Use of ceramides in promoting in vitro maturation of oocytes

Abstract

The application provides use of ceramide in promoting in-vitro maturation of oocytes. By culturing oocytes in maturation liquid containing ceramide, the in-vitro maturation rate of oocytes can be effectively improved, and the development rate of in-vitro fertilized early embryos can be improved. The application also provides a preparation for promoting in-vitro maturation of oocytes, and the core component of the preparation is an effective amount of ceramide. The application breaks through the low-efficiency bottleneck of traditional culture systems by regulating the maturation microenvironment of oocytes, and provides an innovative solution for assisted reproductive technology. The method has a good application prospect.

Description

Technical Field

[0001] This invention relates to the field of reproduction. Specifically, it relates to the use of ceramide Cer(d18:1 / 26:0) in promoting in vitro maturation of oocytes. Background Technology

[0002] The establishment of the In Vitro Production (IVP) technology system provides an innovative solution for the efficient utilization of high-quality germplasm resources. This technology integrates the genetic advantages of superior bull breeds with the germplasm characteristics of core cows, enabling the large-scale preparation of high-quality embryos. Its application value is reflected in three aspects: first, deeply exploring the reproductive potential of superior female animals; second, accelerating the propagation process of germplasm resources; and third, constructing a biological germplasm resource protection technology system. In Vitro Maturation (IVM) of oocytes is a crucial step in IVP. By performing in vitro maturation culture on ovarian follicle oocytes, it not only effectively solves the key problem of insufficient oocyte supply in embryo engineering but also provides technical support for innovation in modern bio-breeding technology.

[0003] Oocyte maturation involves two synergistic processes: nuclear maturation and cytoplasmic maturation, encompassing a series of complex nucleoplasmic remodeling events. These intrinsic changes lay the crucial molecular and cellular biological foundation for post-fertilization embryonic development and zygotic genome activation. The core marker of nuclear maturation is the oocyte's recovery and completion of the first meiotic division, ultimately arresting at metaphase II (MII). Key events include germinal vesicle rupture (GVBD), chromosome condensation, spindle assembly, separation of homologous chromosomes, and expulsion of the first polar body. Recovery at MII requires activation of fertilization signals, a process accompanied by a series of epigenetic reprogramming events that ensure chromatin is in a fertile state and supports early embryonic development. The core of cytoplasmic maturation lies in the accumulation of maternal factors (such as specific mRNAs, proteins, and metabolic substrates) to ensure autonomous development of the early embryo after fertilization. Simultaneously, various organelles (such as mitochondria, endoplasmic reticulum, and microtubules) synergistically promote the synchronization of cytoplasmic and nuclear maturation by regulating key processes such as protein synthesis, energy supply, substance transport, and precise chromosome separation. As an important indicator for assessing the cytoplasmic maturation status, cortical granules undergo dynamic changes: in the early stages of synthesis, they are widely distributed in the cytoplasm, and as the maturation process progresses, they gradually migrate to the cortical region, eventually becoming evenly distributed in the cortical region of MII oocytes, forming a high-density distribution zone adjacent to the plasma membrane.

[0004] In in vitro fertilization (IVP) systems, the quality of oocyte maturation is crucial, directly impacting fertilization success rates and the potential for subsequent development into transferable blastocysts. However, in vitro-acquired oocytes are typically in an immature stage. While meiosis can spontaneously resume after detachment from the physiologically inhibitory microenvironment of high cAMP concentrations within the follicle, disruption of cytoplasmic maturation regulatory pathways often leads to a significant asynchronous process between nuclear and cytoplasmic maturation, negatively impacting embryonic development. Studies show that the blastocyst rate after fertilization of in vivo mature oocytes can reach 80%, while the rate after fertilization of in vitro mature oocytes is only around 35%, a nearly 2.3-fold difference in developmental efficiency. This highlights the importance of achieving coordinated nuclear-cytoplasmic maturation in in vitro culture systems. The follicle is the basic functional unit for oocyte development and contains granulosa cells, membrane cells, secretory factors, and various substances permeating from plasma. The presence of these components allows the follicular microenvironment to function as a natural medium, providing a substrate for oocyte metabolism and influencing oocyte growth and development. Multiple studies have shown that the developmental potential of oocytes obtained from pre-ovulatory follicles varies, partly due to changes in certain components of the follicular microenvironment. However, the impact of the follicular microenvironment on oocyte developmental capacity requires further investigation.

[0005] Cer(d18:1 / 26:0), also known as N-hexadecyl-sphingosine-4-enine, is a ceramide molecule. Ceramides are central molecules in sphingolipid metabolism and precursors to sphingolipids such as sphingomyelin, ceramide-1-phosphate, and glucosylglycerolamide. As a key structural component of the cell membrane, ceramides, with their unique saturated long-chain fatty acid groups, significantly alter the physical properties of the cell membrane (such as fluidity, microdomain reorganization, and permeability) by enhancing intermolecular forces and promoting the orderly arrangement of lipids, thereby precisely regulating conformational changes of membrane proteins and the efficiency of transmembrane transport. Furthermore, ceramides also act as important lipid second messengers, directly participating in various cell fate determinations. High levels of ceramides often act as stress response signals (such as oxidative stress, DNA damage, and cytokine stimulation), inducing cell cycle arrest, cell senescence, and programmed cell death (apoptosis). It is worth noting that its downstream metabolite, sphingosine-1-phosphate (S1P), often has antagonistic functions against ceramides (such as promoting proliferation, anti-apoptosis, and promoting migration). This "ceramide / S1P bidirectional metabolic switch" dynamically maintains the homeostasis of the intracellular environment by regulating the balance of sphingolipid metabolic flux.

[0006] Ceramides have been studied and used in various diseases, including skin barrier-related diseases, metabolic diseases, cardiovascular diseases, neurodegenerative diseases, and cancer, showing great application prospects and development potential. Currently, patents related to ceramides mainly focus on their extraction and preparation technologies and disease intervention applications, such as the application of ceramides in cosmetics (CN119792132A), the application of ceramides in anti-tumor treatment (CN119792335A), the application of ceramides in Parkinson's disease (CN119355184A), the use of ceramides as biomarkers for the diagnosis of drug-induced liver injury (CN119881183A), the application of ceramides in skin repair (CN117653567B), and the application of ceramides in the treatment of bacterial and fungal infections (AU6856401A). However, the application of Cer(d18:1 / 26:0) in in vitro mature oocytes has not yet been reported. Summary of the Invention

[0007] The first objective of this invention is to provide new uses for Cer(d18:1 / 26:0).

[0008] A second objective of this invention is to provide a culture medium that promotes the in vitro maturation of oocytes.

[0009] This invention utilizes metabolomics analysis of follicular fluid to screen for a small ceramide lipid molecule, Cer(d18:1 / 26:0). Studies have shown that Cer(d18:1 / 26:0) is present in high concentrations in the follicular fluid of oocytes with high developmental potential. Further research revealed that Cer(d18:1 / 26:0) can enhance the developmental potential of bovine oocytes matured in vitro.

[0010] Accordingly, the present invention first provides the use of Cer(d18:1 / 26:0) in enhancing the developmental potential of in vitro mature oocytes.

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

[0012] The present invention further provides a method for promoting in vitro maturation of oocytes, which involves maturing and culturing oocytes in an in vitro maturation medium containing Cer(d18:1 / 26:0).

[0013] Further, the concentration of Cer(d18:1 / 26:0) is 0.1~10 μg / mL. Preferably, the concentration of Cer(d18:1 / 26:0) is 1 μg / mL. The effective amount of Cer(d18:1 / 26:0) can be determined by the enhancement of the developmental potential of in vitro mature oocytes. In practice, the effective amount of Cer(d18:1 / 26:0) can be determined using the method provided in the embodiments of the present invention.

[0014] Furthermore, the oocytes include, but are not limited to, other mammals such as cattle, horses, donkeys, and sheep.

[0015] Furthermore, the present invention also provides the use of Cer(d18:1 / 26:0) in the preparation of preparations that promote in vitro maturation of oocytes.

[0016] Furthermore, the present invention also provides a formulation comprising an effective amount of Cere(d18:1 / 26:0). Preferably, the concentration of Cere(d18:1 / 26:0) is 0.1~10 μg / mL. More preferably, the concentration of Cere(d18:1 / 26:0) is 1 μg / mL.

[0017] In one embodiment of the present invention, the formulation further includes TCM199 (1×), 0.01 IU / mL FSH, 0.01 IU / mL LH, 1 μg / mLE2, 10% FBS, and 10 μg / mL heparin.

[0018] This invention, for the first time, adds Cerebrolysin (d18:1 / 26:0) to the in vitro maturation culture medium of oocytes, promoting the in vitro maturation rate and in vitro development capacity of oocytes, and providing a new use for Cerebrolysin (d18:1 / 26:0). Adding Cerebrolysin (d18:1 / 26:0) can significantly improve the quality of nucleocytoplasmic maturation in bovine oocytes. The method and formulation of this invention have promising application prospects. Attached Figure Description

[0019] Figure 1 The effect of adding 1 μg / mL Cer(d18:1 / 26:0) to the maturation solution 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) indicate 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, cumulus cells (COCs) were gently removed by repeated pipetting, with continuous observation under a stereomicroscope. Once most COCs had been removed, the fertilized eggs were washed three times with pre-embryo culture medium and transferred to pre-embryo culture medium that had been equilibrated for at least 2 hours in an incubator. 20-25 fertilized eggs were placed per 100 μL of pre-embryo culture medium. Cleavage rate was calculated after 48 hours. All cleaved embryos were transferred to post-embryo culture medium and cultured for another 7 days, with half the medium changed every other day. Blastocyst development rate was calculated after 7 days of in vitro culture.

[0032] The above-mentioned pre-embryo culture medium consisted of: 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 galactobionate, 6 mg / mL BSA, 50× essential amino acids, 100× non-essential amino acids, and 0.15 mg / mL glutamine.

[0033] The late embryo culture medium 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 galactobionate, 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): Weigh 2 g of paraformaldehyde and 50 mg of PVA and dissolve them in 50 mL of calcium- and magnesium-free DPBS. Heat in a water bath to promote dissolution. After complete dissolution, store at 4°C or room temperature for later use. (2) Immunostaining permeabilization solution: 1% Triton X-100, diluted with PBS. (3) Immunostaining washing solution: 0.1% Tween 20 and 0.01% Triton X-100, diluted with PBS. (4) Immunostaining blocking solution: 3% BSA, diluted with immunostaining washing solution.

[0035] Methods: (1) Fixation: Take the embryos to be stained, wash them once with DPBS, fix them with 4% PFA for 30 min, transfer the embryos to the washing solution, and wash them 3 times (5 min / time) on a horizontal shaker at 120 rpm. (2) Permeabilization: Permeabilize the embryos with 0.5% Triton X-100 for 15 min to ensure that the antibody can reach the antigen site. Remove the permeabilization solution, add the washing solution and wash them 3 times (5 min / time) on a horizontal shaker at 120 rpm. (3) Blocking: Add 3% BSA blocking solution and block on a shaker for 1 h at 120 rpm. (4) Primary antibody binding: Dilute the primary antibody according to the appropriate ratio, incubate overnight on a shaker at 4°C, and then wash them 3 times (5 min / time) on a horizontal shaker with the washing solution at 120 rpm. The primary antibody can be diluted with the blocking solution. Under normal circumstances, the diluted primary antibody can be reused three times. (5) Secondary antibody binding. From this step onwards, all operations must be performed in the dark. Dilute the secondary antibody with washing buffer according to the species of the primary antibody, incubate at room temperature on a horizontal shaker for 1 hour at a shaking speed of 50 rpm, and then wash 3 times (5 min / time) on a horizontal shaker with washing buffer at a shaking speed of 120 rpm. Note: The secondary antibody should be centrifuged before use, and the upper layer of antibody should be taken out and diluted. Otherwise, non-specific impurities are likely to be present, which are extremely difficult to remove and will affect the final observation. (6) Add DAPI diluted 1:10000 to stain the cell nuclei. Stain on a horizontal shaker for 3 min before observation.

[0036] 5. Data Statistics Experimental data were analyzed using SAS statistical software. One-way ANOVA and Duncan's test were used to determine the significance of differences between different treatments. A p-value < 0.05 was considered significant.

[0037] 6. Adding Cer (d18:1 / 26:0) to the maturation solution 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 solution, and the oocyte first polar body extrusion rate was recorded (Table 1). The results showed that, compared with the control group, 1 μg / mL Cer(d18:1 / 26:0) significantly increased the oocyte first polar body extrusion rate, while the 5 μg / mL and 10 μg / mL Cer(d18:1 / 26:0) groups significantly decreased the oocyte first polar body extrusion rate (P < 0.05); there was no significant difference between the 0.5 μg / mL Cer(d18:1 / 26:0) group and the control group (P > 0.05).

[0038] Table 1. Effects of different concentrations of Cere (d18:1 / 26:0) on in vitro maturation of bovine oocytes. Note: First polar body expulsion rate = number of oocytes expelled from the first polar body / number of oocytes; different letters (ac) in the same column represent significant differences (P < 0.05).

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

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

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

[0042] 9. Adding 1 μg / mL Cer (d18:1 / 26:0) to the maturation solution can improve the quality of bovine in vitro fertilization blastocysts. The effect of adding 1 μg / mL Cer (d18:1 / 26:0) to the in vitro maturation medium of oocytes on the quality of in vitro fertilized blastocysts was assessed. Figure 2 The results showed that, compared with the control group, the total number of cells and trophoblast cells in the blastocyst group supplemented with 1 μg / mL Cer(d18:1 / 26:0) did not change significantly (P>0.05), but the number of inner cell mass cells and the ratio of inner cell mass to trophoblast cells were significantly higher than those in the control group (P<0.05). The results indicate that adding 1 μg / mL Cer(d18:1 / 26:0) to the in vitro maturation solution can significantly improve the quality of in vitro fertilized blastocysts.

[0043] 10. Conclusion The results above show that adding 1 μg / mL Cer(d18:1 / 26:0) to the maturation solution can promote the maturation of the nucleus and cytoplasm of bovine oocytes, improve the development rate of in vitro fertilized blastocysts, and improve embryo quality.

Claims

1. Application of ceramide Cer (d18:1 / 26:0) in promoting in vitro maturation of bovine oocytes.

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

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

4. A method for promoting in vitro maturation of bovine oocytes, wherein bovine oocytes are cultured in an in vitro maturation medium containing ceramide Cer (d18:1 / 26:0) for maturation.

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 ceramide Cer (d18:1 / 26:0) in the preparation of a formulation to promote in vitro maturation of bovine oocytes.

8. An in vitro maturation-promoting preparation for oocytes, comprising an effective amount of ceramide Cer (d18:1 / 26:0) for promoting in vitro maturation of oocytes, and further comprising TCM199, 0.01 IU / mL FSH, 0.01 IU / mL LH, 1 μg / mL E2, 10% FBS, and 10 mg / mL heparin.

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

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