哺乳动物腔前卵泡体外培养研究进展

哺乳动物卵巢中含有数以万计的腔前卵泡（preantral follicles）,仅不足1％能够发育至预排卵卵泡。建立哺乳动物腔前卵泡有效分离及体外培养技术体系,能够大量利用腔前卵泡,增加体外成熟卵母细胞数量,对哺乳动物胚胎体外生产、克隆及转基因动物生产等胚胎工程技术的研究与应用,以及在体外条件下揭示哺乳动物卵泡发育机理,都具有重要的理论意义和实用价值。鉴于卵巢质地与卵泡划、发生周期的固有差异,不同物种腔前卵泡分离方法与培养方式亦有所不同。同时,培养基类型、卵泡问相互作用、促性腺激素与细胞因子等因素均会对卵泡的体外发育产生影响。系统阐述了腔前卵泡的分离方法、培养方式以及相关因素对卵泡发育的影响,期望为从事相关研究的学者提供参考。     

塔里木马鹿腔前卵泡分离方法比较

以分离获得的腔前卵泡数量、正常卵泡率和72h体外培养存活率为指标,比较了酶消化法、梳刮法和剪碎法3种不同方法分离塔里木马鹿Cervus elaphus yarkandensis腔前卵泡的效果。结果表明:在卵巢数目相同的情况下,以酶消化法平均获得的腔前卵泡数最多,梳刮法次之,剪碎法最少,3种不同分离方法获得腔前卵泡数量之间存在极显著差异(P<0.01);梳刮法和剪碎法分离获得的腔前卵泡正常率均极显著高于酶消化法(P<0.01);梳刮法和剪碎法分离获得的腔前卵泡在体外培养24h、48h和72h后的存活率极显著和显著高于酶消化法(培养24h、48h后,P<0.01;培养72h后,P<0.05)。由此可见,梳刮法是塔里木马鹿腔前卵泡更有效的分离方法。     

水牛腔前卵泡的分离方法

为确定水牛腔前卵泡的有效分离方法 ,作者取用来自屠宰场 31头水牛的 6 1个卵巢 ,先用剪刀去掉髓质部 ,然后分别用梳刮法、剪碎法和显微分离法分离回收腔前卵泡。结果发现 ,使用梳刮法时 ,平均每个水牛卵巢回收所得的腔前卵泡数 (15 2 35± 4 4 81)明显高于剪碎法 (32 6 2± 14 81)和显微分离法 (8 95± 3 4 4 ) ,且其平均每个卵巢的处理时间 (39 0 5± 4 2 7min)明显少于剪碎法 (4 6 4 3± 4 15min)和显微分离法 (4 4 5 5± 7 82min)。梳刮法分离所得的腔前卵泡中 ,以原始卵泡最多 (占 4 1 2 5 % ) ,其次为初级卵泡 (占 38 79% ) ,而次级卵泡最少 (仅占 19 95 % )。用梳刮法获得的腔前卵泡在体外培养 72h后 ,存活率为 5 6 38% ,与剪碎法(4 8 91% )及显微分离法 (5 9 34% )没有显著差异。用梳刮法处理 10头黄牛的 2 0个卵巢 ,平均每个卵巢可分离获得 1195 2 0± 6 85 0 0个腔前卵泡 ,明显多于水牛的 15 2 35± 4 4 81个腔前卵泡。由此可见 ,梳刮法是水牛腔前卵泡的有效分离方法     

水牛腔前卵泡的体外培养方法

本研究旨在建立一套适合水牛腔前卵泡体外生长发育的培养体系.取用来自本地屠宰场的中国沼泽型水牛卵巢,采用梳刮法回收腔前卵泡,以McCoys 5a作为基础培养液,分别用微孔板培养法、二维培养法、三维培养法进行体外培养.结果表明:不同培养方法对水牛腔前卵泡的体外发育能力有显著差异.培养至10 d,三维培养法的卵泡存活率显著高于微孔板培养法和二维培养法的卵泡存活率(65.05% vs 33.08%,49.52%,P<0.05);二维培养法的卵泡成腔率为1.91%(2/105),三维培养法的卵泡成腔率为1.94%(2/103),而微孔培养法的卵泡未发现成腔;三维培养法的卵泡直径平均增长显著高于微孔板培养法和二维培养法的卵泡直径增长(13.03±5.37 μm vs 7.53±2.26 μm,10.27±4.24 μm,P<0.05).由此可见,三维培养法是水牛腔前卵泡的有效体外培养方法.     

小鼠腔前卵泡体外培养中BMP-6和GDF-9的表达

目的观察BMP-6及GDF-9蛋白在小鼠体外培养卵泡中的定位和定量表达,探讨二者与卵泡发育的关系。方法采用免疫荧光和western blot技术观察BMP-6及GDF-9在体外培养的第6、10天卵泡的定位和定量表达情况。结果在体外培养中,腔前卵泡和有腔卵泡的卵母细胞和颗粒细胞中均检测到BMP-6和GDF-9蛋白的表达;western blot定量显示,在卵泡体外发育的不同阶段,BMP-6和GDF-9蛋白的表达水平不同。结论 BMP-6及GDF-9蛋白存在于体外培养卵泡的卵母细胞和颗粒细胞中;二者的表达水平随卵泡的发育成熟而发生变化。     

FSH对腔前卵泡生长发育的影响

随着腔前卵泡体外培养体系的发展,对其影响因子的研究也逐渐深入,促性腺激素(FSH)在卵泡的生长和发育过程中发挥了重要的作用。本实验方法是以昆明小鼠为模型,机械分离并选择120~140μm的腔前卵泡,以添加10%血清和1%ITS的α-MEM为基础培养,分为正常添加FSH和不添加FSH组,以及在培养的0 d、2 d、4 d、6d、8 d添加FSH(100 mIU/ml)组,探讨腔前卵泡的生长发育情况。结果表明:正常添加组其卵泡的存活率、出腔率、GVBD率和2-cell胚胎率(78.7%、55.0%、35.0%和7.5%)显著高于培养液中未添加FSH的结果(分别为13.7%、8.8%、6.3%和0)(P<0.05);6 d之前添加FSH的培养组腔前卵泡能够正常生长和发育;2~4 d添加FSH可能更利于卵母细胞的成熟,以及E2的产生依赖于FSH的存在。     

人重组卵泡刺激素和表皮生长因子对性未成熟小鼠卵母细胞和颗粒细胞复合体体外发育的作用(英文)

卵泡刺激素(FSH)对有腔卵泡和排卵前卵泡的促生长作用已被普遍接受,但关于其对腔前卵泡发育的作用报道结果不尽相同。关于表皮生长因子(EGF)对腔前卵泡的作用尚不确切。本研究目的在于探讨人重组卵泡刺激素(rechFSH)和EGF对早期卵泡发育的作用。利用胶原酶消化法从12日龄的小鼠卵巢中分离得到卵母细胞-颗粒细胞复合体(OGCs)(Fig.1)。体外每孔30~40个培养物并分别添加胎牛血清(FBS)、rechFSH和EGF。培养物每4天测量卵母细胞和OGCs直径,并每天照相。结果显示rechFSH显著促进小鼠OGCs及其卵母细胞的体外发育,这一作用可被EGF进一步增强(p<0.05)(Fig.2)。但到第八天培养结束时,培养后的OGCs卵母细胞要显著小于体内同期生长对照组(p<0.05)(Fig.3)。说明FSH和EGF在卵泡早期发育中起重要作用。     

人重组卵泡刺激素和表皮生长因子对性未成熟小鼠卵母细胞和颗粒细胞复合体体外发育的作用

卵泡刺激素（FSH）对有腔卵泡和排卵前卵泡的促生命作用已被普遍接受,但关于其对腔前卵泡发育的作用报道结果不尽相同,关于表皮生长因子（EGF）对腔前卵泡的作用尚不确切,本研究目的在于探讨人重组卵泡刺激素（rechFSH)和EGF对早期卵泡发育的作用,利用胶原酶消化法从12日龄的小鼠卵巢中分离得到卵母细胞－颗粒细胞复合体（OGCs)(Fig.1)。体外每孔30－40个培养物并分别添加胎牛血清（FBS）,rechFSH和EGF。培养物每4天测量卵母细胞和OGCs直径,并每天照相,结果显示,rechFSH显著促进小鼠OGCs 及其卵母细胞的体外发育,这一作用可被EGF进一步增强（P＜0．05）（Fig.2),但到第八天培养结束时,培养后的OGCs卵母细胞要显著小于体内期生长对照组（P＜0．05）（Fig.3),说明FSH和EGF在卵泡早期发育中起重要作用。     

中国荷斯坦奶牛卵巢卵泡细胞的免疫组织化学研究

目的和方法应用七种特异性抗体(sGCα、sGCβ、nNOS、FOXO1、PDE4、PKB/Akt和Inhibinα)对中国荷斯坦奶牛卵巢卵泡细胞进行免疫组织化学定位。结果表明PKB主要存在于原始卵泡、腔前卵泡和有腔卵泡颗粒细胞,黄体细胞中没有检测到;FoxO1主要位于原始卵泡、腔前卵泡和有腔卵泡颗粒细胞;PDE4仅位于部分有腔卵泡的颗粒细胞;抑制素α、nNOS、sGCα和β存在于各级卵泡的颗粒细胞层,其中sGCα和β主要存在于原始卵泡和腔前卵泡的颗粒细胞。结论这七种蛋白的阶段和细胞特异性表达表明它们与中国荷斯坦卵巢卵泡的发育、闭锁和黄体化过程有着密切的关系。     

人腔前卵泡分离及培养

采用机械吹打、胶原酶消化、镜下显微解剖及胶原酶消化加镜下显微解剖4种卵泡提取方式并相互对比.将分离得到的腔前卵泡在含FSH0.5IU/mL、1.0IU/mL和2.0IU/mL的培养液中培养,检测其E2分泌量.与机械吹打、胶原酶消化相比,胶原酶消化加镜下显微解剖法不仅提取卵泡多(P＜0.01),而且可以得到原始、初级、次级各级卵泡.但操作时间较长(P＜0.01).得到的腔前卵泡在FSH为0.5IU/mL、1.0IU/mL和2.01IU/mL的培养液中培养,所分泌的E2分别为10.86pg±4.11pg、31.55pg±9.34pg和43.82pg±18.76pg,较之对照组的4.99pg±2.09pg有显著性差异(P＜0.01),E2的分泌量与FSH浓度呈剂量依赖性关系.FSH有促进腔前卵泡分泌E2的作用.     

Quantitative and qualitative analysis of the effectiveness of a mechanical method for the isolation of preantral follicles from ovine ovaries

The preantral follicles are the major source of oocytes and its utilization has been investigated as an important tool to store large numbers of female gametes for further utilization in reproductive programs. The aim of the present study was to perform quantitative and qualitative analyses of the efficacy of a mechanical method for isolating of preantral follicles from the ovaries of fetuses and from nonpregnant and pregnant ewes, using as reference the population of preantral follicles in situ. In the isolation method the ovaries were cut into fragments in the tissue chopper. Then, the suspension was filtered through nylon mesh filters. The number of isolated follicles per ovary was 1655, 4735 and 4770, respectively, for the fetus, nonpregnant ewe and pregnant ewe. The number of in situ preantral follicles per ovary was 32961, 16627 and 17794, respectively, for the fetus, nonpregnant ewe and pregnant ewe. The follicle recovery rate (number of isolated preantral follicles/number of in situ preantral follicles x 100) was higher in adult ewes (26 and 28%, respectively, for nonpregnant and pregnant ewes) than in fetuses (5%). Histological analysis showed that very few preantral follicles (less than 0.26% in situ and 0.46% after the isolation procedure) were degenerated. In conclusion, this study showed that a mechanical method could be used effectively to isolate a large number of intact ovine preantral follicles. In the future, with improvements in culture systems, the isolation of a great number of oocytes enclosed in preantral follicles will make a valuable contribution to the rare breeds and endangered species, agricultural efficiency and basic research in folliculogenesis.     

Development of a combined new mechanical and enzymatic method for the isolation of intact preantral follicles from fetal, calf and adult bovine ovaries

The isolation of preantral follicles from the ovaries of bovine fetuses, calves and adult cows was performed using a simple, rapid mechanical and enzyme method. The ovaries were cut into small pieces with a tissue chopper. Then, the suspension was filtered successively through 500 and 100 mum nylon mesh filters. This simple mechanical procedure resulted in large numbers of isolated preantral follicles: 2,142 +/- 254; 512 +/- 92 and 298 +/- 54 from the ovaries of bovine fetuses, calves and cows, respectively. In addition, the ovarian fragments between 100 and 500 mum were suspended in 10 ml of M199 Hepes medium plus 5% FCS and divided into 2 equal parts: one portion was used for collagenase treatment (200 U/ml) for 20 minutes, while the other served as a control. Collagenase treatment resulted in 841 +/- 161; 216 +/- 51 and 52 +/- 17 preantral follicles from fetuses, calves and cows, respectively, compared with 312 +/- 86; 52 +/- 15 and 10 +/- 2 in the control group. The use of collagenase with ovarian fragments selected by filtration as a method for increasing the rate of recovery of preantral follicles is described here.     

Study of preantral follicle population in situ and after mechanical isolation from caprine ovaries at different reproductive stages.

The purposes of this study were to estimate the population of caprine preantral follicles, and to evaluate quantitatively and qualitatively the efficiency of a specific mechanical method for the isolation of preantral follicles from mixed breed goats at different reproductive stages. On average, 37,646+/-4277 preantral follicles were present in goat ovaries, and 13,631+/-2399 preantral follicles were obtained after isolation. The number of preantral follicles isolated or in situ was not significantly affected by the reproductive stage. The mean recovery rate per ovary ([number of isolated follicles/number of in situ follicles] x 100) of isolated follicles was 36.2%. The distribution of follicles in situ was 67.8% primordial, 25.8% primary and 6.4% secondary; the respective distribution after isolation was 93.8%, 5.2% and 1.0%. In this study, many polyovular follicles were also observed, mainly in prepubertal goat ovaries. Histological analysis showed that few preantral follicles were atretic in situ (4.83%+/-0.35) or after the isolation procedure (4.67%+/-0.65) in the three reproductive stages. The percentage of atretic follicles was not affected either by the mechanical method or by the reproductive stage. It is concluded that a large number of preantral follicles can be successfully isolated mechanically, with a high recovery rate and a low rate of follicular atresia, irrespective of the reproductive stage of the caprine female.     

Effect of fetal age and method of recovery on isolation of preantral follicles from bovine ovaries

The objective of this study was to compare enzymatic and mechanical methods, at distinct fetal ages, on isolation of different developmental stages of preantral follicles from bovine ovaries. Fetal ovaries were obtained from pregnant cattle at 150 to 270 d of gestation, and 135,521 preantral follicles at different stages of development were studied. The dissociation of ovaries with a mechanical procedure resulted in an average of 938.16 prenatral follicles. In contrast, 3,715.56 follicles were obtained when enzymatic digestion was used (P = 0.0001). Histological evaluation confirmed follicular stages and demonstrated that both mechanical and mechanical-enzymatic procedure did not affect the cellular integrity of the follicles. Granulosa cell-oocyte complexes surrounded by a basal membrane, were considered preantral follicles in this study. The ratio of different stages of isolated preantral follicles was significantly (P = 0.0001) correlated to fetal age. The earliest fetal age at which tertiary follicles were identified was at 210 d of gestation. The results confirm previous observation that follicular development and atresia are initiated during fetal development. These data provide information on methodologies to isolate intact bovine preantral follicles for investigating the control and regulation of follicular development and the growth of preantral follicles in vitro.     

Efficient isolation and long-term viability of bovine small preantral follicles in vitro

Summary A comparison of isolation techniques for small preantral follicles (30–70 μm) from bovine ovaries using a mechanical method with a grating device or collagenase treatment was performed. The mean number (157.0) of intact follicles per ovary isolated by the mechanical method was significantly greater (P<0.05) than that (26.0) of follicles isolated by the enzymatic method. Isolated morphologically normal follicles (MNF) were cultured for up to 30 d either in control cultures (non-coculture) or in coculture with bovine ovary mesenchymal cells (BOM), fetal bovine skin fibroblasts (FBF), and/or bovine granulosa cells (BGC). In control cultures, most of the follicles degenerated and only a few MNF (1.2%) were present after 30 d in culture. In contrast, the cocultures with BOM, FBF, and BGC resulted in 50.7, 46.6, and 21.4% viable MNF, respectively. Trypan blue and Hoechst 33258 staining were used for a quick and sensitive assessment of oocyte and granulosa cell viability during follicle isolation and culture in vitro. After 30 d, percentages of viable follicles in coculture with BOM (18.6%) and FBF (17.1%) were significantly greater than those of follicles in the control cultures (0%) or in coculture with BGC (10.0%). There was a gradual increase in the average diameter of the MNF during culture. The mean diameter of the follicles increased by 15.4 and 30.0% in coculture with BOM and FBF, respectively, by day 30. In conclusion, small bovine preantral follicles were efficiently isolated using a mechanical method that utilizes a grating device, and could be maintained for up to 30 d in the presence of mesenchymal cell cocultures such as BOM and FBF. This in vitro culture system that supports long-term survival of bovine preantral follicles should be beneficial for studying follicle growth and development.     

Isolation of preantral follicles from nondomestic cats—viability and ultrastructural investigations

Large scale isolation of small preantral follicles (40–90 μm) from nondomestic cats (lion, puma, cheetah, jaguar, and three kinds of tigers) is described and compared with domestic cats. The viability of preantral follicles was estimated by trypan blue staining of granulosa cells and/or by 5-bromo-2′-deoxy-uridine (BrdU) incorporation into oocytes and granulosa cells during short term culture. Native and isolated preantral follicles were compared ultrastructurally. In nondomestic cats the mechanical dissection of ovaries provided 0–12 500 follicles per ovary with a viability of 20–50%, estimated by trypan blue staining. Even the follicles recovered from domestic cats, whose ovaries are considerable smaller than ovaries from all other felids, are characterised by only 28.7% viable follicles. The follicles from one Siberian tiger and three Indian lions were cultivated and their in vitro viability assessed by BrdU labelling. Lion follicles were comparable to domestic cat follicles with respect to BrdU incorporation. Tiger follicles were characterised by a decreased staining of granulosa cells.The ultrastructure of feline oocytes appears similar to that of most mammalian species and was only slightly affected during the isolation procedure. A central vesicular body was only observed in tiger oocytes.     

Zebu (Bos indicus) ovarian preantral follicles: morphological characterization and development of an efficient isolation method

Preantral follicles are a major source of oocytes, and their utilization as an important tool to store large number of female gametes for future use in reproductive programs has been investigated. The increasing importance of studies in this subject, together with the important role of Zebu cattle in the economy of tropical and subtropical countries as well as their well-known differences from European cattle, led to this research. The present study aims to determine the best size interval for sectioning ovarian tissue to isolate preantral follicles from Zebu cows using a tissue chopper and to evaluate the follicular quality after isolation. Furthermore, it aims to provide information about the Zebu cow preantral follicle population and use this data (as a control) to evaluate the effectiveness of the tested isolation method. Testing eight different tissue sectioning size intervals, it was possible to conclude that the 125-microm-section interval is shown to be better than the intervals of 25, 50, 175 and 200 microm to isolate preantral follicles from Zebu cow ovaries. The 125-microm interval allowed the recovery of 26,050+/-1611 (mean +/- S.E.M.) preantral follicles per one-half ovary, while the number of preantral follicles in situ estimated by evaluation of histological sections was 35,288+/-2342 per one-half ovary. Thus, the mean (+/-S.E.M.) recovery rate (=[number of preantral follicles isolated/number of preantral follicles in situ in the same ovary] x 100) was 74.3+/-4.3%. The morphometrical analysis showed that Bos indicus preantral follicles are similar to B. taurus preantral follicles based on previous reports. In conclusion, this study showed that a simple, mechanical method can be used effectively to isolate a large number of intact preantral follicles from Zebu cow ovaries, with a high recovery rate.     

Effect of coconut water and Braun-Collins solutions at different temperatures and incubation times on the morphology of goat preantral follicles preserved in vitro

Preservation of preantral follicles becomes very important to ensure follicle quality at the onset of cryopreservation or in vitro culture. However, for domestic animals, the ovarian donor of preantral follicles for in vitro studies is commonly encountered far away from reproduction laboratories. We investigated the effectiveness of coconut water and Braun-Collins solutions on the preservation of goat preantral follicles. At the slaughterhouse, the ovarian pair of each animal was divided into 19 fragments. One ovarian fragment was immediately fixed (Control - Time 0). The other 18 fragments were randomly distributed into tubes containing 2 mL of coconut water or Braun-Collins solution at 4 degrees, 20 degrees or 39 degrees C and then stored for 4, 12 or 24 h. Histological analysis showed that the storage of ovarian fragments in coconut water and Braun-Collins solutions at 20 degrees or 39 degrees C for 12 or 24 h significantly reduced (P < 0.05) the percentage of morphologically normal preantral follicles when compared with the control. However, storage in coconut water at 20 degrees C for 4 h and in both solutions at 4 degrees C kept the percentage at control values. Ultrastructural analysis of follicles exposed to the stated conditions confirmed the integrity of preantral follicles stored at 4 degrees C in Braun-Collins and coconut water solutions for up to 12 and 24 h, respectively. Reduced cellular metabolism at 4 degrees C may explain why the best preservation of preantral follicles was at 4 degrees C, which may suggest a useful method for ovary transport in the future.     

Effect of the interval of serial sections of ovarian tissue in the tissue chopper on the number of isolated caprine preantral follicles.

The present work investigated the effect of the interval of serial sections of ovarian tissue on the number of isolated preantral follicles in the goat. Goat ovaries were cut in the tissue chopper at eight different intervals. The quality of isolated follicles were evaluated by histology and transmission electron microscopy. Best results were obtained when the ovaries were cut in the tissue chopper at intervals of 75.0 microm (9664 preantral follicles per ovary). Histochemical and ultrastructural analysis showed that the follicular morphology was preserved after mechanical isolation as demonstrated by the normality of oocytes and granulosa cells as well as by preservation of basement membrane. The percentages of isolated primordial, primary and secondary follicles were 96.3%, 2.5%, and 1.2% and their average diameters were 21.5, 34.7 and 65.3 microm, respectively. It was concluded that the interval of serial sections of ovarian tissue in the tissue chopper affects the number of isolated preantral follicles, and that the follicles remained intact after mechanical isolation in goats.