An additive, culture medium for promoting the quality of in vitro matured bovine oocytes and its application

Abstract

The present invention relates to the field of in vitro maturation culture of oocytes, and particularly to an additive, a culture medium for promoting the quality of in vitro matured bovine oocytes and their applications; the additive for promoting the in vitro maturation of oocytes is NAD+, and the concentration of the additive NAD+ is 50 μmol / L. A culture medium for promoting the in vitro maturation of oocytes, the components of the culture medium are: M199 medium, 10 mmol / L Hepes, 10% FBS, 0.01 μg / mL FSH, 1 μg / mL LH, 0.01 μg / mL E2, 10-100 μmol / L NAD+. Adding NAD+ to the in vitro maturation culture medium of oocytes can effectively increase the number of mature oocytes and improve the quality of in vitro matured oocytes. By adding NAD+ to the in vitro maturation solution of oocytes, the in vitro maturation efficiency of oocytes can be improved, the existing oocyte maturation culture system can be improved, and then it can be applied to technical fields such as in vitro fertilization, parthenogenetic activation, somatic cell cloning, etc., so as to improve its production and application value.

Description

Technical Field

[0001] The present invention relates to the field of in vitro maturation culture of oocytes, and particularly to an additive, a culture medium, and their applications for promoting the quality of in vitro matured bovine oocytes. Background Art

[0002] In vitro maturation (IVM) of oocytes refers to the process of taking cumulus-oocyte complexes at the germinal vesicle (GV) stage from ovarian follicles, placing them in an oocyte maturation culture medium for maturation culture, thereby entering the metaphase of the second meiosis, extruding the first polar body, and enabling them to have normal fertilization ability. At present, mammalian embryo engineering technology has developed rapidly, and in vivo embryos can be obtained by superovulation. However, when studying large animals, the number of embryos obtained by this method is small, and the human and material resources consumed are large. Therefore, obtaining immature oocytes from the ovaries discarded at the slaughterhouse and further culturing them for maturation is currently the best solution to this problem. However, there are significant differences between in vivo and in vitro embryo culture, and a large number of factors can affect the quality of in vitro maturation of oocytes, such as ovarian transportation temperature and time, ovarian quality, follicle size, culture medium composition, culture time, etc. Therefore, understanding the important biological events during oocyte maturation and improving the existing in vitro maturation system of oocytes are of great significance for improving the efficiency of oocyte maturation and subsequent fields such as parthenogenetic activation, in vitro fertilization, and in vitro production of cloned embryos.

[0003] Nicotinamide adenine dinucleotide (NAD + ), a key coenzyme in cellular energy metabolism and oxidative stress adaptation responses, exists in all cells and is an important cofactor or cosubstrate. It mediates redox reactions through electron transfer between NAD + (oxidized form) and NADH (reduced form), and can be used in a variety of enzymatic processes, including glycolysis, the TCA cycle, oxidative phosphorylation, DNA repair, and protein deacetylation, etc. Summary of the Invention

[0004] The purpose of the present invention is to provide an additive for promoting the in vitro maturation of oocytes in a culture medium, and this additive is NAD + . By adding NAD + to the existing in vitro maturation culture medium of oocytes, the in vitro maturation of oocytes is promoted to solve the above problems.

[0005] In one aspect of the present invention, an additive for an in vitro maturation culture medium of oocytes is provided, and this additive is NAD +, it can significantly improve the maturation rate and quality of oocytes, significantly increase the cleavage rate and blastocyst rate of in vitro fertilized embryos, and reduce the ROS content and apoptosis of oocytes.

[0006] In some embodiments, the concentration of NAD in the additive for in vitro maturation culture medium of oocytes + is 10 - 100 μmol / L. The in vitro maturation culture medium containing NAD within this concentration range + has a higher maturation rate and better quality of oocytes cultured in vitro.

[0007] In some embodiments, the concentration of NAD in the additive for in vitro maturation culture medium of oocytes + is 50 μmol / L. Thus, the in vitro maturation culture medium supplemented with NAD at a concentration of 50 μmol / L + has the highest maturation rate and best quality of oocytes cultured in vitro.

[0008] Another aspect of the present invention provides an in vitro maturation culture medium for oocytes, which comprises NAD + ; preferably, the culture medium contains 10 - 100 μmol / L of NAD + ; more preferably, the culture medium contains 50 μmol / L of NAD + . Thus, by using the in vitro maturation culture medium for oocytes containing 10 - 100 μmol / L NAD + or 50 μmol / L NAD + , the in vitro maturation rate and quality of oocytes can be significantly improved.

[0009] In some embodiments, the in vitro maturation culture medium for oocytes comprises: M199 medium + 10 mmol / L Hepes + 10% FBS + 0.01 μg / mL FSH + 1 μg / mL LH + 0.01 μg / mL E2 + 10 - 100 μmol / L NAD + . This culture medium can improve the in vitro maturation rate and in vitro maturation quality of oocytes.

[0010] In some embodiments, the in vitro maturation culture medium for oocytes comprises: M199 medium + 10 mmol / L Hepes + 10% FBS + 0.01 μg / mL FSH + 1 μg / mL LH + 0.01 μg / mL E2 + 50 μmol / L NAD + . This culture medium has the best effect on improving the in vitro maturation rate and quality of oocytes.

[0011] Compared with the prior art, the present invention has at least the following beneficial effects:

[0012] Provided is a culture medium additive for promoting in vitro maturation of oocytes, and its application. The additive is NAD + , and adding it to the in vitro maturation culture medium of oocytes can effectively increase the number of mature oocytes and improve the quality of in vitro maturation of oocytes. By adding NAD + to the in vitro maturation solution of oocytes, the in vitro maturation efficiency of oocytes can be improved, the existing in vitro maturation culture system of oocytes can be improved, and then it can be applied to technical fields such as in vitro fertilization, parthenogenetic activation, somatic cell cloning, etc., so as to improve its production and application value. Description of the Drawings

[0013] Figure 1 Figure showing oocytes matured in the control group's maturation culture medium.

[0014] Figure 2 Figure showing oocytes matured in the experimental group's maturation culture medium.

[0015] Figure 3 Figure showing the oocyte maturation rates of the experimental group and the control group.

[0016] Figure 4 Fluorescence image showing the ROS content in oocytes of the experimental group and the control group.

[0017] Figure 5 Fluorescence image showing the apoptosis situation of oocytes in the experimental group and the control group.

[0018] Figure 6 Figure showing the cleavage rates of oocytes after in vitro fertilization in the experimental group and the control group.

[0019] Figure 7 Figure showing the blastocyst rates of oocytes after in vitro fertilization in the experimental group and the control group. Detailed Embodiments

[0020] To make the technical problems, technical solutions and advantages to be solved by the present invention clearer, the following will be described in detail with reference to the drawings and specific embodiments.

[0021] Example 1

[0022] 1. In vitro maturation of oocytes

[0023] (1) Obtaining of oocytes

[0024] Retrieve bovine ovaries from the slaughterhouse, place them in physiological saline, and transport them back using a thermos flask. Wash them 3 - 5 times with alcohol and physiological saline respectively until the liquid is clear and transparent. Pour oocyte collection fluid into a domestic large dish, and use a sterile scalpel to cut the follicles so that the cumulus oocyte complexes (COCs) flow out with the follicular fluid. Under a stereomicroscope, use a self-made oocyte pick-up needle to pick out COCs with uniform cytoplasm and more than three layers of dense cumulus cells.

[0025] (2)In vitro maturation of oocytes

[0026] Wash the picked COCs 4 - 6 times with oocyte washing fluid, transfer the COCs to a four-well plate containing 500 μL of maturation culture medium in the following different groups and that has been equilibrated for 2 h, with 50 COCs per well, and incubate them for 24 h under the conditions of 38.5 °C and 5% CO 2 .

[0027] (3)Preparation of required liquids:

[0028] Oocyte collection fluid: M199 medium + 2.2 g / L NaHCO 3 + 2.38 g / L Hepes (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) + 1% FBS (fetal bovine serum) + 0.2% heparin + 1% double antibody.

[0029] In vitro maturation culture medium for oocytes: M199 medium + 10 mmol / L Hepes + 10% FBS + 0.01 μg / mL FSH (follicle-stimulating hormone) + 1 μg / mL LH (luteinizing hormone) + 0.01 μg / mL E2 (estradiol). The maturation culture of COCs is divided into four groups: control group (maturation culture medium without adding NAD + ), 10 μmol / L group (maturation culture medium added with 10 μmol / L NAD+), 50 μmol / L group (maturation culture medium added with 50 μmol / L NAD + ), 100 μmol / L group (maturation culture medium added with 100 μmol / L NAD + ).

[0030] 2. Cumulus cell removal and maturation rate statistics

[0031] After 22 - 24 h of maturation culture, add hyaluronidase to the four-well plate and pipette 40 - 60 times until the cumulus cells fall off. Under a stereomicroscope, pick out oocytes with uniform cytoplasm and extruded first polar body, place them in an incubator at 38.5 °C and 5% CO 2 to wait for subsequent experiments, and record the maturation rate.

[0032] 3. Detection of ROS level in mature oocytes

[0033] The ROS level of mature oocytes was detected using an active oxygen detection kit (Solarbio). DCFH-DA was diluted with M199 at a ratio of 1:1000 to a final concentration of 10 μmol / L. The mature oocytes were placed in DCFH-DA and incubated in an incubator at 37 °C for 20 min. After washing 3 times with PBS, images were observed and collected using a laser confocal microscope.

[0034] 4. Detection of apoptosis in mature oocytes

[0035] The early apoptosis of mature oocytes was detected using an ANNEXIN-FITC apoptosis detection kit (Solarbio). Binding Buffer was diluted with deionized water at a ratio of 1:9, and the oocytes were added to 100 μL of Binding Buffer for washing. 5 μL of Annexin V / FITC was added, and the mixture was incubated at room temperature in the dark for 5 min. After washing 3 times with PBS, images were observed and collected using a laser confocal microscope.

[0036] 5. In vitro fertilization

[0037] (1) Sperm treatment

[0038] Bovine frozen semen was taken out from the liquid nitrogen tank and thawed in a 37 °C water bath. The two ends of the tube were cut off to allow the semen to flow into a 15 mL centrifuge tube containing 5 mL of washing semen. The tube was placed at 45° in a 37 °C incubator for 10 min to wait for the sperm to float. 4 mL of the supernatant was taken and added to a 15 mL centrifuge tube containing 1 mL of washing semen, and centrifuged at 1000 rpm for 5 min. The supernatant was discarded, and the sperm was resuspended with washing semen.

[0039] (2) In vitro fertilization

[0040] The matured oocytes were washed 2 times in fertilization medium and placed in a 50 μL drop of fertilization medium that had been equilibrated for 2 h. 15 - 20 oocytes were placed in each drop. Sperm were transferred into the fertilization medium drop using a mouth pipette, and fertilization was carried out at 38.5 °C and 5% CO 2 for 6 h, and then transferred to sof + BSA culture medium. After 48 h, the embryos were transferred to sof + FBS culture medium, and half of the culture medium was replaced using a mouth pipette every 48 h.

[0041] (3) Preparation of required liquids

[0042] BO solution: 6.702 g / L NaCl + 0.30004 g / L KCl + 0.33 g / L CaCl 2 ·2H 2 O + 0.1276 g / L NaH 2 PO 4 ·2H2 0 + 0.09688 g / L MgCl 2 ·6H 2 0 + 2.5 g / L glucose (glucose) + 0.1376 g / L sodium pyruvate (sodium pyruvate) + 3.1048 g / L NaHCO 3 + 1% double antibody + phenol red washing semen: 30 mL BO solution + 0.0583 g caffeine.

[0043] Fertilization solution: 6 mL BO solution + 40 μL heparin + 0.12 g BSA.

[0044] Sof + BSA: 6.9685 g / L NaCl + 0.5885 g / L KCl + 0.176 g / L KH 2 PO 4 + 0.198 g / L MgSO 4 ·7H 2 0 + 2.1 g / L NaHCO 3 + 0.08 g / L sodium pyruvate + 2.62 g / L CaCl 2 ·2H 2 O + 0.5 g / L myo-Inositol (inositol) + 0.1% double antibody + 2% essential amino acids (50×) + 1% non-essential amino acids (100×) + 0.1% Glutamax (glutamine) + 0.1% Na-lactate (sodium lactate) + 6 g / L BSA.

[0045] Sof + FBS: 6.9685 g / L NaCl + 0.5885 g / L KCl + 0.176 g / L KH 2 PO 4 + 0.198 g / L MgSO 4 ·7H 2 0 + 2.1 g / L NaHCO 3 + 0.08 g / L sodium pyruvate + 2.62 g / L CaCl 2 ·2H 2 O + 0.5 g / L myo-Inositol (inositol) + 0.1% double antibody + 2% essential amino acids (50×) + 1% non-essential amino acids (100×) + 0.1% Glutamax (glutamine) + 0.1% Na-lactate (sodium lactate) + 0.05% FBS.

[0046] Example 2

[0047] Test Results and Analysis:

[0048] 1. Adding NAD to the maturation medium during IVM + can improve the quality of oocytes

[0049] Compared with the control group, the extrusion rate of the first polar body of cells in the 50 μmol / L group was significantly increased (P < 0.05). Adding 50 μmol / L NAD + to the maturation medium can significantly increase the maturation rate of oocytes (P < 0.05). The results are shown in Table 1 and Appendix Figure 1-3 .

[0050] Table 1 Effects of the culture medium on oocytes

[0051] Group Number of oocytes (pieces) Number of mature oocytes (pieces) Maturation rate (%) Control group 265 173 65.28 10 μmol / L group 274 195 71.17 50 μmol / L group 302 247 81.79 100 μmol / L group 296 223 75.34

[0052] 2. Adding NAD to the maturation medium during IVM + can reduce the ROS level in oocytes

[0053] Compared with the control group, adding NAD + to the maturation medium can significantly reduce the ROS content in oocytes. Among them, adding 50 μmol / L NAD + has the best effect. It can be seen that NAD + can effectively reduce the content of ROS in oocytes, thereby promoting the maturation of oocytes and improving the quality of oocytes. The results are shown in Appendix Figure 4 .

[0054] 3. Adding NAD to the maturation medium during IVM + can reduce oocyte apoptosis

[0055] Compared with the control group, adding NAD + to the maturation medium can significantly reduce the number of apoptotic oocytes. Among them, adding 50 μmol / L NAD + has the best effect. It can be seen that NAD + can effectively reduce the apoptosis of oocytes, thereby improving the quality of oocytes. The results are shown in Appendix Figure 5 .

[0056] 4. Adding NAD to the maturation medium during IVM + can improve the quality of in vitro fertilized embryos

[0057] Compared with the control group, the cleavage rate and blastocyst rate of embryos in the 50 μmol / L group were significantly increased (P < 0.05). Adding 50 μmol / L NAD +The maturation medium can significantly improve the quality of in vitro fertilized embryos (P < 0.05), and the results are shown in Table 2 and the appendix Figure 6-7 .

[0058] Table 2 Effects of the culture medium on oocytes

[0059] Group Number of embryos (pieces) Cleavage rate (pieces / %) Blastocyst rate (pieces / %) Control group 173 115（66.47） 25（14.45） 10 μmol / L group 195 138（70.77） 32（16.41） 50 μmol / L group 247 198（80.16） 52（21.05） 100 μmol / L group 223 169（75.78） 38（17.04）

[0060] In summary, adding 50 μmol / L NAD + to the in vitro maturation medium of oocytes can effectively improve the quality of oocytes and the developmental potential of in vitro fertilized embryos, and has potential application value in improving the developmental ability of bovine in vitro matured oocytes.

[0061] The above is the preferred embodiment of the present invention. It should be pointed out that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. A culture medium for promoting in vitro maturation of bovine oocytes, characterized in that: The culture medium components are: M199 culture medium, 10mmol / L Hepes, 10% FBS, 0.01μg / mL FSH, 1μg / mL LH, 0.01μg / mL E2, 50μmol / L NAD + .

2. Use of the culture medium as claimed in claim 1 in promoting in vitro maturation of bovine oocytes.

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