All‐trans retinoic acid improves goat oocyte nuclear maturation and reduces apoptotic cumulus cells during in vitro maturation

All‐trans retinoic acid (t‐RA) is a natural component and representative physiologically active metabolite of vitamin A, having multiple physiologic functions. The objective of this study was to evaluate the effect of t‐RA on goat oocyte maturation and cumulus cell apoptosis during in vitro maturation (IVM). Immature goat cumulus‐oocyte complexes (COCs) were matured in vitro in the absence or presence of t‐RA at concentrations of 10 nmol/L, 100 nmol/L and 1000 nmol/L. Oocyte maturation and embryo development were assessed by polar body formation and parthenogenetic activation, respectively. Oocyte survival was checked by Trypan blue staining. Apoptosis of cumulus cells was analyzed by terminal deoxynucleotidyl transferase nick end labeling staining and quantitative real‐time PCR. In comparison with the control group, 100 nmol/L and 10 nmol/L t‐RA significantly improved goat nuclear oocyte maturation and survival (P < 0.05). Addition of 1000 nmol/L t‐RA improved nuclear maturation (P < 0.05), but had no effect on survival of goat oocytes. t‐RA had no positive effect on goat parthenogenetic embryonic cleavage, blastocyst formation or total cell numbers. However, t‐RA inhibits the apoptosis of cumulus cells (P < 0.01). t‐RA treatment up‐regulated the expression of B‐cell lymphoma 2 (BCL‐2), catalase (CAT) (P < 0.05) and down‐regulated the expression of Caspase‐8 (P < 0.05). In conclusion, t‐RA has positive effects on goat oocyte nuclear maturation and reduces apoptotic cumulus cells during IVM.     

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。...     

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     

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。     

牦牛卵母细胞的体外成熟

以屠宰场牦牛卵巢为材料,比较了抽吸加切割法和抽吸法2种卵母细胞离体采集方法的效率和5种成熟培养液的培养效果,并研究了卵泡位置、形态对卵母细胞体外成熟的影响。结果表明:在牦牛乏情期,平均每个卵巢用抽吸加切割法回收卵数极显著高于抽吸法(9.33±4.30VS4.70±2.62,P<0.01),可用卵数也极显著高于抽吸法(5.63±4.19VS4.37±2.32,P<0.01)。将牦牛卵丘卵母细胞复合体(COCs)分别置于5种成熟培养液中培养,其中M199(缓冲体系为Earles盐) 10% FBS 5.0mg/LLH 1.0mg/LE2 双抗(青霉素100IU/mL和链霉素100mg/L)的成熟液效果最好,成熟率为81.33%,卵裂率为49.33%。来自卵巢表面卵泡的COCs的成熟率和卵裂率均高于来自卵巢内卵泡的COCs(分别为81.33%VS69.33%,P>0.05;49.33%VS34.67%,P<0.05)。来自明亮卵泡的COCs的成熟率和卵裂率均极显著高于来自浑浊卵泡的COCs(分别为81.33%VS33.33%,P<0.01;49.33%VS3.33%,P<0.01)。     

In vitro maturation and fertilization of prepubertal and pubertal black Bengal goat oocytes

Oocytes retrieval, in vitro maturation (IVM) and fertilization (IVF) efficiency are inevitable steps towards in vitro production of embryos. In the present study, these parameters were investigated in the ovaries of prepubertal (n = 31) and pubertal (n = 61) black Bengal goats obtained from a slaughterhouse. Nuclear maturation was evaluated upon aspiration and following IVM in TCM-199 (Earle''s salt with L-glutamine and sodium bicarbonate) for 27 h at 39℃ under 5% CO₂ in humidified air. The oocytes retrieval and efficiency (mean ± SD) per prepubertal and pubertal goats were 5.2 ± 0.6 and 6.8 ± 0.6, and 77.3 ± 0.1% and 80.5 ± 0.6%, respectively. Anaphase I - telophase I stages differed significantly (7.3 ± 0.8 vs. 2.6 ± 0.2, p < 0.05) between the two groups of goats. After IVM, the percentages of metaphase II were significantly higher (66.3 vs. 60.3, p < 0.05) in pubertal goats than in their prepubertal counterparts. The percentages of normal in vitro fertilization (IVF) in Fert-Tyrode''s albumin lactate pyruvate of pubertal goat oocytes did not differ between Percoll and swim-up sperm separation methods (36.7 ± 0.9% vs. 32.7 ± 1.3%, p > 0.05). Furthermore, sperm capacitation by heparin alone or in combination with ionomycin did not lead to a significant increase in the normal fertilization rate (34.8 ± 1.7 vs. 32.2 ± 1.5%, respectively) in the oocytes of pubertal goats. In conclusion, the ovaries of pubertal black Bengal goats obtained from the slaughterhouse could be used for in vitro embryo production. However, further optimization of the IVM and IVF techniques are necessary for satisfactory in vitro embryo production.     

牦牛卵巢卵母细胞体外培养成熟条件的建立

研究了不同采集方法和不同培养液对牦牛卵母细胞体外成熟的培养效果。结果:在牦牛乏情期,每个卵巢平均回收卵数为(9.33±4.30),可用卵数为(5.63±4.19)。将牦牛卵丘卵母细胞复合体分别置于5种成熟培养液中培养,成熟率分别为75.56%、71.11%、81.33%、77.33%和77.78%,卵裂率分别为42.22%、33.33%、49.33%、46.67%和42.22%,其中C的成熟液效果最好。来自卵巢表面卵泡的COCs的成熟率(81.33(vs69.33(,P>0.05)高于来自卵巢内卵泡的COCs,卵裂率(49.33%vs34.67%,P<0.05)显著高于来自卵巢内卵泡的COCs。来自明亮卵泡的COCs的成熟率(81.33%vs33.33%,P<0.01)和卵裂率(49.33%vs3.33%,P<0.01)极显著高于来自混浊卵泡的COCs。     

In vitro maturation using αMEM with reduced NaCl enhances maturation and developmental competence of pig oocytes after somatic cell nuclear transfer

BackgroundCompared to medium containing 108 mM sodium chloride (NaCl), in vitro maturation (IVM) using a simple medium with reduced (61.6 mM) NaCl increases the cytoplasmic maturation and embryonic development of pig oocytes.ObjectivesThis study determines the effect of a complex medium containing reduced NaCl on the IVM and embryonic development of pig oocytes.MethodsPig oocytes were matured in Minimum Essential Medium Eagle-alpha modification (αMEM) supplemented with 61.6 (61αMEM) or 108 (108αMEM) mM NaCl, and containing polyvinyl alcohol (PVA) (αMEMP) or pig follicular fluid (PFF) (αMEMF). Medium-199 (M199) served as the control for conventional IVM. Cumulus cell expansion, nuclear maturation, intra-oocyte glutathione (GSH) contents, size of perivitelline space (PVS), and embryonic development after parthenogenesis (PA) and somatic cell nuclear transfer (SCNT) were evaluated after IVM.ResultsRegardless of PVA or PFF supplementation, oocytes matured in 61αMEM showed increased intra-oocyte GSH contents and width of PVS (p < 0.05), as well as increased blastocyst formation (p < 0.05) after PA and SCNT, as compared to oocytes matured in 108αMEMP and M199. Under conditions of PFF-enriched αMEM, SCNT oocytes matured in 61αMEMF showed higher blastocyst formation (p < 0.05), compared to maturation in 108αMEMF and M199, whereas PA cultured oocytes showed no significant difference.ConclusionsIVM in αMEM supplemented with reduced NaCl (61.6 mM) enhances the embryonic developmental competence subsequent to PA and SCNT, which attributes toward improved oocyte maturation.     

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.     

冷冻方法和冷冻保护液对山羊卵母细胞发育的影响

比较了不同冷冻方法和冷冻保护液对山羊卵母细胞冷冻-解冻后体外受精和激活后的发育效果.结果表明,开放式拉长塑料细管(OPS)法的效果好于玻璃化细管法和常规冷冻法;在OPS法中,20%EG 20%DMSO对卵母细胞的冷冻保护效果好于EDFS40,而在玻璃化细管法冷冻中,则是EDFS40好于20%EG 20%DMSO.对于常规冷冻法而言,则是1.5mol/L EG的冷冻效果好于1.5mol/L PROH.     

CK1 inhibitor affects in vitro maturation and developmental competence of bovine oocytes

The objectives of present study were to evaluate the effect of casein kinase 1 (CK1) inhibition D4476 on in vitro maturation (IVM) and developmental competence of bovine oocytes. The cumulus oocyte complexes (COCs) were cultured in maturation medium with D4476 (0, 2, 5, 10, 20 μM) for 24 hr. After IVM and in vitro fertilization, through expansion average scores of cumulus cells (CCs), oocyte maturation efficiency, cleavage rate and blastocyst rate of zygote, we found 5 μM D4476 could increase the development potential of oocytes. After the COCs were treated with 5 μM D4476, the results of quantitative real‐time PCR analysis, Lichen red staining and PI staining showed that under without affecting germinal vesicle breakdown and nuclear morphology, D4476 could significantly decrease CK1 and upregulate TCF‐4 in oocytes. Furthermore, without influencing the level of Bad and CTSB, D4476 could significantly increase the expression of β‐catenin, TCF‐4, Cx43, MAPK, PTGS‐2, PTX‐3, TGS‐6, Bax and Bcl‐2 in CCs. Western blot analysis revealed that the addition of 5 μM D4476 during the maturation of COCs resulted in a lower level of Cx43 protein at 12 hr and a higher expression of Cx43 protein at 24 hr compared to the group without D4476. These results indicate that adding optimum D4476 (5 μM) to maturation medium is beneficial to maturity efficiency and development competence of bovine oocytes.     

Modification of maturation condition improves oocyte maturation and in vitro development of somatic cell nuclear transfer pig embryos

This study examined effects on the developmental competence of pig oocytes after somatic cell nuclear transfer (SCNT) or parthenogenetic activation (PA) of : 1) co-culturing of oocytes with follicular shell pieces (FSP) during in vitro maturation (IVM); 2) different durations of maturation; and 3) defined maturation medium supplemented with polyvinyl alcohol (PVA; control), pig follicular fluid (pFF), cysteamine (CYS), or β-mercaptoethanol (β-ME). The proportion of metaphase II oocytes was increased (p < 0.05) by co-culturing with FSP compared to control oocytes (98% vs. 94%). However, blastocyst formation after SCNT was not improved by FSP coculture (9% vs. 12%). Nuclear maturation of oocytes matured for 39 or 42 h was higher (p < 0.05) than that of oocytes matured for 36 h (95-96% vs. 79%). Cleavage (83%) and blastocyst formation (26%) were significantly higher (p < 0.05) in oocytes matured for 42 h than in other groups. Supplementation of a defined maturation medium with 100 µM CYS or 100 µM β-ME showed no stimulatory effect on oocyte maturation, embryo cleavage, or blastocyst formation after PA. β-ME treatment during IVM decreased embryo cleavage after SCNT compared to pFF or PVA treatments, but no significant difference was found in blastocyst formation (7-16%) among the four treatment groups. The results indicated that maturation of oocytes for 42 h was beneficial for the development of SCNT embryos. Furthermore, the defined maturation system used in this study could support in vitro development of PA or SCNT embryos.     

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.     

牛有腔卵泡卵母细胞体外成熟的研究

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 )。     

Kaempferol alleviates the reduction of developmental competence during aging of porcine oocytes

Kaempferol (KAE) is a natural flavonoid present in different plant species and exhibits anti‐inflammatory, antioxidant, and anticancer therapeutic properties. In the present study, we investigated the influence and underlying mechanisms of KAE supplementation on porcine oocytes during in vitro aging. The results show that KAE treatment can alleviate the aging‐related reduction of developmental competence. We observed that the blastocyst production rate in aged oocytes treated with 0.1 μM KAE was significantly higher than in untreated aging oocytes (36.78 ± 0.86% vs. 27.55 ± 2.60%, respectively, p < .05). The KAE‐treated aging oocytes had significantly reduced levels of reactive oxygen species (p < .05). Furthermore, the mRNA levels of the embryonic pluripotency‐related genes Oct4, NANOG, and ITGA5 were significantly increased in blastocysts derived from KAE‐treated oocytes (p < .05). During excessive oocyte culture, KAE treatment maintained the mitochondrial membrane potential and reduced apoptosis; however, this was not observed in untreated aging oocytes. In conclusion, our results suggest that KAE treatment can alleviate the aging of porcine oocytes by reducing oxidative stress and improving mitochondrial function.     

水牛卵母细胞在不同培养液中成熟与凋亡的比较研究

为了解卵母细胞体外成熟与凋亡过程,进而提高卵母细胞体外成熟率,本试验研究了在培养液中添加不同浓度的表皮生长因子(EGF)、胰岛素样生长因子-1(IGF-1)对水牛卵母细胞体外成熟和凋亡的影响。结果表明:(1)添加各种浓度的EGF(10,20,30,50,100 ng/mL)均可以提高水牛卵母细胞的成熟率,降低卵母细胞的凋亡率,其中50 ng/mL EGF有显著影响(P<0.05);(2)添加各种浓度的IGF-1(10,30,50,100 ng/mL)均能提高水牛卵母细胞体外成熟率,降低卵母细胞的凋亡率,以30 ng/mL效果明显(P<0.05);(3)添加20 ng/mL EGF+30ng/mL IGF-1组卵母细胞体外成熟率和凋亡率分别高于和低于单独添加IGF-1和EGF组。     

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.     

PS48 promotes in vitro maturation and developmental competence of porcine oocytes through activating PI3K/Akt signalling pathway

Oocyte maturation plays a vitally important role in porcine reproduction. Regrettably, the quality of oocytes matured in vitro is weaker than that of in vivo matured oocytes. We collected and cultivated porcine cumulus oocyte complexes (COCs) in vitro with phosphoinositide-dependent kinase 1 (PDK1) activator 5-(4-chloro-phenyl)-3-phenyl-pent-2-enoic acid (PS48), whose concentrations were 0, 2, 5, 10 and 20 µM to investigate whether the phosphatidylinositol-3-kinase/protein kinase B (PI3K/Akt) signalling pathway would impact the oocyte quality. The results showed that 10 µM PS48 increased the oocyte proportion of metaphase II (MII) stage and improved the expansion of cumulus cells (CCs). What's more, the activation of PI3K/Akt signalling pathway could regulate the expression of maturation-related genes and proteins. The results of quantitative real-time PCR showed that 10 µM PS48 increased the mRNA and protein levels of Akt and regulated maturation-related genes, including cyclin B1, MOS, BMP15, GDF9, CDC2, mTOR, BAX, BCL2 and caspase-3. The results of Western blot indicated that 10µM PS48 increased the protein abundance of Akt, phosphorylation of Akt Thr308 (p-Akt^Thr308) and cyclin B1, but decreased the protein abundance of pro-apoptotic BAX. These results suggested that adding 10 µM PS48 to mature culture medium could promote the maturation of porcine oocytes, potentially through activating the PI3K/Akt signalling pathway.