CN114045256B - Culture solution and method for improving quality of mother-source diabetic ovum - Google Patents
Culture solution and method for improving quality of mother-source diabetic ovum Download PDFInfo
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- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
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- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0681—Cells of the genital tract; Non-germinal cells from gonads
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- C12N2501/30—Hormones
- C12N2501/31—Pituitary sex hormones, e.g. follicle-stimulating hormone [FSH], luteinising hormone [LH]; Chorionic gonadotropins
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Abstract
The invention discloses a culture solution and a method for improving the quality of parent-source diabetic ova, which relate to the technical field of reproductive biology. The tea polyphenol used in the culture is from tea extract, belongs to natural active substances, has no toxic or side effect on human body, animals and the like, and has good safety, easy obtainment and low cost. The culture solution provided by the invention can improve the quality of the parent-source diabetic ovum and improve the fertility problem of the parent-source diabetic patient.
Description
Technical Field
The invention relates to the technical field of reproductive biology, in particular to a culture solution and a method for improving the quality of parent-source diabetic ova.
Background
With the development of socioeconomic performance, the problem of infertility of human beings is also increasing. Currently, about 15% of worldwide sterile couples, up to about 30% in some countries and regions, with 40-50% of infertility being caused by maternal factors. In infertility caused by maternal factors, the quality of ova is reduced as one of the most major causes. In the maturation process of the ovum, many in-vivo and in-vitro factors can cause the low quality of the ovum, such as abnormal spindle body, unequal chromosome separation, reduced mitochondrial function and the like. Low quality ova have reduced fertilization rates and reduced embryo development potential, and can also easily lead to a higher risk of illness in newborns and even in adulthood. Among the factors that lead to the deterioration of ovum quality, maternal metabolic abnormalities such as diabetes, obesity, etc. are one of the most important causes. Furthermore, metabolic diseases such as diabetes have become the most important diseases threatening human health. The incidence of diabetes in China is as high as 9 tens of millions, wherein the prevalence rate of teenagers is the first in the world, and the prevalence rate of teenagers is rapidly increased. Maternal diabetes can lead to meiosis abnormalities such as spindle shape abnormalities, chromosomal arrangement abnormalities, increased chromosomal uneven segregation rates, decreased fertilization rates and embryo developmental potential, increased abortion rates, abnormal mitochondrial distribution and function of ova, and the like during ovum maturation.
Disclosure of Invention
The invention mainly aims to provide a culture solution and a method for improving the quality of maternal diabetic ova, and aims to improve the quality of maternal diabetic ova.
In order to achieve the above object, the present invention provides a culture solution for in vitro culture of a mother-source diabetic cumulus-oocyte complex, the culture solution including tea polyphenol.
Optionally, the concentration of tea polyphenol in the culture solution is 25-50 mu M.
Alternatively, the concentration of tea polyphenols in the culture broth is 50. Mu.M.
Optionally, the culture broth further comprises inactivated fetal bovine serum, sodium pyruvate, 100 x green streptomycin, FSH, and alpha-MEM medium.
Optionally, the volume fraction of the inactivated fetal bovine serum in the culture solution is 4% -6%; and/or the number of the groups of groups,
the concentration of sodium pyruvate in the culture solution is 0.20-0.30 mM; and/or the number of the groups of groups,
the volume fraction of 100 Xof the green streptomycin in the culture solution is 0.7-0.8%; and/or the number of the groups of groups,
the concentration of FSH in the culture solution is 0.4-0.6U/ml.
The invention further provides a method for improving the quality of the parent-source diabetes ovum, which comprises the following steps:
s10, constructing a maternal diabetes animal model, and separating a cumulus-oocyte complex from the maternal diabetes animal model;
s20, placing the cumulus-oocyte complex in a culture solution for culture to obtain the MII-stage oocyte, wherein the culture solution is the culture solution.
Optionally, in step S10, the maternal diabetes animal model has a blood glucose concentration of 17.0mM or more.
Optionally, in step S10, the step of isolating a cumulus-oocyte complex from the maternal diabetes animal model includes:
injecting pregnant mare serum gonadotropin into the abdominal cavity of the maternal diabetes animal model, taking out the ovary after 46-48 hours, and collecting the cumulus-oocyte complex.
Alternatively, in step S20, the culture conditions are: the volume concentration of the carbon dioxide is 4-6%, the temperature is 36-38 ℃, and the culture is carried out for 15-17 h.
Optionally, in step S20, the surface of the culture solution is covered with paraffin oil.
The culture solution provided by the invention is used for in vitro culture of mother-source diabetes cumulus-oocyte complex, comprises tea polyphenol, and is used for in vitro culture in a culture solution containing the tea polyphenol, so that the quality of ova is effectively improved. The tea polyphenol used in the culture is from tea extract, belongs to natural active substances, has no toxic or side effect on human body, animals and the like, and has good safety, easy obtainment and low cost. The culture solution provided by the invention can improve the quality of the parent-source diabetic ovum and improve the fertility problem of the parent-source diabetic patient.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are required in the embodiments or the description of the prior art will be briefly described, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and other related drawings can be obtained according to the drawings without inventive effort for a person skilled in the art.
FIG. 1 is a flow chart of a method for improving quality of maternal diabetic ova according to an embodiment of the present invention;
FIG. 2 is a schematic view of a cumulus-oocyte complex obtained in example 3 of the present invention;
FIG. 3 is a schematic diagram of a culture dish and an apparatus for culturing COCs in example 3 of the present invention;
FIG. 4 is a schematic diagram of a cumulus-oocyte complex after 16h of culture obtained in example 3 of the present invention and an MII-stage oocyte;
FIG. 5 shows spindle shapes and chromosome profiles of MII phase oocytes of example 3, example 4, comparative example 1 and control group according to the present invention;
FIG. 6 is a graph showing the ROS content of the MII phase oocytes of example 3, example 4, comparative example 1 and the control group of the present invention;
FIG. 7 is a graph showing the results of mitochondrial membrane potential in MII phase oocytes of example 3, example 4, comparative example 1 and control group according to the present invention.
Detailed Description
The following description of the embodiments of the present invention will be made clearly and fully with reference to the accompanying drawings, in which it is evident that the embodiments described are only some, but not all embodiments of the invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
It should be noted that, if directional indications (such as up, down, left, right, front, back, outer, inner … …) are included in the embodiments of the present invention, the directional indications are merely used to explain the relative positional relationship, movement conditions, etc. between the components in a specific posture (as shown in the drawings), and if the specific posture is changed, the directional indications are correspondingly changed.
In addition, if there is a description of "first", "second", etc. in the embodiments of the present invention, the description of "first", "second", etc. is for descriptive purposes only and is not to be construed as indicating or implying a relative importance or implicitly indicating the number of technical features indicated. Thus, a feature defining "a first" or "a second" may explicitly or implicitly include at least one such feature. In addition, the meaning of "and/or" as it appears throughout includes three parallel schemes, for example "A and/or B", including the A scheme, or the B scheme, or the scheme where A and B are satisfied simultaneously. In addition, the technical solutions of the embodiments may be combined with each other, but it is necessary to base that the technical solutions can be realized by those skilled in the art, and when the technical solutions are contradictory or cannot be realized, the combination of the technical solutions should be considered to be absent and not within the scope of protection claimed in the present invention.
In order to make the objects, technical solutions and advantages of the embodiments of the present invention more clear, the technical solutions of the embodiments of the present invention will be clearly and completely described below. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.
Maternal diabetes can lead to meiosis abnormalities such as spindle shape abnormalities, chromosomal arrangement abnormalities, increased chromosomal uneven segregation rates, decreased fertilization rates and embryo developmental potential, increased abortion rates, abnormal mitochondrial distribution and function of ova, and the like during ovum maturation.
In view of the above, the invention provides a culture solution for improving the quality of parent-source diabetic ova. In the drawings, FIG. 1 is a schematic flow chart of an embodiment of a method for improving quality of a mother-source diabetic ovum according to the present invention; FIG. 2 is a schematic view of a cumulus-oocyte complex obtained in example 3 of the present invention;
FIG. 3 is a schematic diagram of a culture dish and an apparatus for culturing COCs in example 3 of the present invention; FIG. 4 is a schematic diagram of a cumulus-oocyte complex after 16h of culture obtained in example 3 of the present invention and an MII-stage oocyte; FIG. 5 shows spindle shapes and chromosome profiles of MII phase oocytes of example 3, example 4, comparative example 1 and control group according to the present invention; FIG. 6 is a graph showing the ROS content of the MII phase oocytes of example 3, example 4, comparative example 1 and the control group of the present invention; FIG. 7 is a graph showing the results of mitochondrial membrane potential in MII phase oocytes of example 3, example 4, comparative example 1 and control group according to the present invention.
The invention provides a culture solution for in vitro culture of mother-source diabetes cumulus-ooyte complexes (COCs), wherein the culture solution comprises tea polyphenol.
The culture solution provided by the invention is used for in vitro culture of mother-source diabetes cumulus-oocyte complex, comprises tea polyphenol, and is used for in vitro culture in a culture solution containing the tea polyphenol, so that the quality of ova is effectively improved. The tea polyphenol used in the culture is from tea extract, belongs to natural active substances, has no toxic or side effect on human body, animals and the like, and has good safety, easy obtainment and low cost. The culture solution provided by the invention can improve the quality of parent-source diabetic ova.
The present invention is not limited as to the concentration of tea polyphenols in the culture solution, and preferably the concentration of tea polyphenols in the culture solution is 25 to 50. Mu.M. When the concentration is too low, the tea polyphenol has no obvious effect on improving the quality of the ovum, and when the concentration is too high, the tea polyphenol is not beneficial to improving the quality of the ovum, and in the range, the improvement effect is good. More preferably, the concentration of tea polyphenols in the culture broth is 50. Mu.M. Research shows that under the above concentration, the spindle structure of the ovum is abnormal, the chromosome arrangement is obviously reduced, the ROS (intracellular reactive oxygen species) level of the ovum is obviously reduced, the mitochondrial function of the ovum is restored, and the ovum has no obvious difference with a normal ovum.
Preferably, in an embodiment of the invention, the culture broth further comprises inactivated fetal bovine serum, sodium pyruvate, penicillin, FSH and alpha-MEM medium. Wherein FSH is follicle stimulating hormone, has effects of promoting follicular development and maturation, and can promote estrogen secretion together with luteinizing hormone. The components and tea polyphenol act together to improve the quality of the parent-source diabetic ovum.
The concentration of the above components is not limited, and preferably, the volume fraction of the inactivated fetal bovine serum in the culture solution is 4% -6%, for example, may be 4%, 5%, 6%, etc., and the inactivated fetal bovine serum is heat-inactivated fetal bovine serum; the concentration of sodium pyruvate is 0.20 to 0.30mM, for example, 0.20mM, 0.22mM, 0.25mM, 0.28mM, 0.30mM, etc.; 100 Xgreen streptomycin is a mixed solution of penicillin and streptomycin, wherein the content of penicillin is 10kU/ml, the content of streptomycin is 10mg/ml, the volume fraction of 100 Xgreen streptomycin is 0.7% -0.8%, such as 0.7%, 0.75%, 0.8%, and the like, and the volume fraction is the sum of the volumes of penicillin and streptomycin accounting for the volume fraction of the mixed solution; the concentration of FSH is 0.4 to 0.6U/ml, for example 0.4U/ml, 0.5U/ml, 0.6U/ml, etc. More preferably, the volume fraction of inactivated fetal bovine serum in the culture broth is 5%; the concentration of sodium pyruvate in the culture solution is 0.25mM; the volume fraction of 100 x of the streptomycin in the culture solution is 0.75%; the concentration of FSH in the culture solution is 0.5U/ml, under the concentration, the spindle structure abnormality and the chromosome arrangement abnormality of the ovum are obviously reduced, the ROS (intracellular reactive oxygen species) level of the ovum is obviously reduced, the mitochondrial function of the ovum is restored, and the ovum has no obvious difference from a normal ovum.
Referring to fig. 1, the invention further provides a method for improving the quality of a parent-source diabetic ovum, which comprises the following steps:
s10, constructing a maternal diabetes animal model, and separating a cumulus-oocyte complex from the maternal diabetes animal model.
In the step, a maternal diabetes animal model is established by the following method: the animal was injected with 220mg/kg body weight of STZ (streptozotocin) into the abdominal cavity in a single injection, blood was taken through the tail tip after 4 days, the blood glucose concentration was measured by a Rogowski glucometer, and if the blood glucose concentration was 17.0mM/L or more, the maternal diabetes animal model was considered to be successfully constructed. Streptozotocin is a drug for inducing an animal model of diabetes, and after a certain amount of STZ is injected into animals, the animal model of diabetes can be obtained and used for researching diabetes. Therefore, the maternal diabetes animal model has a blood glucose concentration of 17.0mM or more.
Further, the step of isolating a cumulus-oocyte complex from the maternal diabetes animal model includes:
injecting Pregnant Mare Serum Gonadotropin (PMSG) into the abdominal cavity of the maternal diabetes animal model, taking out the ovary after 46-48h, and collecting the cumulus-oocyte complex.
Specifically, on the 10 th day after the successful construction of the maternal diabetes animal model, 8IU of PMSG is injected into the abdominal cavity of an animal, the ovary is taken from the abdominal cavity after 46-48 hours, and in the M2 culture solution containing milrinone, the follicle is pierced by a 1mL syringe needle, COCs are collected, and the collection process is carried out in the M2 culture solution containing milrinone, so that the separation of the cumulus-oocyte complex is completed.
S20, placing the cumulus-oocyte complex in a culture solution for culture to obtain the MII-stage oocyte, wherein the culture solution is the culture solution.
In this step, the isolated cumulus-oocyte complex is cultured in a culture solution containing tea polyphenol to obtain a stage MII oocyte, specifically, milrinone is removed by washing with the culture solution, and then COCs are transferred to culture drops covered with paraffin oil subjected to night equilibration, each culture drop being 30 to 50. Mu.L. Preferably, the culture conditions are: the volume concentration of the carbon dioxide is 4-6%, the temperature is 36-38 ℃, and the culture is carried out for 15-17 h. More preferably, the culture conditions are: the carbon dioxide volume concentration is 5%, the temperature is 37 ℃, and the culture is carried out for 16 hours. In addition, the humidity is preferably saturated at the temperature, and under the conditions, the quality of the obtained MII-stage oocyte is good.
In addition, preferably, during the culture, the surface of the culture solution is covered with paraffin oil, so that the evaporation of the culture solution and the change of pH value in the culture process can be prevented, and the stability of the culture environment is ensured.
In the examples of the present invention, both steps S10 and S20 were carried out in plastic dishes of 3.5cm diameter, and further, the step S20 cultivation was carried out with a plurality of culture droplets of 30 to 50. Mu.l each.
The method for improving the quality of the maternal-source diabetes ovum is simple and easy, can improve the quality of the maternal-source diabetes ovum and can improve the fertility problem of maternal-source diabetes patients.
The technical scheme of the present invention will be described in further detail with reference to the specific examples and the accompanying drawings, and it should be understood that the following examples are only for explaining the present invention and are not limited thereto.
EXAMPLE 1 culture solution
The culture solution comprises the following components: tea polyphenols (50. Mu.M), heat-inactivated fetal bovine serum (volume fraction 5%), sodium pyruvate (0.25 mM), 100 Xgreen streptomycin (volume fraction 0.75%), FSH (0.5U/ml) and alpha-MEM medium.
EXAMPLE 2 culture solution
The formulation and concentration were the same as in example 1 except that the tea polyphenol concentration was 25. Mu.M.
Example 3 method for improving quality of maternal diabetic ovum
(1) Constructing a maternal diabetes animal model, and separating a cumulus-oocyte complex from the maternal diabetes animal model: the maternal diabetes animal model was established by intraperitoneal injection of 220mg/kg body weight of STZ. The STZ dose was determined by first selecting 7-8 week old mice body weight. STZ was dissolved in ice protected from light with citric acid-sodium citrate buffer, and the amount of liquid injected per mouse was no more than 100 μl. Blood was collected from the tail tip after 4 days, and the blood glucose concentration was measured with a blood glucose meter, which was 17.0mM/L or more, and the model construction was considered successful. 10 days after the model was successfully constructed, PMSG 8IU was intraperitoneally injected, ovaries were taken after 46-48 hours, follicles were punctured with a 1ml syringe needle under a dissecting scope, and COCs were collected, as shown in fig. 2, which is high quality COCs. The whole collection process is carried out in M2 culture solution added with milrinone, and the culture solution is preheated for at least 2 hours in advance.
(2) Placing the cumulus-oocyte complex in a culture solution for culture: the COCs of (1) were transferred to overnight equilibrated paraffin-covered drops (broth of example 1) and washed three times to remove milrinone. As shown in FIG. 3, the washed COCs were cultured in an incubator under conditions of a carbon dioxide concentration of 5%, a temperature of 37℃and a saturation humidity, the culture solution used was the culture solution of example 1, and the culture was performed in vitro for 16 hours to obtain MII-stage oocytes.
After culturing, the obtained oocyte in MII stage is shown in the left graph of FIG. 4, and it can be seen that COCs develop well after culturing for 16 hours, the ovum after removing granulosa cells discharges a first polar body and develops and matures, then the matured COCs are transferred into an M2 culture solution containing 1% hyaluronidase, incubated for 5 minutes at room temperature, and the liquid is blown by a pipetting gun to separate granulosa cells from the ovum; the naked eggs were then collected and washed with M2 medium until clean, as shown in the right panel of fig. 4.
Example 4 method for improving quality of maternal diabetic ovum
The procedure and conditions were the same as in example 3, except that the culture broth of example 2 was used.
Comparative example 1
The procedure and conditions were the same as in example 3, except that tea polyphenol was not added to the culture broth.
The following test was performed on the MII-stage oocytes obtained in examples 3 and 4 and comparative example 1 using a normal ovum as a Control:
using normal ovum as Control, fixing the cleaned ovum with 4% paraformaldehyde for 40 min at room temperature, and cleaning with PBS cleaning solution containing 0.1% Tween20 and 0.01% Triton X-100 for 3 times each for 5 min; then placing in PBS containing 0.01% Triton X-100 for 20 minutes at room temperature, and washing with washing liquid for 3 times each for 5 minutes; the washed ova were transferred to a washing solution (blocking solution) containing 1% BSA (bovine serum), and blocked at room temperature for 1 hour or at 4℃overnight; then transferring into a blocking solution containing a tubulin antibody (an antibody with a fluorescent label, or else a secondary antibody is required to be dyed) for four times overnight; then, washing with a washing liquid for 3 times, each time for 5 minutes; then placing the sample in PI containing quencher, sealing, and observing spindle morphology and chromosome distribution by using a fluorescence inverted microscope to obtain FIG. 5. As can be seen from FIG. 5, compared with comparative example 1 in which tea polyphenol was not added to the culture solution, the spindle structure abnormality and chromosome arrangement abnormality of the MII stage oocyte obtained in examples 3 and 4 of the present invention were significantly reduced, and the MII stage oocyte of example 3 was closer to a normal ovum (Control) similarly to the normal ovum.
The ovum ROS content was detected with a ROS detection kit, purchased from Biyun Tian Biotechnology Co., ltd, using a normal ovum as a Control, and operated as per the instructions to give FIG. 6. As can be seen from FIG. 6, the MII-stage oocytes obtained in examples 3 and 4 of the present invention showed significantly reduced ROS levels compared to comparative example 1, in which tea polyphenol was not added to the culture solution, and had no significant difference from the normal ovum, the MII-stage oocyte of example 3 was closer to the normal ovum.
The mitochondrial membrane potential was detected with a membrane potential detection kit, purchased from Biyun biotechnology Co., ltd, using normal ova as a Control, and the procedure was performed according to the instruction to obtain FIG. 7. As can be seen from FIG. 7, the MII-stage oocytes obtained in examples 3 and 4 of the present invention were recovered in mitochondrial function compared to comparative example 1 in which tea polyphenol was not added to the culture broth, and the MII-stage oocytes of example 3 were closer to normal ova without difference from normal ova.
In conclusion, after the culture solution provided by the invention is used for in vitro culture of the mother-source diabetic cumulus-oocyte complex, the quality of the mother-source diabetic ovum can be obviously improved, the spindle structure of the ovum is abnormal, the arrangement of chromosomes is obviously reduced, the ROS (intracellular reactive oxygen species) level of the ovum is obviously reduced, the mitochondrial function of the ovum is restored, and the ovum has no obvious difference from a normal ovum. Namely, the culture solution provided by the invention can improve the quality of the parent-source diabetic ovum and improve the fertility problem of the parent-source diabetic patient.
The foregoing is merely a preferred embodiment of the present invention and is not intended to limit the scope of the present invention, but various modifications and variations will be apparent to those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the scope of the present invention.
Claims (7)
1. A culture solution for in vitro culture of mother-source diabetes cumulus-oocyte complex is characterized in that the culture solution comprises tea polyphenol, the concentration of the tea polyphenol in the culture solution is 25-50 mu M,
the culture solution also comprises inactivated fetal bovine serum, sodium pyruvate, 100 Xgreen streptomycin, FSH and alpha-MEM culture medium, wherein the volume fraction of the inactivated fetal bovine serum in the culture solution is 4-6%, the concentration of sodium pyruvate in the culture solution is 0.20-0.30 mM, the volume fraction of the 100 Xgreen streptomycin in the culture solution is 0.7-0.8%, and the concentration of FSH in the culture solution is 0.4-0.6U/ml.
2. The culture broth of claim 1, wherein the concentration of tea polyphenols in the broth is 50 μm.
3. The method for improving the quality of the parent-source diabetic ovum is characterized by comprising the following steps of:
s10, constructing a maternal diabetes animal model, and separating a cumulus-oocyte complex from the maternal diabetes animal model;
s20, placing the cumulus-oocyte complex in a culture solution for culturing to obtain the MII-stage oocyte, wherein the culture solution is the culture solution according to claim 1 or 2.
4. The method for improving the quality of a parent-diabetic ovum according to claim 3, wherein the blood sugar concentration of the parent-diabetic animal model is 17.0mM or more in step S10.
5. The method for improving the quality of a maternal diabetic egg according to claim 3, wherein in step S10, the step of isolating a cumulus-oocyte complex from the maternal diabetes animal model comprises:
injecting pregnant mare serum gonadotropin into the abdominal cavity of the maternal diabetes animal model, taking out the ovary after 46-48 hours, and collecting the cumulus-oocyte complex.
6. The method for improving the quality of parent-diabetic ova according to claim 3, wherein in step S20, the culture conditions are as follows: the volume concentration of the carbon dioxide is 4-6%, the temperature is 36-38 ℃, and the culture is carried out for 15-17 h.
7. The method for improving the quality of parent-diabetic ova according to claim 3, wherein in step S20, the surface of the culture broth is covered with paraffin oil.
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| CN1904038A (en) * | 2006-07-28 | 2007-01-31 | 浙江大学 | Cow ovum cell in vitro ripening culturing liquid containing tea polyphenol and its culturing method |
| CN1904039A (en) * | 2006-07-28 | 2007-01-31 | 浙江大学 | Ox embryo in vitro culturing liquid containing tea polyphenol and its culturing method |
| CN104312971A (en) * | 2014-11-04 | 2015-01-28 | 广西大学 | Method for promoting in-vitro buffalo oocyte maturation |
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