牛体外受精早期胚胎发育基因差异表达的研究

应用mRNA差异显示技术,对牛卵母细胞、体外培养的牛早期8细胞期和囊胚期胚胎的基因表达进行了研究。选取4条mRNA表达量存在差异的基因条带进行测序和同源性分析,并利用RT-PCR技术粗略检测其在卵母细胞、8细胞和囊胚中的表达水平。结果表明:4个基因分别与核糖体蛋白S4,Y连接1(RPS4Y1)、末端脱氧核苷酰转移酶作用因子2(DNTTIP2)、剪接和多聚腺苷酸化特异因子3(CPSF3)和转录因子AP-2γ(TFAP2C)高度同源。RPS4Y1、DNTTIP2、CPSF3和TFAP2C在牛卵母细胞和早期胚胎发育过程中mRNA表达量存在时间性差异,可能与其参与不同的生理活动有关。     

Genetic diagnosis of band 3 deficiency and sexing in bovine preimplantation embryos

Band 3 deficiency with hereditary spherocytosis and hemolytic anemia in Japanese black cattle, band 3(Bov.Yamagata), is caused by a total lack of band 3 protein with an autosomal dominant inheritance. Genotyping for band 3 deficiency and sexing were successfully achieved in biopsied embryo cells with efficiencies of 98.4% and 97.4%, respectively. Transfer of the embryo that was determined as homozygous for the mutant allele into a recipient cow resulted in the production of a fetus exhibiting the genotype and red cell phenotypes characteristic of band 3(Bov.Yamagata). These results demonstrate that our procedure is reliable and applicable to produce animals free from or homozygous for the mutant allele by breeding carrier animals.     

Evaluation of bovine viral diarrhea virus uptake by preimplantation embryos

Bovine embryos were exposed to bovine viral diarrhea (BVD) virus in vitro. An uptake of BVD virus by the embryos could not be detected by several assay systems. A significant decrease in the titer of BVD virus was found to occur when the virus was incubated in saline solution + 5% goat serum or minimal essential medium + 5% goat serum for 24 hours at 37 C. Since there was significant inactivation of the BVD virus during the incubation period, lack of viral infectivity of the embryos may have been due to adverse effects of the experimental environmental conditions on the virus or the embryos or upon viral-embryo interaction.     

Potential of preimplantation genomic selection using the blastomere separation technique in bovine in vitro fertilized embryos

Preimplantation genomic selection combined with an in vitro embryo production system is expected as a means of accelerating genetic improvement in cattle. While micromanipulation-based biopsy approaches are often used to collect embryonic cells for genetic testing, they require expensive equipment and sophisticated skills, hindering the adoption of this system. In the present study, to develop a simple method for preimplantation genomic selection using the blastomere separation (BS) technique in bovine in vitro fertilized embryos, we examined the accuracy of single nucleotide polymorphism (SNP) genotyping and optimal cryopreservation method in demi-blastocysts produced by the BS technique. We demonstrated reliable SNP genotyping using DNA derived from demi-blastocysts. We indicated a suitable equilibrium time in vitrification solution for demi-blastocysts and succeeded obtaining pregnancies by the transfer of vitrified demi-blastocysts. In conclusion, our findings suggest that the BS technique provides a simple method for preimplantation genomic selection in bovine in vitro fertilized embryos.     

Resistance of preimplantation bovine embryos to infection with Brucella abortus

Preimplantation bovine embryos were exposed in vitro to Brucella abortus to determine if the bacteria would adhere to zona pellucida (ZP)-intact embryos or adhere to or infect ZP-free embryos. Brucella abortus was not isolated from ZP-intact or ZP-free groups of embryos after 10 sequential antibiotic-free washings. Brucella abortus was isolated from all groups containing ZP-defective embryos after the exposure period and washing. Detrimental effects on healthy in vitro development of embryos were not observed.     

Co-culture of in vitro fertilized bovine embryos with different cell monolayers.

The effects of different cell monolayers on in vitro development of early bovine embryos derived from in vitro maturation and fertilization were examined in this study. Early embryos (four to eight cells) were randomly allocated to bovine granulosa cell (GC), oviductal cell (OC), or uterine cell (UC) monolayers in Exp. 1 and to GC, skin cell (SC; from 10-d-old chicken embryos), testicular cell (TC; from 10-d-old mouse), and liver cell (LC; from 10-d-old chicken embryos) monolayers in Exp. 2, and cultured for 6 d at 38.6 degrees C in a humidified atmosphere of 5% CO2 in air. The culture medium was 12.5 mM HEPES TCM 199 supplemented with 1% calf serum and 1 mM sodium pyruvate. In Exp. 1, the percentage of four- to eight-cell embryos that developed to blastocysts on GC, OC, and UC monolayers was 26.9 (28/104), 37.5 (39/104), and 39.2 (40/102), respectively. In Exp. 2, the percentage of four- to eight-cell embryos that developed to blastocysts on GC, SC, TC, and LC monolayers was 53.3 (40/75), 42.9 (33/77), 49.3 (37/75), and 44.3 (35/79), respectively. There were no significant differences in development among groups in either experiment.(ABSTRACT TRUNCATED AT 250 WORDS)     

Genetic diagnosis of claudin-16 deficiency and sex determination in bovine preimplantation embryos

Renal tubular dysplasia is an autosomal recessively inherited disorder in Japanese black cattle that is due to deletion mutations in the claudin-16 gene and causes chronic renal failure and death of affected animals. Here, we report a multiplex-PCR procedure to determine the genotype for claudin-16 deficiency in preimplantation embryos. The presence or absence of the wild-type and mutant allele(s) was precisely detected with the multiplex-PCR using as little as 5 pg of genomic DNA from leukocytes. When biopsied embryo cells were examined for claudin-16 deficiency, 97.2% of genotypes were consistent with the PCR results obtained for the corresponding embryos. In addition, sexing of embryos by PCR was performed using an aliquot of DNA extracted from biopsied embryo cells, and determination of claudin-16 genotype and sex was successfully achieved with an efficiency of 91.7% for claudin-16 genotyping and 83.3% for sexing. The production of a 100-day fetus that was male and homozygous for claudin-16 deficiency, as expected from the analysis of biopsied embryo cells, gave evidence of the reliability and applicability of this procedure for preventing the transmission of this disease and for enabling advances in animal breeding.     

Evaluation of bovine embryos produced in high performance serum-free media

This review evaluates the quality of bovine embryos developed from in vitro-matured (IVM) and -fertilized (IVF) oocytes cultured in either serum-free or serum-containing media. Bovine embryos cultured in serum-supplemented medium contain numerous cytoplasmic lipid droplets and immature mitochondria compared to those cultured in serum-free medium. The accumulation of cytoplasmic lipids in embryos developed in serum-containing medium may be a result of incorporation of lipoproteins from the serum and may result in impaired function of mitochondria. The improved serum-free media (IVMD101 and IVD101) offer several advantages over culture in serum-containing medium, including increased rates of blastocyst formation and higher cell numbers. Additionally, the survival and hatching rates of embryos produced in serum-free media after post-thaw culture were superior to those of embryos produced in the serum-containing medium, suggesting that the abnormal accumulation of cytoplasmic lipids in embryos may have a negative effect on the sensitivity of embryos to chilling and freezing. These serum-free culture systems have proven to be beneficial for the production of good quality embryos from IVM-IVF bovine oocytes. Furthermore, recent studies have shown a correlation between mitochondrial function (oxygen consumption) and embryo quality. A new method using scanning electrochemical microscopy may be capable of assessing the viability and developmental potential of bovine embryos.     

Proteomic profile of pre-implantational ovine embryos produced in vivo

The present study was conducted to decipher the proteome of in vivo-produced pre-implantation ovine embryos. Ten locally adapted Morana Nova ewes received hormonal treatment and were inseminated 12 hr after ovulation. Six days later, 54 embryos (morula and blastocyst developmental state) were recovered from eight ewes and pooled to obtain sufficient protein for proteomic analysis. Extracted embryo proteins were analysed by LC-MS/MS, followed by identification based on four database searches (PEAKS, Proteome Discoverer software, SearchGUI software, PepExplorer). Identified proteins were analysed for gene ontology terms, protein clusters and interactions. Genes associated with the ovine embryo proteome were screened for miRNA targets using data sets of TargetScan ( http://www.targetscan.org ) and mIRBase ( http://www.mirbase.org ) servers. There were 667 proteins identified in the ovine embryos. Biological processes of such proteins were mainly related to cellular process and regulation, and molecular functions, to binding and catalytic activity. Analysis of the embryo proteins revealed 49 enriched functional clusters, linked to energy metabolism (TCA cycle, pyruvate and glycolysis metabolism), zona pellucida (ZP), MAPK signalling pathway, tight junction, binding of sperm to ZP, translation, proteasome, cell cycle and calcium/phospholipid binding. Sixteen miRNAs were related to 25 pre-implantation ovine embryo genes, all conserved in human, bovine and ovine species. The interaction network generated by miRNet showed four key miRNAs (hsa-mir-106b-5p; hsa-mir-30-5p; hsa-mir-103a-5p and hsa-mir-106a-5p) with potential interactions with embryo-expressed genes. Functional analysis of the network indicated that miRNAs modulate genes related to cell cycle, regulation of stem cell and embryonic cell differentiation, among others. Retrieved miRNAs also modulate the expression of genes involved in cell signalling pathways, such as MAPK, Wnt, TGF-beta, p53 and Toll-like receptor. The current study describes the first major proteomic profile of 6-day-old ovine embryos produced in vivo, setting a comprehensive foundation for our understanding of embryo physiology in the ovine species.     

Method for obtaining bovine zygotes produced in vivo

A superovulatory and surgical protocol was developed for recovery of bovine zygotes. Holstein cows and heifers were given follicle-stimulating hormone and cloprostenol to induce superovulation. Surgical cannulation and lavage of the uterine tube was performed 40 to 48 hours after the start of standing estrus. In general, cows had more corpora hemorrhagica than did heifers, but a higher percentage (P less than 0.05) of ova recovered from cows were infertile. Several heifers were subjected to the procedure twice, and embryo recovery rates were equivalent both times.     

间隙连接蛋白Cx43和Cx45在体外受精绵羊早期胚胎中的表达

为了研究间隙连接蛋白Cx43和Cx45在体外受精绵羊胚胎早期发育过程各阶段的表达情况，利用RT—PCR扩增得到绵羊。Cx43（43ku）及Cx45（45ku）基因的mRNA，并进行序列测定。收集绵羊未成熟和成熟卵母细胞以及体外受精早期胚胎各发育阶段的卵裂球细胞，通过RT—PCR半定量检测Cx43和Cx45基因的mRNA表达量；通过免疫组织化学方法结合激光扫描共聚焦显微镜检测Cx43和Cx45蛋白在绵羊体外受精的早期胚胎发育过程中的表达情况及表达区域。结果表明，在绵羊卵母细胞成熟过程以及早期胚胎发育的各个时期，Cx43及Cx45在mRNA水平和蛋白水平均有表达；Cx43、Cx45蛋白主要分布在细胞膜表面，在细胞质和细胞核中表达微弱。研究结果为进一步探索Cx43和Cx45在绵羊早期胚胎发育过程中的作用提供了试验数据。     

Relationship between group II caspase activity of bovine preimplantation embryos and capacity for hatching

The occurrence of apoptosis in a fraction of blastomeres in the preimplantation embryo is well known but the consequences of this phenomenon for the developmental potential of the blastocyst has not been well established. Here we demonstrate that blastocysts with low amounts of activated group II caspase activity have increased potential for development to the hatched blastocyst stage. Bovine blastocysts produced in vitro were assayed using a non-invasive fluoregenic substrate that is cleaved by activated group II caspases (i.e., caspase-2, -3 and -7). Subsequently, blastocysts were cultured until Day 10 post-insemination and the proportion undergoing hatching determined. In Experiment 1, blastocysts were cultured without respect to stage of development (expanded or non-expanded); blastocysts classified as having low caspase activity had higher hatching rates than blastocysts with medium or high caspase activity. In Experiment 2, embryos were categorized as nonexpanded or expanded blastocysts. Caspase activity was lower and hatching rate higher for expanded blastocysts than for nonexpanded blastocysts. For nonexpanded blastocysts, embryos classified as having low caspase activity had higher hatching rates as compared to embryos with medium or high caspase activity. In conclusion, the capacity for blastocysts to undergo further development is related to degree of group II caspase activity. Conditions that enhance the incidence of apoptosis in blastocysts may reduce developmental competence. In addition, determination of caspase activity may be useful for selection of embryos for transfer into recipients.     

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。