牛卵母细胞体外成熟

卵母细胞体外成熟培养研究主要集中在提高体外成熟卵母细胞支持胚胎发育的能力上。本文主要从成熟机理、卵母细胞的来源、卵母细胞的成熟培养环境及培养时间等方面进行了综述,介绍了目前卵母细胞体外成熟培养的进展情况。     

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.     

牛卵泡液对牛卵母细胞体外成熟及受精胚发育力的影响

研究了牛卵母细胞体外成熟液和胚胎培养液中添加不同浓度的牛卵泡液对其体外成熟率和受精胚发育力的影响。结果表明:添加10%牛卵泡液的实验组,卵母细胞的成熟率与血清对照组没有显著差异(P>0.05);添加10%卵泡液的实验组与血清对照组相比,卵裂率和囊胚率没有显著差异,却显著高于添加5%和20%牛卵泡液的实验组(P<0.01),且各实验组囊胚内细胞数差异不大(P>0.05)。因此,用10%的牛卵泡液可以取代成熟液和胚胎培养液中的血清,并可降低实验成本。     

The influence of follicular factors on the in vitro maturation of bovine oocytes]

Immature oocytes from antral follicles of cattle were tested for the effect of follicular factors on maturation. In vitro maturation was accomplished by use of follicular fluid from small (2--5 mm) and large (above 15 mm) follicles and by addition to the medium of a granulose factor (GF) which had been isolated from the surface of granulosa cells. The parent material, with 84% (72/86) of oocytes at the germinal vesicle stage (GV-S) at the beginning of culturing, could be rated immature. 46% of all oocytes (41/89) had reached telophase I or metaphase II (full maturation) after 24 hours of maturation in hormone-free control medium (TCM 199 + 10% of foetal calf serum). 36% of oocytes (53/84), on the other hand, stayed between GV breakdown (GVBD) and anaphase I (incipient maturation). Full maturation was reached by as little as 14%. GF and follicular fluid from small antral follicles were found to inhibit GVBD in the oocytes. 59% (36/61) or 48% (61/127) of oocytes were blocked at GV stage. Positive determination of maturation inhibiting action of the above follicular components may provide a chance for their target-oriented use in control of the maturation process. The pool of immature oocytes of the ovaries, under such circumstances, might be more systematically utilised for in vitro manipulations.     

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.     

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 )。     

影响黄牛小腔卵泡卵母细胞体外受精及早期胚胎发育的因素

从屠宰场收集黄牛卵巢,取皮质深层卵母细胞进行体外成熟、体外受精和早期胚胎体外培养,分析了影响其效果的因素。结果表明,在成熟培养液中添加FSH(10IU/mL)、HCG(20IU/mL)和17β-E2(1mg/L)对卵母细胞受精后早期胚胎发育能力有极显著促进作用;等量牛卵泡液(BFF)与新生牛血清(NCS)对体外受精胚胎发育效果影响不显著,以15?F为宜;颗粒细胞与输卵管上皮细胞均能显著提高卵母细胞体外成熟受精后早期胚胎的发育率,颗粒细胞 输卵管上皮细胞对克服胚胎阻滞现象效果显著。     

激素和血清对牛卵母细胞体外成熟的影响

卵母细胞的体外成熟直接关系到体外受精胚胎的数量和质量,从而影响到胚胎移植的成功率。本文就促性腺激素、类固醇激素和血清对牛卵母细胞体外成熟的影响,进行了较为详尽的阐述。     

Leptin enhances maturation and development of calf oocytes in vitro

We investigated the effects of leptin on the in vitro maturation (IVM) and development of calf oocytes. Cumulus-oocyte complexes were matured in IVM medium containing 0-100 ng/ml leptin. Experiment 1 showed that exposure of calf oocytes to IVM medium containing 1 or 10 ng/ml leptin significantly increased rates of development to the metaphase II stage compared with the control (81.7 ± 3.0% and 83.3 ± 2.1% for 1 and 10 ng/ml leptin, respectively, vs 64.1 ± 5.1% for control; p < 0.05). Experiment 2 showed that 1 or 10 ng/ml leptin significantly improved cleavage rates after in vitro fertilization when compared to control (58.6 ± 3.3% and 59.3 ± 2.9% for 1 and 10 ng/ml leptin, respectively, vs 48.5 ± 2.6% for control; p < 0.05); in addition, when compared to control medium, the addition of 10 ng/ml leptin to the IVM medium resulted in more presumptive zygotes reaching the 4- to 8-cell stage after 48 h of in vitro culture (30.3 ± 2.3% vs 20.1 ± 2.3%; p < 0.05) and developing into blastocysts after 8 days of culture (20.4 ± 1.6% vs 11.7 ± 1.7%; p < 0.05). Experiment 3 showed that the addition of 1 or 10 ng/ml leptin significantly increased the total number of blastocyst cells on day 8 of culture (114.6 ± 7.8 and 117.4 ± 5.9 for 1 and 10 ng/ml leptin, respectively, vs 92.7 ± 8.3 for control; p < 0.05) and trophectoderm (TE) cells (88.5 ± 5.5 and 90.6 ± 3.7 for 1 and 10 ng/ml leptin, respectively, vs 70.1 ± 5.9 for control; p < 0.05). In summary, these results indicate that the addition of leptin to IVM medium enhances meiotic maturation and embryo development from calf oocytes and improves the quality of embryos derived from these oocytes.     

Exogenous paraoxonase‐1 during oocyte maturation improves bovine embryo development in vitro

Paraoxonase‐1 (PON1) is an enzyme found in serum and follicular fluid that protects cell membrane and circulating lipids against oxidative damage. The aims of this study were to measure the direct effects of recombinant PON1 (rPON1) on bovine oocyte maturation at the molecular level (gene expression) and to measure the carry‐over effects of PON1 on pre‐implantation embryo development in vitro. COCs were submitted to IVM with the addition of 0.0, 0.02, 0.04 and 0.08 mg ml^?1 of rPON1, corresponding to an average PON1 arylesterase enzyme activity of 2.2 ± 0.4, 15.5 ± 1.5, 30.2 ± 3.0 and 57.9 ± 5.0 U ml^?1, respectively. The results indicated that addition of rPON1 during IVM improved embryo development in a dose‐dependent manner as D7 embryo development was 22.2%, 29.4%, 32.2% and 37.0% for the treatment groups, respectively (p = 0.02). In conclusion, addition of PON1 enzyme during IVM exerted dose‐related positive effects on embryo development rates to blastocysts.     

Breed influences on in vitro development of abattoir-derived bovine oocytes

Background

There is a discrepancy in the reproductive performance between different cattle breeds. Using abattoir-derived ovaries and data base information we studied the effects of breed on in vitro fertilization and early embryo development.

Methods

The in vitro developmental competence of oocytes from cattle (n = 202) of Swedish Red (SR), Swedish Holstein (SH) and mixed beef breeds was compared, retrospectively tracing donors of abattoir-derived ovaries using a combination of the national animal databases and abattoir information. Age was significantly lower and carcass conformation score was higher in the beef breeds than in the dairy breeds.Cumulus oocyte complexes (n = 1351) were aspirated from abattoir-derived ovaries from animals of known breed (visual inspection confirmed through databases), age (databases), and abattoir information. Oocytes were matured, fertilized (frozen semen from two dairy bulls) and cultured according to conventional protocols. On day 8, blastocysts were graded and the number of nuclei determined.

Results

Cleavage rate was not different between the breeds but was significantly different between bulls. The percentage of blastocysts on day 8 was significantly higher when the oocyte donor’s breed was beef or SR than SH. There was no significant difference in blastocyst grades or stages between the breeds, but the number of nuclei in day 8 blastocysts was significantly lower in SH compared to the beef.

Conclusions

The use of abattoir-derived ovaries from animals whose background is traceable can be a valuable tool for research. Using this approach in the present study, oocyte donor breed was seen to affect early embryo development during in vitro embryo production, which may be a contributing factor to the declining fertility in some dairy breeds seen today.     

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.     

聚乙烯醇、聚乙烯吡咯烷酮和牛血清白蛋白对牛卵母细胞成熟及体细胞克隆胚胎发育的影响

为了比较不同浓度的聚乙烯醇(PVA)、聚乙烯吡咯烷酮(PVP)、牛血清白蛋白(BSA)对牛卵母细胞体外成熟率、重组胚卵裂率和发育率的影响,试验采用抽吸法回收卵母细胞,用不同的无血清成熟液进行卵母细胞的体外成熟培养,然后对成熟卵母细胞进行核移植、融合、激活以及胚胎的体外培养.结果表明,适合延边黄牛卵母细胞成熟及体细胞克隆胚胎发育的PVA、PVP和BSA的最佳浓度分别是1.0 mg/mL,0.3%和0.4%.     

Effect of modification of ovary preservation solution by adding glucose on the maturation and development of pig oocytes after prolonged storage

Oocytes lose their developmental competence during prolonged storage of the ovary. In the present study, we supplemented the preservation solution for pig ovaries (phosphate buffered saline, PBS) with glucose and preserved the ovaries for 6 h at 25 C. Subsequently, we examined the glucose concentration of the follicular fluid (FF), pH of the FF, survival rate of the granulosa cells, and maturation and developmental competence of oocytes after storage. During storage, the glucose concentration of the FF (2.1 mM), pH of the FF (7.4), and survival rate of the granulosa cells (69.5%) rapidly decreased (glucose concentration: under 1.1 mM; pH: 6.8; and survival rate: 43%). On the other hand, when the preservation solution was supplemented with glucose (15 mM), the glucose concentration of the FF increased and the survival rate of the granulosa cells improved, although the pH of the FF decreased further (from 6.8 to 6.6). In addition, supplementation with glucose significantly improved the rates of oocytes at metaphase II (0 h: 65.0%; 6 h without glucose: 23.8%; and 6 h with glucose: 43.8%) and attenuated the decline in the rates of fertilization and development that resulted from prolonged storage, although there were no significant differences. In conclusion, modification of the preservation solution by the addition of glucose increased the glucose concentration of the FF and improved the rate of maturation of pig oocytes.     

In vitro fertilization and development of in vitro matured bovine follicular oocytes

Bovine follicular oocytes matured in vitro were fertilized in vitro using epididymal spermatozoa from five different bulls and then cultured to the blastocyst stage in vitro. The fertilization rate, based on one pair of pronuclei and presence of one sperm tail, ranged from 55.2 to 64.3%. Embryo development (cleavage to blastocyst stage) ranged from 21.4 to 31.0% of the cultured ova reaching 8 cells at 3 to 4 d after insemination to 1.3 to 3.7% reaching hatched blastocysts at 9 to 10 d. It is concluded that individual variation among bulls is not a significant factor in fertilization and development rates of bovine follicular oocytes when epididymal spermatozoa are used.     

Transporting bovine oocytes in a medium supplemented with different macromolecules and antioxidants: Effects on nuclear and cytoplasmic maturation and embryonic development in vitro

We investigated whether supplementing the medium used to transport bovine oocytes with different macromolecules [foetal calf serum (FCS) or bovine serum albumin (BSA)] or a mixture of antioxidants (cysteine, cysteamine and catalase) affects their nuclear and cytoplasmic maturation and thereby affects their subsequent embryonic development and cryotolerance. Oocytes were transported for 6 hr in a portable incubator and then subjected to standard in vitro maturation (IVM) for 18 hr. The oocytes in the control groups were cultured (standard IVM) for 24 hr in medium containing 10% FCS (Control FCS) or 10% FCS and the antioxidant mixture (Control FCS+Antiox). The intracellular concentrations of reactive oxygen species (ROS) at the end of IVM period were lower in the oocytes subjected to simulated transport in the presence of a macromolecular supplement or the antioxidant mixture than that of the control group (FCS: 0.62 and BSA: 0.66 vs. Control FCS: 1.00, p < .05; and Transp: 0.58 and Transp Antiox: 0.70 vs. Control FCS: 1.00, p < .05). After IVM, the mitochondrial membrane potentials of the transported oocytes were lower than those of the non‐transported oocytes (FCS: 0.41 and BSA: 0.57 vs. Control FCS: 1.00, p < .05; and Transp: 0.48 and Transp Antiox: 0.51 vs. Control FCS: 1.00 and Control Antiox: 0.84, p < .05). The blastocyst formation rates (36.9% average) and the re‐expansion rates of vitrified‐warmed blastocysts (53%, average) were unaffected (p > .05) by the treatments. In conclusion, supplementing the medium in which bovine oocytes are transported with antioxidants or different macromolecules did not affect their in vitro production of embryos or their cryotolerance.     

Long-term effects of in vitro growth of mouse oocytes on their maturation and development

Since very few oocytes grow completely in vivo, in vitro growth (IVG) of ovarian oocytes may provide a new source of functional oocytes. The long-term effects of in vitro maturation (IVM) of oocytes and in vitro culture of fertilized eggs have been reported; however, the effects of IVG of oocytes are unknown. Here in, we report the long-term effects of IVG of oocytes. Ovaries from 1-day-old mice containing non-growing oocytes were cultured for 10 days; the isolated follicles were then cultured for 11 days. Secondary follicles from 10-day-old mice were also cultured for 11 days. The nuclei of oocytes collected from the IVG and Graafiais follicles of adult mice were transferred to enucleated oocytes grown in vivo, respectively. Developmental competence was examined following IVM of the reconstituted oocytes. Chronologically, oocytes of 1-day-old, 10-day-old and adult mice were cultured for 22, 12 and 1 day(s). The result showed that the reconstituted eggs developed into pups at high rates after nuclear transfer and in vitro fertilization (IVF) in all the experimental groups (29-45%). However, the pups from reconstituted eggs containing the nuclei of 22-day cultured oocytes were heavier than the control pups (P<0.05). We concluded that long-term culture of oocytes did not affect their nuclear ability to develop to term; however, fetal growth was affected by the culture duration or culture conditions during the initial phase of follicular growth.     

Recovery of bovine oocytes from small vesicular follicles for in vitro maturation and fertilization

Five dairy and four beef breed, mature cows were used as oocyte donors to develop a system of multiple recovery of oocytes for in vitro maturation and fertilization. The animals were alternately treated with either 20 mg of follicle stimulating hormone (FSH) in four equal intramuscular injections or saline at 12 h intervals starting between days 9 and 13 of the oestrous cycle, and the procedure was repeated at three-week intervals for up to four collections. Eighteen collections resulted in the recovery of 124 oocytes from 181 follicles (69%). No serious side-effects were observed. Recovery was equally successful in both breeds and was not reduced in repeat attempts upon the same animal. Treatment with FSH only marginally increased the recovery rate (p<0.07) and did not affect the number of follicles aspirated (p>0.05), which varied significantly (p<0.05) between cows.From 110 oocytes matured and fertilized in vitro, 70 embryos were recovered after culture in the rabbit oviduct or with trophoblastic vesicles in vitro, of which 30 had cleaved and 5 had progressed to an advanced stage of development. Hormone treatment did not affect zygote development (p>0.05). Four non-surgical transfers of embryos obtained in these studies have resulted in two pregnancies determined ultrasonographically and the birth of a heifer calf. This suggests that the procedure for multiple oocyte recovery is safe and that it can be used sucessfully for obtaining oocytes for in vitro maturation and fertilization.