Testicular and ovarian gonocytes from 20-day incubated chicken embryos contribute to germline lineage after transfer into bloodstream of recipient embryos

The present study was conducted to elucidate whether testicular and ovarian gonocytes obtained from 20-day incubated chicken embryos (stage 45) have the ability to migrate to the germinal ridges and contribute to germline lineage after transfer into the bloodstream of recipient embryos. Testicular and ovarian gonocytes were first identified as relatively large cells in a population of gonadal cells. The proportions of testicular and ovarian gonocytes in the total gonadal cells were 0.94 and 0.75% respectively, recognised as chicken vasa homologue-positive cells. Then, the dissociated gonadal cells obtained from 20-day incubated embryos containing testicular or ovarian gonocytes, with or without transfection, were transferred into recipient embryos. Expression of the introduced GFP gene was observed in the gonads of 6.5-day cultured recipient embryos (stage 30) in males and females, suggesting that the transferred testicular and ovarian gonocytes have the ability to migrate to the germinal ridges and enter the gonads. Furthermore, the presence of the donor-derived DNA was detected in the gonads of 20-day cultured recipient embryos in males and females, and also in the sperm samples obtained from the hatched male putative chimaeric chickens, suggesting that the transferred testicular and ovarian gonocytes were incorporated into the germline of chimaeric embryos and chickens. It is concluded that testicular and ovarian gonocytes obtained from 20-day incubated embryos have the ability to migrate to the germinal ridges after transfer into the bloodstream of recipient embryos, enter the gonads and contribute to the germline lineage of chimaeric embryos and chickens.     

Effect of in vitro fertilization on gene expression and development of mouse preimplantation embryos

In vitro culture (IVC) of preimplantation mouse embryos is associated with changes in gene expression. It is however, not known if the method of fertilization affects the global pattern of gene expression. We compared gene expression and development of mouse blastocysts produced by in vitro fertilization (IVF) versus blastocysts fertilized in vivo and cultured in vitro from the zygote stage (IVC) versus control blastocysts flushed out of the uterus on post coital day 3.5. The global pattern of gene expression was assessed using the Affymetrix 430 2.0 chip. It appears that each method of fertilization has a unique pattern of gene expression and development. Embryos cultured in vitro had a reduction in the number of trophoblastic cells (IVF 33.5 cells, IVC 39.9 cells, and 49.6 cells in the in vivo group) and, to a lesser degree, of inner cell mass cells (12.8, 11.7, and 13.8 respectively). The inner cell mass nuclei were larger after culture in vitro (140 microm(2), 113 microm(2), and 86 microm(2) respectively). Although a high number of genes (1912) was statistically different in the IVF cohort when compared with the in vivo control embryos, the magnitude of the changes in gene expression were low and only a minority of genes (29 genes) was changed more than fourfold. Surprisingly, IVF embryos were different from IVC embryos (3058 genes were statistically different, but only three changed more than fourfold). Proliferation, apoptosis, and morphogenetic pathways are the most common pathways altered after IVC. Overall, IVF and embryo culture have a profound effect on gene expression pattern and phenotype of mouse preimplantation embryos.     

Delayed and stage specific phosphorylation of H2AX during preimplantation development of gamma-irradiated mouse embryos

Within minutes of the induction of DNA double-strand breaks in somatic cells, histone H2AX becomes phosphorylated in the serine 139 residue at the damage site. The phosphorylated H2AX, designated as gamma-H2AX, is visible as nuclear foci in the irradiated cells which are thought to serve as a platform for the assembly of proteins involved in checkpoint response and DNA repair. It is known that early stage mammalian embryos are highly sensitive to radiation but the mechanism of radiosensitivity is not well understood. Thus, we investigated the damage response of the preimplantation stage development by analyzing focus formation of gamma-H2AX in mouse embryos gamma-irradiated in utero. Our analysis revealed that although H2AX is present in early preimplantation embryos, its phosphorylation after 3 Gy gamma-irradiation is hindered up to the two cell stage of development. When left in utero for another 24-64 h, however, these irradiated embryos showed delayed phosphorylation of H2AX. In contrast, phosphorylation of H2AX was readily induced by radiation in post-compaction stage embryos. It is possible that phosphorylation of H2AX is inefficient in early stage embryos. It is also possible that the phosphorylated H2AX exists in the dispersed chromatin structure of early stage embryonic pronuclei, so that it cannot readily be detected by conventional immunostaining method. In either case, this phenomenon is likely to correlate with the lack of cell cycle arrest, apoptosis and high radiosensitivity of these developmental stages.     

Increased TNF-alpha expression in cultured mouse embryos exposed to teratogenic concentrations of glucose

Diabetes-induced early embryonic death is accompanied by an increased expression of tumour necrosis factor alpha (TNF-alpha) in the embryonic microenvironment. The aim of the present study was to evaluate whether diabetes-induced embryopathic stress may also alter the expression of TNF-alpha produced by the embryo itself. As a model, whole postimplantation embryos were cultured for 24 h in a medium with high concentrations of glucose, one of the main diabetes-associated teratogenic metabolites. An anomaly such as an open neural tube was used as an end-point characterizing the glucose-induced teratogenic effect and the number of somites was counted to evaluate growth retardation induced by glucose. The expression of TNF-alpha (by immunohistochemistry), apoptosis (by TdT-mediated dUTP nick-end labelling; TUNEL) and the activity of caspases 3 and 8 (by a fluorometric assay) were evaluated in normal and malformed embryos. Ninety-seven per cent of the embryos exposed to 1300 mg glucose dl(-1) exhibited an open neural tube. The percentage of malformed embryos was smaller in media containing 800 and 500 mg glucose dl(-1) (68 and 37%, respectively) but it still exceeded significantly the value registered in embryos developing in a normoglycaemic medium (12%). In addition, a significant decrease in the number of somites was observed in embryos developing in media containing 1300 and 800 mg glucose dl(-1). Malformed embryos exhibited a greater number of nuclei that were positive in the TUNEL assay as well as a higher amount of active caspase 8 compared with normal embryos (with closed neural folds). TNF-alpha expression was detected in the neuroepithelial layer of the neural tube of the malformed embryos, whereas the expression of this cytokine was weak, if detectable, in normal embryos. Together, these findings indicate that TNF-alpha produced by the embryo may be involved in regulating the response of embryos to diabetes-generated embryopathic stress.     

Role of facilitative glucose uptake in the glucose-inorganic phosphate-mediated retardation and inhibition of development in different strains of mouse embryos

Mouse embryos from different strains develop differently in vitro depending on the composition of the culture medium, and in particular on the presence or absence of glucose and inorganic phosphate. Glucose is both stimulatory and inhibitory in certain conditions. Glucose uptake by cells can be passive, down concentration gradients, or active, through sodium driven pumps, or can occur through facilitative transport. This study investigated the effects of inhibition of facilitative glucose transport on the glucose-inorganic phosphate-mediated blocks in development in three different strains of mouse embryo, CF-1, CD-1 and an F2 hybrid. Development of CF-1 and CD-1 embryos is blocked in medium containing glucose and inorganic phosphate but not in medium containing glucose alone, and F2 embryos are not affected. Inhibition of facilitated glucose transport to the eight-cell-morula stage in CF-1 and CD-1 embryos resulted in development in medium containing both glucose and inorganic phosphate, indicating that the prevention of facilitative glucose uptake can overcome the developmental block. Removal of inhibition before the eight-cell-morula stage resulted in total arrest of CF-1 embryos and minimum development of CD-1 embryos. F2 embryos are not affected by inorganic phosphate and glucose and showed no response to the transporter inhibitor at any stage. These data support the contention that facilitated glucose transport is active in embryos, is phosphate-dependent and that its inhibition can overcome the glucose-inorganic phosphate-mediated developmental blocks in mouse embryos.     

ZFP57 regulates DNA methylation of imprinted genes to facilitate embryonic development of somatic cell nuclear transfer embryos in Holstein cows

Aberrant epigenetic nuclear reprogramming, especially imprinting pattern disorders, is one of the major causes of failure of clone development from somatic cell nuclear transfer (SCNT). Previous studies showed that ZFP57 is a key protein required for imprint maintenance after fertilization. In this study, we found that imprinting control regions in several imprinted genes were significantly hypomethylated in cloned embryos compared with in vitro fertilization embryos, indicating a loss of imprinted gene methylation. The ZFP57 expression was capable of maintaining the correct degree of methylation at several imprinting control regions and correcting abnormal hypomethylation. Moreover, we successfully obtained bovine fetal fibroblasts overexpressing ZFP57, which were used as donors for SCNT. Our results demonstrated that overexpression of ZFP57 increased total and trophectoderm cell numbers and the ratio of inner cell mass to total cells, reduced the apoptosis rate and significantly enhanced the development of SCNT blastocysts in vitro, ultimately achieving a degree of methylation similar to that in in vitro fertilization embryos. We concluded that overexpression of ZFP57 in donor cells provided an effective method for enhancing nuclear reprogramming and developmental potential in SCNT embryos. The ZFP57 protein played a key role in maintaining the methylation of imprinted genes during early embryonic development, which may be effective for enhanced SCNT in cattle.     

The 'GO' system--a novel method of microculture for in vitro development of mouse zygotes to the blastocyst stage

A novel system of in vitro culture termed the 'glass oviduct' or 'GO' culture system is described. Mouse zygotes were cultured in pairs to the blastocyst stage in open-ended 1 microl glass capillaries. 'GO' culture supported the development of significantly more hatching or hatched blastocysts than did a standard microdroplet (10 zygotes per 20 microl) control culture (48.3 versus 3.3%, respectively). 'GO' bslastocysts contained significantly larger populations of cells (92+/-3 versus 75+/-3), and inner cell mass (25+/-1 versus 21+/-1) and trophectoderm (68+/-2 versus 53+/-3) subpopulations, compared with microdroplet-derived blastocysts. Before blastulation, 'GO'-derived morulae were found to contain significantly more cells than microdroplet-derived morulae (27+/-0.7 versus 14+/-0.5). After implantation, 'GO' blastocysts formed fetuses at a similar rate to microdroplet-derived blastocysts (55 versus 62%), but at a lower rate than blastocysts derived in vivo (80%). 'GO'- and microdroplet-derived fetuses were similar in wet weight to each other (0.412 and 0.415 g, respectively) but were heavier than fetuses derived from flushed blastocysts (0.390 g). An additional experiment investigated whether the beneficial effect of 'GO' culture was due to the significantly increased embryo density. Proportions of hatching or hatched blastocysts after 'GO' culture (50%) were higher than after standard microdroplet culture (7.6%), but were not different from culture in high embryo density microdroplets (20 zygotes per 10 microl; 42%). 'GO' blastocysts contained more cells (79.6+/-2.1) than did standard microdroplet-derived blastocysts (68.7+/-2.0), but were similar to high density microdroplet-derived blastocysts (85.8+/-2.7). Similarly, 'GO' blastocysts contained more trophectoderm cells (62.2+/-2.0) than did standard microdroplet-derived blastocysts (52.7+/-1.7), but were similar to the high density microdroplet blastocysts (68.8+/-2.5). Numbers of inner cell mass cells ('GO', standard microdroplet and high density microdroplet culture) were not different from each other (17.4+/-0.5, 16+/-0.5 and 17+/-0.4, respectively). In conclusion, the 'GO' culture system represents an alternative method to the microdroplet system for small numbers of preimplantation embryos, without detriment to implantation potential.