Application of ceramide in promoting in-vitro maturation of oocytes

Abstract

The invention provides application of ceramide in promoting in-vitro maturation of oocytes. By carrying out maturation culture on the oocytes in the maturation liquid containing ceramide, the in-vitro maturation rate of the oocytes can be effectively improved, and the development rate of in-vitro fertilization early embryos can be improved. The invention also provides a preparation for promoting in-vitro maturation of oocytes. The core component of the preparation is ceramide with an effective amount. By regulating and controlling the maturation microenvironment of the oocytes, the bottleneck of low efficiency of a traditional culture system is broken through, and an innovative solution is provided for an assisted reproduction technology. The method has a good application prospect.

Description

TECHNICAL FIELD

[0001] The present invention relates to the field of propagation. In particular, it relates to the use of ceramide Cer(d18:1 / 26:0) for promoting in vitro maturation of oocytes. BACKGROUND

[0002] The establishment of in vitro embryo production (IVP) technology system provides an innovative solution for efficient use of high-quality germplasm resources. This technology integrates the genetic advantages of good bull and the characteristics of core cow germplasm, enabling the large-scale preparation of high-quality embryos. Its application value lies in three aspects: first, it deeply excavates the reproductive potential of excellent cows; second, it accelerates the process of germplasm resource propagation; and third, it builds a biological germplasm resource protection technology system. In vitro maturation of oocytes (IVM) is an important part of IVP. Through in vitro maturation culture of ovarian antral follicle oocytes, not only does it effectively solve the key problem of insufficient egg supply in embryo engineering, but also provides technical support for modern biological breeding technology innovation.

[0003] Oocyte maturation involves two coordinated processes, nuclear maturation and cytoplasmic maturation, involving a series of complex nuclear-cytoplasmic remodeling events. These intrinsic changes lay the foundation for molecular and cellular biology for post-fertilization embryo development and zygotic genome activation. The core marker of nuclear maturation is the restoration and completion of the first meiotic division in oocytes, ultimately arresting at the second meiotic metaphase (MII phase). Key events include germinal vesicle breakdown (GVBD), chromosome condensation, spindle assembly, homologous chromosome separation, and first polar body extrusion. MII phase restoration requires the activation of fertilization signals, accompanied by a series of epigenetic reprogramming events to ensure that chromatin is in a state that can be fertilized and support early embryo development. The core of cytoplasmic maturation is the accumulation of maternal factors (such as specific mRNAs, proteins, and metabolic substrates) to ensure the autonomous development of early embryos after fertilization. At the same time, various organelles (such as mitochondria, endoplasmic reticulum, microtubules, etc.) coordinate to promote the synchronization of cytoplasmic maturation and nuclear maturation by regulating protein synthesis, energy supply, material transport, and accurate chromosome separation. As an important indicator for evaluating the state of cytoplasmic maturation, cortical granules undergo dynamic changes: initially widely distributed in the cytoplasm, they gradually migrate to the cortical region as maturation progresses, and finally uniformly distributed in the MII phase oocyte cortical region, forming a high-density distribution band adjacent to the plasma membrane.

[0004] In vitro maturation (IVM) is a critical step for oocyte maturation, which directly affects the success rate of fertilization and the potential of subsequent development to the transplantable blastocyst. However, oocytes obtained in vitro are usually immature, and their meiosis can resume spontaneously after leaving the high-concentration cAMP physiological inhibition microenvironment in the follicle. However, the asynchronous phenomenon between nuclear maturation and cytoplasmic maturation often occurs due to the disorder of cytoplasmic maturation regulation pathway, which further affects the developmental outcome of embryos. Studies have shown that the blastocyst rate of in vivo matured oocytes after fertilization can reach 80%, while the blastocyst rate of in vitro matured oocytes after fertilization is only about 35%, nearly 2.3 times the difference in development efficiency, revealing the importance of achieving nuclear-cytoplasmic maturation in in vitro culture system. Follicle is the basic functional unit of oocyte occurrence and development, which contains granulosa cells, membrane cells, secretory factors, and various substances permeated by plasma. The presence of the above components makes the follicular microenvironment function like a natural medium, providing a substrate for oocyte metabolism and affecting the growth and development of oocytes. Many studies have shown that the developmental potential of oocytes obtained from pre-ovulatory follicles is different, and one of the reasons for this difference is that some components of the follicular microenvironment have changed. However, the influence of follicular microenvironment on the developmental capacity of oocytes still needs further research.

[0005] Cer(d18:1 / 26:0) is also known as N-dodecosyl-sphingosine-4-ene, a ceramide molecule. Ceramide is a central molecule in sphingolipid metabolism and is the precursor of sphingomyelin, ceramide-1-phosphate, and glucosylceramide. As a key structural component of the cell membrane, ceramide significantly changes the physical properties of the cell membrane (such as fluidity, microdomain reorganization, and permeability) by enhancing intermolecular forces and promoting lipid ordering, thereby precisely regulating the conformational changes of membrane proteins and the efficiency of material transport across the membrane. In addition, ceramide can also act as an important lipid second messenger, directly involved in various cell fate decisions. High levels of ceramide often act as a stress response signal (such as oxidative stress, DNA damage, and cytokine stimulation), inducing cell cycle arrest, cell senescence, and programmed cell death (apoptosis). Notably, its downstream metabolite sphingosine-1-phosphate (S1P) often has an antagonistic function to ceramide (such as promoting proliferation, anti-apoptosis, and promoting migration). This "ceramide / S1P two-way metabolic switch" regulates the balance of sphingolipid metabolism flow to dynamically maintain the homeostasis of the intracellular environment.

[0006] Ceramides have been studied and used in a variety of diseases, including skin barrier-related diseases, metabolic diseases, cardiovascular diseases, neurodegenerative diseases and cancers, etc., and show great application prospects and development potential. At present, the patents related to ceramides are mainly concentrated in the extraction and preparation technology and the application of disease intervention, such as the application of ceramides in cosmetics (CN119792132A), the application of ceramides in anti-tumor (CN119792335A), the application of ceramides in Parkinson's disease (CN119355184A), ceramides as biomarkers for drug-induced liver injury diagnosis (CN119881183A), the application of ceramides in skin repair (CN117653567B), the application of ceramides in the treatment of bacterial and fungal infections (AU6856401A) and the like. However, the application of Cer(d18:1 / 26:0) in in vitro matured oocytes has not been reported so far. SUMMARY

[0007] The first object of the present application is to provide a new use of Cer(d18:1 / 26:0).

[0008] The second object of the present application is to provide a culture solution for promoting in vitro maturation of oocytes.

[0009] The present application obtains a ceramide lipid small molecule Cer(d18:1 / 26:0) through metabolomics detection and screening of follicular fluid. It is found that the content of Cer(d18:1 / 26:0) in the follicular fluid corresponding to the oocyte with higher development potential is higher. Further research shows that Cer(d18:1 / 26:0) can improve the development potential of bovine in vitro matured oocytes.

[0010] Accordingly, the present application first provides the use of Cer(d18:1 / 26:0) in improving the development potential of in vitro matured oocytes.

[0011] Further, the development potential includes at least one of the in vitro maturation rate of oocytes and the development rate of in vitro fertilized early embryos.

[0012] The present application further provides a method for promoting in vitro maturation of oocytes, which is to mature and culture oocytes in an in vitro maturation solution containing Cer(d18:1 / 26:0).

[0013] Further, the concentration of the Cer(d18:1 / 26:0) is 0.1-10 μg / mL. Preferably, the concentration of the Cer(d18:1 / 26:0) is 1 μg / mL. The in vitro maturation potential of oocytes can be used as the effective amount of the Cer(d18:1 / 26:0). In practice, the effective amount of the Cer(d18:1 / 26:0) can be determined according to the method provided in the embodiments of the present application.

[0014] Further, the oocytes include, but are not limited to, bovine, equine, donkey, sheep and other mammals.

[0015] Further, the present application also provides the use of the Cer(d18:1 / 26:0) in the preparation of a preparation for promoting the in vitro maturation of oocytes.

[0016] Further, the present application also provides a preparation comprising an effective amount of the Cer(d18:1 / 26:0). Preferably, the concentration of the Cer(d18:1 / 26:0) is 0.1-10 μg / mL. More preferably, the concentration of the Cer(d18:1 / 26:0) is 1 μg / mL.

[0017] In one embodiment of the present application, the preparation further comprises TCM199 (1×), 0.01 IU / mL FSH, 0.01 IU / mL LH, 1 μg / mL E2, 10% FBS, 10 μg / mL heparin.

[0018] The present application first adds the Cer(d18:1 / 26:0) in the in vitro maturation culture medium of oocytes, promotes the in vitro maturation rate and in vitro development ability of oocytes, and provides a new use of the Cer(d18:1 / 26:0). The addition of the Cer(d18:1 / 26:0) can significantly improve the quality of the nuclear and cytoplasmic maturation of bovine oocytes. The method and the preparation of the present application have good application prospects. BRIEF DESCRIPTION OF DRAWINGS

[0019] Figure 1 Effect of adding 1 μg / mL Cer(d18:1 / 26:0) in the maturation medium on the cytoplasmic maturation of bovine oocytes; red fluorescence represents the expression and distribution of F-actin, and green fluorescence represents the expression and distribution of cortical granules. Different letters (a, b) represent significant differences (P<0.05).

[0020] Figure 2The effect of adding 1 μg / mL Cer(d18:1 / 26:0) to the maturation solution on the quality of bovine in vitro fertilized blastocysts was investigated. Green fluorescence represents the expression of SOX2, a marker protein of the inner cell mass, and red fluorescence represents the expression of CDX2, a marker protein of the trophoblast. Different letters (a, b) indicate significant differences (P<0.05). Detailed Implementation

[0021] The following embodiments are used to further illustrate the present invention, but should not be construed as limiting the present invention. Any modifications or refinements to the technical solutions of the present invention without departing from the spirit and substance of the present invention shall fall within the scope of the present invention.

[0022] The ovaries used in the experiment were collected from a slaughterhouse in Dachang County, Langfang City, Hebei Province. TCM199 and fetal bovine serum used in this invention were purchased from Gibco, and other reagents, unless otherwise specified, were purchased from Sigma-Aldrich.

[0023] Example 1 1. In vitro maturation of oocytes (1) Oocyte collection Ovaries were collected from the slaughterhouse and placed in sterile saline containing antibiotics at 28-30°C. They were then transported back to the laboratory within 2 hours. The ovaries were washed 2-3 times with preheated sterile saline at approximately 38°C. Follicular fluid was extracted from follicles 2-8 mm in diameter on the ovaries using a 10 mL syringe. Under a stereomicroscope, cumulus-oocyte complexes (COCs) with uniform cytoplasm and at least two layers of dense cumulus cells were selected using an oral pipette for in vitro maturation.

[0024] (2) In vitro maturation of oocytes The selected COCs were washed twice in the egg washing solution and then three times in the maturation solution. They were then transferred into four-well plates (750 μL of maturation solution per well, 50 COCs) that had been equilibrated in a CO2 incubator for at least 2 h and were matured in a CO2 incubator at 38.5℃, 5% CO2, and saturated humidity for 23 h.

[0025] The egg washing solution described in (2) is TCM199 (10×), 0.22 g / L sodium pyruvate, 3.58 g / L Hepes, 0.146 g / L L-glutamine, 0.168 g / L NaHCO3, 10 mg / L heparin, 2% FBS, and 1% penicillin and streptomycin.

[0026] (2) The oocyte in vitro maturation solution described in the text is: TCM199 (1×), 0.01 IU / mL FSH, 0.01 IU / mL LHLH, 1 μg / mL E2, 10% FBS, 10 μg / mL heparin, 1% penicillin and streptomycin, and different concentrations (0, 0.5 μg / mL, 1 μg / mL, 5 μg / mL and 10 μg / mL) of Cer (d18:1 / 26:0).

[0027] 2. In vitro fertilization (1) Semen processing The frozen bovine semen was removed from the liquid nitrogen tank and thawed in a water bath at 38.5°C. The semen was then placed in a 15 mL centrifuge tube containing 6 mL of semen wash solution and centrifuged at 1800 rpm for 5 min. The supernatant was discarded, and the washing process was repeated once. The sperm were resuspended in the wash solution, and the sperm density was adjusted to 2 × 10⁻⁶ cells / mL using a hemocytometer under a microscope. 6 Quantity / mL, incubate in an incubator for later use.

[0028] (2) In vitro fertilization Cocci maturing in vitro for 23 hours were washed three times with fertilization solution and then transferred into microdroplets of fertilization solution that had been equilibrated in an incubator for at least 2 hours, with 15-20 cocci per 50 μL of fertilization solution. 50 μL of diluted sperm suspension was added to the fertilization droplets containing cocci, and the sperm and eggs were incubated for 13 hours in a CO2 incubator at 38.5℃, 5% CO2, and saturated humidity.

[0029] The semen washing solution described in (1) consists of 112 mM NaCl, 4.02 mM KCl, 2.25 mM CaCl2·2H2O, 0.83 mM NaH2PO4·H2O, 0.52 mM MgCl2·6H2O, 37 mM NaHCO3, 1.25 mM sodium pyruvate, 10 μg / mL heparin, 10 mM caffeine, and 4 mg / mL bovine serum albumin (BSA).

[0030] (2) The fertilization fluid described is 112 mM NaCl, 4.02 mM KCl, 2.25 mM CaCl2·2H2O, 0.83 mM NaH2PO4·H2O, 0.52 mM MgCl2·6H2O, 37 mM NaHCO3, 1.25 mM sodium pyruvate, 10 μg / mL heparin, and 4 mg / mL BSA.

[0031] 3. In vitro embryo culture After fertilization, the COCs were repeatedly and gently blown with a pipette to remove the cumulus cells, and the removal was observed under a stereomicroscope. After most of the COCs were removed, the fertilized eggs were washed three times with the pre-culture medium, and then were transferred into the pre-culture medium in a culture box which had been balanced for at least 2 hours. 20-25 fertilized eggs were placed in 100 μL of the pre-culture medium. The cleavage rate was calculated after 48 hours. All the cleaved embryos were transferred into the post-culture medium, and the medium was changed every other day. After 7 days of in vitro culture, the blastocyst development rate was calculated.

[0032] The pre-culture medium for the embryos was as follows: 109.5 mM NaCl, 3.1 mM KCl, 26.2 mM NaHCO3, 0.8 mM MgCl2·6H2O, 1.19 mM KH2PO4, 0.4 mM sodium pyruvate, 1.5 mM glucose, 5 mM calcium lactobionate, 6 mg / mL BSA, 50× essential amino acids, 100× non-essential amino acids, and 0.15 mg / mL glutamine.

[0033] The post-culture medium for the embryos was as follows: 109.5 mM NaCl, 3.1 mM KCl, 26.2 mM NaHCO3, 0.8 mM MgCl2·6H2O, 1.19 mM KH2PO4, 0.4 mM sodium pyruvate, 1.5 mM glucose, 5 mM calcium lactobionate, 10% FBS, 50× essential amino acids, 100× non-essential amino acids, and 0.15 mg / mL glutamine.

[0034] 4. Immunofluorescence Reagent preparation: (1) 4% paraformaldehyde (PFA): 2 g of PFA and 50 mg of PVA were weighed and dissolved in 50 mL of DPBS without calcium and magnesium in a water bath to promote dissolution. After complete dissolution, the solution was stored at 4°C or room temperature for later use. (2) Immunostaining permeation solution: 1% Triton X-100 diluted with PBS to volume. (3) Immunostaining washing solution: 0.1% Tween 20 and 0.01% Triton X-100 diluted with PBS to volume. (4) Immunostaining blocking solution: 3% BSA diluted with the immunostaining washing solution to volume.

[0035] Method: (1) Fixation: Take the embryos to be dyed, wash them once with DPBS, fix the embryos with 4% PFA for 30 min, transfer the embryos into the washing solution, and wash them 3 times (5 min each time) on a horizontal shaker at a speed of 120 rpm. (2) Permeation: Permeate the embryos with 0.5% Triton X-100 for 15 min to ensure that the antibodies can reach the antigen sites. Remove the permeation solution, add the washing solution, and wash the embryos 3 times (5 min each time) on a horizontal shaker at a speed of 120 rpm. (3) Blocking: Add 3% BSA blocking solution, and block the embryos on a shaker for 1 h at a speed of 120 rpm. (4) Primary antibody binding. Dilute the primary antibody according to the corresponding proportion, incubate the embryos on a shaker at 4°C overnight, then wash the embryos 3 times (5 min each time) on a horizontal shaker at a speed of 120 rpm. The primary antibody can be diluted with the blocking solution, and the diluted primary antibody can be used repeatedly for 3 times under normal circumstances. (5) Secondary antibody binding. From this step, all operations are carried out in the dark. Dilute the secondary antibody with the washing solution according to the species of the corresponding antibody of the primary antibody, incubate the embryos on a horizontal shaker at room temperature for 1 h at a speed of 50 rpm, then wash the embryos 3 times (5 min each time) on a horizontal shaker at a speed of 120 rpm. Note: The secondary antibody should be centrifuged before use, and the upper antibody should be aspirated for dilution, otherwise non-specific impurities may be easily generated, which are difficult to remove and affect the final observation. (6) Add 1:10,000 diluted DAPI to stain the cell nuclei, and observe after staining for 3 min on a horizontal shaker.

[0036] 5. Data statistics The experimental data were analyzed by SAS statistical software, and the significance of differences between different treatments was judged by one-way ANOVA and Duncan's test, and P<0.05 was considered to be significant.

[0037] 6. The addition of Cer(d18:1 / 26:0) in the maturation medium can significantly improve the in vitro maturation rate of bovine oocytes Different concentrations (0, 0.5 μg / mL, 1 μg / mL, 5 μg / mL, and 10 μg / mL) of Cer(d18:1 / 26:0) were added to the maturation medium, and the first polar body extrusion rate of oocytes was counted (Table 1). The results showed that, compared with the control group, 1 μg / mL Cer(d18:1 / 26:0) significantly improved the first polar body extrusion rate of oocytes, and 5 μg / mL and 10 μg / mL Cer(d18:1 / 26:0) significantly reduced the first polar body extrusion rate of oocytes (P<0.05); the difference between the 0.5 μg / mL Cer(d18:1 / 26:0) addition group and the control group was not significant (P>0.05).

[0038] Table 1 Effects of different concentrations of Cer(d18:1 / 26:0) on bovine oocyte in vitro maturation Note: The first polar body discharge rate = the number of oocytes that discharged the first polar body / the number of oocytes; different letters (a-c) in the same column represent significant differences (P<0.05).

[0039] 7. The addition of 1 μg / mL Cer(d18:1 / 26:0) in the maturation solution can promote the cytoplasmic maturation of bovine oocytes The addition of 1 μg / mL Cer(d18:1 / 26:0) in the maturation solution was evaluated for its effect on oocyte cytoplasmic maturation (). Figure 1 The results showed that the expression levels of F-actin and cortical granules in the 1 μg / mL Cer(d18:1 / 26:0) addition group were significantly higher than those in the control group (P<0.05). The results showed that the addition of 1 μg / mL Cer(d18:1 / 26:0) in the maturation solution can promote the cytoplasmic maturation of oocytes.

[0040] 8. The addition of Cer(d18:1 / 26:0) in the maturation solution can improve the development rate of bovine in vitro fertilization embryos By counting the cleavage rate and blastocyst rate of in vitro fertilization embryos, the effects of different concentrations of Cer(d18:1 / 26:0) added in the maturation solution on the development rate of in vitro fertilization embryos were compared and analyzed (Table 2). The results showed that the cleavage rate and blastocyst rate of in vitro fertilization in the 1 μg / mL Cer(d18:1 / 26:0) addition group were significantly higher than those in the control group (P<0.05), the cleavage rate and blastocyst rate in the 5 μg / mL and 10 μg / mL Cer(d18:1 / 26:0) addition groups were significantly lower than those in the control group (P<0.05), and there was no significant difference between the 0.5 μg / mL Cer(d18:1 / 26:0) addition group and the control group (P>0.05).

[0041] Table 2 Effects of different concentrations of Cer(d18:1 / 26:0) on bovine in vitro fertilization embryo development Concentration (pg / mL) Number of embryos cultured Number of oocytes (% ± SEM) Number of blastocysts (% ± SEM) 0 160 127 (79.3 ± 2.7 b )]) 51 (31.9 ± 1.5 b ])]] 0.5 139 113 (81.2 ± 1.3 b )]) 47 (33.8 ± 0.8 b ) 1 152 133 (87.5 ± 2.3 a )]) 57 (37.4 ± 1.8 a ) <!-- 5 -->]]> 5 127 84 (66.1 ± 0.4 c ])]] 36 (28.3 ± 1.3 c )]]> 10 163 81 (49.6 ± 1.2 d ])]] 32 (19.6 ± 2.1 d )]]> ​ Note: Cleavage rate = number of oocytes x 100 / number of embryo culture, blastocyst rate = number of blastocysts x 100 / number of embryo culture; different letters (a-d) in the same column represent significant differences (P<0.05).

[0042] 9. The addition of 1 μg / mL Cer(d18:1 / 26:0) in the maturation solution can improve the quality of bovine in vitro fertilization blastocysts To evaluate the effect of 1 μg / mL Cer(d18:1 / 26:0) on the quality of in vitro fertilized blastocysts Figure 2 The results showed that the total cell number and trophoblast cell number of the group with 1 μg / mL Cer(d18:1 / 26:0) added in the maturation medium were not significantly different from the control group (P>0.05), but the inner cell mass cell number and the ratio of inner cell mass / trophoblast were significantly higher than the control group (P<0.05). The results indicated that the addition of 1 μg / mL Cer(d18:1 / 26:0) in the maturation medium could significantly improve the quality of in vitro fertilized blastocysts.

[0043] 10、Conclusion The results showed that the total cell number and trophoblast cell number of the group with 1 μg / mL Cer(d18:1 / 26:0) added in the maturation medium were not significantly different from the control group (P>0.05), but the inner cell mass cell number and the ratio of inner cell mass / trophoblast were significantly higher than the control group (P<0.05). The results indicated that the addition of 1 μg / mL Cer(d18:1 / 26:0) in the maturation medium could significantly improve the quality of in vitro fertilized blastocysts.

Claims

1. Application of ceramide in promoting in vitro maturation of oocytes.

2. The application as described in claim 1, characterized in that, The promotion of in vitro oocyte maturation includes promoting the in vitro maturation rate of oocytes.

3. The application as described in claim 1, characterized in that, The promotion of in vitro oocyte maturation includes increasing the development rate of early embryos fertilized in vitro.

4. A method for promoting in vitro maturation of oocytes, which involves maturing and culturing oocytes in an in vitro maturation medium containing ceramides.

5. The method as described in claim 4, characterized in that, The concentration of the ceramide is 0.1~10 μg / mL.

6. The method as described in claim 5, characterized in that, The concentration of the ceramide is 1 μg / mL.

7. Use of ceramides in the preparation of formulations that promote in vitro maturation of oocytes.

8. An in vitro maturation-promoting agent for oocytes, comprising an effective amount of ceramide for promoting in vitro maturation of oocytes.

9. The formulation as described in claim 8, characterized in that, The concentration of the ceramide is 1 μg / mL.

10. The formulation as described in claim 8, characterized in that, The formulation also includes TCM199, 0.01 IU / mL FSH, 0.01 IU / mL LH, 1 μg / mL E2, 10% FBS, and 10 μg / mL heparin.

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