JP4122425B2 - Oocyte culture method and growth method - Google Patents

Description

[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a technique for culturing animal oocytes. More specifically, the present invention relates to a technique for growing an oocyte by culturing a developing oocyte in a complex state with surrounding somatic cells.
[0002]
[Prior art]
An oocyte is a progenitor cell that becomes an egg that can be fertilized through its developmental stage and maturation process, and all the oocytes are formed in the fetal period and before entering the developmental stage or in the developing egg. It is known that mother cells do not divide and perform "meiosis" for the first time at the stage of development and maturation. “Oocyte development” refers to a process in which the volume of the oocyte increases significantly and does not mean that the cell population increases due to cell division. The diameter of the oocyte, which is a standard for achieving growth, is about 75 μm for small animals such as mice, and 120 to 130 μm for medium and large animals such as pigs and cows, excluding the thickness of the zona pellucida that wraps outside the cell membrane. is there.
[0003]
Each oocyte volume is 220,000 μm ^Three And 1,020,000 μm ^Three The volume of oocytes in medium and large animals is about 4.6 times that of small animals. The difference in size is also reflected in the time required for development, and the period from the start of development of mouse oocytes to completion is 16-18 days, although in cattle and pigs it has not been examined as closely as mice , Approximately 1.5 to 2 months.
[0004]
In the ovary of an animal, there are many oocytes before the development period and developing oocytes. Many of them are lost in the ovaries, but if such oocytes can be grown using culture techniques, they can increase production of good livestock and supply oocytes to biotechnology that requires oocytes. Is possible. Although such techniques have been studied, it has been extremely difficult to obtain mature oocytes except for mice, and it has not left the range of trial and error.
[0005]
Communication with surrounding somatic cells is essential for the survival and development of oocytes in the ovary. Even in culture, “naked” oocytes that have lost surrounding cells cannot develop. For example, in the method developed by Eppig et al. (See, for example, Non-Patent Documents 1 and 2), somatic cells are separated while adhering so as to wrap the developing oocytes of the mouse, and then separated on a culture dish or insert membrane. By adhering and culturing, the oocyte is developed to obtain a mature egg. Therefore, the minimum configuration that enables the growth of oocytes in in vitro culture is the oocyte and the somatic cell layer that encloses it. This culture system is composed only of a granulosa cell layer that wraps the oocyte, and does not include the basement membrane that naturally wraps the oocyte and the surrounding cells. In an example using such a simple culture method, although there has been a report using rats (for example, see Non-Patent Document 3), the results of mice are not far, and other animal species (for example, pigs and cows) are used. There are no reports that the oocytes have grown and matured (such as medium and large animals).
[0006]
When culturing oocytes of medium-sized or large animals such as pigs and cows using conventional culture medium by the above-mentioned method, somatic cells prefer to spread on the surface of the culture dish or insert membrane and migrate, Mother cells often become naked and die. Alternatively, in order to prevent nakedness, when cultured under conditions that allow the oocyte to be wrapped in a large cell mass, the oocyte is killed inside the enlarged cell mass. In any case, the failure of the culture is that the functional and appropriate three-dimensional structure centering on the oocyte is not maintained.
[0007]
In addition to the above-described method, there is a method called follicular culture in which follicular tissue is isolated and cultured as a whole. In animals other than mice such as hamsters, pigs, cows, sheep, and humans, this follicular culture has been attempted exclusively (see, for example, Non-Patent Documents 4 to 10). In the follicular structure, the innermost oocyte is wrapped with a granulosa cell layer, the outer side thereof is covered with a basement membrane, and the outer side is surrounded by a follicular membrane cell layer. When the system for culturing an oocyte with a tissue composed of the above-described oocyte and granulosa cell is an open culture system, this follicle culture is closed at the deepest part of the follicular tissue for the oocyte. Culture system. In view of the time required for growth in vivo, it is clear that culture for growing oocytes generally has to be long. There are reports of long-term culture of bovine and porcine follicles for 2 to 4 weeks, but there is little information on oocytes after such long-term culture, and the ability of the obtained oocytes is also mature oocytes It does not reach the stage of using as (for example, refer nonpatent literatures 5-8).
[0008]
In addition, as described above, in follicular culture, the oocyte is retained in the deep part of the culture system, so it is not possible to directly know the developmental stage of the oocyte inside the follicle, and it is also possible to judge the health condition. Have difficulty. Therefore, in follicular culture, the culture is terminated when the follicle is opened, and it is rather rare that the oocyte matures at the appropriate time. In addition, this problem becomes more serious when the culture takes a long time.
Furthermore, this method has a great limitation in that the culture can be performed only when the follicle is collected in a complete state in the first operation stage.
[0009]
As a method similar to follicular culture, a follicular follicle is formed by breaking the follicle once, collecting a complex of the inner developing oocyte and granulosa cell group, and embedding it in a collagen gel and culturing. The method of making it report is also reported (for example, refer nonpatent literature 11). In this method, matured ova are obtained after 2 weeks of culture in cattle, but the follicular cavity-like structure is formed again, so it is impossible to directly know the developmental stage of the oocyte inside, The constraint that it is difficult to determine the state remains. Therefore, if such problems can be avoided and the oocytes can be directly observed without being embedded in the collagen gel until the final stage, it should be a more useful culture system.
[0010]
Therefore, if the oocytes under development of various animal species can be cultured with a minimal functional cell configuration, the above problems are solved and a simple culture system for oocytes is provided. .
[0011]
[Non-Patent Document 1]
Eppig and Schroeder, Biology of Reproduction (USA), 1989, 41, 268-276.
[Non-Patent Document 2]
Eppig and O'Brien, Biology of Reproduction (USA), 1996, Vol. 54, p.197-207
[Non-Patent Document 3]
Daniel et al., Gamete Research (USA), 1989, 24, p.109-121
[Non-Patent Document 4]
Roy and Greenwald, Journal of Reproduction and Fertility (UK), 1989, 87, p.103-114
[Non-Patent Document 5]
Hirao et al., Journal of Reproduction and Fertility (UK), 1994, 100, p.333-339.
[Non-Patent Document 6]
Shuttleworth et al., Reproduction (UK), 2002, 123, 807-818
[Non-Patent Document 7]
Gutierres et al., Biology of Reproduction (USA), 2000, 62, p.1322-1328
[Non-Patent Document 8]
Telfer et al., Molecular and Cellular Endocrinology (IR), 2000, 163, p.117-123
[Non-patent document 9]
Cecconi et al., Biology of Reproduction (USA), 1999, Vol. 60, p.594-601
[Non-Patent Document 10]
Wu et al., Human Reproduction (UK), 1998, Vol. 13, p. 2562-2563
[Non-Patent Document 11]
Yamamoto et al., Theriogenology (USA), 1999, Vol. 52, p.81-89
[0012]
[Problems to be solved by the invention]
As described above, almost all animal species (particularly medium-sized and large animals) have not succeeded in growing oocytes in a tissue culture system containing oocytes. Accordingly, an object of the present invention is to provide a simple culture method capable of growing such an animal oocyte in vitro.
[0013]
[Means for Solving the Problems]
As a result of intensive studies to solve the above-mentioned problems, the present inventors have found that animal oocytes and surrounding somatic cells are good by adding a polymer compound such as PVP to the culture solution at a very high concentration. It was found that the cells were cultured. Furthermore, the present inventor, when the developing oocyte is cultured under the above conditions in a complex state with the surrounding somatic cells, the oocyte grows normally and is obtained. Was further matured and found to be usable as an egg, and the present invention was completed.
[0014]
That is, the present invention provides an egg comprising culturing a complex of an oocyte and surrounding somatic cells in a culture solution containing a polymer compound at a concentration of 1 to 12% (w / v). This is a method for culturing mother cells.
[0015]
In the above culture method, as the polymer compound, a polymer selected from the group consisting of synthetic polymers, polysaccharide polymers, proteins, proteoglycans, and combinations thereof can be used. The polymer compound is preferably selected from the group consisting of polyvinylpyrrolidone (PVP), Ficoll, hydroxypropylmethylcellulose, serum albumin, and combinations thereof.
[0016]
When using PVP alone as the polymer compound, it is particularly preferable to use a culture solution containing PVP at a concentration of about 4 to 6% (w / v). When Ficoll alone is used as the polymer compound, it is particularly preferable to use a culture solution containing Ficoll at a concentration of about 2 to 6% (w / v). In the case of hydroxypropylmethylcellulose, it is preferable to use a culture solution containing hydroxypropylmethylcellulose at a concentration of about 1-2% (w / v).
[0017]
In the culture method, the complex can be derived from a mammal, for example. Mammals include pigs, cows, sheep, goats, dogs, cats, rabbits, hamsters, rats, mice, or humans.
[0018]
In the above culture method, the oocyte may be a developing one. Furthermore, examples of the surrounding somatic cell include cumulus cells, granulosa cell mass, and / or granulosa cell layer.
The present invention is also an oocyte obtained by the above culture method.
[0019]
Furthermore, the present invention is characterized in that a complex of a developing oocyte and its surrounding somatic cells is cultured in a culture solution containing a polymer compound at a concentration of 1 to 12% (w / v). This is a method for developing oocytes.
[0020]
The present invention also provides an oocyte grown by the above growth method.
The present invention further relates to a method for producing a mature egg, characterized in that the grown oocyte is matured.
[0021]
DETAILED DESCRIPTION OF THE INVENTION
The present invention relates to an oocyte culture method and a growth method, wherein a complex of an oocyte and a surrounding somatic cell is mixed with a polymer compound at a concentration of 1 to 12% (w / v). It culture | cultivates with the culture solution containing.
[0022]
The “oocyte” to be cultured in the present invention refers to a germ cell that becomes an oocyte that can be fertilized through the developmental stage / maturation process. All oocytes are formed in the fetal period, and the oocytes that are in the developmental stage or in the developing stage do not divide, and are in the first stage of “meiosis”. It is known to perform “meiosis” for the first time at the stage of maturity, and completion of oocyte development is an essential requirement for maturation into an egg. “Oocyte development” refers to a process in which the volume of the oocyte increases significantly and does not mean that the cell population increases due to cell division. The diameter and volume of an oocyte that serves as a standard for achieving growth differs between a small animal and a large animal. For example, the volume of an oocyte in a large animal is about 4.6 times that of a small animal. Since this difference in size is also reflected in the time required for development, the number of culture days necessary and appropriate for the completion of development is determined by the size of the developing oocyte. Conventionally, artificial culture and growth have been successful in small animal oocytes with a short development period, but favorable results have not been obtained with large animal oocytes with a long development period.
[0023]
In the present invention, the term “developing oocyte” or “developing oocyte” is defined as that the size does not reach the maximum oocyte diameter specific to the animal species, and the ability is healthy. Nonetheless, it has no ability to resume meiosis while being stopped in the first meiotic prophase (the germinal vesicle stage), or has the potential to resume meiosis immediately, but has the second potential It is defined as having no potential to progress to the medium term. In general, developing oocytes are not accompanied by embryogenic ability even after in vitro fertilization.
“Maturation” of an oocyte means that meiosis is resumed, division proceeds to the second middle meiosis stage, and a process after fertilization becomes possible.
[0024]
Furthermore, the “follicle” refers to a structure that wraps an oocyte from the developmental stage to the mature stage, and the volume of the follicle increases with the development of the oocyte. Follicles differ from developing oocytes that do not undergo cell division in that they develop by cell proliferation. First, the follicle has a structure in which a single layer to several granule membrane cell layers enclose the oocyte, the basement membrane covers the outside, and the outside is surrounded by the follicle membrane cell layer. In the second half of the follicular development period, gaps appearing in the layer of granulosa cells that have increased significantly develop to become follicular cavities. The follicular cavity is filled with a fluid called follicular fluid that gradually grows and eventually occupies most of the follicular volume. When the follicular cavity is formed, the oocyte is enveloped in several layers of granulosa cells, and is attached to one point of the granulosa cell layer constituting the inner wall of the follicle. At this time, the cells forming a layer around the oocyte are particularly called cumulus cells, and are distinguished from other granulosa cells constituting the inner wall of the follicle.
[0025]
In the present invention, a portion assumed to be a “functional minimum cell configuration” centering on an oocyte is cultured. This minimum cell structure refers to the elements that constitute the follicle, that is, the inner part partitioned by the basement membrane, particularly a group of cells around the oocyte. For example, a complex of an oocyte and cumulus cell, or a complex of an oocyte and cumulus cell with the granule membrane cell population remaining on the wall side (oocyte, cumulus cell, granulosa cell) Complex).
[0026]
The operation of the oocyte culture method and growth method according to the present invention will be described in detail below. All of the following operations have good results when working in a room having a room temperature of about 25 ° C., but are not limited thereto as long as they do not damage cells or tissues.
[0027]
First, the ovaries are collected to collect the complex of the oocyte and the surrounding somatic cells.
The ovaries are collected from the animals. In the present invention, the “animal” is not limited as long as it is an animal having an oocyte, but in particular, mammals such as cows, pigs, sheep, goats, dogs, cats, rabbits, hamsters, rats, Mouse or human is preferred.
[0028]
To collect ovaries from animals, the animals are usually opened and separated, regardless of whether the animals are alive or dead. Since the inside of the ovary goes to an ischemic state after the animal is killed or separated from the living body, it is preferable to move to the following operation as soon as possible in order to reduce damage to the ovarian tissue.
[0029]
After the ovary is washed with a physiological buffer phosphate buffer (PBS), a tissue piece is cut from the surface with a thickness of 1 to 2 mm and transferred to a tissue culture solution. The tissue culture solution can be used in the present invention as long as it is used for normal tissue culture such as Waymouth's medium, Minimum Essential Medium, and 199 medium (Medium 199). Moreover, the culture solution used for the operation on the laboratory bench is HEPES (2- [4- (2-hydroxyethyl) -1-piperazinyl] ethanesulfone so that its pH is stabilized at around 7 in the gas phase in the atmosphere. What is buffered with a buffer solution such as acid) is desirable. The cutting of the tissue may be performed using any blade, and may be performed using a generally used replacement knife for surgical operation (attached to a knife handle) or a scalpel.
[0030]
Subsequently, the tissue in the culture medium is further minced to cut out follicular tissue. The follicular tissue should be excised with care so as not to be injured by excessive pressure, and any follicles that show obvious signs of regression, such as cell shedding, should not be used. Sorting the follicles by size is convenient for separating the oocytes by size (see, for example, Hirao et al., Zygote (UK), 1995, Vol. 3, pages 325-332).
[0031]
Next, a cut reaching the inside of the basement membrane is made at one location of the cut follicular tissue. From this incised portion, a complex of the oocyte and the surrounding somatic cells is collected. In the present invention, “complex of an oocyte and its surrounding somatic cells” or “complex” refers to a complex tissue composed of an oocyte and its surrounding somatic cells. Examples include complexes of cells and cumulus cells, and complexes of oocytes, cumulus cells, granulosa cells, and the like. Accordingly, “surrounding somatic cells” include, but are not limited to cumulus cells and granulosa cells. When follicles are collected by enzymatic treatment of ovaries (tissue fragments thereof) to form a complex in the culture dish, follicular membrane cells may be contaminated.
[0032]
In addition to the above, collecting the complex of the oocyte and its surrounding somatic cells omits the process of isolating the follicle, cut the ovary with a razor or the like, and then rinses it with the culture solution. You may carry out by the method of collect | recovering the composite_body | complex released in the inside directly. In collecting the complex, a method of sucking the complex from the follicle in the ovary using an injection needle and a syringe can be used. Further, a method of sucking a complex from a ovary in a living body using a technique called in vivo ovum suction can also be employed.
[0033]
As described above, the physical separation method for collecting the complex of the oocyte and the surrounding somatic cells has been described. However, in the present invention, the method of collecting the complex is not limited to the above method. .
[0034]
In addition to the physical method described above, the follicles are separated by treating the ovary fragment with an enzyme (eg, collagenase), or the ovary is cultured with the enzyme so that the oocyte and its surrounding somatic cells The complex of the oocyte and the surrounding somatic cells can also be collected.
[0035]
Furthermore, in the present invention, the “complex” is temporarily transplanted to an organ of another animal species that has been temporarily cultured (for example, follicular culture or ovarian organ culture) before being used in the present invention. Also included after collection. In addition, culturing separately prepared somatic cells together with the “complex” is also included. Accordingly, the “complex” of the present invention includes any form and state as long as it is composed of oocytes and surrounding somatic cells. For example, the “complex” to be cultured in the present invention includes the entire follicle.
[0036]
After collecting the complex, it is preferable to examine the state of the oocyte at the time of collection. Usually, the diameter of the oocyte at the time of collection, the viability of the oocyte, and its health state are important information in the subsequent culture.
[0037]
The method for measuring the diameter of the oocyte (the portion not including the zona pellucida) is not limited to the following, but a method for measuring the diameter using a microscopic eyepiece connected to a microscope, and a method that is visible through a microscope There is a method of taking a screen into a computer and measuring it with image analysis software.
[0038]
There is a correlation between the diameter of the developing oocyte and the diameter of the follicle (see, for example, Hirao et al., Zygote (UK), 1995, Vol. 3, pages 325-332), and the direct oocyte It is also possible to infer the diameter of the oocyte from the diameter of the follicle before measuring the cell diameter. For example, it is estimated that bovine developing oocytes having a diameter of about 90 to 99 μm used in Example 4 in the present invention are present in follicles having a diameter of about 0.5 to 0.7 mm. Such correlation is well known in the art, and the diameter of the oocyte can be easily determined by utilizing the correlation.
[0039]
In addition, the viability and health status of the oocyte can be determined by observing under a microscope whether the cytoplasm or cell membrane of the cell is broken.
The diameter measurement and observation methods described above are also applied to cultured oocytes.
[0040]
Subsequently, a complex of the oocyte collected as described above and its surrounding somatic cells, preferably a complex containing an oocyte in the developmental stage, is prepared in a culture dish or Introduce onto the insert membrane attached to the culture dish.
[0041]
The culture medium for complex culture is used by modifying the basic tissue culture medium. The basic tissue culture medium that can be used in the present invention is not particularly limited as long as it is used for normal cell culture. Examples thereof include Weymouth medium, minimum essential medium, and 199 medium.
[0042]
The most important modification added to the culture medium used for culturing the complex is the addition of a polymer compound. In the present invention, the “polymer compound” is an organic substance having a molecular weight of tens of thousands to millions, and includes both natural polymers (such as biopolymers) and synthetic polymers. The “polymer compound” used in the present invention is particularly easy to dissolve in water, has extremely low cytotoxicity, does not have the property of destabilizing the pH of the culture solution during culture, In addition, it is preferable to satisfy the conditions such that the initial characteristics are stably maintained for a long time. For example, examples of the polymer compound that can be used in the present invention include synthetic polymers, polysaccharide polymers, proteins, proteoglycans, and the like. Examples of synthetic polymers include polyvinyl pyrrolidone (PVP; molecular weight of about 360,000), polyvinyl alcohol (PVA; molecular weight of about 70,000 to 100,000), and the like. Examples of the polysaccharide polymer include dextran, hydroxyethylated starch, and derivatives of cellulose (for example, hydroxypropylmethylcellulose). Examples of the polysaccharide polymer include sucrose synthetic polymer Ficoll (molecular weight 400,000), hyaluronic acid and chondroitin sulfate glycosaminoglycan. Examples of proteins include serum albumin (molecular weight of about 69,000). Examples of proteoglycans include chondroitin sulfate proteoglycans.
[0043]
Particularly preferred polymer compounds in the present invention include, but are not limited to, PVP, Ficoll, hydroxypropyl methylcellulose, and serum albumin.
[0044]
In addition to those described above, the high molecular compounds usable in the present invention include compounds that are presently known to exhibit equivalent functions, or compounds that will be found or synthesized in the future. In order to confirm whether or not a certain compound is a compound exhibiting an “equivalent function” that can be used in the present invention, when used in culture, it does not impair the viability of the oocyte. It is effective to cultivate the "complex" for a long time, keeping in mind that somatic cells do not fall off and that the structure centered on the oocyte is not lost (irregular and extensive cell proliferation does not occur) . It is also important to ensure that good conditions are maintained for a sufficient period of time so that the oocyte develops to the final stage.
[0045]
The concentration of the polymer compound added to the culture solution is about 1 to 12% (w / v), preferably 2 to 8% (w / v), more preferably 4 to 6% (w / v) with respect to the culture solution. v), most preferably about 4% (w / v).
[0046]
If the concentration of the polymer compound is within the above-mentioned range, the object of the present invention can be achieved. However, the object of the present invention may be achieved even when the concentration is in other ranges. Specifically, this is a case where a compound having a high viscosity is added even if the concentration is low, such as hydroxypropylmethylcellulose and glycosaminoglycan. For example, when a certain kind of hydroxypropylmethylcellulose is added to the culture solution at a concentration of 1% (w / v), the form of the complex of oocyte and somatic cell is 4% (w / v) PVP (molecular weight 360,000). It was observed that the effect of the present invention was achieved.
[0047]
There is a fact that polymer compounds such as PVP and PVA are conventionally used in egg cultures. In particular, it is often added as a substitute for a polymer to a culture solution not containing fetal bovine serum or serum albumin. In this case, it is said that it is useful for preventing the oocyte from being attached to the bottom surface or wall surface of the culture dish and the outer surface or inner surface of the ultrafine glass tube for embryo manipulation. However, a concentration of about 0.1% to 0.4% is generally sufficient to achieve such a purpose, which is a concentration that is usually used. Further, when serum or the like is added to the basic culture solution, the polymer compound is often not added. In contrast, in the present invention, it was derived that a completely different effect can be achieved by increasing the concentration to about 1 to 12%. On the other hand, when it is added at a conventionally used concentration of 0.1% to 0.4%, the intended effect of the present invention is not exhibited. Therefore, in the present invention, it is important to add the polymer compound at a concentration of about 2.5 times or more, preferably about 10 to 15 times the conventional concentration of 0.4%.
[0048]
In addition to the addition of the above-described polymer compound, the culture medium is also modified in general cell culture, but is not limited to a specific modification. For example, addition of 5% fetal bovine serum and antibiotics.
[0049]
In addition, the culture solution may be modified to be a culture solution suitable for cell culture such as granulosa cells and oocytes. For example, but not limited thereto, sodium pyruvate is not limited to specific animal species, and is generally added to the culture medium of oocytes.
[0050]
In addition, for example, in the culture of a complex containing bovine granulosa cells, cell growth may be good when 2 mM to 4 mM hypoxanthine and 10 to 100 ng / ml estradiol 17β are added. Are known. In addition, in the culture of a complex containing pig cumulus cells, when a low concentration of follicle stimulating hormone (FSH) (for example, 10 ng / ml) is further added, cell growth may be good. Are known. In the present invention, it is preferable to appropriately modify the culture solution according to such animal species and cell types contained in the complex to be cultured.
The culture dish to be used is not particularly limited as long as it is generally used for culturing animal cells.
[0051]
In addition, the culture dish is preferably provided with a substrate for cell attachment, whereby a cell layer is provided on the culture substrate, and a complex of the oocyte and surrounding somatic cells is disposed thereon. It is possible to maintain the state of being present, and the object of the present invention can be satisfactorily achieved. As the substrate to which such cells adhere, it is preferable to use a substrate that does not interfere with the adhesion and proliferation of the cell group, for example, various insert membranes. A culture dish with a collagen coat is also suitable. However, as long as the condition of cell growth is satisfied, the culture substrate is not particularly limited.
[0052]
The number of oocytes per culture dish can be changed according to the source of the oocyte, the amount of the culture medium, the type of the culture dish, and the like. In the case of about 4.5 ml / well), it is preferable to culture 10 to 20 large complexes to which granulosa cell mass adheres, and 20 to 50 for complexes composed of cumulus cells and oocytes. The complex can be cultured. On the other hand, in a 24-well or smaller culture dish with a small volume per well, in the case of a large complex to which granulosa cell mass adheres, the amount of the culture solution per complex is about 200 to 500 μl, from cumulus cells and oocytes. In the complex, the culture is preferably performed at about 50 to 150 μl. The above example is an example, and the number of complexes per culture solution can be increased in consideration of changes in pH due to cell morphology and cell metabolism.
[0053]
The incubator in which the culture dish is installed may be any one that is generally used for animal cell culture. The culture conditions are, for example, a gas phase of 37 ° C. to 39 ° C., 5% carbon dioxide 95% air, and a high humidity environment, and an incubator capable of setting such conditions is used. The above culture conditions are described as an example, and can be changed within a range that does not impede cell growth and survival in consideration of the type of culture solution, pH adjustment method, and the like. Preferred culture conditions for culturing oocytes are known in the art.
[0054]
After the culture is started, the oocyte continues to grow in a state of being wrapped by surrounding somatic cells. According to the present invention, in the culturing process, the binding with the somatic cell layer closest to the oocyte is well maintained.
[0055]
It is common sense to exchange a part of the culture solution with a fresh one once every 2 to 4 days during the culture period, and it is preferable to change the culture solution as appropriate in the present invention. The culture period varies depending on the animal species to be used and the state of the collected oocyte, but is generally several days to about 4 weeks for cattle and pigs, and can be appropriately set by those skilled in the art.
[0056]
According to the present invention, it is possible to recover the developed oocyte while maintaining the binding state between many oocytes and surrounding somatic cells. In the complex recovered after culturing, the ratio of oocytes wrapped in somatic cells is significantly increased (for example, about 2 to 5 times) by using the present invention.
[0057]
The diameter of the oocyte after culture is measured by the method described above. Thereby, the degree of oocyte development during the culture period can be known. Development may be considered complete when the diameter of the oocyte reaches the maximum value specific to the animal species, but further confirming that the oocyte and surrounding somatic morphology are normal, The culture may be continued for several days to about 1 week.
[0058]
According to the present invention, it is possible not only to promote the growth of an oocyte but also to develop an oocyte in a complex state with a somatic cell. This means that in the subsequent maturation process of the oocyte, it remains encased in somatic cells. It is extremely important in terms of the effect of protecting the oocyte from various stresses such as
[0059]
This culture method is also easier to operate than the start of culture, observation during culture, and recovery of oocytes after culture compared to conventional culture methods and follicular culture embedded in collagen gel. Is.
[0060]
Although the present invention enables long-term culture, since the growth of the oocyte can be observed, the culture period can be changed according to the size of the oocyte, that is, the degree of development. It is. In addition, since the oocytes that have developed during the culture period are still alive in a state of maintaining the association with somatic cells, this culture method is not limited to the developing oocytes. It is also effective for later oocytes.
[0061]
Another object of the present invention is to maintain a functional and minimal three-dimensional structure of a complex of an oocyte and its surrounding somatic cells, and a method for culturing a complex embedded in a collagen gel. Or the culture solution used by this invention can be applied also to follicular culture, and culture | cultivation can be performed effectively.
[0062]
The oocytes obtained as described above can be subjected to physical operations such as culturing after microinjection of proteins such as nucleic acids, chemical substances or antibodies. Therefore, the oocyte obtained by the present invention is useful for oocyte research.
[0063]
In addition, the oocytes obtained as described above can be used by allowing them to mature into oocytes by performing a culture method for in vitro maturation of commonly used immature oocytes.
[0064]
Conversion to mature culture is usually performed by recovering the complex of oocytes and somatic cells from the culture dish that has undergone growth culture, washing it with the maturation medium, and then transferring it to the final maturation medium. Do. The tissue culture solution for maturation is used with a 199 medium or the like as a basic medium to which essential elements such as sodium pyruvate and antibiotics are added, and gonadotropin, growth factor, serum, follicular fluid and the like are appropriately added. Preferred culture conditions for such oocyte maturation culture have been extensively investigated and are known in the art. The incubator in which the culture dish is installed is used with the same settings as those normally used for growth culture.
[0065]
Eggs matured in this way can be used for normal in vitro fertilization, and can also be used for recipient eggs in the production of parthenogenetic embryos and cloned animals.
[0066]
【Example】
Hereinafter, the present invention will be described by way of examples. However, the technical scope of the present invention is not limited to these examples.
[0067]
[Example 1] Culture of porcine oocytes using PVP-added culture medium
Porcine ovaries collected at the slaughterhouse were washed with PBS, and tissue pieces were cut from the surface to a thickness of 1 to 2 mm, and transferred to a culture dish (diameter 100 mm) containing a tissue washing medium. A HEPES buffer 199 culture medium (Medium 199; manufactured by Sigma) (pH 7.2) containing 0.1% bovine serum albumin was used as the tissue washing culture medium. A developing follicle was searched under a stereomicroscope, and a follicle with a diameter of 0.5 to 1 mm was cut out using a disposable surgical blade attached to a handle and transferred to a fresh tissue washing culture medium. Next, a notch reaching the inside of the basement membrane was made, and the opposite side was pushed with tweezers to collect an oocyte / cumulus cell complex from the opening.
[0068]
Each oocyte / cumulus cell complex is transferred to a small drop of the washing medium, and the oocytes are individually collected at a magnification of 200 times using a microscopic eyepiece connected to an inverted microscope. Diameter was measured. At this stage, oocytes that clearly showed signs of regression were removed, and the remaining healthy oocyte / cumulus cell complex was subjected to culture.
[0069]
To the culture solution, 5% fetal calf serum, 4 mM hypoxanthine, 10 ng / ml FSH, 100 μg / ml sodium pyruvate, 80 μg / ml kanamycin and 0.1 μg / ml estradiol 17β were added as a basic tissue culture solution. Weymouth culture solution (Gibco) was used. For PVP (manufactured by Sigma, average molecular weight 360,000), those added at a concentration of 0% to 10% were used (0, 1, 2, 4, 6, 8 and 10%).
[0070]
As a culture dish, a 6-well or 24-well culture plate was used with an insert membrane (Biocoat, manufactured by Becton Dickinson) provided with a collagen coat. In such a culture dish, the culture medium is added in advance (in the case of 6 wells, 2 ml of the inside of the insert, 2.5 ml of the outside, in the case of 24 wells, 500 μl of the inside, 600 μl of the outside), ₂ ・ Equilibrated in an incubator (manufactured by Sanyo Electric Co., Ltd.) with 95% air conditions and introduced the oocyte / cumulus cell complex, which was observed before culture, onto the membrane of the culture dish did. Thereafter, it was immediately transferred to an incubator and culture was started. The culture was performed for 12 days, and once every four days, half of the culture solution was replaced with fresh one.
[0071]
Changes in the morphology of the complex as the culture progresses are shown in FIGS. 1A to f and FIGS. 1B to g. In FIG. 1, a, c, e, g and i show the forms after 1 day, 3 days, 6 days, 9 days and 12 days of culture in the PVP-free group. On the other hand, b, d, f, h, and j in FIG. 1 show the forms when the same number of days as in the PVP non-added section has elapsed in the PVP 4% added section. As can be seen from a and b in FIG. 1A, the influence of the presence or absence of addition of PVP is not so much observed in the form of the complex after 1 day of culture. However, after 3 days of culturing, an orderly somatic proliferation around the oocyte is clearly observed in the group with 4% PVP added (d in FIG. 1A), whereas a sufficient and uniform thickness is obtained in the non-added group. Thus, the basic structure of the cell layer covering the oocyte is already disturbed (c in FIG. 1A). The tendency becomes more remarkable after 6 days of culture (e and f in FIG. 1A). As can be seen from g and i in FIG. 1B, in the PVP-free group, somatic cells proliferated, but grew in a non-regular manner in a manner independent of the presence of oocytes. On the other hand, as is apparent from h and j in FIG. 1B, in the group with 4% PVP added, a tendency was observed in which the oocyte proliferated so as to surround the cell layer having a stable thickness.
[0072]
It was clear from the form of the oocytes collected after the culture that the communication between the oocytes and the somatic cells was suitably maintained in the PVP 4% addition group (FIGS. 2A and B). In the PVP-free group (FIG. 2A), there were many oocytes that became naked and regressed during the culture, and those that were finally recovered were oocytes that lost many cumulus cells. On the other hand, the oocytes collected in the PVP 4% addition group (FIG. 2B) were more enriched in somatic cells and the degree of attachment and form compared to the PVP-free group (FIG. 2B).
[0073]
In addition to examining the viability of the recovered oocyte-somatic complex, 10 IU / ml of pregnant mare serum gonadotropin (PMSG) and human chorionic gonadotropin (hCG) were respectively added to the complex. For 48 hours of maturation culture. Thereafter, the oocytes were fixed and stained, and the maturation rate to the second metaphase was examined. The proportion of oocytes collected in the PVP-added group was higher than that in the PVP-free group, showing a significantly higher value at 4% (FIG. 3A). The percentage of oocytes that reached the second meiotic metaphase after maturation culture was also high. When the PVP concentration was 8% or more, the proliferation of granulosa cells tended to be suppressed. When the diameter of the oocyte shown in FIG. 3A was examined, it was much larger than that at the start of culture, and it had grown to the final size (FIG. 3B).
[0074]
[Example 2] Culture of pig oocytes using ficoll-added culture medium
In the same manner as in Example 1, a developing oocyte / cumulus cell complex was collected and subjected to culture. Ficoll (Ficoll (registered trademark) 400, average molecular weight 400,000, manufactured by Sigma) was added to the same culture broth as in Example 1 at a concentration of 0 to 10% (w / v) instead of PVP. (0, 1, 2, 4, 6, 8 and 10%). The setting of the culture dish and the incubator was the same as in Example 1.
[0075]
FIG. 4A shows a form in the case where 12 days have passed in the Ficoll 4% (w / v) addition group. As observed in the PVP 4% addition group of Example 1, the oocyte tends to proliferate so as to be surrounded by a cell layer having a stable thickness, and the communication between the oocyte and the somatic cell is suitably maintained. It was known that
[0076]
When mature culture was performed in the same manner as in Example 1, cumulus cells were swollen (FIG. 4B, photograph after 24 hours of mature culture). FIG. 4B shows that hyaluronic acid was synthesized in cumulus cells as a result of responding to stimulation of PMSG and hCG, and in the complex after long-term culture, receptors for these hormones were placed on cumulus cells. It was shown that the process leading to hyaluronic acid synthesis after stimulation was functioning. At the same time, maturation of the oocyte was confirmed, indicating that the cumulus cell and oocyte communicate normally, and as a result, it is possible to induce maturation of the oocyte.
[0077]
FIG. 5 shows the ratio of the complex of porcine oocytes and somatic cells in normal form obtained after 12 days of culture in the areas where Ficoll 0%, 1%, 2% and 4% (w / v) were added, and The maturation rate to the second meiotic metaphase when 48-hour maturation culture is performed is shown. Normally, Ficoll is not used as an additive in long-term culture of oocytes, but if used, it is not within the range of 0.1% to less than 1%, which is the range of common polymer additions. As a result, it was revealed that a good form in which the oocyte is surrounded by a cell layer having a stable thickness cannot be obtained. On the other hand, it was found that as a result of increasing the concentration of Ficoll to a high concentration (particularly 4% or more), the survival rate of the complex of oocytes and somatic cells and the maturation rate of the oocytes were increased.
[0078]
[Example 3] Culture of porcine oocytes using a culture solution supplemented with hydroxypropylmethylcellulose
In the same manner as in Example 1, a developing oocyte / cumulus cell complex was collected and subjected to culture. Hydroxypropylmethyl cellulose ((Hydroxypropyl) methyl cellulose) (Sigma, H9262) was added to the same culture broth as in Example 1 at a concentration of 1% instead of PVP. The setting of the culture dish and the incubator was the same as in Example 1. Due to its molecular properties, the used hydroxypropylmethylcellulose exhibited different viscosities in the solution even at the same concentration as the PVP of Example 1. As is clear from the photograph after 12 days of culture (FIG. 6), the oocytes tended to proliferate so as to be surrounded by a cell layer having a stable thickness.
[0079]
[Example 4] Culture of bovine oocytes using PVP
After washing bovine ovaries collected at the slaughterhouse with PBS, tissue pieces were cut from the surface with a thickness of 1 to 2 mm, and follicles were cut out in the same manner as in Example 1. Among them, one having a diameter of 0.5 to 0.7 mm was selected, transferred to a fresh tissue washing culture medium, and a cut reaching the inside of the basement membrane was made. As in Example 1, a complex was obtained by pressing the opposite side of the cut with tweezers. In Example 4, a complex of oocytes, cumulus cells, and granulosa cells was collected and used for culture. did. Immediately after collection, the diameter of the oocyte was measured and a healthy complex was selected by the method shown in Example 1.
[0080]
As a basic tissue culture medium, the culture medium is a way mouse culture medium (5% fetal bovine serum, 4 mM hypoxanthine, 100 μg / ml sodium pyruvate, 80 μg / ml kanamycin and 0.1 μg / ml estradiol 17β added). Gibco) or 199 culture solution (Nissui Pharmaceutical Co., Ltd.) and finally added PVP at a concentration of 0% to 12% (0, 0.25, 1, 2, 4, 6 and 12%). The setting of the culture dish and the incubator was the same as in Example 1.
[0081]
As in Example 1 in pigs, the complex of oocytes and cumulus cells can be recovered with stable results after 14 days in culture as the concentration of PVP added is increased to about 4% or more. Became. Even in the group with the highest concentration of 12%, the ovum was recovered as a complex without being naked, and after 24 hours of maturation culture, the oocytes that released the first polar body could be confirmed. The proliferation of somatic cells and the health condition of their appearance are best in the case of the complex with 4-6% added, and the specific gravity of the culture solution is excessive due to the operability when introducing the complex. It is particularly preferable to add 4 to 6% of PVP to the culture medium from the viewpoint of preventing the weight from becoming heavy. On the other hand, in the 0.25% addition group, the effect was not recognized, but in the 1% addition group, although the form of the composite was slightly improved, it did not reach the effect observed in the addition group of 2% or more. The 4% added group showed the best value, and the 2% added group showed the result between 4% and 0%.
[0082]
7A, 7B, and 7C show changes in the form of the complex in the PVP 4% addition group as the culture progresses. It is clear that after 9 days in culture, the oocytes are proliferating and spreading as the somatic cells surround them, after 2 days in culture. FIG. 7C shows a typical complex form after 14 days of culture in the PVP 4% addition group. It can be seen that a tight cell layer surrounds the oocyte closest to it, centered on the large oocyte in the center, and is arranged radially around it.
[0083]
A comparison of the ratio of the complex of normal oocytes / cumulus cells recovered after 14 days of culture, as compared with the experiment in Example (Example 1) in swine, compared to the group without PVP and the group with 2% addition. In addition, a high value was shown in the PVP 4% addition group (FIG. 8A). Moreover, the diameter of the oocyte became much larger than that at the start of the culture, and it was observed that it developed to the final size (FIG. 8B).
[0084]
[Example 5] Maturation and fertilization into eggs
A 199 culture solution (manufactured by Gibco) was used as a basic culture solution, and bovine oocytes were grown and cultured in a culture solution supplemented with 0% or 4% PVP for 14 days in the same manner as in Example 3. After the growth culture, the complex of the oocyte and cumulus cell was collected and matured and in vitro fertilized.
[0085]
In vitro maturation is performed using a culture solution obtained by adding 5% fetal bovine serum, sodium pyruvate and 20 mAU / ml follicle stimulating hormone (Antoline, manufactured by Denka Pharmaceutical Co., Ltd.) to a 199 culture solution (manufactured by Gibco). Cultivation was performed for 24 hours in a high humidity environment at 5 ° C. in a gas phase of 5% carbon dioxide gas and 95% air. After maturation culture, oocytes wrapped in swollen cumulus cells were collected and washed with a basic medium for medium (BO solution) containing caffeine, heparin and bovine serum albumin to obtain an egg.
[0086]
On the other hand, frozen bovine semen was thawed and subjected to sperm washing treatment, and in addition to the culture solution containing eggs prepared as described above, it was subjected to in vitro fertilization. Six hours after the nymph, the ovum was transferred to a fertilized egg culture solution (IVD-101, manufactured by Functional Peptide Institute), in a gas phase of 38.5 ° C., 5% oxygen 5% carbon dioxide 90% nitrogen, and high humidity In an incubator (manufactured by Hirasawa Co., Ltd.) adjusted to the above conditions, the cells were cultured for 7 to 8 days, and the proportion of those that advanced to the blastocyst stage was examined. As a result, it was found that the embryo development ability can actually be acquired (FIG. 9). Of the 28 eggs grown in vitro in the PVP-free group, one (4%) developed to the blastocyst stage after in vitro fertilization, whereas the egg 60 grown in vitro in the PVP-added group 60 Of these, 7 (12%) developed to the blastocyst stage.
[0087]
【The invention's effect】
The present invention provides a simple culture method and growth method for oocytes. In this culturing method, a complex of an oocyte and surrounding somatic cells can be cultured while maintaining a good morphological and functional relationship. In addition, even when the developing oocyte is subjected to culture, the oocyte can be grown to the final stage. In the main culture method or the growth method, the binding between the oocyte and the surrounding somatic cells is well maintained even after the culture, and the oocyte that has succeeded in the growth is matured to an oocyte and It can be used.
[Brief description of the drawings]
FIG. 1A is a photograph of a complex of a developing porcine oocyte and a somatic cell in culture in a PVP 0% addition group (no addition group) or PVP 4% (w / v) addition group.
a: PVP 0% addition group, after 1 day of culture. The scale indicates 200 μm.
b: PVP 4% addition group, 1 day after culture
c: PVP 0% addition group, after 3 days of culture
d: PVP 4% addition group, 3 days after culture
e: PVP 0% addition group, 6 days after culture
f: PVP 4% addition group, 6 days after culture
FIG. 1B shows a photograph of the complex of porcine oocytes and somatic cells during culture in a group with 0% PVP added (no addition group) or a group with 4% PVP added.
g: PVP 0% addition group, after 9 days of culture
h: PVP 4% addition group, 9 days after culture
i: PVP added at 0%, after 12 days of culture
j: PVP 4% addition group, after 12 days of culture
FIG. 2 is a photograph of a complex of porcine oocytes and somatic cells obtained after 12 days of culture in a group with 0% PVP added (A) and a group with 4% PVP added (B). All scales indicate 200 μm.
FIG. 3A: Percentage of normal form porcine oocyte-somatic complex obtained after 12 days of culture in the 0%, 1%, 2% and 4% (w / v) groups of PVP. In addition, the maturation rate to the second meiotic metaphase when a further 48 hours of maturation culture is applied is shown.
B: Shows the diameter (B) of porcine oocytes before culturing and after culturing for 12 days.
[Fig. 4] Culture 12 of a complex of developing porcine oocytes and somatic cells in a group with 4% (w / v) addition of Ficoll (registered trademark) 400, average molecular weight of 400,000, manufactured by Sigma. The photograph after a day (A) and the swelling (B) of the cumulus cell in a mature culture are shown. All scales indicate 200 μm.
FIG. 5 shows the proportion of normal form porcine oocyte / somatic complex obtained after 12 days of culture in the Ficoll 0%, 1%, 2% and 4% (w / v) addition groups, and Furthermore, the maturation rate to the 2nd meiosis metaphase at the time of carrying out the mature culture for 48 hours is shown.
FIG. 6 is a photograph of a complex of porcine oocytes and somatic cells after 12 days of culture in a group containing 1% (w / v) hydroxypropylmethylcellulose ((hydroxypropyl) methyl cellulose, Sigma H9262). The scale indicates 200 μm.
FIG. 7 shows photographs after 2 days (A), 9 days (B), and 14 days (C) of culture of a complex of developing bovine oocytes and somatic cells in a PVP 4% (w / v) addition group. Show. All scales indicate 200 μm.
FIG. 8 shows the ratio (A) of the complex of bovine oocytes and somatic cells in normal form obtained after 14 days of culture in the PVP 0%, 2% and PVP 4% (w / v) addition groups, and bovine. Oocyte diameter (B) is shown.
FIG. 9 shows photographs of embryos obtained by in vitro fertilization of matured eggs derived from bovine oocytes / somatic cell complexes cultured for 14 days in a PVP 4% (w / v) addition group. The scale indicates 100 μm.

Claims (6)

Culturing a complex developmental developing oocyte and the surrounding somatic cells in culture at a concentration of polyvinylpyrrolidone (PVP) or Ficoll (Ficoll (registered trademark)) and 2~8% (w / v) A method for developing an oocyte, comprising: A method for growing an oocyte, comprising culturing a complex of a developing oocyte and surrounding somatic cells in a culture solution containing hydroxypropylmethylcellulose at a concentration of 1% (w / v). . The method according to claim 1 or 2 , wherein the complex is derived from a mammal. 4. The method according to claim 3 , wherein the mammal is a pig, cow, sheep, goat, dog, cat, rabbit, hamster, rat, or mouse . The method according to any one of claims 1 to 4 , wherein the surrounding somatic cells are cumulus cells, granulosa cell mass and / or granulosa cell layer. A method for producing a mature egg, wherein the oocyte developed by the method according to any one of claims 1 to 5 is matured.

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