水牛卵泡颗粒细胞对卵母细胞体外成熟的影响

为了系统研究颗粒细胞对水牛卵母细胞体外成熟的影响,使用颗粒细胞条件液处理或单层颗粒细胞和卵母细胞共培养的方法,探讨颗粒细胞共培养对水牛卵母细胞体外成熟和早期胚胎发育的影响.结果显示,添加颗粒细胞传代接种第2天收集的20％颗粒细胞条件液到水牛卵母细胞成熟液中能显著提高水牛卵母细胞体外成熟率和囊胚发育率(P＜0.05);然...     

Chlorogenic acid supplementation during in vitro maturation improves maturation,fertilization and developmental competence of porcine oocytes

Chlorogenic acid (CGA) is a quinic acid conjugate of caffeic acid, and a phytochemical found in many fruits and beverages that acts as an antioxidant. The present study investigated the effects of CGA supplementation during in vitro maturation (IVM), on in vitro development of porcine oocytes, to improve the porcine in vitro production (IVP) system. Oocytes were matured either without (control) or with CGA (10, 50, 100 and 200 μM). Subsequently, the matured oocytes were fertilized and cultured in vitro for 7 day. The rates of maturation, fertilization and blastocyst formation of oocytes matured with 50 μM CGA were significantly (p < .05) higher than those of the control oocytes. Hydrogen peroxide (H₂O₂) is one of the reactive oxygen species and induces DNA damage in porcine oocytes. When oocytes were matured with 1 mM H₂O₂ to assess the protective effect of CGA, 50 μM CGA supplementation improved the maturation rate and the proportion of DNA‐fragmented nuclei in oocytes compared with control oocytes matured without CGA. Moreover, when oocytes were matured with either 50 μM CGA (control) or caffeic acid (10, 50 and 100 μM), the rates of maturation, fertilization and the blastocyst formation of oocytes matured with 50 μM CGA were similar to those of oocytes matured with 10 and 50 μM caffeic acid. Our results suggest that CGA has comparable effects to caffeic acid, and IVM with 50 μM CGA is particularly beneficial to IVP of porcine embryos and protects oocytes from DNA damage induced by oxidative stress. Supplementation of CGA to the maturation medium has a potential to improve porcine IVP system.     

单层细胞共培养对猪去卵丘卵母细胞体外成熟和孤雌发育的影响

本试验比较观察第一极体(The first polar body,PbⅠ)、Oosight imaging system观察和hocchst33342染色法对第2次减数分裂中期(Metaphase Ⅱ,MⅡ)卵母细胞判定结果的相关性分析,并探讨卵巢皮质细胞(porcine ovarian cortex cells,pOCCs)、猪输卵管上皮细胞(porcine oviductal epithelial cells,pOECs)和猪卵丘颗粒细胞(porcine cumulus cells,pCCs)等3种单层细胞体外共培养体系对猪去卵丘卵母细胞(cumulus cells denuded oocytes,Dos)体外成熟(in vitro maturation,IVM)和孤雌发育的影响。结果显示:(1)Oosight imaging system判定卵母细胞成熟的结果与hocchst33342染色法判定的结果有很强相关性(R=0.973,P<0.01,N=90);(2)pOCCs单层细胞共培养体系中猪去卵丘卵母细胞成熟率显著高于pOECs((52.5±0.30)%vs(43.8±2.18)%,P<0.05),且...     

卵泡大小和颗粒细胞对山羊卵泡卵母细胞体外成熟和体外受精的影响

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。     

Phosphorylation of inositol 1,4,5-triphosphate receptor 1 during in vitro maturation of porcine oocytes

During fertilization in mammalian species, a sperm-induced intracellular Ca²⁺ signal ([Ca²⁺]_i) mediates both exit of meiosis and oocyte activation. Recently, we demonstrated in mouse oocytes that the phosphorylation levels of inositol 1,4,5 trisphosphate receptor type1 (IP₃R1), the channel responsible for Ca²⁺ release and oscillations during fertilization, changed during maturation and fertilization. Therefore, we examined the expression and phosphorylation of IP₃R1 during in vitro maturation of pig oocytes. Here, our present study shows that expression of IP₃R1 protein did not change during maturation, although the phosphorylation status of the receptor, specifically at an MPM-2 epitope, did. We found that while at the beginning of maturation IP₃R1 lacked MPM-2 immunoreactivity, it became MPM-2 reactive by 24 h and reached maximal reactivity by 36 h. Interestingly, the acquisition of MPM-2 reactivity coincided with the activation of p34^cdc2 kinase and mitogen-activated protein kinase (MAPK), which are involved in meiotic progression. Following completion of maturation, inactivation of MAPK by U0126 did not affect IP₃R1 phosphorylation, although inactivation of p34^cdc2 kinase by roscovitine dramatically reduced IP₃R1 phosphorylation. Neither inhibitor affected total expression of IP₃R1. Altogether, our results show that IP₃R1 undergoes dynamic phosphorylation during maturation and this might underlie the generation of oscillations at fertilization.     

Effects of exposure to zearalenone on porcine oocytes and sperm during maturation and fertilization in vitro

The influence of acute exposure to zearalenone (ZEN) on porcine oocyte maturation, fertilization or sperm penetration ability during both in vitro maturation and fertilization was evaluated. First, oocytes were cultured in ZEN-containing (0-1000 μg/l) maturation medium and then fertilized. The oocytes maturing in vitro without ZEN were then fertilized in ZEN-containing fertilization medium. The maturation rates of oocytes and penetration ability of sperm decreased significantly in the presence of 1000 μg/l of ZEN. However, neither increases in the rates of degeneration and DNA fragmentation of oocytes nor reductions in normal and polyspermic fertilization were observed. ZEN did not affect the sperm penetration rates; however, 1000 μg/l ZEN had positive effects on normal and polyspermic fertilization rates. Therefore, it can be suggested that an acute exposure of porcine oocytes during maturation and of oocytes and sperm during fertilization to ZEN up to 1000 μg/l may not affect the fertility of the oocytes.     

叶酸对猪卵母细胞体外成熟的影响

将体外收集的猪卵母细胞随机分成对照组和试验组(0、1、10、50μg/L),研究叶酸对猪卵母细胞GVBD率、成熟率和孤雌激活发育潜能的影响。结果表明,10μg/L叶酸能有效的促进GVBD发生、成熟率和激活胚的发育能力,与对照组相比具有上升趋势,其中成熟率显著提高(P0.05)。当叶酸质量浓度为50μg/L时,GVBD率、成熟率和激活胚的发育能力呈下降趋势,其中GVBD率(P0.05)和成熟率(P0.01)显著低于其他处理组。综上所述,叶酸能提高猪卵母细胞体外成熟效果,在本研究中以添加10μg/L叶酸效果最明显。     

BMP-6对猪卵母细胞体外成熟的影响

为研究BMP-6在猪卵泡发育过程中的作用,采用免疫荧光和Western blot技术检测BMP-6在猪排卵前卵泡卵母细胞、颗粒细胞以及卵泡液中的表达;在猪卵母细胞体外成熟培养过程中分别添加5、25及50 ng/mL BMP-6蛋白,观察其对猪卵母细胞在体外成熟过程中的影响。结果表明,猪排卵前卵泡的卵母细胞和颗粒细胞以及卵泡液中均存在BMP-6蛋白,体外添加不同浓度的BMP-6蛋白均能明显促进猪卵母细胞体外成熟,说明BMP-6在猪卵泡发育及猪卵母细胞的成熟过程中发挥作用。     

Changes in follicular endocrinology during final maturation of porcine oocytes

Thirty-one gilts were ovariectomized between 21 and 34 hr after the onset of estrus to compare changes in follicular endocrinology with stages of oocyte maturation. Oocytes were recovered from 6 to 8 mm follicles and classified by stage of meiosis. Remaining follicular fluid was assayed for steroids and dermatan sulfate. Amounts of prostaglandin F2 alpha (PGF2 alpha) and E2 (PGE2) were measured in intramural tissues. Coincident with germinal vesicle breakdown, the follicular content of all steroids except testosterone decreased (P less than .05). As oocytes approached metaphase II, the amount of progesterone within follicles increased (P less than .05), and estradiol continued to decrease (P less than .05). The pattern of dermatan sulfate content was biphasic and peaked at germinal vesicle breakdown and anaphase stages. Amounts of PGF2 alpha and PGE2 within intramural tissues increased (P less than .05) throughout oocyte maturation. Follicular atresia was evident during estrus; however, more (P less than .05) atretic follicles were recovered at germinal vesicle than metaphase II stages (20 vs 3%, respectively). Follicular development, within a gilt, was skewed (P less than .05) and classification of follicles by hormone content demonstrated that a majority were more mature than a minority of less mature follicles. These data suggest that follicular maturation and oocyte development are highly correlated in swine. Furthermore, partitioning the follicular variability by hour and stage of oocyte maturation allowed for more precise assessment of follicular endocrinology than previously reported.     

Growth and maturation of follicles and oocytes following xenotransplantation of porcine ovarian tissues and in vitro maturation

This is the first report to show morphological evidence of in vitro maturation of oocytes recovered from xenotransplanted antral follicles. To develop a suitable tool for studing the growth and maturation of follicles and oocytes, we xenotransplanted small pieces of ovarian cortical tissue from sows, which contained small preantral follicles (primordial, primary, and secondary follicles; less than 0.05, 0.1 and 0.3 mm in diameter, respectively), under the capsules of kidneys of adult female severe combined immunodeficient (SCID) mice for 2 and 8 weeks, and then recovered cumulus-oocyte complexes from the growing tertiary follicles in xenografted tissues. The distribution of processes from cumulus cells to oocytes and the follicular growth, development, and maturation during xenotransplantation were histochemically analyzed. Tertiary follicles, 0.5 to 3.0 mm in diameter, were obtained from grafted tissues 2 (85%: 52 follicles/61 grafted tissues) and 8 (50%: 15/30) weeks after xenotransplantation, and then oocytes, which were tightly attached to cumulus cells, were collected from each tertiary follicle and cultured to assess their quality. At 2 weeks after grafting, 17.6% of the oocytes had matured to the metaphase II stage, but no such maturation was observed 8 weeks after grafting. Thus, in the 2 weeks group, preantral follicles rapidly grew in xenotransplanted porcine ovarian tissues to the tertiary stage, and oocytes could be recovered and matured from them by in vitro culture.     

Changes in the amount of proteins, glycogen and lipids in porcine oocytes during in vitro meiotic maturation

Changes in the cytoplasmic inclusions during meiotic maturation were histochemically examined in cultured porcine oocytes. The oocytes contained a small amount of protein and glycogen granules throughout the maturation culture, as well as Sudanophilic lipids composed of small, medium and large droplets. Soon after collection, the amount of Sudanophilic lipid droplets of small and medium size was small and there were 167 ± 11.2 large droplets. After being cultured for 22 h, the number of large lipid droplets decreased remarkably, while the number of small and medium ones increased. There were no differences in the number of Sudanophilic lipid droplets of different sizes between ovulated oocytes and the oocytes cultured for 44 h. The oocytes always contained a large amount of neutral fats and lipoids, but not cholesterols. In the oocytes cultured for 22 h with olomoucine, both the resumption of nuclear maturation and the decrease in the size of the Sudanophilic lipid droplets were inhibited. From the present findings, it appears that the change in the size of the Sudanophilic lipid droplets in the cytoplasm of porcine oocytes is closely related to nuclear maturation.     

Cytoskeletal abnormalities in relation with meiotic competence and ageing in porcine and bovine oocytes during in vitro maturation

We investigated the frequencies of cytoskeletal anomalies in metaphase-II (M-II) and incompetent [arrested at an immature metaphase (IM) stage] porcine and bovine oocytes during in vitro maturation (IVM) in relation with ageing by immunostaining and confocal microscopy. In porcine oocytes, meiotic arrest at the IM stage was associated with abnormalities of cortical actin but not with abnormal spindles. Prolongation of IVM culture to 52 h did not affect microfilament and spindle abnormalities, but reduced the microfilament-rich area overlaying the spindle. Meiotic arrest of bovine oocytes at the IM stage was associated with degenerations of microfilaments, and the frequencies of abnormal spindles were also higher than those of M-II oocytes. Ageing of bovine oocytes (IVM for 30 h) did not affect cortical microfilaments but increased the frequency of spindle alterations in both M-II and IM bovine oocytes. These results suggest that, in both species, altered ability of oocytes to polymerize F-actin might be a possible reason for the failure of polar body extrusion during IVM. Also, there seem to be differences between the two species in the sensitivity of oocytes to suffer ageing-related spindle damages.     

绵羊卵母细胞和卵丘细胞体外成熟过程中ghrelin mRNA的表达

运用实时定量RT-PCR技术检测了不同成熟培养时间绵羊卵母细胞和卵丘细胞ghrelin mRNA的相对表达量。结果表明,卵母细胞ghrelin mRNA相对表达量在16、24h呈现显著升高,而卵丘细胞ghrelin mRNA则随着成熟时间的延长表现显著降低。绵羊卵母细胞和卵丘细胞ghrelin mRNA的持续表达以及动力学变化提示这一新型分子在生殖功能上有潜在调控作用。     

在成熟过程中添加牛血清和猪卵泡液对猪卵母细胞核成熟及体外受精后早期胚胎发育的影响

研究目的在于探讨在成熟过程中添加牛血清和猪卵泡液对猪卵母细胞核成熟、卵丘细胞扩散及体外受精后早期胚胎发育的影响。卵母细胞·卵丘细胞复合体在含FSH和LH的以下处理组的成熟液中成熟培养 2 3～ 2 4h :(1)对照组-改良TCM - 199+0 .1%PVA ;(2 )试验组 1-改良TCM - 199+10 %新生牛血清 ;(3)试验组 2 -改良TCM - 199+10 %猪卵泡液 ,再移至无FSH和LH的不同处理组的成熟液中成熟培养 2 3～ 34h。试验 1中 ,卵母细胞在 4 6～ 4 8h成熟培养后 ,观察卵丘细胞扩散情况 ,并对卵母细胞进行固定和染色 ,鉴定卵母细胞减数分裂情况 :试验 2中 ,对在不同处理组的成熟液中成熟培养 4 6～ 4 8h的卵母细胞进行体外受精 ,再培养 8d。受精后第 2天检查分裂率、第 6天检查桑椹胚 /囊胚率、第 8天检查囊胚率。 4 6～ 4 8h成熟培养后试验组 1和试验组 2的大部分卵母细胞 -卵丘细胞复合体的卵丘细胞完全扩散 ,而对照组的卵丘细胞只有 5 0 %扩散。试验组 1和试验组 2的卵母细胞核成熟率分别为 39.9% (77/ 193)和 4 4 .3% (93/ 2 10 ) ,与对照组的卵母细胞核成熟率 4 8.1% (99/ 2 0 6 )相比没有显著差异 (P <0 .0 5 )。卵母细胞分裂率试验组 1(5 0 .0± 1.8) %和试验组 2 (49.9± 2 .6 ) %与对照组的卵母细胞分裂率 (49.0± 2     

The effect of nerve growth factor on nuclear progression of porcine oocytes during in vitro maturation and embryo development

The present study examined the effect of nerve growth factor (NGF) on in vitro maturation (IVM), in vitro fertilisation (IVF) and subsequent embryonic development of porcine oocytes. Cumulus-oocyte complexes were cultured with or without 1.0 ng/ml NGF for 40 h. After IVF, they were cultured in vitro for 6 days. After 10 and 20 h of IVM, there was no difference in nuclear status between the NGF-treated and control oocytes. Significant differences were detected in nuclear progression of oocytes matured in the presence or absence of NGF at 30 h of culture. A higher proportion of NGF-treated oocytes were at M-II stage compared to the control. Nevertheless, at the end of the 40-h IVM period, there was no difference in the proportion of M-II stage oocytes between the NGF-treated and control groups. NGF in IVM medium did not influence the developmental competence of putative embryos. Most embryos remained at the 2- to 4-cell stage; however, a significant amount of embryos reached the morula stage both in the NGF and the control groups. These results suggest that NGF during IVM accelerates nuclear progression of porcine oocytes by enhancing the post-diakinetic events of meiosis.     

Presence of chlorogenic acid during in vitro maturation protects porcine oocytes from the negative effects of heat stress

Chlorogenic acid (CGA) is known to protect oocytes from oxidative stress. Here we investigated the effects of CGA on porcine oocyte maturation under heat stress and subsequent embryonic development after parthenogenetic activation. For in vitro maturation (IVM) at 41.0°C (hyperthermic condition), supplementation of the maturation medium with 50 μM CGA significantly improved the percentage of matured oocytes and reduced the rate of apoptosis relative to oocytes matured without CGA (p < .05). CGA treatment of oocytes during IVM under hyperthermia tended to increase (p < .1) percentage of blastocyst formation after parthenogenesis and significantly increased (p < .05) the total cell number per blastocyst relative to oocytes matured without CGA. For IVM at 38.5°C (isothermic condition), CGA significantly improved the rate of blastocyst development compared with oocytes matured without CGA (p < .05), but did not affect oocyte maturation, apoptosis rate or the number of cells per embryo. Omission of all antioxidants from the IVM medium significantly reduced the rate of oocyte maturation, but the rate was restored upon addition of CGA. These results demonstrate that CGA is a potent antioxidant that protects porcine oocytes from the negative effects of heat stress, thus reducing the frequency of apoptosis and improving the quality of embryos.     

Treatment of porcine oocytes with MEM vitamins during in vitro maturation improves subsequent blastocyst development following nuclear transfer

This study was carried out to investigate the effects of minimum essential medium (MEM) vitamins during in vitro maturation (IVM)/in vitro culture (IVC) of porcine nuclear transfer (NT) embryos on subsequent developmental capacity in vitro. Porcine cumulus-oocyte complexes (COCs) were divided into five groups, matured for 44 h in maturation medium with various concentrations of MEM vitamins (0, 0.05, 0.1, 0.2 and 0.4%), and observed for maturation rate. Also, COCs were matured in NUSU-23 media without MEM vitamins for 44 h and cultured in PZM-3 media with various concentrations of MEM vitamins (0, 0.05, 0.4 and 1.0%) for 6 days following nuclear transfer. Factorial (IVM/IVC) experiments were also performed in NCSU-23 medium with or without 0.05% MEM vitamins and PZM-3 medium with or without 0.4% MEM vitamins. They were then tested by examining in vitro development of the porcine reconstructed embryos. The maturation rates of the COCs treated with the MEM vitamins did not differ significantly among the MEM vitamin-treated groups. Addition of vitamins to culture medium did not affect development of porcine reconstructed embryos in vitro. However, addition of low concentrations of MEM vitamins only to maturation medium increased (P<0.05) the proportion of NT embryos developing into blastocysts compared with the control group. Addition of MEM vitamins to IVC medium did not enhance the developmental rate compared with the control group. Thus, addition of MEM vitamins to IVM medium could improve subsequent blastocyst development of porcine NT embryos.     

高浓度葡萄糖对猪卵母细胞体外成熟和早期胚胎发育的影响

试验旨在探究高浓度葡萄糖对猪卵母细胞体外成熟及早期胚胎发育能力的影响。取体外分离处于生发泡期的猪卵丘卵母细胞复合体(COCs),分为3个处理组。分别用含葡萄糖浓度为5.6 mmol/L(C组)、10 mmol/L(G-1组)、15 mmol/L(G-2组)的培养液,进行体外成熟(IVM)处理,42 h后观察,并统计卵丘细胞扩散情况和第一极体排出率;对体外成熟42 h后的卵母细胞孤雌激活,统计2-细胞、4-细胞和第7天囊胚发育。结果发现,G-1组和G-2组卵丘细胞扩散度显著低于C组(P<0.05);G-1组和G-2组的MII期卵母细胞死亡率和存活率与C组相比无显著差异(P>0.05),但G-1组极体率显著降低(P<0.05),G-2组极体率极显著低于C组(P<0.01)。孤雌激活后,与C组相比,G-1组和G-2组的2-细胞分裂率显著降低(P<0.05),4-细胞分裂率以及囊胚发育率均极显著降低(P<0.01),但G-1、G-2组囊胚细胞数量与C组相比无显著性差异(P>0.05)。进一步线粒体染色发现,G-1组和G-2组的线粒体与C组相比分布不均。...     

玉米赤霉烯酮对体外培养猪卵巢颗粒细胞的影响

以原代培养的猪卵巢颗粒细胞为模型,设立玉米赤霉烯酮(ZEA)对照组(0.0 mg/L)及高(80.0 mg/L)、中(20.0,40.0mg/L)、低(0.2,2.0mg/L)剂量组,比较不同质量浓度ZEA对体外培养猪卵巢颗粒细胞的影响。形态学观察发现,随着ZEA质量浓度的增加,颗粒细胞呈散在生长,体积缩小变圆,数量减少;高剂量组(80.0mg/L)中,76.96%细胞均已脱壁死亡。MTT法表明,ZEA对卵巢颗粒细胞具有生长抑制作用;免疫荧光结果显示随ZEA质量浓度的增加,细胞凋亡率逐渐升高,高质量浓度组细胞增殖率仅为1.01%;Western blot检测结果显示经ZEA处理,Fas、FasL、FADD、Caspase-10的表达量均明显上调,且存在质量浓度-效应关系。结果表明,ZEA能诱导卵巢颗粒细胞凋亡,并显著上调Fas、FasL、FADD、Caspase-10的表达,可通过死亡受体通路介导卵巢颗粒细胞发生凋亡。     

Steroidogenesis and the influence of MAPK activity during in vitro maturation of porcine cumulus oocyte complexes

The aim of this study was to investigate steroidogenesis within porcine cumulus oocyte complexes during in vitro maturation and to examine the possible influence of the mitogen-activated protein kinase (MAPK). Porcine cumulus oocyte complexes were matured in vitro with and without the MAPK kinase inhibitor U0126 for 0, 5, 26 and 46 h. The 17β-estradiol and progesterone concentration in the culture medium were then determined. In addition, the mRNA levels of StAR, Cyp11A1, 3β-HSD and Cyp19A1 in cumulus cells were analysed by RT-PCR. Using an immunoblot, the MAPK phosphorylation in cumulus cells and oocytes was examined. During the first 26 h of in vitro maturation, 17β-estradiol secretion was predominant, whereas, after a culture period of 46 h, the progesterone secretion decreased conspicuously. Under the influence of U0126, the secretion of 17β-estradiol increased progressively during the complete maturation period, while progesterone secretion was completely inhibited. The mRNA levels of StAR and Cyp11A1 were not altered by U0126; however, corresponding to the hormone secretion, the gene expression of Cyp19A1 was up-regulated and the expression of 3β-HSD down-regulated. The results suggested an influence of the MAPK on steroidogenesis in cumulus cells comparable to a luteinization factor. Hormone synthesis in cumulus cells during oocyte maturation seems to be regulated by altering expression of Cyp19A1 and 3β-HSD.