CN107460161B - Culture medium for promoting in-vitro maturation of immature oocyte and application thereof - Google Patents
Culture medium for promoting in-vitro maturation of immature oocyte and application thereof Download PDFInfo
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Abstract
The invention relates to a culture medium for promoting in-vitro maturation of immature oocyte and application thereof. According to the invention, through researching the effect of different additives on the immature oocyte, a culture medium capable of promoting the in vitro maturation of the immature oocyte is screened, the culture medium is prepared by adding 20-100 mu M BAPTA-AM into a conventional oocyte in vitro maturation culture solution, the culture medium is safe and has no toxic or side effect, the immature oocyte, especially the oocyte in a three-level cumulus-oocyte complex, can be subjected to meiosis, the in vitro developmental capacity of the cells is improved, the cell maturation is promoted, the cleavage rate and blastocyst rate after parthenogenesis activation after the cell maturation are improved, and the embryo quality is further improved. The application of the culture medium provides favorable support for the in vitro propagation technology of the animal embryo, and has good application prospect.
Description
Technical Field
The invention relates to a culture medium for promoting in-vitro maturation of immature oocyte and application thereof, belonging to the technical field of reproductive biology.
Background
In reproductive biology, a large amount of egg sources are needed for embryo production, generally, oocytes obtained directly from slaughtered animal ovaries are adopted, or oocytes are obtained from living animal ovaries in a living egg collecting mode, however, because the oocytes obtained from the ovaries are not completely mature, the immature oocytes need to be cultured in vitro for a period of time to be mature, and then the subsequent stages of in vitro fertilization, embryo development and the like can be completed, so that the reproductive production is really realized.
The mammalian ovary contains a large amount of immature oocytes, so that the direct taking of the immature oocytes from the ovary for in vitro maturation culture is the most effective and economic way to obtain a large amount of mature oocytes, the large-scale commercial production of good variety genetic materials can be realized, a plurality of oocyte in vitro culture systems are also established, and the relationship between the form of the oocytes and the capabilities of maturation, fertilization, development and the like of the oocytes is clear. However, oocytes matured in vitro still have a great difference from those matured in vivo, because most of the oocytes obtained from ovaries do not mature, immature oocytes obtained from ovaries conventionally are also called cumulus-oocyte complexes, i.e., COCs, and are classified into four grades, wherein the primary oocytes are uniform in cytoplasm and are surrounded by 5 or more layers of granulocytes; the cytoplasm of the secondary COCs is uniform, and 2-4 layers or more of granular cells are arranged around the secondary COCs; the third-level COCs have nonuniform cytoplasm and less than 2 layers or partially naked granulocytes around the third-level COCs; quaternary COCs are not formed. The COCs can be subjected to in vitro maturation culture through a current culture system and can develop into usable mature oocytes, about 80% of primary COCs can be subjected to conventional in vitro maturation culture to become usable mature oocytes, about 40% of secondary COCs can be subjected to conventional in vitro maturation culture to become usable mature oocytes, and the tertiary COCs are one of the most extracted oocytes, but only about 30% of conventional in vitro maturation culture systems can be subjected to maturation, and the quaternary COCs cannot be subjected to maturation development and can only be discarded. However, only a small number of primary and secondary COCs were collected, the largest number being less than 2 or partially naked tertiary COCs of generally considered unusable cytosolic inhomogeneities, surrounding granulocytes.
If the three-stage cumulus-oocyte compound with the largest quantity obtained by taking eggs each time can be developed into mature oocytes through in-vitro maturation culture, rich mature egg sources can be provided for breeding, cloning, sex control, nuclear transplantation, transgenic animal production and the like, and waste is changed into valuable. The propagation of excellent genes of high-value breeding stock can be expanded, the pain of egg taking can be relieved for people, and the hope of utilizing the ovum to breed offspring is seen for many old women with ovarian function decline.
BAPTA-AM (1, 2-bis (2-aminophenoxy) ethane-N, N, N ', N' -tetraacetic acid tetraacetoxymethyl ester) is a selectively permeable membrane Ca2+Chelating agents, which release BAPTA rapidly with Ca upon entry into the cell by lipase2+Complexing, controlling intracellular calcium levels, is a study of Ca2+The standard scientific research tool medicine for regulating the cell function has the following advantages: for Ca2+The selectivity is strong, the affinity is strong, and the combination and dissociation speed is high; is stable and is little influenced by pH; simple stoichiometry with Ca2+The binding ratio is 1: 1; has high safety to cells, is easy to enter cells without damaging the cells, and has no toxicity in an effective dosage range. Has research proved that Ca2+Activation of endonuclease is responsible for DNA fragmentation during apoptosis, BAPTA-AM complex Ca2+Then, the activity of endonuclease is inhibited, and the cells are protected. However, it is not yet determined whether BAPTA-AM can promote the in vitro maturation culture of the tertiary cumulus-oocyte complex and is used for embryo production, and no research report in the aspect is available at present.
Disclosure of Invention
The invention aims to provide a culture medium for promoting in-vitro maturation of immature oocyte. Another object of the present invention is a method for promoting maturation of cumulus-oocyte complexes in vitro using the culture medium.
The culture medium for promoting in-vitro maturation of immature oocyte provided by the invention contains BAPTA-AM.
Further, the final concentration of BAPTA-AM in the in vitro maturation medium is 20-100. mu.M.
Further, the final concentration of BAPTA-AM in the in vitro maturation medium is 30-80. mu.M.
Preferably, the final concentration of BAPTA-AM in the in vitro maturation medium is 50-60. mu.M.
More preferably, the final concentration of BAPTA-AM in the in vitro maturation medium is 50. mu.M.
In one embodiment of the invention, BAPTA-AM is added to conventional oocyte in vitro maturation medium TCM199 with FBS 10%, FSH 0.02IU/m L, L H0.02 IU/m L, E2 1mg/m L by volume of the total final system.
The immature oocyte is a primary, secondary or tertiary cumulus-oocyte complex.
Preferably, the immature oocyte is a secondary or tertiary cumulus-oocyte complex.
The invention provides application of the culture medium in improving the cleavage rate and/or the embryo development rate of the parthenogenetic activation of the oocyte of the animal.
The invention provides application of the culture medium in promoting in-vitro maturation of immature oocyte of an animal.
The invention provides an application of BAPTA-AM in promoting in-vitro maturation of immature oocyte of an animal. The animal is a fetal mammal.
The fetal mammals include cattle, sheep, horse, mouse, panda, tiger, and primate.
The invention also provides a method for promoting in-vitro maturation of immature oocyte, which comprises the following steps:
(1) washing immature oocyte in an egg extracting solution, and then washing by using the in vitro maturation culture medium;
(2) after washing, the immature oocyte is placed in the in vitro maturation medium and treated with CO2Balancing in an incubator for 2-3h, at 38-39 ℃, 5% of CO2 and 100% of humidity;
(3) number of oocytes: culturing the culture solution at a ratio of 1:10-1:15 in an incubator for 20-28 hr under the conditions of 38-39 deg.C, 5% CO2, and 100% humidity. Preferably, the incubation time should be 24 h.
Preferably, the immature oocyte of step (1) is a secondary or tertiary cumulus-oocyte complex.
The invention has the advantages that:
1) the application of BAPTA-AM to promote the immature oocyte to be cultured and matured in vitro: the invention adopts a one-step in vitro maturation culture method for the first time, and uses BAPTA-AM to promote the meiosis of immature oocyte, especially the oocyte in the largest number of tertiary cumulus-oocyte complexes, thereby improving the in vitro culture and development capacity of the tertiary cumulus-oocyte complexes, and by adopting the technical scheme, about 60 percent of the tertiary cumulus-oocyte complexes of the cattle can be cultured in vitro to develop and mature, so that the final available number of the oocytes extracted from one ovary is greatly increased. The invention can also be used for the in vitro maturation culture of the secondary cumulus-oocyte complex, the in vitro culture maturation probability of the oocytes collected each time is increased to the maximum extent, so that the oocytes which can be matured at any time are cultured and matured, waste is changed into valuable, and the best use is achieved.
2) The cleavage rate and blastocyst development rate of the bovine tertiary cumulus-oocyte complex after parthenogenetic activation after in-vitro culture and maturation are improved, and the embryo quality is improved: according to the in vitro maturation culture method, after the bovine tertiary cumulus-oocyte complex is matured by in vitro culture, the cleavage rate after parthenogenetic activation is 60.10 +/-0.72%, which is nearly two times that of a control group, the blastocyst development rate is 24.22 +/-0.27%, which is nearly 5 times that of the control group, the embryo quality is improved, and the potential utilization value of the bovine tertiary cumulus-oocyte complex is improved.
3) Provides support for embryo biotechnology of other animals: the culture medium provided by the invention can be used for powerfully promoting the success rate of in-vitro maturation culture of the immature oocyte. The result provides support for the development of breeding technology for other animals, such as sheep, horses, pigs, pandas, primates, and humans.
4) Simple operation, safety and nontoxicity: BAPTA-AM has high safety to cells, selective permeability, easy entry into cells without damage to cells, and no toxicity within effective dosage range. The operation is simple, and only a certain amount of BAPTA-AM needs to be added into the conventional oocyte in-vitro maturation culture solution.
Detailed Description
The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention. Modifications or substitutions to methods, procedures, or conditions of the invention may be made without departing from the spirit and scope of the invention.
Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art.
The ovary for experiment was collected from slaughterhouse of county of great factory in Hebei province, BAPTA-AM from Sigma company in USA.
EXAMPLE 1 in vitro maturation culture of immature oocyte of cattle
(1) Collection of oocytes
Collecting cow ovaries from slaughter houses, placing the cows in a vacuum flask containing physiological saline with double antibodies (0.024 g/L penicillin +0.02 g/L streptomycin), keeping the temperature at 35-37 ℃, and returning to the laboratory within 6h, washing the ovaries with the physiological saline with the double antibodies for 2-3 times, extracting oocyte-follicle fluid complexes of follicles of 3-8 mm by using a 10ml syringe with an 18-gauge needle containing 2ml of an oocyte-extracting fluid (9.5 g/L M199+ 10% FBS + 0.042% NaHCO3+ 0.2383% Hepes + 0.2603% Hepes-Na + 0.001% heparin sodium + 0.005% penicillin + 0.0065% streptomycin), placing the oocyte-extracted oocyte into a domestic culture dish with a diameter of 9cm, and selecting primary COCs (homogeneous oocytes, surrounding granulocytes having 5 layers and more), secondary oocytes (homogeneous oocytes-cytoplasm complexes, mature oocytes having 2-2 layers and more than 2-stage granulocytes surrounding oocytes), and culturing the oocytes in vitro.
(2) In vitro maturation culture of oocytes
In vitro maturation medium was prepared by adding 10% by volume of FBS (fetal bovine serum), 0.02IU/M L FSH (follicle stimulating hormone), 0.02IU/M L L H (luteinizing hormone), 1mg/M L E2(β -estradiol), and 50 μ M BAPTA-AM to TCM 199.
Cleaning the selected three-stage COCs in an egg extracting solution for 3-4 times, then cleaning in an in vitro maturation culture solution for 2-3 times, and then putting in CO in advance2Balancing in an incubator for 2-3h, placing 50-75 μ L in each well of a four-well plate containing the in vitro maturation culture solution, covering with mineral oil, placing 50-75 COCs in each well, and culturing in the incubator under 39 deg.C and 5% CO2And 100% humidity, the incubation time was 24 h.
After maturation for 24h, COCs were enucleated with DPBS solution containing 0.1% (w/v) hyaluronidase, examined microscopically for first polar body expulsion and the maturation rate was counted (oocytes cultured in maturation solution without conventional BAPTA-AM addition were used as control). The results are shown in Table 1. The culture conditions of the following control group and experimental group were the same except whether BAPTA-AM was added or not.
TABLE 1 Effect of BAPTA-AM addition on oocyte meiosis in bovine three-stage COCs in vitro
| Grouping | Number of COCs | Maturation Rate (%) |
| Primary COCs | 360 | 87.10±2.14a |
| Secondary COCs | 345 | 47.28±1.65b |
| Three-stage COCs | 359 | 31.83±1.58c |
| Three-stage COCs + BAPTA-AM | 362 | 59.42±2.20d |
Note: the above table was statistically analyzed by t-test. Different letters in the same column indicate significant difference (P <0.05), as follows.
The experimental results in Table 1 show that after the three-stage COCs are cultured in the maturation culture solution added with 50 mu M BAPTA-AM for 24 hours, the maturation rate is greatly improved compared with that in the conventional maturation culture solution, and the statistical significance is obvious (P is less than 0.05).
(3) Parthenogenetic activation and embryo culture of mature oocytes
Further experiments were performed using the above-described in vitro culture of mature oocytes.
Parthenogenetic activation: continuously culturing the oocytes for 24h to obtain mature (MII stage) oocytes, removing granular cells by using DPBS (docosamide phosphate buffer) liquid containing 0.1% (w/v) hyaluronidase, activating the oocytes for 5min by using an egg pumping liquid added with 5 mu M ionomycin, and then culturing the oocytes in a 6-DMAP (Dimethylacetamide) culture solution for 4-6 h, wherein the 6-DMAP culture solution is a traditional CR1aa embryo culture solution added with 2mM 6-DMAP; and counting the number of MII-stage oocytes for parthenogenetic activation.
Embryo culture: washing the parthenogenetic activated oocytes with CR1aa embryo culture solution for 2-3 times, transferring into four-well plate containing embryo culture solution, and culturing (covering with mineral oil, 10ul embryo culture solution/plate, 50-75) under the conditions of 39 deg.C and 5% CO2And 100% humidity culture; the in vitro culture time is 8 days, the cleavage rate is counted after 48h of activation, 5% FBS is added into the culture solution, and the blastocyst rate is counted at the 8 th day. The results are shown in tables 2 and 3.
TABLE 2 influence of BAPTA-AM addition on cleavage rate of bovine tertiary COCs oocytes matured in vitro after parthenogenetic activation
| Grouping | Number of COCs | Number of activations | Cleavage rate% |
| Primary COCs | 360 | 316 | 82.98±1.47a |
| Three-stage COCs | 359 | 114 | 27.40±1.50b |
| Three-stage COCs + BAPTA-AM | 362 | 215 | 60.10±0.72c |
Note: the above table was statistically analyzed by t-test. Different letters in the same column indicate significant differences (P < 0.05).
TABLE 3 influence of BAPTA-AM addition on blastocyst Rate of bovine tertiary COCs oocytes matured in vitro after parthenogenetic activation
| Grouping | Number of COCs | Number of activations | Percentage of blastocyst |
| Primary COCs | 360 | 316 | 42.96±2.07a |
| Three-stage COCs | 359 | 114 | 5.16±0.72b |
| Three-stage COCs + BAPTA-AM | 362 | 215 | 24.22±0.27c |
Note: the above table was statistically analyzed by t-test. Different letters in the same column indicate significant differences (P < 0.05).
The experimental result shows that after the bovine tertiary COCs are cultured in the in vitro maturation culture solution added with 50 mu M BAPTA-AM for 24 hours, the cleavage rate (table 2) of the mature oocytes after 48 hours of parthenogenetic activation and the blastocyst rate (table 3) of the mature oocytes at the 8 th day of culture are both obviously higher than those of the conventional maturation culture solution treatment group (P < 0.05). The cleavage rate of the strain reaches 60.10 percent, although the cleavage rate is still lower than 82.98 percent of the first-level COCs, the cleavage rate is improved by 32.70 percent compared with 27.40 percent of the third-level COCs treated by the conventional mature culture solution, the blastocyst rate is 5 times higher than that of the third-level COCs treated by the conventional mature culture solution, and the utilization rate of the third-level COCs is greatly increased.
In the step (2), the inventors also try to add FBS (fetal bovine serum) with the volume ratio of 10%, FSH (follicle stimulating hormone) with the volume ratio of 0.02IU/M L, L H (luteinizing hormone) with the volume ratio of 0.02IU/M L, E2(β -estradiol) with the volume ratio of 1mg/M L and BAPTA-AM with the volume ratio of 1-120 μ M except 50 μ M into TCM199, and find that adding BAPTA-AM with the volume ratio of 20-100 μ M into the conventional in vitro maturation culture medium can obviously promote meiosis of oocytes in bovine tertiary COCs, promote in vitro maturation of oocytes in bovine tertiary COCs, improve the cleavage rate and the blastocyst development rate after parthenogenetic activation, improve the embryo quality, improve the in vitro developmental capacity of oocytes in bovine tertiary COCs, and have potential utilization value in the aspect of improving the success rate of in vitro culture of oocytes of other animals.
The BAPTA-AM can be expected to promote the success rate of in vitro maturation culture of bovine secondary COCs, meanwhile, as the oocytes of mammals have the same mechanism of occurrence and development, the in vitro maturation culture solution has little difference and can be used universally sometimes, the BAPTA-AM can be expected to promote the in vitro maturation culture of immature oocytes of different kinds of mammals, including cows, sheep, horses, mice, pandas, tigers, primates and the like, and the development of the breeding technology of the mammals is supported.
Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.
Claims (3)
1. The application of a culture medium for promoting in vitro maturation of immature oocytes in improving the cleavage rate of the bovine oocytes after parthenogenetic activation is characterized in that FBS accounting for 10% of the volume of the whole final system, FSH accounting for 0.02IU/M L, L H accounting for 0.02IU/M L and E2 accounting for 1mg/M L are added into TCM199, and the final concentration of BAPTA-AM in the culture medium is 20-100 mu M.
2. Use of a medium comprising 10% FBS, 0.02IU/M L FSH, 0.02IU/M L L H, 1mg/M L E2 to TCM199 based on the total final system volume to promote in vitro maturation of bovine immature oocytes, wherein the final concentration of BAPTA-AM in the medium is 20-100 μ M.
The application of BAPTA-AM in promoting the in vitro maturation of bovine immature oocyte, characterized in that the final concentration of BAPTA-AM is 50-100 μ M.
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| CN108384751A (en) * | 2018-03-12 | 2018-08-10 | 山大生殖研发中心有限公司 | In-vitro maturation culture method for oocyte and culture medium |
| CZ307943B6 (en) * | 2018-06-20 | 2019-09-04 | Výzkumný Ústav Živočišné Výroby V.V.I. | A method of eliminating glyphosate A-induced maturation defects in mammalian oocytes |
| CZ2018320A3 (en) * | 2018-06-30 | 2019-09-04 | Výzkumný Ústav Živočišné Výroby V.V.I. | A method of eliminating glyphosate B-induced maturation defects in mammalian oocytes |
| CZ2018344A3 (en) * | 2018-07-10 | 2019-09-04 | Výzkumný Ústav Živočišné Výroby V.V.I. | A method of eliminating glyphosate C-induced maturation defects in mammalian oocytes |
| CZ2018351A3 (en) * | 2018-07-11 | 2019-09-04 | Výzkumný Ústav Živočišné Výroby V.V.I. | A method of eliminating glyphosate C-induced maturation defects in mammalian oocytes. |
| CN109628385B (en) * | 2019-01-18 | 2022-12-23 | 周桦 | Human oocyte in-vitro maturation culture solution and preparation method and culture method thereof |
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