免疫荧光检测绵羊体外受精胚胎Oct4、Cdx2表达研究

Oct4基因和Cdx2基因是附植前胚胎发育的重要调控基因,并最终调控了胎儿和胎盘的发育,也在胚胎妊娠识别和附植时调控了干扰素(interferon tau,IFNT)的表达.本研究通过体外成熟、体外受精和体外培养获得了不同发育时期的绵羊胚胎,应用免疫荧光染色探讨了Oct4在早期胚胎的表达规律,结果表明:受精卵和早期卵裂...     

Embryo gene expression in pig pregnancy

Pregnancy is a complex process in which significant changes occur continually in both the corpora lutea and in the endometrium of the females and varies depending on the embryonic, pre-implantation or foetal stages. In the embryonic stages, the majority of genes expressed in the pig embryo correspond to the loss of cellular pluripotency. In contrast, the implantation consists of three phases: elongation of the conceptus, adhesion and union of the embryo to the endometrial epithelium. During these phases, many factors are expressed, including growth factors, molecules that facilitate adhesion and cytokines. All these changes are ultimately regulated by different lipid and hormonal substances, specifically by progesterone, oestradiol and prostaglandins, which regulate the expression of many proteins necessary for the development of the embryo, endometrial remodelling and embryo–maternal communication. This paper is a review of primary gene regulatory mechanisms in pigs during different stages of implantation.     

Buffalo (Bubalus bubalis) Embryonic Stem Cell‐Like Cells and Preimplantation Embryos Exhibit Comparable Expression of Pluripotency‐Related Antigens

In this study, inner cell mass (ICM) cells were isolated from in vitro produced buffalo blastocysts and were cultured on mitomycin‐C treated buffalo foetal fibroblast feeder layer for producing embryonic stem (ES) cells. Among different sources (hatched vs expanded blastocysts) or methods (enzymatic vs mechanical), mechanical isolation of ICM from hatched blastocysts resulted in the highest primary colony formation rate and the maximum passage number up to which ES cells survived. Putative ES cells expressed alkaline phosphatase and exhibited a normal karyotype up to passage 7. Putative ES cells and embryos at 2‐ to 4‐cell, 8‐ to 16‐cell, morula and blastocyst stages strongly expressed stage‐specific embryonic antigen (SSEA)‐4 but lacked expressions of SSEA‐1 and SSEA‐3. Putative ES cells also expressed tumour rejection antigen (TRA)‐1‐60, TRA‐1‐81 and Oct4. Whereas in all early embryonic stages, TRA‐1‐60 was observed only in the periplasmic space, and TRA‐1‐81 expression was observed as small spots at a few places inside the embryos, both these markers were expressed by ICM. Oct4 expression, which was observed at all the embryonic stages and also in the trophectoderm, was the strongest in the ICM. Buffalo putative ES cells possess a unique pluripotency‐related surface antigen phenotype, which resembles that of the ICM.     

Mitochondrial activity and morphology in developing porcine oocytes and pre-implantation non-cultured and cultured embryos

Mitochondria are important determinants of developmental competence for oocytes and embryos owing to their central role in cellular metabolism, yet mitochondrial activity and morphometry during early porcine development have not been quantified. In this study, we examined the membrane potential Δψ(m) and the surface density Sv(in,m) of the inner mitochondrial membrane in pig oocytes and pre-implantation embryos using fluorescent probes and confocal microscopy. Mitochondria and their cristae were also examined by transmission electron microscope. Δψ(m) was consistently low from immature oocytes up to morulae and increased significantly in the early blastocyst before decreasing at the expanded blastocyst stage. This stage-dependent pattern of Δψ(m) changes differs from that reported for other mammals. We also determined that Δψ(m) is lower in cultured when compared to non-cultured porcine early blastocysts. Sv(in,m) was higher in immature oocytes than mature oocytes and remained constant up to the 4- to 8-cell embryo stage. It increased significantly at morula and early blastocyst stages. No differences in Sv(in,m) were found between developmentally matched non-cultured and cultured embryos. These results indicate that the inner mitochondrial membrane potential and surface density change significantly during pre-implantation porcine development in relation to metabolic alterations of the embryo. It is possible that modification of Δψ(m) by manipulating culture conditions may improve the performance of embryos that develop in vitro.     

Telomerase Activity in Swamp Buffalo (Bubalus bubalis) Oocytes and Embryos Derived from In Vitro Fertilization, Somatic Cell Nuclear Transfer and Parthenogenetic Activation

The objective of this study was to examine the telomerase activity in swamp buffalo oocytes and pre-implantation stage embryos derived from in vitro fertilization (IVF), somatic cell nuclear transfer (NT) and parthenogenetic activation (PA). Immature and mature oocytes, and embryos at the 2-4 cell, 8-16 cell, morula and blastocyst stages produced by IVF, NT and PA were collected and the telomerase activity was assayed by using a Telomerase PCR ELISA kit. Telomerase activity was detected in all developmental stages evaluated from immature oocytes to blastocyst stage embryos. Telomerase activity was detected in higher amounts in immature as compared with mature oocytes (p < 0.05). Embryos derived from NT showed a profile of telomerase activity similar to that of IVF. In IVF and NT embryos, telomerase activity was low in the 2-4 cell and 8-16 cell stages, but the activity significantly increased (p < 0.05) at the morula stage, reaching its highest level at the blastocyst stage. In PA embryos, low levels of telomerase activity were detected from the 2-4 cell to the morula stage, and the highest level of telomerase activity was found at the blastocyst stage. Telomerase activity in NT blastocysts is higher than that derived from IVF and the activity is highest in PA blastocysts. These results suggest that the successful reprogramming of telomerase activity in buffalo NT embryos follow a pattern similar to that in embryos derived from IVF and PA.     

胚胎质量和发育阶段对奶牛胚胎移植妊娠率的影响

[目的]为了评估胚胎质量和发育阶段对奶牛胚胎移植妊娠率的影响。[方法]使用63头青年奶牛作为供体进行超数排卵,评估回收胚胎质量和发育阶段。选择334头青年奶牛作为受体鲜胚移植不同质量和发育阶段胚胎。对胚胎质量分布、发育阶段分布、不同质量胚胎和不同发育阶段胚胎移植30 d妊娠率进行统计分析。[结果]可用胚胎中A级胚胎比例(60.78%)显著高于B级和C级胚胎比例(36.70%和2.52%)(P<0.05);致密桑椹胚比例(54.36%)显著高于早期囊胚,囊胚和扩张囊胚比例(18.35%,25.0%和2.29%)(P<0.05)。A级和B级胚胎移植30 d妊娠率(63.55%和64.35%)显著高于C级胚胎移植30 d妊娠率(44.44%)(P<0.05);致密桑椹胚、早期囊胚、囊胚和扩张囊胚移植30 d妊娠率差异不显著(P<0.05),早期囊胚、囊胚移植30 d妊娠率高于致密桑椹胚、扩张囊胚移植30 d妊娠率(P<0.05)。[结论]选择不同发育阶段的A级和B级胚胎能够获得较高胚胎移植妊娠率,增加早期囊胚和囊胚阶段胚胎移植数量能够提高胚胎移植妊娠率。     

Influence of Apoptosis in Bovine Embryo's Development

The correlation between apoptosis and early bovine embryonic loss is still not fully elucidated. In the present study, the relationship between the arrest of bovine embryos at the different stages of development and apoptosis was evaluated. We used embryos 7 days after in vitro maturation and fertilization, and morphologic and biochemical apoptotic analyses were performed by using a phase contrast microscope and by the terminal transferase dUTP nick end‐labelling respectively. For the statistic, the apoptotic cell ratio (ACR) was determined as the percentage of apoptotic cells per embryo. To evaluate the relation between ACR and fragmentation pattern, embryos were divided into five groups, groups I–V. To assess the relation between ACR and cytoplasmatic fragmentation, embryos were divided into three groups, according to the fragmentation percentage (<5%; 5–15% and >15%). Of the total 139 embryos included, 65 arrested at 2–8 cells; 14 arrested at 9–16 cells; 18 compacted morula and 42 were non‐arrested blastocysts. The average number of embryonic fragmentation at different stages of the development, 2–8 cells, 9–16 cells, compacted morula and blastocyst, was 16.0 ± 1.5, 28.7 ± 4.4, 4.4 ± 2.4 and 1 ± 0.3 respectively. The embryos at the stage of arrested 9–16 cells and compacted morula had higher ACR than those at the blastocyst stage, excluding the stage of 2–8 cells (the genome is not yet active). The correlation detected between embryonic development and ACR was 0.92 (p < 0.01). It was observed that embryos possessing high fragmentation showed the higher ACR value (r = 0.98, p < 0.05). Comparing the results between fragmentation percentage and ACR, it was observed that the embryos with higher percentage of fragmentation corresponded to higher ACR (r = 0.97, p < 0.01). These results clearly demonstrated that bovine embryonic arrest at different stages of development is correlated with the apoptotic mechanisms.     

排卵不同阶段氯胺酮麻醉对昆明小鼠卵母细胞和早期胚胎的影响

主要研究排卵不同阶段氯胺酮III期麻醉深度对昆明小鼠卵母细胞和早期胚胎的影响。结果表明:排卵前麻醉(150mg/kg体重)造成卵母细胞和早期胚胎数量的极显著下降(P<0.01),而排卵期间麻醉及排卵后麻醉对排卵及早期胚胎数量影响不显著(P>0.05),排卵不同阶段麻醉均造成一定数量异常卵母细胞和非正常胚胎出现。因此,排卵期间麻醉小鼠相比排卵前及排卵后麻醉小鼠对卵母细胞和早期胚胎的影响最小,但仍有可能造成卵母细胞异常、胚胎畸形的发生。     

Function of berberine on porcine in vitro fertilization embryo development and differential expression analysis of microRNAs

The effect of berberine (Ber) on in vitro fertilization (IVF) embryo development in pigs and the associated differential expression of microRNAs (miRNAs) in the embryo were investigated. NCSU‐23 embryonic culture medium was used for a control group, while NCSU‐23 embryonic culture medium added with Ber was used for a Ber group. The embryo development rates in these groups were determined, and the zygotes, 4‐ and 8‐cell embryos, and blastocysts were collected for cDNA microarray analysis. The development rates of 2‐, 4‐, 8‐cell embryos and blastocysts were significantly higher in the Ber group than those in the control group (p < 0.01). The differentially expressed miRNAs in the 8‐cell versus the 4‐cell stage in control group as well as in the 8‐cell Ber group versus the 8‐cell control group overlapped, and it was found that nine miRNAs were commonly upregulated and two of them were downregulated, while there was no overlap among the other groups. The target genes of Ber‐regulated miRNAs at the 8‐cell stage were mainly associated with the molecular pathway of nucleic acid and protein synthesis. These findings suggest that Ber may regulate the expression of miRNAs at the 8‐cell stage, which is beneficial to provide material reserves for the maternal to zygote transition of porcine embryos, thereby increasing the porcine IVF embryo development rate.     

DNA甲基化在猪胚胎发育过程中的研究进展

猪的胚胎发育需要经历受精、卵裂、孵化、形态转变、附植、器官分化等一系列重要的生理阶段。虽然在胚胎发育过程中基因的严格表达与正确指导是胚胎能否正常发育的决定性条件,但研究表明DNA甲基化修饰对胚胎的发育也起着必不可少的作用。DNA甲基化是一种常见且重要的表观遗传修饰,虽然不改变DNA的一级序列,但也包含可遗传信息,并在基因的转录调控中起重要作用。在猪的胚胎发育中,DNA甲基化呈现出高度动态的过程,这一过程受孕期母体营养和发育环境条件影响。本文将从胚胎早期发育、体细胞核移植和孕期母体营养三个方面来阐述DNA甲基化对胚胎发育的影响,为进一步研究猪胚胎在发育过程中的DNA甲基化机制和提高体细胞核移植的成功率提供参考。     

Expression Analysis of Autophagy Regulators Atg5 and Beclin1 during Early Embryonic Development of Mouse (Mus musculus) from Different Sources

This study aimed to explore the expression patterns of autophagy regulators Atg5 and Beclin1 in the early embryonic development and the effects of different embryonic production methods on the expression of the two factors. Female mice aged 6-8 weeks were subjected to superovulation and divided into 2 groups. The mouse oocytes of one group were collected, and cultured in vitro after parthenogenetic activation. The other group of female mice were caged with male mice (1:1), and the next day, the mouse fertilized eggs were collected for in vitro culture. Parthenogenetic activated embryos and naturally fertilized embryos were collected at 2 cell stage, 4-8 cell stage, mulberry embryo stage and blastocyst stage, respectively. RNA and protein were extracted, real-time fluorescence quantitative PCR, Western blot and other methods were used to detect the expression of key autophagy factors Atg5 and Beclin1. And indirect immunofluorescence was used to detect the expression and location of Atg5 and Beclin1 in mouse blastocysts. The results showed that Atg5 and Beclin1 were expressed in all development stages of naturally fertilized and parthenogenetic activated embryos in mice, and showed a high level in the early stage of embryonic development. The expression of Atg5 and Beclin1 were gradually reduced from the 2 cell stage in mouse naturally fertilized embryos. The expression levels of Atg5 and Beclin1 in parthenogenetic activated embryos were the highest in the 4-8 cell stage, which was extremely significantly different from the naturally fertilized embryos of the same period (P<0.01). From the 4 cell stage, the expression levels of Atg5 and Beclin1 in parthenogenetic activated embryos were higher than naturally fertilized embryos at all subsequent stages, the difference was extremely significantly different (P<0.01). In mouse blastocysts, the fluorescence of Atg5 and Beclin1 protein could be detected in the trophoblast cells and the inner cell mass, but the fluorescence intensity in the inner cell mass was higher than that in the trophoblast cells. In addition, the fluorescence intensity of Beclin1 protein in the inner cell mass of parthenogenetic activated embryos was higher than that in naturally fertilized embryos. Atg5 and Beclin1, the key autophagy factors, are expressed at different levels in the early development of mouse embryos from different sources. It is suggested that the regulation of autophagy on early embryonic development is related to embryo production modes. The results will provide a theoretical basis for further exploring the role of autophagy in the physiological regulation of mammalian embryo development.     

Ultrastructural Study of the Early Development of the Sheep Embryo

An ultrastructural study of the different stages of pre-implantation in sheep was carried out, analysing the changes brought about mainly in the morula and blastocyst stages. The analysis of the embryos obtained showed a series of common characteristics in all stages, most noticeable being the presence of a high number of vesicles distributed in a uniform way in the cytoplasm, and also the presence of numerous electron-dense mitochondria in many varied forms. The most important ultrastructural modifications took place at the 16-cell stage and affected, principally, the nucleus, which presented numerous condensations of chromatin distributed along the nucleoplasm. The nucleoli adopted a reticular morphology, abandoning the compact aspects presented in the previous stage. These changes might be involved in the synthesis of embryonic RNA, and, accordingly, in the activation of the genome of this species. These data indicate that this stage is critical to the embryonic development and might be related to the blockage produced in the development of cultivated sheep embryos at the point of transition from 8 to 16 cells. Nevertheless, it should be pointed out that the first signs of modifications in the aspect of the nucleus are observed at the four-cell stage, being characterized by the appearance of vacuolated areas in the nucleolus, indicating the first signs of embryonic nucleic activity, which would anticipate the main change in the control of the protein synthesis.