Transcriptome analysis of pigeon pituitary gland: expression changes of genes encoding protein and peptide hormones at different breeding stages.

Unlike other poultry, parent pigeons produce "pigeon milk" in their crops to nurture their squabs, which is mainly controlled by prolactin (PRL). Exception for PRL, the pituitary gland may also release various other peptide and protein hormones. However, whether these hormones change during pigeon crop lactation and their potential physiological functions remain unclear. Here, to identify potential peptide or protein hormone genes that regulate crop lactation, we conducted transcriptome analysis of pigeon pituitary glands at 3 different breeding stages (the ceased stage-nonincubation and non-nurturing stage, the 11th d of the incubation, and the 1st d of the nurturing stage) using RNA sequencing (RNA-Seq). Our analysis identified a total of 15,191 mRNAs and screened out 297 differentially expressed genes (DEG), including PRL, VIP, etc. The expression abundance of PRL mRNA on the 1st d of the nurturing stage was respectively 4.93 and 3.62 folds higher when compared to the ceased stage and the 11th d of the incubation stage. Additionally, the expression abundance of VIP is higher in the 1st d of the nurturing stage than in the ceased stage. Protein-protein interaction (PPI) network and Molecular Complex Detection (MCODE) analysis identified several vital DEGs (e.g., GHRHR, VIP, etc.), being closely linked with hormone and enriched in neuropeptide signaling pathway and response to the hormone. Expression pattern analysis revealed that these DEGs exhibited 4 distinct expression patterns (profile 10, 16, 18, 19). Genes in profile 10 and 19 presented a trend with the highest expression level on 1st d of the nurturing stage, and functional enrichment analysis indicated that these genes are involved in neuropeptide hormone activity, receptor-ligand activity, and the extracellular matrix, etc. Taken together, being consistent with PRL, some genes encoding peptide and protein hormones (e.g., VIP) presented differentially expressed in different breeding stages. It suggests that these hormones may be involved in regulation of the crop lactation process or corresponding behavior in domestic pigeons. The results of this study help to gain new insights into the role of pituitary gland in regulating pigeon lactation.

Transcriptome analysis revealed the roles of long non-coding RNA and mRNA in the bursa of Fabricius during pigeon (Columba livia) development.

The bursa of Fabricius (BF) is the critical humoral immune organ to birds, playing an essential role in B lymphocyte differentiation. However, unlike other poultries, surgical removal of pigeon BF did not limit humoral immune responsiveness. To investigate the expression profiles and the potential role of mRNA and long non-coding RNA (LncRNA) in squab BFs, transcriptome analysis was performed by RNA-Sequencing (RNA-Seq) over three developmental stages (1-day, 13 and 26 days old). We identified 13,072 mRNAs and 19,129 lncRNAs, of which 2,752 mRNAs and 1,515 lncRNAs were differential expressed (DE) in pigeon BFs over three developmental stages. Cluster analysis presented different expression patterns in DE mRNAs and lncRNAs. Functional enrichment analysis revealed that DE lncRNAs and mRNAs with distinct expression patterns might play crucial roles in the immune system process and tissue morphogenesis. In particular, some DE genes and lncRNAs with higher expression levels in 13D or 26D are related to lymphocyte activation and differentiation, adaptive immune response, positive regulation of immune response, leukocyte migration, etc. Protein-protein interaction (PPI) network and Molecular Complex Detection (MCODE) analysis sreened six significant modules containing 37 genes from immune-related DE gene cluster, which is closely linked in B cell activation, lymphocyte differentiation, B cell receptor signaling pathway, etc. Our study characterizes mRNA and lncRNA transcriptomic variability in pigeon BFs over different developmental stages and enhances understanding of the mechanisms underlying physiological functions of pigeon BF.

[Scanning electron microscopic observation of erythrocytes and endothelial cells of electrified death rabbits].

OBJECTIVE: To identify electrified death of animals without electric marks. METHODS: Two animal models (model A and model B) of electrified death without electric marks were established. Model A: right anterior limb and left hind limb of rabbits were soddened by 0.1 mol/L PBS, and two pieces of wet gause which were soaked in the 0.1 mol/L PBS were winded on the right anterior limb and left hind limb of them, respectively, and then two nake wires were winded outside of the gause; then, the two wires were connected to the alternating current of 220 voltage (A1 group) or 110 voltage (AZ group) electrified. Model B: two nake wires were fixed at the bilaterals of an oblong container, then a small quantities water were infunded into it, about 1.5 cm deep, and then, one rabbit was put in each time; finally, the two wires were connected to the alternating current of 220 voltage (B1 group) or 110 voltage (B2 group) electrified. The erythrocytes and endothelium of aorta and pulmonary artery of the animals were observed under scanning electron microscopes. RESULTS: Pores were found on the surface of the erythrocytes and endothelial cells of aorta and pulmonary artery of the electrified animals, which was absent in the control animals. The erythrocytes had the greatest perforated cell index among the three cells, followed by the pulmonary endothelium. CONCLUSION: The perforation phenomena can serve as an evidence of electrified death when no electric mark is observable.

[Postmortem changes of liver phosphofructokinase-2 level in rats following different causes of death].

OBJECTIVE: To study the changes of liver phosphofructokinase-2 (PFK-2) level at different postmortem intervals as well as due to different causes of death. METHODS: One hundred and five rats were randomly divided into 3 groups and the rats were sacrificed by either bleeding, suffocating, and neck breaking, respectively. The content of liver PFK-2 at 0, 1, 2, 4, 8, 12 and 24 hours following death were studied using immunohistochemishtry and image analysis. RESULTS: PFK-2 content of the rat's liver in all 3 groups showed a linear decrease at different postmortem intervals with no significant statistical differences found between the different groups. CONCLUSION: PFK-2 level in rat liver appears to decrease linearly in correlation with prolonged PMI.

[A study on expression of caspase-8 in organs of rats after electrical injury at antemortem or postmortem].

OBJECTIVE: This study was conducted to detect the expression of caspase-8 in organs of rats after The electrical injury so as to elucidate whether caspase-8 is useful in identifying electrical lesion. METHODS: experiment included two parts. In the first part (the antemortem electrical injury part), thirty-five healthy male SD rats were randomly divided into seven groups (n = 5 per group), i. e. the group of rats subjected to instantaneous electrothanasia; the groups of rats subjected to cervical dislocation at 1 h, 2 h, 4 h, 8 h after electrical injury; the sham group and the normal control group. In the second part (the postmortem electrical injury part), twenty-five healthy male SD rats wererandomly divided into five groups (n=5 per group), i. e. the groups of rats electrically injured just after death, and at 15 min, 30 min, 60 min after death; and the postmortem sham group. All experimental rats were given respectively an electric shock with two metal clamps that were connected with two poles of 220 V alternating current by clamping the rats' left hind limbs and right forelimbs. The rats of sham group after death were clamped but not electrified. The brain, lung, heart, liver, spleen, kidney, the muscle of electrified limb, the cutis of electrified limb of all experimental rats and those organs of control groups were dissected to detect the expression of caspase-8 by immunohistochemistry staining, and the staining intensities were assessed by image analysis system. RESULTS: In the antemortem electrical injury groups, the expression of caspase-8 was positive in brain, heart, liver and kidney; the strongest staining intensity appeard at 4 h after electrical injury and decreased at 8 h after electrical injury. In the group of rats electrically injuryed just after death, the expression of caspase-8 was faint, and the expression of caspase-8 in spleen, lung, muscle and cutis was negative in the other groups. The expression of caspase-8 in all detected organs was negative in the other rats that were electrified after death. CONCLUSION: Caspase-8 can be regarded as an index in identifying electrical injury and distinguishing between antemortem and postmortem electrical injuryies.

[Expression of caspase-8 after brain injury from fluid percussion in rats].

OBJECTIVE: To provide the evidence of the relationship between brain injury and the time of injury. METHODS: Rats were contused on brain by fluid percussion, then were killed after injury for 15 min, 30 min, 1,3,6,12 h, and 1,4,7,14 d respectively. The expression of caspase-8 were detected by immunohistochemical technology on rat brain section and the results were assessed by image analysis system in the cerebral cortex, thalamus, and hippocampus. RESULTS: The expression of caspase-8 in cortex and hippocampus could be detected in 30 min after injury, increased significantly in 3h, reached apex in 1d after injury, remained 4d before decreased. In addition, the expression of caspase-8 can be detected in 1h after injury and reached apex in 1d after injury, and remained 4d then reduced. CONCLUSION: It seems that the expression of caspase-8 should be a useful target for diagnosis of early brain injury.

[Expression of c-fos protein after muscle contusion in experimental rats].

OBJECTIVE: To study the relationship between the expression of c-Fos protooncogene and skeleton muscle contusion of rats, and to search for a sensitive marker of timing for skeleton muscle contusion. METHODS: Fifty Sprague-Dawley rats were randomly distributed into control group and experimental groups. The expression of c-fos was microscopically observed by immunohistochemical method. RESULTS: With the time prolonged the c-fos positive intensity and area were increased. Positive expression of c-fos protein appeared at 15 min after skeleton muscle contusion, and reached to the peak at 1h after skeleton muscle contusion, then decreased gradually and returned to the normal level on 1d after skeleton muscle contusion. CONCLUSION: The detection of the expression of c-fos protein could be a sensitive marker for timing skeleton muscle contusion.

[The expression of Fas after fluid percussion brain injury in rats].

OBJECTIVE: To inquire into the pathologic diagnosis and the dating of injuries of light closed encephalon injury. METHODS: Wistar rats were hurt by fluid percussion, and were killed respectively at 15 min, 30 min, 1 h, 3 h, 6 h, 12 h, 1 d, 4 d, 7 d, and 14 d after injury. The expression of Fas in the cerebral cortex, thalamus, and hippocampi was detected by immunohistochemistry and the results were assessed by image analysis system. RESULTS: The results showed that the expression of Fas could be detected in thirty minutes after injury, and then it increased significantly in three hours, reached apex in twelve hours after injury, decreased gradually in four days after injury, and returned to normal in 14 days after injury. CONCLUSION: This research demonstrated that Fas-mediated apoptosis appeared not only around brain trauma but also in the brain tissue far away from the traumatic area. It indicted that Fas expression is a useful target for diagnosis of early brain injury and the regularity of Fas expression could be used as one of indications in deducing the time of brain injury.

[Changes of connexin 43 in rabbit with early myocardial ischemia].

OBJECTIVE: To reveal gap junction connexin 43 (Cx43) in rabbit with early myocardial ischemia. METHODS: The left coronary arteries were ligated to create early myocardial ischemia. Cx43 was visualized by use of SP immunohistochemical method (IHC) and Colloidal gold of the electron microscopy, and a comparison was made between the experiment group and the control group. The semi-quantitation assessment was performed. RESULTS: The positive areas of Cx43 gap junctions by IHC and Colloidal gold particles of the electron microscopy were found decreased by about 40% at 30 minutes of acute myocardial ischemia. The distributions were not obviously modified. CONCLUSION: The decrease of Cx43 is characterized by the time during myocardial ischemia, and the early stage of acute myocardial ischemia in rat is of positive significance.

[A study on expression of hsp70 mRNA in the early stage of diffuse brain injury in rats].

OBJECTIVE: To evaluate the changes of hsp70 mRNA in neuron and glia of rat suffering early diffuse brain injury. METHODS: SD rats were used in the replication of the animal pattern of diffuse brain injury. In situ hybridization technique and image analysis technique were applied in detecting the hsp70 mRNA in the rat's cerebral cortex, thalamus and brain stem. RESULTS: The signal intensity of hsp70 mRNA could be detected in 5 minutes after brain injury; subsequently, it attained to the summit at 6 hours and declined at 12 hours after brain injury. CONCLUSION: The above data suggest that hsp70 mRNA is useful for the diagnosis of early diffuse brain injury and for distinguishing the postmortem injury from antemortem injury.

[A study of the expression of Fas after fluid percussion brain injury in rats].

OBJECTIVE: To study the pathologic diagnosis and the injury time estimation in light closed encephalon injury. METHODS: Mice were hurt by fluid percussion, and were killed at 15, 30 min, 1, 3 , 6, 12 h, 1, 4, 7, 14 d respectively after injury. The expression of Fas-L in the cerebral cortex, thalamus, and hippocampi was detected by immunohistochemistry and the results were assessed by image analysis system. RESULTS: It is showed that the expression of Fas-L could be detected in 1 h after injury, and increased significantly in three hours, and it reached apex 12 h after injury, and decreased gradually four days after injury, and returned normal 14 days after injury. CONCLUSION: This research demonstrated that Fas-L mediated apoptosis appeared not only around brain trauma but also in the brain tissue far away from the traumatic area. It indicted that the expression of Fas-L is a useful target for diagnosis of early brain injury; the regularity of Fas-L expression could be used as one of indication to date the time of brain injury.