共查询到19条相似文献，搜索用时 453 毫秒
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目前 ,动物腔前卵泡的体外培养正日益受到重视 ,并已取得了较大进展 ,已建立的培养体系可成功地使腔前卵泡发育到有腔阶段 ,猪、山羊等已可实现腔前卵泡卵母细胞体外成熟 ,体外受精并发育至囊胚阶段。但有关腔前卵泡体外成熟的机制仍不明了。本文通过对体内发育与体外培养之卵泡及其卵母细胞超微结构进行比较 ,从微细结构上客观评定体外培养卵泡的形态、活力、代谢状况及功能完整性 ,界定体外生长卵泡所处发育阶段 ,从而为确立和完善腔前卵泡体外培养体系并最终选择高质量的卵母细胞进行体外受精提供可靠的理论依据。1 体内发育卵泡及其卵… 相似文献
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为了解水牛卵巢卵泡中颗粒细胞（GCs）和卵母细胞在体外培养过程中发生凋亡的变化特征,通过体外培养、血清撤除法诱导水牛卵泡GCs凋亡,并应用苏木精伊红（HE）染色和DNA原位末端标记的TUNEL方法观察其病理特征;通过水牛腔前卵泡体外培养、诱导或等待其中的卵母细胞及卵泡细胞发生凋亡,然后应用透射电镜技术检查其超微结构改变。结果显示：凋亡卵泡GCs的HE染色特征包括细胞体积缩小,染色较深呈紫黑色,且多成簇分布。DNA原位末端标记检查的特征是DNA发生降解,其降解碎片呈TUNEL阳性。原始卵泡体外培养中调亡卵母细胞的超微结构表现为胞核碎裂成电子密度高的大碎片,或出现形态奇特的凋亡小体;初级卵泡中的调亡GCs,细胞内外均出现凋亡小体,细胞内还见有典型的螺层状内质网结构,其中包涵部分核质。 相似文献
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为探究不同直径有腔卵泡中猪卵巢颗粒细胞的生理机能差异,以期为后续生殖毒理学研究奠定基础。采用剖剪法获取猪卵巢内三种直径范围的有腔卵泡（小型〈2mm,中型2-5mm,大型≥5mm）并获得颗粒细胞,台盼蓝染色方法计算细胞存活率,倒置显微镜下观察细胞形态并计算细胞纯度,实时定量方法检测促卵泡刺激素受体基因（FSHR）及促黄体生成素受体基因（LHR）的表达进一步鉴定颗粒细胞,四甲基偶氮唑蓝（MTT）法测定细胞生长曲线。结果显示从大中小三种不同直径有腔卵泡中获得的猪卵巢颗粒细胞存活率分别为68%、75%、72%,纯度分别为97%、95%、90%;细胞生长曲线表明,从24h开始进入对数生长期,并于120h达到最大值,且中型卵泡中获得的颗粒细胞生长增殖状态最佳;实时定量结果显示FSHR在猪三种直径的有腔卵泡颗粒细胞中均表达阳性,且呈现差异。 相似文献
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为了解水牛有腔卵泡闭锁的起因和有腔卵泡中是否存在卵泡颗粒细胞凋亡，本研究选择青春期前水牛和成年（发情间期和发情期）水牛的卵巢，采用石蜡组织切片、HE染色技术进行光镜观察，并采用DNA原位末端标记（TUNEL）技术和透射电镜观察研究了卵泡颗粒细胞凋亡的特征。结果表明：青春期前水牛的健康有腔卵泡上无或仅有极个别凋亡的卵泡颗粒细胞（GCs），而闭锁有腔卵泡上存在有多量凋亡的GCs，凋亡的形式：GCs层内出现单个或多个凋亡细胞，卵泡腔内出现凋亡小体群。在成年水牛发情间期的闭锁大卵泡上，GCs层弯曲皱折，存在多量凋亡的GCs。发情期水牛成熟卵泡的GCs层也存在GCs凋亡，排卵前的GCs层细胞全部脱落到卵泡腔内。本研究首次在同一组织切片上先后分别应用HE染色和TUNEL检查细胞凋亡，且证实了水牛有腔卵泡GCs凋亡的存在。超微结构显示了GCs凋亡的核边集化、核浓缩及凋亡小体的形态。上述结果提示：在水牛闭锁有腔卵泡中存在着GCs凋亡，GCs凋亡是启动水牛有腔卵泡闭锁的潜在机理。 相似文献
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体外培养牛腔前卵泡卵母细胞的超微结构 总被引：1，自引：1，他引：0
直径约100μm牛腔前卵泡在无血清下体外培养15d。超微结构研究显示，培养前腔前卵泡卵母细胞微绒毛短小，分布均匀。胞质内细胞器致密而均匀，线粒体多为长或圆形，嵴较少，高尔基体、脂滴稀少，未见有皮质颗粒出现。培养6d时，微绒毛略变粗长，但数量减少。胞质内细胞器，由近核分布向胞质中央区转移，线粒体形态丰富，基质电子密度极高。培养15d腔前卵泡卵母细胞微绒毛增多，变长，斜向或垂直插入已经形成的透明带中。线粒体增多，大小不等，形态各异，胞质中区有溶酶体样物质及零星的皮质颗粒出现。电镜研究表明，体外培养与体内正常发育腔前卵泡卵母细胞超微结构变化规律基本相似。 相似文献
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R.M.P. Rocha L.F. Lima A.M.C.V. Alves J.J.H. Celestino M.H.T. Matos I.B. Lima-Verde M.P. Bernuci C.A.P. Lopes S.N. Báo C.C. Campello A.P.R. Rodrigues J.R. Figueiredo 《Domestic animal endocrinology》2013
The aim of this study was to investigate the effects of melatonin and follicle-stimulating hormone (FSH) on the in vitro culture of goat preantral follicles. Ovarian fragments were cultured for 7 d in α-minimum essential medium (α-MEM+) containing melatonin (100, 250, 500, or 1,000 pM), FSH (50 ng/mL), or a combination of the 2 hormones and further analyzed by histology and transmission electron and fluorescent microscopy. The results showed that after 7 d of culture, tissues cultured in α-MEM+ alone or supplemented with FSH alone, melatonin (500 and 1,000 pM), or the combination of FSH and melatonin (1,000 pM) maintained percentages of normal preantral follicles similar to the fresh control. In contrast to the noncultured tissues, the percentage of developing follicles was increased under all culture conditions after 7 d (P < 0.05). The addition of 1,000 pM melatonin associated with FSH to the culture medium increased follicular and oocyte diameters compared with α-MEM+ alone after 7 d of culture (P < 0.05). Ultrastructural and fluorescent analyses confirmed the integrity of follicles cultured with 1,000 pM of melatonin plus FSH for 7 d. In conclusion, this study demonstrated that the interaction between melatonin and FSH maintains ultrastructural integrity and stimulates further growth of cultured caprine preantral follicles. 相似文献
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Almeida AP Saraiva MV Alves Filho JG Silva GM Gonçalves RF Brito IR Silva AW Lima AK Cunha RM Silva JR Figueiredo JR 《Reproduction in domestic animals》2012,47(1):20-25
This study quantified Fibroblast growth factor 2 (FGF-2) mRNA and localized FGF-2 protein in different categories of follicles isolated from goat ovaries. In addition, we verified the effects of this factor on the in vitro culture of preantral follicles isolated from goats. For mRNA quantification, we performed real-time PCR using primordial, primary and secondary follicles, as well as cumulus-oocyte complexes (COCs) and mural granulosa and theca cells of small and large antral follicles. For FGF-2 protein localization, the ovaries were subjected to conventional immunohistochemical procedures. Preantral follicles were isolated and cultured in vitro for 12 days in either control (basic) or supplemented with FGF-2 medium. The expression of FGF-2 mRNA was detected in all categories of follicles and there was no difference in preantral follicles and COCs or granulosa/theca cells from small and large antral follicles. However, in large antral follicles, COCs showed expression levels significantly lower than in granulosa/theca cells (p < 0.05). We observed moderate expression of FGF-2 protein in preantral follicles but not in granulosa cells of primordial follicles and theca cells of secondary follicles. In both small and large antral follicles, strong, moderate and weak staining was observed in oocytes, granulosa and theca cells, respectively. The addition of FGF-2 caused a significant increase in the daily follicular growth rate compared to the control group. We conclude that FGF-2 mRNA is expressed throughout follicular development and that its protein can be found in different patterns in preantral and antral follicles. Furthermore, FGF-2 increases the follicular growth rate in vitro. 相似文献
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Hashimoto S Ohsumi K Tsuji Y Harauma N Miyata Y Fukuda A Hosoi Y Iritani A Morimoto Y 《The Journal of reproduction and development》2007,53(2):379-384
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
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In vitro culture of mouse preantral follicles using membrane inserts and developmental competence of in vitro ovulated oocytes 总被引：3，自引：0，他引：3
Adam AA Takahashi Y Katagiri S Nagano M 《The Journal of reproduction and development》2004,50(5):579-586
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
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Amanda Michelle torres Panta Ana Flvia Bezerra da Silva Rodrigo Tenrio Padilha Hudson Henrique Vieira Correia Davide Rondina Jos Ricardo Figueiredo Deborah de Melo Magalhes Padilha 《Reproduction in domestic animals》2019,54(3):480-485
This study aimed to examine the in vitro culture of secondary preantral follicles, using reused ovaries, to compare both the 2D and 3D methods of in vitro culture of preantral follicles, and the system of medium replacement. Twenty‐five pairs of ovaries from mixed‐breed goats were used for the experiment. Follicular puncture of antral follicles was performed for in vitro production. After this procedure, the secondary preantral follicles were submitted to a microdissection procedure. The isolated preantral follicles were randomly divided into three treatments: (a) Two‐dimensional culture with partial replacement of medium during culture (2D PR), (b) Three‐dimensional culture with addition of medium during culture (3D AD) and (c) Three‐dimensional culture with partial replacement of medium (3D PR). The culture period was 18 days. All treatments at the end of the in vitro culture period (18 days) presented a follicular survival rate which ranged from 59% to 70%, demonstrating that it was possible to perform an experiment with preantral follicles using ovaries that had previously been used in another reproductive biotechnique. The 3D AD treatment showed a survival percentage and follicular diameter higher than the 2D PR treatment, however, it did not differ from the 3D PR treatment. In conclusion, experiments employing the use of preantral follicles can be performed with success after the ovaries have been used for experiments with antral follicles. Moreover, the three‐dimensional system with the addition of medium is recommended for in vitro culture of preantral follicles, since this system is more practical and financially feasible. 相似文献
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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
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卵巢大小及发育状况与牛腔前卵泡采集数量的关系 总被引：3，自引：1，他引：2
用简单机械分离法处理了 12 7枚成年牛卵巢。结果显示 ,在外观正常的卵巢中 ,腔前卵泡的采集数量与卵巢的大小成正相关关系 ,而有无黄体与腔前卵泡的采集数量无明显关系 ;卵巢上不同大小的可见卵泡的数量和分布与腔前卵泡的采集量有关。卵巢上可见卵泡分布均衡 ,大、中、小卵泡均有分布 ,小卵泡不过多以及无大卵泡 ,但中、小卵泡较多的 ,无论是否有黄体存在 ,均可获得较多腔前卵泡。而卵巢表面脂肪化、卵巢充血、有弥散性片状黄体及幼稚卵巢的 ,则腔前卵泡分离很少或几乎分离不到 相似文献
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Fibrin–alginate hydrogel supports steroidogenesis,in vitro maturation of oocytes and parthenotes production from caprine preantral follicles cultured in group 
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下载免费PDF全文 IR Brito GM Silva AD Sales CH Lobo GQ Rodrigues RF Sousa AAA Moura CEM Calderón M Bertolini CC Campello J Smitz JR Figueiredo 《Reproduction in domestic animals》2016,51(6):997-1009
This study aimed to establish a culture system that improves the in vitro development of caprine preantral follicles. In a first experiment, follicles were encapsulated as a single unit per bead and cultured singly or in groups or with five follicles in the same alginate (ALG) bead for 18 days. In a subsequent experiment, the “five follicles per bead” design was chosen to culture in ALG, fibrin–alginate (FA) or hyaluronate (HA) for 18 days. In a third experiment, we chose the five follicles per bead in FA to culture for 30 days. The culture set‐up of five follicles per ALG bead increased antrum formation and follicle diameter compared to the other culture designs (p < .05). Moreover, under this condition, 44.44% of the oocytes from in vitro cultured preantral follicles reached meiotic resumption. A significant increase of follicle diameter occurred in attachment system and FA (p < .05), but the ALG condition reached the highest among all groups on day 18 (p < .05). Follicles encapsulated in matrix produced more estradiol and progesterone than attachment system (p < .05). The expression of MMP‐9 mRNA was higher in FA than in other groups (p < .05) and similar to antral follicles from in vivo control (p > .05). Only FA group resulted in oocytes matured. After 30 days, oocytes from preantral follicles in vitro grown in FA developed to eight‐cell parthenotes. In conclusion, a culture system using FA supported the development of caprine preantral follicles cultured in group and included in the same bead of hydrogel, improving the oocyte maturation and producing parthenotes. 相似文献
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H Iwata H Tanaka T Kanke Y Sakaguchi K Shibano T Kuwayama Y Monji 《Reproduction in domestic animals》2010,45(5):888-895
Follicle growth, oocyte quality or oocyte growing environment (follicular fluid) were evaluated in cows with severe liver damage (haemorrhage, telangiectasis, cholangitis and abscess) that were visually diagnosed at the slaughterhouse. Holstein cows aged 40–90 months with either a healthy liver (HL cow) or damaged liver (DL cow) were selected as donors. Follicle development kinetics was evaluated by counting the follicles at various developmental stages. In addition, the biochemical characteristics of the follicular fluids, developmental competence of preantral follicles cultured for 16 days in vitro and the ability of oocytes to develop to the blastocyst stage 8 days after fertilization were examined. DL cows had fewer secondary follicles than HL cows, and the correlation between the number of secondary follicles and the number of primary follicles differed among DL and HL cows. The follicular fluid of DL cows contained significantly lower levels of albumin and a higher total protein content than that of HL cows. Oocyte nuclear maturation assessed at 5, 16 and 21 h after beginning of culture was slower in DL cows than in HL cows, although the final maturation rates did not differ. The rate of polyspermic fertilization was significantly higher and the proportion of cleavage at 48 h after insemination and blastulation lower in DL cows compared with HL cows. When preantral follicles were cultured in vitro, the rate of follicles with normal morphology was lower in DL cows than in HL cows. These findings suggest that the kinetics of folliculogenesis differ among DL and HL cows and the developmental ability of preantral follicles and oocytes is lower in DL cows than in HL cows. 相似文献