Study Of Prolactin Regulating Pigeon Crop Epithelial Proliferation Through Hippo Pathway

During the period of crop milk formation,significant morphological changes occur in the crop of breeding pigeons,which specifically manifested by a significant increase in the relative weight,thickness of the crop,and the prolactin content in the serum of breeding pigeons.Some studies have shown that injection of a certain amount of prolactin in vitro can significantly increase the relative weight of pigeon crops.Therefore,exploring the mechanism of prolactin regulating crop proliferation in pigeons has certain effect on the understanding of crop milk formation and physiological mechanism of birds.Hippo pathway has been confirmed to play an important role in the development of various tissues and organs.However,whether it participates in the proliferation of pigeon crops has not been reported.In the present study,RT-PCR and Western Blot techniques were used to detected the expression of Hippo pathway related genes in the crop of pigeons at different breeding stages,and immunohistochemical techniques were used to localize the Hippo pathway effector protein YAP in crop cells;In addition,in vivo injection experiments were conducted to verify whether prolactin participates in crop tissue proliferation by regulating the Hippo pathway.The main research results are as follows:(1)The crop tissue of pigeons significantly proliferated on incubation day 17(117)and rearing day 1(R1).Through HE staining,it was observed that the "spike process" structure in the pigeon crop epithelium continued to grow and bifurcate,and it was filled with proliferating and about to shed epithelial cells.The thickness of the crop epithelium gradually increases with the progress of the breeding period,and recovers to the level of the rest period at the end of the feeding period.(2)The mRNA levels of related genes of the Hippo pathway in the crop tissue of male and female pigeons,including SAV1,STK3,LATS1,YAP,TEAD3,CTGF,SOX2,SNAI2,and C-Myc,were significantly upregulated compared to the non-proliferative phase(I4 and R25).Correlation analysis showed that there was a significant positive correlation between the gene expression of LATS1,STK3,TEAD3,C-Myc,SOX2 and the thickness of the crop epidermis in male pigeons(P<0.05).In females,a significant positive correlation was observed between crop epidermal thickness and STK3 gene expression(P<0.05).The results suggest that the Hippo pathway may be involved in crop tissue proliferation.(3)Compared with I4 and R25,the expression levels of LATS1,YAP,and CTGF proteins in the crop of both male and female pigeons were significantly increased at 117 and R1,and the level of pYAP were decreased significantly at 117.The enrichment of YAP in crop epithelium of male and female pigeons increased significantly at I17 and R1(P<0.05).It is further demonstrated that the Hippo pathway plays an important role in crop proliferation.(4)Inject 5μg prolactin into the pigeon crops(injected continuously for 6 days)can significantly promote crop epithelial proliferation,and induce a decrease in the phosphorylation level of the Hippo pathway effector molecule YAP protein.It significantly promotes the transcription and protein expression of Hippo pathway downstream factor CTGF.On this basis,injection of 1 mg(continuous injection for 6 days)of Hippo pathway inhibitor Verteporfin(VP)can inhibit prolactin induced crop proliferation,significantly inhibiting the transcription of YAP and the protein level of the downstream factor CTGF,but does not affect the total protein level of YAP;The EdU test results showed that the EdU positive rate of pigeon crop epithelial cells in the injection group of prolactin was significantly higher than that in the control group and the prolactin+VP group,indicating that prolactin may regulate the proliferation of pigeon crop epithelial cells through the Hippo pathway.In summary,the process of pigeon crop proliferation may be influenced by the Hippo pathway effector molecule YAP and transcription factors C-Myc,CTGF,and SOX2.PRL may participate in the formation of pigeon milk by inhibiting gene transcription of the upstream kinases LATS1 and SAV1 of the Hippo signaling pathway,promoting the expression of YAP and the downstream transcription factor CTGF,thereby promoting the proliferation of crop epithelial cells.