of the two is, for example, 1 to 10:1 to 10, for example, 1:0.5 to 5, for example, 1:1. In one embodiment of the present invention, the amount added is a final concentration of 0.01 mM melatonin and 0.01 mM nuclear receptor protein RORα agonist. In specific applications, the final addition amount can be tested according to the method in the embodiment of the present invention. For the preparation, the preparation concentration can be set to, for example, 3.4 mM melatonin and 3.4 mM melatonin nuclear receptor protein RORα agonist.

[0020] The invention proposes that the quality of frozen semen after thawing can be further improved by increasing the expression of melatonin nuclear receptor RORα and then adding melatonin. The method of the present invention adds melatonin to the frozen semen after thawing and adds a melatonin nuclear receptor RORα agonist for incubation, and detects sperm motility and acrosome integrity. The results show that melatonin improves semen quality through the nuclear receptor RORα. The method and preparation of the present invention have good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 The expression of melatonin nuclear receptor gene RORα in bovine frozen sperm;

[0022] Figure 2 The expression of melatonin nuclear receptor gene RORα in bovine frozen sperm before and after adding melatonin and after co-incubation with agonists and inhibitors. DETAILED DESCRIPTION

[0023] The following examples are used to further illustrate the present invention, but should not be construed as limiting the present invention. Without departing from the spirit and essence of the present invention, modifications or modifications to the technical solutions of the present invention all fall within the scope of the present invention.

[0024] Example 1

[0025] 1.1 Materials and methods

[0026] 1.1.1 Experimental design

[0027] Eight frozen semen samples from three cows were collected and divided into control group, melatonin, melatonin + agonist, and melatonin + inhibitor group. After incubation for 2 hours, semen quality test, RNA and protein extraction were performed, and fluorescence quantitative PCR and Western blot were performed, respectively. Each group was tested three times.

[0028] 1.1.2 Main instruments and equipment

[0029] 1.1.3 Preparation of main reagents and solutions

[0030]

[0031]

[0032] 1.2 Test methods

[0033] 1.2.1 Thawing of semen

[0034] Use tweezers to quickly remove the sex-controlled frozen semen from liquid nitrogen, place it in a 37℃ constant temperature water bath and shake to thaw for 30 seconds. After thawing, wipe the capillary with absorbent paper, collect the semen in a 1.5mL centrifuge tube, centrifuge at 378×g at room temperature for 5 minutes, discard the supernatant, add 1mL 37℃ PBS again to resuspend the sperm, centrifuge at 378×g at room temperature for 5 minutes, discard the supernatant, and repeat twice.

[0035] 1.2.2 Incubation with melatonin, agonists and antagonists

[0036] The control group was treated by adding 170 μL PBS and 170 μL semen diluent to the semen; the melatonin group was treated by adding 0.01 mM Melatonin to the control group; the agonist group was treated by adding 0.01 mM Melatonin + 0.01 mM CGP52608 to the control group; the inhibitor group was treated by adding 0.01 mM Melatonin + 0.01 mM TO901317 to the control group. The following experiments were performed after incubation at room temperature for 2 hours.

[0037] 1.2.3 Semen quality determination

[0038] Sperm motility test: Take 100μL of the incubated semen mixture, add 5μL PI and 5μL Hochest33342, and incubate in a 37℃ water bath away from light for 10 minutes. Take out 10μL of the preparation, observe it under a fluorescence microscope at 400×, count the number of dead sperm (red) and total sperm (blue) in the field of view, and calculate the sperm motility. Take 5 fields of view for each sample to calculate the average value. The calculation formula is:

[0039] Sperm motility (%) = (total sperm count - dead sperm count) / total sperm count × 100%

[0040] Sperm acrosome integrity test: Take 100μL of the incubated semen mixture, add 5μL PI, 5μL Hochest33342 and 10μL FITC-PNA, and incubate in a 37℃ water bath in the dark for 30min. Take out 10μL of the preparation, place it under a fluorescence microscope at 400× for observation, count the number of dead sperm (red) and total sperm (blue) in the field of view, as well as the number of sperm with acrosome reaction or acrosome damage (green), and calculate the proportion of sperm without acrosome reaction in the live sperm to the total number of sperm. Take 5 fields of view for each sample to calculate the average value. The calculation formula is:

[0041] Acrosome integrity rate (%) = (number of sperm stained with Hochest 33342 - number of green sperm - number of sperm not stained green but stained red) / number of sperm stained with Hochest 33342 × 100%

[0042] 1.2.4 Sperm RNA extraction

[0043] Place the centrifuge tube on ice. Add 300 μL RNAstore to the sperm pellet, pipette the pellet evenly, and place it in a 4°C refrigerator overnight. Take the sperm out of the refrigerator and place it on ice, add 2 times the volume of pre-cooled PBS, centrifuge at 4°C 5000g for 5 minutes, discard the supernatant, and place the sperm pellet on ice for later use.

[0044] Add 500 μL RL lysis buffer to the sperm precipitate (add β-mercaptoethanol to the RL lysis buffer to a final concentration of 1% according to the kit), place in a 60°C water bath for 3 min until the solution is transparent, and observe under a microscope that there is no complete sperm morphology. Add 500 μL TRIzol to the centrifuge tube, vortex for 15 s, let stand for 3 min, centrifuge at 4°C 15000×g for 15 min, take 500 μL supernatant, add one volume of 100% anhydrous ethanol, and mix gently. Transfer to RNase-Free adsorption column CR3 for adsorption, centrifuge at 13,400×g for 45S to remove waste liquid, transfer RNase-Free adsorption column CR3 to a collection tube, add 700μL of deproteinization solution to RNase-Free adsorption column CR3, centrifuge at 13,400×g for 45S to remove waste liquid, add 500μL of rinsing solution to RNase-Free adsorption column CR3, let stand at room temperature for 2min, centrifuge at 13,400×g for 45S to remove waste liquid, repeat rinsing once, let stand to dry, transfer RNase-Free adsorption column CR3 to a new RNase-Free centrifuge tube, add RNase-Free ddH2O, let stand at room temperature for 2min, and centrifuge at 13,400×g for 2min to obtain RNA solution.

[0045] 1.2.5 Sperm protein extraction

[0046] Place the centrifuge tube on ice. Add 300 μL RNAstore to the sperm pellet, pipette the pellet evenly, and place it in a 4°C refrigerator overnight. Take the sperm out of the refrigerator and place it on ice, add 2 times the volume of pre-cooled PBS, centrifuge at 4°C 5000g for 5 minutes, discard the supernatant, and place the sperm pellet on ice for later use.

[0047] Add 40 μL RIPA, 10 μL 10% SDS and 50 μL 4x Loading buffer, 1 μL protease inhibitor, 1 μL PMSF to the sperm pellet, vortex and mix. Boil the mixture in a boiling water bath for 10 min, centrifuge at 4°C 15000g for 10 min, and take the supernatant.

[0048] 1.2.6 Fluorescence quantitative PCR

[0049] Using iScript TM cDNA Synthesis Kit Cdna reverse transcription kit was used for genomic DNA removal and first-strand complementary DNA synthesis. With reference to domestic and foreign literature, specific primers were designed according to the sequences of bovine GAPDH and RORα genes in the NCBI database (Table 1) and synthesized by Shanghai Shenggong Biotechnology Co., Ltd. The Bio-Rad CFX96 real-time fluorescence quantitative PCR system was used for detection, and iTaq TM Universal Green Supermix fluorescent quantitative reagent was used for the test. The total volume of each sample was 20 μL and there were 3 replicates. The PCR amplification cycle conditions included initial denaturation at 95°C for 10 min, followed by 40 cycles of denaturation at 95°C for 10 s, annealing at 60°C for 20 s, and extension at 72°C for 25 s. This was followed by a final extension cycle at 72°C for 5 min. -ΔΔCT The relative levels of mRNA transcriptome were determined by the PCR method, and the housekeeping gene (GAPDH) was used for correction to normalize the relative values. The CT value of the control group was taken as a reference to calculate the relative expression level of mRNA transcripts of each component.

[0050] Table 1 Primer sequences used in this study

[0051]

[0052] 1.2.7 Western blot

[0053] Proteins were separated by polyacrylamide gel electrophoresis. 20 μL of protein solution (2×10 5 sperm), RORα receptor protein was electrophoresed using 10% separation gel, working conditions: constant voltage 80V 30min, 120V 90min. The protein was transferred to PVDF membrane, working conditions: constant current 250mA 2 hours ice bath. After washing the membrane with PBS for 5min, add blocking solution, block at room temperature for 1 hour, add RORα protein primary antibody (1:2000), incubate at 37℃ for 2 hours. Wash the membrane with TBST 5 times, 5min each time. Goat anti-mouse secondary antibody (1:4000), incubate at room temperature for 2 hours, wash the membrane with TBST 5 times, 5min each time. Incubate with ECL for 3min, and expose the detection band.

[0054] 1.3 Statistical methods

[0055] SPSS 25.0 statistical software was used for one-way analysis of variance and Tukey's multiple comparison method to analyze the data and compare the significance of differences. The results were expressed as mean ± standard deviation (Mean ± SD), P < 0.05 indicated a significant difference, and P > 0.05 indicated an insignificant difference. Origin statistical drawing software was used to generate a bar graph. Image J image analysis software was used to calculate the optical density of the protein bands.

[0056] 2 Results

[0057] 2.1 Expression of nuclear receptor genes in frozen sperm

[0058] like Figure 1 As shown, the expression of nuclear receptor gene RORα mRNA and protein was present in frozen sperm.

[0059] 2.2 Effects of different treatments on frozen semen quality

[0060] After 2 hours of incubation with the four different treatments, the sperm motility and acrosome integrity were highest in the nuclear receptor agonist group, which was significantly different from the other groups (P<0.05) (Table 2).

[0061] Table 2 Effects of different treatments on semen quality

[0062]

[0063] Note: In the same column, different letters indicate significant differences (P<0.05), and the same letters indicate no significant differences (P>0.05).

[0064] 2.3 Effects of different treatments on nuclear receptor gene expression

[0065] Compared with the control group, after melatonin treatment, the expression of melatonin receptor gene RORα mRNA in sperm was significantly reduced, and the protein content was significantly increased; after adding nuclear receptor gene agonists, the expression of melatonin receptor gene RORα mRNA was significantly reduced, and the protein content was significantly increased; and after adding nuclear receptor inhibitors, the protein content of melatonin receptor gene RORα was significantly reduced. Figure 2 shown.

[0066] Table 3 Effects of different treatments on the relative expression of RORα

[0067]

[0068] It can be seen from this that the melatonin nuclear receptor gene promotes the protective effect of melatonin on sperm and improves the quality of frozen semen.

Claims

1. The use of melatonin nuclear receptor protein RORα agonist and melatonin in improving the quality of frozen semen, characterized in that: The method for improving semen quality includes improving sperm motility and improving sperm acrosome integrity, and the melatonin nuclear receptor protein RORα agonist is CGP52608.

2. The use according to claim 1, which improves semen quality by increasing the expression of melatonin nuclear receptor protein RORα and adding melatonin to semen.

3. The use according to claim 2, characterized in that After the frozen semen is thawed and sperm is recovered, PBS and semen diluent are added to resuspend the sperm, and then melatonin nuclear receptor protein RORα agonist and melatonin are added and incubated.

4. The use according to claim 3, characterized in that The melatonin nuclear receptor protein RORα agonist is added to a final concentration of 0.001-0.1 mM.

5. The use according to claim 4, characterized in that The melatonin nuclear receptor protein RORα agonist was added to a final concentration of 0.01 mM.

6. The use according to claim 3, characterized in that The melatonin is added to a final concentration of 0.001-0.1 mM.

7. The use according to claim 6, characterized in that The melatonin was added to a final concentration of 0.01 mM.

8. Use of melatonin and melatonin nuclear receptor protein RORα agonist in the preparation of a preparation for improving the quality of frozen semen, characterized in that: The molar ratio of melatonin to melatonin nuclear receptor protein RORα agonist in the preparation is 1-10:1-10; improving semen quality includes improving sperm motility and improving sperm acrosome integrity, and the melatonin nuclear receptor protein RORα agonist is CGP52608.

9. The use according to claim 8, characterized in that The molar ratio of melatonin to melatonin nuclear receptor protein RORα agonist is 1:1.

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