促性腺激素对山羊卵母细胞体外成熟的影响

为了建立针对山羊卵母细胞的理想体外成熟培养体系,本试验研究了在成熟培养液中添加不同浓度的促卵泡素(follicle stimulating hormone,FSH)、促黄体素(luteinizing hormone,LH),不同来源的FSH+LH对山羊卵母细胞体外培养成熟效果的影响。结果表明,采用TCM199添加10%FCS,1～2mg/L17β-E2,10mmol/LHepes,0.055mg/mL丙酮酸钠,2.2mg/mL NaHCO3,500μg/mL链霉素,500μg/mL青霉素,10mg/L FSH及20mg/LLH的培养体系,有利于山羊卵母细胞的体外成熟;进口的和国产FSH和LH对山羊卵母细胞成熟有相同促进效果。     

牛卵母细胞的体外成熟

在卵母细胞体外成熟液（含FSH）中分别添加10、30、50μg/L表皮生长因子（EGF）,24 h检查成熟率。结果表明,添加10、30μg/L EGF组牛卵母细胞成熟率为79.8%、71.5%与对照组（未添加EGF）成熟率（70.4%）无明显差异（P〉0.05）,但EGF添加至50μg/L时牛卵母细胞第一极体（PB1）排出率显著提高,达85.4%（P〈0.01）;在此基础上,比较了成熟液中添加尿促性素（HMG）对牛卵母细胞体外成熟的影响,结果发现在含FSH和EGF的成熟液中添加HMG未能显著提高牛卵母细胞体外成熟效果（P〉0.05）;最后,比较了HMG对牛卵母细胞体外成熟的效果,结果发现单独添加HMG成熟效果显著高于FSH（P〈0.05）,表明成熟液中如不含其他生长因子,则单独添加HMG较FSH对牛卵母细胞体外成熟的效果好。     

人尿促性腺激素、雌二醇及表皮生长因子对牛卵母细胞体外成熟质量的影响

试验旨在研究不同激素配比及表皮生长因子(EGF)浓度对牛卵母细胞体外成熟及卵母细胞质量的影响。将随机分组的卵丘-卵母细胞复合体于添加FSH+LH、HMG、FSH+LH+E₂、HMG+E₂ 4种不同激素组合配比的成熟基础液中培养,对比其体外成熟率,比较了EGF对牛卵母细胞体外成熟率和孤雌胚胎体外发育的影响,并采用TUNEL法检测添加不同浓度EGF的牛孤雌激活囊胚细胞凋亡情况。结果表明,添加HMG的成熟试验结果稳定,E₂对牛卵母细胞成熟有一定的促进作用,HMG+E₂联合使用可以得到高效稳定的成熟结果;在此基础上,在成熟液中添加30 ng/mL EGF对牛卵母细胞的成熟质量、胚胎发育及降低胚胎细胞凋亡都有明显的促进作用。因此,在体外成熟培养液中添加0.075 IU/mL HMG、1 μg/mL E₂和30 ng/mL EGF对牛卵母细胞的成熟和质量较为有益。     

不同培养液对牛卵母细胞成熟及核移植效率的影响

在牛卵母细胞成熟液中分别添加促卵泡素(FSH)10μg/L、胰岛素-转铁蛋白-硒(ITS)10μL/mL、表皮生长因子(EGF)10μg/L,配成3种不同体外成熟液对牛卵母细胞进行成熟培养,结果FSH组卵母细胞成熟率84.2%显著高于ITS组75.9%和EGF组70.2%。卵母细胞成熟后构建牛体细胞核移植胚胎,结果FSH、ITS组卵裂率分别为87.2%、81.9%,显著高于EGF组(73.6%),但三组间囊胚率差异不显著(P0.05)。     

激素及生长因子对牛卵母细胞体外成熟的影响

在卵母细胞体外成熟液(含FSH)中分别添加10, 30, 50ng/mL表皮生长因子(EGF),24h检查其成熟率.结果表明:添加 EGF50ng/mL时牛卵母细胞第一极体排出率显著提高,达85.4%(P＜0.01);同时发现在含FSH和EGF的成熟液中添加HMG(尿促性素)并不能提高牛卵母细胞体外成熟效果(P>0.05);同时还发现单独添加HMG成熟效果显著好于FSH(P＜0.05).     

EGF和IGF-1对山羊卵母细胞体外成熟的影响

研究了在培养液中添加不同浓度的EGF、IGF-1以及EGF联合IGF-1对山羊有腔卵母细胞体外成熟的影响。结果表明: (1)10, 20, 30μg/LEGF对山羊卵母细胞体外成熟无显著影响(P>0.05)。(2) 10μg/L的IGF-1对山羊卵母细胞体外成熟无显著影响(P>0.05); 20, 30μg/L的IGF-1能显著提高山羊卵母细胞体外成熟率 (P<0.05)。(3)添加 10μg/LEGF+20μg/LIGF-1组卵母细胞体外成熟率显著高于添加 20μg/L的IGF-1组(P<0.05),极显著高于添加 10μg/LEGF组和对照组(P<0.01)。可见,EGF和IGF-1对山羊卵母细胞体外成熟有协同作用。     

不同培养液及不同培养时间对犬卵母细胞体外成熟的影响

试验采集犬卵巢,用切割法回收卵泡中的卵母细胞,选取胞质均匀、2层或2层以上颗粒细胞、直径≥110μm的卵母细胞移入不同的培养液中:(A)M199+10%FBS+0.5μg/mL FSH+10 IU/mL hCG,(B)M199+0.1%PVA进行体外成熟培养。在培养不同时间(0,48,72 h)用1%醋酸地衣红染色,在显微镜下观察卵母细胞核成熟情况,探索适宜犬卵母细胞体外成熟的培养体系。结果表明:卵母细胞在A液中培养48 h MII期比例(7.84%)高于B液中的比例(P<0.01);在培养72 h MII期比例也是A液中(8.00%)高于B液,虽然二者差异不显著(P>0.05),但72 h卵母细胞退化比例高于48 h(P<0.01)。因此培养液中添加FBS,FSH和hCG有利于犬卵母细胞体外成熟,且在此培养液中培养48 h为最佳成熟时间。     

FSH对牦牛卵母细胞EGF、EGFR表达及其细胞凋亡的影响

旨在研究促卵泡素(FSH)对牦牛卵母细胞体外成熟,以及对表皮生长因子(EGF)、表皮生长因子受体(EGFR)的表达影响和与细胞凋亡的关系。在牦牛卵母细胞体外成熟培养液中添加不同浓度FSH(终浓度分别为0、2、5、10μg·mL~(-1)),体外成熟培养后,运用实时荧光定量PCR(qRT-RCR)和免疫荧光染色技术检测EGF、EGFR表达情况,采用qRT-RCR和蛋白免疫印迹法检测Bax、Bcl-2表达变化。结果表明:1)成熟培养液中加入FSH可提高COCs成熟率,当成熟液中FSH为5μg·mL~(-1)时,成熟率最高,达到76.84%,显著高于其他组(P0.05);2)随着FSH浓度的增加,EGF和EGFR表达逐渐升高,其中在5μg·mL~(-1) FSH组中,EGF和EGFR的表达最高,显著高于其他组(P0.05),而在10μg·mL~(-1)FSH组中,EGF和EGFR的表达水平降低;EGF主要位于卵丘细胞中,而EGFR在卵丘细胞和卵母细胞均有表达;3)随着FSH浓度的增加,Bax和Bcl-2的表达显示出明显的逆向模式,Bax的表达水平逐渐降低,Bcl-2的表达水平逐渐升高,在5μg·mL~(-1) FSH组中Bax的表达量最低,Bcl-2的表达量最高,而在10μg·mL~(-1)FSH组中,Bax的表达水平升高。综上表明,在牦牛卵母细胞体外成熟过程中,FSH提高了卵母细胞发育能力,诱导EGF和EGFR表达,而且可能通过调控Bax、Bcl-2等凋亡相关因子的表达,抑制卵母细胞凋亡。     

不同基础培养液对猪卵母细胞体外成熟的影响

为了研究不同体外基础培养液对猪卵母细胞体外成熟的影响,采用回归分析和方差分析的方法。结果表明:在NCSU-23+10%胎牛血清(FBS)和NCSU-23+10%胎牛血清(FBS)+10%猪卵泡液(PFF)中培养的卵母细胞成熟效果最好,PFF与2种成熟液组合的成熟效果明显高于FBS和空白对照组(P0.05)。以NCSU-23作为基础培养液成熟效果要比TCM-199好,但差异不显著(P0.05);在成熟液中加入EGF+IGF-1的成熟率(84.11%)最高,表皮生长因子(EGF)对猪卵母细胞的成熟效果比IGF-1明显,但是不如两者的联合使用效果好。     

多种激素对小鼠卵母细胞体外成熟的影响

为了探讨促卵泡素(FSH)、人绒毛膜促性膜激素(HCG)、雌二醇(E_2)三种激素和丙酮酸钠在卵母细胞体外成熟培养液中的最佳添加浓度,深入研究小鼠卵母细胞体外成熟的规律,进而完善小鼠卵母细胞体外成熟培养体系,试验对小鼠卵母细胞进行体外培养,在完全培养基中单独或组合添加FSH、HCG、E_2三种激素,并在此基础上添加不同浓度的丙酮酸钠,分别测定第一极体(PB1)排出率和自然裸卵细胞(NO)死亡率。结果表明:小鼠卵母细胞经体外培养14 h后,在完全培养基中只添加FSH,1 IU/mL FSH试验组的卵母细胞体外成熟率最高,PB1排出率为52.50%, NO死亡率为15.00%;在只添加HCG以及1 IU/mL FSH和HCG组合添加试验中, 1 IU/mL HCG试验组卵母细胞体外成熟率最高,PB1排出率分别为35.70%和53.83%,NO死亡率分别为67.50%和25.00%;在只添加E_2试验中,1μg/mL E_2试验组卵母细胞体外成熟率最高,PB1排出率为47.00%,NO死亡率为0;在添加1 IU/mL FSH、1 IU/mL HCG和1μg/mL E_2的条件下添加不同浓度的丙酮酸钠,0.04 mg/mL丙酮酸钠试验组卵母细胞体外成熟率最高,PB1排出率为75.80%,NO死亡率为11.37%。说明在完全培养基中添加1 IU/mL FSH、1 IU/mL HCG、1μg/mL E_2和0.04 mg/mL丙酮酸钠,对小鼠卵母细胞PB1排出的促进作用最好,对NO体外成熟培养效果最好。     

Intracytoplasmic Glutathione Levels in Heifer Oocytes Cultured in different Maturation Media and its Effect on Embryo Development

The present study was carried out to study the effect of different maturation media on embryo development of heifer oocytes and on their glutathione (GSH) synthesis during in vitro maturation (IVM). Immature heifer oocytes were matured in parallel in one of four maturation media: (i) Tissue Culture Medium (TCM)-199 supplemented with 10 ng/ml of epidermal growth factor (EGF); (i) TCM-199 supplemented with 10 ng/ml of EGF plus 1 microg/ml of FSH; (iii) TCM-199 supplemented with 10% of foetal bovine serum (FBS) and (iv) TCM-199 supplemented with 10% of FBS plus 1 microg/ml of FSH. Cow oocytes were used as control and were matured in TCM-199 supplemented with 10 ng/ml of EGF. No differences were observed in blastocyst rate among the different heifer oocyte groups (8.8, 7.5. 8.4 and 6.8%, respectively) however, the percentage of blastocysts obtained from cow oocytes was significantly higher (30%; p < 0.01) than those obtained from heifer oocytes. De novo GSH synthesis during oocyte maturation of heifer and cow oocytes was detected. No significant differences in intracytoplasmic GSH levels were observed among the experimental heifer oocyte groups or between heifer and cow oocytes both before and after IVM. In conclusion, the blastocyst yield obtained from heifer oocytes was lower than that from cow oocytes and this fact could not be explained by significant differences in intracytoplasmic GSH contents of oocytes before or after IVM.     

Effect of Human Menopausal Gonadotropin,17β-estradiol and Epidermal Growth Factor on the Quality of in vitro Maturation Bovine Oocytes

For optimizing in vitro maturation system of bovine oocytes,we firstly examined the influence of four different hormonal regimes(FSH+LH,HMG,FSH+LH+E₂ and HMG+E₂) on oocyte maturation rates.Then we studied the effects of epidermal growth factor (EGF) in the above defined medium on bovine oocyte maturation,in vitro development and quality of parthenogenetic embryos.The cell apoptotic index of parthenogenetic blastocysts was detected by TUNEL.No significant difference was observed in maturation rates in four groups supplemented with different hormones.However,human menopausal gonadotropin (HMG) provided steady maturation results in replicates.Maturation of oocytes was promoted by supplementation with 17β-estradiol (E₂).Combination of HMG and E₂ gave rise to steady and efficient mature results.The presence of EGF at 30 ng/mL concentration significantly increased maturation rate and blastocyst rate and reduced apoptotic cells in parthenogenetic blastocysts.Therefore,the optimal oocyte maturation solution could be supplemented with 0.075 IU/mL HMG,1 μg/mL E₂ and 30 ng/mL EGF.     

Changes in Zona Pellucida Surface after in vivo and in vitro Maturation of Caprine Oocytes

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The zona pellucida (ZP) surface features of ovulated, inmature and in-vitro -matured goat oocytes were evaluated by scanning electron microscopy. The in vitro maturation (IVM) process of the ZP surface of oocytes from prepubertal and adult goats were also compared. Ovulated oocytes were collected from superovulated adult goats. Immature oocytes were recovered from slaughterhouse ovaries of prepubertal and adult goats. In-vitro -matured oocytes from adult and prepubertal goats were obtained after culture in TCM199 supplemented with 20% oestrous goat serum + 10 μg/ml FSH + 10 μg/ml LH + 1 μg/ml estradiol 17β for 27 h at 38.5°C in 5% CO₂ in air. All oocytes were fixed in 2.5% glutaraldehyde and postfixed in 1% osmium tetroxide. Before IVM, the ZP surface of immature oocytes showed a rough surface with tight holes (Type I ZP). After the maturation process, the ZP surface acquired a lattice-like appearance with the outermost layer characterized by the presence of shallower large holes (Type II ZP) . A higher percentage of oocytes showing the mature type II ZP surface was observed in ovulated than in in-vitro -matured oocytes (82.6 versus 56.7%, respectively, p < 0.05). No significant differences were observed in ZP surface features when the IVM process of oocytes (immature and in-vitro -matured oocytes) from adult and prepubertal females was compared. These results show that the morphology of the ZP surface is related to the oocyte maturity in caprine. The IVM process gives rise to an adequate and similar development of the ZP surface in oocytes from adult and prepubertal goats.     

In Vitro Maturation of Dromedary (Camelus dromedarius) Oocytes: Effect of Different Protein Supplementations and Epidermal Growth Factor*

The present experiment was aimed to compare the effect of different protein supplementation sources, foetal calf serum (FCS), oestrous dromedary serum (EDS) and BSA, in experiment 1, and the effect of different concentrations of epidermal growth factor (EGF), in experiment 2, on in vitro nuclear maturation of the dromedary oocytes. Cumulus oocyte complexes (COCs) were harvested from the ovaries collected from a local slaughterhouse by aspirating the visible follicles in PBS supplemented with 5% FCS. Pooled COCs were randomly distributed to 4‐well culture plates containing 500 μl of the maturation medium and cultured at 38.5°C in an atmosphere of 5% CO₂ in air for 32–36 h. The basic maturation medium consisted of TCM‐199 supplemented with 0.1 mg/ml L‐glutamine, 0.8 mg/ml sodium bicarbonate, 0.25 mg/ml pyruvate, 50 μg/ml gentamicin, 10 μg/ml bFSH, 10 μg/ml bLH and 1 μg/ml estradiol. In experiment 1, this medium was supplemented with 10% FCS, 10% EDS or 0.4% BSA, whereas in experiment 2, it was supplemented with 0.4% BSA and 0, 10, 20 or 50 ng/ml of EGF. The oocytes were fixed, stained with 1% aceto‐orcein stain and their nuclear status was evaluated. Oocytes were classified as germinal vesicle, diakinesis, metaphase‐I, anaphase‐I (A‐I), metaphase‐II (M‐II) and those with degenerated, fragmented, scattered, activated or without visible chromatin as others. There was no difference (p > 0.05) observed in the proportion of oocytes reaching M‐II stage between the media supplemented with FCS (71.5 ± 4.8), EDS (72.8 ± 2.9) and BSA (72.7 ± 6.2). In experiment 2, a higher proportion (p < 0.05) of oocytes reached M‐II stage when the medium was supplemented with 20 ng/ml of EGF (81.4 ± 3.2) when compared with the media supplemented with 10 ng/ml (66.9 ± 4.1) and control (67.2 ± 7.1) groups. It may be concluded that the maturation media for dromedary camel oocytes can be supplemented with any of the three protein sources, i.e. FCS, EDS and BSA without any significant differences on the maturation rates. Also, a supplementation of 20 ng/ml of EGF in the maturation medium seems to be optimal and improves the nuclear maturation of dromedary camel oocytes.     

Optimizing the Conditions for In Vitro Maturation and Artificial Activation of Sika Deer (Cervus nippon hortulorum) Oocytes

With the goal of establishing experimental protocols for cloning sika deer, various conditions for in vitro maturation (IVM) and artificial activation of sika deer oocytes were examined. In vitro maturation was evaluated in seven different culture media. The highest rate of oocyte maturation was 75.4% in 10 μg/ml follicle‐stimulating hormone (FSH), 1 μg/ml LH, 0.2 mm cysteamine and 50 ng/ml epidermal growth factor (EGF) after 24 h of IVM. The efficiency after 24 h of IVM did not differ significantly (p > 0.05) from that observed after 20 h. Cysteamine (0.2 mm ) significantly increased the maturation rates after 20 h (from 59.1% to 67.2%, p < 0.05) and after 24 h (from 63.2% to 71.6%, p < 0.05) of IVM. The IVM rates of oocytes collected during the oestrous season (75.4%) and the anoestrous season (23.3%) were significantly different at 24 h. The 20 μg/ml FSH, 2 μg/ml LH, 0.4 mm cysteamine and 100 ng/ml EGF significantly increased the maturation rates (from 23.3% to 54.2%, p < 0.01) at 24 h during the anoestrous season. For the activation experiments, the most effective method was chemical activation [ionomycin + 6‐dimethylaminopurine (6‐DMAP)], which promoted the development of sika deer oocytes to the blastocyst stage (32.4%). Our results indicate that in vitro matured sika deer oocytes are good candidates for parthenogenetic activation and that chemical treatment is needed for relatively efficient activation of the oocytes. These optimized conditions for IVM and parthenogenetic activation may be useful for efforts to restore populations of the endangered sika deer using the somatic cell nuclear transfer technique.     

The efficiency of in vitro ovine embryo production using an undefined or a defined maturation medium is determined by the source of the oocyte

In vitro oocyte maturation can be influenced by oocyte source and maturation media composition. The aim of the present study was to compare the efficiency of a defined in vitro maturation medium (TCM199 supplemented with cysteamine and epidermal growth factor; Cys + EGF) with an undefined medium (TCM199 supplemented with follicle-stimulating hormone and follicular fluid; FSH + FF) for in vitro production (IVP) of ovine embryos, using oocytes obtained by laparoscopic ovum pick-up from FSH-stimulated [n=11; 158 cumulus-oocyte complexes (COCs)] and non-stimulated (n=16; 120 COCs) live ewes, as well as abattoir-derived oocytes (170 COCs). The produced blastocysts were vitrified and some of them were transferred to synchronized recipients. The best and the worst final yields of embryo IVP observed in this study were obtained using oocytes from FSH-stimulated ewes matured in FSH + FF (41.3%; 33/80) and in Cys + EGF (19.2%; 15/78) medium, respectively (p<0.01). No significant differences between both media were attained in the blastocyst development rate or in the final yield of embryo IVP using oocytes from non-stimulated ewes or abattoir-derived oocytes. The overall in vivo survival rate of the transferred vitrified blastocysts was 13.1% (8/61), without significant differences between oocyte sources or maturation media. In conclusion, under the experimental conditions of the present study, TCM199 supplemented with cysteamine and EGF is a convenient defined maturation medium for IVP of embryos from oocytes of live non-stimulated ewes or from oocytes of abattoir-derived ovaries. However, the best final yield of embryo IVP observed in this study was attained when oocytes came from FSH-stimulated donors and TCM199 was supplemented with FSH and follicular fluid.     

Time Course of In Vitro Maturation of Compact Cumulus Horse Oocytes after Roscovitine‐Induced Meiotic Inhibition: Effects on the Coordination Between Nuclear and Cytoplasmic Maturation

This study was carried out to evaluate the usefulness of a pre‐maturation step in improving the coordination between cytoplasmic and nuclear maturation of horse compact cumulus oocytes by the addition of roscovitine (ROSC). Oocytes were collected by scraping and pre‐cultured for 18 h in a maturation medium TCM199 supplemented with pyruvate, LH, FSH, insulin growth factor (IGF), epidermal growth factor (EGF), insulin, transferrin and selenium (IVM‐ROSC) or in a simple medium (M199‐ROSC). After pre‐maturation, oocytes from both the groups were in part denuded and fixed‐stained and in part in vitro matured to assess the kinetic of in vitro maturation (IVM). The nuclear progression and the cytoskeletal organization of microfilaments and cortical granules (CG) of treated and untreated oocytes were assessed by fluorescent probes. Oocytes immediately fixed after recovery and oocytes pre‐cultured in M199‐ROSC for 18 h did not show metaphase II (MII) plates, whereas in IVM‐ROSC group, 6/69 oocytes (8.7%) showed MII plates. After inhibition, during maturation kinetics at 11, 18 and 29 h, maturation rate of M199‐ROSC group progressively increased and at 29 h of IVM, reached the maturation rate of control group (13/66, 19.7% vs 31/125, 24.8%). No statistically significant differences in cytoplasmic maturation were found. The number of MII plates after 29 h of IVM, was significantly higher (p < 0.05) in IVM‐ROSC group (34/90) compared with M199‐ROSC (13/66) and control groups (31/125) as well as the number of oocytes with microfilaments and CG distributed in cortical region (25/34 vs 3/13 and 7/31 respectively). Our results showed that pre‐culturing in the presence of Roscovitine in a fully supplemented maturation medium containing gonadotropins and growth factors partially suppressed the meiotic maturation, but established a more suitable environment for improving cytoplasmic maturation of horse compact cumulus oocytes as defined by microfilaments and CG configuration.     

Oocyte holding in the Iberian red deer (Cervus elaphus hispanicus): Effect of initial oocyte quality and epidermal growth factor addition on in vitro maturation

Current in vitro embryo production protocols in the Iberian red deer (Cervus elaphus hispanicus) need to be optimized; oocyte harvesting in situ followed by overnight holding could reduce the human effort and shipping costs. In our work, post‐mortem ovaries were retrieved, and the oocytes were harvested and allocated to G1 group (good quality) or G2 + G3 group (low quality). The oocytes were separately subjected to immediate in vitro maturation (IVM) or held overnight in a holding medium composed of 40% of TCM 199 with Earle's salts, 40% TCM 199 with Hanks' salts and 20% fetal bovine serum (FBS), at room temperature (16 hr). In vitro maturation was carried out in a basal medium supplemented or not with 50 ng/ml of epidermal growth factor (EGF). Our data showed that addition of EGF to the maturation medium increases the percentage of G1 oocytes reaching metaphase II (3.9% vs. 50%, basal vs. EGF; p < .001) and decreased their degeneration rate (69.9% vs. 22.2%, basal vs. EGF; p < .01) when oocytes were immediately matured. Overnight holding increased the meiotic competence of G1 oocytes (37.5% matured in basal medium) and EGF increased prophase arrest in G2 + G3 oocytes (16.1% vs. 38.8% in germinal vesicle [GV] stage in basal medium vs. EGF added medium; p < .05). Our data demonstrate that oocyte holding can be used in Iberian red deer oocytes. Interestingly, EGF addition increases the oocytes' meiotic competence in immediately matured oocytes but not after oocyte holding depending upon initial oocyte quality.