Preparation for in-vitro maturation of oocytes and application thereof

Abstract

The invention belongs to the technical field of biology, and particularly relates to a preparation for in-vitro maturation of oocytes and application of the preparation. According to the preparation for in-vitro maturation of the oocytes, a new choice which is stable, efficient, convenient, low in cost and safe is provided for in-vitro maturation culture of the oocytes by innovatively adding the cell lysis solution of the granular cells, the preparation is simple and convenient to use, and the preparation can be used for improving the maturation quality of the oocytes / the development potential of embryos and improving the survival rate of the oocytes. The batch-to-batch difference and the pollution risk are remarkably reduced, the repeatability and the result reliability of experiment and clinical application are ensured, and the application prospect is wide.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology, and particularly relates to a preparation for in vitro maturation of oocytes and application thereof. BACKGROUND

[0002] In in vitro embryo culture, the production process of in vitro embryos starts from the collection of oocytes, mainly including three stages of in vitro maturation of oocytes, in vitro fertilization and in vitro culture of fertilized eggs. The in vitro maturation of oocytes as a key link in this process has a huge impact on the efficiency of subsequent in vitro fertilization and embryo development potential. The in vitro maturation medium of oocytes as one of the core elements in this stage plays a key role in the quality of oocyte maturation. At present, the optimization of the formula of the in vitro maturation medium of oocytes is mainly through the addition of various growth factors, hormones and special components simulating the in vivo environment, so as to better simulate the microenvironment required for in vivo oocyte maturation, and thus improve the efficiency and quality of in vitro maturation of oocytes.

[0003] Ovarian granulosa cells play a crucial role in the growth, development and maturation of oocytes. At present, for the application of ovarian granulosa cells in in vitro maturation of oocytes and preparation of in vitro maturation medium of oocytes, the skilled person in the art has developed two ways. One is to add granulosa cell culture medium to the in vitro maturation medium of oocytes, expecting to improve the maturation environment of oocytes by means of the rich nutritional components and active factors in the granulosa cell culture medium. The other is to co-culture oocytes with ovarian granulosa cells, and then to construct a cell interaction microenvironment closer to the in vivo physiological state to promote the maturation process of oocytes. However, the above two application methods have the problems of poor batch stability, low repeatability, high cost, complex operation, tedious steps, easy pollution and the like, which seriously affect the actual application effect.

[0004] Therefore, it is of great significance to develop a new application method of ovarian granulosa cells in in vitro maturation of oocytes and preparation of in vitro maturation medium of oocytes for further improving the quality and efficiency of in vitro maturation of oocytes. SUMMARY

[0005] The present application aims to at least partly solve at least one of the technical problems existing in the prior art. To this end, the present application provides a preparation for in vitro maturation of oocytes and application thereof. The preparation for in vitro maturation of oocytes of the present application provides a new stable, efficient, convenient, low-cost and safe option for in vitro maturation culture of oocytes by innovatively adding a cell lysate of granulosa cells, and the preparation is simple to use, significantly reduces batch-to-batch variation and contamination risk while improving the maturation quality of oocytes / development potential of embryos, ensures the repeatability and reliability of experimental and clinical applications, and has a wide application prospect.

[0006] In a first aspect of the present application, a preparation for in vitro maturation of oocytes is provided. According to an embodiment of the present application, the preparation contains a cell lysate of granulosa cells. The preparation according to the embodiment of the present application significantly improves the in vitro maturation quality of oocytes and the development efficiency of embryos after fertilization by adding a cell lysate of granulosa cells, is simple to use, has small batch-to-batch variation and contamination risk, has high repeatability and reliability of experimental and clinical applications, provides a new way for efficient use of granulosa cells, and has a wide application prospect.

[0007] According to an embodiment of the present application, the preparation for in vitro maturation of oocytes described above can have the following additional technical features:

[0008] According to an embodiment of the present application, the granulosa cells are derived from the ovary of a mammal.

[0009] According to an embodiment of the present application, the granulosa cells are derived from follicles with a diameter of 5 mm or more.

[0010] According to an embodiment of the present application, the cell lysate is obtained by crushing and centrifuging the granulosa cells.

[0011] According to an embodiment of the present application, the centrifugation is performed at a speed of 3000-200000g for 10-30min.

[0012] According to an embodiment of the present application, the granulosa cells are obtained by in vivo oocyte collection or in vitro oocyte collection.

[0013] According to an embodiment of the present application, the in vitro oocyte collection includes obtaining from the ovary of a dead animal or from in vitro cultured ovarian tissue, wherein the dead animal is a mammal and the ovarian tissue is derived from a mammal.

[0014] According to an embodiment of the present application, the granulosa cells are used in an amount of 33-33000 per oocyte.

[0015] According to an embodiment of the present application, the oocyte is derived from a mammal.

[0016] According to an embodiment of the present application, the preparation further comprises one or more of basal medium, hormone supplement, energy metabolism supplement, growth factor, other supplement, buffer, and antibiotic.

[0017] In a second aspect, the present application provides an in vitro culture method of oocyte. According to an embodiment of the present application, the in vitro culture method comprises: culturing an oocyte in the preparation of the first aspect; wherein the oocyte is derived from a mammal. According to the in vitro culture method of the embodiment of the present application, the maturation quality of the oocyte and the development potential of the in vitro embryo are greatly improved by using the aforementioned preparation for in vitro culture of the oocyte, and the method has the advantages of good oocyte culture effect, simple operation, small batch difference and low pollution risk, and has a wide application prospect.

[0018] In a third aspect, the present application provides an application of the granulosa cell lysate or the preparation of the first aspect in in vitro embryo production, wherein the granulosa cell is derived from the ovary of a mammal.

[0019] It can be understood by those skilled in the art that the features and advantages described above for the preparation for in vitro maturation of oocytes also apply to this application, which will not be described here.

[0020] According to an embodiment of the present application, the above-mentioned application in in vitro embryo production can further have the following additional technical features:

[0021] According to an embodiment of the present application, the granulosa cell lysate or the preparation of the first aspect is used for at least one of the following: improving the maturation quality of in vitro oocyte; improving the development potential of in vitro embryo; wherein the improving the development potential of in vitro embryo comprises improving the development efficiency and / or development quality of in vitro embryo.

[0022] According to an embodiment of the present application, the granulosa cell lysate or the preparation of the first aspect is used for at least one of the following: improving the maturation rate of in vitro oocyte; improving the cleavage rate of in vitro embryo; improving the blastocyst rate of in vitro embryo.

[0023] Additional aspects and advantages of the present application will be in part apparent and in part pointed out hereinafter. BRIEF DESCRIPTION OF DRAWINGS

[0024] The above and / or additional aspects and advantages of the present application will become apparent and be readily appreciated from the following description, including the appended drawings.

[0025] Figure 1 The flowchart of different ways of in vitro maturation culture of oocytes in the embodiments of the present application is shown. DETAILED DESCRIPTION

[0026] Embodiments of the present application are described in detail below. The embodiments described below are exemplary only and are not intended to be limiting of the present application.

[0027] It should be noted that the terms "first", "second" are used only for descriptive purposes and should not be construed as indicating or implying relative importance or an indicated number of technical features. Thus, features defined with "first", "second" can explicitly or implicitly include one or more of the features. Further, in the description of the present application, the meaning of "a plurality of" is two or more, unless otherwise specified.

[0028] The endpoints of the ranges and any values described herein are not limited to the precise values stated. The ranges and values should be construed to be approximations that allow for significant variation. Within the ranges and values, endpoints are included. Other values not explicitly mentioned can be included from the endpoints of the ranges.

[0029] In this document, the terms "comprise" or "comprising" are open-ended, that is, they mean including, but not limited to, what is specifically recited.

[0030] In this document, the terms "optionally", "optional" or "optional" generally mean that the subsequently described event or circumstance can or can not occur, and the description includes situations in which the event or circumstance occurs, as well as situations in which it does not.

[0031] Terms and definitions

[0032] In this document, the term "in vitro maturation medium" refers to a culture medium used for in vitro maturation culture of oocytes, i.e. in vitro maturation medium for oocytes, which contains basic nutrients necessary for the growth and maturation of oocytes, such as amino acids, vitamins, minerals, energy sources, buffers and antibiotics, etc.

[0033] In this document, the term "follicle" refers to the basic functional unit of the ovary, which is a structure formed by oocytes and their surrounding cells in the ovary, including granulosa cells, which are tightly attached to oocytes and secrete various factors to promote the development of oocytes (in this document, "granulosa cells" are synonymous with "follicular granulosa cells").

[0034] In the present specification, the term "FSH" refers to follicle stimulating hormone (a glycoprotein hormone secreted from the anterior pituitary), which is added to the in vitro maturation medium to promote maturation of oocytes.

[0035] In the present specification, the term "hCG" refers to human chorionic gonadotropin (a glycoprotein hormone secreted from the trophoblast cells of the placenta), which is added to the in vitro maturation medium to induce oocytes to complete meiosis.

[0036] In the present specification, the term "penicillin-streptomycin" refers to a mixture of penicillin and streptomycin, which is used to prevent contamination of the in vitro maturation medium by microorganisms, thereby ensuring the normal progress of oocyte culture and the reliability of experimental results.

[0037] In the present specification, the term "TCM199 medium" refers to a commercially available basal medium that has been widely used for cell culture, which is used as a basal medium for in vitro maturation and culture of oocytes, providing essential nutrients such as amino acids and vitamins for oocyte growth.

[0038] In the present specification, the term "DMEM / F12 medium" refers to a commonly used cell culture medium, which is used for culture of follicular granulosa cells in the present embodiment.

[0039] In the present specification, the term "BME amino acid solution" refers to a minimum essential amino acid solution containing essential amino acids required for cell growth.

[0040] In the present specification, the term "1wt% MEM non-essential amino acid solution" refers to a minimum non-essential amino acid solution in the form of 1% by volume, which is mainly used to provide a source of non-essential amino acids for cells.

[0041] In the present specification, the term "number of cleaved oocytes" refers to the number of fertilized oocytes that can be observed to have undergone significant cell division after in vitro fertilization, which reflects the early developmental potential of the fertilized oocyte and the success rate of embryo culture.

[0042] In the present specification, the term "maturation rate" refers to the percentage of oocytes that have reached a mature state among the total number of oocytes cultured after in vitro maturation culture, which is an important indicator for evaluating the effectiveness of in vitro maturation culture of oocytes and the effectiveness of the culture system.

[0043] In the present specification, the term "cleavage rate" refers to the percentage of cleaved oocytes among the total number of fertilized oocytes after in vitro fertilization, which reflects the developmental potential of the fertilized oocyte in the early stage and the effectiveness of the embryo culture process, and is one of the key indicators for assessing the quality of fertilization and the potential of embryo development.

[0044] In the present document, the term "blastocyst rate" refers to the percentage of the number of embryos successfully developed to the blastocyst stage in the total number of fertilized oocytes during the in vitro culture of embryos, which is an important indicator for measuring the quality of embryo development and the effectiveness of the culture system.

[0045] Preparation for in vitro maturation of oocytes

[0046] The present application provides a preparation for in vitro maturation of oocytes. According to an embodiment of the present application, the preparation contains a cell lysate of granulosa cells. The preparation according to the embodiment of the present application significantly improves the quality of in vitro maturation of oocytes and the efficiency of embryo development after fertilization by adding the cell lysate of granulosa cells, and has the advantages of simple use, small batch-to-batch difference and pollution risk, high repeatability and reliability of experimental and clinical application, provides a new way for efficient use of granulosa cells, and has a wide application prospect.

[0047] According to an embodiment of the present application, the granulosa cells are derived from the ovary of a mammal. In this way, by obtaining granulosa cells from the ovary of a mammal, it is ensured that the cell lysate of the granulosa cells prepared is highly matched with the development needs of mammalian oocytes, thereby better playing its promoting role in the in vitro maturation of oocytes.

[0048] It should be noted that in the present application, the mammal is a non-human mammal, including one or more of non-human primates, cows, sheep, pigs, horses and mice, etc.; as long as the ovary of this part of animal can be obtained by in vitro fertilization, cloning, parthenogenesis or other in vitro techniques, it shall belong to the protection scope of the present application.

[0049] According to an embodiment of the present application, the granulosa cells are derived from follicles with a diameter of ≥5mm. In this way, the relative maturity of follicles with a diameter of ≥5mm is higher, and the granulosa cells thereof have more advantages in physiological functions and activities, etc. The performance of the preparation for in vitro maturation of oocytes according to the present application is more optimal when using granulosa cells derived from follicles with a diameter of ≥5mm, and the effect of improving the maturation quality of oocytes and the development potential of subsequent embryos is better.

[0050] According to an embodiment of the present application, the cell lysate is obtained by crushing and centrifuging the granulosa cells. In this way, by first crushing the granulosa cells (such as ultrasonic crushing), the cell contents (including various proteins, nucleic acids, enzymes and other biomolecules beneficial to the maturation of oocytes) in the granulosa cells are fully released, and then by centrifuging, the cell debris and other impurities are removed to obtain a granulosa cell lysate with pure texture, which is added to the preparation for in vitro maturation of oocytes according to the present application, so that the use effect is better and more stable.

[0051] It should be noted that before the granulosa cells are subjected to the crushing treatment, the cell membrane of the granulosa cells can be completely destroyed by operations such as repeated freezing and thawing to further release the bioactive components in the cells, thereby further improving the lysis efficiency and the purity of the granulosa cell lysate. Through this pretreatment method, the subsequent crushing treatment effect can be further improved to ensure that the granulosa cell lysate is rich in sufficient effective components. Such operations should all fall within the protection scope of the present application.

[0052] According to an embodiment of the present application, the centrifugal treatment has a rotation speed of 3000-200000g and a time of 10-30min. In this way, by optimizing the rotation speed and time of the centrifugal treatment, effective separation and enrichment of different components are achieved. Suitable centrifugal conditions can retain key active components in the granulosa cell lysate while removing macromolecular impurities or cell debris that may adversely affect oocytes, thereby further improving the purity and use effect of the preparation for in vitro maturation of oocytes and enhancing the promotion effect on in vitro maturation of oocytes. Exemplarily, the centrifugal treatment has a rotation speed of 3000g, 4000g, 5000g, 6000g, 7000g, 8000g, 9000g, 10000g, 20000g, 30000g, 40000g, 50000g, 60000g, 70000g, 80000g, 90000g, 100000g, 150000g, 200000g, preferably 5000-40000g, and more preferably 10000-30000g. The centrifugal treatment has a time of 10min, 15min, 20min, 25min, 30min, preferably 10-25min, and more preferably 15-20min.

[0053] According to an embodiment of the present application, the granulosa cells are obtained by in vivo oocyte collection or in vitro oocyte collection. In this way, two flexible granulosa cell acquisition methods are provided, both of which can provide a stable and sustainable source of granulosa cells for the preparation for in vitro maturation of oocytes. In vivo oocyte collection can directly obtain fresh granulosa cells from the ovary of a living animal, ensuring the activity and physiological state of the granulosa cells. In vitro oocyte collection obtains granulosa cells from the ovary of a dead animal or in vitro cultured ovarian tissue, further broadening the source channel of granulosa cells and reducing the cost and difficulty of obtaining granulosa cells.

[0054] According to an embodiment of the present application, the in vitro oocyte collection includes obtaining from the ovary of a dead animal or obtaining from in vitro cultured ovarian tissue. The dead animal is a mammal, and the ovarian tissue is derived from a mammal. In this way, the specific implementation of in vitro oocyte collection is further clarified, providing the possibility of adjusting the source channel of granulosa cells according to different application requirements.

[0055] According to the embodiment of the present application, the granulosa cells are used in an amount of 33-33000 per oocyte. In this way, by optimizing the amount of granulosa cells used, the concentration of active ingredients in the granulosa cell lysate contacted by the oocyte during in vitro maturation is further precisely adjusted. The lysate obtained from the appropriate amount of granulosa cells can provide an appropriate concentration of growth factors, nutrients and signaling molecules, thereby more effectively promoting the maturation of oocytes and avoiding problems such as insufficient or excessive maturation of oocytes caused by excessively high or low concentrations, and further improving the maturation quality of oocytes and the developmental potential of in vitro embryos. It should be noted that "the granulosa cells are used in an amount of 33-33000 per oocyte" refers to the appropriate range of the number of granulosa cells from the granulosa cell lysate used for a single oocyte when the oocyte is cultured. Exemplarily, the granulosa cells are used in an amount of 33 per oocyte, 100 per oocyte, 500 per oocyte, 1000 per oocyte, 5000 per oocyte, 8000 per oocyte, 10000 per oocyte, 15000 per oocyte, 20000 per oocyte, 25000 per oocyte, 30000 per oocyte, 33000 per oocyte, preferably 33-33000 per oocyte, and more preferably 3300-33000 per oocyte.

[0056] According to the embodiment of the present application, the oocyte is derived from a mammal. In this way, the specificity and effectiveness of the preparation for in vitro maturation of oocytes of the present application in promoting in vitro maturation of mammalian oocytes are further clarified.

[0057] According to an embodiment of the present application, the preparation further comprises one or more of basal medium, hormone supplement, energy metabolism supplement, growth factor, other supplement, buffer and antibiotic. Thus, by adding basal medium and other components to the preparation for in vitro maturation of oocytes of the present application, the components work together to improve the quality and efficiency of in vitro maturation of oocytes, further enhancing the use effect of the preparation; for example, the basal medium provides basic nutrients and can be TCM199 medium, alpha-modified Eagle's minimum essential medium or other common basal medium in the art; the hormone supplement helps to regulate the physiological state of oocytes and can include one or more of pregnant mare serum gonadotropin, luteinizing hormone, estrogen, follicle-stimulating hormone and human chorionic gonadotropin; the energy metabolism supplement provides energy for cell activity and can include one or more of glucose, sodium pyruvate, pyruvic acid and adenosine triphosphate; the growth factor promotes the growth and development of cells and can include epidermal growth factor, insulin-like growth factor-1, etc.; the other supplement can include one or more of follicular fluid, human serum albumin and amino acids; the buffer maintains acid-base balance and can include one or more of sodium bicarbonate, 4-hydroxyethylpiperazine ethanesulfonic acid and tris-hydroxymethyl aminomethane; and the antibiotic avoids microbial contamination and can include one or more of penicillin, streptomycin, gentamicin, kanamycin, tetracycline and ampicillin.

[0058] Method for in vitro culture of oocytes

[0059] The present application provides a method for in vitro culture of oocytes. According to an embodiment of the present application, the method for in vitro culture comprises: culturing oocytes in the aforementioned preparation; wherein the oocytes are derived from mammals. According to the method for in vitro culture of oocytes of the present application, the maturation quality of oocytes and the development potential of in vitro embryos are greatly improved by using the aforementioned preparation for in vitro culture of oocytes, and the method has the advantages of good oocyte culture effect, simple operation, small batch-to-batch difference and low risk of contamination, and has a wide application prospect.

[0060] It should be noted that, compared to adding granulosa cell culture medium to the in vitro maturation medium of oocytes, co-culturing oocytes and granulosa cells, the method of the present application directly adds granulosa cell lysate, which significantly improves the maturation quality of oocytes, the efficiency of embryo development and the stability of batch culture effect while avoiding contamination and reducing the complexity of operation.

[0061] The mammals include one or more of non-human primates, cows, sheep, pigs, horses and mice; as long as the oocytes of these animals can be obtained by in vitro fertilization, cloning, parthenogenesis or other techniques, they should be within the protection scope of the present application.

[0062] Use in in vitro embryo production

[0063] The present application provides use of a cell lysate of granulosa cells or the aforementioned preparation in in vitro embryo production, wherein the granulosa cells are derived from ovaries of mammals.

[0064] It is understood by those skilled in the art that the features and advantages described above for the preparation for in vitro maturation of oocytes also apply to this use, which will not be repeated here.

[0065] According to an embodiment of the present application, the cell lysate of granulosa cells or the aforementioned preparation is used for at least one of the following: improving the maturation quality of in vitro oocytes; improving the developmental potential of in vitro embryos; wherein the improving the developmental potential of in vitro embryos includes improving the developmental efficiency and / or developmental quality of in vitro embryos.

[0066] According to an embodiment of the present application, the cell lysate of granulosa cells or the aforementioned preparation is used for at least one of the following: improving the maturation rate of in vitro oocytes; improving the cleavage rate of in vitro embryos; improving the blastocyst rate of in vitro embryos.

[0067] The solutions of the present application will be explained below with reference to examples. Those skilled in the art will understand that the following examples are only used to illustrate the present application and should not be regarded as limiting the scope of the present application. If specific techniques or conditions are not specified in the examples, they are carried out according to the techniques or conditions described in the literature in the art or according to the product instructions. If the manufacturers of the reagents or instruments are not specified, they are all conventional products that can be obtained commercially.

[0068] Example 1: Collection, culture of follicular granulosa cells and preparation of granulosa cell lysate

[0069] 1. Collection of follicular granulosa cells

[0070] The ovaries of slaughtered sheep or ovaries obtained by hormone-induced in vivo ovum collection are placed in physiological saline at 30-35°C; they are washed 3 times with physiological saline, and the follicles are divided into <5 mm and ≥5 mm in diameter, and then a 20G needle disposable syringe containing 5 mL of ovum suction solution is used to puncture the follicles, and the follicles are sucked back and forth 2-3 times, and the granulosa cells are collected in a 15 mL centrifuge tube, and the granulosa cells are allowed to settle at the bottom in a 37°C water bath for 20-30 min to obtain in vivo ovum collection ovary-derived granulosa cells / slaughtered ovary-derived granulosa cells, follicle diameter <5 mm follicle-derived granulosa cells / follicle diameter ≥5 mm follicle-derived granulosa cells.

[0071] 2. Collection of follicular granulosa cell culture solution

[0072] The supernatant after removing the cell precipitate in step 1 was removed, and each cell was washed with PBS buffer for 3 times, and then DMEM / F12 culture medium (purchased from Thermo Fisher, item number 11320033) containing 15% fetal bovine serum was used, and 10 mIU / mL FSH (purchased from MERCK, item number 869001-M), 10 IU / mL hCG (purchased from MERCK, item number 230734), and 1 mM sodium pyruvate were added to promote adhesion and proliferation, and the cell inoculation density was 1×10 cells / mL, and the cells were cultured at 37°C and 5% CO to a cell confluence of 80%-90%, and then the cells were digested with 0.25% trypsin-EDTA for 3-5 min, and then the digestion was terminated with DMEM / F12 culture medium containing 15% fetal bovine serum, and the cells were subcultured at a ratio of 1:2-1:3, and the cell culture liquid was collected in a 15 mL centrifuge tube, centrifuged at 12000 g for 20 min, and the supernatant was taken and aliquoted to obtain follicular granulosa cell culture liquid, which was stored at -80°C for standby.

[0073] 3. Preparation of follicular granulosa cell lysate

[0074] The supernatant after removing the cell precipitate in step 1 was removed, and each cell was resuspended in a centrifuge tube containing PBS buffer containing 5% penicillin-streptomycin, centrifuged at 1000-1500 g for 3 min, resuspended, centrifuged, and washed twice, and then each cell was resuspended in 500 μL TCM199 culture medium (purchased from Thermo Fisher, item number 11150059) and transferred to a 1.5 mL centrifuge tube; then each 1.5 mL centrifuge tube was placed in liquid nitrogen for freezing and thawing operation, repeated 3 times, and finally placed in an ultrasonic disruptor to completely break the cells to obtain a lysate; then the lysate was centrifuged at 1000 g, 3000 g, 5000 g, 8000 g, 10000 g, 12000 g, 15000 g, 20000 g, 30000 g, 40000 g, 60000 g, 100000 g, 200000 g for 20 min, and the supernatant was aliquoted to obtain follicular granulosa cell lysate, which was stored at -80°C for standby.

[0075] Example 2: In vitro maturation of oocytes, in vitro fertilization, and embryo culture

[0076] 1. In vitro maturation of oocytes

[0077] Collect sheep ovary, put in 30-35℃ physiological saline, 2h in the laboratory, and use physiological saline to wash 3 times, and then use a 20G needle of 5mL suction liquid to puncture the follicle, release the cumulus oocyte complex, collect the sheep cumulus oocyte complex (COCs) with uniform cytoplasm and more than 3 layers of cumulus cells under the stereoscope, and then transfer the selected sheep cumulus oocyte complex to a four-well plate containing 600μL of different in vitro maturation culture medium, 300μL of mineral oil, and 30 sheep cumulus oocyte complexes per well, and then place the four-well plate in a 5% CO2, 38.5℃ saturated humidity incubator for 22-24h to obtain mature COCs, and calculate the maturation rate, wherein the calculation method of the maturation rate is shown in formula 1.

[0078] Mature rate = mature oocyte number / total number of cultured oocytes * 100% formula 1

[0079] Among them, the above-mentioned "different in vitro maturation culture medium" specifically refers to: the control group uses oocyte in vitro maturation basic culture medium; the culture medium used in the treatment group is oocyte in vitro maturation basic culture medium added with follicular granulosa cells obtained in step 1 of example 1, or oocyte in vitro maturation basic culture medium added with follicular granulosa cell culture medium prepared in step 2 of example 1, or oocyte in vitro maturation basic culture medium added with follicular granulosa cell lysate prepared in step 3 of example 1.

[0080] The preparation method of the suction liquid is as follows:

[0081] 49mL TCM199 culture medium + 1mL FBS (fetal bovine serum).

[0082] The preparation method of the oocyte in vitro maturation basic culture medium is as follows:

[0083] 90wt% TCM199 culture medium, 10IU / mL pregnant mare serum gonadotropin, 10IU / mL luteinizing hormone, 10ng / mL estrogen, 100IU / mL penicillin, 100μg / mL streptomycin, 10wt% follicular fluid, 0.50mg / mL glucose, 2.10mg / mL sodium bicarbonate, 0.06mg / mL sodium pyruvate, 0.57mg / mL cysteine.

[0084] 2, in vitro fertilization

[0085] The mature COCs obtained in step 1 were placed in 0.5% hyaluronidase, most of the cumulus cells were removed by gently repeated pipetting, and then the mature oocytes were obtained by washing 3 times with sperm solution, and then were transferred into a four-well plate pre-equilibrated in an incubator for more than 2 hours and containing 400 μL of sperm solution, 300 μL of mineral oil per well for culture, 30 mature oocytes per well, for in vitro fertilization.

[0086] The frozen sheep semen was taken out from liquid nitrogen and quickly thawed in a 38°C water bath, and then the thawed sperm was transferred into 600 μL of sperm upstream solution (purchased from IVF Bioscience, item number 73671004) and incubated in an incubator for 30 min to allow the sperm to fully float; then 100 μL of supernatant was taken and added to the four-well plate containing the mature oocytes, and the sperm-mature oocyte was co-incubated for 19-21 h, with the fertilization conditions set as saturated humidity, 38.5°C, 5% CO2, 95% air.

[0087] The preparation method of the sperm solution is as follows:

[0088] mSOFaa culture solution + 2 wt% fetal bovine serum + 100 μg / mL streptomycin + 100 IU / mL penicillin.

[0089] The preparation method of mSOFaa is as follows (all reagents required for preparation are purchased from Sigma-Aldrich):

[0090] Each 1 L contains 6.34 g of NaCl, 0.54 g of KCl, 0.23 g of CaCl2·2H2O, 0.01 g of MgSO4·7H2O, 0.17 g of KH2PO4, 0.24 g of NaHCO3, 0.30 g of glucose, 0.04 g of sodium pyruvate, 5.8 mL of sodium lactate, 2 wt% BME amino acid solution and 1 wt% MEM non-essential amino acid solution.

[0091] 3. Embryo culture

[0092] After the sperm-mature oocyte was co-incubated for 19-21 h, the oocytes were aspirated, the residual cumulus cells and sperm were removed by gentle pipetting, and then washed 3 times with mSOFaa culture solution, and then transferred into a four-well plate pre-equilibrated in an incubator for more than 2 hours and containing 500 μL of mSOFaa culture solution per well for culture, and cultured in a saturated humidity incubator at 38.5°C, 5% O2, 5% CO2, 90% N2; after 2 days of culture, the cleavage rate was counted, and the culture was continued to the 7th day, and the blastocyst rate was counted, wherein the calculation method of the cleavage rate is shown in formula 2, and the calculation method of the blastocyst rate is shown in formula 3.

[0093] Cleavage rate = (number of oocytes with cleavage / total number of fertilized oocytes) * 100% formula 2

[0094] Blastocyst rate = (number of embryos developed to blastocyst stage / total number of zygotes) * 100% Formula 3

[0095] Example 3: Effect of granulosa cell lysate obtained by adding different centrifugal speeds on oocyte maturation and development

[0096] 1. Control group / treatment group condition setting

[0097] According to the reference example 2 step 1, the immature oocytes were cultured in vitro for 24 hours using the oocyte in vitro maturation basic culture solution (500 μL) to obtain mature COCs; wherein the only difference between the culture conditions of the control group and each treatment group is whether to additionally add the follicular granulosa cell lysate obtained in example 1 step 3, and the only difference between the culture conditions of each treatment group is to add the follicular granulosa cell lysate obtained by different centrifugal speeds in example 1 step 3 (the follicular granulosa cell lysate of each treatment group is obtained by referring to example 1 and the treatment conditions are consistent except for the different centrifugal speeds), and the addition of follicular granulosa cell lysate of the control group and each treatment group is shown in Table 1.

[0098] Table 1 Addition of follicular granulosa cell lysate of the control group and each treatment group

[0099]

[0100]

[0101] The maturation rate was counted, and then in vitro fertilization and embryo culture were carried out according to the reference example 2 steps 2 and 3, and the cleavage rate and blastocyst rate were counted.

[0102] 2. Test results

[0103] The investigation of the effect of follicular granulosa cell lysate obtained by different centrifugal speeds on oocytes is shown in Table 2.

[0104] Table 2 Investigation of the effect of follicular granulosa cell lysate obtained by different centrifugal speeds on oocytes

[0105]

[0106]

[0107] In Table 2, the upper right corner of each result value is marked with a, b, c, d, indicating that there is a significant difference p < 0.05 between each group.

[0108] The results show that: the blastocyst rates of oocytes added with follicular granulosa cell lysate obtained by centrifugation at 5000-40000g for 20min (treatment groups 3-10) are 25.25±3.25%, 34.02±3.38%, 39.31±3.72%, 43.46±3.29%, 43.37±3.56%, 42.63±3.83%, 39.58±3.48%, 32.12±3.23%, which are significantly higher than the blastocyst rates of the control group and other treatment groups (p<0.05); the blastocyst rates of oocytes added with follicular granulosa cell lysate obtained by centrifugation at 60000, 100000, 200000g for 20min (treatment groups 12-13) are 19.99±2.11%, 20.24±2.34%, 17.65±2.86%, which are higher than that of the control group (16.98±3.47%), but there is no significant difference (p>0.05)

[0109] The above results show that: the follicular granulosa cell lysate obtained by centrifugation at 5000-40000g for 20min is the most effective for the development of oocytes and has the best culture effect when added to the culture of immature oocytes.

[0110] Example 4: Effect of follicular granulosa cell lysate from follicles of different sizes on oocyte maturation and development

[0111] 1. Control group / treatment group condition setting

[0112] According to step 1 of Reference Example 2, immature oocytes were cultured in vitro for 24h using oocyte in vitro maturation basic culture medium (500μL) to obtain mature COCs; wherein the only difference between the culture conditions of the control group and each treatment group is whether to additionally add follicular granulosa cell lysate obtained in step 3 of Example 1, and the only difference between the culture conditions of each treatment group is the addition of follicular granulosa cell lysate obtained in step 1 of Example 1 (except that the follicles from which the follicular granulosa cell lysate is obtained are different, the steps for obtaining the follicular granulosa cell lysate are referred to Example 1 and the treatment conditions are consistent), and the addition of follicular granulosa cell lysate in the control group and each treatment group is shown in Table 3.

[0113] Table 3 Addition of follicular granulosa cell lysate in the control group and each treatment group

[0114]

[0115]

[0116] The maturation rate was counted, and then in vitro fertilization and embryo culture were performed according to steps 2 and 3 of Reference Example 2, and the cleavage rate and blastocyst rate were counted.

[0117] 2. Test results

[0118] The influence of follicular granulosa cell lysate from follicles with different diameters on oocyte development is shown in Table 4.

[0119] Table 4 Influence of follicular granulosa cell lysate from follicles with different diameters on oocyte development

[0120] Group Oocyte number Maturity rate (%) cleavage rate (%) blastocyst rate (%) Control group 277 80.71 ± 6.02 b ]] 72.66 ± 5.33 b ]] 21.38 ± 2.31 b ]] Treatment group 1 295 80.33 ± 5.67 b ]] 75.31 ± 6.12 b ]] 22.47 ± 2.54 b ]] Treatment group 2 283 92.84 ± 6.18 a ]] 85.19 ± 7.02 a ]] 45.63 ± 2.68 a ]]

[0121] In Table 4, the upper right corner of each result value is marked with a, b, and c, indicating that there is a significant difference between each group (p < 0.05).

[0122] The results show that the oocyte maturation rate (92.84 ± 6.18%), cleavage rate (78.9 ± 7.1%), and blastocyst rate (45.63 ± 2.68%) of the treatment group 2, which added follicular granulosa cell lysate from follicles with a diameter of ≥5 mm, were significantly higher than those of the control group and treatment group 1 (80.71 ± 6.02%; 80.33 ± 5.67%; 72.66 ± 5.33%; 75.31 ± 6.12%; 21.38 ± 2.31%; 22.47 ± 2.54%) (p < 0.05), and there was no difference between treatment group 1 and the control group (p > 0.05).

[0123] The above results show that adding follicular granulosa cell lysate from follicles with a diameter of ≥5 mm is the most effective for the development of immature oocytes and has the best culture effect.

[0124] Example 5: Influence of follicular granulosa cell lysate from different sources on oocyte maturation and development

[0125] 1. Control group / treatment group condition setting

[0126] According to step 1 of Reference Example 2, immature oocytes were cultured in vitro for 24 h using oocyte in vitro maturation basic culture medium (500 μL) to obtain mature COCs. The difference between the control group and each treatment group was only whether to additionally add follicular granulosa cell lysate obtained in step 3 of Example 1. The difference between each treatment group was only the addition of follicular granulosa cell lysate obtained in step 1 of Example 1 (except for the different follicle sources, the steps for obtaining follicular granulosa cell lysate of each treatment group were based on Example 1, and the treatment conditions were consistent). The addition of follicular granulosa cell lysate in the control group and each treatment group is shown in Table 5.

[0127] Table 5 Addition of follicular granulosa cell lysate in the control group and each treatment group

[0128]

[0129]

[0130] Statistical maturation rate, then refer to Example 2 step 2, 3 for in vitro fertilization and embryo culture, and statistics cleavage rate and blastocyst rate.

[0131] 2、Test results

[0132] The effect of follicular granulosa cell lysate obtained from different sources of ovaries on oocytes is shown in Table 6.

[0133] Table 6 The effect of follicular granulosa cell lysate obtained from different sources of ovaries on oocytes

[0134] Group Oocyte number Maturity rate (%) cleavage rate (%) blastocyst rate (%) Control group 193 78.44 ± 5.76 b ]] 71.52 ± 6.08 b ]] 22.06 ± 2.74 c ]] Treatment group 1 208 90.15 ± 6.24 a ]] 86.92 ± 6.73 a ]] 46.13 ± 3.05 b ]]> Treatment group 2 225 93.36 ± 6.58 a ]] 87.34 ± 6.81 a ]] 54.85 ± 3.89 a ]]

[0135] In Table 6, the upper right corner of each result value is marked with a, b, c, indicating that there is a significant difference between groups p < 0.05.

[0136] The results show that the oocyte maturation rate (90.15 ± 6.24%; 93.36 ± 6.58%) and cleavage rate (86.92 ± 6.73%; 87.34 ± 6.81%) of the follicular granulosa cell lysate obtained from slaughterhouse ovaries (treatment group 1) and the follicular granulosa cell lysate obtained from live oocyte collection (treatment group 2) are significantly higher than the maturation rate (78.44 ± 5.76%) and cleavage rate (71.52 ± 6.08%) of the control group (p < 0.05), and there is no significant difference between treatment group 1 and 2 (p > 0.05); However, the blastocyst rate of treatment group 2 (54.85 ± 3.89%) is significantly higher than that of the control group and treatment group 1 (22.06 ± 2.74%; 46.13 ± 3.05%) (p < 0.05).

[0137] The above results show that compared with the follicular granulosa cell lysate obtained from slaughterhouse ovaries, the addition of follicular granulosa cell lysate obtained from live oocyte collection is the most effective for immature oocyte culture, and the culture effect is better.

[0138] Example 6 Effect of follicular granulosa cell lysate with different amounts of granulosa cells on oocyte maturation and development

[0139] 1、Control group / treatment group condition setting

[0140] Reference Example 2 Step 1, the immature oocytes were cultured in vitro for 24 h using oocyte in vitro maturation basic medium (500 μL) to obtain mature COCs; wherein the only difference between the culture conditions of the control group and each treatment group was whether 10 wt% follicular granulosa cell lysate obtained in Example 1 Step 3 was additionally added, and the only difference between the culture conditions of each treatment group was the addition of follicular granulosa cell lysate obtained in Example 1 Step 3 (the steps for obtaining follicular granulosa cell lysate of each treatment group were all with reference to Example 1, except that the number of granulosa cells was different, the follicle diameter was ≥5 mm, and the centrifugal conditions were 12000 g, 20 min), and the addition of follicular granulosa cell lysate of the control group and each treatment group is shown in Table 7.

[0141] Table 7 Addition of follicular granulosa cell lysate of the control group and each treatment group

[0142] Group Oocyte number Maturity rate (%) cleavage rate (%) blastocyst rate (%) Add 10 wt% of the lysate obtained from 1 x 10 4 follicular granulosa cells Control group Add 10 wt% of the lysate obtained from 1 x 10 5 follicular granulosa cells Treatment group 1 Add 10 wt% of the lysate obtained from 1 x 10 6 follicular granulosa cells Treatment group 2 Add 10 wt% of the lysate obtained from 1 x 10 7 follicular granulosa cells Group Add 10 wt% of the lysate obtained from 1 x 10 8 follicular granulosa cells

[0143] The maturation rate was counted, and then in vitro fertilization and embryo culture were performed according to Reference Example 2 Steps 2 and 3, and the cleavage rate and blastocyst rate were counted.

[0144] 2. Test results

[0145] The influence of follicular granulosa cell lysate obtained from different numbers of follicular granulosa cells on oocytes is shown in Table 8.

[0146] Table 8 Influence of follicular granulosa cell lysate obtained from different numbers of follicular granulosa cells on oocytes

[0147] Oocyte number Maturity rate (%) cleavage rate (%) blastocyst rate (%) Control group Treatment group 1 232 82.34 ± 7.11 b ]] 72.45 ± 4.67 b ]] 18.69 ± 3.42 d ]] Treatment group 2 264 87.65 ± 5.31 a ]] 76.32 ± 4.26 b ]] 23.22 ± 2.34 c ]] Treatment group 3 235 92.12 ± 6.24 a ]]> 84.93 ± 5.13 a ]]> 36.30 ± 2.75 b ]] Treatment group 4 267 93.91 ± 5.83 a ]] 85.67 ± 5.46 a ]] 43.15 ± 3.29 a ]] Treatment group 5 241 91.78 ± 5.36 a ]] 87.14 ± 5.52 a ]]> 43.02 ± 3.38 a ]] Group 258 84.53 ± 4.75 b ]] 75.32 ± 3.94 b ]] 18.01 ± 4.57 d ]]

[0148] In Table 8, the upper right corner of each result value is marked with a, b, c, d, indicating that there is a significant difference p < 0.05 between each group.

[0149] The results show that: the addition of 10 wt% follicular granulosa cell lysate obtained from 1 × 10 4 ~ 1 × 10 7The maturation rate of oocytes (87.65±5.31%; 92.12±6.24%; 93.91±5.83%; 91.78±5.36%), cleavage rate (76.32±4.26%; 84.93±5.13%; 85.67±5.46%; 87.14±5.52%), and blastocyst rate (23.22±2.34%; 36.30±2.75%; 43.15±3.29%; 43.02±3.38%) of the follicular granulosa cell lysate obtained from 10×106 follicular granulosa cells (treatment groups 1-4) were significantly higher than the maturation rate, cleavage rate, and blastocyst rate of the control group and treatment group 5 (82.34±7.11%, 72.45±4.67%, 18.69±3.42%; 84.53±4.75%, 75.32±3.94%, 18.01±4.57%) (p<0.05).

[0150] The above results show that: the addition of 10wt% of 1×106 follicular granulosa cells to the follicular granulosa cell lysate is the most effective for the development of immature oocytes, has obvious promotion effect, and has better culture effect. 4 ~1×106 7 The follicular granulosa cell lysate obtained from 10×106 follicular granulosa cells has the most effective, obvious promotion effect, and better culture effect on the development of immature oocytes.

[0151] Example 7: Comparison of the effects of different granulosa cell treatment methods on in vitro maturation of oocytes

[0152] 1. Control group / treatment group condition setting

[0153] In step 1 of the reference example 2, immature oocytes were cultured in vitro for 24 hours using oocyte in vitro maturation basic culture solution (500 μL) to obtain mature COCs; wherein, the only difference between the culture conditions of the control group and each treatment group is whether to additionally add follicular granulosa cell culture solution, follicular granulosa cells, or follicular granulosa cell lysate. The culture conditions of the control group and each treatment group are shown in Table 9.

[0154] Table 9 Treatment conditions of the control group and each treatment group

[0155]

[0156] The maturation rate was calculated, and then in vitro fertilization and embryo culture were performed according to steps 2 and 3 of the reference example 2, and the cleavage rate and blastocyst rate were calculated. The experiment was repeated for 20 batches, and the batch error rate was calculated. The calculation method of the batch error rate is shown in formula 4.

[0157] Batch error rate = (absolute error / true value) × 100% formula 4

[0158] 2. Test results

[0159] The influence of different granulosa cell treatment methods on oocyte development is shown in Table 10, the influence of different granulosa cell treatment methods on oocyte culture batch error is shown in Table 11, and the influence of different granulosa cell treatment methods on oocyte contamination is shown in Table 12.

[0160] Table 10 Influence of different granulosa cell treatment methods on oocyte development

[0161] Oocyte number Maturity rate (%) cleavage rate (%) blastocyst rate (%) Control group Treatment group 1 202 81.09 ± 5.97 b ]] 70.31 ± 4.68 b ]] 19.36 ± 2.05 c ]] Treatment group 2 216 80.06 ± 12.39 b ]] 71.42 ± 8.11 b ]] 21.12 ± 9.48 c ]]> Treatment group 3 223 83.60 ± 8.71 b ]] 71.27 ± 5.63 b ]] 25.18 ± 3.83 b ]] Treatment group 4 239 93.15 ± 4.52 a ]] 88.09 ± 4.87 a ]] 48.55 ± 4.07 a ]]

[0162] In Table 10, the upper right corner of each result value is marked with a, b, and c, indicating that there is a significant difference between each group (p < 0.05).

[0163] The results show that the oocyte maturation rate (93.15 ± 4.52%), cleavage rate (88.09 ± 4.87%), and blastocyst rate (48.55 ± 4.07%) of the additional follicular granulosa cell lysate (treatment group 3) are significantly higher than those of the control group, treatment group 1, and treatment group 2 (maturation rate: 81.09 ± 5.97%; 80.06 ± 12.39%; 83.60 ± 8.71%; cleavage rate: 70.31 ± 4.68%; 71.42 ± 8.11%; 71.27 ± 5.63%; blastocyst rate: 19.36 ± 2.05%; 21.12 ± 9.48%; 25.18 ± 3.83%) (p < 0.05).

[0164] The above results show that compared with the additional follicular granulosa cell culture solution and co-culture with follicular granulosa cells, the addition of follicular granulosa cell lysate in the oocyte in vitro maturation culture solution is the most effective way to improve the development efficiency of oocytes.

[0165] Table 11 Influence of different granulosa cell treatment methods on oocyte culture batch error

[0166] Treatment group 5 Group Oocyte number Maturity rate (%) 20 3.37 c <!-- 14 -->]]> cleavage rate (%) 20 21.69 a ]]> blastocyst rate (%) 20 10.36 b ]]> Control group 20 3.11 c ]]>

[0167] In Table 11, the upper right corner of each result value is marked with a, b, and c, indicating that there is a significant difference between each group (p < 0.05).

[0168] The results show that the batch error rate of the additional follicular granulosa cell culture solution (treatment group 1) is 21.69%, and the batch error rate of the co-culture with follicular granulosa cells (treatment group 2) is 10.36%, both of which are significantly higher than the batch error rate of the additional follicular granulosa cell lysate (treatment group 3) (3.11%) (p < 0.05).

[0169] The above results show that, compared with the two ways of adding follicular granulosa cell culture solution and co-culturing with follicular granulosa cells, the method of adding follicular granulosa cell lysate to the in vitro maturation culture solution of oocytes has the smallest batch error on the development efficiency of oocytes, that is, the cell culture effect is the most stable.

[0170] Table 12 Investigation of the influence of different granulosa cell treatment methods on oocyte contamination

[0171] Treatment group 1 Treatment group 2 Treatment group 3 Group Number of repeated batches 20 0 0.00 c ]]> Batch error rate (%) 20 3 15.00 b ]]> Control group 20 9 45.00 a ]]> Treatment group 1 Treatment group 2 Treatment group 3 Group Number of repeated batches Number of contaminated batches Contamination rate (%) Control group Treatment group 1 Treatment group 2 Treatment group 3 20 0 0.00 c ]]>

[0172] In Table 11, the upper right corner of each result value is marked with a, b, and c, indicating that there is a significant difference between each group (p < 0.05).

[0173] The results show that the contamination rate of co-culturing with follicular granulosa cells (treatment group 2) is 45.00%, and the contamination rate of adding follicular granulosa cell culture solution (treatment group 1) is 15.00%, both of which are significantly higher than the contamination rate of adding follicular granulosa cell lysate (treatment group 3) (0%) (p < 0.05).

[0174] The above results show that, compared with the two ways of adding follicular granulosa cell culture solution and co-culturing with follicular granulosa cells, the method of adding follicular granulosa cell lysate to the in vitro maturation culture solution of oocytes has the smallest batch error on the development efficiency of oocytes, that is, the cell culture effect is the most stable.

[0175] In the description of the present specification, the description of the terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" and the like means that the specific features, structures, materials or characteristics described in connection with the embodiment or example are included in at least one embodiment or example of the present application. In the present specification, the illustrative description of the above terms does not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any appropriate manner in any one or more embodiments or examples. In addition, different embodiments or examples described in the present specification and the features of different embodiments or examples can be combined and combined by those skilled in the art without contradiction.

[0176] Although the embodiments of the present application have been shown and described above, it should be understood that the above embodiments are exemplary and should not be construed as limiting the present application, and those skilled in the art can make changes, modifications, replacements and variations to the above embodiments within the scope of the present application.

Claims

1. A preparation for in vitro maturation of oocytes, characterized by, The preparation contains: A cell lysate of granulosa cells.

2. The preparation according to claim 1, characterized in that, The granulosa cells are derived from the ovary of a mammal.

3. The preparation according to claim 1, characterized in that, The granulosa cells are derived from follicles with a diameter of ≥5mm.

4. The preparation according to claim 1, characterized in that, The cell lysate is obtained by crushing and centrifuging the granulosa cells.

5. The preparation according to claim 4, characterized in that, The centrifugation is performed at a speed of 3000-200000g for 10-30min.

6. The formulation of claim 1, wherein, The granulosa cells are obtained by in vivo or in vitro oocyte collection.

7. The preparation according to claim 6, characterized in that The in vitro oocyte collection includes obtaining from the ovary of a dead animal or from in vitro cultured ovarian tissue. The dead animal is a mammal and the ovarian tissue is derived from a mammal.

8. The preparation according to claim 4, characterized in that, The granulosa cells are used in an amount of 33-33000 per oocyte.

9. The formulation of claim 1, wherein, The oocyte is derived from a mammal.

10. The formulation of claim 1, wherein, The preparation further comprises one or more of basal medium, hormone supplement, energy metabolism supplement, growth factor, other supplement, buffer and antibiotic.

11. A method for in vitro culture of oocytes, characterized by, The in vitro culture method comprises: Placing the oocyte in the preparation of any one of claims 1-10 for culture. The oocyte is derived from a mammal.

12. Use of a cell lysate of granulosa cells or a preparation according to any one of claims 1 to 10 in the production of embryos in vitro, wherein, The granulosa cells are derived from the ovary of a mammal.

13. Use according to claim 12, characterized in that, The cell lysate of granulosa cells or the preparation of any one of claims 1-10 is used for at least one of: Improving the maturation quality of in vitro oocytes; Improving the developmental potential of in vitro embryos; The improvement of the developmental potential of in vitro embryos includes improving the developmental efficiency and / or quality of in vitro embryos. The cell lysate of granulosa cells or the preparation of any one of claims 1-10 is used for at least one of:

14. The use according to claim 12, characterized in that, Improving the maturation rate of in vitro oocytes; Improving the cleavage rate of in vitro embryos; Improving the blastocyst rate of in vitro embryos. ​

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