WO2014043835A1 - 卵母细胞体外成熟方法 - Google Patents

卵母细胞体外成熟方法 Download PDF

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WO2014043835A1
WO2014043835A1 PCT/CN2012/001633 CN2012001633W WO2014043835A1 WO 2014043835 A1 WO2014043835 A1 WO 2014043835A1 CN 2012001633 W CN2012001633 W CN 2012001633W WO 2014043835 A1 WO2014043835 A1 WO 2014043835A1
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oocytes
oocyte
vitro
maturation
culture medium
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PCT/CN2012/001633
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French (fr)
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田见晖
贾振伟
张家新
安磊
吴中红
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中国农业大学
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Publication of WO2014043835A1 publication Critical patent/WO2014043835A1/zh
Priority to US14/860,814 priority Critical patent/US10011818B2/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0608—Germ cells
    • C12N5/0609—Oocytes, oogonia
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/11—Epidermal growth factor [EGF]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/30—Hormones
    • C12N2501/31—Pituitary sex hormones, e.g. follicle-stimulating hormone [FSH], luteinising hormone [LH]; Chorionic gonadotropins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2517/00—Cells related to new breeds of animals
    • C12N2517/10—Conditioning of cells for in vitro fecondation or nuclear transfer

Definitions

  • the invention relates to a novel method for in vitro maturation of oocytes, in particular to the application of a C-type natriuretic peptide for preparing an in vitro maturation system of an oocyte, which can be used for obtaining an ovary oocyte producing a transgenic clone by using a slaughterhouse. Protection of livestock, livestock breeding and endangered species. It belongs to the field of applied embryo biotechnology. Background technique
  • ovarian-derived oocytes are widely used to produce animal embryos in vitro, so in vitro oocyte maturation is an important platform technology for animal breeding, cloning and transgenic animal production.
  • bovine oocyte in vitro maturation technology is widely used, this process is completely achieved under unnatural conditions.
  • in vitro maturation relative to in vivo maturation.
  • oocytes are concentrated in small and medium follicles, and the in vivo capacitation process is not fully realized, because the oocytes gradually acquire meiosis and developmental ability during follicular formation.
  • bovine oocytes actively synthesize mRNA.
  • the later stage of oocyte development is considered to be "encapacity of oocytes", at which stage oocytes acquire cytoplasmic maturation and support abrontive embryo development. Therefore, in vitro maturation of small and medium follicle-derived oocytes interferes with the process of capacitation. After spontaneous maturation in vitro, there is a lack of cytoplasmic maturation events and the ability of the material to support the complete development of oocytes. On the other hand, the oocyte moves out of the follicle and loses its natural meiotic inhibition environment, leading to spontaneous meiosis maturation in vitro.
  • in vitro maturation process Despite the addition of gonadotropins, spontaneous meiotic recovery is independent of hormonal effects, and oocytes lack stimulation in vivo to induce mature endocrine and paracrine cascade signals. Therefore, the in vitro oocyte maturation process is non-physiological. Due to the immature cytoplasm of the oocyte, the embryo development, attachment and birth rate are lower than the traditional in vitro fertilization technology, which greatly limits the application of in vitro maturation technology.
  • the C-type natriuretic peptide is a member of the sodium peptide family and consists of 22 amino acids [H. S ang, K., and Scheit, K. H (1994) cDNA cloning identified a calmodulin-binding protein in bovine seminal plasma as bovine C-type natriuretic peptide. DNA. Cell. Biol. 13: 409417.], generally considered Autocrine and paracrine modes regulate the cardiovascular, nervous, endocrine, and reproductive properties of animals.
  • mice C-type natriuretic peptides are secreted by parietal granules through receptors acting on cumulus granule cells, producing cGMP into oocytes, inhibiting oocytes by maintaining high levels of cA P Meiosis [Zhang, ⁇ , Su, YQ, Sugiura, ⁇ , Xia, G., and Eppig, J. J, (2010) Granulosa cell ligand NPPC and its receptor NPR2 maintain meiotic arrest in mouse oocytes.
  • a first object of the present invention is to provide a novel use of c-type natriuretic peptide in the in vitro maturation of oocytes.
  • a second object of the present invention is to provide a culture solution for in vitro maturation of oocytes.
  • a third object of the present invention is to provide a method for in vitro maturation of oocytes.
  • c-type natriuretic peptide can inhibit oocyte meiosis in vitro, thereby prolonging the time of material and information exchange between granulosa cells and oocytes through gap junction, and promoting oocyte-rich mRNA accumulation. And protein. Further studies have shown that, unlike chemical methods, the use of C-type sodium peptide inhibits oocyte meiosis in vitro, and does not cause damage to cells, and actually improves the in vitro development of oocytes. Thus, the C-type sodium peptide can be applied to the in vitro maturation of the oocyte.
  • the present invention provides an oocyte culture solution containing the C-type sodium peptide. It is a C-type natriuretic peptide as an oocyte meiosis inhibitor for pre-mature culture of oocytes.
  • the inventors also found that the inhibition of oocyte meiosis by C-type natriuretic peptides is not higher and stronger.
  • the C-type natriuretic peptide is 100-400 nM. At the concentration, it exhibited a better inhibitory treatment effect, especially at a concentration of 200 nM, and when it was lower than ⁇ or a concentration higher than 400 nM, the inhibitory effect was remarkably lowered.
  • the invention also provides an optimized oocyte culture solution for pre-mature, which is a TCM199 culture solution containing C-type sodium peptide 100-400 nM and BSA 2.5-3.5 mg/ml, more specifically for bovine oocytes.
  • a TCM199 culture solution containing a C-type sodium peptide of 200 nM and a BSA of 3 mg/ml.
  • the present invention provides a method for in vitro maturation of an oocyte by prematurely culturing an oocyte in an oocyte culture solution containing a C-type natriuretic peptide, followed by ripening culture in the mature solution.
  • the mature solution is a TCM199 culture solution containing FSH 8 to 12 g/ml, LH 0.8 to 1.2 g/ml, E2 0.8 to 1.2 g/ml, EGF 8 to 1.2 ng/ml, and 8 to 12% FBS.
  • the bovine oocytes are prematurely matured in the oocyte culture solution for 5 to 7 hours, and then matured in the mature solution for 24 to 28 hours to obtain excellent mature oocytes.
  • the present invention provides a method for producing an oocyte in vitro, which comprises in vitro maturation of an oocyte by the above method, followed by fertilization of the mature oocyte and in vitro embryo culture.
  • the invention is based on the mechanism that CNP can inhibit the meiosis of oocytes in vitro, and the toxic effect of physiologically active substances on oocytes should be less than that of chemically synthesized meiotic inhibitors, and pretreatment of oocytes in vitro can promote cytoplasm Mature, improve developmental ability, make full use of ovarian oocyte resources in slaughterhouses, accelerate the breeding system of improved varieties and expand the breeding system, and greatly improve breeding efficiency and technical level.
  • the "oocyte” used for culture is an oocyte obtained from a follicle in a germinal vesicle (GV) phase, and a "mature oocyte” is obtained by two meiosis. product.
  • GV germinal vesicle
  • C-type natriuretic peptide is a member of the urinary sodium peptide family. It was originally isolated and purified in the brain of pigs in 1990. It is widely distributed in tissues such as the brain, kidney, heart and blood vessels of animals, and has diastolic blood vessels and inhibition. Smooth muscle cell proliferation, antithrombotic and bone growth.
  • the "C-type natriuretic peptide” also includes any polypeptide derived therefrom having such a function, for example, by substitution of amino acids of similar nature, so that it still has or enhances the activity of inhibiting oocyte meiosis; Due to species factors, it has an approximate structure with CNP and has an equivalent of inhibiting oocyte meiotic activity.
  • premature mature liquid means a cell culture liquid having inhibition of oocyte meiosis.
  • C-type natriuretic oocyte culture solution of the present invention is sometimes also referred to as “pre-mature solution”.
  • mature liquid means an in vitro mature liquid of oocytes containing hormones.
  • Example 1 In vitro maturation and embryo production of bovine oocytes 1.
  • a 3-8 mm diameter follicle was taken from the surface of the bovine ovary obtained from the slaughterhouse using a 10 ml syringe containing egg extract.
  • the oviposited liquid after the oocyte was aspirated was placed in 100 mL of domestically cultured JE, and a grade A (cytoplasmic uniform multi-layered tight cumulus granule cell wrap) and B grade (cytoplasmic uniformity less than 4) were selected under a stereo microscope. 3 layers of tight cumulus granule cells are wrapped or partially exposed) Oocytes are used for pre-mature culture in vitro.
  • the selected A and B grade COCs were washed 3 times in the egg washing liquid (TCM199-Hepes), and the pre-mature solution containing CNP (containing C-type sodium peptide 50, 100, 200 and 400 nM, respectively; BSA 3 mg/ml TCM199 culture solution) was washed twice, and then cultured in a pre-mature culture solution which was previously equilibrated in a C0 2 incubator for 2 hours or more (four-well plate culture, 500 ⁇ l pre-mature solution, 50 or so oocytes) culture condition was 5 % C0 2 air, temperature 39 ° C, saturated humidity, incubation time 6 h.
  • the bovine oocytes were matured in vitro for 6 h before the pre-mature solution containing 200 nM CNP, and the three culture time treatments were 24 h, 26 h and 28 h, respectively, and the oocytes without pre-maturation treatment were set as controls. , Set 3 culture time treatments for 24h, 26h and 28h respectively.
  • Maturation medium was: TCM199 added FSHlO g / ml, LH ⁇ g / ml, ⁇ 21 ⁇ ⁇ / ⁇ 1, EGF10ng / ml, 10% FBS.
  • the culture conditions were 5% C0 2 air, temperature 39 ° C, and saturated humidity.
  • the mature oocytes are washed 2-3 times in the fertilized liquid and then put into the balanced fertilization solution (50 ⁇ 1 fertilization solution, 15 oocytes);
  • the frozen semen is treated by floating method, and the sperm is floated on the washing liquid for 20-30 min.
  • the supernatant is taken 600-800 ⁇ l and placed in a 1.5 ml centrifuge tube for centrifugation (1500 rpm, centrifugation for 5 min) twice. After centrifugation, the supernatant is removed, and the washing solution is added.
  • the final volume is 250 ⁇ 1, and the 50 ⁇ 1 treated semen is added to the fertilized solution that has been placed in the oocyte.
  • the final sperm concentration is 1 ⁇ 10 6 sperm/ml, and the culture condition is 5% C0 2 air, the temperature is 39 ° C. Saturated humidity, fertilization time is 8h.
  • the fertiliser is BO liquid (Wako Pure Chemical Industries, Ltd.).
  • the zygote was washed 3 times in the pre-CR1 developmental fluid (2 ml of developmental fluid) and then cultured in two-step microdroplet method: firstly, cultured in ⁇ pre-developmental solution (15 or so zygote) for 2 days, culture condition was 5% CO 2 : 5% O 2 : 90% N 2 , temperature 39 ° C, saturated humidity. After counting the cleavage embryos, they were transferred to ⁇ . The late developmental culture was cultured for 5 days, and the blastocysts were counted on the 7th day of embryonic development. The culture conditions were 5% C0 2 air, temperature 39 ° C, and saturated humidity. Second, the results (1) Results of CNP inhibition of oocyte meiosis
  • the in vitro maturation culture was carried out 6 hours after the mature treatment of CNP.
  • the cleavage rate and blastocyst rate increased with the prolongation of ripening time.
  • the cleavage rate (Table 1) and blastocyst rate (Table 2) were significantly higher after 28 h of maturation. Control ( ⁇ 0 ⁇ 05).
  • the invention uses the C-type sodium peptide as a meiotic inhibitor to establish an in vitro maturation system of the oocyte, which can be applied to the in vitro embryo production of livestock, realizes an ultra-conventional rapid expansion of important scarce varieties, reduces the cost of improved varieties, effectively promotes income increase, and reduces breeding. The total amount, to effectively alleviate ecological pressure.

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Abstract

一种新型的卵母细胞体外成熟方法，该方法以C型钠肽作为减数分裂抑制剂在卵母细胞培养液中进行前成熟培养，然后在成熟液中进行成熟培养。本发明方法体外处理卵母细胞能够促进胞质成熟，提高发育能力，充分利用屠宰场卵巢卵母细胞资源，加速良种选育及扩繁技术体系，提高育种效率和技术水平。

Description

卵母细胞体外成熟方法 技术领域
本发明涉及一种新型的卵母细胞体外成熟方法， 具体地说， 涉及 C 型钠 肽在用于制备卵母细胞体外成熟体系的应用，可用于利用屠宰场获取卵巢的卵 母细胞生产转基因克隆家畜、家畜育种及濒危物种的保护。属于应用型胚胎生 物技术领域。 背景技术
在家畜繁殖上，广泛釆用卵巢来源卵母细胞体外成熟后生产动物胚胎， 因 此体外卵母细胞成熟对于动物育种、克隆及转基因动物生产是一个重要的平台 技术。尽管牛卵母细胞体外成熟技术广泛应用，但是此过程完全在非自然的条 件下实现的。体外成熟相对于体内成熟存在两方面差异。 一方面卵母细胞釆集 于中小卵泡， 没有完全实现体内获能过程， 因为在卵泡形成期间卵母细胞逐渐 获得减数分裂及发育能力。 在有腔卵泡发育的早期， 牛卵母细胞积极的合成 mRNA, 随着卵泡生长至排卵大小， 卵母细胞转录能力逐渐较少， 直至静止状 态。 因此， 卵母细胞发育的后期阶段被认为是"卵母细胞的获能"， 此阶段卵母 细胞获得了胞质成熟并支持附植胚胎发育。 因此， 中小卵泡来源卵母细胞体外 成熟干扰了获能过程，体外自发成熟后， 缺少胞质成熟事件及物质支持卵母细 胞完全发育能力。 另一方面卵母细胞移出卵泡， 失去了自然的减数分裂抑制环 境， 导致体外自发的减数分裂成熟。 体外成熟过程尽管添加促性腺激素， 但是 自发的减数分裂恢复独立于激素的作用，卵母细胞缺少体内诱导成熟的内分泌 及旁分泌级联信号的刺激。 因此， 体外卵母细胞成熟过程是非生理的， 由于卵 母细胞胞质不成熟， 导致胚胎发育、 附植及出生率低于传统的体外受精技术， 极大地限制了体外成熟技术的应用。
鉴于以上原因， 为了提高牛卵母细胞体外发育能力， 许多学者模拟体内环 境开发了卵母细胞体外两段成熟培养方法，即通过体外使用减数分裂抑制剂暂 时可逆的阻止卵母细胞减数分裂恢复， 同时促进卵母细胞生长发育，增强胞质 成熟， 然后移出减数分裂抑制环境， 进行体外成熟。 这种方法目的延长颗粒细 胞与卵母细胞通过间隙连接进行物质和信息交流的时间，促进卵母细胞积累丰 富的 mRNA和蛋白质。 但是， 这些研究结果显示通过使用减数分裂抑制剂并 没有提高牛卵母细胞体外发育能力， 甚至产生了不利的影响。 因而， 开发具有 抑制卵母细胞减数分裂并能提高卵母细胞体外发育能力的方法及试剂是亟待 解决的问题。
C型钠肽为钠肽家族成员， 由 22个氨基酸组成 [H。Sang， K.， and Scheit, K. H (1994) cDNA cloning identified a calmodulin-binding protein in bovine seminal plasma as bovine C-type natriuretic peptide. DNA. Cell. Biol. 13: 409417.]，一般认为以自分泌和旁分泌的 方式调控动物心血管、 神经系统、 内分泌系统及繁殖性能。 值得注意的是， 近 年在小鼠上研究发现 C 型钠肽由壁层颗粒分泌通过作用于卵丘颗粒细胞上的 受体， 产生 cGMP进入卵母细胞， 通过维持高水平 cA P抑制卵母细胞减数 分裂 [Zhang, Μ·， Su, Y. Q., Sugiura, Κ·, Xia, G.， and Eppig, J. J, (2010) Granulosa cell ligand NPPC and its receptor NPR2 maintain meiotic arrest in mouse oocytes. Science 330, 36^369.] 然而， C型钠肽在体外是否具有抑制卵母细胞减数分裂， 特别是抑 制牛等大家畜卵母细胞的减数分裂， 并提高卵母细胞的体外发育能力， 目前尚 无研究报道。 发明内容
本发明的第一个目的在于提供 c型钠肽在卵母细胞体外成熟中的新用途。 本发明的第二个目的在于提供一种用于卵母细胞体外成熟的培养液。
本发明的第三个目的在于提供一种卵母细胞体外成熟的方法。
本发明通过大量研究发现， c型纳肽在体外能够抑制卵母细胞减数分裂， 从而能够延长颗粒细胞与卵母细胞通过间隙连接进行物质和信息交流的时间， 促进卵母细胞积累丰富的 mRNA和蛋白质。 进一步研究表明， 与化学方式不 同， 釆用 C 型钠肽在体外抑制卵母细胞减数分裂的同时， 未见对细胞造成损 伤， 并切实提高了卵母细胞的体外发育能力。 从而可以将 C 型钠肽应用于卵 母细胞体外成熟。 C型钠肽的所述用途优选应用于哺乳动物卵母细胞， 更优选 为诸如牛等大家畜卵母细胞的体外成熟。 进而， 本发明还提供含有所述 C型钠肽的卵母细胞培养液。 其是将 C型 钠肽作为卵母细胞减数分裂抑制剂，用于卵母细胞的前成熟培养。发明人研究 还发现 C 型纳肽作为抑制剂对卵母细胞减数分裂的抑制并非越高越强， 在对 牛卵母细胞的前成熟处理的实验中， C型钠肽为 100~400nM的浓度时， 表现 出较佳的抑制处理作用， 尤其是 200nM的浓度时表现出更加的抑制效果， 而 当低于 ΙΟΟηΜ或者高于 400nM的浓度时， 抑制作用显著降低。
针对牛卵母细胞，本发明还提供了一种优化的用于前成熟的卵母细胞培养 液， 其为含有 C型钠肽 100~400nM、 BSA 2.5〜3.5mg/ml的 TCM199培养液， 更优选为含有 C型钠肽 200nM、 BSA 3mg/ml的 TCM199培养液。
进一步本发明提供一种卵母细胞体外成熟的方法，该方法是将卵母细胞在 含有 C 型钠肽的卵母细胞培养液中进行前成熟培养， 然后在成熟液中进行成 熟培养。 如前所述， 对于牛卵母细胞， 优选釆用上述卵母细胞培养液。 优选， 所述成熟液为含有 FSH 8〜12 g/ml、 LH 0.8〜1.2 g/ml、 E2 0.8〜1.2 g/ml、 EGF 8~1.2ng/ml、 8〜12%FBS的 TCM199培养液。 该方法将所述牛卵母细胞在所述 卵母细胞培养液中前成熟处理 5〜7小时，然后在所述成熟液中成熟 24~28小时， 即可得到优良的成熟卵母细胞。
进一步， 本发明还提供卵母细胞体外生产方法， 其包括釆用上述的方法进 行卵母细胞体外成熟， 然后对成熟的卵母细胞受精， 并进行体外胚胎培养。
本发明基于 CNP体外能够抑制卵母细胞减数分裂的机制， 且为生理活性 物质对卵母细胞的毒害作用应小于化学合成的减数分裂抑制物质，体外前处理 卵母细胞将能够促进胞质成熟，提高发育能力， 充分利用屠宰场卵巢卵母细胞 资源， 加速良种选育及扩繁技术体系， 大幅度提高育种效率和技术水平。
在本发明中，用于培养的 "卵母细胞"是从卵泡中取得的处于 GV( germinal vesicle, GV )期的卵母细胞， "成熟卵母细胞" 是其经过两次减数分裂得到的 产物。
"C型钠肽" 属于尿钠肽家族成员， 最初于 1990年在猪的大脑中被分离 提纯， 广泛分布于动物大脑、 肾脏、 心脏及血管等组织， 具有舒张血管、 抑制 平滑肌细胞增殖、 抗血栓形成及促进骨骼生长等作用。 在本发明中， "C型钠 肽"还包括任何由其衍生的具有该功能的多肽，例如通过性质相近氨基酸的替 换，使其仍然具有或增强抑制卵母细胞减数分裂的活性； 或者是由于物种的因 素， 与 CNP具有近似结构且具有抑制卵母细胞减数分裂活性的等同物。
在本发明中， "前成熟液"是指具有抑制卵母细胞减数分裂的细胞培养液。 本发明中的 "C型钠肽的卵母细胞培养液" 有时也表述为 "前成熟液" 。
在本发明中， "成熟液" 是指含有激素的卵母细胞体外成熟液。 具体实施方式
以下实施例用于说明本发明， 但不应理解为对本发明的限制。 在不背离本 发明精神和实质的情况下， 对本发明方法、步驟或条件所作的修改或润饰均属 于本发明的范围。 实施例 1 牛卵母细胞体外成熟培养及胚胎生产 一、 实验方法
( 1 ) 卵母细胞釆集：
使用含有抽卵液的 10ml注射器从屠宰场获取的牛卵巢表面抽取直径 3-8 mm卵泡。 将吸取卵母细胞后的吸卵液放入 100 mL国产培养 JE中， 在体视显 微镜下挑选出 A级（胞质均匀多层紧密卵丘颗粒细胞包裹）和 B级（胞质均 匀少于 3 层紧密卵丘颗粒细胞包裹或部分裸露） 卵母细胞用于体外前成熟培 养。
( 2 ) 卵母细胞前成熟处理：
将挑选出的 A、 B级 COCs 在洗卵液（TCM199- Hepes )中清洗 3次， 含 有 CNP的前成熟液（分别含 C型钠肽 50、 100、 200及 400 nM; BSA 3mg/ml 的 TCM199培养液） 清洗 2次， 然后放入预先在 C02培养箱中平衡 2h 以上 的前成熟培养液中培养（四孔板培养， 500μ1前成熟液， 50枚左右卵母）培养 条件为含 5% C02的空气， 温度 39°C， 饱和湿度， 培养时间为 6h。 前成熟后 一部分卵母细胞脱去颗粒细胞， 然后 DAPI染色在显微镜下观察 CNP对牛卵 母细胞减数分裂抑制情况（未添加 CNP的前成熟液培养的卵母细胞为对照）， 确定最佳的前成熟 CNP浓度。
( 3 ) 卵母细胞成熟培养：
牛卵母细胞在含有 200nM CNP的前成熟液前成熟处理 6h后进行体外成熟 培养， 设 3个培养时间处理分别为 24h、 26h和 28h, 同时没有经过前成熟处 理的卵母细胞设定为对照， 设定 3个培养时间处理分别为 24h、 26h和 28h。 成熟培养液为： TCM199添加 FSHlO g/ml, LH^g/ml, Ε21μβ/ηι1, EGF10ng/ml, 10%FBS。 培养条件为 5% C02的空气， 温度 39°C , 饱和湿度。
( 4 )体外受精：
釆用培养皿 BO液微滴法， 首先将成熟的卵母细胞在受精液中清洗 2-3次 后放入平衡好的受精液中 （50μ1受精液， 15枚卵母细胞）； 然后釆用上浮法 处理冷冻精液，精子在洗精液上浮 20-30min，之后取上清 600-800μ1放入 1.5ml 离心管离心洗涤（ 1500转， 离心 5min ) 2次， 离心后除去上清， 加入洗精液 使最终体积为 250μ1, 取 50μ1处理后的精液加入已放入卵母细胞的受精液中， 精子终浓度为 1 X 106精子 /ml， 培养条件为 5% C02的空气， 温度 39°C , 饱和 湿度， 受精时间为 8h。 其中受精液为 BO液 （ Wako Pure Chemical Industries, Ltd )。
( 5 )体外胚胎生产：
受精后的合子在 CR1前期发育液中洗涤 3次 （发育液 2ml )后釆用微滴 两步法培养： 首先在 ΙΟΟμΙ前期发育液 （ 15枚左右合子）培养 2d, 培养条件 为 5% CO2: 5%O2: 90%N2, 温度 39°C， 饱和湿度。 计数卵裂胚胎后移入 ΙΟΟμΙ 后期发育液培养 5d， 间隔 2d半量换液， 胚胎发育第 7天计数囊胚。 培养条件 为 5% C02的空气， 温度 39°C， 饱和湿度。 二、 结果 (1) CNP对卵母细胞减数分裂抑制结果
实验结果表明不同浓度 CNP处理 6h后， 随着浓度的增加 CNP抑制牛卵 母细胞减数分裂的效果增强， 其中 200nM浓度抑制效果最好， 卵母细胞维持 在生发泡 （GV) 阶段的比例显著高于与对照 （P<0.01)， 浓度继续增加抑制 效果有下降趋势。 表 1C型钠肽前处理后对体外牛卵母细胞减数分裂的影响 处理 卵母细胞数 GV数 （％) GVBD数 (%)
Control 91 51(56±2.6C) 40(44 ±2.6 c)
CNP (50nM) 101 64(63.4±3.1bc) 37(21.3±3.1bc)
CNP (ΙΟΟηΜ) 98 68(69.4±2.6abc) 30(30.6±2.6abc)
CNP (200nM) 97 79(81.4±2. ) 18(19.6±2.1a)
CNP (400nM) 106 79(74.5±2.8ab) 27(25.5±2.8ab)
注： 同列不同字母（a、 b)表示差异显著（P<0.01); GVBD是指生发泡破裂（Germinal vesicle breakdown) 。
(2) CNP前处理后卵母细胞发育结果
CNP前成熟处理 6h后进行体外成熟培养， 随着成熟时间的延长卵裂率和 囊胚率逐渐增加， 在成熟 28h后卵裂率 （表 1 ) 和囊胚率 （表 2 )均显著高于 对照 （Ρ<0·05)。
表 2C型钠肽前处理后对体外牛卵母细胞受精后胚胎卵裂率的影响 注： 同列不同字母 （a、 b) 表示差异显著 （PO.01) 卵母细胞数 卵裂数(％)
处理
IVM24h IV 26h IVM28h IVM24h IVM 26h IV 28h 对照 251 216 198 190(75.7±2.7b) 168(77.8±3.0b) 143(72.2±3.4b)
CNP(6h) 193 217 214 157(81.3±2.7ab) 89(87.1 ±0.75 a) 186(88.3±3.0a)
表 3 C型钠肽前处理后对体外牛卵母细胞受精后囊胚率的影响 卵母细胞数 囊胚数 （％)
处理
IV 24h IVM26h IVM28h IVM24h IV 26h IVM 28h 对照 251 216 198 58(23.1±2.3) 52(24.7±1.9) 42(21.2±2.4b)
CNP(6h) 193 217 214 53(27.5±1.3) 62(28.6±0.7) 80(37.4±2.5a) 注： 同列不同字母 （a、 b) 表示差异显著 （PO.01) 工业实用性
本发明以 C 型钠肽作为减数分裂抑制剂建立卵母细胞体外成熟体系， 能 够应用于家畜体外胚胎生产中， 实现超常规快速扩繁重要稀缺品种， 降低良种 成本， 有效促进增收， 减少饲养总量， 切实缓解生态压力。

Claims

权 利 要 求 书
1、 c型钠肽在卵母细胞体外成熟中的应用。
2、 根据权利要求 1所述的应用， 其特征在于， 所述卵母细胞为哺乳动物 卵母细胞。
3、 根据权利要求 2所述的应用， 其特征在于， 所述卵母细胞为牛卵母细 胞。
4、 含有 C型钠肽的卵母细胞培养液。
5、 根据权利要求 4所述的卵母细胞培养液， 其特征在于， 所述卵母细胞 培养液中 C型钠肽的含量为 100~400nM。
6、 根据权利要求 5所述的卵母细胞培养液， 其特征在于， 所述卵母细胞 培养液为含有 C型钠肽 100〜400nM、 BSA 2.5-3.5mg/ml的 TCM199培养液。
7、 根据权利要求 6所述的卵母细胞培养液， 其特征在于， 所述卵母细胞 培养液为含有 C型钠肽 200nM、 BSA 3mg/ml的 TCM199培养液。
8、 一种卵母细胞体外成熟的方法， 该方法是将卵母细胞在权利要求 4~7 任一项所述的卵母细胞培养液中进行前成熟培养，然后在成熟液中进行成熟培 养。
9、 根据权利要求 8所述的方法， 其特征在于， 该方法将所述牛卵母细胞 在所述卵母细胞培养液中前成熟处理 5~7 小时， 然后在所述成熟液中成熟培 养。
10、 牛卵母细胞体外生产方法， 其包括釆用杈利要求 8或 9所述的方法进 行卵母细胞体外成熟， 然后对成熟的卵母细胞受精， 并进行体外胚胎培养。
11、 C型钠肽在作为减数分裂抑制剂中的应用。
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