Molecular and Signal Signatures in the Dormant Avian Embryo

Abstract

In most of the avian species, the early embryo suspends development after oviposition because of the low ambient temperature, which does not meet the thermal requirements for development. A periodic developmental layoff in the embryo is known as cold torpor, and it enables to reduce energy expenditure during periods of stressful events such as cold exposure. Immediately after oviposition, the early avian embryo undergoes cold torpor before the major development occurs, but it is able to endure some period of cold exposure. At the cellular level, it is known that shortly after exposure to thermal stress, various protective or destructive molecular signals are activated. Some examples of the representative molecular changes that occur under stressful conditions are unfolded protein response (UPR) triggered by endoplasmic reticulum (ER) stress and stress-activated protein kinase (SAPK) signaling. Multiple cellular process, such as apoptosis or cell cycle arrest occur in response to the activation of the ER stress or SAPK signaling initiation, thus promoting the cell death. In spite of the understanding of stress activated molecular mechanisms in various types of cells, the exact molecular signatures occurring in the avian embryo during cold torpor are not clearly elucidated to date. Here we investigated the molecular signaling signatures that occur in the dormant avian embryo, especially the mechanisms related to stress response and energy conservation. To investigate the level of ER stress and SAPK signaling, and its effect on the cellular integrity in the post-ovipositional avian embryo, molecular analysis was conducted using the avian embryos that were ovipositioned and cooled at 16°C during several time periods. First of all the internal egg temperature was measured to observe the timing of cold stress initiation in the blastoderm. This indicated that hypothermia occur with in 4 hours post-oviposition, which is caused by at least 20°C decline in the egg internal temperature, and enough to initiate stress-activated molecular signals. To conduct studies on the degree of ER stress and SAPK signaling during avian embryo dormancy, the mRNA expression pattern was screened in various time points after oviposition. As a result, the mRNA of ER stress, SAPK signaling and apoptosis related genes were gradually up-regulated during the post-ovipositional periods, especially the gene expression was significantly up-regulated after 7 days of storage, indicating the strong activation of ER stress and SAPK signaling related genes. Furthermore, to investigate the degree of protein expression change of ER stress and SAPK signaling related molecules, western blot analysis was conducted with proteins obtained from blastoderm that were storaged at 16°C for several time points post-oviposition. Western blot results indicated that SAPK signal phosphorylation, especially JNK and p38 signaling pathway, was stronger as the duration of cold torpor increases in the avian embryo, and is strongest at 7 days of storage. Similarly, the ER stress effector protein IRE-1 phosphorylation increased during the cold torpor period. Identical analysis was performed with duck blastodermal proteins, and it has shown identical results as chicken. To investigate the cellular process that occur in response to the activation of ER stress and SAPK signaling, the degree of cell cycle progression was measured by gene expression profiling and propidium iodide (PI) staining. The results indicated that shortly after oviposition, genes related to the progression of cell cycle were significantly down regulated, especially at 1 day of storage. Additionally, the distribution of cells throughout the cell cycle phases were investigated by PI staining, accordingly the results indicated that shortly after oviposition, most of the blastodermal cells are in the G0/G1 phase. However, the cell cycle was arrested at the G2/M phase at 7 days of storage, which shows that the blastodermal cells suspend the progression of cell cycle during cold torpor. Accordingly, the mRNA expression pattern of the purine/pyrimidine metabolism related genes were investigated to observe the extent of nucleotide metabolism in the dormant embryo, and the results indicated that the expression of nucleotide metabolism related genes are down-regulated at 7 days of storage. Another cellular process that occurs in response to stress is apoptosis, which is a form of programmed cell death induced by various cellular signals as ER stress and SAPK signaling. AnnexinV-PI staining was conducted to investigate the progression of apoptotic cell death in the blastoderm and measure proportion of apoptotic cells. The results shown that early apoptotic cells rise after 7 day of storage at 16°C, while late apoptotic cells does not. Nonetheless, after incubation of the embryos at 37.5°C for 4 hours, the early and late apoptotic cells were cleared and amount of live cells increased. The TUNEL assay results show that there is little damage to the DNA during storage, indicating that the early apoptotic cells in the blastoderm does not affect DNA integrity. In conclusion, after oviposition, various stress-related molecular signals are active, which affects the molecular status of the blastoderm by arresting the cell cycle and inducing early apoptosis. However, the avian embryo is able to prohibit the progression of apoptosis to late phase, and minimize damage to genetic information. This result suggests that the avian embryo is able to actively protect itself from external stress during the dormant period.