Chromatin Configurations in the Ferret Germinal Vesicle that Reflect Developmental Competence for In Vitro Maturation

In several mammalian species, the configuration of germinal vesicle (GV) chromatin correlates with the developmental competence of oocytes. Yet, no study has been published on the configuration of GV chromatin in ferret, nor is it known whether a specific configuration predicts meiotic competence in this species, in spite of the potential importance of ferret cloning to the study of human disease and to species conservation efforts. Here, we report on an analysis of the chromatin configuration in ferret GV oocytes and on how they correlate with meiotic development. Three distinct configurations were identified based on the degree of chromatin condensation: (1) fibrillar chromatin (FC), featuring strands of intertwined chromatin occupying most of the visible GV region; (2) intermediate condensed chromatin (ICC), characterized by dense, irregular chromatin masses throughout the GV; and (3) condensed chromatin (CC), which is highly compact and centered around the nucleolus. We also found that chromatin configuration was related to the extent of association with cumulus cells in cumulus–oocyte complexes; CC-configured oocytes were most often surrounded by a compact cumulus layer and also a compact corona but FC-configured oocytes were associated with neither. In addition, increasing chromatin condensation corresponded to an increase in oocyte diameter. Finally, following in vitro culture, significantly more CC-configured oocytes underwent maturation to meiotic metaphase II than did FC- or ICC-configured oocytes. We conclude that, in ferret, chromatin condensation is related to the sequential achievement of meiotic competencies during oocyte growth and differentiation, and thus can be used as a predictor of competence.     

Cryopreservation of Immature Bovine Oocytes to Reconstruct Artificial Gametes by Germinal Vesicle Transplantation

Joining immature gamete cryopreservation and germinal vesicle transplantation (GVT) technique could greatly improve assisted reproductive technologies in animal breeding and human medicine. The present work was aimed to assess the most suitable cryopreservation protocol between slow freezing and vitrification for immature denuded bovine oocytes, able to preserve both nuclear and cytoplasmic competence after thawing. In addition, the outcome of germinal vesicle transfer procedure and gamete reconstruction was tested on the most effective cryopreservation system. Oocytes, isolated from slaughterhouse ovaries, were stored after cumulus cells removal either by slow freezing or by vitrification in open pulled straws. After thawing, oocytes were matured for 24 h in co-culture with an equal number of just isolated intact cumulus enclosed oocytes, and fixed in order to evaluate the stage of meiotic progression and cytoskeleton organization. Our results showed that after warming, vitrified oocytes reached metaphase II (MII) in a percentage significantly higher than oocytes cryopreserved by slow freezing (76.2% and 36.5% respectively, p < 0.05). Moreover, vitrification process preserved the organization of cytoskeleton elements in a higher proportion of oocytes than slow freezing procedure. Therefore vitrification has been identified as the elective method for denuded immature oocytes banking and it has been applied in the second part of the study. Our results showed that 38.3% of oocytes reconstructed from vitrified gametes reached the MII of meiotic division, with efficiency not different from oocytes reconstructed with fresh gametes. We conclude that vitrification represents a suitable method of GV stage denuded oocyte banking since both nuclear and cytoplasmic components derived from cryopreserved immature oocytes can be utilized for GVT.     

甘氨酸对水貂GV期卵母细胞冷冻保存效率的影响

试验旨在探究玻璃化冷冻及培养过程中添加甘氨酸（glycine,Gly）对水貂GV期卵母细胞冷冻解冻后存活率、核发育、线粒体和皮质颗粒分布的影响。试验分为3组：对照组（没有进行冷冻处理）、冷冻组和Gly添加处理组（1 mmol/L Gly）。对玻璃化冷冻解冻后的水貂GV期卵母细胞分别进行平衡恢复3 h和体外成熟培养,采用免疫荧光标记法检测各组GV期卵母细胞线粒体分布的差异及MⅡ期皮质颗粒分布的变化。结果显示,Gly添加处理组卵母细胞在解冻后3 h的存活率与冷冻组相比差异不显著（P>0.05）,但显著低于对照组（P<0.05）;Gly添加处理组卵母细胞的减数分裂恢复率显著高于冷冻组（P<0.05）,但与对照组相比差异不显著（P>0.05）。免疫荧光结果显示,Gly添加处理组的GV期卵母细胞线粒体正常分布率显著高于冷冻组（P<0.05）,但Gly添加处理组和冷冻组的GV期卵母细胞线粒体正常分布率均显著低于对照组（P<0.05）。皮质颗粒分布结果显示,水貂GV期卵母细胞在冷冻后体外成熟培养至MⅡ期时,Gly添加处理组皮质颗粒的正常皮质区分布比例显著高于冷冻组（P<0.05）,但Gly添加处理组与冷冻组的正常皮质区分布比例均显著低于对照组（P<0.05）。结果表明,添加Gly可以提高冻融后水貂卵母细胞的减数分裂恢复率,降低冷冻对其线粒体及皮质颗粒的损失。     

Calves Born after Direct Transfer of Vitrified Bovine In Vitro-produced Blastocysts Derived from Vitrified Immature Oocytes

Vitrification has been the method of choice for the cryopreservation of bovine oocytes, as rapid cooling decreases chilling sensitivity. The aim of this study was to determine the in vitro and in vivo survival and the viability of immature oocytes vitrified using super‐cooled liquid nitrogen. Immature oocytes were randomly allocated to three groups: (i) non‐vitrified control group, (ii) vitrified in normal (?196°C) liquid nitrogen (LN₂) and (iii) vitrified in super‐cooled LN₂ (≤?200°C). Open‐pulled glass micropipettes were used as vitrification containers. Immature oocytes were in vitro‐matured, fertilized and cultured to the blastocyst stage. In vitro viability was assessed by cleavage and blastocyst rates on days 2 and 7 of culture respectively. Vitrified blastocysts derived from the immature vitrified oocytes were directly transferred to synchronous recipients. The in vitro embryo development of vitrified immature oocytes was not influenced by the LN₂ state. After direct transfer (one embryo per recipient) of 16 embryos obtained from immature vitrified oocytes (eight from each vitrified group), two healthy calves were born in each group. These results indicated that vitrification of immature bovine oocytes using glass micropipettes under normal or super‐cooled LN₂, resulted in viable blastocysts and live calves following in vitro embryo production.     

山羊卵母细胞体外成熟的研究

在38℃和39℃不同温度条件下培养山羊卵母细胞,结果表明:在38℃和39℃下成熟率分别为63.6%和57.3%,二者之间差异不显著,说明只要培养温度在山羊正常体温的变动范围内,对其卵母细胞的成熟率没有影响;在38℃温度条件下用10?S的M199培养液成熟率为62.5%,而10 mg/ml BSA的M199培养液成熟率为58.7%,二者之间差异不显著,说明无血清培养可以代替血清培养.     

Effect of Post-IVF Developmental Kinetics on In Vitro Survival of Vitrified-warmed Domestic Cat Blastocysts

A limited number of reports is available on cryopreservation of in vitro fertilization (IVF)‐derived cat blastocysts. In the present study, IVF‐derived domestic cat embryos which reached the blastocyst stage either on day 6 or day 7 were cryopreserved by vitrification using Cryotop as a cryodevice. Fresh control and post‐warm surviving blastocysts were examined by differential cell staining with Hoechst 33342 and propidium iodide to determine total cell number and inner cell mass (ICM) ratio, and the post‐warm survival rate was determined by re‐expansion of the blastocoel during 24 h of in vitro culture. In fresh control, the mean number of total cells of day 7 blastocysts (61.4 cells) tended to be smaller than that of day 6 blastocysts (81.9 cells, p = 0.096). The post‐warm survival rates of day 6 and day 7 blastocysts were not statistically different (73.8%; 31 of 42 vs 66.7%; 18 of 27). There were no significant differences in the total cell number and ICM ratio between fresh control and vitrified blastocysts, although the ICM ratio of surviving day 7 blastocysts was significantly smaller than that of fresh controls (stained at day 8, 18.9% vs 28.9%, p < 0.05). These results indicate that IVF‐derived domestic cat embryos that reached the blastocyst stage earlier can survive the Cryotop vitrification without a reduction in the parameters studied.     

Beneficial Effect of Melatonin on Blastocyst In Vitro Production from Heat‐Stressed Bovine Oocytes

Melatonin may play an important role in protecting gametes and embryos from the potential harmful effects of oxidative stress. In this study, we first examined two different heat stress (HS) treatments for in vitro oocyte maturation (Experiment 1: 38.5 vs 41.0°C, during the first 20 h; Experiment 2: 38.5 vs 41.5°C, during the entire period) on bovine oocyte maturation and embryo development. Second, we tested different melatonin concentrations added to the maturation and culture medium (Experiment 3: 0, 10^?12, 10^?9, 10^?4 m ; Experiment 4: 0, 10^?3 m ), both with and without HS (38.5 or 41.5°C, respectively). In Experiment 1, the HS treatment resulted in a lower maturation rate and number of cells/blastocyst (C/B) and a higher blastocyst rate than that in the control group. In Experiment 2, oocytes/embryos from heat‐stressed oocytes (HSO) had a lower maturation, cleavage and blastocyst rates, as well as a lower C/B compared with the control. In Experiment 3, in HSO groups, 10^?4 m melatonin resulted in an increased blastocyst rate compared with 0 m melatonin, with a similar blastocyst rate to the non‐HSO without melatonin. Melatonin did not have any effect in embryos from non‐HSO groups compared with the control. In Experiment 4, 10^?3 m melatonin produced lower cleavage and blastocyst rates in HSO and lower blastocyst rate in non‐HSO when compared to melatonin‐untreated oocytes/embryos. In conclusion, 10^?4 m melatonin was found to alleviate bovine oocytes from the harmful effects of HS.     

Ultrastructural Characteristics of Non-matured and In Vitro Matured Oocytes Collected from Pre-pubertal and Adult Domestic Cat Ovaries

The present study describes the ultrastructural characteristics of cat oocytes before maturation and after 12- and 24-h in vitro maturation (IVM). Oocytes were recovered from pre-pubertal and adult queen ovaries after ovariohysterectomy and a proportion were stored in glutaraldehyde at 4°C until examination by transmission electronic microscopy (TEM). Those selected for maturation were cultured before TEM in DMEM for 12 and 24 h at 38°C in a humidified environment of 5% O₂, 5% CO₂ and 90% N₂. Specimens were divided into six groups: non-matured oocytes from pre-pubertal queens (PP0), non-matured oocytes from adult queens (A0), 12-h in vitro matured oocytes from pre-pubertal queens (PP12), 12-h in vitro matured oocytes from adult queens (A12), 24-h in vitro matured oocytes from pre-pubertal queens (PP24) and 24-h in vitro matured oocytes from adult queens (A24). Across the treatment groups, it was possible to observe differences in the thickness of the perivitelline space, the penetration of cumulus cell projections forming a junctional complex, distribution and density of small vesicles, lipid droplets, microvilli, mitochondria and cortical granules and variable degrees of development of Golgi complexes. These findings demonstrated that ultrastructural analysis of oocytes matured in vitro is a valuable tool to evaluate oocyte cytoplasmic maturation and that this IVM protocol was efficient in inducing gradual morphological changes necessary for cytoplasmic maturation of pre-pubertal and adult cat oocytes.     

Hematoxylin Staining Reveals a Decrease in Nucleolar Diameter of Pig Oocytes Before Germinal Vesicle Breakdown

During oocyte growth, the morphology of the nucleolus changes into a compact and homogenous structure. The compact nucleoli in full-grown oocytes are not stained by aceto-orcein staining or immunofluorescence staining. In this study, we developed a hematoxylin staining method for pig oocytes in whole-mount preparations to visualize the nucleoli. Nucleoli of growing and full-grown oocytes were stained blue with hematoxylin. Using this staining method, the changes in the oocyte nucleolus during maturation were examined. The nucleolar diameter gradually decreased in maturing oocytes (10.7 ± 0.1 μm to 9.0 ± 0.7 μm, P<0.05) before germinal vesicle breakdown (GVBD). The results suggest that the nucleolar volume of oocytes decreases before GVBD.     

Developmental Capacity In Vitro of Prepubertal Oocytes

A 2-year comparative study was carried out to evaluate the effect of ovary size, follicle size and oocyte quality of 3-month-old Simmental calves and the efficiency of using calf ovaries in an in vitro fertilization (IVF) programme. We evaluated the effects of different concentrations of follicle-stimulating hormone (FSH) and oestradiol-17beta (E-17beta) in the maturation medium on the in vitro development of calf oocytes into morula and blastocysts. The proportion of recovered oocytes (62.1%; 42.8%; 25.3%) and the percentage of good quality cumulus oocyte complexes (84.2%: 59.8%; 45.9%) decreased significantly (P < 0.01) with decreasing ovary size (L, M and S). The rates of two or more cells on Day 2 and of blastocysts on Day 7 and Day 9 were significantly lower (P < 0.01) for calf oocytes (61.5%; 18.9%: 15.9%) compared with those from sexually matured females (70.1%: 32.3%; 22.2%). Calf oocytes. matured in medium supplemented with 20 microg/ml or 10 microg/ml FSH plus 2 microg/ml E-17beta had higher rates of cleavage on Day 2 (64.1% and 64.7%) and blastocysts on Day 7 (24.5% and 22.4%) than the control supplemented with 10 microg/ml FSH (55.6% and 19.2%, respectively). Groups supplemented with 20 microg/ml FSH plus 2 microg/ml E-17beta and 10 mg/ml plus 4 mg/ml E-17beta showed a significantly lower developmental rate of blastocysts on Day 7 (14.6% and 14.5%). High concentrations of E-17beta (4 microg/ml) resulted in a significantly lower development of blastocysts on Day 9 (8.1%) and hatched blastocysts on Day 13 (3.5%) (P < 0.01). We conclude that the proportion of calf oocytes obtained from immature animals and their suitability for IVF are lower than those of cows. Thus, the use of oocytcs from sexually immature females would decrease the relative efficiency of IVF programmes. Supplementation with high concentrations of FSH can improve the maturation and developmental capacity of oocytes from prepubertal calves.     

海狸鼠卵母细胞体外成熟培养初探

实验用ＰＭＳＧ或ＰＭＳＧ＋ＨＣＧ处理或未经激素处理的海狸鼠８只，共获卵巢卵母细胞１３８枚。激素处理对获取卵巢卵母细胞的数量没有影响，而对体外成熟发育至卵丘扩展和半成熟阶段有促进作用。三种不同培养液（Ｗｈｉｔｅｎ＋ＦＣＳ；ＴＣＭ１９９＋ＰＭＳＧ＋ＦＣＳ；ＴＣＭ１９９＋ＨＣＧ＋ＦＣＳ）共培养１２５枚卵母细胞，培养后卵丘扩展率及半成熟率分别为５６．５％，４５．７％，４７．６％和２１．７％，１２．３％，９．５％，以Ｗｈｉｔｅｎ液较高（分别为５６．５％和２１．７％），但只有ＴＣＭ１９９＋ＰＭＳＧ＋ＦＣＳ组有２枚卵母细胞出现第一极体。结果表明海狸鼠卵母细胞与其它啮齿动物的卵母细胞一样，能够在体外培养成熟，完成第一次减数分裂，排出第一极体     

Ghrelin Accelerates In Vitro Maturation of Bovine Oocytes

Ghrelin, apart from its metabolic role, is nowadays considered as a basic regulator of reproductive functions of mammals, acting at central and gonadal levels. Here, we investigated for possible direct actions of ghrelin on in vitro maturation of bovine oocytes and for its effects on blastocyst yield and quality. In experiment 1, cumulus oocyte complexes (COCs) were matured in the presence of four different concentrations of ghrelin (0, 200, 800 and 2000 pg/ml). In vitro fertilization and embryo culture were carried out in the absence of ghrelin, and blastocyst formation rates were examined on days 7, 8 and 9. In experiment 2, only the 800 pg/ml dose of ghrelin was used. Four groups of COCs were matured for 18 or 24 h (C18, Ghr18, C24 and Ghr24), and subsequently, they were examined for oocyte nuclear maturation and cumulus layer expansion; blastocysts were produced as in experiment 1. The relative mRNA abundance of various genes related to metabolism, oxidation, developmental competence and apoptosis was examined in snap‐frozen cumulus cells, oocytes and day‐7 blastocysts. In experiment 1, ghrelin significantly suppressed blastocyst formation rates. In experiment 2, more ghrelin‐treated oocytes matured for 18 h reached MII compared with controls, while no difference was observed when maturation lasted for 24 h. At 18 and 24 h, the cumulus layer was more expanded in ghrelin‐treated COCs than in the controls. The blastocyst formation rate was higher in Ghr18 (27.7 ± 2.4%) compared with Ghr24 (17.5 ± 2.4%). Differences were detected in various genes’ expression, indicating that in the presence of ghrelin, incubation of COCs for 24 h caused over‐maturation (induced ageing) of oocytes, but formed blastocysts had a higher hatching rate compared with the controls. We infer that ghrelin exerts a specific and direct role on the oocyte, accelerating its maturational process.     

影响动物卵母细胞体外成熟的因素

文章就卵母细胞体外成熟的影响因素做了简要的论述。主要介绍了卵母细胞来源、培养系统、激素和生长因子等因素对卵母细胞体外成熟的影响。     

Influence of Cysteamine on In Vitro Maturation, In Vitro and In Vivo Fertilization of Equine Oocytes

The effect of cysteamine on in vitro nuclear and cytoplasmic maturation of equine oocytes collected by transvaginal ultrasound guided follicular aspiration was assessed. Oocytes were matured in vitro with (cysteamine group) or without (control group) cysteamine. The nuclear stage after DNA Hoechst staining, penetration rates after two different in vitro fertilization (IVF) techniques (IVF media with ionophore and Hepes buffer with heparin) and the embryo yield following oocyte intra-oviductal transfer were used as a criterion for assessing nuclear and cytoplasmic maturation, respectively. Contrary to the data described in other domestic species, there was no effect of cysteamine on in vitro nuclear maturation, IVF or in vivo embryonic development under our conditions. Ovum pick up yields (52%) and maturation rates (control group: 47% and cysteamine group: 55%) were similar to those previously reported. From 57 oocytes transferred to the oviduct in each group, the number of embryos collected was 10 (17%) in the control group and five in the cysteamine group (9%). Those two percentages were not statistically different (p > 0.05). No effect of IVF technique was seen on the success rate (6%) in each group.     

马卵母细胞体外成熟的研究进展

随着全球马产业的发展,马发挥的经济价值越来越大。辅助生殖技术有利于发挥优良马匹的潜在价值。马卵母细胞体外成熟(IVM)是辅助生殖技术重要的组成部分,卵母细胞的获取是体外成熟的前提,切刮法能从离体卵巢中获得较多的马卵母细胞,而活体采卵技术(OPU)则能持续地获得卵母细胞,并能较好的保存马卵母细胞的发育能力。扩张型卵母细胞的成熟率高于紧密型卵母细胞,母马的年龄会影响到其卵母细胞的质量。马卵母细胞体外存放较长时间不会影响其发育能力,现在已有较为成熟的体系能使马卵母细胞在体外保存24 h以上而不影响其成熟率。在马卵母细胞成熟体系中常用的基础培养液是M199,添加胎牛血清(FBS)、促卵泡素(FSH)、促黄体生成素(LH)、胰岛素样生长因子-1(IGF-1)等物质能显著提高成熟率,常用培养环境为38~39℃,5%CO2饱和湿度下培养,培养时间30 h。成熟的卵母细胞有扩张的卵丘细胞和极体,且成熟的卵母细胞的细胞骨架及微管结构也会发生变化。本文针对马卵母细胞的采集和体外成熟培养的相关研究进行总结,重点阐述了不同采集技术的回收率以及影响马卵母细胞体外成熟率的关键因素,以期对今后马卵母细胞体外成熟的进一步研究及后期体外受精技术的发展提供借鉴与参考。     

促卵泡素对奶牛卵母细胞体外成熟的影响

为研究促卵泡素（FSH）在奶牛卵母细胞的成熟过程中的促进作用，在牛卵母细胞成熟液中添加0μg／mL、10μg／mL、25／μg／mL、50μg／mL、100μg／mLFSH统计成熟率。结果表明，添加FSH各组与对照组相比，卵母细胞的成熟率均显著提高。而添加10μg／mL、25μg／mL、100μg／mL组卵母细胞的成熟率之间差异不显著，50μg／mL组卵母细胞的成熟率最高。无论是否添加FSH都不会影响到孤雌激活的卵裂率。说明FSH具有促进奶牛卵母细胞成熟的功能。     

影响猪卵母细胞体外成熟的相关物理性因素

猪的研究是生物新技术中新的热点和经济增长点,猪的体外成熟是体外受精、性别控制、克隆及转基因等许多新技术成功与否的前提和关键,有许多物理方面的因素影响到猪的体外成熟。     

不同促性腺激素添加方式对猪卵母细胞生发泡染色质构型和卵母细胞成熟能力的影响

在卵母细胞体外成熟培养过程中,培养基中添加激素与否及其激素添加的先后顺序是影响猪卵母细胞核成熟和质成熟的一个重要因素.本试验将猪卵母细胞分别在FSH→不含激素、FSH→LH、FSH LH不含激素中培养48 h(培养第20～22 h后换液),并于成熟培养的第24 h(未换液)、48 h将卵母细胞进行荧光染色,观察其生发泡内染色质构型及卵母细胞核成熟情况.实验表明:(1)在IVM的前24 h,添加FSH LH组的GVIV期卵母细胞比例低于只添加FSH组,但差异不显著(8.99%比17.19%,P＞0.05);(2)在FSH存在的情况下,IVM的前期和后期添加LH能促进卵母细胞发生GVBD;(3)FSH LH培养24 h后转入不含激素培养基组,卵母细胞的核成熟比率显著高于添加FSH组和先添加FSH培养24 h后转入添加LH组(P＜0.05).     

Energy Status Characteristics of Porcine Oocytes During In Vitro Maturation is Influenced by Their Meiotic Competence

The characteristics of energy status in porcine oocytes as related to their meiotic competence and in vitro maturation were studied. Cycling pubertal gilts in the early luteal to early follicular phases of the ovarian cycle were used as oocyte donors. The oocytes recovered from medium (MF) or small follicles (SF) were considered meiotically more or less competent, respectively. A half of oocytes from each category was matured by the standard protocol. The oocytes were examined before or after maturation by confocal microscopy, a bioluminescent cell assay and Western blotting. Four experiments, each in triplicate, were performed to assess both SF and MF oocytes in terms of metabolic units formed by mitochondria and lipids, ATP and lipid consumption and lipid droplets with adipose differentiation‐related protein (ADRP) expression. The proportion of oocytes with metabolic units, the mean ATP content and the number of lipid droplets per oocyte, and the relative number of lipid droplets with ADRP expression were significantly higher in the MF compared to SF oocytes before maturation. On the other hand, after maturation, there was an increase in the proportion of oocytes with metabolic units and the relative number of lipid droplets with ADRP expression in the SF compared to MF oocytes. In conclusion, specific differences in energy characteristics between porcine oocytes with different meiotic competence were found. Meiotically more competent oocytes are more advanced in terms of energy reserves before maturation, while meiotically less competent oocytes are more active in replenishing energy stores during maturation.     

Addition of Recombinant Human Growth Hormone to In Vitro Maturation Medium of Bovine Oocytes

The aim of this study was to increase the bovine embryonic development rate, adding recombinant human growth hormone (rhGH) to maturation medium of bovine oocytes. Oocytes were matured for 24 h in TCM 199 Earle's salts and five treatments were developed: T1, 0.01 IU/ml of recombinant human follicle stimulating hormone (rhFSH); T2, 0.01 IU/ml of rhFSH + 100 ng/ml of rhGH; T3, 0.01 IU/ml of rhFSH + 1000 ng/ml of rhGH; T4, 100 ng/ml of rhGH; and T5, 1000 ng/ml of rhGH at 39 degrees C and 5% of CO(2) in air and saturated humidity. In vitro fertilization from cumulus-oocyte complexes was conducted in TALP-Fert medium (18-22 h) and spermatozoa were selected by Percoll gradient. Zygotes were incubated in SOFaaci medium in 5% of CO(2) in air, 5% of O(2) at 39 degrees C and saturated humidity for 11 days. There was no statistical difference in cleavage rate and embryo production on day 7 and day 9 among treatments. However, the hatching rate increased significantly in the T4 and T5 treatments (11.0 and 12.8%, respectively), compared with the T1 treatment (4.6%) (p < 0.05). Therefore, the rhGH addition to the oocyte maturation medium showed beneficial effects on the hatching rate of in vitro-produced bovine embryos.