Isolation of DNA from embryo and chorio-allantoic membranes and sexing by PCR in Japanese quail

1. The aim of this study was to evaluate the amount and quality of genomic DNA isolated from embryos and their chorioallantoic membranes (CAM) and to investigate the utility of different PCR methods for identifying the sex of Japanese quail embryos.

2. Fertilised eggs were incubated at 37°C for 120?h and DNA was isolated from samples of embryos and CAM. Target regions of the CHD-W gene or XhoI repeat sequence were amplified by PCR and examined on agarose gels or by using a capillary electrophoresis system.

3. DNA samples from embryos had significantly higher OD260 values than those from CAM, while OD260/280 values were not significantly different between embryos and CAM.

4. Gender identification was not possible by PCR amplification of the CHD gene region or XhoI repeat sequences examined on agarose gels, whereas males and females of Japanese quail were distinguishable when PCR products of the CHD gene were separated by capillary electrophoresis.

5. The results showed that high molecular weight DNA could be isolated from both embryo and CAM of Japanese quail. DNA isolated from CAM could be used for molecular genetic studies where embryos would be used for other purposes, such as in situ hybridisation. A capillary electrophoresis system could be used for identifying the gender of Japanese quail embryos.     

Developmental stages of the blue‐breasted quail (Coturnix chinensis)

Chickens and Japanese quail (Coturnix japonica) have traditionally been the primary avian models in developmental biology research. Recently, the blue‐breasted quail (Coturnix chinesis), the smallest species in the order Galliformes, has been proposed as an excellent candidate model in avian developmental studies owing to its precocious and prolific properties. However, data on the embryonic development of blue‐breasted quail are scarce. Here, we developed a normal developmental series for the blue‐breasted quail based on developmental features. The blue‐breasted quail embryos take 17 days to reach the hatching period at 37.7°C. We documented specific periods of incubation in which significant development occurred, and created a 39‐stage developmental series. The developmental series for the blue‐breasted quail was almost identical to that for chickens and Japanese quail in the earlier stages of development (stages 1–16). Our staging series is especially useful at later stages of development (stages 34–39) of blue‐breasted quail embryos as a major criterion of staging in this phase of development was the weight of embryos and the length of third toes.     

Skeletal development in blue‐breasted quail embryos

The blue‐breasted quail (Coturnix chinensis), the smallest species of quail with short generation interval and excellent reproductive performance, is a potential avian research model. A normal series of skeletal development of avian embryos could be served as a reference standard in the fields of developmental biology and teratological testing as well as in the investigation of mutation with skeletal abnormalities and in the study of the molecular mechanisms of skeletal development through genome manipulation. Furthermore, ossification sequence shows a species‐specific pattern and has potential utility in phylogeny. However, data on the skeletal development of blue‐breasted quail embryos are scarce. Here, we established a series of normal stages for the skeletal development of blue‐breasted quail embryos. Cartilage and ossified bones of blue‐breasted quail embryos were stained blue and red with Alcian blue 8GX and Alizarin red S, respectively. The time and order of chondrification and calcification of their skeletons were documented every 24 hr from 3 to 17 days of incubation, and a 15‐stage series of skeletal development was created. Moreover, a comparative study with the Japanese quail (Coturnix japonica) demonstrated that ossification sequence differed significantly between these two species.     

Expression and Characterization of Constitutive Heat Shock Protein 70.1 (HSPA‐1A) Gene in In Vitro Produced and In Vivo‐Derived Buffalo (Bubalus bubalis) Embryos

Cells are blessed with a group of stress protector molecules known as heat shock proteins (HSPs), amongst them HSP70, encoded by HSPA‐1A gene, is most abundant and highly conserved protein. Variety of stresses hampers the developmental competence of embryos under in vivo and in vitro conditions. Present work was designed to study the quantitative expression of HSPA‐1A mRNA in immature oocytes (IMO), matured oocytes (MO), in vitro produced (IVP) and in vivo‐derived (IVD) buffalo embryos to assess the level of stress to which embryos are exposed under in vivo and in vitro culture conditions. Further, HSPA‐1A gene sequence was analysed to determine its homology with other mammalian sequences. The mRNA expression analysis was carried out on 72 oocytes (40 IMO; 32 MO), 76 IVP and 55 IVD buffalo embryos. Expression of HSPA‐1A was found in oocytes and throughout the developmental stages of embryos examined irrespective of the embryo source; however, higher (p < 0.05) expression was observed in 8–16 cell, morula and blastocyst stages of IVP embryos as compared to IVD embryos. Phylogenetic analysis of bubaline HSPA‐1A revealed that it shares 91–98% identity with other mammalian sequences. It can be concluded that higher level of HSPA‐1A mRNA in IVP embryos in comparison with in vivo‐derived embryos is an indicator of cellular stress in IVP system. This study suggests need for further optimization of in vitro culture system in which HSPA‐1A gene could be used as a stress biomarker during pre‐implantation development.     

Early development of porcine parthenogenetic embryos and reduced expression of primed pluripotent marker genes under the effect of lysophosphatidic acid

To further promote the early development of porcine embryos and capture “naïve” pluripotent state within blastocyst, the experiment explored the effects of lysophosphatidic acid (LPA) on the early development of porcine parthenogenetic embryos and the expression of pluripotency relevant genes. The results showed that the addition of 50 μM LPA significantly improved parthenogenetic embryo cleavage rate (82.7% vs. 74.7%, p < 0.05), blastocyst rate (24.5% vs. 11.3%, p < 0.05) and blastocyst cell count (56 ± 7.9 vs. 42 ± 1.0, p < 0.05) than that of the control group. In addition, immunostaining experiment determined that the fluorescence intensity of OCT4 was also significantly higher than that of the control group. The quantitative real‐time polymerase chain reaction (qRT–PCR) test revealed that addition of 50 μM LPA could significantly enhance the expression level of pluripotent gene OCT4 and trophoblast marker genes CDX2, however, decrease the expression of primitive hypoblast marker gene GATA4. The results also indicated that LPA might decrease the expression of GATA4 through the ROCK signalling pathway. For further investigating the effect of the addition of LPA on the expression of “primed” and “naïve” genes, we also detected the expression of those pluripotency‐related genes by qRT–PCR. The results showed addition of LPA had no significant effect on the expression of “naïve” pluripotent genes, but it was able to significantly decrease the expression of “primed” pluripotent genes, NODAL and Activin‐A; furthermore, it also could significantly improve the expression of OCT4 and c‐Myc which act as two important ES cell renewal factors. Above all, the addition of LPA can facilitate the early development of porcine parthenogenetic embryos, which may be able to benefit for capturing “naïve” pluripotency in vitro through inhibiting “primed” pluripotency.     

Effect of sex differences in donor foetal fibroblast on the early development and DNA methylation status of buffalo (Bubalus bubalis) nuclear transfer embryos

Low efficiency of somatic cell nuclear transfer (SCNT) embryos is largely attributable to imperfect reprogramming of the donor nucleus. The differences in epigenetic reprogramming between female and male buffalo cloned embryos remain unclear. We explored the effects of donor cell sex differences on the development of SCNT embryos. We and then compared the expression of DNA methylation (5‐methylcytosine‐5mC and 5‐hydroxymethylcytosine‐5hmC) and the expression level of relevant genes, and histone methylation (H3K9me2 and H3K9me3) level in SCNT‐♀ and SCNT‐♂ preimplantation embryos with in vitro fertilization (IVF) counterparts. In the study, we showed that developmental potential of SCNT‐♀ embryos was greater than that of SCNT‐♂ embryos (p < 0.05). 5mC was mainly expressed in SCNT‐♀ embryos, whereas 5hmC was majorly expressed in SCNT‐♂ embryos (p < 0.05). The levels of DNA methylation (5mC and 5hmC), Dnmt3b, TET1 and TET3 in the SCNT‐♂ embryos were higher than those of SCNT‐♀ embryos (p < 0.05). In addition, there were no significant differences in the expression of H3K9me2 at eight‐stage of the IVF, SCNT‐♀ and SCNT‐♂embryos (p < 0.05). However, H3K9me3 was upregulated in SCNT‐♂ embryos at the eight‐cell stage (p < 0.05). Thus, KDM4B ectopic expression decreased the level of H3K9me3 and significantly improved the developmental rate of two‐cell, eight‐cell and blastocysts of SCNT‐♂ embryos (p < 0.05). Overall, the lower levels of DNA methylation (5mC and 5hmC) and H3K9me3 may introduce the greater developmental potential in buffalo SCNT‐♀ embryos than that of SCNT‐♂ embryos.     

Detection of the EGFP sequence in breast muscle of 3‐year‐old chicken after transfection using sonoporation

In our continuing effort to generate transgenic chickens, sonoporation was chosen to insert an exogenous gene into the chicken genome. An EGFP expression vector (pCAG‐EGFPac) and microbubbles were injected into the central disc of stage‐X blastoderm or the germinal crescent of stage‐4 embryos, followed by ultrasonic vibration. Nineteen chicks out of 108 treated embryos hatched, six females and six males out of these 19 chicks grew to sexual maturity and two females and three males lived for 3 years. Genomic DNA from 17 out of 35 gonads from embryos and chicks that died before sexual maturity was EGFP‐positive by PCR. No EGFP sequence was detected in the genomic DNA of 322 embryos from six sexually mature females and the semen from four sexually mature males by PCR. When genomic DNA was obtained from various tissues of five 3‐year‐old chickens, the EGFP sequence was amplified from the genomic DNA of the breast muscle of a female (No. 85). The above sequence was subjected to DNA sequencing and verified to be the EGFP sequence. These results showed that sonoporation is an effective tool for the transduction of exogenous genes into chicken embryos for the generation of transgenic chickens.     

Expression patterns in mouse embryos of neuroleukin/glucose-6-phosphate isomerase and autocrine motility factor receptor

The pattern of expression by using in situ hybridization in whole mouse embryos of the neuroleukin/glucose-6-phosphate isomerase (NK/GPI) gene and its receptor (AMF-R) is reported. NK/GPI expression was first seen at embryonic day 9 whereas AMF-R was detected at embryonic day 8; both were detected up to day 12 with NK/GPI showing peaking in the limbs around day 11. The main regions of expression are limb buds, spinal cord and brain. This work contributes to understanding how both proteins act in the development of somatosensory and motoric neural structures.     

Pluripotency‐associated genes reposition during early embryonic developmental stages in pigs

We examined the allelic expression and positioning of two pluripotency‐associated genes, OCT4 and SOX2, and two housekeeping genes, ACTB and TUBA, in 4‐ and 8‐cell porcine embryos utilizing RNA and DNA fluorescence in situ hybridization (FISH) in single blastomeres. The proportion of blastomeres expressing SOX2 bi‐allelically increased from 45% at the 4‐cell stage to 60% at the 8‐cell stage. Moreover, in 8‐cell embryos, SOX2 was expressed bi‐allelically in significantly more blastomeres than was the case for OCT4, and this was associated with a tendency for SOX2 alleles to move toward the nuclear interior during 4‐ to 8‐cell transition. However, the radial location of OCT4 alleles did not change significantly during this transition. The locations of active and inactive alleles based on DNA and RNA FISH signals were also calculated. Inactive OCT4 alleles were located in very close proximity to the nuclear membrane, whereas active OCT4 alleles were more centrally disposed in the nucleus. Nevertheless, the nuclear location of active and inactive SOX2 alleles did not change in either 4‐ or 8‐cell blastomeres. Our RNA and DNA FISH data provide novel information on the allelic expression patterns and positioning of pluripotency‐associated genes, OCT4 and SOX2, during embryonic genome activation in pigs.     

Effects of trichostatin A on in vitro development and transgene function in somatic cell nuclear transfer embryos derived from transgenic Clawn miniature pig cells

The present study was carried out to examine the effects of post‐activation treatment of trichostatin A (TSA), a histone deacetylase inhibitor, on in vitro development and transgene function of somatic cell nuclear transfer (SCNT) embryos derived from Clawn miniature pig embryonic fibroblast (PEF) transfected with a bacterial endo‐β‐galactosidase C gene (removal of the α‐galactosyl (Gal) epitope). SCNT embryos were incubated with or without TSA (50 or 100 nmol/L) after activation, cultured in vitro and assessed for cleavage, blastocyst formation and transgene function. The rate of blastocyst formation was significantly higher in SCNT embryos treated with 50 nmol/L TSA than that in control (P < 0.05), whereas the rate of cleavage and cell number of blastocyst did not differ. Following labelling with fluorescein isothiocyanate‐labelled BS‐I‐B₄ isolectin, the intensity of fluorescence observed on cell‐surface was dramatically reduced in transgenic SCNT blastocyst in comparison with non‐transgenic SCNT blastocyst. However, the reduction of α‐Gal epitope expression in transgenic SCNT blastocyst was not affected by TSA treatment. The results of this study showed that post‐activation treatment with 50 nmol/L TSA is effective to improve in vitro developmental capacity of transgenic SCNT miniature pig embryos without the modification of transgene function.     

OB‐cadherin cloning and expression in a model of wound repair in horses

Reason for performing study: Horses suffer from a debilitating impediment in repairing wounds located on the lower limb that leads to the development of a fibroproliferative disorder (exuberant granulation tissue). This condition is a source of wastage since it often forces retirement from competition. Treatments that resolve or prevent this condition are still lacking, maybe due to deficient knowledge of the underlying molecular mechanisms. Fibroblast‐to‐myofibroblast conversion is an essential step allowing contraction during wound repair and is accompanied by an increase in OB‐cadherin expression. Objectives: To clone equine cadherin‐11 (CDH11) cDNA and to study its spatiotemporal expression profile during the repair of body and limb wounds, thereby contributing to a better understanding of the repair process. Methods: Cloning was by a PCR technique. Expression was studied in intact skin and in 1, 2, 3, 4 and 6‐week‐old wounds of the body and limb. Temporal CDH11 gene expression was determined by RT‐PCR while OB‐cadherin protein expression was mapped immunohistochemically. Results: Equine CDH11 is a highly conserved gene and protein. mRNA was not expressed in equine skin whereas the wound repair process was characterised by a significantly higher expression in the thorax than in limb samples. mRNA expression pattern was paralleled by protein data as confirmed by immunohistochemistry. Conclusions: The data suggest that deficient OB‐cadherin expression in the first phases of wound repair contributes to the excessive proliferative response seen in horse limb wounds. Potential relevance: Future studies should verify the quantitative, temporal expression of this protein in order to provide the basis for targeted therapies that might prevent the development of EGT in horse wound repair.     

Relationship between abdominal fat content and avian uncoupling protein gene expression in skeletal muscle of Japanese quail Coturnix japonica

1. The genetic architecture of the avian uncoupling protein (avUCP) was investigated and the relationship between avUCP gene expression and the amount of abdominal fat of Japanese quail was determined by quantitative real-time PCR.

2. The Japanese quail avUCP gene consists of six exons and five introns. Sequences of nucleotides and amino acids were 94·6% and 86·0% identical to those of the chicken avUCP gene, and phylogenetic analysis showed that the Japanese quail avUCP gene consists of the same clusters as the chicken and turkey avUCP.

3. Expression of the avUCP gene was significantly higher in the Pectoralis major (1·28?±?0·24) than in the Biceps femoris (0·63?±?0·14).

4. A positive correlation coefficient between the avUCP gene expression in the Pectoralis major and Biceps femoris was observed (r?=?0·79, P?=?0·02), whereas a negative correlation coefficient was observed between the abdominal fat percentage (AFP) and gene expression in both the Pectoralis major (r?=??0·82, P?=?0·01) and Biceps femoris (r?=??0·61, P?=?0·11).

5. The avUCP gene was associated with the accumulation of abdominal fat in Japanese quail and it was concluded that modulation of avUCP gene expression could be utilised to control abdominal fat accumulation in poultry.     

Effect of short‐term exposure of cumulus–oocyte complex to 3‐morpholinosydnonimine on in vitro embryo development and gene expression in cattle

Short‐term exposure of gametes to different types of stress might induce stress tolerance in mammalian embryos. The aim of this study was to evaluate the effect of short‐term exposure of bovine mature cumulus–oocyte complex (COC) to 3‐morpholinosydnonimine (SIN‐1) on subsequent in vitro embryo development, embryo quality and relative gene expression. Matured COCs were incubated with SIN‐1 (0, 0.1, 1, 10 and 100 μM SIN‐1) for 1 hr before in vitro fertilization and zygotes were cultured until Day 7. The cleavage rate at 72 hr did not show any differences among groups. However, the blastocyst rate on Day 7 decreased with all treatments evaluated, with the embryos generated with 10 μM SIN‐1 showing the lowest embryo production rate. Embryo quality analysis did not show any differences in total cell number (TCN) or inner cell mass (ICM) among groups. Relative gene expression analysis showed a downregulation of eNOS expression and an upregulation of nNOS expression in all treatments evaluated compared to the control group. Also, a downregulation was observed in some treatments: SOD2 at 0.1 μM; SOD1 at 0.1 and 100 μM; PRDX5 at 0.1, 10 and 100 μM; and NANOG at 10 and 100 μM; and an upregulation of CDX2 expression was observed at 100 μM. The other genes (OCT4, HIF1A, HSPA1A, BCL2A and iNOS) did not show any differences in the relative gene expression. These results suggest that the short‐term exposure of mature bovine COCs to SIN‐1 does not induce stress tolerance and has no beneficial effect on bovine in vitro embryo production.     

Significance of CCN2 expression in bovine preimplantation development

In mammalian preimplantation development, the first cell lineage segregation occurs during the blastocyst stage, when the inner cell mass and trophectoderm (TE) differentiate. Species‐specific analyses are essential to elucidate the molecular mechanisms that underlie this process, since they differ between various species. We previously showed that the reciprocal regulation of CCN2 and TEAD4 is required for proper TE differentiation in bovine blastocysts; however, the function of CCN2 during early embryogenesis has remained otherwise elusive. The present study assessed the spatiotemporal expression dynamics of CCN2 in bovine embryos, and evaluated how changes to CCN2 expression (using a CCN2 knockdown (KD) blastocyst model) regulate the expression of pluripotency‐related genes such as OCT4 and NANOG. The conducted quantitative PCR analysis revealed that CCN2 mRNA was expressed in bovine oocytes (at the metaphase stage of their second meiosis) and embryos. Similarly, immunostaining detected both cytoplasmic and nuclear CCN2 at all analyzed oocyte and embryonic stages. Finally, both OCT4 and NANOG expression levels were shown to be significantly reduced in CCN2 KD blastocysts. Together, these results demonstrate that bovine CCN2 exhibits unique expression patterns during preimplantation development, and is required for the proper expression of key regulatory genes in bovine blastocysts.     

鸡、鹌鹑及其杂交种早期胚胎bcl-2基因甲基化的分析

采用甲基化特异性PCR(MSP)方法进行鸡、鹌鹑及其属间杂交种早期胚胎发育期60、66、72、84、96、108、120 h 7个不同胚龄胚胎组织bcl-2基因启动子区CpG岛甲基化状态的对比分析,探讨bcl-2基因甲基化对鸡与鹌鹑属间杂交种早期胚胎发育的影响。结果显示,正常发育的鸡胚和鹌鹑胚龄在60、66、72、120 h均呈高甲基化状态,84和96 h呈非甲基化状态,而鸡与鹌鹑属间杂交种胚胎在60、66、72、96、108和120 h则与鸡、鹌鹑不同,呈现出甲基化或非甲基化无规律性并存,甚至检测不到甲基化状态;84 h则只检测到非甲基化状态。鸡与鹌鹑杂交种早期胚胎组织中bcl-2 基因启动子区CpG岛的异常甲基化有可能是引起鸡与鹌鹑属间杂交种胚胎早期死亡的一个重要影响因素。     

Effects of Addition of Tissue‐Type Plasminogen Activator in In Vitro Fertilization Medium on Bovine Embryo Development and Quality

Plasminogen activators/Plasmin system plays pivotal role in regulating reproductive functions of mammals. Here, we examined the effects of modification of in vitro fertilization medium (IVF medium) with the addition of tissue‐type plasminogen activator (t‐PA), on bovine embryo development and quality, assessed by quantification of expression of various genes related to metabolism, oxidation, implantation and apoptosis. In addition, plasminogen activator activity (PAA) and plasminogen activator inhibition (PAI) were measured in the spent media. After conventional IVM, 2016 cumulus‐oocyte complexes (COCs) were divided into four groups with modified composition of the IVF medium containing t‐PA and/or its inhibitor epsilon‐aminocaproic acid (control, t‐PA, t‐PA+ε‐ACA, ε‐ACA). Presumptive zygotes were cultured for 8 days in synthetic oviductal fluid (SOF) medium; gene expression studies were carried out on morulae and blastocysts. t‐PA alone significantly suppressed cleavage and blastocyst formation rates, but this effect was neutralized by the addition of ε‐ACA. PAA in the treated group was significantly reduced by ε‐ACA, but without total elimination. Significant differences were detected in the expression of genes related to apoptosis and/or cell cycle arrest (BAX, BCL2L1, KAT2B) between embryos produced in t‐PA‐modified media and controls, giving an overall notion that the inferior developmental competence of treated embryos may be attributed to apoptotic phenomena induced by t‐PA. In conclusion, it appears that excessive t‐PA content in the IVF media, suppresses blastocyst formation rate, possibly due to induction of apoptotic phenomena.