Method for improving in-vitro maturation quality and cryopreservation effect of porcine oocytes

Abstract

The invention discloses a method for improving in-vitro maturation quality and cryopreservation effect of porcine oocytes, and relates to the technical field of biology. By adding the NMN, the comprehensive efficiency of in-vitro maturation and cryopreservation of the porcine oocytes is remarkably improved. Research finds that by adding NMN into an IVM culture medium, maturation of cell nucleuses is promoted, discharge of first polar bodies of oocytes is improved, oxidative stress and lipid peroxidation of the oocytes are reduced, mitochondrial activity is improved, and cytoplasm maturation of the oocytes is facilitated. Meanwhile, after the NMN is used for pretreatment, the survival rate of the vitrified and frozen oocytes is remarkably improved, and the active oxygen level and the DNA breakage level are reduced. Therefore, the addition of the NMN in the IVM culture solution may become a new method for improving and optimizing the IVM technology, and an innovative solution is provided for pig germplasm resource preservation and excellent breeding stock breeding.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a method for improving the in vitro maturation quality and cryopreservation effect of pig oocytes. Background Art

[0002] In recent years, with the development and application of biotechnology, in vitro maturation and cryopreservation of oocytes have become important research areas, possessing significant theoretical and practical significance for in vitro production and the conservation of germplasm resources. In vitro maturation of oocytes involves the maturation of immature oocytes from the ovary under in vitro culture conditions. However, compared with in vivo maturation, the in vitro maturation of porcine oocytes is susceptible to various factors, including the environment in which the oocytes are collected, the season, and the in vitro culture system. This results in maturation rates and quality often failing to meet practical application requirements, thereby impacting the quality of cryopreserved germplasm. Cryopreservation is a commonly used method for preserving biological materials and holds broad application prospects. Cryopreservation can effectively extend the storage life of biological materials such as oocytes, embryos, and semen, providing a strong foundation for the development of animal husbandry. However, cryopreservation of porcine oocytes remains at the experimental stage. Due to their high lipid content and sensitivity to low temperatures, porcine oocytes are highly susceptible to cryoprotectant toxicity, osmotic stress, oxidative stress, and other damage during vitrification, resulting in damage to the cytoskeleton and subcellular organelles, ultimately leading to reduced cell developmental capacity or even cell death. The freezing and thawing process of oocytes can cause mitochondrial damage and affect mitochondrial function, manifesting as increased oxidative damage, decreased mitochondrial function, and increased production of reactive oxygen species (ROS). This leads to changes in cellular oxidative metabolism, damage to the oocyte's endogenous antioxidant system, and further leads to loss of oocyte viability and developmental potential. Therefore, improving the quality of oocyte in vitro maturation, reducing oocyte lipid droplet content, and enhancing anti-oxidative stress capacity are important approaches to enhance the efficacy of porcine oocyte cryopreservation.

[0003] Nicotinamide Mononucleotide (NMN) is a naturally occurring biologically active nucleotide and a derivative of vitamin B. It is a + ) and Nicotinamide Adenine Dinucleotide Phosphate (NADP +NMN is a natural nucleotide precursor in the biosynthesis pathway and is endogenous in all mammalian tissues. It has antioxidant, DNA damage reduction, mitochondrial homeostasis maintenance, and cytoskeletal maintenance functions. However, there is a lack of systematic research on the effects of NMN on in vitro maturation and vitrification of porcine oocytes. Summary of the Invention

[0004] In order to overcome the shortcomings and deficiencies of the prior art in the in vitro maturation quality and cryopreservation effect of porcine oocytes, the purpose of the present invention is to provide an application of nicotinamide mononucleotide in improving the in vitro maturation quality and cryopreservation effect of porcine oocytes.

[0005] Another object of the present invention is to provide a method for improving the in vitro maturation quality and cryopreservation effect of porcine oocytes.

[0006] The purpose of the present invention is achieved through the following technical solutions: Application of nicotinamide mononucleotide in improving the quality of in vitro maturation and cryopreservation of porcine oocytes.

[0007] Furthermore, the final concentration of nicotinamide mononucleotide is 0.1 to 10 μM; preferably 0.1 to 1 μM; more preferably 0.1 μM.

[0008] A method for improving the in vitro maturation quality and cryopreservation effect of porcine oocytes, comprising the following steps: (1) In vitro maturation of porcine oocytes: porcine oocytes are placed in a maturation culture medium containing nicotinamide mononucleotide to obtain in vitro matured oocytes; (2) Freezing of porcine oocytes: The in vitro matured porcine oocytes obtained in step (1) are vitrified and frozen.

[0009] Furthermore, in step (1), The final concentration of the nicotinamide mononucleotide is 0.1 to 10 μM, preferably 0.1 to 1 μM, and more preferably 0.1 μM.

[0010] The maturation culture medium is further supplemented with gonadotropin, growth factors, fetal bovine serum and L-cysteine.

[0011] The gonadotropins are pregnant mare serum gonadotropin and human chorionic gonadotropin; the growth factor is epidermal growth factor; Preferably, the concentration of pregnant mare serum gonadotropin in the maturation culture medium is 100 IU / mL; the concentration of human chorionic gonadotropin is 100 IU / mL; the concentration of epidermal growth factor is 10 ng / mL; and the concentration of L-cysteine ​​is 0.1 mg / mL.

[0012] The culture conditions are 39° C., 5% CO 2 , and saturated humidity for 42 to 44 hours.

[0013] Furthermore, in step (2), the freezing liquid used for vitrification includes ethylene glycol, propylene glycol and sucrose.

[0014] Preferably, the freezing solution contains 17.5% v / v ethylene glycol, 17.5% v / v propylene glycol and 0.3 mol / L sucrose.

[0015] More preferably, the freezing solution is a maintenance medium supplemented with 17.5% v / v ethylene glycol, 17.5% v / v propylene glycol and 0.3 mol / L sucrose; and the maintenance medium is TCM199+20% w / v fetal bovine serum.

[0016] The specific steps include: 1) In vitro maturation of porcine oocytes Cumulus-oocyte complexes (COCs) from follicles with a diameter of 2-8 mm were collected from porcine ovaries. After washing, 50-60 COCs / well were placed in 500 μL of maturation medium containing nicotinamide mononucleotide in a four-well plate and cultured at 39°C, 5% CO2, and saturated humidity to obtain in vitro matured oocytes. The maturation medium was TCM199 supplemented with 10% v / v porcine follicular fluid, 10% w / v fetal bovine serum, 100 IU / mL pregnant mare serum gonadotropin, 100 IU / mL human chorionic gonadotropin, 10 ng / mL epidermal growth factor, and 0.1 mg / mL L-cysteine. The final concentration of nicotinamide mononucleotide was 0.1-10 μM, preferably 0.1-1 μM, and more preferably 0.1 μM. 2) Freezing of porcine oocytes The in vitro matured oocytes obtained in step 1) were equilibrated in equilibration solution for 12–15 min, then transferred to freezing solution for equilibration for 25–30 s, aspirated into an OPS tube, and directly placed in liquid nitrogen for storage. The equilibration solution consisted of maintenance medium supplemented with 2% v / v ethylene glycol, 2% v / v propylene glycol, and 7.5 µg / mL cytochalasin B. The freezing solution consisted of maintenance medium supplemented with 17.5% v / v ethylene glycol, 17.5% v / v propylene glycol, and 0.3 mol / L sucrose. The maintenance medium consisted of TCM199 supplemented with 20% w / v fetal bovine serum.

[0017] The present invention has the following advantages and effects compared to the prior art: The present invention significantly improves the overall efficiency of in vitro maturation and cryopreservation of porcine oocytes by adding NMN. Given the importance of promoting the synchronous development of the nucleus and cytoplasm during in vitro oocyte maturation (IVM), cytoplasmic maturation is crucial for ensuring adequate metabolic reserves, mitochondrial function, and molecular regulatory capacity in oocytes, directly impacting fertilization success rates and embryonic developmental potential. The present invention's research found that the addition of NMN to IVM culture medium promoted nuclear maturation, aided in the extrusion of the first polar body, reduced oxidative stress and lipid peroxidation in oocytes, and enhanced mitochondrial activity, thus promoting cytoplasmic maturation. Furthermore, pretreatment with NMN significantly improved the survival rate of oocytes after vitrification, while reducing reactive oxygen species levels and DNA fragmentation. Therefore, the addition of NMN to IVM culture medium may represent a novel approach to improve and optimize IVM technology, providing an innovative solution for the preservation of porcine germplasm resources and the breeding of high-quality breeding stock. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 It is the effect of NMN on the rate of first polar body extrusion of oocytes.

[0019] Figure 2 The effect of NMN on the level of reactive oxygen species in oocytes. Note: Scale bar: 100 µm.

[0020] Figure 3 The effect of NMN on the lipid droplet content in oocytes; Note: Scale bar: 100 µm.

[0021] Figure 4 The effect of NMN on the mitochondrial membrane potential of oocytes. Note: Scale bar: 100 µm.

[0022] Figure 5 The effect of NMN on the survival rate of oocytes after freezing and thawing.

[0023] Figure 6 The effect of NMN on the reactive oxygen species level in oocytes after cryo-thawing. Note: Scale bar: 100 µm.

[0024] Figure 7 The effect of NMN on the degree of DNA damage in oocytes after cryo-thawing. Note: Scale bar: 20 µm.

[0025] Among them, * P < 0.05, ** P < 0.01, *** P < 0.001. DETAILED DESCRIPTION

[0026] The present invention is described in further detail below with reference to the examples and accompanying drawings, but the present invention is not limited thereto. Experimental methods in the following examples, where specific experimental conditions are not specified, generally followed conventional experimental conditions or those recommended by the manufacturer. Materials and reagents used were commercially available unless otherwise specified.

[0027] Example 1 1. Oocyte collection After pig ovaries were collected from the slaughterhouse, they were placed in a thermos containing 37°C saline (with penicillin and streptomycin) and transported to the laboratory within 2-4 hours. The ovaries were then washed three to four times with 37°C saline containing 1%-2% double-streptomycin (penicillin and streptomycin) and placed in a 38°C water bath until ready for use. Follicles with a diameter of 2-8 mm were aspirated from the pig ovaries using a 10 mL sterile syringe. The aspirated follicular fluid was slowly injected into a 50 mL sterile centrifuge tube preheated in a 38°C water bath. The tubes were allowed to settle naturally for 10 minutes, after which the supernatant was removed. The tubes were then poured into a preheated 37°C DPBS-PVA solution, gently shaken, and allowed to settle for another 10 minutes before the supernatant was removed. This was repeated two to three times. Under a stereomicroscope, quickly select cumulus-oocyte complexes (COCs) that are wrapped in multiple layers of cumulus, have no exposed zona pellucida, and have uniform cytoplasm. Wash them 2-3 times with 37°C DPBS-PVA solution, and then wash them 2-3 times with maturation culture medium that has been equilibrated overnight in advance. After washing and collection, prepare for the next step of culture.

[0028] The DPBS-PVA solution is prepared by dissolving 1 g of PVA and 9.6 g of DPBS in ddH2O, diluting to 1 L, filtering with a 0.22 µm filter, and aliquoting. The solution is then stored at 4°C for later use.

[0029] 2. Oocyte maturation culture in vitro Different concentrations of NMN (0.1, 1, and 10 μM) were added to the maturation medium. Groups of 50–60 COCs were cultured in four-well plates containing various concentrations of NMN. Each well contained 500 μL of maturation medium and covered with 200 μL of paraffin oil. The cells were incubated in a 39°C, 5% CO2, saturated humidity incubator for 44 hours. The oocytes were then repeatedly pipetted in 0.1% hyaluronidase to remove cumulus cells. Oocytes with homogeneous cytoplasm and the extrusion of the first polar body (MII stage) were selected for subsequent experiments.

[0030] The NMN was purchased from Sigma (N3501, ≥95% HPLC), and 3.3422 mg of NMN was dissolved in 2 mL of TCM199 culture medium. After sufficient dissolution, the solution was filtered through a 0.22 µm filter and aliquoted, and stored at -80°C for later use.

[0031] The maturation culture medium is TCM199 supplemented with 10% (v / v) porcine follicular fluid (pFF), 10% (w / v) fetal bovine serum, 100 IU / mL pregnant mare serum gonadotropin (eCG), 100 IU / mL human chorionic gonadotropin (hCG), 10 ng / mL epidermal growth factor (EGF), and 0.1 mg / mL L-cysteine.

[0032] 3. Vitrification and thawing The freezing carrier is an OPS tube, which is softened by the outer flame of an alcohol lamp and then drawn.

[0033] Freezing process: The equilibration solution and freezing solution are placed in the incubator for equilibration for more than 3 hours in advance. The prepared MII stage oocytes are transferred to the equilibration solution for 12-15 minutes and then transferred to the freezing solution. The cell volume can be seen to shrink rapidly and become a crescent shape. The oocytes in the freezing solution are aspirated with an OPS tube within 30 seconds to avoid excessive dehydration of the cells. The liquid in the tube is controlled within 1 μL. After loading, the oocytes are quickly placed in liquid nitrogen for storage. The oocytes should be stored in liquid nitrogen for at least one day.

[0034] Thawing process: During thawing, use pointed tweezers to pick up the end of the OPS tube, and move half of the total length of the tube out of the liquid nitrogen level to allow the gas in the tube to escape from the end. After no gas escapes, remove the tube from the liquid nitrogen, and transfer the oocytes in the tube into the thawing solution and maintenance medium (HM) balanced overnight for 2 minutes each. Wash with HM 2-3 times, transfer to a culture dish containing HM, and recover in the incubator for 2 hours before subsequent experiments.

[0035] The maintenance medium (HM) is TCM199 + 20% (w / v) fetal bovine serum (FBS), the equilibration solution is the maintenance medium supplemented with 2% (v / v) ethylene glycol, 2% (v / v) propylene glycol, and 7.5 µg / mL cytochalasin B (cB), the freezing solution is the maintenance medium supplemented with 17.5% (v / v) ethylene glycol, 17.5% (v / v) propylene glycol, and 0.3 mol / L sucrose, and the thawing solution is the sucrose thawing solution prepared in the maintenance medium with concentrations of 0.4, 0.2, 0.1, and 0.05 mol / L.

[0036] 4. Oocyte Lipid Droplet Content Detection Cells were fixed overnight in PBS containing 4% (w / v) paraformaldehyde at 4°C and then washed three times in DPBS-PVA solution. Concentrated BODIPY (boron dipyrrolidone) stock dye was diluted 1:1000 in PBS and the cells were transferred to the staining solution. The cells were incubated at room temperature for 25 minutes and then washed three times in DPBS-PVA solution. The stained cells were imaged using an inverted fluorescence microscope, and fluorescence intensity was analyzed using Image J software.

[0037] 5. Detection of Oocyte Reactive Oxygen Species Levels Oocyte reactive oxygen species (ROS) levels were measured using a Beyotime assay kit. Oocytes were transferred to a DCFH-DA probe dye solution diluted 1:1000 in DPBS-PVA. After incubation in a CO2 incubator for 20 minutes, the cells were washed three times in DPBS-PVA to remove excess dye. The stained cells were imaged using an inverted fluorescence microscope, and fluorescence intensity was analyzed using Image J software.

[0038] 6. Oocyte mitochondrial membrane potential detection Changes in oocyte mitochondrial membrane potential were measured using a Beyotime assay kit. Oocytes, free of cumulus granulosa cells and expelled from the first polar body, were transferred to JC-1 staining solution and incubated in a CO2 incubator for 30 minutes. The cells were then washed three times with JC-1 buffer to remove excess dye. The stained cells were imaged using an inverted fluorescence microscope, and fluorescence intensity was analyzed using Image J software.

[0039] 7. Oocyte immunofluorescence detection Oocytes were fixed in 4% (w / v) paraformaldehyde overnight at 4°C and then permeabilized with 0.2% Triton X-100 in PBS for 30 minutes. Cells were then transferred to 1% (w / v) bovine serum albumin (BSA) in PBS and blocked for 1 hour at room temperature. Oocytes were incubated with the primary antibody γ-H2A.X (1:200, 9718T, CST, USA) overnight at 4°C. Following incubation, the cells were washed by pipetting in DPBS-PVA. Samples were incubated with the secondary antibody goat anti-rabbit IgG H&L (1:200, #S0008, Affinity, China) for 2 hours and then washed by pipetting in DPBS-PVA. Cells were transferred to slides coated with an anti-quencher solution and observed using laser confocal microscopy.

[0040] 8. Experimental Results (1) The sign of oocytes entering the MII stage is the extrusion of the first polar body. 0 (control), 0.1, 1, and 10 µM NMN were added to the oocyte maturation culture medium. After 44 h of in vitro culture, the number of cells that extruded the first polar body was counted to evaluate the effect of NMN on the extrusion rate of the first polar body of porcine oocytes. The results were ( Figure 1 ) showed that the first polar body extrusion rate after adding NMN to the mature culture medium was significantly increased compared with the control group (the three concentration groups were 84.23±1.49% respectively). vs. 84.87±1.32% vs.The difference between the experimental and control groups was 82.43±0.65% (P<0.001), but there was no difference between the experimental groups. Therefore, the addition of NMN to the maturation medium helps to increase the rate of first polar body extrusion in oocytes.

[0041] (2) Insert fluorescent probes to detect ROS levels in different groups of cells and analyze the fluorescence intensity. The results are shown in Figure 2 It can be seen that the reactive oxygen content in the NMN-treated group showed a downward trend compared with the control group, but there was no significant difference between the 10 µM NMN group and the control group, and there was a significant difference between the 1 µM NMN group and the control group (P<0.05). Among them, the reactive oxygen content in the 0.1 µM NMN group was the lowest (P<0.01), indicating that NMN treatment reduced the accumulation of reactive oxygen species in oocytes during in vitro maturation.

[0042] (3) Pig oocytes contain a large amount of lipids. To explore whether NMN can improve the lipid metabolism of oocytes, fluorescent probes were inserted into oocytes to detect the intracellular lipid droplet content. The fluorescence intensity of lipid droplets in pig oocytes after treatment with different concentrations of NMN was compared. The results were ( Figure 3 ) It can be seen that after NMN treatment, the lipid droplet content in oocytes decreased significantly (P<0.05), among which the lipid droplet content in the 1 and 10 µM NMN groups was extremely significantly lower than that in the control group (P<0.001).

[0043] (4) Insert fluorescent probes to detect the mitochondrial membrane potential difference of different groups of cells. Results ( Figure 4 ) showed that the mitochondrial activity of the NMN-treated group increased compared with the control group, but the difference between the 10 µM NMN group and the control group was not significant, while the difference between the 1 µM NMN group and the control group was significant (P<0.05), among which the mitochondrial activity of the 0.1 µM NMN group was the highest (P<0.01).

[0044] (5) The oocytes were vitrified and cultured for 2 h after thawing to calculate their survival rate. The results are shown in Figure 5 The survival rate of MII stage oocytes cultured with 0.1 µM NMN after thawing was significantly higher than that of the control group and other treatment groups (43.83±4.46% vs. 36.28±5.53% for the two concentration groups, and 32.68±3.45% for the control group) (P<0.01).

[0045] (6) The content of reactive oxygen species in thawed oocytes was significantly higher than that in fresh oocytes (Control) (P<0.001) ( Figure 6), indicating that the vitrification process causes oxidative stress in oocytes and accumulation of excessive reactive oxygen species. The reactive oxygen species content in oocytes treated with 0.1 µM NMN was significantly reduced compared with the frozen control group (Control-V) (P<0.05), and the reactive oxygen species content in the 1 µM NMN treatment group was extremely significantly reduced compared with the frozen oocyte control group (P<0.01) ( Figure 6 ). This indicates that adding NMN to oocytes during in vitro maturation can alleviate the oxidative stress caused by the vitrification process on cells.

[0046] (7) Immunofluorescence staining of fresh oocytes (Control), frozen oocytes (Control-V) and frozen oocytes treated with NMN (0.1, 1 μM NMN-V) was performed using γH2A.X. The levels of DNA double-strand breaks in different treatment groups were compared based on the fluorescence intensity. The results were ( Figure 7 ) showed that the abundance of DNA double-strand breaks in frozen-thawed oocytes was extremely significantly increased after freezing (P<0.001), while the abundance of DNA double-strand breaks in frozen-thawed oocytes treated with 0.1 µM NMN was significantly reduced compared with the Control-V group (P<0.05), and extremely significantly reduced in the 1 µM NMN treatment group (P<0.001).

[0047] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be considered as equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. Application of nicotinamide mononucleotide in improving the in vitro maturation quality and cryopreservation effect of porcine oocytes.

2. The use according to claim 1, characterized in that: The final concentration of nicotinamide mononucleotide is 0.1 to 10 μM; further 0.1 to 1 μM; further 0.1 μM.

3. A method for improving the in vitro maturation quality and cryopreservation effect of porcine oocytes, characterized in that: The steps include: (1) In vitro maturation of porcine oocytes: porcine oocytes are placed in a maturation culture medium containing nicotinamide mononucleotide to obtain in vitro matured oocytes; (2) Freezing of porcine oocytes: The in vitro matured porcine oocytes obtained in step (1) are vitrified and frozen.

4. The method according to claim 3, wherein: In step (1), the final concentration of the nicotinamide mononucleotide is 0.1 to 10 μM; further 0.1 to 1 μM; further 0.1 μM.

5. The method according to claim 3, wherein: In step (1), gonadotropin, growth factor, fetal bovine serum and L-cysteine ​​are also added to the maturation culture medium; further, the gonadotropin is pregnant mare serum gonadotropin and human chorionic gonadotropin; and the growth factor is epidermal growth factor.

6. The method according to claim 5, characterized in that: The concentration of pregnant mare serum gonadotropin in the maturation culture medium is 100 IU / mL; the concentration of human chorionic gonadotropin is 100 IU / mL; the concentration of epidermal growth factor is 10 ng / mL; and the concentration of L-cysteine ​​is 0.1 mg / mL.

7. The method according to any one of claims 3 to 6, characterized in that: In step (1), the culture conditions are 39° C., 5% CO 2 , and saturated humidity for 42 to 44 hours.

8. The method according to any one of claims 3 to 6, characterized in that: In step (2), the freezing liquid used for vitrification includes ethylene glycol, propylene glycol and sucrose; further, the freezing liquid contains 17.5% v / v ethylene glycol, 17.5% v / v propylene glycol and 0.3 mol / L sucrose.

9. The method according to claim 8, characterized in that: The freezing solution is a maintenance medium supplemented with 17.5% v / v ethylene glycol, 17.5% v / v propylene glycol and 0.3 mol / L sucrose; the maintenance medium is TCM199+20% w / v fetal bovine serum.

10. The method according to claim 9, characterized in that The specific steps include: 1) In vitro maturation of porcine oocytes Cumulus-oocyte complexes (COCs) from follicles with a diameter of 2-8 mm were collected from porcine ovaries. After washing, 50-60 COCs / well were placed in 500 μL of maturation medium containing nicotinamide mononucleotide in a four-well plate and cultured at 39°C, 5% CO2, and saturated humidity to obtain in vitro matured oocytes. The maturation medium was TCM199 supplemented with 10% v / v porcine follicular fluid, 10% w / v fetal bovine serum, 100 IU / mL pregnant mare serum gonadotropin, 100 IU / mL human chorionic gonadotropin, 10 ng / mL epidermal growth factor, and 0.1 mg / mL L-cysteine. The final concentration of nicotinamide mononucleotide was 0.1-10 μM; further 0.1-1 μM; and further 0.1 μM. 2) Freezing of porcine oocytes The in vitro matured oocytes obtained in step 1) were equilibrated in equilibration solution for 12–15 min, then transferred to freezing solution for equilibration for 25–30 s, aspirated into an OPS tube, and directly placed in liquid nitrogen for storage. The equilibration solution consisted of maintenance medium supplemented with 2% v / v ethylene glycol, 2% v / v propylene glycol, and 7.5 µg / mL cytochalasin B. The freezing solution consisted of maintenance medium supplemented with 17.5% v / v ethylene glycol, 17.5% v / v propylene glycol, and 0.3 mol / L sucrose. The maintenance medium consisted of TCM199 supplemented with 20% w / v fetal bovine serum.

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