WO2022007897A1 - 小分子化合物用于激活卵泡的方法及其制剂 - Google Patents

小分子化合物用于激活卵泡的方法及其制剂 Download PDF

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WO2022007897A1
WO2022007897A1 PCT/CN2021/105248 CN2021105248W WO2022007897A1 WO 2022007897 A1 WO2022007897 A1 WO 2022007897A1 CN 2021105248 W CN2021105248 W CN 2021105248W WO 2022007897 A1 WO2022007897 A1 WO 2022007897A1
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medium
ovarian
vinylcyclohexene diepoxide
ovary
follicles
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French (fr)
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马金龙
刘洪彬
曹连宝
吴克良
苏献伟
路钢
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青岛卓云海智医疗科技有限公司
山东大学
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Priority to US18/005,035 priority Critical patent/US20230330055A1/en
Publication of WO2022007897A1 publication Critical patent/WO2022007897A1/zh

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    • C12N5/0602—Vertebrate cells
    • C12N5/0681—Cells of the genital tract; Non-germinal cells from gonads
    • C12N5/0682—Cells of the female genital tract, e.g. endometrium; Non-germinal cells from ovaries, e.g. ovarian follicle cells
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Definitions

  • the present invention relates to the field of reproduction in mammals, including humans. Specifically, the present invention relates to methods for improving the developmental capacity of ovaries or ovarian tissue in mammals, including humans, as well as methods and media for in vitro cultivation of ovaries or ovarian tissue.
  • the number of follicles contained in the ovaries of female mammals is determined at the time of birth. As the age increases, a large part of the follicles will go into apoptosis and atresia during the development of the ovary, and only a few follicles will eventually experience Primordial follicles - primary follicles - secondary follicles - pre-ovulatory follicles - ovulation this process eventually matures and discharges. As mammals age, the functional follicular reserve (FOR) and the ability to mature decline. In some cases, even if there are primary follicles or small secondary follicles, it is difficult to continue to grow to maturity. . At the same time, with the increase of age, the body's ability to respond to endogenous or exogenous hormones such as follicle-stimulating hormone (FSH) also decreases.
  • FSH follicle-stimulating hormone
  • Egg maturation is critical in human reproduction, including assisted reproduction, and plays an important role in the development of animal husbandry.
  • human females older women with reproductive requirements have diminished ovarian reserve, which is insufficient for a successful pregnancy.
  • Clinically some patients, such as those who need radiation therapy, will have some ovarian tissue removed and preserved for possible future reproductive use.
  • the continuation of the genes of livestock with good genes is especially important, even in the stage of old age and declining fertility, there is still the need to reproduce offspring. In these situations, the protection of the ovary and the maintenance and promotion of its follicular developmental potential are extremely important.
  • VCD 4-vinylcyclohexene diepoxide
  • VCD 4-vinylcyclohexene diepoxide
  • POI chemically induced premature ovarian insufficiency
  • the present invention discovers and proves for the first time that low concentration and/or short time 4-vinylcyclohexene diepoxide (VCD) treatment has hormone-like effect on ovary or cultured ovarian tissue, which is beneficial to the ovary of female mammals.
  • VCD 4-vinylcyclohexene diepoxide
  • the maintenance or even enhancement of developmental and reproductive potential in particular, promotes the development of early follicles and the further maturation of secondary follicles to the final ovulatory state.
  • the present invention thus provides methods and media for in vivo treatment of mammalian, including human, ovaries with 4-vinylcyclohexene diepoxide, or for in vitro culture of isolated ovaries or ovarian tissue.
  • the present invention provides a medium for culturing a mammalian ovary or ovarian tissue, the medium comprising 4-vinylcyclohexene diepoxide.
  • the content of 4-vinylcyclohexene diepoxide in the medium is about 0.1 nM-5000 nM, preferably about 1 nM-1000 nM, more preferably about 1 nM-20 nM. In one aspect of the present invention, the content of 4-vinylcyclohexene diepoxide in the medium is less than 1000 nM.
  • the mammal can be any mammal, including and not limited to rodents (such as mice and rats), lagomorphs (rabbits), carnivores (felines and canines), cloven hoofs Orders (bovines and porcines), Odd-hoofs (equines), or primates and simian (humans or monkeys).
  • rodents such as mice and rats
  • lagomorphs such as bobits
  • carnivores felines and canines
  • cloven hoofs Orders bovines and porcines
  • Odd-hoofs equines
  • primates and simian humans or monkeys
  • the ovary or ovarian tissue used for culture may be an intact ovary or a part of an ovary, or an ovarian tissue such as ovarian cortical tissue.
  • ovary or ovarian tissue can be the isolated ovary of animal body or its part or isolated ovarian tissue, also can be the ovary or ovarian tissue resuscitated after cryopreservation, also can be cultured in cell culture fluid or medium.
  • ovarian tissue In the present invention, the ovary or ovarian tissue contains follicles of different developmental stages.
  • the purpose of the culture medium and culture method provided by the present invention is to maintain/promote the activity, developmental potential and ovulation of follicles in the ovary or ovarian tissue of mammals.
  • the content of the 4-vinylcyclohexene diepoxide refers to the working concentration, that is, the concentration in the organ/tissue culture environment.
  • the 4-vinylcyclohexene diepoxide in the medium of the present invention is present in an amount that is a multiple of the working concentration.
  • the components of the medium are provided at their 5-fold or 10-fold working concentration, and are diluted with water/solution/culture medium when used.
  • the culture medium provided by the present invention also contains one or more of the following other compounds: inorganic salts, energy sources, amino acids, proteins, cytokines, chelating agents, antibiotics, hyaluronic acid, growth factors, hormones or vitamins.
  • the inorganic salt may be an inorganic salt that dissociates into inorganic ions in an aqueous solution.
  • the inorganic salt may be an inorganic salt comprising one or more of the following inorganic ions: Na + , K + , Cl ⁇ , Ca 2+ , Mg 2+ , SO 4 2 ⁇ , or PO 4 3 ⁇ .
  • the energy source can be pyruvic acid, lactic acid or glucose and the like.
  • the protein source can be albumin or synthetic serum.
  • Suitable sources for protein supplementation include human serum, human umbilical cord serum (HCS), human serum albumin (HSA), fetal calf serum (FCS) or bovine serum albumin (BSA).
  • the one or more additional compounds may be a buffered solution.
  • Suitable buffer solutions include, for example, HEPES buffer or MOPS buffer.
  • the one or more additional compounds may be the background medium. That is, in the medium provided by the present invention, 4-vinylcyclohexene diepoxide is added to the background medium.
  • Background medium refers to an available medium suitable for culturing an organ or tissue, such as a commercially available minimal medium, simple medium, or supplemented medium.
  • the present invention provides a method for in vitro culture of mammalian ovaries or ovarian tissue, wherein 4-vinylcyclohexene diepoxide is added to the culture medium.
  • the content of 4-vinylcyclohexene diepoxide added therein is about 0.1 nM-5000 nM, preferably about 1-1000 nM, more preferably about 1-20 nM. In one aspect of the present invention, the content of the 4-vinylcyclohexene diepoxide added therein is less than 1000 nM.
  • the culturing time in the medium added with the 4-vinylcyclohexene diepoxide is not more than 6 days, preferably not more than 4 days, most preferably not more than 1 day .
  • the above-described in vitro culture method is used to treat the ovary or ovarian tissue of a juvenile or young mammal.
  • the ovary or ovarian tissue is cultured in the medium to which the 4-vinylcyclohexene diepoxide is added for no more than 4 days, most preferably no more than 1 day.
  • the above-mentioned in vitro culture method is used to treat the ovary or ovarian tissue of a middle-aged or aged mammal.
  • the ovary or ovarian tissue is cultured in the medium to which the 4-vinylcyclohexene diepoxide is added for no more than 6 days, most preferably no more than 4 days.
  • "juvenile”, “young”, “middle age”, and “old age” of a mammal are defined from the perspective of the life cycle of the mammal.
  • the life cycle is closely linked to the reproductive cycle of mammals.
  • "Juvenile” and “young” generally refer to before, during normal reproductive age (especially early normal reproductive age) of the mammal in question.
  • "Middle age” and “old age” generally refer to the late stage of or after the normal reproductive age of the mammal.
  • the present invention provides a method of improving ovarian function in a mammal, wherein 4-vinylcyclohexene diepoxide is administered to the ovary of the mammal.
  • about 5-200 mg/kg body weight, preferably about 10-160 mg/kg body weight, more preferably about 20-100 mg/kg body weight of 4-vinylcyclohexane is administered to the ovary of the mammal alkene diepoxide.
  • the above method promotes follicular maturation in the mammal, including but not limited to promoting primordial follicle activation, primordial to primary transition or primary to secondary transition.
  • the above method promotes ovulation, ie increases the number of mature egg cells excreted, in said mammal.
  • the above method wherein 4-vinylcyclohexene diepoxide is administered to the mammal at intervals.
  • the 4-vinylcyclohexene diepoxide is administered at intervals of about 12-48 hours, preferably at intervals of about 20-24 hours.
  • 4-vinylcyclohexene diepoxide is administered to the mammal for a short period of time in the above method.
  • the 4-vinylcyclohexene diepoxide is administered for no more than 10 days, preferably no more than 5 days, and most preferably no more than 3 days.
  • the method of the present invention is suitable for the action of 4-vinylcyclohexene diepoxide on the ovary.
  • the above-described method is used for intraperitoneal administration of 4-vinylcyclohexene diepoxide.
  • the above method is used to treat patients whose ovaries have been removed clinically, for example, patients whose ovaries are partially or completely removed for ex vivo preservation due to the need for radiotherapy.
  • the above method is used for the treatment of ovarian-related reproductive disorders in mammals, especially humans, in particular reproductive disorders due to a decrease in the number or activity of follicles in the ovary, such as premature ovarian failure (POI) or ovarian reserve Decreased function (DOR), etc.
  • POI premature ovarian failure
  • DOR ovarian reserve Decreased function
  • the above method is used in the field of human assisted reproduction.
  • the use of 4-vinylcyclohexene diepoxide in the preparation of a medicament for improving ovarian function in mammals is provided.
  • the 4-vinylcyclohexene diepoxide is formulated at a dose of about 5-200 mg/kg body weight, preferably 10-160 mg/kg body weight, more preferably 20-100 mg/kg body weight administered to the mammal.
  • the medicament in the above use, is formulated for administration of 4-vinyl ring at a dose of about 5-200 mg/kg body weight, preferably 10-160 mg/kg body weight, more preferably 20-100 mg/kg body weight
  • the dosage form of hexene diepoxide is formulated for administration of 4-vinyl ring at a dose of about 5-200 mg/kg body weight, preferably 10-160 mg/kg body weight, more preferably 20-100 mg/kg body weight.
  • the medicament in the above-mentioned use, is in a dosage form for multiple administration.
  • the medicament is formulated for administration at intervals of about 12-48 hours, preferably at intervals of about 20-24 hours.
  • the medicament in the above use, is a medicament for short-term administration.
  • the medicament is administered for no more than 10 days, preferably no more than 5 days, for 4-vinylcyclohexene diepoxide.
  • the medicament is for the treatment of ovarian-related reproductive disorders in mammals, such as premature ovarian failure (POI) or diminished ovarian reserve (DOR).
  • POI premature ovarian failure
  • DOR diminished ovarian reserve
  • the medicament is in a dosage form for intraperitoneal administration.
  • VCD 4-vinylcyclohexene diepoxide
  • the maintenance or even enhancement of reproductive potential is due to low concentrations and/or short durations of 4-vinylcyclohexene diepoxide treatment Hormone-like effect on ovary or cultured ovarian tissue, can transiently activate PI3K-akt and mTOR pathways; in addition, low-concentration and/or short-term 4-vinylcyclohexene diepoxide treatment also increases ovarian granules Expression of cellular aromatase promotes estrogen production.
  • the present invention thus provides methods and media for in vivo treatment of mammalian, including human, ovaries with 4-vinylcyclohexene diepoxide, or for in vitro culture of isolated ovaries or ovarian tissue.
  • Figure 1 shows that 4-vinylcyclohexene diepoxide (VCD) culture promotes primordial follicle activation in vitro.
  • Figure 1A shows staining of ovarian sections from different treatment groups.
  • Figure 1B shows the count results of follicles at all levels in ovarian tissue of different treatment groups.
  • Figure 1C Western experiment observed changes in protein phosphorylation levels related to follicle activation in the ovary.
  • Figure 2 shows that short-term VCD culture promotes the transition of primordial follicles to primary follicles in vitro.
  • Figure 2A shows staining of ovarian sections for different treatment groups.
  • Figure 2B shows the count results of secondary follicles in ovarian tissue of different treatment groups.
  • Figure 3 shows that VCD promotes follicular development in neonatal mice in vivo.
  • Figure 3A shows staining of ovarian sections for different treatment groups.
  • Figure 3B shows the count results of follicles in ovarian tissue of different treatment groups.
  • Figure 4 shows that VCD promotes follicular development and more mature egg release in aged mice in vivo.
  • Figure 4A shows staining of ovarian sections for different treatment groups.
  • Figure 4B shows the count results of different follicles in ovarian tissue of different treatment groups.
  • Figure 4C shows the MII eggs obtained by ovulation (the arrows indicate the MII eggs).
  • Figure 4D shows the results of MII egg counts in the experimental group and the control group.
  • Figure 5 shows that VCD promotes the expression of aromatase and estrogen production in ovarian granulosa cells.
  • Figures 5A-B show that the expression level of aromatase in mouse granulosa cells was significantly increased after VCD treatment.
  • Figure 5C-D showed that the expression level of aromatase in KGN was significantly increased after VCD treatment.
  • VCD 4-vinylcyclohexene diepoxide
  • the 2-day (PD2) or 12 (PD12)-day-old C57bl/6 female mice were sacrificed by decapitation, and then the ovaries and surrounding tissues were dissected and separated under a dissecting microscope.
  • Periovarian adipose tissue was dissected in Leibovitz's L-15 medium (containing 10% fetal bovine serum + 0.5% penicillin + 0.5% streptomycin). The isolated ovaries were washed three times in PBS.
  • ovarian medium add 1mg/ml bovine serum albumin (BSA, Cat#A4161-1G, Sigma) to DMEM/F12 (Cat#11039-021, Gibco) medium, 1mg/ml AbluMAX II Lipid-Rich BSA (Cat#11021029, Gibco), 5% Insulin-Transferrin-Selenium (ITS-G) (Cat#41400-045, Gibco), 0.5% Penicillin + 0.5% Streptomycin and 100 nM VCD. After mixing the prepared medium, add it to the six-well plate, and add 1.5 ml of medium to each empty space. A mesh (Cat#PICMORG50, Millipore) was added to the medium and placed in an incubator to preheat.
  • BSA bovine serum albumin
  • ITS-G Insulin-Transferrin-Selenium
  • mice in the same litter were divided into experimental group and control group according to their body weight. After weighing the mice in the experimental group, they were given 20 mg/kg or 160 mg/kg of VCD by intraperitoneal injection, and the mice in the control group were given the same volume of saline. The mice were sacrificed after ten consecutive days of administration, and the ovaries were dissected and the surrounding excess adipose tissue was isolated under a microscope and fixed in 4% PFA. Dehydrated sections were used to observe and count the development of ovarian follicles in different periods.
  • mice 10-12 months old C57bl/6 mice were divided into experimental group and control group according to their body weight.
  • the experimental group was weighed and injected with 80 mg/kg of VCD, and the control group was given the same volume of normal saline. After continuous administration for 5 days, the experimental group and the control group were injected with 5 units of pregnant horse serum gonadotropin (PMSG) respectively, and 10 IU of hCG was injected 48 hours later to induce secondary egg excretion.
  • the mice were sacrificed 16 hours after the injection of hCG, and both fallopian tubes were collected and placed in M2 dissection solution (Cat#M7167, Sigma).
  • the cumulus granulosa cell complexes on both sides of the mouse were transferred to a drop of hyaluronidase (Cat#90101, Fujifilm Irvine Scientific), and after the granulosa cells around the eggs were digested, the number of eggs in each mouse was counted under a microscope .
  • mice After PD2 mice were sacrificed, they were dissected in dissection solution (Leibovitz's L-15 medium, Cat#41400-045, Gibco; +10% fetal bovine serum (FBS)), washed twice in ovarian medium and then placed in ovarian culture. cultured in the base. After 3 minutes of incubation in medium with or without the addition of VCD, ovarian tissue was harvested for lysis digestion in RIPA lysis buffer (Cat#89900, Thermo). Proteins were denatured and separated by SDS PAGE and transferred to PVDF membranes (Millipore). Membranes were incubated with primary antibodies followed by HRP-conjugated secondary antibodies and bands were examined using an enhanced chemiluminescence detection kit (Bio-Rad).
  • dissection solution Leibovitz's L-15 medium, Cat#41400-045, Gibco; +10% fetal bovine serum (FBS)
  • FBS +10% fetal bovine serum
  • anti-rabbit aromatase (Cat#NBP1-45360, Novus, 1:2000), anti-rabbit phosphor-AKT (Serine 473) (D9E) (Cat#4060, Cell Signaling, 1:500), anti-rabbit phosphor-FoxO1 (Threonine 24)/FoxO3a(Threonine 32)(Cat#9464,Cell Signaling,1:500),anti-rabbit phosphor-P-S6(Serine 235/236)(Cat#4858,Cell Signaling,1:1000), anti-rabbit phosphor-P-S6(Serine 240/244)(Cat#2215,Cell Signaling,1:1000),anti-rabbit phosphor-P70S6k(Threonine 389)(Cat#9205,Cell Signaling,1:1000), anti-rabbit(Cat#7074S,CST)or anti-mouse(Cat#7076S,CST).
  • E2 detection kit Human Estradiol (E2) ELISA Kit, Cat#KAQ0621, Invitrogen.
  • two-day-old (PD2) mouse ovaries were isolated and cultured in vitro.
  • VCD 4-vinylcyclohexene diepoxide
  • FSH 50 ng/ml follicle-stimulating hormone
  • FIG. 1A the arrows indicate primary follicles.
  • Figure 1B shows the count results of follicles at all levels in ovarian tissue of different treatment groups. The results demonstrated that the number of primordial follicles and early primary follicles was significantly increased in the ovaries treated with VCD compared to the control group.
  • Figure 1C shows that the ovaries of PD2 mice were treated with 10 nM VCD in vitro to collect tissue proteins for 3 minutes, and the changes in protein phosphorylation levels related to follicle activation in the ovaries were observed by Western assay. The results showed that VCD treatment for 2-3 minutes could significantly activate the PI3K-Akt pathway, and the expression of the involved proteins was significantly increased. It can be seen that low-concentration VCD treatment promoted the activation of primordial follicles.
  • Example 3 Short-term VCD culture promotes the transformation of primordial follicles to primary follicles in vitro
  • Example 4 VCD promotes follicle development in neonatal mice in vivo experiments
  • the mice were sacrificed.
  • the collected ovarian sections were stained to observe the morphology.
  • the results are shown in Figure 3A (the yellow arrows indicate early antral follicles, and the red arrows indicate antral follicles).
  • Figure 3B shows the results of counting follicles in ovarian tissue of different treatment groups after continuous administration for 5 days. The results showed that compared with the control group, there were more follicles close to pre-ovulation in the ovaries of the mice in the administration group.
  • VCD treatment can promote the transition of primary and secondary follicles to mature eggs.
  • Example 5 VCD promotes follicle development and more mature egg release in aged mice in vivo experiments
  • mice Using 10-12 months old mice as the research object, the effect of VCD on the development of ovarian follicles and the final egg release was observed.
  • mice 10-12 month old C57bl/6 mice were divided into experimental group and control group according to their body weight.
  • the experimental group and the control group were injected with 5 units of PMSG respectively, and 48 hours later, 10 IU of hCG was injected to promote the secondary egg excretion.
  • Mice were sacrificed 16 hours after hCG injection, and both fallopian tubes were removed and MII eggs in the fallopian tubes were collected.
  • FIG. 4A the arrows indicate preovulatory follicles, and CL indicates the corpus luteum.
  • Figure 4B shows the count results of different follicles in ovarian tissue of different treatment groups. The follicle count results showed that there were more antral follicles and newly formed corpus luteum in the ovaries of the mice in the VCD group.
  • Figure 4C shows the MII eggs obtained by ovulation (the arrows indicate the MII eggs).
  • Figure 4D shows the results of MII egg counts in the experimental group and the control group. The results of ovulation induction showed that the mice in the VCD group could induce more eggs after 5 days of administration. It can be seen that VCD can improve the ovarian function of aged mice and further promote egg excretion.
  • Example 6 VCD promotes the expression of ovarian granulosa cells aromatase and the production of estrogen
  • the primary granulosa cells of PD20 mice were extracted, and the effect of VCD treatment on the expression of aromatase in granulosa cells was observed.
  • VCD has the effect of promoting the expression of granulosa cell aromatase and its downstream estrogen production.
  • the present invention discovers and proves for the first time that low concentration and/or short time 4-vinylcyclohexene diepoxide (VCD) treatment is beneficial to the maintenance or even enhancement of the development and reproductive potential of female mammalian ovary or ovarian tissue, In particular, it can promote the development of early follicles and the further maturation of secondary follicles to reach the final ovulation state.
  • VCD 4-vinylcyclohexene diepoxide
  • the present inventors believe that part of this effect is due to the hormone-like effect of low-concentration and/or short-duration 4-vinylcyclohexene diepoxide treatment on the ovary or cultured ovarian tissue, which can transiently activate PI3K-Akt and mTOR pathway; in addition, low-concentration and/or short-duration 4-vinylcyclohexene diepoxide treatment also promoted estrogen production by increasing ovarian granulosa cell aromatase expression.
  • the present invention thus provides methods and media for in vivo treatment of mammalian, including human, ovaries with 4-vinylcyclohexene diepoxide, or for in vitro culture of isolated ovaries or ovarian tissue.

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Abstract

4-乙烯基环己烯二环氧对哺乳动物包括人类的卵巢进行在体处理，或是对离体卵巢或卵巢组织进行体外培养的方法和培养基。所提供的方法或培养基有利于哺乳动物的卵巢或卵巢组织的发育和生殖潜力的保持和增强，特别是能够促进卵泡成熟和增加排卵数目。

Description

小分子化合物用于激活卵泡的方法及其制剂
本申请要求2020年7月10日提交的、申请号为202010662524.2、发明名称为“小分子化合物用于激活卵泡的方法及其制剂”的中国专利申请的优先权，其全部内容通过引用结合在本申请中。
技术领域
本发明涉及哺乳动物包括人的生殖领域。具体的，本发明涉及改进哺乳动物包括人的卵巢或卵巢组织发育能力的方法，以及对卵巢或卵巢组织的体外培育方法和培养基。
背景技术
雌性哺乳动物的卵巢含有的卵泡的数量在其出生之时即已被决定，随着年龄的增长，会有很大一部分卵泡在卵巢发育过程中走向凋亡闭锁，只有为数不多的卵泡最终经历原始卵泡-初级卵泡-次级卵泡-排卵前卵泡-排卵这个过程最终成熟并排出。随着哺乳动物年龄的增大，功能性卵泡储备(functional ovarian reserve，FOR)跟成熟能力下降，在某些情况下，即便有初级卵泡或者小的次级卵泡存在，也很难继续生长至成熟。同时，随着年龄的增加，机体对促卵泡生成素(Follicle-stimulating hormone,FSH)等内源的或者外源的激素的反应能力也随之下降。
卵子成熟在人的生殖，包括辅助生殖领域至关重要，在畜牧业的发展中 也有重要作用。在人类女性中，有生育要求的高龄妇女卵巢储备减少，不足以成功妊娠。另外，部分需要借助辅助生殖来满足生育要求的患者很难从卵巢取到足够的高质量的卵子。在临床上，某些患者，例如需要做放射性治疗的患者会将部分卵巢组织移除和保存，用作未来可能的生殖用途。在畜牧业中，具有优良基因的牲畜的基因的延续尤其重要，即使在其老年和生育力下降的阶段，依然存在繁殖后代的需要。在这些情况中，对卵巢的保护以及对其卵泡发育潜力的维持和促进都极其重要。
4-乙烯基环己烯二环氧(4-vinylcyclohexene diepoxide，VCD)是一种工业原料，作为中间体用于制造阻燃剂，调味剂，农药，粘合剂，香料和合成橡胶等。已经被发现对哺乳动物卵巢内卵泡发育构成威胁。已有的研究一致表明，在大剂量和长时间暴露下，VCD诱发了啮齿动物原始卵泡和初级卵泡的闭锁性退化。已经发现VCD诱导的卵毒性与Bcl-2促凋亡信号通路以及KIT/KITL信号通路相关，以及与Nrf2介导的氧化应激反应途径有关。在研究领域，4-乙烯基环己烯二环氧(VCD)被用作化学诱导的卵巢早衰(premature ovarian insufficiency，POI)动物模型中使用的诱导物。卵巢早衰是女性不育的常见原因之一；在该动物模型中，VCD耗尽了功能性卵泡储备，研究者将这种动物模型用于生殖系统疾病的病因的研究。
在哺乳动物包括人的辅助生殖领域，还需要对卵巢的发育有更多的研究，并且需要有利于卵巢的发育，特别是保持和改善卵巢中胚泡转化为卵母细胞的潜力和能力的方法，以及可用于体外培养卵巢或卵巢组织的培养基。
发明内容
本发明首次发现和证明了低浓度和/或短时间的4-乙烯基环己烯二环氧(VCD)处理对卵巢或培养的卵巢组织具有类激素的作用，有利于雌性哺乳动物的卵巢的发育和生殖潜力的保持甚至是加强，特别是能够促进早期卵泡的发育以及次级卵泡的进一步成熟并达到最终排卵状态。本发明由此提供了用4-乙烯基环己烯二环氧对哺乳动物包括人类的卵巢进行在体处理，或是对离体卵巢或卵巢组织进行体外培养的方法和培养基。
具体的，本发明提供了一种用于培养哺乳动物的卵巢或卵巢组织的培养基，所述培养基中包含4-乙烯基环己烯二环氧。在本发明的其中一个方面，其中所述培养基中的4-乙烯基环己烯二环氧的含量为约0.1nM-5000nM，优选为约1nM-1000nM，更优选为约1nM-20nM。在本发明的其中一个方面，所述培养基中的4-乙烯基环己烯二环氧的含量为小于1000nM。
4-乙烯基环己烯二环氧(4-vinylcyclohexene diepoxide，VCD)，其具有如下结构式：
Figure PCTCN2021105248-appb-000001
在本发明中，所述哺乳动物可以为任何哺乳动物，包括和不限于啮齿目(如小鼠和大鼠)，兔形目(兔子)、食肉目(猫科动物和犬科动物)、偶蹄目(牛科动物和猪科动物)、奇蹄目(马科动物)，或为灵长目和猿猴亚目(人或猴)。所述哺乳动物优选为人或小鼠。
在本发明中，用于培养的卵巢或卵巢组织可为完整的卵巢或卵巢的部分，或为卵巢的组织，例如卵巢皮质组织。所述卵巢或卵巢组织可以为动物体的离体卵巢或其部分或离体的卵巢组织，也可以为冷冻保存后复苏的卵巢或卵巢组织，也可为在细胞培养液或培养基中培养的卵巢组织。在本发明中，所述卵巢或卵巢组织含有不同发育阶段的卵泡。本发明提供的培养基和培养方法的目的在于维持/促进哺乳动物的卵巢或卵巢组织中的卵泡的活性、发育潜力以及排卵。
在本发明的其中一个方面，所述4-乙烯基环己烯二环氧的含量是指工作浓度，即在器官/组织培养环境中的浓度。在有些情况下，本发明的培养基中的4-乙烯基环己烯二环氧以工作浓度倍数的含量存在。例如，为了便于储存或操作，培养基的组分以其5倍或10倍工作浓度提供，在使用时加入水/溶液/培养液进行稀释使用。
本发明提供的培养基还含有以下的一种或多种其它化合物：无机盐、能量源、氨基酸、蛋白质、细胞因子、螯合剂、抗生素、透明质酸、生长因子、激素或维生素。
其中，无机盐可以是在水溶液中解离成无机离子的无机盐。合适地，无机盐可以是包含一种或多种以下无机离子的无机盐：Na +、K +、Cl -、Ca 2+、Mg 2+、SO 4 2-、或PO 4 3-。
其中，能量源可以是丙酮酸、乳酸或葡萄糖等。
其中，蛋白质来源可以是白蛋白或合成血清。用于蛋白质补充的合适的来源包括人血清、人脐带血清(HCS)、人血清白蛋白(HSA)、胎牛血清(FCS)或牛血清白蛋白(BSA)。
在本发明的其中一个方面，一种或多种另外的化合物可以是缓冲溶液。合适的缓冲溶液例如包括HEPES缓冲液或MOPS缓冲液。
在本发明的其中一个方面，一种或多种另外的化合物可以是背景培养基。即本发明提供的培养基是在背景培养基中增加4-乙烯基环己烯二环氧。背景培养基是指可用的适合于培养器官或组织的培养基，例如商业上提供的基本培养基、简单培养基或补充培养基。
在本发明的另一个方面，本发明提供了哺乳动物的卵巢或卵巢组织的体外培养方法，其中在培养基中加入4-乙烯基环己烯二环氧。在本发明的其中又一个方面，其中加入的4-乙烯基环己烯二环氧的含量为约0.1nM-5000nM，优选为约1-1000nM，更优选为约1-20nM。在本发明的其中一个方面，其中加入的4-乙烯基环己烯二环氧的含量小于1000nM。
在本发明的其中一个方面，上述体外培养方法中在加入所述4-乙烯基环己烯二环氧的培养基培养的时间不超过6天，优选不超过4天，最优选不超过1天。
在本发明的其中一个方面，上述体外培养方法用于处理幼年或年轻哺乳动物的卵巢或卵巢组织。在本发明的其中又一个方面，所述卵巢或卵巢组织在加入所述4-乙烯基环己烯二环氧的培养基培养的时间不超过4天，最优选不超过1天。
在本发明的其中一个方面，上述体外培养方法用于处理中年或老年哺乳动物的卵巢或卵巢组织。在本发明的其中又一个方面，所述卵巢或卵巢组织在加入所述4-乙烯基环己烯二环氧的培养基培养的时间不超过6天，最优选不超过4天。
在本发明中，哺乳动物的“幼年”、“年轻”、“中年”、“老年”是从该哺乳动物的生命周期的角度定义。在本发明的其中一个方面，所述生命周期与哺乳动物的生殖周期密切联系。“幼年”、“年轻”通常是指所述哺乳动物的正常育龄之前，正常育龄期间(尤其是正常育龄早期)。“中年”、“老年”通常是指所述哺乳动物的正常育龄的晚期或正常育龄之后。
在本发明的另一方面，本发明提供了改善哺乳动物的卵巢功能的方法，其中对所述哺乳动物的卵巢给予4-乙烯基环己烯二环氧。在本发明的其中又一个方面，对所述哺乳动物的卵巢给予约5-200mg/kg体重，优选约10-160mg/kg体重，更优选约20-100mg/kg体重的4-乙烯基环己烯二环氧。
在本发明的其中一个方面，上述方法促进所述哺乳动物的卵泡成熟，包括而不限于促进原始卵泡的激活、促进原始卵泡向初级卵泡转变或促进初级卵泡向次级卵泡转变。
在本发明的其中一个方面，上述方法促进所述哺乳动物的排卵，即增加成熟卵细胞排出的数目。
在本发明的其中一个方面，上述方法中4-乙烯基环己烯二环氧为间隔给药与所述哺乳动物。在其中一种实施方式中，4-乙烯基环己烯二环氧以约12-48小时一次的间隔给药，优选为以约20-24小时一次的间隔给药。
在本发明的其中一个方面，上述方法中将4-乙烯基环己烯二环氧短期给予所述哺乳动物。在其中一种实施方式中，给予4-乙烯基环己烯二环氧的时间不超过10天，优选不超过5天，最优选不超过3天。
本发明的方法适合将4-乙烯基环己烯二环氧在卵巢上起作用。在本发明的其中一个方面，上述方法中4-乙烯基环己烯二环氧通过腹腔给药。
在本发明的其中一个方面，上述方法用于处理临床上卵巢被摘除的患者，例如因需要做放射性治疗而预先将部分或完整卵巢摘除做离体保存的患者。
在本发明的其中一个方面，上述方法用于治疗哺乳动物特别是人的卵巢相关生殖障碍，特别是由于卵巢中的卵泡数量减少或活性衰退造成的生殖障碍，例如卵巢早衰(POI)或卵巢储备功能减退(DOR)等。在本发明的其中又一个方面，上述方法用于人的辅助生殖领域。
在本发明的其中一个方面，提供了4-乙烯基环己烯二环氧在用于制备改善哺乳动物的卵巢功能的药物中的用途。在本发明的其中又一个方面，所述4-乙烯基环己烯二环氧配置为以约5-200mg/kg体重，优选10-160mg/kg体重，更优选20-100mg/kg体重的剂量给予所述哺乳动物。
在本发明的其中一个方面，上述用途中，所述药物配制为以约5-200mg/kg体重，优选10-160mg/kg体重，更优选20-100mg/kg体重的剂量给予4-乙烯基环己烯二环氧的剂型。
在本发明的其中一个方面，上述用途中，所述药物为多次给药的剂型。在其中一种实施方式中，所述药物配制为以约12-48小时一次的间隔给药的剂型，优选为以约20-24小时一次的间隔给药的剂型。
在本发明的其中一个方面，上述用途中，所述药物为短期给药的药物。在其中一种实施方式中，所述药物给予4-乙烯基环己烯二环氧的时间不超过10天，优选不超过5天。
在本发明的其中一个方面，所述药物用于治疗哺乳动物的卵巢相关生殖障碍，例如卵巢早衰(POI)或卵巢储备功能减退(DOR)。
在本发明的其中又一个方面，其中所述药物为腹腔给药的剂型。
不受理论限制的，申请人认为本发明的发现，即低浓度和/或短时间的4-乙烯基环己烯二环氧(VCD)处理有利于雌性哺乳动物的卵巢或卵巢组织的发育和生殖潜力的保持甚至是加强，特别是能够促进早期卵泡的发育以及次级卵泡的进一步成熟并达到最终排卵状态，是由于低浓度和/或短时间的4-乙烯基环己烯二环氧处理对卵巢或培养的卵巢组织具有类激素的作用，能瞬时地激活PI3K-akt和mTOR通路；另外，低浓度和/或短时间的4-乙烯基环己烯二环氧处理还通过增加卵巢颗粒细胞芳香化酶的表达促进了雌激素的生成。本发明由此提供了用4-乙烯基环己烯二环氧对哺乳动物包括人类的卵巢进行在体处理，或是对离体卵巢或卵巢组织进行体外培养的方法和培养基。
在本文中，在提供数值范围的情况下，应该理解，除非上下文清楚地另外指明，否则在所述范围内的任何规定值或中间值与该规定范围内的任何其他规定值或中间值之间的每个较小范围包含在本公开内。本文中的“约”表示其描述的数值包括了在其领域技术人员理解的范围内的正常浮动。一般而言，“约”表示±10％。在某些情况下，“约”表示±5％。在某些特定情况下，“约”表示±1％。
附图说明
图1显示4-乙烯基环己烯二环氧(VCD)培养在体外实验中促进原始卵泡的激活。图1A显示不同处理组的卵巢切片染色。图1B显示不同处理组的卵巢组织中各级卵泡的计数结果。图1C Western实验观察卵巢中与卵 泡激活相关的蛋白磷酸化水平变化情况。
图2显示短时间VCD培养在体外实验中促进原始卵泡向初级卵泡转变。图2A显示不同处理组的卵巢切片染色。图2B显示不同处理组的卵巢组织中次级卵泡的计数结果。
图3显示VCD在新生小鼠体内实验中促进卵泡发育。图3A显示不同处理组的卵巢切片染色。图3B显示不同处理组的卵巢组织中卵泡的计数结果。
图4显示VCD在老年小鼠体内实验中促进卵泡发育以及更多成熟卵子排出。图4A显示不同处理组的卵巢切片染色。图4B显示不同处理组的卵巢组织中不同卵泡的计数结果。图4C显示促排得到的MII卵子(箭头所指为MII卵子)。图4D显示实验组与对照组的MII卵子计数结果。
图5显示VCD促进卵巢颗粒细胞芳香化酶的表达以及雌激素的生成。图5A-B显示VCD处理后小鼠颗粒细胞中芳香化酶的表达水平明显升高。图5C-D显示VCD处理后KGN中芳香化酶的表达水平明显升高。
具体实施方式
下面将结合实施例进一步说明本发明的实质内容和有益效果，该实施例仅用于说明本发明而非对本发明的限制。
实施例1实验方法和试剂
4-乙烯基环己烯二环氧(4-vinylcyclohexene diepoxide，VCD)购自Sigma Aldrich(Cat#94956-100ML)。在体外培养实验中，VCD溶于PBS缓冲液(Cat#10010-0323,Gibco)。在腹腔注射实验，VCD溶于生理盐水。
新生鼠卵巢体外培养
小鼠卵巢分离
新生2天(PD2)或者12(PD12)天的C57bl/6小雌鼠(山东大学实验动物中心提供)采取断头的方式处死后在解剖显微镜下解剖分离卵巢以及周围组织，切下的卵巢置于Leibovitz's L-15培养基中(包含10％胎牛血清+0.5％青霉素+0.5％链霉素)，解剖分离卵巢周围脂肪组织。分离后的卵巢在PBS中清洗三遍。
小鼠卵巢培养
配置卵巢培养基：在DMEM/F12(Cat#11039-021,Gibco)培养基中加入1mg/ml牛血清白蛋白(BSA,Cat#A4161-1G,Sigma)，1mg/ml AbluMAX II Lipid-Rich BSA(Cat#11021029,Gibco)，5％Insulin-Transferrin-Selenium(ITS-G)(Cat#41400-045,Gibco)，0.5％青霉素+0.5％链霉素以及100nM VCD。将配置好的培养基混匀后加入六孔板中，每空加入1.5ml培养基。培养基上加入筛网(Cat#PICMORG50,Millipore)后放入培养箱中预热。
将分离好的卵巢用移液器转移到卵巢培养基中，清洗三遍后置于预热的培养基的上层筛网上，在筛上层的卵巢周围加一滴培养基，保持卵巢周围湿润。将六孔板置于37℃，5％二氧化碳培养箱中连续培养4天，每天更换下层培养基。对于PD2的小鼠卵巢，仅在第一天培养时培养基中加入VCD。对于PD12的小鼠卵巢，连续暴露在VCD中四天。
新生鼠体内注射给药，观察卵巢变化
同一窝PD12 C57bl/6小雌鼠按照体重分为实验组与对照组，实验组小鼠称重后，经腹腔注射给与20mg/kg或160mg/kg的VCD，对照组小鼠给与 相同体积的生理盐水。连续给药十天后处死老鼠，解剖卵巢在显微镜下分离周围多余的脂肪组织，固定于4％PFA中。脱水切片观察并计数不同时期卵巢内卵泡的发育情况。
老年鼠促排卵实验
10-12月龄C57bl/6老鼠按照体重分为实验组与对照组，实验组称重后腹腔注射80mg/kg的VCD,对照组给与相同体积的生理盐水。连续给药5天后，实验组对照组分别注射5单位孕马血清促性腺激素(PMSG)，48小时后注射10IU的hCG来促使次级卵子排出。注射hCG后16小时处死小鼠，收集两侧输卵管置于M2解剖液(Cat#M7167,Sigma)中，解剖显微镜下挑开膨大的输卵管壶腹部可见卵丘颗粒细胞复合物流出，将每只小鼠的两侧卵丘颗粒细胞复合物转移至透明质酸酶(Cat#90101,Fujifilm Irvine Scientific)液滴中，待卵子周围的颗粒细胞被消化掉以后置于显微镜下计数每只老鼠的卵子数目。
Western blot实验观察卵巢中与卵泡激活相关的蛋白磷酸化
PD2小鼠处死后，于解剖液(Leibovitz's L-15 medium,Cat#41400-045,Gibco；+10％胎牛血清(FBS))中解剖，在卵巢培养基中清洗两遍以后置于卵巢培养基中培养。在加入或者不加入VCD的培养基中培养3分钟后，收集卵巢组织于RIPA裂解液(Cat#89900,Thermo)中裂解消化。蛋白变性后通过SDS PAGE分离并转移至PVDF膜(Millipore)。将膜与一抗孵育，然后与HRP缀合的二抗一起孵育，并使用增强化学发光检测试剂盒(Bio-Rad)检查条带。
抗体信息：
anti-rabbit aromatase(Cat#NBP1-45360,Novus,1:2000),anti-rabbit phosphor-AKT(Serine 473)(D9E)(Cat#4060,Cell Signaling,1:500),anti-rabbit phosphor-FoxO1(Threonine 24)/FoxO3a(Threonine 32)(Cat#9464,Cell Signaling,1:500),anti-rabbit phosphor-P-S6(Serine 235/236)(Cat#4858,Cell Signaling,1:1000),anti-rabbit phosphor-P-S6(Serine 240/244)(Cat#2215,Cell Signaling,1:1000),anti-rabbit phosphor-P70S6k(Threonine 389)(Cat#9205,Cell Signaling,1:1000),anti-rabbit(Cat#7074S,CST)or anti-mouse(Cat#7076S,CST)。
细胞培养上清中雌二醇(E2)水平的检测(ELISA)
KGN细胞培养24小时后收集细胞上清用于检测E2水平，具体检测方法流程参照E2检测试剂盒(Human Estradiol(E2)ELISA Kit，Cat#KAQ0621,Invitrogen)说明书。
统计分析
数据表示为来自至少三次独立实验的平均值±标准偏差。统计比较采用单因素方差分析，统计学差异设为P<0.05。
实施例2短时间VCD培养在体外实验中促进原始卵泡的激活
实验流程和结果如图1所示。
根据实施例1描述的方法，分离得到出生两天(PD2)的小鼠卵巢，在体外进行培养。培养第一天在培养基中加入不同浓度的4-乙烯基环己烯二环氧(VCD)(0,1nM,10nM,100nM,1000nM),从培养第二天开始换为普通培养基继续培养3天，然后收集卵巢；对照组于培养第一天在培养基中加 入50ng/ml促卵泡生成素FSH(Sigma#F4021-2UG)；其中每组N＝9。收集的卵巢切片后染色观察形态，结果如图1A所示(箭头所指为初级卵泡)。图1B显示不同处理组的卵巢组织中各级卵泡的计数结果。结果证明，与对照组比较，用VCD处理的卵巢中原始卵泡和早期初级卵泡数目明显增加。图1C显示PD2小鼠的卵巢在体外用10nM VCD处理3分钟后收集组织蛋白，通过Western实验观察卵巢中与卵泡激活相关的蛋白磷酸化水平变化情况。结果显示，VCD处理2-3分钟可以明显激活PI3K-Akt通路，其中涉及的蛋白的表达明显升高。可见，低浓度VCD处理促进了原始卵泡的激活。
实施例3短时间VCD培养在体外实验中促进原始卵泡向初级卵泡转变
根据实施例1描述的方法，分离得到出生12天(PD12)的小鼠卵巢，在体外进行培养。在培养基中加入不同浓度的4-乙烯基环己烯二环氧(VCD)(0,1nM,10nM,100nM,1000nM)，培养4天后收集卵巢；对照组在培养基中加入50ng/ml促卵泡生成素FSH(Sigma#F4021-2UG)；其中每组N＝9。收集的卵巢切片后染色观察形态，结果如图2A所示(箭头所指为正常次级卵泡)。图2B显示不同处理组的卵巢组织中次级卵泡的计数结果。结果证明，与对照组比较，用VCD处理的卵巢中次级卵泡数目明显增加。
实施例4 VCD在新生小鼠体内实验中促进卵泡发育
根据实施例1描述的方法，对同一窝PD12 C57bl/6小雌鼠按照体重分为实验组与对照组，经腹腔注射给与20mg/kg或160mg/kg的VCD，对照组 小鼠给与相同体积的生理盐水。其中每组N＝6。连续5天给药，然后处死老鼠，收集的卵巢切片后染色观察形态，结果如图3A所示(黄色箭头所指为早期窦卵泡，红色箭头所指为窦卵泡)。图3B显示连续给药5天后不同处理组的卵巢组织中卵泡的计数结果。结果证明，与对照组比较，给药组小鼠卵巢中有更多接近排卵前的卵泡。
与实施例3观察的现象一起，可以得出结论，VCD处理可以促进初级卵泡和次级卵泡向成熟卵子的转变。
实施例5 VCD在老年小鼠体内实验中促进卵泡发育以及更多成熟卵子排出
用10-12个月龄的老年鼠作为研究对象，观察VCD对其卵巢内卵泡发育以及最终卵子排出的影响。
根据实施例1描述的方法，10-12月龄C57bl/6老鼠按照体重分为实验组与对照组，实验组称重后腹腔注射80mg/kg的VCD,对照组给与相同体积的生理盐水。其中每组N＝15。连续给药5天后，实验组对照组分别注射5单位PMSG，48小时后注射10IU的hCG来促使次级卵子排出。hCG注射16小时后处死小鼠，取两侧输卵管并收集输卵管内的的MII卵子。收集的卵巢切片后染色观察形态，结果如图4A所示(箭头所指为排卵前卵泡，CL指黄体)。图4B显示不同处理组的卵巢组织中不同卵泡的计数结果。卵泡计数结果表明，给VCD组小鼠卵巢中有更多的窦卵泡以及新形成黄体。图4C显示促排得到的MII卵子(箭头所指为MII卵子)。图4D显示实验组与对照组的MII卵子计数结果。促排卵结果表明，给VCD组小鼠在给 药5天后可以促排出更多的卵子。可见，VCD可以改善老年鼠卵巢功能，并进一步促进卵子排出。
实施例6 VCD促进卵巢颗粒细胞芳香化酶的表达以及雌激素的生成
提取PD20小鼠原代颗粒细胞，并观察VCD处理对颗粒细胞中芳香化酶的表达产生的影响。
根据Reddy P等，Science 2008,319(5863):611-613和Liu YX等Biol Reprod 1986,35(1):27-36描述的方法从PD20小鼠的卵巢中分离颗粒细胞，由此获得原代颗粒细胞系。用不同浓度的VCD(0,1nM,10nM,100nM,1000nM)处理24小时，以50ng/ml FSH为对照。结果显示，VCD处理后小鼠颗粒细胞中芳香化酶的表达水平明显升高(图5A-B)
人来源颗粒细胞样细胞系KGN(日本福冈大学Toshihiko Yanase教授提供)在DMEM/F12培养基中培养。用不同浓度的VCD(0,1nM,10nM,100nM,1000nM)处理24小时，以50ng/ml FSH为对照。结果显示，VCD处理后KGN中芳香化酶的表达水平明显升高(图5C-D)。用ELISA进一步观察KGN培养上清中雌激素(E2)的产生情况，发现VCD处理组细胞上清中雌激素的水平明显高于对照组(图5E)。
以上数据表明VCD具有促进颗粒细胞芳香化酶的表达以及其下游雌激素产生的作用。
本发明首次发现和证明了低浓度和/或短时间的4-乙烯基环己烯二环氧(VCD)处理有利于雌性哺乳动物的卵巢或卵巢组织的发育和生殖潜力 的保持甚至是加强，特别是能够促进早期卵泡的发育以及次级卵泡的进一步成熟并达到最终排卵状态。本发明认为这个效果的部分成因是由于低浓度和/或短时间的4-乙烯基环己烯二环氧处理对卵巢或培养的卵巢组织具有类激素的作用，能瞬时地激活PI3K-Akt和mTOR通路；另外，低浓度和/或短时间的4-乙烯基环己烯二环氧处理还通过增加卵巢颗粒细胞芳香化酶的表达促进了雌激素的生成。本发明由此提供了用4-乙烯基环己烯二环氧对哺乳动物包括人类的卵巢进行在体处理，或是对离体卵巢或卵巢组织进行体外培养的方法和培养基。
上面是对本发明进行的说明，不能将其看成是对本发明进行的限制。除非另外指出，本发明的实践将使用有机化学、聚合物化学、生物技术等的常规技术，显然除在上述说明和实施例中所特别描述之外，还可以别的方式实现本发明。其它在本发明范围内的方面与改进将对本发明所属领域的技术人员显而易见。根据本发明的教导，许多改变和变化是可行的，因此其在本发明的范围之内。

Claims (20)

  1. 一种用于培养哺乳动物的卵巢或卵巢组织的培养基，其包含4-乙烯基环己烯二环氧或其类似物，其中，所述培养基中的4-乙烯基环己烯二环氧的含量为约0.1nM-5000nM，优选为约1nM-1000nM，更优选为约1nM-20nM。
  2. 根据权利要求1所述的培养基，其中所述培养基用于培养离体卵巢或其部分。
  3. 根据权利要求1所述的培养基，其中所述培养基用于培养卵巢组织，例如卵巢皮质组织。
  4. 根据前述权利要求中任一项所述的培养基，其中所述培养基还含有背景培养基，例如基本培养基、简单培养基或补充培养基。
  5. 根据前述权利要求中任一项所述的培养基，其还含有以下的一种或多种其它化合物：无机盐、能量源、氨基酸、蛋白质、细胞因子、螯合剂、抗生素、透明质酸、生长因子、激素或维生素。
  6. 根据前述权利要求中任一项所述的培养基，其中所述哺乳动物为啮齿目(如小鼠和大鼠)，兔形目(兔子)、食肉目(猫科动物和犬科动物)、偶蹄目(牛科动物和猪科动物)、奇蹄目(马科动物)，或为灵长目和猿猴亚目(人或猴)，优选的，所述哺乳动物为人或小鼠。
  7. 哺乳动物的卵巢或卵巢组织的体外培养方法，所述方法包括在培养基中加入4-乙烯基环己烯二环氧或其类似物，其中加入的4-乙烯基环己烯二环氧的含量为约0.1nM-5000nM，优选为约1nM-1000nM，更优选为约 10nM-200nM。
  8. 根据权利要求7所述的体外培养方法，其中所述卵巢或卵巢组织在加入所述4-乙烯基环己烯二环氧的培养基培养的时间不超过6天，优选不超过4天，最优选不超过1天。
  9. 根据权利要求7或8所述的体外培养方法，其用于处理幼年或年轻哺乳动物的卵巢或卵巢组织，优选的，所述卵巢或卵巢组织在加入所述4-乙烯基环己烯二环氧的培养基培养的时间不超过4天，最优选不超过1天。
  10. 根据权利要求7或8所述的体外培养方法，其用于处理中年或老年哺乳动物的卵巢或卵巢组织，优选的，所述卵巢或卵巢组织在加入所述4-乙烯基环己烯二环氧的培养基培养的时间不超过6天，最优选不超过4天。
  11. 改善哺乳动物的卵巢功能的方法，其中给予所述哺乳动物约5-200mg/kg体重，优选约10-160mg/kg体重，更优选约20-100mg/kg体重的4-乙烯基环己烯二环氧。
  12. 根据权利要求11所述的方法，其中所述方法促进卵泡成熟，例如促进原始卵泡的激活、促进原始卵泡向初级卵泡转变或促进初级卵泡向次级卵泡转变。
  13. 根据权利要求11所述的方法，其中所述方法增加成熟卵细胞排出的数目。
  14. 根据权利要求中11-13中任一项所述的方法，其中4-乙烯基环己烯二环氧间隔给药，例如4-乙烯基环己烯二环氧以约12-48小时一次的间隔给药，优选为以约20-24小时一次的间隔给药。
  15. 根据权利要求中11-14中任一项所述的方法，其中将4-乙烯基环己 烯二环氧短期给予所述哺乳动物，例如给予所述4-乙烯基环己烯二环氧的时间不超过10天，优选不超过5天。
  16. 根据权利要求中11-15中任一项所述的方法，其中4-乙烯基环己烯二环氧通过腹腔给药。
  17. 根据权利要求中11-16中任一项所述的方法，其中所述方法用于治疗哺乳动物的卵巢相关生殖障碍，例如卵巢早衰(POI)或卵巢储备功能减退(DOR)。
  18. 4-乙烯基环己烯二环氧在用于制备改善哺乳动物的卵巢功能的药物中的用途。
  19. 权利要求18所述的用途，其中所述4-乙烯基环己烯二环氧配置为以约5-200mg/kg体重，优选10-160mg/kg体重，更优选20-100mg/kg体重的剂量给予所述哺乳动物。
  20. 权利要求18或19所述的用途，其中所述药物为腹腔给药的剂型。
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