Application of a regulator in improving the maturation quality of frozen porcine oocytes

Abstract

The present invention relates to an application of a regulator in improving the maturation quality of frozen pig oocytes. The regulator is N-(2-(2-(4-chlorophenyl)thiazol-4-yl)ethyl)butyryl. The regulator improves the maturation quality of frozen pig oocytes by improving the mitochondrial function after in vitro maturation of frozen pig oocytes. The present invention provides a new application of N-(2-(2-(4-chlorophenyl)thiazol-4-yl)ethyl)butyramide, which is used for developing a new type of oocyte vitrification freezing solution and in vitro maturation culture medium. The obtained high-quality oocytes can be beneficial to embryo biotechnology research such as in vitro fertilization and somatic cell nuclear transplantation.

Description

Technical Field

[0001] The invention relates to the technical field of animal reproduction, and in particular to application of a regulator in improving the maturation quality of frozen pig oocytes. Background Art

[0002] Oocyte vitrification technology can help to rescue and protect endangered local breeds, efficiently utilize improved varieties, and accelerate genetic improvement. It can also make the research of embryo biotechnology such as somatic cell nuclear transfer and gene editing not restricted by time and space, and has broad application value in animal husbandry, biotechnology and biomedicine.

[0003] At present, the vitrification technology of porcine oocytes has developed rapidly, especially the freezing survival rate of immature germinal vesicle (GV) stage oocytes has been greatly improved, and the in vitro nuclear maturation ability can also reach the level of fresh oocytes, but the early embryonic development ability is still low, indicating that frozen GV stage oocytes still have different degrees of damage at the subcellular organelle function, physiological or molecular level after in vitro maturation. Therefore, how to reduce or eliminate these damages is the key to improving the quality of in vitro maturation of frozen porcine oocytes.

[0004] In recent years, endoplasmic reticulum stress has begun to attract attention in the field of reproductive biology. Studies have found that oocytes or embryos will experience increased endoplasmic reticulum stress levels in adverse environments during in vitro culture. Tauroursodeoxycholic acid (TUDCA) is often used as an endoplasmic reticulum stress inhibitor to improve the quality of oocytes or embryos cultured in vitro. It has been reported in the literature that adding TUDCA to balancing solution and vitrification freezing solution can reduce endoplasmic reticulum stress in frozen mouse mature oocytes ( Endoplasmic reticulum stress inhibition is a valid therapeutic strategy in vitrifying oocytes, Cryobiology 2015; 70: 48-52. ). On the other hand, Salubrinal is a selective eIF2α dephosphorylation inhibitor that can also inhibit endoplasmic reticulum stress. However, the addition of Salubrinal to in vitro maturation of bovine oocytes failed to improve the cryopreservation ability of the formed blastocysts (Does the Addition of Salubrinal to in vitro Maturation Medium Enhance Bovine Blastocyst Yields and Embryo Cryotolerance?, Cryo Letters 2018; 39: 219-226.). In short, the use of endoplasmic reticulum stress inhibitors disclosed in the prior art cannot improve the maturation quality of frozen pig oocytes, and another approach must be found. Summary of the invention

[0005] To solve the above problems, the present invention proposes the use of a regulator in improving the maturation quality of frozen pig oocytes, and its purpose is to provide a method for improving the maturation quality of frozen pig oocytes and solve the problem of unsatisfactory in vitro maturation quality of frozen pig oocytes.

[0006] The present invention provides an application of a regulator in improving the maturation quality of frozen pig oocytes. The regulator is N-(2-(2-(4-chlorophenyl)thiazol-4-yl)ethyl)butyramide (Azoramide). The regulator improves the maturation quality of frozen pig oocytes by improving the mitochondrial function of frozen pig oocytes after in vitro maturation.

[0007] Furthermore, in the two processes of vitrification and in vitro maturation of porcine oocytes, the regulator is added to one or more of the basic solution, balancing solution, freezing solution, thawing solution or in vitro maturation solution used.

[0008] Furthermore, the regulator is added to the basal solution, balanced solution, frozen solution, thawed solution and in vitro maturation solution at a final concentration of 5-50 μM.

[0009] Furthermore, the final concentration of the regulator added to the basic solution, balanced solution, frozen solution, thawed solution and in vitro maturation solution is 10 μM.

[0010] Furthermore, the base solution is Dulbecco's phosphate buffer supplemented with 20% fetal bovine serum.

[0011] Furthermore, the balancing solution is the base solution with 5% ethylene glycol added.

[0012] Furthermore, the vitrification freezing solution is the base solution with 5% polyvinyl pyrrolidone, 0.6 mol / L sucrose and 5% ethylene glycol added.

[0013] Furthermore, the thawing solution is a sucrose thawing solution prepared using the base solution with concentrations of 1 mol / L, 0.5 mol / L and 0.25 mol / L respectively.

[0014] Furthermore, the in vitro maturation fluid includes 90% volume fraction TCM199, 10% volume fraction porcine follicular fluid, 0.1 g / L sodium pyruvate, 0.07 g / L cysteine, 0.55 g / L glucose, 10 ng / mL epidermal growth factor, 0.065 g / L penicillin, 0.05 g / L streptomycin, 0.5 μg / mL follicle-stimulating hormone, and 0.5 μg / mL luteinizing hormone.

[0015] Furthermore, the porcine oocyte is a porcine GV stage oocyte.

[0016] The beneficial effects of the present invention are:

[0017] 1. For the first time, it was clarified that N-(2-(2-(4-chlorophenyl)thiazol-4-yl)ethyl)butyramide (Azoramide) can improve the maturation quality of frozen pig oocytes. Azoramide is an effective small molecule regulator of unfolded protein response, which can improve the folding of endoplasmic reticulum proteins and enhance the ability of endoplasmic reticulum chaperone proteins, and promote endoplasmic reticulum homeostasis. There have been no reports on the application of Azoramide in the field of animal breeding technology, and there have been no research reports on whether Azoramide can be used as an endoplasmic reticulum stress inhibitor to improve the quality of in vitro cultured oocytes or embryos. The present invention uses the endoplasmic reticulum stress inhibitor TUDCA reported in existing technical literature as a reference group for evaluating the efficacy of Azoramide. The method for improving the maturation quality of frozen pig oocytes with Azoramide provided by the present invention, by adding 10 μM Azoramide to various solutions required for oocyte vitrification and in vitro maturation, has a better ability to improve the maturation quality of frozen oocytes than TUDCA with an optimized concentration. The embryo in vitro culture experiment found that the blastocyst formation rate of the parthenogenetically activated embryos of the mature oocytes obtained by the technical solution of the present invention exceeded that of the conventional solution by about 13 percentage points on the 7th day, while TUDCA had no obvious effect on improving the subsequent embryo development.

[0018] 2. Using the vitrification and in vitro maturation culture scheme containing Azoramide provided by the present invention, there are 506 differentially expressed genes in porcine oocytes, and the top 20 most significantly enriched GO entries include NADH dehydrogenase activity, mitochondrial electron transport, mitochondrial respiratory chain complex I assembly, ATP synthesis coupled electron transport, mitochondrial respiratory chain complex I, redox process, respiratory chain complex IV. In addition, Azoramide can improve the mitochondrial activity and ATP level of frozen porcine GV stage oocytes after in vitro maturation. The above results show that Azoramide has a significant improvement effect on the mitochondrial function of frozen porcine oocytes after in vitro maturation.

[0019] 3. Azoramide provided by the present invention can regulate mitochondrial function during the in vitro maturation of frozen porcine GV-stage oocytes. Given that the endoplasmic reticulum stress inhibitor TUDCA cannot improve the early embryonic development ability of frozen porcine GV-stage oocytes, Azoramide may improve the maturation quality of frozen porcine oocytes mainly by acting on mitochondria rather than the endoplasmic reticulum. The present invention provides a new use of Azoramide, namely, for developing a new type of oocyte vitrification freezing solution and in vitro maturation culture medium, and the obtained high-quality oocytes can be beneficial to embryonic biotechnology research such as in vitro fertilization and somatic cell nuclear transplantation. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1The volcano plot of differentially expressed genes in mature porcine oocytes of the frozen control group and the Azoramide group in Example 3;

[0021] Figure 2 This is the GO term enrichment map of differentially expressed genes in the frozen control group and the Azoramide group of pig mature oocytes in Example 3;

[0022] Figure 3 The fluorescence-labeled images of mitochondrial activity and ATP level of mature porcine oocytes in the frozen control group and the Azoramide group in Example 4;

[0023] Figure 4 This is a bar graph of the relative fluorescence intensity of mitochondrial activity of mature porcine oocytes in the frozen control group and the Azoramide group in Example 4;

[0024] Figure 5 This is a bar graph of the relative fluorescence intensity of ATP levels in mature porcine oocytes in the frozen control group and the Azoramide group in Example 4. DETAILED DESCRIPTION

[0025] The technical solution of the present invention is further illustrated by specific examples below.

[0026] Unless otherwise specified, the technical means used in the embodiments are conventional technical means well known to those skilled in the art.

[0027] Example 1: Azoramide method for improving the quality of in vitro maturation of frozen porcine oocytes:

[0028] (1) Pig oocyte collection:

[0029] Porcine ovaries were collected from a local slaughterhouse, placed in 37°C saline containing penicillin and streptomycin, and transported back to the laboratory within 4 hours. The ovaries were washed 2-3 times with the above saline, and follicular fluid from 3-8 mm diameter follicles on the surface of the ovaries was extracted using a 20 mL disposable syringe with an 18-gauge needle, and then placed in a 50 mL centrifuge tube to allow the cumulus-oocyte complexes (COCs) to settle naturally. The precipitate was washed twice in TL-Hepes-PVA solution and transferred to a culture dish. COCs with uniform oocyte cytoplasm and more than three layers of granulosa cells were selected under a stereomicroscope using a mouth pipette, i.e., GV-stage oocytes.

[0030] The TL-Hepes-PVA solution is 6.662 g / L sodium chloride, 0.2386 g / L potassium chloride, 0.168 g / L sodium bicarbonate, 0.046 g / L potassium dihydrogen phosphate, 2.383 g / L HEPES, 0.022 g / L sodium pyruvate, 0.856 mL sodium lactate, 2.186 g / L sorbitol, 0.102 g / L magnesium chloride hexahydrate, 0.294 g / L calcium chloride dihydrate, 0.065 g / L penicillin, 0.05 g / L streptomycin, and 3 g / L bovine serum albumin, which is fully dissolved in ultrapure water, fixed to 1 L, adjusted to a pH value of 7.2-7.4, filtered and packaged using a 0.22 μm filter, and stored in a refrigerator at 4° C. for standby use.

[0031] (2) Freezing and thawing of porcine oocytes:

[0032] At room temperature of 25℃, Cryotop was used as a cryocarrier for oocyte vitrification. First, the porcine GV-stage oocytes obtained in step 1) were washed in the basal solution for 3 minutes, then transferred to the equilibration solution for 10 minutes, and then transferred to the vitrification freezing solution for 30-40 seconds, and finally neatly arranged on the tip of the Cryotop and stored in liquid nitrogen. The time of the oocytes in the vitrification freezing solution does not exceed 1 minute. When thawing, the Cryotop carrier was removed from the liquid nitrogen, and the part containing the oocytes was quickly immersed directly in the 1mol / L sucrose thawing solution at 42℃ for 1 minute, and then transferred to 0.5 and 0.25mol / L sucrose thawing solutions for 2.5 minutes each, and then placed in the basal solution for 5 minutes to complete the thawing of the oocytes.

[0033] The base solution is Dulbecco's phosphate buffer supplemented with 20% fetal bovine serum, the balanced solution is the base solution supplemented with 5% ethylene glycol, the freezing solution is the base solution supplemented with 5% polyvinyl pyrrolidone, 0.6 mol / L sucrose and 5% ethylene glycol, and the thawing solution is a sucrose thawing solution prepared using the base solution with concentrations of 1 mol / L, 0.5 mol / L and 0.25 mol / L.

[0034] (3) In vitro maturation of porcine oocytes:

[0035] First, add the in vitro maturation solution to each well of the 24-well plate, with a volume of 500 μL, cover with 300 μL paraffin oil and equilibrate in a carbon dioxide incubator at 39°C, 5% CO2 and 100% humidity for more than 3 hours. After washing the thawed porcine GV-stage oocytes obtained in step 2) three times in the in vitro maturation solution, place them in the culture wells, about 50 oocytes per well, and culture them in the carbon dioxide incubator under the above conditions for 42-44 hours. After completing the in vitro maturation culture, place the oocytes in a 0.5 mL centrifuge tube containing 0.1% (w / v) hyaluronidase solution, use a 200 μL pipette to gently blow off the cumulus granulosa cells, and then wash them three times with in vitro operating solution. Finally, select mature oocytes with uniform cytoplasm and excretion of the first polar body under a stereomicroscope for use.

[0036] The in vitro maturation fluid includes 90% volume fraction TCM199, 10% volume fraction porcine follicular fluid, 0.1g / L sodium pyruvate, 0.07g / L cysteine, 0.55g / L glucose, 10ng / mL epidermal growth factor, 0.065g / L penicillin, 0.05g / L streptomycin, 0.5μg / mL follicle-stimulating hormone, and 0.5μg / mL luteinizing hormone.

[0037] The in vitro operating solution includes TCM199 supplemented with 2.383 g / L HEPES, 2.603 g / L HEPES-Na, 0.065 g / L penicillin, 0.05 g / L streptomycin, and 0.5% (v / v) fetal bovine serum.

[0038] (4) Parthenogenetic activation and in vitro embryo culture:

[0039] The mature oocytes obtained in step 3) were placed in the electro-activation solution for washing for about 30 seconds, and then quickly transferred to the 1mm fusion tank covered with electro-activation solution for electro-activation between two electrodes. The conditions were electric field strength of 1.3kV / cm, DC pulse duration of 80μs, and 1 pulse. After electro-activation, the oocytes continued to be cultured in the auxiliary activation solution for 4 hours, and then transferred into 50μL PZM-3 droplets in groups of 15, and embryos were cultured in vitro at 39°C, 5% CO2 and 100% humidity. Both the auxiliary activation solution and the PZM-3 solution need to be balanced in the carbon dioxide incubator under the above conditions for more than 3 hours. The cleavage rate and blastocyst formation rate of the embryos were observed at 48h and 168h, respectively.

[0040] The electro-activation solution is 0.3 mol / L mannitol, 0.1 mmol / L calcium chloride dihydrate, 0.1 mmol / L magnesium sulfate heptahydrate, and 0.1 g / L polyvinyl alcohol, which is filtered and packaged through a 0.22 μm filter and stored in a -20°C refrigerator for later use.

[0041] The PZM-3 solution is 6.312 g / L sodium chloride, 2.106 g / L sodium bicarbonate, 0.746 g / L potassium chloride, 0.048 g / L potassium dihydrogen phosphate, 0.022 g / L sodium pyruvate, 0.048 g / L magnesium sulfate, 0.616 g / L L-lactic acid hemicalcium salt, 0.292 g / L L-glutamine, 0.05 g / L gentamicin, 0.546 g / L sodium hypotaurine, 4 g / L BSA, 20 mL non-essential amino acid solution, 40 mL essential amino acid solution, the volume is fixed to 1 L, the pH value is adjusted to 7.2-7.4, the osmotic pressure is 272-288 mOsm, the solution is filtered and packaged through a 0.22 μm filter, and stored in a refrigerator at 4°C for later use.

[0042] The auxiliary activation solution is PZM-3 solution with 5 μg / mL cytochalasin B and 10 μg / mL cycloheximide added.

[0043] (5) Statistics:

[0044] Unless otherwise specified, SPSS 20 software was used for statistical analysis of the data. Duncan multiple comparisons were used to determine the significance of the differences. P < 0.05 was considered significant. The results are expressed as mean ± standard error.

[0045] (6) Experimental group design:

[0046] Fresh control group: The in vitro maturation medium used in the in vitro maturation culture of fresh oocytes does not contain Azoramide.

[0047] Frozen control group: The base solution, balancing solution, freezing solution, thawing solution used in the vitrification and thawing process of fresh oocytes and the in vitro maturation solution used in the in vitro maturation culture process did not contain Azoramide.

[0048] The experimental group was divided into 4 groups: the basal solution, balancing solution, freezing solution, thawing solution used in the vitrification and thawing of fresh oocytes and the in vitro maturation solution used in the in vitro maturation culture process all contained 5μM, 10μM, 20μM or 40μM final concentration of Azoramide.

[0049] Preparation of Azoramide: Weigh 4 mg of Azoramide and dissolve it in 0.3238 mL of dimethyl sulfoxide to prepare a 40 mM concentrated stock solution, which is then stored at -80° C. When used, add the Azoramide concentrated stock solution to the above-mentioned solutions and dilute to the desired final concentration.

[0050] Test results:

[0051] During vitrification and in vitro maturation, the effects of different concentrations of Azoramide on the early embryonic development of frozen oocytes were evaluated. As shown in Table 1, compared with the frozen control group, the blastocyst formation rate in the 10μM and 20μM Azoramide test groups was significantly increased (P<0.05), among which 10μM Azoramide had the best efficiency in promoting the development of parthenogenetic embryos into blastocysts, with an increase of more than 10 percentage points.

[0052] Table 1 Development of parthenogenetic embryos in frozen porcine GV-stage oocytes after treatment with different concentrations of Azoramide

[0053]

[0054] Note: Data in the same column with different letters on their shoulders represent significant differences (P<0.05), and data without letters represent no significant differences (P>0.05).

[0055] Example 2: Comparison of the application effects of endoplasmic reticulum stress inhibitors:

[0056] A method for improving the in vitro maturation quality of frozen porcine oocytes by using Azoramide, wherein the dosage of Azoramide is 10 μM. To compare the application effect of Azoramide, the control group uses the endoplasmic reticulum stress inhibitor TUDCA. Other operations are the same as in Example 1.

[0057] Test results:

[0058] As shown in Table 2, compared with the frozen control group, the blastocyst formation rate of the Azoramide group was significantly increased (P<0.05), while there was no significant difference in the blastocyst formation rate of the different concentrations of TUDCA groups. It can be seen that the blastocyst formation rate of the mature oocytes obtained by the technical solution of the present invention, the parthenogenetic activated embryos on the 7th day exceeded that of the conventional solution by about 13 percentage points, while TUDCA had no significant effect on the subsequent embryonic development.

[0059] Table 2 Development of parthenogenetic embryos in frozen porcine GV-stage oocytes after treatment with Azoramide and TUDCA

[0060]

[0061] Example 3, Smart-seq2 single-cell transcriptome sequencing:

[0062] The sequencing company is Hangzhou Lianchuan Biotechnology Co., Ltd. Referring to Example 1, porcine GV-stage oocytes were treated with vitrification and in vitro maturation culture schemes without or with 10 μM Azoramide, and mature oocytes were collected as frozen control group and Azoramide group, respectively. The oocyte samples were placed in cell lysate and stored at -80 ° C for standby use. First, the polyA RNA in the sample was used as a template, and the oligo (dT) sequence with a connector was used as a primer to obtain a complete single-stranded cDNA sequence with connectors at both ends under the action of SMART reverse transcriptase. PCR amplification was performed using the connectors at both ends of the cDNA sequence as primers, and finally a double-stranded cDNA sufficient for library construction and sequencing was obtained. The SMART pre-amplification product was fragmented by enzyme digestion, and then the magnetic beads were used to screen the fragments of appropriate size (150-300 bp). After the ends were filled and A was added to the 3' end, a connection reaction was performed with the Y-type sequencing connector. PCR amplification was performed using the connection product as a template to obtain a library that can be sequenced. After the library passed the quality inspection, Illumina Novaseq was used TM 6000 sequencing, with a read length of 2x150 bp (PE150) at both ends. The sequencing data were aligned to the reference genome using Hisat software, and the transcripts were assembled and the expression levels were calculated using StringTie software, and then differential expression analysis was performed using edgeR. Differential gene screening was performed based on P < 0.05 and differential expression fold ≥ 2 or ≤ 0.5. Functional annotation and enrichment analysis were performed using BLAST2GO and KEGG pathway databases.

[0063] Test results:

[0064] Depend on Figure 1 It can be seen that compared with the frozen control group, there are 506 differentially expressed genes in the mature oocytes of pigs in the Azoramide group, of which 188 are significantly upregulated and 318 are significantly downregulated. The top 20 most significantly enriched GO terms include NADH dehydrogenase activity, mitochondrial electron transport, mitochondrial respiratory chain complex I assembly, ATP synthesis coupled electron transport, mitochondrial respiratory chain complex I, redox process, respiratory chain complex IV, all of which are related to mitochondrial energy production ( Figure 2 ). It can be seen that Azoramide plays an important role in regulating the mitochondrial function during the in vitro maturation of frozen porcine GV-stage oocytes.

[0065] Example 4, mitochondrial function detection:

[0066] According to the method for improving the in vitro maturation quality of frozen porcine oocytes using Azoramide in Example 1, the mitochondrial function was detected after obtaining mature oocytes to further clarify the regulatory effect of Azoramide on mitochondria.

[0067] Mitochondrial activity detection: Oocytes were washed three times with DPBS-PVA solution, transferred to 20nM MitoTracker™ RedCMXRos fluorescent probe solution and incubated in a carbon dioxide incubator for 30 minutes, then washed three times with DPBS-PVA, and then observed under a laser scanning confocal microscope with an excitation light of 561nm. TIFF format images were collected and analyzed with ImageJ software to obtain the intracellular fluorescence intensity value, which represents the activity of live mitochondria.

[0068] ATP level detection: oocytes were fixed in 4% paraformaldehyde solution for 2 h, washed three times with DPBS-PVA, and then incubated in 500 nmol / L BODIPY FL ATP fluorescent probe in the dark for 1 h, and then washed three times with DPBS-PVA. The oocytes were observed and scanned using a laser confocal microscope with an excitation wavelength of 488 nm. The fluorescence intensity value, which represents the ATP level, was analyzed using ImageJ software.

[0069] Test results:

[0070] The fluorescence intensity of oocytes in the frozen control group was set as 1, and the fluorescence intensity of oocytes in the Azoramide group was the relative value to that of the control group. Figure 3-5 It can be seen that compared with the frozen control group, the mitochondrial activity of oocytes in the Azoramide group was significantly enhanced (P<0.05), and the ATP level was also significantly increased (P<0.05). It was further confirmed that Azoramide can improve the mitochondrial function of frozen porcine oocytes after in vitro maturation, which may be the main reason for improving the maturation quality of frozen porcine oocytes.

[0071] The above is a detailed introduction to the application of the small molecule regulator Azoramide provided by the present invention in improving the quality of frozen pig oocyte maturation. This article describes the principles and implementation methods of the present invention through specific implementation methods, and the above description is only used to help understand the method of the present invention and its core idea. It should be pointed out that for ordinary technicians in this technical field, without departing from the principles of the present invention, the present invention can also be improved and modified in a number of ways, and these improvements and modifications also fall within the scope of protection of the claims of the present invention.

Claims

1. Use of a regulator in improving the maturation quality of frozen pig oocytes, characterized in that: The regulator is N-(2-(2-(4-chlorophenyl)thiazol-4-yl)ethyl)butyramide. In the two processes of vitrification and in vitro maturation culture of pig oocytes, the regulator is added to the basic solution, balanced solution, freezing solution, thawing solution and in vitro maturation solution. The regulator improves the maturation quality of frozen pig oocytes by improving the mitochondrial function after in vitro maturation of frozen pig oocytes. The pig oocytes are GV stage oocytes. The final concentration of the regulator in the basic solution, balanced solution, freezing solution, thawing solution and in vitro maturation solution is 10-40 μM.

2. The use of the regulator according to claim 1 in improving the maturation quality of frozen pig oocytes, characterized in that: The final concentration of the regulator added to the basal solution, balanced solution, frozen solution, thawed solution and in vitro maturation solution is 10 μM.

3. The use of the regulator according to claim 1 in improving the maturation quality of frozen pig oocytes, characterized in that: The base solution is Dulbecco's phosphate buffer supplemented with 20% fetal bovine serum.

4. The use of the regulator according to claim 1 in improving the maturation quality of frozen pig oocytes, characterized in that: The balanced solution is the base solution with 5% ethylene glycol added.

5. The use of the regulator according to claim 1 in improving the maturation quality of frozen pig oocytes, characterized in that: The vitrification freezing solution is the base solution with 5% polyvinyl pyrrolidone, 0.6 mol / L sucrose and 5% ethylene glycol added.

6. The use of the regulator according to claim 1 in improving the maturation quality of frozen pig oocytes, characterized in that: The thawing solutions are sucrose thawing solutions prepared using the base solution with concentrations of 1 mol / L, 0.5 mol / L and 0.25 mol / L respectively.

7. The use of the regulator according to claim 1 in improving the maturation quality of frozen pig oocytes, characterized in that: The in vitro maturation fluid includes 90% volume fraction TCM199, 10% volume fraction porcine follicular fluid, 0.1g / L sodium pyruvate, 0.07g / L cysteine, 0.55g / L glucose, 10ng / mL epidermal growth factor, 0.065g / L penicillin, 0.05g / L streptomycin, 0.5μg / mL follicle-stimulating hormone, and 0.5μg / mL luteinizing hormone.

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