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1.
The objectives of the present study were to investigate the relationship between the morphological status of cumulus cells surrounding canine oocytes after maturation culture and the meiotic stage of the oocytes. In addition, the effect of the removal of cumulus cells from canine cumulus-oocyte complexes (COCs) during maturation culture on their meiotic competence was examined. Canine COCs were collected from bitches at the anoestrous and dioestrous stages and only COCs with >110 microm in vitelline diameter were cultured in medium 199 with 10% canine serum for 72 h. In the first experiment, the relation between the morphological status of cumulus cells surrounding oocytes cultured for 72 h and their meiotic stages was examined. At the end of maturation culture, the proportions of intact, partially nude and completely nude oocytes were 65.2%, 22.9% and 11.9%, respectively. The proportion of maturation to metaphase II of completely nude oocytes was highest among the oocytes with different morphological status of cumulus cells. In the second experiment, the cumulus cells were partially or completely removed from COCs at 48 h after the start of maturation culture and the oocytes were cultured for a further 24 h. The proportion of oocytes reaching metaphase II in the completely denuded oocytes was significantly higher than that in the control oocytes without the removal treatment of cumulus cells. The results indicate that morphological status of cumulus cells surrounding oocytes may be related to the nuclear maturation of canine oocytes, and the removal of cumulus cells from COCs during maturation culture can promote the completion of oocyte meiotic maturation.  相似文献   

2.
本研究探讨绵羊卵丘细胞对裸卵体外成熟和后期发育的影响。实验分为4组,对照组为卵丘-卵母细胞复合体(COCs);裸卵(DO)组;COCs与DO共培养组(CODO);卵丘细胞(CC)与DO共培养组(CCDO)。体外成熟培养18 h后,检测各组卵子成熟质量并进行孤雌发育研究。结果表明:CODO和CCDO组中裸卵活率显著高于DO组(P<0.05),但显著低于COCs组(P<0.05),且COCs组中卵子活率极显著高于DO组(P<0.01);在极体排出率方面,CODO、CCDO和DO3组差异不显著(P>0.05),但是它们都显著低于COCs组(P<0.05)。COCs、CODO、CCDO和DO 4组卵裂率差异均不显著(P>0.05)。然而,DO组的囊胚率显著低于CODO和CCDO组(P<0.05),极显著低于COCs组(P<0.01),但CODO和CCDO 2组中裸卵的囊胚率差异不显著(P>0.05)。总之,无论散在的卵丘细胞还是COCs均能提高裸卵体外成熟及后期发育潜力。  相似文献   

3.
The aim of this study was to induce the cold‐inducible RNA‐binding protein (CIRBP) expression on cumulus–oocyte complexes (COCs) through exposure to a sub‐lethal cold shock and determine the effects of hypothermic temperatures during the in vitro maturation of bovine oocytes. Nuclear maturation, cortical granule redistribution and identification of cold‐inducible RNA‐binding protein (CIRBP) were assessed after 24 hr of in vitro maturation of control (38.5°C) and cold‐stressed oocytes (33.5°C). The presence of CIRBP was assessed by Western blot in COCs or denuded oocytes and their respective cumulus cells. Based on the odds ratio, cold‐stressed oocytes presented higher abnormal cytoplasmic distribution of cortical granules and nuclear maturation than the control group. Although CIRBP was detected in both control and cold‐stressed groups, cold‐stressed COCs had 2.17 times more expression of CIRBP than control COCs. However, when denuded oocytes and cumulus cells were assessed separately, CIRBP only was detected in cumulus cells in both groups. In conclusion, cold shock induced CIRBP expression, but it negatively affected nuclear maturation and cortical granule distribution of bovine oocytes. Moreover, the expression of CIRBP was only identified in cumulus cells but not in oocytes.  相似文献   

4.
Ultrastructural morphological injuries and maturation rates were investigated in equine oocytes exposed to vitrification solutions (VS) containing synthetic ice blockers (SIBs) during different exposure times. In experiment 1, compact cumulus-oocyte complexes (COCs; n = 30) were randomly allocated to treatments: (1) fresh fixed (control); (2) VS-1 (1.4 M dimethyl sulfoxide [DMSO] + 1.8 M ethylene glycol [EG] + 1% SIB) for 3 minutes of equilibrium time and VS-2 (2.8 M DMSO + 3.6 M EG + 0.6 M sucrose + 1% SIB) for 1 minute (Eq-long); and (3) VS-1 for 1.5 minutes and VS-2 for 30 seconds (Eq-short). In experiment 2, compact (n = 248) and expanded (n = 264) COCs were evenly distributed to the following treatments: (1) immediate maturation in vitro (control); (2) vitrification using the Eq-short protocol as in experiment 1; and (3) vitrification using a stock solution containing 2.8 M formamide, 2.8 M DMSO, 2.7 M EG, 7% polyvinylpyrrolidone, and 1% SIB (Eq-short-mod). More (P < .02) oocytes with normal ultrastructural morphology were seen in fresh control and Eq-short groups than in Eq-long group. Metaphase-II (MII) rates were higher (P < .05) for oocytes with expanded cumulus than compact cumulus in the control group, and higher (P < .05) for oocytes with expanded cumulus than compact cumulus in Eq-short and Eq-short-mod groups. No difference in MII rates was detected among groups within each type of COC. In conclusion, reduction of exposure time to VS better preserved oocyte ultrastructural features, and MII rates were higher for vitrified oocytes with expanded cumulus. This study advances our knowledge on potential alternatives for vitrification of immature equine oocytes.  相似文献   

5.
为了进一步探索辽宁绒山羊卵母细胞体外成熟的方法,本试验以屠宰场绒山羊卵巢为材料,采用抽吸法收集直径大于2 mm卵泡的卵母细胞,研究卵丘细胞对卵母细胞体外成熟和孤雌发育的影响。试验1,将一部分COCs经机械吹打脱除卵丘细胞成为机械裸卵(DOs),然后以4种方式培养,即COCs单独培养、DOs与COCs共培养(DOs(COCs))、DOs与卵丘颗粒细胞共培养(DOs(CCs)),以及DOs单独培养。试验2,根据包裹卵母细胞的卵丘细胞的完整性分为3组,有3层卵丘细胞紧密包围的卵母细胞复合体COCs3,有1-3层卵丘细胞包围的卵母细胞COCs1-3,无卵丘细胞包围的裸露卵母细胞NOs,3组各自单独培养。结果表明:COCs组的成熟率、孤雌卵裂率和囊胚率最高,分别为83.25%、41.75%和29.25%,卵丘细胞的存在有利于卵母细胞体外成熟和随后的发育,COCs3组成熟率、孤雌卵裂率和囊胚率分别为83.25%、42.50%和29.75%,显著高于COCs1-3和NOs组,卵丘细胞的完整性也影响着卵母细胞的体外发育,自然裸卵已失去体外发育能力。  相似文献   

6.
The relationship between the peripheral concentrations of estradiol-17β (E(2)) and the preovulatory characteristics of cumulus oocyte complexes (COCs) during superovulation treatment was investigated in Japanese Black cows. A superovulation regimen with FSH treatment in a descending manner was commenced on day 7 (n=3) or day 10 (n=2) of the estrous cycle (day 0=estrus). Peripheral blood was collected to measure E(2) concentrations twice a day throughout the treatment. Ovariectomies were performed at 100 h after the initial FSH treatment in five cows. Every follicle more than 8 mm in diameter was isolated from the ovaries, and cumulus-oocyte complexes (COCs) were gently aspirated. The COCs were then separated into three groups based on the characteristics of the cumulus (compact, expanded and denuded) and subgrouped based on the stage of the nucleus in the oocytes (GV, GVBD). Plasma E(2) concentrations tended to increase gradually and reached the peak level at around 84 h (E(2)-84: n=3) or 96 h (E(2)-96: n=2) after the initial FSH treatment. The ratio of COCs with expanded cumulus was significantly higher in E(2)-84 than in E(2)-96 (P<0.01). However, there was no difference in the ratio of oocytes showing GVBD between E(2)-84 and E(2)-96 (P=0.73), and the characteristics of the cumulus did not affect the stage of the nucleus in the oocytes in either groups (compact, expanded and nude; P=0.61, 0.81 and 1.00). It was possible that the time until the peak plasma E(2) concentrations after the FSH treatment could become an indicator for the maturation of follicles and oocytes in preovulatory follicles during superovulation treatment in Japanese Black cows.  相似文献   

7.
牛卵丘细胞对卵母细胞体外成熟与孤雌发育的影响   总被引：1，自引：0，他引：1  
试验以牛卵泡内卵丘-卵母细胞复合体(COCs)为材料,探讨了牛卵丘细胞包裹程度对卵母细胞体外成熟(IVM)和孤雌胚胎(PAEs)发育的影响。试验1,将COCs随机分为2组,在开展IVM前,将其中一组COCs的卵丘细胞机械吹打去除成为机械裸卵(DOs),研究卵丘细胞层存在与否对卵母细胞体外核成熟(排出第一极体)和孤雌激活后发育能力的影响;试验2,根据COCs卵丘细胞包裹层数将COCs分为3组,即卵丘细胞层少(1～4层)的COCs为A组、卵丘细胞层多(5层以上)的COCs为B组及包裹5层以上卵丘细胞且附带卵泡壁颗粒细胞层(FSP)的COCs为C组,研究卵丘细胞层包裹程度对卵母细胞的体外核成熟和孤雌激活后胚胎发育能力的影响。结果表明:卵丘细胞的存在更有利于卵母细胞体外成熟和孤雌胚胎发育;COCs中卵丘细胞包裹层数对卵母细胞体外核成熟率没有显著影响,但包裹卵丘细胞层数多且附带FSP的卵母细胞,其PAEs的囊胚率显著高于包裹卵丘细胞少的卵母细胞PAEs。  相似文献   

8.
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9.
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10.
In this work, we studied the incidence of DNA fragmentation, interpreted as apoptotic changes and assessed by the TUNEL assay, in cumulus cells and oocytes of immature Grade 1 cumulus‐oocyte complexes (COCs) obtained from healthy bitches (n = 27) of three age groups: young (1–3 years; n = 13), adult (4–6 years; n = 8) and elderly (7–10 years; n = 6). Age affected (p < 0.05) Grade 1 COCs recovery rates, with young animals yielding more (p < 0.01) Grade 1 COCs than the other two age groups. Conversely, no differences were observed in the incidence of DNA fragmentation (TUNEL‐positive) in cumulus cells or oocytes between the three age groups. Overall, more than 80% of Grade 1 COCs presented <15% of TUNEL‐positive cumulus cells and enclosed TUNEL‐negative (intact DNA) oocytes. Despite a higher proportion of TUNEL‐negative oocytes being found in the germinal vesicle stage, most of the oocytes with nuclear material compatible with meiosis resumption (MR) or with non‐identifiable nuclear material (ND) did not present DNA fragmentation. No correlation was observed between DNA fragmentations in oocytes and in cumulus cells. We concluded that the morphological parameters used to classify canine Grade 1 COCs are reliable to select a homogeneous population of COCs with low incidence of DNA fragmentation. Furthermore, these results indicate that DNA fragmentation can only explain a minor proportion of the incidence of MR and degeneration in canine oocytes at collection.  相似文献   

11.
Unfertilized oocytes are one of the most desired germ cell stages for cryopreservation because these cryopreserved oocytes can be used for assisted reproductive technologies, including in vitro fertilization (IVF) and intracytoplasmic sperm injection. However, in general, the fertility and developmental ability of cryopreserved oocytes are still low. The aim of the present study was to improve vitrification of mouse oocytes. First, the effects of calcium and cryoprotectants, dimethyl sulfoxide and ethylene glycol (EG), in vitrification medium on survival and developmental ability of vitrified oocytes were evaluated. Oocytes were vitrified by a minimal volume cooling procedure using different cryoprotectants. Most of the vitrified oocytes were morphologically normal after warming, but their fertility and development were low independently of calcium and cryoprotectants. Second, the effect of cumulus cells on ability of oocytes to be fertilized and develop in vitro was examined. The fertility and developmental ability of denuded oocytes (DOs) after IVF were reduced compared with cumulus-oocyte complexes (COCs) both in fresh and cryopreserved groups. Vitrified COCs showed significantly (P<0.05) higher fertility and ability to develop to the 2-cell and blastocyst stages than those of vitrified DOs with cumulus cells and vitrified DOs alone. The vitrified COCs developed to term at a high success rate equivalent to the rate obtained with IVF using fresh COCs. Taken together, the current results clearly demonstrate that, in the presence of surrounding cumulus cells, matured mouse oocytes vitrified using calcium-free media and EG retain their developmental competence. These findings will contribute to improve oocyte vitrification in not only experimental animals but also clinical application for human infertility.  相似文献   

12.
In the case of high valuable individuals with very precious genetic material, widening the genetic pool including gametes with poor morphological characteristics, as cumulus‐denuded oocytes (CDOs), could be an option. To improve the in vitro culture of low‐competence feline CDOs, an enriched three‐dimensional (3D) system in association with competent cumulus–oocyte complexes (COCs) was developed. For this purpose, domestic cat CDOs were cultured with or without companion COCs in the 3D barium alginate microcapsules. The overall viability and the meiotic progression of feline CDOs cocultured with COCs or cultured separately in 3D or in 2D (traditional microdrops) system were compared. The 3D system was able to support viability and meiotic resumption of the feline oocytes, as well as the 2D microdrops. In 3D microcapsules, the presence of COCs resulted in a higher viability of CDOs (91.1%, p < .05), than that obtained without COCs or in 2D microdrops (71.2% and 67.3%, respectively), but the percentages of meiotic resumption were similar of those of CDOs cultured separately (55.4% vs. 40.4%, p > .05). It is notable that the presence of CDOs seemed to enhance the meiotic progression of the associated COCs. In conclusion, the 3D barium alginate microcapsules are a suitable system for feline oocytes in vitro culture, but more specific enriched conditions should be developed to improve the CDOs full competence in vitro.  相似文献   

13.
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14.
Co‐culture of cumulus‐oocyte complexes (COCs) with denuded oocytes (DOs) during in vitro maturation (IVM) was reported to improve the developmental competence of oocytes via oocyte‐secreted factors in cattle. The aim of the present study was to investigate if addition of DOs during IVM can improve in vitro fertilization (IVF) and in vitro culture (IVC) results for oocytes in a defined in vitro production system in pigs. The maturation medium was porcine oocyte medium supplemented with gonadotropins, dbcAMP and β‐mercaptoethanol. Cumulus‐oocyte complexes were matured without DOs or with DOs in different ratios (9 COC, 9 COC+16 DO and 9 COC+36 DO). Consequently; oocytes were subjected to IVF as intact COCs or after denudation to examine if DO addition during IVM would affect cumulus or oocyte properties. After fertilization, penetration and normal fertilization rates of zygotes were not different between all tested groups irrespective of denudation before IVF. When zygotes were cultured for 6 days, no difference could be observed between all treatment groups in cleavage rate, blastocyst rate and cell number per blastocyst. In conclusion, irrespective of the ratio, co‐culture with DOs during IVM did not improve fertilization parameters and embryo development of cumulus‐enclosed porcine oocytes in a defined system.  相似文献   

15.
The aim of this work was to examine the influence of the cumulus and gonadotropins on the metabolic profile of porcine cumulus oocyte complexes (COCs) during in vitro maturation. Immature COCs were assigned to morphological classes A1 (with a dense cumulus), A2 (with a translucent cumulus), B1 (with the corona radiata), B2 (with only some remaining cumulus cells) and matured with or without gonadotropins. Glycolysis and ammonia production were higher in the A class COCs; gonadotropins increased both, especially in the A1 COCs (p < 0.05). The A class COCs had the highest initial protein contents and at the end of in vitro maturation. Furthermore, hormonal stimulation induced a similar increase in protein contents of both A classes (p < 0.05). The neutral lipid content and reactive oxygen species (ROS) levels were similar in the immature oocytes of the COCs of all classes. A reduction was seen in both these variables when maturation proceeded either in the presence or absence of gonadotropins. The cumulus type surrounding the oocyte is related to the metabolism of carbohydrates and amino acids by the COC during in vitro maturation under gonadotropic stimulation. Oocyte lipolytic activity and ROS production appear to be independent of the surrounding cumulus and the presence of gonadotropins.  相似文献   

16.
The aim of this work was to examine the influence of the cumulus and gonadotropins on the metabolic profile of porcine cumulus oocyte complexes (COCs) during in vitro maturation. Immature COCs were assigned to morphological classes A(1) (with a dense cumulus), A(2) (with a translucent cumulus), B(1) (with the corona radiata), B(2) (with only some remaining cumulus cells) and matured with or without gonadotropins. Glycolysis and ammonia production were higher in the A class COCs; gonadotropins increased both, especially in the A(1) COCs (p?相似文献   

17.
用切割法采集卵泡液,收集卵丘一卵母细胞复合体（Cumulus oocytes comlexs,COCs）和自然裸卵,将部分COCs去除卵丘细胞获得机械裸卵,COCs放入体外成熟培养液中培养为成熟卵母细胞,加入获能的精子液,进行体外受精。结果表明：卵母细胞的体外成熟率和卵裂率与卵泡直径密切相关,大卵泡（80．95％,P〈0．01）和中等卵泡（75．50％,P〈0．05）的卵母细胞成熟率高于小卵泡（50．27％）;犬卯泡（53．53％）和中等卵泡（47．13％）的卵裂率显著高于小卵泡的32．26％（P〈0．05）。COCs、机械裸卵和自然裸卵的体外成熟率分别为75．0％、54．2％和10．5％,差异极显著（P〈0．01）,卵裂率分别为53．8％、10．8％和0％,差异极显著（P〈0．01）。对照组和1×10^5、1×10^6个／mL颗粒细胞组卵母细胞体外成熟率分别为68．6％、69．6％和67．8％,无显著差异（P〉0．05）,但均显著高于1×10^7个／mL（51．5％,P〈0．05）和1×10^10个／mL（35．5％,P〈0．05）颗粒细胞组,但各组间的体外受精率无显著差异（P〉0．05）。结果提示,大卵泡和中卵泡的卵母细胞的体外成熟率和卵裂率显著高于小卵泡,体外成熟培养液中添加高浓度的颗粒细胞能显著抑制卵母细胞的体外成熟。  相似文献   

18.
胰岛素对犬卵母细胞体外成熟的影响   总被引：2，自引：2，他引：0  
研究不同浓度胰岛素及不同培养时间对犬卵母细胞体外成熟率的影响,为改善犬卵母细胞体外培养体系提供参考,采用切割法收集卵巢表面卵丘—卵母细胞复合体(cumulus oocyte complexes, COCs),在含有0.6%葡萄糖的TCM199中添加不同浓度的胰岛素（0、3、6、9 IU/mL）,38.5 ℃、5% CO2培养箱内成熟培养,观察卵丘扩散程度,剥离卵丘细胞获得裸卵后,室温下固定15 min;Hoechst 33342染色,压片,荧光显微镜下观察核形态,用SPSS 14.0软件统计试验数据。不同浓度胰岛素培养48 h后,各组卵丘细胞扩散效果都不明显;卵母细胞核成熟期没有达到减数分裂中期（MⅡ）,但是6 IU/mL胰岛素组生发泡破裂期（GVBD）比率（35.88%±14.63%）显著高于对照组（11.25%±9.75%）;6 IU/mL胰岛素组延长培养时间至72和96 h后,MⅠ-MⅡ期卵母细胞成熟率分别为13.33%±1.5%、20.8%±1.9%。以上结果表明,犬体外成熟培养基中添加胰岛素既没有提高犬卵母细胞核成熟到MⅡ期,也没有改善犬卵母细胞卵丘扩散效果。但是,在6 IU/mL浓度下延长培养时间,相对增加了成熟率。  相似文献   

19.
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20.
The maturation and developmental potential on cumulus-cell-free oocytes is of great importance theoretically and practically. The present study was to investigate the effects of l -ascorbic acid, α-tocopherol and co-culture on in vitro developmental potential of porcine denuded oocytes (DOs). Porcine DOs were cultured in maturation medium supplemented with vitamin C (0, 50, 100, 250, 500, 750 μM) and vitamin E (0, 10, 20, 50, 100, 250 μ m ), respectively. And they were also co-cultured with dispersed cumulus cells (group CCscoculture), intact cumulus cells oocyte complexes (COCs) (group COCscoculture), and COCs whose oocytes were removed (group OOXcoculture), respectively. After 44 h incubation, the maturation rates, cleavage rates and blastocyst rates after parthenogenetic activation in three experiments mentioned above were collected and analysed, respectively. L -Ascorbic acid promoted porcine DOs in vitro maturation and blastocyt development after parthenogenetic activation while α-tocopherol did not increase the in vitro maturation rates, but improved the blastocyst rate. None of the three co-culture manner promoted the in vitro maturation and the cleavage of porcine DOs after parthenogenetic activation, but all the co-culture manners improved the blastocyst rates. Both Vitamin C and E enhance the in vitro developmental potential of porcine DOs. Co-culture increases the developmental potential of porcine DOs.  相似文献   

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