| The ion channel TRPV6is one member that belongs to the TRP super-family of cation channel, and distributed in calcium-transporting tissues such as intestine, kidney, placenta and bone of mammals, all of which are characterized by high Ca2+transport requirements. TRPV6is known as highly Ca+-selective channels, and serves as an important rate-limiting step in facilitating the entry of Ca2+into the epithelial cells. However, the presence of TRPV6in avian tissues is unclear. Therefore, the objective of the present study was to determine the distribution and expression patterns of TRP V6for different tissues in the laying hens, and investigate the role of TRPV6during the eggshell calcification. This information is important to fully understand the physiological role of this Ca2+channel in maintaining Ca2+homeostasis during eggshell mineralization in laying hens.Experiment I The mRNA expression of proteins involved in the Ca2+transcellular transport in the different intestinal segments and kidney in laying hensTen ISA laying hens at the peak stage (220days old) were decapitated for sampling. To obtain the encoding gene of gallus TRPV6, CaBP-D28K and PMCA lb from the different intestinal segments and kidney in laying hen, the RT-PCR method was carried out. Then these special DNA fragments were cloned into pMD18-T vector and the sequence was determined. In addition, TRPV6, CaBP-D28K and PMCA1b mRNA expression in the kidney and different intestinal segments were quantified by Real-time PCR. The results were as follows: standard RT-PCR with TRPV6, CaBP-D28K and PMCA1b primers yielded three single amplified fragments of the expected size of426bp,789bp and644bp from all intestinal segments and the kidney, respectively. Sequence analysis revealed a100%homology at the nucleotide level with the predicted mRNA sequence of Gallus Gallus TRPV6、CaBP-D28K and PMCA lb. Real-time PCR analysis demonstrated that, among these tissues examined, the highest level of TRPV6expression was in the duodenum, then jejunum (P<0.05), kidney (P<0.05) and ileum (P<0.01). The lowest level of expression was found in the cecum and rectum samples (P<0.01). Furthermore, the levels of CaBP-D28K and PMCA1b were high in duodenum, jejunum and ileum, but the lower levels of expression were showed in the rectal and renal samples compared with duodenum (P<0.05), respectively. In conclusion, three calcium-transporters involved in the Ca2+transcellular transport were expressed in the intestine and kidney in laying hens, suggestting that the upper intestine (duodenum and jejunum) is the main site of Ca2+absorption in the birds, and the expression of TRPV6supplys new basis to do research in mechanism of calcium transport in laying hen.Experiment Ⅱ Localization and expression of TRPV6in different intestinal segments and kidney in laying hensThe experimental animals were the same with the above one. Immunohistochemical analysis was used to investegate the TRPV6localization and expression in different intestinal segments and kidney in laying hens during peak laying. In addition, changes of concentration of TRPV6protein among these tissues were obtained by the western-blot analysis. The results are as follows:TRPV6was localized to the brush-border membranes of the duodenum, jejunum, ileum, cecum and rectum. Expression was weaker in the rectum and little or no expression was found in crypt and goblet cells. In the kidney, distinct immunopositive staining for TRPV6was detected at the apical domain of the proximal convoluted tubules (PCT), distal convoluted tubules (DCT) and medullary connecting tubules (CNT). Furthermore, western-blot analysis indicated that the duodenum expressed the greatest amount of TRPV6and the rectum the least, the other segments expressing intermediate levels. Moreover, the kidney expressed lower TRPV6protein levels compared to duodenum. In conclusion, the epithelial Ca2+channel TRPV6is strongly expressed in the apical cells of the entire intestine and the renal tubules suggesting that active Ca2+transcellular transport plays a crucial role in dietary calcium (re)absorption in laying hens.Experiment Ⅲ The expression of TRPV6in the different reproductive organs in laying hensThe experimental animals were the same with the above one. Immunohistochemical analysis and RT-PCR were used to investegate the TRPV6localization and expression in the ovarium, magnum, isthmus and uterus in laying hens. In addition, quantitation of mRNA level and protein concentration of TRPV6in these tissues were carried out by Real-time PCR and western-blot analysis, respectively. The results are as follows:positive expression of the TRPV6protein and the TRPV6mRNA was observed in the ovarium, magnum, isthmus and uterus in laying hens. TRPV6was localized to the granulose cells and theca cells and the oocytes in the ovarium. Furthermore, intense staining was observed in villus epithelial cell (face to lumen) in mucous epithelium of the magnum, isthmus and uterus, and the weaker staining was also found in the basilar layer of secretory cell of endometrium in the uterus. Moreover, the levels of TRPV6mRNA in the magnum and isthmus were significantly decreased compared with that in the ovarium (P<0.05). The expression of TRPV6in the magnum was also lower than that in the ovarium (P<0.05). However, the expression of TRPV6mRNA in the uterus was lower than that in the ovarium, but the change of protein concentration was in contrast, and the difference was not significent. In conclusion, the expression of TRPV6in these tissues implyed that the TRPV6is not only involved in the development and maturation, but also plays an important role in albumen secretion and eggshell calcification.Experiment IV Dynamic expression of TRPV6, CaBP-D28K and PMC A lb in the ESG during the oviposition cycle in laying hensForty ISA laying hens at the peak stage (220days old) were assigned to five reproductive stages for sampling. The samples of ESG were obtained by decapitated at0,2,4.5,8and16h after post-oviposition, respectively. Thus, the aim of this study is to investigate the effect of active Ca2+transcellular transport on the eggshell calcification. In addition, quantitation of mRNA level and protein concentration of TRPV6, CaBP-D28K and PMCA lb at different stages in the ESG were carried out by Real-time PCR and western-blot analysis, respectively. The results are as follows:the expression levels of TRPV6, CaBP-D28K and PMCA lb mRNA in the ESG were retained very low until the egg movement into the shell gland (0-4.5h after ovulation), then significantly increased at16h during eggshell calcification. In addition, TRPV6, and CaBP-D28K indicated significant statistical difference (P<0.01), respectively. Furthermore, western blotting showed that the expression of TRPV6and PMCA1b reached the maximum at16h after ovulation, but the statistical difference was not significant. The change of CaBP-D28K expression was very similar to that of TRPV6, but the concentration was significantly increased at16h than that at0h after ovulation (P<0.05). In conclusion, the expression of TRPV6, CaBP-D28K and PMCA lb in the ESG was regulated by the oviposition cycle, suggestting that active Ca2+transcellular transport exerted significant effects in calcium delivering in the ESG.Experiment V Dynamic changes of levels of plasma total Ca, P and calcium-regulated hormones during the oviposition cycleForty ISA laying hens at the peak stage (220days old) were assigned to five reproductive stages for sampling. Production records were kept before the study. Blood were obstained by decapitated at0,2,4.5,8and16h after post-oviposition, respectively. Thus, the aim of this study is to investigate the concentration changes of plasma total calcium, phosphorus, alkaline phosphatase (ALP), parathyroid hormone (PTH), calcitionin (CT) and osteocalcin (BGP) during the oviposition cycle. The results are as follows:there were not significant changes of total calcium and phosphorus after oviposition. The level of plasma CT was the highest at0h and the lowest at8h (P<0.05) after oviposition, the change of plasma PTH level was antithesis to that of plasma CT, which was constant slightly increase after oviposition, and reached the maximum at16h during the eggshell calcification (P<0.05). In addition, the level of plasma ALP activity was notablely increased at8h after oviposition (P<0.05). Moreover, the concentration of plasma BGP reached the maximum at4.5h after oviposition. In conclusion, the calcium-regulated hormones and biochemical indicators involved in the metabolism of calcium and phosphorus revealed the dynamic changes during the oviposition cycle. |
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