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1.
Since very few oocytes grow completely in vivo, in vitro growth (IVG) of ovarian oocytes may provide a new source of functional oocytes. The long-term effects of in vitro maturation (IVM) of oocytes and in vitro culture of fertilized eggs have been reported; however, the effects of IVG of oocytes are unknown. Here in, we report the long-term effects of IVG of oocytes. Ovaries from 1-day-old mice containing non-growing oocytes were cultured for 10 days; the isolated follicles were then cultured for 11 days. Secondary follicles from 10-day-old mice were also cultured for 11 days. The nuclei of oocytes collected from the IVG and Graafiais follicles of adult mice were transferred to enucleated oocytes grown in vivo, respectively. Developmental competence was examined following IVM of the reconstituted oocytes. Chronologically, oocytes of 1-day-old, 10-day-old and adult mice were cultured for 22, 12 and 1 day(s). The result showed that the reconstituted eggs developed into pups at high rates after nuclear transfer and in vitro fertilization (IVF) in all the experimental groups (29-45%). However, the pups from reconstituted eggs containing the nuclei of 22-day cultured oocytes were heavier than the control pups (P<0.05). We concluded that long-term culture of oocytes did not affect their nuclear ability to develop to term; however, fetal growth was affected by the culture duration or culture conditions during the initial phase of follicular growth.  相似文献   

2.
刘海军 《猪业科学》2001,18(1):30-31
哺乳动物卵巢中绝大多数卵母细胞以无腔形式存在，有腔卵泡所占比例很少。通过建立腔前卵泡的培养体系，获取大量的具有成熟和受精能力的卵母细胞，将极大地促进体外受精、核移植等胚胎工程技术的发展，并有利于研究卵泡和卵母细胞的发育规律。  相似文献   

3.
Mammalian oogenesis occurs concomitantly with folliculogenesis in a coordinated manner in the ovaries. In vitro growth (IVG) culture systems of the oocytes have been developed as a new technology for utilizing incompetent oocytes in the ovary as a source of mature oocytes as well as for studying oogenesis, folliculogenesis, and oocyte-somatic cell interactions. The results of IVG experiments have suggested that direct association of oocytes and surrounding granulosa cells supports oocyte viability and growth through the gap junctions, which are efficient conduits for low molecular weight substances. It has been revealed that granulosa cells metabolize some molecules which are in turn transported into the oocytes. IVG systems have also provided evidence that FSH promotes the development of follicles at secondary or later stages by its stimulation of proliferation and differentiation of granulosa cells, and perhaps by its anti-apoptotic effects. In addition, interactions between granulosa cell-derived KIT ligands and oocyte KIT receptors have been suggested as initiating oocyte growth and follicular development. Furthermore, recent findings suggest there are growth factors derived from oocytes such as GDF-9 and BMP-15. With such factors, oocytes participate in follicular development by regulating the differentiation of surrounding somatic cells. These bidirectional communications between oocytes and somatic cells are important for oocyte growth and follicular development. IVG systems should provide further information regarding oogenesis and folliculogenesis in the ovary.  相似文献   

4.
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5.
The availability of cow ovaries from the slaughterhouse has been very limited in Taiwan. To maximize the use of cow ovaries for research purposes, whole ovary dissection was performed and the developmental competence of the oocytes derived from different sizes of follicles was assessed by the rates of in vitro maturation (IVM) and parthenogenetic activation of the oocytes in Experiment 1 (Exp 1). Cumulus-oocyte complexes (COCs) derived from small (1-2 mm) and large (3-8 mm) follicles were subjected to standard IVM culture for 24 h. Mature oocytes were selected and then parthenogenetically activated using A23187 (5 microm, 5 min) or thimerosal (200 microm, 10 min) alone or combined with 6-dimethylaminopurine (2.5 mm and 3.5 h, respectively). Activation rates of the oocytes, neither from the large nor small follicles, were affected by different activation treatments (single or combined stimuli). Whereas maturation rates for the oocytes from large follicles were superior to those from small follicles in both the single (59% vs 45%) and combined treatments (76% vs 40%; p < 0.05). To understand how prolonged heat shock (HS) influences cytoskeletal configurations of mature bovine oocytes, in Experiment 2 (Exp 2), matured oocytes derived from large follicles were randomly allocated to different durations of HS treatments at 41.5 degrees C for 0 (C0h, control, n = 12), 1 (HS1h, n = 28), 2 (HS2h, n = 31), and 4 h (HS4h, n = 30). An additional control group was cultured for 4 h without HS (38.5 degrees C, 4 h, n = 35). Alterations in nuclear structures, microtubules (MTs), and microfilaments (MFs) of the oocytes were examined. Abnormalities in the chromosomes, spindle MTs and the percentages of oocytes with cytoplasmic MTs increased with time of HS treatment. The intensity of the MF distribution in the HS oocytes was also altered. Significant changes in the cytoskeleton after HS may be associated with the reduced development under hyperthermia and, perhaps, with the low pregnancy rates of the animals during hot seasons.  相似文献   

6.
Two groups of mouse preantral follicles with diameters of 125-150 and 151-175 microm were cultured individually for 6 days in a medium supplemented with FSH and fetal calf serum to determine their in vitro growth characteristics. Their oocyte capacity for maturation and development to the blastocyst stage following in vitro fertilization was also assessed. Antral formation rate at the end of culture was higher in the follicles of 151-175 microm (89%) than 125-150 microm (76%). The timing of antrum formation was different between the two follicle categories: most 151-175 microm follicles formed antra earlier than 125-150 microm follicles (days 4 and 5 vs. 5 and 6). However, follicle diameters at the time of antrum formation were the same regardless of the initial size and the culture period. Maturation rates of the oocytes derived from both categories of in vitro grown follicles (70 and 62%) were not different from those of oocytes from in vivo grown follicles (74%). The in vitro derived oocytes, however, showed less cleavage (30 and 35%) than the in vivo derived oocytes (89%). Although the oocytes from both follicle categories developed to the morula stage after in vitro fertilization, blastocysts were only obtained from oocytes derived from the 151-175 microm category. These results demonstrate that an individual follicle culture system using a medium with FSH and fetal calf serum supports in vitro growth of mouse preantral follicles with diameters of 151-175 microm to the preovulatory stage, and that their oocytes have the capability to develop to the blastocyst stage.  相似文献   

7.
This study was undertaken to examine pre- and postimplantation developmental potency of cryopreserved embryos that had undergone in vitro growth (IVG), maturation (IVM) and fertilization (IVF) of oocytes from the preantral follicle stage. An oocyte culture system for IVG and IVM was used in oocyte-granulosa cell complexes (OGCs) derived from preantral follicles in 12-day-old mice. The rate of oocyte maturation was improved by the addition of gonadotropins (FSH/LH) and cytokines (IGF-I/SCF) to culture medium for IVG. During culture for IVG, estradiol-17β and progesterone concentrations increased progressively to the latter period of culture. This culture system enabled IVG, IVM, IVF and pre- and postimplantation development. From 90 cryopreserved 2-cell stage embryos transferred into recipients after warming, 10 live pups were produced. Cryopreservation of embryos by vitrification at the 2-cell stage showed no harmful effect on development to the blastocyst stage or on the cell numbers of the inner cell mass (ICM) and trophectoderm (TE). This study demonstrated that embryos derived from oocytes grown in vitro have tolerance for vitrification and competence to develop to term after warming. This IVG-IVM-IVF technology combined with embryo cryopreservation might be useful for assisted reproduction in mice.  相似文献   

8.
Primordial, pre-vitellogenic and vitellogenic follicles were present in the ovary of the immature ostrich. The oocytes of these follicles were composed of a nucleus surrounded by ooplasm. Central, intermediate and cortical regions formed the ooplasm. The organelles present in these ooplasmic regions varied depending on the stage of follicular development. In primordial and small pre-vitellogenic (100-150 microm in diameter) follicles the central region of the ooplasm was dominated by an accumulation of organelles, which formed Balbiani's vitelline body. In contrast, the central region in vitellogenic follicles was filled with numerous large yolk spheres, many of which contained lining bodies. Numerous lipid droplets interspersed with mitochondria and small yolk spheres formed the intermediate ooplasmic region in primordial and small pre-vitellogenic follicles. In large pre-vitellogenic (150-400 microm in diameter) and vitellogenic follicles the intermediate region contained a greater density of mitochondria and small yolk spheres. Small yolk spheres were observed in the cortical region of pre-vitellogenic follicles. An interesting feature of the cortical region in vitellogenic follicles was the frequent occurrence of Golgi complexes. The results of the study indicate that although the ovarian follicles in the immature ostrich are not ovulated, the components and composition of the ooplasm are similar to those observed in the mature follicles of other avian species.  相似文献   

9.
Mammalian ovaries are endowed with a huge number of small oocytes (primordial oocytes) in primordial follicles. A small number of primordial oocytes start to grow, while others remain quiescent. Little is known about the mechanism regulating the activation of primordial oocytes. Recently, we found that primordial follicles in mature cows and prepubertal pigs took longer to initiate growth in xenografts compared with those in neonatal animals. We think that primordial oocytes in adult mammals are different from those in neonatal mammals. In this review, we summarize the results regarding the activation of primordial oocytes in neonatal and adult ovaries of different species and propose a model in which ovaries of neonatal mammals contain a mixed population of both quiescent and activated primordial oocytes, while almost all primordial oocytes are quiescent in adult females. The dormancy of primordial oocytes may be required to reserve the non-growing oocyte pool for the long reproductive life in mammals. FOXO3 is considered one of the molecules responsible for the dormancy of primordial oocytes in adult ovaries. These quiescent primordial oocytes are activated, perhaps by certain mechanisms involving the interaction between stimulatory and inhibitory factors, to enter the growth phase.  相似文献   

10.
The objective of this study was to clarify the effects of prematurational culture (pre-IVM) supplemented with 3-isobutyl-1-methylxanthine (IBMX) on nuclear and cytoplasmic maturation of in vitro-grown bovine oocytes. In experiment 1, oocytes (95 μm in diameter) derived from early antral follicles (0.5–1 mm in diameter) were cultured for 12 days for in vitro growth (IVG). IVG oocytes with a normal appearance were subjected to examinations of diameter and chromatin structure in the germinal vesicle (GV) before IVM. In addition, percentages of metaphase II (M II) were examined after IVM. Regardless of pre-IVM, the mean diameters of IVG oocytes were about 115 μm. The proportions of GV3 (50.0%) and M II stages (80.1%) of IVG oocytes with pre-IVM were higher than those without pre-IVM (28.0 and 49.4%, respectively). In experiment 2, the fertilizability and developmental competence of IVG oocytes were examined. Regardless of pre-IVM, the normal fertilization rates of IVG oocytes were similar (around 70%) but were lower than that of in vivo-grown oocytes (88.0%). Cleavage and blastocyst rates of IVG oocytes with pre-IVM (63.0 and 26.1%, respectively) were higher than those without pre-IVM (45.8 and 12.7%, respectively). The blastocyst rate based on cleaved IVG oocytes with pre-IVM (41.7%) was similar to that of in vivo-grown oocytes (48.7%), although the cleavage rate of IVG oocytes with pre-IVM was lower than that of in vivo-grown oocytes. In conclusion, pre-IVM with IBMX improved the maturational and developmental competences of IVG oocytes, probably due to promotion of their chromatin transition and synchronization of meiotic progression.  相似文献   

11.
We previously found that bovine oocytes 90-99 microm in diameter in early antral follicles grew to nearly their final size in serum-free medium, with some of the oocytes acquiring the nuclear competence to reach the second metaphase. In the present study, we examined the competence of the fertilization and pre-implantational development of the oocytes grown in serum-free medium. Bovine early antral follicles, 0.4-0.7 mm in diameter, were collected mechanically using fine forceps, embedded in collagen gels, and cultured in serum-free medium for 16 days. Grown oocytes which were enclosed by granulosa cells and did not show disintegrated ooplasm were recovered as normal oocytes, were transferred to the maturation medium, and then inseminated with spermatozoa. Ten to 12 h after insemination, 28% (41/145) of the oocytes were penetrated by spermatozoa. Of the penetrated oocytes, 18 (12%) formed a female and a male pronuclei, and 10 (7%) had a female pronucleus and an enlarged sperm head. Among the abnormally penetrated oocytes (13/41), 10 were penetrated by multiple spermatozoa and 3 were penetrated by a spermatozoon at the first metaphase stage. Of the 106 inseminated oocytes grown under serum-free conditions, 8 oocytes had cleaved and developed to the 2-cell stage 48 h after insemination, and 3-4-cell embryos and 5-8-cell embryos were observed after 72-96 h. However, no embryo developed to the blastocyst stage within 8 days. These results indicate that bovine oocytes grown in serum-free medium can be fertilized, but acquire insufficient embryonic development competence under the employed culture conditions.  相似文献   

12.
The aim of this study was to establish a culture system to improve the meiotic competence of porcine oocyte-granulosa cell complexes (OGCs) obtained from preantral or early antral follicles. Porcine OGCs were recovered from follicles with diameters of 230-300 (preantral follicles), 300-500, and 500-700 mum (early antral follicles) using scalpels. The OGCs were cultured for 2 weeks in culture medium. We examined the effects of the sizes of the follicles from which OGCs were recovered, the concentrations of polyvinylpyrrolidone (PVP, 0-8%) in the culture medium, and 2 types of culture dish (Falcon 3002 vs 1007) on formation of the antrum of OGCs. After culture, the oocytes were matured for 44 h to assess their meiotic competence. OGCs recovered from small follicles (230-500 microm) required longer (P<0.05) than larger follicles to form the antrum structure. The percentage of OGCs forming the antrum structure that were cultured in 2% PVP (31%) was higher (P<0.05) than for those cultured in other PVP concentrations (0-11%). The percentages of antrum-structure formation for OGCs cultured on Falcon 3002 (83% for 2% PVP and 60% for 4% PVP) were higher (P<0.05) than those cultured on Falcon 1007 (47% for 2% PVP and 9% for 4% PVP). Furthermore, all of the intact oocytes that were obtained from culture of OGCs and that formed an antrum were in the GV stage (n=28). When these immature oocytes were cultured for 44 h, the percentage of oocytes that reached the metaphase II stage (25%, n=68) was higher (P<0.0001) than that of oocytes matured without culture (0.7%, n=137). The results of the present study show that porcine OGCs obtained from preantral or early antral follicles acquire meiotic competence in vitro.  相似文献   

13.
This study was conducted to culture in vitro caprine pre-antral follicles for determining the competence of growth and maturation of oocytes and establishing a suitable culture system for oocyte maturation from pre-antral follicles. Two different culture methods (microdrop and agar gel clot) were employed to culture caprine pre-antral follicles. The pre-antral follicles were isolated from prepubertal goat ovaries by treatment with collagenase and DNase. The isolated pre-antral follicles were cultured in basic culture medium for 9 days (for growth). And oocytes were cultured in maturation culture medium for another 2 days for maturation. The result demonstrated that the growth rate of oocytes cultured in microdrops was significantly (p < 0.05) higher than that in agar gel clots, whereas the viability of oocytes in microdrops was considerably (p < 0.05) lower than that in agar gel clots. The oocytes grew over 150 microm in diameter, and two of 151 oocytes cultured in microdrops yielded morphologically abnormal first polar bodies. However, the size of oocytes cultured in agar gel approached to 120 microm in diameter and no polar body was produced.  相似文献   

14.
Xenografting of ovarian tissue into immunodeficient mice has been used as a model to study the dynamics of follicular development and provides an alternative method for the production of mature oocytes. In a previous experiment, we demonstrated that xenografted bovine secondary follicles developed to the antral stage in severe combined immunodeficient (SCID) mice. In the present study, we examined the development of bovine secondary follicles (140-190 microm in diameter) grafted into ovariectomized mice in comparison with intact female mice as a control. At 4 weeks after grafting, several antral follicles ranging from 350 to 550 microm (457.6 +/- 50.8 microm) in diameter were found in the control mice, while a single large (larger than 2.5 mm) antral follicle and other small follicles were observed in every ovariectomized mouse. At 6 weeks after grafting, the mean diameter of morphologically normal follicles had further increased in the control group (591.8 +/- 132.0 microm). In ovariectomized mice, however, the mean diameter of follicles decreased (4 weeks: 864.2 +/- 988.2 microm; 6 weeks: 496.5 +/- 137.6 microm), since the single large antral follicle observed at 4 weeks had degenerated by 6 weeks. In control mice, more than 70% of follicles were morphologically normal and formed an antrum, and most of the follicles contained morphologically normal oocytes which grew to 122.5 +/- 2.2 microm. In ovariectomized mice, morphologically normal oocytes also grew larger than before grafting, but their survival rate was significantly lower than that in control mice. These results suggest that ovariectomy of host mice alters the developmental pattern of xenografted bovine secondary follicles to accelerate a single follicle to develop in the graft.  相似文献   

15.
A technique for in vitro maturation of oocytes from small ovarian follicles of marmoset monkeys (Callithrix jacchus) has been developed. We employed a two‐step culture system for primary follicles (45–85 μm) and a one‐step culture technique for secondary follicles (>85 μm). The two‐step technique started with the culture of stromal tissue fragments for 2 days. Thereafter, mechanically isolated follicles were transferred to a culture system where they attached to the culture surface and grew for up to a further 12 days. Significant growth of the small follicles and their oocytes was only achieved with gonadotrophins in the medium. Oocytes with a mean diameter of 39 μm from follicles <85 μm reached a mean diameter of 90 μm by the end of the two‐step culture. After in vitro maturation, 19% of oocytes from these follicles had progressed to the germinal vesicle breakdown (GVBD). Follicles between 85 and 170 μm in diameter were isolated from the stroma and placed directly in the culture. Oocytes from these follicles had a mean diameter of 64 μm. The maximum size the oocytes reached in culture was related to the age of the females (pre‐pubertal females: 102 ± 1.3 μm; adults: 96 ± 1.4 μm). Twenty‐seven per cent of oocytes from pre‐pubertal ovaries achieved GVBD and nearly two‐thirds of these progressed to polar body stage. From adult ovaries, only 12% progressed to GVBD and one‐third of these to polar body stage. It is possible to develop mature oocytes in vitro from marmoset secondary pre‐antral follicles (>85 μm). From primary follicles, although near full size oocytes were developed, maturation capacity was incomplete.  相似文献   

16.
To develop a reliable follicle culture system, mouse preantral follicles 150-200 microm in diameter were cultured individually for 5 or 6 days in membrane inserts or in droplets, and then induced to ovulate with hCG (Experiment 1). The nuclear maturation and developmental competence of the oocytes that ovulated from the follicles cultured in inserts were determined (Experiment 2). There was no significant difference between the two culture systems in the survival rate (83 and 77%). However, follicles cultured in inserts showed a higher ovulation rate (63%) than those cultured in droplets (39%, P<0.05). About 80% of the oocytes that ovulated from the follicles cultured in inserts were at the metaphase II stage. After in vitro fertilization, 75 and 48% of in vitro ovulated oocytes cleaved and developed into blastocysts, respectively. These results demonstrate that the insert culture system is superior to the droplet culture system in terms of follicular growth and ovulation, and can be used to investigate the growth and ovulation of follicles in vitro.  相似文献   

17.
试验从屠宰场收集了 4 8头青年黄牛、34头青年水牛的卵巢共 16 4个 ,共回收可用卵母细胞 86 4枚。水牛平均每个卵巢可回收 3.2 2枚可用卵母细胞 ,相当于黄牛 (6 .72枚 )的一半。试验结果表明 :水牛卵巢生长卵泡少 ,卵母细胞产量少、质量差 ;3种采集黄牛卵泡卵母细胞的方法 ,用抽吸加切割法平均从每个卵巢回收的可用卵数 (7.71枚 )都极显著高于抽吸法 (6 .19枚 )和切割法 (6 .4 4枚 ) (P <0 .0 1) ,但是该法手续繁杂 ,适于一次且只能收集少量卵巢时使用 ;在黄牛和水牛卵母细胞成熟培养液中用 0 .3%PVP代替 10 %FBS ,牛卵母细胞的体外成熟率无显著差异 (P >0 .0 5 )。  相似文献   

18.
研究休情期银黑狐卵巢形态和卵泡的显微结构,以揭示银黑狐卵巢发育的一般规律。本试验于2012年12月份采集5只健康一岁龄银黑狐卵巢10枚,用游标卡尺测量其长、宽、厚,用电子天平测量其重量,并对其表面可见卵泡数量进行统计,然后利用光学显微镜对各级卵泡分别观察1~3个,共计原始卵泡30个,初级卵泡20个,次级卵泡15个,三级卵泡12个,成熟卵泡10个,并进行拍照。结果表明:随银黑狐卵巢体积不断增大,其中80%的卵巢重量也随之增大;可见卵泡数量与卵巢体积及重量没有相关性;卵巢由被膜、皮质和髓质构成,髓质位于卵巢内层,分布着较多血管,皮质位于卵巢外层,内有不同发育阶段的卵泡;原始卵泡由卵母细胞和颗粒细胞构成,初级卵泡开始出现透明带物质,到次级卵泡阶段发育完整,三级卵泡出现卵泡腔,卵泡及卵母细胞直径在有腔卵泡阶段比腔前卵泡阶段增长速度快,成熟卵泡的直径及透明带厚度达到最大,各级卵泡均有闭锁现象。  相似文献   

19.
随着体外受精、克隆与转基因动物等基础研究快速发展,对卵母细胞的需求越来越多,科学家们不得不寻求能够获得大量优质卵母细胞的新途径。原始卵泡作为生长卵泡的来源,其初始容量影响哺乳动物的繁殖性能,是整个雌性生殖期中各阶段卵泡及卵子发育的源头。哺乳动物的卵巢在出生时由一定数量的卵泡组成,大多数原始卵泡处于休眠状态,只有极少数的原始卵泡被激活并进入生长卵泡池中。因此合理开发卵巢里尚未激活的原始卵泡对提高动物繁殖率、加速优良牛种的遗传改良、阐明动物卵泡发生的分子机理具有重要意义。本文将目前哺乳动物原始卵泡体外激活的国内外进展做一综述,为研究动物和人类卵泡激活的生物学过程提供理论基础。  相似文献   

20.
Bone morphogenetic protein‐4 (BMP‐4) inhibits luteinization of granulosa cells during in vitro growth (IVG) culture of bovine oocytes; however, oocytes derived from a 12 day IVG were less competent for development than in vivo‐grown oocytes. We herein investigated whether an extended IVG culture with BMP‐4 improves oocyte growth and development to blastocysts after in vitro fertilization. Oocyte‐granulosa cell complexes (OGCs) were cultured for 14 or 16 days with BMP‐4 (10 ng/mL), while a 12 day culture with BMP‐4 served as the in vitro control. OGC viability was maintained for the 16 day culture with BMP‐4 (83.2%), but was significantly lower without BMP‐4 (58.9%) than the control (83.0%). Prolong‐cultured oocytes at 16 days had statistically greater diameter (114.6 μm) than the control (111.7 μm). IVG oocytes with BMP‐4 for the 16 day culture had a similar nuclear maturation rate to the control (approximately 67%); however, blastocyst rates in BMP‐4 treated oocytes of 14 (1.8%) and 16 day (0%) IVG were statistically lower than that of 12 day IVG (9.0%). In conclusion, BMP‐4 maintained OGC viability and promoted oocyte growth in a prolonged culture, but impaired the developmental competence of oocytes. Prolonged culture may not be an appropriate strategy for enhancing the developmental competence of IVG oocytes.  相似文献   

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