牛腔前卵泡体外无血清培养及超微结构研究
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摘要
本研究建立了一种简便快速的牛腔前卵泡机械分离方法，探讨了卵巢大小及发育状况与腔前卵泡采集数量的关系。初步确立了牛腔前卵泡体外培养过程中的超微结构评定标准。通过研究McCOY’s 5a培养液(含3mM L-谷氨酰胺、0.1％BSA、29ng／mL睾酮、2.5ng／mL转铁蛋白、100ng／mL胰岛素和4ng／mL硒)中有、无血清对腔前卵泡体外发育的影响，确立了牛腔前卵泡的无血清培养体系，在该体系中研究了促性腺激素(FSH、LH)、牛卵泡液(BFF)对腔前卵泡的体外生长及激素分泌的影响，以及体外培养过程中超微结构变化。结果如下：
1．建立了皮肤移植刀切割、剪碎、过滤直接检卵的腔前卵泡简易机械分离技术，平均每卵巢采卵数为46.9±18.2个，卵泡回收效率可达99.5个／h，采集腔前卵泡绝大部分是直径为60～150μm的次级卵泡。
2．腔前卵泡的采集数量与牛卵巢大小成正相关关系(r=0.4475)，而有、无黄体腔前卵泡的采集数量无明显差异；卵巢上不同大小的可见卵泡数量分布也与腔前卵泡的采集量有关。卵巢上可见卵泡(大、中、小)分布均衡者无论是否有黄体存在，均可获得较多的腔前卵泡。而卵巢表面脂肪化、血体化、弥散性片状黄体及幼稚卵巢等则分离很少或几乎分离不到腔前卵泡。根据卵巢发育状况在分离前进行预选择，可显著提高腔前卵泡的回收效率。
3．通过研究基础培养液中不添加血清(FCS)及添加不同比例FCS对腔前卵泡体外发育和激素分泌的影响，结果显示：在本研究系统添加血清对腔前卵泡体外生长无促进作用，对腔前卵泡的体外发育、存活及激素分泌也影响不大。腔前卵泡在无血清条件下可持续生长、分泌E_2，超微结构研究也表明腔前卵泡在该体系中发育正常。该体系可作为进一步研究激素和生长因子等对卵泡早期发育调控作用的一个良好的研究模型。
4．卵泡活力评定结果表明：台盼蓝染色和相差显微镜观察检测卵泡活力有一定的误检率，超微结构检测能够客观、真实地反映腔前卵泡健康状况，可作为评定腔前卵泡培养系统优劣的一个可靠手段。在实际应用中。相差显微镜观察与超微结构评定结合使用，可发挥良好的效果。
5．培养液中单独添加LH对腔前卵泡生长无促进作用。添加50、100ng／ml FSH组腔前卵泡
及其卵母细胞培养不同时间的直径增长值都较对照组有所提高。当培养液中有 10nghl LH
存在时，两种促性腺激素协同刺激卵泡生长的作用更加明显。添加 10ng／ml LH＋50nghl FSH
对腔前卵泡体外生长及存活具有显著的促进作用。
6．体外培养腔前卵泡时，添加一定比例BFF有提高卵泡发育率和存活率的作用。在促性腺
激素存在下，培养液中加入10％＊F于可显著刺激卵泡的体外生长、发育和存活：体外培养
12d腔前卵泡的直径增长值为 33．7士4．8 u m，显著高于其它组门％BFF组：23对土3．8 u m，
20％BFF组：20．4上3．4 u m）及对照组（25．6士4．3 u m）．
7在基础无血清培养液条件下卵泡不论大小在整个培养过程中均可测到一定量的EZ，且大
卵泡分泌比较多，O＞100 p 2卵泡在培养第6d EZ分泌量最高（3．sl士1．62 pg／ml）；慢性
腺激素、叮s及＊w都有刺激腔前卵泡分泌己的作用。巾＞1皿pm卵泡在体外培养6d时
10Dglffil LH＋100wtlFSH组、10O血清组及 10％牛卵泡液组已分泌分别为 5．76士0．2 us加1，
4石2士0．87 pg／inl和 26．72土8．23 pghl。在 10llghlLH＋100ng／ffilFSH存在下，10％BFF组培
养6d时的E。分泌达最大值N8．96土11．54pg／inl入表明培养液中添加一定比例牛卵泡液（BFP）
可显著刺激体外培养腔前卵泡EZ的分泌，促性腺激素存在下更加强了卵泡液对E。分泌的刺
激作用。
8．透射电镜下，简易机械法分离得到的腔前卵泡有卜 层颗粒细胞，卵泡基膜完整，外无卵
泡膜，其颗粒细胞及卵母细胞超微结构与卵巢皮质片中的腔前卵泡一致。培养过程中超微
结构的变化与体内发育腔前卵泡类似。培养 6d时观察到颗粒细胞增殖现象，培养 15d时，
个别卵泡的内膜细胞开始形成。超微结构研究结果表明本培养体系适于小腔前卵泡的体外
培养。
A new meachanical method for the isolation of preantral follicles from bovine ovaries was developed and the relationships between the size and developmental status of bovine ovaries and the number of isolated preantral follicles from them were disscussed. A serum-free culture system has been established by comparing the growth .development and secretion of E2 of the preantral follicles cultured in vitro in the presence or absence of FCS. By this system, effects of gonadotrophins (FSH and LH) and bovine follicular fluid(BFF) on in vitro development of preantral follicles, secretion of E2 , and the ultrastructural changes during culture were studied. The results showed that:
1 A simple, rapid mechanical method can be used effectively to isolate intact preantral follicles from adult cow ovaries. The mean numerber of preantral follicles isolated from each ovary of cows was 46.9 18.8. The processing time was 25.4?.7 min for every follicle . The diameters of the preantral follicles isolated were 60~150, most of which were secondary follicles and could be cultured in vitro.
2 The number of preantral follicles was positively correlated with the ovarian weight in the ovaries with normal external appearance. The presence or absence of corpus lutea did not affect the number and distribution status of isolated preantral follicles.The distribution of superficial antral follicles from ovaries was associated with the number of preantral follicle isolated. Large number of preantral follicles could be aquired from ovaries with balanced distribution of superficial antral follicles, whether corpus lutea existed or not. But seldom or no preantral follicles could be collected from fatly bloodly ovaries, and immature ovaries, or ovaries with fat corpus lutea.
3 There was no obvious difference on growth and development of preantral follicles in the
presence or absence of of FCS in culture medium. In the serum-free culture system ,the follicles could grow continuously, synthesizing measurable level of oestradiol and maintaining their normal ultrastructral morphology.
4 The quality evalution for preantral follicles before or after culture can be done by contrast phase microscope combined withTEM.
5 Culture medium supplemented with gonadotrophins (FSH and LH) was helpful for the in vitro development and growth of preantral follicles. The addition of BFF in the presence of gonadotrophins promoted the in vitro development and growth of preantral follicles and the secretion of E2 significiantly. The level of oestradiol arrived the highest value(48.24pg/ml) in the sixth day of culture of follicles with a diameter of 100 m above.
6 The transmission electron microscope(TEM) analysis showed that the mechanically isolated preantral follicles were with 1 to 3 layers of granulose cells tightly enclosing the oocyte and intact basement membrane. The ultrastructural characteristics of the preantral follicles fresh isolated or cultured in vitro were mostly similar to those presented in situ.
1．建立了皮肤移植刀切割、剪碎、过滤直接检卵的腔前卵泡简易机械分离技术，平均每卵巢采卵数为46.9±18.2个，卵泡回收效率可达99.5个／h，采集腔前卵泡绝大部分是直径为60～150μm的次级卵泡。
2．腔前卵泡的采集数量与牛卵巢大小成正相关关系(r=0.4475)，而有、无黄体腔前卵泡的采集数量无明显差异；卵巢上不同大小的可见卵泡数量分布也与腔前卵泡的采集量有关。卵巢上可见卵泡(大、中、小)分布均衡者无论是否有黄体存在，均可获得较多的腔前卵泡。而卵巢表面脂肪化、血体化、弥散性片状黄体及幼稚卵巢等则分离很少或几乎分离不到腔前卵泡。根据卵巢发育状况在分离前进行预选择，可显著提高腔前卵泡的回收效率。
3．通过研究基础培养液中不添加血清(FCS)及添加不同比例FCS对腔前卵泡体外发育和激素分泌的影响，结果显示：在本研究系统添加血清对腔前卵泡体外生长无促进作用，对腔前卵泡的体外发育、存活及激素分泌也影响不大。腔前卵泡在无血清条件下可持续生长、分泌E_2，超微结构研究也表明腔前卵泡在该体系中发育正常。该体系可作为进一步研究激素和生长因子等对卵泡早期发育调控作用的一个良好的研究模型。
4．卵泡活力评定结果表明：台盼蓝染色和相差显微镜观察检测卵泡活力有一定的误检率，超微结构检测能够客观、真实地反映腔前卵泡健康状况，可作为评定腔前卵泡培养系统优劣的一个可靠手段。在实际应用中。相差显微镜观察与超微结构评定结合使用，可发挥良好的效果。
5．培养液中单独添加LH对腔前卵泡生长无促进作用。添加50、100ng／ml FSH组腔前卵泡
及其卵母细胞培养不同时间的直径增长值都较对照组有所提高。当培养液中有 10nghl LH
存在时，两种促性腺激素协同刺激卵泡生长的作用更加明显。添加 10ng／ml LH＋50nghl FSH
对腔前卵泡体外生长及存活具有显著的促进作用。
6．体外培养腔前卵泡时，添加一定比例BFF有提高卵泡发育率和存活率的作用。在促性腺
激素存在下，培养液中加入10％＊F于可显著刺激卵泡的体外生长、发育和存活：体外培养
12d腔前卵泡的直径增长值为 33．7士4．8 u m，显著高于其它组门％BFF组：23对土3．8 u m，
20％BFF组：20．4上3．4 u m）及对照组（25．6士4．3 u m）．
7在基础无血清培养液条件下卵泡不论大小在整个培养过程中均可测到一定量的EZ，且大
卵泡分泌比较多，O＞100 p 2卵泡在培养第6d EZ分泌量最高（3．sl士1．62 pg／ml）；慢性
腺激素、叮s及＊w都有刺激腔前卵泡分泌己的作用。巾＞1皿pm卵泡在体外培养6d时
10Dglffil LH＋100wtlFSH组、10O血清组及 10％牛卵泡液组已分泌分别为 5．76士0．2 us加1，
4石2士0．87 pg／inl和 26．72土8．23 pghl。在 10llghlLH＋100ng／ffilFSH存在下，10％BFF组培
养6d时的E。分泌达最大值N8．96土11．54pg／inl入表明培养液中添加一定比例牛卵泡液（BFP）
可显著刺激体外培养腔前卵泡EZ的分泌，促性腺激素存在下更加强了卵泡液对E。分泌的刺
激作用。
8．透射电镜下，简易机械法分离得到的腔前卵泡有卜 层颗粒细胞，卵泡基膜完整，外无卵
泡膜，其颗粒细胞及卵母细胞超微结构与卵巢皮质片中的腔前卵泡一致。培养过程中超微
结构的变化与体内发育腔前卵泡类似。培养 6d时观察到颗粒细胞增殖现象，培养 15d时，
个别卵泡的内膜细胞开始形成。超微结构研究结果表明本培养体系适于小腔前卵泡的体外
培养。
A new meachanical method for the isolation of preantral follicles from bovine ovaries was developed and the relationships between the size and developmental status of bovine ovaries and the number of isolated preantral follicles from them were disscussed. A serum-free culture system has been established by comparing the growth .development and secretion of E2 of the preantral follicles cultured in vitro in the presence or absence of FCS. By this system, effects of gonadotrophins (FSH and LH) and bovine follicular fluid(BFF) on in vitro development of preantral follicles, secretion of E2 , and the ultrastructural changes during culture were studied. The results showed that:
1 A simple, rapid mechanical method can be used effectively to isolate intact preantral follicles from adult cow ovaries. The mean numerber of preantral follicles isolated from each ovary of cows was 46.9 18.8. The processing time was 25.4?.7 min for every follicle . The diameters of the preantral follicles isolated were 60~150, most of which were secondary follicles and could be cultured in vitro.
2 The number of preantral follicles was positively correlated with the ovarian weight in the ovaries with normal external appearance. The presence or absence of corpus lutea did not affect the number and distribution status of isolated preantral follicles.The distribution of superficial antral follicles from ovaries was associated with the number of preantral follicle isolated. Large number of preantral follicles could be aquired from ovaries with balanced distribution of superficial antral follicles, whether corpus lutea existed or not. But seldom or no preantral follicles could be collected from fatly bloodly ovaries, and immature ovaries, or ovaries with fat corpus lutea.
3 There was no obvious difference on growth and development of preantral follicles in the
presence or absence of of FCS in culture medium. In the serum-free culture system ,the follicles could grow continuously, synthesizing measurable level of oestradiol and maintaining their normal ultrastructral morphology.
4 The quality evalution for preantral follicles before or after culture can be done by contrast phase microscope combined withTEM.
5 Culture medium supplemented with gonadotrophins (FSH and LH) was helpful for the in vitro development and growth of preantral follicles. The addition of BFF in the presence of gonadotrophins promoted the in vitro development and growth of preantral follicles and the secretion of E2 significiantly. The level of oestradiol arrived the highest value(48.24pg/ml) in the sixth day of culture of follicles with a diameter of 100 m above.
6 The transmission electron microscope(TEM) analysis showed that the mechanically isolated preantral follicles were with 1 to 3 layers of granulose cells tightly enclosing the oocyte and intact basement membrane. The ultrastructural characteristics of the preantral follicles fresh isolated or cultured in vitro were mostly similar to those presented in situ.
引文
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