WO2018056461A1 - 卵子、受精卵又は胚の質改善剤 - Google Patents
卵子、受精卵又は胚の質改善剤 Download PDFInfo
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Definitions
- the present invention relates to an egg, fertilized egg and / or embryo quality improving agent comprising a substance that inhibits signal transmission from CXCL5.
- the cells that make up the human body repeat division and proliferation as they grow, but in the process, they are exposed to various stresses associated with aging and accumulate damage. This is considered to be caused by oxidative damage due to accumulation of active oxygen, DNA damage due to ultraviolet rays, etc. As a result, mitochondrial dysfunction and intracellular metabolic dysfunction are caused, leading to canceration and cell death.
- the deterioration of cell quality with aging is a phenomenon common to all living organisms including human beings. At present, the deterioration of cell quality with aging is inevitable, and studies for delaying, stopping, and improving them have been widely conducted.
- CXCL5 is a type of chemokine and is known to contribute to neutrophil chemotaxis / activation and to participate in inflammatory reactions (Patent Document 1).
- an object of the present invention is to provide a means for improving the deterioration of the quality of the pre-implantation embryo due to aging or the like.
- the present inventors have intensively studied to solve the above problems. As a result, we found that CXCL5, a type of chemokine, induces senescence of the embryo, and we found that inhibiting the signal transduction from CXCL5 can improve the deterioration of the quality of the preimplantation embryo due to aging, etc. . Based on this finding, the present invention has been completed. That is, the present invention relates to the following.
- An egg, fertilized egg and / or embryo quality improving agent comprising a substance that inhibits signal transmission from CXCL5.
- the improving agent according to [1], wherein the substance that inhibits signal transmission from CXCL5 is a substance that inhibits the interaction between CXCL5 and CXCR2.
- the improving agent according to [2], wherein the substance that inhibits the interaction between CXCL5 and CXCR2 is an anti-CXCL5 antibody or a fragment thereof, an anti-CXCR2 antibody or a fragment thereof, or a CXCR2 antagonist.
- a method for improving the quality of an ovum, a fertilized egg or an embryo comprising a step of bringing an ovum, a fertilized egg or an embryo into contact with a substance that inhibits signal transmission from CXCL5.
- a method for culturing an ovum, a fertilized egg or an embryo comprising culturing an ovum, a fertilized egg or an embryo in a culture solution containing a substance that inhibits signal transmission from CXCL5.
- the quality of eggs, fertilized eggs or embryos that have decreased due to aging etc. is improved, and the pregnancy rate / birth rate is increased in in vitro fertilized embryo transfer etc. Can do.
- aging of an egg, a fertilized egg, or an embryo can be predicted by using the CXCL5 concentration in blood or ovarian tissue as a biomarker.
- FIG. 1 is a diagram showing the results of a biological function analysis of a gene whose expression has changed by comparing gene expression in blastocysts of young and infertile patients.
- FIG. 2 is a diagram showing the results of the blastocyst arrival rate of embryos obtained by CXCL5 neutralizing antibodies, CXCR2 antagonists, and combinations thereof.
- FIG. 3 is a diagram showing the results of embryo transfer of embryos obtained by the combined use of a CXCL5 neutralizing antibody and a CXCR2 antagonist.
- A shows the implantation rate of each group of mice.
- B shows the offspring acquisition rate of each group of mice.
- FIG. 1 is a diagram showing the results of a biological function analysis of a gene whose expression has changed by comparing gene expression in blastocysts of young and infertile patients.
- FIG. 2 is a diagram showing the results of the blastocyst arrival rate of embryos obtained by CXCL5 neutralizing antibodies, CXCR2 antagonists, and
- FIG. 4 is a diagram showing the results of embryo transfer of embryos obtained by adding a CXCL5 neutralizing antibody and a CXCR2 antagonist alone.
- A is a figure which shows the implantation rate of a CXCL5 neutralizing antibody addition group.
- B is a diagram showing the litter acquisition rate of the CXCL5 neutralizing antibody addition group.
- C is a graph showing the implantation rate of the CXCR2 antagonist addition group.
- D is a diagram showing the litter acquisition rate in the CXCR2 antagonist addition group.
- FIG. 5 is a diagram showing the results of the blastocyst arrival rate of embryos obtained by adding CXCL5 to young mice.
- FIG. 6 shows the results of embryo transfer of embryos obtained by adding CXCL5 to young mice.
- A shows the implantation rate of each group of mice.
- FIG. 7 is a diagram showing the results of expression levels of aging markers in embryos obtained by adding CXCL5 to young mice.
- A shows the expression level of p21.
- B shows the expression level of p53.
- C shows the expression level of PAI-1.
- D shows the expression level of IL-6.
- FIG. 8 is a diagram showing the measurement results of CXCL5 concentration in serum and ovary of aging mice and young mice. A indicates the concentration in serum. B indicates the concentration in the ovary.
- One embodiment of the present invention relates to an egg, fertilized egg and / or embryo quality improving agent (hereinafter sometimes simply referred to as “quality improving agent”), which comprises a substance that inhibits signal transduction from CXCL5. That is, the present invention is characterized in that a substance that inhibits signal transduction from CXCL5 is used for the purpose of improving the quality of eggs, fertilized eggs and / or embryos. “Improving the quality of eggs, fertilized eggs and embryos” can be restated as “improving the ability of eggs, fertilized eggs and embryos to become pregnant”.
- CXCL5 concentration in the ovary increases with aging and the like. It was also found that CXCL5 induces senescence of eggs, fertilized eggs, embryos, etc., resulting in a decrease in the quality of the embryos obtained. Furthermore, it has been found that the quality of an egg, a fertilized egg or an embryo that has been lowered by CXCL5 can be improved by inhibiting signaling from CXCL5 via CXCR2, which is a chemokine receptor. Based on this finding, the present invention has been completed.
- the ovum, fertilized egg and / or embryo quality improving agent of the present invention is a substance that inhibits signal transmission from CXCL5, and is not particularly limited as long as it has an effect of improving the quality of the egg, fertilized egg or embryo. .
- examples thereof include substances that inhibit the interaction between CXCL5 and CXCR2.
- substances that inhibit the interaction between CXCL5 and CXCR2 include an anti-CXCL5 antibody or a fragment thereof, an anti-CXCR2 antibody or a fragment thereof, or a CXCR2 antagonist. These can be used alone or in combination of two or more.
- anti-CXCL5 antibody examples include an antibody (neutralizing antibody) or a fragment thereof that inhibits the interaction between CXCL5 and CXCR2 by binding to CXCL5 or CXCR2.
- anti-CXCL5 antibodies and anti-CXCR2 antibodies can be used.
- examples of the anti-CXCL5 antibody include Anti-CXCL5 antibody (abcam, R and D systems, LifeSpan Biosciences, ThermoFisher SCIENTIFIC).
- anti-CXCR2 antibodies include, for example, Anti-CXCR2 antibody (abcam), Human CXCR2 / IL-8RB Mab (R and D systems), Mouse Monoclonal CXCR2 / IL-8 RB Antibody (Novuss Biologicals), CXCR2 / IL8RB antibody (ThermoFisher SCIENTIFIC) and the like.
- An anti-CXCL5 antibody can be prepared by a conventional method using CXCL5 or a fragment thereof as an antigen, and such an antibody can also be used in the present invention.
- An anti-CXCR2 antibody can be prepared by a conventional method using CXCR2 or a fragment thereof as an antigen, and such an antibody can also be used in the present invention.
- the antibody may be a polyclonal antibody or a monoclonal antibody. Further, it may be a complete antibody molecule, and an antibody fragment capable of specifically binding to an antigen, for example, Fab (fragment of antigen binding), F (ab ′) 2 , Fab ′, Fv, scFv (single chain Fv), dsFv (Disulfide stabilized Fv), CDR (complementarity determining region), etc. may be used.
- the antibody can also be a human chimeric antibody or a humanized antibody.
- Examples of the CXCR2 antagonist include substances that inhibit the interaction between CXCL5 and CXCR2 by competing with CXCL5 and binding to CXCR2, but themselves do not have the ability to activate CXCR2.
- CXCR2 antagonist known ones can be used. Although not particularly limited, for example, SB225002 (TOCRIS) and the like can be mentioned.
- the antibodies and antagonists used in the present invention can be selected by a known method using CXCL5 and CXCR2 as indicators of whether or not their binding is inhibited. By inhibiting the binding between CXCL5 and CXCR2, signaling through CXCR2 from CXCL5 is inhibited, and the quality of the egg, fertilized egg or embryo that has decreased due to aging or the like can be improved.
- the antibodies and antagonists used in the present invention can also be selected by a known method using CXCL5 and CXCR2 as indicators of whether or not signal transmission from CXCL5 via CXCR2 is inhibited.
- the quality improving agent of the present invention By contacting the ovum, fertilized egg and / or embryo quality-improving agent of the present invention with the ovum, fertilized egg or embryo, signal transmission via CXCR2 from CXCL5 is inhibited, and the egg and fertilized egg decreased by CXCL5. Or improve the quality of the embryo.
- the quality improving agent of the present invention is added to the culture solution, and the egg, The method of culturing a fertilized egg or embryo is mentioned.
- the quality of the embryo is improved by improving the quality of the egg and / or fertilized egg by bringing the quality improving agent of the present invention into contact with the egg and / or fertilized egg. As well as contacting the embryo with the quality improving agent of the present invention to improve the quality of the embryo.
- the quality of the fertilized egg is improved by bringing the quality improving agent of the present invention into contact with the egg to improve the quality of the egg, and the quality of the present invention. It includes contacting the fertilized egg with an improving agent to improve the quality of the fertilized egg.
- the egg, fertilized egg or embryo quality-improving effect of the present invention is an evaluation item related to pregnancy / childbirth, in the quality-improving agent-added group of the present invention, in the commonly used in vitro culture system, etc. For example, it can be confirmed by improving any one of the blastocyst arrival rate, the implantation rate, the litter (child) acquisition rate, the miscarriage rate, etc., or a plurality of them.
- the application target of the ovum, fertilized egg or embryo quality improving agent of the present invention is not particularly limited as long as the quality of the ovum, fertilized egg or embryo is lowered due to aging or the like and improvement is required.
- the subject is a mammal, and the mammal is not particularly limited, but is commercially available for meat and dairy production such as humans, non-human animals such as pigs, cattle, obidos, horses, buffalos, etc. Including animals with great value.
- One aspect of the present invention is an egg, fertilized egg and / or embryo culture medium (hereinafter simply referred to as an egg, fertilized egg and / or embryo quality improving agent comprising a substance that inhibits signal transduction from CXCL5. (Sometimes referred to as "culture medium").
- the culture solution of the egg, fertilized egg and / or embryo of the present invention contains the egg, fertilized egg and / or embryo quality improving agent of the present invention, and during the culture of the egg, fertilized egg or embryo, It can be used to improve the quality of an egg, a fertilized egg or an embryo that has decreased due to the secretion and increased secretion of CXCL5 accompanying aging and the like.
- the culture solution of the present invention is not particularly limited as long as it contains the egg, fertilized egg and / or embryo quality improving agent of the present invention as an active ingredient.
- the ovum, fertilized egg and / or embryo quality improving agent of the present invention can be added to a known ovum, fertilized egg or embryo culture solution to produce the culture solution of the present invention.
- a culture solution having a different composition can be used depending on the stage of development. Conventionally, it is not particularly limited as long as it is a culture solution used for culturing eggs, fertilized eggs or embryos, and the culture solution of the present invention is obtained by adding the egg, fertilized egg and / or embryo quality improving agent of the present invention. Can be manufactured. In addition, the form which added the quality improving agent of this invention to the culture solution used in order to culture
- the ovum, fertilized egg or embryo culture solution used for in vitro fertilization etc. is used for culturing embryos from the sperm and ovum after fertilization and fertilization (pronuclear stage) to the 16 cell stage.
- the first culture solution, the second culture solution used for culturing embryos from the mulberry stage to the blastocyst stage, and the like are used.
- the first culture solution and the second culture solution may be the same culture solution.
- the first culture solution can be used for sperm collection and egg collection.
- Sydney IVF Fertilization medium Cook medical
- Sydney IVF Cleavage medium Cook medical
- Sequential Fert (ORIGIO)
- Sequential Cleav (ORIGIO)
- Universal IVF medium (ORIGIO)
- G-IVF TM Vitrolife
- G-1 TM v5 Vitrolife
- the culture solution of the present invention can be produced using a prepared culture solution by dissolving inorganic salts, saccharides, amino acids, cytoprotective substances, antibiotics, physiologically active substances, etc. in ultrapure or distilled water. .
- the content of the ovum, fertilized egg and / or embryo quality-improving agent of the present invention in the culture solution is usually 0.01 to 100 ⁇ g / ml, preferably 0.1 to 10 ⁇ g / ml in the case of an antibody or a fragment thereof as the concentration in the culture solution. May be ml. If it is an antagonist, it may be usually 1 to 100 nM, preferably 10 to 30 nM. When an antibody or a fragment thereof is used in combination with an antagonist, it can be usually 28.6: 1 to 2857.1: 1, preferably 28.6: 1 to 1000: 1 (mass ratio).
- Oocytes, fertilized eggs or embryos can be cultured according to known culture methods except that the culture solution of the present invention is used.
- the culture conditions are exemplified below, but are not limited to the same conditions, and can be appropriately changed according to the application target, required effects, and the like.
- the culture temperature can usually be 37 ° C or higher.
- the culture gas phase can be usually 5% CO 2 and 95% O 2 .
- Culture time is usually 1 to 2 hours for pre-cultured eggs for in vitro fertilization, usually 5 to 72 hours for fertilized eggs, and usually 72 to 144 hours for embryos after the mulberry stage. be able to.
- the quality improving agent of the present invention can be brought into contact with an ovum, a fertilized egg or an embryo during at least one of the preculture period of the ovum, the culture period of the fertilized egg, and the culture period of the embryo after the mulberry stage. .
- the quality improving agent of the present invention may be brought into contact with an egg, a fertilized egg or an embryo over a plurality of culture periods.
- the time for contacting the quality improving agent of the present invention with an egg, a fertilized egg or an embryo may be all in each period or a part of the period.
- the ovum, fertilized egg and / or embryo culture solution of the present invention is improved in ovum, fertilized egg or embryo quality due to aging, etc., and in vitro fertilization / microscopic observation with a reduced quality egg, fertilized egg or embryo
- mammalian eggs, fertilized eggs, and embryos that require improvement in the decrease in the success rate of fertilization.
- Mammals include, but are not limited to, humans and non-human animals such as pigs, cows, obidos, horses, buffalos and the like.
- One embodiment of the present invention relates to a method for predicting aging of an egg, a fertilized egg, and / or an embryo, wherein the presence or absence of aging of the egg, fertilized egg, and / or embryo is determined using the CXCL5 concentration in serum separated from the subject as an index. .
- the aging of an egg, a fertilized egg and / or an embryo is judged using the CXCL5 concentration in an ovarian tissue separated from a subject as an index to determine whether the egg, fertilized egg and / or embryo is aging.
- the present invention relates to a method (hereinafter, these may be simply referred to as “aging prediction method”).
- the CXCL5 concentration in the ovary and the CXCL5 concentration in the serum are correlated. Therefore, in addition to the CXCL5 concentration in the ovary as an index, the CXCL5 concentration in the ovary is used as an index to determine whether the ovary, fertilized egg or embryo is induced by aging. Can be predicted.
- blood used as a specimen includes serum, and serum can be appropriately obtained by processing blood collected from a subject according to a conventional method.
- examples of ovarian tissues used as specimens include ovarian tissues themselves, ovarian tissue preparations, ovarian tissue-derived components (ovarian cells, follicular fluid, granulosa cells, etc.), and these were collected from subjects. It can be appropriately obtained by treating ovarian tissue according to a conventional method.
- the CXCL5 concentration in the ovarian tissue is not limited to the CXCL5 protein concentration, and may be a concentration of a substance related to CXCL5 expression.
- expression means transcription of the gene of CXCL5 or translation from the transcription product of the gene.
- the method for measuring the CXCL5 concentration in serum or ovarian tissue is not particularly limited, and examples thereof include immunochemical methods and various chromatographic methods such as HPLC methods.
- examples of the method for measuring CXCL5 concentration include PCR methods such as RT-PCR. In view of convenience and the like, measurement is preferably performed by an immunochemical method using an antibody recognizing CXCL5.
- the immunochemical method used for measuring the CXCL5 concentration in serum or ovarian tissue is not particularly limited, and known methods such as enzyme immunoassay (EIA method), latex agglutination method, immunochromatography method, radiation Uses immunoassay (RIA), fluorescence immunoassay (FIA), luminescence immunoassay, spin immunoassay, turbidimetric method for measuring turbidity associated with antigen-antibody complex formation, and antibody solid phase membrane electrode Examples thereof include an enzyme sensor electrode method, an immunoelectrophoresis method, and a Western blot method for detecting a potential change due to binding to an antigen. From the viewpoint of simplicity, it is preferable to measure by EIA method, latex agglutination method or immunochromatography method.
- the EIA method includes a competitive method in which the enzyme-labeled antigen and the antigen in the sample are allowed to compete with each other, and a non-competitive method that does not allow competition.
- sandwich enzyme-linked immunoassay which is a non-competitive method using two types of antibodies.
- a phase measurement method (sandwich ELISA method) is preferable from the viewpoint of ease of operation.
- the cutoff value is the CXCL5 concentration in the serum or ovarian tissue of the same type of control (eg, healthy subject, young subject, etc.) or based on the same concentration.
- the cut-off value can be appropriately set based on a conventional method according to the specimen, required effect, etc. as a value based on the CXCL5 concentration in the control serum or ovarian tissue or the same concentration.
- CXCL5 concentration in serum or ovarian tissue separated from the subject is higher than the cut-off value or the already set cut-off value, it is judged that the egg, fertilized egg or embryo is aged and separated from the subject
- the CXCL5 concentration in serum or ovarian tissue is the same as or lower than the cut-off value, it is determined that the egg, fertilized egg or embryo is not senescent, and senescence can be predicted.
- the degree of aging can be predicted by comparing the CXCL5 concentration in serum or ovarian tissue separated from the subject with a cut-off value.
- the application target of the aging prediction method of the present invention is a mammal, and is not particularly limited. Mammals include, but are not limited to, humans and non-human animals such as pigs, cows, obidos, horses, buffalos and the like.
- the present invention includes a method for improving the quality of an ovum, a fertilized egg or an embryo, which comprises a step of bringing an ovum, a fertilized egg or an embryo into contact with a substance that inhibits signal transmission from CXCL5.
- the present invention also includes a method of improving the quality of an egg, a fertilized egg, or an embryo, comprising a step of contacting an egg, a fertilized egg, or an embryo with a substance that inhibits signal transmission from CXCL5, and increasing a pregnancy rate / birth rate including.
- the contents of the preceding section can be referred to.
- the present invention includes a method for culturing an ovum, a fertilized egg or an embryo, comprising a step of culturing an ovum, a fertilized egg or an embryo in a culture solution containing a substance that inhibits signal transmission from CXCL5.
- a method for culturing an ovum, a fertilized egg or an embryo comprising a step of culturing an ovum, a fertilized egg or an embryo in a culture solution containing a substance that inhibits signal transmission from CXCL5.
- the contents of the preceding section can be referred to.
- Example 1 We compared gene expression profiles of early-implantation embryos in young infertility patients and those in older infertility patients over 39 years old, and comprehensively analyzed genes that showed differences in expression. Among them, secretory factors and those that are highly expressed in elderly patients were searched for and used as candidate factors involved in the deterioration of embryo quality due to aging. Specifically, the experiment was performed as follows.
- CXCL5 which is a secretory factor and highly expressed in elderly patients, was selected from among the expression variation genes, and CXCL5, which is 202.8 times higher in expression in blastocysts of aging patients than in young patients, was the final candidate factor. .
- Example 2 Whether or not the candidate factor CXCL5 obtained in Example 1 can improve the quality of embryos that have decreased due to aging in the in vitro culture system of aging mouse embryos by the addition of neutralizing antibodies or antagonists of specific receptors Investigated about.
- In vitro fertilization by collecting ovulated mature eggs from a 42-week-old mouse, and zygote obtained by adding a neutralizing antibody of a candidate factor or an antagonist of a specific receptor In vitro culture was performed using the solution, and the blastocyst arrival rate was determined in order to verify embryo development ability.
- the obtained blastocysts were transplanted into pseudopregnant mice, the implantation rate and the offspring acquisition rate were examined, and the quality of the embryos was further evaluated. Specifically, the experiment was performed as follows.
- mice (Experimental animal) ICR female mice (CLEA Japan, Tokyo, Japan) were used as experimental animals. The mice were raised in a light and dark environment every 12 hours at a room temperature of 22 ° C. and a humidity of 55%. The mouse feed was MF control feed (Oriental Yeast, Tokyo, Japan), 6 g / day per mouse. Water was allowed to be freely taken at any time. Three-week-old mice were used as young mice, and 42-week-old mice were used as aging mice. Each group had 5 animals. The handling and breeding of all mice was in accordance with the standards of the laboratory animal facility of St. Marianna University School of Medicine.
- a part of the epididymis tail was incised to collect the sperm inside.
- the collected sperm mass was submerged in a 1.5 mL microtube containing 400 ⁇ l TYH medium and swim up for 10 minutes in a CO 2 incubator (37 ° C., 5% CO 2 , 95% in air).
- sperm was removed from 30 ⁇ l KSOM Medium (Merck Millipore, Darmstadt, Germany), and the number of embryos at the 2-cell stage was calculated. Thereafter, the young mouse control and the aging mouse control were transferred to another 30 ⁇ l KSOM Medium and cultured in a CO 2 incubator for 4 days.
- KSOM Medium supplemented with CXCL5 neutralizing antibodies (ab135203, abcam) at final concentrations of 0.1 ⁇ g / ml, 1 ⁇ g / ml, and 10 ⁇ g / ml, final concentrations of 10 nM, and 30 nM of CXCR2 antagonist (2725, TOCRIS, respectively)
- 30 ml KSOM Medium supplemented with CXCL5 neutralizing antibody and CXCR2 antagonist, and cultured in a CO 2 incubator for 4 days.
- the blastocyst arrival rate was calculated by the number of blastocysts / two-cell stage embryos.
- the uterus was fixed with tweezers, a hole was made in the oviduct junction with a 30G injection needle (Dentronics, Tokyo, Japan), a glass capillary that sucked the blastocyst was inserted, and the embryo was implanted inside the uterus. After transplantation, the uterus was carefully returned to the body, and the retroperitoneum and skin were sutured. At the time of transplantation, blastocysts obtained from each group of mice were transplanted individually into Recipient mice. The day after the egg collection was the first day, and a cesarean section was performed on the 19th day.
- Recipient mice were euthanized and laparotomized, and the uterus was removed and the fetus removed.
- the implantation rate was calculated by the number of implantation marks / number of embryo transfer, and the litter acquisition rate was calculated by the number of litters / implantation marks.
- Example 3 In addition, in the in vitro culture system of young mouse embryos, the same method as in Example 2 was used to determine whether the blastocyst arrival rate, implantation rate, and litter acquisition rate were changed by the addition of the candidate factor. investigated. In addition, the expression of p16, p21, p53, PAI-1, and IL-6, which are senescence markers, in the embryo was measured to examine whether cell senescence was induced. Specifically, the experiment was performed as follows.
- p16 showed no difference in the expression level, but p21, p53, PAI-1, and IL-6 significantly increased expression (T-test, * P ⁇ 0.05 vs control, Fig. 7).
- Example 4 The concentrations of candidate factors in serum and ovarian tissue in young and aged mice were measured to evaluate whether the candidate factors are useful as biomarkers that can predict embryo aging. Specifically, the experiment was performed as follows.
- CXCL5-CXCR2 signaling in aging embryos has been shown to improve the quality of ova, fertilized eggs, and embryos that have decreased due to aging, and to increase the pregnancy rate and fertility rate in in vitro fertilized embryo transfer etc. It was. CXCL5 induced embryo senescence, and its serum concentration was shown to be useful as an aging marker for eggs, fertilized eggs, and embryos.
- the present invention can be applied to pharmaceuticals, medical devices, research reagents, and the like.
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Abstract
Description
すなわち、本発明は以下に関する。
[2] CXCL5からのシグナル伝達を阻害する物質が、CXCL5とCXCR2との相互作用を阻害する物質である、[1]に記載の改善剤。
[3] CXCL5とCXCR2との相互作用を阻害する物質が、抗CXCL5抗体若しくはその断片、抗CXCR2抗体若しくはその断片、又はCXCR2アンタゴニストである、[2]に記載の改善剤。
[4] 卵子、受精卵及び/又は胚の質が、加齢により低下したものである、[1]~[3]の何れかに記載の改善剤。
[6] 対象より分離した血清中のCXCL5濃度を指標として、卵子、受精卵及び/又は胚の老化の有無を判断する、卵子、受精卵及び/又は胚の老化の予測方法。
[7] 対象より分離した卵巣組織中のCXCL5濃度を指標として、卵子、受精卵及び/又は胚の老化の有無を判断する、卵子、受精卵及び/又は胚の老化の予測方法。
[9] CXCL5からのシグナル伝達を阻害する物質を含有する培養液中で、卵子、受精卵又は胚を培養する工程を含む、卵子、受精卵又は胚の培養方法。
本発明の一形態は、CXCL5からのシグナル伝達を阻害する物質からなる、卵子、受精卵及び/又は胚の質改善剤(以下、単に「質改善剤」と称することがある)に関する。すなわち、本発明は、CXCL5からのシグナル伝達を阻害する物質を、卵子、受精卵及び/又は胚の質改善の用途に用いることを特徴とする。なお、「卵子、受精卵及び胚の質改善」とは「卵子、受精卵及び胚の妊娠する能力の改善」と言い換えることができる。
抗CXCR2抗体は、CXCR2又はそのフラグメントを抗原として、常法により作製することも可能であり、そのような抗体を本発明に用いることも可能である。
本発明の一形態は、CXCL5からのシグナル伝達を阻害する物質からなる、卵子、受精卵及び/又は胚の質改善剤を含有する、卵子、受精卵及び/又は胚の培養液(以下、単に「培養液」と称することがある)に関する。本発明の卵子、受精卵及び/又は胚の培養液は、本発明の卵子、受精卵及び/又は胚の質改善剤を含有することを特徴とし、卵子、受精卵又は胚の培養中に、加齢等に伴うCXCL5の分泌・分泌増加により低下した卵子、受精卵又は胚の質を改善するために用いることができる。
培養温度は、通常37℃以上で行うことができる。培養気相は、通常5% CO2、95% O2の気相とすることができる。
培養時間は、体外受精のための卵子の前培養であれば通常1時間~2時間、受精卵であれば通常5~72時間、桑実期以降の胚であれば通常72~144時間とすることができる。
本発明の質改善剤を、卵子の前培養期間、受精卵の培養期間、及び桑実期以降の胚の培養期間の少なくともいずれかの期間に、卵子、受精卵又は胚と接触させることができる。
複数の培養期間に渡り、本発明の質改善剤を卵子、受精卵又は胚と接触させてもよい。本発明の質改善剤を卵子、受精卵又は胚と接触させる時間は、各期間中の全て又は期間中の一部の期間であってよい。
本発明の一形態は、対象より分離した血清中のCXCL5濃度を指標として、卵子、受精卵及び/又は胚の老化の有無を判断する、卵子、受精卵及び/又は胚の老化の予測方法に関する。さらなる本発明の一形態は、対象より分離した卵巣組織中のCXCL5濃度を指標として、卵子、受精卵及び/又は胚の老化の有無を判断する、卵子、受精卵及び/又は胚の老化の予測方法(以下、これらを単に「老化の予測方法」と称することがある)に関する。
本発明において、検体として使用される卵巣組織としては、卵巣組織自体、卵巣組織調製物、卵巣組織由来成分(卵巣細胞、卵胞液、顆粒膜細胞等)等が挙げられ、これらは対象から採取した卵巣組織を常法に従って処理することで、適宜、得ることができる。
血清又は卵巣組織中のCXCL5濃度を測定する方法としては、特に限定されないが、例えば、免疫化学的方法、HPLC法等の各種クロマトグラフィー法などが挙げられる。関連する物質が核酸である場合には、CXCL5濃度を測定する方法としては、RT-PCR法等のPCR法などが挙げられる。簡便性等の点から、CXCL5を認識する抗体を使用する免疫化学的方法により測定するのが好ましい。
同カットオフ値又は既に設定されたカットオフ値よりも、対象より分離した血清又は卵巣組織中のCXCL5濃度が高い場合、卵子、受精卵又は胚が老化していると判断し、対象より分離した血清又は卵巣組織中のCXCL5濃度がカットオフ値と同じ若しくは低い場合、卵子、受精卵又は胚が老化していないと判断し、老化を予測することができる。また、対象より分離した血清又は卵巣組織中のCXCL5濃度をカットオフ値と比較することにより、老化の程度を予測することができる。
さらに、本発明は、CXCL5からのシグナル伝達を阻害する物質に、卵子、受精卵又は胚を接触させる工程を含む、卵子、受精卵又は胚の質改善方法を含む。また、本発明は、CXCL5からのシグナル伝達を阻害する物質に、卵子、受精卵又は胚を接触させる工程を含む、卵子、受精卵又は胚の質を改善し、妊娠率・出産率を高める方法を含む。接触方法等については、前述の項の内容を参照できる。
着床前期胚を若年不妊症患者のものと39歳以上の高齢不妊症患者のもので遺伝子発現プロファイルを比較し、発現に差が認められる遺伝子を網羅的に解析した。その中で、分泌因子かつ高齢患者で高発現となるものを探索し、加齢による胚の質の低下に関与する候補因子とした。具体的には以下のように実験を行った。
実施例1で得られた候補因子CXCL5について、加齢マウス胚の体外培養系において、その中和抗体や特異的受容体のアンタゴニスト添加により、加齢によって低下した胚の質が改善されるかどうかについて調べた。42週齢の加齢マウスより、排卵した成熟卵子を回収して体外受精を行い、得られた受精卵(zygote)に対し、候補因子の中和抗体や特異的受容体のアンタゴニストを添加した培養液を用いて体外培養を行い、胚発育能を検証するため胚盤胞到達率を求めた。また、得られた胚盤胞を偽妊娠マウスに胚移植し、着床率、産仔獲得率を調べ、胚の質をさらに評価した。具体的には以下のように実験を行った。
ICR雌マウス(日本クレア, Tokyo, Japan)を実験動物として用いた。マウスは室温22℃、湿度55%、12時間ごとの明暗環境下で飼育された。マウス飼料はMFコントロール飼料(オリエンタル酵母, Tokyo, Japan)マウス一匹あたり一日6 gを与えた。水分は随時自由に摂取できる状態とした。若年マウスとして3週齢のマウスを、加齢マウスとして42週齢のマウスを用いた。各群は5匹とした。
全てのマウスの取り扱いや飼育に関しては聖マリアンナ医科大学の実験動物施設の基準に準じた。
3週齢及び42週齢を過ぎたマウスは膣スメアにより性周期を確認した。発情前期のタイミングでゴナトロピン(ASKA Pharmaceutical. Co., Tokyo, Japan) 10 IUを腹腔内投与した。投与後15時間で卵子卵丘細胞複合体(COCs:Cumulus Oocyte Complexes)を卵管内から回収し、100μl TYH medium(LSI Medience corporation, Tokyo, Japan)内で30分間前培養した。その間にICR雄マウス(10-12週齢)にソムノペンチル麻酔薬(共立製薬, Tokyo, Japan)で全身麻酔を施し、精巣上体尾部を摘出した。精巣上体尾部の一部を切開し内部の精子を押し出すように回収した。回収した精子塊を400μl TYH mediumが入った1.5 mLマイクロチューブに沈めてCO2 incubator 内で10分間swim upした(37℃、5% CO2、95% in air)。Swim up後の精子をCOCsが入った100μl TYH mediumに最終濃度2-3×105/mlとなるように加え、CO2 incubator (37℃、5% CO2、95% in air)内で5-6時間培養した。培養後の卵子は30μl KSOM Medium(Merck Millipore, Darmstadt, Germany)内で精子の除去を行い、その際に2細胞期胚数を算出した。その後、若年マウス対照、加齢マウス対照については、別の30μl KSOM Mediumに移し、CO2 incubator 内で4日間培養した。薬剤処理群については、それぞれ最終濃度0.1μg/ml、1μg/ml、10μg/mlのCXCL5中和抗体(ab135203, abcam)を添加した30μl KSOM Medium、最終濃度10nM、30nMのCXCR2アンタゴニスト(2725, TOCRIS)を添加した30ml KSOM Medium、並びに、CXCL5中和抗体及びCXCR2アンタゴニストを添加した30μl KSOM Mediumに移し、CO2 incubator 内で4日間培養した。胚盤胞到達率は、胚盤胞数/二細胞期胚数で算出した。
4日間の培養後、胚盤胞期胚に到達した胚を胚移植した。Recipientマウスは6-10週齢のICR雌マウスで、採卵日の前日に精管結紮されたICR雄マウスと交配させ、翌日膣栓が確認できた個体を使用した。ソムノペンチル麻酔薬で全身麻酔を施し、後背部切開により子宮を露出させた。子宮をピンセットで固定し30G の注射針(Dentronics, Tokyo, Japan)で卵管接合部に穴を開け、胚盤胞を吸引したガラスキャピラリーを挿入し子宮内部に胚を移植した。移植後、子宮を慎重に体内に戻し、後腹膜及び皮膚を縫合した。移植に際しては、各群のマウスから得られた胚盤胞を個体毎にそれぞれRecipientマウスに移植した。採卵の翌日を1日目として、19日目で帝王切開を実施した。Recipientマウスを安楽死させ開腹、子宮を摘出して胎仔を取り出した。着床率は着床痕数/胚移植数、産仔獲得率は産仔数/着床痕数で算出した。
中和抗体(10μg/ml)のみの添加群、アンタゴニスト(10nM)のみの添加群でも薬剤処理群で着床率、産仔獲得率ともに高い傾向を示した(図4)。
また、若年マウス胚の体外培養系において、その候補因子の添加により、胚盤胞到達率、着床率、産仔獲得率に変化があるかどうかについて、実施例2と同様の方法を用いて検討した。また、老化マーカーである、p16, p21, p53, PAI-1, IL-6の胚における発現を測定し、細胞老化が誘導されるかどうかについて調べた。具体的には以下のように実験を行った。
若年マウスの受精卵にCXCL5及び熱変性CXCL5を添加し培養したところ、胚盤胞到達率は、非添加対照群との間に有意な差は認められなかった(図5)。
若年マウス及び加齢マウスにおける候補因子の血清中及び卵巣組織における濃度を測定し、候補因子が胚の老化を予測可能なバイオマーカーとしての有用か否かを評価した。具体的には以下のように実験を行った。
Claims (7)
- CXCL5からのシグナル伝達を阻害する物質からなる、卵子、受精卵及び/又は胚の質改善剤。
- CXCL5からのシグナル伝達を阻害する物質が、CXCL5とCXCR2との相互作用を阻害する物質である、請求項1に記載の改善剤。
- CXCL5とCXCR2との相互作用を阻害する物質が、抗CXCL5抗体若しくはその断片、抗CXCR2抗体若しくはその断片、又はCXCR2アンタゴニストである、請求項2に記載の改善剤。
- 卵子、受精卵及び/又は胚の質が、加齢により低下したものである、請求項1~3の何れか一項に記載の改善剤。
- 請求項1~4の何れか一項に記載の改善剤を含有する、卵子、受精卵及び/又は胚の培養液。
- 対象より分離した血清中のCXCL5濃度を指標として、卵子、受精卵及び/又は胚の老化の有無を判断する、卵子、受精卵及び/又は胚の老化の予測方法。
- 対象より分離した卵巣組織中のCXCL5濃度を指標として、卵子、受精卵及び/又は胚の老化の有無を判断する、卵子、受精卵及び/又は胚の老化の予測方法。
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KAWAGOE,Y ET AL.: "Inhibition of CXCL5-CXCR2 signaling improves the implantation of aging embryos for pregnancy", FERTILITY AND STERILITY, vol. 108, no. 3, 1 September 2017 (2017-09-01), pages E372, XP085159241, ISSN: 0015-0282, DOI: 10.1016/j.fertnstert.2017.07.1086 * |
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